Application of lycium barbarum glycopeptide in preparation of medicine for preventing or treating ischemic encephalopathy

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of lycium barbarum glycopeptide in preparation of a medicine for preventing or treating ischemic encephalopathy. Specifically, the invention relates to application of lycium barbarum glycopeptide in preparation of drugs for preventing or treating cerebral arterial thrombosis. Pharmacological experiments show that the lycium barbarum glycopeptide has an obvious treatment effect on dyskinesia and neurological impairment of focal ischemic stroke model rats caused by middle cerebral artery ischemia reperfusion; the compound also has a remarkable improvement effect on learning and memory cognitive impairment of the model rat, so that the compound can be used for preparing medicines for preventing or treating related ischemic encephalopathy.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to the application of Lycium barbarum glycopeptide in preparing a drug for preventing or treating ischemic encephalopathy. Background Art

[0002] Ischemic encephalopathy is a common disease in the elderly caused by insufficient blood supply to the brain due to stenosis or occlusion of the cerebral blood supply arteries, which leads to brain tissue necrosis. Common clinical symptoms include unconsciousness, difficulty speaking, facial paralysis, limb numbness, weakness, sensory impairment, dizziness, ataxia, etc. The incidence of ischemic encephalopathy is high, and the mortality and disability rates of this type of disease are also high. Because this type of disease has many pathogenic factors, slow pathological progression, long course of disease, and high treatment costs, it has brought a heavy burden to society and families, and has developed into one of the public health problems that need to be solved urgently in China and even the world.

[0003] At present, the treatment of ischemic encephalopathy is mainly based on prevention (control of risk factors such as blood pressure, blood sugar, smoking, obesity, education, etc.), supplemented by treatment (treatment of symptoms). In clinical practice, the means of treating ischemic encephalopathy include intravenous thrombolytic drugs, mechanical thrombectomy, antiplatelet aggregation drugs, lipid-lowering drugs, neuroprotective agents, etc. However, due to the complexity and diversity of the pathological mechanism of ischemic encephalopathy and the large individual differences among patients, the efficacy of these clinical drugs is very limited. my country is rich in traditional Chinese medicine resources, and has unique advantages in screening and developing single Chinese medicines, effective ingredients of Chinese medicines, and compound preparations that are effective, safe, and non-toxic for the prevention and treatment of ischemic encephalopathy from natural products.

[0004] Lycium barbarum, a Chinese medicine, is a plant of the Solanaceae family. It is the mature fruit of Ningxia Lycium barbarum. It has been praised as a precious Chinese herbal medicine since ancient times and has been used in a variety of tonic prescriptions. "Shennong's Herbal Classic" records: "Lycium barbarum treats internal evil, thirst in heat, rheumatism, long-term use of it strengthens the bones and muscles, lightens the body and prevents aging, and is resistant to cold and heat." It tastes sweet and flat, and its pharmacological effects are to nourish the kidney, moisten the lungs, nourish the liver, dispel wind, improve eyesight, and treat liver and kidney yin deficiency, soreness of the waist and knees, dizziness, and vertigo. Lycium barbarum has been included in the list of Chinese herbal medicines for both medicinal and edible purposes by the Ministry of Health of the People's Republic of China since 2002. The homology of medicine and food is the advantage of Lycium barbarum, which has a good development prospect. Modern pharmacological results show that Lycium barbarum has the effects of neuroprotection, scavenging free radicals, and anti-inflammatory, and is widely used in health preservation and health care.

[0005] Lycium barbarum glycopeptide (LbGp) is a glycoconjugate and is considered to be the most promising monomer component in wolfberry fruit. It includes LbGp1 (88kDa), LbGp2 (68.2kDa), LbGp3 (92.5kDa), LbGp4 (214.8kDa), and LbGp5 (23.7kDa). It is mainly composed of sugar chains and covalently bound to polypeptides. Its molecular weight is 88kDa, and its monosaccharide composition is arabinose (Ara), galactose (Gal) and glucose (Glc), with a molar ratio of 2.5:1.0:1.0. Its protein content is 30%, and the linkage between the sugar group and the core protein skeleton is O-linked. It also contains 18 other natural amino acids, which can ensure the quantification and reproducibility of experimental drugs. Studies have shown that LbGp has the effects of lowering blood sugar, lowering blood lipids, reducing depression and anxiety behaviors, and preventing the occurrence and progression of acute colitis.

[0006] However, there has been no relevant report on the preventive or therapeutic effects of Lycium barbarum glycopeptide on ischemic encephalopathy. In view of this, the present inventors have developed a new medical use of Lycium barbarum glycopeptide for preventing or treating ischemic encephalopathy. Summary of the invention

[0007] The purpose of the present invention is to solve the deficiencies of the prior art, to develop and utilize natural products using modern drug research methods, and to provide new medical uses of wolfberry glycopeptide in combination with pharmacodynamic experiments, namely, the use of wolfberry glycopeptide in preventing or treating ischemic encephalopathy.

