TW201542219A - Use of yangxueqingnao formulation in preparation of medicine for treating alzheimer's disease - Google Patents

Abstract

Use of a traditional Chinese medicine composition in preparation of medicine for treating Alzheimer's disease (senile dementia). The traditional Chinese medicine composition is prepared from Radix Angelicae Sinensis 6.75%, Rhizoma Chuanxiong 6.75%, Radix Paeoniae Alba 5.4%, Ramulus Uncariae Cum Uncis 13.5%, Caulis Spatholobi 13.5%, Radix Rehmanniae Preparata 5.4%, Semen Cassiae 13.5%, Spica Prunellae 13.5%, Herba Asari 1.34%, Rhizoma Corydalis 6.75% and Concha Margaritifera Usta 13.5%.

Description

Application of serum-sparing brain preparation in preparing medicine for treating Alzheimer's disease

The present invention relates to a novel use of a formulation of a traditional Chinese medicine composition, in particular to the use of a serum-sparing brain preparation for the preparation of a medicament for the treatment of 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. A granule prepared by a preparation process such as mixing granulation. The preparation has the advantages of quick dissolution of active ingredients and high bioavailability. Yangxue Qingnao Granule has 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 (AD), also known as Alzheimer's disease, is a degenerative progressive neurological degenerative disease 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, Selegiline, vitamin E, melatonin, ginkgo extract (gingko bilobi), piracetam (piracetam), aniracetam (aniracetam aniracetam, San Lexi), Naftiracetam and the like.

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.

In particular, 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 invention is prepared from the following weight percentages: 6.75% of Angelica sinensis, 6.75% of 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 is as follows: pretreatment of the medicinal material → water extraction → concentration → ethanol precipitation Precipitate → recover ethanol → concentrate into a paste → mix to prepare a preparation.

The preparation includes any one which can be administered, preferably an oral preparation such as a granule, a pill, a tablet, a capsule, or an oral solution.

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 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 acetylcholine transferase 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 β protein in the brain and reduce the production of A β protein in the brain.

The application of the present invention resides in that the traditional Chinese medicine composition can inhibit the pathological cleavage of APP by inhibiting the pathological cleavage of APP, which inhibits the expression level of the pathogenic γ-secretase presenilin PS1, and promotes the physiological α-cleavage of APP.

Figure 1 shows the genotypic identification of APPswe/PS1dE9 transgenic mice.

Figure 2 is a Y-maze experiment demonstrating that Yangxuenao granules have a significant effect on improving memory and cognitive ability in mice with early and mid-term Alzheimer's disease (compared with saline group *p<0.05, ** p<0.01).

Figure 3 is a Congo red staining of the frontal cortex of the brain of each drug group.

Figure 4 is a Congo red staining of the frontal cortex of the brain of male transgenic mice of each drug group (the pink color indicated by the arrow is a β-amyloid plaque).

Figure 5 is a Congo red staining of the frontal cortex of the brain of female transgenic mice of each drug group (the pink color indicated by the arrow is a β-amyloid plaque).

Figure 6 is a Congo red staining of the hippocampus of transgenic mice of each drug group (the pink color indicated by the arrow is a β-amyloid plaque).

Figure 7 is the number of β-amyloid plaques in the brain tissue of transgenic mice of each drug group (* p < 0.05; ** p < 0.01 compared with the saline group).

Figure 8 is the area of β-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 β α segment (1-16 aa of A β 42) (immunized histochemical brain particles were confirmed by immunohistochemical staining of APP β α specific antibody recognizing insoluble protein).

Figure 10 shows the number and morphology of β-amyloid plaques in the cerebral cortex of transgenic mice in each drug group. The arrows in the figure indicate the β-amyloid plaques positive for APP staining, which are dark brown.

Figure 11 shows the number and morphology of A β-amyloid plaques in the hippocampus of male transgenic mice in each drug group. The arrows in the figure indicate that APP staining positive β- Amyloid plaque, dark brown.

Figure 12 shows the number and morphology of A β-amyloid plaques in the hippocampus of female transgenic mice in each drug group. The arrows in the figure indicate the β-amyloid plaques positive for APP staining, which are dark brown.

Figure 13 shows the number of strongly positive Aβ-amyloid plaques in the brain tissue of transgenic mice of each drug group, **p<0.01 compared with the saline group.

Figure 14 is a graph showing the effect of each drug group on key splicing enzymes and splicing products of APP in the brain of transgenic mice.

Figure 15 is a molecular mechanism of the effect of Yangxue brain particles on the significant elimination of senile plaques in the brain of early and mid-term Alzheimer's disease animal models.

Figure 16 is a graph showing the spontaneous activity of SAMP8 mice. A: blank control group, B: model group, C: doxorubicin hydrochloride group, D: nourishing serum brain particles 935mg/kg group, E: nourishing serum brain particles 1870mg/kg group, F: nourishing serum brain particles 3740mg/ Kg group.

Figure 17 is the effect of Yangxue brain particles on the total number of times the YAMP maze of SAMP8 mice was advanced (n = 23-28, mean ± SD).

Figure 18 is the effect of Yangxue brain particles on the spontaneous alternating rate of Y maze in SAMP8 mice (n=23-28, mean±SD). Compared with the blank control group, ###p<0.001; compared with the model group, ***p<0.001.

Figure 19 is the effect of Yangxue brain particles on the 1h priority index of new object discrimination in SAMP8 mice (n=22-28, mean±SD). ###p<0.001 compared to the blank control group; ***p<0.001 or **p<0.01 or *p<0.05 compared to the model group.

Figure 20 is the identification of new objects in SAMP8 mice by Yangxue brain particles for 24h. The effect of the priority index (n = 22-28, mean ± SD). ###p<0.001 compared to the blank control group; ***p<0.001 or **p<0.01 compared to the model group.

Figure 21 is the effect of Yangxue brain particles on the escape latency of water maze in SAMP8 mice (n=18-26, mean±SD). Compared with the blank control group, ###p<0.001; compared with the model group, ***p<0.001 or **p<0.01 or *p<0.05

Figure 22 is the effect of Yangxue brain particles on the water maze swimming distance of SAMP8 mice (n=18-26, mean±SD). Compared with the blank control group, ###p<0.001; compared with the model group, ***p<0.001 or **p<0.01.

Figure 23 is the effect of Yangxue brain granule on the swimming trajectory of SAMP8 mice in space exploration experiments.

Figure 24 is the effect of Yangxue brain granules on the swimming time of target quadrants of SAMP8 mice in space exploration experiments (n=18-26, mean±SD). ###p<0.001 compared to the blank control group; ***p<0.001 or **p<0.01 or *p<0.05 compared to the model group.

Figure 25 is the effect of Yangxue brain granules on the percentage of target quadrants in the space exploration experiment of SAMP8 mice (n=18-26, mean±SD). Compared with the blank control group, ###p<0.001; compared with the model group, ***p<0.001 or **p<0.01.

Figure 26 is a graph showing the effect of Yangxue brain particles on the pathological changes of hippocampal neurons in SAMP8 mice (x40).

Figure 27 is a graph showing the effect of Yangxue brain particles on the ultrastructure of neurons in the hippocampal CA1 region of SAMP8 mice (n=4, ×6000).

Figure 28 is a synapse in the hippocampal CA1 region of SAMP8 mice by Yangxue brain particles. The effect of ultrastructure (n=4, ×10000).

Figure 29 is a graph showing the effect of Yangxue brain particles on the expression of brain-derived nerve growth factor (BDNF) in hippocampus of SAMP8 mice (x4).

Figure 30 is a graph showing the effect of Yangxuenao Granule on the expression of brain-derived nerve growth factor (BDNF) in the hippocampal CA1 region of SAMP8 mice (x40).

Figure 31 is a graph showing the effect of Yangxuenao Granule on the expression of brain-derived nerve growth factor (BDNF) in the hippocampal CA1 region of SAMP8 mice (n=6, mean±SD). Compared with the blank control group, ###p<0.001; compared with the model group, ***p<0.001, **p<0.01 or *p<0.05.

Figure 32 is a graph showing the effect of Qingnao Granule on the expression of brain-derived nerve growth factor (BDNF) in the cerebral cortex of SAMP8 mice (×40).

Figure 33 is the effect of Qingnao Granule on the expression of brain-derived nerve growth factor (BDNF) in the cerebral cortex of SAMP8 mice (n=6, mean±SD). ###p<0.001 compared to the blank control group; **p<0.01 or *p<0.05 compared to the model group.

Figure 34 is a graph showing the effect of Yangxue brain particles on the expression of hippocampal nerve growth factor (NGF) in SAMP8 mice (x4).

Figure 35 is a graph showing the effect of Yangxue brain particles on the expression of nerve growth factor (NGF) in the hippocampal CA1 region of SAMP8 mice (x40).

Figure 36 is the effect of Yangxuenao Granule on the expression of nerve growth factor (NGF) in the hippocampal CA1 region of SAMP8 mice (n=6, mean±SD) compared with the blank control group, ###p<0.001; with the model group In comparison, ***p<0.001 or **p<0.01 or *p<0.05.

Figure 37 is the serum of cerebral cortex in the cerebral cortex of SAMP8 mice. The effect of long factor (NGF) expression (x40).

Figure 38 is a graph showing the effect of Yangxue brain particles on the expression of nerve growth factor (NGF) in the cerebral cortex of SAMP8 mice (n=6, mean±SD). Compared with the blank control group, ##p<0.01; compared with the model group, **p<0.01.

Figure 39 is a graph showing the effect of Yangxue brain granules on the expression of TrkA in the hippocampal CA1 region of SAMP8 mice (x4).

Figure 40 is a graph showing the effect of Yangxue brain particles on the expression of TrkA in the hippocampal CA1 region of SAMP8 mice (x40).

Figure 41 is the effect of Yangxuenao Granule on the expression of TrkA in the hippocampal CA1 region of SAMP8 mice (n=6, mean±SD) compared with the blank control group, ###p<0.001; compared with the model group, ** *p<0.001 or **p<0.01 or *p<0.05.

Figure 42 is a graph showing the effect of Yangxue brain particles on the expression of TrkA in the cerebral cortex of SAMP8 mice (x40).

Figure 43 is the effect of Yangxuenao Granule on the expression of TrkA in the cerebral cortex of SAMP8 mice (n=6, mean±SD) compared with the blank control group, ###p<0.001; compared with the model group, *** p < 0.001 or ** p < 0.01.

Figure 44 is a graph showing the effect of Yangxue brain particles on hippocampal synaptophysin (SYP) expression in SAMP8 mice (n=3, mean ± SD). Compared with the blank control group, ##p<0.01; compared with the model group, *p<0.05.

Figure 45 is a graph showing the effect of Yangxue brain particles on the expression of hippocampal synapse growth associated protein (GAP-43) in SAMP8 mice (n=3, mean±SD).

Figure 46 is a graph showing the effect of Yangxue brain particles on the expression of hippocampal postsynaptic density (PSD-95) in SAMP8 mice (n=3, mean±SD). With blank control In comparison, ##p<0.01; compared with the model group, **p<0.01.

Figure 47 is the effect of Yangxue brain particles on synaptophysin (SYP) expression in cerebral cortex of SAMP8 mice (n=3, mean ± SD). Compared with the blank control group, ##p<0.01; compared with the model group, **p<0.01.

Figure 48 is a graph showing the effect of Yangxue brain particles on the expression of synaptic growth-associated protein (GAP-43) in the cerebral cortex of SAMP8 mice (n=3, mean ± SD).

Figure 49 is a graph showing the effect of Yangxue brain particles on the expression of postsynaptic compacts (PSD-95) in the cerebral cortex of SAMP8 mice (n=3, mean±SD). Compared with the blank control group, ###p<0.001; compared with the model group, ***p<0.001 or *p<0.05.

Figure 50 is a representation of the spontaneous activity trajectory of each group of rats.

Figure 51 is a graph showing the effect of Yangxue brain particles on the total number of times of quinolinic acid-induced dementia in rats in the Y-maze experiment (n=16-18, ±SD).

Figure 52 is the effect of Yangxuenao Granule on the spontaneous alternation rate of quinolinic acid-induced dementia in NBM-induced dementia rats (n=16-18, ±SD). Compared with the sham operation group, ###p<0.001; compared with the model group, *p<0.05, ***p<0.001.

Figure 53 is the effect of Yangxuenao Granule on the 1h priority index of new object discrimination in NBM-induced dementia rats (Fig. ±SD). Compared with the sham operation group, ###p<0.001; compared with the model group, **p<0.01, ***p<0.001.

Figure 54 is a graph showing the effect of Yangxuenao Granule on the 24h priority index of new object discrimination in NBM-induced dementia rats in the presence of quinoline acid (n=16-18, ±SD). Compared with the sham operation group, ###p<0.001; compared with the model group, *p<0.05, ***p<0.001.

Figure 55 is the effect of Yangxuenao Granule on swimming time during water maze training of NBM-induced dementia rats in quinolinic acid (n=15-18, ±SD). Compared with the sham operation group, ###p<0.001; compared with the model group, *p<0.05, **p<0.01, ***p<0.001.

Figure 56 is the effect of Yangxuenao Granule on the swimming distance during the water maze training of NBM-induced dementia rats in the destruction of quinolinic acid (n=15-18, ±SD). Compared with the sham operation group, ###p<0.001; compared with the model group, *p<0.05, **p<0.01, ***p<0.001.

Fig. 57 is a swimming trajectory diagram of a space exploration experiment of each group of rats.

Figure 58 is the effect of Yangxuenao Granule on the swimming time of NBI-induced dementia in the fourth quadrant of quinolinic acid (n=15-18, ±SD). Compared with the sham operation group, ###p<0.001; compared with the model group, *p<0.05, ***p<0.001.

Figure 59 is the effect of Yangxuenao Granule on the percentage of swimming in the fourth quadrant of NBM-induced dementia rats in the presence of quinoline acid (n=15-18, ±SD). Compared with the sham operation group, ###p<0.001; compared with the model group, *p<0.05, **p<0.01.

