Use of water-soluble tomato concentrate in preparation of composition for treating cerebral ischemia-reperfusion injury

Abstract

A use of water-soluble tomato concentrate in the preparation of a drug for preventing or treating cerebral ischemia-reperfusion injury or in the preparation of a composition for preventing or treating cerebral ischemia-reperfusion injury. After long-term research, on the basis of neurological function evaluation, tetrazolium red staining and HE staining, the researchers first found and proved that water-soluble tomato concentrate has a significant repair effect on ischemic stroke reperfusion injury, can significantly reduce the cerebral infarction area, lower the neurological deficits score and improve the brain tissue pathology, and can be applied to the preparation of a composition for preventing or treating cerebral ischemia-reperfusion injury.

Description

Application of water-soluble tomato concentrate in preparing composition for treating cerebral ischemia-reperfusion injury

[0001] Related applications

[0002] This application claims priority to Chinese patent application No. 2023107389753, filed with the Patent Office of China on June 21, 2023, entitled “Application of water-soluble tomato concentrate in the preparation of a composition for treating cerebral ischemia-reperfusion injury”, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present application relates to the field of biomedicine technology, and in particular to the use of a water-soluble tomato concentrate in preparing a composition for treating cerebral ischemia-reperfusion injury. Background Art

[0004] The statements herein merely provide background information related to the present application and do not necessarily constitute prior art.

[0005] Stroke, also known as cerebral infarction, is an acute cerebrovascular disease caused by ruptured or blocked blood vessels in the brain, leading to a lack of blood supply and consequent brain damage. It is characterized by high morbidity, disability, and recurrence rates. Depending on the pathogenesis, it can be divided into hemorrhagic and ischemic strokes. Ischemic strokes, which result from stenosis or occlusion of important intracranial blood supply arteries, disrupt cerebral blood circulation, damage brain tissue, and cause focal neurological dysfunction. They account for approximately 80% of all strokes.

[0006] Currently, there are few treatment strategies for ischemic stroke. Recanalization and restoration of blood supply are the primary treatment options for ischemic stroke. Clinically, these strategies include intravenous thrombolysis and vascular interventional therapy. Their mechanisms of action are to rapidly restore blood oxygen supply to the ischemic site and mitigate ischemic brain damage. For example, the anticoagulant (tissue plasminogen activator, t-PA) is used to dissolve blood vessels, thereby restoring blood supply and alleviating neuronal damage. Because the brain is a specialized organ with extremely high energy demands and is extremely sensitive to ischemia and hypoxia, brain damage caused by ischemia progresses rapidly, resulting in a very short window of time during which recanalization therapy can be beneficial. Recanalization beyond this window is likely to result in secondary reperfusion injury, termed cerebral ischemia-reperfusion injury (CIRI).

[0007] The mechanisms involved in CIRI include excitotoxicity, free radical damage, neuroinflammation, and neuronal apoptosis. Studies have shown that ischemia-reperfusion can cause ischemia-reperfusion injury through a series of pathophysiological pathways, including mitochondrial dysfunction, cellular calcium overload, excitotoxicity, and apoptosis, further aggravating neuronal structural damage and dysfunction, leading to greater neuronal necrosis and apoptosis, and increased cerebral infarction area.

[0008] CIRI is an important factor leading to severe disability and death in stroke patients. Treatment of CIRI has always been an inevitable challenge in the treatment of ischemic stroke. Clinically, treatment of post-reperfusion injury in ischemic stroke remains an urgent and unmet need, and the development of new compositions is urgently needed.

[0009] Summary of the Invention

[0010] Based on this, the present application provides a new use of a water-soluble tomato concentrate, which relates to the use of the water-soluble tomato concentrate in preparing a composition for preventing, alleviating or treating cerebral ischemia-reperfusion injury.

[0011] The specific technical solutions are as follows:

[0012] In a first aspect of the present application, a water-soluble tomato concentrate is provided for use in preparing a medicament for preventing or treating cerebral ischemia-reperfusion injury, or in preparing a composition for preventing or treating cerebral ischemia-reperfusion injury.

[0013] In one embodiment, the prevention or treatment of cerebral ischemia-reperfusion injury includes improving neurological function damage.

[0014] In one embodiment, the prevention or treatment of cerebral ischemia-reperfusion injury includes reducing the area of ​​cerebral infarction.

[0015] In one embodiment, the prevention or treatment of cerebral ischemia-reperfusion injury includes improving brain tissue pathology.

[0016] In one embodiment, the composition for preventing or treating cerebral ischemia-reperfusion injury is in the form of an oral or injectable dosage form, and the oral or injectable dosage form includes a solution, powder, tablet, granule, capsule, emulsion and suspension.

[0017] In one embodiment, the composition for preventing or treating cerebral ischemia-reperfusion injury is in the form of a solution, mainly consisting of a water-soluble tomato concentrate and a solvent.

[0018] In one embodiment, in the composition for preventing or treating cerebral ischemia-reperfusion injury, the dosage of the water-soluble tomato concentrate is 15 mg / kg to 150 mg / kg.

[0019] In one embodiment, the composition further comprises Danshen extract and / or Ginkgo biloba extract.

[0020] In one embodiment, the composition is a health food composition or a pharmaceutical composition.

