WO2015039619A1 - 养血清脑制剂在制备治疗阿尔茨海默症的药物中的应用 - Google Patents

养血清脑制剂在制备治疗阿尔茨海默症的药物中的应用 Download PDF

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WO2015039619A1
WO2015039619A1 PCT/CN2014/086915 CN2014086915W WO2015039619A1 WO 2015039619 A1 WO2015039619 A1 WO 2015039619A1 CN 2014086915 W CN2014086915 W CN 2014086915W WO 2015039619 A1 WO2015039619 A1 WO 2015039619A1
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brain
group
compared
mice
composition
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吴迺峰
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天士力制药集团股份有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56—Materials from animals other than mammals
    • A61K35/618—Molluscs, e.g. fresh-water molluscs, oysters, clams, squids, octopus, cuttlefish, snails or slugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/23—Apiaceae or Umbelliferae (Carrot family), e.g. dill, chervil, coriander or cumin
    • A61K36/232—Angelica
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/23—Apiaceae or Umbelliferae (Carrot family), e.g. dill, chervil, coriander or cumin
    • A61K36/236—Ligusticum (licorice-root)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/26—Aristolochiaceae (Birthwort family), e.g. heartleaf
    • A61K36/264—Aristolochia (Dutchman's pipe)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/26—Aristolochiaceae (Birthwort family), e.g. heartleaf
    • A61K36/268—Asarum (wild ginger)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A61K36/482—Cassia, e.g. golden shower tree
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A61K36/486—Millettia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/53—Lamiaceae or Labiatae (Mint family), e.g. thyme, rosemary or lavender
    • A61K36/536—Prunella or Brunella (selfheal)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/65—Paeoniaceae (Peony family), e.g. Chinese peony
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/66—Papaveraceae (Poppy family), e.g. bloodroot
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/71—Ranunculaceae (Buttercup family), e.g. larkspur, hepatica, hydrastis, columbine or goldenseal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/74—Rubiaceae (Madder family)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/80—Scrophulariaceae (Figwort family)
    • A61K36/804—Rehmannia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • the invention relates to a new use of a preparation of a traditional Chinese medicine composition, in particular to the application of a serum-sparing brain preparation in the preparation of a medicament for treating Alzheimer's disease (Alzheimer's disease).
  • Yangxue Brain Granule is a modern Chinese medicine preparation developed by Tianjin Tianshili Pharmaceutical Co., Ltd., and obtained the National New Drug Certificate in 1996. It was listed in the National Essential Drugs List in 1999 and was listed in the National Medical Insurance Drug List in 2000. Yangxue Brain Granules are made from Angelica sinensis, Rhizoma Chuanxiong, Radix Paeoniae Alba, Uncaria sinensis, Radix Rehmanniae, Radix Rehmanniae, Radix Scutellariae, Prunella vulgaris, Asarum, Corydalis and Mother of Pearl. After being extracted by modern high-tech means, appropriate auxiliary ingredients are added.
  • the preparation has the advantages of quick dissolution of active ingredients and high bioavailability.
  • Yangxue serum granules have the effect of nourishing blood and calming the liver, promoting blood circulation and collaterals. It can be used for headaches caused by blood deficiency and liver sputum, dizziness, irritability, insomnia and other dreams. It has significant clinical effects.
  • the serum-sparing brain preparation is a prior art, and the formulation and preparation method thereof are described in Chinese Patent No. 93100050.5.
  • the formulation described in the patent can also be prepared into different dosage forms such as tablets, capsules, oral liquids and the like according to conventional techniques of preparation.
  • Alzheimer disease also known as Alzheimer's disease, is a progressive degenerative neurological degenerative disease. Clinically, it is characterized by memory impairment, aphasia, misuse, loss of recognition, visual spatial impairment, executive dysfunction, and personality and behavioral changes such as personality dementia. The etiology has not been known so far. Before the age of 65, the disease is called Alzheimer's disease; after the age of 65, the disease is called Alzheimer's disease.
  • Current drugs for the treatment of Alzheimer's disease include: donepezil, Rivastigmine, Galantamine, Huperzine A, Memantine, Syracuse Selegiline, vitamin E, melatonin, ginkgo extract (gingko bilobi), piracetam (piracetam), aniracetam (aniracetam aniracetam, San Lexi), naphthocil Tan et al.
  • the present inventors have unexpectedly discovered that the serum-supplemented brain preparation has an effect of treating Alzheimer's disease.
  • the invention provides a new use of a traditional Chinese medicine composition, in particular to a new medical use of a serum-supplemented brain preparation.
  • the present invention provides a use of a traditional Chinese medicine composition for the preparation of a medicament for treating Alzheimer's disease.
  • the application is that the traditional Chinese medicine composition can be used to remove senile plaques in the brain of Alzheimer's patients.
  • the traditional Chinese medicine composition of the present invention is prepared from the following weight percentages: 6.75% of Angelica sinensis, 6.75% of Rhizoma Chuanxiong, 5.4% of Radix Paeoniae, 13.5% of Uncaria, 13.5% of spatholobus, 5.4% of Rehmannia glutinosa, 13.5% of cassia seed. , Prunella 13.5%, Asarum 1.34%, Corydalis 6.75% and Mother of Pearl 13.5%.
  • the preparation process comprises the following steps: preparing the medicinal material by pretreatment, water extraction, concentration, ethanol precipitation, recovery of ethanol, concentration into a paste, and mixing.
  • the formulation includes any of the dosage forms that can be administered, preferably oral preparations such as granules, pills, tablets, capsules, oral solutions.
  • the application of the present invention resides in that the traditional Chinese medicine composition significantly improves memory cognitive ability.
  • the application of the invention is that the traditional Chinese medicine composition can improve the expression of brain-derived nerve growth factor, nerve growth factor and its receptor TrkA in the cerebral cortex and hippocampus, and improve the ultrastructure of hippocampal neurons.
  • the application of the invention is that the traditional Chinese medicine composition can increase the total antioxidant capacity and GSH content of the cerebral cortex, increase the activity of SOD, GSH-px and reduce the MDA content.
  • the application of the present invention is that the traditional Chinese medicine composition can improve the expression of synaptophysin and hippocampal synaptophysin 95 in the cerebral cortex and hippocampus, and improve the abnormality of hippocampal synaptic structure.
  • the application of the present invention is that the traditional Chinese medicine composition can increase the expression of acetylcholine in the cerebral cortex and hippocampus, and increase the expression of choline acetyltransferase and cerebral cortex M1 choline receptor.
  • the application of the present invention resides in the use of the traditional Chinese medicine composition for the removal of senile plaques in the brain of Alzheimer's patients.
  • the application of the present invention resides in that the traditional Chinese medicine composition can clear A ⁇ protein in the brain and reduce the production of A ⁇ protein in the brain.
  • the application of the present invention lies in the application that the traditional Chinese medicine composition can inhibit the pathological cleavage of APP by inhibiting the pathological cleavage of APP, and inhibiting the physiological alpha cleavage of APP.
  • the preferred extraction method of the traditional Chinese medicine composition of the present invention is:
  • Extract 3 Rehmannia glutinosa, Uncaria, Spatholobus suberectus, Prunella vulgaris, mother-of-pearl, and asarum were boiled, filtered, concentrated, added with ethanol, filtered, and the ethanol was recovered and concentrated. ;
  • Another preferred method of preparation is:
  • Extract 3 Rehmannia glutinosa, Uncaria, Spatholobus suberectus, Prunella vulgaris, mother-of-pearl, and asarum are added to 4-10 times the amount of water for 2-3 times, the first 0.5-3 hours, the first Two or three times each for 1-3 hours, filtered, concentrated to a relative density of 1.06-1.10 (75-85 ° C), add ethanol to make the alcohol content 60-85%, let stand for 12-24 hours, filtered, and recover ethanol , concentrated to a relative density of 1.270-1.350 (75-85 ° C), spare;
  • the most preferred preparation method is:
  • Extract 3 Rehmannia glutinosa, Uncaria, Spatholobus suberectus, Prunella vulgaris, mother-of-pearl, and asarum were added to 5 times the amount of water for 2 times, the first 2 hours, the second 1 hour, and filtered. Afterwards, concentrate to a relative density of 1.06-1.10 (80 ° C), add ethanol to make the alcohol content 65-70%, let stand for 12-24 hours, filter, recover ethanol, and concentrate to a relative density of 1.320-1.325 (79-81 °C), spare;
  • the present invention when describing the percentage concentration of ethanol, refers to the volume specific concentration of the aqueous ethanol solution.
  • the alcohol content referred to in the present invention is the ethanol content (v/v).
  • the application of the present invention is obtained through experimental observation, and one of the preparations of the traditional Chinese medicine composition is used, that is, "nutrient serum brain particles" produced by Tasly Pharmaceutical Co., Ltd. as an experimental medicine, and any of the serum brain particles have The extracts extracted by the same prescription all have the same use as the serum-suppressing brain particles.
  • the experimental results are as follows:
  • the APPswe/PSldE9 double transgenic mice are internationally recognized Alzheimer's Disease (AD) model mice. Age-related plaques began to appear in the 6-month-old APPswe/PSldE9 transgenic brain, mimicking mild Alzheimer's disease in humans. 4 months old APPswe/PSldE9 double transgenic Alzheimer's model mice for 60 consecutive days, oral administration of serum brain particles 48g crude drug / kg (high dose) and 16g crude drug / kg (low dose), or control drug An Li Shen 1.03mg /kg to 6 months of age in mice, it was found that after one month and two months of oral administration of serum brain particles, the memory cognitive ability of mice was significantly improved, and the effect was better than that of the positive drug An Lishen group.
  • AD Alzheimer's Disease
  • Congo red staining and immunohistochemical staining of brain tissue sections demonstrated that the serum-sparing brain particles cleared the brain's ⁇ -amyloid plaques (senile plaques) in the early and mid-term Alzheimer's disease model mice, from the hippocampus and cortex of the mouse brain.
  • the clearance rate of senile plaques in the brain was 60-90%, which was significantly higher than that of the positive drug Anritsu group.
  • Yangxue Brain Granule is a traditional Chinese medicine compound preparation produced by Tianshili Group Co., Ltd. It is mainly used for treating headache and improving chronic cerebral ischemia. In long-term clinical observations, the drug was found to have a certain effect on improving Alzheimer's disease. The laboratory performed the drug on the memory and cognitive ability of the early and mid-term APPswe/PSldE9 double transgenic Alzheimer's disease model mice, especially for the characteristic lesion of Alzheimer's disease – ⁇ amyloid A ⁇ in the brain.
  • the deposition of the A ⁇ senile plaques in the brain of early and mid-term APPswe/PSldE9 double transgenic Alzheimer's disease mice was studied by means of specific staining of insoluble A ⁇ and Congo red staining of amyloid recognition.
  • the study of protein cleavage pathway reveals its target and mechanism, and provides experimental basis for the treatment of mild to moderate Alzheimer's disease.
  • APPswe/PSldE9 double transgenic AD mice were purchased from the Institute of Model Animals, Nanjing University.
  • the APPswe/PSldE9 double transgenic mouse is an internationally recognized Alzheimer's model rat.
  • APPswe/PSldE9 double transgenic mice were transfected with human Swedish mutant APPswe and exon 9 deletion mutant PSldE92 genes, which can express human mutant APP molecules, and abnormal shear production is excessive.
  • the inventors performed genotypic identification of APPswe/PSldE9 double transgenic AD mice for drug testing.
  • the tail of the rat tail was taken, and the lysate and protein kinase K10 mg/ml were added, and the tail was digested overnight at 56 °C.
  • the protein was removed by adding phenol chloroform, and the DNA was precipitated by isopropanol, and washed with 70% ice ethanol to obtain rat tail DNA.
  • a PCR reaction for identifying the APPswe gene was carried out using APPswe primers: 5'-GACTGACCACTCGACCAGGTTCTG-3' and 5'-CTGACTGGTGAGCTGGTCCAAGAC-3'.
  • Paraformaldehyde (Sigma), Pentobarbital Sodium (Genview), Congo Red, Hematoxylin, Mercury Oxide (Sigma), DAB Colorimetric Kit (Beijing Dingguo), Rat Rabbit Universal Immunological Antibody (Proteintech) , concentrated ammonia, anhydrous ethanol, concentrated hydrochloric acid, glycerin, methanol, citric acid, trisodium citrate, xylene (Beijing Chemical).
  • TEMED acrylamide, methylidene bisacrylamide, Tris, Gly, Coomassie Brilliant Blue R250, BSA (Sigma), Triton X-100 (Genview), protein standard molecular weight (Beijing full gold), Tween-20 (BBI), Rabbit anti-mouse ⁇ -actin antibody (Wuhan Dr.), HRP-labeled goat anti-rabbit IgG (Beijing full-scale gold), ECL photochemical kit (Biyuntian).
  • Congo red staining solution and hematoxylin staining solution 0.5 g of Congo red powder and 20 ml of glycerin were added to 80 ml of methanol to prepare a 0.2% Congo red staining solution.
  • 20 g of potassium aluminum sulfate was dissolved in 200 ml of distilled water and dissolved at 100 ° C.
  • 1 g of hematoxylin was dissolved in 10 ml of absolute ethanol, added to an aqueous solution of potassium aluminum sulfate, boiled, 0.5 g of mercury oxide was added, heating was continued and stirred until the solution was dark purple, and immediately cooled and filtered to prepare a hematoxylin staining solution.
  • Y-maze system (RD1102-YM), vertical electrophoresis system and power system (Beijing Liuyi Factory), semi-dry film transfer machine (BIO-RAD).
  • the DP70 upright microscope and imaging system (OLYMPUS) collects images using the microscope image processing software DP Controller.
  • the experimental animals APPswe/PSldE9 double transgenic AD mice were randomly divided into normal saline control group, low serum concentration group (sometimes referred to as low blood concentration group or low blood dose group), and high serum concentration group.
  • This article is sometimes referred to as the high-concentration group of nourishing blood or high-dose group of nourishing blood) and the An Lishen group, male and female, each group, 16 in each group.
  • Conventional feeding, free drinking water, 4 months old APPswe/PSldE9 transgenic mice were administered intragastrically, and the administration volume was 0.5 ml per 25 g body weight. Administered by gavage to 6 months of age.
  • the Y labyrinth instrument consists of three arms of equal length, each with an angle of 120°, in a Y shape. Mice in the closed space (Y-maze) will instinctively find a way out, and mice with normal cognitive and spatial memory will explore each path in the search process, rather than repeating the path that has already been explored. Specifically, the mouse is placed in the maze from the Y-maze with a phylum, and the three arms are arbitrarily numbered as the A arm, the B arm, and the C arm. The mouse is free to move for 8 minutes, and the mouse is recorded. For example, if the path of the mouse can be cycled sequentially (no repetition in the three paths), it is recorded as correct.
  • correct rate (%) n / (total number of arms - 2) ⁇ 100%, n is the correct number of paths.
  • mice were deeply anesthetized with sodium pentobarbital. After anesthesia, the thoracic cavity of the mice was cut, and the perfusion needle was inserted into the left ventricle of the mouse. The right atrial appendage was also cut and filled with 0.01 M PBS (pH 7.4) 50-100 ml. Until the perfusate is clear. At the same time, refill 4% paraformaldehyde 50-100ml. After the perfusion was completed, the brain was taken and immersed in a 4% paraformaldehyde solution overnight. Paraffin sections were taken after the embedded machine was embedded. The sheet thickness is 1.5 ⁇ m.
  • paraffin tablets After the paraffin tablets were taken out, they were baked on a baking machine at 60-65 ° C for 30 min.
  • the sheets were removed from the roaster and soaked in xylene for 30 min.
  • the mixture was sequentially dehydrated in the order of absolute ethanol, absolute ethanol, 95% ethanol, 95% ethanol, 90% ethanol, 80% ethanol, water, and distilled water. Hematoxylin staining for 1 min, ammonia blue.
  • the alkaline differentiation solution was differentiated and dehydrated in the order of 80% ethanol, 90% ethanol, 95% ethanol, 95% ethanol, absolute ethanol, and absolute ethanol. Soak in xylene for 1 min, air dry, and seal the gel.
  • the A ⁇ senile plaque in the brain is amyloid, which is red after Congo red staining.
  • the number and area of red A ⁇ amyloid plaques in the cortex and hippocampus were measured and calculated for each section, and the average value was analyzed.
  • Paraffin section was immersed in xylene for 30 min. Then, it is sequentially dehydrated in the order of absolute ethanol, absolute ethanol, 95% ethanol, 95% ethanol, 90% ethanol, 80% ethanol, water, and distilled water. pH6.0 citric acid high pressure 120 ° C repair 2-2.5min, 3% medical disinfection H 2 O 2 to remove endogenous active substances.
  • the APP ⁇ antibody was added for 4 h, and the mouse rabbit universal type was incubated with a secondary antibody for 50 min at room temperature. DAB staining for 3 min. Hematoxylin counterstaining, ammonia bluening, dehydration. Soak in xylene for 1 min, air dry, and seal the gel.
  • Each immunohistochemical staining section was used to measure three visual fields in the cortex and hippocampus, and the number of strongly positive and weakly positive ⁇ -amyloid plaques in each field was counted, and the average value was analyzed.
  • the mouse brain tissue was dissected, and the protein was quantified by BCA protein quantification kit after lysis, and then the protein amount of the sample was subjected to SDS-PAGE and then transferred to the PVDF membrane.
  • the cells were blocked with 5% skim milk powder for 1 h, and various primary anti- ⁇ APPs, ADAM10 or PS1 were added dropwise, and the cells were washed 3 times with PBS at room temperature for 2 h.
  • Goat anti-rabbit IgG2HRP reacted for 1 h at room temperature, and washed 3 times with PBS.
  • the signal was detected by ECL photochemical kit (Biyuntian), and the X-ray film was exposed and developed.
  • ADAM10, PS1, ⁇ -actin antibodies were purchased from Boosen.
  • the APP ⁇ segment antibody was independently developed, and the 16 amino acid short peptide DAEFRHDSGYEVHHQK between APP ⁇ cleavage and ⁇ -cleavage was selected (this peptide is a fibrous insoluble peptide, which is a key region that determines the brain insolubility of A ⁇ 42) as an antigen, through gene recombination and sequencing. Protein expression and purification, animal immunization and ProteinA/G purification yield high titers of APP ⁇ antibodies.
  • the data was applied to SPSS Statistics 17.0 for statistical analysis.
  • the results were represented by scatter plots combined with mean and mean ⁇ standard error, and data were compared by group analysis using one-way ANOVA combined with LSD method.
  • the transgenic mice When the transgenic mice were 4 months old, symptoms of mental injury had already occurred. Compared with the background mice, the food intake was reduced, the autonomic activity was reduced, the movement was slow, the spirit was wilting, and the fur was not shiny. After the administration, the transgenic mice in the Yangxuenao granule group had different degrees of improvement, the food intake increased, and the spirit improved.
  • the Y-maze experiment is a simple and widely used behavioral tool for detecting spatial cognitive ability and short-term memory ability in mice.
  • Table 2 and Figure 2 the Y-maze experiment demonstrated that Yangxue brain granules have a significant effect on improving the memory and cognitive ability of mice in the early and mid-term Alzheimer's disease model.
  • the correct rate of normal saline was 79.59%
  • the average correct rate of Y-maze in the low-concentration group and the high-concentration group were 86.10% and 87.12%, respectively. It is 80.67%. Both the low-concentration group and the high-concentration group were significantly different from the control group.
  • the correct rate of normal saline was 77.52%, and the average correct rate of Y maze in the low-concentration group and the high-concentration group were 83.86% and 85.64%, respectively.
  • the control group was 82.19%. The effect of improving the cognitive ability of the high-concentration group and the low-concentration group was significantly higher than that of the control group.
  • the inventors' results showed that the correct rate of Y maze in the low-concentration group and the high-concentration group was increased by nearly 10% compared with the control, which was significant for one month and two months of administration; Although the positive rate of the Y maze was increased in the positive control group at the two months of administration, the statistical difference was not reached. Therefore, the results demonstrate that Yangxue Brain Granule has a significant effect on improving the memory and cognitive ability of mice in the early and mid-term Alzheimer's disease model.
  • AD patients present with central nervous system degenerative lesions in the frontotemporal and hippocampus, and A ⁇ amyloid plaques are characteristic lesions of Alzheimer's disease.
  • the agglutination and accumulation of A ⁇ are the initial factors in the pathogenesis and development of AD, while other pathological changes such as neurofibrillary tangles in the brain, dysfunction and loss of neurons, etc. are considered to be due to the dissociation and aggregation of A ⁇ . Cleared and caused by the imbalance caused.
  • a characteristic lesion of Alzheimer's disease is the deposition of A ⁇ amyloid in the brain.
  • Methanol Congo red staining is used to show amyloid,
  • the deposition of amyloid A ⁇ protein in the brain can be well recognized.
  • the inventors found that the number and area of A ⁇ amyloid plaques in the cerebral cortex of mice fed the serum-enriched brain granules for 2 months were significantly reduced, and there was a dose-related effect, and the lower concentration group of the high-concentration group was more effective.
  • Figure 3 to Figure 7 ).
  • Figure 3 to Figure 5 show Congo red staining of amyloid plaques in the cerebral cortex of the control and experimental groups.
  • the number of A ⁇ amyloid plaques in the brain of the saline group and the Anrisheng group was large, and the number of amyloid plaques in the high concentration and low concentration groups of the serum-supplemented brain particles was small.
  • Fig. 4 the brain frontal cortex of male mice (Fig. 4) and female mice (Fig. 5)
  • a certain number of extracellular red chrysanthemum-like plaques stained with Congo red were detected in the saline group and the Ariase group.
  • amyloid plaque of AD It is a characteristic amyloid plaque of AD, while the number of amyloid plaques in the high concentration and low concentration groups of the serum-lowering brain particles is small, and the plaque area is small. It can be seen that the low-concentration group and the high-concentration group have significant effects on the amyloid plaque deposition in the frontal cortex, while the control drug An Lishen has no significant effect on amyloid plaque clearance.
  • the hippocampus of the brain is a key part of storing short-term memory, and it is also a brain region where humans deal with long-term learning and memory spatial localization.
  • Figure 6 shows Congo red staining in the hippocampus of each group of mice.
  • the saline group and the Anritsu group can detect the deposition of amyloid plaques, while the high concentration and low concentration groups of the serum-sparing brain particles detect the surrounding area of the hippocampus. Less than the deposition of amyloid plaques.
  • the inventors quantified the number of congo red-stained amyloid plaques throughout the brain. As shown in Table 3 and Figure 7, from the number of senile plaques, 6 months old APPswePSEN1dE9 transgenic mice showed significant senile plaques in the brain of the saline control group. In comparison, the number of senile plaques in the positive control group was reduced by 37.5. %, but no significant difference. Significantly, compared with the saline control group, the senile plaques in the low concentration group of Yangxue brain granules decreased by 62.2% (p ⁇ 0.05), and the senile plaques in the high concentration group of serum cerebral cerebral granules decreased by 70.1% (p ⁇ 0.01). ). It is suggested that Yangxue granules have the effect of significantly eliminating the number of senile plaques in the cerebral cortex of early and middle mice.
  • the inventors quantified the area of the whole brain of Congo red stained amyloid plaques. As shown in Table 3 and Figure 8, from the area of senile plaques, Anritsu acts as an acetylcholinesterase inhibitor, thus reducing the role of ⁇ -amyloid plaque deposition in the brain or hippocampus.
  • the age of senile plaques in the positive control group was 17.8% lower than that in the control group, but there was no significant difference.
  • the area of senile plaques in the low-concentration group was reduced by 61.4% (p ⁇ 0.01), and the area of senile plaques in the high-concentration group was reduced by 72.4% (p ⁇ 0.01).
  • Yangxue Qingnao Granule has the effect of clearing amyloid senile plaques in the brain of early and middle mice, and the clearance rate is more than 60-70%.
  • the effect of high-dose serum-sparing brain particles is more significant.
  • the commercially available drug for the treatment of Alzheimer's disease, Anritsu acts as an acetylcholinesterase inhibitor, thus reducing the effect of ⁇ -amyloid plaque deposition in the brain or hippocampus.
  • the therapeutic effect of Yangxue Qingnao Granule on amyloid senile plaque clearance was significantly stronger than that of the positive drug An Lishen.
  • Human amyloid precursor APP is a key protein in the pathogenesis of Alzheimer's disease.
  • the deposition of amyloid A ⁇ in the brain by ⁇ -shear formation is one of the main pathological features of Alzheimer's disease.
  • the APP secretes the ⁇ -secretase cleavage pathway under normal conditions, producing extracellular soluble sAPP ⁇ protein.
  • APP is cleaved under the action of two proteases, ⁇ -secretase and ⁇ -secretase, to produce 42 amino acid A ⁇ peptide, which is the main toxic substance of AD.
  • Large amounts of neurons and synaptic sites in the brain tissue form amyloid senile plaques, which can impair brain function, leading to memory and cognitive impairment.
  • the inventors selected the 16 peptide between the N-segment ⁇ -cleavage and ⁇ -cleavage in the A ⁇ 42 peptide as an antigen, which has a unique advantage in detecting pathological shear of Alzheimer's disease.
  • the brain insoluble peptide of A ⁇ 42 peptide is composed of 16 peptides, so this 16-peptide antibody is displayed.
  • This antibody is described in the Methods section. The inventors used this antibody to detect the deposition of A ⁇ peptide in the brain of Alzheimer's disease transgenic mice by serum-sparing brain particles.
  • the inventors statistically analyzed the total number of ⁇ -amyloid plaques in the cerebral cortex of each experimental group. As shown in Table 4 and Figure 13, from the number of strong positive A ⁇ senile plaques in the mouse brain, each experimental group was compared with the saline control group. There was a significant difference. The number of senile plaques in the strong positive control group was 67.7% lower than that in the control group. Compared with the saline control group, the senile plaques in the low-concentration group were reduced by 81.4%, and the senile plaques in the high-concentration group. Reduced by 87.5%.
  • Yangxue serum granules can significantly promote the physiological ⁇ -cleavage of APP in the brain of early and mid-term Alzheimer's disease animal model, and promote the production of soluble neurotrophic sAPP ⁇ protein.
  • Human amyloid precursor APP is a key protein in the pathogenesis of Alzheimer's disease, and the shear mechanism of APP is divided into two types.
  • the normal body APP undergoes physiological non-amyloid alpha-secretase ADAM10 cleavage and produces a neurotrophic sAPP ⁇ protein.
  • ADAM10 physiological non-amyloid alpha-secretase
  • amyloid-like pathological cleavage is induced by ⁇ - and ⁇ -secretase.
  • the deposition of amyloid A ⁇ in the brain by ⁇ and ⁇ shearing of APP is one of the main pathological features and the most important molecular mechanism of Alzheimer's disease.
  • the physiological ⁇ -cleaved cleavage product sAPP ⁇ of APP not only has important neurotrophic effects, but also hinders the production of A ⁇ , which is of great significance for the treatment of AD.
  • Western blot analysis of antibody-stained soluble physiological sAPP ⁇ demonstrated that Yangxue brain particles significantly promoted physiological ⁇ -cleavage and promoted the role of soluble and neurotrophic sAPP ⁇ protein.
  • the extracellular soluble fragment sAPP ⁇ produced by ⁇ -cleavage of APP has a size of 66kD.
  • the brain of AD model rats rarely shows physiological shear under physiological saline conditions, and the level of sAPP ⁇ is very low, which is compared with saline group.
  • the sAPP ⁇ of the Anritsu group has increased.
  • the high-dose group of nourishing blood was significantly higher than the positive Anritsu control group.
  • the ⁇ -secretase cleavage pathway produces extracellular soluble APP peptide, which has neurotrophic effect.
  • physiological ⁇ -cleavage of sAPP ⁇ has a significant antagonistic effect on pathological ⁇ -cleavage.
  • the alpha secretase ADAM10 is a key enzyme that performs physiological cleavage of APP.
  • the inventors examined the expression levels of ADAM10 in the brain of each drug group.
  • the expression level of ADAM10 in the brain of 6-month-old AD transgenic mice was very low under physiological saline conditions, indicating that physiological shearing rarely occurred.
  • the expression of ADAM10 in the Anrisheng group was not significantly changed.
  • the expression level of ADAM10 was significantly increased in the low-concentration group, especially in the high-concentration group (Fig. 14).
  • Yangxueqingnao granules can promote the physiological ⁇ -shearing of APP, and the serum-raising brain granules can significantly increase the expression level of ⁇ -secreting cleavage enzyme ADAM10 in the rat brain and the soluble neurotrophic sAPP ⁇ protein in the brain.
  • ADAM10 ⁇ -secreting cleavage enzyme
  • the formation by significantly improving the physiological alpha shear of APP in the brain, antagonized the pathological shear pathway, and its effect was more significant than the positive drug An Li Shen.
  • the role of serum-sparing brain particles in clearing senile plaques in the brain is achieved by inhibiting the pathogenic ⁇ -secretase presenilin PS1.
  • the transgenic mouse is the PSldE9 gene of the exon 9 deletion mutation of PS1.
  • the mutation of presenilin PS1 gene is the key link of A ⁇ formation and AD treatment.
  • the expression level of presenilin PSldE9 in the brain of 6-month-old AD transgenic model rats was high under physiological saline conditions, but compared with the control group, the expression of PSldE9 in the Anritsu group was not significant.
  • the inventors have demonstrated that the serum-suppressing brain granules have a significant effect of eradicating senile plaques in the brain of early and mid-term Alzheimer's disease animal models.
  • This molecular mechanism is mainly achieved by two aspects, as shown in Figure 15.