Figure 60 is the effect of Yangxuenao Granule on the number of shocks in the dark-avoidance test of NBM-induced dementia in rats with quinolinic acid damage (n=15-18, ±SD). Compared with the sham operation group, ###p<0.001; compared with the model group, **p<0.01, ***p<0.001.

Figure 61 is a graph showing the effect of Yangxuenao Granule on the pathological changes of hippocampal neurons in the rat model of NBM-induced dementia induced by quinolinic acid ((40).

Figure 62 is the effect of Yangxue brain particles on the destruction of NBM-induced dementia by quinolinic acid. The effect of neuronal cell ultrastructure on the hippocampal CA1 region (n=4, ×6000).

Figure 63 is a graph showing the effect of Yangxuenao Granule on synaptic ultrastructure of hippocampal CA1 region in rats with dementia induced by quinolinic acid (n=4, ×10000).

Figure 64 is the effect of Yangxuenao Granule on the expression of synaptophysin (SYP) in hippocampus of rats with dementia induced by quinolinic acid (n=3, ±SD). Compared with the sham operation group, ##p<0.01; compared with the model group, *p<0.05, **p<0.01.

Figure 65 is the effect of Yangxuenao Granule on the expression of postsynaptic compacts (PSD-95) in hippocampus of NBM-induced dementia rats (Fig. ±SD). Compared with the sham operation group, ##p<0.01; compared with the model group, *p<0.05, **p<0.01.

Figure 66 is the effect of Yangxuenao Granule on the expression of synaptic growth-associated protein (GAP-43) in hippocampus of NBM-induced dementia rats (n=3, ±SD). #p<0.05 compared to the sham operation group; *p<0.05 compared to the model group.

Figure 67 is the effect of Yangxuenao Granule on the expression of synaptophysin (SYP) in cerebral cortex of NBM-induced dementia rats (n=3, ±SD). Compared with the sham operation group, ##p<0.01; compared with the model group, *p<0.05, **p<0.01.

Figure 68 is the effect of Yangxuenao Granule on the expression of postsynaptic dense material (PSD-95) in cerebral cortex tissue of rats with dementia induced by quinolinic acid (n=3, ±SD). Compared with the sham operation group, ###p<0.001; compared with the model group, **p<0.01.

Figure 69 is the effect of Yangxuenao Granule on the expression of synaptic growth-associated protein (GAP-43) in the cerebral cortex of rats with dementia induced by quinolinic acid (n=3, ±SD).

Figure 70 is the effect of Yangxuenao Granule on the content of acetylcholine (Ach) in hippocampus of rats with dementia induced by quinolinic acid (n=6, ±SD). Compared with the sham operation group, ###p<0.001; compared with the model group, *p<0.05, ***p<0.001.

Figure 71 is the effect of Yangxuenao Granule on the content of acetylcholine (Ach) in the cerebral cortex of NBM-induced dementia rats (n=6, ±SD). Compared with the sham operation group, #p<0.05; compared with the model group, *p<0.05

Figure 72 is the effect of Yangxuenao Granule on the content of choline acetyltransferase (ChAT) in hippocampus of rats with dementia induced by quinolinic acid (n=6, ±SD). Compared with the sham operation group, ###p<0.001; compared with the model group, **p<0.01, ***p<0.001.

Figure 73 is the effect of Yangxuenao Granule on the activity of choline acetyltransferase (ChAT) in hippocampus of rats with dementia induced by quinolinic acid (n=6, ±SD).

Figure 74 is the effect of Yangxuenao Granule on the expression of M1 receptor (CHRM1) in hippocampus of NBM-induced dementia rats induced by quinolinic acid (n=3, ±SD).

Figure 75 is the effect of Yangxuenao Granule on the expression of M1 receptor (CHRM1) in cerebral cortex tissue of rats with dementia induced by quinolinic acid (n=6, ±SD). #p<0.05 compared to the sham operation group; *p<0.05 compared to the model group.

The preferred extraction method of the traditional Chinese medicine composition of the present invention is as follows: according to the above-mentioned weight percentage, the prescription materials of the traditional Chinese medicine composition are reserved for use: (1) Preparation of the extract 1: Angelica, Chuanxiong, Corydalis, Cassia Add ethanol to reflux extraction, filter, remove impurities, recover ethanol and concentrate, and reserve; (2) Preparation of extract 2: white peony and ethanol are heated and refluxed for extraction, filtered, ethanol is recovered and concentrated, and used; (3) extract 3 Preparation: Rehmannia glutinosa, Uncaria, Spatholobus suberectus, Prunella vulgaris, mother-of-pearl, asarum and water to cook, filter, concentrate, add ethanol to stand, filter, recover ethanol and concentrate, spare; (4) preparation of the preparation: Take the above three kinds of extracts, add appropriate amount of excipients, and prepare the preparation.

Another preferred preparation method is as follows: (1) preparation of extract 1: Angelica, Chuanxiong, Corydalis, Cassia are added 3 to 6 times the amount of 50 to 80% ethanol and refluxed for 2 to 3 times, the first 0.5 to 2.5 Hours, second and third times each 0.5 to 2 hours, filtered, decontaminated, recovered ethanol and concentrated to a relative density of 1.250-1.350 (70 to 80 ° C), spare; (2) preparation of extract 2: white peony added 3 to 6 times the amount of 50 to 80% ethanol, immersed, heated and refluxed for 2 to 3 times, the first 0.5 to 2.5 hours, the second and third times each 0.5 to 2 hours, filtered, and the ethanol was recovered and concentrated to a relative density of 1.10 to 1.35 ( 55 to 65 ° C), spare; (3) Preparation of extract 3: Rehmannia glutinosa, Uncaria, Spatholobus suberectus, Prunella vulgaris, mother-of-pearl, asarum, 4 to 10 times the amount of water to cook 2 to 3 times, the first 0.5 to 3 hours at a time, 1 to 3 hours for the second and third times, filtered, concentrated to a relative density of 1.06-1.10 (75 to 85 ° C), ethanol added to make the alcohol content 60 to 85%, and allowed to stand 12 to 24 Hour, filtered, recovered ethanol, concentrated to phase For the density of 1.270-1.350 (75 to 85 ° C), spare; (4) preparation of the preparation: take the above extract, add appropriate amount of excipients, preparation, that is.

The best preparation method is as follows: (1) Preparation of extract 1: Angelica, Chuanxiong, Corydalis, and Cassia are added to 4 times 70% ethanol and heated to reflux for 2 times, the first time is 2 hours, the second time is 1 hour, and filtered. In addition to impurities, ethanol is recovered and concentrated to a relative density of 1.300-1.310 (74 to 76 ° C), standby; (2) preparation of extract 2: white peony is added 4 times the amount of 60% ethanol, impregnated, heated and refluxed for 2 times, the first 2 hours at a time, 1 hour at the second time, filtered, recovered ethanol and concentrated to a relative density of 1.23-1.33 (65 ° C), spare; (3) Preparation of extract 3: Rehmannia glutinosa, Uncaria, spatholobus, summer Blight, mother-of-pearl, and asarum were added to 5 times the amount of water for 2 times. The first time was 2 hours, the second time was 1 hour, filtered, concentrated to a relative density of 1.06-1.10 (80 ° C), and ethanol was added to make the alcohol content 65 to 70%, let stand for 12 to 24 hours, filter, recover ethanol, concentrate to a relative density of 1.320-1.325 (79 to 81 ° C), spare; (4) preparation of the preparation: take the above extract, add appropriate amount of excipients, preparation That's it.

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).

Example

Example 1

Raw material weight ratio: Take Angelica 253.5g, Chuanxiong 253.5g, white peony 202.7g, rehmannia 202.7g, Uncaria 506.8g, spatholobus 506.8, Prunella vulgaris 506.8g, cassia seed 506.8g, mother-of-pearl 506.8g, Corydalis 253.5g, Asarum 50.5g.

Preparation of 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 to 76 ° C), get the extract, spare.

Preparation of extract 2: white peony was added to 4 times the amount of 60% ethanol, immersed, heated and refluxed for 2 times, the first 2 hours, the second time 1 hour, filtered, and the ethanol was recovered and concentrated to a relative density of 1.23-1.33 (65 °C), get the extract, spare.

Preparation of extract 3: Rehmannia glutinosa, Uncaria, Spatholobus suberectus, Prunella vulgaris, mother-of-pearl, 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 Relative density 1.06-1.10 (80 ° C), add ethanol to make the alcohol content 65-70%, let stand for 12-24 hours, filter, recover ethanol, concentrate to a relative density of 1.320-1.325 (79 to 81 ° C), dipping Cream, spare.

300 g of dextrin was taken, opened with purified water, 3.0 g of stevioside was added, and the mixture was sufficiently 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 and 1.23 (42 to 50 ° C). Filter from 60 mesh to 100 mesh.

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 at Between 30-60 ° C. Dry and allow the temperature of the material to rise to 80 to 90 ° C to dry thoroughly.

Whole sieving, total mixing, granules, aluminum-plastic composite film pillow bag, size 4 g / bag.

Example 2

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%.

Preparation of 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 to 76 ° C), get the extract, spare.

Preparation of extract 2: white peony was added to 4 times the amount of 60% ethanol, immersed, heated and refluxed for 2 times, the first 2 hours, the second time 1 hour, filtered, and the ethanol was recovered and concentrated to a relative density of 1.23-1.33 (65 °C), get the extract, spare.

Preparation of extract 3: Rehmannia glutinosa, Uncaria, Spatholobus suberectus, Prunella vulgaris, mother-of-pearl, 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 Relative density 1.06-1.10 (80 ° C), add ethanol to make the alcohol content 65 to 72%, let stand for 12-24 hours, filter, recover ethanol, concentrate to a relative density of 1.320-1.325 (79 to 81 ° C), dipping Cream, spare.

The prepared extract is prepared by a conventional preparation method to obtain a serum-supplemented brain pill.

Example 3

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%.

Preparation of 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 to 74 ° C), get the extract, spare.

Preparation of extract 2: white peony was added to 4 times the amount of 80% ethanol, immersed, heated and refluxed for 2 times, the first 2 hours, the second 2 hours, filtered, and the ethanol was recovered and concentrated to a relative density of 1.15 - 1.25 (65 °C), get the extract, spare.

Preparation of extract 3: Rehmannia glutinosa, Uncaria, Spatholobus suberectus, Prunella vulgaris, mother-of-pearl, 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 Relative density 1.06-1.10 (80 ° C), add ethanol to make the alcohol content 60 to 65%, let stand for 12-24 hours, filter, recover ethanol, concentrate to a relative density of 1.27-1.320 (75 to 80 ° C), dipping Cream, spare.

The prepared extract is prepared by a conventional preparation method to obtain a serum-removing brain drop pellet.

Example 4

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%.

Preparation of extract 1: Angelica, Chuanxiong, Yanhusuo, Cassia 4 times the amount of 80% ethanol heated and refluxed for 2 times, the first 2.5 hours, the second time 1 hour, filtered, decontaminated, recovered ethanol and concentrated to a relative density of 1.30-1.350 (75 to 80 ° C), to obtain extract, spare.

Preparation of extract 2: white peony was added to 6 times the amount of 60% ethanol, immersed, heated and refluxed for 3 times, the first 2 hours, the second time 1 hour, the third time 0.5 hour, filtered, ethanol was recovered and concentrated to relative Density 1.20-1.35 (60 ° C), get the extract, spare.

Preparation of 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, and concentrated to Relative density 1.06-1.10 (80 ° C), add ethanol to 80-85% alcohol content, let stand for 12-24 hours, filter, recover ethanol, concentrate to a relative density of 1.30-1.350 (80 to 85 ° C), dipping Cream, spare.

The prepared extract is prepared by a conventional preparation method to obtain a serum-free brain drop oral solution.

Example 5

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%.

Preparation of 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 to 78 ° C), get the extract, spare.

Preparation of extract 2: white peony was added to 5 times the amount of 70% ethanol, impregnated, The mixture was heated and refluxed for 2 times, the first time for 1 hour, the second time for 1 hour, filtered, and the ethanol was recovered and concentrated to a relative density of 1.23-1.35 (65 ° C) to obtain an extract for use.

Preparation of 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, and concentrated to Relative density 1.06-1.10 (80 ° C), add ethanol to make the alcohol content 63-70%, let stand for 12-24 hours, filter, recover ethanol, concentrate to a relative density of 1.290-1.330 (78 to 83 ° C), dipping Cream, spare.

The prepared extract is prepared by a conventional preparation method to obtain a serum-enriched brain capsule.

Example 6

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.

Preparation of 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, filtration, impurity removal, recovery Ethanol was concentrated to a relative density of 1.29-1.340 (73 to 78 ° C) to obtain an extract.

Preparation of extract 2: white peony was added to 4 times the amount of 80% ethanol, immersed, heated and refluxed for 3 times, the first 2 hours, the second time 1 hour, the third time 1 hour, filtered, ethanol was recovered and concentrated to relative Density 1.18-1.33 (65 ° C), get the extract, spare.

Preparation of extract 3: Rehmannia glutinosa, Uncaria, Spatholobus suberectus, Prunella vulgaris, Mother-of-pearl and asarum are added to 7 times the amount of water for 2 times. The first time is 2 hours, the second time is 1 hour, filtered, concentrated to a relative density of 1.06-1.10 (80 ° C), and ethanol is added to make the alcohol content 70. 75%, let stand for 12-24 hours, filter, recover ethanol, concentrate to a relative density of 1.310-1.330 (77 to 82 ° C), get the extract, spare.

84 g of dextrin was taken, and it was opened with purified water, 3 g of stevioside was added, and it melted by stirring well, and 780 g of the above-mentioned prepared extract was added to the above-mentioned slurry in steps, and it stirred. Adjust the specific gravity of the slurry between 1.12 and 1.23 (42 to 50 ° C). Filter from 60 mesh to 100 mesh.

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.

