Application of penicillin tenuiplicatum in preparation of product for preventing or treating ischemic encephalopathy

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of penicillin tenuipes in preparation of a product for preventing or treating ischemic encephalopathy, cell experiments and animal experiments show that penicillin tenuipes has a good treatment effect on cerebral thrombosis and middle artery embolism, can obviously improve behavioral scores after ischemia, and can be used for preparing a medicine for preventing or treating ischemic encephalopathy. And cerebral thrombus and blood vessel diameter conditions. The penicillin tenuipes also has an anti-inflammatory effect. Therefore, the compound can be used for preparing medicines for preventing or treating ischemic encephalopathy.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to a new use of penicillin, specifically the application of penicillin in the preparation of products for the prevention or treatment of ischemic encephalopathy. Background Technology

[0002] Ischemic encephalopathy (IE) is a common age-related disease caused by insufficient blood supply to the brain due to narrowing or blockage of arteries supplying blood to the brain, leading to brain tissue necrosis. Ischemic encephalopathy includes ischemic stroke, cerebral thrombosis, cerebral embolism, lacunar ischemic stroke, multiple ischemic stroke, and mini-stroke. Ischemic encephalopathy has a high incidence, and its mortality and disability rates are also high. Studies show that 75% of patients will die or be left with varying degrees of disability. Because this type of disease is characterized by multiple causative factors, slow pathological progression, long course, and high treatment costs, it places a heavy burden on society and families, and has become one of the most pressing public health challenges to address in China and even the world. Current treatment methods include a method for preparing and applying a drug for ischemic encephalopathy such as cerebral embolism and cerebral vasospasm, as well as cerebral arteriosclerosis, as disclosed in Chinese patent CN200610041875.1. The raw materials consist of: 1400-2000g of kudzu root, 1400-2000g of ginkgo leaves, and 5.5-8.5g of borneol. The kudzu root and ginkgo leaves are selected, washed, and dried separately, then mixed and pulverized into coarse powder. 70% ethanol is added, and the mixture is soaked and then refluxed twice, each time for 2 hours, with the ethanol amount being 8 times the weight of the medicinal materials. The extracts are combined, and the ethanol is recovered under reduced pressure to concentrate the extract. The relative density was reduced to 1.06 to 1.10 at 60℃; half the volume of water was added to the concentrate, stirred evenly, and allowed to stand for 24 hours before filtration; the filtrate was loaded onto a D-101 macroporous adsorption resin column, eluted with water until colorless and discarded, then eluted with 80% ethanol; the ethanol was recovered from the eluent, and the concentrate was concentrated to 1.06 to 1.10 at 60℃; spray drying or vacuum drying was then performed. For spray drying, the inlet air temperature was 160℃ to 180℃, the outlet air temperature was 80℃ to 100℃, and the atomizer speed was 400 rpm to 60 rpm; for vacuum drying, the vacuum degree was 0.09. Dry at 50℃ for 15 to 25 hours at a pressure of MPa to obtain a dry extract powder or powder. Separately, dissolve borneol in anhydrous ethanol and add or spray the borneol solution dissolved in anhydrous ethanol into the dry extract powder or powder, stir evenly, and granulate. Alternatively, obtain a dry extract powder by spray drying, add 1% to 1.5% starch by weight, and then add a borneol solution dissolved in anhydrous ethanol to granulate. Dry the granules, sizing them, and prepare them into capsules, tablets, or granules using existing mature technologies. It has the functions of promoting blood circulation, removing blood stasis, and clearing the meridians and collaterals, and is clinically used for cerebral thrombosis and thrombotic cerebral infarction. Chinese patent CN202210344731.2 discloses the application of tamarind extract in the preparation of drugs for the prevention or treatment of ischemic encephalopathy. Tamarind extract has a significant therapeutic effect on motor disorders and neurological function damage in rats with focal ischemic stroke model caused by middle artery ischemia-reperfusion injury; it also has a significant effect on improving learning and cognitive impairment in this model rats.

[0003] Ruggulosin is an organic compound produced by a specific fungus, with CAS registry number 23537-16-8 and molecular formula C30H22O10. To date, there are no reports regarding the preventive or therapeutic effects of ruggulosin on ischemic encephalopathy. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the existing technology and provide a new use for fine-wrinkle penicillin in the preparation of products for the prevention or treatment of ischemic encephalopathy.

