CN115737697A

CN115737697A - Application of blueberry extract

Info

Publication number: CN115737697A
Application number: CN202211397738.7A
Authority: CN
Inventors: 张伟; 王饶琼; 朱国元; 姜志宏; 王彩云
Original assignee: Macau University of Science and Technology
Current assignee: Macau University of Science and Technology
Priority date: 2022-11-09
Filing date: 2022-11-09
Publication date: 2023-03-07

Abstract

The blueberry extract can be used for preparing medicines for treating cerebrovascular diseases, particularly ischemic stroke, can improve symptoms of the ischemic stroke, can inhibit release of excitatory amino acid after the ischemic stroke occurs, reduces excitotoxicity of an organism, promotes release of acetylcholine (Ach), reduces neurotoxic substances, and treats the ischemic stroke by protecting a central nervous system; in addition, the blueberry extract can also increase beneficial bacteria and reduce harmful bacteria, and intestinal metabolic disorder caused by ischemic stroke is treated by regulating the pathway of tryptophan metabolism kynurenine, so that the structure of intestinal flora of the ischemic stroke is optimized, and the ischemic stroke is treated by changing the intestinal flora.

Description

Application of blueberry extract

Technical Field

The invention relates to the technical field of biological medicines, and particularly relates to application of a blueberry extract.

Background

Ischemic Stroke (AIS) is the second leading cause of death worldwide, accounting for 25% of the global lifetime risk. Cerebral ischemic stroke caused by focal occlusion of the brain or stenosis of cerebral arteries is a central nervous system disease that seriously endangers human life. Ischemic stroke has complex pathological features, including calcium overload, blood brain barrier destruction, neuronal apoptosis, encephalitis and the like, and often causes a series of syndromes, such as local blood circulation obstruction, brain tissue necrosis, cerebral edema, neurological dysfunction, cognitive dysfunction and the like. Clinically, recombinant tissue plasminogen activator (rt-PA) is used for intravenous thrombolysis to treat ischemic stroke. However, the narrow available time window for thrombolysis (4.5 hours), the increased risk of intracranial bleeding due to activation of the fibrinolytic system, and the possibility of reperfusion injury limit its clinical use. Therefore, there is a need to develop a drug that can effectively treat ischemic stroke.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the blueberry extract provided by the invention can be used for treating cerebrovascular diseases and improving intestinal flora, and can be used for treating cerebrovascular diseases, especially ischemic stroke, by optimizing the intestinal flora structure of the cerebrovascular diseases and further changing the intestinal flora.

The invention provides an application of a blueberry extract in a first aspect.

In particular to application of a blueberry extract in preparing a medicament for improving intestinal flora of patients with cerebrovascular diseases.

Application of blueberry extract in preparing medicine for treating cerebrovascular diseases by changing intestinal flora.

In the prior art, blueberry extracts are generally utilized to play health care effects of resisting radiation, protecting eyesight, resisting oxidation, eliminating in-vivo inflammation, resisting cancer, enhancing immunity, delaying senescence and the like, and researches show that wild low-plex blueberries can protect neurons from damage caused by stroke by down-regulating iNOS/TNF-alpha and miR-146a/miR-21 (Moradi et al, 2021) (Sweeney et al, 2002), and have a neuroprotective effect on rat whole brain ischemia/reperfusion injury, while most of other researches mainly adopt dietary supplements or traditional Chinese medicine compositions containing blueberries (Yasuhara et al, 2008, wang et al, 2004), and the application of the single blueberry extracts in the regulation of intestinal flora and metabolites thereof of patients with cerebrovascular diseases, particularly ischemic stroke. The blueberry extract provided by the invention is rich in (poly) phenol, especially anthocyanin, is related to reducing the risk of cerebrovascular diseases, can improve intestinal flora, and can treat ischemic stroke by changing the intestinal flora. Ischemic stroke is accompanied by gastrointestinal symptoms, dysfunctional central nervous system affects gastrointestinal function through vagus nerve signals, neurotransmitters, endocrine system and immune pathways, intestinal flora may participate in the pathogenesis of ischemic stroke through chronic inflammation, autonomic nervous system and metabolism, and there is two-way communication between the intestinal tract and its microbiota and the brain, which is called microbiota-gut brain axis. Thus, the intestinal flora plays a key role in the development and progression of ischemic stroke. In one aspect, top-down signaling (brain → intestinal tract), the intestinal wall communicates directly via parasympathetic and sympathetic fibers, or indirectly after stimulation of the enteric nervous system (a highly developed neuronal junction system located in the intestinal submucosa and mesenteric plexus), which can affect intestinal motility, intestinal permeability, microbiota composition and immune cell activation of resident, including neurotransmitter release (e.g., dopamine, 5-hydroxytryptamine), stress response (e.g., cortisol release), mucus secretion and motility control. On the other hand, bottom-up signaling (gut → brain) is thought to occur by a number of different mechanisms, first, the vagus nerve, which consists of 80% of afferent fibers and 20% of efferent fibers, which may be stimulated by microbial compounds and metabolites released by enteroendocrine cells of the gut epithelium and hormones (e.g., serotonin, cholecystokinin, glucagon-like peptide-1, peptide YY) to initiate bottom-up signaling, including barrier integrity maintenance, immune responses (e.g., immunoglobulin a secretion), neurotransmitter and neuropeptide release, short chain fatty acid (e.g., butyric acid) release, and vagal nerve activation, plays a dual role in signaling between the gut and the brain, these afferent projections of stimulation signal throughout the brain, including the hypothalamic neurons that regulate pituitary secretion and the downstream projected solar nucleus. In addition, immunogenic endotoxins from the microbiota, such as lipopolysaccharide endotoxin (LPS), can induce neuroinflammation directly, or can be activated by activating peripheral immune cells and then migrate to the brain.

Preferably, the cerebrovascular disease is stroke.

More preferably, the stroke is an ischemic stroke.

Preferably, the blueberry extract comprises 1300-1500ug/g of chlorogenic acid, 50-70ug/g of cyano-3-O-glucoside, 40-60ug/g of rutin, 20-40ug/g of procyanidin B, 10-20ug/g of D-catechin, 10-20ug/g of L-epicatechin, and 1-3ug/g of quercetin.

More preferably, the blueberry extract comprises 1411.68ug/g chlorogenic acid, 59.40ug/g cyano-3-O-glucoside, 53.72ug/g rutin, 29.63ug/g procyanidin B, 14.13ug/g D-catechin, 12.26ug/g L-epicatechin, and 1.65ug/g quercetin.

Preferably, the preparation method of the blueberry extract comprises the following steps:

extracting fructus Myrtilli with organic solvent to obtain extractive solution, concentrating, dispersing with solvent, and eluting to obtain fructus Myrtilli extract.

Preferably, the solvent is water.

Preferably, the organic solvent is an ethanol solution.

Preferably, the elution is performed with a resin.

Preferably, the elution is with an eluent comprising a water and/or ethanol solution.

Preferably, the ethanol solution comprises 1-10% by volume of ethanol solution and/or 60-80% by volume of ethanol solution.