[0008] Specifically, the present invention is realized through the following technical solutions:

[0009] The present invention provides the use of Lycium barbarum glycopeptide in preparing a medicine for preventing or treating ischemic encephalopathy.

[0010] As an optional mode, in the above application, the present invention provides the use of Lycium barbarum glycopeptide as the sole active ingredient in the preparation of a drug for preventing or treating ischemic encephalopathy.

[0011] As an option, in the above application, the ischemic encephalopathy is ischemic stroke, ischemic cerebral infarction or cerebral ischemic reperfusion.

[0012] As an optional manner, in the above application, the ischemic stroke is selected from one or both of focal ischemic stroke and diffuse ischemic stroke.

[0013] As an optional manner, in the above application, the ischemic stroke is selected from one or more of transient ischemic attack (TIA), reversible infarction (RIND), progressive infarction (SIE) and complete stroke (CS).

[0014] As an optional mode, in the above application, the Lycium barbarum glycopeptide reduces the iron content of brain tissue after ischemia and inhibits ferroptosis.

[0015] As an optional method, in the above application, the Lycium barbarum glycopeptide reduces the volume of cerebral infarction, reduces neuronal death, and improves learning and memory ability and limb coordination and fine movement.

[0016] As an optional method, in the above application, the preparation method of the wolfberry glycopeptide mainly includes the steps of washing, soaking, crushing and beating, leaching, double-pass beating, two-stage separation, membrane separation, single-effect concentration, and freeze-drying. Among them, wolfberry seeds are separated in the double-pass beating step, wolfberry pulp is separated in the two-stage centrifugation step, and oligosaccharides are separated in the membrane separation step, and the final product obtained is wolfberry glycopeptide.

[0017] As an optional manner, in the above application, the Lycium barbarum glycopeptide comprises one or more of the following: LbGp1, LbGp2, LbGp3, LbGp4 or LbGp5.

[0018] Among them: the protein content in LbGp1 is 28.87%, the protein content in LbGp2 is 9.25%, the protein content in LbGp3 is 5.19%, the protein content in LbGp4 is 10.75% and the protein content in LbGp5 is 59.87%.

[0019] As an optional manner, in the above application, the wolfberry glycopeptide contains LbGp1, LbGp2, LbGp3, LbGp4 and LbGp5, wherein the mass ratio of LbGp1, LbGp2, LbGp3, LbGp4 and LbGp5 is (0.01-0.02): (0.005-0.01): (0.01-0.02): (0.02-0.03): (0.01-0.02).

[0020] Preferably, in the wolfberry glycopeptide, the mass ratio of LbGp1, LbGp2, LbGp3, LbGp4 and LbGp5 is 0.018:0.0077:0.018:0.029:0.014.

[0021] As an optional mode, in the above-mentioned use, the medicine comprises Lycium barbarum glycopeptide and a pharmaceutically acceptable carrier.

[0022] As an optional mode, in the above-mentioned use, the dosage form of the drug is tablets, pills, oral liquid, capsules, syrups, pellets, granules, injections, powder injections, sprays, aerosols, powder sprays or suppositories.

[0023] As an optional mode, in the above-mentioned use, the drug further comprises other active ingredients, wherein the other active ingredients are selected from: one or more of urokinase, streptokinase, recombinant tissue-type plasminogen activator, reteplase, rivastigmine, memantine, ginkgo biloba extract, ginsenosides, huperzine A, distilbene glycosides, levodopa, carbidopa, benserazide, dimenhydrinate, benztropine, procyclidine, profenofibrate, butylphthalide, dipyridamole, low molecular weight dextran, heparin, urokininogenase, citicoline, edaravone, nimodipine or aspirin.

[0024] More preferably, the other active ingredients are selected from: huperzine A, levodopa or nimodipine.

[0025] Preferably, in the medicine, the weight ratio of Lycium barbarum glycopeptide to other active ingredients is 10:1-1:10.

[0026] More preferably, in the medicine, the weight ratio of Lycium barbarum glycopeptide to other active ingredients is selected from 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10.

[0027] Most preferably, in the medicine, the weight ratio of Lycium barbarum glycopeptide to other active ingredients is selected from 1:3, 1:4, 1:5 or 1:6.

[0028] Among them, in the medicine, the combination of Lycium barbarum glycopeptide and other active ingredients (especially, huperzine A, levodopa or nimodipine) has a significant synergistic effect in preventing or treating ischemic encephalopathy (especially ischemic stroke).

[0029] The experimental method used in the detailed description of the present application can be used to verify the synergistic effect of the combination of the above-mentioned Lycium barbarum glycopeptide and other active ingredients, which is easy for those skilled in the art.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The inventors have discovered for the first time the use of Lycium barbarum glycopeptide in preparing a drug for preventing or treating ischemic encephalopathy.