[0021] In one embodiment, the method for preparing the composition for preventing or treating cerebral ischemia-reperfusion injury comprises the following steps: mixing a water-soluble tomato concentrate with a solvent to prepare the composition for preventing or treating cerebral ischemia-reperfusion injury.

[0022] In a second aspect of the present application, a composition for preventing or treating cerebral ischemia-reperfusion injury is provided, comprising a water-soluble tomato concentrate and at least one of a salvia miltiorrhiza extract and a ginkgo biloba leaf extract.

[0023] Researchers in this application evaluated the therapeutic efficacy of WSTC on cerebral ischemia-reperfusion injury using a rat middle cerebral artery occlusion / reperfusion (MACO / R) model. Key indicators examined included neurological function assessment, infarct size, and brain tissue pathology. The results demonstrated that water-soluble tomato concentrate significantly repaired ischemic stroke-reperfusion injury, significantly reducing infarct size, lowering neurological function scores, and improving brain tissue pathology. The study suggests that it could be used to prepare a composition for the prevention or treatment of cerebral ischemia-reperfusion injury.

[0024] The details of one or more embodiments of the present application are set forth in the following drawings and description. Other features, objects, and advantages of the present application will become apparent from the description, drawings, and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the conventional technology, the following briefly introduces the drawings required for use in the embodiments or the conventional technology descriptions. Obviously, the drawings described below are merely embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on the disclosed drawings without any creative work.

[0026] FIG1 is a graph showing the neurological function scores of each drug-administered group in Example 1, wherein ## indicates p<0.01 vs SHAM group; * indicates p<0.05 vs MCAO / R group; ** indicates p<0.01 vs MCAO / R group;

[0027] FIG2 is a TTC staining image and a cerebral infarction area image of each drug-administered group in Example 1, wherein ## indicates p<0.01 vs SHAM group; ** indicates p<0.01 vs MCAO / R group;

[0028] FIG3 is a HE staining image of brain tissues of each drug-administered group in Example 1;

[0029] FIG4 is an observation diagram of the gastric mucosa of each drug-administered group in Example 1. DETAILED DESCRIPTION

[0030] The following will be combined with the drawings in the embodiments of this application to clearly and completely describe the technical solutions in the embodiments of this application. Obviously, the embodiments described are only part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0031] The optional scope of the terms "and / or", "or / and", and "and / or" used in this document includes any one of two or more related listed items, and also includes any and all combinations of the related listed items, and the said any and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items.

[0032] In this application, terms such as "first aspect" and "second aspect" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor as implicitly indicating the importance or quantity of the technical features indicated. Furthermore, terms such as "first" and "second" serve only as non-exhaustive enumeration and description and should not constitute a closed-ended limitation on quantity.

[0033] In this application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0034] In this application, when referring to numerical ranges, unless otherwise specified, the numerical ranges are considered continuous and include the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe a feature or characteristic, the ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges subsumed therein.

[0035] The temperature parameters in this application, unless otherwise specified, allow for both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows for temperature fluctuations within the precision range of instrument control.

[0036] Cerebral ischemia-reperfusion injury refers to the situation where, after cerebral ischemia occurs and blood supply is restored within a certain period of time, not only does the brain tissue and function not improve, but more serious neurological dysfunction and tissue damage occur.

[0037] Neurological function encompasses multiple aspects, including consciousness, movement, sensation, vision, coordination, and reflexes, all of which are reflected in clinical neurological function examinations. In animal studies of the middle cerebral artery occlusion / reperfusion (MCAO / R) model, neurological impairment and recovery can be assessed through motor, sensory, consciousness, and reflexes, based on the animal's physiological and pathological characteristics. Neurological function assessment not only serves as a criterion for model success but also provides a basis for observing the progression of pathophysiological development, standardizing models, and conducting research on combined interventions.

[0038] Tetrazolium red (TTC) staining is used to observe cerebral infarction foci. TTC is a lipid-soluble, light-sensitive compound that acts as a proton receptor for the pyridine-nucleoside structural enzyme system in the respiratory chain. It reacts with dehydrogenases in normal tissues to produce deep red, lipid-soluble formazan, which causes normal tissue to appear rose-red. Mitochondrial damage and decreased dehydrogenase activity in ischemic tissue prevent the staining reaction and result in a pale appearance.

[0039] HE staining, also known as hematoxylin-eosin staining (HE), is a commonly used staining method in paraffin sectioning. Hematoxylin, an alkaline stain, primarily stains chromatin within the cell nucleus and nucleic acids within the cytoplasm purplish-blue; eosin, an acidic dye, primarily stains components of the cytoplasm and extracellular matrix red. HE staining is a fundamental and widely used technique in teaching and research in histology, embryology, and pathology.

[0040] Water-soluble tomato concentrate (WSTC) is extracted from ripe tomatoes. It is obtained by physically removing the fat-soluble components in tomatoes and concentrating the water-soluble active ingredients. Its preparation method is known to those skilled in the art and includes, for example, the method described in patent "202210372407.1". WSTC mainly contains components that are beneficial to the human body, such as nucleosides, phenols and flavonoids. Studies have shown that it has anti-platelet aggregation and thus has the effect of improving blood microcirculation. Based on this, water-soluble tomato concentrate provides a new option for people to prevent cardiovascular diseases. The use of water-soluble tomato concentrate in the prior art is mainly focused on preventing thrombotic cardiovascular diseases, and there are no reports on its use as an active ingredient in a composition for preventing or treating cerebral ischemia-reperfusion injury.