  • Yangxue brain granules inhibit the Pathogenic Shear A ⁇ process of APP by inhibiting the pathogenic ⁇ -secretase presenilin PS1-dE9, and reduce the age spots in the brain.
  • Yangxue brain granules promote the production of neurotrophic sAPP ⁇ by promoting the level of physiological secretase alpha secretion cleavage enzyme ADAM10, thereby promoting the physiological shearing of APP and antagonizing the production of A ⁇ in the brain. Make the age spots in the brain further reduced.
  • the inventors have demonstrated that the serum-supplemented brain particles have a remarkable effect of treating mild to moderate Alzheimer's disease. Reflected in two aspects:
  • the behavioral Y-maze test results showed that the mice were memorized and recognized after oral administration of high-concentration nourishing serum brain particles (48g crude drug/kg) and low-concentration nourishing serum brain particles (16g crude drug/kg) one month and two months later.
  • the knowledge ability was significantly improved compared with the saline control group, and the effect was better than the positive drug An Lishen. That is, Yangxue brain granules have a significant effect on improving the memory and cognitive ability of early and mid-stage AD model mice.
  • ⁇ -amyloid plaques is formed by the deposition of A ⁇ protein, a characteristic lesion of Alzheimer's disease, which plays an extremely significant therapeutic role.
  • the Congo red staining and immunohistochemical staining experiments were used to demonstrate the hippocampus and cortex of the mouse brain.
  • the number of ⁇ -amyloid plaques (age spots) was significantly reduced from the number, coverage and coloration (clearance rate reached 60-90%), and the effect was much higher than that of the Anritsu group.
  • the protein extracted from mouse brain tissue was analyzed by Western blot, which proved that Yangxue brain particles can promote the physiological ⁇ -cleavage of ⁇ -amyloid precursor APP and inhibit the pathological shear of APP, thereby reducing the A ⁇ in the brain. It is produced to play a significant role in clearing A ⁇ -amyloid plaques in the brain.
  • Yangxue Brain Granules can increase the dementia model dose-dependently in the range of 935-3740mg/kg in mice and 647-2588mg/kg in rats (1-4 times the clinical equivalent of headache in patients).
  • the spontaneous alternation rate of mice and rats, the priority index and the discrimination coefficient in the new object discrimination experiment shorten the time and distance of reaching the safety platform in the water maze experiment, and prolong the swimming time and distance in the quadrant of the original platform in the water labyrinth space exploration experiment. Percentage, prolong the avoidance period in the darkness test and reduce the number of electric shocks.
  • Yangxue Qingnao Granule can significantly improve non-spatial learning and memory disorders such as working memory disorder, spatial learning and memory impairment and visual discrimination memory in dementia model animals.
  • the effect of high dose group and clinical equivalent amount of donepezil hydrochloride The results of HE staining showed that Yangxueqingnao can improve the pathological changes of mouse and rat neurons.
  • Transmission electron microscopy showed that Yangxue brain particles can improve the ultrastructure and synaptic structure of hippocampal neurons in the brain.
  • Yangxue brain granules may improve neurotrophic factor expression, protect against free radical damage, inhibit lipid peroxidation, protect neurons and synaptic structures, improve brain Ach, ChAT, and improve central cholinergic energy. Neurological function, which in turn improves learning and memory obstacle.
  • mice with SAMP8 rapid aging were used to investigate the improvement of learning and memory impairment in mice with SAMP8 rapid aging by using Yangxue brain particles.
  • the hippocampus of mouse brain was observed by HE staining. Changes in tissue structure; changes in hippocampal neurons and synaptic ultrastructure were observed by transmission electron microscopy; brain-derived nerve growth factor (BDNF) in the cerebral cortex and hippocampus of SAMP8 mice was examined by immunohistochemistry.
  • BDNF brain-derived nerve growth factor
  • NGF nerve growth factor
  • TrkA nerve growth factor
  • GAP-43 synaptic growth-associated protein 43
  • SYP synaptophysin
  • PSD-95 postsynaptic density 95
  • MDA lipid peroxidation products malondialdehyde
  • GSH total antioxidant capacity
  • SOD superoxide dismutase
  • GSH-px glutathione peroxidase
  • Yangxue Brain Granules can increase the spontaneous alternation rate of SAMP8 mice and the new object discrimination experiment in a dose-dependent manner within the range of 935-3740mg/kg (1-4 times the clinical equivalent of headache treatment).
  • the medium priority index and the discrimination coefficient shorten the time and distance of reaching the safety platform in the water maze test of SAMP8 mice, prolong the swimming time and distance percentage in the quadrant of the original platform in the water labyrinth space exploration experiment, prolong the escape latency in the darkness avoidance experiment, and reduce The number of electric shocks.
  • Yangxuenao Granule can significantly improve non-spatial learning and memory impairment in SAMP8 mice, such as working memory disorder, spatial learning and memory impairment, and visual ability to distinguish memory.
  • the effect of high-dose group and clinical equivalent amount of donepezil hydrochloride The results of HE staining showed that Yangxuenao Granule could improve the pathological changes of neurons in SAMP8 mice.
  • Yangxue brain granules may improve the learning and memory disorders by improving the expression of neurotrophic factors, combating free radical damage, inhibiting lipid peroxidation, etc., and protecting neurons and synaptic structures.
  • Yangxue brain particles extract, supplied by Tianjin Tianshili Pharmaceutical Group Co., Ltd., batch No. 20120502W, stored at 4 °C-8 °C.
  • the daily dose of human is 45g, and each gram of extract is equivalent to 6.26g of medicinal material.
  • the clinical equivalent amount converted into mice is 935mg (extract)/kg.
  • Donepezil hydrochloride tablets Eisai (China) Pharmaceutical Co., Ltd., batch number 110707A, stored, stored at room temperature below 30 °C.
  • the maximum recommended clinical dose is 10 mg/d/person, and the clinical equivalent amount converted to mice is about 1.3 mg/kg.
  • Disodium hydrogen phosphate Shantou Xiqiao Chemical Factory Co., Ltd., batch number 100802;
  • Absolute ethanol Tianjin Hengxing Chemical Reagent Manufacturing Co., Ltd., batch number 20100327;
  • Paraformaldehyde Tianjin Bodi Chemical Co., Ltd., batch number 20111028;
  • Glutaraldehyde Tianjin Bodi Chemical Co., Ltd., batch number 20110906;
  • Xylene Tianjin Bodi Chemical Co., Ltd., batch number: 20100505;
  • Citric acid Tianjin Bodi Chemical Co., Ltd., batch number: 20100321;
  • Hydrochloric acid Shenyang Economic and Technological Development Zone Reagent Factory, batch number: 20110612;
  • PVDF membrane Pall Corporation of the United States
  • Mouse anti- ⁇ -actin Santa Cruz Biotechnology, product number: sc-41478;
  • Tris-base BIOSHARP Amresco 0497;
  • Horseradish-labeled goat anti-mouse IgG Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.;
  • mouse Y maze new object identification, and dark-avoidance devices were developed by Shenyang Pharmaceutical University;
  • Paraffin embedded slicer Leica, Germany
  • Ultrasonic cell pulverizer JY92-II, Ningbo Xinzhi Biotechnology Co., Ltd.;
  • Constant temperature water bath device BCFCO Changfeng Electronic Technology Co., Ltd.;
  • Electrothermal constant temperature incubator DNP-9272, Shanghai Precision Experimental Equipment Co., Ltd.;
  • High-speed refrigerated centrifuge HC-3018R, HKUST Innovation Co., Ltd.;
  • Microwave oven Galanz company
  • SAMP8 mice were randomly divided into model group, serum granules 935mg/kg (human clinical equivalent), 1870mg/kg (2 times clinical equivalent), 3740mg/kg (4 times clinical equivalent) dose group And donepezil hydrochloride 1.3mg/kg group, 30 in each group, another 25 sets of SAMR1 blank control group.
  • the Y-maze and new object discrimination experiments were started 2 months after intragastric administration in each group. Morris water maze and dark-avoidance experiments were performed after 2 and a half months of administration. Dosing continued during the behavioral experiment, once a day until the end of the experiment.
  • the experimental device is composed of four rectangular activity boxes, built-in infrared probes and cameras, and the spontaneous activity video analysis system is provided by Shanghai Jiliang Software Technology Co., Ltd.
  • the animals were placed in four movable boxes, one for each box. Set the collection time to 5 minutes, record the total distance of animal activities, activity time and average speed.
  • the device consists of three equal length wooden arms at an angle of 120°, namely the three arms A, B and C of the labyrinth. Each arm is 40cm long, 12cm high, 10cm wide and 5cm wide.
  • the mice were placed at the end of the A-arm, and they were allowed to freely enter and exit the three arms. The number and arm entries of each mouse entering the three arms within 5 minutes were recorded to continuously enter three different arms. For a successful alternation, record the number of alternations. The spatial working memory ability was reflected by the spontaneous alternation rate (%).
  • the experimental device is a wooden square open field, and the experiment is divided into an adaptation phase and a test phase. 2-3 mice were placed in the open field and freely explored for 3 min to suit the environment twice a day for 2 days. On the day of the test, the animals were placed in the experimental field for 3 min to re-adapt to the environment. The animals were removed and the two identical objects (A1, A2) that the animals had never seen were placed equidistant from the edge of the device. On the other hand, place the mouse at the same position from the two objects, record the time to explore the two objects within 5 minutes (tA1, tA2), then remove the mouse and return it to the cage for 1h, then replace one object in the device with one.
  • mice For new objects (B) with different colors, shapes, and materials, the mice were placed again, and the time taken to explore the two objects (tA1, tB) was recorded, and the mice were taken out and returned to the squirrel cage. After 24 hours, the object B was replaced with a new object (C), and the mice were placed again, and the time (tA1, tC) used to explore the two objects was recorded. Calculate the priority index and discrimination coefficient of each group for new objects,
  • Priority index (1h) tB / (tA1 + tB)
  • Priority index (24h) tC / (tA1 + tC)
  • Discrimination coefficient (24h) (tC-tA1) / (tA1 + tC)
  • Positioning navigation experiment Morris water maze training was performed once a day in the afternoon and afternoon for 5 consecutive days.
  • the platform is placed at the midpoint of the fourth quadrant. Select two points at the opposite side of the platform as the water inlet point, put the mouse into the water in the pool wall, collect 60s, record the time from the water entering the platform to find the platform (escape latency) and swimming distance Then, let the mice rest on the platform for 10 s. If the platform is not found in 60s, the incubation period is recorded as 60 s and the mice are placed on the platform for 10 s. Each time, two water intake points were performed once a day, and the arithmetic mean of the two incubation periods was used as a statistical analysis of the results of the one day.
  • the experimental device is distinct and dark. Both chambers are 15 cm x 10 cm x 11 cm in size, and there is a semi-circular door with a diameter of 3 cm between the two chambers.
  • the bottom of both chambers is covered with a copper grid, and the bottom of the dark chamber is energized by a fourth copper grid, and the voltage intensity is controlled by a voltage regulator.
  • This method uses the mouse's dark habits, puts the face of the mouse back to the doorway into the bright room, adapts to the environment for 3 minutes, and then passes the voltage of 30V.
  • the mice were immediately shocked by entering the darkroom, then fed back to the bright room from the door, trained for 5 minutes, and recorded the number of times the mice were shocked, that is, the number of errors, as a result of academic achievement. After 24 hours, the test recorded the latency of entering the darkroom for the first time and the number of errors within 5 minutes as a memory score.
  • the mouse cerebral cortex and hippocampus tissue samples were stored in a refrigerator at -80 ° C for use.
  • the protein lysate was added at a ratio of 1:10, and 5 ⁇ l of PMSF was added per 1 ml of the protein lysate.
  • BCA working solution was prepared by adding 50 volumes of BCA reagent A plus 1 volume of BCA reagent B (50:1), and thoroughly mixed.
  • the protein standard was 5 mg/ml BSA, and the protein standard was completely dissolved. 10 ⁇ L was diluted to 100 ⁇ L to a final concentration of 0.5 mg/ml, and the standard was diluted with PBS. 2 Add the standard to 0, 1, 2, 4, 8, 12, 16, 20 ⁇ l to the standard well of a 96-well plate, add 20 ⁇ l of standard dilution (PBS), and repeat each concentration three times.
  • SDS-PAGE 1SDS-polyacrylamide gel
  • Assembly of plastic glass plates Prepare the separation glue, mix well, immediately inject into the gap of the glass plate, inject it to about 3cm from the top of the glass plate, seal it with deionized water, polymerize at room temperature for about 30-60min, and pour off the deionized water. Drain the water from the surface of the separation gel as much as possible.
  • Electrophoresis The proteins in the sample were separated by electrophoresis on an SDS-polyacrylamide gel (SDS-PAGE). According to the results of BCA protein quantification, the protein concentration of the sample was adjusted to be consistent before loading, and 5 ⁇ SDS-PAGE protein loading buffer was added, and heated at 100 ° C or a boiling water bath for 5 min to fully denature the protein. The gel was placed in an electrophoresis tank, and 1 ⁇ running buffer was added, and 30 ⁇ g of total protein was added to each well. At the beginning of the electrophoresis, the voltage was 80V. After the dye entered the separation gel, it increased to 180V, and the dye was turned off when it reached the bottom of the separation gel.
  • SDS-PAGE SDS-polyacrylamide gel
  • An ice box is added to both sides of the transfer film to prevent overheating during film transfer.
  • the current is turned on (the gel is connected to the negative electrode and the PVDF film is connected to the positive electrode), and the constant current is transferred for 2 hours, and the current is 100 mA. After the transfer is completed, turn off the power and take out the film.
  • the membrane was placed in a blocking solution (5% skim milk powder was placed in PBS buffer), shaken at room temperature for 2 h, and the primary antibody (SYP, GAP-43, PSD-95, ⁇ -muscle) was prepared with blocking solution. Actin), put in the refrigerator at 4 ° C overnight. The membrane was washed three times with PBS at room temperature, and the membrane was washed once with Tris-NaCl at room temperature for 10 min each time. Add a horseradish peroxidase-labeled secondary antibody (1:3000) with blocking solution and incubate for 2 h at room temperature. The membrane was washed three times with Tris-NaCl at room temperature for 10 min each.
  • mice Each group of mice was killed by decapitation 2 hours after the last administration, and the cerebral cortex tissue was quickly exfoliated, washed with pre-cooled saline, blood stains were removed, the filter paper was dried, and after weighing, the brain tissue was quickly immersed in cold saline (salt quality)
  • 10% brain homogenate was prepared in an ultrasonic cell pulverizer, centrifuged at 3000-4000 r/min for 20 min, the supernatant was taken, and biochemical detection was performed according to the kit instructions.
  • chloral hydrate 350 mg/kg was anesthetized by intraperitoneal injection, fixed on the operating table in the supine position, and the heart was exposed by thoracotomy.
  • the perfusion needle is inserted into the left ventricle from the apex of the apex, while the right atrial ear is cut into a small mouth, firstly perfused with about 200-300 ml of normal saline, and the liquid flowing out of the right atrial appendage becomes colorless and clarified.
  • the replacement is 300- 400 ml 4 ° C 4% paraformaldehyde buffer (0.1 mol / L) perfusion until the body is stiff.
  • the whole brain was removed by decapitation and fixed in 4% paraformaldehyde at 4 °C. After 24 h, conventional paraffin-embedded, coronal sections, thickness 5 ⁇ m, were used for routine HE staining and immunohistochemical staining.
  • Four mice in each group 4% paraformaldehyde and 2.5% glutaraldehyde 1:1 mixture were perfused, and 1 mm 3 hippocampal CA1 area tissue was fixed, 2.5% glutaraldehyde was fixed, and the nerve cells were observed by transmission electron microscope. Ultrastructure.
  • Immunohistochemical immunohistochemistry was performed using the streptomycin avidin-peroxidase (SP) ligation method.
  • SP streptomycin avidin-peroxidase
  • the antigen in the tissue was determined using a SP immunohistochemical staining kit with a biotin-labeled secondary antibody and a streptomycin avidin-linked peroxidase and matrix dye mixture.
  • the specific experimental steps are as follows: tissue section dewaxing to water; microwave antigen retrieval: the slice is placed in a container containing the antigen repair solution, and the antigen repair solution is a citrate buffer (0.01M, pH 6.0), and the microwave is placed in a high fire.
  • Paraffin sections were subjected to routine HE staining. The procedure was as follows: paraffin sections were routinely dewaxed to water; distilled water was washed for 2 min, hematoxylin staining was carried out for 3 min, washed with water for 5 min, 1% hydrochloric acid alcohol was differentiated for 30 s, tap water returned to blue for 8 min, eosin stained for 1 min, tap water was washed for 3 min. Gradient alcohol dehydration, xylene transparent 2 ⁇ 5min, neutral gum seal. Histopathological changes were observed under an optical microscope.
  • mice in each group entered the three arms of the Y maze (see Figure 17, Table 6), suggesting that the serum-free brain particles did not significantly affect the spontaneous activity of the mice.
  • the spontaneous alternation rate of the Y maze in the model group was significantly decreased.
  • the serum-sparing brain particles dose-dependently increased the spontaneous alternation rate of the mouse Y maze (see Figure 18, table). 6), suggesting that serum brain particles can improve working and memory impairment in mice.
  • the results of HE staining showed that the neurons in the hippocampal CA1 region of the blank control group had clear and tightly arranged cells.
  • the neurons in the mice were loosely arranged and the chromatin was reduced.
  • the neurons in the 1870 mg/kg group, 3740 mg/kg group and the donepezil hydrochloride group were closely arranged, and no edema and nuclear pyknosis were observed (see Figure 26).
  • Electron microscopy showed that the neurons in the blank control group had clear outlines, the nucleus was elliptical, the chromatin distribution in the nucleus was relatively uniform, the nuclear membrane and nucleolus were clear, and the cytoplasm was rich in ribosomes, rough endoplasmic reticulum, mitochondria and Organelles such as lysosomes, ultrastructure are normal; the neuron cells in the model group are irregular in shape, have notch, see nucleoli, partial nucleus membrane is blurred, heterochromatin in the nucleus has edge set, and endoplasmic reticulum in cytoplasm The Golgi complex is slightly dilated, and the mitochondrial outer membrane is partially damaged.
  • the neuron cell membrane of the donepezil group is clear, the chromatin distribution in the nucleus is uniform, and the cytoplasm is rich in ribosomes, mitochondria, rough endoplasmic reticulum, Golgi complex and Lysosomes, etc.; the neuron nucleus of the 3740mg/kg serum brain granules is elliptical, the contour of the nuclear membrane is clear, the nucleolus is clear, the chromatin in the nucleus is evenly distributed, and the cytoplasm is rich in ribosome and rough endoplasmic reticulum.
  • Organelles such as mitochondria, Golgi complex and lysosome, but the rough endoplasmic reticulum has edema, and the mitochondrial outer membrane is damaged.
  • the neuron cell line of the 1870mg/kg serum brain granule is clear, and there are some parts in the nucleus. Heterochromatin condensation, abundant ribosomes and mitochondria in the cytoplasm, mild expansion of the rough endoplasmic reticulum; nuclear membranes in the 935mg/kg group of serum-raised brain particles are blurred, and some heterologous chromatin in the nucleus
  • organelles such as ribosome, rough endoplasmic reticulum, mitochondria, lysosome and Golgi complex in the cytoplasm. Some mitochondria are reduced or the outer membrane is damaged, and the rough endoplasmic reticulum is expanded (see Figure 27).
  • BDNF can promote the survival and growth of a variety of neurons, increase the biological activity of neurons, increase the density of synaptic terminals and promote the growth of dendrites and axons.
  • the positive result of BDNF immunohistochemistry is the appearance of brownish yellow particles in the cytoplasm of neurons.
  • the results showed that the cytoplasm staining of the hippocampal CA1 area and cortical neurons was deeper in the blank control group; the cytoplasmic staining of the neurons in the model group was significantly shallower, the donepezil group, the serum cerebral granules 1870 mg/kg group and 3740 mg/kg.
  • Group neuronal staining was significantly deeper than the model group (see Figures 29 to 33, Table 13).
  • BDNF brain-derived nerve growth factor
  • Nerve growth factor is a classical neurotrophic factor of the nerve growth factor family, which has the dual biological functions of neurotrophic nutrition and promotion of neuronal processes.
  • Positive results of NGF immunohistochemistry showed brownish yellow particles in the cytoplasm of neurons.
  • the results showed that the cytoplasm of CA1 and cortical neurons in the hippocampus of the control group was darker in the cerebral cortex of the brain.
  • the coloration of the neurons in the model group was significantly shallower.
  • the donepezil group and the serum granules were 1870 mg/kg and 3740 mg.
  • the neuronal staining in the /kg group was significantly deeper than in the model group (see Figures 34 to 38, Table 14).
  • TrkA is a transmembrane protein with a molecular weight of 140 kD.
  • NGF binds to TrkA, it induces cell proliferation, differentiation and survival, inhibits apoptosis, increases neuronal excitability and induces cell release mediators expressing TrkA.
  • the positive result of TrkA receptor immunohistochemistry showed brownish yellow particles in the cytoplasm of neurons.
  • the results showed that the mice in the blank control group had darker cytoplasmic staining in the hippocampal CA1 area and cerebral cortex neurons.
  • the coloration of the neurons in the model group was significantly shallower in the cytoplasm of the mice, and the donepezil group and the serum cerebral granules were 1870 mg/kg and 3740 mg.
  • the neuronal staining in the /kg group was significantly deeper than in the model group (see Figures 39 to 43, Table 15).
  • the experimental results are shown in Table 16.
  • MDA malondialdehyde
  • GSH glutathione
  • T-AOC total antioxidant capacity
  • SOD superoxide dismutase
  • GSH-px glutathione peroxidase
  • Synaptophysin also known as synaptophysin (SYP)
  • SYP synaptophysin
  • SYP synaptophysin
  • Development which plays a role in synaptic plasticity, is an important molecule in the process of learning and memory.
  • Synaptophysin is specifically localized to the synaptic vesicle membrane at the terminal axon, so it can be used to label synaptic structures and reflect the distribution of axonal terminal structures.
  • Postsynaptic density refers to a semi-circular band in the cytoplasmic surface of the postsynaptic membrane.
  • PSD-95 also known as SAP90
  • SAP90 is the first identified backbone protein in PSD, which belongs to membrane-associated guanylate kinase protein. A member of the super family.
  • GAP-43 (Growth associated protein-43) is a axonal membrane protein, a neuron-specific protein involved in extracellular growth and triggering and neuronal regeneration.
  • Yangxue brain particles were within the dose range of 935-3740mg/kg (1-4 times equivalent of clinical treatment headache) It can dose-dependently improve non-spatial learning and memory impairment in SAMP8 mice, such as working memory disorder, spatial learning and memory impairment, and visual discrimination.
  • the effect of high dose group is similar to donepezil hydrochloride.
  • NNF nerve growth factor
  • TrkA receptor TrkA
  • Yangxue brain granules can increase the total antioxidant capacity and GSH content of cerebral cortex in SAMP8 mice in a dose-dependent manner, increase SOD, GSH-px activity and reduce MDA content.
  • Yangxue brain granules can increase the expression of synaptophysin and post-synaptic density 95 in the cerebral cortex and hippocampus of SAMP8 mice, and improve the synaptic structure of hippocampus.
  • the behavioral experimental methods such as Y-maze, new object discrimination, Morris water maze and avoidance of darkness were used to investigate the effect of Yangxuenao Granule on learning and memory impairment in rats with dementia caused by quinolinic acid-damaged Meyert basal ganglia (NBM nucleus).
  • NBM nucleus quinolinic acid-damaged Meyert basal ganglia
  • the changes of hippocampal tissue structure were observed by HE staining.
  • the changes of hippocampal neurons and synaptic ultrastructure were observed by transmission electron microscopy.
  • the immunostaining (Western blot) method was used to detect the damage of quinolinic acid by Yangxue brain particles.
  • Hippocampus and cerebral cortex synaptophysin SYP
  • GAP-43 synapse growth associated protein 43
  • PSD-95 postsynaptic compact 95
  • Hippocampus and cerebral cortex synaptophysin SYP
  • GAP-43 synapse growth associated protein 43
  • PSD-95 postsynaptic compact 95
  • Hippocampus and cerebral cortex synaptophysin SYP
  • GAP-43 synapse growth associated protein 43
  • PSD-95 postsynaptic compact 95
  • hippocampal muscarinic acetylcholine receptor Hippocampus and cerebral cortex synaptophysin (SYP), synapse growth associated protein 43 (GAP-43), postsynaptic compact 95 (PSD-95) and hippocampal muscarinic acetylcholine receptor in NBM-induced dementia rats
  • CHRM1 The effect of CHRM1 expression; the effects of acetylcholine (Ach) content in hippo
  • Yangxuenao Granule can significantly improve the non-spatial learning and memory impairment of quinolinic acid-induced dementia in rats with NBM-induced dementia, spatial learning and memory impairment, and visual discrimination.
  • Donepezil is similar.
  • Yangxue Qingnao Granule can improve the abnormalities of ultrastructure and synaptic structure of hippocampal neurons; improve the pathological changes of neurons in dementia model rats; increase the expression of SYP and PSD-95 in hippocampus and cerebral cortex in a dose-dependent manner. There was no significant effect on the expression of GAP-43 in the cerebral cortex and hippocampus.
  • Yangxuenao granules may improve the expression of SYP and PSD-95 in the brain, improve synaptic abnormalities, increase the content of Ach and its synthetase ChAT in the brain, and improve the function of the central cholinergic nervous system. Improve quinolinic acid damage to learning and memory impairment in rats with dementia induced by NBM.
  • SD rats Healthy Sprague-Dawley (SD) rats, 120 rats, male and female, weighing 180-220g, purchased from Liaoning Changsheng Biotechnology Co., Ltd. Division certificate number: scxk2010-0001; raised in SPF laboratory animal center of Shenyang Pharmaceutical University, free drinking water, 12h cycle lighting.
  • Yangxue brain particles extract, supplied by Tianjin Tianshili Pharmaceutical Group Co., Ltd., batch No. 20120502W, stored at 4 °C-8 °C.
  • the human daily dose of human is 45 g, and each 1 g of extract is equivalent to 6.26 g of the medicinal material, and the clinical equivalent amount converted into a rat is 647 mg (extract)/kg.
  • Donepezil hydrochloride tablets Eisai (China) Pharmaceutical Co., Ltd., batch number 110707A, stored, stored at room temperature below 30 °C.
  • the maximum recommended clinical dose is 10 mg/d/person, and the clinical equivalent amount converted to rats is about 0.95 mg/kg.
  • Chloral hydrate Tianjin Ruijinte Chemical Co., Ltd., batch number: 20101124;
  • Fire cotton glue Tianjin Hengxing Chemical Reagent Manufacturing Co., Ltd., batch number: 20110421;
  • Disodium hydrogen phosphate Shantou Xiqiao Chemical Co., Ltd., batch number: 110802;
  • Paraformaldehyde Tianjin Bodi Chemical Co., Ltd., batch number: 20121028;
  • Glutaraldehyde Tianjin Bodi Chemical Co., Ltd., batch number: 20110906;
  • Xylene Tianjin Bodi Chemical Co., Ltd., batch number: 20121205;
  • Citric acid Tianjin Bodi Chemical Co., Ltd., batch number: 20100321;
  • Hydrochloric acid Shenyang Economic and Technological Development Zone Reagent Factory, batch number: 20120304;
  • Polylysine Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., batch number: 050M4339;
  • PVDF membrane Pall Corporation, USA, LOT: K1BA3015GK;
  • Mouse anti- ⁇ -actin Santa Cruz Biotechnology, product number: sc-41478;
  • Mouse anti-GAP-43 Santa Cruz Biotechnology, product number: sc-17790;
  • Rabbit Anti-CHRM1 Doctor De Biotechnology Co., Ltd., Product Code: BA1544;
  • Rat acetylcholine enzyme-linked immunosorbent assay kit Shanghai Huole Biotechnology Co., Ltd., LOT: 20130 1,4-8 ° C;
  • Rat choline acetyltransferase content enzyme-linked immunoassay kit Shanghai Live Music Biotechnology Co., Ltd., LOT: 201303, 4-8 ° C;
  • Rat choline acetyltransferase activity enzyme-linked immunoassay kit Shanghai Huole Biotechnology Co., Ltd., LOT: 20130 1,4-8 ° C;
  • Rat muscarinic acetylcholine M1 enzyme-linked immunoassay kit Shanghai Huole Biotechnology Co., Ltd., LOT: 201304, 4-8 ° C;
  • BCA protein concentration determination kit Biyuntian Biotechnology Research Institute
  • Protein marker Thermo scitific26616, Exp: 2014/02;
  • Tris-base BIOSHARP Amresco 0497, LOT: 2012/10;
  • Horseradish-labeled goat anti-mouse IgG Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., batch number: 107724;
  • Horseradish-labeled goat anti-rabbit IgG Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., batch number: 101964;
  • Skim milk powder Yili, batch number: 66196131T;
  • Rat brain stereotaxic instrument NARISHIGE SR-5N, Tokyo, Japan;
  • Rat Y maze, new object identification, and dark-avoidance devices are all produced by Shenyang Pharmaceutical University;
  • Paraffin slicer Leica, Germany.
