Stem cell PQQ cordyceps sinensis ganoderma lucidum anthocyanin compound preparation for preventing aging and application of stem cell PQQ cordyceps sinensis ganoderma lucidum anthocyanin compound preparation

Abstract

The invention belongs to the field of health care products and biological medicines, and particularly discloses an anti-aging stem cell PQQ cordyceps sinensis ganoderma lucidum anthocyanin compound preparation and application thereof.The preparation is composed of apple stem cell extract, PQQ, cordyceps militaris extract, ganoderma lucidum extract, procyanidine and pharmaceutic adjuvants and can be made into capsules or pills or tablets. The preparation method comprises the following steps: extracting the raw material components, uniformly mixing, adding the procyanidine and the auxiliary materials, performing high-pressure homogenization treatment, and finally preparing different dosage forms. Wherein the preparation of the apple stem cell extract, the cordyceps militaris extract and the lucid ganoderma extract comprises detailed steps. The compound preparation can be used for preparing health care products or medicines for preventing / treating senile nervous system diseases, especially medicines for preventing / improving Alzheimer's symptoms. In addition, the invention also provides a pharmaceutical composition containing the compound preparation and an acetylcholin esterase inhibitor.

Description

Technical Field

[0001] The invention belongs to the field of health care products and biomedicine, and specifically discloses a stem cell PQQ cordyceps ganoderma anthocyanin compound preparation for anti-aging and application thereof. Background Art

[0002] With the aging of the global population, the incidence of geriatric diseases, especially neurological diseases such as Alzheimer's Disease (AD), has increased significantly, becoming a major social problem threatening human health. Alzheimer's disease not only seriously affects the quality of life of patients, but also brings heavy economic and mental burdens to families and society. At present, although there are some drugs and health products for geriatric neurological diseases on the market, these products often have many shortcomings.

[0003] On the one hand, although many drugs can temporarily relieve the symptoms of Alzheimer's disease, long-term use may cause serious side effects and even lead to drug dependence. These drugs usually work through a single target or mechanism, and it is difficult to fully cope with the complex pathological process of Alzheimer's disease. On the other hand, although health products on the market claim to have multiple health functions, their effects often lack sufficient scientific verification and may pose safety risks. These products have complex ingredients and lack scientific formulas and preparation processes, resulting in unclear interactions between ingredients, and may even produce antagonistic effects, thereby reducing the overall effect.

[0004] In addition, existing products for elderly neurological diseases are often too single and lack comprehensive treatment and health care solutions. Many products only focus on the protection of the nervous system, while ignoring anti-aging and other health care needs. This one-sided treatment strategy is difficult to meet the growing and diversified health needs of the elderly population.

[0005] In summary, the development of a safe, effective and comprehensive anti-aging and neuroprotective product, especially for the prevention and treatment of elderly neurological diseases such as Alzheimer's disease, has important social significance and economic value. Summary of the invention

[0006] In view of the above problems, the present invention discloses a stem cell PQQ cordyceps ganoderma anthocyanin compound preparation for anti-aging and its application. The compound preparation combines a variety of ingredients with anti-aging and neuroprotective effects through scientific formula and reasonable preparation process, aiming to provide a safe, effective and convenient health product or medicine for preventing or improving elderly nervous system diseases, especially Alzheimer's symptoms. At the same time, the present invention also provides the application of the compound preparation in the preparation of health products or medicines, and a pharmaceutical composition comprising the compound preparation and an acetylcholinesterase inhibitor, so as to further improve its prevention and treatment effects on Alzheimer's disease.

[0007] The purpose of the present invention is achieved through the following technical solutions.