Preferably, the elution is performed by sequentially adopting water, ethanol solution with the volume fraction of 1-10% and ethanol solution with the volume fraction of 60-80%.

Preferably, after the elution, a drying step is further included.

Preferably, the drying is drying under reduced pressure.

Preferably, the temperature of the drying is 30-50 ℃.

Preferably, the medicament further comprises pharmaceutically acceptable auxiliary materials.

Preferably, the dosage form of the medicament is a pharmaceutically acceptable dosage form.

More preferably, the dosage form of the medicament is one of tablets, powders or capsules.

Compared with the prior art, the invention has the following beneficial effects:

(1) The blueberry extract can be used for preparing medicines for improving the intestinal flora and metabolites thereof of patients with cerebrovascular diseases, can be used for preparing medicines for treating the cerebrovascular diseases, and can improve the symptoms of ischemic stroke;

(2) The blueberry extract is obtained by extracting blueberries with an organic solvent and then eluting, the preparation method is simple and easy to operate, and the prepared blueberry extract can be further used for preparing medicines for treating cerebrovascular diseases.

Drawings

FIG. 1 is a graph showing the results of monitoring the body weight of rats in each group in example 2 of the present invention;

FIG. 2 is a graph showing the results of behavioral testing of rats in each group according to example 2 of the present invention;

FIG. 3 is a graph showing the results of the staining experiment with 2,3,5-triphenyltetrazolium chloride (TTC) for rats in each group according to example 2 of the present invention;

FIG. 4 is a diagram of the intestinal morphology of rats in each group according to example 2 of the present invention;

FIG. 5 is a graph showing the results of the intestinal flora shift test of rats in each group according to example 2 of the present invention;

FIG. 6 is a graph showing the results of the tight junction protein assay in each group of rats in example 2 of the present invention;

FIG. 7 is a graph showing the results of inflammatory factor changes in rats of each group in example 2 of the present invention;

FIG. 8 is a graph showing the results of a fecal flora transplantation (FMT) experiment performed on various groups of rats in example 2 of the present invention;

FIG. 9 is a graph showing the results of the composition of intestinal microorganisms at phylum and genus levels in various groups of rats according to example 3 of the present invention;

FIG. 10 is a graph of Alpha diversity analysis of gut microbiota composition according to example 3 of the present invention;

FIG. 11 is a graph showing the diversity analysis of Beta in the intestinal microbiota of rats in each group according to example 3 of the present invention;

FIG. 12 is a LEfSe analysis chart of intestinal microbiota composition of rats in each group according to example 3 of the present invention;

FIG. 13 is a score chart of an orthogonal partial least squares discriminant analysis (OPLS-DA) model in example 4 of the present invention;

fig. 14 is a hierarchical clustering heatmap of positive and negative ion pattern significant difference metabolites for the blueberry (bg) and model (mg) groups of example 4 of the present invention;

FIG. 15 is a graph of the KEGG enrichment pathway (bubble map) for the bg and mg groups of example 4 of the invention.

Detailed Description

In order to make the technical solutions of the present invention more apparent to those skilled in the art, the following examples are given for illustration. It should be noted that the following examples are not intended to limit the scope of the claimed invention.

The starting materials, reagents or apparatuses used in the following examples are conventionally commercially available or can be obtained by conventionally known methods, unless otherwise specified.

The blueberry extract used in the examples below was produced at australia university of science. Reagents and instruments used in the following examples:

TTC reagent: batch number: g3005-100, beijing Sorleibao technologies, inc.

And (3) dehydrating reagent: absolute ethanol (AR grade), batch No.: 220112 1, specification: 2.5L/barrel, sichuan Kanglong science, inc. The preparation method comprises the following steps: the absolute ethyl alcohol is diluted by purified water to prepare 75 percent, 85 percent and 95 percent ethanol solution.

Transparent reagent: xylene (AR grade), batch No.: 2201111, specification: 500 mL/bottle, szechwan Ringgaku GmbH.

Hematoxylin staining solution: batch number: CR2109146, specification: 500 mL/vial, wuhan Severe Biotech, inc.

Eosin dye liquor: batch number: CR2011064, specification: 500 mL/vial, wuhan Severe Biotech, inc.

Hydrochloric acid-ethanol differentiation solution: hydrochloric acid (AR grade), batch number: 200630, specification: 500 mL/bottle, kyoto Shigaku GmbH; absolute ethanol (AR grade), lot number: 220112 1, specification: 2.5L/barrel, szechwan science, inc.; the preparation method comprises the following steps: pouring 416mL of absolute ethyl alcohol and 178mL of purified water into a beaker, uniformly mixing, slowly adding 6mL of concentrated hydrochloric acid, and uniformly stirring to obtain the product.

Mounting a reagent: neutral gum, batch number: 21217111, specification: 100 g/vial, biosharp Bio Inc.

Rat DAO ELISA KIT: the goods number is: ZC-36566, specification: 48Test, manufactured by Shanghai color-thriving Biotech, inc.

Rat IL-1 β ELISA KIT: the goods number is: ZC-36391, specification: 48Test, manufactured by Shanghai color-thriving Biotech, inc.

Rat IL-6 ELISA KIT: the goods number is: ZC-36404, specification: 48Test, manufactured by Shanghai color-thriving Biotech, inc.

Rat LPS ELISA KIT: the goods number is: ZC-37600, specification: 48Test, manufactured by Shanghai color-producing Biotechnology, inc.

Rat TNF-alpha ELISA KIT: the goods number is: ZC-37624, specification: 48Test, manufactured by Shanghai color-thriving Biotech, inc.

Rat ZO-1 ELISA KIT: the goods number is: ZC-36832, specification: 48Test, manufactured by Shanghai color-producing Biotechnology, inc.

Rat Claudins-5 ELISA KIT: the goods number is: HS629-Ra, specification: 48Test, manufactured by Shanghai Hengyuan Biotech, inc.

Rat D-LA ELISA KIT: the goods number is: HS1217-Ra, specification: 48Test, manufactured by Shanghai Hengyuan Biotech, inc.;

rat JAM1 ELISA KIT: the goods number is: HS1215-Ra, specification: 48Test, manufactured by Shanghai Hengyuan Biotech, inc.;

rat NF- κ B ELISA KIT: the goods number is: HS1213-Ra, specification: 48Test, manufactured by Shanghai Hengyuan Biotech, inc.;

rat Occludin-1 ELISA KIT: the goods number is: HS1216-Ra, specification: 48Test, manufactured by Shanghai Hengyuan Biotech, inc.

And (3) chromatographic column: waters, ACQUITY UPLC BEH Amide 1.7 μm,2.1 mm. Times.100 mm column.

Acetonitrile: merck,1499230-935.

Ammonium acetate: sigma,70221.

Methanol: fisher, A456-4.

Ammonia water: fisher, A470-500.

Example 1

A preparation method of a blueberry extract comprises the following steps:

adding 1500mL of 80% ethanol into 375g of blueberry (fresh) to perform reflux extraction for 2 times, each time for 30min, filtering, combining filtrates, recovering solvent, then obtaining 300mL of water solution, dispersing the water solution to 1000mL by using pure water, passing through D101 macroporous resin, sequentially eluting by using 1000mL of pure water, 500mL of 10% ethanol solution in volume fraction and 1000mL of 70% ethanol solution in volume fraction, recovering 70% ethanol solution elution solution, and performing reduced pressure drying at 40 ℃ to obtain 2.207g of blueberry extract.

