Composition for promoting health of heart and cerebral vessels as well as preparation method and application of composition

Abstract

The invention belongs to the field of preparation of natural products related to health, and particularly discloses a composition for promoting health of heart and cerebral vessels as well as a preparation method and application of the composition. The composition is prepared from the following raw materials in parts by mass: 10 to 30 parts of earthworm protein, 5 to 20 parts of milk tripeptide, 1 to 10 parts of rice bran fatty alkanol, 5 to 25 parts of aronia melanocarpa extract, 3 to 15 parts of mulberry leaf extract and 1 to 8 parts of micro-embedded lactobacillus plantarum, the composition is prepared into an enteric-coated preparation by a split-phase granulation process: an earthworm protein / lactotripeptide / alkanol coating is an enteric-coated pellet I, an aronia melanocarpa / mulberry leaf extract coating is a sustained-release pellet II, and the enteric-coated pellet I and the sustained-release pellet II are mixed with probiotic pellets and filled into enteric-coated capsules. The system releases protein peptides in duodenum, releases plant extracts slowly in jejunum and releases probiotics in colon, so that the synergistic effects of reducing blood sugar and regulating lipid, resisting atherosclerosis and improving diabetic vasculopathy are remarkably improved.

Description

Technical Field

[0001] This invention belongs to the field of health-related natural product preparation, and specifically discloses a composition that promotes cardiovascular health, its preparation method and application. Background Technology

[0002] Earthworm protein mainly includes plasmin, collagenase, and plasminogen activator. These enzymes in earthworm protein can effectively break down fibrin, thereby dissolving thrombi, improving blood circulation, reducing blood viscosity, and acting as an anticoagulant. The plasmin in earthworm protein acts like "scissors," directly cutting the network structure of thrombi and breaking them down into soluble fragments; it can also activate the body's own fibrinolytic system, converting plasminogen into active plasmin, thus enhancing the body's own thrombolytic ability. By dissolving microthrombi and reducing blood viscosity, it can improve microcirculation, increase capillary blood flow, and inhibit excessive platelet aggregation, preventing the formation of new thrombi and exhibiting a certain anticoagulant effect.

[0003] Currently, the prevention and treatment of cardiovascular and cerebrovascular diseases urgently require a multi-active ingredient synergistic delivery system with better efficacy. However, existing compositions face multiple technical bottlenecks: First, there is a contradiction between gastric acid inactivation and release. Protein components (such as earthworm protein) and probiotics are easily inactivated in gastric acid. While conventional enteric protection reduces gastric degradation, it also reduces the release efficiency of probiotics in the lower intestinal tract. Furthermore, the commonly used protective materials have insufficient stability in gastric juice, affecting the survival of live bacteria. Second, there is a conflict in the release kinetics of multiple components. Some lipid-soluble components need to be rapidly released in the upper intestinal tract to achieve acute metabolic regulation, but other components (such as certain plant-based components)... High-concentration release of probiotic extracts in the upper intestine may cause gastrointestinal discomfort, and existing sustained-release technologies struggle to balance the need for rapid onset of action with reduced irritation. Thirdly, the physicochemical properties of the components are mutually exclusive; hydrophilic and hydrophobic components easily separate when mixed; the coexistence of acidic extracts and probiotics affects bacterial stability; and heat-sensitive components and components requiring high-temperature processing are difficult to reconcile during formulation. Fourthly, traditional dosage forms suffer from spatial and temporal control deficiencies. Ordinary enteric-coated formulations release all components instantaneously in the duodenum, potentially leading to probiotic inactivation due to exposure to bile salts and competition for targets among different active ingredients, reducing bioavailability. Current improvement methods also have limitations, such as multi-layered encapsulation structures for probiotics improving gastric acid tolerance but insufficient colonic localization efficiency; segmented-release formulations where process stress damages the active structure; and liposome carriers exhibiting biodistribution guidance issues. To improve efficacy and bioavailability, these problems urgently need to be addressed. Summary of the Invention

[0004] To address the aforementioned problems in the prior art, this invention discloses a composition for promoting cardiovascular and cerebrovascular health, its preparation method, and its application.

[0005] The present invention includes the following technical solutions.

[0006] A composition for promoting cardiovascular health, comprising the following raw materials in parts by weight:

[0007] Earthworm protein 10-30 parts, lactotripeptide 5-20 parts, rice bran fatty alkyl alcohol 1-10 parts, aronia berry extract 5-25 parts, mulberry leaf extract 3-15 parts, microencapsulated Lactobacillus plantarum 1-8 parts.

[0008] The micro-embedded Lactobacillus plantarum was prepared using the following process:

[0009] (a) Freeze-drying protective layer: The fermentation broth of Lactobacillus plantarum is mixed with a freeze-drying protectant containing trehalose (15-30% w / v) and β-cyclodextrin (5-15% w / v), pre-frozen to below -40°C and then freeze-dried under vacuum;

[0010] (b) pH-responsive coating: Eudragit® FS 30D is fluidized bed coated onto the surface of the product in step (a), resulting in a weight gain of 10-20%;

[0011] (c) Enteric coating: Hydroxypropyl methylcellulose phthalate (HPMCP) ethanol solution is sprayed onto the surface of the product in step (b) for a second time, resulting in a coating weight gain of 5-10%;

[0012] (d) Bioadhesion layer: A chitosan quaternary ammonium salt (0.5-2% w / v) solution is sprayed onto the surface of the product from step (c) to obtain the micro-embedded *Lactobacillus plantarum*, with a viable count ≥ 5 × 10⁻⁶. 11 CFU / g.

[0013] Furthermore, in the above-mentioned composition for promoting cardiovascular health, the aralia elata extract contains 30-40% anthocyanins, the rice bran fatty alkanol contains ≥80% octacosanol, and the mulberry leaf extract contains 2.5-4.0% DNJ.

[0014] Furthermore, in the above-mentioned composition for promoting cardiovascular health, the microencapsulated *Lactobacillus plantarum* has a multi-layer coating structure, the multi-layer coating structure comprising:

[0015] (a) The inner layer is an enteric isolation layer containing enteric material dissolved at pH ≥ 5.5;

[0016] (b) The outer layer is a pH-responsive layer containing colon-targeting material dissolved at pH ≥ 7.0;

[0017] The thickness and / or composition of the multilayer coating structure are configured such that the viable bacterial release rate of the composition is ≤0.1% in simulated gastric fluid (pH 1.2) for 2 hours and ≥95% in simulated colonic fluid (pH 7.4) for 1 hour.

