Application of Yangxue oral liquid in preparing neuroprotective drugs for Alzheimer's disease and / or Parkinson's disease

Abstract

The present invention provides the use of Yangxue Oral Liquid in the preparation of drugs for neuroprotection against Alzheimer's disease and / or Parkinson's disease, and belongs to the field of biopharmaceutical technology. The present invention provides the use of Yangxue Oral Liquid in the preparation of drugs for neuroprotection against Alzheimer's disease and / or Parkinson's disease. By verifying the effects of Yangxue Oral Liquid on the amyloid beta-induced neuronal apoptosis model, the cobalt chloride-induced SH‑SY5Y cell hypoxia model, and the LPS-induced SH‑SY5Y cell inflammation model, the results showed that Yangxue Oral Liquid has a neuroprotective effect against Alzheimer's disease and / or Parkinson's disease. Therefore, the application provided by the present invention provides a new means for neuroprotection against Alzheimer's disease and / or Parkinson's disease.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceuticals, and particularly relates to the use of a blood-nourishing oral liquid in the preparation of a neuroprotective drug for Alzheimer's disease and / or Parkinson's disease. Background Art

[0002] Alzheimer's disease (AD) and Parkinson's disease (PD) are two of the most common neurodegenerative diseases with a significant impact on the aging population worldwide. Alzheimer's disease is characterized by progressive memory loss, cognitive decline, and the accumulation of amyloid beta (Aβ) plaques and neurofibrillary tangles in the brain. On the other hand, Parkinson's disease is characterized by motor symptoms such as tremor, rigidity, and bradykinesia, as well as the loss of dopaminergic neurons in the substantia nigra. Although the etiology of AD and PD remains complex and multifactorial, emerging evidence suggests that hypoxia is detrimental to the brain and plays a crucial role in the etiology of these diseases. Hypoxia can be caused by multiple factors, including impaired blood flow, mitochondrial dysfunction, and oxidative stress, all of which have been implicated in the development and progression of AD and PD. Understanding the mechanisms of neuroprotection is crucial for the treatment of AD and PD and the development of new drugs.

[0003] The active ingredients of Yangxue Oral Liquid include alum, hawthorn, sea buckthorn, and jujube, which mainly play the role of iron supplementation, blood tonification, and qi invigoration, and also have antioxidant and anti-ischemic effects (CN102309572A). However, there are currently no reports on the neuroprotective effects of Yangxue Oral Liquid. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide a blood-nourishing oral liquid for use in the preparation of a neuroprotective drug for Alzheimer's disease and / or Parkinson's disease, wherein the blood-nourishing oral liquid has a protective effect on amyloid beta-induced nerve cell damage, cobalt chloride-induced SH-SY5Y cell hypoxia damage, and LPS-induced SH-SY5Y cell inflammatory damage.

[0005] The present invention provides an application of a blood-nourishing oral liquid in preparing a neuroprotective drug for Alzheimer's disease and / or Parkinson's disease.

[0006] Preferably, the neuroprotection for Alzheimer's disease and Parkinson's disease includes at least one of the following: reducing inflammatory damage, apoptosis, hypoxic damage and oxidative stress levels in nerve cells or neurons.

[0007] Preferably, the hypoxic injury to nerve cells includes hypoxic injury to nerve cells induced by cobalt chloride.

[0008] Preferably, the inflammatory damage to nerve cells includes inflammatory damage to nerve cells induced by lipopolysaccharide.

[0009] Preferably, the apoptosis of nerve cells includes apoptosis of nerve cells induced by amyloid β.

[0010] The present invention provides an application of a blood-nourishing oral liquid in the preparation of a nerve cell protective agent.

[0011] Preferably, the neural cells include SH-SY5Y cells.

[0012] Preferably, the neuron protective agent comprises at least one of the following: reducing inflammatory damage, apoptosis, hypoxic damage and oxidative stress levels in neuron cells.

[0013] Preferably, the hypoxic injury to the nerve cells comprises induction by cobalt chloride;

[0014] The induction concentration of the cobalt chloride is 750-850 μM.