Identification of blueberry extract components: the main compounds were selected for quantification based on Mass Spectrometry (MS) response. These components were quantified by Multiple Reaction Monitoring (MRM). The Thermo TSQ QQQ LC-MS system was used. Chromatography was performed on a Sepax GP-C18 column (2.1 mm. Times.150mm, 1.8 μm). The column was maintained at 35 ℃ and the flow rate was 0.2mL/min. The mobile phase consisted of a volume fraction of 0.1% formic acid (a) and acetonitrile (B). The column was eluted with a linear gradient system: 0-8min,15% -30% B;8-9 minutes, 30% -95% b;9-15 minutes, 95% -95% b;15-17 minutes, 95% -15% b. The autosampler was set to 4 ℃ and the sample size was 10. Mu.L. The MS equipped with a heated ESI source was run in positive and negative mode using the following optimization parameters: the ion spraying voltage is 3500V in positive mode and 2800V in negative mode; the evaporator temperature is 280 ℃; sheath gas pressure 50psi; the capillary temperature was 320 ℃ and the assist gas pressure was 15psi. MS/MS fragmentation was performed with different collision energies. Through detection, the prepared blueberry extract comprises the following components: chlorogenic acid 1411.68ug/g, cyano-3-O-glucoside 59.40ug/g, rutin 53.72ug/g, procyanidin B1.63 ug/g, D-catechin 14.13ug/g, L-epicatechin 12.26ug/g, and quercetin 1.65ug/g.

Grouping and administration of drugs

A Middle Cerebral Artery Occlusion (MCAO) model is established and randomly divided into: the method comprises the following steps of performing pseudo-operation on a group (non-MCAO, normal saline gavage for short so), a model group (MCAO, normal saline gavage for short mg), a positive control group (or nimodipine tablet group, western medicine group, po or wm) (nimodipine tablet: 20 mg/tablet, western medicine produced in Shanxi Bao company), a blueberry high-dose group (rat gavage concentration is 400mg/kg, bh for short) and a blueberry low-dose group (rat gavage concentration is 100mg/kg, bl for short), wherein the blueberry high-dose group and the blueberry low-dose group are collectively called as a blueberry group (bg). The positive control group and the blueberry group are collectively called the drug group. Each group contained 10 rats. Body weight was monitored weekly until the end of the study.

And (3) weight monitoring results: as shown in figure 1, the weight of each group of rats is about 200g-220g on average, and after 1 week of molding, the weight of each group of rats is reduced; after the blueberry extract and western medicines are fed for 4 weeks, the weight of rats in each medicine group is gradually increased, and the weight of rats in the blueberry high-dose group is larger than that of rats in the blueberry low-dose group and that in the western medicine group (P is less than 0.05). The results show that: ischemic stroke causes weight loss in rats; and the weight gain of rats fed the high dose blueberry extract was faster than that fed nimodipine and the low dose blueberry extract. Wherein, po in figure 1 represents a positive control group (nimodipine group), # represents a significant difference from so ratio, and P is less than 0.05; * The difference from the mg ratio is significant, and P is less than 0.05; delta represents the significant difference from bh, and P is less than 0.05.

Rat cerebral ischemia injury MCAO model

A Middle Cerebral Artery Occlusion (MCAO) rat model was prepared. After the anesthetized rats were injected intraperitoneally, the dorsal position was fixed, a longitudinal incision was made at the midline of the neck to dissect the subcutaneous muscle, and the right Common Carotid Artery (CCA), the External Carotid Artery (ECA) and the Internal Carotid Artery (ICA) were isolated. Then, ligation of CCA and ECA is performed at the proximal end, the proximal bifurcation part of the CCA distal end is clamped and closed, then a small opening is cut at the position, close to the CCA, of the proximal end of the CCA, a thrombus thread is inserted into the CCA, the CCA enters the ICA from the bifurcation of the blood vessel, ligation is performed at the distal end of the CCA from the bifurcation, and the part of the thrombus thread is pulled out after 2h for reperfusion. Sham group rats were not inserted with a tether and the procedure was followed in the other groups. Grading the model-made rats by a neurology (Zea Longa five-score) grading method, selecting 1-3 scores, bringing in the rats meeting the model success standard, removing the rats which do not meet the model success standard, supplementing the number of animals by a random sampling principle, and re-modeling.

Example 2 pharmacodynamic study of blueberry extract in treating ischemic stroke by intestinal flora

1. Behavioral testing

The behavioral testing mainly adopts a Zea Longa five-score scoring method to evaluate motor nerve functional defects caused by the stroke. The scoring standard is as follows: 0 minute, normal walking without any nerve defect symptom; 1 minute, the contralateral forepaw can not be fully extended; 2min, turning to the opposite side; 3 min, pouring towards the opposite side; 4 points, spontaneous walking and loss of consciousness.

Results as shown in figure 2, groups of rats were scored on the Zea Long five point scale 4 weeks after feeding. The score of the model group is larger than that of the sham operation group, which indicates that the model building of the rat is successful; the behavior score of the medicine group is lower than that of the model group, the blueberry high-dose group has a lower score (P < 0.05) than that of the western medicine group and the blueberry low-dose group, and the western medicine group has a lower score (P > 0.05) than that of the blueberry low-dose group. The results show that: the blueberry and the nimodipine can effectively restore the nerve function of the rats with ischemic stroke; and the effect of the blueberry high-dose group is superior to that of the nimodipine group and the blueberry low-dose group.

2. Fecal flora transplantation (FMT)

To test the causal relationship between gut flora and blueberry extract-mediated neuroprotection, FMT experiments were performed by transferring the feces of the blueberry extract group rats to the model group. It was observed whether the FMT group of microbiota of the rats receiving blueberry treatment performed better in the neurobehavioral test than the model group to demonstrate that the gut flora was involved in the blueberry-mediated neuroprotection. Specific experiments are as follows: feces from blueberry-treated rats were collected under sterile conditions, and 0.1g of feces from donor rats were pooled and suspended in 1mL of sterile phosphate buffered saline PBS. The solution was mixed vigorously for 15s using a bench Vortex (Vortex-Genie 2, scientific Industries, USA). After centrifugation at 800g for 3 minutes at room temperature, the supernatant was collected and administered to MCAO rats by gavage (500. Mu.L/mouse). Fresh faeces were prepared within 15 minutes prior to oral gavage on the day of faecal transplantation. Neurobehavioral indicators, inflammatory cytokine levels, gut flora translocation indicators, and tight junction protein levels were evaluated and compared between the model group and FMT rats.