[0008] A stem cell PQQ cordyceps ganoderma anthocyanin compound preparation for anti-aging, comprising the following raw materials in parts by weight:

[0009] 5-15 parts of apple stem cell extract, 0.1-3 parts of PQQ, 10-25 parts of Cordyceps militaris extract, 8-20 parts of Ganoderma lucidum extract, 5-18 parts of proanthocyanidins, and appropriate amount of pharmaceutical excipients;

[0010] The compound is in the form of capsules, pills or tablets;

[0011] The preparation method of the above-mentioned compound preparation comprises the following steps:

[0012] a) preparing apple stem cell extract, cordyceps militaris extract and ganoderma lucidum extract respectively;

[0013] b) Put the extract obtained in step a) into a three-dimensional motion mixer, add PQQ powder with a particle size of D90 ≤ 20 μm, mix for 15-45 minutes at 20-25°C and relative humidity < 30%, and the mixing uniformity RSD ≤ 5%;

[0014] c) adding proanthocyanidins and pharmaceutical excipients to the mixed material obtained in step b), injecting purified water to adjust the solid-liquid ratio to 1:3-5, and using a high-pressure homogenizer to circulate the mixture for 3-5 times at a pressure of not less than 50 MPa to control the slurry particle size D50 ≤ 10 μm;

[0015] d) Preparation into capsules, pills or tablets:

[0016] Capsules: The slurry is spray-dried to obtain powder, which is then filled into the shell of the HPMC capsule, and the moisture content of the capsule is controlled to be ≤5%;

[0017] Pills: The homogenized slurry is made into pills by dripping method or extrusion spheronization method;

[0018] Tablets: Tablets are compressed by a rotary tablet press at a pressure of 10-15 kN and coated with a film coat.

[0019] Furthermore, in the above-mentioned composite preparation, the preparation of the apple stem cell extract in step a) comprises the following steps:

[0020] Take young apple fruit tissue, wash it with phosphate buffer at pH 4.5-5.5, add cellulase and pectinase, stir it into a paste, hydrolyze it at 38-42℃ for 6-8h, centrifuge it at 3000-8000g for 10-15min, take the supernatant and separate it through a 50kDa ultrafiltration membrane, collect the filtrate and freeze-dry it to control the water content to less than 8%.

[0021] Preferably, the apples are selected from Swiss Uttwiler Spätlauber apples.

[0022] Furthermore, in the above-mentioned compound preparation, the added amount of cellulase is 30-50 U / g, and the added amount of pectinase is 50-100 U / g.

[0023] Furthermore, in the above-mentioned compound preparation, the preparation of the Cordyceps militaris extract in step a) comprises:

[0024] The fruiting bodies of Cordyceps militaris were crushed to less than 80 mesh, 60-70% ethanol was added at a solid-liquid ratio of 1:15-25, and reflux extraction was performed at 70-80°C for 3-5 times, each time for 1-2 hours. The combined extracts were concentrated under reduced pressure to a relative density of 1.08-1.15, and purified by AB-8 macroporous resin, first eluted with 3-5BV water, then eluted with 5-8BV 50% ethanol to extract the effective ingredients, and concentrated and dried to a water content of less than 5%.

[0025] Furthermore, in the above-mentioned compound preparation, the preparation of the Ganoderma lucidum extract in step a) comprises:

[0026] After the Ganoderma lucidum fruiting body is dried, it is crushed to less than 100 mesh, and 50-60% ethanol is added at a solid-liquid ratio of 1:20-30. Reflux extraction is performed at 60-70°C for 2-4 times, each time for 1.5-2.5 hours. The extracts are combined and concentrated under reduced pressure to a relative density of 1.10-1.20. Purification is performed by D101 macroporous resin, first eluting with 4-6BV water to remove impurities, and then eluting the effective ingredients with 6-10BV 70% ethanol. The extract is concentrated and dried to a water content of less than 3%.

[0027] Furthermore, in the above-mentioned compound preparation, the pharmaceutical excipients in step c) include one or more of fillers, disintegrants, and lubricants;

[0028] The filler is selected from at least one of lactose, starch, and microcrystalline cellulose;

[0029] The disintegrant is selected from at least one of cross-linked sodium carboxymethyl cellulose and sodium carboxymethyl starch;

[0030] The lubricant is selected from at least one of magnesium stearate and talc.

[0031] The invention also discloses the use of the compound preparation in preparing health products or medicines for preventing / treating elderly nervous system diseases.

[0032] Furthermore, in the above application, the drug is a drug for preventing / improving Alzheimer's symptoms.