[0015] Preferably, the inflammatory damage to the nerve cells includes induction by lipopolysaccharide; and the apoptosis of the nerve cells includes induction by amyloid β.

[0016] The present invention provides a use of Yangxue Oral Liquid in the preparation of a drug for neuroprotection against Alzheimer's disease and / or Parkinson's disease. The present invention verifies the effects of Yangxue Oral Liquid on an amyloid beta-induced neuronal apoptosis model, a cobalt chloride-induced SH-SY5Y cell hypoxia model, and an LPS-induced SH-SY5Y cell inflammation model. The results show that serum containing Yangxue Oral Liquid not only significantly improves SH-SY5Y cell viability, but also reduces oxidative stress levels in the hypoxic cell model. At the same time, Yangxue Oral Liquid has a protective effect against amyloid beta-induced SH-SY5Y cell apoptosis. In addition, Yangxue Oral Liquid has a protective effect against LPS-induced SH-SY5Y cell inflammatory damage. It can be seen that the application provided by the present invention provides a new means for neuroprotection against Alzheimer's disease and / or Parkinson's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The cell survival rate results of SH-SY5Y cells induced by different concentrations of cobalt chloride;

[0018] Figure 2 The results show the cell survival rate of SH-SY5Y cells treated with serum containing Yangxue Oral Liquid for 24 hours in the hypoxia model induced by 800μM cobalt chloride.

[0019] Figure 3 The results show the cell survival rate of SH-SY5Y cells treated with different concentrations of Yangxue oral liquid for 6 hours and then subjected to 800μM cobalt chloride-induced hypoxia model for 24 hours.

[0020] Figure 4 The effect of different concentrations of Yangxue oral liquid on ROS levels in SH-SY5Y cell hypoxia model induced by 800 μM cobalt chloride;

[0021] Figure 5 The cell survival rate of SH-SY5Y cells induced by LPS after being treated with different concentrations of Yangxue Oral Liquid for 24 hours;

[0022] Figure 6 This is the result of cell survival rate after SH-SY5Y cells were treated with serum containing Yangxue Oral Liquid for 24 hours under the action of amyloid protein β (Aβ1-42) induced stimulation. DETAILED DESCRIPTION

[0023] The present invention provides an application of a blood-nourishing oral liquid in preparing a neuroprotective drug for Alzheimer's disease and / or Parkinson's disease.

[0024] The present invention provides an application of a blood-nourishing oral liquid in the preparation of a nerve cell protective agent.

[0025] In the present invention, the active ingredients of the blood-nourishing oral liquid include alum, sea buckthorn, hawthorn and jujube. In the embodiment of the present invention, the blood-nourishing oral liquid is purchased from Hubei Phoenix Baiyunshan Pharmaceutical Co., Ltd.

[0026] In the present invention, the neuroprotection for Alzheimer's disease and Parkinson's disease preferably includes at least one of the following: reducing inflammatory damage, apoptosis, hypoxic damage and oxidative stress levels in nerve cells or neurons. Hypoxic damage to nerve cells preferably includes hypoxic damage to nerve cells induced by cobalt chloride. The induction concentration of cobalt chloride is 750-850 μM, more preferably 800 μM. Inflammatory damage to nerve cells preferably includes inflammatory damage to nerve cells induced by lipopolysaccharide. Apoptosis of nerve cells preferably includes apoptosis of nerve cells induced by amyloid beta. The amyloid beta is preferably Aβ1-42. The nerve cells preferably include SH-SY5Y cells.

