CN109985032A - Salvianolic acid D is preparing the application in anti-cerebral apoplexy drug - Google Patents

Abstract

The invention discloses the application of salvianolic acid D in the preparation of anti-ischemic stroke medicines and/or health care products. The present invention finds that salvianolic acid D can significantly improve the neurobehavioral defect symptoms of cerebral ischemia-reperfusion rats, significantly reduce the cerebral infarction volume of cerebral ischemia-reperfusion rats, and at the same time can significantly reduce cerebral ischemia-reperfusion rats The percentage of cerebral edema, reducing the openness and permeability of the blood-brain barrier. Salvianolic acid D can be used to prepare medicines and/or health care products for the treatment of ischemic stroke, reduce neuronal apoptosis in brain tissue, improve neuronal survival rate, reduce the degree of brain tissue damage, and provide a solution for disease prevention and treatment. an effective solution.

Description

Application of salvianolic acid D in the preparation of anti-stroke drugs

technical field

The invention relates to a new application of salvianolic acid D in the preparation of medicines and/or health care products, mainly relates to the application of salvianolic acid D in the preparation of medicines and/or health care products for preventing and treating ischemic stroke, and belongs to medical technology field.

Background technique

Stroke has the characteristics of high morbidity, high disability rate and high fatality rate, and is a global disease that seriously threatens human health. The types of stroke are mainly divided into ischemic stroke, hemorrhagic stroke and transient ischemic attack. Ischemic stroke is mainly caused by insufficient blood supply to brain tissue due to vascular embolism and/or arteriosclerosis, resulting in brain tissue damage. Hemorrhagic stroke is mainly due to the rupture of blood vessels in the brain and the infiltration of blood into the brain, resulting in interruption of blood flow to normal brain tissue, resulting in brain tissue damage. Ischemic stroke accounts for 70-80% of all stroke types. The pathogenesis and pathophysiological process of stroke are complex, in which various mechanisms such as oxidative stress, inflammatory response, calcium overload and excitotoxicity are involved in its occurrence and development.

Currently, the number of anti-stroke drugs available clinically is limited. The FDA-approved tissue plasminogen activator (tPA) is one of the more recognized stroke treatments. However, the thrombolytic therapy of tPA has a narrow time window, high bleeding risk, and severe reperfusion injury, so it can only benefit a small number of clinical patients from thrombolytic therapy. Therefore, it is imperative to develop new drugs for the treatment of stroke.

Salvianolic acid D (SalD) is a monomeric compound extracted from the traditional Chinese medicine Salvia miltiorrhiza. Chemical name is (2R)-2-[(E)-3-[2-(carboxymethyl)-3,4-dihydroxyphenyl]prop-2-enoyl]oxy-3-(3,4-dihydroxyphenyl)propanoic acid, molecular formula is C ₂₀ H ₁₈ O ₁₀ . The content of salvianolic acid D in Salvia miltiorrhiza is very low, and there are few research reports on its pharmacological effects. Its chemical structure is as follows:

So far, there has been no report on the anti-stroke pharmacological effect of salvianolic acid D, and no report on the effect of salvianolic acid D in the preparation of anti-stroke drugs and/or health products.

SUMMARY OF THE INVENTION

The technical problem solved by the present invention is to provide the application of salvianolic acid D in the preparation of drugs and/or health care products for preventing and treating ischemic stroke, thereby providing an effective solution for the treatment of cerebral ischemia.

For this reason, the present invention provides the following technical solutions:

The invention provides the application of salvianolic acid D in the preparation of drugs and/or health care products for preventing and treating ischemic stroke.