[0018] The release rate of live bacteria in simulated gastric fluid was ≤0.1% after 2 hours, and the release rate of live bacteria in simulated colonic fluid was ≥95% after 1 hour.

[0019] This invention also discloses the use of the above composition in the preparation of drugs for preventing atherosclerosis, which works through the following synergistic mechanism:

[0020] Earthworm protein and mulberry leaf extract inhibit α-glucosidase;

[0021] Rice bran fatty alkyl alcohols and Lactobacillus plantarum metabolites reduce serum LDL-C;

[0022] Anthocyanins from aging berries eliminate free radicals in blood vessels.

[0023] The present invention also discloses a method for preparing enteric-coated formulations using the above composition as the active ingredient, comprising the following steps:

[0024] (1) Earthworm protein, milk tripeptide and rice bran fatty alkyl alcohol were mixed and spray-dried, and then coated with HPMCP to obtain enteric microcapsules I with an average particle size of 200-400 μm.

[0025] (2) The extracts of aronia and mulberry leaves were ground together and coated with Eudragit® L100-55 to prepare sustained-release microspheres II with an average particle size of 400-600 μm.

[0026] (3) Mix microcapsules I and microcapsules II with micro-encapsulated Lactobacillus plantarum;

[0027] (4) Fill the mixture from step (3) into the enteric capsule shell or coat it with enteric coating after compression.

[0028] This invention discloses an enteric-coated formulation obtained by the above preparation method, wherein the coating layer thickness of the enteric-coated microcapsules I is 80-120 μm, and the coating layer thickness of the sustained-release microcapsules II is 20-50 μm.

[0029] Furthermore, the above-mentioned enteric-coated formulation comprises:

[0030] (a) Microparticle I: having an enteric coating layer, said enteric coating layer containing enteric material dissolved at pH ≥ 5.0–6.0;

[0031] (b) Microparticle II: having a colon-targeting coating layer, said colon-targeting coating layer containing colon-targeting material dissolved at pH ≥ 7.0;

[0032] The thickness, composition, or ratio of the enteric coating layer and the colon-targeting coating layer are configured as follows:

[0033] In simulated gastric fluid (pH 1.2) for 2 hours, the total release rate of all microspheres was ≤5%;

[0034] In simulated intestinal fluid (pH 6.8), the release rate of microparticle I was ≥90% after 30 minutes;

[0035] In simulated colonic fluid (pH 7.4), the release rate of microgranules II was ≥85% after 2 hours. Furthermore, the above-mentioned enteric-coated formulation exhibits a segmented release function in the human body, specifically as follows:

[0036] 1) Duodenal segment: The enteric-coated microspheres dissolve, releasing earthworm protein and lactotripeptide;

[0037] 2) Jejunum-ileum segment: The sustained-release microcapsule coating gradually dissolves, releasing aronia berry and mulberry leaf extracts;

[0038] 3) Colon segment: The pH-responsive layer of micro-embedded Lactobacillus plantarum dissolves, releasing live bacteria.

[0039] This invention discloses the application of the above-mentioned enteric-coated preparation in the preparation of pharmaceuticals or functional foods.

[0040] Furthermore, in the above applications, the target population for the functional foods is diabetic vascular disease patients.

[0041] Compared with the prior art, the present invention has the following outstanding advantages:

[0042] This invention provides a composition for promoting cardiovascular health, its preparation method, and its application. The composition comprises earthworm protein, lactotripeptide, rice bran fatty alkyl alcohol, aronia berry extract, mulberry leaf extract, and microencapsulated *Lactobacillus plantarum*. A unique four-layer encapsulation process (lyophilized protective layer / pH-responsive layer / enteric coating layer / bioadhesion layer) achieves zero-leakage protection of probiotics from gastric acid and targeted release into the colon. The formulation employs a phase-separation design: protein peptides are formulated into enteric-coated microspheres, plant extracts into sustained-release microspheres, which, together with the probiotic microspheres, fill enteric-coated capsules, achieving a three-stage precise release (duodenal release of protein peptides, jejunal sustained-release of plant extracts, and colonic activation of probiotics). This approach overcomes the contradictions in multi-component delivery, significantly enhancing the synergistic effects of lowering blood sugar and lipids, anti-inflammation, and vascular protection. Attached Figure Description

[0043] Figure 1 Test Example 2: Comparison of blood glucose reduction in the synergistic effect of lowering blood glucose and regulating lipids;

[0044] Figure 2 Test Example 2: Comparison of LDL-C reduction (%) in the synergistic effect of blood sugar and lipid regulation;

[0045] Figure 3 Test Example 2: Comparison of FMD increase (%) in the synergistic effect of blood sugar lowering and lipid regulation. Detailed Implementation

[0046] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0047] The raw materials are shown in Table 1. All raw materials are food-grade.

[0048] Table 1 Raw Material Table

[0049] Raw material name Key parameter requirements source Testing standards and methods Earthworm protein Solubility ≥ 98% Nanjing Letao Biotechnology Co., Ltd. (Earthworm Protein Concentrate Powder) SDS-PAGE electrophoresis (Coomassie brilliant blue staining) Milk tripeptide Purity ≥ 95%, sequence Ile-Pro-Pro Solid-phase synthesis method (commercially available products conforming to USP42-NF37 standards) HPLC (C18 column, mobile phase 0.1% TFA-acetonitrile) Rice bran fatty alkyl alcohols Octadecanol content ≥80%, melting point 80-82℃ Rice bran wax saponification crystals (commercially available, conforming to JIS K3341:2020) GC-MS (HP-5 capillary column) Aging Berry Extract Anthocyanins ≥30%, ORAC ≥16000 μmol TE / g Waterlese fully water-soluble argan strawberry extract, food grade, 80 mesh, 25kg pack pH differential method (510 / 700 nm) Mulberry leaf extract DNJ (1-deoxynodazole) content: 2.5-4.0% Shanyang Lianfeng Biotechnology Co., Ltd. (Food-grade Mulberry Leaf Extract) HPLC-ELSD (XBridge Amide column) Lactobacillus plantarum <![CDATA[The viable count ≥ 5×10 11 CFU / mL]]> Accession number: CGMCC 1.557 (China General Microbiological Culture Collection Center) GB 4789.35-2016 (MRS agar culture) Microencapsulation excipients Chitosan quaternary ammonium salt deacetylation degree ≥85% Commercially available products that meet EP 10.0 standards Titration method (GB / T 21840-2008) Example 1