[0033] The present invention also discloses a pharmaceutical composition for preventing / improving Alzheimer's symptoms, characterized in that it contains the above-mentioned compound preparation and an acetylcholinesterase inhibitor.

[0034] Compared with the existing technology, the present invention has the following advantages and beneficial effects:

[0035] 1. Comprehensive anti-aging and neuroprotective effects: The present invention uses a scientific formula to organically combine apple stem cell extract, pyrroloquinoline quinone (PQQ), Cordyceps militaris extract, Ganoderma lucidum extract and proanthocyanidins and other ingredients with anti-aging and neuroprotective effects to form a comprehensive health product or medicine. These ingredients work synergistically to comprehensively combat various pathological changes in the aging process, especially for the protection of the nervous system, with significant effects.

[0036] 2. Highly effective anti-Aβ deposition effect: The core pathological feature of Alzheimer's disease is the deposition of β-amyloid protein (Aβ). The compound preparation provided by the present invention can effectively inhibit the fibrosis process of Aβ and reduce its deposition in brain tissue, thereby delaying or preventing the development of Alzheimer's disease.

[0037] 3. Significantly improve cognitive function: Behavioral experiments on transgenic mice confirmed that the compound preparation provided by the present invention can significantly improve cognitive dysfunction in APP / PS1 double transgenic mice, including shortening escape latency, increasing target quadrant residence time, and improving new object recognition index, etc. This indicates that the compound preparation has a potential therapeutic effect on the recovery of cognitive function in patients with Alzheimer's disease.

[0038] 4. Safe and reliable: The raw materials used in the present invention are all natural plant extracts and bioactive substances, which are processed by scientific extraction and purification processes to ensure the safety and reliability of the product. At the same time, through reasonable formula and preparation process, the antagonism between the ingredients is avoided and the overall effect is improved.

[0039] 5. Various dosage forms, easy to take: The compound preparation provided by the present invention can be made into various dosage forms such as capsules, pills or tablets, which is convenient for patients to choose the way of taking according to their personal needs and preferences. At the same time, the preparation process is simple and feasible, and it is easy to mass produce and promote.

[0040] In summary, the stem cell PQQ cordyceps ganoderma anthocyanin compound preparation for anti-aging provided by the present invention has significant comprehensive anti-aging and neuroprotective effects, especially for the prevention and treatment of elderly nervous system diseases such as Alzheimer's disease. The development and application of this product will provide new options and hopes for the health care of the elderly population. DETAILED DESCRIPTION

[0041] A stem cell PQQ cordyceps ganoderma anthocyanin compound preparation for anti-aging, comprising the following raw materials in parts by weight:

[0042] 5-15 parts of apple stem cell extract, 0.1-3 parts of PQQ, 10-25 parts of Cordyceps militaris extract, 8-20 parts of Ganoderma lucidum extract, 5-18 parts of proanthocyanidins

[0043] The preparation method of the above-mentioned compound preparation comprises the following steps:

[0044] a) preparing apple stem cell extract, cordyceps militaris extract and ganoderma lucidum extract respectively;

[0045] b) Put the extract obtained in step a) into a three-dimensional motion mixer, add PQQ powder with a particle size of D90 ≤ 20 μm, mix for 15-45 minutes at 20-25°C and relative humidity < 30%, and the mixing uniformity RSD ≤ 5%;

[0046] c) adding proanthocyanidins and pharmaceutical excipients to the mixed material obtained in step b), injecting purified water to adjust the solid-liquid ratio to 1:3-5, and using a high-pressure homogenizer to circulate the mixture for 3-5 times at a pressure of not less than 50 MPa to control the slurry particle size D50 ≤ 10 μm;

[0047] d) Preparation into capsules, pills or tablets:

[0048] Capsules: The slurry is spray-dried to obtain powder, which is then filled into the shell of the HPMC capsule, and the moisture content of the capsule is controlled to be ≤5%;

[0049] Pills: The homogenized slurry is made into pills by dripping method or extrusion spheronization method;

[0050] Tablets: Tablets are compressed by a rotary tablet press at a pressure of 10-15 kN and coated with a film coat.

