Traditional Chinese medicine formula YA3D3 and preparation method and application of liquid medicine

Abstract

The invention relates to the technical field of traditional Chinese medicine application, and particularly discloses a traditional Chinese medicine formula YA3D3 and a preparation method and application of liquid medicine. The traditional Chinese medicine formula YA3D3 is prepared from the following raw materials in parts by weight: 8.38 to 10.38 parts of folium mori, 6.81 to 8.81 parts of gypsum, 1.19 to 3.19 parts of apricot kernel, 2 to 4 parts of folium eriobotryae, 1.19 to 3.19 parts of ginseng, 9 to 11 parts of radix astragali seu hedysari, 9 to 11 parts of radix ophiopogonis, 9 to 11 parts of rhizoma atractylodis macrocephalae and 9 to 11 parts of radix codonopsis. Through a network pharmacology method, TCMSP, Uniprot and Metascape websites are used for screening traditional Chinese medicine ancient prescriptions, a TCMSP database is used for searching traditional Chinese medicine active ingredients to obtain related targets, a Uniprot database is used for correcting the detection targets to obtain human protein data, a new formula YA3D3 composed of nine traditional Chinese medicines is determined, and the traditional Chinese medicine formula YA3D3 can play a role in preventing the Alzheimer's disease.

Description

Technical Field

[0001] The present invention relates to the technical field of application of traditional Chinese medicine formulas, and in particular to a traditional Chinese medicine formula YA3D3, a preparation method of a medicinal liquid and application thereof. Background Art

[0002] Alzheimer's disease, also known as Alzheimer's disease in English, is a brain disease caused by damage to nerve cells in the brain. The first neurons to be damaged are the parts of the brain responsible for memory, language, and thinking. Therefore, the initial symptoms of AD are usually memory, language, and thinking problems. Although these symptoms are newly discovered for patients, the brain changes they cause are believed to begin 20 years or more before symptoms appear. Studies have shown that people aged 65 and over survive an average of 5 to 8 years after being diagnosed with Alzheimer's disease, but some people live as long as 20 years. The cost of care for AD will become a huge burden, both for society and families.

[0003] As AD research gradually advances, more and more evidence shows that the onset of AD is related to multiple factors, including family genetics, Aβ plaque deposition, neuroinflammation, infectious disease hypothesis, lysosomal acidification disorders, gene mutations, oxidative stress, and intestinal flora imbalance.

[0004] Currently, drugs approved for the treatment of AD, such as cholinesterase inhibitors and N-methyl-D-aspartate, referred to as NMDA receptor antagonists, are only symptomatic treatments. GV-971, sodium oligomannolate, and the new drug aducanumab, which are approved for the treatment of mild to moderate AD, have not yet achieved satisfactory results in clinical trials. Traditional Chinese medicine is low in price and toxicity. Traditional Chinese medicine formulas are composed of a combination of multiple single herbs and contain multiple active ingredients. They can intervene in the disease by acting on multiple drug targets, multiple pathways, and multiple levels. Therefore, it is necessary to develop a traditional Chinese medicine formula for the prevention of Alzheimer's disease. Summary of the invention

[0005] To solve the above problems, the present invention provides a traditional Chinese medicine formula YA3D3, a preparation method of a medicinal solution and application thereof.

[0006] The present invention is achieved through the following technical solutions:

[0007] A Chinese medicine formula YA3D3 is composed of the following raw materials in parts by weight:

[0008] 8.38 to 10.38 parts of mulberry leaves, 6.81 to 8.81 parts of gypsum, 1.19 to 3.19 parts of almonds, 2 to 4 parts of loquat leaves, 1.19 to 3.19 parts of ginseng, 9 to 11 parts of astragalus, 9 to 11 parts of ophiopogon, 9 to 11 parts of atractylodes, and 9 to 11 parts of codonopsis.

[0009] The method for preparing YA3D3 medicinal solution using the traditional Chinese medicine formula YA3D3 comprises the following steps:

[0010] The raw materials in the traditional Chinese medicine formula YA3D3 are prepared according to the weight ratio, the raw materials are crushed into powder respectively, 9 to 11 times the amount of water are added, mixed, and sterilized to obtain a mixture.

[0011] Bacillus subtilis and Aspergillus oryzae powder are inoculated into the mixture respectively to carry out the first fermentation culture to preliminarily change the composition and properties of the raw materials.

[0012] After the first fermentation culture, plant lactobacillus L9 and bifidobacterium longum powder are inoculated into the mixture respectively for a second fermentation culture to further improve the conversion of active substances; sterilization is performed; and the supernatant is collected to obtain the traditional Chinese medicine formula YA3D3 liquid.

[0013] Preferably, the inoculation amount of Bacillus subtilis accounts for 3% to 5% of the volume of the mixture; the inoculation amount of Aspergillus oryzae powder accounts for 0.1% to 0.3% of the volume of the mixture; the inoculation amount of Lactobacillus plantarum L9 accounts for 3% to 5% of the volume of the mixture; the inoculation amount of Bifidobacterium longum powder accounts for 3% to 5% of the volume of the mixture.

[0014] Preferably, the deposit number of the Bacillus subtilis is CMCC63501; the deposit number of the Lactobacillus plantarum L9 is CCTCC NO: M2022169.

[0015] Preferably, the food grade number of the inoculated Aspergillus oryzae powder is 3042; the food grade number of the Bifidobacterium longum powder is HH-BL18.