[0005] To achieve the above objectives, the present invention provides the use of frenulum penicillin in the preparation of products for the prevention or treatment of ischemic encephalopathy.

[0006] The chemical structural formula of the rugulosin is as follows:

[0007] .

[0008] The products described in this invention include pharmaceuticals and food; the food includes functional foods or health products.

[0009] The ischemic encephalopathy described in this invention includes cerebral thrombosis and middle cerebral artery embolism.

[0010] The fine-wrinkle penicillin described in this invention is a commercially available product.

[0011] The dosage forms of the drugs described in this invention include, but are not limited to, injections, capsules, tablets, granules, gels, sustained-release preparations, oral liquids, drop pills, or nano-preparations.

[0012] The drug of the present invention includes pharmaceutically acceptable excipients, including: fillers, disintegrants, lubricants, suspending agents, binders, sweeteners, flavoring agents, preservatives, matrix, etc.

[0013] The present invention demonstrates that the micro-wrinkled penicillin has a significant therapeutic effect on ischemic encephalopathy, and can significantly improve behavioral scores, cerebral thrombosis and vascular diameter after ischemia.

[0014] Compared with existing technologies, this invention is the first to discover that frenuloplastin can be used to prevent or treat ischemic encephalopathy. Cell experiments and animal experiments have shown that frenuloplastin has a good therapeutic effect on cerebral thrombosis and middle cerebral artery embolism, and it also has anti-inflammatory effects. Therefore, it can be used to prepare drugs for the prevention or treatment of ischemic encephalopathy. Attached Figure Description

[0015] Figure 1 This is a schematic diagram illustrating the experimental results of the effect of the wrinkled penicillin on cerebral thrombosis in zebrafish, as per the present invention.

[0016] Figure 2 This is a schematic diagram of the experimental results of the effect of fine-wrinkled penicillin on the movement of zebrafish with cerebral thrombosis, as per the present invention. In the diagram, A is the zebrafish's movement trajectory; B is the total distance traveled; C is the average speed; D is the acceleration; E is the activity level; F is the angular velocity; and G is the tortuosity of the movement. * P<0.05, ** P<0.01, *** P<0.001.

[0017] Figure 3 This is a schematic diagram of the experimental results of the effect of the wrinkled penicillin on the blood vessel diameter of zebrafish involved in this invention. In this diagram, A is a fluorescence image of transgenic zebrafish Tg(lyz: DsRED2); B is a statistical analysis diagram of the fluorescence of zebrafish juvenile Tg(lyz: DsRED2); *P<0.05, **P<0.01, ***P<0.001.

[0018] Figure 4 The present invention relates to a fine-wrinkled penicillin that inhibits the expression of zebrafish inflammatory genes TNF-α, IL-1β and IL-6, wherein A is TNF-α, B is IL-1β and C is IL-6; * P<0.05, ** P<0.01, *** P<0.001.

[0019] Figure 5 The effect of the penicillin in the present invention on the inflammatory response of BV2 microglia is shown in Figure A, where A represents the nitrite content in the supernatant; BD represents the expression of inflammatory factors IL-6, IL-1β, and TNF-α in the supernatant; *P<0.05, **P<0.01, ***P<0.001.

[0020] Figure 6 The present invention relates to the fine-wrinkled penicillin for improving ischemic stroke injury in mice, wherein A is TTC staining; B is neurological behavioral score; C is weight loss index; *P<0.05, **P<0.01, ***P<0.001.

[0021] Figure 7 The figure shows the toxicity test results of the wrinkled penicillin of the present invention on zebrafish, where A is the tail whipping frequency, B is the body length, C is the heart rate, and D is the mortality rate.

[0022] Figure 8 The figure shows the cytotoxicity test results of the fine-wrinkle penicillin involved in this invention.

