Seven-component yang-warming traditional Chinese medicine composition for treating Alzheimer's disease

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to application of a seven-ingredient yang-warming traditional Chinese medicine composition in treatment of Alzheimer's disease. The seven-ingredient yang-warming traditional Chinese medicine composition provided by the invention has a remarkable treatment effect on the Alzheimer's disease in a caenorhabditis elegans Alzheimer's disease pathological model, can remarkably delay the paralysis rate of paralysis phenotype of the caenorhabditis elegans Alzheimer's disease, and has the potential of treating the Alzheimer's disease; the compound can be applied to preparation of medicines for treating the Alzheimer's disease.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to an application of a seven-flavor warming yang traditional Chinese medicine composition in treating Alzheimer's disease. Background Art

[0002] Alzheimer's disease (AD) is a progressive neurodegenerative disease that seriously endangers the quality of life and physical and mental health of the elderly. The disease is characterized by memory loss and cognitive dysfunction. There are about 50 million patients worldwide, and it is expected to reach 150 million by 2050. [1] Therefore, effective prevention and treatment of AD has become another major issue before us.

[0003] At present, Western medicines used clinically to treat Alzheimer's disease mainly include cholinesterase inhibitors (such as donepezil, rivastigmine, galantamine) and N-methyl-D-aspartate (NMDA) receptor antagonists (such as memantine). [2] Although these drugs can improve patients' cognitive function and delay disease progression, the side effects of the drugs themselves cannot be ignored. For example, significant adverse reactions may cause gastrointestinal reactions and cardiovascular toxicity after long-term use; long-term use of Western medicines may also lead to drug resistance, reducing the efficacy of the drugs. [3] The Chinese medicine composition has the characteristics of multiple active components and multiple targets, which makes it promising to screen drug candidates for the treatment of AD from the treasure trove of Chinese medicine compositions.

[0004] Caenorhabditis elegans is a very useful and versatile platform for drug screening and drug mechanism research. The establishment of a humanized Caenorhabditis elegans AD pathological model is to introduce human Aβ 1-42 The gene was transferred to the downstream of the myosin promoter and expressed under temperature control. The nematode strain could grow normally at 15°C. When it was transferred to 25°C, Aβ 1-42 The protein is expressed and accumulated in the muscles, causing the nematodes to lose their ability to move and become paralyzed. Adding the test drug can delay the muscle paralysis phenotype, and the result is expressed as the percentage of muscle paralyzed nematodes to the number of test nematodes. The higher the ratio of non-paralyzed nematodes, the more significant the drug's anti-AD effect. This model has long been used to study the pathogenesis of anti-AD diseases and the mechanism of action of anti-AD drugs and candidates. [4] .

[0005] Qiwei Wenyang Capsule is a compound Chinese medicine, composed of deer antler glue, deer antler frost, salt dodder seed, prepared rehmannia root, cypress seed, poria, and psoralea corylifolia. It is mainly used to nourish the kidney and warm yang, nourish essence and blood, and is suitable for middle-aged and elderly people with kidney yang deficiency caused by soreness of waist and knees, chills and cold limbs, dizziness, tinnitus, frequent urination, etc. In previous studies, it has been found to have anti-inflammatory and analgesic effects. [5] .

[0006] The present invention unexpectedly discovered the application of the seven-flavor warming Yang Chinese medicine composition in the treatment of Alzheimer's disease. The seven-flavor warming Yang Chinese medicine composition showed significant therapeutic effects in the Caenorhabditis elegans Alzheimer's disease pathological model, and can significantly delay the paralysis rate of the Alzheimer's disease Caenorhabditis elegans paralysis phenotype. It has the potential to treat Alzheimer's disease and can be used in the preparation of drugs for the treatment of Alzheimer's disease.

[0007] References

[0008] [1] Fu Yue. Research on medical visualization design and display application of Alzheimer's disease[D]. Jianghan University, 2023.

[0009] [2] Jia Jianping, Chen Shengdi. Guidelines for the diagnosis and treatment of Alzheimer's disease (2018)[J]. Chinese Journal of Neurology, 2019, 52(1): 51-62. [3] Chen Chunni. Exploring the mechanism of action of total flavonoids of Scutellaria baicalensis in improving Alzheimer's disease based on metabolomics and network pharmacology technology[D]. Shanxi University, 2023.

[0010] [4]Link C D.Transgenic invertebrate models of age-associated neurodegenerative diseases[J].Mechanisms of Aging&Development,2001,122(14):1639-1649.