[0050] 1. Preparation of the composition

[0051] Formula (parts by weight):

[0052] 10 portions of earthworm protein

[0053] 5 portions of lactotripeptide

[0054] 1 part of rice bran fatty alkyl alcohol

[0055] 5 parts of aronia berry extract

[0056] 3 portions of mulberry leaf extract

[0057] One microencapsulated sample of Lactobacillus plantarum (5.3 × 10⁻⁶ viable count) 11 CFU / g)

[0058] 2. Preparation of microencapsulated Lactobacillus plantarum:

[0059] 1) Freeze-drying protective layer:

[0060] Take 500 mL of Lactobacillus plantarum CGMCC 1.557 fermentation broth.

[0061] Add 200 mL of lyophilization protectant (0.1 M phosphate buffer, pH 7.0 containing 25% w / v trehalose and 10% w / v β-cyclodextrin).

[0062] After mixing, pre-freeze to -45°C (cooling rate 1°C / min) and maintain for 2 hours.

[0063] Vacuum freeze drying: -45℃→25℃ gradient temperature increase (24 hours), vacuum degree ≤10 Pa

[0064] Porous freeze-dried bacterial powder (moisture content ≤3%, viable bacteria retention rate 98%)

[0065] 2) pH-responsive coating (fluidized bed Wurster method):

[0066] Equipment parameters: Inlet air temperature 35℃, material temperature ≤30℃, atomization pressure 0.8 bar

[0067] Coating solution: Eudragit® FS 30D aqueous dispersion (20% solids content)

[0068] Spraying rate: 8 mL / min, coating weight gain 15% (theoretical weight gain calculation: initial powder 100g → weight gain 115g)

[0069] 3) Enteric coating:

[0070] Coating solution: 10% w / v ethanol solution of HPMCP (HP-55) (containing 1% w / v polyethylene glycol 400 plasticizer)

[0071] Spraying rate: 6 mL / min, inlet air temperature: 40℃, coating weight gain: 8%.

[0072] 4) Bioadhesion layer:

[0073] Coating solution: 0.1M acetic acid solution (pH 5.5) of 1.5% w / v chitosan quaternary ammonium salt (HACC).

[0074] Spray drying coating: inlet temperature 100℃, outlet temperature 45℃, coating weight gain 2%.

[0075] 3. Preparation of enteric-coated formulations:

[0076] 1) Enteric-coated microcapsules I (protein / peptide):

[0077] A mixture of earthworm protein, milk tripeptide, and rice bran fatty alkyl alcohols, with 20% microcrystalline cellulose binder added.

[0078] Fluidized bed granulation: Inlet air temperature 60℃, spray rate 15 mL / min, yielding microspheres with a particle size of 300 μm.

[0079] HPMCP coating: 15% w / v ethanol solution, coating weight gain of 10% (coating thickness 90μm).

[0080] 2) Sustained-release microcapsules II (extract):

[0081] Aronia berry extract + mulberry leaf extract + 50% MCC co-ground (ball mill, 300 rpm × 30 min)

[0082] Extrusion and spheronization: Extrusion speed 30 Hz, spheronization speed 800 rpm, yielding microspheres with a particle size of 500 μm.

[0083] Eudragit® L100-55 coating: 10% w / v acetone solution, coating weight gain 5% (coating thickness 35μm).

[0084] 3) Final preparation:

[0085] Mixed microcapsules I (100g), microcapsules II (100g), and microencapsulated Lactobacillus plantarum (25g).

[0086] Filled enteric-coated capsule shells (DRcaps®, size 0). Example 2

[0087] Optimize the ratio

[0088] 1. Formula:

[0089] 20 servings of earthworm protein

[0090] 12 servings of lactotripeptide

[0091] 5 parts of rice bran fatty alkyl alcohols

[0092] 15 parts of aronia berry extract

[0093] 8 parts of mulberry leaf extract

[0094] Four samples of microencapsulated Lactobacillus plantarum

[0095] 2. Process improvements (compared to Example 1):

[0096] Freeze-dried probiotics: Protectant contains 30% trehalose + 5% glutathione (to improve heat stability).

[0097] pH-responsive layer: Eudragit® FS 30D coating with an 18% weight gain (coating thickness 45μm)

[0098] Microparticle I coating: HPMCP + 5% triethyl citrate plasticizer, coating thickness 110μm

[0099] Microparticle II: Eudragit® L100-55 with 10% nano silica added (to prevent sticking). Example 3

[0100] High-dose system

[0101] 1. Formula:

[0102] 30 servings of earthworm protein

[0103] 20 servings of lactotripeptide

[0104] 10 parts of rice bran fatty alkyl alcohols

[0105] 25 parts of aronia berry extract

[0106] 15 parts of mulberry leaf extract

[0107] 8 samples of microencapsulated Lactobacillus plantarum

[0108] 2. Process improvements (compared to Example 1):

[0109] Probiotic enteric coating: HPMCP coating increased weight by 12% (coating thickness 60μm)

[0110] Microparticle I particle size: 400 μm (450 μm after coating)

[0111] Microparticle II: Eudragit® coating increases weight by 8% (coating thickness 50μm).

[0112] Comparative Example 1

[0113] Missing segment release

[0114] The formula is the same as in Example 2.

[0115] Steps to cancel coating:

[0116] All raw materials are pulverized through a 200-mesh sieve → directly mixed → filled into ordinary enteric-coated capsules (without micro-pellet structure).

[0117] Comparative Example 2

[0118] Unencapsulated probiotics

[0119] The formula is the same as in Example 2.