  • Ultrasonic cell pulverizer JY92-II, Ningbo Xinzhi Biotechnology Co., Ltd.;
  • Constant temperature water bath device BCFCO Changfeng Electronic Technology Co., Ltd.;
  • Electrothermal constant temperature incubator DNP-9272, Shanghai Precision Experimental Equipment Co., Ltd.;
  • High-speed refrigerated centrifuge HC-3018R, HKUST Innovation Co., Ltd.;
  • Rats were anesthetized by intraperitoneal injection of 3.5% chloral hydrate (350 mg/kg body weight). The hair in the cranial incision region was cut, and after iodophor disinfection, it was fixed on the brain stereotaxic instrument. According to the brain stereotaxic map of the rat, the NBM nuclear position (AP: B-1.4mm, ML: ⁇ 2.4mm, DV: -7.5mm) was determined. The skull was drilled with a dental drill and the needle was inserted vertically with a micro syringe. 2 ⁇ l (containing 120 nmol) of quinolinic acid (dissolved in 0.1 mol/L, pH 7.4 PBS buffer) was slowly injected into the left NBM core, and the needle was left for 5 min. The right injection was consistent with the left side. The sham group was injected with an equal amount of PBS buffer.
  • AP B-1.4mm
  • ML ⁇ 2.4mm
  • DV -7.5mm
  • SD rats were randomly divided into sham operation group (negative control group), model group, donepezil hydrochloride 0.95 mg/kg group, Qingnao Yangxue granules 647 mg/kg group (human clinical equivalent), 1294 mg/kg group (2 Double clinical equivalent), 2588mg/kg group (4 times human clinical equivalent), 17-21 per group.
  • Each group began intragastric administration on the next day after modeling, and once every day until the behavioral experiment was completed.
  • the rat Y maze experiment aims to investigate the effects of test drugs on working memory in rats through the indicators of spontaneous alternation.
  • the device consists of three wooden arms with an angle of 120°, which are three arms of A, B and C respectively.
  • the rats were placed at the end of the A-arm, and they were allowed to freely enter and exit the three arms.
  • the total number of pieces of arm entries (N) and the sequence of the in-arms of each rat entering the three arms within 8 minutes were recorded to continuously enter three
  • the different arms are a successful alternation, recording the number of alternations N.
  • the spontaneous alternating reaction rate Alternation behavior (%) number of alternation / (N-2) ⁇ 100 was used.
  • the experimental device was made of black plastic with a diameter of 1.5 meters. Two days before the test, 2-3 rats were placed in the experimental device facing the device wall for 5 min, twice daily. On the day of the test, the rats were placed in the experimental apparatus and allowed to freely explore for 3 minutes to adapt to the environment and then taken out. Place two identical objects (A1, A2) at the same distance from the wall of the device, put the rats into the device again, and record the time (tA1, tA2) of exploring the two objects within 5 minutes, each group is half larger. The color of the objects A1, A2 was changed during the test to exclude the rat's preference for color.
  • Priority index (1h) tB / (tA1 + tB) formula (1)
  • Priority index (24h) tC / (tA1 + tC) formula (2)
  • Discrimination coefficient (24h) (tC-tA1/(tA1+tC) formula (4)
  • the spontaneous activity experiment in rats was designed to investigate the effects of test drugs on the excitability of rat cerebral cortex.
  • the experimental device is composed of four rectangular movable boxes (45 ⁇ 45 ⁇ 13cm) with built-in infrared probe and camera.
  • the spontaneous activity video analysis system is provided by Shanghai Jiliang Software Technology Co., Ltd. During the experiment, the animals were placed in four movable boxes, one for each box. Set the collection time for 15 minutes, and record the total distance of animal activities, activity time and average speed.
  • the rat Morris water maze experiment was designed to investigate the effects of test drugs on spatial learning and memory in rats.
  • Positioning navigation experiment Morris water maze training was performed once a day in the afternoon and afternoon for 4 consecutive days.
  • the platform is placed at the midpoint of the fourth quadrant. Two points equal to the distance are selected as the water inlet points on the opposite side of the platform.
  • the rats are placed in the water facing the pool wall for 90s, and the time from the water entering the platform to the platform is recorded. Escape latency, and then let the rats rest on the platform for 10s. If the platform is not found in 90s, the incubation period is recorded as 90s and the rats are placed on the platform for 10s. Train at different water intake points every day.
  • the rat darkness test was designed to investigate the effects of test drugs on long-term memory in rats.
  • the device is a dark box (divided into a bright room and a dark room, the upper part of the bright room is illuminated by tungsten light, the rear part of the dark room can be connected to alternating current, and there is a door hole with a diameter of 6cm ⁇ 4cm between the two rooms).
  • the experiment is divided into two parts: training and testing. This method utilizes the dark-skin habits of rats.
  • the rats are placed in the bright room with their backs to the doorway. After 3 minutes of free movement, the animals enter the darkroom, and the gates are closed and energized.
  • the door was opened and the animal entered the bright room due to the shock of the electric shock. If it re-enters the darkroom, the shock is continued until the rat reaches the bright room for 2 minutes and is trained for 5 minutes. After 24 hours, the test was carried out, and the rats were placed in a dark box, and the number of times they entered the dark room within 5 minutes was recorded as the number of errors, and the time from the time when the rat was placed in the darkroom to the first time entered the dark room as the incubation period (s ), as a memory score.
  • Rat hippocampal and cortical tissue samples were stored in a -80 ° C refrigerator for later use.
  • the protein lysate was added at a ratio of 1:10, and 5 ⁇ l of PMSF was added per 1 ml of the protein lysate.
  • BCA working solution was prepared by adding 50 volumes of BCA reagent A plus 1 volume of BCA reagent B (50:1), and thoroughly mixed.
  • the protein standard was 5 mg/ml BSA, and the protein standard was completely dissolved, and 20 ⁇ L was diluted to 100 ⁇ L to give a final concentration of 1 mg/ml.
  • the standard was diluted with PBS. 2 Add the standard to 0, 1, 2, 4, 8, 12, 16, 20 ⁇ l to the standard well of a 96-well plate, add 20 ⁇ l of standard dilution (PBS), and repeat each concentration three times.
  • SDS-PAGE 1SDS-polyacrylamide gel
  • Assembly of plastic glass plates Prepare 12% separation glue, mix well, immediately inject into the gap of the glass plate, inject it to about 3cm from the top of the glass plate, seal with gelatinized water, polymerize at room temperature for about 30-60min, and separate the deionized Water, as much as possible to absorb the water on the surface of the separation gel.
  • Electrophoresis The proteins in the samples were separated by electrophoresis on a 12% SDS-polyacrylamide gel (SDS-PAGE). According to the results of BCA protein quantification, the protein concentration of the sample was adjusted to be consistent before loading, and 5 ⁇ SDS-PAGE protein loading buffer was added, and heated at 100 ° C or a boiling water bath for 5 min to fully denature the protein. The gel was placed in an electrophoresis tank and 1X running buffer was added. 30 ⁇ g of total protein was added to each well. At the beginning of the electrophoresis, the voltage was 80V. After the dye entered the separation gel, it increased to 180V, and the dye was turned off when it reached the bottom of the separation gel.
  • SDS-PAGE SDS-polyacrylamide gel
  • An ice box is added to both sides of the transfer film to prevent overheating during film transfer.
  • the current is turned on (the gel is connected to the negative electrode and the PVDF film is connected to the positive electrode), and the constant current is transferred for 2 hours, and the current is 100 mA. After the transfer is completed, turn off the power and take out the film.
  • the membrane was placed in a blocking solution (5% skimmed milk powder was placed in PBS buffer) and gently shaken at room temperature for 2 h.
  • the primary antibody (SYP, GAP-43, PSD-95, CHMR1) was prepared with a blocking solution and placed in a refrigerator at 4 ° C overnight.
  • the membrane was washed three times with PBS at room temperature, and the membrane was washed once with Tris-NaCl at room temperature for 10 min each time.
  • Add a horseradish peroxidase-labeled secondary antibody (1:3000) with blocking solution and incubate for 2 h at room temperature.
  • the membrane was washed three times with Tris-NaCl at room temperature for 10 min each.
  • chloral hydrate 350 mg/kg was anesthetized by intraperitoneal injection, fixed on the operating table with the supine position, and the heart was exposed by thoracotomy.
  • the perfusion needle is inserted into the left ventricle from the apex of the apex, while the right atrial ear is cut into a small mouth, firstly perfused with about 200-300 ml of normal saline, and the liquid flowing out of the right atrial appendage becomes colorless and clarified.
  • the replacement is 300- 400 ml 4 ° C 4% paraformaldehyde buffer (0.1 mol / L) perfusion until the body is stiff.
  • the whole brain was removed by decapitation and fixed in 4% paraformaldehyde at 4 °C. After 24 h, conventional paraffin-embedded, coronal sections, 5 ⁇ m thick, were used for routine HE staining.
  • Four rats in each group were treated with 4% paraformaldehyde and 2.5% glutaraldehyde 1:1 mixture.
  • the hippocampal CA1 area was fixed and 2.5% glutaraldehyde was fixed for transmission electron microscopy. structure.
  • Paraffin sections were subjected to routine HE staining. The procedure was as follows: paraffin sections were routinely dewaxed to water; distilled water was washed for 2 min, hematoxylin staining was carried out for 3 min, washed with water for 5 min, 1% hydrochloric acid alcohol was differentiated for 30 s, tap water returned to blue for 8 min, eosin stained for 1 min, tap water was washed for 3 min. Gradient alcohol dehydration, xylene transparent 2 ⁇ 5min, neutral gum seal. Histopathological changes were observed under an optical microscope.
  • Electron microscopy showed that the neurons in the sham operation group had clear outlines, the nucleus was elliptical, the chromatin distribution in the nucleus was uniform, the nuclear membrane and nucleolus were clear, and the cytoplasm was rich in ribosome, rough endoplasmic reticulum, and more.
  • the mitochondria and lysosomes are organelles; the nucleus of the model group is irregular in nucleus, the heterochromatin in the nucleus has obvious cohesion, the edge set, the cytoplasm is partially dissolved, and the remaining mitochondrial outer membrane is blurred; the donepezil group and the serum-suppressed brain In the 2588mg/kg group, the nucleus of the neuron was nearly round, the chromatin in the nucleus was evenly distributed, the contour of the nuclear membrane was clear, and the cytoplasm was rich in ribosomes, mitochondria, rough endoplasmic reticulum and lysosomes.
  • the nucleus of the neuron was round, the nuclear membrane and nucleolus were clear, the chromatin in the nucleus was evenly distributed, there were more ribosomes in the cytoplasm, the rough endoplasmic reticulum expanded, the mitochondria or the outer membrane was partially missing. See also lysosome and other organelles; the serum cerebral granules in the 647 mg/kg group have a nearly round neuron nucleus, a uniform distribution of chromatin in the nucleus, and more ribosomes, mitochondria, lysosomes, and mild expansion in the cytoplasm. Rough surface Interstitial reticulum (see Figure 62).
  • the synaptic ultrastructure of neurons was visible, and the synaptic structure of the sham operation group was clear, and the presynaptic membrane, posterior membrane and gap were visible. There are more synaptic vesicles in the presynaptic membrane, and the thickness of the dense substance in the synaptic membrane is relatively uniform.
  • the synaptic structure of the model group is disordered, the presynaptic membrane and the posterior membrane are fused; part of the presynaptic membrane is seen.
  • Acetylcholine (Ach) is the earliest discovered neurotransmitter closely related to learning and memory. Choline acetyltransferase (ChAT) catalyzes the binding of acetyl group of acetyl-CoA to choline in the cytosol to form Ach. After Ach synthesis, it is transported into the vesicles by the synaptic vesicle vector. When activated, Ach diffuses into the postsynaptic membrane and binds to the postsynaptic membrane receptor, exerting its biological effects. Acetylcholine receptors are classified into nicotinic acetylcholine receptor (nAChR) and muscarinic acetylcholine receptor (mAChR).
  • nAChR nicotinic acetylcholine receptor
  • mAChR muscarinic acetylcholine receptor
  • mAChR is a G protein-coupled neurotransmitter with five subtypes: M1-M5, and the M1 receptor located in the postsynaptic membrane is mainly present in the brain.
  • the results of ELISA showed that the expression of Ach in the cerebral cortex and hippocampus of the model group was significantly reduced compared with the sham operation group. Compared with the model group, the serum-induced brain granules increased the expression of Ach in a dose-dependent manner, and the donepezil hydrochloride group was also significant. Increase Ach expression (see Figure 70, Figure 71, and Table 29).
  • Non-spatial learning and memory impairments such as working memory impairment, spatial learning and memory impairment, and object discrimination ability are similar in the high-dose group to donepezil hydrochloride.
  • Yangxuenao granules can significantly increase the expression of synaptophysin and postsynaptic density 95 in cerebral cortex and hippocampus of NBM-induced dementia rats, and improve hippocampal synaptic structure abnormalities.
  • Yangxuenao Granule can significantly increase the expression of acetylcholine in the cerebral cortex and hippocampus of NBM-induced dementia rats, and increase the expression of choline acetyltransferase and cerebral cortex M1 choline receptor in hippocampus.
  • Figure 2Y maze experiments demonstrate that nourishing has a significant effect on improving memory and cognitive ability in early and mid-term Alzheimer's animal models (compared with saline group * p ⁇ 0.05, ** p ⁇ 0.01).
  • Figure 7 Number of ⁇ -amyloid plaques in brain tissue of transgenic mice of each drug group (compared with saline group, * p ⁇ 0.05; ** p ⁇ 0.01).
  • Figure 8 Areas of ⁇ -amyloid plaque coverage in brain tissue of transgenic mice of each drug group ( ** p ⁇ 0.01 compared with saline group).
  • Figure 9 is a design of a specific antibody against the APP[beta][alpha] (1-16 aa of A[beta]42) (immunohistochemical staining with APP[beta] specific antibodies recognizing insoluble proteins to demonstrate serum-sparing brain particles).
  • Figure 10 The number and morphology of ⁇ -amyloid plaques in the cerebral cortex of transgenic mice in each drug group.
  • the arrows in the figure indicate the ⁇ -amyloid plaques positive for APP staining, which are dark brown.
  • Figure 11 The number and morphology of A ⁇ -amyloid plaques in the hippocampus of male transgenic mice in each drug group.
  • the arrows in the figure indicate the ⁇ -amyloid plaques positive for APP staining, which are dark brown.
  • Figure 12 The number and morphology of A ⁇ -amyloid plaques in the hippocampus of female transgenic mice in each drug group.
  • the arrows in the figure indicate the ⁇ -amyloid plaques positive for APP staining, which are dark brown.
  • Figure 13 shows the number of strongly positive A ⁇ -amyloid plaques in the brain tissue of transgenic mice of each drug group, compared with the saline group, ** p ⁇ 0.01.
  • Figure 14 Effect of each drug group on key splicing enzymes and splicing products of APP in the brain of transgenic mice.
  • Figure 15 shows the molecular mechanism by which serum brain granules significantly clear the role of senile plaques in the brain of early and mid-term Alzheimer's disease models.
  • FIG. 16 Spontaneous activity trace of SAMP8 mice.
  • F nourishing serum brain particles 3740 mg/kg.
  • Fig. 29 Effect of Yangxuenao Granule on the expression of brain-derived nerve growth factor (BDNF) in hippocampus of SAMP8 mice ( ⁇ 4).
  • BDNF brain-derived nerve growth factor
  • Fig. 30 Effect of Yangxuenao Granule on the expression of brain-derived nerve growth factor (BDNF) in hippocampal CA1 region of SAMP8 mice ( ⁇ 40).
  • BDNF brain-derived nerve growth factor
  • BDNF brain-derived nerve growth factor
  • FIG 32 Effect of Qingnao Granule on the expression of brain-derived nerve growth factor (BDNF) in the cerebral cortex of SAMP8 mice ( ⁇ 40).
  • BDNF brain-derived nerve growth factor
  • BDNF brain-derived nerve growth factor
  • FIG. 34 Effect of Yangxue Brain Granule on the expression of hippocampal nerve growth factor (NGF) in SAMP8 mice ( ⁇ 4).
  • Fig. 35 Effect of Yangxuenao Granule on the expression of nerve growth factor (NGF) in hippocampal CA1 region of SAMP8 mice ( ⁇ 40).
  • NGF nerve growth factor
  • NGF nerve growth factor
  • FIG 37 Effect of Yangxuenao Granule on the expression of nerve growth factor (NGF) in the cerebral cortex of SAMP8 mice ( ⁇ 40).
  • NGF nerve growth factor
  • NGF nerve growth factor
  • Fig. 39 Effect of Yangxue brain granule on TrkA expression in hippocampal CA1 region of SAMP8 mice ( ⁇ 4).
  • Fig. 40 Effect of Yangxue brain granule on TrkA expression in hippocampal CA1 region of SAMP8 mice ( ⁇ 40).
  • Figure 50 is a representation of the spontaneous activity trajectory of each group of rats.
  • Fig. 61 Effect of Yangxuenao Granule on the pathological changes of hippocampal neurons in rat model of dementia induced by quinolinic acid ( ⁇ 40).
  • Raw material weight ratio 253.5g of Angelica sinensis, 253.5g of Rhizoma Chuanxiong, 202.7g of Radix Paeoniae Alba, 202.7g of Rehmannia glutinosa, 506.8g of Uncaria, 506.8g of Spatholobus suberectus, 506.8g of Prunella vulgaris, 506.8g of Cassia, 506.8g of cassia seed, 506.8g of mother-of-pearl, 253.5g g, Asarum 50.5g.
  • Extract 1 Angelica, Chuanxiong, Corydalis, Cassia seed were added to 4 times 70% ethanol and heated to reflux for 2 times, the first 2 hours, the second time 1 hour, filtered, decontaminated, recovered ethanol and concentrated to relative Density 1.300-1.310 (74-76 ° C), get the extract, spare.
  • Extract 3 Rehmannia glutinosa, Uncaria, Spatholobus suberectus, Prunella vulgaris, mother-of-pearl, and asarum were added to 5 times the amount of water for 2 times, the first 2 hours, the second time 1 hour, filtered, concentrated To a relative density of 1.06-1.10 (80 ° C), add ethanol to make the alcohol content 65-70%, let stand for 12-24 hours, filter, recover ethanol, and concentrate to a relative density of 1.320-1.325 (79-81 ° C). Get the extract, spare.
  • the remaining dextrin 250.0g was put into the granulator to adjust the granulation parameters such as fan frequency, inlet air temperature, infusion frequency and atomization pressure, so that the materials in the bed were in a good fluidized state.
  • Spray granulation the granulation process controls the temperature of the material between 30-60 °C. Dry and let the temperature of the material rise to 80-90 ° C and dry thoroughly.
  • Raw material weight ratio Angelica 6.75%, Chuanxiong 6.75%, Baizhu 5.4%, Uncaria 13.5%, Spatholobus chinensis 13.5%, Rehmannia glutinosa 5.4%, Cassia 13.5%, Prunella 13.5%, Asarum 1.34%, Corydalis 6.75 % and mother-of-pearl 13.5%.
  • Extract 1 Angelica, Chuanxiong, Corydalis, Cassia seed were added to 4 times 70% ethanol and heated to reflux for 2 times, the first 2 hours, the second time 1 hour, filtered, decontaminated, recovered ethanol and concentrated to relative Density 1.300-1.310 (74-76 ° C), get the extract, spare.
  • Extract 3 Rehmannia glutinosa, Uncaria, Spatholobus suberectus, Prunella vulgaris, mother-of-pearl, and asarum were added to 5 times the amount of water for 2 times, the first 2 hours, the second time 1 hour, filtered, concentrated To a relative density of 1.06-1.10 (80 ° C), add ethanol to make the alcohol content 65-72%, let stand for 12-24 hours, filter, recover ethanol, and concentrate to a relative density of 1.320-1.325 (79-81 ° C). Get the extract, spare.
  • the prepared extract is prepared by a conventional preparation method to obtain a serum-supplemented brain pill.
  • Raw material weight ratio Angelica 6.75%, Chuanxiong 6.75%, Baizhu 5.4%, Uncaria 13.5%, Spatholobus chinensis 13.5%, Rehmannia glutinosa 5.4%, Cassia 13.5%, Prunella 13.5%, Asarum 1.34%, Corydalis 6.75 % and mother-of-pearl 13.5%.
  • Extract 1 Angelica, Chuanxiong, Corydalis, and Cassia seed were added to 5 times 70% ethanol and heated to reflux for 2 times, the first 2.5 hours, the second time 1 hour, filtered, decontaminated, recovered ethanol and concentrated to relative Density 1.250-1.310 (70-74 ° C), get the extract, spare.
  • Extract 3 Rehmannia glutinosa, Uncaria, Spatholobus suberectus, Prunella vulgaris, mother-of-pearl, and asarum were added to 5 times the amount of water for 2 times, the first 2 hours, the second time 1 hour, filtered, concentrated To a relative density of 1.06-1.10 (80 ° C), add ethanol to make the alcohol content 60-65%, let stand for 12-24 hours, filter, recover ethanol, and concentrate to a relative density of 1.27-1.320 (75-80 ° C), Get the extract, spare.
  • the prepared extract is prepared by a conventional preparation method to obtain a serum-removing brain drop pellet.
  • Raw material weight ratio Angelica 6.75%, Chuanxiong 6.75%, Baizhu 5.4%, Uncaria 13.5%, Spatholobus chinensis 13.5%, Rehmannia glutinosa 5.4%, Cassia 13.5%, Prunella 13.5%, Asarum 1.34%, Corydalis 6.75 % and mother-of-pearl 13.5%.
  • Extract 1 Angelica, Chuanxiong, Corydalis, Cassia seed were added to 4 times 80% ethanol and heated to reflux for 2 times, the first 2.5 hours, the second time 1 hour, filtered, decontaminated, recovered ethanol and concentrated to relative Density 1.30-1.350 (75-80 ° C), get the extract, spare.
  • Extract 3 Rehmannia glutinosa, Uncaria, Spatholobus suberectus, Prunella vulgaris, mother-of-pearl, and asarum were added to 8 times the amount of water for 2 times, the first 3 hours, the second 2 hours, filtered, concentrated To a relative density of 1.06-1.10 (80 ° C), add ethanol to 80-85% alcohol content, let stand for 12-24 hours, filter, recover ethanol, and concentrate to a relative density of 1.30-1.350 (80-85 ° C), Get the extract, spare.
  • the prepared extract is prepared by a conventional preparation method to obtain a serum-free brain drop oral solution.
  • Raw material weight ratio Angelica 6.75%, Chuanxiong 6.75%, Baizhu 5.4%, Uncaria 13.5%, Spatholobus chinensis 13.5%, Rehmannia glutinosa 5.4%, Cassia 13.5%, Prunella 13.5%, Asarum 1.34%, Corydalis 6.75 % and mother-of-pearl 13.5%.
  • Extract 1 Angelica, Chuanxiong, Corydalis, Cassia seed were added to 4 times 50% ethanol and heated to reflux for 2 times, the first 2 hours, the second 2 hours, filtered, decontaminated, recovered ethanol and concentrated to relative Density 1.300-1.350 (73-78 ° C), get the extract, spare.
  • Extract 3 Rehmannia glutinosa, Uncaria, Spatholobus suberectus, Prunella vulgaris, mother-of-pearl, and asarum were added to 10 times the amount of water for 2 times, the first 2 hours, the second 2 hours, filtered, concentrated To a relative density of 1.06-1.10 (80 ° C), add ethanol to make the alcohol content of 63-70%, let stand for 12-24 hours, filter, recover ethanol, and concentrate to a relative density of 1.290-1.330 (78-83 ° C). Get the extract, spare.
  • the prepared extract is prepared by a conventional preparation method to obtain a serum-enriched brain capsule.
  • Raw material weight ratio 300g of Angelica sinensis, 300g of Rhizoma Chuanxiong, 400g of Radix Paeoniae Alba, 400g of Rehmannia glutinosa, 650g of Uncaria, 650g of spatholobus, 650g of Prunella vulgaris, 650g of Cassia, 650g of mother-of-pearl, 300g of Corydalis, 300g of Corydalis, and 50g of Asarum.
  • Extract 1 Angelica, Chuanxiong, Corydalis, and Cassia are added to 3 times 60% ethanol and heated to reflux for 3 times, the first 2 hours, the second 1 hour, the third 0.5 hour, filtered, and impurities. The ethanol was recovered and concentrated to a relative density of 1.29-1.340 (73-78 ° C) to obtain an extract for use.
  • Extract 3 Rehmannia glutinosa, Uncaria, Spatholobus suberectus, Prunella vulgaris, mother-of-pearl, and asarum were added to 7 times the amount of water for 2 times, the first 2 hours, the second time 1 hour, filtered, concentrated To a relative density of 1.06-1.10 (80 ° C), add ethanol to make the alcohol content 70-75%, let stand for 12-24 hours, filter, recover ethanol, and concentrate to a relative density of 1.310-1.330 (77-82 ° C). Get the extract, spare.
  • the remaining dextrin 336g was put into the granulator to adjust the granulation parameters such as fan frequency, inlet air temperature, infusion frequency and atomization pressure, so that the materials in the bed were in a good fluidized state.
  • Spray granulation the granulation process controls the temperature of the material between 30-60 °C. Dry and let the temperature of the material rise to 70-90 ° C to dry thoroughly.
  • Raw material weight ratio 338g of Angelica sinensis, 338g of Rhizoma Chuanxiong, 300g of Radix Paeoniae Alba, 300g of Rehmannia glutinosa, 413g of Uncaria, 413g of Spatholobus suberectus, 413g of Prunella vulgaris, 413g of Cassia, 413g of mother-of-pearl, 337g of Corydalis, and 75g of Asarum.
  • Extract 1 Angelica, Chuanxiong, Corydalis, and Cassia seed were added to 6 times 70% ethanol and heated to reflux for 2 times, the first 2 hours, the second 0.5 hour, filtered, decontaminated, recovered ethanol and concentrated to relative Density 1.260-1.310 (74-76 ° C), get the extract, spare.
  • Extract 3 Rehmannia glutinosa, Uncaria, Spatholobus suberectus, Prunella vulgaris, mother-of-pearl, and asarum were added to 6 times the amount of water for 2 times, the first 2 hours, the second time 1 hour, filtered, concentrated To a relative density of 1.06-1.10 (80 ° C), add ethanol to make the alcohol content 65-75%, let stand for 12-24 hours, filter, recover ethanol, and concentrate to a relative density of 1.300-1.300 (79-81 ° C). Get the extract, spare.
  • the remaining dextrin 163g was put into the granulator to adjust the granulation parameters such as fan frequency, inlet air temperature, infusion frequency and atomization pressure, so that the materials in the bed were in a good fluidized state.
  • Spray granulation the granulation process controls the temperature of the material between 30-60 °C. Dry and let the temperature of the material rise to 70-90 ° C to dry thoroughly.
  • Raw material weight ratio Take Angelica 450g, Chuanxiong 450g, Radix Paeoniae Radix 350g, Radix Rehmanniae Radix 350g, Uncaria 570g, Spatholobus 570g, Prunella vulgaris 570g, Cassia 570g, Mother of Pearl 570g, Corydalis 450g, Asarum 100g.
  • Extract 1 Angelica, Chuanxiong, Corydalis, Cassia are added to 6 times 80% ethanol and heated to reflux for 2 times, the first time 1 Hour, the second time 1 hour, filtered, decontaminated, ethanol was recovered and concentrated to a relative density of 1.29-1.340 (73-78 ° C), the extract was obtained, and used.
  • Extract 3 Rehmannia glutinosa, Uncaria, Spatholobus suberectus, Prunella vulgaris, mother-of-pearl, asarum were added to 9 times the amount of water for 2 times, the first 3 hours, the second 3 hours, filtered, concentrated To a relative density of 1.06-1.08 (80 ° C), add ethanol to make the alcohol content 65-75%, let stand for 12-22 hours, filter, recover ethanol, and concentrate to a relative density of 1.310-1.330 (77-82 ° C). Get the extract, spare.
  • the remaining dextrin 256g was put into the granulator to adjust the granulation parameters such as fan frequency, inlet air temperature, infusion frequency and atomization pressure, so that the materials in the bed were in a good fluidized state.
  • Spray granulation the granulation process controls the temperature of the material between 30-60 °C. Dry and let the temperature of the material rise to 70-90 ° C to dry thoroughly.
  • Raw material weight ratio 253.5g of Angelica sinensis, 253.5g of Rhizoma Chuanxiong, 202.7g of Radix Paeoniae Alba, 202.7g of Rehmannia glutinosa, 506.8g of Uncaria, 506.8g of Spatholobus suberectus, 506.8g of Prunella vulgaris, 506.8g of Cassia, 506.8g of cassia seed, 506.8g of mother-of-pearl, 253.5g g, Asarum 50.5g.
  • Extract 1 Angelica, Chuanxiong, Corydalis, Cassia seed were added to 4 times 70% ethanol and heated to reflux for 2 times, the first 2 hours, the second time 1 hour, filtered, decontaminated, recovered ethanol and concentrated to The relative density of 1.250-1.310 (70-74 ° C), the extract, spare.
  • Extract 3 Rehmannia glutinosa, Uncaria, Spatholobus suberectus, Prunella vulgaris, mother-of-pearl, and asarum were added to 5 times the amount of water for 2 times, the first 2 hours, the second time 1 hour, filtered, concentrated To a relative density of 1.06-1.10 (80 ° C), add ethanol to make the alcohol content 65-75%, let stand for 12-24 hours, filter, recover ethanol, and concentrate to a relative density of 1.27-1.320 (75-80 ° C). Get the extract, spare.
  • the remaining soluble starch 250.0g was put into the granulator to adjust the granulation parameters such as fan frequency, inlet air temperature, infusion frequency and atomization pressure, so that the materials in the bed were in a good fluidized state.
  • Spray granulation the granulation process controls the temperature of the material between 30-60 °C. Dry and let the temperature of the material rise to 80-90 ° C and dry thoroughly.
