Application of compound lactobacillus acidophilus tablets in preparation of drugs for treating hypertension

Abstract

The application belongs to the technical field of medicine, and particularly relates to application of a compound lactobacillus acidophilus tablet in preparation of a medicine for treating hypertension. The compound lactobacillus acidophilus tablet can significantly improve the gastrointestinal tolerance and intestinal colonization capacity of the compound lactobacillus acidophilus, realizes a safe and effective antihypertensive effect by regulating intestinal flora and promoting the release of endothelial diastolic factor NO, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and in particular relates to the application of compound Lactobacillus acidophilus tablets in the preparation of drugs for treating hypertension. Background Technology

[0002] Hypertension is a cardiovascular syndrome characterized primarily by elevated systemic arterial pressure, and its pathogenesis results from the interaction of genetic and environmental factors. In recent years, the prevalence and related mortality rates of hypertension have been on a continuous upward trend, making it a major global public health problem.

[0003] Currently, first-line antihypertensive drugs in clinical practice mainly include five classes: angiotensin-converting enzyme inhibitors (ACEIs), angiotensin II receptor blockers (ARBs), calcium channel blockers, beta-blockers, and diuretics. However, hypertension is a chronic disease requiring long-term medication, and existing drugs inevitably bring various adverse reactions during long-term use. For example, diuretics can cause hypokalemia, ACEIs can easily induce dry cough, ARBs pose a risk of hyperkalemia, calcium channel blockers can cause peripheral edema, and beta-blockers may cause bradycardia. In addition, while combining antihypertensive drugs with different mechanisms of action can enhance efficacy, it also increases the risk of cumulative adverse reactions, seriously affecting patient medication adherence and quality of life.

[0004] In recent years, the relationship between gut microbiota and the development of hypertension has received increasing attention. Existing studies have confirmed that gut microbiota dysbiosis can participate in blood pressure regulation through influencing short-chain fatty acid metabolism, oxidative stress, inflammatory responses, and the accumulation of toxins in enterogenic uremic diseases. Lactobacillus acidophilus, as a common probiotic strain, has been widely used in the treatment of intestinal dysfunctional diseases such as diarrhea and functional dyspepsia; however, its application in the treatment of hypertension has not yet been reported. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, the present invention aims to provide the application of compound Lactobacillus acidophilus tablets in the preparation of drugs for treating hypertension. The compound Lactobacillus acidophilus tablets can significantly improve the gastrointestinal tolerance and intestinal colonization ability of compound Lactobacillus acidophilus. By regulating the intestinal flora and promoting the release of endothelial relaxing factor NO, it achieves a safe and effective antihypertensive effect and has broad application prospects.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] Application of compound Lactobacillus acidophilus tablets in the preparation of drugs for treating hypertension.

[0008] Furthermore, the preparation method of the compound Lactobacillus acidophilus tablets is as follows:

[0009] (1) Soybean lecithin and modified cholesterol were added to anhydrous ethanol, and then vanillic acid methyl ester glycoside was added to prepare a modified lipid film.

[0010] (2) The modified lipid film is ultrasonically hydrated with the compound Lactobacillus acidophilus solution, filtered, and freeze-dried to obtain compound Lactobacillus acidophilus liposome powder; pharmaceutically acceptable excipients are added to the compound Lactobacillus acidophilus liposome powder to make tablets.

[0011] The structural formula of the modified cholesterol mentioned in step (1) is as follows:

[0012] .

[0013] Furthermore, the method for preparing the modified cholesterol described in step (1) is as follows:

[0014] (a) Hydrazine hydrate and triethylamine were added to anhydrous ethanol to obtain a mixed solution; the mixed solution was added to an anhydrous ethanol solution of 2,4,6-trichloro-5-pyrimidinecarboxaldehyde, and purified after reaction to obtain intermediate 1;

[0015] The structural formula of intermediate 1 is as follows:

[0016]

[0017] (b) Under inert gas protection, cholesterol was added to 1,4-dioxane, sodium hydride was added and stirred, and then intermediate 1 was added. After reaction, the mixture was purified to obtain the modified cholesterol.

[0018] The preparation principle of the modified cholesterol of this invention is as follows:

[0019] First, 2,4,6-trichloro-5-pyrimidinecarboxaldehyde undergoes a condensation reaction with hydrazine hydrate to obtain intermediate 1 containing a pyrazolopyrimidine structure. Then, intermediate 1 undergoes a nucleophilic substitution reaction with cholesterol to obtain the modified cholesterol.