[0041] In one embodiment, the water-soluble tomato concentrate is a water-soluble component extracted from tomatoes at low temperature using physical methods such as centrifugation, membrane filtration, and evaporation. Water-soluble tomato concentrate was approved by the European Food Safety Authority (EFSA) in 2009. The EFSA Scientific Assessment Panel concluded that "water-soluble tomato concentrate helps maintain normal platelet aggregation."

[0042] In one embodiment, the method for preparing the water-soluble tomato concentrate comprises the following steps:

[0043] (1) Prepare mixture A of tomato homogenate.

[0044] (2) separating and removing water-insoluble solids having a particle size greater than 500 μm from mixture A to obtain a water-soluble component B; the separation comprises at least one of centrifugal separation and natural sedimentation separation, and the water-soluble component B is the supernatant obtained after the separation; the operating temperature for the separation is not higher than 60°C.

[0045] (3) Filter the water-soluble component B to obtain filtrate C.

[0046] (4) Purify the filtrate C to obtain the tomato water-soluble active ingredient D.

[0047] (5) Concentrating the water-soluble active ingredient D of tomatoes to obtain a water-soluble tomato concentrate.

[0048] In some embodiments, the consistency of the tomato puree is adjusted by diluting it with water or an aqueous solution, optionally demineralized water, to form "Mixture A" during the preparation of the tomato puree in step (1).

[0049] In some embodiments, the solid content of the mixture A in step (1) is less than 33%.

[0050] In some embodiments, the solid content of the mixture A in the above step (1) is 10% to 15%.

[0051] In some embodiments, the temperature of the mixture A in step (1) is not higher than 35°C.

[0052] In some embodiments, the temperature of the mixture A in step (1) is not higher than 30°C.

[0053] In some embodiments, the pH of the mixture A in step (1) above does not exceed 5.5.

[0054] In some embodiments, the pH of the mixture A in step (1) above does not exceed 4.2, and the pH of the mixture A can be adjusted by adding citric acid.

[0055] In some embodiments, the pore size of the filter membrane used for filtration in step (3) is no greater than 1 μm.

[0056] In some embodiments, the pore size of the filter membrane used for filtration in step (3) is no greater than 0.2 μm.

[0057] In some embodiments, the purification in step (4) above can be carried out by alcohol precipitation or column chromatography.

[0058] In some embodiments, when the purification in step (4) is performed by alcohol precipitation, ethanol is added to the filtrate C to make the ethanol concentration in the filtrate C higher than 70%, more preferably higher than 90%.

[0059] In some embodiments, when the purification in step (4) is performed by column chromatography, the column filler of the chromatography column used is a food-grade resin, more preferably Amberlite FPX66 macroporous adsorption resin.

[0060] In some embodiments, the concentration operation temperature in the above step (5) is not higher than 60°C.

[0061] In some embodiments, the concentration in the above step (5) can be carried out by membrane filtration concentration, low-temperature evaporation concentration, freeze-drying concentration, or spray-drying concentration.

[0062] In one embodiment, the water-soluble tomato concentrate (WSTC) used above is mainly composed of heat-stable water-soluble compounds that have the activity of preventing platelet aggregation.

[0063] In one embodiment, the water-soluble compounds in the water-soluble tomato concentrate may be derived from the pulp of the tomato fruit and / or the juice surrounding the seeds of the tomato fruit, followed by further processing.

[0064] Optionally, the water-soluble compounds in the water-soluble tomato concentrate include nucleoside compounds, flavonoid compounds and phenolic acid compounds.

[0065] In one embodiment, the nucleoside compound includes one or more of uridine, adenosine, and guanosine, and adenosine can be further selected.

[0066] In one embodiment, the flavonoid compound includes a glycosylated flavonoid compound, and further selected from quercetin-3-O-glucoside or rutin.

[0067] In one embodiment, the phenolic acid compounds include phenolic acid compounds themselves and glycosylation products thereof, and the phenolic acid compounds are selected from cinnamic acid, caffeic acid, coumaric acid, ferulic acid, benzoic acid and chlorogenic acid.

[0068] In one embodiment, the total content of the nucleoside compounds, flavonoid compounds and phenolic acid compounds in the water-soluble tomato concentrate is ≥14 mg / g.

[0069] In one embodiment, the water-soluble tomato concentrate is lycopene-free or substantially lycopene-free. "Substantially lycopene-free" means that the water-soluble tomato concentrate contains substantially none of the lycopene found in most tomato juices. This means that the water-soluble tomato concentrate contains less than 10% w / w lycopene, optionally less than 5% w / w, and further optionally less than 1% w / w lycopene, including but not limited to 0.75%, 0.5%, 0.25%, or 0.1%, less than the amount found in an equivalent weight of tomato fruit or tomato juice. Optionally, the water-soluble tomato concentrate contains no or only trace amounts of lycopene.