[0051] The preparation of the apple stem cell extract in step a) comprises the following steps:

[0052] Take young apple fruit tissue, wash it with phosphate buffer at pH 4.5-5.5, add cellulase and pectinase, stir it into a paste, hydrolyze it at 38-42℃ for 6-8h, centrifuge it at 3000-8000g for 10-15min, take the supernatant and separate it through a 50kDa ultrafiltration membrane, collect the filtrate and freeze-dry it to control the water content to less than 8%.

[0053] The added amount of the cellulase is 30-50 U / g, and the added amount of the pectinase is 50-100 U / g.

[0054] The preparation of the Cordyceps militaris extract in step a) comprises:

[0055] The fruiting bodies of Cordyceps militaris were crushed to less than 80 mesh, 60-70% ethanol was added at a solid-liquid ratio of 1:15-25, and reflux extraction was performed at 70-80°C for 3-5 times, each time for 1-2 hours. The combined extracts were concentrated under reduced pressure to a relative density of 1.08-1.15, and purified by AB-8 macroporous resin, first eluted with 3-5BV water, then eluted with 5-8BV 50% ethanol to extract the effective ingredients, and concentrated and dried to a water content of less than 5%.

[0056] The preparation of the ganoderma lucidum extract in step a) comprises:

[0057] After the Ganoderma lucidum fruiting body is dried, it is crushed to less than 100 mesh, and 50-60% ethanol is added at a solid-liquid ratio of 1:20-30. Reflux extraction is performed at 60-70°C for 2-4 times, each time for 1.5-2.5 hours. The extracts are combined and concentrated under reduced pressure to a relative density of 1.10-1.20. Purification is performed by D101 macroporous resin, first eluting with 4-6BV water to remove impurities, and then eluting the effective ingredients with 6-10BV 70% ethanol. The extract is concentrated and dried to a water content of less than 3%.

[0058] The pharmaceutical excipients in step c) include one or more of fillers, disintegrants, and lubricants;

[0059] The filler is selected from at least one of lactose, starch, and microcrystalline cellulose;

[0060] The disintegrant is selected from at least one of cross-linked sodium carboxymethyl cellulose and sodium carboxymethyl starch;

[0061] The lubricant is selected from at least one of magnesium stearate and talc.

[0062] To make the purpose, technical solutions and advantages of the present invention clearer, the present invention is further described in detail below. However, it should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the scope of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention. All raw materials in the embodiments of the present invention can be obtained through commercial channels.

[0063] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0064] Example 1

[0065] Capsules

[0066] 1. Raw material composition:

[0067] Apple Stem Cell Extract 12 Servings

[0068] PQQ 2 parts

[0069] Cordyceps militaris extract 20 parts

[0070] Ganoderma Lucidum Extract 15 parts

[0071] 15 parts of proanthocyanidins

[0072] 30 parts of microcrystalline cellulose

[0073] 5 parts of cross-linked carboxymethyl cellulose sodium

[0074] 1 part magnesium stearate

[0075] 2. Preparation process of extract:

[0076] 1) Extraction of apple stem cell extract:

[0077] Take 200 g of young apple fruit, wash it three times with pH 5.0 phosphate buffer, add cellulase (40 U / g) and pectinase (80 U / g), enzymolyze it at 40℃ for 7 h, centrifuge it at 6000g for 12 min, treat the supernatant with a 50 kDa ultrafiltration membrane (transmembrane pressure 0.3 MPa), and freeze-dry it to a water content of 6.2% to obtain a light yellow powder.