Further, the effect of salvianolic acid D includes the therapeutic effect on the occurrence of cerebral ischemia, and also includes the preventive effect;

Further, the treatment of cerebral ischemia is the treatment of acute ischemic stroke, transient ischemic attack and chronic cerebral insufficiency caused by various factors;

Further, the prevention of cerebral ischemia is the prevention of acute ischemic stroke, transient ischemic attack and chronic cerebral insufficiency caused by various factors;

Further, the therapeutic effect of salvianolic acid D on cerebral ischemia is characterized by treatment at any stage after acute attack, including acute phase, subacute phase and recovery phase;

Further, the preventive effect of salvianolic acid D on cerebral ischemia is characterized for high-risk groups without acute symptoms, as well as patients who have been treated for cerebral ischemia, the purpose of which is to avoid recurrence;

Further, the salvianolic acid D can be prepared into various dosage forms of anti-stroke drugs with pharmaceutically acceptable excipients according to conventional preparation methods.

Further, the salvianolic acid D can be made into various anti-stroke health products according to the conventional preparation method with health products including health food and functional food acceptable auxiliary materials.

The present invention therefore also relates to pharmaceutical compositions comprising the compounds of the present invention as active ingredients. The pharmaceutical composition can be prepared according to methods known in the art. The salvianolic acid D described in the invention can be combined with one or more pharmaceutically acceptable solid or liquid excipients and/or adjuvants to prepare any dosage form for human or animal use. The content of the compound of the present invention in its pharmaceutical composition is usually 0.1-99%.

Further, the present invention also relates to a health care product composition using the compound of the present invention as an active ingredient. The composition can be prepared according to methods well known in the art. The salvianolic acid D described in the invention can be combined with one or more solid or liquid excipients and/or adjuvants acceptable for health care products and foods to prepare any dosage form for human or animal use. The content of the compound of the present invention in its pharmaceutical composition is usually 0.1-99%.

The compound of the present invention or the pharmaceutical composition containing it can be administered in unit dosage form, and the route of administration can be gastrointestinal or parenteral, such as oral, intravenous, intramuscular, subcutaneous, nasal cavity, oral mucosa, eye, lung and respiratory tract, skin, vagina, rectum, etc.

The pharmaceutical combination dosage forms include oral preparations, injection dosage forms and skin and mucous membrane route dosage forms.

The oral preparations include tablets, capsules, sustained-release preparations, controlled-release preparations, drop pills, powders, granules, solutions, emulsions, and suspensions; the injection dosage forms include intramuscular injection, intravenous injection , Intravenous infusion; the dosage forms of the skin and mucous membrane route include topical solutions, lotions, liniments, ointments, plasters, pastes, patches, eye drops, nasal drops, eye ointments, containing Gargle, sublingual tablet, patch, patch.

The dosage form for administration can be a liquid dosage form, a solid dosage form or a semi-solid dosage form. Liquid dosage forms can be solutions (including true solutions and colloidal solutions), emulsions (including o/w, w/o and double emulsion), suspensions, injections (including water injection, powder injection and infusion), eye drops solid dosage forms can be tablets (including ordinary tablets, enteric-coated tablets, buccal tablets, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets), capsules ( Including hard capsules, soft capsules, enteric-coated capsules), granules, powders, pellets, drop pills, suppositories, films, patches, gas (powder) aerosols, sprays, etc.; semi-solid dosage forms can be ointments, Gels, pastes, etc.

The compounds of the present invention can be prepared into common preparations, as well as sustained-release preparations, controlled-release preparations, targeted preparations and various microparticle drug delivery systems.

In order to formulate the compounds of the present invention into tablets, various excipients well known in the art can be widely used, including diluents, binders, wetting agents, disintegrating agents, lubricants, glidants. The diluent can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; Propanol, etc.; the binder can be starch slurry, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, acacia mucilage, gelatin slurry, sodium carboxymethyl cellulose, methyl cellulose, carboxypropyl methyl cellulose Base cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, etc.; disintegrants can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polymer Vinylpyrrolidone, croscarmellose sodium, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfonate, etc.; lubricants and flow aids The agent may be talc, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol and the like.

The compounds of the present invention can be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer and multi-layer tablets.

In order to make the dosing unit into capsules, salvianolic acid D can be mixed with diluent and glidant, and the mixture can be directly placed in hard capsules or soft capsules. Salvianolic acid D can also be made into granules or pellets with diluents, binders and disintegrating agents, and then placed in hard capsules or soft capsules. The various diluents, binders, wetting agents, disintegrants, and glidants used to prepare tablets of the compounds of the present invention can also be used to prepare capsules of the compounds of the present invention.