[0016] Preferably, the temperature of the first fermentation culture and the second fermentation culture are both 36° C. to 38° C., and the time is both 23 h to 25 h.

[0017] Preferably, the sterilization temperature is 75°C to 85°C, and the time is 25min to 35min.

[0018] Preferably, the particle size of the powder is 24 mesh to 65 mesh.

[0019] The invention discloses a decoction of a traditional Chinese medicine formula YA3D3, which is obtained by adding water to raw materials in the traditional Chinese medicine formula YA3D3 and then decocting the raw materials.

[0020] The application of the traditional Chinese medicine formula YA3D3, the prepared traditional Chinese medicine YA3D3 liquid or the traditional Chinese medicine formula YA3D3 decoction in the preparation of drugs for preventing Alzheimer's disease.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The invention provides a traditional Chinese medicine formula YA3D3, which is composed of the following raw materials in parts by weight: 8.38 to 10.38 parts of mulberry leaves, 6.81 to 8.81 parts of gypsum, 1.19 to 3.19 parts of almonds, 2 to 4 parts of loquat leaves, 1.19 to 3.19 parts of ginseng, 9 to 11 parts of astragalus, 9 to 11 parts of ophiopogon, 9 to 11 parts of atractylodes and 9 to 11 parts of codonopsis. The compatibility principle is to use ginseng as the main medicine, which can greatly replenish vital energy, soothe the mind and improve intelligence, restore the pulse and consolidate the body, directly act on the spleen and lung meridians, and strengthen the body's foundation; use astragalus as the assistant medicine, which complements ginseng, replenishes qi and raises yang, consolidates the exterior and stops sweating, so as to enhance the body's defense ability and promote qi and blood circulation; it is assisted by white atractylodes to strengthen the spleen and replenish qi, dry dampness and promote diuresis; codonopsis to replenish the spleen and benefit the lungs, nourish blood and produce body fluid; mulberry leaves to dispel wind-heat, clear the lungs and moisten dryness; ophiopogon to nourish yin and produce body fluid, moisten the lungs and clear the heart; gypsum to clear heat and purge fire, eliminate restlessness and quench thirst, and assist the main and assistant medicines to better play their roles; loquat leaves and bitter almonds are used as guiding medicines to harmonize the various medicines to ensure the harmony and unity of the entire prescription. The whole prescription comprehensively regulates the body through multiple pathways such as replenishing qi and nourishing blood, strengthening the spleen and benefiting the kidneys, soothing the mind and improving intelligence, so as to improve the cognitive dysfunction and quality of life of patients with Alzheimer's disease.

[0023] In addition, the present invention also improves the utilization rate of effective ingredients in traditional Chinese medicine through the preparation method of the medicinal solution transformed by microorganisms, and it is found that the spatial memory ability and memory impairment of APP / PS1 transgenic model mice after medication are significantly improved, which is significantly better than that without medication, indicating that YA3D3 can play a preventive role in the treatment of Alzheimer's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 This is a graph showing the effect of YA3D3 of the present invention on the total antioxidant capacity;

[0026] Figure 2 This is a result diagram showing the effect of YA3D3 of the present invention on the scavenging ability of hydroxyl free radicals in vitro;

[0027] Figure 3 This is a graph showing the effect of YA3D3 of the present invention on promoting PC12 cell activity in vitro;

[0028] Figure 3In the figure, a is the activity of PC12 cells after different concentrations of YA3D3-HF acted for 24 h; b is the activity of PC12 cells after different concentrations of YA3D3-MHF acted for 24 h; c is the activity of PC12 cells after different concentrations of YA3D3-HF acted for 48 h; d is the activity of PC12 cells after different concentrations of YA3D3-MHF acted for 48 h; *: P < 0.05 vs Control group;

[0029] Figure 4 The figure is a result diagram of the influence of YA3D3 of the present invention on the trajectory of APP / PS1 transgenic mice in the space exploration experiment; wherein, the red dot represents the starting position of the mouse swimming, and the blue dot represents the end position of the mouse swimming; Control is the normal control group; Model is the APP / PS1 blank control group; PC is the donepezil positive group; HL is the low dose of YA3D3-HF; HM is the medium dose of YA3D3-HF; HH is the high dose of YA3D3-HF; ML is the low dose of YA3D3-MHF; MM is the medium dose of YA3D3-MHF; MH is the high dose of YA3D3-MHF;

[0030] Figure 5 The figure is the result of the effect of YA3D3 of the present invention on the escape latency of APP / PS1 transgenic mice; wherein: Control is the normal control group; Model is the APP / PS1 blank control group; PC is the donepezil positive group; HL is the low dose of YA3D3-HF; HM is the medium dose of YA3D3-HF; HH is the high dose of YA3D3-HF; ML is the low dose of YA3D3-MHF; MM is the medium dose of YA3D3-MHF; MH is the high dose of YA3D3-MHF;

[0031] Figure 5 In the figure, a is the result of the effect of YA3D3 on the escape latency of APP / PS1 transgenic mice on the first day;

[0032] b is the effect of YA3D3 on the escape latency of APP / PS1 transgenic mice on the second day; c is the third day

[0033] The results of the effect of YA3D3 on the escape latency of APP / PS1 transgenic mice; d is the effect of YA3D3 on the escape latency of APP / PS1 transgenic mice on the fourth day.