[0078] 2) Cordyceps militaris extract:

[0079] The fruiting bodies of Cordyceps militaris were crushed to 80 mesh, and 65% ethanol was added at a solid-liquid ratio of 1:20; reflux extraction was performed at 75°C for 4 times, each time for 1.5 hours, and the extracts were combined; the extracts were concentrated under reduced pressure to a density of 1.12 (60°C), eluted with AB-8 resin (column volume 500 mL) with water for 4 BV, and then eluted with 50% ethanol for 7 BV, and concentrated and dried to obtain a brown powder;

[0080] 3) Ganoderma Lucidum Extract:

[0081] Ganoderma lucidum fruiting bodies were crushed to 100 mesh, 55% ethanol was added at a solid-liquid ratio of 1:25, and refluxed at 65°C for 3 times, each time for 2 hours. After concentrating to a density of 1.15, the mixture was passed through D101 resin (column volume 800 mL), eluted with water for 5 BV, eluted with 70% ethanol for 8 BV, and dried to obtain a light brown powder.

[0082] Mixing and homogenization:

[0083] The above three extracts and PQQ were put into a three-dimensional mixer (speed 15 rpm), mixed for 35 min (RSD = 3.8%) at 22 ° C and 25% humidity, and proanthocyanidins and auxiliary materials were added. Purified water was added to a solid-liquid ratio of 1:4, and high-pressure homogenization (55 MPa) was cycled 4 times, and the slurry D50 = 8.2 μm;

[0084] 3. Capsule preparation process:

[0085] The flowable powder was obtained by spray drying (inlet air temperature 180°C, outlet air temperature 85°C), and the capsule shell (model 00#) was used with a filling amount of 500 mg / capsule.

[0086] Example 2

[0087] tablet

[0088] Apple Stem Cell Extract 15 Servings

[0089] PQQ 3 parts

[0090] Cordyceps militaris extract 25 parts

[0091] Ganoderma Lucidum Extract 20 parts

[0092] 18 parts of proanthocyanidins

[0093] Lactose 40 parts

[0094] 8 parts of sodium carboxymethyl starch

[0095] 2 parts talcum powder

[0096] 2. Preparation process of extract:

[0097] Same as Example 1.

[0098] 3. Tablet preparation process

[0099] Nitrogen protection was used during mixing (oxygen content < 0.5%), tableting pressure was 12kN, tablet weight was 1000mg,

[0100] Coating liquid formula: Opadry II (weight gain 3%).

[0101] Example 3

[0102] pill

[0103] 1. Raw material composition:

[0104] Apple Stem Cell Extract 8 Servings

[0105] PQQ 0.5 parts

[0106] 15 parts of Cordyceps militaris extract

[0107] Ganoderma Lucidum Extract 10 parts

[0108] 8 parts of proanthocyanidins

[0109] Microcrystalline cellulose 20 parts

[0110] Mannitol 15 parts

[0111] 2. Preparation process of extract:

[0112] Same as Example 1.

[0113] 3. Pill preparation process

[0114] Pellet making by extrusion-spheronization method:

[0115] After homogenization, the slurry was mixed with the auxiliary materials (wet granulation, L / S ratio 0.8), the extrusion speed was 30 rpm, the mesh size was 1.0 mm, the spheronization time was 8 min, and the pellets with a diameter of 1.2 mm were obtained, which were then fluidized bed dried (40°C) to a moisture content of ≤3%.

[0116] Comparative Example 1

[0117] Except for the apple stem cell extract, the remaining components are the same as those in Example 1, and the preparation process is the same as that in Example 1.

[0118] Comparative Example 2

[0119] Except for PQQ, the remaining components and the preparation process are the same as those in Example 1.

[0120] Comparative Example 3

[0121] Meanwhile, the apple stem cell extract and PQQ were removed, and the remaining components and the preparation process were the same as those in Example 1.

[0122] Example 4

[0123] Drug combination

[0124] One portion of donepezil (added together with proanthocyanidins) was added to the active ingredients in Example 1, and the remaining components were the same as in Example 1, and the preparation process was the same as in Example 1.

[0125] Test Case

[0126] Active ingredient effectiveness testing.

[0127] The active ingredients of Example 1 include 12 parts of apple stem cell extract, 2 parts of PQQ, 20 parts of Cordyceps militaris extract, 15 parts of Ganoderma lucidum extract, and 15 parts of proanthocyanidins.

[0128] The active ingredients of Comparative Example 1 include 2 parts of PQQ, 20 parts of Cordyceps militaris extract, 15 parts of Ganoderma lucidum extract, and 15 parts of proanthocyanidins.