[0070] Ginkgo biloba leaf extract is prepared by extracting and purifying the leaves of Ginkgo biloba L. using the following method: dried ginkgo leaves are ground and placed in an extraction tank for extraction using water and / or ethanol as a solvent. Ethanol can be used as the extraction solvent, and the mass ratio of ginkgo leaves to ethanol is 1:(1-20), preferably 1:10. The extraction temperature is 70°C to 100°C, the extraction time is 30 minutes to 120 minutes, preferably 60 minutes, and the number of extractions is 1 to 5 times, preferably 3 times. The extract is collected, filtered through a 200-300 mesh filter cloth, and then concentrated to obtain an extract. The resulting extract is then further extracted with ethyl acetate three times to obtain an ethyl acetate extract. The ethyl acetate extract is then concentrated and spray-dried to obtain the ginkgo biloba leaf extract.

[0071] The ginseng extract is prepared by extracting the fleshy root of Panax ginseng CA Meyer (commonly known as ginseng), a plant of the Araliaceae family. The specific method is as follows: coarsely crushing the dried ginseng and placing it in an extraction tank, extracting it with water and / or ethanol as a solvent, wherein ethanol can be used as the extraction solvent, and the mass ratio of ginseng to ethanol is 1:(1-20), optionally 1:10. The extraction temperature is 70°C to 100°C, the extraction time is 1 hour to 3 hours, optionally 2 hours, and the number of times is 1 to 5, optionally 3 times. After collecting the extract, the extract is filtered through a 200-300 mesh filter cloth and then concentrated to obtain a concentrated solution. An appropriate amount of maltodextrin is added, wherein the maltodextrin can account for 10% to 20% of the ginseng extract, and the extract is spray-dried to obtain the ginseng extract.

[0072] The salvia miltiorrhiza extract is prepared by extracting the dried roots and rhizomes of the Lamiaceae plant Salvia miltiorrhiza Bge., and the specific method is as follows: the dried salvia miltiorrhiza is coarsely ground and placed in an extraction tank for extraction with water and / or ethanol as a solvent, wherein a 20% ethanol aqueous solution can be used as the extraction solvent, the mass ratio of salvia miltiorrhiza to the extraction solution is 1:(1-20), and can be 1:10. The extraction temperature is 70°C to 100°C, the extraction time is 1 hour to 3 hours, and can be 2 hours, and the number of times is 1 to 5, and can be 3 times. After collecting the extract, the extract is filtered through a 200-300 mesh filter cloth and concentrated to obtain a concentrated solution. An appropriate amount of maltodextrin is added, and the maltodextrin can be 10% to 20% of the salvia miltiorrhiza extract. The extract is then spray-dried to obtain the salvia miltiorrhiza extract.

[0073] The present application provides the use of a water-soluble tomato concentrate in preparing a medicament for preventing or treating cerebral ischemia-reperfusion injury, or in preparing a composition for preventing or treating cerebral ischemia-reperfusion injury.

[0074] In one embodiment, preventing or treating cerebral ischemia-reperfusion injury includes improving neurological function damage.

[0075] In one specific example, preventing or treating cerebral ischemia-reperfusion injury includes reducing cerebral infarct size.

[0076] In one embodiment, preventing or treating cerebral ischemia-reperfusion injury includes ameliorating brain tissue pathology.

[0077] In one specific example, the composition for preventing or treating cerebral ischemia-reperfusion injury is in the form of oral or injectable administration.

[0078] Specifically, the oral or injectable dosage forms of the composition for preventing or treating cerebral ischemia-reperfusion injury include solutions, powders, tablets, granules, capsules, emulsions, and suspensions. It is understood that the composition for preventing or treating cerebral ischemia-reperfusion injury can be administered by subcutaneous injection, intracerebral administration, intravenous injection, oral administration, etc.

[0079] In one embodiment, the composition for preventing or treating cerebral ischemia-reperfusion injury may only contain water-soluble tomato concentrate without adding other ingredients.

[0080] In one embodiment, the medicament comprises a water-soluble tomato concentrate powder for use by dilution in a liquid or encapsulation in a capsule.

[0081] Optionally, the water-soluble tomato concentrate in the composition for preventing or treating cerebral ischemia-reperfusion injury can be formulated with health food or pharmaceutically acceptable excipients to improve its commercial attributes, including improving delivery, shelf life and taste.

[0082] In one embodiment, the oral dosage form of the composition for preventing or treating cerebral ischemia-reperfusion injury is a solution, such as a beverage solution.

[0083] In one embodiment, the components of the beverage solution include one or more auxiliary materials selected from the group consisting of sugar, vitamins, flavorings, colorants, preservatives, and thickeners; the components of the beverage solution also include an osmotic pressure regulator, such as sodium chloride or sugar, to provide a solution with a specific osmotic strength; the components of the beverage solution also include a pH regulator, such as a buffer; the buffer includes one or more of sodium citrate / citric acid buffer and phosphate buffer.

[0084] In one embodiment, the water-soluble tomato concentrate can be formulated into a powder and provided to the subject as a premix for a beverage.