[0034] Figure 2. Effect of escape latency on APP / PS1 transgenic mice;

[0035] Figure 6The figure is the result of the effect of YA3D3 of the present invention on the number of times of APP / PS1 transgenic mice crossing the platform; wherein: Control is the normal control group; Model is the APP / PS1 blank control group; PC is the donepezil positive group; HL is the low dose of YA3D3-HF; HM is the medium dose of YA3D3-HF; HH is the high dose of YA3D3-HF; ML is the low dose of YA3D3-MHF; MM is the medium dose of YA3D3-MHF; MH is the high dose of YA3D3-MHF; *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001 vs Model group; ###: P < 0.001, ####: P < 0.0001 vs Control group;

[0036] Figure 7 This is a graph showing the effect of YA3D3 of the present invention on Aβ plaque deposition in the brain tissue of APP / PS1 transgenic mice;

[0037] Figure 7 In the figure, a is the result of YA3D3 on Aβ plaque deposition in the brain tissue of APP / PS1 transgenic mice; among them: Control is the normal control group; Model is the APP / PS1 blank control group; Donepezil is the donepezil positive group; HF-H is the high dose of YA3D3-HF; HF-M is the medium dose of YA3D3-HF; HF-L is the low dose of YA3D3-HF; MHF-H is the high dose of YA3D3-MHF; MHF-M is the medium dose of YA3D3-MHF; MHF-L is the low dose of YA3D3-MHF; b is the effect of YA3D3 on APP / PS1 transgenic mice Statistical graphs corresponding to the results of Aβ plaque deposition in the brain tissue of S1 transgenic mice; among them: Control is the normal control group; Model is the APP / PS1 blank control group; PC is the donepezil positive group; HL is the low dose of YA3D3-HF; HM is the medium dose of YA3D3-HF; HH is the high dose of YA3D3-HF; ML is the low dose of YA3D3-MHF; MM is the medium dose of YA3D3-MHF; MH is the high dose of YA3D3-MHF; *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001 vs Model group; ####: P < 0.0001 vs Control group;

[0038] Figure 8The figure is the result of the effect of YA3D3 of the present invention on neurotransmitters in the brain tissue of APP / PS1 transgenic mice; wherein: Control is the normal control group; Model is the APP / PS1 blank control group; PC is the donepezil positive group; HL is the low dose of YA3D3-HF; HM is the medium dose of YA3D3-HF; HH is the high dose of YA3D3-HF; ML is the low dose of YA3D3-MHF; MM is the medium dose of YA3D3-MHF; MH is the high dose of YA3D3-MHF; *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001 vs Model group; ####: P < 0.0001 vs Control group;

[0039] Figure 8 In the figure, a is the bar graph of 5-HT, b is GABA, and c is the result bar graph of Glu;

[0040] Fig. 9 The figure is the result of the effect of YA3D3 of the present invention on the diversity of intestinal flora in feces of APP / PS1 transgenic mice; wherein: NM is normal C57BL / 6 mouse, which is the Control group; BC is APP / PS1 transgenic mouse, which is the Model group; PC is the positive control group, which is the Donepezil group; MH is the YA3D3-MHF high-dose administration group; MM is the YA3D3-MHF medium-dose administration group; ML is the YA3D3-MHF low-dose administration group; HM: is the YA3D3-HF medium-dose administration group; HL is the YA3D3-HF low-dose administration group; different colors represent different metabolic species, corresponding to the legend on the right; the horizontal axis represents different samples or groups, and the vertical axis represents the relative abundance of different species;

[0041] Fig. 9 In, a: Phylum; b: Class; c: Order; d: Family;

[0042] Fig.10 This is a graph showing the effect of YA3D3 of the present invention on short-chain fatty acids in feces of APP / PS1 transgenic mice. DETAILED DESCRIPTION

[0043] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively below, and preferred embodiments of the present invention are provided. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0045] The inventive concept of the present invention is as follows:

[0046] Currently, drugs approved for the treatment of AD, such as cholinesterase inhibitors and N-methyl-D-aspartate, referred to as NMDA receptor antagonists, are only symptomatic treatments. GV-971, sodium oligomannolate, and the new drug aducanumab, which are approved for the treatment of mild to moderate AD, have not yet achieved satisfactory results in clinical trials. Traditional Chinese medicine is low in price and toxicity. Traditional Chinese medicine formulas are composed of a combination of multiple single herbs and contain multiple active ingredients. They can intervene in the disease by acting on multiple drug targets, multiple pathways, and multiple levels. Therefore, it is necessary to develop a traditional Chinese medicine formula for the prevention of Alzheimer's disease.

[0047] The Chinese medicine formula YA3D3 is composed of the following raw materials by weight:

[0048] Mulberry leaves 8.38g~10.38g, gypsum 6.81g~8.81g, almond 1.19g~3.19g, loquat leaves 2g~4g, ginseng 1.19g~3.19g, astragalus 9g~11g, ophiopogon 9g~11g, atractylodes 9g~11g and codonopsis 9g~11g.

