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

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of a compound lactobacillus acidophilus tablet in preparation of a medicine for treating insomnia. According to weight parts, the compound lactobacillus acidophilus tablet comprises the following raw materials: 5-7 parts of probiotic powder, 25-35 parts of diluent, 15-20 parts of protective agent, 7-10 parts of coating material, 4-7 parts of disintegrating agent, 4-6 parts of adhesive, and 0.8-1.2 parts of lubricant. The tablet has excellent storage stability and gastrointestinal tolerance, and has a significant therapeutic effect on insomnia.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of compound Lactobacillus acidophilus tablets in the preparation of drugs for treating insomnia. Background Technology

[0002] Insomnia is the most common sleep disorder and the second most common mental disorder, characterized by difficulty initiating sleep, difficulty maintaining sleep, and poor sleep quality. Currently, clinical treatment for insomnia primarily involves chemical drugs, but long-term use can easily lead to significant side effects such as nerve damage and drug dependence.

[0003] In recent years, numerous studies have demonstrated a strong correlation between gut microbiota dysbiosis and sleep disorders. Therefore, regulating the gut microbiota through probiotics to improve sleep, promote restful sleep, and alleviate fatigue has become a potential health intervention strategy with broad market application prospects.

[0004] Lactobacillus acidophilus is a widely studied and applied probiotic in the human body. It produces lactic acid in the intestines, inhibiting the colonization of harmful bacteria and helping to maintain the microecological balance of the digestive and reproductive systems. It is often used to improve abdominal discomfort such as diarrhea and bloating. Enterococcus faecalis is a Gram-positive coccus and one of the normal flora of the human gut, with strong environmental adaptability. Bacillus subtilis is widely found in soil, plants, and the human gut and is often used as a probiotic in food, health products, and pharmaceuticals to regulate the intestinal microecology. 2,3,5,6-Tetramethylpyrazine is commonly used as a flavoring agent and sweetener in alcoholic beverages; however, there are currently no reports on its use for probiotic protection or its combination with probiotics. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, one of the objectives of this invention is to provide a compound Lactobacillus acidophilus tablet, which has both excellent storage stability and gastrointestinal tolerance, and has a significant therapeutic effect on insomnia.

[0006] The second objective of this invention is to provide a method for preparing compound Lactobacillus acidophilus tablets, which is simple in process and suitable for industrial production.

[0007] The third objective of this invention is to provide an application of compound Lactobacillus acidophilus tablets, which has broad application prospects.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] A compound Lactobacillus acidophilus tablet, by weight, comprises the following raw materials: 5-7 parts probiotic powder, 25-35 parts diluent, 15-20 parts protectant, 7-10 parts coating material, 4-7 parts disintegrant, 4-6 parts binder, and 0.8-1.2 parts lubricant;

[0010] The structural formula of the protective agent is:

[0011] .

[0012] Preferably, the coating material is composed of modified acrylic resin and hydroxypropyl methylcellulose phthalate in a mass ratio of 1:1.

[0013] Preferably, the preparation process of the modified acrylic resin is as follows:

[0014] An initiator and methyl methacrylate were added to acetonitrile, followed by the addition of methacrylic acid. The mixture was refluxed, and then evodiamine, di-tert-butyl dicarbonate, 4-dimethylaminopyridine, and 2,6-dimethylpyridine were added. After heating and reaction, the mixture was purified to obtain the modified acrylic resin.

[0015] The preparation principle of the modified acrylic resin of this invention is as follows:

[0016] Acrylic resin was prepared using methyl methacrylate and methacrylic acid as raw materials and potassium persulfate as an initiator. Then, modified acrylic resin was obtained by amidation reaction between the carboxyl groups of methacrylic acid retained in the acrylic resin and the secondary amine of evodiamine indole ring.

[0017] Preferably, the molar ratio of methyl methacrylate, methacrylic acid, initiator, evodiamine, di-tert-butyl dicarbonate, 4-dimethylaminopyridine, and 2,6-dimethylpyridine is 1:(1.05-1.10):(0.002-0.003):(1.08-1.12):(1.08-1.12):(0.01-0.02):(0.02-0.03); the initiator is potassium persulfate; the reflux reaction time is 2-4 h; and the heating reaction temperature is 35-40 °C for 10-16 h.