[0011] [5] Lü Dong, Du Yajuan, Ma Yingchun, et al. Experimental study on analgesic and anti-inflammatory effects of Qiwei Wenyang Capsule [J]. Chinese Journal of Rational Drug Use, 2017, 14(12): 59-61. Summary of the invention

[0012] In view of the above technical problems, the present invention discloses an application of a Chinese medicine composition in preparing a drug for treating Alzheimer's disease, which specifically includes the following contents:

[0013] In a first aspect, the present invention provides a use of a seven-flavor warming yang Chinese medicine composition in the preparation of a drug for treating Alzheimer's disease.

[0014] Preferably, the active ingredients in the seven-flavor warming yang traditional Chinese medicine composition are composed of deer antler glue, deer antler frost, cypress seed, salt dodder seed, rehmannia root, poria, and psoralea corylifolia.

[0015] Preferably, the seven-flavor warming yang Chinese medicine composition is composed of the following raw materials in parts by weight: 83.3 parts of deer antler glue, 83.3 parts of deer antler frost, 83.3 parts of cypress seeds, 83.3 parts of salt dodder seeds, 83.3 parts of rehmannia root, 41.7 parts of poria, and 41.7 parts of psoralea corylifolia.

[0016] Preferably, the seven-flavor warming yang Chinese medicine composition is a water extract of a combination of seven-flavor warming yang Chinese medicines.

[0017] Preferably, the preparation process of the seven-flavor warming yang Chinese medicine composition comprises the following steps:

[0018] S1: Weigh antler glue, antler cream, cypress seed, salt dodder seed, rehmannia root, poria, and psoralea corylifolia according to weight;

[0019] S2: Add 10 times the amount of water to soak, heat to reflux, and centrifuge to collect the supernatant solution;

[0020] S3: Add 8 times the amount of water to the drug residue precipitate after centrifugation in S2, heat and reflux, and centrifuge to collect the supernatant drug solution;

[0021] S4: The supernatant solutions in S2 and S3 are combined and concentrated, the concentrated solution is freeze-dried, and the freeze-dried powder is collected.

[0022] Preferably, S2 is: adding 10 times the amount of water, soaking for 2 hours, heating and reflux in a condensation reflux device for 2 hours, centrifuging at 12000 rpm for 10 minutes, and collecting the supernatant.

[0023] Preferably, S3 is: adding 8 times the amount of water to the medicinal residue precipitate after centrifugation in S2, heating and reflux in a condensation reflux device for 2 hours, centrifuging at 12000 rpm for 10 minutes, and collecting the supernatant medicinal liquid.

[0024] Preferably, S4 comprises: combining the supernatant solutions in S2 and S3, and concentrating the solution using a rotary evaporator, then freezing the concentrated solution in a -80°C refrigerator for 24 hours, placing it in a freeze dryer for freeze drying, collecting the freeze-dried powder, and storing it at -20°C.

[0025] Preferably, the seven-flavor Yang-warming Chinese medicine composition is added with pharmaceutically acceptable excipients to form any pharmaceutically acceptable dosage form.

[0026] Preferably, the dosage forms include powders, granules, powders, pills, tablets, capsules, pills, and oral liquids.

[0027] The beneficial effects of the present invention are:

[0028] The present invention conducts experiments on a Caenorhabditis elegans Alzheimer's disease pathological model, and the results show that the water extract of the seven-flavor warming yang Chinese medicine composition has a significant therapeutic effect on the Caenorhabditis elegans Alzheimer's disease pathological model. Among them, (1) compared with each single herb and other single herb compositions, the water extract of the seven-flavor warming yang Chinese medicine composition has a more significant therapeutic effect on the Caenorhabditis elegans Alzheimer's disease pathological model; (2) the water extract of the seven-flavor warming yang Chinese medicine composition has a higher safety.

[0029] The following specific embodiments are provided to implement the technical solutions of the present invention, but the protection scope of the present invention is not limited to the following embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The therapeutic effect of the aqueous extract of the seven-flavor warming yang Chinese medicine composition on the Caenorhabditis elegans model of Alzheimer's disease; wherein, Control is a blank control group; P is a positive drug memantine (50 μM) group; 0.01 is a 0.01 mg / mL (raw drug amount 0.026 mg / mL) aqueous extract group of the seven-flavor warming yang Chinese medicine composition; 0.1 is a 0.1 mg / mL (raw drug amount 0.256 mg / mL) aqueous extract group of the seven-flavor warming yang Chinese medicine composition; 1 is a 1 mg / mL (raw drug amount 2.556 mg / mL) aqueous extract group of the seven-flavor warming yang Chinese medicine composition;