[0129] The active ingredients of Comparative Example 2 include 12 parts of apple stem cell extract, 20 parts of Cordyceps militaris extract, 15 parts of Ganoderma lucidum extract, and 15 parts of proanthocyanidins.

[0130] The active ingredients of Comparative Example 3 include 20 parts of Cordyceps militaris extract, 15 parts of Ganoderma lucidum extract, and 15 parts of proanthocyanidins.

[0131] The active ingredients of Example 4 (drug combination) include 12 parts of apple stem cell extract, 2 parts of PQQ, 20 parts of Cordyceps militaris extract, 15 parts of Ganoderma lucidum extract, 15 parts of proanthocyanidins, and 1 part of donepezil.

[0132] Positive control: donepezil

[0133] Negative control: pure water

[0134] The above active ingredients are added into ultrapure water to prepare active solution.

[0135] 1. In vitro neuronal cell protection experiment

[0136] 1. Cell culture and model establishment

[0137] Cell line: PC12 cells (rat pheochromocytoma cell line).

[0138] Culture medium: RPMI 1640 + 10% FBS + 1% penicillin / streptomycin.

[0139] Induced injury: Aβ25-35 peptide (20 μM, pre-treated and incubated at 37°C for 48 h to form aggregates).

[0140] 2. Experimental Grouping

[0141] Treatment conditions of groups (final concentration)

[0142] Negative control group pure water treatment

[0143] Model group Aβ25-35 (20 μM)

[0144] Example 1 Group Aβ25-35 + 50 μg / mL Example 1 Active Solution

[0145] Example 4 Group Aβ25-35 + 50 μg / mL Example 4 Active Solution

[0146] Comparative Example 1 Group Aβ25-35 + 50μg / mL Comparative Example 1 Active Solution

[0147] Comparative Example 2 Group Aβ25-35 + 50μg / mL Comparative Example 2 Active Solution

[0148] Comparative Example 3 Group Aβ25-35 + 50μg / mL Comparative Example 3 Active Solution

[0149] The positive control group was Aβ25-35 + 10 μM donepezil.

[0150] 3. Detection indicators

[0151] Cell viability: CCK-8 reagent (absorbance at 450 nm).

[0152] Oxidative stress: ROS levels (DCFH-DA fluorescent probe).

[0153] Mitochondrial function: membrane potential (JC-1 staining), ATP content.

[0154] The experimental results are shown in Table 1.

[0155]

[0156] 4. Experimental results analysis

[0157] Cell Viability:

[0158] The cell survival rate in the model group was significantly reduced to 42.5%, indicating that the Aβ25-35 peptide fragment had a significant damaging effect on nerve cells.

[0159] The cell survival rate of the Example 1 group was increased to 84.7%, which was significantly higher than that of the model group, indicating that the compound preparation has a significant protective effect on nerve cells.

[0160] The cell survival rate of Example 4 group (containing donepezil) was higher, reaching 93.2%, indicating that the combination of the compound preparation and the acetylcholinesterase inhibitor can further enhance the protective effect.

[0161] The cell survival rates of Comparative Examples 1, 2, and 3 were all lower than those of Example 1, indicating that apple stem cell extract and PQQ played an important role in the compound preparation.

[0162] Oxidative stress (ROS levels):

[0163] The ROS level in the model group was significantly increased, indicating that the Aβ25-35 peptide induced oxidative stress.

[0164] The ROS levels in the Example 1 group and the comparative example group to which the compound preparation was added were both reduced, and the reduction in the Example 1 group was the most significant, indicating that the compound preparation can effectively inhibit oxidative stress.

[0165] Mitochondrial function (membrane potential, ATP content):

[0166] The mitochondrial membrane potential and ATP content in the model group decreased, indicating that the Aβ25-35 peptide fragment damaged mitochondrial function.

[0167] The mitochondrial membrane potential and ATP content of the Example 1 group were significantly restored, indicating that the compound preparation can improve mitochondrial function.