[0018] Preferably, the preparation process of the protective agent is as follows:

[0019] The protective agent was prepared by adding 3,6-dimethyl-2,5-pyrazinic acid, 1,4-dialkyl-2,5-diol, and p-toluenesulfonic acid to dioxane, stirring, and then purifying.

[0020] The preparation principle of the protective agent of this invention is as follows:

[0021] The present invention provides a protective agent by esterification of 3,6-dimethyl-2,5-pyrazinedicarboxylic acid with the hydroxyl group of 1,4-dialkyl-2,5-diol.

[0022] Preferably, the molar ratio of 3,6-dimethyl-2,5-pyrazinic acid, 1,4-dialkyl-2,5-diol, and p-toluenesulfonic acid is 1:(2-3):(0.5-0.8); the stirring temperature is 50-70℃, and the stirring time is 3-5h.

[0023] Preferably, the effective viable bacteria count in the compound Lactobacillus acidophilus tablets is (1-5) × 10⁻⁶. 9 CFU / g; the probiotic powder is composed of Chinese strain of Lactobacillus acidophilus powder, Japanese strain of Lactobacillus acidophilus powder, Enterococcus faecalis powder, and Bacillus subtilis powder in a mass ratio of 1:1:1:0.002; the diluent is composed of dextrin, microcrystalline cellulose, and mannitol in a mass ratio of 1:1.5:0.9; the binder is hydroxypropyl methylcellulose; the disintegrant is croscarmellose sodium or low-substituted hydroxypropyl cellulose; and the lubricant is magnesium stearate or calcium stearate.

[0024] The preparation method of the above-mentioned compound Lactobacillus acidophilus tablets includes the following steps:

[0025] S1. Mix probiotic powder, protectant, diluent, and disintegrant evenly, then add binder to prepare soft material, granulate, dry, granulate, add lubricant, mix evenly, compress into tablets, and obtain plain tablets;

[0026] S2. Add the coating material to anhydrous ethanol to prepare a coating solution;

[0027] S3. Coat the raw film with the coating solution and dry it.

[0028] Preferably, the concentration of the coating solution in step S2 is 0.2-0.3 g / mL; the coating temperature in step S3 is 40-50℃, and the coating time is 2-4 h.

[0029] The aforementioned compound Lactobacillus acidophilus tablets are used in the preparation of drugs for treating insomnia.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0031] 1. This invention provides a compound Lactobacillus acidophilus tablet, which has excellent storage stability and gastrointestinal tolerance, and has a significant therapeutic effect on insomnia.

[0032] 2. This invention enhances the storage stability of probiotic tablets by adding a protective agent with a specific structure. The ester bonds in the protective agent molecule can be slowly hydrolyzed in the storage microenvironment, continuously releasing glycolaldehyde as the smallest sugar molecule carbon source, providing the probiotics with the energy substrate required for metabolism, effectively maintaining their survival rate and biological activity during long-term storage. Simultaneously, the free hydroxyl groups on the dioxane ring can form a hydrogen bond network with water molecules, constructing a moisturizing microenvironment on the surface of the probiotics. The steric hindrance layer formed by the cyclic skeleton and the pyrazine ring can block direct attack from external oxygen, moisture, and temperature fluctuations on the bacteria, thus playing a physical barrier protective role. Furthermore, the protective agent retains the neuroprotective activity of the parent compound 2,3,5,6-tetramethylpyrazine, forming a synergistic effect with the probiotics and enhancing the application value of probiotics in the preparation of treatments for insomnia.

[0033] 3. This invention uses modified acrylic resin as one of the raw materials for the coating material, thereby improving the gastrointestinal tolerance of the bacterial tablets. In this modified acrylic resin, the indolinequinazolinone five-membered fused-ring skeleton of evodiamine has a highly sterically hindered rigid planar structure, which can effectively restrict the free rotation of the acrylic resin molecular chains, enhance the rigidity of the molecular chains, and thus improve the mechanical strength of the coating film. Simultaneously, this rigid fused-ring skeleton can improve the heat resistance of the resin, allowing the coating film to maintain good structural integrity and density under humid and hot environments, acting as a physical barrier to maintain the activity of probiotics. Furthermore, this enteric coating film works synergistically with enteric-coated cellulose derivatives to resist the erosion of gastric acid and bile, ensuring that probiotics successfully reach specific sites in the intestine for release, achieving effective treatment. Attached Figure Description

[0034] Figure 1 The infrared spectrum of the acrylic resin obtained in Comparative Example 2 is shown below.