To prepare the compounds of the present invention into injections, water, ethanol, isopropanol, propylene glycol or their mixtures can be used as solvents and appropriate amount of solubilizers, cosolvents, pH adjusters and osmotic pressure adjusters commonly used in the art can be added. The solubilizer or cosolvent can be poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc.; the pH adjuster can be phosphate, acetate, hydrochloric acid, sodium hydroxide, etc.; the osmotic pressure adjuster can be It is sodium chloride, mannitol, glucose, phosphate, acetate, etc. For example, in the preparation of freeze-dried powder injection, mannitol, glucose, etc. can also be added as proppant.

In addition, colorants, preservatives, fragrances, flavors, or other additives can also be added to the pharmaceutical preparations, if desired. In order to achieve the purpose of medication and enhance the therapeutic effect, the medicament or pharmaceutical composition of the present invention can be administered by any known administration method. The dosage of the pharmaceutical composition of the compound of the present invention may vary widely according to the nature and severity of the disease to be prevented or treated, the individual condition of the patient or animal, the route of administration and the dosage form. In general, a suitable daily dose of a compound of the present invention will range from 0.001 to 400 mg/kg body weight. The above doses may be administered in a single dosage unit or divided into several dosage units, depending on the clinical experience of the physician and the dosing regimen including the use of other therapeutic means.

The compounds or compositions of the present invention may be administered alone or in combination with other therapeutic or symptomatic drugs. When the compound of the present invention has a synergistic effect with other therapeutic drugs, its dosage should be adjusted according to the actual situation.

The beneficial effects of the present invention are as follows: the present invention adopts a cerebral ischemia-reperfusion rat model to investigate the anti-cerebral ischemia effect of salvianolic acid D. The results showed that injection of salvianolic acid D could significantly improve the symptoms of neurobehavioral defects in rats with cerebral ischemia, reduce the volume of cerebral infarction and the percentage of cerebral edema in rats with cerebral ischemia, and reduce the openness and permeability of the blood-brain barrier in rats. At the same time, it can significantly reduce the apoptosis of nerve cells in the brain tissue, improve the vitality of nerve cells, and provide an effective solution for the prevention and treatment of stroke.

Description of drawings

Figure 1. The effect of salvianolic acid D on the symptoms of neurobehavioral defect in cerebral ischemia-reperfusion rats ( n=12). Sham: sham operation control group; I/R: model group; I/R+1mg/kg SalD: Salvianolic acid D low-dose group (1mg/kg); I/R+3mg/kg SalD: Salvianolic acid D medium Dose group (3mg/kg); I/R+15mg/kg SalD: Salvianolic acid D high dose group (15mg/kg); I/R+Nimodipine: Nimodipine positive drug group; ^### and sham operation control P<0.001 compared with group; ^* P<0.05 compared with model group; ^** P<0.01 compared with model group; ^*** P<0.001 compared with model group.

Figure 2. The effect of salvianolic acid D on the volume of cerebral infarction in rats with cerebral ischemia-reperfusion ( n=6). Sham: sham operation control group; I/R: model group; I/R+15mg/kg SalD: salvianolic acid D treatment group (15mg/kg); I/R+Nimodipine: nimodipine positive drug group; ^{## #P} <0.001 compared with the sham-operated control group; ^* P<0.05 compared with the model group; ^** P<0.01 compared with the model group.

Figure 3. The effect of salvianolic acid D on the percentage of cerebral edema in rats with cerebral ischemia-reperfusion ( n=6). Sham: sham operation control group; I/R: model group; I/R+1mg/kg SalD: Salvianolic acid D low dose group (1mg/kg); I/R+3mg/kgSalD: Salvianolic acid D medium dose Group (3mg/kg); I/R+15mg/kg SalD: Salvianolic acid D high dose group (15mg/kg); I/R+Nimodipine: Nimodipine positive drug group; ^### and sham operation control group P<0.001 compared to; ^* P<0.05 compared to model group; ^** P<0.01 compared to model group.