[0020] Furthermore, the molar ratio of 2,4,6-trichloro-5-pyrimidinecarboxaldehyde, hydrazine hydrate, and triethylamine in step (a) is 1:(1.2-1.3):(1.5-1.6).

[0021] Furthermore, the concentration of the anhydrous ethanol solution of 2,4,6-trichloro-5-pyrimidinecarboxaldehyde in step (a) is (0.3-0.35) mmol / mL; the concentration of triethylamine in the mixed solution is (0.45-0.5) mmol / mL; the reaction is first carried out at -78~-75℃ for 1-2 h, and then at 20-22℃ for 1-2 h.

[0022] Furthermore, in step (b), the molar ratio of intermediate 1, cholesterol, and sodium hydride is 1:(2.25-2.5):(2.25-2.5); the stirring time is 30-45 min; the reaction temperature is 95-100℃, and the reaction time is 3-5 h.

[0023] Furthermore, the total number of viable bacteria in the compound Lactobacillus acidophilus tablets is (1-5) × 10⁻⁶. 9 CFU / g.

[0024] Furthermore, in step (1), the mass ratio of soybean lecithin, modified cholesterol, and vanillic acid methyl glycoside is 1:(0.15-0.2):(0.16-0.25); in step (2), the ratio of compound Lactobacillus acidophilus solution to modified lipid film is 1mL:(2-3)g; the ultrasonic power is 150-180W and the time is 12-15min; the hydration temperature is 40-50℃ and the time is 1.5-2.5h.

[0025] Furthermore, the preparation method of the compound Lactobacillus acidophilus solution is as follows:

[0026] The Chinese strain of Lactobacillus acidophilus powder, the Japanese strain of Lactobacillus acidophilus powder, Enterococcus faecalis powder, and Bacillus subtilis powder were mixed evenly to prepare compound Lactobacillus acidophilus; the compound Lactobacillus acidophilus was added to a phosphate buffer solution to prepare the compound Lactobacillus acidophilus solution.

[0027] Furthermore, the mass ratio of the Chinese strain of Lactobacillus acidophilus powder, the Japanese strain of Lactobacillus acidophilus powder, the fecal streptococcus powder, and the Bacillus subtilis powder is 1:1:1:0.002; the total concentration of each bacterial powder in the compound Lactobacillus acidophilus solution is 4-8 mg / mL; and the pH of the phosphate buffer solution is 7-7.2.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] 1. This invention provides the application of compound Lactobacillus acidophilus tablets in the preparation of drugs for treating hypertension. These compound Lactobacillus acidophilus tablets can significantly improve the gastrointestinal tolerance and intestinal colonization ability of compound Lactobacillus acidophilus, and achieve a safe and effective antihypertensive effect by regulating the intestinal flora and promoting the release of endothelial relaxing factor NO, thus showing broad application prospects.

[0030] 2. This invention utilizes modified cholesterol with a specific structure as a liposome material to encapsulate compound Lactobacillus acidophilus, significantly improving the protective performance and intestinal colonization ability of the liposome encapsulation system, and endowing it with an auxiliary blood pressure lowering function. Specifically, the modified cholesterol molecule possesses two cholesterol hydrophobic backbones, which can act as molecular cross-linking rivets embedded in the phospholipid bilayer formed by soybean lecithin, significantly improving the density and mechanical strength of the lipid membrane and reducing vesicle permeability, thereby effectively protecting the internally encapsulated compound Lactobacillus acidophilus from damage by gastric acid and bile salts in the gastrointestinal tract; the pyrimidine-pyrazole aromatic heterocycle in the middle of the molecule can further stabilize the lipid membrane structure through π-π interactions, and its NH polar site can regulate the hydrophilicity-hydrophobicity balance, increasing the adhesion and retention time of liposomes on the intestinal mucosa, which is beneficial to the colonization of compound Lactobacillus acidophilus. In addition, the modified cholesterol itself also has an auxiliary blood pressure lowering effect.