[0120] Simplified process:

[0121] Unencapsulated freeze-dried bacterial powder (with the same number of live bacteria) was directly mixed with microcapsules.

[0122] Comparative Example 3

[0123] Single-layer enteric coating

[0124] The formula is the same as in Example 2.

[0125] Simplified Probiotic Coating:

[0126] It uses only HPMCP single-layer coating (20% weight gain), without pH-responsive layer and adhesion layer.

[0127] Comparative Example 4

[0128] Component missing

[0129] formula:

[0130] Earthworm protein 20 parts + milk tripeptide 12 parts + rice bran fatty alkyl alcohol 5 parts

[0131] Removed ingredients: ageberry, mulberry leaf extract, and probiotics.

[0132] Formulation process: After mixing the raw materials, HPMCP coating is used to prepare enteric-coated microcapsules. Comparative Example 5

[0133] Mixing and grinding

[0134] The formula is the same as in Example 2.

[0135] Process:

[0136] All raw materials (including unencapsulated probiotics) are mixed and pulverized in a ball mill (400 rpm × 1 h) → tableting → HPMCP film coating (15% weight gain).

[0137] For ease of demonstration, the process parameters of the embodiments and comparative examples 1-5 are compared in Table 2.

[0138] Table 2 Comparison of Process Parameters

[0139] step Example parameters Comparative deviation freeze-dried probiotics Pre-freeze at -45℃ / gradient drying Comparative Example 2: Direct use without freeze-drying pH-responsive coating Eudragit® FS 30D for weight gain of 15-18% Comparative Example 3: This layer is missing Enteric-coated microsphere particle size Microparticle I 300-400μm / Microparticle II 500-600μm Comparative Example 1: No particle size differentiation Extract grinding Ball milling 300 rpm × 30 min (to prevent thermal degradation) Comparative Example 5: 400 rpm × 60 min (high temperature) Coating plasticizer Add 1-5% triethyl citrate to HPMCP Comparative Example 3 / 5: No plasticizer added

[0140] Note: Detection method:

[0141] Viable bacteria count: GB 4789.35-2016 Lactic acid bacteria count

[0142] DNJ content: HPLC-ELSD (column: XBridge Amide 3.5μm).

[0143] The chromatographic conditions are shown in Table 3 below.

[0144] Table 3 Chromatographic conditions

[0145] parameter Setting value illustrate chromatographic column XBridge Amide (4.6×250 mm, 3.5 μm) Hydrophilic interaction chromatographic columns are suitable for polar compounds. mobile phase Acetonitrile:water = 80:20 (v / v) Isocratic elution Flow rate 1.0 mL / min constant flow rate Column temperature 40°C Temperature control reduces baseline drift Injection volume 10 μL -

[0146] Dissolution rate: Chinese Pharmacopoeia 2020 Edition, Method IV (pH 1.2→6.8→7.4 gradient medium).

[0147] Test Example 1

[0148] Probiotic gastric acid tolerance and colonic release rate

[0149] Objective: To verify the protective effect of microencapsulation technology on live bacteria (corresponding to claim 3). Method:

[0150] Simulated digestion experiment:

[0151] Simulated gastric juice: 0.1M HCl + 3.2 g / L pepsin (pH 1.2)

[0152] Simulated intestinal fluid: 6.8 mg / mL KH2PO4 + 10 g / L pancreatic enzyme (pH 6.8)

[0153] Simulated colonic fluid: 0.5 g / L bile salts + 2 g / L mucin (pH 7.4)

[0154] Sample preparation: Take microencapsulated probiotic pellets (containing 1×10⁻⁶) from Examples 1-3 and Comparative Examples 2-4. 10 CFU), respectively placed in:

[0155] Gastric juice (37℃, shaken, 120 min) → Take 1 mL to determine the viable bacterial count.

[0156] Transfer to intestinal fluid (120 min) → Transfer to colonic fluid (60 min)

[0157] viable cell count:

[0158] MRS agar plate culture (37℃ anaerobic, 48 h)

[0159] Release rate = (Number of viable bacteria in each segment / Initial number of viable bacteria) × 100%

[0160] The results are shown in Table 4:

[0161] Table 4 Simulated Digestion Experiment

[0162] Group Gastric juice release rate (%) Colonic release rate (%) Example 1 0.08±0.02 96.5±2.3 Example 2 0.05±0.01 98.1±1.8 Example 3 0.07±0.01 97.3±2.1 Comparative Example 2 79.7±3.2 10.3±1.5 Comparative Example 3 12.5±1.8 58.4±3.0 Comparative Example 4 0.11±0.03 72.6±2.7

[0163] analyze:

[0164] The gastric juice release rate in the example was ≤0.1% (P<0.01 vs. the comparative example), attributed to the synergistic protection of the four-layer coating:

[0165] HPMCP isolates stomach acid (dissolves at pH ≥ 5.5)

[0166] Eudragit® FS 30D Controlled Colonic Release (Dissolves at pH ≥ 7.0)

[0167] Comparative Example 3 showed that less than 60% of the colon was released from a single-layer coated colon, demonstrating the necessity of a pH-responsive layer.

[0168] Comparative Example 2 (Probiotics not encapsulated): High gastric release (~80%) was due to the lack of any protection, resulting in the large-scale inactivation of live bacteria by gastric acid. Very low colonic release (~10%) was due to further loss of the remaining live bacteria.

[0169] Comparative Example 4 (component missing): Low gastric release (~0.1%) demonstrates the effectiveness of enteric isolation, but insufficient colonic release (~73%) is due to the lack of probiotic synergy and adhesion layer, resulting in decreased colonization efficiency.

[0170] Test Example 2

[0171] Synergistic effect of lowering blood sugar and regulating blood lipids

[0172] Objective: To verify the component synergistic mechanism. Methods:

[0173] 1. Animal models:

[0174] Type 2 diabetic SD rats induced by a high-fat, high-sugar diet (blood glucose ≥11.1 mmol / L, TC ≥6.2 mmol / L)

[0175] Groups were administered gavage for 28 days (n=10):

[0176] Control group: physiological saline

[0177] Examples 1-3: 200 mg / kg / d

[0178] Comparative Examples 1, 3, and 5: 200 mg / kg / day

[0179] Positive drug group: Metformin (150 mg / kg / day)

[0180] 2. Detection indicators:

[0181] Fasting blood glucose (glucose oxidase method)

[0182] Serum LDL-C (enzyme colorimetric method)

[0183] Vascular endothelial function (FMD, ultrasound detection of brachial artery diastolic changes)

[0184] The results are shown in Table 5, and Figure 1-3 As shown.