Figure 4. Effect of salvianolic acid D on neuronal apoptosis in brain tissue of rats with cerebral ischemia-reperfusion ( n=4). A: Relative expression level of Bax/Bcl-2; B: Relative expression level of Cleaved caspase-3; C: Relative expression level of Cleaved caspase-9; D: Relative expression level of Cytochrome C; E: Immunofluorescence staining analysis of TUNEL positive cell rate; F: Statistical graph of TUNEL-positive cell rate. Sham: sham operation control group; I/R: model group; I/R+1mg/kg SalD: Salvianolic acid D low-dose group (1mg/kg); I/R+3mg/kg SalD: Salvianolic acid D medium Dose group (3mg/kg); I/R+15mg/kg SalD: Salvianolic acid D high dose group (15mg/kg); ^### Compared with sham operation control group, P<0.001; ^* Compared with model group ratio, P<0.05; ^** P<0.01 compared with the model group; ^*** P<0.001 compared with the model group.

Figure 5. The effect of salvianolic acid D on the opening of the blood-brain barrier in rats with cerebral ischemia-reperfusion ( n=5). Sham: sham operation control group; I/R: model group; I/R+1mg/kg SalD: Salvianolic acid D low-dose group (1mg/kg); I/R+3mg/kg SalD: Salvianolic acid D medium Dose group (3mg/kg); I/R+15mg/kg SalD: Salvianolic acid D high-dose group (15mg/kg); ### Compared with the sham operation control group, P<0.001; ** Compared with the model group P<0.01 compared to; ***P<0.001 compared to model group.

Figure 6. The effect of salvianolic acid D on the viability of BV2 cells injured by hypoglycemia, hypoxia, complexation and reoxygenation ( n=4). Control: control group; OGD/R: model group; OGD/R+0.1 μM SalD: low concentration group of salvianolic acid D (0.1 μM SalD); OGD/R+1 μM SalD: medium concentration group of salvianolic acid D (1 μM SalD ); OGD/R+5μM SalD: Salvianolic acid D high concentration group (5μMSalD); OGD/R+NAC: Positive drug NAC group; ###Compared with the control group, P<0.001; *Compared with the model group , P<0.05; *** Compared with the model group, P<0.001.

Detailed ways

In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings.

Example 1: The effect of salvianolic acid D on the symptoms of neurobehavioral defects in cerebral ischemia-reperfusion rats

Experimental materials: SPF male SD rats, weighing 240-260 grams, were purchased from Beijing Weitong Lihua Animal Technology Co., Ltd., certificate number: SCXK (Beijing) 2014-0004. Salvianolic acid D was prepared by the Institute of Materia Medica, Chinese Academy of Medical Sciences. Nimodipine was obtained from the Crystal Form Center, Institute of Materia Medica, Chinese Academy of Medical Sciences.

Experimental grouping: SD rats were randomly divided into sham operation control group, model group, salvianolic acid D low-dose group (1 mg/kg), medium-dose group (3 mg/kg), high-dose group (15 mg/kg), nimo Dipine group (20mg/kg, positive control), 12 in each group.

Establishment of cerebral ischemia-reperfusion animal model: The middle cerebral artery occlusion/reperfusion (MCAO/R) model was established by suture method. Rats were anesthetized with isoflurane, fixed in the supine position, the midline of the neck was incised, and blunt dissection was performed. ) and the internal carotid artery (ICA). Use No. 1 suture to tighten the proximal end of the CCA and the ICA, ligate the distal end of the ECA, and bury the suture under the ECA to fix the suture. Cut an oblique incision on the ECA, insert the suture, and enter the 18mm or so to fix the suture. bolt. After 1.5 hours of ischemia, the suture was pulled out to achieve reperfusion, and the drug was administered by tail vein injection at the same time. After the wounds were sutured layer by layer, the rats were returned to their cages.