Whole sieving, total mixing, granules, aluminum-plastic composite film pillow bag, size 3 g / bag.

Example 7

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.

Preparation of extract 1: Angelica, Chuanxiong, Corydalis, and Cassia were added to 6 times 70% ethanol and heated to reflux for 2 times, the first 2 hours, the second 0.5 hours, filtered, decontaminated, recovered ethanol and concentrated to relative density. 1.260-1.310 (74 to 76 ° C), get the extract, spare.

Preparation of extract 2: white peony was added to 6 times the amount of 60% ethanol, impregnated, The mixture was heated under reflux for 2 times, the first 2 hours, the second 2 hours, filtered, and the ethanol was recovered and concentrated to a relative density of 1.21-1.34 (55 ° C) to obtain an extract for use.

Preparation of 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, and concentrated to Relative density 1.06-1.10 (80 ° C), add ethanol to make the alcohol content 65-75%, let stand for 12-24 hours, filter, recover ethanol, concentrate to a relative density of 1.300-1.300 (79 to 81 ° C), dipping Cream, spare.

40 g of dextrin was taken, and it was opened with purified water, 3 g of stevioside was added, and it melted by stirring well, and 794 g of the above-mentioned prepared extract was added to the above-mentioned slurry in steps, and it stirred. Adjust the specific gravity of the slurry between 1.12 and 1.23 (42 to 50 ° C). Filter from 60 mesh to 100 mesh.

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 allow the temperature of the material to rise to 70 to 90 ° C to dry thoroughly.

Whole sieving, total mixing, granules, aluminum-plastic composite film pillow bag, size 4 g / bag.

Example 8

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.

Preparation of extract 1: Angelica, Chuanxiong, Yanhusuo, Cassia 6 times 80% ethanol was heated and refluxed for 2 times, the first time was 1 hour, the second time was 1 hour, filtered, and the impurities were removed. The ethanol was recovered and concentrated to a relative density of 1.29-1.340 (73 to 78 ° C) to obtain an extract. spare.

Preparation of extract 2: White peony was added to 3 times 60% ethanol, immersed, and heated under reflux for 2 times, the first 2.5 hours, the second 2 hours, filtered, and the ethanol was recovered and concentrated to a relative density of 1.17 - 1.33 (65 °C), get the extract, spare.

Preparation of extract 3: Rehmannia glutinosa, Uncaria, Spatholobus suberectus, Prunella vulgaris, mother-of-pearl, and asarum were added to 9 times the amount of water for 2 times, the first 3 hours, the second 3 hours, filtered, and concentrated to Relative density 1.06-1.08 (80 ° C), add ethanol to make the alcohol content 65 to 75%, let stand for 12-22 hours, filter, recover ethanol, concentrate to a relative density of 1.310-1.330 (77 to 82 ° C), dipping Cream, spare.

110 g of dextrin was taken, and it was opened with purified water, 3 g of stevioside was added, and it melted by stirring well, and 840 g of the above-mentioned prepared extract was added to the above-mentioned slurry in steps, and it stirred. Adjust the specific gravity of the slurry between 1.12 and 1.23 (42 to 50 ° C). Filter from 60 mesh to 100 mesh.

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.

Whole sieving, total mixing, granules, aluminum-plastic composite film pillow bag, size 3 g / bag.

Example 9

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.

Preparation of extract 1: Angelica, Chuanxiong, Corydalis, and 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 1.250-1.310 (70 to 74 ° C), get the extract, spare.

Preparation of extract 2: white peony was added to 4 times the amount of 60% ethanol, immersed, heated and refluxed for 2 times, the first 2 hours, the second time 1 hour, filtered, and the ethanol was recovered and concentrated to a relative density of 1.23-1.33 (65 °C), get the extract, spare.

Preparation of extract 3: Rehmannia glutinosa, Uncaria, Spatholobus suberectus, Prunella vulgaris, mother-of-pearl, 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 Relative density 1.06-1.10 (80 ° C), add ethanol to make the alcohol content 65-75%, let stand for 12-24 hours, filter, recover ethanol, concentrate to a relative density of 1.27-1.320 (75 to 80 ° C), dipping Cream, spare.

300 g of soluble starch was taken, opened with purified water, and 3.0 g of stevioside was added, and the mixture was thoroughly stirred to dissolve, and the prepared extract was added to the above slurry in portions and stirred. Adjust the specific gravity of the slurry between 1.12 and 1.23 (42 to 50 ° C). Filter from 60 mesh to 100 mesh.

Put the remaining soluble starch 250.0g into the granulator, adjust the granulation parameters such as fan frequency, inlet air temperature, infusion frequency and atomization pressure to make the bed The material is in a good fluidized state. Spray granulation, the granulation process controls the temperature of the material between 30 and 60 °C. Dry and allow the temperature of the material to rise to 80 to 90 ° C to dry thoroughly.

Whole sieving, total mixing, granules, aluminum-plastic composite film pillow bag, size 4 g / bag.

Example 10

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.

Preparation of 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 to 80 ° C), get the extract, spare.

Preparation of extract 2: white peony was added to 4 times the amount of 60% ethanol, immersed, heated and refluxed for 2 times, the first 2 hours, the second time 1 hour, filtered, and the ethanol was recovered and concentrated to a relative density of 1.23-1.33 (65 °C), get the extract, spare.

Preparation of extract 3: Rehmannia glutinosa, Uncaria, Spatholobus suberectus, Prunella vulgaris, mother-of-pearl, 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 Relative density 1.06-1.10 (80 ° C), add ethanol to make the alcohol content 60 to 65%, let stand for 12-24 hours, filter, recover ethanol, concentrate to a relative density of 1.315-1.320 (76 to 79 ° C), dipping ,spare.

80 g of microcrystalline cellulose 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 and 1.23 (42 to 50 ° C). Filter from 60 mesh to 100 mesh.

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 and 60 °C. Dry and allow the temperature of the material to rise to 70 to 90 ° C to dry thoroughly.

Whole sieving, total mixing, granules, aluminum-plastic composite film pillow bag, size 3 g / bag.

Example 11

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.

Preparation of 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 to 77 ° C), get the extract, spare.

Preparation of extract 2: white peony was added to 4 times the amount of 80% ethanol, immersed, heated and refluxed for 2 times, the first 2 hours, the second 2 hours, filtered, and the ethanol was recovered and concentrated to a relative density of 1.15 - 1.25 (65 °C), get the extract, spare.

Preparation of extract 3: Rehmannia glutinosa, Uncaria, Spatholobus suberectus, Prunella vulgaris, Mother-of-pearl and asarum are added to 5 times the amount of water for 2 times. The first time is 2 hours, the second time is 1 hour, filtered, concentrated to a relative density of 1.06-1.10 (80 ° C), and ethanol is added to make the alcohol content 65. 70%, let stand for 12-24 hours, filter, recover ethanol, concentrate to a relative density of 1.310-1.315 (79 to 82 ° C), get the extract, spare.

231 g of lactose 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 and 1.23 (42 to 50 ° C). Filter from 60 mesh to 100 mesh.

The remaining 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 and 60 °C. Dry and let the temperature of the material rise to 70-90 ° C to dry thoroughly.

Whole sieving, total mixing, granules, aluminum-plastic composite film pillow bag, size 4 g / bag.

Example 12

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.

Preparation of 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, ethanol was recovered and concentrated to relative density. 1.280-1.300 (75 to 77 ° C), dipped, spare.

Preparation of extract 2: white peony was added to 6 times the amount of 60% ethanol, impregnated, The mixture was heated and refluxed for 3 times, the first time was 2 hours, the second time was 1 hour, and the third time was 0.5 hours. After filtration, the ethanol was recovered and concentrated to a relative density of 1.20 to 1.35 (60 ° C) to obtain an extract for use.

Preparation of 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, and concentrated to Relative density 1.06-1.10 (80 ° C), add ethanol to 80 to 85% alcohol content, let stand for 12-24 hours, filter, recover ethanol, concentrate to a relative density of 1.280-1.330 (75 to 80 ° C), dipping Cream, spare.

80 g of dextrin was taken, opened with purified water, and 3 g of aspartame was added, 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 and 1.23 (42 to 50 ° C). Filter from 60 mesh to 100 mesh.

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 and 60 °C. Dry and allow the temperature of the material to rise to 70 to 90 ° C to dry thoroughly.

Whole sieving, total mixing, granules, aluminum-plastic composite film pillow bag, size 3 g / bag.

Example 13

Formula for raising serum brain formula: The weight ratio of raw materials is: 6.75% for Angelica, 6.75% for Chuanxiong, 5.4% for Radix Paeoniae, 13.5% for Uncaria, 13.5% for Spatholobus, 5.4% for Rehmannia glutinosa, 13.5% for Cassia, 13.5% for Prunella, and 13.5% for Prunella vulgaris. Asarum 1.34%, Corydalis 6.75% and Pearl Mother 13.5%

The preparation method comprises the following steps: pretreatment of the medicinal material, water extraction, concentration, ethanol precipitation, recovery of ethanol, concentration into a paste, mixing and granulation, and packaging of the finished product. 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.

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 same as the serum-retaining brain particles The extracts obtained by the prescription have the same use as the serum-suppressing brain particles. The experimental results are as follows:

Effect Experiment I. Effect of Yangxuenao Granule on the Scavenging Effect and Memory Cognition of β-Amyloid Plaque in Brain of Mild to Moderate Alzheimer's Disease Model Rats

The APPswe/PS1dE9 double transgenic mice are internationally recognized Alzheimer's Disease (AD) model mice, and senile plaques began to appear in the 6-month-old APPswe/PS1dE9 transgenic brain, mimicking mild Alzheimer's disease in humans. 4 months old APPswe/PS1dE9 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 Amway After applying 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. Congo red staining and immunohistochemical staining experiments of brain tissue sections demonstrated that the serum-sparing brain particles cleared the brain's β-amyloid plaques (senile plaques) in the early and mid-term Alzheimer's disease model mice, from the mouse hippocampus and In terms of the number, coverage and coloration of β-amyloid plaques (age spots) in the cortex, the clearance rate of senile plaques in the brain was 60 to 90%, which was significantly higher than that of the positive drug Anritsu group. Abnormal splicing of human amyloid precursor protein (APP) is the key to the pathogenesis of Alzheimer's disease. It is further proved by the detection of key splicing enzymes and splicing products of APP in the brain of transgenic mice. Serum brain granules reduce the formation and deposition of β-amyloid plaques in the brain by increasing the physiological scission pathway of APP in the brain and inhibiting the pathological cleavage pathway of APP. This study fully proves that Yangxue Brain Granule has a good therapeutic effect on mild to moderate Alzheimer's disease.

1 Experimental purpose

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/PS1dE9 double transgenic Alzheimer's disease model mice, especially for the characteristic lesion of Alzheimer's disease-β amyloid A β in the brain. Sedimentation, using the specific staining of insoluble Aβ, Congo red staining for amyloid recognition, etc. The scavenging effect of Aβ senile plaques in the brain of APPswe/PS1dE9 double transgenic Alzheimer's disease model, and its target and mechanism through the study of protein cleavage pathway, for the treatment of mild to moderate aerogen Alzheimer's disease provides an experimental basis.

2 experimental materials 2.1 Tested drugs and preparation

2.1.1 Tested drugs: Yangxue brain granule extract (referred to as serum brain), dark brown extract, provided by Tianshili Pharmaceutical Group, batch number: 20120514, the creaming rate is 13%, equivalent to 7.7 per gram of extract g crude drug, prepared into 2.4g, 0.8g crude drug / ml concentration solution with physiological saline, for the mice to be administered by gavage (the dose in the text is based on the amount of raw drug), the dosage volume is 20ml / kg. The dose of the mouse is a high concentration of 48 g of crude drug/kg, and a low concentration of 16 g of crude drug/kg.

Positive control An Lishen, commercially available, 5mg per tablet, Eisai (China) Co., Ltd., batch number: 111030A, used as a suspension with normal saline at 0.0515mg/ml for oral administration. The administration volume was 20 ml/kg. The dose of the drug for mice was 1.03 mg/kg.

2.2 Experimental animals and genotype identification

Experimental animals APPswe/PS1dE9 double transgenic AD mice were purchased from the Institute of Model Animals, Nanjing University. The APPswe/PS1dE9 double transgenic mouse is an internationally recognized Alzheimer's model rat. As shown in Figure 1, APPswe/PS1dE9 double transgenic mice were transfected with human Swedish mutant APPswe and exon 9 deletion mutant PS1dE9 genes, which expressed human mutant APP molecules and abnormally over-producing A β 42 polypeptide, a beta plaque in the brain and other tissue diseases during AD lesions The change in behavior, the corresponding behavioral abnormal changes in AD. It has been reported that some of the Congo red plaques (in the early stage of senile plaques) appear in the amygdala and hippocampus when the mice are 4 to 5 months old, and a large number of amyloid plaques form in the cerebral cortex, hippocampus and amygdala at 6 months of age. Simulates mild Alzheimer's disease in humans.

The inventors performed genotypic identification of APPswe/PS1dE9 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'. Reaction conditions: 94 ° C for 30 sec, 69 ° C for 60 sec, 72 ° C for 60 sec, 30 cycles. After PCR amplification, a specific band of APP gene appeared in the transgenic mice, and the size was 350 bp, while the background mice did not have a band. All the mice were genotyped by PCR, which ensured that all the mice were positive homozygous transgenic mice, as shown in Figure 1.

2.3 Experimental reagents and preparation 2.3.1 Reagents

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, metformin, Tris, Gly, Coomassie Brilliant Blue R250, BSA (Sigma), Triton X-100 (Genview), protein Standard molecular weight (Beijing full-size gold), Tween-20 (BBI), rabbit anti-mouse β-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.

2.4 Experimental main instruments

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) uses the microscope image processing software DP Controller to capture images.