[0185] Table 5 Blood Sugar Lowering Experiment

[0186] Group Blood glucose reduction (%) LDL-C reduction (%) FMD increase (%) Example 1 18.5±1.9* 30.2±2.8* 42.7±3.6* Example 2 22.7±2.1* 35.8±3.2* 52.3±4.5* Example 3 20.3±2.0* 33.1±3.0* 48.2±4.0* Comparative Example 1 13.5±1.8 18.6±2.7 28.4±3.1 Comparative Example 3 8.2±1.2 24.9±2.3 15.7±2.0 Comparative Example 5 6.5±1.0 12.3±1.8 10.2±1.5 Positive drug group 25.3±2.4 46.8±4.0 5.8±0.9

[0187] (*P<0.01 vs Comparative Example 1)

[0188] Mechanism Analysis:

[0189] 1. Dosage effect in Examples 1-3

[0190] Blood glucose regulation: Example 2 (medium dose) showed the best effect (reduction of 22.7%), because the combination of lactopeptide (12 parts) and mulberry leaf DNJ (3.7%) achieved the optimal ratio for α-glucosidase inhibition.

[0191] Lipid-regulating effect: The LDL-C reduction in Example 2 (35.8%) was significantly higher than that in Examples 1 (30.2%) and 3 (33.1%), because rice furfuranol (5 parts) and probiotic metabolites synergistically activated LDL receptor expression.

[0192] Vascular protection: FMD increase was positively correlated with the dose of aroniaberry anthocyanin (the highest anthocyanin content in Example 3 → FMD↑48.2%), but excessive DNJ (4.2% in Example 3) slightly inhibited the eNOS pathway, so it was weaker than in Example 2.

[0193] 2. Analysis of comparative defects, see Table 6.

[0194] Table 6 Comparative Defect Analysis

[0195] Comparative Example Key defects This leads to a decrease in effectiveness. 1 Missing segment release Component competition: Mulberry leaf DNJ and earthworm protein are released simultaneously in the duodenum, competing for α-glucosidase binding sites → blood sugar lowering efficiency ↓40% (vs. Example 2) 3 Lack of probiotic synergy Rice furfuranol alone cannot adequately upregulate LDL receptors → lipid-regulating effect decreased by 30% (vs. Example 2); lack of anti-inflammatory effects of gut microbiota → insufficient improvement in FMD. 5 Mixed grinding destructive activity Anthocyanin thermal degradation (high temperature ball milling) + live bacteria inactivation → all indicators fail (lowest levels in blood sugar / lipid regulation / FMD). Test Example 3

[0196] Objective: To monitor the segmented release behavior of enteric-coated formulations and verify the regulatory effect of phase-separated granulation process on the release sequence of components.

[0197] Methods: Gradient dissolution test (Chinese Pharmacopoeia 2020 Edition, Method IV):

[0198] Medium timing:

[0199] 0-2 h: Simulated gastric juice (pH 1.2, containing 0.1M HCl + 3.2 g / L pepsin)

[0200] 2-4 h: Simulated intestinal fluid (pH 6.8, containing 6.8 mg / mL KH2PO4 + 10 g / L pancreatic enzymes)

[0201] 4-6 h: Simulated colonic fluid (pH 7.4, containing 0.5 g / L bile salts + 2 g / L mucin)

[0202] sample:

[0203] Example 2 (Segmented release design: containing enteric-coated microcapsules I + sustained-release microcapsules II + probiotic microcapsules)

[0204] Comparative Example 1 (lacking segmented release: all raw materials were mixed, pulverized, and directly filled into enteric-coated capsules)

[0205] Testing indicators:

[0206] Lactopeptide: HPLC (C18 column, mobile phase 0.1% TFA-acetonitrile, detection wavelength 220 nm)

[0207] Anthocyanins: pH differential method (510 nm & 700 nm, calculate ΔA = A) 510 -A 700 )

[0208] Viable count: Plate count method (MRS agar, anaerobic culture at 37°C for 48 h)

[0209] The results are shown in Table 7.

[0210] Table 7: Gradient dissolution experiment

[0211] Group Time point Milk tripeptide dissolution rate Anthocyanin dissolution rate Live bacteria release rate Example 2 2 h (stomach) 5.2±0.3% 8.1±0.5% 0.06±0.01% Example 2 4 h (intestine) 94.7±2.1% 45.3±3.2% 12.5±1.8% Example 2 6 h (colon) - 88.6±3.8% 97.3±2.5% Comparative Example 1 2 h (stomach) 4.8±0.4% 7.5±0.6% 85.2±4.3% Comparative Example 1 4 h (intestine) 68.5±3.7%* 92.4±4.1%* 14.3±2.2% Comparative Example 1 6 h (colon) - 95.1±3.5% 15.8±2.5%

[0212] **P<0.01 vs. Example 2 at the same time point*

[0213] The data analysis in Table 7 is as follows:

[0214] Segmented release logic in Example 2:

[0215] Duodenal targeting: Enteric-coated microcapsules I (HPMCP coating) dissolve rapidly at pH 6.8 → Lactopeptide release rate >94% after 4 hours (meets the needs of acute lipid regulation).

[0216] Jejunal sustained release: Sustained-release microcapsules II (Eudragit® L100-55 coating) gradually dissolve → anthocyanin release rate of 88.6% after 6 hours (avoiding gastrointestinal irritation).

[0217] Precise colon delivery: Probiotic microcapsules (four-layer encapsulation) are released at pH 7.4 → live bacteria release rate of 97.3% (maximizing colonization efficiency).

[0218] The defects of Comparative Example 1:

[0219] Leakage of live bacteria in gastric juice: Due to the lack of probiotic encapsulation layer, the release rate of live bacteria in gastric juice is as high as 85.2% → which is inactivated by gastric acid (echoing the background technology of "survival rate ↓70%").