[0085] In another embodiment, the oral dosage form of the composition for preventing or treating cerebral ischemia-reperfusion injury is an oral tablet. In tablet formation, the water-soluble tomato concentrate is typically mixed with a diluent, which includes a sugar, a sugar alcohol, or a modified cellulose or cellulose derivative; optionally, the sugar includes sucrose and lactose, the sugar alcohol includes xylitol, sorbitol, and mannitol, and the modified cellulose includes powdered cellulose, microcrystalline cellulose, or carboxymethyl cellulose.

[0086] In one embodiment, the tablet further comprises one or more excipients selected from the group consisting of granules, binders, lubricants, and disintegrants.

[0087] In one embodiment, the disintegrant includes starch and starch derivatives and other swellable polymers, such as cross-linked polymer disintegrants; alternatively, the disintegrant includes cross-linked carboxymethyl cellulose, cross-linked polyvinyl pyrrolidone and starch sugar alcohol.

[0088] In one embodiment, the lubricant comprises stearate; alternatively, the lubricant comprises magnesium stearate and stearic acid.

[0089] In one embodiment, the binder comprises polyvinyl pyrrolidone.

[0090] In one embodiment, when the diluent itself is not very sweet, a sweetener may be added; the sweetener includes ammonium glycyrrhizate or an artificial sweetener, and the artificial sweetener includes aspartame or sodium saccharin.

[0091] In one embodiment, the oral dosage form of the composition for preventing or treating cerebral ischemia-reperfusion injury is a capsule, and the water-soluble tomato concentrate is formulated into powder, granules, gel or semisolid to be incorporated into the capsule.

[0092] In one embodiment, when the water-soluble tomato concentrate is used in powder form, the water-soluble tomato concentrate may be formulated with any one or more of the excipients defined above with respect to tablets, or may be presented in undiluted form.

[0093] In one embodiment, when the water-soluble tomato concentrate is used in a gel or semisolid form, the water-soluble tomato concentrate can be dissolved or suspended in a viscous liquid, a semisolid carrier, a liquid carrier, a vegetable oil, or a fish oil. Alternatively, the water-soluble tomato concentrate can be dissolved or suspended in one or more of polyethylene glycol, propylene glycol, glycerin, olive oil, sunflower oil, safflower oil, evening primrose oil, soybean oil, cod liver oil, or herring oil. The water-soluble tomato concentrate and excipients can be filled into hard or soft gelatin capsules. For viscous liquid or semisolid fills, soft gelatin or gelatin-equivalent capsules can be used. Alternatively, the water-soluble tomato concentrate can be provided in powder form, optionally with an optional solid excipient, for incorporation into a capsule, such as a hard gelatin capsule.

[0094] In one embodiment, the capsule comprises a size 00 Vegecaps (LGA, La Seyne-sur-Mer, France) capsule containing 150 mg of a water-soluble tomato concentrate and a suitable carrier / filler; the final weight of each capsule is 600 mg based on the combined weight of the water-soluble tomato concentrate and the filler; optionally, the filler comprises tapioca starch.

[0095] In a specific example, the composition for preventing or treating cerebral ischemia-reperfusion injury is in the form of a solution, which is mainly composed of a water-soluble tomato concentrate and a solvent.

[0096] Specifically, the solvent is water, but is not limited thereto.

[0097] In one specific example, the dosage of the water-soluble tomato concentrate in the composition for preventing or treating cerebral ischemia-reperfusion injury is 15 mg / kg to 150 mg / kg. Alternatively, the dosage of the water-soluble tomato concentrate in the composition for preventing or treating cerebral ischemia-reperfusion injury is 47.25 mg / kg. It will be appreciated that the specific concentration can be adjusted based on the condition or individual circumstances.

[0098] In one embodiment, the composition further comprises Danshen extract and / or Ginkgo biloba extract.

[0099] In one embodiment, the composition further comprises one or more of a binder, a lubricant, a glidant, a diluent and a disintegrant.

[0100] In one embodiment, the binder includes one or more of povidone K30, copovidone, sodium carboxymethylcellulose, hydroxypropyl cellulose and hypromellose.

[0101] In one embodiment, the lubricant includes one or more of magnesium stearate, talc and polyethylene glycol.

[0102] In one embodiment, the glidant includes one or more of silicon dioxide, micro-powdered silica gel, calcium hydrogen phosphate, tricalcium phosphate and milk mineral salt.

[0103] In one embodiment, the diluent comprises one or more of microcrystalline cellulose, isomalt, starch, dextrin, lactose, pregelatinized starch, mannitol, sorbitol and erythritol.

[0104] In one embodiment, the disintegrant comprises one or more of crospovidone, sodium starch glycolate, croscarboxymethyl cellulose sodium, low-substituted hydroxypropyl cellulose and dry starch.

[0105] In some embodiments, the above composition is a health food composition or a pharmaceutical composition.

[0106] In one specific example, the method for preparing the aforementioned composition for preventing or treating cerebral ischemia-reperfusion injury comprises the following steps: mixing a water-soluble tomato concentrate with a solvent to prepare the composition for preventing or treating cerebral ischemia-reperfusion injury. It will be appreciated that the preparation method may be adjusted as needed depending on the dosage form or formulation.