[0032] Pharmacological experiments have shown that Lycium barbarum glycopeptide has a significant therapeutic effect on movement disorders and neurological damage in rats with focal ischemic stroke model caused by middle artery ischemia-reperfusion; it also has a significant improvement effect on learning and memory cognitive disorders in the model rats, so it can be used to prepare drugs for preventing or treating related ischemic encephalopathy. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0034] Figure 1 :Effects of different ischemic time on the volume of cerebral infarction and mortality in rats (n=4). Figure 1 A: Representative photographs of TTC-stained brain sections (2 mm) at day 3 after MCAO. Figure 1 B: Different ischemic time resulted in different infarct volumes in rat brain. Figure 1 C: The survival rate of rats with different ischemia times within three days. ** P<0.01Compared with the Vehicle group, ## P<0.01 compared with the 1.5h group.

[0035] Figure 2 : Effects of Lycium barbarum glycopeptide on the volume of cerebral infarction after MCAO (n=6). Figure 2 A MRI shows the representative images of the size of the brain infarct lesions in rats 24h and 30d after MCAO; Figure 2 B: Changes of infarct volume in each group at 24 h; Figure 2 C: Changes of infarct volume in each group at 30 days. * P<0.05, ** P<0.01 compared with the Vehicle group; - indicates no LBGP administration, + indicates LBGP administration.

[0036] Figure 3 : Effects of different concentrations of Lycium barbarum glycopeptide on the behavior of MCAO rats (n=6). Figure 3 A. Time spent in the central area of ​​the open field; Figure 3 B. Time required to find the platform every day during the water maze training period. ** P<0.01 compared with the Vehicle group.

[0037] Figure 4 : Effects of LBGP on brain tissue iron content, MDA, SOD and GSH (n=3). Figure 4 A. Changes in total iron content in each group; Figure 4 B. Changes of MDA content in each group; Figure 4 C. Changes of SOD content in each group; Figure 4 D. Changes of GSH content in each group. ** P < 0.01 compared with the sham group; ## P < 0.01 compared with the Vehicle group; && P<0.01 compared with LBGP+Erastin.

[0038] Figure 5 : Effects of LBGP or combined with ferroptosis inhibitor (Lip-1) or ferroptosis activator (Erastin) on the size of cerebral infarction in MCAO rats (n=6). Figure 5 A MRI shows the changes in cerebral infarction area of ​​the rats in each group 24h and 30d after MCAO. Figure 5 B. The percentage change of cerebral infarction area in the brains of rats in each group 24 hours after MCAO. Figure 5 C. Percentage of changes in cerebral infarction area in rats of each group 30 days after MCAO * P<0.05Compared with the Vehicle group, ** P < 0.01 compared with the Vehicle group; # P<0.05, ## P<0.01 compared with LBGP+Era; - indicates no MCAO, + indicates MCAO.

[0039] Figure 6 : Lycium barbarum glycopeptide reduces neuronal death (n=3). Figure 6 A Representative micrographs of Nissl-stained brain sections. The infarct core (IC) is indicated by a dashed outline, and the area adjacent to the infarct core is marked by a rectangle with an area of ​​500 × 1500 μm; Figure 6 B. Changes in the number of neuronal cells in the cortex. ** P < 0.01 compared with the sham group; ## P < 0.01 compared with Vehicle; && P<0.01 compared with LBGP+Era.

[0040] Figure 7 : Effects of Lycium barbarum glycopeptide, Lip-1 and Erastin on neurological function (n=6). Figure 7 A Neurological function score 24 h after MCAO; Figure 7 B Neurological function scores 30 days after MCAO. ** P < 0.01 compared with the Vehicle group; ## P<0.01 compared with LBGP+Era.

[0041] Figure 8 :Lycium barbarum glycopeptide improves fine motor skills and limb motor endurance (n=6). Figure 8 A The time taken to tear off the tape on the left forelimb; Figure 8 B Rotarod rat persistence time. ** P < 0.01 compared with the Sham group; ## P < 0.01 compared with Vehicle; &&P<0.01 compared with LBGP+Era group. DETAILED DESCRIPTION

[0042] The present invention uses modern drug research methods to develop and utilize natural products, combines pharmacodynamic experiments, and through a large number of screenings, finds for the first time that Lycium barbarum glycopeptide has the effect of preventing or treating ischemic encephalopathy (especially ischemic stroke). On this basis, the present invention was completed.

[0043] As used herein, the dosage form of the pharmaceutical preparation of the present invention is a tablet, capsule, granule, oral solution or injection. Preferably, the dosage form of the present invention is a tablet or capsule.

[0044] As used herein, the "pharmaceutically acceptable carrier" of the present invention refers to a conventional drug carrier in the field of pharmaceutical preparations, selected from one or more of fillers, binders, disintegrants, lubricants, suspending agents, wetting agents, pigments, flavoring agents, solvents, and surfactants.