3 experimental methods 3.1 Experimental grouping and administration

The experimental animals APPswe/PS1dE9 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 concentration group of nourishing serum brain particles. (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, half male and half female, each group has 16. In the course of the experiment, there are 2 deaths in each of the saline group and the An Lishen group. Groups 14 to 16 and weigh 20 to 40 g. Conventional feeding, free drinking water, 4 months old APPswe/PS1dE9 transgenic mice were administered intragastrically, and the administration volume was 0.5 ml per 25 g body weight. Oral administration to 6 Month age.

3.2 Y maze test to detect memory cognitive behavior in mice

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. If the wrong path occurs, it is recorded as an error, and the path is recalculated from the beginning. In the final calculation formula: correct rate (%) = n / (total number of arms - 2) × 100%, n is the correct number of paths. For example, the mouse activity route is ABCABCBACBACACBABCA, where the bottom line is the wrong path, n=16, the total number of arms is 19, and the correct rate (%)=16/(19-2)×100%=94.11%.

3.3 mouse perfusion fixation and embedding slice

The 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 μm .

3.4 Congo red staining for detection of amyloid in the brain

After the paraffin tablets were taken out, they were baked on a baking machine at 60 to 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. After 1 h of Congo red staining, 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 β senile plaque in the brain is amyloid, which is red after Congo red staining. The number and area of red A β amyloid plaques in the cortex and hippocampus were measured and calculated for each section, and the average value was analyzed.

3.5 Immunohistochemical staining

1 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 H2O2 to remove endogenous active substances. The APP β α antibody was added for 4 h, and the murine 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.

3.6 β-amyloid plaque count

Random observation of cortical and hippocampus by immunohistochemical staining of each section 3 The visual field was measured, and the number of strongly positive and weakly positive β-amyloid plaques in each field of view was counted, and the average value was analyzed.

3.7 Western Bloting method for detecting protein levels of α APPs, ADAM10 and PS1 in mouse brain tissue

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% non-fat milk powder for 1 h, and various anti-α 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.

Antibodies: ADAM10, PS1, β-actin antibodies were purchased from Boosen. The APP β α segment antibody was independently developed, and the 16 amino acid short peptide DAEFRHDSGYEVHHQK between APP β cleavage and α cleavage was selected (this peptide is a fibrous insoluble peptide, which is a key region determining the brain insolubility of A β 42 ) as an antigen. Highly priced APP β α antibodies were obtained by gene recombination, sequencing, protein expression and purification, animal immunization and ProteinA/G purification.

3.8 statistics

Statistical analysis was performed using SPSS Statistics 17.0. The results were represented by scatter plots combined with mean and mean ± standard error. Data were analyzed by one-way ANOVA combined with LSD method for comparison between groups.

4 experimental results 4.1 General observation

At 4 months of age, transgenic mice have developed symptoms of mental injury and Compared with the reduction in food intake, the reduction of voluntary activities, slow movements, lack of energy, and lack of luster. After the administration, the transgenic mice in the Yangxuenao granule group had different degrees of improvement, the food intake increased, and the spirit improved.

4.2 Y labyrinth test proves that Yangxuenao granules can significantly improve the memory and cognitive ability of mice with Alzheimer's disease in early and mid-term.

The Y-maze experiment is a simple and widely used behavioral tool for detecting spatial cognitive ability and short-term memory ability in mice. As shown in 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. For example, in the mid-month of the administration, the correct rate of normal saline was 79.59%, and 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. At the end of the two-month behavioral test, 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.

Therefore, the inventors' results showed that the correct rate of Y maze of the low-concentration group and the high-concentration group was increased by nearly 10% compared with the control for one month and two months of administration, and was significant; 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 animal model.

Note: Compared with saline group *p<0.05, **p<0.01

4.3 Congo red staining for specific identification of amyloid plaques demonstrates that Yangxueqingnao has a significant role in eradicating senile plaques in the brain of early and middle mice.

AD patients present with central nervous system degenerative lesions in the frontotemporal and hippocampus, and A beta amyloid plaque is a characteristic lesion of Alzheimer's disease. Aβ agglutination and accumulation 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 condensation of Aβ. Caused by the elimination and elimination of imbalances.

A characteristic lesion of Alzheimer's disease is the deposition of A beta amyloid in the brain. Methanol Congo red staining is used to display amyloid and can well recognize the deposition of amyloid A beta protein in the brain. The inventors found that the number and area of A β 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 relationship. The lower concentration group of the high-concentration group was more effective. (Figures 3 to 7).

Figure 3 to Figure 5 show Congo red staining of amyloid plaques in the cerebral cortex of the control and experimental groups. Intracerebral A β in saline group and Ariase group The number of amyloid plaques was high, and the number of amyloid plaques in the high concentration and low concentration of serum brain particles was small. In 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. 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 removal of amyloid plaque deposits in the frontal cortex, while the control drug Anritsu 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. Compared with the positive control group, the number of senile plaques in the Anritsu group decreased 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 age spots, As a kind of acetylcholinesterase inhibitor, Lishen has no effect on reducing the deposition of β-amyloid plaque 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. Compared with the control group, 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).

The above results prove that 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% to 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 β-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.

Note: compared with saline group, **p<0.01

4.4 Immunohistochemical staining of APP β α specific antibody for recognizing insoluble A β protein in brain, which proves that Yangxue Qingnao Granule can significantly eliminate A β senile plaque in brain of early and middle AD mice.

Human amyloid precursor APP is a disease of Alzheimer's disease The bond protein, the deposition of amyloid A β formed by β-shear in the brain, is one of the main pathological features of Alzheimer's disease. The APP secretes the α-secretase cleavage pathway under normal conditions, producing an extracellular soluble sAPP α protein. In the process of Alzheimer's disease, APP is cleaved under the action of two proteases, β-secretase and γ-secretase, to produce 42 amino acid A β peptides, which is the main toxicity of AD. The accumulation of amyloid senile plaques between neurons and synaptic sites in the brain tissue can impair brain function, leading to memory and cognitive impairment. Most of the current antibodies are antibodies to the A β segment (β-cleavage and γ-cleaved 42 peptide), and the specific β-cleavage is not recognized (detection of C-segment protein containing both β-cleavage protein and α-cleavage protein), especially It is difficult to distinguish between soluble peptides (physiological) and insoluble peptides (pathology) in the brain.

The inventors selected the 16 peptide between the N-segment β-cleavage and α-cleavage in the A β 42 peptide as an antigen, which has a unique advantage in detecting pathological shear of Alzheimer's disease. As shown in Fig. 9, firstly, the brain insoluble peptide of the Aβ42 peptide is composed of the 16 peptides, so the 16-peptide antibody significantly enhances the specific detection efficiency of recognizing the insoluble protein (Aβ) in the brain, and insoluble in the brain. The detection of peptides has been improved to a new stage. The preparation and purification of the antibody are described in the method section. The inventors used this antibody to detect the deposition of Aβ peptide in the brain of the transgenic mouse of Alzheimer's disease.

Immunohistochemical staining using APP β α specific antibodies recognizing insoluble proteins demonstrated that the serum-suppressing brain particles have a significant effect of eliminating A β protein in mouse brain. As shown in Fig. 10 to Fig. 12, the deposition of Aβ-insoluble protein in the brain of the saline control group and the Ariase group was recognized by the APP-specific antibody recognizing the insoluble protein. Low blood concentration group and high blood concentration group See the deposition of Aβ insoluble protein. Male AD model mice (Fig. 11) and female AD model mice (Fig. 12) showed significant differences in immunostaining of Aβ protein in the hippocampus after 2 months of different drug effects. Deep-stained insoluble Aβ senile plaques were detected in the hippocampus of the control group and the Anrisheng group, while Aβ deposition was difficult to detect in the low-concentration group and the high-concentration group.

The inventors statistically analyzed the total number of β-amyloid plaques in the cerebral cortex of each experimental group. As shown in Table 4 and Figure 13, the experimental group was compared with the saline control group from the number of strong positive Aβ senile plaques in the mouse brain. There were significant differences. 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 high-concentration group was in the brain. Age spots were reduced by 87.5%. Compared with the An Lishen group, the therapeutic effect of Yangxuenao Granules was more obvious, suggesting that high-dose Yangxuenao Granules can significantly clear the Aβ protein in the brain of early and middle mice, and is superior to the An Lishen group.

Note: compared with saline group, **p<0.01

4.5 Yangxue serum granules can significantly promote the physiological α-shock of APP in the brain of Alzheimer's disease in early and mid-term, and promote the production of soluble neurotrophic sAPP α 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 alpha protein. In the development of Alzheimer's disease, amyloid-like pathological cleavage is induced by β- and γ-secretase. The deposition of amyloid A β formed by β and γ shear in the brain is one of the main pathological features and the most important molecular mechanism of Alzheimer's disease.

The physiological α-cleaved cleavage product sAPP α of APP not only has important neurotrophic effects, but also hinders the production of A β, which is of great significance for the treatment of AD. First, the Western blot analysis of the soluble physiological sAPP α by the antibody demonstrated that the serum-supplemented brain particles significantly promoted the physiological α-cleavage and promoted the action of soluble and neurotrophic sAPP α protein. The extracellular soluble fragment sAPP α produced by α-cleavage of APP has a size of 66kD. As shown in Fig. 14, the brain of AD model rats rarely exhibits physiological shear under physiological saline conditions, and the level of sAPP α is very low. Compared with the group, the sAPP α of the Anritsu group increased. The concentration of sAPP α in the low concentration of nourishing blood, especially in the high concentration group of nourishing blood, was significantly increased. And the high-dose group of nourishing blood was significantly higher than the positive Anritsu control group. Under normal conditions, the α-secretase cleavage pathway produces extracellular soluble APP peptide, which has neurotrophic effect. At the same time, physiological α-cleavage of sAPP α has a significant antagonistic effect on pathological β-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. June The expression level of ADAM10 in the brain of the aged AD transgenic model rats was very low under physiological saline conditions, indicating that physiological shearing rarely occurred. Compared with the control group, 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).

Therefore, the inventors found that Yangxue brain granules have the effect of promoting physiological α-shearing of APP. The serum-raising brain granules significantly increase the expression level of α-secreting cleavage enzyme ADAM10 in the rat brain and the soluble neurotrophic sAPP α protein in the brain. The formation of antagonizes the pathological shear pathway by significantly increasing the physiological alpha shear of APP in the brain, and its effect is more significant than that of the positive drug Anritsu.

The role of serum-sparing brain particles in clearing senile plaques in the brain is achieved by inhibiting the pathogenic γ-secretase presenilin PS1. The transgenic mouse is the PS1dE9 gene of the exon 9 deletion mutation of PS1. The mutation of presenilin PS1 gene is the key link of Aβ formation and AD treatment. As shown in Figure 14, the expression level of presenilin PS1dE9 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 PS1dE9 in the Anritsu group was not significant. However, the expression level of presenilin PS1dE9 with low concentration of nourishing blood and high concentration of nourishing blood decreased significantly and was difficult to detect. It is indicated that Yangxue Naobi Granule inhibits the pathological shearing of APP by inhibiting the expression level of proinflammatory γ-secretase presenilin PS1 in the brain, thereby reducing the deposition of Aβ senile plaques in the brain.

Therefore, 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. On the one hand, Yangxue brain granules inhibit by inhibiting the pathogenic gamma secretase presenilin PS1-dE9 The pathological process of APP cuts the Aβ process, which reduces the age spots in the brain. On the other hand, Yangxue brain granules promote the production of neurotrophic sAPP α by promoting the level of physiological secretase α secreting cleavage enzyme ADAM10, thereby promoting the physiological shearing of APP and antagonizing the Aβ in the brain. Generated to further reduce the age spots in the brain.

5 summary

From the above results, the inventors have demonstrated that the serum-supplemented brain particles have a remarkable effect of treating mild to moderate Alzheimer's disease. It is reflected in two aspects: First, the behavioral Y-maze test results show that oral high-concentration serum brain particles (48g crude drug / kg) and low-concentration serum brain particles (16g crude drug / kg) one month and two months later The memory and cognitive ability of the mice were significantly improved compared with the saline control group, and the effect was better than the positive drug An Lishen. That is, Yangxue brain particles have a significant effect on improving the memory and cognitive ability of AD mice in the early and middle stages.

More importantly, the formation of β-amyloid plaques for the characteristic lesions of Alzheimer's disease-A β protein deposition, the serum-enriched brain particles play an extremely significant therapeutic effect. The brain tissue of the Alzheimer's disease model mice, which was administered for 2 months, was sliced. The Congo red staining and immunohistochemical staining experiments were used to demonstrate the hippocampus and cortex of the mouse brain. The number of β-amyloid plaques (age spots) was significantly reduced from the number, coverage and coloration (clearance rate reached 60 to 90%), and the effect was much higher than that of the Anritsu group. At the same time, the protein extracted from mouse brain tissue was analyzed by Western blot, which proved that Yangxue brain particles can promote the physiological α-cleavage of β-amyloid precursor APP and inhibit the pathological shear of APP, thereby reducing the brain. The production of A β in the brain plays a significant role in clearing A β-amyloid plaques in the brain.

In summary, this study fully demonstrates that Yangxue Brain Granule has a good therapeutic effect on mild to moderate Alzheimer's disease.

Effect Experiment 2, Yangxue Brain Granules in the Treatment of Alzheimer's Dementia

In this experiment, Y-maze, new object discrimination, Morris water maze and avoidance of darkness behavioral experiments were used to investigate the effects of Yangxuenao Granule on SAMP8 rapidly aging mice and quinolinic acid-induced Meyert basal ganglia (NBM nucleus)-induced dementia rats. The improvement of learning and memory impairment; and the possible mechanisms of improving learning and memory impairment from the aspects of cerebral cortex and hippocampal neuronal cells and synaptic morphology, cholinergic nervous system function, neurotrophic factor and free radical oxidative damage.