Neurobehavioral defect score: 24h after reperfusion, the neurobehavioral defect score was performed before the animals were sacrificed. Specific operation: lift the tail of the rat off the ground, observe the extension of the two forelimbs of the rat, then place the rat on the level ground, observe its crawling, push its shoulders, and observe whether there is any difference in resistance between the two sides. Using a four-point scale (0-4 points), the higher the score, the more serious the neurobehavioral damage.

Results: As shown in Figure 1, the neurobehavioral defect score of the rats in the model group was 3.417±0.669. The neurobehavioral defect score of the salvianolic acid D low-dose group (1mg/kg) was 3.000±0.739, and there was no significant difference compared with the model group. The neurobehavioral defect score of the rats in the salvianolic acid D medium dose group (3 mg/kg) was 2.417±0.669, which was significantly lower than that in the model group (P<0.01). The neurobehavioral defect score of the rats in the salvianolic acid D high-dose group (15mg/kg) was 2.083±0.669, and the neurobehavioral defect symptoms were significantly improved compared with the model group (P<0.001). This suggests that salvianolic acid D has the effect of improving neurological damage induced by cerebral ischemia-reperfusion.

Example 2: The effect of salvianolic acid D on cerebral infarct volume in cerebral ischemia-reperfusion rats

Experimental materials: TTC (2,3,5-triphenyltetrazolium chloride) was purchased from Sigma-Aldrich. Other relevant experimental materials are the same as in Example 1.

Experimental grouping: SD rats were randomly divided into sham operation control group, model group, salvianolic acid D treatment group (15mg/kg), nimodipine group (20mg/kg, positive control), 6 rats in each group. Other relevant experimental protocols are the same as those in Example 1.

Determination of cerebral infarction volume: The rat brain tissue was placed in a -20°C refrigerator, taken out after 15 minutes, and placed in a rat brain slice mold. The brain slices were then quickly placed in 0.5% TTC solution and incubated at 37°C for 20 min in the dark. After TTC staining, normal tissue was rose red, while ischemic infarct tissue was pale. Brain slices were removed and fixed in 4% paraformaldehyde solution. Finally, the brain slices of each rat were arranged neatly and photographed for preservation. Image J image analysis software was used to calculate the total area and infarct area of each brain slice, and then the cerebral infarction volume was converted according to the thickness of the brain slice and the measured area.

Results: TTC is a lipid-soluble light-sensitive complex, which reacts with succinate dehydrogenase in the mitochondria of living cells to generate red formazan, which is used to reflect the vitality of tissue cells. Decreased, could not react with TTC, and was pale. As shown in Figure 2, the percentage of cerebral infarction volume of rats in the model group was 39.98±9.73%. The percentage of cerebral infarction volume in the salvianolic acid D treatment group (15mg/kg) was 21.96±6.01%, which was significantly lower than that in the model group (P<0.01). This suggests that salvianolic acid D can inhibit the death of cells in the damaged brain region of rats with cerebral ischemia-reperfusion, thereby reducing the volume of cerebral infarction in rats.

Example 3: The effect of salvianolic acid D on the percentage of cerebral edema in cerebral ischemia-reperfusion rats

Experimental grouping: SD rats were randomly divided into sham operation control group, model group, salvianolic acid D low, medium and high dose groups, nimodipine group (20 mg/kg, positive control), 6 rats in each group. Other relevant experimental materials and experimental protocols are the same as those in Example 1.

Brain edema detection: After the rat brain tissue was taken out, the wet weight was weighed, and then the brain tissue was placed in a constant temperature drying box at 100 °C, and dried for 24 hours to constant weight. Percent brain edema = (wet weight - dry weight)/wet weight x 100%.