[0031] 3. This invention enhances the stability of liposomes and strengthens the antihypertensive effect by adding methyl vanillic acid glycoside. Specifically, methyl vanillic acid glycoside can further improve the structural stability of liposomes and enhance the proliferation activity of Akkermansia muciniphila, thereby further strengthening the regulatory effect of compound Lactobacillus acidophilus tablets on intestinal flora, promoting the release of endothelial relaxing factor NO, and amplifying the vasodilatory effect. Attached Figure Description

[0032] Figure 1 This is a graph showing the cytotoxicity test results for the blank control group;

[0033] Figure 2 This is a graph showing the cytotoxicity test results of the compound Lactobacillus acidophilus liposome powder in Example 4;

[0034] Figure 3 This is a graph showing the cytotoxicity test results for the positive control group. Detailed Implementation

[0035] The present invention will now be further described with reference to the accompanying drawings and specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments. Specific conditions not specified in the embodiments are performed according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, all reagents or instruments used are conventional products obtained through commercial channels.

[0036] The Chinese strain of Lactobacillus acidophilus powder, the Japanese strain of Lactobacillus acidophilus powder, the fecal enterococcus powder, and the Bacillus subtilis powder are all commercially available products. Among them, the Chinese strain of Lactobacillus acidophilus powder, the Japanese strain of Lactobacillus acidophilus powder, and the fecal enterococcus powder were purchased from Changchun Yuetong Technology Co., Ltd., and the Bacillus subtilis powder was purchased from Changchun Xianji Technology Co., Ltd.

[0037] Example 1

[0038] The preparation method of modified cholesterol is as follows:

[0039]

[0040] (a) 2,4,6-trichloro-5-pyrimidinecarboxaldehyde was added to anhydrous ethanol and stirred at -78°C for 15 min to prepare a 0.32 mmol / mL solution of 2,4,6-trichloro-5-pyrimidinecarboxaldehyde for later use; hydrazine hydrate (80 wt%) and triethylamine were added to anhydrous ethanol to prepare a mixed solution with a triethylamine concentration of 0.47 mmol / mL for later use; the mixed solution was added dropwise to the 2,4,6-trichloro-5-pyrimidinecarboxaldehyde, hydrazine hydrate, and triethylamine in a molar ratio of 1:1.25:1.55, and the mixture was reacted at -76°C for 1.5 h, followed by a reaction at 21°C for 1.5 h. The solvent was removed by concentration, and the crude product was purified by column chromatography (V 石油醚 V 乙酸乙酯 Intermediate 1 was prepared by mixing a ratio of 4:1 (yield 71.6%). The NMR and mass spectrometry results of intermediate 1 are as follows:

[0041] 1 H NMR: (C5H2N4Cl2, 400MHz, DMSO-d6) δ: 7.58 (s, 1H), 13.98 (s, 1H). MS (ESI) m / z=187.97 [M].

[0042] (b) With intermediate 1, cholesterol, sodium hydride, and 1,4-dioxane in a ratio of 2 mmol:4.8 mmol:4.8 mmol:55 mL, cholesterol was dissolved in 1,4-dioxane under nitrogen protection. Sodium hydride (60% mineral oil suspension) was added at 4 °C, and the mixture was stirred for 40 min. Then, intermediate 1 was added, and the reaction was carried out at 98 °C for 4 h. After the reaction was completed, the mixture was cooled to 45 °C, and 5 drops of anhydrous ethanol were carefully added to neutralize the excess sodium hydride. The solvent was removed by concentration under reduced pressure. The residue was diluted with chloroform, and the organic phase was washed with 10 wt% hydrochloric acid solution. The organic phases were combined, dried over anhydrous magnesium sulfate, concentrated, and purified by column chromatography (V). 乙酸乙酯 V 正己烷 =1:6), yielding modified cholesterol (yield 77.3%); the NMR and mass spectrometry results of the modified cholesterol are as follows:

[0043] 1 H NMR: (C 59 H 92N4O2, 400MHz, DMSO-d6) δ: 0.80-2.50 (m, 86H), 3.54-3.60 (m, 2H), 5.25-5.30 (t, 2H), 7.58 (s, 1H), 13.98 (s, 1H). MS (ESI) m / z=888.72 [M].

[0044] Example 2

[0045] The preparation method of modified cholesterol is as follows:

[0046] (a) 2,4,6-trichloro-5-pyrimidinecarboxaldehyde was added to anhydrous ethanol and stirred at -78°C for 20 min to prepare a solution of 0.35 mmol / mL 2,4,6-trichloro-5-pyrimidinecarboxaldehyde for later use; hydrazine hydrate (80 wt%) and triethylamine were added to anhydrous ethanol to prepare a mixed solution of 0.5 mmol / mL triethylamine for later use; the mixed solution was added dropwise to the solution of 2,4,6-trichloro-5-pyrimidinecarboxaldehyde, hydrazine hydrate, and triethylamine in a molar ratio of 1:1.3:1.6, and reacted at -75°C for 1 h, followed by a reaction at 22°C for 1 h. The solvent was removed by concentration, and the crude product was purified by column chromatography (V 石油醚 V 乙酸乙酯 Intermediate 1 was prepared by mixing a ratio of 4:1 (yield 69.1%). The NMR and mass spectrometry results of intermediate 1 were consistent with those of Example 1.