[0027] In the present invention, SH-SY5Y cells induced by Aβ1-42 were used as research subjects to illustrate the neuroprotective effects of Yangxue Oral Liquid on Alzheimer's disease and Parkinson's disease. The prior art has disclosed that neurodegenerative diseases are associated with the misfolding and aggregation of specific polypeptides and neuronal loss (Chiti and Dobson, 2006). The accumulation of β-amyloid protein is considered to be the cause of the pathogenesis of Alzheimer's disease (AD) and the progressive increase in the disease. Protofibrillary tangles (NFTs) and amyloid plaques are the pathological hallmarks of this disease (Selkoe, 1997). Amyloid plaques are formed by Aβ, which is a 39-43 peptide fragment produced by the cleavage of β-amyloid protein precursor (APP) (Wilquet and De Strooper, 2004), and also from Aβ25-35 oligomers (Gruden et al., 2007). Among Aβ peptides, those that undergo extracellular release depend on specific cleavage sites, of which the 42-residue long form (Aβ42) is the most pathogenic (O'brien and Wong, 2011). Since Aβ is abnormally elevated in patients with familial AD, in vitro and in vivo studies have shown the neurotoxic effects of Aβ, and Aβ may play a central role in the pathogenesis of AD. Therefore, promoting the degradation and clearance of endogenous Aβ may be an effective strategy to reduce Aβ levels (Inhibition of tauphosphorylation and Aβaccumulation by S.cerevisiae-derived vacuoles in LPS-induced SH-SY5Y cells Journal of Biotechnology Vol. 376, No. 10, November 2023, pp. 45-52.). In addition, the prior art also reports that the SH-SY5Y neuronal cell line induced by β-amyloid protein (Aβ1-42) was used as a research object to illustrate the role of effective active ingredients in treating and improving Alzheimer's disease (AD) (The influence of Schisandrin B on a model of Alzheimer's disease using β-amyloid protein Aβ1-42-mediated damage in SH-SY5Y neuronal cell line and underlying mechanisms, 2017, Journal of Toxicology and Environmental Health Part A 80(22):1-7).

[0028] In the examples of the present invention, a neural apoptosis model was induced by Aβ1-42, and this was used as the experimental subject to carry out a neuroprotective effect verification experiment of Yangxue Oral Liquid. The results showed that the serum containing Yangxue Oral Liquid had a protective effect on SH-SY5Y cells stimulated by 2μM Aβ1-42 amyloid protein, showing a significant difference.

[0029] In the present invention, lipopolysaccharide-induced SH-SY5Y cells are used as a model of neuronal inflammation to illustrate the neuroprotective effect of Yangxue Oral Liquid on Alzheimer's disease and Parkinson's disease. The prior art discloses the differentiation of human neuroblastoma cells SH-SY5Y into DA neuron-like cells. Terminally differentiated neurons are exposed to lipopolysaccharide to simulate acute or chronic inflammation in order to screen for viable candidate biomarkers for acute and chronic neuroinflammation (Robust Dopaminergic Differentiation and Enhanced LPS-Induced Neuroinflammatory Response in Serum-Deprived Human SH-SY5Y Cells: Implication for Parkinson's Disease, Journal of Molecular Neuroscience, Volume 71, 13 August 2020, Nos. 565-582). In addition, the prior art also discloses the use of LPS-induced SH-SY5Y cells as research subjects to identify gene expression related to human neurogenesis (Evaluation of dihydrotestosterone and dihydroprogesterone levels and gene expression of genes involved in neurosteroidogenesis in the SH-SY5Y Alzheimer disease cell model Neuroscience 23 May 2023 Segment II neurodegenerative disease).

[0030] In the examples of the present invention, the neuroprotective effect of Yangxue Oral Liquid was tested in a model of neuronal inflammation induced by lipopolysaccharide (LPS). The results demonstrated that Yangxue Oral Liquid exhibited a dose-dependent protective effect against SH-SY5Y cell inflammation induced by 100 μg LPS. The concentration of Yangxue Oral Liquid was 0.5 mg / ml, more preferably 1-2 mg / ml.