The study found that 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 to 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. The above behavioral results indicate that Yangxuenao Granule can significantly improve non-spatial learning and memory impairments such as working memory impairment, spatial learning and memory impairment, and visual discrimination memory in dementia model animals. The effect of doxorubicin hydrochloride was similar; HE staining results showed that Yangxuenao Granule could improve the pathological changes of mouse and rat neurons in model mice; transmission electron microscopy showed that Yangxue brain particles can improve the hippocampal neurons in the brain. Structural abnormalities and abnormalities in synaptic structures; Western blot results showed that Yangxue brain granules can significantly increase the expression of SYP and PSD-95 in the cerebral cortex and hippocampus of dementia model mice and rats, and to the cerebral cortex and hippocampus The expression of GAP-43 was not significantly affected by immunohistochemistry. The results of immunohistochemistry showed that Yangxue Brain Granule could increase the expression of BDNF, NGF and its receptor TrkA in cerebral cortex and hippocampus of SAMP8 mice in a dose-dependent manner. The results of biochemical tests showed that Yangxue brain particles could increase the total antioxidant capacity, SOD and GSH-px activities, increase the GSH content and reduce the MDA content of lipid peroxidation products in a dose-dependent manner. Significantly increased the content of acetylcholine in the cerebral cortex and hippocampus of NBM-induced dementia rats, and increased the expression of choline acetylcholine transferase and cerebral cortex M1 choline receptor.

In summary, 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 nerves. Systematic functions to improve learning and memory impairment.

Part I: Effect and mechanism of Yangxue Qingnao Granule on learning and memory impairment in SAMP8 mice

In this experiment, Y-maze, new object discrimination, Morris water maze and avoidance of dark behavioral experiments were used to investigate the effect of Yangxue Qingnao Granule on learning and memory impairment in SAMP8 rapidly aging mice. The changes of hippocampal tissue structure in mouse brain were observed. The changes of hippocampal neurons and synaptic ultrastructure were observed by transmission electron microscopy. The brain-derived neurons in the cerebral cortex and hippocampus of SAMP8 mice were detected by immunohistochemistry. The effects of growth factor (BDNF), nerve growth factor (NGF) and its receptor TrkA expression; Western blot analysis of the serum growth brain protein on synaptic growth-related proteins in the cerebral cortex and hippocampus of SAMP8 mice 43 Effects of (GAP-43), synaptophysin (SYP) and postsynaptic density 95 (PSD-95) expression; biochemical methods for detection of lipid peroxidation products malondialdehyde (MDA), reduced valleys in cerebral cortex The content of glutathione (GSH), the total antioxidant capacity (T-AOC), and the activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-px).

The study found that Yangxue Brain Granules can dose-dependently increase the spontaneous alternation rate of SAMP8 mice and the new object discrimination experiment in the range of 935-3740mg/kg (1 to 4 times the clinical equivalent of human headache). 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. The above behavioral results indicate that Yangxuenao Granule can significantly improve non-spatial learning and memory impairment in SAMP8 mice, such as working memory impairment, spatial learning and memory impairment, and visual discrimination. The high-dose group has a clinically equivalent amount of hydrochloric acid. The results of HE staining showed that Yangxueqingnao can improve the pathological changes of neurons in SAMP8 mice. Transmission electron microscopy showed that Yangxue brain particles can improve the ultrastructural abnormalities and synaptic structure of hippocampal neurons in the brain. different Often; immunohistochemistry results showed that Yangxue Brain Granule could increase the expression of BDNF, NGF and its receptor TrkA in cerebral cortex and hippocampus of SAMP8 mice in a dose-dependent manner; Western blot results showed that Yangxue brain particles 3740mg The /kg group can significantly increase the expression of SYP and PSD-95 in the cerebral cortex and hippocampus of SAMP8 mice, and has no significant effect on the expression of GAP-43 in the cerebral cortex and hippocampal synaptic neonatal marker; biochemical test results show that serum Brain particles can increase total antioxidant capacity and SOD, GSH-px activity, increase GSH content, and reduce lipid peroxidation MDA content in a dose-dependent manner.

In summary, 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.

One. Experimental Materials Experimental animal

Male SAMP8 mice of 7 months old, 150 male mice, SAMR1 mice of the same age, 25 clean, graded by the Animal Center of the First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine, certificate number: scxk (Tian) 2008-0001, breeding At the SPF laboratory animal center of Shenyang Pharmaceutical University, free drinking water, 12h cycle lighting.

2. Drugs and preparation

Yangxue brain particles: extract, supplied by Tianjin Tianshili Pharmaceutical Group Co., Ltd., batch No. 20120502W, stored at 4 °C to 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. Preparation: precision weighing The serum brain granule extract was added with distilled water to prepare a solution of 93.5 mg/ml, 187 mg/ml and 374 mg/ml, respectively, to determine the drug concentration of the low, medium and high dose groups of the serum brain particles.

Donovan 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. Preparation: Take donate tablets of hydrochloric acid, add distilled water, and prepare a solution of 0.13 mg / ml.

3. Reagent

Sodium chloride: Tianjin Bodi Chemical Co., Ltd., batch number 20120221; disodium hydrogen phosphate: Shantou Xiqiao Chemical Co., Ltd., batch number 100802; anhydrous ethanol: Tianjin Hengxing Chemical Reagent Manufacturing Co., Ltd., batch number 20100327; Formaldehyde: 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; sodium citrate: Tianjin Bodi Chemical Co., Ltd., batch number: 20100719; sodium hydroxide: Tianjin Bodi Chemical Co., Ltd., batch number: 20111102; hydrochloric acid: Shenyang Economic and Technological Development Zone reagent Factory, batch number: 20110612; neutral gum: China Shanghai specimen model factory, batch number: 20101201; methanol: Tianjin Fuyu Fine Chemical Co., Ltd., batch number: 120306; BCA protein quantification kit: Biyuntian Biotechnology Research Institute; cell lysis Liquid: Biyuntian Biotechnology Research Institute; PMSF: Biyuntian Biotechnology Research Institute; PVDF membrane: Pall, USA; developer powder, fixing powder: Biyuntian Biotechnology Research Institute; mouse anti-SYP (D-4): Santa Cruz Biotechnology, product number: sc-17750; Anti-GAP-43: Santa Cruz Biotechnology, product number: sc-17790 rabbit anti-PSD-95: Abcam (Hong Kong) Ltd LOT: GR76077-1 mouse anti-β-actin: Santa Cruz Biotechnology, product number: sc-41478 Rabbit anti-BDNF: Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., product number: E0112; rabbit anti-NGF-β: Boster Biological Technology CO., Ltd. product number: 3574102; rabbit anti-TrkA: Boster Biological Technology CO., Ltd, product number :9H121C; SP immunohistochemical staining kit: Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.; ZLI-9018 concentrated DAB kit: Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.; Tris-base: BIOSHARP Amresco 0497; Glycine: BIOSHARP Amresco 0617; SDS: BIOSHARP Sigma L-5750 LOT 2012/09; Acrylamide: BIOSHARP Amresco 0341 LOT 2011/11; Methylene bis acrylamide: BIOSHARP Amresco; horseradish-labeled goat anti-mouse IgG: Beijing Zhongshang Jinqiao Biotechnology Co., Ltd.; skim milk powder: Heilongjiang Province Wandashan Dairy Co., Ltd.; high sensitivity chemiluminescence detection kit: Beijing Kangwei Century Biotechnology Co., Ltd.; Development Fixing Kit: Biyuntian Biotechnology Research Institute; T-AOC Total Antioxidant Capacity Kit: Nanjing Jiancheng Bioengineering Research Institute Lot No.: 20121220; SOD Kit: Nanjing Jiancheng Bioengineering Research Institute Batch number: 20121217GSH-px test box: Nanjing Institute of Bioengineering, batch number: 20121220; micro malondialdehyde test kit: Nanjing Institute of Bioengineering, batch number: 20121212; GSH test box: Nanjing Institute of Bioengineering, batch number :20121212.

4. Experimental equipment

Morris Water Maze Video Analysis System: Institute of Materia Medica, China Academy of Medical Sciences; Spontaneous Activity Video Analysis System: Shanghai Jiliang Software Technology Co., Ltd.; Other Behavioral Experimental Devices: Mouse Y Maze, New Object Recognition, and Dark Avoidance Devices Development of Shenyang Pharmaceutical University; paraffin-embedded slicer: Leica, Germany; optical microscope: OLYMPUS; DP72-Color Video Camera: OLYMPUS; U-PMTVC 7C06559: OLYMPUS company; Image-pro6.3 image analysis system: OLYMPUS company; ultrasonic cleaner: KQ5200B, Kunshan Ultrasonic Instrument Co., Ltd.; ultrasonic cell pulverizer: JY92-II, Ningbo Xinzhi Biotechnology Co., Ltd. Company; TECAN enzyme standard quantitative tester: SPECTRA CLASSIC; -80 °C low temperature refrigerator: Zhongke Meiling; one ten thousand balance: Sartorius Scientific Instruments (Beijing) Co., Ltd.; electronic balance: Changzhou Shuangjie Test Instrument Factory; Constant temperature water bath device: BCFCO Changfeng Electronic Technology Co., Ltd.; H-1 micro vortex mixer: Shanghai Jingke Industrial Co., Ltd.; electric heating constant temperature incubator: DNP-9272, Shanghai Precision Experimental Equipment Co., Ltd.; vertical automatic electrothermal pressure steam sterilization Device: LDZX-40AI, Shanghai Shenan Medical Instrument Factory; High Speed Refrigerated Centrifuge: HC-3018R, HKUST Innovation Co., Ltd.; Microwave oven: Galanz Company.

Second, the experimental method 1. Animal grouping and administration

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 the group of 1.3 mg/kg of donepezil hydrochloride, each group of 30, and another 25 of the 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. Enter Dosing continued during the line of behavioral experiments, once a day until the end of the experiment.

2. Behavioral experiment 2.1 mouse spontaneous activity experiment

The experimental device is composed of four rectangular activity boxes 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 to 5 minutes, record the total distance of animal activities, activity time and average speed.

2.2 mouse Y maze experiment

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. During the experiment, 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 (%). Spontaneous alternating reaction rate (%) = number of alternating reactions / (N-2) × 100 (for example, the order of entering the three arms within 5 min is ABCACBACCAB, then N is 11, and a correct alternating reaction is: ABC, BCA, ACB , CBA, BAC, CAB. Spontaneous alternating reaction rate (%) = [6 / (11-2)] × 100 = 66.7%)

2.3 mouse new object discrimination experiment

The experimental device is a wooden square open field, and the experiment is divided into an adaptation phase and a test phase. Put 2-3 mice into the open field and explore for 3 minutes. To adapt to 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. 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. The formula is as follows: The formula for calculating the priority index is as follows: priority index (1h) = tB / (tA1 + tB)

Priority index (24h) = tC / (tA1 + tC)

The formula for determining the coefficient is as follows:

Discrimination coefficient (1h)=(tB-tA1)/(tA1+tB)

Discrimination coefficient (24h) = (tC-tA1) / (tA1 + tC)

2.4 Mouse Morris water maze experiment

(1) 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, and two points equal to the distance are selected as the water inlet points on the opposite side of the platform. The mice are placed in the water facing the pool wall for 60 seconds, and the time from the entry of the water to the finding of the platform is recorded. Escape latency, swimming, and then let the mice rest on the platform for 10s. If the platform is not found in 60s, the incubation period is recorded as 60s, and The mice were 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.

(2) Space exploration experiment: After positioning the navigation experiment, remove the platform and place the mouse in the water at the midpoint of the opposite side of the original platform for free swimming for 60s. The maze system automatically records the time the mouse stays in the original platform quadrant and the percentage of the platform quadrant.

2.5 mouse darkening experiment

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 fled 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.

3. Western blotting method 3.1 Tissue protein extraction

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 μl of PMSF was added per 1 ml of the protein lysate. After homogenization by ultrasonic cell pulverizer, it was allowed to stand in an ice bath for 30 min, 12000×g, and centrifuged at 4 ° C for 20 min. The supernatant was taken, dispensed, and stored in a refrigerator at -80 ° C for use. A small portion of the supernatant was taken for protein quantification.

3.2 Determination of protein concentration - BCA method

According to the number of samples, 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. ① protein standards of 5mg / ml BSA, was completely dissolved protein standards, take 10 μ L was diluted to 100 μ L, final concentration of 0.5mg / ml, diluted with PBS standards. ② The standard press 0,1,2,4,8,12,16,20 μ l of each standard was added to 96-well plate wells, add Standard dilution solution (PBS) made up to 20 μ l, each The concentration was repeated three times. 3 After the above protein extract was diluted 10-fold with PBS, 20 μl of the protein dilution was added to the sample well of a 96-well plate, and each sample was repeated three times. 4 Add 200 μl BCA working solution to each well and let stand at 37 °C for 30 min. 5 The absorbance of the sample at a wavelength of 540 nm was measured with a microplate reader. 6 Calculate the protein concentration in the sample based on the standard curve.

3.3 Western blot analysis

Preparation of 1SDS-polyacrylamide gel (SDS-PAGE): Assembly of made glass plates. Prepare the separation glue, mix well, immediately inject into the gap of the glue 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 to 60 minutes, and separate the gel to solidify and pour off the deionized water. Soak the water on the surface of the separation gel as much as possible. Prepare 5% concentrated glue, mix well, immediately inject into the gap of the glass plate, insert the comb, avoid mixing bubbles, place and polymerize at room temperature for about 60min. After the concentrated gel is polymerized, the comb is removed, and the comb hole is rinsed with deionized water, directly electrophoresed or placed in a refrigerator at 4 ° C for use.

2 Electrophoresis: The proteins in the samples were separated by electrophoresis on 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 the electrophoresis tank electrophoresis buffer added to 1 × per well were added to 30 μ g total protein. 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.

3 Transfer: Transfer the protein to the PVDF membrane in transfusion buffer. The PVDF membrane was activated, and the cut membrane was sequentially immersed in 100% methanol (10 s) → deionized water (5 min) → transfer buffer (greater than 10 min), while the filter paper and sponge pad were immersed in the transfer buffer (greater than 10 min). The concentrated gel that ends the electrophoresis is removed. Install the film transfer device, from the positive (red) to the negative (black) to white side box → porous gasket → (2 sheets) filter paper → PVDF membrane → gel → (2 sheets) filter paper → porous gasket → black side box , put the shackle into the film transfer slot. 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.