[0047] (b) Using intermediate 1, cholesterol, sodium hydride, and 1,4-dioxane in a ratio of 2 mmol:5 mmol:5 mmol:60 mL, under nitrogen protection, cholesterol was dissolved in 1,4-dioxane. Sodium hydride (60% mineral oil suspension) was added at 5 °C, and the mixture was stirred for 30 min. Then, intermediate 1 was added, and the reaction was carried out at 100 °C for 3 h. After the reaction was completed, the mixture was cooled to 50 °C, and 3 drops of anhydrous ethanol were carefully added to neutralize the excess sodium hydride. The solvent was removed by concentration under reduced pressure. The residue was diluted with chloroform, and the organic phase was washed with 10 wt% hydrochloric acid solution. The organic phases were combined, dried over anhydrous magnesium sulfate, concentrated, and purified by column chromatography (V). 乙酸乙酯 V 正己烷 =1:6), to obtain modified cholesterol (yield 75.8%); the NMR and mass spectrometry results of the modified cholesterol were consistent with those of Example 1.

[0048] Example 3

[0049] The preparation method of modified cholesterol is as follows:

[0050] (a) 2,4,6-trichloro-5-pyrimidinecarboxaldehyde was added to anhydrous ethanol and stirred at -78°C for 10 min to prepare a solution of 2,4,6-trichloro-5-pyrimidinecarboxaldehyde with a concentration of 0.3 mmol / mL for later use; hydrazine hydrate (80 wt%) and triethylamine were added to anhydrous ethanol to prepare a mixed solution with a triethylamine concentration of 0.45 mmol / mL for later use; the mixed solution was added dropwise to the solution of 2,4,6-trichloro-5-pyrimidinecarboxaldehyde, hydrazine hydrate, and triethylamine in a molar ratio of 1:1.2:1.5, and the mixture was reacted at -78°C for 2 h, then at 20°C for 2 h. The solvent was removed by concentration, and the crude product was purified by column chromatography (V 石油醚 V 乙酸乙酯 Intermediate 1 was prepared by mixing a ratio of 4:1 (yield 67.4%). The NMR and mass spectrometry results of intermediate 1 were consistent with those of Example 1.

[0051] (b) Using intermediate 1, cholesterol, sodium hydride, and 1,4-dioxane in a ratio of 2 mmol:4.5 mmol:4.5 mmol:50 mL under nitrogen protection, cholesterol was dissolved in 1,4-dioxane. Sodium hydride (60% mineral oil suspension) was added at 0 °C, and the mixture was stirred for 45 min. Then, intermediate 1 was added, and the reaction was carried out at 95 °C for 5 h. After the reaction was completed, the mixture was cooled to 40 °C, and 5 drops of anhydrous ethanol were carefully added to neutralize the excess sodium hydride. The solvent was removed by concentration under reduced pressure. The residue was diluted with chloroform, and the organic phase was washed with 10 wt% hydrochloric acid solution. The organic phases were combined, dried over anhydrous magnesium sulfate, concentrated, and purified by column chromatography (V). 乙酸乙酯 V 正己烷 =1:6), to obtain modified cholesterol (yield 74.0%); the NMR and mass spectrometry results of the modified cholesterol were consistent with those of Example 1.

[0052] Example 4

[0053] The preparation method of compound Lactobacillus acidophilus tablets is as follows:

[0054] (1) Mix the above bacterial powders evenly according to the mass ratio of Chinese strain of Lactobacillus acidophilus powder, Japanese strain of Lactobacillus acidophilus powder, fecal streptococcus powder and Bacillus subtilis powder of 1:1:1:0.002 to obtain compound Lactobacillus acidophilus; add compound Lactobacillus acidophilus to a phosphate buffer solution with pH 7.1 to prepare a compound Lactobacillus acidophilus solution with a total bacterial powder concentration of 6 mg / mL;

[0055] (2) With a mass ratio of soybean lecithin, modified cholesterol, and vanillic acid methyl ester glycoside of 1:0.18:0.2, soybean lecithin and modified cholesterol from Example 1 were dissolved in anhydrous ethanol, and then vanillic acid methyl ester glycoside was added. The mixture was stirred evenly, and then dried by rotary evaporation and vacuum to obtain a modified lipid film.