[0031] Figure 2 Comparison of the therapeutic effects of the water extract of the seven-flavor warming yang Chinese medicine composition and each single medicine on the Caenorhabditis elegans model of Alzheimer's disease; wherein, Control is a blank control group; P is a positive drug memantine (50 μM) group; M is a 0.1 mg / mL (raw drug amount 0.256 mg / mL) water extract of the seven-flavor warming yang Chinese medicine composition group; A is a raw drug amount 0.043 mg / mL antler glue group; B is a raw drug amount 0.043 mg / mL antler cream group; C is a raw drug amount 0.043 mg / mL salt dodder group; D is a raw drug amount 0.043 mg / mL prepared rehmannia group; E is a raw drug amount 0.043 mg / mL pine nuts group; F is a raw drug amount 0.021 mg / mL tuckahoe group; G is a raw drug amount 0.021 mg / mL psoralea group;

[0032] Figure 3Comparison of the therapeutic effects of the water extract of the seven-flavor warming yang Chinese medicine composition and other single-flavor medicine compositions on the Caenorhabditis elegans model of Alzheimer's disease; wherein C is a blank control group; P is a positive drug memantine (50 μM) group; M is a 0.1 mg / mL (raw drug amount 0.256 mg / mL) water extract group of the seven-flavor warming yang Chinese medicine composition; I is a raw drug amount 0.150 mg / mL of Rehmannia glutinosa, Poria cocos, Platycladus orientalis, and Cuscuta chinensis composition group; J is a raw drug amount 0.107 mg / mL of Rehmannia glutinosa, antler glue, and Poria cocos composition group; K is a raw drug amount 0.107 mg / mL of Rehmannia glutinosa, Platycladus orientalis, and Psoralea corylifolia composition group;

[0033] Figure 4 Effect of the aqueous extract of the seven-flavor warming yang Chinese medicine composition on the body length of nematodes; wherein, Control is the blank control group; 0.01 is the group of the aqueous extract of the seven-flavor warming yang Chinese medicine composition with a concentration of 0.01 mg / mL (raw drug amount 0.026 mg / mL); 0.1 is the group of the aqueous extract of the seven-flavor warming yang Chinese medicine composition with a concentration of 0.1 mg / mL (raw drug amount 0.256 mg / mL); 1 is the group of the aqueous extract of the seven-flavor warming yang Chinese medicine composition with a concentration of 1 mg / mL (raw drug amount 2.556 mg / mL). DETAILED DESCRIPTION

[0034] Example 1 Preparation method of water extract of seven-flavor warming yang Chinese medicine composition

[0035] Weigh 83.3 parts of antler glue, 83.3 parts of antler cream, 83.3 parts of cypress seeds, 83.3 parts of salt dodder seeds, 83.3 parts of Rehmannia glutinosa, 41.7 parts of Poria cocos, and 41.7 parts of psoralea corylifolia by weight; add 10 times the amount of water, soak for 2 hours, heat and reflux in a condensation reflux device for 2 hours, centrifuge at 12000rpm for 10 minutes, take the supernatant solution, and set aside. Add 8 times the amount of water to the centrifuged residue precipitate, heat and reflux in a condensation reflux device for 2 hours, centrifuge at 12000rpm for 10 minutes, take the supernatant solution, and set aside; combine the two supernatant solutions, and use a rotary evaporator to concentrate the solution, then freeze the concentrated solution in a -80℃ refrigerator for 24 hours, place it in a freeze dryer for freeze drying, collect the freeze-dried powder, store it at -20℃, and set aside.

[0036] Example 2 Therapeutic effect of the aqueous extract of the seven-flavor warming Chinese medicine composition on the Caenorhabditis elegans model of Alzheimer's disease 1. Biomaterials

[0037] (1) Caenorhabditis elegans CL4176 was purchased from Caenorhabditis Genetics Center (CGC); it is a transgenic strain that specifically expresses human Aβ in muscle under 25°C temperature induction. 1-42 , Aβ 1-42Aggregation in muscle tissue eventually leads to paralysis of nematodes. In this example, the CL4176 strain of Caenorhabditis elegans was used as a pathological model for screening anti-AD drugs.

[0038] (2) Escherichia coli OP50 (uracil leaky mutant), purchased from Caenorhabditis Genetics Center (CGC), was used as food for C. elegans.

[0039] 2. Reagents

[0040] (1) The composition and preparation of solid NGM (Nematode Growth Medium) culture medium (taking 1000 mL as an example) are shown in Table 1:

[0041] Table 1 Solid NGM medium

[0042] Element content NaCl 3.00g <![CDATA[K 2 HPO 4 ]]> 2.34g <![CDATA[KH 2 AFTER 4 ]]> 17.23g Peptone 2.50g Agar 17.00g <![CDATA[Supplementary H 2 O to]]> 1000mL

[0043] After the solid NGM medium was prepared, it was sterilized at 121°C under high pressure for 20 min. 1 mL of 5 mg / mL cholesterol and 1 M MgSO 4 1mL, 1M CaCl 2 1mL, shake well, and pour into sterilized 9cm culture plates while hot, about 20mL / plate. Let stand and wait for the culture medium to solidify, and set aside.