[0031] In the present invention, SH-SY5Y cells induced by cobalt chloride are used as a neuronal hypoxia model to illustrate the neuroprotective effects of Yangxue Oral Liquid on Alzheimer's disease and Parkinson's disease. Hypoxia not only promotes the accumulation of Aβ and increases the expression of phosphorylated Tau, but also causes synaptic damage, which is an important pathogenic factor of AD; hypoxia promotes the modification, aggregation and propagation of α-Syn. Overexpression of α-syn will lead to impaired mitochondrial function, ultimately leading to decreased respiratory function and neuronal death, thereby driving the progression of PD ([1] Burtscher, J., et al., Hypoxia and brain aging: Neurodegeneration or neuroprotection? Ageing Res Rev, 2021. 68: p. 101343., [2] March-Diaz, R., et al., Hypoxia compromises the mitochondrial metabolism of Alzheimer's disease microglia via HIF1. Nat Aging, 2021. 1(4): p. 385-399., [3] Guo, M., X. Ji, and J. Liu, Hypoxia and Alpha-Synuclein: Inextricable Link Underlying the Pathologic Progression of Parkinson's Disease. Front Aging Neurosci, 2022.14:p.919343.). Cobalt chloride (hypoxia)-induced neural cell (SH-SY5Y) injury model (AD and PD) ([4] Deng Ting, Yu Zhijie, Xu Ying, Li Jinsong, Sun Tao. Research progress on the establishment and evaluation methods of AD cell models. Chinese Pharmacological Bulletin, 2020, 36(4):470-474. [5] Song MS, Ryu PD, Lee SY. Kv3.4 is modulated by HIF-1α to protect SH-SY5Y cells against oxidative stress-induced neural cell death. SciRep. 2017May 18; 7(1):2075. doi:10.1038 / s41598-017-02129-w. PMID:28522852; PMCID:PMC5437029.).

[0032] In the present embodiment, a neuronal hypoxia model induced by cobalt chloride was used as the experimental subject to conduct an experiment to verify the neuroprotective effect of Yangxue Oral Liquid. The results showed that serum containing Yangxue Oral Liquid significantly increased cell viability by 67%. In addition, serum containing Yangxue Oral Liquid reduced ROS levels in the hypoxia model. As can be seen, Yangxue Oral Liquid not only restored the damage to SH-SY5Y cells caused by cobalt chloride but also reduced ROS levels.

[0033] The application of the Yangxue oral solution provided by the present invention in the preparation of neuroprotective drugs for Alzheimer's disease and / or Parkinson's disease will be described in detail below with reference to the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0034] The reagents involved in the embodiments of the present invention are shown in Table 1

[0035] Table 1 Reagents involved in this experiment

[0036]

[0037] The instruments involved in the embodiments of the present invention are shown in Table 2.

[0038] Table 2 Instruments involved in this test

[0039]

[0040] Example 1

[0041] Concentration screening experiment of cobalt chloride treatment on SH-SY5Y cells

[0042] 1. Materials

[0043] 1.1 Cells: Human neuroblastoma SH-SY5Y cells

[0044] 2. Experimental Methods

[0045] 1) Take SH-SY5Y cells in the logarithmic growth phase, wash twice with PBS, add 1 ml of trypsin, shake and mix well, and place in an incubator with 5% CO2 for 5 minutes.

[0046] 2) Add 2 ml of DMEM medium containing 10% FBS and 1% penicillin-streptomycin to terminate the digestion.

[0047] 3) Transfer to a 15 ml centrifuge tube and centrifuge at 1200 rpm for 5 min at room temperature.

[0048] 4) After centrifugation, discard the supernatant, resuspend the cells in DMEM medium, and count the cells.

[0049] 5) Take 10 μL of cell suspension and add it to a cell counting plate for cell counting. Dilute the cells and add them to a 96-well plate, 100 μL per well, and make each well contain 1×10 4 cells.

[0050] 6) Place the cells in a cell culture incubator containing 5% CO2 overnight.

[0051] 7) On the next day, the drug concentration was first diluted according to the experimental design. The cobalt chloride concentration was set to: 0 μM, 25 μM, 50 μM, 100 μM, 200 μM, 400 μM, 500 μM, 600 μM, 800 μM, and 1000 μM. The Yangxue Oral Solution was gradiently diluted using DMEM medium.

[0052] 8) Take out the 96-well plate from the incubator, add the diluted drug into the 96-well plate, and then put it back into the cell culture incubator containing 5% CO2.