[0045] The filler of the present invention includes but is not limited to starch, microcrystalline cellulose, sucrose, dextrin, lactose, powdered sugar, glucose, etc.; the lubricant includes but is not limited to magnesium stearate, stearic acid, sodium chloride, sodium oleate, sodium lauryl sulfate, poloxamer, etc.; the binder includes but is not limited to water, ethanol, starch slurry, syrup, hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, sodium alginate, polyvinyl pyrrolidone, etc.; the disintegrant includes but is not limited to starch effervescent mixture, i.e. sodium bicarbonate and citric acid, tartaric acid, low-substituted hydroxypropyl cellulose, etc.; the suspending agent includes but is not limited to polysaccharides such as acacia gum, agar, alginic acid, cellulose ether and carboxymethyl chitosan, etc.; the solvent includes but is not limited to water, a balanced salt solution, etc.

[0046] Preferably, the drug of the present invention can be made into various solid oral preparations, liquid oral preparations, etc. Pharmaceutically acceptable oral solid preparations include: ordinary tablets, dispersible tablets, enteric-coated tablets, granules, capsules, dripping pills, powders, etc., and oral liquid preparations include oral liquids, emulsions, etc.

[0047] In addition, the drug of the present invention can also be made into injections, including water injections or freeze-dried powder injections.

[0048] The above-mentioned various dosage forms can be prepared according to conventional processes in the field of pharmaceutical preparations.

[0049] As used herein, the "Lycium Barbarum Glycopeptide (LbGp)" powder used in the present invention is provided by Ningxia Tianren Lycium Barbarum Biotechnology Co., Ltd.

[0050] The preparation of the wolfberry glycopeptide of the present invention includes extracting and separating the water extract of wolfberry by pure physical centrifugation, membrane filtration purification, vacuum freeze drying and other process technologies to obtain a polysaccharide and a covalently bonded glycoconjugate of a peptide chain. It contains LbGp1, LbGp2, LbGp3, LbGp4 and LbGp5. Among them: the protein content in LbGp1 is 28.87%, the protein content in LbGp2 is 9.25%, the protein content in LbGp3 is 5.19%, the protein content in LbGp4 is 10.75% and the protein content in LbGp5 is 59.87%. In the wolfberry glycopeptide, the mass ratio of LbGp1, LbGp2, LbGp3, LbGp4 and LbGp5 is 0.018:0.0077:0.018:0.029:0.014.

[0051] In the medical uses described above, the administration time, number of administrations and frequency of administration of "Lycium barbarum Glycopeptide" and other active ingredients need to be determined according to the specific diagnosis results of the disease, which is within the technical scope of those skilled in the art. For example, the treatment plan for mice or rats is applied to humans, and the effective doses of all drugs for humans can be converted by the effective doses of the drugs for mice or rats, which is also easy for ordinary technicians in this field to achieve.

[0052] The present invention will be further described with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0053] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0054] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0055] Unless otherwise specified, all percentages and parts in the present invention are percentages and parts by weight.

[0056] Example:

[0057] 1. Preparation method of wolfberry glycopeptide

[0058] The preparation process of Lycium barbarum glycopeptide has the following steps: raw materials (dried wolfberries) - washing - soaking - crushing - extraction - filtration - concentration - freeze drying - finished product. The main overview is as follows: After picking, the wolfberries need to be dried, washed, and chopped, and ether and acetone are used to extract the fat-soluble and partially water-soluble pigments in the wolfberries, and then soaked in neutral water. After soaking, the liquid is filtered and centrifuged to extract the supernatant, and the supernatant is precipitated with anhydrous ethanol. Then the precipitate is dissolved with water and the protein in the solution is freed with a mixture of chloroform and n-butanol. After multiple dialysis and centrifugation to remove the precipitate, the supernatant is freeze-dried to obtain LBP. The extracted crude polysaccharide LBP is then purified again. First, LBP is subjected to column chromatography using DEAE-Cellulose and eluted with 0.05 mol / L NaHCO3 solution to obtain LBP-1. Next, LBP-1 needs to be dialyzed, desalted, and concentrated, and then subjected to column chromatography using Sephadex G100, followed by elution with 0.10 M NaCl solution. The eluted solution can be freeze-dried to obtain Lycium barbarum glycopeptide (LbGp).

[0059] 2. Experimental Methods

[0060] 2.1 Experimental animals

[0061] Male Sprague-Dawley rats (200-250 g, 8-10 weeks) were used in this experiment and purchased from Liaoning Changsheng. All animals were housed in the Experimental Animal Center of Jinan University School of Medicine, which is SPF grade and has stable conditions of 23-25°C, 12 h light / 12 h dark cycle, and free access to food and water. The use of experimental animals was in accordance with the management regulations of the Experimental Animal Ethics Committee of Jinan University, and the experimental protocol was approved by the Ethics Committee.

[0062] 2.2 Preparation of focal cerebral ischemia-reperfusion model (MCAO)

[0063] (1) Preoperative preparation:

[0064] Disinfect the surgical work surface with disinfectant and prepare sterile surgical kits including surgical instruments, gauze, sutures, and surgical blades by autoclaving. Wear a surgical mask, hair cap, and sterile gloves and surgical gowns during the operation. Use a constant temperature heating pad placed under the rat to prevent hypothermia during the operation.