[0056] (3) The modified lipid film was mixed with the compound Lactobacillus acidophilus solution and the modified lipid film in a ratio of 1 mL: 2.5 g. The mixture was sonicated at 170 W for 13 min, then hydrated at 45 °C for 2 h. After filtration through a 0.22 μm sterile filter membrane and freeze-drying, the compound Lactobacillus acidophilus liposome powder was obtained.

[0057] (4) Using compound Lactobacillus acidophilus liposome powder, microcrystalline cellulose, polyvinylpyrrolidone / vinyl acetate copolymer, cross-linked carboxymethyl cellulose sodium, and magnesium stearate in a mass ratio of 1:5.5:3.5:4.5:0.55, microcrystalline cellulose, polyvinylpyrrolidone / vinyl acetate copolymer, and cross-linked carboxymethyl cellulose sodium are added to the above compound Lactobacillus acidophilus liposome powder, mixed evenly, and then an appropriate amount of ethanol is added to form a soft mass. After granulation, drying, and granulation, magnesium stearate is added, mixed, and tableted to obtain the product. The total number of effective viable bacteria in this compound Lactobacillus acidophilus tablet is 4×10⁻⁶. 9 CFU / g.

[0058] Example 5

[0059] The preparation method of compound Lactobacillus acidophilus tablets is as follows:

[0060] (1) Mix the above bacterial powders evenly according to the mass ratio of Chinese strain of Lactobacillus acidophilus powder, Japanese strain of Lactobacillus acidophilus powder, fecal streptococcus powder and Bacillus subtilis powder of 1:1:1:0.002 to obtain compound Lactobacillus acidophilus; add compound Lactobacillus acidophilus to a phosphate buffer solution with pH 7.2 to prepare a compound Lactobacillus acidophilus solution with a total bacterial powder concentration of 8 mg / mL;

[0061] (2) With a mass ratio of soybean lecithin, modified cholesterol and vanillic acid methyl ester glycoside of 1:0.2:0.25, soybean lecithin and modified cholesterol from Example 2 were dissolved in anhydrous ethanol, and then vanillic acid methyl ester glycoside was added. The mixture was stirred evenly, and then dried by rotary evaporation and vacuum to obtain a modified lipid film.

[0062] (3) The modified lipid film was mixed with the compound Lactobacillus acidophilus solution and the modified lipid film in a ratio of 1 mL: 3 g. The mixture was sonicated at 180 W for 12 min, then hydrated at 50 °C for 1.5 h. After filtration through a 0.22 μm sterile filter membrane and freeze-drying, compound Lactobacillus acidophilus liposome powder was obtained.

[0063] (4) Using compound Lactobacillus acidophilus liposome powder, microcrystalline cellulose, polyvinylpyrrolidone / vinyl acetate copolymer, cross-linked carboxymethyl cellulose sodium, and magnesium stearate in a mass ratio of 1:6:4:5:0.6, microcrystalline cellulose, polyvinylpyrrolidone / vinyl acetate copolymer, and cross-linked carboxymethyl cellulose sodium are added to the above compound Lactobacillus acidophilus liposome powder. After mixing evenly, an appropriate amount of ethanol is added to form a soft mass. After granulation, drying, and granulation, magnesium stearate is added, mixed, and tableted to obtain the product. The total number of effective viable bacteria in this compound Lactobacillus acidophilus tablet is 5×10⁻⁶. 9 CFU / g.

[0064] Example 6

[0065] The specific preparation method of Compound Lactobacillus Acidophilus Tablets is as follows:

[0066] (1) Mix the above bacterial powders evenly according to the mass ratio of Chinese strain of Lactobacillus acidophilus powder, Japanese strain of Lactobacillus acidophilus powder, fecal streptococcus powder and Bacillus subtilis powder of 1:1:1:0.002 to obtain compound Lactobacillus acidophilus; add compound Lactobacillus acidophilus to a phosphate buffer solution with pH 7 to prepare a compound Lactobacillus acidophilus solution with a total bacterial powder concentration of 4 mg / mL;

[0067] (2) With a mass ratio of soybean lecithin, modified cholesterol, and vanillic acid methyl ester glycoside of 1:0.15:0.16, soybean lecithin and modified cholesterol from Example 3 were dissolved in anhydrous ethanol, and then vanillic acid methyl ester glycoside was added. The mixture was stirred evenly, and then dried by rotary evaporation and vacuum to obtain a modified lipid film.