[0168] II. Anti-Aβ deposition (in vitro) experiment

[0169] 1. Aβ42 fibrosis inhibition experiment

[0170] Aβ42 treatment: dissolved in hexafluoroisopropanol (HFIP), vacuum dried and resuspended in PBS (50 μM).

[0171] Group processing:

[0172] Treatment conditions of groups (final concentration)

[0173] Negative control group pure water treatment

[0174] Model group Aβ42 (50 μM)

[0175] Example 1 Group Aβ42 + 100 μg / mL Example 1 Active Solution

[0176] Example 4 Group Aβ42 + 100 μg / mL Example 4 Active Solution

[0177] The positive control group was Aβ42 + 10 μM donepezil.

[0178] 2. Detection Method

[0179] Thioflavin T fluorescence method: Ex / Em=440 / 490nm.

[0180] 3. Test results are shown in Table 2

[0181]

[0182] 4. Experimental results analysis

[0183] Experimental results analysis:

[0184] Fluorescence intensity and fiber length:

[0185] The model group showed a high degree of Aβ42 fibrillation, strong fluorescence intensity, and long fiber length.

[0186] The fluorescence intensity of the Example 1 group was significantly reduced, and the fiber length was significantly shortened, indicating that the compound preparation can effectively inhibit the fibrosis of Aβ42.

[0187] The inhibitory effect of Example 4 group was better, indicating that the combination of the compound preparation and the acetylcholinesterase inhibitor can further enhance the anti-Aβ deposition effect.

[0188] Inhibition rate and EC50:

[0189] The inhibition rate of Example 1 group was high and the EC50 value was low, indicating that the compound preparation had a strong inhibitory effect on Aβ42 fibrosis.

[0190] 3. Behavioral Experiments of Transgenic Mice

[0191] Experimental procedures

[0192] 1. Animal Model

[0193] Strain: APP / PS1 double transgenic mice (6 months old, n=10 / group).

[0194] Grouping and treatment (gavage, once a day × 8 weeks)

[0195] Group treatment conditions (200 mg / kg)

[0196] Wild-type group pure water

[0197] Model group: pure water

[0198] Example 1 Group Example 1 Active Liquid

[0199] Example 4 Group Example 4 Active Liquid

[0200] The positive control group was given donepezil (2 mg / kg).

[0201] 2. Behavioral testing

[0202] Morris water maze: Escape latency and target quadrant residence time were recorded.

[0203] New object recognition: Calculate the recognition index.

[0204] 3. Experimental results

[0205] The experimental results are shown in Table 3.

[0206]

[0207] 4. Experimental results analysis:

[0208] Morris water maze:

[0209] The escape latency in the model group was prolonged and the residence time in the target quadrant was reduced, indicating that APP / PS1 double transgenic mice had cognitive dysfunction.

[0210] The escape latency of the Example 1 group was shortened and the target quadrant residence time was increased, indicating that the compound preparation can improve the cognitive function of mice.

[0211] The improvement effect of Example 4 group was better, which further proved the advantage of the combination of the compound preparation and the acetylcholinesterase inhibitor.

[0212] New object recognition:

[0213] The recognition index of the model group decreased, indicating that the mice had impaired memory.

[0214] The recognition index of the Example 1 group was improved, indicating that the compound preparation can enhance the memory of mice.

[0215] Based on the above embodiments and test examples, the following conclusions can be drawn:

[0216] The stem cell PQQ cordyceps ganoderma anthocyanin compound preparation for anti-aging provided by the present invention has significant neuroprotective effect and anti-Aβ deposition effect, and is expected to become a safe, effective and convenient health product or medicine for preventing or improving elderly nervous system diseases, especially Alzheimer's symptoms.

[0217] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Therefore, based on the innovative concept of the present invention, changes and modifications to the embodiments described herein, or equivalent structures or equivalent process transformations made using the contents of the present invention specification, directly or indirectly applying the above technical solutions to other related technical fields are all included in the protection scope of the patent of the present invention.