[0065] (2) Preparation of rat MCAO model:

[0066] The SD rats were placed in an anesthesia box and induced with 5% isoflurane (the anesthesia machine was set to 1.0L / min O2 and 1.0L / min N2O). When the rats lost consciousness, the flow of the anesthesia machine was reduced to 1-2% isoflurane to maintain anesthesia. The hair on the neck of the rats was shaved with a shaver. The skin was disinfected with medical iodine tincture, and the skin was wiped spirally outward from the center of the surgical area, and repeated two or three times. The rats were placed on a sterile heating pad and covered with sterile surgical cloth. Under a microscope, the skin and superficial fascia were incised in the midline of the ventral neck. In the triangle formed by the right sternocleidomastoid muscle and the infrahyoid group of muscles, the external carotid artery (ECA), internal carotid artery (ICA) and common carotid artery (CCA) were bluntly separated. Be careful not to injure the vagus nerve. Use 5-0 silk thread to tie the separated CCA and ECA about 1-1.5cm away from the bifurcation of the artery; tie the ECA tightly near the bifurcation of the artery; and tie another knot between this knot and the distal knot to fix the inserted thread plug; use vascular scissors to cut a small incision on the external carotid artery at the knot near the distal end of the ECA; insert the thread plug with a silicone head through the incision, loosen the distal knot of the ECA, cut the ECA from the outside of the distal knot, turn the ECA downward so that it is in a straight line with the ICA, and slowly send the thread plug into the middle cerebral artery (MCA). If you feel resistance, stop sending the thread plug immediately. Use 4-0 silk thread to quickly suture the incision, and carefully put the rat into the recovery cage to move freely. After 1.5h of MCA occlusion, re-anesthetize the rat, disinfect the incision site with iodine, and reopen the incision; pull the thread plug out of the ICA and tie the knot at the stump to prevent blood from flowing out. Untie the CCA knot and restore MCA blood flow; drip a small amount of sterile saline into the incision and suture the incision with 4-0 silk sutures; after the rat wakes up, grab the rat's tail and observe whether the rat's head is deflected to preliminarily determine whether the MCAO model is successful.

[0067] 2.3 TTC staining to observe changes in cerebral infarction volume

[0068] Use conventional methods.

[0069] 2.4 Neurological function score

[0070] The Modified neurological severity scores (mNSS) scoring system was used to score movement, sensation, reflexes, balance and neurological function on the 1st and 28th day after MCAO. The scoring criteria are as follows: 0 points for normal state. 1-6 points for mild damage. 7-12 points for moderate damage. 13-18 points for severe damage, and the highest score is 18 points. The detailed scoring criteria are shown in Table 1 below:

[0071] Table 1: mNSS rating scale

[0072]

[0073]

[0074] 2.5 Iron, MDA, SOD and GSH content in tissues

[0075] The levels of iron ions, MDA, SOD, and GSH in brain tissue were measured using conventional methods using a kit.

[0076] 2.6 MRI

[0077] The infarction of rat brain tissue was examined using a 9.4T small animal magnetic resonance imaging system. The rats were first anesthetized (using 5% isoflurane gas anesthesia) and placed on a scanning platform. The infarction area of ​​each brain image was measured and the percentage of cerebral infarction volume was calculated according to the Image-J data analysis program of the image analyzer.

[0078] 2.7 Rat brain tissue processing and staining

[0079] The rat brain tissues were processed and sliced ​​using conventional methods, and then subjected to Nissl staining and immunofluorescence staining.

[0080] 2.8 Conduct behavioral tests on rats to observe behavioral changes

[0081] The open field test, adhesive removal test, rotarod test, sugar water preference test, and Morris water maze test were performed using conventional methods.

[0082] 2.9 Data Analysis

[0083] GraphPad Prism8 software was used for statistical analysis of data and drawing statistical graphs, and ImageJ and Image-Pro Plus 6.0 were used to analyze immunofluorescence images. Data were expressed as mean ± standard deviation (X ± SD), and one-way ANOVA was used for comparison between multiple groups. Independent sample Tukey's HSD test was used for comparison between two independent groups, and P < 0.05 was considered statistically significant.