  • Raw material weight ratio 338g of Angelica sinensis, 338g of Rhizoma Chuanxiong, 270.3g of Radix Paeoniae Alba, 270.3g of Radix Rehmanniae, 675.7g of Uncaria, 675.7g of Spatholobus, 675.7g of Prunella vulgaris, 675.7g of Prunella vulgaris, 675.7g of cassia seed, 675.7g of mother-of-pearl, 338g of Corydalis Asarum 67.3g.
  • Extract 1 Angelica, Chuanxiong, Corydalis, Cassia seed were added to 4 times 70% ethanol and heated to reflux for 2 times, the first 2 hours, the second time 1 hour, filtered, decontaminated, recovered ethanol and concentrated to relative Density 1.280-1.320 (75-80 ° C), get the extract, spare.
  • Extract 3 Rehmannia glutinosa, Uncaria, Spatholobus suberectus, Prunella vulgaris, mother-of-pearl, and asarum were added to 5 times the amount of water for 2 times, the first 2 hours, the second time 1 hour, filtered, concentrated To a relative density of 1.06-1.10 (80 ° C), add ethanol to make the alcohol content 60-65%, let stand for 12-24 hours, filter, recover ethanol, and concentrate to a relative density of 1.315-1.320 (76-79 ° C). Dip, spare.
  • microcrystalline cellulose 80 g was taken and opened with purified water, and 3.0 g of aspartame was added thereto, and the mixture was thoroughly stirred to dissolve, and the prepared extract was added to the above slurry stepwise and stirred. Adjust the specific gravity of the slurry between 1.12.1.23 (42-50 °C). 60 mesh - 100 mesh online filtering.
  • 320 g of the remaining microcrystalline cellulose was put into a granulator to adjust the granulation parameters such as the fan frequency, the inlet air temperature, the infusion frequency and the atomization pressure, so that the materials in the bed were in a good fluidized state.
  • Spray granulation the granulation process controls the temperature of the material between 30-60 °C. Dry and let the temperature of the material rise to 70-90 ° C to dry thoroughly.
  • Raw material weight ratio 150g of Angelica, 150g of Rhizoma Chuanxiong, 150g of Radix Paeoniae Alba, 225g of Radix Rehmanniae, 225g of Rehmannia glutinosa, 551g of Uncaria, 551g of Spatholobus suberectus, 551g of Prunella vulgaris, 551g of Cassia, 551g of mother-of-pearl, 225g of Corydalis, and 19g of Asarum.
  • Extract 1 Angelica, Chuanxiong, Corydalis, and Cassia seed were added to 5 times 70% ethanol and heated to reflux for 2 times, the first 2.5 hours, the second time 1 hour, filtered, decontaminated, recovered ethanol and concentrated to relative Density 1.290-1.300 (75-77 ° C), get the extract, spare.
  • Extract 3 Rehmannia glutinosa, Uncaria, Spatholobus suberectus, Prunella vulgaris, mother-of-pearl, and asarum were added to 5 times the amount of water for 2 times, the first 2 hours, the second time 1 hour, filtered, concentrated To a relative density of 1.06-1.10 (80 ° C), add ethanol to make the alcohol content 65-70%, let stand for 12-24 hours, filter, recover ethanol, and concentrate to a relative density of 1.310-1.315 (79-82 ° C). Get the extract, spare.
  • lactose 151g was put into the granulator to adjust the granulation parameters such as fan frequency, inlet air temperature, infusion frequency and atomization pressure, so that the materials in the bed were in a good fluidized state.
  • Spray granulation the granulation process controls the temperature of the material between 30-60 °C. Dry and let the temperature of the material rise to 70-90 ° C to dry thoroughly.
  • Raw material weight ratio Take Angelica 250g, Chuanxiong 250g, Radix Paeoniae 250g, Radix Rehmanniae 250g, Uncaria 740g, Spatholobus 740g, Prunella vulgaris 740g, Cassia 740g, Mother of Pearl 740g, Corydalis 250g, Asarum 50g.
  • Extract 1 Angelica, Chuanxiong, Corydalis, Cassia seed were added to 4 times 80% ethanol and heated to reflux for 2 times, the first 2.5 hours, the second time 1 hour, filtered, decontaminated, recovered ethanol and concentrated to relative Density 1.280-1.300 (75-77 ° C), dipped, spare.
  • Extract 3 Rehmannia glutinosa, Uncaria, Spatholobus suberectus, Prunella vulgaris, mother-of-pearl, and asarum were added to 8 times the amount of water for 2 times, the first 3 hours, the second 2 hours, filtered, concentrated To a relative density of 1.06-1.10 (80 ° C), add ethanol to 80-85% alcohol content, let stand for 12-24 hours, filter, recover ethanol, and concentrate to a relative density of 1.280-1.330 (75-80 ° C), Get the extract, spare.
  • the remaining dextrin 330g is put into the granulator to adjust the granulation parameters such as the fan frequency, the inlet air temperature, the infusion frequency and the atomization pressure, so that the materials in the bed are in a good fluidized state.
  • Spray granulation the granulation process controls the temperature of the material between 30-60 °C. Dry and let the temperature of the material rise to 70-90 ° C to dry thoroughly.
  • Serum serum brain formula 1
  • the weight ratio of raw materials was: 6.75% of Angelica, 6.75% of Chuanxiong, 5.4% of Radix Paeoniae, 13.5% of Uncaria, 13.5% of Spatholobus, 5.4% of Radix Rehmanniae, 13.5% of Cassia, 13.5% of Prunella vulgaris, 13.5% of Asarum, 1.34% of Asarum 6.75% and mother-of-pearl 13.5%
  • the medicinal materials are processed by pretreatment, water extraction, concentration, ethanol precipitation, recovery of ethanol, concentration into a paste, mixing and granulation, and packaging of finished products. That is to take the above drugs in proportion, add water to boil three times, each time for 1 hour, combine the decoction, concentrate the appropriate amount, add 2 times the amount of ethanol, let stand for 24 hours to precipitate, take the supernatant to concentrate into a paste, the relative density is 1.3- 1.4, the creaming rate is 10%, and other suitable dosage forms are prepared according to a conventional process.

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Abstract

一种中药组合物用于制备治疗阿尔茨海默氏症（老年痴呆）药物的应用，该中药组合物由当归6.75%、川芎6.75%、白芍5.4%、钩藤13.5%、鸡血藤13.5%、熟地黄5.4%、决明子13.5%、夏枯草13.5%、细辛1.34%、延胡索6.75%和珍珠母13.5%制成。

Description

养血清脑制剂在制备治疗阿尔茨海默症的药物中的应用 技术领域
本发明涉及一种中药组合物制剂的新用途，特别涉及养血清脑制剂在制备治疗阿尔茨海默症(老年痴呆症)的药物中的应用。
背景技术
养血清脑颗粒是天津天士力制药股份有限公司研制的现代中药制剂，并于1996年获得国家新药证书，并于1999年被列入国家基本药物目录，2000年被列入国家医保药品目录。养血清脑颗粒是由当归、川芎、白芍、钩藤、鸡血藤、熟地黄、决明子、夏枯草、细辛、延胡索和珍珠母11味中药，经现代高科技手段提取后，加入适当辅料，经混合制粒等制剂过程制成的一种颗粒剂。该制剂具有有效成分溶出快、生物利用度高等优点。养血清脑颗粒具有养血平肝，活血通络的功效，可用于血虚肝亢所致的头痛、眩晕眼花、心烦易怒、失眠多梦等病症，在临床上具有显著的疗效。
养血清脑制剂为现有技术，中国专利93100050.5描述了其配方及制备方法，该专利描述的配方还可以根据制剂学常规技术制备成不同的剂型，如片剂、胶囊、口服液等。
阿尔茨海默症(Alzheimer disease，AD)，也称为老年痴呆症，是一种起病隐匿的进行性发展的神经系统退行性疾病。临床上以记忆障碍、失语、失用、失认、视空间技能损害、执行功能障碍以及人格和行为改变等全面性痴呆表现为特征，病因迄今未明。65岁以前发病者，称早老性痴呆；65岁以后发病者称老年性痴呆。
目前治疗阿尔茨海默症的药物包括：多奈哌齐(donepezil)、利斯的明(Rivastigmine)、加兰他敏(Galantamine)、石杉碱甲(Huperzine A)、美金刚(Memantine)、司来吉兰(Selegiline)、维生素E、褪黑素、银杏提取物(gingko bilobi)、吡拉西坦(piracetam脑复康)、茴拉西坦(aniracetam阿尼西坦，三乐喜)、萘非西坦等。
本发明人经过研究，意外地发现养血清脑制剂具有治疗阿尔茨海默症的作用。
发明内容
本发明提供一种中药组合物的新用途，特别涉及养血清脑制剂的新的医药用途。
具体而言，本发明提供一种中药组合物在制备治疗阿尔茨海默症的药物中的应用。所述应用在于所述中药组合物可以用于清除阿尔茨海默症患者脑内的老年斑。
本发明所述中药组合物是由如下重量百分比的药材制备而成：当归6.75％、川芎6.75％、白芍5.4％、钩藤13.5％、鸡血藤13.5％、熟地黄5.4％、决明子13.5％、夏枯草13.5％、细辛1.34％、延胡索6.75％和珍珠母13.5％。
制备工艺为：药材经前序处理→水提→浓缩→乙醇沉淀→回收乙醇→浓缩成膏→混合制成制剂。
所述制剂包括任何一种可服用的剂型，优选口服制剂，如：颗粒剂、丸剂、片剂、胶囊剂、口服液。
本发明所述的应用在于该中药组合物使记忆认知能力显著提高。
本发明所述的应用在于该中药组合物能够提高大脑皮层及海马组织中脑源性神经生长因子、神经生长因子及其受体TrkA的表达，改善海马神经元超微结构的异常。
本发明所述的应用在于该中药组合物能够增加大脑皮层总抗氧化能力及GSH含量，提高SOD、GSH-px活性，减少MDA含量。
本发明所述的应用在于该中药组合物能够提高大脑皮层及海马突触素及突触后致密物95的表达，改善海马突触结构的异常。
本发明所述的应用在于该中药组合物能够提高大脑皮层及海马组织中乙酰胆碱的表达，提高海马胆碱乙酰转移酶及大脑皮质M1胆碱受体的表达。
本发明所述的应用在于该中药组合物用于清除阿尔茨海默患者脑内的老年斑。
本发明所述的应用在于该中药组合物能够清除脑内Aβ蛋白，降低脑内Aβ蛋白的生成。
本发明所述的应用在于所述应用在于该中药组合物能够抑制致病性γ分泌酶早老素PS1的表达水平抑制APP的病理性剪切，促进APP发生生理性α剪切的作用。
具体实施方式
本发明所述该中药组合物的优选提取方法为：
按照上文所述的重量百分比，取中药组合物处方量的各药材备用：
(1)提取物1的制备：当归、川芎、延胡索、决明子加乙醇加热回流提取，滤过，除杂，回收乙醇并浓缩，备用；
(2)提取物2的制备：白芍加乙醇加热回流提取，滤过，回收乙醇并浓缩，备用；
(3)提取物3的制备：熟地黄、钩藤、鸡血藤、夏枯草、珍珠母、细辛加水煎煮，滤过，浓缩，加乙醇静置，滤过，回收乙醇并浓缩，备用；
(4)制剂的制备：取以上3种提取物，加入适量辅料，制剂，即得。
另一优选的制备方法为：
(1)提取物1的制备：当归、川芎、延胡索、决明子加入3-6倍量50-80％乙醇加热回流提取2-3次，第一次0.5-2.5小时，第二、三次各0.5-2小时，滤过，除杂，回收乙醇并浓缩至相对密度1.250-1.350(70-80℃)，备用；
(2)提取物2的制备：白芍加入3-6倍量50-80％乙醇，浸渍，加热回流提取2-3次，第一次0.5-2.5小时，第二、三次各0.5-2小时，滤过，回收乙醇并浓缩至相对密度1.10-1.35(55-65℃)，备用；
(3)提取物3的制备：熟地黄、钩藤、鸡血藤、夏枯草、珍珠母、细辛加入4-10倍量水煎煮2-3次，第一次0.5-3小时，第二、三次各1-3小时，滤过，浓缩至相对密度1.06-1.10(75-85℃)，加乙醇使含醇量为60-85％，静置12-24小时，滤过，回收乙醇，浓缩至相对密度1.270-1.350(75-85℃)，备用；
(4)制剂的制备：取以上提取物，加入适量辅料，制剂，即得。
最优选的制备方法为：
(1)提取物1的制备：当归、川芎、延胡索、决明子加入4倍量70％乙醇加热回流提取2次，第一次2小时，第二次1小时，滤过，除杂，回收乙醇并浓缩至相对密度1.300-1.310(74-76℃)，备用；
(2)提取物2的制备：白芍加入4倍量60％乙醇，浸渍，加热回流提取2次，第一次2小时，第二次1小时，滤过，回收乙醇并浓缩至相对密度1.23-1.33(65℃)，备用；
(3)提取物3的制备：熟地黄、钩藤、鸡血藤、夏枯草、珍珠母、细辛加入5倍量水煎煮2次，第一次2小时，第二次1小时，滤过，浓缩至相对密度1.06-1.10(80℃)，加乙醇使含醇量为65-70％，静置12-24小时，滤过，回收乙醇，浓缩至相对密度1.320-1.325(79-81℃)，备用；
(4)制剂的制备：取以上提取物，加入适量辅料，制剂，即得。
本发明在描述乙醇的百分比浓度时，是指乙醇水溶液的体积比浓度。本发明所称的含醇量为乙醇含量(v/v)。
本发明所述应用是经过实验观察获得的，其使用了该中药组合物制剂中的一种，即天士力制药股份有限公司生产的“养血清脑颗粒”作为实验药品，任何与养血清脑颗粒具有相同处方经提取得到的提取物均具有与养血清脑颗粒相同的用途。实验结果如下：
效果实验一、养血清脑颗粒对轻中度阿尔茨海默症模型鼠脑内β-淀粉样斑块的清除作用和记忆认知能力改善作用研究
APPswe/PSldE9双转基因小鼠是国际公认的阿尔茨海默症(Alzheimer’s Disease，AD)模型鼠，6月龄APPswe/PSldE9转基因脑内开始出现了老年斑，模拟人类中轻度阿尔茨海默症。4月龄APPswe/PSldE9双转基因阿尔茨海默症模型小鼠连续60天，口服养血清脑颗粒48g生药/kg(高剂量)和16g生药/kg(低剂量)，或对照药物安理申1.03mg/kg至小鼠6月龄，发现口服养血清脑颗粒一个月和二个月后，小鼠记忆认知能力显著提高，且作用效果优于阳性药物安理申组。通过脑组织切片的刚果红染色和免疫组织化学染色实验证明养血清脑颗粒清除早中期阿尔茨海默症模型鼠大脑β-淀粉样斑块(老年斑)的显著作用，从小鼠大脑海马区和皮质区β-淀粉样斑块(老年斑)的个数、覆盖面积和着色程度方面，养血清脑颗粒对脑内老年斑的清除率达60-90％，显著高于阳性药物安理申组。人淀粉样蛋白前体APP(β-amyloid precursor protein)的异常剪切是阿尔茨海默症发病的关键，进一步通过转基因小鼠脑内APP的关键剪切 酶和剪切产物的检测，证明养血清脑颗粒通过提高脑内APP的生理性剪切途径并抑制APP病理性剪切途径，从而降低了β-淀粉样斑块在脑内的形成和沉积。本研究充分证明养血清脑颗粒对轻中度阿尔茨海默症有很好的治疗效果。
1实验目的
养血清脑颗粒是天士力集团有限公司生产的中药复方制剂，临床上主要用于治疗头痛，改善慢性脑缺血。在长期临床观察中，发现该药还有一定的改善阿尔茨海默症的作用。本实验室进行了该药对早中期APPswe/PSldE9双转基因阿尔茨海默症模型小鼠记忆、认知能力影响，特别是针对阿尔茨海默症特征性病变–β淀粉样蛋白Aβ在脑内的沉积，利用不溶性Aβ的特异染色、淀粉蛋白识别的刚果红染色等手段，研究了养血清脑颗粒对早中期APPswe/PSldE9双转基因阿尔茨海默症模型小鼠脑内Aβ老年斑的清除作用，并通过蛋白剪切途径的研究揭示其作用靶点和机制，为养血清脑颗粒用于治疗轻中度阿尔茨海默症提供实验依据。
2实验材料
2.1受试药品及配制
2.1.1受试药物：养血清脑颗粒浸膏(简称养血清脑)，黑褐色浸膏，由天士力药业集团提供，批号：20120514，出膏率13％，相当于每克浸膏含7.7g生药，用生理盐水配制成2.4g、0.8g生药/ml浓度的溶液，供小鼠灌胃给药用(文中剂量按生药量计)，给药体积为20ml/kg。小鼠用药剂量为高浓度48g生药/kg，低浓度16g生药/kg。
阳性对照安理申，市售，每片5mg，卫材(中国)有限公司，批号：111030A，临用时用生理盐水按0.0515mg/ml制成混悬液，供小鼠灌胃给药用，给药体积为20ml/kg。小鼠用药剂量为安理申1.03mg/kg。2.2实验动物与基因型鉴定
实验动物APPswe/PSldE9双转基因AD小鼠购自南京大学模式动物研究所。APPswe/PSldE9双转基因小鼠是国际公认的阿尔茨海默症模型鼠。如图1所示，APPswe/PSldE9双转基因小鼠转入了人的瑞典型突变APPswe和第9外显子缺失突变的PSldE92个基因，可表达人的突变型APP分子，异常剪切产生过量的Aβ42多肽，在脑内形成Aβ斑块及其他AD病变过程中的组织病理变化，出现AD相应的行为学异常改变。据报道该品系4-5月龄小鼠时开始在杏仁体和海马出现一些嗜刚果红的斑块(老年斑初期)，6月龄开始在大脑皮质、海马和杏仁体形成大量淀粉样斑块，模拟人类中轻度阿尔茨海默症。
发明人对用于药物测试的APPswe/PSldE9双转基因AD小鼠进行了基因型鉴定。取鼠尾尾尖，加入裂解液和蛋白激酶K10mg/ml，56℃隔夜消化鼠尾。加入酚氯仿去除蛋白,异丙醇沉淀DNA,70％冰乙醇洗涤干燥获得鼠尾DNA。采用APPswe引物：5’-GACTGACCACTCGACCAGGTTCTG-3’和5’-CTGACTGGTGAGCTGGTCCAAGAC-3’进行鉴定APPswe基因的PCR反应。反应条件：94℃30sec，69℃60sec，72℃60sec，30个循环。经PCR扩增，转基因鼠中出现APP基因特异条带，大小为350bp，而背景鼠无条带产生。利用PCR方法对所有实验鼠进行了基因型鉴定，保证了所有实验鼠为阳性纯合子的转基因鼠，如图1所示。
2.3实验试剂及配制
2.3.1试剂
多聚甲醛(Sigma)、戊巴比妥钠(Genview)、刚果红、苏木精、氧化汞(Sigma)、DAB显色试剂盒(北京鼎国)、鼠兔通用型免疫二抗(Proteintech)、浓氨水、无水乙醇、浓盐酸、甘油、甲醇、柠檬酸、柠檬酸三钠、二甲苯(北京化工)。TEMED、丙烯酰胺、甲叉双丙烯酰胺、Tris、Gly、考马斯亮蓝R250、BSA(Sigma)、Triton X-100(Genview)、蛋白质标准分子量(北京全式金)、Tween-20(BBI)、兔抗鼠β-肌动蛋白抗体(武汉博士德)、HRP标记山羊抗兔IgG(北京全式金)、ECL光化学试剂盒(碧云天)。
刚果红染色液与苏木精染色液：取0.5g刚果红粉末和20ml甘油加至80ml甲醇中，配制成0.2％刚果红染色液。取20g硫酸铝钾溶于200ml蒸馏水，100℃溶解。取苏木精1g溶于10ml无水乙醇，加至硫酸铝钾水溶液中煮沸，加入0.5g氧化汞，继续加热并搅拌至溶液为深紫色，立即冷却过滤，配制成苏木精染色液。
2.4实验主要仪器
Y型迷宫系统(RD1102-YM)，垂直电泳仪和电源系统(北京六一厂)，半干转膜仪(BIO-RAD)。DP70正置显微镜和摄影系统(OLYMPUS)，应用显微镜图像处理软件DP Controller采集图片。
3实验方法
3.1实验分组及给药
实验动物APPswe/PSldE9双转基因AD小鼠随机分为生理盐水对照组、养血清脑颗粒低浓度组(本文有时简称养血低浓度组或养血低剂量组)、养血清脑颗粒高浓度组(本文有时简称养血高浓度组或养血高剂量组)和安理申组，雌雄各半，各组16只，在实验过程中，生理盐水组和安理申组各有2只死亡，每组14-16只，体重20-40g。常规饲养，自由饮水，4月龄APPswe/PSldE9转基因鼠连续灌胃给药，给药容积每25g体重灌胃0.5ml。灌胃给药至6月龄。
表1 试验分组及给药量
Figure PCTCN2014086915-appb-000001
3.2Y迷宫实验检测小鼠记忆认知行为
Y迷宫仪器包括3个等长的臂，各夹角120°，呈Y字形。小鼠在封闭的空间内(Y迷宫)会本能的寻找出路，认知和空间记忆能力正常的小鼠会在寻找过程中探寻每一条路径，而非总是重复已经探寻过的路径。具体为将小鼠自Y迷宫的带有小门的臂放入迷宫，将三个臂任意编号为A臂、B臂和C臂，小鼠在其内自由活动8分钟，记录小鼠的路线，如小鼠的路径能够依次循环(三个路径中无重复)则记为正确，如出现错误路径，则记为错误，并以该处为起点重新计路径。最终计算公式中：正确率(％)＝n/(总进臂次数-2)×100％，n是指正确的路径数。例如：小鼠活动路线为ABCABCBACBACACBABCA，其中下划线即为错误的路径，n＝16，总进臂次数为19，正确率(％)＝16/(19-2)×100％＝94.11％。
3.3小鼠心脏灌流固定及包埋切片
小鼠通过戊巴比妥钠深度麻醉，麻醉后剪开小鼠的胸腔，将灌流针插入小鼠左心室内，同时剪开右心耳，灌入0.01M PBS(pH7.4)50-100ml，直到灌流液清亮为止。同时再灌入4％的多聚甲醛50-100ml。灌流完成后，取脑，在4％多聚甲醛溶液中浸泡固定过夜。自动包埋机包埋后进行石蜡切片。片厚1.5μm。
3.4刚果红染色检测大脑内淀粉样物质
石蜡片子取出后，在60-65℃烤片机上烤片30min。片子从烤片机上取下后浸泡于二甲苯中30min。按照无水乙醇、无水乙醇、95％乙醇、95％乙醇、90％乙醇、80％乙醇、水、蒸馏水的顺序依次脱水。苏木精染色1min，氨水蓝化。刚果红染色1h后，碱性分化液分化，按照80％乙醇、90％乙醇、95％乙醇、95％乙醇、无水乙醇、无水乙醇的顺序脱水。二甲苯浸泡1min，风干后，树胶封片。脑内Aβ老年斑为淀粉样物质，刚果红染色后呈红色。每张切片对皮质和海马的红色Aβ淀粉样斑块数量和面积进行测量和计算，取其平均值进行分析。