[0023] Figure 9 The figure shows the toxicity test results of the wrinkled penicillin involved in this invention on mice. Detailed Implementation

[0024] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0025] Example 1:

[0026] This embodiment involves an experiment on the effect of fine-wrinkle penicillin on cerebral thrombosis, specifically:

[0027] I. Effects of fine-wrinkled penicillin on thrombus area and motor injury in zebrafish with cerebral thrombosis

[0028] 1. Establishing a zebrafish cerebral thrombosis model

[0029] Zebrafish are excellent model organisms for studying thrombosis. A zebrafish cerebral ischemia model was prepared using ponatinib. Specifically, 2-day-fiber zebrafish embryos were cultured in 6-well plates with 15-20 embryos per well. After developing into juveniles, the zebrafish juveniles were treated with 1 μg / ml ponatinib (pon) for 24 hours to induce cerebral thrombosis.

[0030] 2. Effects of fine-wrinkle penicillin on cerebral thrombosis in zebrafish

[0031] Model zebrafish were co-incubated with fine-wrinkle penicillin (1 μg / ml) for 24 hours to form the experimental group; normal zebrafish larvae served as the normal control group. Zebrafish were stained with o-dianisidine. Each group of zebrafish was placed in a 0.6 mg / ml o-dianisidine staining solution containing 10 mM sodium acetate and 4 v / v% ethanol and incubated in the dark at 28 ℃ for 15 min, followed by washing three times with 100% DMSO. The zebrafish were observed and photographed under a stereomicroscope. The results are as follows: Figure 1 As shown.

[0032] from Figure 1 It can be seen that stimulation of zebrafish juveniles with 1 μg / ml ponatinib can induce significant cerebral thrombosis in the brain and significantly reduce cardiac blood perfusion, suggesting that ponatinib can obstruct systemic circulation, indicating successful modeling. Compared with the model group, the experimental group, after intervention with penicillin, showed a significant reduction in thrombosis and a significant increase in myocardial hemoglobin staining intensity, indicating that penicillin has a significant therapeutic effect on zebrafish thrombosis.

[0033] 3. Zebrafish sports injury assessment

[0034] Zebrafish from the normal control group, the model group, and the experimental group treated with different concentrations (2 μg / ml, 1 μg / ml, and 0.5 μg / ml) of penicillin as described above were placed in 96-well plates, one fish per well. The zebrafish were acclimatized in the 96-well plates for 10 min at 28°C before movement was recorded. All experiments were conducted in bright or dark environments (10 min bright, 10 min dark alternation) for a total of 60 min. The total distance, average speed, acceleration, angular velocity, and activity level of the zebrafish were recorded and analyzed using a viewpoint behavior analyzer. The zebrafish behavior trajectory was tracked and quantified using the Nordas zebrafish behavior trajectory tracking system. The results are as follows: Figure 2 As shown.

[0035] from Figure 2 As can be seen, compared with the normal control group, the zebrafish in the model group exhibited significantly reduced total distance of movement, average speed, acceleration, and activity frequency, as well as significantly increased flexure and angular velocity, indicating that cerebral thrombosis can cause damage to the overall behavior and neurobehavioral behavior of zebrafish. Compared with the model group, the three experimental groups, especially the experimental groups treated with 2 μg / ml and 1 μg / ml concentrations, could significantly reverse these motor indicators, indicating that Rugulosin can improve the motor damage in zebrafish caused by cerebral ischemia.

[0036] 4. Effects of fine-wrinkle penicillin on the expression of inflammation-related genes in zebrafish

[0037] Inflammation-related genes were detected in zebrafish from the normal control group, the model group, and the experimental group treated with 1 μg / ml of penicillin. The expression of inflammatory-related genes TNF-α, IL-1β, and IL-6 in zebrafish was detected by RT-qPCR. Specifically, total RNA was extracted from homogenates of 50 zebrafish in each group using Trizol reagent, and three replicates of RNA extracted from zebrafish were analyzed. The quality of RNA samples was assessed by agarose gel electrophoresis. Approximately 2 μg of total RNA from each sample was used for cDNA synthesis using the FastQuant RT Kit (With gDNase) (Tiangen), and qPCR amplification was performed using the iTaq Universal SYBR Green Supermix (Biorad) on the CFX Connect detection system (Biorad). Melting curve analysis was performed to check primer specificity. Gene expression data were normalized to β-actin levels and analyzed using 2... -△△Ct The method involved calculating the relative quantification of mRNA levels for each gene between groups. Results are as follows: Figure 4 As shown. Primer sequences are shown in Table 1.