[0053] 9) After the drug reaction for 24 hours, the 96-well plate was removed, 10 μl of enhanced CCK-8 solution was added to each well, and the plate was placed in a 5% CO 2 cell culture incubator for 2 hours.

[0054] 10) After the reaction is complete, the 96-well plate is removed and placed in a microplate reader for reading at an absorbance of 450 nm for data analysis.

[0055] 11) One-way ANOVA was performed using Grapahad. P > 0.5 indicated no difference, P < 0.05 indicated a difference, and P < 0.001 indicated a significant difference.

[0056] Table 3 Cell survival rate of SH-SY5Y cells treated with different concentrations of cobalt chloride for 24 h

[0057]

[0058] The cell survival rates of SH-SY5Y cells after treatment with different cobalt chlorides are shown in Tables 3 and Figure 1 The experimental results showed that at a concentration of 200 μM cobalt chloride, SH-SY5Y cells did not show any signs of cell death. At a concentration of 400 μM cobalt chloride, cells began to show significant cell death, and at a concentration of 800 μM, the survival rate was even lower. Therefore, the concentration of 800 μM will be used in subsequent studies.

[0059] Example 2

[0060] Protective effect of Yangxue oral liquid on SH-SY5Y cell hypoxia model induced by cobalt chloride

[0061] 1. Reagents:

[0062] 1.1 Preparation of blood-nourishing oral liquid-containing serum

[0063] The original concentration of Yangxue Oral Liquid is 0.4g / ml. The rat dosage of Yangxue Oral Liquid is 2mg / ml. Each rat is gavaged twice daily, once in the morning and once in the afternoon, for 5 consecutive days. Blood is collected from the abdominal aorta 1 hour after the last dose, centrifuged at 3000 rpm, and serum is separated. The serum is inactivated by incubation at 56°C for 30 minutes, sterilized by filtration through a 0.22 μm filter, and stored at -20°C until use. Dilute with culture medium to the desired concentration for experimental use.

[0064] 2. Experimental methods:

[0065] 1) Take SH-SY5Y cells in the logarithmic growth phase, wash twice with PBS, add 1 ml of trypsin, shake and mix well, and place in an incubator with 5% CO2 for 5 minutes.

[0066] 2) Add 2 ml of DMEM medium containing 10% FBS and 1% penicillin-streptomycin to terminate the digestion.

[0067] 3) Transfer to a 15 ml centrifuge tube and centrifuge at 1200 rpm for 5 min at room temperature.

[0068] 4) After centrifugation, discard the supernatant, resuspend the cells in DMEM medium, and count the cells.

[0069] 5) Take 10 μL of cell suspension and add it to a cell counting plate for cell counting. Dilute the cells and add them to a 96-well plate, 100 μL per well, and make each well contain 1×10 4 cells.

[0070] 6) Place the cells in a cell culture incubator containing 5% CO2 overnight.

[0071] 7) The next day, the serum containing Yangxue Oral Liquid was mixed with the culture medium to a final concentration of 10%, and the same volume of blank rat serum was used as a parallel control.

[0072] 8) Take out the 96-well plate, add the diluted drug into the 96-well plate, and put it back into the cell culture incubator containing 5% CO2.

[0073] 9) After the drug reaction for 24 hours, the 96-well plate was removed, 10 μl of enhanced CCK-8 solution was added to each well, and the plate was placed in a 5% CO 2 cell culture incubator for 2 hours.

[0074] 10) After the reaction is complete, the 96-well plate is removed and placed in a microplate reader for reading at an absorbance of 450 nm for data analysis.

[0075] 11) One-way ANOVA was performed using Grapahad. P > 0.5 indicated no difference, P < 0.05 indicated a difference, and P < 0.001 indicated a significant difference.

[0076] The results of the determination of blood serum containing Yangxue oral liquid are shown in Table 4 and Figure 2 The results showed that the serum containing Yangxue Oral Liquid had a certain protective effect on the SH-SY5Y cell hypoxia model induced by 800μM cobalt chloride.