[0220] Competitive release of components:

[0221] The release rate of the lactotripeptide in intestinal fluid was only 68.5% (vs. 94.7% in Example 2), because mixing with hydrophobic rice furfuryl alcohol hindered dissolution;

[0222] Anthocyanins are released prematurely in intestinal fluid at a rate of 92.4% (vs. 45.3% in Example 2) → increasing the risk of gastrointestinal irritation (Background Art: incidence >15%).

[0223] Probiotic colonization failed: the colon release rate was only 15.8% (because the live bacteria were almost completely lost in the gastric juice).

[0224] in conclusion:

[0225] The necessity of segmented release design:

[0226] Comparative Example 1 (mixed pulverized formulation) demonstrates that the absence of the phase separation granulation process will result in:

[0227] ① Live bacteria are inactivated by gastric acid (colonic colonization rate <16%).

[0228] ② Protein peptide release is inhibited (liquid tripeptide dissolution ↓27%);

[0229] ③ Premature release of plant extracts can cause side effects.

[0230] Test Example 4

[0231] Accelerated stability testing of formulation

[0232] Objective: To verify the effect of component isolation process on stability.

[0233] method:

[0234] 1. Acceleration conditions: 40℃±2℃ / 75%±5% RH, 6 months

[0235] 2. Test Samples: Example 2 vs. Comparative Example 5 (Mixed Powdered Formulation)

[0236] 3. Detection indicators:

[0237] viable cell retention rate: MRS plate count

[0238] Anthocyanin retention rate: HPLC (520 nm)

[0239] Layering phenomenon: Microscopic observation (100×)

[0240] The results are shown in Table 8:

[0241] Table 8: Accelerated Stability Tests of Agents

[0242] Group viable bacteria retention rate Anthocyanin retention rate Physical stratification Example 2 86.7±3.5% 92.1±2.1% none Comparative Example 5 15.2±2.3% 68.4±4.7% Clearly layered

[0243] Key attribution:

[0244] Example 2: Avoiding component mutual exclusion through phase separation granulation:

[0245] Hydrophobic rice furfuranol (microcapsule I) and hydrophilic anthocyanins (microcapsule II) are physically isolated;

[0246] Acidic mulberry leaf extract is kept away from probiotic microcapsules.

[0247] Test Example 5

[0248] Validation of the mechanism of improvement of vascular endothelial inflammation and component synergy

[0249] Purpose

[0250] Verify the core mechanism of the composition in preventing atherosclerosis (inhibition of inflammation + anti-apoptosis).

[0251] Revealing the necessity of multi-component synergy (comparing the shortcomings of missing key components)

[0252] method

[0253] Cell model:

[0254] Human umbilical vein endothelial cells (HUVECs) were induced to undergo damage (simulating diabetic vascular lesions) by high glucose (30 mM) + ox-LDL (50 μg / mL).

[0255] Group intervention (n=6):

[0256] Control group: No damage, routine culture;

[0257] Damage group: Damage model + no intervention;

[0258] Example 2 group: Damage + Example 2 full component extract (50 μg / mL);

[0259] Comparative Example 4: Damage + Comparative Example 4 extract (50 μg / mL, lacking argan berry / mulberry leaf / probiotics).

[0260] Testing indicators:

[0261] Inflammatory factors: TNF-α, IL-6 (ELISA kit);

[0262] Apoptosis rate: Annexin V / PI double staining flow cytometry detection;

[0263] Key pathway proteins: NF-κB p65 nuclear translocation (immunofluorescence), Bcl-2 / Bax ratio (Western blot).

[0264] The results are shown in Table 9.

[0265] Table 9 Comparison of improvement in vascular endothelial inflammation and apoptosis

[0266] Group TNF-α (pg / mL) IL-6 (pg / mL) Apoptosis rate (%) NF-κB activation inhibition rate* Bcl-2 / Bax ratio Blank group 12.3±1.5 15.2±1.8 5.2±0.8 - 2.8±0.3 Damage group 85.6±6.2 92.7±7.1 38.7±3.1 0% 0.6±0.1 Example 2 group 29.4±2.8*▲ 36.5±3.3*▲ 11.5±1.2*▲ 78.2±5.1% 2.1±0.2▲ Comparative Example 4 Groups 62.8±4.7* 71.3±5.9* 29.6±2.5* 32.5±4.3% 1.0±0.1

[0267] *P<0.01 vs. the injury group; ▲P<0.01 vs. the control group (4 groups)

[0268] *NF-κB activation inhibition rate = (1 - number of translocated cells in the intervention group / number of translocated cells in the damage group) × 100%

[0269] Key Mechanism Analysis

[0270] 1. Synergistic advantages of Example 2

[0271] Anti-inflammatory:

[0272] Anthocyanins from aronia significantly inhibited NF-κB nuclear translocation (inhibition rate > 78%) → TNF-α ↓ 65.6% and IL-6 ↓ 60.7%.

[0273] Compared with control group 4 (anthocyanins): the NF-κB inhibition rate was only 32.5%, and the reduction of inflammatory factors was less than 30%.

[0274] Anti-apoptosis:

[0275] Earthworm protein and mulberry leaf DNJ synergistically upregulated the Bcl-2 / Bax ratio (from 0.6 to 2.1) → apoptosis rate ↓70.3%.

[0276] Compared with control group 4 (DNJ without mulberry leaves): Bcl-2 / Bax was only 1.0, and the improvement in apoptosis rate was limited (↓23.5%).

[0277] 2. Root causes of defects in Comparative Example 4

[0278] The absence of argan anthocyanins leads to insufficient inhibition of the NF-κB pathway, resulting in residual inflammation (TNF-α > 60 pg / mL).

[0279] The absence of mulberry leaf DNJ → failure to coordinate with earthworm protein to regulate the apoptosis pathway → impaired endothelial repair.

[0280] The lack of probiotic metabolites indirectly weakens the anti-inflammatory effect (the background technology mentions that bacterial metabolites reduce LDL-C inflammatory factors).

[0281] in conclusion

[0282] Multi-component synergy is irreplaceable:

[0283] Compared to Comparative Example 4, the composition provided in Example 2 of this invention has unexpected technical effects such as significant inhibition of inflammation (TNF-α) and endothelial protection (improved apoptosis rate).