[0044] (2) The formula of M9 liquid is shown in Table 2:

[0045] Table 2M9 liquid formula

[0046]

[0047]

[0048] (3) Preparation of lysis solution: 6.4% NaClO solution and 1 M NaOH solution were mixed in a volume ratio of 1:1.

[0049] 3. Preparation of NGM plates containing the aqueous extract of the seven-flavor warming yang Chinese medicine composition

[0050] Preparation of the aqueous extract of the seven-flavor warming yang Chinese medicine composition: 0.1 g of the freeze-dried powder of the aqueous extract of the seven-flavor warming yang Chinese medicine composition is added with water to make up to 10 mL, filtered and sterilized, and set aside.

[0051] Prepare NGM plates containing the water extract of the seven-flavor warming-yang Chinese herbal composition: take a solution of the water extract of the seven-flavor warming-yang Chinese herbal composition with an appropriate dilution, add it to the NGM culture medium, and pour it onto the plates.

[0052] The final concentration of the water extract of the seven-flavor warming yang Chinese medicine composition is 10 times, 100 times, and 1000 times of the initially prepared medicinal solution. The culture medium is left to stand and wait for solidification. Escherichia coli OP50 is evenly spread on the culture medium as food for nematodes.

[0053] 4. Implementation steps

[0054] (1) Cultivation of nematodes

[0055] The nematodes were inoculated on solid NGM plates coated with E. coli OP50 and then cultured in an incubator at 15°C. When the nematodes grew into adults, they were synchronized.

[0056] (2) Synchronization of nematodes

[0057] Select NGM culture medium containing a large number of adult worms and some nematode eggs that have already hatched, wash the nematodes off the culture medium with M9 solution, transfer to a centrifuge tube, let the nematodes settle freely to the bottom of the tube, and discard the supernatant. Add lysis solution to the centrifuge tube according to the amount of nematodes, oscillate on a vortex for 5-7 minutes, stop vortexing when all the nematodes are broken, and divide into 1.5mL centrifuge tubes, and wash the nematode eggs three times with M9 solution.

[0058] (3) Experimental group settings

[0059] Blank control group (Control): C. elegans were cultured using ordinary NGM medium;

[0060] Positive drug memantine (50 μM) group (P): nematodes were cultured in NGM medium containing 50 μM memantine;

[0061] 0.01, 0.1, 1 mg / mL (the amount of crude drug is 0.026, 0.256, 2.556 mg / mL, respectively) Seven-flavor warming yang Chinese medicine composition water extract group: Nematodes were cultured using NGM medium containing 0.01, 0.1, 1 mg / mL (the amount of crude drug is 0.026, 0.256, 2.556 mg / mL, respectively) Seven-flavor warming yang Chinese medicine composition water extract;

[0062] (4) Therapeutic effect of the aqueous extract of the seven-flavor warming Chinese medicine composition on the Caenorhabditis elegans Alzheimer's disease model CL4176

[0063] Nematode drug treatment: The incubated L1 stage nematodes were transferred to NGM plates coated with OP50 and mixed with different concentrations of the aqueous extract of the seven-flavor warming yang Chinese medicine composition. 60 nematodes were plated on each plate, and three parallel plates were set for each drug concentration. The plates were cultured at 15°C until the L3 stage.

[0064] In order to make the nematodes express Aβ, the L3 stage nematodes were transferred to 25°C for constant temperature induction, and the number of paralyzed nematodes was counted after 34 hours (paralysis means that the nematodes cannot move or only the head moves when the body of the nematode is mechanically stimulated). Count once every two hours for a total of 10 hours.

[0065] 5. Experimental Results

[0066] The results are as follows Figure 1 As shown, the ordinate represents the percentage of C. elegans without paralysis. At the same time point, the larger this value is, the larger the proportion of C. elegans without paralysis in the treatment group is, that is, the stronger the effect of the drug in delaying paralysis. The results show that the positive drug memantine (50 μM) significantly delays the paralysis rate of the paralysis phenotype of Alzheimer's disease C. elegans compared with the blank control group. Compared with the blank control group, the 0.01, 0.1, and 1 mg / mL (raw drug dosage is 0.026, 0.256, and 2.556 mg / mL) water extract groups of the seven-flavor warming yang Chinese medicine composition significantly delay the paralysis rate of the paralysis phenotype of Alzheimer's disease C. elegans. It shows that the water extract of the seven-flavor warming yang Chinese medicine composition has a significant therapeutic effect on the pathological model of C. elegans Alzheimer's disease.