FMT results: the results are shown in FIG. 8, in which FIG. 8A is a graph showing the results of behavioral tests on rats in each group, FIG. 8B is a graph showing the results of diamine oxidase (DAO) tests on rats in each group, FIG. 8C is a graph showing the results of endotoxin (LPS) tests on rats in each group, FIG. 8D is a graph showing the results of D-lactic acid (D-LA) tests on rats in each group, FIG. 8E is a graph showing the results of tight junction protein tests on rats in each group, and FIG. 8F is a graph showing the results of inflammatory factor tests on rats in each group. The FMT group of microbiota of rats receiving blueberry treatment performed better in neurobehavioral testing than the model group, demonstrating that the gut flora is involved in blueberry-mediated neuroprotection (fig. 8A); the FMT group of microbiota of rats receiving blueberry treatment performed better in DAO, LPS and D-LA than the model group, demonstrating that intestinal flora was involved in intestinal flora translocation reversed by blueberry extract (fig. 8B); the FMT group of microbiota of rats treated with blueberry extract showed better performance in the tight-chain proteins ZO-1, claudins-5, occludin-1 and JAM-1 than the model group, which proved that the intestinal flora was involved in the repair of the brain and intestinal barrier by blueberry (FIG. 8C); the FMT group of the microbiota of the rats treated with blueberry showed better performance in the inflammatory factors IL-1 beta, IL-6 and TNF-alpha than the model group, demonstrating that the intestinal flora is involved in the action of blueberry in reducing the inflammatory factors (FIG. 8D).

TTC staining experiment

Rats were anesthetized, intact brains were rapidly harvested, cut into 2mm tissue sections, stained with 2% TTC for 5min, and fixed in 4% formaldehyde for 6 hours. Brain sections were arranged in order and photographed. The cerebral infarction area was calculated using Image J1.41 software. Infarct size is the area of the non-ischemic hemisphere minus the non-infarct size of the ischemic hemisphere. Infarct volume = infarct area x thickness (2 mm). The cerebral infarction percentage calculation formula is as follows: percent cerebral infarction = infarct volume/non-ischemic hemisphere volume x 100%.

TTC staining results are shown in FIG. 3, wherein FIG. 3A is a graph of TTC staining results and FIG. 3B is a graph of percentage of cerebral infarction area (dark area indicates no infarction; light area indicates infarction), and it can be seen that the proportion of cerebral infarction area in the model group is increased (P < 0.05) compared with that in the sham operation group; compared with the model group, the cerebral infarction area proportion of each medicine group is reduced (P is less than 0.05); compared with the western medicine group, the proportion of cerebral infarction area of the blueberry high-dose group is reduced (P < 0.05), and the proportion of blueberry low-dose group is increased (P < 0.05); compared with the blueberry low dose group, the blueberry high dose group has a reduced cerebral infarction area ratio (P < 0.05). The results show that: the blueberry extract and the nimodipine can relieve cerebral infarction of rats with ischemic stroke; and the effect of the blueberry high-dose group is superior to that of the nimodipine group and the blueberry low-dose group.

4. Determination of villus length, width and crypt depth

Observing the section by using a Digital three-eye camera microscope (BA 210Digital, miaodi industry group, inc.), opening image analysis software dynamic Images Advanced 3.2 in a computer, opening an acquisition window, selecting a better area in the section to acquire a picture, guiding the acquired picture into dynamic Images Advanced 3.2, selecting measurement options in a toolbar, adjusting the multiple (4X) of an objective lens and the unit (micrometer) of measurement data, and selecting a broken line tool to measure required data.

Fluff length and width results: the small intestine, as an important component of the digestive system, is responsible for the digestion and absorption of most of the nutrients required by the body. The villus is the place where the small intestine is most mainly used for digesting and absorbing nutrient substances, the longer the villus is, the wider the absorption area is, the narrower the villus is, the smaller the absorption area is, the lower the nutrient utilization rate is, and the harmful bacteria can be well prevented from being planted in the intestinal tract. As shown in fig. 4, the length and width of the duodenal villi of each group of drugs are greater than those of the model group (P < 0.05); the length of the fuzz of the blueberry high-dose group is larger than that of the blueberry low-dose group (P is less than 0.05), but the width of the fuzz of the blueberry high-dose group is larger than that of the blueberry low-dose group (P is more than 0.05); the blueberry low dose group is larger than the western medicine group (P > 0.05). The results show that: the blueberry extract and nimodipine can improve the length and width of villus of duodenum and can well prevent harmful bacteria from being planted in the intestinal tract; and the effect of the blueberry high-dose group is superior to that of the nimodipine group.

Crypt depth results: the depth of the crypts is increased, which can weaken the absorption capacity of nutrient substances; the shallower the crypt, the better the cell maturity, the better the secretory function. The crypt depth is increased and the secretory function is deteriorated. As shown in fig. 4, the depth of the duodenal crypt of each group of drugs was smaller than that of the model group (P < 0.05); the blueberry high dose group is smaller than the blueberry low dose group (P < 0.05); the low dose group of blueberry is smaller than the western medicine group (P >0.05, with no statistical significance). The results show that: the blueberry and nimodipine can reduce the depth of a duodenal crypt and increase the secretion function and the absorption capacity of nutrient substances; and the effect of the blueberry high-dose group is superior to that of the nimodipine group and the blueberry low-dose group.

5. Determination of inflammatory factors and tight junction proteins

Detecting indexes for detecting intestinal flora translocation, namely diamine oxidase (DAO), endotoxin (LPS) and D-lactic acid (D-LA) according to an ElISA kit scheme; inflammatory factors: IL-1b, IL-6, TNF-a and NF-KB; closely linked proteins: ZO-1, occludin-1, claudins-5 and JAM-1.

Translocation of intestinal flora: given the increased intestinal permeability and impaired intestinal barrier, the gut flora and its metabolites may have migrated from the gut into the systemic circulation and cause a range of complications such as endotoxemia and infections. This phenomenon is called intestinal flora translocation. The present invention investigates indicators of intestinal flora translocation, namely DAO, LPS and D-LA. As shown in FIG. 5, in which FIG. 5A is a graph showing diamine oxidase (DAO) results of various groups of rats, FIG. 5B is a graph showing endotoxin (LPS) results of various groups of rats, and FIG. 5C is a graph showing D-lactic acid (D-LA) results of various groups of rats, the results show that increased serum DAO, LPS, and D-LA levels in the model group indicate that cerebral ischemic stroke disrupts the intestinal barrier, thereby causing translocation of intestinal microorganisms. The levels of DAO, LPS and D-LA in the blueberry high and low dose groups are reduced, which shows that the blueberry extract can effectively protect intestinal barriers, so that translocation of bacteria is weakened, and the blueberry high dose group has a better effect. The effect of the nimodipine group is reduced compared with the model group, but the P is more than 0.05, and the difference is not statistically significant, which indicates that the nimodipine can not protect the intestinal barrier and weaken the translocation of bacteria. The results show that: the blueberry extract reverses the intestinal barrier disruption and the intestinal flora shift caused by cerebral ischemic stroke; the effect of the blueberry high-dose group is superior to that of the blueberry low-dose group; however, nimodipine does not reverse gut barrier disruption and gut flora translocation.