[0035] Figure 2 This is the infrared spectrum of the modified acrylic resin obtained in Example 1. Detailed Implementation

[0036] The present invention will be further described in conjunction with 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 based on conventional conditions or product instructions. Unless otherwise specified, all reagents or instruments used are conventional products obtained through commercial channels.

[0037] The Chinese strain of Lactobacillus acidophilus powder, the Japanese strain of Lactobacillus acidophilus powder, Enterococcus faecalis powder, and Bacillus subtilis powder of this invention are all commercially available products. Among them, the Chinese strain of Lactobacillus acidophilus powder, the Japanese strain of Lactobacillus acidophilus powder, and the Enterococcus faecalis powder were purchased from Changchun Yuetong Technology Co., Ltd., and the Bacillus subtilis powder was purchased from Changchun Xianji Technology Co., Ltd.

[0038] In this invention, the probiotic powder is composed of Chinese strain of Lactobacillus acidophilus powder, Japanese strain of Lactobacillus acidophilus powder, Enterococcus faecalis powder, and Bacillus subtilis powder in a mass ratio of 1:1:1:0.002; the diluent is composed of dextrin, microcrystalline cellulose, and mannitol in a mass ratio of 1:1.5:0.9.

[0039] The preparation process of 3,6-dimethyl-2,5-pyrazinic acid is as follows:

[0040] Potassium permanganate (0.1 mol) was added to deionized water (150 mL), stirred until homogeneous, and then heated to 70 °C. 2,3,5,6-Tetramethylpyrazine (20 mmol) was added, stirred until homogeneous, and then heated to 90 °C for 4 h. After the reaction was completed, the reaction solution was filtered, the filter cake was washed with deionized water, and the filtrate was concentrated to obtain the residue. The pH of the residue was adjusted to 1 with 3 mol / L sulfuric acid solution, filtered, the filter cake was washed with deionized water, and dried to obtain 3,6-dimethyl-2,5-pyrazinedicarboxylic acid (CAS: 43015-44-7).

[0041] Preparation Example 1

[0042] The preparation process of modified acrylic resin is as follows:

[0043] With a molar ratio of methyl methacrylate, methacrylic acid, initiator, evodiamine, di-tert-butyl dicarbonate, 4-dimethylaminopyridine, and 2,6-dimethylpyridine of 1:1.07:0.0025:1.11:1.11:0.015:0.025, potassium persulfate and methyl methacrylate were added sequentially to acetonitrile. After stirring until homogeneous, methacrylic acid was added, and the mixture was refluxed for 3 hours. After cooling to room temperature, evodiamine, di-tert-butyl dicarbonate, 4-dimethylaminopyridine, and 2,6-dimethylpyridine were added to the reaction system, and the mixture was reacted at 38°C for 14 hours. After the reaction was completed, an appropriate amount of water was added to the reaction system to allow the resin to precipitate completely. The mixture was filtered, the filter cake was washed with deionized water, then pulped with deionized water for 30 minutes, filtered, and dried to obtain the modified acrylic resin.

[0044] Preparation Example 2

[0045] The preparation process of modified acrylic resin is as follows:

[0046] With methyl methacrylate, methacrylic acid, initiator, evodiamine, di-tert-butyl dicarbonate, 4-dimethylaminopyridine, and 2,6-dimethylpyridine in a molar ratio of 1:1.10:0.003:1.12:1.12:0.02:0.03, potassium persulfate and methyl methacrylate were added sequentially to acetonitrile. After stirring until homogeneous, methacrylic acid was added, and the mixture was refluxed for 4 hours. After cooling to room temperature, evodiamine, di-tert-butyl dicarbonate, 4-dimethylaminopyridine, and 2,6-dimethylpyridine were added to the reaction system, and the mixture was reacted at 40°C for 10 hours. After the reaction was completed, an appropriate amount of water was added to the reaction system to allow the resin to precipitate completely. The mixture was filtered, the filter cake was washed with deionized water, then pulped with deionized water for 30 minutes, filtered, and dried to obtain the modified acrylic resin.