Results: As shown in Figure 3, the percentage of cerebral edema in the model group was 19.23±2.38%. The percentage of brain edema in the salvianolic acid D low-dose group (1 mg/kg) was 14.72±4.27%, which was significantly lower than that in the model group (P<0.05). The percentage of cerebral edema in the salvianolic acid D medium dose group (3 mg/kg) was 13.44±2.72%, which was significantly lower than that in the model group (P<0.01). The percentage of brain edema in the salvianolic acid D high-dose group (15 mg/kg) was 12.93±1.85%, which was also significantly lower than that in the model group (P<0.01). This suggests that salvianolic acid D can inhibit cerebral edema in rats with cerebral ischemia-reperfusion and reduce brain tissue damage.

Example 4: Effect of salvianolic acid D on neuronal apoptosis in brain tissue of rats with cerebral ischemia-reperfusion

Experimental materials: TUNEL apoptosis kit was purchased from Roche, Bax, Bcl-2, Cleaved Caspase-3, Cleaved Caspase-9, GAPDH antibodies were purchased from Cell Signaling Technology Inc., NeuN, CytochromeC antibodies were purchased from Abcam, DAPI was purchased from Sigma-Aldrich , BCA Protein Assay Kit was purchased from Pulilai geneTechnology Co., Ltd., HRP conjugated Goat Anti-Mouse IgG, HRP conjugated Goat Anti-Rabbit IgG and cECL Western Blot Kit were purchased from CWBIO. Other relevant experimental materials are the same as in Example 1.

Experimental grouping: SD rats were randomly divided into sham operation control group, model group, salvianolic acid D low, medium and high dose groups, 8 in each group, including 4 in Western blot experiment and 4 in TUNEL fluorescence experiment. Other relevant experimental protocols are the same as those in Example 1.

Western blot was used to detect the changes of protein expression: protein extraction kits were used to extract cellular proteins, and BCA protein quantification kits were used for quantification. Add SDS sample buffer, bath in boiling water for 10 min, conduct SDS-PAGE electrophoresis with the same protein content, transfer to PVDF membrane, then block with 5% BSA for 2 h at room temperature, add diluted primary antibody, incubate at 4°C overnight; wash 3 times with TBST , each time for 10min; add the corresponding secondary antibody diluent, incubate at room temperature for 1h, wash 3 times with TBST for 10min each time; use ECL luminescence method to detect the expression of various proteins in different samples.

TUNEL method: Incubate brain tissue sections with proteinase K solution for 30 min at room temperature. Wash three times with PBS, add buffer solution and incubate for 20 min at room temperature. Next, the reaction solution was added and incubated for 60 min. Subsequently, 2× sodium citrate solution was added and incubated for 15 min. After washing three times with PBS, the brain tissue sections were incubated in 0.5% Triton X-100 solution for 20 min. 10% goat serum was added and incubated for 30 min. The primary antibody against NeuN was then added and incubated overnight. After washing three times with PBS, the corresponding secondary antibodies were added and incubated at room temperature for 1 h. Add DAPI and incubate for 10 min. Finally, fluorescence images are acquired with a fluorescence microscope.

Results: During the process of apoptosis, many proteins were involved in its signal transduction pathway, including the anti-apoptotic protein Bcl-2, the pro-apoptotic proteins Bax, Caspase-3, Caspase-9 and Cytochrome C. Western blotting was used. Detection of changes in protein expression can well reflect the level of apoptosis in tissue cells. TUNEL method, also known as in situ end labeling of DNA breaks, is a classical method that can reflect the level of apoptosis by fluorescence intensity. As shown in Figure 4, the expression levels of the pro-apoptotic proteins Bax, Caspase-3, Caspase-9 and Cytochrome C in the brain tissue of the model group were significantly increased, while the expression level of the anti-apoptotic protein Bcl-2 was significantly decreased. Salvianolic acid D group can reduce the expression levels of pro-apoptotic proteins Bax, Caspase-3, Caspase-9 and Cytochrome C, and at the same time increase the expression level of anti-apoptotic protein Bcl-2. The results of TUNEL assay showed that the rate of TUNEL positive cells in the damaged brain area of the model group was 46.22±12.29% (P<0.001). The rate of TUNEL positive cells in the damaged brain area of the salvianolic acid D low-dose group (1 mg/kg) was 39.21±5.16%, and there was no significant difference compared with the model group. The rate of TUNEL positive cells in the injured brain area of the rats in the salvianolic acid D medium dose group (3 mg/kg) was 28.29±3.69%, which was significantly lower than that in the model group (P<0.01). The rate of TUNEL positive cells in the damaged brain area of the rats in the high-dose salvianolic acid D group (15 mg/kg) was 22.03±3.92%, which was also significantly lower than that in the model group (P<0.001). This suggests that salvianolic acid D can significantly reduce the apoptosis of nerve cells in the damaged brain region and play a protective role in cerebral ischemia.