[0084] 3. Experimental results

[0085] 3.1 Neuroprotective Effect of Lycium barbarum Glycopeptide on MACO Rats

[0086] (1) Establishment of rat MCAO model

[0087] In order to explore the protective effect of Lycium barbarum glycopeptide on cerebral ischemia-reperfusion injury, a MCAO rat model was established. The middle cerebral artery of the rats was ligated for 1, 1.5 and 2 hours respectively by thread occlusion. TTC staining was performed on the 3rd day after surgery to determine the infarct volume ( Figure 1 A). The results showed that after 1 hour of ischemia, the infarct volume was 9.8±2.5% (n=4); after 1.5 hours of ischemia, the infarct volume was 25.8±2.8% (n=4); after 2 hours of ischemia, the infarct volume was 45.5±2.2% (n=4) ( Figure 1 B), while the survival rate of rats after MCAO was 100% on the first day, on the second day, the survival rate of the ischemic 0h and 1h groups remained at 100%, the survival rate of the ischemic 1.5h group dropped to 90%, and the survival rate of the ischemic 2h group dropped to 80%; on the third day, the survival rate of the ischemic 0h group was 100%, the survival rate of the ischemic 1h group dropped to 90%, the survival rate of the ischemic 1.5h group dropped to 80%; the survival rate of the ischemic 2h group dropped to 60%. Based on the above results, the infarct lesions of ischemic 1h are smaller and easier to recover, making it difficult to reflect the effect of the drug; while the infarct lesions of ischemic 2h are larger, the survival rate is low, which increases the difficulty of the experiment, and it is difficult to accurately judge the efficacy of the drug. Therefore, SD rats with ischemic 1.5h were selected for subsequent research.

[0088] (2) Effects of different concentrations of Lycium barbarum glycopeptide on the volume of infarct after MCAO

[0089] The cerebral infarction volume of rats was measured by 9.4T small animal magnetic resonance imaging system at 24h and 30d after MCAO. The experimental SD rats were divided into five groups: sham group, vehicle group (oral administration of normal saline), 20mg / kg LBGP group (oral administration), 40mg / kg LBGP group (oral administration), and 60mg / kg LBGP group (oral administration).

[0090] like Figure 2 As shown in A and 2B, no infarct was detected in the sham group. The infarct volume of the Vehicle group increased significantly 24 hours after MCAO, reaching 25.8±1.8%. 30 days after MCAO, the infarct volume remained at 21.3±0.9%. The infarct volume of the 20mg / kg LBGP group was 27.4±2.5% 24 hours after MCAO and 16.5±2.0% 30 days after MCAO. The infarct volume of the 40mg / kg LBGP group was 28.9±4.1% 24 hours after MCAO and 19.3±1.8% 30 days after MCAO. The infarct volume of the 60mg / kg LBGP group was 27.5±3.6% 24 hours after MCAO and 18.1±4.5% 30 days after MCAO. Based on the above data, it is believed that Lycium barbarum glycopeptide can effectively reduce the infarct volume of the infarct after transient cerebral ischemia reperfusion, and the recovery effect of the 20mg / kg LBGP dosage is more significant.

[0091] (3) Effects of different concentrations of Lycium barbarum glycopeptide on the behavior of MCAO rats

[0092] On the 14th day after MCAO, the rats were subjected to an open field test. The experimental results showed that ( Figure 3 A) After MCAO, the time that rats (Vehicle group) stayed in the central area decreased significantly (10.3s±1.3), which was significantly different from the sham group (64.4s±4.5), P<0.01. The 20mg / kg LBGP group (40.8s±7.1), 40mg / kg LBGP group (39.6s±6.7) and 60mg / kg LBGP group (31.8s±11.2) also showed significant differences compared with the Vehicle group, P<0.01. In addition, the 20mg / kg LBGP group spent more time in the central area and had milder anxiety symptoms than the 40mg / kg LBGP group and the 60mg / kg LBGP group.

[0093] Next, the learning and memory abilities of the rats were tested on the 21st day after MCAO. Figure 3 B), on the first day of training, the escape latency of the five groups of animals did not change significantly and remained at 60 seconds; on the second day, the escape latency of the sham group animals decreased significantly, reaching 42.3s±4.6; while the Vehicle (58.9s±0.1), 20mg / kg LBGP (55.8s±1.0), 40mg / kg LBGP (59.3s±1.2) and 60mg / kg LBGP groups (58.1s±4.3) did not change much compared with the first day, and the sham group was compared with the other groups, P<0.05. On the third day, the sham group (36.9s±7.1) shortened the time to find the platform compared with the Vehicle (54.1s±0.9), 20mg / kg LBGP (45.4s±3.5), 40mg / kg LBGP (48.4s±4.1) and 60mg / kg LBGP groups (50.6s±1.5), P<0.05. On the fourth day, the time difference between the sham group (23.51s±1.2), 2mg / kg LBGP (33.9s±2.3), 40mg / kg LBGP (39.1s±4.1) and 60mg / kg LBGP group (44.62s±3.5) and the Vehicle group (47.8±2.2) was even greater. On the fifth day, the time to find the platform in the 20mg / kg LBGP group (20.2s±1.1), 40mg / kg LBGP group (35.1s±3.8) and 60mg / kg LBGP group (27.67s±3.9) was closer to that in the sham group (12.71s±1.6), which was significantly different from that in the Vehicle group (46.28s±3.2), P<0.01( Figure 3 B).

[0094] Therefore, combined with the results of pathological and behavioral tests, 20 mg / kg LBGP is considered to be an ideal dosing concentration and will be used in subsequent studies.