Claims

1. A stem cell PQQ cordyceps ganoderma anthocyanin compound preparation for anti-aging, characterized in that: It is composed of the following raw materials in parts by weight: 5-15 parts of apple stem cell extract, 0.1-3 parts of PQQ, 10-25 parts of Cordyceps militaris extract, 8-20 parts of Ganoderma lucidum extract, 5-18 parts of proanthocyanidins, and appropriate amount of pharmaceutical excipients; The compound is in the form of capsules, pills or tablets; The preparation method of the above-mentioned compound preparation comprises the following steps: a) preparing apple stem cell extract, cordyceps militaris extract and ganoderma lucidum extract respectively; b) Put the extract obtained in step a) into a three-dimensional motion mixer, add PQQ powder with a particle size of D90 ≤ 20 μm, mix for 15-45 minutes at 20-25°C and relative humidity < 30%, and the mixing uniformity RSD ≤ 5%; c) adding proanthocyanidins and pharmaceutical excipients to the mixed material obtained in step b), injecting purified water to adjust the solid-liquid ratio to 1:3-5, and using a high-pressure homogenizer to circulate the mixture for 3-5 times at a pressure of not less than 50 MPa to control the slurry particle size D50 ≤ 10 μm; d) Preparation into capsules, pills or tablets: Capsules: The slurry is spray-dried to obtain powder, which is then filled into the shell of the HPMC capsule, and the moisture content of the capsule is controlled to be ≤5%; Pills: The homogenized slurry is made into pills by dripping method or extrusion spheronization method; Tablets: Tablets are compressed by a rotary tablet press at a pressure of 10-15 kN and coated with a film coat.

2. The compound preparation according to claim 1, characterized in that: The preparation of the apple stem cell extract in step a) comprises the following steps: Take young apple fruit tissue, wash it with phosphate buffer at pH 4.5-5.5, add cellulase and pectinase, stir it into a paste, hydrolyze it at 38-42℃ for 6-8h, centrifuge it at 3000-8000g for 10-15min, take the supernatant and separate it through a 50kDa ultrafiltration membrane, collect the filtrate and freeze-dry it to control the water content to less than 8%.

3. The compound preparation according to claim 2, characterized in that: The added amount of the cellulase is 30-50 U / g, and the added amount of the pectinase is 50-100 U / g.

4. The compound preparation according to claim 2, characterized in that: The preparation of the Cordyceps militaris extract in step a) comprises: The fruiting bodies of Cordyceps militaris were crushed to less than 80 mesh, 60-70% ethanol was added at a solid-liquid ratio of 1:15-25, and reflux extraction was performed at 70-80°C for 3-5 times, each time for 1-2 hours. The combined extracts were concentrated under reduced pressure to a relative density of 1.08-1.15, and purified by AB-8 macroporous resin, first eluted with 3-5BV water, then eluted with 5-8BV 50% ethanol to extract the effective ingredients, and concentrated and dried to a water content of less than 5%.

5. The compound preparation according to claim 2, characterized in that: The preparation of the ganoderma lucidum extract in step a) comprises: After the Ganoderma lucidum fruiting body is dried, it is crushed to less than 100 mesh, and 50-60% ethanol is added at a solid-liquid ratio of 1:20-30. Reflux extraction is performed at 60-70°C for 2-4 times, each time for 1.5-2.5 hours. The extracts are combined and concentrated under reduced pressure to a relative density of 1.10-1.

20. Purification is performed by D101 macroporous resin, first eluting with 4-6BV water to remove impurities, and then eluting the effective ingredients with 6-10BV 70% ethanol. The extract is concentrated and dried to a water content of less than 3%.

6. The compound preparation according to claim 2, characterized in that: The pharmaceutical excipients in step c) include one or more of fillers, disintegrants, and lubricants; The filler is selected from at least one of lactose, starch, and microcrystalline cellulose; The disintegrant is selected from at least one of cross-linked sodium carboxymethyl cellulose and sodium carboxymethyl starch; The lubricant is selected from at least one of magnesium stearate and talc.

7. A pharmaceutical composition for preventing / improving Alzheimer's symptoms, characterized in that: Contains the compound preparation according to any one of claims 1 to 6 and an acetylcholinesterase inhibitor.

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