3.5免疫组织化学染色
1石蜡切片浸泡于二甲苯中30min。然后按照无水乙醇、无水乙醇、95％乙醇、95％乙醇、90％乙醇、80％乙醇、水、蒸馏水的顺序依次脱水。pH6.0柠檬酸高压120℃修复2-2.5min，3％医用消毒H2O2去除内源活性物质。加入APPβα抗体4h，鼠兔通用型即用二抗室温孵育50min。DAB染色3min。苏木精复染，氨水蓝化，脱水。二甲苯浸泡1min，风干后，树胶封片。
3.6β-淀粉样斑块计数
每张免疫组织化学染色切片对皮质和海马随机观察3个视野进行测量，计数每个视野下强阳性与弱阳性β-淀粉样斑块数量，取其平均值进行分析。
3.7Western Bloting方法检测小鼠大脑组织内αAPPs、ADAM10和PS1的蛋白水平
剖离小鼠大脑组织，裂解超声后用BCA蛋白定量试剂盒对其进行蛋白定量，之后将样品等蛋白量进行SDS-PAGE，再电转移至PVDF膜上。以5％脱脂奶粉封闭1h，依次滴加各种一抗αAPPs、ADAM10或PS1，室温2h，PBS洗3次。山羊抗兔IgG2HRP，室温反应1h，PBS洗涤3次。后用ECL光化学试剂盒(碧云天)检测信号，X光片曝光显影。
抗体：ADAM10、PS1、β-肌动蛋白抗体购自博奥森公司。APPβα段抗体自主研发，选择APPβ剪切与α剪切之间的16氨基酸短肽DAEFRHDSGYEVHHQK(此肽段为纤维状不溶性肽，是决定Aβ42脑不溶性的关键区域)作为抗原，通过基因重组、测序，蛋白表达与纯化，动物免疫和ProteinA/G纯化获得高效价的APPβα抗体。
3.8统计
将数据应用SPSS Statistics 17.0进行统计学分析。结果用散点图结合平均值和平均值±标准误表示，数据采用单因素方差分析结合LSD法进行组间比较。
4实验结果
4.1一般观察
转基因小鼠4月龄时，已经出现精神损伤症状，与背景鼠相比进食量减少、自主活动减少，行动迟缓，精神萎靡，皮毛欠光泽等。给药后，养血清脑颗粒组转基因小鼠均有不同程度的改善，食量增加，精神好转。
4.2Y迷宫实验证明养血清脑颗粒具有显著改善早中期阿尔茨海默症动物模型小鼠记忆、认知能力的作用
Y迷宫实验是用于检测小鼠空间认知能力和短期记忆能力的一种操作简单、应用广泛的行为学手段。如表2和图2所示，Y迷宫实验证明养血清脑颗粒具有显著改善早中期阿尔茨海默症动物模型小鼠记忆、认知能力的作用。如给药中期一个月的检测结果，生理盐水正确率最低为79.59％，养血低浓度组和高浓度组的小鼠Y迷宫正确率平均值分别为86.10％和87.12％，安理申阳性对照组为80.67％。养血低浓度组和高浓度组都与对照组有显著差别。给药末期两个月的行为学检测结果，生理盐水正确率最低为77.52％，养血低浓度组和高浓度组的小鼠Y迷宫正确率平均值分别为83.86％和85.64％，安理申阳性对照组为82.19％。养血高浓度组和养血低浓度组改善认知能力的作用效果显著高于对照组。
所以，发明人的结果证明，给药一个月和给药两个月，养血低浓度组和高浓度组的小鼠Y迷宫正确率与对照相比提高了近10％，具有显著性；安理申阳性对照组虽然给药两个月时Y迷宫正确率有所提高，但未达到统计学差别。所以，结果证明养血清脑颗粒具有显著改善早中期阿尔茨海默症动物模型小鼠记忆、认知能力的显著作用。
表2 Y迷宫实验检测连续给药2个月对AD模型小鼠记忆、认知能力的影响
Figure PCTCN2014086915-appb-000002
注：与生理盐水组比较*p<0.05，**p<0.01
4.3特异识别淀粉样斑块的刚果红染色证明养血清脑颗粒具有显著清除早中期小鼠脑内老年斑的作用
AD患者表现为额颞叶和海马区等部位的中枢神经系统退行性的病变，Aβ淀粉样斑块是阿尔茨海默症特征性的病变。Aβ的凝集和聚积是AD病理发生、发展的起始因素，而其他的病理改变如脑内神经纤维缠结、神经元的功能紊乱和丢失等，均被认为是由于Aβ的解离与凝聚、清除与产生的失衡所引发的。
阿尔茨海默症的特征性病变是Aβ淀粉样蛋白在脑内的沉积。甲醇刚果红染色用于显示淀粉样物质， 可以很好识别脑内淀粉样Aβ蛋白的沉积。发明人发现通过2个月口服养血清脑颗粒小鼠的大脑皮层中Aβ淀粉样斑块个数和面积显著减少，并且存在剂量相关性，养血高浓度组较低浓度组治疗效果更加明显(图3至图7)。
如图3至图5显示对照组和实验组大脑皮质区淀粉样斑块的刚果红染色情况。生理盐水组和安理申组脑内Aβ淀粉样斑块数目多，养血清脑颗粒高浓度和低浓度组淀粉样斑块数目少。在雄性小鼠(图4)和雌性小鼠(图5)的大脑额叶皮质区，生理盐水组和安理申组可以检测到一定数目的嗜刚果红染色的细胞外红色的菊花样斑块，为AD特征性淀粉斑块，而养血清脑颗粒高浓度和低浓度组淀粉样斑块数目少，斑块面积小。可见养血低浓度组和高浓度组具有显著清除额叶皮质区淀粉样斑块沉积的作用，而对照药物安理申对淀粉样斑块清除作用不显著。
大脑海马区是存储短期记忆关键部位，也是人类处理长期学习与记忆空间定位的大脑区域。图6是各组小鼠大脑海马区的刚果红染色，生理盐水组和安理申组可检测到淀粉样斑块的沉积，而养血清脑颗粒高浓度和低浓度组清脑颗粒海马区周围检测不到淀粉样斑块的沉积。
发明人对全大脑的嗜刚果红染色淀粉样斑块的数目进行了量化。如表3和图7所示，从老年斑的个数来看，6月龄APPswePSEN1dE9转基因鼠生理盐水对照组脑内出现了显著的老年斑，与之比较，阳性对照安理申组老年斑个数下降了37.5％，但是无显著差别。非常显著的是，与生理盐水对照组比较，养血清脑颗粒低浓度组脑内老年斑减少了62.2％(p<0.05)，养血清脑颗粒高浓度组脑内老年斑减少了70.1％(p<0.01)。提示养血清脑颗粒具有显著清除早中期小鼠大脑皮质区老年斑的个数的作用。
发明人对全大脑的嗜刚果红染色淀粉样斑块的面积进行了量化。如表3和图8所示，从老年斑的面积来看，安理申作为一种乙酰胆碱酯酶抑制剂发挥疗效，因而降低大脑或海马区β-淀粉样斑块沉积的作用不明显。阳性对照安理申组老年斑面积比对照组下降了17.8％，但是无显著差别。而与对照组比较，养血低浓度组脑内老年斑面积减少了61.4％(p<0.01)，养血高浓度组脑内老年斑面积减少了72.4％(p<0.01)。
以上结果证明，养血清脑颗粒具有显著清除早中期小鼠脑内淀粉样老年斑的作用，清除率达60-70％以上，高剂量养血清脑颗粒的作用更为显著。市售的治疗阿尔茨海默症的阳性药物安理申作为一种乙酰胆碱酯酶抑制剂发挥疗效，因而降低大脑或海马区β-淀粉样斑块沉积的作用不明显。养血清脑颗粒对淀粉样老年斑清除的治疗效果明显强于阳性药物安理申。
表3 连续给药2个月后不同给药组AD模型小鼠大脑组织β-淀粉样斑块的个数和覆盖面积
Figure PCTCN2014086915-appb-000003
注：与生理盐水组比较，**p<0.01
4.4利用识别脑内不溶Aβ蛋白的APPβα特异抗体的免疫组化染色，证明养血清脑颗粒具有显著清除早中期AD小鼠脑内Aβ老年斑的作用
人淀粉样蛋白前体APP是一种阿尔茨海默症发病的关键蛋白质，其发生β剪切形成的淀粉样蛋白Aβ在脑内的沉积是阿尔茨海默症的主要病理特征之一。APP正常情况下发生α分泌酶剪切途径，产生胞外可溶性sAPPα蛋白。而在阿尔茨海默症的发生过程中APP在两种蛋白酶即β-分泌酶和γ-分泌酶的先后作用下被剪切而产生42个氨基酸的Aβ肽，是AD的主要毒性物，至脑组织中神经元之间及突触部位大量聚集，形成淀粉样老年斑,可使大脑功能受损，导致记忆和认知障碍。目前的抗体多为Aβ段(β剪切与γ剪切的42肽)的抗体，无法识别特异性β剪切(检测C段蛋白中同时包含β剪切蛋白和α剪切蛋白)，特别是对脑内可溶肽(生理)和不溶肽(病理)区分困难。
发明人选择了Aβ42肽中的N段β剪切与α剪切之间的16肽作为抗原，在检测阿尔茨海默症病理剪切具有独特的优势。如图9所示，首先Aβ42肽的脑不溶解肽段发生由此16肽组成，所以此16肽抗体显 著提高识别脑内不溶蛋白(Aβ)的特异性检测效率，将脑内不溶肽的检测研究手段提高到新阶段。抗体的制备和纯化见方法部分，发明人应用此抗体检测了养血清脑颗粒清除阿尔茨海默症转基因鼠脑内Aβ肽沉积作用。
利用识别不溶性蛋白的APPβα特异抗体的免疫组化染色证明养血清脑颗粒具有清除小鼠脑内Aβ蛋白的显著作用。如图10至图12所示，生理盐水对照组和安理申组大脑内有Aβ不溶性蛋白的沉积，可被识别不溶性蛋白的APP特异抗体所识别。养血低浓度组和养血高浓度组都少见Aβ不溶性蛋白的沉积。雄性AD模型鼠(图11)和雌性AD模型鼠(图12)在不同药物作用2个月后，海马区的Aβ蛋白的免疫染色出现显著差别。对照组与安理申组海马区能够检测到深染的不溶性Aβ老年斑，而养血低浓度组和养血高浓度组难以检测到Aβ沉积。
发明人对各实验组大脑皮层β-淀粉样斑块总的数量统计分析，如表4和图13所示，从小鼠大脑强阳性Aβ老年斑数目来看，各实验组与生理盐水对照组比较都有显著性差别，强阳性对照安理申组老年斑个数比对照组下降了67.7％，相对于生理盐水对照组，养血低浓度组脑内老年斑减少了81.4％，养血高浓度组脑内老年斑减少了87.5％。相对于安理申组，养血清脑颗粒的治疗效果更明显，提示了高剂量养血清脑颗粒具有显著清除早中期小鼠脑内Aβ蛋白的作用，并优于安理申组。
表4 各药物组早中期AD模型小鼠脑组织中强阳性Aβ-淀粉样斑块的个数
Figure PCTCN2014086915-appb-000004
注：与生理盐水组比较，**p<0.01
4.5养血清脑颗粒具有显著促进早中期阿尔茨海默症动物模型脑内APP的生理性α剪切，并促进了可溶性神经营养性sAPPα蛋白的生成
人淀粉样蛋白前体APP是一种阿尔茨海默症发病的关键蛋白质，APP的剪切机制分为两种。正常机体APP发生生理性非淀粉样的α分泌酶ADAM10剪切，并产生具有神经营养作用的sAPPα蛋白。而在阿尔茨海默病发生中，由β-和γ分泌酶介导，APP发生淀粉样的病理性剪切。APP发生β和γ剪切形成的淀粉样蛋白Aβ在脑内的沉积是阿尔茨海默症的主要病理特征之一和最重要的分子机制。
APP的生理性α剪切的裂解产物sAPPα，不仅具有重要的神经营养作用，而且可阻碍Aβ的生成，对AD的治疗具有重要意义。首先，通过抗体识别可溶性生理sAPPα的Western blot检测证明了养血清脑颗粒具有显著促进了生理性α剪切，并促进可溶性和神经营养性sAPPα蛋白的作用。APP发生α剪切产生的胞外可溶片段sAPPα，大小为66kD，如图14所示，AD模型鼠脑内在生理盐水条件下很少出现生理性剪切，sAPPα水平很低，与生理盐水组相比，安理申组的sAPPα有所增加。养血低浓度、特别是养血高浓度组的sAPPα增加显著。且养血高剂量组显著高于阳性安理申对照组。APP正常情况下发生α分泌酶剪切途径产生胞外可溶性APP肽具有神经营养作用，同时生理性α剪切产生的sAPPα与病理性β剪切具有显著的拮抗作用。
α分泌酶ADAM10是执行APP生理剪切的关键酶。发明人检测了各药物组大脑内ADAM10的表达水平。6月龄AD转基因模型鼠在生理盐水条件下大脑内ADAM10表达水平很低，说明很少出现生理性剪切。与对照组相比，安理申组ADAM10的表达变化不显著。养血低浓度组、特别是养血高浓度组的ADAM10表达水平显著增高(图14)。
所以，发明人发现了养血清脑颗粒具有促进APP发生生理性α剪切的作用，养血清脑颗粒显著提高鼠脑内α分泌剪切酶ADAM10的表达水平和脑内可溶性神经营养性sAPPα蛋白的生成，通过显著提高了脑内APP的生理性α剪切，拮抗了病理性剪切途径，其作用效果较阳性药物安理申更为显著。
养血清脑颗粒清除脑内老年斑的作用是通过抑制致病性γ分泌酶早老素PS1来实现的。转基因鼠是PS1第9外显子缺失突变的PSldE9基因，早老素PS1基因突变是Aβ形成和AD治病的关键环节。如图 14所示，6月龄AD转基因模型鼠在生理盐水条件下大脑内早老素PSldE9表达水平很高，而与对照组相比，安理申组PSldE9表达变化不显著。而养血低浓度、养血高浓度的早老素PSldE9表达水平显著下降，难以被检测到。说明养血清脑颗粒通过抑制脑内致病性γ分泌酶早老素PS1的表达水平抑制APP的病理性剪切，从而减少脑内Aβ老年斑的沉积。
所以，发明人证明养血清脑颗粒具有显著清除早中期阿尔茨海默症动物模型脑内老年斑的作用。这种分子机制主要通过双方面来实现，如图15所示。一方面，养血清脑颗粒通过抑制致病性γ分泌酶早老素PS1-dE9，从而抑制APP的病理剪切Aβ过程，使脑内老年斑减少。另一方面，养血清脑颗粒通过促进生理性分泌酶α分泌剪切酶ADAM10的水平，促进了神经营养性sAPPα的生成，从而促进了APP的生理性剪切，拮抗了脑内Aβ的生成，使脑内老年斑进一步减少。
5小结
由上述结果，发明人证明了养血清脑颗粒具有显著地治疗轻中度阿尔茨海默症的作用。体现为两个方面：
首先，行为学上Y迷宫实验结果表明，口服高浓度养血清脑颗粒(48g生药/kg)和低浓度养血清脑颗粒(16g生药/kg)一个月和二个月后，小鼠记忆、认知能力均较生理盐水对照组有明显提高，且作用效果优于阳性药物安理申。即，养血清脑颗粒具有显著地改善早中期AD模型小鼠的记忆、认知能力的作用。
更重要的是针对阿尔茨海默症的特征性病变—Aβ蛋白沉积形成β-淀粉样斑块，养血清脑颗粒发挥了极其显著的治疗作用。将给药2个月的早中期阿尔茨海默症模型小鼠脑组织制成切片，应用刚果红染色和免疫组织化学染色实验均证明了口服养血清脑颗粒的小鼠大脑海马区和皮质区β-淀粉样斑块(老年斑)从个数、覆盖面积和着色程度上都显著降低(清除率达到60-90％)，效果远远高于安理申组。同时，提取小鼠脑组织的蛋白进行Western blot分析，证明养血清脑颗粒能够促进β-淀粉样蛋白前体APP的生理性α剪切，并抑制APP病理性剪切，从而降低脑内Aβ的生成，从而发挥清除脑内Aβ-淀粉样斑块的显著作用。
综上，本研究充分说明了养血清脑颗粒对轻中度阿尔茨海默症具有很好的治疗效果。
效果实验二、养血清脑颗粒治疗阿尔茨海默氏痴呆药效学研究
本实验采用Y迷宫、新物体辨别、Morris水迷宫及避暗等行为学实验方法，考察了养血清脑颗粒对SAMP8快速老化小鼠及喹啉酸损毁Meyert基底核(NBM核)致痴呆大鼠学习记忆障碍的改善作用；并从大脑皮层及海马神经细胞及突触的形态学、胆碱能神经系统功能、神经营养因子、自由基氧化损伤等方面探讨了其改善学习记忆障碍的可能机制。
研究发现，养血清脑颗粒在小鼠935-3740mg/kg剂量、大鼠647-2588mg/kg(1-4倍人治疗头痛等症的临床等效量)范围内能够剂量依赖性地增加痴呆模型小鼠及大鼠自发交替反应率、新物体辨别实验中优先指数及辨别系数，缩短水迷宫实验中到达安全台的时间及路程，延长水迷宫空间探索实验中在原平台所在象限的游泳时间和路程百分比，延长避暗实验中逃避潜伏期，减少电击次数。以上行为学结果表明，养血清脑颗粒能够显著改善痴呆模型动物工作记忆障碍、空间学习记忆障碍以及视觉辨别记忆能力等非空间学习记忆障碍，其高剂量组的作用与临床等效量的盐酸多奈哌齐相近；HE染色结果表明，养血清脑颗粒能改善模型小鼠及大鼠神经元的病理改变；透射电镜观察发现，养血清脑颗粒能够改善大脑海马神经元超微结构的异常及突触结构的异常；Western blot实验结果显示，养血清脑颗粒能够显著提高痴呆模型小鼠及大鼠大脑皮层及海马组织中SYP及PSD-95的表达，对大脑皮层及海马突触新生标志物GAP-43的表达未见显著影响；免疫组化实验结果表明，养血清脑颗粒能够剂量依赖性地提高SAMP8小鼠大脑皮层及海马组织中BDNF、NGF及其受体TrkA的表达；生化检测结果表明，养血清脑颗粒能够剂量依赖性地提高总抗氧化能力及SOD、GSH-px活性，增加GSH含量，减少脂质过氧化产物MDA含量。显著提高喹啉酸损毁NBM核致痴呆大鼠大脑皮层及海马组织中乙酰胆碱的含量，提高海马胆碱乙酰转移酶及大脑皮层M1胆碱受体的表达。
综上所述，养血清脑颗粒可能通过改善神经营养因子表达，对抗自由基损伤、抑制脂质过氧化反应等，保护神经元及突触结构，提高脑内Ach、ChAT，改善中枢胆碱能神经系统功能，进而改善学习记忆 障碍。
第一部分：养血清脑颗粒对SAMP8小鼠学习记忆障碍的改善作用及机制探讨
本实验采用Y迷宫、新物体辨别、Morris水迷宫及避暗等行为学实验方法，考察了养血清脑颗粒对SAMP8快速老化小鼠学习记忆障碍的改善作用；用HE染色方法观察小鼠大脑海马组织结构的改变；采用透射电镜观察海马神经细胞和突触超微结构的变化；用免疫组化方法考察养血清脑颗粒对SAMP8小鼠大脑皮层及海马组织中脑源性神经生长因子(BDNF)、神经生长因子(NGF)及其受体TrkA表达的影响；用免疫印迹(Western blot)方法考察养血清脑颗粒对SAMP8小鼠大脑皮层及海马组织中突触生长相关蛋白43(GAP-43)、突触素(SYP)及突触后致密物95(PSD-95)表达的影响；用生化方法检测大脑皮层组织中脂质过氧化产物丙二醛(MDA)、还原型谷胱甘肽(GSH)的含量、总抗氧化的能力(T-AOC)及超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-px)的活性。
研究发现，养血清脑颗粒在935-3740mg/kg剂量(1-4倍人治疗头痛等症的临床等效量)范围内能够剂量依赖性地增加SAMP8小鼠自发交替反应率、新物体辨别实验中优先指数及辨别系数，缩短SAMP8小鼠水迷宫实验中到达安全台的时间及路程，延长水迷宫空间探索实验中在原平台所在象限的游泳时间和路程百分比，延长避暗实验中逃避潜伏期，减少电击次数。以上行为学结果表明，养血清脑颗粒能够显著改善SAMP8小鼠工作记忆障碍、空间学习记忆障碍以及视觉辨别记忆能力等非空间学习记忆障碍，其高剂量组的作用与临床等效量的盐酸多奈哌齐相近；HE染色结果表明，养血清脑颗粒能改善SAMP8小鼠神经元的病理改变；透射电镜观察发现，养血清脑颗粒能够改善大脑海马神经元超微结构的异常及突触结构的异常；免疫组化实验结果表明，养血清脑颗粒能够剂量依赖性地提高SAMP8小鼠大脑皮层及海马组织中BDNF、NGF及其受体TrkA的表达；Western blot实验结果显示，养血清脑颗粒3740mg/kg组能够显著提高SAMP8小鼠大脑皮层及海马组织中SYP及PSD-95的表达，对大脑皮层及海马突触新生标志物GAP-43的表达未见显著影响；生化检测结果表明，养血清脑颗粒能够剂量依赖性地提高总抗氧化能力及SOD、GSH-px活性，增加GSH含量，减少脂质过氧化产物MDA含量。
综上所述，养血清脑颗粒可能通过改善神经营养因子表达，对抗自由基损伤、抑制脂质过氧化反应等，保护神经元及突触结构，进而改善学习记忆障碍。
一.实验材料
1.实验动物
7月龄雄性SAMP8小鼠，150只，同龄空白对照鼠SAMR1小鼠25只，清洁级，由天津中医药大学第一附属医院动物中心提供，合格证号：scxk(津)2008-0001，饲养于沈阳药科大学SPF级实验动物中心，自由饮水摄食，12h循环光照。
2.药物及配制
养血清脑颗粒：浸膏状，由天津天士力制药集团股份有限公司提供，批号20120502W，4℃-8℃保存。人的日服生药量为45g，每一克浸膏相当于6.26g药材，换算成小鼠的临床等效量为935mg(浸膏)/kg。配制：精密称取养血清脑颗粒浸膏，加入蒸馏水，分别配成93.5mg/ml、187mg/ml及374mg/ml的溶液，为养血清脑颗粒的低、中、高剂量组药物浓度。
盐酸多奈哌齐片：卫材(中国)药业有限公司，批号110707A，保存，30℃以下室温环境贮存。临床推荐最大剂量为10mg/d/人，换算成小鼠的临床等效量约为1.3mg/kg。配制：取盐酸多奈哌齐片，加入蒸馏水，配成0.13mg/ml的溶液。
3.试剂
氯化钠：天津博迪化工股份有限公司，批号20120221；
磷酸氢二钠：汕头市西陇化工厂有限公司，批号100802；
无水乙醇：天津市恒兴化学试剂制造有限公司，批号20100327；
多聚甲醛：天津市博迪化工股份有限公司，批号20111028；
戊二醛：天津市博迪化工股份有限公司，批号20110906；
二甲苯：天津市博迪化工有限公司，批号：20100505；
柠檬酸：天津市博迪化工有限公司，批号：20100321；
柠檬酸钠：天津市博迪化工有限公司，批号：20100719；
氢氧化钠：天津市博迪化工有限公司，批号：20111102；
盐酸：沈阳经济技术开发区试剂厂，批号：20110612；
中性树胶：中国上海标本模型厂，批号：20101201；
甲醇：天津市富宇精细化工有限公司，批号：120306；
BCA蛋白定量试剂盒：碧云天生物技术研究所；
细胞裂解液：碧云天生物技术研究所；
PMSF：碧云天生物技术研究所；
PVDF膜：美国Pall公司；
显影粉、定影粉：碧云天生物技术研究所；
鼠抗SYP(D-4)：Santa Cruz Biotechnology，产品编号：sc-17750；
鼠抗GAP-43：Santa Cruz Biotechnology，产品编号：sc-17790
兔抗PSD-95：Abcam(Hong Kong)Ltd LOT:GR76077-1
鼠抗β-肌动蛋白：Santa Cruz Biotechnology，产品编号：sc-41478；
兔抗BDNF:北京中杉金桥生物技术有限公司,LOT:E0112
兔抗NGF-β：Boster Biological Technology CO.,Ltd LOT NO:3574102
兔抗TrkA：Boster Biological Technology CO.,Ltd LOT NO:9H121C
SP免疫组化染色试剂盒：北京中杉金桥生物技术有限公司；
ZLI-9018浓缩型DAB试剂盒：北京中杉金桥生物技术有限公司；
Tris-base：BIOSHARP Amresco 0497；
Glycine：BIOSHARP Amresco 0617；
SDS：BIOSHARP Sigma L-5750LOT 2012/09；
Acrylamide：BIOSHARP Amresco 0341LOT 2011/11；
亚甲双丙烯酰胺：BIOSHARP Amresco；
辣根酶标记山羊抗小鼠IgG：北京中杉金桥生物技术有限公司；
脱脂奶粉：黑龙江省完达山乳业股份有限公司；
高灵敏度化学发光检测试剂盒：北京康为世纪生物科技有限公司；
显影定影试剂盒：碧云天生物技术研究所；
T-AOC总抗氧化能力试剂盒南京建成生物工程研究所批号：20121220
SOD试剂盒南京建成生物工程研究所批号：20121217
GSH-px测试盒南京建成生物工程研究所批号：20121220
微量丙二醛测试盒南京建成生物工程研究所批号：20121212
GSH测试盒南京建成生物工程研究所批号：20121212
4.实验仪器
Morris水迷宫视频分析系统：中国医学科学研究院药物研究所；
自发活动视频分析系统：上海吉量软件科技有限公司；
其他行为学实验装置：小鼠Y迷宫、新物体辨别、避暗装置均为沈阳药科大学研制；
石蜡包埋切片机：德国莱卡公司；
光学显微镜：OLYMPUS公司；
DP72-Color Video Camera：OLYMPUS公司；
U-PMTVC 7C06559：OLYMPUS公司；
Image-pro6.3图像分析系统：OLYMPUS公司；
超声波清洗器：KQ5200B，昆山市超声仪器有限公司；
超声波细胞粉碎机：JY92-Ⅱ，宁波新芝生物科技股份有限公司；
TECAN酶标定量测试仪：SPECTRA CLASSIC；
-80℃低温冰箱：中科美菱；
万分之一天平：赛多利斯科学仪器(北京)有限公司；
电子天平：常州市双杰测试仪器厂；
恒温水浴装置：BCFCO长风电子科技有限公司；
H-1微型漩涡混合器：上海精科实业有限公司；
电热恒温培养箱：DNP-9272，上海精密实验设备有限公司；
立式自动电热压力蒸汽灭菌器：LDZX-40AI，上海申安医疗器械厂；
高速冷冻离心机：HC-3018R，科大创新股份有限公司；
微波炉：格兰仕公司；
二、实验方法
1.动物分组及给药
将SAMP8小鼠随机分为模型组、养血清脑颗粒935mg/kg(人临床等效量)、1870mg/kg(2倍临床等效量)、3740mg/kg(4倍临床等效量)剂量组及盐酸多奈哌齐1.3mg/kg组，每组30只，另设SAMR1空白对照组25只。各组小鼠灌胃给药2个月后开始进行Y迷宫及新物体辨别实验，给药2个半月后进行Morris水迷宫及避暗实验。进行行为学实验时继续给药，每天一次，直至实验结束。
2.行为学实验
2.1小鼠自发活动实验
实验装置为四个长方形活动箱，内置红外线探头及摄像头，自发活动视频分析系统由上海吉量软件科技有限公司提供。实验时，将动物分别面壁放入四个活动箱中，每箱一只。设置采集时间5分钟，记录动物活动总路程、活动时间以及平均速度等指标。
2.2小鼠Y迷宫实验
装置由三个成120°夹角的等长木制支臂组成，即迷宫的A、B、C三臂。每臂长40cm，高12cm，上宽10cm，下宽5cm。实验时将小鼠放入A臂末端，让其自由出入三个臂，记录5min内每只小鼠进入三个臂的顺序及总次数(number of arm entries)，以连续进入三个不同的臂为一次正确交替反应(successive alternation)，记录正确交替反应次数(number of alternation)。用自发交替反应率(alternation behavior；％)反映空间工作记忆能力。Alternation behavior(％)＝number of alternation/(N-2)×100(例如动物5min内进入三个臂的顺序依次为ABCACBACCAB，则N为11，successive alternation为：ABC、BCA、ACB、CBA、BAC、CAB。Alternation behavior(％)＝[6/(11-2)]×100＝66.7％)
2.3小鼠新物体辨别实验
实验装置为一木制的正方形开放场，实验分为适应阶段和测试阶段。将2-3只小鼠放入开放场中自由探索3min以适应环境，每天两次，进行2天。测试当天，先将动物放入实验场中自由探索3min以再次适应环境后，取出动物，将动物未曾见过的两个完全相同的物体(A1、A2)置于装置内距边缘等距离的位置上，将小鼠置于距两物体相等的位置，记录5min内探索两物体的时间(tA1、tA2)，之后，将小鼠取出放回鼠笼1h后，将装置中一个物体换成一个在颜色、形状及材质方面均不相同的新物体(B)，将小鼠再次放入，记录探索两物体所用时间(tA1、tB)，将小鼠取出放回鼠笼。24h后，将物体B换成一个新物体(C)，将小鼠再次放入，记录探索两物体所用时间(tA1、tC)。计算各组对新物体的优先指数及辨别系数，
计算公式如下：
优先指数计算公式如下：
优先指数(1h)＝tB/(tA1+tB)
优先指数(24h)＝tC/(tA1+tC)
辨别系数计算公式如下：
辨别系数(1h)＝(tB-tA1)/(tA1+tB)
辨别系数(24h)＝(tC-tA1)/(tA1+tC)
2.4小鼠Morris水迷宫实验
(1)定位航行实验：每天上下午各进行1次Morris水迷宫训练，连续5天。平台置于第四象限中点， 在平台对侧选两个与之距离相等的点作为入水点，将小鼠面向池壁放入水中，采集60s，记录小鼠从入水至找到平台的时间(逃避潜伏期,escape latency)及游泳路程，然后，让小鼠在平台上休息10s。如果60s未找到平台，潜伏期记为60s，并将小鼠置于平台上休息10s。每天在2个入水点各进行1次，以两次潜伏期的算术均值作为这1天的成绩进行统计分析。
(2)空间探索实验：定位航行实验后，撤除平台，将小鼠放入原平台对侧象限中点的水中，自由游泳60s。迷宫系统自动记录小鼠在原平台象限停留的时间及平台象限路程百分比等参数。
2.5小鼠避暗实验
实验装置分明、暗两室。两室大小均为15cm×10cm×11cm，两室之间有一直径为3cm大小的半圆形门。两室底部均铺以铜栅，暗室底部由第四根铜栅起可以通电，电压强度由一稳压器控制。此法系利用鼠类的嗜暗习性，将小鼠面部背向门口放入明室，适应环境3min，然后通以30V电压。小鼠进入暗室立即遭到电击，然后从门口逃回明室，如此训练5min，并记录小鼠受到电击的次数即错误次数，以此作为学习成绩。24h后测验，记录第一次进入暗室的潜伏期、5min内的错误次数，以此作为记忆成绩。
3.Western blotting方法
3.1组织蛋白质的提取
小鼠大脑皮层及海马组织样本放于-80℃冰箱储存备用。按1：10比例加入蛋白裂解液，每1ml蛋白裂解液加5μl PMSF。用超声细胞粉碎机匀浆后，冰浴中静置30min，12000×g，4℃离心20min，取上清液，分装，于-80℃冰箱储存备用。取少部分上清液用于蛋白定量。
3.2蛋白浓度的测定-BCA法