[0049] The compatibility principle of the traditional Chinese medicine formula YA3D3 is as follows:

[0050] Ginseng is the main drug, which can greatly replenish vital energy, soothe the mind and improve intelligence, restore the pulse and consolidate the body, directly act on the spleen and lung meridians, and strengthen the body's foundation; Astragalus is the auxiliary drug, which complements ginseng, replenishes qi and raises yang, consolidates the exterior and stops sweating, so as to enhance the body's defense ability and promote qi and blood circulation; Atractylodes is used to strengthen the spleen and replenish qi, dry dampness and promote diuresis; Codonopsis is used to replenish the spleen and benefit the lungs, nourish blood and produce fluid; mulberry leaves disperse wind-heat, clear the lungs and moisten dryness; Ophiopogon nourishes yin and produces fluid, moistens the lungs and clears the heart; Gypsum clears heat and purges fire, eliminates restlessness and quenches thirst, and assists the main and auxiliary drugs to better play their roles; loquat leaves and bitter almonds are used as guiding drugs to harmonize the various drugs to ensure the harmony and unity of the entire prescription. The whole prescription comprehensively regulates the body through multiple pathways such as replenishing qi and nourishing blood, strengthening the spleen and benefiting the kidneys, soothing the mind and improving intelligence, so as to improve the cognitive dysfunction and quality of life of patients with Alzheimer's disease.

[0051] Based on the same inventive concept, the present invention provides a method for preparing YA3D3 liquid medicine by using the traditional Chinese medicine formula YA3D3, comprising the following steps: preparing raw materials in the traditional Chinese medicine formula YA3D3 according to a weight ratio, crushing the raw materials into powders respectively, adding 9 to 11 times the amount of water to mix, sterilizing for the first time to obtain a mixture; inoculating Bacillus subtilis and Aspergillus oryzae powder into the mixture respectively, and performing a first fermentation culture; the inoculation amount of the Bacillus subtilis accounts for 3% to 5% of the volume of the mixture; the inoculation amount of the Aspergillus oryzae powder accounts for 0.1 to 0.3% of the volume of the mixture; after the first fermentation culture, inoculating plant lactobacillus L9 and long bifidobacterium powder into the mixture respectively; the inoculation amount of the plant lactobacillus L9 accounts for 3% to 5% of the volume of the mixture; the inoculation amount of the long bifidobacterium powder accounts for 3% to 5% of the volume of the mixture, and performing a second fermentation culture; second sterilization; collecting the supernatant to obtain the traditional Chinese medicine formula YA3D3 liquid medicine. This method adds microbial flora to the preparation of the medicinal solution to improve the utilization rate of the active ingredients in the traditional Chinese medicine, thereby intervening in AD and achieving a more effective effect.

[0052] The present invention uses experimental raw materials:

[0053] 1. Chinese medicinal raw materials: mulberry leaves, gypsum, almonds, loquat leaves, ginseng, astragalus, ophiopogon, atractylodes, codonopsis: purchased from Bozhou Chujianhuakai E-commerce Co., Ltd.

[0054] 2. The deposit number of Bacillus subtilis is CMCC63501, which is disclosed in “Du X, Lu C, Li Y, Zheng Z, Su W, Shen Y. Three new antimicrobial metabolites of Phomopsis sp. J Antibiot (Tokyo). 2008 Apr; 61(4): 250-3. doi: 10.1038 / ja.2008.37. PMID: 18503206.”.

[0055] 3. The food grade number of Aspergillus oryzae powder is 3042, which was purchased from Jining Xiangyuan Biotechnology Co., Ltd.

[0056] 4. The deposit number of Lactobacillus plantarum L9 is CCTCC NO: M2022169, which is disclosed in “Wang B, Chen J, Wang S, Zhao X, Lu G, Tang X. Lactobacillus plantarum L9 but not Lactobacillus acidophilus LA reduces tumour necrosis factor induced bacterial translocation in Caco-2 cells. Benef Microbes. 2017 May 30; 8(3): 497-505. doi: 10.3920 / BM2016.0019. Epub 2017 Apr 26. PMID: 28441885.”.

[0057] 5. The food grade number of Bifidobacterium longum powder is HH-BL18, which was purchased from Xi'an Mixianer Biotechnology Co., Ltd.

[0058] 6. PC12 cells: preserved by the research team.

[0059] 7. APP / PS1 transgenic mice: bred by the research team.

[0060] PC12 cell culture: cultured in complete medium containing 1640 medium + 10% FBS + 5% HS + 1% 100U / mL penicillin and 100μg / mL streptomycin, in a cell culture incubator with 5% CO2 at 37°C. When the cells grew to the logarithmic growth phase, they were digested with 0.25% trypsin for subsequent experiments.

[0061] The technical solution of the present invention will be further described below in conjunction with specific embodiments.

[0062] Example 1, a Chinese medicine formula YA3D3

[0063] Composed of the following raw materials by weight:

[0064] 9.38g mulberry leaf, 7.81g gypsum, 2.19g almond, 3g loquat leaf, 2.19g ginseng, 10g astragalus, 10g ophiopogon, 10g atractylodes, 10g codonopsis.

[0065] Example 2: Method for preparing YA3D3 medicinal solution from Chinese medicine formula YA3D3

[0066] The raw materials in the Chinese medicine formula YA3D3 of Example 1 were prepared according to the weight ratio, and the raw materials were crushed into powders with a particle size of 24 mesh respectively, and 9 times the amount of water was added to mix, and the mixture was placed in a culture bottle and sterilized at 75° C. for 35 minutes to obtain a mixture.