Inflammatory factor changes results: as shown in figure 7, the inflammatory factors IL-1 beta, IL-6 and TNF-alpha in the model group are obviously increased, the dosage of each group is reduced, and the blueberry high-dosage group is lower than that of the blueberry low-dosage group and the nimodipine group (P is less than 0.05). The results show that: the cerebral ischemic stroke can cause obvious increase of inflammatory factors, and the blueberry extract and the nimodipine can reduce the level of the inflammatory factors; and the effect of the blueberry high-dose group is superior to that of the nimodipine group and the blueberry low-dose group.

Tight junction protein results: since claudin plays an important role in maintaining the integrity, permeability and function of the brain-intestinal barrier, the present inventors further investigated claudin, i.e., occludin-5, ZO-1 and JAM-1, to investigate whether the intestinal barrier was disrupted. As shown in FIG. 6, the model groups occludin, claudin-5, ZO-1 and JAM-1 were decreased, while the blueberry high and low dose groups were increased, and the blueberry high dose group was significantly increased compared to the low dose group, while the western medicine group was increased, but P >0.05, and the difference was not statistically significant. The results show that: the blueberry extract can effectively repair the brain and intestine barrier damage caused by cerebral ischemic stroke; the effect of the blueberry high-dose group is superior to that of the blueberry low-dose group; however, nimodipine does not reverse the breakdown of the gut barrier.

Example 3 analysis of intestinal flora of blueberry for treating ischemic stroke

All feces analyzed by 16S rRNA sequencing were collected at fixed time periods (9 am). The abdomen massage avoids the day and night oscillation of intestinal flora and the pollution of exogenous bacteria. In addition, the tubes and forceps used for collection were also autoclaved and sterilized beforehand. The DNA was extracted using PowerSoil DNA Isolation kit (Mobio, USA), and the quality and concentration of the DNA were tested using a Nanodrop ND-1000 spectrophotometer (Thermo Electron Corporation, USA). PCR amplification of the 16S rRNA sequence was performed using a primer set specific for the V3-V4 region. The final PCR product was purified from unincorporated nucleotides and primers using the Qiaquick PCR purification kit (Qiagen, valencia, USA). Purified samples were normalized to equal DNA concentrations and sequenced using Illumina Miseq sequencer PE250 (Illumina, usa).

In the study of the present invention, intestinal microbiota compositions of five groups of rats were compared, namely, a blueberry high dose group (bh, sample size of 5), a blueberry low dose group (bl, sample size of 4), a model group (mg, sample size of 4), a sham operation group (so, sample size of 4), and a western medicine group (wm, sample size of 4). A series of analyses were performed to investigate the effect of blueberry extract on intestinal microbiota.

1. Multi-level analysis of differences in gut flora composition

And (4) counting the community composition of each sample at different classification levels of the gate and the genus. And drawing a corresponding histogram according to the OTU abundance table and the annotation information on the classification thereof, and observing the composition and abundance changes of one or more samples at each classification level. The species composition and relative abundance distribution of the enteric bacteria in each sample at phylum and genus levels is shown in fig. 9. Wherein, FIG. 9A is the composition chart of rat Phylum level (Phyum) microorganism of each group, and FIG. 9B is the composition chart of rat Genus level (Genus) microorganism of each group. Analysis at each level only shows the top 10 gates and genera. In FIG. 9, relative Absundance is the Abundance of the element.

The dominant flora in the sample is listed in fig. 9, and as the bacterial classification is further refined, the species in the sample exhibit a high degree of diversity. Gut microbiota analysis showed that, at the phylum level, firmicutes and bacteroidetes (bacteroidetes) were most abundant in the five groups; at the genus level, the genus of the genus muribacterium (muribacteriaceae) is most abundant in the five groups. The results show that: the blueberry extract can improve the microbial diversity of intestinal flora of rats with ischemic stroke.

The Lactobacillus (Lactobacillus) group is different from one group to another, and the Lactobacillus group is the most abundant in the blueberry high-dose group, the blueberry low-dose group and the western medicine group, but the number of the Lactobacillus group in the model group and the sham operation group is remarkably reduced; the number of Prevotella (Prevotella) in the model group and the sham-operated group was significantly higher than that of the blueberry high dose group, the blueberry low dose group and the western medicine group. Therefore, prevotella (revatella) and Lactobacillus (Lactobacillus) may be related to the efficacy of blueberry extracts and western medicines.

2. Alpha diversity analysis of intestinal flora composition

Alpha diversity analysis is a quantitative measure of microbiome diversity, with emphasis on a single sample. In the present study, four measurements were used to quantify the abundance, uniformity and dominance of species in gut microbiota samples.

The Kruskal-Wallis test is used in the present invention to analyze the variance between five groups of rats as a non-parametric method of testing the distribution of samples. The results are shown in fig. 10, in which fig. 10A is a graph of observed features of each sample group (observed features) as a measure of richness, fig. 10B is a graph of Faith phylogenetic diversity (Faith pd), fig. 10C is a graph of Shannon (Shannon) diversity index, and fig. 10D is a graph of uniformity index (Evennes). As can be seen from fig. 10A: 1) The features observed in the blueberry high dose group were significantly lower than in the sham group; 2) The characteristics observed in the blueberry low dose group are obviously lower than those in the sham operation group but higher than those in the western medicine group; 3) The characteristics observed by the model group are obviously higher than those of the western medicine group; 4) The characteristics observed in the sham group were significantly higher than those observed in the western group.

Faith's phylogenetic diversity, also known as Faith's PD, is a biodiversity measure based on phylogeny, which is the sum of the branch lengths connecting all species in a phylogenetic tree. In FIG. 10B, it is noted that 1) Faith's PD was significantly lower in the blueberry high dose group than in the sham group; 2) The Faith's PD of the blueberry low-dose group is obviously lower than that of a sham operation group but higher than that of a western medicine group; 3) The Faith's PD in the sham group was significantly higher than that in the western group.

Shannon (Shannon) diversity index: also known as the shannon index or shannon wiener index, is used to measure the diversity of species in a community. The Evenness (Evenness) index is used primarily to measure the relative differences in abundance of species in a population. As can be seen from the statistical analysis results in fig. 10C and 10D, only the model group and the western medicine group showed significant differences, while the other groups had no statistical significance from each other in terms of diversity. It should be noted, however, that for intra-set analysis, the shannon index and the uniformity index were very different within the same set, except for the uniformity index of the model set.

Alpha diversity analysis results suggest that the blueberry can change the intestinal flora structure of rats with ischemic stroke, and although the uniformity of the intestinal flora of rats is not obviously changed, the richness and diversity of the intestinal flora are changed.

3. Analysis of the diversity between habitats of the intestinal flora composition (Beta diversity)

Beta diversity analysis was used to measure diversity variation between populations and was first proposed by r.h. whitetake. The present invention uses two algorithms to calculate the Beta diversity of five groups, principal coordinate analysis (PCoA) and partial least squares discriminant analysis (PLS-DA). The results are shown in FIG. 11, in which FIG. 11A is a graph of the results of principal coordinate analysis (PCoA) based on the Bray-Curtis dissimilarity algorithm, and FIG. 11B is a graph of the results of partial least squares discriminant analysis (PLS-DA). According to the results of Beta diversity analysis, it is obvious that the rest groups except the blueberry low-dose group and the blueberry high-dose group can be well distinguished, and the change of the intestinal flora composition of the ischemic stroke rat is shown, the blueberry extract has the effect of improving the intestinal flora composition of the ischemic stroke rat, and the influence on the intestinal flora is not dose-dependent.