[0077] Table 4 Cell survival rate of SH-SY5Y cells treated with serum containing Yangxue oral liquid for 24 h in the hypoxia model induced by 800 μM cobalt chloride

[0078] Grouping Blank serum Blank serum + 800 μM cobalt chloride Drug-containing serum + 800 μM cobalt chloride Repeat one 87.58％ 54.21％ 61.31％ Repeat 2 96.77％ 61.66％ 67.79％ Repeat three times 108.22％ 54.35％ 68.67％ Repeat four times 103.60％ 61.21％ 69.75％ Repeat five times 97.90％ 52.41％ 70.10％ Repeat six 105.94％ 59.33％ 63.19％

[0079] Example 3

[0080] Preventive and protective effects of Yangxue oral liquid and isorhamnetin on cobalt chloride-induced hypoxia model in SH-SY5Y cells

[0081] 1. Materials:

[0082] 1.1 Cells: Human neuroblastoma SH-SY5Y cells

[0083] 1.2 Reagents:

[0084] Preparation of medicated serum for Yangxue oral liquid

[0085] The original concentration of Yangxue Oral Liquid is 0.4g / ml. The rat dosage of Yangxue Oral Liquid is 2mg / ml. Each rat is gavaged twice daily, once in the morning and once in the afternoon, for 5 consecutive days. Blood is collected from the abdominal aorta 1 hour after the last dose, centrifuged at 3000 rpm, and serum is separated. The serum is incubated at 56°C for 30 minutes to inactivate the serum, filtered through a 0.22 μm filter, and stored at -20°C until use. Dilute to the desired concentration in culture medium for experimental use.

[0086] 2. Experimental methods:

[0087] 1) Take SH-SY5Y cells in the logarithmic growth phase, wash twice with PBS, add 1 ml of trypsin, shake and mix well, and place in an incubator with 5% CO2 for 5 minutes.

[0088] 2) Add 2 ml of DMEM medium containing 10% FBS and 1% penicillin-streptomycin to terminate the digestion.

[0089] 3) Transfer to a 15 ml centrifuge tube and centrifuge at 1200 rpm for 5 min at room temperature.

[0090] 4) After centrifugation, discard the supernatant, resuspend the cells in DMEM medium, and count the cells.

[0091] 5) Take 10 μL of cell suspension and add it to a cell counting plate for cell counting. Dilute the cells and add them to a 96-well plate, 100 μL per well, and make each well contain 1×10 4 cells.

[0092] 6) Place the cells in a cell culture incubator containing 5% CO2 overnight.

[0093] 7) The next day, the serum containing Yangxue Oral Liquid was mixed with the culture medium to a final concentration of 10%, and the same volume of blank rat serum was used as a parallel control.

[0094] 8) Take out the 96-well plate, add the diluted drug into the 96-well plate, and put it back into the cell culture incubator containing 5% CO2.

[0095] 9) After the drug reaction for 24 hours, remove the 96-well plate, add 10 μl of enhanced CCK-8 solution to each well, and place in a 5% CO2 cell culture incubator for 2 hours.

[0096] 10) After the reaction is complete, the 96-well plate is removed and placed in a microplate reader for reading at an absorbance of 450 nm for data analysis.

[0097] 11) One-way ANOVA was performed using Grapahad. P > 0.5 indicated no difference, P < 0.05 indicated a difference, and P < 0.001 indicated a significant difference.

[0098] The results are shown in Table 5 and Figure 3 The experimental results showed that Yangxue Oral Liquid and isorhamnetin had no preventive and protective effect on the SH-SY5Y cell hypoxia model induced by 800 μM cobalt chloride.