[0067] Bacillus subtilis and Aspergillus oryzae powder were inoculated into the mixture respectively, and the first fermentation culture was carried out at 36° C. for 23 hours; the inoculation amount of the Bacillus subtilis accounted for 3% of the volume of the mixture; the inoculation amount of the Aspergillus oryzae powder accounted for 0.1% of the volume of the mixture.

[0068] After the first fermentation and cultivation, plant lactobacillus L9 and long bifidobacterium powder are inoculated into the mixture respectively; the inoculation amount of plant lactobacillus L9 accounts for 3% of the volume of the mixture; the inoculation amount of long bifidobacterium powder accounts for 3% of the volume of the mixture, and the second fermentation and cultivation is carried out at 36°C for 23h; sterilized at 75°C for 35min; the supernatant is collected to obtain the traditional Chinese medicine formula YA3D3 liquid. Freeze-dried in a freeze dryer, stored at -20°C for standby use. It is called YA3D3-MHF.

[0069] Example 3: Method for preparing YA3D3 medicinal solution from Chinese medicine formula YA3D3

[0070] The raw materials in the Chinese medicine formula YA3D3 of Example 1 were prepared according to the weight ratio, and the raw materials were crushed into powders with a particle size of 45 mesh respectively, and 9 times the amount of water was added to mix, and the mixture was placed in a culture bottle and sterilized at 80° C. for 30 min to obtain a mixture.

[0071] Bacillus subtilis and Aspergillus oryzae powder were inoculated into the mixture respectively, and the first fermentation culture was carried out at 37° C. for 24 hours; the inoculation amount of the Bacillus subtilis accounted for 4% of the volume of the mixture; the inoculation amount of the Aspergillus oryzae powder accounted for 0.2% of the volume of the mixture.

[0072] After the first fermentation culture, plant lactobacillus L9 and long bifidobacterium powder are inoculated into the mixture respectively; the inoculation amount of plant lactobacillus L9 accounts for 4% of the volume of the mixture; the inoculation amount of long bifidobacterium powder accounts for 4% of the volume of the mixture, and the second fermentation culture is carried out at 37°C for 24 hours; sterilized at 80°C for 30 minutes; the supernatant is collected to obtain the traditional Chinese medicine formula YA3D3 liquid. Freeze-dried in a freeze dryer, stored at -20°C for standby use. It is called YA3D3-MHF.

[0073] Example 4: Method for preparing YA3D3 medicinal solution from Chinese medicine formula YA3D3

[0074] The raw materials in the Chinese medicine formula YA3D3 of Example 1 were prepared according to the weight ratio, and the raw materials were crushed into powders with a particle size of 65 mesh respectively, and 9 times the amount of water was added to mix, and the mixture was placed in a culture bottle and sterilized at 85° C. for 25 minutes to obtain a mixture.

[0075] Bacillus subtilis and Aspergillus oryzae powder were inoculated into the mixture respectively, and the first fermentation culture was carried out at 38° C. for 25 hours; the inoculation amount of the Bacillus subtilis accounted for 5% of the volume of the mixture; the inoculation amount of the Aspergillus oryzae powder accounted for 0.3% of the volume of the mixture.

[0076] After the first fermentation culture, plant lactobacillus L9 and long bifidobacterium powder are inoculated into the mixture respectively; the inoculation amount of plant lactobacillus L9 accounts for 5% of the volume of the mixture; the inoculation amount of long bifidobacterium powder accounts for 5% of the volume of the mixture, and the second fermentation culture is carried out at 38°C for 25h; sterilized at 85°C for 25min; the supernatant is collected to obtain the traditional Chinese medicine formula YA3D3 liquid. Freeze-dried in a freeze dryer, stored at -20°C for standby use. It is called YA3D3-MHF.

[0077] Example 5: A Chinese medicine formula YA3D3 decoction

[0078] Prepare the raw materials in the Chinese medicine formula YA3D3 in Example 1 according to the weight ratio, according to the raw material amount: water = 1kg: 10L, first fry the raw gypsum for 30 minutes, then fry all the medicines together, turn down the fire and fry for 40 minutes after the water boils, collect the first liquid medicine; add water 8 times the weight of the medicinal materials for the second decocting, collect the second liquid medicine, combine the first liquid medicine with the second liquid medicine, collect the supernatant liquid freeze-drying machine, seal to obtain the medicine, and store at -20°C. It is called YA3D3-HF.

[0079] Experimental Example 1: Effects of YA3D3 on PC12 cells and total antioxidant content and hydroxyl free radical scavenging ability

[0080] In this experimental example, different concentrations of YA3D3-MHF and YA3D3-HF were prepared using the YA3D3-MHF and YA3D3-HF prepared above and ultrapure water, respectively.

[0081] 1. Effect of YA3D3 on total antioxidant content and hydroxyl radical scavenging ability

[0082] Weigh 0.1g YA3D3-MHF and YA3D3-HF powders respectively and dissolve them in 1mL ultrapure water to obtain 100mg / mL YA3D3-MHF solution and YA3D3-HF solution, dilute them in multiples to obtain 2.5, 5, 10, 20, 40mg / mL YA3D3-MHF solution and YA3D3-HF solution for in vitro total antioxidant test, dilute them in multiples to obtain 0.625mg / mL, 1.25mg / mL, 2.5mg / mL, 5mg / mL, 10mg / mL, 20mg / mL YA3D3-MHF solution and YA3D3-HF solution for in vitro hydroxyl radical scavenging ability test, and each sample was measured in parallel 3 times. Fe 3+ The total antioxidant content and hydroxyl radical scavenging ability were determined by reduction kit.