Example 5: The effect of salvianolic acid D on the opening degree of the blood-brain barrier in rats with cerebral ischemia-reperfusion

Experimental materials: Evans Blue was purchased from Sigma-Aldrich. Other relevant experimental materials are the same as in Example 1.

Experimental grouping: SD rats were randomly divided into sham-operated control group, model group, salvianolic acid D low-dose, medium-dose and high-dose groups, with 5 rats in each group. Other relevant experimental protocols are the same as those in Example 1.

At the end of the experiment, rats were intravenously injected with 4% Evans blue (0.2 mL/100 g), followed by 20 mL of 10 U/mL heparin sodium to flush out the blood. The brains were then isolated, weighed, and homogenized with 50% trichloroacetic acid solution. After centrifugation at 400 × g for 20 min, the measurement was performed spectrophotometrically at 620 nm. The content of Evans blue in cerebral ischemic hemisphere was expressed in ng/g.

Results: Evans blue is a commonly used azo dye reagent. Its molecular weight is similar to that of plasma albumin, and it has a high affinity with plasma albumin in blood. Under normal conditions, plasma albumin cannot penetrate the blood-brain barrier (BBB), so Evans blue cannot color it. During a stroke, the blood-brain barrier is disrupted, and Evansblue enters the nervous system and colors it. As shown in Figure 5, the penetration of Evans blue in the rats in the model group was significantly increased, indicating that the blood-brain barrier was severely damaged and the opening of the blood-brain barrier was increased. Compared with the model group, the salvianolic acid D low-dose group (1 mg/kg) had a significant decrease in the penetration of Evans blue (P<0.01), and the opening of the blood-brain barrier was reduced. Compared with the model group, the salvianolic acid D medium and high dose groups (3, 15 mg/kg) had significantly lower Evans blue penetration (P<0.001), and the degree of blood-brain barrier damage was significantly reduced. This suggests that salvianolic acid D can inhibit the damage of the blood-brain barrier in rats with cerebral ischemia, reduce its openness and permeability, and play a protective role in the blood-brain barrier.

Example 6: The effect of salvianolic acid D on the viability of BV2 cells injured by hypoglycemia, hypoxia, reglucose and reoxygenation

Experimental materials: NAC (N-Acetyl-L-cysteine), polylysine and MTT were purchased from Sigma-Aldrich, Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum was purchased from Thermo Fisher Scientific.

BV2 cell culture: BV2 cells were cultured in culture flasks or 96-well plates. The high-glucose DMEM medium used for the culture contained 10% inactivated fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin. The incubator contains 95% air and 5% _CO2 . Flasks or 96-well plates were coated with polylysine at 37°C for 2h before use.

The establishment and grouping of the hypoglycemia hypoxia-reoxygenation (OGD/R) model: when the model was established, Earle's balanced salt solution (116mmol/L NaCl, 5.4mmol/L KCl, 0.8mmol/L MgSO ₄ , 1mmol/ L NaH ₂ PO ₄ , 0.9 mmol/LCaCl ₂ and 10 mg/L phenol red) to replace the medium, and then immediately transfer the BV2 cells from the normal incubator to the Thermo Scientific Series 8000WJ/IR/N2oxygen-free incubator (94% _N2 , 5% _CO2 , 1% _O2 ). After 2 h of OGD, complete medium with or without salvianolic acid D or NAC was re-added to the cells, and the cells were transferred to a normal incubator for 24 h. The experiment was divided into 6 groups: normal control group, OGD/R model group, low-dose group of salvianolic acid D (OGD/R+0.1 μM SalD), medium-dose group of salvianolic acid D (OGD/R+1 μM SalD), and salvianolic acid D group. Phenolic acid D high dose group (OGD/R+5μM SalD) and positive control NAC group (OGD/R+50μM NAC).