[0099] Table 5 Cell survival rate of SH-SY5Y cells treated with different concentrations of Yangxue oral liquid for 6 hours in the hypoxia model induced by 800 μM cobalt chloride for 24 hours

[0100]

[0101] Example 4

[0102] Effects of Yangxue Oral Liquid on ROS Levels in SH-SY5Y Cells Induced by Cobalt Chloride

[0103] 1. Materials

[0104] Cells: Human neuroblastoma SH-SY5Y cells

[0105] 2. Reagents: Reactive Oxygen Detection Kit Biyuntian S0033S

[0106] 3. Experimental methods:

[0107] First, dilute DCFH-DA at a ratio of 1:1000 in serum-free culture medium according to the kit manufacturer's instructions, to a final concentration of 10 μmol / L. Next, remove the cells, remove the cell culture medium, wash twice with PBS, and add an appropriate volume of diluted DCFH-DA. The volume added should be sufficient to cover the cells; generally, add at least 1 ml of diluted DCFH-DA to each well of a six-well plate. Incubate in a 37°C cell culture incubator for 20 minutes. Wash the cells three times with serum-free culture medium to fully remove any DCFH-DA that has not entered the cells. Analyze by flow cytometry.

[0108] See the results Figure 4 The results showed that serum containing Yangxue Oral Liquid could reduce the ROS level in the SH-SY5Y cell hypoxia model induced by 800 μM cobalt chloride.

[0109] Example 5

[0110] Protective effect of Yangxue oral liquid on LPS-induced SH-SY5Y cell inflammation model

[0111] 1. Materials

[0112] 1.1 Cells

[0113] Human neuroblastoma SH-SY5Y cells.

[0114] 2. Experimental Methods

[0115] 1) Take SH-SY5Y cells in the logarithmic growth phase, wash twice with PBS, add 1 ml of trypsin, shake and mix well, and place in an incubator with 5% CO2 for 5 minutes.

[0116] 2) Add 2 ml of DMEM medium containing 10% FBS and 1% penicillin-streptomycin to terminate the digestion.

[0117] 3. Transfer to a 15 ml centrifuge tube and centrifuge at 1200 rpm for 5 min at room temperature.

[0118] 4. After centrifugation, discard the supernatant, resuspend the cells in DMEM medium, and count the cells.

[0119] 5. Take 10 μL of cell suspension and add it to a cell counting plate for cell counting. Dilute the cells and add them to a 96-well plate, 100 μL per well, and make each well contain 1×10 4 cells.

[0120] 6. Place the cells in a cell culture incubator containing 5% CO2 overnight.

[0121] 7. On the next day, use DMEM medium to perform gradient dilution of the Yangxue Oral Liquid. The LPS concentration is set to 100 μg / ml according to the reported literature, and the crude drug concentration of the Yangxue Oral Liquid is set to 0 mg / ml, 0.125 mg / ml, 0.25 mg / ml, 0.5 mg / ml, 1 mg / ml, and 2 mg / ml.

[0122] 8. Take out the 96-well plate from the incubator, add the diluted drug into the 96-well plate, and then put it back into the cell culture incubator containing 5% CO2.

[0123] 9. After the drug reaction for 24 hours, remove the 96-well plate, add 10 μl of enhanced CCK-8 solution to each well, and place it in a 5% CO2 cell culture incubator for 2 hours.

[0124] 9. After the reaction is completed, take out the 96-well plate and place it in a microplate reader to read the absorbance at 450 nm for data analysis.

[0125] 10. One-way ANOVA was performed using Grapahad. P>0.5 indicated no difference, P<0.05 indicated a difference, and P<0.001 indicated a significant difference.

[0126] The experimental results are shown in Table 6 and Figure 5 The crude drug of Yangxue Oral Liquid showed a protective effect on the SH-SY5Y cell inflammation model induced by 100 μg LPS in a dose-dependent manner. Differences were observed at 0.5 mg / ml, and significant differences were observed at the effective concentration of 1 mg / ml Yangxue Oral Liquid.

[0127] Table 6 Effect of different concentrations of Yangxue oral liquid on cell survival rate in SH-SY5Y cell inflammation model induced by 800 μM LPS for 24 h

[0128]

[0129] Example 6

[0130] Protective effect of Yangxue oral liquid on SH-SY5Y cells induced by Aβ1-42 amyloid protein

[0131] 1. Materials

[0132] Human neuroblastoma SH-SY5Y.