[0083] The total antioxidant capacity T-AOC was calculated according to formula (1). The standard curve was drawn as follows:2+ The final concentration is the abscissa x, and the standard curve is drawn with ΔA as the ordinate y, and the linear regression equation y=kx+b is obtained. ΔA is substituted into the equation to obtain xμmol / mL; its unit is defined as: the antioxidant capacity of the sample is expressed in μmol / mL of the standard solution ion concentration required to achieve the same absorbance change value ΔA.

[0084] AOC (μmol / mL) = x × V total ÷ V sample = 34 × x (1)

[0085] Where: Vreaction: total reaction volume; Vsample: sample volume in the reaction.

[0086] The Fenton reaction kit method was used to measure the absorbance of the sample at 536 nm, and each sample was measured three times in parallel. The ability of the sample to scavenge hydroxyl free radicals was calculated according to formula (2). Hydroxyl free radical scavenging rate.

[0087] D% = (A test - A pair) ÷ (A empty - A pair) × 100% (2)

[0088] In the formula: Atest: the absorbance value of the measuring tube; Apair: the absorbance value of the control tube; Ablank: the absorbance value of the blank tube.

[0089] The experimental results are as follows Figure 1 and Figure 2 As shown in the figure, the in vitro total antioxidant capacity and hydroxyl radical scavenging capacity of YA3D3-MHF and YA3D3-HF were concentration-dependent, and the total antioxidant activity of YA3D3-MHF at the same concentration was strong, indicating that YA3D3 after microbial transformation, i.e. YA3D3-MHF, had a higher content of antioxidant active ingredients, and the utilization rate of the active ingredients of traditional Chinese medicine was improved.

[0090] Experimental Example 2: Effect of YA3D3 on promoting PC12 cell activity in vitro

[0091] After PC12 cells were cultured to 90% confluence in a culture flask, 3 × 10 3 Cells were cultured in an incubator for 24 h. The medium was replaced with 100 μL of complete medium containing YA3D3-HF and YA3D3-MHF at concentrations of 0 mg / mL, 0.03125 mg / mL, 0.0625 mg / mL, 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, and 2 mg / mL, respectively. After 24 and 48 h of treatment, the cell survival rate was calculated by CCK8.

[0092] The experimental results are as follows Figure 3After 24 hours of action of different concentrations of YA3D3-HF and YA3D3-MHF, compared with the control group, the cell activity within the concentration range selected in the experiment was greater than 80%, showing an effect of promoting cell proliferation; the trend of different concentrations of YA3D3-HF and YA3D3-MHF action for 48 hours was the same as that for 24 hours, and no cytotoxic effect was shown.

[0093] Experimental Example 3: Determination of the anti-AD effect of YA3D3

[0094] In this experimental example, different concentrations of YA3D3-MHF and YA3D3-HF were prepared using the YA3D3-MHF and YA3D3-HF prepared above and ultrapure water, respectively.

[0095] 1. Experimental Methods

[0096] (1) Mouse breeding and reproduction: Breeding mice were mated at a ratio of 2:1 between male and female, that is, one male APP / PS1 transgenic mouse was housed with two female C57 mice in the same cage, and pups were born about 20 days after mating. Mice generally gave birth to 5 to 10 pups each time, with a gestation period of 18 to 21 days. They were generally weaned at 3 weeks of age and started to be raised in separate cages at 4 weeks of age; the growth time of each generation was: 21 days of gestation + 21 days of separation + 21 days of sexual maturity.

[0097] (2) Experimental grouping: The 3-month-old experimental mice that met the experimental requirements were divided into groups of 8. Detailed grouping information is shown in Table 1.

[0098] Table 1

[0099]

[0100]

[0101] Note: “ / ” means there is no such item.

[0102] (3) Mouse water maze behavior experiment

[0103] After the drug intervention, mice in each group will be subjected to a navigation experiment in the first 4 days and a spatial exploration experiment on the fifth day to ensure that the mice are trained at the same time every day. In each navigation experiment, the time taken by the mice from entering the water maze to finding the escape platform is recorded as the escape latency. Each mouse has 60 seconds to find the escape platform. Mice that fail to find the platform within the specified time are manually guided to the platform and stay for 10 seconds; on the fifth day, the escape platform will be removed, and the mice will be placed in any quadrant and swim freely. The number of times the mice cross the original platform in 60 seconds and the time they stay in the original platform quadrant are recorded. During the Morris water maze experiment, experimental conditions such as water temperature, water quality, light, noise, etc. need to be strictly controlled to ensure the stability and reliability of the experimental results. At the same time, the selection, feeding and training of experimental animals are also key factors affecting the experimental results.

[0104] (4) Collection and preservation of mouse specimens

[0105] At the end of the behavioral experiment, mice in each group were fasted but not watered, and injected with 1% sodium pentobarbital to anesthetize the mice. After complete anesthesia, the brain and organs were removed by perfusion with normal saline for later use. The cranial nerves at the base of the skull were cut off from the removed mouse brain, and the left and right hemispheres were separated on ice; one half of the brain was quickly frozen in liquid nitrogen and transferred to a -80℃ refrigerator for storage, and the other half was immediately placed in 4% paraformaldehyde and fixed at 4℃ for 24 hours, then dehydrated with sucrose, embedded in 10%~20%~30% embedding agent, and set aside.