[0068] (3) The modified lipid film was mixed with the compound Lactobacillus acidophilus solution and the modified lipid film in a ratio of 1 mL: 2 g. The mixture was sonicated at 150 W for 15 min, then hydrated at 40 °C for 2.5 h. After filtration through a 0.22 μm sterile filter membrane and freeze-drying, compound Lactobacillus acidophilus liposome powder was obtained.

[0069] (4) Using compound Lactobacillus acidophilus liposome powder, microcrystalline cellulose, polyvinylpyrrolidone / vinyl acetate copolymer, cross-linked carboxymethyl cellulose sodium, and magnesium stearate in a mass ratio of 1:5:3:4:0.5, add microcrystalline cellulose, polyvinylpyrrolidone / vinyl acetate copolymer, and cross-linked carboxymethyl cellulose sodium to the above compound Lactobacillus acidophilus liposome powder, mix evenly, add an appropriate amount of ethanol to make a soft mass, granulate, dry, and granulate, add magnesium stearate, mix, and compress into tablets to obtain the product; the total number of effective viable bacteria in this compound Lactobacillus acidophilus tablet is 1×10⁻⁶. 9 CFU / g.

[0070] Comparative Example 1

[0071] The difference between this comparative example and Example 4 is that the modified cholesterol in Example 1 in step (2) is replaced with cholesterol.

[0072] Comparative Example 2

[0073] The difference between this comparative example and Example 4 is that vanillic acid methyl glycoside in step (2) is omitted.

[0074] Experimental Example 1

[0075] 1.1 Simulated Gastric Fluid Acid Resistance Test

[0076] Take 2g of the bacterial tablets obtained in Examples 4-6 and Comparative Examples 1-2, add them to 30mL of simulated gastric juice preheated to 37℃, vortex for 5s, adjust the pH to 2.0 with 1mol / L hydrochloric acid solution, add pepsin and gastric lipase, and digest at 37℃ for 2h. Take samples at 1h and 2h of gastric digestion. For each sampling, add 1mL of digestion solution to 10mL of 0.1mol / L phosphate buffer, vortex for 5min to completely dissolve the bacterial tablets, perform serial dilutions, and determine the viable cell count using the plate counting method. Take another batch of bacterial tablets from the same batch, dissolve them using the same method, and determine the initial viable cell count. Calculate the survival rate according to the following formula: Survival rate (%) = (Number of viable cells after simulated digestion / Initial number of viable cells) × 100%. The results are shown in Table 1 below.

[0077] 1.2 Simulated artificial intestinal fluid bile salt tolerance test

[0078] Take 2g of the bacterial tablets obtained in Examples 4-6 and Comparative Examples 1-2, add them to 30mL of simulated intestinal fluid (containing bile salts and pancreatic digestive enzymes) preheated to 37℃, and digest at 37℃ for 2h. Take samples at 1h and 2h of intestinal digestion, respectively. The sampling, viable cell count and survival rate calculation methods are the same as in 1.1. Calculate the intestinal fluid tolerance survival rate, and the results are shown in Table 1.

[0079] Table 1

[0080]

[0081] As shown in Table 1, in the tolerance tests using simulated gastric and intestinal fluids, the survival rates of the compound Lactobacillus acidophilus tablets prepared in Examples 4-6 after 1 hour and 2 hours of digestion in gastric and intestinal fluids were significantly higher than those in Comparative Examples 1-2. This indicates that the liposome encapsulation system constructed by combining modified cholesterol and methyl vanillic acid glycoside exhibits excellent gastrointestinal tolerance to compound Lactobacillus acidophilus. In contrast, Comparative Example 1, which used ordinary cholesterol instead of modified cholesterol, showed the lowest survival rates at all time points. Comparative Example 2, which omitted methyl vanillic acid glycoside, had a survival rate between that of Examples 1 and Comparative Example 1. This suggests that modified cholesterol, as a liposome membrane material, can effectively resist the damage from gastric acid, pepsin, bile salts, and pancreatic enzymes. The addition of methyl vanillic acid glycoside can further stabilize the liposome structure and synergistically enhance the intestinal colonization potential of probiotics. The combination of these two components can achieve efficient gastrointestinal delivery of compound Lactobacillus acidophilus.

[0082] Experimental Example 2

[0083] 2.1 Experimental Animals

[0084] Seventy 8-week-old Wistar rats were used. Housing conditions: room temperature 20-24℃, humidity 50%-60%, 12-hour light-dark cycle, free access to food and water.