[0107] Example

[0108] Below, the embodiment of the present application is described. The embodiment described below is exemplary, is only used to explain the present application, and is not to be construed as limiting the present application. Where the technology or conditions are not specified in the embodiment, the technology or conditions described in the literature in this area or the product instructions are used. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0109] Example 1

[0110] 1. Test materials

[0111] 1.1 Test reagents and instruments

[0112] Silicone-coated nylon sutures were purchased from Beijing Xinong Technology Co., Ltd.; 4-0 non-absorbable surgical sutures were purchased from Yangzhou Yuankang Medical Device Co., Ltd.; tetrazolium red (TTC) dye was purchased from Beijing Solaibao Technology Co., Ltd.; and butylphenol soft capsules were purchased from Baixing Pharmacy. A microplate reader was purchased from Tecan (Shanghai). A laser blood flow imaging system (model: Moor FLPI-2) was purchased from Moore Instruments. A flow cytometer (model: Bricyte E6) was purchased from Shenzhen Mindray Bio-Medical Electronics Co., Ltd.

[0113] The preparation method of a water-soluble tomato concentrate comprises the following steps: homogenizing ripe tomatoes into a slurry, diluting the slurry with 5 parts by weight of ultrapure water to obtain a mixture A. Citric acid is added to the mixture A (initial temperature <30°C) to a pH of 4.2, and the mixture A is centrifuged at 8000×g for 20 minutes at 4°C. The supernatant (water-soluble component B) is then ultrafiltered using a ceramic membrane with a pore size of 0.2 μm (operating temperature <60°C), and the filtrate C is collected.

[0114] A chromatographic column was packed with 3 times the volume of the filtrate, using food-grade macroporous adsorption resin FPX66, and equilibrated with ultrapure water. The ultrafiltrate was loaded onto the column, washed with 1 column volume of distilled water, and then eluted with 1 column volume of 80% ethanol. The 80% ethanol eluate (water-soluble active ingredient D) was collected.

[0115] The eluate was concentrated by reverse osmosis using a Trisep ACM5 membrane (operating temperature <60°C). The ethanol / water solvent passed through the membrane, while all dissolved compounds were retained within. When the eluate volume was concentrated to 1 / 10, ultrapure water was added to the retentate at the same rate of concentration until the volume of the added ultrapure water reached twice the volume of the 80% ethanol eluate.

[0116] The retentate is collected and spray-dried to obtain a fine powder with a water content of less than 6%, which is a water-soluble tomato concentrate.

[0117] 1.2 Animals and housing conditions

[0118] 1.2.1 Experimental animals

[0119] Healthy male SD rats (weighing 280 g to 300 g) were purchased from Beijing Weitonglihua Laboratory Animal Company with license number SCXK (Beijing) 2022-0011.

[0120] 1.2.2 Rearing conditions

[0121] Animals were housed at Beijing Pony Medical Laboratory Co., Ltd. The animal experimental facilities maintained barrier environment standards. Key environmental parameters were controlled within the following ranges: room temperature 20°C–26°C, daily temperature range ≤4°C; relative humidity 40%–70%; minimum air changes 15 times / hour; and light conditions: dark = 12 hours, light = 12 hours. Animals were housed in standard enclosures, 5 per enclosure, in accordance with the minimum space requirements for experimental animals as specified in the National Standard of the People's Republic of China, GB14925-2010. All animals were maintained by trained personnel, and free movement and feeding were maintained throughout the entire enclosure.

[0122] 1.2.3 Quarantine and acclimation process

[0123] Newly received animals should be quarantined for 3-5 days. During quarantine, observe the animals' drinking water, food intake, and health, as well as any signs of illness or death. Healthy animals have smooth fur, are responsive, and have no abnormal secretions. Any abnormalities must be reported to the project leader and clinical veterinarian. Under the guidance of the veterinarian and with the approval of the project leader, sick animals should be removed and replaced with healthy ones. Animals should only be used after they are confirmed to be healthy and disease-free. Experiments may only begin after the animals have been acclimated to the animal's care for one week.

[0124] 1.2.4 Animal Welfare

[0125] The animals used in this study and related treatments should comply with animal welfare requirements and must be reviewed and approved by the Institutional Animal Care and Welfare Committee (IACUC) of the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences / Peking Union Medical College before the experiment is carried out.

[0126] 2. Test methods

[0127] 2.1 Animal grouping and drug treatment

[0128] Animal Grouping:

[0129] i. Sham operation (SHAM) group;

[0130] ii. cerebral ischemia-reperfusion injury (MCAO / R) model group;

[0131] iii. water-soluble tomato concentrate (WSTC, 15.75 mg / kg) group;

[0132] iv. water-soluble tomato concentrate (WSTC, 47.25 mg / kg) group;

[0133] v. Water-soluble tomato concentrate (WSTC, 141.75 mg / kg) group;

[0134] vi. N-butylphenol soft capsule (NBP, 54 mg / kg, clinical equivalent dose) group.

[0135] Combining the safety data and the experimental results of the preliminary experiment, this experiment designed three dosage groups of water-soluble tomato concentrate: low, medium and high. According to the body surface area method in pharmacological experimental methodology (converted according to 60 kg of human), the recommended human dosage is 150 mg / day.

[0136] Drug treatment:

[0137] The water-soluble tomato concentrate and butylphenol soft capsule treatment groups were gavaged daily for 5 days before MCAO / R surgery and continued to be gavaged for 14 consecutive days after surgery. The rats in the SHAM group and MCAO / R model group were gavaged with an equal volume of solvent.