[0067] Example 3 Comparison of the therapeutic effects of the aqueous extract of the seven-flavor warming Chinese medicine composition and each single herb on the Caenorhabditis elegans model of Alzheimer's disease

[0068] 1. Preparation of the extracts of each single herb in the seven-herb warming yang Chinese medicine composition

[0069] 0.1 mg / mL (raw drug amount 0.256 mg / mL) Seven-flavor warming yang Chinese medicine composition water extract group (M): prepared as shown in Example 1

[0070] Drug group A (deer antler glue water extract with a crude drug amount of 0.043 mg / mL): 2g of deer antler glue, add 10 times the amount of water, soak for 2h, heat and reflux for 2h, centrifuge at 12000rpm for 10min, add 8 times the amount of water to the drug residue, heat and reflux for 2h, centrifuge at 12000rpm for 10min. Combine the two drug solutions, make up to 50mL, filter and sterilize, and set aside. Dilute to 0.430mg / mL mother solution when using, and dilute 10 times when preparing drug-containing NGM culture medium.

[0071] Drug group B (antler frost water extract with a crude drug amount of 0.043 mg / mL): 2g of antler frost, add 10 times the amount of water, soak for 2h, heat and reflux for 2h, centrifuge at 12000rpm for 10min, add 8 times the amount of water to the residue, heat and reflux for 2h, centrifuge at 12000rpm for 10min. Combine the two drug solutions, make up to 50mL, filter and sterilize, and set aside. Dilute to 0.430mg / mL mother solution when using, and dilute 10 times when preparing drug-containing NGM culture medium.

[0072] Drug group C (water extract of salt dodder with a crude drug amount of 0.043 mg / mL): 2g of salt dodder, add 10 times the amount of water, soak for 2h, heat and reflux for 2h, centrifuge at 12000rpm for 10min, add 8 times the amount of water to the drug residue, heat and reflux for 2h, centrifuge at 12000rpm for 10min. Combine the two drug solutions, make up to 50mL, filter and sterilize, and set aside. Dilute to 0.430mg / mL mother solution when using, and dilute 10 times when preparing drug-containing NGM culture medium.

[0073] Drug group D (0.043 mg / mL of Rehmannia glutinosa water extract): 2g of Rehmannia glutinosa, add 10 times the amount of water, soak for 2h, heat and reflux for 2h, centrifuge at 12000rpm for 10min, add 8 times the amount of water to the residue, heat and reflux for 2h, centrifuge at 12000rpm for 10min. Combine the two drug solutions, make up to 50mL, filter and sterilize, and set aside. Dilute to 0.430mg / mL mother solution when using, and dilute 10 times when preparing drug-containing NGM culture medium.

[0074] Drug group E (water extract of cypress seeds with a crude drug amount of 0.043 mg / mL): 2g of cypress seeds, add 10 times the amount of water, soak for 2h, heat and reflux for 2h, centrifuge at 12000rpm for 10min, add 8 times the amount of water to the residue, heat and reflux for 2h, centrifuge at 12000rpm for 10min. Combine the two drug solutions, make up to 50mL, filter and sterilize, and set aside. Dilute to 0.430mg / mL mother solution when using, and dilute 10 times when preparing drug-containing NGM culture medium.

[0075] Drug group F (poria water extract with a crude drug amount of 0.021 mg / mL): 2g of Poria, add 10 times the amount of water, soak for 2h, heat and reflux for 2h, centrifuge at 12000rpm for 10min, add 8 times the amount of water to the drug residue, heat and reflux for 2h, centrifuge at 12000rpm for 10min. Combine the two drug solutions, make up to 50mL, filter and sterilize, and set aside. Dilute to 0.210mg / mL mother solution when using, and dilute 10 times when preparing drug-containing NGM culture medium.

[0076] G drug group (psoralea corylifolia water extract with a crude drug amount of 0.021 mg / mL): 2g of psoralea corylifolia, add 10 times the amount of water, soak for 2h, heat and reflux for 2h, centrifuge at 12000rpm for 10min, add 8 times the amount of water to the drug residue, heat and reflux for 2h, centrifuge at 12000rpm for 10min. Combine the two drug solutions, make up to 50mL, filter and sterilize, and set aside. Dilute to 0.210mg / mL mother solution when using, and dilute 10 times when preparing drug-containing NGM culture medium.