4 blocking and immune reaction: the membrane was placed in a blocking solution (5% skimmed milk powder was placed in PBS buffer), shaken at room temperature for 2 h, and the primary antibody was prepared with blocking solution (SYP, GAP-43, PSD-95, β- Actin), placed 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.

5 Development: The bands obtained by the immunoreaction were displayed with ECL hypersensitive luminescent liquid. After development, use the primary anti-secondary anti-dead solution to regenerate the membrane, re-close, and add Primary antibody, secondary antibody, development.

6 Image scanning and quantitative analysis: X-rays were scanned in grayscale, and analyzed by Quantity One 4.6.2 image analysis software to quantify the target protein in the sample. Β-actin was used as an internal reference to determine differences and changes in target protein expression between groups.

1 biochemical indicator test

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 was removed, the filter paper was dried, and after weighing, the brain tissue was quickly immersed in cold physiological saline (salt quality) For the brain tissue quality 9 times), 10% brain homogenate was prepared in an ultrasonic cell pulverizer, centrifuged at 3000 to 4000 r/min for 20 min, the supernatant was taken, and biochemical detection was performed according to the kit instructions.

2 histopathological observation 5.1 taking materials

At the end of the mouse behavioral experiment, 3.5% 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. Insert the perfusion needle into the left ventricle from the apex, while the right atrial ear cuts a small opening, first perfusion with about 200-300ml saline, until the liquid flowing out of the right atrial appendage becomes colorless and clear, when the tail is bloodless, use 300-400ml 4 °C 4% paraformaldehyde buffer (0.1mol / 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 μm thick, 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 1mm3 hippocampal CA1 area tissue was fixed, 2.5% glutaraldehyde was fixed, and the nerve cells were observed by transmission electron microscope. microstructure.

5.2 Immunohistochemical staining

Immunohistochemical immunohistochemistry was performed using the streptomycin avidin-peroxidase (SP) ligation method. Principle: 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. After heating to boiling, after natural cooling, the distilled water was washed for 3 min×3; 3% H2O2 was incubated at 37 ° C for 20 min. Wash PBS for 3 min × 3; add goat serum blocking solution at 37 ° C for 30 min, aspirate excess serum, add primary antibody (BDNF, NGF, TrkA) to incubate overnight at 4 ° C, negative control with 0.01 M PBS buffer instead of primary antibody; Wash PBS for 5 min × 3; add biotinylated secondary antibody working solution (goat anti-rabbit IgG-HRP), incubate at 37 ° C for 15 min, wash with PBS for 5 min × 3; add horseradish enzyme labeled streptavidin working solution, 37 ° C Incubate for 15 min, wash with PBS for 5 min×3; DAB coloration, observe the staining intensity under microscope to control the reaction time, see brownish color appearing in the slice, rinse immediately with tap water, stop dyeing; hematoxylin counterstaining for 3 min; tap water washing; hydrochloric acid alcohol differentiation In a few seconds, the tap water is fully washed; the gradient alcohol is dehydrated, the xylene is transparent, and the neutral gum is sealed. The expression of positive protein in each field was observed by optical microscope. The integrated optical density (IOD) of positive protein neurons in each slice of each group was determined by computer image analysis system to reflect the intracellular neurons. The relative amount of positive protein expression.

5.3 HE staining

The 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, and 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.

6. Statistical methods

Experimental data in mean ± standard deviation ( ±SD) representation. Statistical analysis was performed using SPSS 17.0 statistical software. Differences between groups were compared by one-way or two-way analysis of variance (Morris water maze test) and Dunnett's t-test. p<0.05 was considered to be significant difference. III. Experimental results 1 Yangxue serum granules improved the learning and memory impairment in SAMP8 mice 1.1 Spontaneous activity experiment

The experimental results showed that there was no significant difference in the total distance of activity, total activity time and average speed of the mice in the spontaneous activity experiment, suggesting that the serum-free brain particles do not interfere with the follow-up behavior by affecting the excitability of the central nervous system. Experiment (see Table 5, Figure 16).

1.2 Y lab experiment

The experimental results showed that there was no significant difference between the total number of times the 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. Compared with the blank control group, the spontaneous alternation rate of the Y maze in the model group was significantly decreased. Compared with the model group, 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.

Compared with the blank control group, ###p<0.001; compared with the model group, ***p<0.001.

1.3 New object discrimination experiment

The experimental results showed that the model group mice were 1 h compared with the blank control group. And the 24h test period significantly reduced the priority index and discrimination coefficient of new objects; compared with the model group, the serum-sparing brain particles dose-dependently increased the priority index and discrimination coefficient of the new object in the 1h and 24h test phases of the model mice ( See Figure 19, Figure 20, Table 7, Table 8).

Compared with the blank control group, ###p<0.001; compared with the model group, ***p<0.001, **p<0.01, *p<0.05.

Compared with the blank control group, ###p<0.001; compared with the model group, ***p<0.001 or **p<0.01.

1.4 Morris water maze experiment 1.4.1 Directional Navigation Experiment

The experimental results showed that compared with the blank control group, the swimming time and distance of the model group mice reaching the platform in the water maze experiment were significantly prolonged; compared with the model group, the serum-sparing brain particles dose-dependently shortened the mouse arrival platform. Swimming time and distance (see Figure 21, Figure 22, Table 9, Table 10).

Note: A: blank control group B: model group C: donepezil hydrochloride group D: nourishing serum brain particle group

Compared with the blank control group, ###p<0.001; compared with the model group, ***p<0.001, **p<0.01, *p<0.05. .

Note: A: blank control group B: model group C: donepezil hydrochloride group D: nourishing serum brain particle group

Compared with the blank control group, ###p<0.001; compared with the model group, ***p<0.001, **p<0.01.

1.4.2 Space Exploration Experiment

The experimental results showed that compared with the blank control group, the swimming time and the fourth quadrant distance of the model group mice in the quadrant of the original platform (fourth quadrant) decreased significantly; compared with the model group, the serum brain particles 3740, 1870mg / The kg dose group significantly prolonged the fourth swimming time and the fourth quadrant distance of the mice (see Figure 24, Figure 25, Table 11), and the space exploration experimental trajectory map of each group of mice (see Figure 23).

Compared with the blank control group, ###p<0.001; compared with the model group, ***p<0.001, *p<0.05.

1.5 avoiding dark test results

The experimental results show that the model group test phase is compared with the blank control group. The latency was significantly shortened and the number of errors increased significantly; compared with the model group, the serum-sparing brain particles dose-dependently prolonged the latency and reduced the number of errors. See Table 12.

Compared with the blank control group, ###p<0.001; compared with the model group, ***p<0.001 or *p<0.05.

2 HE staining to observe the pathological changes of hippocampal neurons

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. In the model group, the neurons in the mice were loosely arranged and the chromatin was reduced. The neurons in the 1870mg/kg group, 3740mg/kg group and the donepe group hydrochloride group were closely arranged, and no edema and nuclear pyknosis were observed (see Figure 26).

3 Electron microscopic observation of neuronal and synaptic ultrastructural changes in hippocampal CA1 region 3.1 Electron microscopic observation of neuronal cell ultrastructure

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 is normal; the nucleus of neurons in the model group is irregular, Incision, see nucleolus, partial nucleus membrane, partial heterochromatin in the nucleus, slight expansion of the endoplasmic reticulum and Golgi complex in the cytoplasm, partial destruction of the mitochondrial outer membrane; The nuclear membrane is clear, the chromatin in the nucleus is evenly distributed, and the cytoplasm is rich in ribosomes, mitochondria, rough endoplasmic reticulum, Golgi complex and lysosome. The neuron nuclei in the 3740mg/kg group of serum brain particles are elliptical. The shape of the nuclear membrane is clear, the nucleolus is clear, the chromatin in the nucleus is evenly distributed, and the cytoplasm is rich in ribosomes, rough endoplasmic reticulum, mitochondria, Golgi complex and lysosomes, but in the rough surface. The reticular mass has edema, and the mitochondrial outer membrane is damaged. The neuron cell line of the 1870mg/kg serum brain granule is clear, some heterochromatin is condensed in the nucleus, and the cytoplasm is rich in ribosomes and mitochondria. Partially mild expansion; the neuron cell nucleus membrane in the 935mg/kg group of serum-raising brain granules is blurred, and there are some edge sets in the nuclear heterochromatin. There are ribosome, rough endoplasmic reticulum, mitochondria, lysosome and Golgi complex in the cytoplasm. Body cell , Or outer membrane of mitochondria ridges reduce breakage, rough endoplasmic reticulum (see Figure 27).

3.2 Electron microscopic observation of neuronal synaptic ultrastructure in hippocampal CA1 region

Multiple synapses were observed in the blank control group, and the structure was normal. Clear presynaptic membrane, synaptic space and postsynaptic membrane were visible. More synaptic vesicles were seen in the anterior membrane; multiple synaptic membrane fusions in the model group. The gap disappeared, part of the posterior membrane thickened, a small amount of synaptic cleft was observed; the synapse of the Donaiqiqi group was almost normal, and there were clear synaptic membranes before and after, there were more synaptic vesicles in the anterior membrane; serum brain particles 3740mg The synapse in the /kg group was almost normal, and there were clear anterior and posterior membranes. There were more synaptic vesicles in the anterior membrane, and some of the anterior and posterior membranes were partially fused. The serum of the brain granules in the 1870 mg/kg group showed more post-synaptic membrane compacts. Thick, synaptic gap The width and width are different, and some have been fused; the serum cerebral granules in the 935mg/kg group have partial synaptic membrane fusion or synaptic gap narrowing (see Figure 28).

4 neurotrophic related protein expression Effect of 4.1 Yangxue Qingnao Granule on Expression of BDNF in Cerebral Cortex and Hippocampus of SAMP8 Mice

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, and the Danaqiqi group and the serum cerebral granules 1870 mg/kg group and Neuronal coloration in the 3740 mg/kg group was significantly deeper than in the model group (see Figures 29 to 33, Table 13).

Compared with the blank control group, ###p<0.001; compared with the model group, ***p<0.001, **p<0.01, *p<0.05.

Effects of nourishing serum brain granules on the expression of NGF in cerebral cortex and hippocampus of SAMP8 mice

Nerve growth factor (NGF) 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 mice in the blank control group had darker cytoplasmic staining in the hippocampal CA1 area and cortical neurons. The coloration of the neurons in the model group was significantly shallower in the cytoplasm of the mice, and the Danaqi group and the serum brain particles were 1870 mg/kg. The coloration of neurons in the 3740 mg/kg group was significantly deeper than that in the model group (see Figure 34 to Figure 38, Table 14).

Compared with the blank control group, ##p<0.01, ###p<0.001; compared with the model group, ***p<0.001, **p<0.01, *p<0.05.

4.3Effect of Yangxue Qingnao Granule on the Expression of TrkA Receptor in SAMP8 Mice Brain

TrkA is a transmembrane protein with a molecular weight of 140 kD. When 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 cytoplasm of the control group in the hippocampal CA1 area and cerebral cortex was darker in the cytoplasm of the hippocampus. The coloration of the neurons in the model group was significantly shallower. The Donaiqiqi group and the serum granules were 1870mg/kg. The neuronal coloration of the group and the 3740 mg/kg group was significantly deeper than that of the model group (see Fig. 39 to Fig. 43, Table 15).

Compared with the blank control group, ###p<0.001, ##p<0.01, #p<0.05; compared with the model group, ***p<0.001, **p<0.01, *p<0.05.

Effect of 5 Yangxue Qingnao Granules on Antioxidant Capacity of SAMP8 Mice

The experimental results are shown in Table 16. Compared with the blank control group, the content of malondialdehyde (MDA) in the cerebral cortex of the model group was significantly increased, and the content of reduced glutathione (GSH) was significantly reduced. Antioxidant capacity (T-AOC) was significantly reduced, and the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-px) were significantly reduced. Compared with the model group, the serum-promoting brain particles were dose-dependently Increase T-AOC capacity and GSH content, increase SOD, GSH-px activity, and reduce MDA content.

Compared with the blank control group, ###p<0.001, ##p<0.01 or #p<0.05; compared with the model group, ***p<0.001, **p<0.01 or *p<0.05.

Effects of 6 Yangxue Qingnao Granules on the expression of synaptophysin, synaptophysin and postsynaptic compacts in cerebral cortex and hippocampus of SAMP8 mice

Synaptophysin, also known as synaptophysin (SYP), is a vesicle-adsorbing protein closely related to synaptic structure and function. It regulates neurotransmitter release and participates in triggering by its phosphorylation and dephosphorylation. Thus, it plays a role in synaptic plasticity and 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 (PSD) refers to a semi-circular band in the cytoplasmic surface of the postsynaptic membrane, which is very sensitive to synaptic sites. The susceptibility structure is the structural basis for information transmission between neurons and neurons, and plays an important role in synaptic plasticity. PSD-95, also known as SAP90, is the first to be identified as rich in PSD. The skeletal protein, a member of the superfamily of membrane-associated guanylate kinase proteins. GAP-43 (Growth associated protein-43) is a axonal membrane protein, a neuron-specific protein involved in extracellular growth and triggering and neuronal regeneration. Western blot results showed that compared with the blank control group, the expression of synaptophysin (SYP) and postsynaptic density (PSD-95) in the hippocampus and cerebral cortex of the model group was significantly reduced; compared with the model group, The expressions of SYP and PSD-95 in the hippocampus and cerebral cortex tissues of Naiqiqi group and Yangxuenao Granules were significantly increased, and there was no significant difference in GAP-43 expression between the groups (see Figure 44 to Figure 49). Table 17, Table 18).

Compared with the blank control group, ###p<0.01; compared with the model group, **p<0.01, *p<0.05.