In order to better understand the clustering effect during Beta diversity analysis by the Bray-Curtis dissimilarity algorithm, P values of pairwise comparisons of five groups were calculated by the similarity Analysis (ANOMIS) method, and the results are shown in Table 1. Wherein the five rat groups are data of 5 rat experimental groups in whole.

Table 1 results of the pairing comparison of each group of rats by ANOMIS method

Group (Group)	P value (P value)	R value
			five rat groups	0.001	0.5844
bh vs bl	0.813	-0.1063
			bh vs wm	0.010	0.5063
bl vs wm	0.024	0.7708
			mg vs bh	0.036	0.2938
mg vs bl	0.028	0.8854
			mg vs wm	0.039	0.9583
so vs bh	0.010	0.7250
			so vs bl	0.034	1.0000
so vs mg	0.020	0.6771
			so vs wm	0.029	1.0000

As can be seen from the statistical results of Table 1, all groups except the blueberry high dose group (bh) and the blueberry low dose group (bl) showed significant differences, which is consistent with the results in FIG. 11.

4. Linear discriminant analysis Effect size (LEfSe) analysis of intestinal flora composition

LEfSe analysis: is an algorithm for high-dimensional biomarker discovery and identification, originally proposed by Segata et al. The method may determine factors or characteristics through organisms, clades and OTUs to account for differences between 2 or more groups. In this project, the present invention performed LEfSe analysis, showing differentially distributed bacteria by using standard LDA (linear discriminant analysis) scores > 4. Since the blueberry high dose group, the blueberry low dose group did not show significant differences in the Beta diversity analysis, the two groups were merged together and named the blueberry group (bb) for LEfSe analysis.

Fig. 12 is a graph showing the results of LEfSe analysis (LDA SCORE) > 4) of intestinal microbiota composition of rats in each group, in which fig. 12A is a graph showing the comparison results between the blueberry group and the western medicine group, fig. 12B is a graph showing the comparison results between the sham operation group and the model group, fig. 12C is a graph showing the comparison results between the model group and the blueberry group, and fig. 12D is a graph showing the comparison results between the model group and the western medicine group. Based on the results showing the difference between the blueberry group and the western group in FIG. 12A, only 3 bacteria were identified, which were Proteus (g-Prevotella), prevotella (f-Prevotella), and Brucella (g-Blautia), respectively. Compared with the western medicine group, the composition of three groups of bacteria in the blueberry group is remarkably reduced; comparing the sham-operated group and the model group, the model group showed significantly increased levels in the murine (muribacteriaceae), clostridia (clostridiales), firmicutes (friimicutes), murius (muribacteriaceae), lachnospiraceae (Lachnospiraceae), oscillatoria (Oscillospira), ruminomycetaceae (ruminococcus), bacteroides (Bacteroides), while the level in the prevoteriaceae (Prevotellaceae) NK3B31 group was significantly decreased (fig. 12B). In fig. 12C and 12D, the blueberry group and the western group were compared to the model group, respectively, to reveal how the two treatment modalities affected the composition of the gut microbiota. In particular, it can be seen from the results that the levels of firmicutes (fricutes) increased significantly after treatment, while the levels of plagiolide Wo Junshu (Alloprevotella) and bacteroides (bacteroides) decreased. Wherein, compared with the model group, the levels of the phylum of firmicutes (Frimictes), the genus of oscillatoria (Oscillospira) and the family of ruminococcaceae (ruminococcaceae) are obviously increased in the blueberry group; reduced levels of Morganella (Morganella), proteus (poteus), prevotella tanaceti (Alloprovella), prevotella (prevotella), bacteroides (Bacteroides). According to the comparison of the sham operation group, the model group and the blueberry group, the result shows that the effect of the blueberry extract on treating ischemic stroke can be related to changing the content of intestinal microorganisms of rats with ischemic stroke, mainly increasing the microorganisms of firmicutes (Frimiucts), oscillatoria (Oscillospira) and ruminococcaceae (ruminococcaceae), and reducing the microorganisms of Prevotella (prevotella) and Bacteroides (Bacteroides).

5. Discussion of the related Art

Multi-level analysis of differences in intestinal flora compositions indicates that Prevotella (Prevotella) and Lactobacillus (Lactobacillus) may be related to the drug effects of blueberry extract and western medicines. The diversity analysis of intestinal flora Alpha (in-sample) shows that the blueberry extract can change the intestinal flora structure of rats with ischemic stroke, and although the uniformity of the intestinal flora of the rats is not obviously changed, the richness and diversity of the intestinal flora are changed; according to analysis of Beta (sample-to-sample) diversity PCA (defined based on species abundance matrix) and PCoA (defined based on distance matrix between samples), the blueberry extract can obviously change the intestinal flora structure of the ischemic stroke rats, but the influence on the intestinal flora is not dose-dependent.

The four major bacterial phyla in the human gut are: firmicutes (including lactobacilli), bacteroidetes, actinomycetes and proteobacteria (including escherichia). The gut microbiota is a trillions of bacteria and other microorganisms living in the human gut, the resulting effect of which is a variable risk factor associated with the risk of stroke and the prognosis of the nervous system after stroke. Firmicutes are the main dominant beneficial bacteria in the human intestinal tract, a large group of bacteria, most of which are gram-positive. Under healthy conditions, bacteroidetes and firmicutes account for more than 90% of the flora in the intestinal tract. Species of Bacteroides account for 30% of all intestinal bacteria, with markedly reduced abundance of Bacteroides vulgatus (Bacteroides vulgatus) and Bacteroides dorei (Bacteroides dorei) in patients with coronary artery disease. The general bacteroides and the bacteroides dorsalis can relieve the formation of atherosclerotic lesions of mice susceptible to atherosclerosis by gastric lavage, remarkably improve endotoxemia, reduce the generation of intestinal microbial lipopolysaccharides and effectively inhibit proinflammatory immune response. In addition, bacteroides can cause infections of the central nervous system, head, neck and soft tissues. Research shows that the Prevotella can be used as a conditional pathogen to cause diseases such as obesity, diabetes, insulin resistance, hypertension, non-alcoholic fatty disease and the like.

The research result of the invention shows that the blueberry extract can increase beneficial bacteria such as firmicutes (including lactobacillus) and the like, reduce harmful bacteria such as Prevotella (prevotella), bacteroides (Bacteroides), proteus and the like, and supposing that the blueberry extract can enrich the beneficial bacteria and inhibit the harmful bacteria, thereby optimizing the flora structure.

Example 4 serum high resolution non-target metabolomics analysis of blueberries for treatment of ischemic stroke

1. Sample extraction method

After the sample is slowly thawed in an environment at 4 ℃, an appropriate amount of the sample is added into a precooled methanol/acetonitrile/water mixed solution (2.