[0077] 2. Preparation of drug-containing tablets for each drug group

[0078] 0.1 mg / mL (raw drug amount 0.256 mg / mL) Seven-flavor warming yang Chinese medicine composition water extract group (M): prepared as shown in Example 1

[0079] Blank control group (Control): C. elegans were cultured using ordinary NGM medium;

[0080] Positive drug memantine (50 μM) group (P): nematodes were cultured in NGM medium containing 50 μM memantine;

[0081] 0.1 mg / mL (crude drug amount 0.256 mg / mL) of the aqueous extract of the seven-flavor warming yang Chinese medicine composition group (M): nematodes were cultured using NGM medium containing 0.1 mg / mL (crude drug amount 0.256 mg / mL) of the aqueous extract of the seven-flavor warming yang Chinese medicine composition;

[0082] Drug group A: C. elegans was cultured in NGM medium containing the antler glue extract prepared in the previous step (raw drug amount 0.043 mg / mL);

[0083] Drug group B: C. elegans was cultured in NGM medium containing the antler frost water extract (raw drug amount 0.043 mg / mL) prepared in the previous step;

[0084] Drug group C: C. nematodes were cultured in NGM medium containing the water extract of Cuscuta salinarum (raw drug amount 0.043 mg / mL) prepared in the previous step;

[0085] Drug group D: C. elegans were cultured in NGM medium containing the aqueous extract of Rehmannia glutinosa (raw drug amount 0.043 mg / mL) prepared in the previous step;

[0086] Drug group E: C. elegans were cultured in NGM medium containing the water extract of Semen Platycladi prepared in the previous step (raw drug amount 0.043 mg / mL);

[0087] Drug group F: C. elegans was cultured in NGM medium containing the Poria cocos aqueous extract (raw drug amount 0.021 mg / mL) prepared in the previous step;

[0088] G drug group: C. elegans were cultured in NGM medium containing the aqueous extract of Psoralea corylifolia prepared in the previous step (raw drug amount 0.021 mg / mL);

[0089] 2. Experimental Procedure

[0090] The specific implementation steps are the same as those in Example 2.

[0091] 3. Experimental results

[0092] The results are as follows Figure 2As shown, the ordinate represents the percentage of C. elegans without paralysis. At the same time point, the larger this value is, the larger the proportion of C. elegans without paralysis in the treatment group is, that is, the stronger the effect of the drug in delaying paralysis. The results showed that compared with the blank control group, the 0.1 mg / mL (raw drug amount 0.256 mg / mL) water extract group of the seven-flavor warming yang Chinese medicine composition (M), the positive drug memantine (50 μM) group (P), the antler glue (raw drug amount 0.043 mg / mL) group (A), the salt dodder seed (raw drug amount 0.043 mg / mL) group (C), the prepared rehmannia root (raw drug amount 0.043 mg / mL) group (D), the tuckahoe (raw drug amount 0.021 mg / mL) group (F), and the psoralea corylifolia (raw drug amount 0.021 mg / mL) group (G) can all significantly delay Alzheimer's disease. The paralysis rate of the paralysis phenotype of Alzheimer's disease in Caenorhabditis elegans (p<0.001), the antler frost (raw drug amount 0.043 mg / mL) group (B) was able to delay the paralysis rate of the paralysis phenotype of Alzheimer's disease in Caenorhabditis elegans (p<0.05), while the cypress seed (raw drug amount 0.043 mg / mL) group (E) failed to delay the paralysis rate of the paralysis phenotype of Alzheimer's disease in Caenorhabditis elegans; and the 0.1 mg / mL (raw drug amount 0.256 mg / mL) seven-flavor warming yang Chinese medicine composition water extract group (M) had a better therapeutic effect on the Caenorhabditis elegans Alzheimer's disease pathological model than each single drug.

[0093] Example 4 Comparison of the therapeutic effects of the aqueous extract of the seven-herb warming Chinese medicine composition and other single-herb medicine compositions on the Alzheimer's disease nematode model

[0094] 1. Preparation of water extracts of each single herb composition of the seven-flavor warming yang Chinese medicine composition

[0095] The present invention designs a comparative experiment based on the retrieved compositions with anti-Alzheimer's disease efficacy, further proving that the seven-flavor warming yang Chinese medicine composition has a more significant anti-Alzheimer's disease efficacy.

[0096] 0.1 mg / mL (raw drug amount 0.256 mg / mL) Seven-flavor warming yang Chinese medicine composition water extract group (M): prepared as shown in Example 1

[0097] I drug group (raw drug amount 0.150mg / mL Radix Rehmanniae Preparata + Poria + Semen Platycladi + Semen Cuscutae water extract): Radix Rehmanniae Preparata 0.6g, Semen Cuscutae Salt 0.6g, Poria 0.3g, Semen Platycladi 0.6g, add 10 times of water, soak for 2h, heat and reflux for 2h, centrifuge at 12000rpm for 10min, add 8 times of water to the residue, heat and reflux for 2h, centrifuge at 12000rpm for 10min. Combine 2 times of medicinal solution, set to 50mL, filter and sterilize, and set aside. Dilute to 1.500mg / mL mother liquor when used, and dilute 10 times when preparing drug-containing NGM culture medium.