[0047] Preparation Example 3

[0048] The preparation process of modified acrylic resin is as follows:

[0049] With a molar ratio of methyl methacrylate, methacrylic acid, initiator, evodiamine, di-tert-butyl dicarbonate, 4-dimethylaminopyridine, and 2,6-dimethylpyridine of 1:1.05:0.002:1.08:1.08:0.01:0.02, potassium persulfate and methyl methacrylate were added sequentially to acetonitrile. After stirring until homogeneous, methacrylic acid was added, and the mixture was refluxed for 2 hours. After cooling to room temperature, evodiamine, di-tert-butyl dicarbonate, 4-dimethylaminopyridine, and 2,6-dimethylpyridine were added to the reaction system, and the mixture was reacted at 35°C for 16 hours. After the reaction was completed, an appropriate amount of water was added to the reaction system to allow the resin to precipitate completely. The mixture was filtered, the filter cake was washed with deionized water, and then pulped with deionized water for 30 minutes. After filtration and drying, the modified acrylic resin was obtained.

[0050] Preparation Example 4

[0051] The preparation process of the protective agent is as follows:

[0052]

[0053] 3,6-Dimethyl-2,5-pyrazinic acid (43015-44-7, 10 mmol), 1,4-dialkyl-2,5-diol (CAS: 23147-58-2, 26 mmol), and p-toluenesulfonic acid (7 mmol) were added to dioxane (50 mL), stirred thoroughly at room temperature, and then heated to 60 °C and stirred for 4 h. The reaction solution was cooled to room temperature, the pH was adjusted to 9 with saturated sodium carbonate solution, and extracted with ethyl acetate. The organic phases were combined, washed successively with deionized water, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the protective agent. The NMR and mass spectrometry results of the protective agent are as follows:

[0054] 1 H NMR (C16 H 20 N2O 10 ,400MHz,CDCl3):δ7.03(t,2H),5.96(t,2H),4.47-3.85(m,8H),2.86(s,6H);HRMS(ESI+):[M+H]+ Calculated to be 401.11, found to be 401.11.

[0055] Preparation Example 5

[0056] The preparation process of the protective agent is as follows:

[0057] 3,6-Dimethyl-2,5-pyrazinic acid (10 mmol), 1,4-dialkyl-2,5-diol (30 mmol), and p-toluenesulfonic acid (8 mmol) were added to dioxane (50 mL), stirred until homogeneous at room temperature, heated to 70 °C, and stirred for 3 h. The reaction solution was cooled to room temperature, the pH was adjusted to 9 with saturated sodium carbonate solution, extracted with ethyl acetate, the organic phases were combined, washed successively with deionized water, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the protective agent. The NMR and mass spectrometry results of the protective agent were consistent with those of Preparation Example 4.

[0058] Preparation Example 6

[0059] The preparation process of the protective agent is as follows:

[0060] 3,6-Dimethyl-2,5-pyrazinic acid (10 mmol), 1,4-dialkyl-2,5-diol (20 mmol), and p-toluenesulfonic acid (5 mmol) were added to dioxane (50 mL), stirred until homogeneous at room temperature, heated to 50 °C, and stirred for 5 h. The reaction solution was cooled to room temperature, the pH was adjusted to 9 with saturated sodium carbonate solution, extracted with ethyl acetate, the organic phases were combined, washed successively with deionized water, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the protective agent. The NMR and mass spectrometry results of the protective agent were consistent with those of Preparation Example 4.

[0061] Example 1

[0062] A compound Lactobacillus acidophilus tablet, by weight, comprises the following raw materials: 6 parts probiotic powder, 32 parts diluent, 18 parts protective agent (Preparation Example 4), 9 parts coating material, 5 parts croscarmellose sodium, 5 parts hydroxypropyl methylcellulose, and 1 part magnesium stearate; the coating material is composed of modified acrylic resin and hydroxypropyl methylcellulose phthalate (Preparation Example 1) in a mass ratio of 1:1; the effective viable count of the compound Lactobacillus acidophilus tablet is 4 × 10⁻⁶. 9 CFU / g.