[0085] 2.2 Grouping and Modeling

[0086] After one week of adaptive feeding, the rats were randomly divided into 7 groups of 10 each: normal control group, model group, Example 4 group, Example 5 group, Example 6 group, Comparative Example 1 group and Comparative Example 2 group.

[0087] The normal control group was fed a regular diet, while the other groups were fed a high-salt diet containing 8% sodium chloride. Simultaneously, each experimental group was administered corresponding bacterial tablets (total viable count 1×10⁻⁶) via gavage daily. 8 CFU / animal / day), normal control group and model group were administered an equal volume of distilled water by gavage. Once daily for 6 consecutive weeks.

[0088] 2.3 Indicator Testing

[0089] 2.3.1 Blood Pressure

[0090] After the last administration, tail artery blood pressure was measured using a rat tail non-invasive blood pressure monitor. The results are shown in Table 2.

[0091] 2.3.2 NO content

[0092] After the last administration, patients were kept on a fasting 12-hour period but allowed free access to water. Blood was drawn from the orbital venous plexus, and the plasma NO content was determined by ELISA. The results are shown in Table 2.

[0093] Table 2

[0094]

[0095] As shown in Table 2, the systolic blood pressure of the rats in the model group was significantly higher than that of the normal control group after the last administration, while the plasma NO content was significantly lower, indicating that the high-salt diet-induced hypertension model was successfully established and accompanied by endothelial dysfunction. Compared with the model group, the systolic blood pressure of groups 4-6 decreased significantly after intervention, with group 4 showing the most outstanding antihypertensive effect, with systolic blood pressure approaching the level of the normal control group. At the same time, the plasma NO content significantly increased, indicating that the compound Lactobacillus acidophilus tablets of the present invention can effectively reduce blood pressure and improve vascular endothelial function. Although the systolic blood pressure of Comparative Example 1 and Comparative Example 2 was lower than that of the model group, the magnitude of the blood pressure reduction and the degree of NO content recovery were significantly less than those of Example 4. In particular, the NO content of Comparative Example 2 was lower than that of Comparative Example 1, showing a trend of best performance in Example 4, followed by Comparative Example 1, and worst performance in Comparative Example 2. This fully demonstrates the key role of modified cholesterol in protecting the gastrointestinal tolerance of bacteria and assisting in blood pressure reduction, as well as the unique contribution of methyl vanillyl glycoside in further improving liposome stability, promoting the release of endothelial relaxing factor NO, and amplifying the vasodilatory effect. The synergy of the two is an important guarantee for the high-efficiency blood pressure reduction of compound Lactobacillus acidophilus tablets.

[0096] Experimental Example 3

[0097] 100 μL of L929 fibroblasts (1 × 10⁻⁶ cells) in logarithmic growth phase were added to a 96-well plate. 5 The cell culture medium in the 96-well plates was DMEM complete medium (cells / mL). After culturing the 96-well plates in a 37°C, 5% CO2 incubator for 12 hours, the cell culture medium in each well was gently aspirated. 200 μL of DMEM complete medium containing 50 μg / mL of the compound Lactobacillus acidophilus liposome powder from Example 4 was added to each well, and the plates were cultured for another 24 hours. The 96-well plates were then removed, and cell morphology was observed under a microscope. Simultaneously, DMEM complete medium was used as a blank control group, and DMEM complete medium supplemented with 5% phenol was used as a positive control group. The results are as follows: Figure 1-3 As shown.

[0098] Figure 1 This is a graph showing the cytotoxicity test results of the blank control group. Figure 2 This is a graph showing the cytotoxicity test results of the compound Lactobacillus acidophilus liposome powder in Example 4. Figure 3 This is a graph showing the cytotoxicity test results for the positive control group. (Observation) Figure 1-3 It can be known that: Figure 1 The cells are typically elongated spindle-shaped, with uniform density and normal morphology. Figure 2 Cell morphology and Figure 1 Similar, with no obvious shrinkage. Figure 3The cells shrank, became rounded, and decreased in density, exhibiting typical toxicity characteristics. The above experimental results demonstrate that the compound Lactobacillus acidophilus liposome powder obtained in this invention has high safety.

[0099] The above embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention shall fall within the scope of protection claimed by the present invention.