根据样品数量，按50体积BCA试剂A加1体积BCA试剂B(50:1)配制BCA工作液，充分混匀。①蛋白标准品为5mg/ml BSA，完全溶解蛋白标准品，取10μL稀释至100μL，使终浓度为0.5mg/ml，用PBS稀释标准品。②将标准品按0、1、2、4、8、12、16、20μl分别加到96孔板的标准品孔中，加标准品稀释液(PBS)补足到20μl，每个浓度重复三次。③上述蛋白提取液用PBS稀释10倍后，将20μl蛋白稀释液加到96孔板的样品孔中，每个样品重复三次。④各孔加入200μl BCA工作液，37℃放置30min。⑤用酶标仪测定样品在波长540nm处的吸光度。⑥根据标准曲线计算出样品中的蛋白浓度。
3.3蛋白印迹分析
①SDS-聚丙烯酰胺凝胶(SDS-PAGE)的配制：组装制胶玻璃板。配制分离胶液，充分混匀后，立即注入制胶玻璃板间隙，灌注至离玻璃板顶端3cm左右，用去离子水封胶，室温大约聚合30-60min分离胶凝固，倒掉去离子水，尽可能吸干分离胶表面的水。配制5％浓缩胶液，充分混匀，立即注入玻璃板间隙，插入梳子，避免混入气泡，放置，室温下聚合约60min。浓缩胶聚合后，拔除梳子，用去离子水冲洗梳孔，直接电泳或放入4℃冰箱备用。
②电泳：将样品中的蛋白在SDS-聚丙烯酰胺凝胶(SDS-PAGE)中进行电泳分离。根据BCA法蛋白定量的结果，上样前调整样品的蛋白浓度使其一致，加入5×SDS-PAGE蛋白上样缓冲液，100℃或沸水浴加热5min，使蛋白充分变性。将凝胶放入电泳槽中，加入1×电泳缓冲液，每孔分别加入30μg总蛋白。电泳开始时电压为80V，染料进入分离胶后，增加到180V，染料抵达分离胶底部时断电。
③转膜：在转膜缓冲液中将蛋白质转移至PVDF膜上。活化PVDF膜，将剪好的膜依次序浸入100％甲醇(10s)→去离子水(5min)→转移缓冲液(大于10min)，同时滤纸和海绵垫浸入转移缓冲液中(大于10min)。将结束电泳的浓缩胶除去。安装转膜装置，从正极(红色)→负极(黑色)依次为白色边盒→多孔垫片→(2张)滤纸→PVDF膜→凝胶→(2张)滤纸→多孔垫片→黑色边盒，扣上吊扣放进转膜槽中。转膜槽内两侧加冰盒，防止转膜时过热。接通电流(凝胶一边接负极，PVDF膜一边接正极)，恒流电转移2h，电流为100mA。转膜结束后，关闭电源，将膜取出。
④封闭和免疫反应：将膜放入封闭液中(用PBS缓冲液配置5％脱脂奶粉)，室温轻摇2h，用封闭液配制一抗(SYP、GAP-43、PSD-95、β-肌动蛋白)，放入冰箱4℃过夜。用PBS室温洗膜三次，Tris-Nacl室温洗膜一次，每次10min。加上用封闭液配制辣根过氧化酶标记的二抗(1：3000)，室温孵育2h。用Tris-Nacl室温洗膜三次，每次10min。
⑤显影：用ECL超敏发光液显示免疫反应得到的条带。显影后用一抗二抗去除液进行膜再生，重新封闭， 加一抗、二抗，显影。
⑥图像扫描及定量分析：对X胶片进行灰度扫描，用Quantity One 4.6.2图像分析软件进行分析，对样品中目标蛋白进行定量。以β-肌动蛋白作为内参来确定组间目标蛋白表达的差异和变化。
①生化指标检测
各组小鼠于末次给药2h后断头处死，快速剥离大脑皮层组织，用预冷生理盐水冲洗，除去血迹，滤纸拭干，称重后，将脑组织迅速浸入冷生理盐水中(盐水质量为脑组织质量的9倍)，在超声波细胞粉碎机中制成10％的脑匀浆，以3000-4000r/min离心20min，取上清液，按照试剂盒说明进行生化检测。
②病理组织学观察
5.1取材
小鼠行为学实验结束后，腹腔注射3.5％水合氯醛(350mg/kg)麻醉，仰位固定于手术台上，开胸暴露心脏。将灌流穿刺针从心尖部位插入左心室，同时右心耳剪一小口，先用约200-300ml生理盐水灌流，待右心耳流出的液体变得无色澄清，剪尾无血时，换用300-400ml 4℃4％多聚甲醛缓冲液(0.1mol/L)灌流，直至躯体僵硬。断头取出整脑，置于4℃4％的多聚甲醛中固定。24h后，常规石蜡包埋，冠状切片，厚度5μm，用于常规HE染色及免疫组织化学染色。每组另取4只小鼠，4％多聚甲醛与2.5％戊二醛1:1混合液心脏灌流后，取1mm3海马CA1区组织，2.5％戊二醛固定，供透射电镜观察神经细胞超微结构。
5.2免疫组织化学染色
用链霉素抗生物素蛋白-过氧化酶(SP)连接法进行免疫组化免疫组化检测。原理：使用SP免疫组化染色试剂盒，用生物素标记的第二抗体与链霉素抗生物素蛋白连接的过氧化酶及基质色素混合液测定组织中的抗原。具体实验步骤如下：组织切片脱蜡至水；微波抗原修复：切片置于盛有抗原修复液的容器中，抗原修复液为枸橼酸盐缓冲液(0.01M，pH 6.0)，置微波炉中高火加热至沸腾，自然冷却后，蒸馏水洗涤3min×3；3％H2O237℃孵育20min。PBS洗3min×3；滴加山羊血清封闭液37℃封闭30min，吸去多余血清，滴加一抗(BDNF、NGF、TrkA)4℃孵育过夜，阴性对照以0.01M PBS缓冲液代替一抗；PBS洗5min×3；滴加生物素化二抗工作液(羊抗兔IgG-HRP)，37℃孵育15min，PBS洗5min×3；滴加辣根酶标记链霉卵白素工作液，37℃孵育15min，PBS洗5min×3；DAB显色，显微镜下观察染色强度以控制反应时间，见切片中出现棕黄色显色，立即自来水冲洗，终止染色；苏木素复染3min；自来水洗；盐酸酒精分化数秒，自来水充分水洗；梯度酒精脱水，二甲苯透明，中性树胶封片。用光学显微镜观察每个视野中阳性蛋白表达，用计算机图像分析系统分别测定各组小鼠每张切片内表达阳性蛋白神经元的整合光密度值(intergrated opticaldensity,IOD)，以反应神经元内阳性蛋白表达的相对含量。
5.3HE染色
将石蜡切片进行常规HE染色，程序如下：石蜡切片常规脱蜡至水；蒸馏水洗2min，苏木素染色3min，水洗5min，1％盐酸酒精分化30s，自来水返蓝8min，伊红染色1min，自来水水洗3min；梯度酒精脱水，二甲苯透明2×5min，中性树胶封片。光学显微镜下观察组织病理学变化。
6.统计学方法
实验数据以均数±标准差
Figure PCTCN2014086915-appb-000005
表示。采用SPSS17.0统计软件进行相关统计学分析。组间差异用单因素或双因素方差分析(Morris水迷宫实验)和Dunnett’s t-test进行比较。p<0.05则认为差异存在显著性差别。
三、实验结果
1养血清脑颗粒对SAMP8小鼠学习记忆障碍的改善作用
1.1自发活动实验
实验结果显示，在自发活动实验中，各组小鼠的活动总路程、活动总时间以及平均速度未见显著差异，提示养血清脑颗粒不会通过影响中枢神经系统兴奋性而干扰后续的行为学实验(见表5、图16)。
表5 养血清脑颗粒对SAMP8小鼠自发活动的影响(n＝18-26，平均值±SD)
Figure PCTCN2014086915-appb-000006
Figure PCTCN2014086915-appb-000007
1.2Y迷宫实验
实验结果表明：各组小鼠进入Y迷宫三个臂的总次数之间未见显著性差异(见图17、表6)，提示养血清脑颗粒未对小鼠自发活动产生明显影响。与空白对照组相比，模型组小鼠Y迷宫自发交替反应率显著下降；与模型组相比，养血清脑颗粒剂量依赖性地增加了小鼠Y迷宫自发交替反应率(见图18、表6)，提示养血清脑颗粒可改善小鼠工作记忆障碍。
表6 养血清脑颗粒对SAMP8小鼠Y迷宫进臂总次数及自发交替反应率的影响(n＝23-28，平均值±SD)。
Figure PCTCN2014086915-appb-000008
与空白对照组相比，###p<0.001；与模型组相比，***p<0.001。
1.3新物体辨别实验
实验结果显示，与空白对照组相比，模型组小鼠1h及24h测试阶段对新物体的优先指数和辨别系数显著降低；与模型组相比，养血清脑颗粒剂量依赖性地增加了模型小鼠1h及24h测试阶段对新物体的优先指数和辨别系数(见图19、图20、表7、表8)。
表7 养血清脑颗粒对SAMP8小鼠新物体辨别1h及24h优先指数的影响(n＝22-28，平均值±SD)。
Figure PCTCN2014086915-appb-000009
与空白对照组相比，###p<0.001；与模型组相比***p<0.001，**p<0.01，*p<0.05。
表8 养血清脑颗粒对SAMP8小鼠新物体辨别系数的影响(n＝22-28平均值±SD)
Figure PCTCN2014086915-appb-000010
与空白对照组相比，###p<0.001；与模型组相比，***p<0.001或**p<0.01。
1.4Morris水迷宫实验
1.4.1定向航行实验
实验结果显示，与空白对照组相比，模型组小鼠在水迷宫实验中到达平台的游泳时间及路程显著延长；与模型组相比，养血清脑颗粒剂量依赖性地缩短了小鼠到达平台的游泳时间及路程(见图21、图22、表9、表10)。
表9养血清脑颗粒对SAMP8小鼠水迷宫逃避潜伏期的影响(n＝18-26平均值±SD)。
Figure PCTCN2014086915-appb-000011
注 A：空白对照组 B：模型组 C：盐酸多奈哌齐组 D：养血清脑颗粒组
与空白对照组相比，###p＜0.001；与模型组相比，***p＜0.001，**p＜0.01，*p＜0.05。
表10 养血清脑颗粒对SAMP8小鼠水迷宫游泳路程的影响(n＝18-26,平均值±SD)。
Figure PCTCN2014086915-appb-000012
注 A：空白对照组 B：模型组 Ｃ：盐酸多奈哌齐组 D：养血清脑颗粒组
与空白对照组相比，###p＜0.001；与模型组相比，***p＜0.001，**p＜0.01。
1.4.2空间探索实验
实验结果表明，与空白对照组相比，模型组小鼠在原平台所在象限(第四象限)的游泳时间和第四象限路程百分比显著下降；与模型组相比，养血清脑颗粒3740、1870mg/kg剂量组可显著延长小鼠第四游泳时间和第四象限路程百分比(见图24、图25、表11)，各组小鼠空间探索实验轨迹图(见图23)。
表11 养血清脑颗粒对SAMP8小鼠空间探索实验目标象限游泳时间及目标象限路程百分比的影响(n＝18-26，平均值±SD)。
Figure PCTCN2014086915-appb-000013
与空白对照组相比，###p＜0.001；与模型组相比，***p＜0.001，**p＜0.01，*p＜0.05。
1.5避暗实验结果
实验结果表明，与空白对照组相比，模型组测试阶段潜伏期显著缩短，错误次数显著增加；与模型组相比，养血清脑颗粒剂量依赖性地延长了潜伏期，并减少了错误次数。见表12。
表12 养血清脑颗粒对SAMP8小鼠长期记忆的影响(n＝18-25，平均值±SD)
Figure PCTCN2014086915-appb-000014
与空白对照组相比，###p<0.001；与模型组相比，***p<0.001或*p<0.05。
2HE染色观察海马神经细胞病理变化
HE染色结果可见，空白对照组小鼠海马CA1区神经元细胞结构清楚，排列紧密。模型组小鼠神经元排列疏松、染色质减少。养血清脑颗粒1870mg/kg组、3740mg/kg组及盐酸多奈哌齐组小鼠神经元排列紧密，未见水肿及核固缩(见图26)。
3电镜观察海马CA1区神经元及突触超微结构变化
3.1电镜观察神经元胞体超微结构
电镜观察可见，空白对照组神经元细胞轮廓清晰，核呈椭圆形，核内染色质分布较均匀，核膜、核仁清楚，胞质内有丰富的核糖体、粗面内质网、线粒体及溶酶体等细胞器，超微结构正常；模型组神经元细胞核形不规则，有切迹，见核仁，核膜部分模糊，核内异染色质有边集，胞质内粗面内质网及高尔基复合体轻度扩张，线粒体外膜部分破损；多奈哌齐组神经元细胞核膜清楚，核内染色质分布均匀，胞质内有丰富的核糖体、线粒体、粗面内质网、高尔基复合体及溶酶体等；养血清脑颗粒3740mg/kg组神经元细胞核呈椭圆形，核膜轮廓清楚，核仁清晰，核内染色质分布均匀，胞质内有丰富的核糖体、粗面内质网、线粒体、高尔基复合体及溶酶体等细胞器，但粗面内质网有水肿，个别线粒体外膜破损；养血清脑颗粒1870mg/kg组神经元细胞轮廓清楚，核内有部分异染色质凝聚，胞质内有丰富的核糖体及线粒体，粗面内质网部分轻度扩张；养血清脑颗粒935mg/kg组神经元细胞核膜模糊，核内异染色质有部分边集，胞质内有核糖体、粗面内质网、线粒体、溶酶体及高尔基复合体等细胞器，部分线粒体嵴减少或外膜破损，粗面内质网扩张(见图27)。
3.2电镜观察海马CA1区神经元突触超微结构
空白对照组可见多个突触，结构正常，可见清晰的突触前膜、突触间隙及突触后膜，前膜可见较多的突触小泡；模型组多个突触前后膜融合，间隙消失，部分后膜增厚，少量可见突触间隙；多奈哌齐组突触近乎正常，可见清晰的突触前后膜，前膜内有较多的突触小泡；养血清脑颗粒3740mg/kg组突触近乎正常，可见清晰的前后膜，前膜内有较多的突触小泡，个别前后膜有部分融合；养血清脑颗粒1870mg/kg组可见多数突触后膜致密物较厚，突触间隙宽窄不一，有的已融合；养血清脑颗粒935mg/kg组部分突触前后膜融合或突触间隙变窄(见图28)。
4神经营养相关蛋白表达
4.1养血清脑颗粒对SAMP8小鼠大脑皮层及海马BDNF表达的影响
BDNF能促进多种神经元的存活和生长发育，可提高神经元的生物活性，增加突触终末的密度和促进树突和轴突的生长。BDNF免疫组化阳性结果为神经元胞浆内出现棕黄色颗粒。结果显示，空白对照组小鼠在海马CA1区和皮层神经元胞浆着色较深；模型组小鼠神经元胞浆着色显著变浅，多奈哌齐组、养血清脑颗粒1870mg/kg组及3740mg/kg组神经元着色较模型组显著加深(见图29至图33、表13)。
表13养血清脑颗粒对SAMP8小鼠海马CA1区及大脑皮层脑源性神经生长因子(BDNF)表达的影响(n＝6，平均值±SD)
Figure PCTCN2014086915-appb-000015
与空白对照组相比，＃＃＃p＜0.001；与模型组相比，***p＜0.001，**p＜0.01，*p＜0.05。
4.2养血清脑颗粒对SAMP8小鼠大脑皮层及海马NGF表达的影响
神经生长因子(nervegrowthfactor，NGF)是一种经典的神经生长因子家族的神经营养因子，具有神经元营养和促进神经元突起生长双重生物学功能。NGF免疫组化阳性结果为神经元胞浆内出现棕黄色颗粒。结果显示，空白对照组小鼠在大脑海马CA1区及皮质神经元胞浆着色较深；模型组小鼠神经元胞浆中着色显著变浅，多奈哌齐组、养血清脑颗粒1870mg/kg组、3740mg/kg组神经元着色较模型组显著加深(见图34至图38、表14)。
表14 养血清脑颗粒对SAMP8小鼠海马CA1区及大脑皮层神经生长因子(NGF)表达的影响(n＝6，平均值±SD)
Figure PCTCN2014086915-appb-000016
与空白对照组相比，##p＜0.01，###p＜0.001；与模型组相比，***p＜0.001，**p＜0.01，*p＜0.05。
4.3养血清脑颗粒对SAMP8小鼠脑内TrkA受体表达的影响
TrkA是一种分子量为140kD的跨膜蛋白质。当NGF与TrkA结合后，诱导细胞的增殖、分化和存活，抑制凋亡，增加神经元的兴奋性并诱导表达TrkA的细胞释放介质。TrkA受体免疫组化阳性结果为神经元胞浆内出现棕黄色颗粒。结果显示，空白对照组小鼠在海马CA1区及大脑皮质神经元胞浆着色较深；模型组小鼠神经元胞浆中着色显著变浅，多奈哌齐组、养血清脑颗粒1870mg/kg组、3740mg/kg组神经元着色较模型组显著加深(见图39至图43、表15)。
表15 养血清脑颗粒对SAMP8小鼠海马CA1区及大脑皮层TrkA表达的影响(n＝6，平均值±SD)
Figure PCTCN2014086915-appb-000017
与空白对照组相比，###p＜0.001；与模型组相比，***p＜0.001或**p＜0.01或*p＜0.05。
5养血清脑颗粒对SAMP8小鼠抗氧化能力的影响
实验结果如表16所示，与空白对照组相比，模型组小鼠大脑皮层脂质过氧化产物丙二醛(MDA)的含量显著增多，还原型谷胱甘肽(GSH)含量显著减少，总抗氧化能力(T-AOC)显著降低，超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-px)的活性显著降低，与模型组相比，养血清脑颗粒能够剂量依赖性地增加T-AOC能力及GSH含量，提高SOD、GSH-px活性，减少MDA含量。
表16 养血清脑颗粒对SAMP8小鼠大脑皮层抗氧化能力的影响(n＝7-8，平均值±SD)
Figure PCTCN2014086915-appb-000018
与空白对照组相比，###p<0.001,##p<0.01或#p<0.05；与模型组相比，***p<0.001,**p<0.01或*p<0.05
6养血清脑颗粒对SAMP8小鼠大脑皮层及海马突触新生蛋白、突触素及突触后致密物表达的影响
突触体素，又称突触素(synaptophysin，SYP)是一种与突触结构和功能密切相关的囊泡吸附蛋白，通过其磷酸化与去磷酸化作用调节神经递质释放、参与突触发育，从而在突触可塑性中发挥作用，是学习记忆过程中的重要分子。突触素因其特异性定位于轴突终末的突触囊泡膜上，故可用于标记突触结构，反应轴突终末结构的分布。突触后致密物(postsynaptic density,PSD)是指位于突触后膜胞浆面电子密度较大的半圆形带状区域，是突触部位非常敏感易变的结构，是神经元与神经元之间进行信息传递的结构基础，在突触的可塑性中发挥了重要作用，PSD-95又称SAP90，是最先被鉴定出的PSD中含量丰富的骨架蛋白，属于膜相关鸟苷酸激酶蛋白超家族的成员。GAP-43(Growth associated protein-43)是一个轴突膜蛋白，是一种神经特异性的蛋白质，参与神经细胞外生长及突触发育形成和神经细胞再生。Western blot结果表明，与空白对照组相比，模型组动物海马及大脑皮层组织突触素(SYP)及突触后致密物(PSD-95)的表达显著减少；而与模型组相比，多奈哌齐组、养血清脑颗粒3740mg/kg组海马及大脑皮层组织SYP及PSD-95的表达显著增加，各组之间GAP-43的表达均未见显著差异(见图44至图49，表17、表18)。
表17 养血清脑颗粒对SAMP8小鼠海马突触素(SYP)、突触生长相关蛋白(GAP-43)及突触后致密物(PSD-95)表达的影响(n＝3，平均值±SD)
Figure PCTCN2014086915-appb-000019
与空白对照组相比，##p＜0.01；与模型组相比，**p＜0.01，*p＜0.05。
表18 养血清脑颗粒对SAMP8小鼠大脑皮层组织突触素(SYP)、突触生长相关蛋白(GAP-43)及突触后致密物(PSD-95)表达的影响(n＝3，平均值±SD)
Figure PCTCN2014086915-appb-000020
与空白对照组相比，##p＜0.01或###p＜0.001；与模型组相比，***p＜0.001，**p＜0.01，*p＜0.05。
四小结
1)Y迷宫、新物体辨别、Morris水迷宫及避暗等行为学实验结果显示，养血清脑颗粒在935-3740mg/kg(临床治疗头痛等症的1-4倍等效量)剂量范围内能够剂量依赖性地改善SAMP8小鼠工作记忆障碍、空间学习记忆障碍以及视觉辨别能力等非空间学习记忆障碍，其高剂量组的作用与盐酸多奈哌齐相近。
2)养血清脑颗粒能够提高SAMP8小鼠大脑皮层及海马组织中脑源性神经生长因子(BDNF)、神经生长因子(NGF)及其受体TrkA的表达，改善海马神经元超微结构的异常。
3)养血清脑颗粒能够剂量依赖性地增加SAMP8小鼠大脑皮层总抗氧化能力及GSH含量，提高SOD、GSH-px活性，减少MDA含量。
4)养血清脑颗粒能够提高SAMP8小鼠大脑皮层及海马突触素及突触后致密物95的表达，改善海马突触结构的异常。
第二部分养血清脑颗粒对喹啉酸毁损基底前脑Meyert基底核致痴呆大鼠学习记忆障碍的改善作用及机制研究
本实验采用Y迷宫、新物体辨别、Morris水迷宫及避暗等行为学实验方法，考察了养血清脑颗粒对喹啉酸损毁Meyert基底核(NBM核)致痴呆大鼠学习记忆障碍的改善作用；用HE染色方法观察大鼠海马组织结构的改变；采用透射电镜观察大鼠海马神经细胞及突触超微结构的变化；用免疫印迹(Western blot)方法考察养血清脑颗粒对喹啉酸损毁NBM核致痴呆大鼠海马及大脑皮层突触素(SYP)、突触生长相关蛋白43(GAP-43)、突触后致密物95(PSD-95)及海马毒蕈碱型乙酰胆碱受体(CHRM1)表达的影响；用酶联免疫(ELISA)方法考察对海马及大脑皮层乙酰胆碱(Ach)含量，海马胆碱乙酰转移酶(ChAT)含量及活性以及大脑皮层CHRM1含量的影响。
实验结果表明，养血清脑颗粒在647-2588mg/kg剂量范围内能够剂量依赖性地增加喹啉酸损毁NBM核致痴呆大鼠自发交替反应率，增加新物体辨别实验中对新物体的优先指数及辨别系数，缩短水迷宫实验中痴呆模型大鼠到达安全台的时间及路程，延长空间探索实验中痴呆模型大鼠在原平台所在象限的游泳时间和路程百分比，减少避暗实验被电击次数。以上行为学结果表明，养血清脑颗粒能够显著改善喹啉酸损毁NBM核致痴呆大鼠工作记忆障碍、空间学习记忆障碍以及视觉辨别能力等非空间学习记忆障碍，其高剂量组的作用与盐酸多奈哌齐相近。养血清脑颗粒能够改善海马神经元超微结构的异常及突触结构的异常；改善痴呆模型大鼠神经元的病理改变；剂量依赖性地提高海马及大脑皮层SYP、PSD-95的表达，而对大脑皮层及海马GAP-43表达未见显著影响。提高大脑皮层及海马Ach的含量及海马ChAT的含量，但对海马ChAT活性及CHRM1的表达未见显著影响。综上所述，养血清脑颗粒可能通过提高脑内SYP、PSD-95的表达，改善突触结构的异常，提高脑内Ach及其合成酶ChAT含量，改善中枢胆碱能神经系统功能，进而改善喹啉酸损毁NBM核致痴呆大鼠学习记忆障碍。
一、实验材料
1、实验动物
健康Sprague-Dawley(SD)大鼠，120只，雌雄各半，体重180-220g，购于辽宁长生生物技术有限公 司合格证号：scxk2010-0001；饲养于沈阳药科大学SPF级实验动物中心，自由饮水进食，12h循环光照。
2、药物及配制
养血清脑颗粒：浸膏状，由天津天士力制药集团股份有限公司提供，批号20120502W，4℃-8℃保存。人的日服生药量为45g，每1g浸膏相当于6.26g药材，换算成大鼠的临床等效量为647mg(浸膏)/kg。配制：精密称取养血清脑颗粒浸膏，加入蒸馏水，分别配成64.7mg/ml，129.4mg/ml及258.8mg/ml的溶液。
盐酸多奈哌齐片：卫材(中国)药业有限公司，批号110707A，保存，30℃以下室温环境贮存。临床推荐最大剂量为10mg/d/人，换算成大鼠的临床等效量约为0.95mg/kg。配制：取盐酸多奈哌齐片，加入蒸馏水，配成0.095mg/ml的溶液。
3、实验试剂
喹啉酸：SIGMA公司，批号：STBB0947V；
水合氯醛：天津市瑞金特化学品有限公司，批号：20101124；
火棉胶：天津市恒兴化学试剂制造有限公司，批号：20110421；
氯化钠：天津博迪化工股份有限公司，批号：20130221；
磷酸氢二钠：汕头市西陇化工厂有限公司，批号：110802；
磷酸二氢钠：汕头市西陇化工厂有限公司，批号：110223；
无水乙醇：天津市恒兴化学试剂制造有限公司，批号：20130327；
多聚甲醛：天津市博迪化工股份有限公司，批号：20121028；
戊二醛：天津市博迪化工股份有限公司，批号：20110906；
二甲苯：天津市博迪化工有限公司，批号：20121205；
柠檬酸：天津市博迪化工有限公司，批号：20100321；
柠檬酸钠：天津市博迪化工有限公司，批号：20100719；
盐酸：沈阳经济技术开发区试剂厂，批号：20120304；
苏木色精：国药集团化学试剂有限公司，批号：20081215；
中性树胶：中国上海标本模型厂，批号：20101201；
切片石蜡：上海华灵康复器械厂，批号：20120206；
多聚赖氨酸：北京中杉金桥生物技术有限公司，批号：050M4339；
甲醇：天津市富宇精细化工有限公司，批号：20130306；
BCA蛋白定量试剂盒：碧云天生物技术研究所；
细胞裂解液：碧云天生物技术研究所；
PMSF：碧云天生物技术研究所；
PVDF膜：美国Pall公司，LOT：K1BA3015GK；
显影粉、定影粉：碧云天生物技术研究所，产品编号：P0019-4；
鼠抗SYP(D-4)：Santa Cruz Biotechnology，产品编号：sc-17750；
鼠抗β-肌动蛋白：Santa Cruz Biotechnology，产品编号：sc-41478；
鼠抗GAP-43：Santa Cruz Biotechnology，产品编号：sc-17790；
兔抗PSD-95：Abcam(Hong Kong)Ltd LOT:GR76077-1；
兔抗CHRM1：博士德生物科技有限公司，产品编号：BA1544；
大鼠乙酰胆碱酶联免疫分析试剂盒：上海活乐生物科技有限公司，LOT：201301，4-8℃保存；
大鼠胆碱乙酰转移酶含量酶联免疫分析试剂盒：上海活乐生物科技有限公司，LOT：201303，4-8℃保存；
大鼠胆碱乙酰转移酶活性酶联免疫分析试剂盒：上海活乐生物科技有限公司，LOT：201301，4-8℃保存；
大鼠毒蕈碱型乙酰胆碱M1酶联免疫分析试剂盒：上海活乐生物科技有限公司，LOT：201304，4-8℃保存；
BCA蛋白浓度测定试剂盒：碧云天生物技术研究所；
蛋白marker：Thermo scitific26616，Exp：2014/02；
Tris-base：BIOSHARP Amresco 0497，LOT：2012/10；
Glycine：BIOSHARP Amresco 0617，LOT：2012/11；
SDS：BIOSHARP Sigma L-5750 Exp：2014/08；
Acrylamide：BIOSHARP Amresco 0341，LOT：2011/11；
亚甲双丙烯酰胺：BIOSHARP Amresco，Exp：2014/08；
辣根酶标记山羊抗小鼠IgG：北京中杉金桥生物技术有限公司，批号：107724；
辣根酶标记山羊抗兔IgG：北京中杉金桥生物技术有限公司，批号：101964；
脱脂奶粉：伊利，批号：66196131T；
高灵敏度化学发光检测试剂盒：北京康为世纪生物科技有限公司，LOT：22912；
Western一抗二抗去除液：Beyotime P0025；
医用X射线胶片：富士胶片株式会社，EXP：2015-07。
4实验仪器
大鼠脑立体定位仪：NARISHIGE SR-5N,日本东京；
自发活动视频分析系统：上海吉量软件科技有限公司；
Morris水迷宫及视频采集分析系统:：中国医学科学研究院药物研究所；
行为学装置：大鼠Y迷宫、新物体辨别、避暗装置均为沈阳药科大学制作；
石蜡切片机：德国莱卡公司。
光学显微镜：OLYMPUS公司；
Image-pro6.3图像分析系统：OLYMPUS公司；
超声波清洗器：KQ5200B，昆山市超声仪器有限公司；
超声波细胞粉碎机：JY92-Ⅱ，宁波新芝生物科技股份有限公司；
TECAN酶标定量测试仪：SPECTRA CLASSIC；
-80℃低温冰箱：中科美菱；
万分之一天平：赛多利斯科学仪器(北京)有限公司；
电子天平：常州市双杰测试仪器厂；
恒温水浴装置：BCFCO长风电子科技有限公司；
H-1微型漩涡混合器：上海精科实业有限公司；
电热恒温培养箱：DNP-9272，上海精密实验设备有限公司；
立式自动电热压力蒸汽灭菌器：LDZX-40AI，上海申安医疗器械厂；
高速冷冻离心机：HC-3018R，科大创新股份有限公司；
二、实验方法
1、喹啉酸损毁双侧基底前脑NBM核大鼠模型的建立
大鼠腹腔注射3.5％水合氯醛(350mg/kg体重)麻醉。剪去颅顶切口区毛,碘伏消毒后，固定在脑立体定位仪上。根据大鼠的脑立体定位图谱确定大鼠NBM核位置(AP:B-1.4mm、ML:±2.4mm、DV:-7.5mm)，用牙科钻钻开颅骨，用微量注射器垂直进针，将2μl(含120nmol)喹啉酸(用0.1mol/L，pH 7.4PBS缓冲液溶解)缓慢注入左侧NBM核，留针5min，右侧注射与左侧保持一致。假手术组注入等量的PBS缓冲液。
2、分组与给药
将SD大鼠随机分为假手术组(阴性对照组)、模型组、盐酸多奈哌齐0.95mg/kg组、清脑养血颗粒647mg/kg组(人临床等效量)、1294mg/kg组(2倍人临床等效量)、2588mg/kg组(4倍人临床等效量)，每组17-21只。各组均于造模后次日开始灌胃给药，每天一次至行为学实验全部结束。
3、行为学实验方法
3.1大鼠Y迷宫实验