[0138] 2.2 Establishment of cerebral ischemia-reperfusion injury (MCAO / R) model

[0139] A suture occlusion method was used to establish a MCAO / R model, with strict aseptic procedures performed during the surgery. The main surgical procedures were as follows: rats were anesthetized with intraperitoneal injection of chloral hydrate, and the common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were isolated. The common carotid artery was clamped with a micro-arterial clamp, and the branches of the external carotid artery were electrocoagulated with a bipolar electrocoagulator. The main trunk of the external carotid artery was freed and ligated 3–4 mm distal to the bifurcation of the common carotid artery. The micro-arterial clamp was used to temporarily clamp the internal carotid artery. A small incision was made in the external carotid artery with ophthalmic scissors, and a silicone-coated nylon suture was inserted. A loose knot was tied at the incision to block blood flow from the internal carotid artery. The micro-arterial clamp was removed, and the nylon suture was gently pushed into the internal carotid artery, reaching the anterior cerebral artery within the skull, and occluding the ostium of the middle cerebral artery (MCA). The insertion depth was approximately 18–22 mm. The carotid artery clip was removed, and the subcutaneous tissue and skin were sutured layer by layer after disinfection. Two hours after surgery, the nylon suture was slowly withdrawn for reperfusion. The sham-operated group (SHAM) underwent the same procedures except that the nylon suture was not inserted. Rectal temperature was maintained at 37°C throughout the experiment for all rats.

[0140] 2.3 Neurological function evaluation

[0141] All rats were assessed for neurological function at 24 hours, 7 days, and 14 days after reperfusion using the Longa 5-point scale. The Longa scale is as follows: 0, normal, no neurological deficit; 1, impaired extension of the left forelimb, decreased function; 2, circling to the left, paralysis of the left forelimb, severe decreased function; after the tail was lifted, the rat repeatedly twisted toward the damaged area; 3, stumbling to the left while walking; 4, paralysis of the limbs, coma.

[0142] 2.4 Determination of cerebral infarction area

[0143] At the end of the experiment, cerebral infarction area was determined using tetrazolium red (TTC) staining. Rats were sacrificed by cervical dislocation, and the whole brain was quickly removed and briefly frozen at -20°C. The olfactory bulb was then removed by placing the brain on an ice tray. The forebrain was then cut into six 2-mm-thick slices along the coronal plane. The slices were incubated in 1.5 mL of 2% TTC solution in the dark for 30 minutes. The slices were then fixed in 10% paraformaldehyde overnight and photographed for analysis. Cerebral infarction area was measured and calculated using Image-J software.

[0144] 2.5 HE staining of brain tissue

[0145] At the end of the experiment, hematoxylin-eosin staining (HE staining) was used to detect the pathological morphological changes of brain tissue.

[0146] 2.6 Gross Observation of Gastric Mucosal Lesions

[0147] At the end of the experiment, the stomach was dissected out and the contents were rinsed with PBS. After being fixed in 10% neutral formalin fixative for 10 min, the stomach was cut open along the greater curvature of the stomach and the gastric mucosa was rinsed with normal saline. The stomach was then unfolded and laid flat on white paper, and the gastric mucosa was photographed with a camera to observe the damage of the gastric mucosa.

[0148] 2.7 Data Processing

[0149] The experimental results are expressed as mean ± standard deviation (X ± SD), and the data were statistically analyzed using SPSS statistical software. One-way ANOVA was used to analyze the measurement data, and P < 0.05 was considered statistically significant.

[0150] 3. Experimental Results

[0151] 3.1 Effect of WSTC on neurological function scores

[0152] Neurological function scores are important indicators of the MCAO / R model, useful for assessing model success and injury severity. This study used the Longa 5-grade scale to assess neurological deficits in rats after MCAO / R. The results showed that neurological function scores in the MCAO / R model group were significantly higher than those in the SHAM group at 24 hours, 7 days, and 14 days after reperfusion (p < 0.01), indicating severe neurological impairment and a successful MCAO / R model. Twenty-four hours after reperfusion, neurological function scores in the WSTC and NBP groups were significantly lower than those in the model group. Neurological function scores in the WSTC 47.25 mg / kg group and the NBP group were significantly lower than those in the WSTC 15.75 mg / kg group; no significant differences were observed among the other treatment groups (Figure 1A). At 7 and 14 days after reperfusion, neurological function scores in all treatment groups were significantly lower than those in the model group; no significant differences were observed among the treatment groups (Figures 1B and 1C). These results suggest that WSTC significantly ameliorates neurological impairment in rats after MCAO / R.

[0153] 3.2 Effect of WSTC on cerebral infarction area

[0154] Measuring cerebral infarct size after cerebral ischemia is a commonly used indicator for assessing the severity of cerebral ischemia. In this study, TTC staining was used to determine cerebral infarct size. The results showed that the infarct size in the MCAO / R model group was significantly greater than that in the SHAM group (p < 0.01). The infarct size in the WSTC 15.75 mg / kg, WSTC 47.25 mg / kg, WSTC 141.75 mg / kg, and NBP groups was significantly lower than that in the model group (p < 0.01). The infarct size in the NBP group was lower than that in the WSTC 15.75 mg / kg group. There were no significant differences among the other treatment groups (Figure 2). Numerically, the WSTC 47.25 mg / kg group was comparable to the NBP group and superior to the WSTC 15.75 mg / kg and WSTC 141.75 mg / kg groups. These results suggest that WSTC has a significant repairing effect on ischemic stroke, significantly reducing cerebral infarct size.