[0063] The preparation method of the above-mentioned compound Lactobacillus acidophilus tablets includes the following steps:

[0064] S1. Probiotic powder, protectant, diluent, and croscarmellose sodium are passed through a 90-mesh sieve, mixed evenly, and hydroxypropyl methylcellulose is added to prepare a soft material. The material is then passed through a 20-mesh sieve to make wet granules, dried at 45°C, granulated through a 16-mesh sieve, magnesium stearate is added, mixed evenly, and compressed into tablets to obtain plain tablets.

[0065] S2. Disperse the coating material in anhydrous ethanol to prepare a coating solution with a concentration of 0.25 g / mL;

[0066] S3. Place the raw film in a coating pan at 45°C, spray the above coating solution into it with a sprayer for coating for 3 hours, and dry it at 55°C for 6 hours to obtain the final product.

[0067] Example 2

[0068] A compound Lactobacillus acidophilus tablet, by weight, comprises the following raw materials: 7 parts probiotic powder, 35 parts diluent, 20 parts protective agent (from Preparation Example 5), 10 parts coating material, 7 parts low-substituted hydroxypropyl cellulose, 6 parts hydroxypropyl methylcellulose, and 1.2 parts calcium stearate; the coating material is composed of modified acrylic resin and hydroxypropyl methylcellulose phthalate (from Preparation Example 2) in a mass ratio of 1:1; the effective viable count of the compound Lactobacillus acidophilus tablet is 5 × 10⁻⁶. 9 CFU / g.

[0069] The preparation method of the above-mentioned compound Lactobacillus acidophilus tablets includes the following steps:

[0070] S1. Probiotic powder, protectant, diluent, and low-substituted hydroxypropyl cellulose are passed through a 100-mesh sieve, mixed evenly, and then hydroxypropyl methylcellulose is added to prepare a soft material. The material is then passed through a 30-mesh sieve to make wet granules, dried at 50°C, granulated through an 18-mesh sieve, calcium stearate is added, mixed evenly, and compressed into tablets to obtain plain tablets.

[0071] S2. Disperse the coating material in anhydrous ethanol to prepare a coating solution with a concentration of 0.3 g / mL;

[0072] S3. Place the uncoated film in a coating pan at 50°C, spray the above coating solution into the film using a sprayer for coating for 2 hours, and dry at 60°C for 5 hours to obtain the final product.

[0073] Example 3

[0074] A compound Lactobacillus acidophilus tablet, by weight, comprises the following raw materials: 5 parts probiotic powder, 25 parts diluent, 15 parts protective agent (Preparation Example 6), 7 parts coating material, 4 parts croscarmellose sodium, 4 parts hydroxypropyl methylcellulose, and 0.8 parts magnesium stearate; the coating material is composed of modified acrylic resin and hydroxypropyl methylcellulose phthalate (Preparation Example 3) in a mass ratio of 1:1; the effective viable count of the compound Lactobacillus acidophilus tablet is 1 × 10⁻⁶.9 CFU / g.

[0075] The preparation method of the above-mentioned compound Lactobacillus acidophilus tablets includes the following steps:

[0076] S1. Probiotic powder, protectant, diluent, and croscarmellose sodium are passed through an 80-mesh sieve, mixed evenly, and hydroxypropyl methylcellulose is added to prepare a soft material. The material is then passed through a 20-mesh sieve to make wet granules, dried at 40°C, granulated through a 16-mesh sieve, magnesium stearate is added, mixed evenly, and compressed into tablets to obtain plain tablets.

[0077] S2. Disperse the coating material in anhydrous ethanol to prepare a coating solution with a concentration of 0.2 g / mL;

[0078] S3. Place the raw film in a coating pan at 40°C, spray the above coating solution into it with a sprayer for coating for 4 hours, and dry it at 50°C for 8 hours to obtain the product.