[0133] 2. Experimental Methods

[0134] 1) Take the long-term SH-SY5Y cells, wash them twice with PBS, add 1 ml of trypsin, shake and mix well, and place them in an incubator with 5% CO2 for 5 minutes.

[0135] 2) Add 2 ml of DMEM medium containing 10% FBS and 1% penicillin-streptomycin to terminate the digestion.

[0136] 3) Transfer to a 15 ml centrifuge tube and centrifuge at 1200 rpm for 5 min at room temperature.

[0137] 4) After centrifugation, discard the supernatant, resuspend the cells in DMEM medium, and count the cells.

[0138] 5) Take 10 μL of cell suspension and add it to a cell counting plate for cell counting. Dilute the cells and add them to a 96-well plate, 100 μL per well, and make each well contain 1×10 4 cells.

[0139] 6) Place the cells in a cell culture incubator containing 5% CO2 overnight.

[0140] 7) The next day, the blood serum containing Yangxue oral solution prepared according to the method described in Example 2 was taken out and administered to the experimental group, and the same volume of blank rat serum was used as a parallel control.

[0141] 8) Add the blood serum containing Yangxue oral liquid into a 96-well plate and mix it with the culture medium to a final concentration of 10%, and place it in a cell culture incubator with 5% CO2.

[0142] 9) After the drug reaction for 24 h and 48 h, the 96-well plate was removed, 10 μl of enhanced CCK-8 solution was added to each well, and the plate was placed in a 5% CO 2 cell culture incubator for 2 h.

[0143] 10) After the reaction is complete, the 96-well plate is removed and placed in a microplate reader for reading at an absorbance of 450 nm for data analysis.

[0144] 11) One-way ANOVA was performed using Grapahad. P > 0.5 indicated no difference, P < 0.05 indicated a difference, and P < 0.001 indicated a significant difference.

[0145] The results are shown in Table 7 and Figure 6 The results showed that the serum containing Yangxue oral liquid had a protective effect on SH-SY5Y cells stimulated by 2μM Aβ1-42 amyloid protein, with a significant difference.

[0146] Table 7 Effect of serum containing Yangxue oral liquid on cell survival rate of SH-SY5Y cells treated with Aβ1-42 amyloid protein for 24 hours

[0147]

[0148]

[0149] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A use of a blood-nourishing oral liquid in the preparation of a drug for neuroprotection against Alzheimer's disease and / or Parkinson's disease, wherein the neuroprotection against Alzheimer's disease and / or Parkinson's disease comprises at least one of the following: reducing inflammatory damage, apoptosis, hypoxic damage, and oxidative stress levels in nerve cells or neurons; the nerve cells are SH-SY5Y cells, and the blood-nourishing oral liquid is prepared from the following Chinese medicinal ingredients: alum, sea buckthorn, hawthorn, and jujube.

2. The application according to claim 1, characterized in that The hypoxic injury to nerve cells includes the hypoxic injury to nerve cells induced by cobalt chloride.

3. The application according to claim 2, characterized in that: Inflammatory damage to nerve cells includes lipopolysaccharide-induced inflammatory damage to nerve cells.

4. The application according to claim 2, characterized in that: The apoptosis of neural cells includes apoptosis of neural cells induced by amyloid β.

5. A use of a blood-nourishing oral liquid in the preparation of a neuron protective agent, wherein the neuron cells include SH-SY5Y cells, and the neuron protection includes at least one of the following: reducing inflammatory damage, apoptosis, hypoxic damage and oxidative stress levels in the neuron cells, and the blood-nourishing oral liquid is made from the following Chinese medicinal raw materials: alum, sea buckthorn, hawthorn and jujube.

6. The application according to claim 5, characterized in that The hypoxic injury to the nerve cells includes induction by cobalt chloride; The induction concentration of the cobalt chloride is 750-850 μM.

7. The use according to claim 5, characterized in that The inflammatory damage to the nerve cells includes induction by lipopolysaccharide; Apoptosis of neurons is induced by amyloid-β.

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