[0106] (5) Frozen sections and immunofluorescence

[0107] The cryostat was precooled in advance, the slice thickness was adjusted to 20 μm, the cut tissue was adhered to the slide, marked, and stored at -20 ° C. The sample was fixed with ice acetone at room temperature for 10 min; 0.3% Trition × 100, 15 min, 37 ° C, washed three times with 1 × PBS, 5 min / time; incubated at 37 ° C for 10 min with 3% H2O2; incubated at 37 ° C for 30 min with 10% goat serum; discarded the blocking solution, added APP working solution (dilution ratio of 1:500), and incubated overnight in a refrigerator at 4 ° C; after rewarming for 30 min, washed three times with 1 × PBS, 5 min / time, added fluorescent secondary antibody working solution, incubated at room temperature for 1 h; washed three times with 1 × PBS, 5 min / time, and sealed with anti-fluorescence quenching sealing medium containing DAPI.

[0108] (6) ELISA to detect neurotransmitter content

[0109] For the extraction of neurotransmitters from mouse tissues, the tissue samples were first completely thawed at 4°C, weighed, and then processed according to the tissue homogenate ratio; the homogenate ratio was selected to be 10%, the homogenate solution was pH = 7.2-7.4, 0.01M PBS; centrifuged at 5000rpm for 15 minutes, and the supernatant was taken for inspection. Add samples to the ELISA plate, set up blank wells, standard wells, and sample wells respectively, add 50μL of sample to each, seal the plate with a sealing film, incubate at 37℃ for 30min, remove the sealing film and discard the liquid to dry, fill each well with 1× washing solution, let it stand for 30s and discard, reread 5 times and pat dry; add 50μL of enzyme labeling reagent to each well, except the blank well, incubate at 37℃ for 30min, wash 5 times and pat dry, add 50μL of color developer A to each well, then add 50μL of color developer B, shake evenly, develop color at 37℃ in the dark for 10min, add 50μL of stop solution, adjust to zero with the blank well, measure absorbance at 450nm, and detect within 15min after adding the stop solution.

[0110] (7) Effects of YA3D3 on the diversity of intestinal flora and short-chain fatty acids in fecal samples of APP / PS1 transgenic AD model mice

[0111] The present invention commissioned Shanghai Zhongke Xinsheng Biotechnology Co., Ltd. to conduct the test. According to the characteristics of the amplified 16S region, a small fragment library was constructed and double-end sequencing was performed based on the sequencing platform using the paired-end sequencing method. By splicing and filtering reads, clustering the full English name of Operational Taxonomic Units, abbreviated as OTUs, species annotation and abundance analysis can reveal the species composition of the sample, and further explore the differences between samples and screen for differential bacterial communities.

[0112] Liquid chromatography tandem mass spectrometry, or LC-MS / MS detection technology, was used, and the basic technical process was as follows: sample collection - sample pretreatment - sample UPLC separation - MS / MS detection - data analysis. The short-chain fatty acids detected mainly included acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, pentanoic acid, and hexanoic acid.

[0113] 2. Experimental Results

[0114] 1. Effects of YA3D3 on learning and memory in APP / PS1 transgenic mice

[0115] Morris water maze navigation test Figure 4 As shown in Figure 2, the effect of YA3D3 on the escape latency of APP / PS1 transgenic mice is as follows Figure 5 As shown, on the first day, compared with the Control group, the Model group had a prolonged escape latency, and P<0.001; compared with the Model group, the Donepezil group, YA3D3-HF low dose, YA3D3-HF medium dose, YA3D3-MHF medium dose, and YA3D3-MHF high dose escape latency were shortened, and the difference was statistically significant, P<0.05. On the second day, compared with the Control group, the Model group had a prolonged escape latency, and P<0.001; compared with the Model group, the Donepezil group, YA3D3-HF low dose, YA3D3-HF medium dose, YA3D3-HF high dose, YA3D3-MHF low dose, YA3D3-MHF medium dose, and YA3D3-MHF high dose escape latency were shortened, and the difference was statistically significant, P<0.001. On the third day, compared with the Control group, the Model group had a prolonged escape latency, and P<0.001; compared with the Model group, the escape latency of the Donepezil group, YA3D3-HF low dose, YA3D3-HF medium dose, YA3D3-MHF low dose, and YA3D3-MHF high dose was shortened, and the difference was statistically significant, P<0.01. On the fourth day, compared with the Control group, the Model group had a prolonged escape latency, and P<0.001; compared with the Model group, the escape latency of the Donepezil group, YA3D3-HF low dose, YA3D3-HF medium dose, YA3D3-MHF medium dose, YA3D3-MHF low dose, and YA3D3-MHF high dose was shortened, and P<0.001, indicating that the escape latency of mice in each group shortened as the experimental days progressed. Effect of YA3D3 on the number of times APP / PS1 transgenic mice cross the platform is shown in Figure 6 As shown, it indicates that the positioning memory ability of the quadrant platform of each group of mice is improved with the increase of training times.