[0079] Comparative Example 1

[0080] The difference between this comparative example and Example 1 is that the protective agent used in Preparation Example 4 is omitted.

[0081] Comparative Example 2

[0082] The difference between this comparative example and Example 1 is that the coating material consists of acrylic resin and hydroxypropyl methylcellulose phthalate in a mass ratio of 1:1. The acrylic resin is prepared as follows:

[0083] With a molar ratio of methyl methacrylate, methacrylic acid, and potassium persulfate of 1:1.07:0.0025, potassium persulfate and methyl methacrylate were added sequentially to acetonitrile, stirred until homogeneous, and then methacrylic acid was added. The mixture was refluxed for 3 hours. An appropriate amount of water was added to the reaction system to allow the resin to precipitate completely. The mixture was filtered, the filter cake was washed with deionized water, and then pulped with deionized water for 30 minutes. After filtration and drying, acrylic resin was obtained.

[0084] Experimental Example 1

[0085] The acrylic resin obtained in Comparative Example 2 and the modified acrylic resin obtained in Preparation Example 1 were analyzed by Fourier Transform Infrared Spectroscopy (FT-IR), and the results are as follows: Figure 1-2 As shown.

[0086] Figure 1 This is the infrared spectrum of the acrylic resin obtained in Comparative Example 2. Figure 2 This is the infrared spectrum of the modified acrylic resin obtained in Example 1. (Observation) Figure 1 It can be seen that: acrylic resin at 1722cm -1 The absorption peak at 1386 cm⁻¹ is attributed to the carbonyl group. -1 1448cm-1 2875-2987cm -1 The absorption peak at 1640 cm⁻¹ is attributed to the characteristic peaks of methylene and methyl groups. -1 The weak absorption peak signal indicates that almost all the double bonds in the monomer have been converted into single bonds, and the acrylic resin has been successfully prepared.

[0087] Depend on Figure 1-2 It can be seen that: compared to acrylic resin, modified acrylic resin has a lower viscosity at 1510 / 1457 cm⁻¹. -1 There were significantly more absorption peaks, which can be attributed to the unsaturated bonds on the benzene ring; 1645 cm⁻¹ -1 An additional, distinct absorption peak signal was observed at the [specific location], attributed to the amide carbonyl bond; other peaks remained almost unchanged. Compared to acrylates, the modified acrylic resin exhibited characteristic peaks of evodiamine, indicating that the modified acrylic resin was successfully prepared.

[0088] Experimental Example 2

[0089] Take 3g of each of the bacterial tablets obtained in the examples and comparative examples, store them in a sealed container at room temperature, and after different periods of storage, use the plate count method to detect the number of viable bacteria in each tablet and calculate the survival rate. The calculation formula is as follows:

[0090] Survival rate (%) = number of viable bacteria after test / number of viable bacteria before test × 100%, see Table 1 for results.

[0091] Table 1

[0092]

[0093] Table 1 shows that the viable bacterial survival rate of the compound Lactobacillus acidophilus tablets prepared in this invention remained above 73% after 9 months of sealed storage at room temperature, demonstrating excellent long-term stability. Comparative Example 1, lacking a protective agent, suffered from insufficient carbon source replenishment, a moisturizing microenvironment, and physical barrier protection, resulting in a 9-month survival rate of only 31.5%, confirming that the protective agent is a key component for maintaining activity. Comparative Example 2, using unmodified acrylic resin coating, had insufficient film density, resulting in a 9-month survival rate of 56.1%, significantly lower than the examples, indicating that the rigid fused ring structure of evodiamine in the modified acrylic resin effectively improved the barrier properties of the coating film.

[0094] Experimental Example 3

[0095] 3.1 Simulated Gastric Fluid Acid Resistance Test

[0096] Take 2g of the bacterial tablets obtained in Examples 1-3 and Comparative Examples 1-2, add them to 30mL of simulated gastric fluid 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 using the following formula: Survival rate (%) = (Number of viable cells after simulated digestion / Initial number of viable cells) × 100%. The results are shown in Table 2.

[0097] 3.2 Simulated artificial intestinal fluid bile salt tolerance test

[0098] Take 2g of the bacterial tablets obtained in Examples 1-3 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 3.1. Calculate the intestinal fluid tolerance survival rate, and the results are shown in Table 2.