[0095] 3.2 Lycium barbarum glycopeptide inhibits ferroptosis and oxidative stress

[0096] Liproxstatin-1 (Lip-1) is a known inhibitor of ferroptosis, while Erastin is a known inducer of ferroptosis. These two reagents were used to explore whether LBGP can protect neurons and improve behavior by inhibiting ferroptosis after ischemia. The rats were divided into 6 groups, namely Sham (sham operation group), Vehicle group (oral administration of normal saline) with MCAO modeling, 20mg / kg LBGP group (oral administration), 5mg / kg Lip-1 group (intraperitoneal injection), 20mg / kg LBGP (oral administration) + 5mg / kg Lip-1 (intraperitoneal injection) group and 20mg / kg LBGP + 25mg / kg Erastin (intraperitoneal injection) group.

[0097] Ferroptosis is caused by an increase in the iron content in cells. The results showed that the iron content in brain tissue of the Vehicle group (14.7±0.8μmol / gprot) increased significantly after MCAO. Figure 4 A) Compared with the sham group (4.1±0.9μmol / gprot), P<0.01; while the iron ion content in the brain tissue of the LBGP group (7.7±1.2μmol / gprot), Lip-1 group (7.0±0.7μmol / gprot) and LBGP+Lip-1 group (7.1±1.4μmol / gprot) was significantly decreased, compared with the Vehicle group, P<0.01. However, after the injection of Erastin, the iron ion content increased significantly, and the iron content in the LBGP+Erastin group was 10.5±0.7μmol / gprot, compared with the LBGP group, Lip-1 group and LBGP+Lip-1 group, P<0.01. From the above results, it can be concluded that LBGP can reduce the iron ion content in the brain tissue of MCAO rats.

[0098] The results showed that after MCAO, the MDA in the brain tissue of the Vehicle group (3.9±0.4nmol / mgprot) increased significantly ( Figure 4B), compared with the sham group (1.9±0.1nmol / mgprot), P<0.01; however, the MDA in the LBGP group (2.4±0.1nmol / mgprot), Lip-1 group (2.3±0.3nmol / mgprot) and LBGP+Lip-1 group (2.3±0.4nmol / mgprot) decreased significantly, compared with the Vehicle group, P<0.01. Erastin injection also weakened the effect of LBGP, causing MDA to increase significantly again to 3.4±0.2nmol / mgprot, compared with the above three groups, P<0.01. SOD( Figure 4 C) In the sham group, it was 2.4±0.2U / mgprot, and it dropped to 1.1±0.1U / mgprot after MCAO modeling, and the difference between the two groups was P<0.01; when LBGP or Lip-1 or both were administered, the SOD value increased to 1.6±0.1U / mgprot and 1.7±0.1U / mgprot, respectively, compared with the Vehicle group, P<0.01; but after Erastin injection, SOD dropped significantly to 1.2±0.1U / mgprot, compared with the first three groups, P<0.01. Changes in GSH ( Figure 4 D) is similar to SOD. GSH was 20.0±0.5μg / mgprot in the sham group, and dropped to 9.1±0.1μg / mgprot (Vehicle group) after MCAO. The two groups were compared, P<0.01; while the GSH in the LBGP group (15.8±1.7μg / mgprot), Lip-1 group (16.3±2.2μg / mgprot) and LBGP+Lip-1 group (17.9±1.8μg / mgprot) was significantly increased compared with the Vehicle group, P<0.01, but after the re-injection of Erastin, it was significantly reduced to 7.6±0.3μg / mgprot, compared with the first three groups, P<0.01. From the above data, it can be seen that the iron ion content in the brain tissue of rats after MCAO increased significantly, and the iron ion content in the brain tissue can be effectively reduced after LBGP administration. In addition, Lycium barbarum glycopeptide has a significant alleviating effect on the oxidative stress in the brain tissue of rats after MCAO.

[0099] 3.3 Pathological detection of LBGP in MCAO rats

[0100] (1) LBGP reduces cerebral infarction in MCAO rats by inhibiting ferroptosis

[0101] The infarct volume of the brain of rats in each group was measured using a 9.4T small animal magnetic resonance imaging system. Figure 5A-5C shows the MRI infarct volume (red box area) of the rat brain in each group. Quantitative analysis found that there was no significant difference in infarct volume among the groups except the sham group after 24 hours, P>0.05. On the 30th day, the percentage of infarct volume in the LBGP group (14.8±2.1%), Lip-1 group (17.32±3.1%), and LBGP+Lip-1 group (15.12±1.8%) was significantly reduced compared with the Vehicle group (20.8±1.1%), P<0.05. However, in the LBGP+Erastin group (19.6±2.6%) with the addition of iron enhancer, LBGP did not significantly reduce the infarct volume, and there was no significant difference in the infarct volume compared with the Vehicle group, P>0.05.