[0098] Drug group J (raw drug amount 0.107mg / mL Rehmannia glutinosa + antler glue + Poria cocos water extract): 0.8g Rehmannia glutinosa, 0.8g antler glue, 0.4g Poria cocos, add 10 times the amount of water, soak for 2h, heat to reflux for 2h, centrifuge at 12000rpm for 10min, add 8 times the amount of water to the residue, heat to reflux for 2h, centrifuge at 12000rpm for 10min. Combine the two drug solutions, make up to 50mL, filter and sterilize, and set aside. Dilute to 1.070mg / mL mother solution when using, and dilute 10 times when preparing drug-containing NGM culture medium.

[0099] Drug group K (raw drug dosage 0.107 mg / mL of Rehmannia glutinosa + Platycladus orientalis + Psoralea corylifolia water extract): 0.8g Rehmannia glutinosa, 0.8g Platycladus orientalis, 0.4g Psoralea corylifolia, add 10 times the amount of water, soak for 2h, heat to reflux for 2h, centrifuge at 12000rpm for 10min, add 8 times the amount of water to the residue, heat to reflux for 2h, centrifuge at 12000rpm for 10min. Combine the two drug solutions, make up to 50mL, filter and sterilize, and set aside. Dilute to 1.070mg / mL mother liquor when used, and dilute 10 times again when preparing drug-containing NGM culture medium. 2. Preparation of drug-containing plates for each drug group

[0100] Blank control group (Control): C. elegans were cultured using ordinary NGM medium;

[0101] Positive drug memantine (50 μM) group (P): nematodes were cultured in NGM medium containing 50 μM memantine;

[0102] 0.1 mg / mL (crude drug amount 0.256 mg / mL) water extract of seven-flavor warming yang Chinese medicine composition group (M): nematodes were cultured using NGM medium containing 0.1 mg / mL (crude drug amount 0.256 mg / mL) water extract of seven-flavor warming yang Chinese medicine composition;

[0103] Drug group I: Cultivate nematodes in NGM medium containing the aqueous extract of Rehmannia glutinosa + Poria cocos + Semen Platycladi + Semen Cuscutae (raw drug amount 0.150 mg / mL) prepared in the previous step;

[0104] Drug group J: C. elegans was cultured in NGM medium containing Rehmannia glutinosa + antler glue + Poria cocos (raw drug amount 0.107 mg / mL) prepared in the previous step;

[0105] Drug group K: C. elegans were cultured in NGM medium containing Rehmannia glutinosa + Semen Platycladi + Psoralea corylifolia (raw drug amount 0.107 mg / mL) prepared in the previous step;

[0106] 2. Experimental Procedure

[0107] The specific implementation steps are the same as those in Example 2.

[0108] 3. Experimental results

[0109] The results are as follows Figure 3 As shown, the ordinate represents the percentage of C. elegans without paralysis. At the same time point, the larger this value is, the larger the proportion of C. elegans without paralysis in the treatment group is, that is, the stronger the effect of the drug in delaying paralysis. The results showed that compared with the blank control group, the 0.1 mg / mL (raw drug amount 0.256 mg / mL) water extract group of the seven-flavor warming yang Chinese medicine composition (M), the positive drug memantine (50 μM) group (P), the Rehmannia glutinosa + Poria cocos + Platycladus orientalis + Cuscuta australis (raw drug amount 0.150 mg / mL) group (I), and the Rehmannia glutinosa + Platycladus orientalis + Psoralea corylifolia (raw drug amount 0.107 mg / mL) group (K) can all significantly delay the paralysis rate of the paralysis phenotype of Alzheimer's disease C. elegans (p < 0.001), and the Rehmannia glutinosa + Deer Horn group (p < 0.001) can significantly delay the paralysis rate of the paralysis phenotype of Alzheimer's disease C. elegans. The glue + Poria cocos (raw drug dosage 0.107 mg / mL) group (J) failed to delay the paralysis rate of the paralysis phenotype of Alzheimer's disease in Caenorhabditis elegans; and the 0.1 mg / mL (raw drug dosage 0.256 mg / mL) seven-flavor warming yang Chinese medicine composition water extract group (M) had a significantly better therapeutic effect on the Caenorhabditis elegans Alzheimer's disease pathological model than the Rehmannia glutinosa + Poria cocos + Platycladus orientalis + Cuscuta australis (raw drug dosage 0.150 mg / mL) group (I) and the Rehmannia glutinosa + Platycladus orientalis + Psoralea corylifolia (raw drug dosage 0.107 mg / mL) group (K).

[0110] The results of the above examples show that the aqueous extract of the seven-flavor warming yang Chinese medicine composition described in the present application has a significant therapeutic effect on the Alzheimer's disease Caenorhabditis elegans pathological model, suggesting that the aqueous extract of the seven-flavor warming yang Chinese medicine composition provided by the present invention has the potential to treat Alzheimer's disease and can be used in the preparation of drugs for the treatment of Alzheimer's disease.