2. chromatography-Mass Spectrometry

2.1 chromatographic conditions

Separating the sample by Agilent 1290Infinity LC ultra-high performance liquid chromatography (UHPLC) and HILIC chromatographic column; the column temperature is 25 ℃; the flow rate is 0.5mL/min; the sample size is 2 mu L; the mobile phase composition is mobile phase A: water +25mM ammonium acetate +25mM ammonia, mobile phase B: acetonitrile; the gradient elution procedure was as follows: 0-0.5min,95% mobile phase B;0.5-7min, the mobile phase B changes linearly from 95% to 65%;7-8min, the mobile phase B changes linearly from 65% to 40%;8-9min, and maintaining the B at 40%;9-9.1min, the mobile phase B changes linearly from 40% to 95%;9.1-12min, and the mobile phase B is maintained at 95%; samples were placed in a4 ℃ autosampler throughout the analysis. In order to avoid the influence caused by the fluctuation of the detection signal of the instrument, the continuous analysis of the samples is carried out by adopting a random sequence. And inserting Quality Control (QC) samples into the sample queue for monitoring and evaluating the stability of the system and the reliability of experimental data.

2.2 quadrupole time of flight (Q-TOF) Mass Spectrometry conditions

And (3) collecting primary and secondary spectrograms of the sample by adopting an AB Triple TOF 6600 mass spectrometer. After being separated by Agilent 1290Infinity LC ultra-high performance liquid chromatography (UHPLC), the sample is subjected to mass spectrometry by a Triple TOF 6600 mass spectrometer (AB SCIEX), and is respectively detected by electrospray ionization (ESI) positive ion mode and negative ion mode. The ESI source set-up parameters are as follows: atomizing Gas-assisted heating Gas1 (Gas 1): 60Psi, auxiliary heating Gas2 (Gas 2): 60Psi, air curtain gas (CUR): 30psi, ion source temperature: spraying Voltage (ISVF) +/-5500V (positive and negative modes) at the temperature of 600 ℃; first-order mass-to-charge ratio detection range: 60-1000Da, and the mass-to-charge ratio detection range of secondary ions: 25-1000Da, first mass spectrum scan accumulation time: 0.20s/spectra, and the second-order mass spectrum scanning accumulation time is 0.05s/spectra; secondary mass spectra were acquired using a data-dependent acquisition mode (IDA), and using a peak intensity value screening mode, declustering voltage (DP): ± 60V (positive and negative modes), collision energy: 35. + -.15eV, IDA was set as follows: dynamic exclusion isotope ion range: 4Da, 10 fragment patterns per scan were acquired.

3. High resolution non-target metabonomics results from serum

3.1. Results of chromatographic, mass and multidimensional statistical analysis

The results of the OPLS-DA analysis are shown in fig. 13, in which fig. 13 (a) is a positive ion mode OPLS-DA score chart, fig. 13 (B) is a positive ion mode displacement test result chart, fig. 13 (C) is a negative ion mode OPLS-DA score chart, and fig. 13 (D) is a negative ion mode displacement test result chart. The results show that: under the positive and negative ion mode, the blueberry group and the model group are well separated, but form a cluster respectively, and the OPLS-DA model is proved to be stable and reliable (fig. 13A and 13C); in order to avoid overfitting of the supervised model in the modeling process, the model is checked by adopting a displacement test (Permutation test) to ensure the effectiveness of the model, and fig. 13B and 13D show displacement test diagrams of two groups of OPLS-DA models, wherein as the displacement retention degree is gradually reduced, R2 and Q2 of the random model are both gradually reduced, which indicates that the original model has no overfitting phenomenon and good model robustness.

3.2. Screening and identification of differential metabolites

Differential metabolites were screened using OPLS-DA VIP >1 and P value (P value) <0.05 as screening criteria. 26 and 42 differential metabolites are respectively screened out from the blueberry group and the model group under the positive and negative ion modes.

The positive ion mode significantly different metabolites are: norepinephrine, cytarabine, (2 r) -3-hydroxyisovalerylcarnitine, 1-methyl-L-histidine, creatinine, norleucine, 2-piperidone, human proline depsipeptide, C17-dihydrosphingosine, β -murine cholic acid, N- α -acetyl-L-lysine, indole-3-acetic acid methyl ester, ethyl 3-hydroxybutyrate, acetylcholine, cytosine, 5-aminopentanoic acid betaine, L-hydroxyarginine, 1-1-hexadecyl-2-octadecadienoyl-sn-glycero-3-phosphocholine, 1,2-hexacosanyl-sn-glycerol, 2-sasanquadine-palmitoyl-sn-glycero-3-phosphocholine, L-hydroxyarginine, artemisinin, phenylpropidine, α. -mannose pentaacetate, and the like (specifically shown in table 2); the hierarchical cluster analysis results of the positive ion pattern analysis are shown in fig. 14A.

Table 2 comparison of positive ion pattern significantly different metabolites between blueberry group and model group

Description of the invention: in table 1, 26 different metabolites were screened in positive ion mode for the blueberry group and the model group. ≈ up-regulation of metabolites in the blueberry groups; ↓ represents the down-regulation of metabolites in the blueberry group.

The significantly different metabolites of the negative ion mode are: oxyinosine, uracil, poncirin, pantothenic acid, inositol, 2-methyl-3-hydroxybutyric acid, L-kynurenine, phenylpropionic acid, 5- (3-carboxybenzoyl) -2- [ [ (5E) -6- (4-methoxyphenyl) -5-hexen-1-yl ] oxy ], D-sorbitol, succinate, pseudouridine, L-asparagine, cytidine, N-acetylglucosamine 1-phosphate, L-glutamine, calyxin, dl-lactic acid, mesaconic acid, quinoline-2-ol, 4-methylphenol, D-ribose 1-phosphate, 2-linoleoyl-1-palmitoyl-sn-glycerol-3-phosphoethanolamine cholesterol sulfate, 1-stearoyl-2-linoleoyl-sn-glycerol-3-phosphoethanolamine, 2-arachidoyl-1-palmitoyl-sn-glycerol-3-phosphoethanolamine, D-mannose and the like (specifically shown in Table 3); the hierarchical cluster analysis result of the negative ion pattern analysis is shown in fig. 14B.

TABLE 3 comparison of metabolites with significant differences in negative ion patterns between blueberry and model groups

Description of the drawings: the blueberry group and the model group in table 2 were screened for 26 different metabolites in positive ion mode. ↓ofthe metabolites up-regulated in the groups of blueberries; ↓ represents the down-regulation of metabolites in the blueberry group.

3.3 kegg enrichment pathway results

Analysis of metabolic pathways by KEGG databases revealed that the pathways identified for the above-mentioned differential metabolites mainly involve Protein digestion and absorption (Protein metabolism and absorption), ABC transporters (ABC transporters), pyrimidine metabolism (pyrimidate metabolism), central carbon metabolism of cancer (Central carbohydrate metabolism in cancer), alanine, aspartate and glutamate metabolism (Alanine, aspartate and glutamate metabolism), mineral absorption (Mineral absorption), glyoxylate and dicarboxylate metabolism (glycine and diacylate metabolism), GABAergic synapses (GABAergic synapse), aminoacyl tRNA Biosynthesis (aminoacyl i-tRNA Biosynthesis), cysteine and methionine metabolism (Cysteine and methionine pathway metabolism), cAMP signaling (threonine), cAMP and methionine Biosynthesis (tyrosine and methionine), and Alanine metabolism (Alanine-methionine) and methionine metabolism (Alanine-methionine metabolism of metabolic pathway), alanine metabolism (metabolic pathway of amino acids), and methionine metabolism (tyrosine metabolism of metabolic pathway), and methionine metabolism (Alanine metabolism of metabolic pathway of tyrosine). In FIG. 15, enrich KEGG Pathways is the abundant KEGG pathway, rich factor is the enrichment factor, metabolite number is the number of metabolites.