[0116] 2. Effect of YA3D3 on Aβ content in brain tissue of APP / PS1 transgenic mice

[0117] The results of immunofluorescence staining experiments showed that compared with the Model group mice, the Aβ deposition content in the brain of mice in each drug intervention group was reduced, and the difference was statistically significant (P<0.0001), including the high-dose water decoction group and YA3D3-HF-H, which increased the escape latency. It is speculated that YA3D3-HF and YA3D3-MHF promoted the clearance of Aβ in brain tissue, among which the medium-dose YA3D3-HF, the low-dose YA3D3-HF and YA3D3-MHF improved the long-term memory of AD mice. Figure 7 shown.

[0118] 3. Effects of YA3D3 on neurotransmitters in brain tissue of APP / PS1 transgenic mice

[0119] The results of ELISA experiments showed that compared with the Control group, the contents of 5-HT, GABA and Glu in the brain tissue of mice in the Model group were reduced, and the difference was statistically significant (P<0.05). Compared with the Model group, the expression of GABA in the brain tissue of mice in the YA3D3-HF high-dose group was increased (P<0.01), the expression of 5-HT, GABA and Glu in the brain tissue of mice in the YA3D3-HF medium and low-dose groups was increased, and the difference was statistically significant (P<0.001, P<0.0001). The expression of 5-HT, GABA, Glu in the brain tissue of mice in the YA3D3-MHF high-, medium- and low-dose groups was increased, and the difference was statistically significant (P<0.05, P<0.001, P<0.0001). Figure 8 shown.

[0120] 4. Effect of YA3D3 on the diversity of intestinal flora in feces of APP / PS1 transgenic mice

[0121] YA3D3 can change the abundance and biological function of intestinal flora in APP / PS1 transgenic mice. YA3D3 can change the content of short-chain fatty acids in APP / PS1 transgenic mice, thereby improving the cognitive function of model mice and the content of neurotransmitters in brain tissue, such as Fig. 9 and Fig.10 shown.

[0122] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0123] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims.

Claims

1. A Chinese medicine formula YA3D3, characterized in that: It is composed of the following raw materials in parts by weight: 8.38 to 10.38 parts of mulberry leaves, 6.81 to 8.81 parts of gypsum, 1.19 to 3.19 parts of almonds, 2 to 4 parts of loquat leaves, 1.19 to 3.19 parts of ginseng, 9 to 11 parts of astragalus, 9 to 11 parts of ophiopogon, 9 to 11 parts of atractylodes, and 9 to 11 parts of codonopsis.

2. A method for preparing YA3D3 liquid medicine using the traditional Chinese medicine formula YA3D3 according to claim 1, characterized in that: The following steps are involved: Prepare the raw materials in the Chinese medicine formula YA3D3 according to claim 1 according to weight parts, mix, crush into powder, add 9 to 11 times the amount of water, mix, sterilize for the first time, and obtain a mixture; Inoculating Bacillus subtilis and Aspergillus oryzae powder into the mixture respectively to carry out the first fermentation culture to preliminarily change the composition and properties of the raw materials; After the first fermentation culture, plant lactobacillus L9 and Bifidobacterium longum powder are inoculated into the mixture respectively for a second fermentation culture to further improve the conversion of active substances; a second sterilization is performed; and the supernatant is collected to obtain the traditional Chinese medicine formula YA3D3 liquid.

3. The method for preparing YA3D3 liquid medicine from the Chinese medicine formula YA3D3 as claimed in claim 2, characterized in that: The inoculation amount of Bacillus subtilis accounts for 3% to 5% of the volume of the mixture; the inoculation amount of Aspergillus oryzae powder accounts for 0.1% to 0.3% of the volume of the mixture; the inoculation amount of Lactobacillus plantarum L9 accounts for 3% to 5% of the volume of the mixture; and the inoculation amount of Bifidobacterium longum powder accounts for 3% to 5% of the volume of the mixture.

4. The method for preparing YA3D3 liquid medicine from the traditional Chinese medicine formula YA3D3 as claimed in claim 2, characterized in that: The deposit number of the Bacillus subtilis is CMCC63501; the deposit number of the Lactobacillus plantarum L9 is CCTCC NO: M2022169.

5. The method for preparing YA3D3 liquid medicine from the traditional Chinese medicine formula YA3D3 as claimed in claim 2, characterized in that: The food grade number of the inoculated Aspergillus oryzae powder is 3042; the food grade number of the Bifidobacterium longum powder is HH-BL18.

6. The method for preparing YA3D3 liquid medicine from the traditional Chinese medicine formula YA3D3 as claimed in claim 2, characterized in that: The temperature of the first fermentation culture and the second fermentation culture are both 36° C. to 38° C., and the time is both 23 h to 25 h.

7. The method for preparing YA3D3 liquid medicine from the traditional Chinese medicine formula YA3D3 as claimed in claim 2, characterized in that: The temperatures of the first sterilization and the second sterilization are both 75° C. to 85° C., and the time is both 25 min to 35 min.

8. The method for preparing YA3D3 liquid medicine from the traditional Chinese medicine formula YA3D3 as claimed in claim 2, characterized in that: The particle size of the powder is 24 mesh to 65 mesh.

9. A Chinese medicine formula YA3D3 decoction, characterized in that: The raw materials in the Chinese medicine formula YA3D3 of claim 1 are added with water and then decocted.

10. Use of the traditional Chinese medicine formula YA3D3 as claimed in claim 1, the traditional Chinese medicine YA3D3 liquid prepared according to claim 3, or the traditional Chinese medicine formula YA3D3 decoction as claimed in claim 9 in the preparation of drugs for preventing Alzheimer's disease.

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