[0099] Table 2

[0100]

[0101] Table 2 shows that the compound Lactobacillus acidophilus tablets prepared in this invention exhibit excellent tolerance in a simulated gastrointestinal environment. Examples 1-3 showed survival rates as high as 83.09-88.46% after 2 hours of simulated gastric digestion and maintained at 88.64-92.35% after 2 hours of simulated intestinal digestion, indicating that the dual barrier constructed by the protectant and modified acrylic resin coating effectively resisted the attack of gastric acid and bile salts. In contrast, Comparative Example 1, lacking a protectant, showed a 2-hour survival rate of only 63.65% in gastric juice and 74.72% in intestinal juice. Comparative Example 2, using unmodified ordinary acrylic resin coating, showed the worst protective effect, with a 2-hour survival rate of only 62.83% in gastric juice and only 70.19% in intestinal juice, indicating insufficient membrane density, easy destruction in a strongly acidic environment, and inability to provide an effective barrier for the bacteria. The above results confirm that the present invention achieves a high survival rate of bacteria in gastrointestinal fluid through the physical barrier and sustained-release effect of the protective agent, as well as the excellent acid resistance and density of the coating film imparted by the rigid fused ring structure of evodiamine in the modified acrylic resin, thus providing a reliable guarantee for the oral delivery of probiotics.

[0102] Test Example 4

[0103] 4.1 Experimental Animals and Grouping

[0104] Seventy SPF-grade ICR mice, weighing 18-22g, were acclimatized for one week under the following conditions: temperature 22±2℃, humidity 50±5%, 12-hour light-dark cycle, and free access to food and water. After the acclimatization period, the mice were randomly divided into seven groups: a blank control group (saline), a model group (saline), Examples 1-3, and Comparative Examples 1-2, with ten mice in each group.

[0105] 4.2 Administration method

[0106] Examples 1-3: with a total viable count of 1×10 8 CFU / animal / day, administer the bacterial tablets from Examples 1-3 by gavage once a day for 15 consecutive days;

[0107] Comparative Examples 1-2: with a total viable count of 1×10⁻⁶ 8 CFU / animal / day, administer 1-2 ratio of bacterial tablets via gavage once daily for 15 consecutive days;

[0108] Blank control group: The same volume of normal saline was administered by gavage once a day for 15 consecutive days.

[0109] The model group was given an equal volume of physiological saline by gavage once a day for 15 consecutive days.

[0110] 4.3 Model Construction

[0111] Starting from day 7 of drug administration, except for the blank control group, the other mice were intraperitoneally injected with p-chlorophenylalanine (PCPA) suspension at a dose of 400 mg / kg for 2 consecutive days. The blank control group was injected with an equal volume of physiological saline.

[0112] 4.4 Detection Methods

[0113] Thirty minutes after the last gavage administration, mice in each group were intraperitoneally injected with sodium pentobarbital solution (45 mg / kg bw, injection volume 0.1 mL / 10 g bw). The criterion for falling asleep was the disappearance of the righting reflex lasting for more than 60 seconds. The time from the disappearance of the righting reflex to its restoration was recorded as the sleep time. The results are shown in Table 3.

[0114] Table 3

[0115]

[0116] Table 3 shows that the compound Lactobacillus acidophilus tablets of this invention exhibit significant therapeutic effects in PCPA-induced insomnia model mice. The sleep time in the blank control group was 52.46 min, while in the model group it was shortened to 34.28 min, indicating successful PCPA modeling. The sleep times in Examples 1-3 recovered to 53.35 min, 50.82 min, and 48.16 min, respectively, approaching or reaching the level of the blank control group, confirming that the tablets of this invention can effectively combat insomnia caused by 5-HT depletion and restore normal sleep. The sleep time in Comparative Example 1 was 41.45 min, slightly longer than the model group, but due to the lack of a protectant, the bacterial survival rate was low, and the improvement effect was limited. The sleep time in Comparative Example 2 was only 38.12 min; due to the poor acid resistance of the ordinary acrylic resin coating, the intestinal delivery efficiency of live bacteria was the lowest, resulting in the weakest efficacy. The above results indicate that the synergistic effect of the protectant and the modified acrylic resin coating is key to ensuring the tablets exert their therapeutic effect on insomnia.