[0284] The core value of Example 2:

[0285] Through the synergistic effect of multiple pathways including anthocyanins (anti-inflammatory), earthworm protein / DNJ (anti-apoptosis), and probiotics (indirect anti-inflammatory), a three-dimensional repair of vascular endothelial damage is achieved, providing an inseparable formulation basis for the prevention of atherosclerosis.

[0286] Test Example 6

[0287] Evaluation of the hypoglycemic and lipid-regulating effects in rats

[0288] Objective: To verify the regulatory efficacy of the composition on blood glucose and blood lipids in an animal model.

[0289] method:

[0290] Animal models:

[0291] SPF-grade SD rats (male, 180-220g) were selected.

[0292] Insulin resistance and dyslipidemia models were induced by feeding the animal with a high-fat, high-sugar diet (containing 60% basal feed, 15% lard, 20% sucrose, and 5% cholesterol) for 8 weeks.

[0293] Successful modeling criteria: fasting blood glucose (FBG) ≥ 7.0 mmol / L and serum total cholesterol (TC) ≥ 5.2 mmol / L.

[0294] Grouping and drug administration (n=10 / group):

[0295] Normal control group: Normal feed + physiological saline by gavage.

[0296] Model control group: High-fat, high-sugar diet + physiological saline by gavage.

[0297] Positive drug group (Metformin): High-fat, high-sugar diet + metformin (150 mg / kg / d) by gavage.

[0298] Example 2 group (Test): High-fat, high-sugar diet + enteric-coated preparation of Example 2 composition (200 mg / kg / d) by gavage.

[0299] Comparative Example 4 (DC4): High-fat, high-sugar diet + Comparative Example 4 composition (lacking argan berry / mulberry leaf / probiotics, equal dose 200 mg / kg / d) by gavage.

[0300] Oral administration volume: 10 mL / kg, once daily, for 6 consecutive weeks.

[0301] Detection indicators and methods:

[0302] Fasting blood glucose (FBG): Blood was collected from the tail vein at the end of weeks 0, 2, 4 and 6 after administration, and measured by glucose oxidase method.

[0303] Oral glucose tolerance test (OGTT): Performed during week 5 of drug administration. After fasting for 12 hours, glucose solution (2 g / kg) was administered by gavage. Blood glucose was measured by tail vein sampling at 0, 30, 60, and 120 min, and the area under the curve (AUC) was calculated.

[0304] Serum lipid profile: Blood was collected from the abdominal aorta at the end of the 6-week period after drug administration, and serum was separated.

[0305] Total cholesterol (TC): CHOD-PAP method.

[0306] Triglycerides (TG): GPO-PAP method.

[0307] Low-density lipoprotein cholesterol (LDL-C): direct method.

[0308] High-density lipoprotein cholesterol (HDL-C): direct method.

[0309] Insulin resistance index (HOMA-IR): Fasting insulin (FINS, ELISA method) was measured at the end of the 6-week period after drug administration. HOMA-IR = (FBG × FINS) / 22.5.

[0310] The results are shown in Table 10.

[0311] Table 10: Effects of the composition on blood glucose and blood lipids in high-fat, high-glucose model rats (X ± SD)

[0312] Group FBG (mmol / L) (Week 6) OGTT AUC (mmol / L·h) TC (mmol / L) TG (mmol / L) LDL-C (mmol / L) HDL-C (mmol / L) HOMA-IR Normal 5.2 ± 0.3**▲▲ 15.8 ± 1.2**▲▲ 1.8 ± 0.2**▲▲ 0.7 ± 0.1**▲▲ 0.5 ± 0.1**▲▲ 1.2 ± 0.1**▲▲ 2.1 ± 0.3**▲▲ Model 9.8 ± 0.6 28.5 ± 2.1 6.5 ± 0.5 1.9 ± 0.3 4.2 ± 0.4 0.8 ± 0.1 8.7 ± 0.9 Metformin 6.5 ± 0.4**▲▲ 19.3 ± 1.5**▲▲ 5.1 ± 0.4** 1.3 ± 0.2**▲ 3.0 ± 0.3**▲▲ 1.0 ± 0.1** 4.5 ± 0.5**▲▲ Test 6.9 ± 0.5**▲▲ 20.1 ± 1.7**▲▲ 4.2 ± 0.3**▲▲ 1.1 ± 0.2**▲▲ 2.4 ± 0.2**▲▲ 1.1 ± 0.1**▲▲ 4.0 ± 0.4**▲▲ DC4 8.2 ± 0.5** 25.0 ± 1.9* 5.7 ± 0.4 1.6 ± 0.2* 3.5 ± 0.3* 0.9 ± 0.1 6.8 ± 0.7*

[0313] Note: Compared with the Model group: P<0.05, ** P<0.01; Compared with the Test group: ▲ P<0.05, ▲▲ P<0.01.

[0314] analyze:

[0315] Significant blood sugar lowering effect:

[0316] In Example 2 (Test), after 6 weeks of administration, FBG significantly decreased to 6.9 mmol / L (vs Model 9.8 mmol / L, P<0.01), a decrease of 29.6%.

[0317] The OGTT-AUC was significantly reduced (20.1 vs 28.5, P<0.01), indicating improved glucose tolerance.

[0318] The HOMA-IR was significantly reduced (4.0 vs 8.7, P<0.01), indicating that insulin resistance was effectively alleviated.

[0319] The efficacy was comparable to that of the positive control drug metformin (FBG: 6.5 vs 6.9, P>0.05; HOMA-IR: 4.5 vs 4.0, P>0.05), and slightly better in improving HOMA-IR (P<0.05 vs Metformin).

[0320] Its lipid-regulating effect is outstanding.

[0321] The test group showed a significant reduction in atherosclerotic lipid markers: TC decreased by 35.4% (4.2 vs 6.5 mmol / L, P<0.01), TG decreased by 42.1% (1.1 vs 1.9 mmol / L, P<0.01), and LDL-C decreased by 42.9% (2.4 vs 4.2 mmol / L, P<0.01).

[0322] At the same time, the protective index HDL-C was significantly increased (1.1 vs 0.8 mmol / L, P<0.01).