[0155] 3.3 Effects of WSTC on brain tissue pathology

[0156] Hematoxylin-eosin (HE) staining is a commonly used staining method in histology and pathology. Low-magnification microscopy (×0.9) of tissue sections revealed intact brain tissue structure in the SHAM group, while severe necrosis and deformation of the injured side of the brain in the MCAO / R group affected most of the cortex and hippocampus, with significant atrophy and tissue loss. The area of ​​cerebral degeneration and necrosis was significantly reduced in all treatment groups. High-magnification microscopy (×10 and ×40) of tissue sections revealed that compared with the SHAM group, the intercellular spaces in the cortex and hippocampal CA1 region of rats in the MCAO / R model group were enlarged, with irregular cell morphology and vacuolated cell bodies. Nuclei were pyknotic and blurred, with unclear nucleoli and a reduced number of nuclei. Pyramidal cell arrangement was disordered. Compared with the MCAO / R model group, all WSTC doses and the NBP 54 mg / kg group significantly ameliorated brain tissue pathology, with significantly improved cell morphology in the cerebral cortex and hippocampal CA1 region, and pyramidal cells were neatly arranged with relatively intact layers (Figure 3). The results of the study showed that WSTC could significantly improve brain tissue pathological damage caused by MCAO / R.

[0157] Effects of WSTC on gastric mucosal injury

[0158] Butylphenol soft capsules are a commonly used drug in the clinical prevention and treatment of cardiovascular and cerebrovascular diseases. Their main adverse reactions are gastric mucosal damage, ulcers, and bleeding. To investigate whether WSTC has a repairing effect on gastric mucosal damage caused by butylphenol soft capsules, this experiment conducted gross observations of the gastric mucosa. The experimental results are shown in Figure 4: The gastric mucosal surface of the three WSTC dose groups and the NBP group was smooth, soft, light red, and had intact folds. No obvious ulcers, bleeding, erosion, or damage were observed. The results suggest that under the experimental conditions, butylphenol soft capsules did not cause gastric mucosal damage, which may be related to the short administration time of the butylphenol soft capsules used in this experiment (5 days + 14 days).

[0159] In summary, under the experimental conditions of this study, water-soluble tomato concentrate has a significant repair effect on MCAO / R injury in rats, significantly reducing cerebral infarction area, lowering neurological function scores, improving brain tissue pathology, and increasing cerebral blood flow on the ischemic side.

[0160] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0161] The above-described embodiments merely represent several implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent application. It should be noted that a person of ordinary skill in the art may make various modifications and improvements without departing from the spirit of the present application, and these modifications and improvements fall within the scope of protection of the present application. Therefore, the scope of protection of the present patent application shall be determined by the appended claims.

Claims

1. Use of a water-soluble tomato concentrate in preparing a drug for preventing or treating cerebral ischemia-reperfusion injury, or in preparing a composition for preventing or treating cerebral ischemia-reperfusion injury.

2. The use according to claim 1, wherein: The prevention or treatment of cerebral ischemia-reperfusion injury includes improving neurological function damage.

3. The use according to claim 1, wherein: The prevention or treatment of cerebral ischemia-reperfusion injury includes reducing the area of ​​cerebral infarction.

4. The use according to claim 1, wherein: The prevention or treatment of cerebral ischemia-reperfusion injury includes improving brain tissue pathology.

5. The use according to any one of claims 1 to 4, wherein: The dosage form of the composition for preventing or treating cerebral ischemia-reperfusion injury is an oral or injectable dosage form, and the oral or injectable dosage form includes a solution, a powder, a tablet, a granule, a capsule, an emulsion and a suspension.

6. The use according to claim 5, wherein: The dosage form of the composition for preventing or treating cerebral ischemia-reperfusion injury is a solution, which is mainly composed of water-soluble tomato concentrate and solvent.

7. The use according to claim 6, wherein: In the composition for preventing or treating cerebral ischemia-reperfusion injury, the dosage of the water-soluble tomato concentrate is 15 mg / kg to 150 mg / kg.

8. The use according to any one of claims 1 to 4, wherein: The composition further comprises a salvia miltiorrhiza extract and / or a ginkgo biloba leaf extract.

9. The use according to any one of claims 1 to 4, wherein: The composition is a health food composition or a pharmaceutical composition.

10. The use according to any one of claims 1 to 4, wherein: The method for preparing the composition for preventing or treating cerebral ischemia-reperfusion injury comprises the following steps: mixing water-soluble tomato concentrate with a solvent to prepare the composition for preventing or treating cerebral ischemia-reperfusion injury.

11. A composition for preventing or treating cerebral ischemia-reperfusion injury, wherein: The invention comprises a water-soluble tomato concentrate and at least one of a salvia miltiorrhiza extract and a ginkgo biloba leaf extract.

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