大鼠Y迷宫实验旨在通过自发交替反应的指标，考察受试药物对大鼠工作记忆的影响。装置由三个夹角为120°的木制支臂组成，分别为A、B、C三臂。实验时将大鼠放入A臂末端，让其自由出入三个臂，记录8min内每只大鼠进入三个臂的总次数number of arm entries(N)及进臂顺序，以连续进入三个不同的臂为一次正确交替反应(successive alternation)，记录正确交替反应次数(number of alternation)N。用自发交替反应率Alternation behavior(％)＝number of alternation/(N-2)×100。
3.2大鼠新物体辨别实验
实验装置由直径为1.5米的黑色塑料制成。测试前两日，将2-3只大鼠面向装置壁放入实验装置中适应5min，每日两次。测试当天，先将大鼠放入实验装置中并允许自由探索3min以适应环境后，取出。将2个完全相同的物体(A1、A2)置于距装置壁等距离处，将大鼠再次放入装置中，分别记录5min内探索两物体的时间(tA1、tA2)，每组另一半大鼠测试时改变物体A1、A2的颜色，以排除大鼠对颜色偏好。1h后，将其中一物体换成一个新物体(B)，将大鼠再次放入，分别记录探索两物体所用时间(tA1、tB)。24h后，将物体B换成一个新物体(C)，将大鼠再次放入，分别记录探索两物体所用时间(tA1、tC)。计算对新物体的优先指数及辨别系数。
优先指数计算公式如下：
优先指数(1h)＝tB/(tA1+tB)公式(1)
优先指数(24h)＝tC/(tA1+tC)公式(2)
辨别系数计算公式如下：
辨别系数(1h)＝(tB-tA1/(tA1+tB)公式(3)
辨别系数(24h)＝(tC-tA1/(tA1+tC)公式(4)
3.3大鼠自发活动实验
大鼠自发活动实验旨在考察受试药物对大鼠大脑皮层兴奋性的影响。实验装置为四个长方形活动箱(45×45×13cm)，内置红外线探头及摄像头，自发活动视频分析系统由上海吉量软件科技有限公司提供。实验时，将动物分别面壁放入四个活动箱中，每箱一只。设置采集时间15分钟，记录动物活动总路程、活动时间以及平均速度等指标。
3.4大鼠Morris水迷宫实验
大鼠Morris水迷宫实验旨在考察受试药物对大鼠空间学习记忆能力的影响。
(1)定位航行实验：每天上下午各进行1次Morris水迷宫训练，连续4天。平台置于第四象限中点，在平台对侧选两个与之距离相等的点作为入水点，将大鼠面向池壁放入水中，采集90s，记录大鼠从入水至找到平台的时间(逃避潜伏期,escape latency)，然后让大鼠在平台上休息10s。如果90s未找到平台,潜伏期记为90s，并将大鼠置于平台上休息10s。每天在不同的入水点进行训练。
(2)空间探索实验：定位航行实验结束后24h，撤除平台，将大鼠放入原平台对侧象限中点的水中，自由游泳90s。水迷宫系统自动记录大鼠在原平台象限停留的时间及路程等参数。
3.5大鼠被动回避反应实验(避暗实验)
大鼠避暗实验旨在考察受试药物对大鼠长期记忆的影响。装置为避暗箱(分为明室和暗室，明室上方以钨灯照明，暗室中后部铜栅可以通交流电，两室间有一直径为6cm×4cm的门洞)。实验分为训练和测试两部分。此法系利用鼠类的嗜暗习性，训练阶段将大鼠背对门口放入明室，自由活动适应3分钟后，待动物进入暗室，将洞门关上后通电。电击大鼠5秒钟后将洞门打开，动物会因为电击疼痛而进入明室。若其再进入暗室，则继续电击，直到大鼠在明室的时间达到2分钟，如此训练5min。24h后进行测试，将大鼠放入避暗箱明室内，记录5分钟内其进入暗室的次数作为错误次数，以及大鼠自放入避暗箱明室至第一次进入暗室的时间作为潜伏期(s)，以此作为记忆成绩。
4Western blotting方法
4.1组织蛋白质的提取
大鼠海马及皮层组织样本放于-80℃冰箱储存备用。按1：10比例加入蛋白裂解液，每1ml蛋白裂解液加5μlPMSF。用超声细胞粉碎机匀浆后，冰浴中静置30min。14000×g，4℃离心20min，取上清液，分装，于-80℃冰箱储存备用。取少部分上清液用于蛋白定量。
4.2蛋白浓度的测定-BCA法
根据样品数量，按50体积BCA试剂A加1体积BCA试剂B(50：1)配制BCA工作液，充分混匀。①蛋白标准品为5mg/ml BSA，完全溶解蛋白标准品，取20μL稀释至100μL，使终浓度为1mg/ml。用PBS稀释标准品。②将标准品按0、1、2、4、8、12、16、20μl分别加到96孔板的标准品孔中，加标准品稀释液(PBS)补足到20μl，每个浓度重复三次。③上述蛋白提取液用PBS稀释10倍后，将20μl蛋白稀释液加到96孔板的样品孔中，每个样品重复三次。④各孔加入200μl BCA工作液，37℃放置30min。⑤用酶标 仪测定样品在波长540nm处的吸光度。⑥根据标准曲线计算出样品中的蛋白浓度。
4.3蛋白印迹分析
①SDS-聚丙烯酰胺凝胶(SDS-PAGE)的配制：组装制胶玻璃板。配制12％分离胶液，充分混匀后，立即注入制胶玻璃板间隙，灌注至离玻璃板顶端3cm左右，用去离子水封胶，室温大约聚合30-60min分离胶凝固，倒掉去离子水，尽可能吸干分离胶表面的水。配制5％浓缩胶液，充分混匀，立即注入玻璃板间隙，插入梳子，避免混入气泡，放置，室温下聚合约60min。浓缩胶聚合后，拔除梳子，用去离子水冲洗梳孔，直接电泳或放入4℃冰箱备用。
②电泳：将样品中的蛋白在12％SDS-聚丙烯酰胺凝胶(SDS-PAGE)中进行电泳分离。根据BCA法蛋白定量的结果，上样前调整样品的蛋白浓度使其一致，加入5×SDS-PAGE蛋白上样缓冲液，100℃或沸水浴加热5min，使蛋白充分变性。将凝胶放入电泳槽中，加入1×电泳缓冲液。每孔分别加入30μg总蛋白。电泳开始时电压为80V，染料进入分离胶后，增加到180V，染料抵达分离胶底部时断电。
③转膜：在转膜缓冲液中将蛋白质转移至PVDF膜上。活化PVDF膜，将剪好的膜依次序浸入100％甲醇(10s)→去离子水(5min)→转移缓冲液(大于10min)。同时滤纸和海绵垫浸入转移缓冲液中(大于10min)。将结束电泳的浓缩胶除去。安装转膜装置，从正极(红色)→负极(黑色)依次为→白色边盒→多孔垫片→(2张)滤纸→PVDF膜→凝胶→(2张)滤纸→多孔垫片→黑色边盒扣上吊扣放进转膜槽中。转膜槽内两侧加冰盒，防止转膜时过热。接通电流(凝胶一边接负极，PVDF膜一边接正极)，恒流电转移2h，电流为100mA。转膜结束后，关闭电源，将膜取出。
④封闭和免疫反应：将膜放入封闭液中(用PBS缓冲液配置5％脱脂奶粉)，室温轻摇2h。用封闭液配制一抗(SYP，GAP-43，PSD-95，CHMR1)，放入冰箱4℃过夜。用PBS室温洗膜三次，Tris-Nacl室温洗膜一次，每次10min。加上用封闭液配制辣根过氧化酶标记的二抗(1：3000)，室温孵育2h。用Tris-Nacl室温洗膜三次，每次10min。
⑤显影：用ECL超敏发光液显示免疫反应得到的条带。显影后用一抗二抗去除液进行膜再生，重新封闭，加一抗、二抗，显影。
⑥图像扫描及定量分析：对X胶片进行灰度扫描，用Quantity One 4.6.2图像分析软件进行分析，对样品中目标蛋白进行定量。以β-肌动蛋白作为内参来确定组间目标蛋白表达的差异和变化。
5病理组织学观察
5.1取材
大鼠行为学实验结束后，腹腔注射3.5％水合氯醛(350mg/kg)麻醉，仰位固定于手术台上，开胸暴露心脏。将灌流穿刺针从心尖部位插入左心室，同时右心耳剪一小口，先用约200-300ml生理盐水灌流，待右心耳流出的液体变得无色澄清，剪尾无血时，换用300-400ml 4℃4％多聚甲醛缓冲液(0.1mol/L)灌流，直至躯体僵硬。断头取出整脑，置于4℃4％的多聚甲醛中固定。24h后，常规石蜡包埋，冠状切片，厚度5μm，用于常规HE染色。每组另取4只大鼠，4％多聚甲醛与2.5％戊二醛1:1混合液心脏灌流后，取海马CA1区组织，2.5％戊二醛固定，供透射电镜观察神经细胞超微结构。
5.2HE染色
将石蜡切片进行常规HE染色，程序如下：石蜡切片常规脱蜡至水；蒸馏水洗2min，苏木素染色3min，水洗5min，1％盐酸酒精分化30s，自来水返蓝8min，伊红染色1min，自来水水洗3min；梯度酒精脱水，二甲苯透明2×5min，中性树胶封片。光学显微镜下观察组织病理学变化。
6统计学方法
实验数据以均数±标准差
Figure PCTCN2014086915-appb-000021
表示。采用SPSS17.0统计软件进行相关统计学分析。组间差异用单因素或双因素方差分析(Morris水迷宫实验)和Dunnett’s t-test进行比较。P<0.05则认为差异存在显著性差别。
三、实验结果
1行为学测试结果
1.1自发活动实验
实验结果表明，在自发活动实验中，各组大鼠的活动总路程、活动总时间以及平均速度未见显著差异， 提示养血清脑颗粒不会通过影响中枢神经系统兴奋性而干扰后续的行为学实验(见表19)。各组大鼠自发活动轨迹图见图50。
表19 养血清脑颗粒对喹啉酸损毁NBM核致痴呆大鼠自发活动的影响(n＝9-10，
Figure PCTCN2014086915-appb-000022
)
Figure PCTCN2014086915-appb-000023
1.2Y迷宫实验
实验结果表明，各组大鼠进入Y迷宫三个臂的总次数之间未见显著性差异(见图51、表20)，提示养血清脑颗粒未对大鼠自发活动产生明显影响。与假手术组相比，模型组大鼠自发交替反应率显著下降；与模型组相比，养血清脑颗粒剂量依赖性地增加了大鼠Y迷宫自发交替反应率，盐酸多奈哌齐组也显著增加自发交替反应率(见图52、表20)，提示养血清脑颗粒可显著改善痴呆大鼠的工作记忆障碍。
表20 Y迷宫实验中养血清脑颗粒对喹啉酸毁损NBM核致痴呆大鼠进臂总次数及自发交替反应率的影响(n＝16-18，
Figure PCTCN2014086915-appb-000024
)。
Figure PCTCN2014086915-appb-000025
与假手术组相比，###p＜0.001；与模型组相比，*p＜0.05，***p＜0.001。
1.3新物体辨别实验
实验结果表明，与假手术组相比，模型组大鼠1h及24h优先指数和辨别系数显著降低；与模型组相比，养血清脑颗粒剂量依赖性地提高1h及24h优先指数和辨别系数，盐酸多奈哌齐组也显著增加优先指数及辨别系数(见图53、图54，表21、表22)。
表21养血清脑颗粒对喹啉酸毁损NBM核致痴呆模型大鼠新物体辨别1h及24h优先指数的影响(n＝16-18，
Figure PCTCN2014086915-appb-000026
)。
Figure PCTCN2014086915-appb-000027
与假手术组相比，###p＜0.001；与模型组相比，*p＜0.05，**p＜0.01，***p＜0.001。
表22 养血清脑颗粒对喹啉酸毁损NBM核致痴呆模型大鼠新物体辨别1h及24h辨别系数的影响(n＝16-18，
Figure PCTCN2014086915-appb-000028
)
Figure PCTCN2014086915-appb-000029
与假手术组相比，###p<0.001；与模型组相比，*p<0.05，**p<0.01，***p<0.001。
1.4Morris水迷宫实验
(1)定向导航实验
实验结果表明，与假手术组相比，模型组大鼠在水迷宫实验中到达平台的游泳时间及路程显著延长；与模型组相比，养血清脑颗粒剂量依赖性地缩短了大鼠到达平台的游泳时间及路程，盐酸多奈哌齐组也显著缩短游泳时间及路程(见图55、图56、表23、表24)。
表23 养血清脑颗粒对喹啉酸毁损NBM核致痴呆大鼠水迷宫训练期间游泳时间的影响(n＝15-18，
Figure PCTCN2014086915-appb-000030
)。
Figure PCTCN2014086915-appb-000031
注 A：空白对照组 B：模型组 C：盐酸多奈哌齐组 D：养血清脑颗粒组
与假手术组相比，###p＜0.001；与模型组相比，*p＜0.05，**p＜0.01，***p＜0.001。
表24 养血清脑颗粒对喹啉酸毁损NBM核致痴呆大鼠水迷宫训练期间游泳路程的影响(n＝15-18，
Figure PCTCN2014086915-appb-000032
)。
Figure PCTCN2014086915-appb-000033
注 A：空白对照组 B：模型组 C：盐酸多奈0齐组 D：养血清脑颗粒组
与假手术组相比，＃＃＃p＜0.001；与模型组相比，*p＜0.05，**p＜0.01，***p＜0.001。
(2)空间探索实验
实验结果表明，与假手术组相比，模型组大鼠在原平台所在象限(第四象限)的游泳时间和第四象限路程百分比显著下降；与模型组相比，养血清脑颗粒2588、1294mg/kg剂量组可显著延长大鼠第四象限游泳时间和第四象限路程百分比，盐酸多奈哌齐组也显著延长第四象限游泳时间及路程百分比(见图58、图59、表25)，各组大鼠空间探索实验轨迹图见图57。
1.5被动回避反应实验
实验结果表明，与假手术组相比，模型组被电击次数显著增加；与模型组相比，养血清脑颗粒剂量依赖性地减少了被电击次数，盐酸多奈哌齐组也显著减少了被电击次数(图60、表26)。
表25 养血清脑颗粒对喹啉酸毁损NBM核致痴呆大鼠第四象限游泳时间及第四象限游泳路程百分比的影响(n＝15-18，
Figure PCTCN2014086915-appb-000034
)。
Figure PCTCN2014086915-appb-000035
与假手术组相比，###p＜0.001；与模型组相比，*p＜0.05，**p＜0.01，***p＜0.001。
表26 养血清脑颗粒对喹啉酸毁损NBM核致痴呆大鼠避暗实验被电击次数的影响(n＝15-18，
Figure PCTCN2014086915-appb-000036
)。
Figure PCTCN2014086915-appb-000037
与假手术组相比，###p＜0.001；与模型组相比，**p＜0.01，***p＜0.001。
2HE染色观察海马神经细胞病理变化
结果可见，假手术组大鼠海马CA1区神经元细胞结构清楚，排列紧密。模型组大鼠神经元排列疏松、水肿，染色质减少，核固缩。养血清脑颗粒2588mg/kg组及盐酸多奈哌齐组大鼠海马神经元排列紧密，未见水肿及核固缩，养血清脑颗粒1294mg/kg组大鼠神经元排列较紧密，有轻微水肿及核固缩(见图61)。3电镜观察海马CA1区神经元及突触超微结构变化
3.1电镜观察神经元胞体超微结构
电镜观察可见，假手术组神经元细胞轮廓清晰，核呈椭圆形，核内染色质分布较均匀，核膜、核仁清楚，胞质内有丰富的核糖体、粗面内质网、较多的线粒体及溶酶体等细胞器；模型组神经元细胞核形不规则，核内异染色质有明显的凝聚、边集，胞质部分溶解，尚存的线粒体外膜模糊；多奈哌齐组及养血清脑颗粒2588mg/kg组神经元细胞核近圆形，核内染色质分布均匀，核膜轮廓清楚，胞质内有丰富的核糖体、线粒体、粗面内质网和溶酶体等细胞器；养血清脑颗粒1294mg/kg组神经元细胞核圆形，核膜及核仁清楚，核内染色质分布均匀，胞质内有较多的核糖体，粗面内质网多扩张、线粒体嵴或外膜部分缺失，还见溶酶体等细胞器；养血清脑颗粒647mg/kg组神经元细胞核近圆形，核内染色质分布均匀，胞质内有较多的核糖体、线粒体、溶酶体和轻度扩张的粗面内质网等细胞器(见图62)。
3.2电镜观察神经元突触超微结构
神经元突触超微结构可见，假手术组突触结构清楚，可见明显的突触前膜、后膜及间隙。突触前膜内有较多的突触小泡，突触后膜内致密物厚度较均匀；模型组部分突触结构紊乱，突触前膜、后膜融合；部分可见突触前膜、后膜及间隙；多奈哌齐组及养血清脑颗粒2588mg/kg组轴-树突触前膜、后膜清楚，结构清晰，前膜内有小泡，后膜致密斑较厚；养血清脑颗粒1294mg/kg组部分突触前膜、后膜融合，突触间隙宽窄不一，部分突触近于正常，可见明显的突触前、后膜及间隙。突触前膜内有较多的突触小泡，突触后膜内致密物厚度较均匀；养血清脑颗粒647mg/kg组突触前后膜融合，后膜深染物质较少，突触数量较少(见图63)。
4突触相关蛋白表达
4.1养血清脑颗粒对喹啉酸毁损NBM核致痴呆大鼠海马组织SYP，PSD-95及GAP-43表达的影响
Western blot结果表明，与假手术组相比，模型组大鼠海马组织的SYP、PSD-95及GAP-43表达显著减少；与模型组相比，养血清脑颗粒剂量依赖性地增加海马组织中SYP、PSD-95的表达，对GAP-43的表达未见显著影响，盐酸多奈哌齐组能显著增加这些突触相关蛋白的表达(见图64、图65、图66、表27)。
表27 养血清脑颗粒对喹啉酸毁损NBM核致痴呆大鼠海马组织突触素(SYP)、突触后致密物(PSD-95)及突触生长相关蛋白(GAP-43)表达的影响(n＝3
Figure PCTCN2014086915-appb-000038
)。
Figure PCTCN2014086915-appb-000039
与假手术组相比，#p<0.05，##p<0.01；与模型组相比，*p<0.05，**p<0.01
4.2养血清脑颗粒对喹啉酸毁损NBM核致痴呆大鼠大脑皮层组织SYP、PSD-95及GAP-43表达的影响
Western blot结果表明，与假手术组相比，模型组大鼠大脑皮层组织SYP、PSD-95的表达显著减少；与模型组相比，盐酸多奈哌齐组及养血清脑颗粒2588mg/kg组大脑皮层组织SYP的表达显著增加；盐酸多奈哌齐组、养血清脑颗粒2588mg/kg及1294mg/kg组PSD-95的表达均显著增加；各组之间GAP-43的表达均未见显著差异(见图67、图68、图69以及表28)。
表28 养血清脑颗粒对喹啉酸毁损NBM核致痴呆大鼠大脑皮层突触素(SYP)、突触后致密物(PSD-95)及突触生长相关蛋白(GAP-43)表达的影响(n＝3，
Figure PCTCN2014086915-appb-000040
)。
Figure PCTCN2014086915-appb-000041
与假手术组相比，##p<0.01，###p<0.001；与模型组相比，*p<0.05，**p<0.01
5中枢胆碱能神经系统功能检测
5.1养血清脑颗粒对喹啉酸损毁NBM核致痴呆大鼠大脑皮层及海马组织Ach含量的影响
乙酰胆碱(Ach)是最早发现的与学习记忆密切相关的神经递质，胆碱乙酰转移酶(ChAT)催化乙酰辅酶A的乙酰基与胆碱在胞浆结合生成Ach。Ach合成后由突触小泡载体转运进入囊泡贮存，当有信号激活时，Ach扩散至突触后膜并与突触后膜受体结合，发挥其生物学作用。乙酰胆碱受体分为烟碱型乙酰胆碱受体(nAChR)与毒蕈碱型乙酰胆碱受体(mAChR)。mAChR属G蛋白偶联的神经递质，有5种亚型：M1-M5，大脑中主要存在位于突触后膜的M1受体。ELISA结果表明，与假手术组相比，模型组大鼠大脑皮层及海马组织Ach的表达显著减少；与模型组相比，养血清脑颗粒剂量依赖性地增加Ach的表达，盐酸多奈哌齐组也显著增加Ach表达(见图70、图71以及表29)。
表29养血清脑颗粒对喹啉酸损毁NBM核致痴呆大鼠海马组织及大脑皮层组织乙酰胆碱(Ach)含量的影响(n＝6，
Figure PCTCN2014086915-appb-000042
)。
Figure PCTCN2014086915-appb-000043
Figure PCTCN2014086915-appb-000044
与假手术组相比，#p<0.05，###p<0.001；与模型组相比，*p<0.05，***p<0.001
5.2养血清脑颗粒对喹啉酸损毁NBM核致痴呆大鼠海马ChAT含量及活性的影响
ELIA结果表明，与假手术组相比，模型组大鼠海马组织中ChAT含量显著减少；与模型组相比，养血清脑颗粒剂量依赖性地增加ChAT的含量，盐酸多奈哌齐组也显著增加其表达；ChAT的活性在各组之间未见显著性差异(见图72、图73以及表30)
表30 养血清脑颗粒对喹啉酸损毁NBM核致痴呆大鼠海马胆碱乙酰转移酶(ChAT)含量及活性的影响(n＝6，
Figure PCTCN2014086915-appb-000045
)。
Figure PCTCN2014086915-appb-000046
与假手术组相比，###p<0.001；与模型组相比，**p<0.01，***p<0.001。
5.3养血清脑颗粒对喹啉酸损毁NBM核致痴呆大鼠大脑皮层及海马组织CHRM1表达的影响
Western blot 结果表明，大鼠海马组织CHRM1表达在各组之间未见显著差异；ELISA结果表明，与假手术组相比，模型组大鼠大脑皮层组织中CHRM1的表达显著减少；与模型组相比，养血清脑颗粒2588mg/kg组及盐酸多奈哌齐组均显著增加其表达(见图74、图75以及表31、表32)。
表31 养血清脑颗粒对喹啉酸损毁NBM核致痴呆大鼠海马组织M1受体(CHRM1)表达的影响(n＝3，
Figure PCTCN2014086915-appb-000047
Figure PCTCN2014086915-appb-000048
)
Figure PCTCN2014086915-appb-000049
表32 养血清脑颗粒对喹啉酸损毁NBM核致痴呆大鼠大脑皮层组织M1受体(CHRM1)表达的影响(n＝6，
Figure PCTCN2014086915-appb-000050
)。
Figure PCTCN2014086915-appb-000051
与假手术组相比，#p<0.05；与模型组相比，*p<0.05
四、小结
1)Y迷宫、新物体辨别、Morris水迷宫及避暗等行为学实验结果显示，养血清脑颗粒在647-2588mg/kg剂量范围内能够剂量依赖性地改善喹啉酸损毁NBM核致痴呆大鼠工作记忆障碍、空间学习记忆障碍以及物体辨别能力等非空间学习记忆障碍，其高剂量组的作用与盐酸多奈哌齐相近。
2)养血清脑颗粒能够显著提高喹啉酸损毁NBM核致痴呆大鼠大脑皮层及海马组织中突触素、突触后致密物95的表达，改善海马突触结构的异常。
3)养血清脑颗粒能够显著提高喹啉酸损毁NBM核致痴呆大鼠大脑皮层及海马组织中乙酰胆碱的表达，提高海马胆碱乙酰转移酶及大脑皮质M1胆碱受体的表达。
附图说明
图1APPswe/PSldE9转基因鼠的基因型鉴定。
图2Y迷宫实验证明养血具有显著改善早中期阿尔茨海默症动物模型小鼠记忆、认知能力的作用(与生理盐水组比较*p<0.05,**p<0.01)。
图3各药物组小鼠大脑额叶皮质区的刚果红染色。
图4各药物组雄性转基因小鼠的大脑额叶皮质区的刚果红染色(箭头所指粉红色β-淀粉样斑块)。
图5各药物组雌性转基因小鼠的大脑额叶皮质区的刚果红染色(箭头所指粉红色β-淀粉样斑块)。
图6各药物组转基因小鼠海马区的刚果红染色(箭头所指粉红色β-淀粉样斑块)。
图7各药物组转基因小鼠的大脑组织β-淀粉样斑块个数(与生理盐水组比较，*p<0.05；**p<0.01)。
图8各药物组转基因小鼠的大脑组织β-淀粉样斑块覆盖面积(与生理盐水组比较，**p<0.01)。
图9针对APPβα段(Aβ42的1-16aa)特异抗体的设计(利用识别不溶性蛋白的APPβα特异抗体的免疫组化染色证明养血清脑颗粒)。
图10各药物组转基因小鼠大脑皮层中β-淀粉样斑块数量及形态，图中箭头所指即为APP染色阳性的β-淀粉样斑块，呈深褐色。
图11各药物组雄性转基因小鼠海马区Aβ-淀粉样斑块数量及形态，图中箭头所指即为APP染色阳性的β-淀粉样斑块，呈深褐色。
图12各药物组雌性转基因小鼠海马区Aβ-淀粉样斑块数量及形态，图中箭头所指即为APP染色阳性的β-淀粉样斑块，呈深褐色。
图13各药物组转基因小鼠脑组织中强阳性Aβ-淀粉样斑块数量，与生理盐水组比较，**p<0.01。
图14各药物组对转基因小鼠脑内APP的关键剪切酶和剪切产物的影响。
图15养血清脑颗粒显著清除早中期阿尔茨海默症动物模型脑内老年斑的作用的分子机制。
图16.SAMP8小鼠自发活动轨迹图。A：空白对照组，B：模型组，C：盐酸多奈哌齐组，D：养血清脑颗粒935mg/kg，E：养血清脑颗粒1870mg/kg，F:养血清脑颗粒3740mg/kg。
图17养血清脑颗粒对SAMP8小鼠Y迷宫进臂总次数的影响(n＝23-28，平均值±SD)。
图18养血清脑颗粒对SAMP8小鼠Y迷宫自发交替反应率的影响(n＝23-28,平均值±SD)。与空白对照组相比，###p<0.001；与模型组相比，***p<0.001。
图19养血清脑颗粒对SAMP8小鼠新物体辨别1h优先指数的影响(n＝22-28，平均值±SD)。与空白对照组相比，###p<0.001；与模型组相比，***p<0.001或**p<0.01或*p<0.05。
图20养血清脑颗粒对SAMP8小鼠新物体辨别24h优先指数的影响(n＝22-28，平均值±SD)。与空白对照组相比，###p<0.001；与模型组相比***p<0.001或**p<0.01。
图21养血清脑颗粒对SAMP8小鼠水迷宫逃避潜伏期的影响(n＝18-26,平均值±SD)。与空白对照组相比，###p<0.001；与模型组相比，***p<0.001或**p<0.01或*p<0.05
图22养血清脑颗粒对SAMP8小鼠水迷宫游泳路程的影响(n＝18-26,平均值±SD)。与空白对照组相比，###p<0.001；与模型组相比，***p<0.001或**p<0.01。
图23养血清脑颗粒对SAMP8小鼠空间探索实验游泳轨迹的影响。
图24养血清脑颗粒对SAMP8小鼠空间探索实验目标象限游泳时间的影响(n＝18-26,平均值±SD)。与空白对照组相比，###p<0.001；与模型组相比，***p<0.001或**p<0.01或*p<0.05。
图25养血清脑颗粒对SAMP8小鼠空间探索实验目标象限路程百分比的影响(n＝18-26，平均值±SD)。 与空白对照组相比，###p<0.001；与模型组相比，***p<0.001或**p<0.01。
图26养血清脑颗粒对SAMP8小鼠海马神经细胞病理变化的影响(×40)。
图27养血清脑颗粒对SAMP8小鼠海马CA1区神经元超微结构的影响(n＝4，×6000)。
图28养血清脑颗粒对SAMP8小鼠海马CA1区突触超微结构的影响(n＝4，×10000)。
图29养血清脑颗粒对SAMP8小鼠海马组织中脑源性神经生长因子(BDNF)表达的影响(×4)。
图30养血清脑颗粒对SAMP8小鼠海马CA1区脑源性神经生长因子(BDNF)表达的影响(×40)。
图31养血清脑颗粒对SAMP8小鼠海马CA1区脑源性神经生长因子(BDNF)表达的影响(n＝6,平均值±SD)。与空白对照组相比，###p<0.001；与模型组相比，***p<0.001，**p<0.01或*p<0.05。
图32清脑颗粒对SAMP8小鼠大脑皮层脑源性神经生长因子(BDNF)表达的影响(×40)。
图33清脑颗粒对SAMP8小鼠大脑皮层脑源性神经生长因子(BDNF)表达的影响(n＝6,平均值±SD)。与空白对照组相比，###p<0.001；与模型组相比，**p<0.01或*p<0.05。
图34养血清脑颗粒对SAMP8小鼠海马神经生长因子(NGF)表达的影响(×4)。
图35养血清脑颗粒对SAMP8小鼠海马CA1区神经生长因子(NGF)表达的影响(×40)。
图36养血清脑颗粒对SAMP8小鼠海马CA1区神经生长因子(NGF)表达的影响(n＝6,平均值±SD)与空白对照组相比，###p<0.001；与模型组相比，***p<0.001或**p<0.01或*p<0.05。
图37养血清脑颗粒对SAMP8小鼠大脑皮层神经生长因子(NGF)表达的影响(×40)。
图38养血清脑颗粒对SAMP8小鼠大脑皮层神经生长因子(NGF)表达的影响(n＝6,平均值±SD)。与空白对照组相比，##p<0.01；与模型组相比，**p<0.01。
图39养血清脑颗粒对SAMP8小鼠海马CA1区TrkA表达的影响(×4)。
图40养血清脑颗粒对SAMP8小鼠海马CA1区TrkA表达的影响(×40)。
图41养血清脑颗粒对SAMP8小鼠海马CA1区TrkA表达的影响(n＝6,平均值±SD)与空白对照组相比,###p<0.001；与模型组相比，***p<0.001或**p<0.01或*p<0.05。
图42养血清脑颗粒对SAMP8小鼠大脑皮层TrkA表达的影响(×40)。
图43养血清脑颗粒对SAMP8小鼠大脑皮层TrkA表达的影响(n＝6,平均值±SD)与空白对照组相比，###p<0.001；与模型组相比，***p<0.001或**p<0.01。
图44养血清脑颗粒对SAMP8小鼠海马突触素(SYP)表达的影响(n＝3，平均值±SD)。与空白对照组相比，##p<0.01；与模型组相比，*p<0.05。
图45养血清脑颗粒对SAMP8小鼠海马突触生长相关蛋白(GAP-43)表达的影响(n＝3，平均值±SD)。
图46养血清脑颗粒对SAMP8小鼠海马突触后致密物(PSD-95)表达的影响(n＝3，平均值±SD),与空白对照组相比，##p<0.01；与模型组相比，**p<0.01。