Determination of cell viability by MTT method: BV2 cells were seeded in a 96-well plate at a density of 2×10 ⁴ per well, and cultured for 24 hours. At the end of the experiment, 20 μL of MTT (5 mg/mL) was added to each well and incubated for 4 h. Finally, the original culture medium was removed, and 100 μL of DMSO was added to each well, followed by shaking for 15 min. Detection was performed at a wavelength of 490 nm using a SpectraMax M5 microplate reader (Molecular Devices).

As shown in Figure 6, the survival rate of BV2 cells in the model group was 52.03±3.72% (P<0.001). The survival rate of BV2 cells in the low-dose salvianolic acid D group (0.1 μM SalD) was 66.40±4.27%, which was significantly higher than that in the model group (P<0.05). The survival rate of BV2 cells in salvianolic acid D medium dose group (1μM SalD) was 73.71±4.65%, which was significantly higher than that in the model group (P<0.001). The survival rate of BV2 cells in the high-dose salvianolic acid D group (5μM SalD) was 81.71±7.38%, and the cell viability was significantly improved compared with the model group (P<0.001). This suggests that salvianolic acid D can improve the survival rate of BV2 cells and play a cytoprotective role.

To sum up, the present invention uses cerebral ischemia-reperfusion rat model to investigate the anti-stroke effect of salvianolic acid D, and the results show that injection of salvianolic acid D can significantly improve the neurobehavioral defects of stroke rats. Symptoms, significantly reduce the volume of cerebral infarction in stroke rats, reduce the percentage of cerebral edema in stroke rats, reduce neuronal apoptosis, improve the survival rate of nerve cells, and reduce the permeability and openness of the blood-brain barrier. Therefore, salvianolic acid D has anti-stroke effect. Using salvianolic acid D as the active substance, used alone or/with other pharmacologically active compounds and/or extracts to form a compound, and prepared into various dosage forms of anti-stroke drugs and/or according to conventional preparation methods in the field of pharmacy Health care products, or compound preparations with other drugs, are used to reduce adverse reactions in drug action while maintaining efficacy, and provide an effective solution for the prevention and treatment of stroke.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described with reference to the preferred embodiments of the present invention, those of ordinary skill in the art should understand that it can be implemented Various changes in the above and in the details may be made therein without departing from the spirit and scope of the present invention as defined by the appended claims.

Claims (6)

1. salvianolic acid D and its pharmaceutically acceptable salt shown in formula I are in preparation prevention and/or treatment cerebral ischemia product Using；

2. application according to claim 1, the cerebral ischemia is acute ischemic cerebral apoplexy, transience brain caused by various factors Ischemic episode and chronic insufficiency of blood supply for brain.

3. a kind of application of pharmaceutical composition in preparation prevention and/or treatment cerebral ischemia product, which is characterized in that containing having the right It is required that 1 salvianolic acid D and its pharmaceutically acceptable salt and pharmaceutically acceptable carrier.

4. application according to claim 1, which is characterized in that the product is selected from drug or health care product.

5. application according to claim 3, which is characterized in that the pharmaceutical composition is selected from oral preparation, is administered to medicament Type and skin and mucosa approach form of administration.

6. application according to claim 5, which is characterized in that the oral preparation includes tablet, capsule, sustained release agent, control Release agent, pill, powder, granule, solution, emulsion, suspension, the injecting medicine-feeding form includes intramuscular injection, quiet Arteries and veins injection, intravenous drip, the skin and mucosa approach form of administration includes externally used solution agent, lotion, liniment, ointment, plaster Agent, paste, patch, eye drops, nasal drop, ophthalmic ointment, gargle, sublingual tablets, adhesive sheet, patch.