Compared with the blank control group, ###p<0.01 or ###p<0.001; compared with the model group, ***p<0.001, **p<0.01, *p<0.05.

Four summary

1) Y-maze, new object discrimination, Morris water maze and avoidance of darkness and other behavioral experiments showed that 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 that of donepezil hydrochloride.

2) Yangxuenao Granule can increase the expression of brain-derived nerve growth factor (BDNF), nerve growth factor (NGF) and its receptor TrkA in cerebral cortex and hippocampus of SAMP8 mice, and improve the ultrastructure of hippocampal neurons. .

3) Yangxue brain granules can increase the total antioxidant capacity and GSH content of cerebral cortex in SAMP8 mice in a dose-dependent manner, and improve SOD and GSH-px Activity, reducing MDA content.

4) 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.

Part II Effect and mechanism of Yangxuenao Granule on learning and memory impairment induced by quinoline acid in dementia rats with Meyert basal ganglia in basal forebrain

In this experiment, 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). 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. Synaptophysin (SYP), synaptic growth associated protein 43 (GAP-43), postsynaptic density 95 (PSD-95) and hippocampus scorpion acetylcholine in hippocampus and cerebral cortex of rats with NBM-induced dementia The effect of receptor (CHRM1) expression; enzyme-linked immunosorbent assay (ELISA) was used to investigate the content of acetylcholine (Ach) in hippocampus and cerebral cortex, the content and activity of hippocampal acetylcholine transferase (ChAT) and the content of CHRM1 in cerebral cortex. Impact.

The results showed that Yangxue Qingnao Granule could increase the rate of spontaneous alternation of NBM-induced dementia rats in a dose-dependent manner in the dose range of 647-2588 mg/kg, and increase the priority index of new objects in the new object discrimination experiment. And discriminating the coefficient, shortening the time and distance of the dementia model rats reaching the safety station in the water maze experiment, and prolonging the large dementia model in the space exploration experiment. The number of swimming time and distance of the mouse in the quadrant of the original platform reduced the number of electric shocks in the dark test. The above behavioral results indicate that 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. The role of high-dose group and hydrochloric acid Donna 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. The content of Ach in hip and hippocampus and the content of ChAT in hippocampus were increased, but there was no significant effect on ChAT activity and CHRM1 expression in hippocampus. In summary, 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.

First, the experimental materials 1. Experimental animals

Healthy Sprague-Dawley (SD) rats, 120 male and female, weighing 180-220g, purchased from Liaoning Changsheng Biotechnology Co., Ltd. Certificate No.: scxk2010-0001; raised at SPF Experimental Animal Center of Shenyang Pharmaceutical University, Free drinking water, 12h cycle of light.

2, drugs and preparation

Yangxue brain particles: extract, supplied by Tianjin Tianshili Pharmaceutical Group Co., Ltd., batch No. 20120502W, stored at 4 °C-8 °C. Daily service The crude drug amount is 45 g, and each 1 g of the extract corresponds to 6.26 g of the medicinal material, and the clinical equivalent amount converted into a rat is 647 mg (extract)/kg. Preparation: Precisely weigh the serum brain granule extract, add distilled water, and prepare a solution of 64.7mg/ml, 129.4mg/ml and 258.8mg/ml respectively.

Donovan 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. Preparation: Take donate tablets of hydrochloric acid, add distilled water, and prepare a solution of 0.095 mg / ml.

3, experimental reagents

Quinolinic acid: SIGMA company, batch number: STBB0947V; chloral hydrate: Tianjin Ruijinte Chemical Co., Ltd., batch number: 20101124; fire cotton glue: Tianjin Hengxing Chemical Reagent Manufacturing Co., Ltd., batch number: 20110421; sodium chloride: Tianjin Bodi Chemical Co., Ltd., batch number: 20130221; disodium hydrogen phosphate: Shantou Xiqiao Chemical Co., Ltd., batch number: 110802; sodium dihydrogen phosphate: Shantou Xiqiao Chemical Co., Ltd., batch number: 110223; absolute ethanol :Tianjin Hengxing Chemical Reagent Manufacturing Co., Ltd., batch number: 20130327; paraformaldehyde: Tianjin Bodi Chemical Co., Ltd., batch number: 20121028; glutaraldehyde: Tianjin Bodi Chemical Co., Ltd., batch number: 20110906; Toluene: Tianjin Bodi Chemical Co., Ltd., batch number: 20121205; Citric acid: Tianjin Bodi Chemical Co., Ltd., batch number: 20100321; sodium citrate: Tianjin Bodi Chemical Co., Ltd., batch number: 20100719; hydrochloric acid: Shenyang Economic and Technological Development Zone Reagent Factory, batch number: 20120304; Sinopharm Chemical Reagent Co., Ltd., batch number: 20081215; neutral gum: China Shanghai specimen model factory, batch number: 20101201; slice paraffin: Shanghai Hualing Rehabilitation Equipment Factory, batch number: 20120206; poly-lysine: Beijing Zhongshan Jinqiao biotechnology Co., Ltd., batch number: 050M4339; Methanol: Tianjin Fuyu Fine Chemical Co., Ltd., batch number: 20130306; BCA protein quantification kit: Biyuntian Biotechnology Research Institute; Cell lysate: Biyuntian Biotechnology Research Institute; PMSF: Biyuntian Biotechnology Research Institute; PVDF membrane: Pall, USA, LOT: K1BA3015GK; developer powder, fixing powder: Biyuntian Biotechnology Research Institute, product number: P0019-4; mouse anti-SYP (D-4): Santa Cruz Biotechnology, product No.: sc-17750; mouse anti-β-actin: Santa Cruz Biotechnology, product number: sc-41478; mouse anti-G AP-43: Santa Cruz Biotechnology, product number: sc-17790; rabbit anti-PSD-95: Abcam (Hong Kong) Ltd LOT: GR76077-1; rabbit anti-CHRM1: Dr. Biotech Co., Ltd., product number: BA1544; Rat Acetylcholine Enzyme-Linked Immunoassay Kit: Shanghai Huole Biotechnology Co., Ltd., LOT: 20130 1,4-8 ° C; Rat cholestyramine transferase enzyme-linked immunosorbent assay kit: Shanghai Huo Le Biotechnology Co., Ltd., LOT: 201303, 4-8 ° C preservation; rat cholestyramine transferase activity enzyme-linked immunoassay kit: Shanghai Live Music Biotechnology Co., Ltd., LOT: 20130 1,4-8 ° C preservation; large Mouse scorpion acetylcholine M1 enzyme-linked immunoassay kit: Shanghai Live Music Biotechnology Co., Ltd., LOT: 201304, 4-8 ° C preservation; BCA protein concentration determination kit: Biyuntian Biotechnology Research Institute; protein 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; Methylene bis acrylamide: BIOSHARP Amresco, Exp: 2014/08; Horseradish-labeled goat anti-mouse IgG: Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., batch number: 107724; Horseradish enzyme Goat anti-rabbit IgG: Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., batch number: 101964; skim milk powder: Yili, batch number: 66196131T; high sensitivity chemiluminescence detection kit: Beijing Kangwei Century Biotechnology Co., Ltd., LOT: 22912; Western one Anti-secondary antibody removal solution: Beyotime P0025; medical X-ray film: Fujifilm Co., Ltd., EXP: 2015-07.

4 experimental instruments

Rat brain stereotaxic instrument: NARISHIGE SR-5N, Tokyo, Japan; spontaneous activity video analysis system: Shanghai Jiliang Software Technology Co., Ltd.; Morris water maze and video acquisition and analysis system: Institute of Materia Medica, China Academy of Medical Sciences; Behavioral Device: Rat Y labyrinth, new object identification, darkening device are all made by Shenyang Pharmaceutical University; paraffin slicer: Leica, Germany.

Optical microscope: OLYMPUS company; Image-pro6.3 image analysis system: OLYMPUS company; ultrasonic cleaner: KQ5200B, Kunshan Ultrasonic Instrument Co., Ltd.; ultrasonic cell pulverizer: JY92-II, Ningbo Xinzhi Biotechnology Co., Ltd.; TECAN enzyme quantitative tester: SPECTRA CLASSIC; -80 °C low temperature refrigerator: Zhongke Meiling; one ten thousand balance: Sartorius Scientific Instruments (Beijing) Co., Ltd.; electronic balance: Changzhou Shuangjie Test Instrument Factory; constant temperature water bath Device: BCFCO Changfeng Electronic Technology Co., Ltd.; H-1 Micro Whirlpool Mixer: Shanghai Jingke Industrial Co., Ltd.; Electrothermal Constant Temperature Incubator: DNP-9272, Shanghai Precision Experimental Equipment Co., Ltd.; Vertical Automatic Electrothermal Pressure Steam Sterilizer: LDZX-40AI, Shanghai Shenan Medical Instrument Factory; High Speed Refrigerated Centrifuge: HC-3018R, HKUST Innovation Co., Ltd.;

Second, the experimental method 1. Establishment of a rat model of NBM nuclear in the bilateral basal forebrain damaged by quinolinic acid

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 μl (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.

2. Grouping and administration

SD rats were randomly divided into sham operation group (negative control group), model group, Donaiqi hydrochloride hydrochloride 0.95 mg/kg group, Qingnao Yangxue granules 647 mg/kg group (human clinical equivalent), 1294 mg/kg Group (2 times human clinical equivalent), 2588 mg/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.

3. Behavioral experimental methods 3.1 Rat Y Maze Experiment

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. At the time of the experiment, 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 Different arms are a successful alternation, recording the correct number of alternating reactions (number of Alternation)N. The spontaneous alternating reaction rate Alternation behavior (%) = number of alternation / (N-2) × 100 was used.

3.2 Rat new object discrimination experiment

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. After 1 h, one of the objects was replaced with a new object (B), and the rats were placed again, and the time (tA1, tB) used to explore the two objects was recorded separately. After 24 hours, the object B was replaced with a new object (C), and the rats were placed again, and the time (tA1, tC) for exploring the two objects was recorded separately. Calculate the priority index and discrimination coefficient for new objects.

The formula for calculating the priority index is as follows: priority index (1h) = tB / (tA1 + tB) formula (1)

Priority index (24h) = tC / (tA1 + tC) formula (2)

The formula for determining the coefficient is as follows: discrimination coefficient (1h) = (tB-tA1/(tA1+tB) formula (3)

Discrimination coefficient (24h) = (tC-tA1/(tA1+tC) formula (4)

3.3 Rat spontaneous activity experiment

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 four rectangular activity boxes (45×45×13cm), built-in infrared probe and camera, spontaneous activity video analysis system 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.

3.4 Rat Morris water maze experiment

The rat Morris water maze experiment was designed to investigate the effects of test drugs on spatial learning and memory in rats.

(1) 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.

(2) Space exploration experiment: 24 hours after the end of the navigation test, the platform was removed, and the rats were placed in the water at the midpoint of the opposite side of the original platform, and swimming for 90 seconds. The water maze system automatically records the time and distance of the rats in the original platform quadrant.

3.5 Rat passive avoidance reaction experiment (dark test)

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 law Using the darkness habits of the rats, the rats were placed in the bright room with the back to the door, and after free movement for 3 minutes, the animals were allowed to enter the dark room, and the door was closed and energized. After 5 seconds of shocking the rat, 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.

4 Western blotting method 4.1 Tissue protein extraction

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 μl of PMSF was added per 1 ml of the protein lysate. After homogenization with an ultrasonic cell pulverizer, it was allowed to stand in an ice bath for 30 min. 14000 × g, centrifuged at 4 ° C for 20 min, the supernatant was taken, dispensed, and stored in a refrigerator at -80 ° C for use. A small portion of the supernatant was taken for protein quantification.

4.2 Determination of protein concentration - BCA method

According to the number of samples, 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. ① protein standards of 5mg / ml BSA, was completely dissolved protein standards, take 20 μ L was diluted to 100 μ L, final concentration of 1mg / ml. The standard was diluted with PBS. ② The standard press 0,1,2,4,8,12,16,20 μ l of each standard was added to 96-well plate wells, add Standard dilution solution (PBS) made up to 20 μ l, each The concentration was repeated three times. 3 After the above protein extract was diluted 10-fold with PBS, 20 μl of the protein dilution was added to the sample well of a 96-well plate, and each sample was repeated three times. 4 Add 200 μl BCA working solution to each well and let stand at 37 °C for 30 min. 5 The absorbance of the sample at a wavelength of 540 nm was measured with a microplate reader. 6 Calculate the protein concentration in the sample based on the standard curve.

4.3 Western blot analysis

Preparation of 1SDS-polyacrylamide gel (SDS-PAGE): Assembly of made 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 it with deionized water, polymerize at room temperature for about 30-60min, and separate it. Ionized water, as much as possible to absorb the water on the surface of the separation gel. Prepare 5% concentrated glue, mix well, immediately inject into the gap of the glass plate, insert the comb, avoid mixing bubbles, place and polymerize at room temperature for about 60min. After the concentrated gel is polymerized, the comb is removed, and the comb hole is rinsed with deionized water, directly electrophoresed or placed in a refrigerator at 4 ° C for use.

2 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. Were added to each well 30 μ g total protein. 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.

3 Transfer: Transfer the protein to the PVDF membrane in transfusion buffer. The PVDF membrane was activated, and the cut membrane was sequentially immersed in 100% methanol (10 s) → deionized water (5 min) → transfer buffer (greater than 10 min). At the same time, the filter paper and sponge pad were immersed in the transfer buffer (greater than 10 min). will The concentrated gel that ends the electrophoresis is removed. Install the film transfer device from positive (red) to negative (black) → white side box → porous gasket → (2 sheets) filter paper → PVDF membrane → gel → (2 sheets) filter paper → porous gasket → black edge The buckle on the buckle is placed in the transfer film slot. 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.

4 Blocking and immune reaction: 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.

5 Development: The bands obtained by the immunoreaction were displayed with ECL hypersensitive luminescent liquid. After development, the membrane is regenerated with a primary anti-secondary solution removal solution, re-blocked, primary antibody, secondary antibody, and developed.