[0323] Verification of the necessity of component synergy:

[0324] The comparative group 4 (DC4, lacking argan berry / mulberry leaf / probiotics) was significantly weaker than the example group 2 (Test) in terms of blood sugar lowering (FBG: 8.2 vs Test 6.9, P<0.01; OGTT-AUC: 25.0 vs 20.1, P<0.01; HOMA-IR: 6.8 vs 4.0, P<0.01) and lipid regulation (TC: 5.7 vs 4.2, P<0.01; LDL-C: 3.5 vs 2.4, P<0.01).

[0325] This confirms the indispensable synergistic effect of aroniaberry anthocyanins (antioxidant, lipid-regulating), mulberry leaf DNJ (hypoglycemic, synergistic lipid-regulating), and Lactobacillus plantarum metabolites (lipid-regulating, improving insulin sensitivity) in the composition.

[0326] This animal experiment confirms that the enteric-coated formulation of the composition described in Example 2 of this invention can effectively reduce fasting blood glucose, improve glucose tolerance, alleviate insulin resistance in high-fat and high-glucose induced insulin resistance model rats, and significantly regulate blood lipid profile (reducing TC, TG, and LDL-C, and increasing HDL-C).

[0327] The above are merely a few preferred embodiments of the present invention, described in a relatively specific and detailed manner, but they should not be construed as limiting the scope of the present 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.

Claims

1. A composition for promoting cardiovascular health, characterized in that, consists of the following raw materials in mass fraction: earthworm protein 10-30 parts, milk tripeptide 5-20 parts, rice bran fatty alcohol 1-10 parts, not old berry extract 5-25 parts, mulberry leaf extract 3-15 parts, micro-embedded lactobacillus 1-8 parts; The micro-embedded lactobacillus is prepared by the following process: (a) Freeze-drying protective layer: mix the lactobacillus fermentation broth with a freeze-drying protective agent containing trehalose 15-30% w / v and β-cyclodextrin 5-15% w / v, pre-freeze to below -40°C, and then vacuum freeze-dry; (b) pH responsive layer coating: fluid bed spray Eudragit L30D-55 on the product of step (a) ® FS 30D, coating weight gain 10-20%; (c) Enteric isolation layer: spray the product of step (b) with a hydroxypropyl methylcellulose phthalate HPMCP ethanol solution on the surface for secondary coating, with a coating weight increase of 5-10%; (d) Bioadhesive layer: spray the surface of the product of step (c) with a solution of chitosan quaternary ammonium salt 0.5-2% w / v, obtaining the microencapsulated Lactobacillus plantarum with a viable count > 5 x 10 11 CFU / g.

2. The composition of claim 1, wherein: consists of the following raw materials in mass fraction: earthworm protein 20 parts milk tripeptide 12 parts rice bran fatty alcohol 5 parts not old berry extract 15 parts mulberry leaf extract 8 parts micro-embedded lactobacillus 4 parts.

3. The composition of claim 1, characterized in that: the not old berry extract contains anthocyanins 30-40%; the content of octacosanol in rice bran fatty alcohol is ≥80%; the content of DNJ in mulberry leaf extract is 2.5-4.0%.

4. The composition of claim 1, characterized in that, the micro-embedded lactobacillus has a multi-layer coating structure, and the multi-layer coating structure comprises: (a) the inner layer is an enteric isolation layer, which comprises an enteric material that dissolves at pH≥5.5; (b) the outer layer is a pH-responsive layer, which comprises a colon-targeting material that dissolves at pH≥7.0; wherein the thickness and / or composition of the multi-layer coating structure are configured so that the live bacteria release rate of the composition is ≤0.1% in simulated gastric fluid for 2 hours and ≥95% in simulated colon fluid for 1 hour.

5. Use of the composition of any one of claims 1-4 in the preparation of a medicine for preventing atherosclerosis, wherein: the earthworm protein and the mulberry leaf extract inhibit α-glucosidase; the rice bran fatty alcohol and the lactobacillus metabolite reduce serum LDL-C; the not old berry anthocyanin scavenges vascular free radicals.

6. A process for the preparation of enteric formulations of the composition of claim 1, characterized in that, comprising the following steps: (1) Mix earthworm protein, milk tripeptide, and rice bran fatty alcohol, spray dry, and coat with HPMCP to obtain enteric micro-pellets I with an average particle size of 200-400 μm; (2) The aralia elata extract and mulberry leaf extract were co-ground and processed with Eudragit. ® L100-55 coating was used to prepare sustained-release microspheres II with an average particle size of 400-600 μm; (3) Mix micro-pellets I, micro-pellets II, and micro-embedded lactobacillus; (4) Fill the mixture of step (3) into enteric capsule shells or press into tablets and then coat with enteric coating.

7. An enteric formulation, characterized in that, obtained by the preparation method of claim 6, the coating layer thickness of the enteric micro-pellets I is 80-120 μm, and the coating layer thickness of the sustained-release micro-pellets II is 20-50 μm.

8. The enteric preparation of claim 7, characterized in that, the enteric preparation comprises: (a) micro-pellets I: having an enteric coating layer comprising an enteric material that dissolves at pH≥5.0-6.0; (b) micro-pellets II: having a colon-targeting coating layer comprising a colon-targeting material that dissolves at pH≥7.0; wherein the thickness, composition, or ratio of the enteric coating layer and the colon-targeting coating layer are configured so that: The total release rate of all the pellets is ≤5% in simulated gastric fluid for 2 hours; The release rate of pellet I is ≥90% in simulated intestinal fluid for 30 minutes; The release rate of pellet II is ≥85% in simulated colonic fluid for 2 hours.

9. The enteric formulation according to claim 7, characterized in that: In human body, the preparation has a segmented release function, which is specifically: 1) Duodenal segment: dissolution of enteric-coated pellet I, releasing earthworm protein and milk tripeptide; 2) Jejunum-ileum segment: gradual dissolution of sustained-release pellet II coating, releasing lingonberry and mulberry leaf extract; 3) Colon segment: dissolution of pH-responsive layer micro-embedded with Lactobacillus plantarum, releasing live bacteria.

10. Use of the enteric preparation according to claim 7 in the preparation of a pharmaceutical product or a functional food.

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