[0038] Table 1. RT-qPCR primer sequences

[0039]

[0040] from Figure 4 As can be seen, compared with the normal control group, the expression of L-6, TNF-α, and IL-1β genes in the model group was significantly upregulated. Compared with the model group, the expression of IL-6, TNF-α, and IL-1β genes in the experimental group was significantly decreased. This indicates that penicillin can significantly inhibit the expression of the above genes.

[0041] II. Anti-inflammatory effects of fine-wrinkled penicillin on BV2 cells

[0042] BV2 microglia were cultured in MEM medium containing 10% inactivated fetal bovine serum, 100 U / ml penicillin, and 100 g / ml streptomycin. BV2 cells were divided into a normal group, an LPS group, and three experimental groups. The normal group received no treatment; the LPS group was treated with 0.1 μg / ml LPS for 24 h; and the three experimental groups were treated with 0.1 μg / ml LPS and different concentrations (0.1 μg / ml, 1 μg / ml, and 10 μg / ml) of penicillin for 24 h. The concentrations of nitrite (which reflects the amount of NO released from the cell supernatant) and inflammatory factors (IL-6, IL-1β, and TNF-α) in the cell supernatant of each group were measured. The results are as follows: Figure 5 As shown.

[0043] from Figure 5 It can be seen that, compared with the normal group, the NO content in the LPS group was significantly increased, and the serum concentrations of IL-6, IL-1β, and TNF-α were also significantly increased, indicating that LPS stimulation can induce cells to produce more NO, mainly reflected in the elevated serum nitrite. LPS intervention significantly increased the serum concentrations of IL-6, IL-1β, and TNF-α. Compared with the LPS group, the NO content in the experimental group was decreased, and the concentrations of IL-6 and IL-1β were also significantly decreased. This indicates that penicillin can significantly reduce the concentrations of nitrite and inflammatory factors IL-6 and IL-1β in the cell supernatant, and has an anti-inflammatory effect on BV2 microglia.

[0044] III. Effects of Penicillin on the Immunity of Zebrafish with Cerebral Thrombosis

[0045] Transgenic zebrafish Tg (lyz: DsRED2) was used as the experimental subject. Juvenile zebrafish were stimulated with ponatinib for 24 hours to form the model group. The model transgenic zebrafish were treated with penicillin (1 μg / ml) for 24 hours to form the experimental group. Normal transgenic zebrafish served as the normal control group. Fifteen juvenile zebrafish from each of the normal control group, model group, and experimental group were randomly selected. After tricaine anesthesia, lyz fluorescence signals were collected using a fluorescence microscope. ImageJ was used to extract a single channel from the acquired images, adjust the threshold, and analyze the average fluorescence intensity of the lyz gene expression region. The analysis formula was: Average fluorescence intensity (Mean) = Sum of fluorescence intensities in the region (IntDen) / Area of ​​the region (Area). The results are as follows: Figure 3 As shown.

[0046] from Figure 3 It can be seen that, compared with the normal control group, the fluorescence intensity of the transgenic zebrafish in the model group was significantly reduced, indicating that the overall immunity of the body was reduced; compared with the model group, the fluorescence intensity of the transgenic zebrafish in the experimental group was significantly increased, indicating that penicillin can improve the body's immunity.

[0047] Example 2:

[0048] This embodiment relates to an experiment on the ameliorative effect of fine-wrinkle penicillin on neurobehavioral damage in mice with middle cerebral artery embolism, specifically:

[0049] 1. Preparation of a mouse middle artery embolism model

[0050] C57BL / 6J mice were weighed, anesthetized with isoflurane, and fixed in a supine position on the operating table. The carotid artery and the origin of the external carotid artery were exposed sequentially under a stereomicroscope. The proximal end of the carotid artery was ligated, and a ligation suture was placed near the bifurcation of the carotid artery. A micro-arterial clamp was placed, and a small incision was made in the carotid artery between the two sutures. The suture plug was quickly inserted, the arterial clamp was released, and insertion was continued. When the suture plug was inserted 1 cm, a slight resistance was felt, and the suture was immediately stopped. At this point, the tip of the suture had just reached the middle cerebral artery. The suture at the bifurcation was ligated, and the excess suture was cut off. The mice were placed in a 37°C incubator and left to be ischemic for 1 hour. The suture plug was then removed, and the soft tissue and skin were sutured. The model preparation was completed.