[0117] 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. A compound Lactobacillus acidophilus tablet, characterized in that, By weight, it includes the following ingredients: 5-7 parts probiotic powder, 25-35 parts diluent, 15-20 parts protectant, 7-10 parts coating material, 4-7 parts disintegrant, 4-6 parts binder, and 0.8-1.2 parts lubricant; The structural formula of the protective agent is: 。 2. The compound Lactobacillus acidophilus tablets according to claim 1, characterized in that, The coating material is composed of modified acrylic resin and hydroxypropyl methylcellulose phthalate in a mass ratio of 1:

1.

3. The compound Lactobacillus acidophilus tablets according to claim 2, characterized in that, The preparation process of the modified acrylic resin is as follows: An initiator and methyl methacrylate were added to acetonitrile, followed by the addition of methacrylic acid. The mixture was refluxed, and then evodiamine, di-tert-butyl dicarbonate, 4-dimethylaminopyridine, and 2,6-dimethylpyridine were added. After heating and reaction, the mixture was purified to obtain the modified acrylic resin.

4. The compound Lactobacillus acidophilus tablets according to claim 3, characterized in that, The molar ratio of methyl methacrylate, methacrylic acid, initiator, evodiamine, di-tert-butyl dicarbonate, 4-dimethylaminopyridine, and 2,6-dimethylpyridine is 1:(1.05-1.10):(0.002-0.003):(1.08-1.12):(1.08-1.12):(0.01-0.02):(0.02-0.03); the initiator is potassium persulfate; the reflux reaction time is 2-4 h; the heating reaction temperature is 35-40 °C, and the time is 10-16 h.

5. The compound Lactobacillus acidophilus tablets according to claim 1, characterized in that, The preparation process of the protective agent is as follows: The protective agent was prepared by adding 3,6-dimethyl-2,5-pyrazinic acid, 1,4-dialkyl-2,5-diol, and p-toluenesulfonic acid to dioxane, stirring, and then purifying.

6. The compound Lactobacillus acidophilus tablets according to claim 5, characterized in that, The molar ratio of 3,6-dimethyl-2,5-pyrazinic acid, 1,4-dialkyl-2,5-diol, and p-toluenesulfonic acid is 1:(2-3):(0.5-0.8); the stirring temperature is 50-70℃ and the stirring time is 3-5h.

7. The compound Lactobacillus acidophilus tablets according to claim 1, characterized in that, The effective viable bacteria count in the compound Lactobacillus acidophilus tablets is (1-5)×10⁻⁶. 9 CFU / g; the probiotic powder is composed of Chinese strain of Lactobacillus acidophilus powder, Japanese strain of Lactobacillus acidophilus powder, Enterococcus faecalis powder, and Bacillus subtilis powder in a mass ratio of 1:1:1:0.002; the diluent is composed of dextrin, microcrystalline cellulose, and mannitol in a mass ratio of 1:1.5:0.9; the binder is hydroxypropyl methylcellulose; the disintegrant is croscarmellose sodium or low-substituted hydroxypropyl cellulose; and the lubricant is magnesium stearate or calcium stearate.

8. A method for preparing compound Lactobacillus acidophilus tablets according to any one of claims 1-7, characterized in that, Includes the following steps: S1. Mix probiotic powder, protectant, diluent, and disintegrant evenly, then add binder to prepare soft material, granulate, dry, granulate, add lubricant, mix evenly, compress into tablets, and obtain plain tablets; S2. Add the coating material to anhydrous ethanol to prepare a coating solution; S3. Coat the raw film with the coating solution and dry it.

9. The method for preparing compound Lactobacillus acidophilus tablets according to claim 8, characterized in that, The concentration of the coating solution in step S2 is 0.2-0.3 g / mL; the coating temperature in step S3 is 40-50℃, and the coating time is 2-4 h.

10. The application of the compound Lactobacillus acidophilus tablets according to any one of claims 1-7, characterized in that, It is used in the preparation of drugs for treating insomnia.

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