图47养血清脑颗粒对SAMP8小鼠大脑皮层突触素(SYP)表达的影响(n＝3，平均值±SD)。与空白对照组相比，##p<0.01；与模型组相比，**p<0.01。
图48养血清脑颗粒对SAMP8小鼠大脑皮层突触生长相关蛋白(GAP-43)表达的影响(n＝3，平均值±SD)。
图49养血清脑颗粒对SAMP8小鼠大脑皮层突触后致密物(PSD-95)表达的影响(n＝3，平均值±SD)。与空白对照组相比，###p<0.001；与模型组相比，***p<0.001或*p<0.05。
图50各组大鼠自发活动轨迹代表图。
图51Y迷宫实验中养血清脑颗粒对喹啉酸毁损NBM核致痴呆大鼠进臂总次数的影响(n＝16-18，
Figure PCTCN2014086915-appb-000052
)。
图52养血清脑颗粒对喹啉酸毁损NBM核致痴呆大鼠自发交替反应率的影响(n＝16-18，
Figure PCTCN2014086915-appb-000053
)。与假手术组相比，###p<0.001；与模型组相比，*p<0.05，***p<0.001。
图53养血清脑颗粒对喹啉酸毁损NBM核致痴呆大鼠新物体辨别1h优先指数的影响(n＝16-18，
Figure PCTCN2014086915-appb-000054
)。与假手术组相比，###p<0.001；与模型组相比，**p<0.01，***p<0.001。
图54养血清脑颗粒对喹啉酸毁损NBM核致痴呆大鼠新物体辨别24h优先指数的影响(n＝16-18，
Figure PCTCN2014086915-appb-000055
)。与假手术组相比，###p<0.001；与模型组相比，*p<0.05，***p<0.001。
图55养血清脑颗粒对喹啉酸毁损NBM核致痴呆大鼠水迷宫训练期间游泳时间的影响(n＝15-18，
Figure PCTCN2014086915-appb-000056
)。与假手术组相比，###p<0.001；与模型组相比，*p<0.05，**p<0.01，***p<0.001。
图56养血清脑颗粒对喹啉酸毁损NBM核致痴呆大鼠水迷宫训练期间游泳路程的影响(n＝15-18，
Figure PCTCN2014086915-appb-000057
)。与假手术组相比，###p<0.001；与模型组相比，*p<0.05，**p<0.01，***p<0.001。
图57各组大鼠空间探索实验游泳轨迹图。
图58养血清脑颗粒对喹啉酸毁损NBM核致痴呆大鼠在第四象限游泳时间的影响(n＝15-18，
Figure PCTCN2014086915-appb-000058
)。与假手术组相比，###p<0.001；与模型组相比，*p<0.05，***p<0.001。
图59养血清脑颗粒对喹啉酸毁损NBM核致痴呆大鼠在第四象限游泳路程百分比的影响(n＝15-18，
Figure PCTCN2014086915-appb-000059
Figure PCTCN2014086915-appb-000060
)。与假手术组相比，###p<0.001；与模型组相比，*p<0.05，**p<0.01。
图60养血清脑颗粒对喹啉酸毁损NBM核致痴呆大鼠避暗实验被电击次数的影响(n＝15-18，
Figure PCTCN2014086915-appb-000061
)。与假手术组相比，###p<0.001；与模型组相比，**p<0.01，***p<0.001。
图61养血清脑颗粒对喹啉酸损毁NBM核致痴呆模型大鼠海马神经细胞病理变化的影响(×40)。
图62养血清脑颗粒对喹啉酸毁损NBM核致痴呆大鼠海马CA1区神经元胞体超微结构的影响(n＝4，×6000)。
图63养血清脑颗粒对喹啉酸毁损NBM核致痴呆大鼠海马CA1区突触超微结构的影响(n＝4，×10000)。
图64养血清脑颗粒对喹啉酸毁损NBM核致痴呆大鼠海马组织突触素(SYP)表达的影响(n＝3，
Figure PCTCN2014086915-appb-000062
)。与假手术组相比，##p<0.01；与模型组相比，*p<0.05，**p<0.01。
图65养血清脑颗粒对喹啉酸毁损NBM核致痴呆大鼠海马组织突触后致密物(PSD-95)表达的影响(n＝3，
Figure PCTCN2014086915-appb-000063
)。与假手术组相比，##p<0.01；与模型组相比，*p<0.05，**p<0.01。
图66养血清脑颗粒对喹啉酸毁损NBM核致痴呆大鼠海马组织突触生长相关蛋白(GAP-43)表达的影响(n＝3，
Figure PCTCN2014086915-appb-000064
)。与假手术组相比，#p<0.05；与模型组相比，*p<0.05。
图67养血清脑颗粒对喹啉酸毁损NBM核致痴呆大鼠大脑皮层组织突触素(SYP)表达的影响(n＝3，
Figure PCTCN2014086915-appb-000065
Figure PCTCN2014086915-appb-000066
)。与假手术组相比，##p<0.01；与模型组相比，*p<0.05，**p<0.01。
图68养血清脑颗粒对喹啉酸毁损NBM核致痴呆大鼠大脑皮层组织突触后致密物(PSD-95)表达的影响(n＝3，
Figure PCTCN2014086915-appb-000067
)。与假手术组相比，###p<0.001；与模型组相比，**p<0.01。
图69养血清脑颗粒对喹啉酸毁损NBM核致痴呆大鼠大脑皮层突触生长相关蛋白(GAP-43)表达的影响(n＝3，
Figure PCTCN2014086915-appb-000068
)。
图70养血清脑颗粒对喹啉酸损毁NBM核致痴呆大鼠海马组织乙酰胆碱(Ach)含量的影响(n＝6，
Figure PCTCN2014086915-appb-000069
)。与假手术组相比，###p<0.001；与模型组相比，*p<0.05，***p<0.001。
图71养血清脑颗粒对喹啉酸损毁NBM核致痴呆大鼠大脑皮层组织乙酰胆碱(Ach)含量的影响(n＝6，
Figure PCTCN2014086915-appb-000070
Figure PCTCN2014086915-appb-000071
)。与假手术组相比，#p<0.05；与模型组相比，*p<0.05
图72养血清脑颗粒对喹啉酸损毁NBM核致痴呆大鼠海马胆碱乙酰转移酶(ChAT)含量的影响(n＝6，
Figure PCTCN2014086915-appb-000072
)。与假手术组相比，###p<0.001；与模型组相比，**p<0.01，***p<0.001。
图73养血清脑颗粒对喹啉酸损毁NBM核致痴呆大鼠海马胆碱乙酰转移酶(ChAT)活性的影响(n＝6，
Figure PCTCN2014086915-appb-000073
Figure PCTCN2014086915-appb-000074
)。
图74养血清脑颗粒对喹啉酸损毁NBM核致痴呆大鼠海马组织M1受体(CHRM1)表达的影响(n＝3，
Figure PCTCN2014086915-appb-000075
Figure PCTCN2014086915-appb-000076
)。
图75养血清脑颗粒对喹啉酸损毁NBM核致痴呆大鼠大脑皮层组织M1受体(CHRM1)表达的影响(n＝6，
Figure PCTCN2014086915-appb-000077
)。与假手术组相比，#p<0.05；与模型组相比，*p<0.05。
实施例
实施例1
原料重量配比：取当归253.5g、川芎253.5g、白芍202.7g、熟地黄202.7g、钩藤506.8g、鸡血藤506.8、夏枯草506.8g、决明子506.8g、珍珠母506.8g、延胡索253.5g、细辛50.5g。
提取物1的制备：当归、川芎、延胡索、决明子加入4倍量70％乙醇加热回流提取2次，第一次2小时，第二次1小时，滤过，除杂，回收乙醇并浓缩至相对密度1.300-1.310(74-76℃)，得浸膏，备用。
提取物2的制备：白芍加入4倍量60％乙醇，浸渍，加热回流提取2次，第一次2小时，第二次1小时，滤过，回收乙醇并浓缩至相对密度1.23-1.33(65℃)，得浸膏，备用。
提取物3的制备：熟地黄、钩藤、鸡血藤、夏枯草、珍珠母、细辛加入5倍量水煎煮2次，第一次2小时，第二次1小时，滤过，浓缩至相对密度1.06-1.10(80℃)，加乙醇使含醇量为65-70％，静置12-24小时，滤过，回收乙醇，浓缩至相对密度1.320-1.325(79-81℃)，得浸膏，备用。
取糊精300g，用纯化水化开，加入甜菊素3.0g，充分搅拌使溶化，将上述备好的浸膏分步加入上述浆料中，搅拌。调整浆料比重在1.12-1.23(42-50℃)之间。60目-100目在线过滤。
将剩余的糊精250.0g投入制粒机，调节风机频率、进风温度、输液频率和雾化压力等制粒参数，使床内物料处于良好的流化状态。喷雾制粒，制粒过程控制物料温度在30-60℃之间。干燥，使物料温度升至80-90℃充分干燥。
整粒过筛，总混，制成颗粒剂，铝塑复合膜枕形袋包装，规格4克/袋。
实施例2
原料重量配比：当归6.75％、川芎6.75％、白芍5.4％、钩藤13.5％、鸡血藤13.5％、熟地黄5.4％、决明子13.5％、夏枯草13.5％、细辛1.34％、延胡索6.75％和珍珠母13.5％。
提取物1的制备：当归、川芎、延胡索、决明子加入4倍量70％乙醇加热回流提取2次，第一次2小时，第二次1小时，滤过，除杂，回收乙醇并浓缩至相对密度1.300-1.310(74-76℃)，得浸膏，备用。
提取物2的制备：白芍加入4倍量60％乙醇，浸渍，加热回流提取2次，第一次2小时，第二次1小时，滤过，回收乙醇并浓缩至相对密度1.23-1.33(65℃)，得浸膏，备用。
提取物3的制备：熟地黄、钩藤、鸡血藤、夏枯草、珍珠母、细辛加入5倍量水煎煮2次，第一次2小时，第二次1小时，滤过，浓缩至相对密度1.06-1.10(80℃)，加乙醇使含醇量为65-72％，静置12-24小时，滤过，回收乙醇，浓缩至相对密度1.320-1.325(79-81℃)，得浸膏，备用。
将上述备好的浸膏用常规制备方法制备得到养血清脑丸。
实施例3
原料重量配比：当归6.75％、川芎6.75％、白芍5.4％、钩藤13.5％、鸡血藤13.5％、熟地黄5.4％、决明子13.5％、夏枯草13.5％、细辛1.34％、延胡索6.75％和珍珠母13.5％。
提取物1的制备：当归、川芎、延胡索、决明子加入5倍量70％乙醇加热回流提取2次，第一次2.5小时，第二次1小时，滤过，除杂，回收乙醇并浓缩至相对密度1.250-1.310(70-74℃)，得浸膏，备用。
提取物2的制备：白芍加入4倍量80％乙醇，浸渍，加热回流提取2次，第一次2小时，第二次2小时，滤过，回收乙醇并浓缩至相对密度1.15-1.25(65℃)，得浸膏，备用。
提取物3的制备：熟地黄、钩藤、鸡血藤、夏枯草、珍珠母、细辛加入5倍量水煎煮2次，第一次2小时，第二次1小时，滤过，浓缩至相对密度1.06-1.10(80℃)，加乙醇使含醇量为60-65％，静置12-24小时，滤过，回收乙醇，浓缩至相对密度1.27-1.320(75-80℃)，得浸膏，备用。
将上述备好的浸膏用常规制备方法制备得到养血清脑滴丸。
实施例4
原料重量配比：当归6.75％、川芎6.75％、白芍5.4％、钩藤13.5％、鸡血藤13.5％、熟地黄5.4％、决明子13.5％、夏枯草13.5％、细辛1.34％、延胡索6.75％和珍珠母13.5％。
提取物1的制备：当归、川芎、延胡索、决明子加入4倍量80％乙醇加热回流提取2次，第一次2.5小时，第二次1小时，滤过，除杂，回收乙醇并浓缩至相对密度1.30-1.350(75-80℃)，得浸膏，备用。
提取物2的制备：白芍加入6倍量60％乙醇，浸渍，加热回流提取3次，第一次2小时，第二次1小时，第三次0.5小时，滤过，回收乙醇并浓缩至相对密度1.20-1.35(60℃)，得浸膏，备用。
提取物3的制备：熟地黄、钩藤、鸡血藤、夏枯草、珍珠母、细辛加入8倍量水煎煮2次，第一次3小时，第二次2小时，滤过，浓缩至相对密度1.06-1.10(80℃)，加乙醇使含醇量为80-85％，静置12-24小时，滤过，回收乙醇，浓缩至相对密度1.30-1.350(80-85℃)，得浸膏，备用。
将上述备好的浸膏用常规制备方法制备得到养血清脑滴口服液。
实施例5
原料重量配比：当归6.75％、川芎6.75％、白芍5.4％、钩藤13.5％、鸡血藤13.5％、熟地黄5.4％、决明子13.5％、夏枯草13.5％、细辛1.34％、延胡索6.75％和珍珠母13.5％。
提取物1的制备：当归、川芎、延胡索、决明子加入4倍量50％乙醇加热回流提取2次，第一次2小时，第二次2小时，滤过，除杂，回收乙醇并浓缩至相对密度1.300-1.350(73-78℃)，得浸膏，备用。
提取物2的制备：白芍加入5倍量70％乙醇，浸渍，加热回流提取2次，第一次1小时，第二次1小时，滤过，回收乙醇并浓缩至相对密度1.23-1.35(65℃)，得浸膏，备用。
提取物3的制备：熟地黄、钩藤、鸡血藤、夏枯草、珍珠母、细辛加入10倍量水煎煮2次，第一次2小时，第二次2小时，滤过，浓缩至相对密度1.06-1.10(80℃)，加乙醇使含醇量为63-70％，静置12-24小时，滤过，回收乙醇，浓缩至相对密度1.290-1.330(78-83℃)，得浸膏，备用。
将上述备好的浸膏用常规制备方法制备得到养血清脑胶囊。
实施例6
原料重量配比：取当归300g、川芎300g、白芍400g、熟地黄400g、钩藤650g、鸡血藤650g、夏枯草650g、决明子650g、珍珠母650g、延胡索300g、细辛50g。
提取物1的制备：当归、川芎、延胡索、决明子加入3倍量60％乙醇加热回流提取3次，第一次2小时，第二次1小时，第三次0.5小时，滤过，除杂，回收乙醇并浓缩至相对密度1.29-1.340(73-78℃)，得浸膏，备用。
提取物2的制备：白芍加入4倍量80％乙醇，浸渍，加热回流提取3次，第一次2小时，第二次1小时，第三次1小时，滤过，回收乙醇并浓缩至相对密度1.18-1.33(65℃)，得浸膏，备用。
提取物3的制备：熟地黄、钩藤、鸡血藤、夏枯草、珍珠母、细辛加入7倍量水煎煮2次，第一次2小时，第二次1小时，滤过，浓缩至相对密度1.06-1.10(80℃)，加乙醇使含醇量为70-75％，静置12-24小时，滤过，回收乙醇，浓缩至相对密度1.310-1.330(77-82℃)，得浸膏，备用。
取糊精84g，用纯化水化开，加入甜菊素3g，充分搅拌使溶化，将上述备好的浸膏共780g分步加入上述浆料中，搅拌。调整浆料比重在1.12-1.23(42-50℃)之间。60目-100目在线过滤。
将剩余的糊精336g投入制粒机，调节风机频率、进风温度、输液频率和雾化压力等制粒参数，使床内物料处于良好的流化状态。喷雾制粒，制粒过程控制物料温度在30-60℃之间。干燥，使物料温度升至70-90℃充分干燥。
整粒过筛，总混，制成颗粒剂，铝塑复合膜枕形袋包装，规格3克/袋。
实施例7
原料重量配比：取当归338g、川芎338g、白芍300g、熟地黄300g、钩藤413g、鸡血藤413g、夏枯草413g、决明子413g、珍珠母413g、延胡索337g、细辛75g。
提取物1的制备：当归、川芎、延胡索、决明子加入6倍量70％乙醇加热回流提取2次，第一次2小时，第二次0.5小时，滤过，除杂，回收乙醇并浓缩至相对密度1.260-1.310(74-76℃)，得浸膏，备用。
提取物2的制备：白芍加入6倍量60％乙醇，浸渍，加热回流提取2次，第一次2小时，第二次2小时，滤过，回收乙醇并浓缩至相对密度1.21-1.34(55℃)，得浸膏，备用。
提取物3的制备：熟地黄、钩藤、鸡血藤、夏枯草、珍珠母、细辛加入6倍量水煎煮2次，第一次2小时，第二次1小时，滤过，浓缩至相对密度1.06-1.10(80℃)，加乙醇使含醇量为65-75％，静置12-24小时，滤过，回收乙醇，浓缩至相对密度1.300-1.300(79-81℃)，得浸膏，备用。
取糊精40g，用纯化水化开，加入甜菊素3g，充分搅拌使溶化，将上述备好的浸膏共794g分步加入上述浆料中，搅拌。调整浆料比重在1.12-1.23(42-50℃)之间。60目-100目在线过滤。
将剩余的糊精163g投入制粒机，调节风机频率、进风温度、输液频率和雾化压力等制粒参数，使床内物料处于良好的流化状态。喷雾制粒，制粒过程控制物料温度在30-60℃之间。干燥，使物料温度升至70-90℃充分干燥。
整粒过筛，总混，制成颗粒剂，铝塑复合膜枕形袋包装，规格4克/袋。
实施例8
原料重量配比：取当归450g、川芎450g、白芍350g、熟地黄350g、钩藤570g、鸡血藤570g、夏枯草570g、决明子570g、珍珠母570g、延胡索450g、细辛100g。
提取物1的制备：当归、川芎、延胡索、决明子加入6倍量80％乙醇加热回流提取2次，第一次1 小时，第二次1小时，滤过，除杂，回收乙醇并浓缩至相对密度1.29-1.340(73-78℃)，得浸膏，备用。
提取物2的制备：白芍加入3倍量60％乙醇，浸渍，加热回流提取2次，第一次2.5小时，第二次2小时，滤过，回收乙醇并浓缩至相对密度1.17-1.33(65℃)，得浸膏，备用。
提取物3的制备：熟地黄、钩藤、鸡血藤、夏枯草、珍珠母、细辛加入9倍量水煎煮2次，第一次3小时，第二次3小时，滤过，浓缩至相对密度1.06-1.08(80℃)，加乙醇使含醇量为65-75％，静置12-22小时，滤过，回收乙醇，浓缩至相对密度1.310-1.330(77-82℃)，得浸膏，备用。
取糊精110g，用纯化水化开，加入甜菊素3g，充分搅拌使溶化，将上述备好的浸膏共840g分步加入上述浆料中，搅拌。调整浆料比重在1.12-1.23(42-50℃)之间。60目-100目在线过滤。
将剩余的糊精256g投入制粒机，调节风机频率、进风温度、输液频率和雾化压力等制粒参数，使床内物料处于良好的流化状态。喷雾制粒，制粒过程控制物料温度在30-60℃之间。干燥，使物料温度升至70-90℃充分干燥。
整粒过筛，总混，制成颗粒剂，铝塑复合膜枕形袋包装，规格3克/袋。
实施例9
原料重量配比：取当归253.5g、川芎253.5g、白芍202.7g、熟地黄202.7g、钩藤506.8g、鸡血藤506.8、夏枯草506.8g、决明子506.8g、珍珠母506.8g、延胡索253.5g、细辛50.5g。
提取物1的制备：当归、川芎、延胡索、决明子加入4倍量70％乙醇加热回流提取2次，第一次2小时，第二次1小时，滤过，除杂，回收乙醇并浓缩至至相对密度1.250-1.310(70-74℃)，得浸膏，备用。
提取物2的制备：白芍加入4倍量60％乙醇，浸渍，加热回流提取2次，第一次2小时，第二次1小时，滤过，回收乙醇并浓缩至相对密度1.23-1.33(65℃)，得浸膏，备用。
提取物3的制备：熟地黄、钩藤、鸡血藤、夏枯草、珍珠母、细辛加入5倍量水煎煮2次，第一次2小时，第二次1小时，滤过，浓缩至相对密度1.06-1.10(80℃)，加乙醇使含醇量为65-75％，静置12-24小时，滤过，回收乙醇，浓缩至相对密度1.27-1.320(75-80℃)，得浸膏，备用。
取可溶性淀粉300g，用纯化水化开，加入甜菊素3.0g，充分搅拌使溶化，将上述备好的浸膏共分步加入上述浆料中，搅拌。调整浆料比重在1.12-1.23(42-50℃)之间。60目-100目在线过滤。
将剩余的可溶性淀粉250.0g投入制粒机，调节风机频率、进风温度、输液频率和雾化压力等制粒参数，使床内物料处于良好的流化状态。喷雾制粒，制粒过程控制物料温度在30-60℃之间。干燥，使物料温度升至80-90℃充分干燥。
整粒过筛，总混，制成颗粒剂，铝塑复合膜枕形袋包装，规格4克/袋。
实施例10
原料重量配比：取当归338g、川芎338g、白芍270.3g、熟地黄270.3g、钩藤675.7g、鸡血藤675.7g、夏枯草675.7g、决明子675.7g、珍珠母675.7g、延胡索338g、细辛67.3g。
提取物1的制备：当归、川芎、延胡索、决明子加入4倍量70％乙醇加热回流提取2次，第一次2小时，第二次1小时，滤过，除杂，回收乙醇并浓缩至相对密度1.280-1.320(75-80℃)，得浸膏，备用。
提取物2的制备：白芍加入4倍量60％乙醇，浸渍，加热回流提取2次，第一次2小时，第二次1小时，滤过，回收乙醇并浓缩至相对密度1.23-1.33(65℃)，得浸膏，备用。
提取物3的制备：熟地黄、钩藤、鸡血藤、夏枯草、珍珠母、细辛加入5倍量水煎煮2次，第一次2小时，第二次1小时，滤过，浓缩至相对密度1.06-1.10(80℃)，加乙醇使含醇量为60-65％，静置12-24小时，滤过，回收乙醇，浓缩至相对密度1.315-1.320(76-79℃)，得浸，备用。
取微晶纤维素80g，用纯化水化开，加入阿司帕坦3.0g，充分搅拌使溶化，将上述备好的浸膏分步加入上述浆料中，搅拌。调整浆料比重在1.12-1.23(42-50℃)之间。60目-100目在线过滤。
将剩余的微晶纤维素320g投入制粒机，调节风机频率、进风温度、输液频率和雾化压力等制粒参数，使床内物料处于良好的流化状态。喷雾制粒，制粒过程控制物料温度在30-60℃之间。干燥，使物料温度升至70-90℃充分干燥。
整粒过筛，总混，制成颗粒剂，铝塑复合膜枕形袋包装，规格3克/袋。
实施例11
原料重量配比：取当归150g、川芎150g、白芍225g、熟地黄225g、钩藤551g、鸡血藤551g、夏枯草551g、决明子551g、珍珠母551g、延胡索225g、细辛19g。
提取物1的制备：当归、川芎、延胡索、决明子加入5倍量70％乙醇加热回流提取2次，第一次2.5小时，第二次1小时，滤过，除杂，回收乙醇并浓缩至相对密度1.290-1.300(75-77℃)，得浸膏，备用。
提取物2的制备：白芍加入4倍量80％乙醇，浸渍，加热回流提取2次，第一次2小时，第二次2小时，滤过，回收乙醇并浓缩至相对密度1.15-1.25(65℃)，得浸膏，备用。
提取物3的制备：熟地黄、钩藤、鸡血藤、夏枯草、珍珠母、细辛加入5倍量水煎煮2次，第一次2小时，第二次1小时，滤过，浓缩至相对密度1.06-1.10(80℃)，加乙醇使含醇量为65-70％，静置12-24小时，滤过，回收乙醇，浓缩至相对密度1.310-1.315(79-82℃)，得浸膏，备用。
取乳糖231g，用纯化水化开，加入阿司帕坦3.0g，充分搅拌使溶化，将上述备好的浸膏分步加入上述浆料中，搅拌。调整浆料比重在1.12-1.23(42-50℃)之间。60目-100目在线过滤。
将剩余的乳糖151g投入制粒机，调节风机频率、进风温度、输液频率和雾化压力等制粒参数，使床内物料处于良好的流化状态。喷雾制粒，制粒过程控制物料温度在30-60℃之间。干燥，使物料温度升至70-90℃充分干燥。
整粒过筛，总混，制成颗粒剂，铝塑复合膜枕形袋包装，规格4克/袋。
实施例12
原料重量配比：取当归250g、川芎250g、白芍250g、熟地黄250g、钩藤740g、鸡血藤740g、夏枯草740g、决明子740g、珍珠母740g、延胡索250g、细辛50g。
提取物1的制备：当归、川芎、延胡索、决明子加入4倍量80％乙醇加热回流提取2次，第一次2.5小时，第二次1小时，滤过，除杂，回收乙醇并浓缩至相对密度1.280-1.300(75-77℃)，得浸，备用。
提取物2的制备：白芍加入6倍量60％乙醇，浸渍，加热回流提取3次，第一次2小时，第二次1小时，第三次0.5小时，滤过，回收乙醇并浓缩至相对密度1.20-1.35(60℃)，得浸膏，备用。
提取物3的制备：熟地黄、钩藤、鸡血藤、夏枯草、珍珠母、细辛加入8倍量水煎煮2次，第一次3小时，第二次2小时，滤过，浓缩至相对密度1.06-1.10(80℃)，加乙醇使含醇量为80-85％，静置12-24小时，滤过，回收乙醇，浓缩至相对密度1.280-1.330(75-80℃)，得浸膏，备用。
取糊精80g，用纯化水化开，加入阿司帕坦3g，充分搅拌使溶化，将上述备好的浸膏分步加入上述浆料中，搅拌。调整浆料比重在1.12-1.23(42-50℃)之间。60目-100目在线过滤。
将剩余的糊精330g投入制粒机，调节风机频率、进风温度、输液频率和雾化压力等制粒参数，使床内物料处于良好的流化状态。喷雾制粒，制粒过程控制物料温度在30-60℃之间。干燥，使物料温度升至70-90℃充分干燥。
整粒过筛，总混，制成颗粒剂，铝塑复合膜枕形袋包装，规格3克/袋。
实施例13
养血清脑制剂配方：
原料重量配比为：当归6.75％、川芎6.75％、白芍5.4％、钩藤13.5％、鸡血藤13.5％、熟地黄5.4％、决明子13.5％、夏枯草13.5％、细辛1.34％、延胡索6.75％和珍珠母13.5％
制备方法：
药材经前序处理→水提→浓缩→乙醇沉淀→回收乙醇→浓缩成膏→混合制粒→成品包装。即按比例取上述各药，加水煮三次，每次1小时，合并煎液，浓缩适量，加2倍量的乙醇，静置24小时沉淀，取上清液浓缩成膏，相对密度为1.3-1.4，出膏率10％，按常规的工艺方法作成其它适当的剂型。

Claims (10)

  1. 一种中药组合物在制备治疗阿尔茨海默症的药物中的应用，其中，所述中药组合物由如下重量配比的药物制备而成：当归6.75％、川芎6.75％、白芍5.4％、钩藤13.5％、鸡血藤13.5％、熟地黄5.4％、决明子13.5％、夏枯草13.5％、细辛1.34％、延胡索6.75％和珍珠母13.5％。
  2. 如权利要求1所述的应用，其特征在于，所述应用在于该组合物用于清除阿尔茨海默患者脑内的老年斑。
  3. 如权利要求2所述的应用，其特征在于，所述应用在于该组合物能够清除脑内Aβ蛋白，降低脑内Aβ蛋白的生成。
  4. 如权利要求3所述的应用，其特征在于，所述应用在于该组合物能够抑制致病性γ分泌酶早老素PS1的表达水平抑制APP的病理性剪切，促进APP发生生理性α剪切的作用。
  5. 如权利要求1所述的应用，其特征在于，所述应用在于该组合物使记忆认知能力显著提高。
  6. 如权利要求5所述的应用，其特征在于，所述应用在于该组合物能够提高大脑皮层及海马组织中脑源性神经生长因子、神经生长因子及其受体TrkA的表达，改善海马神经元超微结构的异常。
  7. 如权利要求5所述的应用，其特征在于，所述应用在于该组合物能够提高大脑皮层及海马突触素及突触后致密物95的表达，改善海马突触结构的异常。
  8. 如权利要求5所述的应用，其特征在于，所述应用在于该组合物能够增加大脑皮层总抗氧化能力及GSH含量，提高SOD、GSH-px活性，减少MDA含量。
  9. 如权利要求5所述的应用，其特征在于，所述应用在于该组合物能够提高大脑皮层及海马组织中乙酰胆碱的表达，提高海马胆碱乙酰转移酶及大脑皮质M1胆碱受体的表达。
  10. 如权利要求1所述的应用，其特征在于，所述该组合物选自颗粒剂、丸剂、片剂、胶囊、口服液中的一种。
PCT/CN2014/086915 2013-09-22 2014-09-19 养血清脑制剂在制备治疗阿尔茨海默症的药物中的应用 WO2015039619A1 (zh)

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CN107917966B (zh) * 2016-10-08 2022-02-18 天士力医药集团股份有限公司 一种养血清脑制剂的检测方法
CN109059117A (zh) * 2018-06-08 2018-12-21 广东轻工职业技术学院 一种多用途的气体检测及净化器
CN109091440A (zh) * 2018-10-23 2018-12-28 扬州工业职业技术学院 一种复方植物提取物的制备方法及作为sod类化妆品添加剂的应用
CN109172452A (zh) * 2018-10-23 2019-01-11 扬州工业职业技术学院 一种白芍和槐花复方提取物及其作为化妆品添加剂的应用
CN109172452B (zh) * 2018-10-23 2020-09-29 扬州工业职业技术学院 一种白芍和槐花复方提取物及其作为化妆品添加剂的应用
CN109091440B (zh) * 2018-10-23 2020-10-30 扬州工业职业技术学院 一种复方植物提取物的制备方法及作为sod类化妆品添加剂的应用
CN116350682A (zh) * 2023-03-09 2023-06-30 上海中医药大学附属岳阳中西医结合医院 一种具有活血补肾功能的中药组合物及其制备方法和应用
CN116350682B (zh) * 2023-03-09 2024-04-23 上海中医药大学附属岳阳中西医结合医院 一种具有活血补肾功能的中药组合物及其制备方法和应用

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