6 Image scanning and quantitative analysis: X-rays were scanned in grayscale, and analyzed by Quantity One 4.6.2 image analysis software to quantify the target protein in the sample. Β-actin was used as an internal reference to determine differences and changes in target protein expression between groups.

5 histopathological observation 5.1 taking materials

At the end of the rat behavioral experiment, 3.5% 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. Insert the perfusion needle into the left ventricle from the apex, while the right atrial ear cuts a small opening, first perfusion with about 200-300ml saline, until the liquid flowing out of the right atrial appendage becomes colorless and clear, when the tail is bloodless, use 300-400ml 4 °C 4% paraformaldehyde buffer (0.1mol / 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 μm 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.

5.2 HE staining

The 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, and 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.

6 statistical methods

Experimental data in mean ± standard deviation ( ±SD) representation. Statistical analysis was performed using SPSS 17.0 statistical software. Differences between groups were compared by one-way or two-way analysis of variance (Morris water maze test) and Dunnett's t-test. P < 0.05 was considered to have a significant difference in the difference.

Third, the experimental results 1 behavioral test results 1.1 spontaneous activity experiment

The experimental results showed that there was no significant difference in the total activity distance, total activity time and average speed of the rats in the spontaneous activity experiment, suggesting that the serum-free brain particles do not interfere with the follow-up behavior by affecting the excitability of the central nervous system. Experiment (see Table 19). The spontaneous activity trajectory of each group of rats is shown in Fig. 50.

1.2 Y lab experiment

The experimental results showed that there was no significant difference between the total number of times the rats in each group entered the three arms of the Y maze (see Figure 51 and Table 20), suggesting that the serum-free brain particles did not significantly affect the spontaneous activity of the rats. Compared with the sham operation group, the spontaneous alternation rate of the model group was significantly decreased. Compared with the model group, the serum-sparing brain particles dose-dependently increased the spontaneous alternation rate of the rat Y maze, and the polydamine hydrochloride group also Significantly increased the rate of spontaneous alternation (see Figure 52, Table 20), suggesting that serum brain particles can significantly improve the working memory impairment in dementia rats.

Table 20 In the Y labyrinth experiment, the serum of the brain granules destroyed the NBM core by quinoline acid

Compared with the sham operation group, ###p<0.01; compared with the model group, *p<0.05, **p<0.01.

1.3 New object discrimination experiment

The experimental results showed that compared with the sham operation group, the 1h and 24h priority index and discrimination coefficient of the model group were significantly lower. Compared with the model group, the serum-enriched brain particles dose-dependently increased the 1h and 24h priority index and discrimination coefficient. The doxorubicin hydrochloride group also significantly increased the priority index and the discrimination coefficient (see Figure 53, Figure 54, Table 21, Table 22).

Compared with the sham operation group, ###p<0.01; compared with the model group, *p<0.05, **p<0.01, ***p<0.001.

Compared with the sham operation group, ###p<0.001; compared with the model group, *p<0.05, **p<0.01, ***p<0.001.

1.4 Morris water maze experiment (1) Directional navigation experiment

The experimental results showed that compared with the sham operation group, the swimming time and distance of the model group rats reaching the platform in the water maze experiment were significantly prolonged; compared with the model group, the serum-sparing brain particles dose-dependently shortened the rat arrival platform. The swimming time and distance, the Donaiqi hydrochloride group also significantly shortened the swimming time and distance (see Figure 55, Figure 56, Table 23, Table 24).

Note: A: blank control group B: model group C: donepezil hydrochloride group D: nourishing serum brain particle group

Compared with the sham operation group, ###p<0.001; compared with the model group, *p<0.05, **p<0.01, ***p<0.001.

Note: A: blank control group B: model group C: donepezil hydrochloride group D: nourishing serum brain particle group

Compared with the sham operation group, ###p<0.001; compared with the model group, *p<0.05, **p<0.01, ***p<0.001.

(2) Space exploration experiment

The experimental results showed that compared with the sham operation group, the swimming time and the fourth quadrant distance of the model group rats in the quadrant of the original platform (fourth quadrant) decreased significantly; compared with the model group, the serum brain particles 2588, 1294mg / The kg dose group significantly prolonged the fourth quadrant swimming time and the fourth quadrant distance percentage in rats. The Donaiqi hydrochloride group also significantly prolonged the fourth quadrant swimming time and the percentage of travel (see Figure 58, Figure 59, Table 25). The trajectory map of the space exploration experiment of the group rats is shown in Fig.

1.5 passive avoidance reaction experiment

The experimental results showed that compared with the sham operation group, the number of electric shocks in the model group was significantly increased. Compared with the model group, the serum-sparing brain particles dose-dependently reduced the number of electric shocks, and the polydamine hydrochloride group also significantly reduced the number of shocks. The number of electric shocks (Figure 60, Table 26).

Compared with the sham operation group, ###p<0.001; compared with the model group, **p<0.01, ***p<0.001.

Compared with the sham operation group, ###p<0.001; compared with the model group, **p<0.01, ***p<0.001.

2 HE staining to observe the pathological changes of hippocampal neurons

The results showed that the neurons in the hippocampal CA1 region of the sham-operated group had clear and tightly arranged cells. In the model group, the neurons were loosely arranged, edematous, with reduced chromatin and nuclear pyknosis. The hippocampal neurons in the 2588mg/kg group and the Danazepine hydrochloride group were tightly arranged, no edema and nuclear pyknosis were observed, and the neurons in the 1294mg/kg group were more tightly arranged and slightly edema. And nuclear pyknosis (see Figure 61).

3 Electron microscopic observation of neuronal and synaptic ultrastructural changes in hippocampal CA1 region 3.1 Electron microscopic observation of neuronal cell ultrastructure

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, the heterochromatin in the nucleus has obvious agglomeration, the edge set, the cytoplasm is partially dissolved, and the remaining mitochondrial outer membrane is blurred; the Donaiqiqi group The neuron nucleus of the 2588mg/kg serum group was nearly round, the chromatin distribution was uniform, the nuclear membrane was clear, and the cytoplasm was rich in ribosome, mitochondria, rough endoplasmic reticulum and lysosome. The neuron nucleus is round, the nuclear membrane and nucleolus are clear, the chromatin in the nucleus is evenly distributed, the cytoplasm has more ribosomes, the rough endoplasmic reticulum expands more, and the mitochondrial ridge Or the outer membrane part is missing, see also lysosome and other organelles; the serum cerebral granules 647mg/kg group neuron nuclei are nearly round, the nuclear chromatin distribution is uniform, there are more ribose in the cytoplasm Organelles such as body, mitochondria, lysosomes, and slightly dilated rough endoplasmic reticulum (see Figure 62).

3.2 Electron microscopy observation of neuronal synaptic ultrastructure

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. Membrane and interstitial; Danaqiqi group and Yangxue brain granules 2588mg/kg group axis-dendritic pre-suppression membrane, posterior membrane clear, clear structure, vesicles in the anterior membrane, dense posterior membrane dense; serum brain Part of the presynaptic membrane and posterior membrane were fused in the 1294mg/kg group. The synaptic gap was wide and narrow, and some synapses were close to normal. The presynaptic and posterior membranes and gaps were obvious. There are more synaptic vesicles in the presynaptic membrane, and the thickness of the dense substance in the synaptic membrane is relatively uniform; the serum of the brain granules in the 647 mg/kg group is fused before and after the membrane, and the post-membrane deep-staining substance is less, the number of synapses Less (see Figure 63).

4 synapse-associated protein expression Effects of 4.1 Yangxue Qingnao Granule on the Expression of SYP, PSD-95 and GAP-43 in Hippocampus of NBM-induced Dementia Rats

Western blot results showed that compared with the sham operation group, the expression of SYP, PSD-95 and GAP-43 in the hippocampus of the model group was significantly reduced; compared with the model group, the serum-sparing brain particles dose-dependently increased in the hippocampus The expression of SYP and PSD-95 had no significant effect on the expression of GAP-43. The doxorubicin hydrochloride group significantly increased the expression of these synaptic-related proteins (see Figure 64, Figure 65, Figure 66, Table 27).

Compared with the sham operation group, #p<0.05, ##p<0.01; compared with the model group, *p<0.05, **p<0.01

Effects of nourishing serum brain granules on the expression of SYP, PSD-95 and GAP-43 in cerebral cortex of rats with dementia induced by quinolinic acid

The results of Western blot showed that compared with the sham operation group, the expression of SYP and PSD-95 in the cerebral cortex of the model group was significantly reduced. Compared with the model group, the Danazepine hydrochloride group and the serum granule 2588 mg/kg group were compared. The expression of SYP in cerebral cortex tissue was significantly increased. The expression of PSD-95 was significantly increased in the group of Dovezin hydrochloride, 2588mg/kg and 1294mg/kg. The expression of GAP-43 was not significant in each group. Differences (see Figure 67, Figure 68, Figure 69, and Table 28).

Table 28 Effect of Yangxuenao Granule on the expression of synaptophysin (SYP), postsynaptic compact (PSD-95) and synaptic growth related protein (GAP-43) in cerebral cortex of NBM-induced dementia rats (n=3, ±SD).

Compared with the sham operation group, ##p<0.01,###p<0.001; compared with the model group, *p<0.05, **p<0.01

5 central cholinergic nervous system function test Effects of 5.1 Yangxue Qingnao Granule on Ach Content in Cerebral Cortex and Hippocampus of NAD-induced Dementia Rats

Acetylcholine (Ach) is the earliest discovered neurotransmitter closely related to learning and memory. Choline acetyltransferase (ChAT) catalyzes the binding of acetylcholine of acetaminophen coenzyme A to choline in the cytosol to form Ach. After Ach synthesis, it is transported into the vesicles by the synaptic vesicle carrier. When a signal is 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). 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-enriched brain particles dose-dependently increased the expression of Ach, and the polydamine hydrochloride The group also significantly increased Ach expression (see Figure 70, Figure 71, and Table 29).

Table 29 Yangxuenao Granules on Quinoline Acid Destroyed NBM Nuclear Dementia Rat Hippocampus

Compared with the sham operation group, #p<0.05, ###p<0.001; *p<0.05, ***p<0.001 compared with the model group.

Effects of 5.2 Yangxue Qingnao Granule on ChAT Content and Activity in Hippocampus of Rats with Dementia Induced by Quinolinic Acid

The ELIA results showed that compared with the sham operation group, the ChAT content in the hippocampus of the model group was significantly reduced. Compared with the model group, the serum-induced brain granules increased the content of ChAT in a dose-dependent manner, and the donepez hydrochloride group was also significant. The expression was increased; the activity of ChAT was not significantly different between the groups (see Figure 72, Figure 73 and Table 30).

Compared with the sham operation group, ###p<0.001; compared with the model group, **p<0.01, ***p<0.001.

Effects of 5.3 Yangxue Qingnao Granule on the Expression of CHRM1 in Cerebral Cortex and Hippocampus of NBM-induced Dementia Rats

The results of Western blot showed that there was no significant difference in the expression of CHRM1 between the hippocampus in the rats. The ELISA results showed that the expression of CHRM1 in the cerebral cortex of the model group was significantly reduced compared with the sham operation group. Compared with the 2588mg/kg group of Yangxuenao Granules and the Danazepa Hydrochloride group, the expression was significantly increased (see Figure 74, Figure 75 and Table 31, Table 32).

#p<0.05 compared to the sham operation group; *p<0.05 compared to the model group.

Fourth, summary

1) Y-maze, new object discrimination, Morris water maze and avoidance of darkness and other behavioral experiments showed that Yangxue brain particles can improve the quantification of NBM core-induced dementia in a dose-dependent manner in the dose range of 647-2588mg/kg. Non-spatial learning and memory impairments such as working memory impairment, spatial learning and memory impairment, and object discrimination ability are similar to those of high-dose group.

2) 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.

3) Yangxuenao Granule can significantly increase the expression of acetylcholine in cerebral cortex and hippocampus of NBM-induced dementia rats, and increase the expression of choline acetylcholine transferase and cerebral cortex M1 choline receptor in hippocampus .

Claims (10)

A traditional Chinese medicine composition for preparing a medicament for treating Alzheimer's disease, which is prepared from the following weight ratio drugs: Angelica 6.75%, Chuanxiong 6.75%, Chalk 5.4%, Uncaria 13.5 %, spatholobus 13.5%, Rehmannia glutinosa 5.4%, cassia seed 13.5%, Prunella vulgaris 13.5%, Asarum 1.34%, Corydalis 6.75% and Mother-of-pearl 13.5%. The application of claim 1, wherein the application is for removing senile plaques in the brain of a patient with Alzheimer's disease. The application of claim 2, wherein the application is capable of removing A β protein in the brain and reducing the production of A β protein in the brain. The application of claim 3, wherein the application is capable of inhibiting the expression level of the pathogenic γ-secretase presenilin PS1, inhibiting pathological shearing of APP, and promoting physiological alpha shearing of APP. The application of claim 1, wherein the application is such that the composition significantly improves memory cognitive ability. The application of claim 5, wherein the application is capable of improving expression of brain-derived nerve growth factor, nerve growth factor and its receptor TrkA in the cerebral cortex and hippocampus, and improving abnormalities of ultrastructure of hippocampal neurons . The application of claim 5, wherein the application is capable of improving expression of the cerebral cortex, hippocampal synaptophysin and postsynaptic compact, and improving abnormalities of the hippocampal synaptic structure. The application of claim 5, wherein the application is capable of increasing the total antioxidant capacity and GSH content of the cerebral cortex, increasing the activity of SOD, GSH-px, and reducing the MDA content. The application of claim 5, wherein the application is capable of increasing the expression of acetylcholine in the cerebral cortex and hippocampus, and increasing the expression of choline acetyltransferase and choline receptor in the cerebral cortex. The application of claim 1, wherein the composition is one selected from the group consisting of granules, pills, tablets, capsules, and oral solutions.