3.4. Discussion of the related Art

(1) Blueberry extract for treating ischemic stroke by protecting central nervous system

Ischemic stroke can result in the accumulation of a large number of amino acid neurotransmitters, with excessive release of Excitatory Amino Acids (EAA) being considered as the pathogenesis of ischemic stroke. EAA is mainly present at the synaptic terminals of neurons, but is also present in the cytoplasm of nerve cells and glial cells. Glutamic acid (Glu) and aspartic acid (Asp) are most abundant in various EAAs of the brain. Neural cells are rich in enzymes, particularly glutamate synthase. Most of the Glu released from the presynaptic membrane enters the intercellular space of the adjacent glial cells, a small portion of which is taken up, and the rest of which binds to the postsynaptic receptors. Glu, which is a major excitatory neurotransmitter in the central nervous system, is involved in rapid excitatory synaptic transmission and plays an important role in maintaining normal signaling in nerve cells. However, in pathological conditions, EAA may have toxic effects on nerve cells. The result of the invention shows that the route of metabolic difference foreign matters between the blueberry group and the model group relates to metabolism of aspartic acid and glutamic acid, and one of the mechanisms of the blueberry extract for treating ischemic stroke is presumed to inhibit release of excitatory amino acid after the ischemic stroke occurs, thereby reducing the excitatory toxicity of an organism.

Acetylcholine is the signal transmitter for cholinergic neurons. The conducted signals are related to brain activities such as cognition and behavior, and the metabolic process is closely related to the occurrence and development of ischemic stroke, so that the signals are widely researched. Inflammatory stimulus signals (IL-1 beta, IL-6 and TNF-alpha) can be projected to brain solitary bundle Nucleus (NTS) from an afferent vagus nerve, excited and efferent to the vagus nerve through a vagal motor dorsal nucleus (DMN) by a signal transduction mechanism of a central muscarinic acetylcholine receptor (mAChR), stimulate nerve endings to release acetylcholine (Ach) and act on an alpha 7 nicotinic acetylcholine receptor (alpha 7 nAChR) on immune cells, and achieve the effects of inhibiting the generation and release of cytokines and chemokines and the like through NF-xB, a protein tyrosine kinase 2/signal transducer and a transcriptional activator 3 (JAK 2/STAT 3) signal channel. The content of acetylcholine in the rat brain of the model group is increased, and the content of the acetylcholine can be adjusted back by the blueberry group, so that the blueberry extract can promote the release of Ach to treat ischemic stroke.

An imbalance in the kynurenine pathway is associated with ischemic stroke. The kynurenine pathway consists of two branches, which can form xanthine acid and kynurenine acid, or produce 3-hydroxyanthranilate and 3-methoxyanthranilate. Canine uric acid is considered neuroprotective as an N-methyl-D-aspartate receptor antagonist, while the other branched metabolites, 3-hydroxyanthranilate and 3-methoxyanthranilate, are considered neurotoxic. Accumulation of 3-hydroxyanthranilate and 3-methoxyanthranilate can lead to apoptosis of astrocytes and some nerve cells, thereby weakening the work of microglial neural network, reducing the synthesis of neurotrophic factors, and damaging the whole nervous system. The blueberry group can reduce the content of kynurenine in serum of rats with ischemic stroke, which indicates that the blueberry extract can protect nerve cells by reducing neurotoxic substances so as to achieve the effect of treating ischemic stroke.

(2) Blueberry for treating ischemic stroke by regulating intestinal microbiota metabolism

Metabolites of intestinal flora of dietary molecules can affect intestinal barrier, blood brain barrier, neuroinflammation, vagus nerve activation, neurogenesis and excitotoxicity, regulate flora-intestine-brain axis and vascular function, and affect the occurrence and development of ischemic stroke diseases. These metabolites include neurotransmitters, bile acids, amino acids, short chain fatty acids, trimethylamine, etc., which communicate with the central nervous system via the gut-liver-brain axis, directly into the blood across the blood-brain barrier or via the vagus nerve; the flora may affect the brain through the kynurenine pathway that regulates tryptophan metabolism.

The gut-brain axis is a bi-directional interaction pathway between the gut and central nervous system. Tryptophan (an essential amino acid) contributes to the normal growth and health of animals and humans, and more importantly, it exerts regulatory functions at multiple levels of the brain-gut axis. Tryptophan is metabolized to kynurenine, which synthesizes host serotonin and oxidatively degrades host tryptophan by the kynurenine pathway, thereby modulating neuroendocrine and intestinal immune responses. The intestinal microorganisms play an important role in regulating tryptophan metabolism through kynurenine. After the blueberry extract is used for treatment, the content of kynurenine metabolites is adjusted back, and the blueberry extract has a treatment effect on intestinal metabolic disorder caused by ischemic stroke.

In conclusion, the non-targeted metabonomics method is adopted, and research results based on ultra-high performance liquid chromatography-quadrupole-time of flight tandem mass spectrometry (UHPLC-Q-TOF-MS) show that the blueberry extract has a certain treatment effect on ischemic stroke; meanwhile, an ischemic stroke rat model is established, and the effect of the blueberry extract on ischemic stroke is preliminarily evaluated through pharmacodynamic investigation; by utilizing a 16S rDNA sequencing technology and based on an intestinal flora-metabonomics regulation and control mechanism, the action mechanism of the blueberry extract for treating ischemic stroke is explained, and a scientific basis is provided for development of blueberry extract series products for treating ischemic stroke.

Claims

1. Application of blueberry extract in preparing medicine for improving intestinal flora and metabolites thereof of patients with cerebrovascular diseases.

2. Application of blueberry extract in preparing medicine for treating cerebrovascular diseases by changing intestinal flora.

3. The use of claim 1, wherein the cerebrovascular disease is stroke.

4. The use according to claim 1, wherein the stroke is an ischemic stroke.

5. The use of claim 1, wherein the blueberry extract comprises chlorogenic acid 1300-1500ug/g, cyano-3-O-glucoside 50-70ug/g, rutin 40-60ug/g, procyanidin B1-40 ug/g, D-catechin 10-20ug/g, L-epicatechin 10-20ug/g, quercetin 1-3ug/g.

6. The application as claimed in claim 1, wherein the preparation method of the blueberry extract comprises the following steps:

7. Use according to claim 6, wherein the organic solvent is an ethanol solution.

8. Use according to claim 6, wherein the elution is with an eluent comprising a water and/or ethanol solution.

9. Use according to claim 6, wherein the elution is carried out with a resin.

10. The use according to claim 6, further comprising a drying step after said elution.