[0102] (2) LBGP reduces neuronal loss after MCAO by inhibiting ferroptosis

[0103] The ischemic penumbra is located between the infarct focus and normal brain tissue in the rat cerebral cortex and is the main observation site for drug effects. Nissl staining was used to observe and analyze the number and morphological changes of neurons in each group of rats. Figure 6 As shown in A, the sham group (1459±71 cells / mm 2 ) rats had full neurons in the ischemic penumbra, darker staining, and more cells; while the neurons in the Vehicle group had lighter staining and fewer cells. 2 ), Lip-1 group (739±81 / mm 2 ) and LBGP+Lip-1 group (901±63 / mm 2 ) had darker staining and more neurons. Quantitative analysis showed that the neurons in these three groups were significantly higher than those in the Vehicle group (470±51 / mm 2 ) and LBGP+Erastin group (513±49 / mm 2 ) were significantly increased compared with those in the control group, P < 0.01.

[0104] 3.4LBGP effectively improves the neurological function and behavioral performance of MCAO rats

[0105] (1) LBGP improves neural function by inhibiting ferroptosis

[0106] Next, neurological function scores were used to analyze the changes in neurological function of rats after drug administration. Figure 7As shown in A-7B, 24h after MCAO, the neurological deficits in each group were severe, with scores ranging from 8 to 12, which were significantly different from the Sham group, P < 0.01, but there was no significant difference between the modeling groups. Neurological function tests were performed again 30d after MCAO. The results showed that the neurological function scores of the LBGP group (3±1), Lip-1 group (3±1) and LBGP+Lip-1 group (4±1) were significantly reduced, compared with the vehicle group (6±1), there were significant differences, P < 0.01, but when LBGP+Erastin (6±1) was co-administered, the neuroprotective function of LBGP was significantly decreased, while the neurological function score increased again, close to the vehicle group.

[0107] (2) LBGP improves limb motor endurance and fine motor skills

[0108] The fine motor skills and motor endurance of the limbs of the rats in each group were evaluated by the tape tearing test and the Rotarod test. The results of the tape tearing test on the 18th day after MCAO showed that ( Figure 8 A), the time for tearing off the tape on the left limb of the rats in the LBGP group (14.2±2.4s) and LBGP+Lip-1 group (15.8±1.8s) was close to that in the sham group (7.2±1.3s), while the time for tearing off the tape on the left limb of the rats in the Vehicle group (39.7±8.3s) and LBGP+Erastin group (37.1±7.3s) was longer, which was significantly different from that in the LBGP group (14.2±2.4s), Lip-1 group (21.5±1.0s), LBGP+Lip-1 group (15.8±1.8s) and sham group, all P<0.01. On the 20th day after MCAO, the results of the Rotarod test showed that the rats in the LBGP group (155.6±11.3s), Lip-1 group (134.3±14.0s) and LBGP+Lip-1 group (170.2±12.9s) had a longer persistence time, which was close to that of the sham group (196.2±21.2s). Figure 8 B), while the rats in the Vehicle group (77±5.6s) and LBGP+Erastin group (103.5±4.1s) persisted for a shorter time, which was significantly different from the first four groups, P<0.01. Through the analysis of the test results of tape tearing and Rotarod, Lycium barbarum glycopeptide can effectively restore the fine motor coordination ability and exercise endurance of rats after MCAO, and this recovery effect will be reduced by the ferroptosis promoter Erastin.

[0109] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Application of Lycium barbarum glycopeptide in the preparation of drugs for preventing or treating ischemic encephalopathy.

2. The use according to claim 1, characterized in that: The ischemic encephalopathy is ischemic stroke, ischemic cerebral infarction or cerebral ischemic reperfusion.

3. The use according to claim 2, characterized in that: The ischemic stroke is selected from one or both of focal ischemic stroke and diffuse ischemic stroke.

4. The use according to claim 2, characterized in that: The ischemic stroke is selected from one or more of transient ischemic attack (TIA), reversible infarction (RIND), progressive infarction (SIE) and complete stroke (CS).

5. The use according to claim 1, characterized in that: The wolfberry glycopeptide reduces the iron content of brain tissue after ischemia and inhibits ferroptosis.

6. The use according to claim 1, characterized in that: The Lycium barbarum glycopeptide reduces the volume of cerebral infarction, reduces neuronal death, and improves learning and memory abilities and limb coordination and refined movement.

7. The use according to claim 1, characterized in that: The Lycium barbarum glycopeptide comprises one or more of the following: LbGp1, LbGp2, LbGp3, LbGp4 or LbGp5.

8. The use according to claim 7, characterized in that: The wolfberry glycopeptide comprises LbGp1, LbGp2, LbGp3, LbGp4 and LbGp5, wherein the mass ratio of LbGp1, LbGp2, LbGp3, LbGp4 and LbGp5 is (0.01-0.02): (0.005-0.01): (0.01-0.02): (0.02-0.03): (0.01-0.02).

9. The use according to any one of claims 1 to 8, characterized in that: The medicine comprises wolfberry glycopeptide and a pharmaceutically acceptable carrier.

10. The use according to claim 9, characterized in that: The dosage form of the drug is tablet, pill, oral liquid, capsule, syrup, pill, granule, injection, powder injection, spray, aerosol, powder spray or suppository.

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