[0111] Example 5 Effect of the aqueous extract of seven-flavor warming yang Chinese medicine composition on the body length of nematodes

[0112] 1. Experimental Group Setup

[0113] Blank control group (Control): C. elegans were cultured using ordinary NGM medium;

[0114] 0.01, 0.1, 1 mg / mL (the amount of crude drug is 0.026, 0.256, 2.556 mg / mL, respectively) Seven-flavor warming yang Chinese medicine composition water extract group: C. elegans were cultured in ordinary NGM medium containing 0.01, 0.1, 1 mg / mL (the amount of crude drug is 0.0256, 0.2556, 2.556 mg / mL, respectively) of Seven-flavor warming yang Chinese medicine composition water extract;

[0115] 2. Experimental Procedure

[0116] (1) Nematode drug treatment: The incubated L1 nematodes were transferred to NGM plates coated with OP50 and mixed with different concentrations of the aqueous extract of the seven warming yang Chinese medicine composition. 600 nematodes were plated on each plate. After culturing at 20°C for 48 h, the nematodes were washed with M9 and transferred to a centrifuge tube.

[0117] (2) Measurement of nematode body length: Each group of nematodes was photographed using the bright field of an Olympus inverted fluorescence microscope (IX73+DP74). Thirty pictures were randomly taken for each group, and the experiment was repeated three times. The pictures were processed using image-J software, and the nematode body length was measured using ROIManager.

[0118] 3. Experimental results

[0119] The results are as follows Figure 4 As shown, within the concentration range of 0.01 mg / mL to 1 mg / mL (crude drug amount 0.026 to 2.556 mg / mL), the water extract of the seven-flavor warming yang Chinese medicine composition had no obvious effect on the body length of the wild-type nematode N2, which indicates that within the concentration range of 0.01 mg / mL to 1 mg / mL (crude drug amount 0.026 to 2.556 mg / mL), the water extract of the seven-flavor warming yang Chinese medicine composition is safe for the wild-type nematode N2.

Claims

1. Application of a seven-flavor warming yang Chinese medicine composition in the preparation of a drug for treating Alzheimer's disease.

2. The use according to claim 1, characterized in that The active components of the seven-flavor yang-warming Chinese medicine composition are composed of deer antler glue, deer antler frost, cypress seed, salt dodder seed, rehmannia root, tuckahoe and psoralea corylifolia.

3. The use according to claim 2, characterized in that The seven-flavor warming yang Chinese medicine composition is composed of the following raw materials in parts by weight: 83.3 parts of deer antler glue, 83.3 parts of deer antler frost, 83.3 parts of cypress seeds, 83.3 parts of salt dodder seeds, 83.3 parts of rehmannia root, 41.7 parts of tuckahoe, and 41.7 parts of psoralea corylifolia.

4. The use according to claim 3, characterized in that The seven-flavor warming yang Chinese medicine composition is a water extract of a combination of seven-flavor warming yang Chinese medicines.

5. The use according to claim 4, characterized in that Preparation process of the seven-flavor yang-warming Chinese medicine composition The following steps are involved: S1: Weigh antler glue, antler cream, cypress seed, salt dodder seed, rehmannia root, poria, and psoralea corylifolia according to weight; S2: Add 10 times the amount of water to soak, heat to reflux, and centrifuge to collect the supernatant solution; S3: Add 8 times the amount of water to the drug residue precipitate after centrifugation in S2, heat and reflux, and centrifuge to collect the supernatant drug solution; S4: The supernatant solutions in S2 and S3 are combined and concentrated, the concentrated solution is freeze-dried, and the freeze-dried powder is collected.

6. The use according to claim 5, characterized in that The S2 is as follows: adding 10 times the amount of water, soaking for 2 hours, heating and reflux in a condensation reflux device for 2 hours, centrifuging at 12000 rpm for 10 minutes, and collecting the supernatant.

7. The use according to claim 5, characterized in that The step S3 is as follows: adding 8 times the amount of water to the medicinal residue precipitate after centrifugation in S2, heating and reflux in a condensation reflux device for 2 hours, centrifuging at 12000 rpm for 10 minutes, and collecting the supernatant medicinal solution.

8. The use according to claim 5, characterized in that The S4 comprises: combining the supernatant solutions in S2 and S3, concentrating the solution using a rotary evaporator, freezing the concentrated solution in a -80°C refrigerator for 24 hours, placing the solution in a freeze dryer for freeze drying, collecting the freeze-dried powder, and storing it at -20°C.

9. The use according to any one of claims 1 to 8, characterized in that: The seven-flavor warming yang traditional Chinese medicine composition is added with pharmaceutically acceptable excipients to prepare any pharmaceutically acceptable dosage form.

10. The use according to claim 9, characterized in that The dosage forms include powders, granules, powders, pills, tablets, capsules, pills, and oral liquids.

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