Claims

1. The application of compound Lactobacillus acidophilus tablets in the preparation of drugs for treating hypertension, characterized in that, The preparation method of the compound Lactobacillus acidophilus tablets is as follows: (1) Soybean lecithin and modified cholesterol were added to anhydrous ethanol, and then vanillic acid methyl ester glycoside was added to prepare a modified lipid film. (2) The modified lipid film is ultrasonically hydrated with the compound Lactobacillus acidophilus solution, filtered, and freeze-dried to obtain compound Lactobacillus acidophilus liposome powder; pharmaceutically acceptable excipients are added to the compound Lactobacillus acidophilus liposome powder to make tablets. The structural formula of the modified cholesterol mentioned in step (1) is as follows: 。 2. The application of the compound Lactobacillus acidophilus tablets according to claim 1 in the preparation of drugs for treating hypertension, characterized in that, The method for preparing the modified cholesterol described in step (1) is as follows: (a) Hydrazine hydrate and triethylamine were added to anhydrous ethanol to obtain a mixed solution; the mixed solution was added to an anhydrous ethanol solution of 2,4,6-trichloro-5-pyrimidinecarboxaldehyde, and purified after reaction to obtain intermediate 1; The structural formula of intermediate 1 is as follows: ； (b) Under inert gas protection, cholesterol was added to 1,4-dioxane, sodium hydride was added and stirred, and then intermediate 1 was added. After reaction, the mixture was purified to obtain the modified cholesterol.

3. The application of the compound Lactobacillus acidophilus tablets according to claim 2 in the preparation of drugs for treating hypertension, characterized in that, The molar ratio of 2,4,6-trichloro-5-pyrimidinecarboxaldehyde, hydrazine hydrate, and triethylamine in step (a) is 1:(1.2-1.3):(1.5-1.6).

4. The application of the compound Lactobacillus acidophilus tablets according to claim 2 in the preparation of drugs for treating hypertension, characterized in that, The concentration of the anhydrous ethanol solution of 2,4,6-trichloro-5-pyrimidinecarboxaldehyde in step (a) is (0.3-0.35) mmol / mL; the concentration of triethylamine in the mixed solution is (0.45-0.5) mmol / mL; the reaction is carried out first at -78~-75℃ for 1-2 h, and then at 20-22℃ for 1-2 h.

5. The application of the compound Lactobacillus acidophilus tablets according to claim 2 in the preparation of drugs for treating hypertension, characterized in that, In step (b), the molar ratio of intermediate 1, cholesterol, and sodium hydride is 1:(2.25-2.5):(2.25-2.5); the stirring time is 30-45 min; the reaction temperature is 95-100℃ and the reaction time is 3-5 h.

6. The application of the compound Lactobacillus acidophilus tablets according to claim 1 in the preparation of drugs for treating hypertension, characterized in that, The total number of viable bacteria in the compound Lactobacillus acidophilus tablets is (1-5)×10⁻⁶. 9 CFU / g.

7. The application of the compound Lactobacillus acidophilus tablets according to claim 1 in the preparation of drugs for treating hypertension, characterized in that, In step (1), the mass ratio of soybean lecithin, modified cholesterol, and methyl vanillic acid glycoside is 1:(0.15-0.2):(0.16-0.25); in step (2), the ratio of compound Lactobacillus acidophilus solution to modified lipid film is 1mL:(2-3)g; the ultrasonic power is 150-180W and the time is 12-15min; the hydration temperature is 40-50℃ and the time is 1.5-2.5h.

8. The application of the compound Lactobacillus acidophilus tablets according to claim 7 in the preparation of drugs for treating hypertension, characterized in that, The preparation method of the compound Lactobacillus acidophilus solution is as follows: The Chinese strain of Lactobacillus acidophilus powder, the Japanese strain of Lactobacillus acidophilus powder, Enterococcus faecalis powder, and Bacillus subtilis powder were mixed evenly to prepare compound Lactobacillus acidophilus; the compound Lactobacillus acidophilus was added to a phosphate buffer solution to prepare the compound Lactobacillus acidophilus solution.

9. The application of the compound Lactobacillus acidophilus tablets according to claim 8 in the preparation of drugs for treating hypertension, characterized in that, The mass ratio of the Chinese strain of Lactobacillus acidophilus powder, the Japanese strain of Lactobacillus acidophilus powder, the fecal streptococcus powder, and the Bacillus subtilis powder is 1:1:1:0.002; the total concentration of each bacterial powder in the compound Lactobacillus acidophilus solution is 4-8 mg / mL; and the pH of the phosphate buffer solution is 7-7.2.

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