[0051] The experimental group was set up. Mice in the experimental group were first administered penicillin by gavage at a dose of 10 mg / kg or 20 mg / kg (10 mg or 20 mg penicillin per kg of mice). Half an hour later, the middle cerebral artery was embolized as described above. The infarct volume was measured 24 hours after ischemia-reperfusion in the model group and the experimental group. Normal mice were used as the control group.

[0052] 2. Neurobehavioral score

[0053] Twenty-four hours after middle cerebral artery embolization surgery, mice underwent neurobehavioral observation. Following the method of Bederson et al., the mice were lifted by their tails approximately one foot off the ground to observe the condition of their forelimbs; then, the mice were placed on a horizontal surface, and their shoulders were pushed to observe any differences in resistance between the two limbs; finally, the mice were placed back on the ground to observe their walking ability. A five-point rating scale (0-4 points) was used, with higher scores indicating more severe neurobehavioral impairment. Results are as follows: Figure 6 As shown in B.

[0054] Those whose behavior is completely normal will receive 0 points. If the rat's tail is lifted off the ground and the forelimb on the opposite side of the operation is internally rotated and adducted, 1 point is awarded. The mouse was placed on the ground and its resistance was checked by squeezing both sides of the mouse. If the resistance on the opposite side of the operation decreased, 2 points were scored. Place the mouse on the ground and observe its walking. If it circles around the side opposite the surgery, score 3 points. The mouse was severely injured and unable to move on its own, so it was scored 4 points.

[0055] from Figure 6 As shown in B, compared with the normal control group, the neurobehavioral scores of the model group were significantly increased; compared with the model group, the neurobehavioral scores of the experimental group mice were significantly decreased.

[0056] 3. Weight Loss Index Calculation

[0057] After observing neurobehavioral scores, the mice were weighed, and the weight loss index (%) was calculated. The results are as follows: Figure 6 As shown in C.

[0058] from Figure 6 As can be seen from C, compared with the normal control group, the weight loss index of the model group was significantly increased; compared with the model group, the weight loss index of the experimental group mice was significantly decreased.

[0059] 4. Measurement of cerebral infarction volume

[0060] 2,3,5-Triphenyltetrazolium chloride (TTC) reacts with succinate dehydrogenase in the mitochondria of living cells to produce red formazan, which is used to detect cell viability and is a commonly used reagent for evaluating cerebral ischemia-reperfusion injury. Mice were euthanized by decapitation after cerebral ischemia. The extracted brain tissue was quickly placed in a -20°C freezer for 10 min, then removed. The olfactory bulb, cerebellum, and lower brainstem were removed, and the brain was cut into six consecutive coronal sections at 2 mm intervals according to the brain localization atlas. The brain slices were then quickly placed in 5 ml of a 2% TTC solution and incubated at 37°C in the dark for 30 min. The slices were turned every 10 min to ensure uniform staining; normal tissue was stained rose-red, and infarcted tissue was stained white. The brain slices were then removed and photographed with a digital camera. The differences in cerebral infarction volume between the groups were compared. The results are as follows: Figure 6 As shown in Figure A.

[0061] from Figure 6 As shown in Figure A, compared to the normal control group, the infarct volume in the model group mice was significantly increased after middle artery embolization. Compared to the model group, the infarct volume in the experimental groups significantly decreased after intervention with penicillin at 10 mg / kg or 20 mg / kg. This indicates that penicillin can significantly reduce the infarct volume induced by middle artery embolization in mice.

[0062] Example 3:

[0063] This embodiment involves toxicological experiments on fine-wrinkle penicillin.

[0064] 1. Toxicity test of penicillin in zebrafish

[0065] Zebrafish larvae from the normal control group were incubated with penicillin (0.5 μg / ml, 1 μg / ml, and 2 μg / ml) for 24 hours, serving as the experimental group; normal zebrafish larvae served as the normal control group. The number of tail flicks, body length, heart rate, and mortality rate of the zebrafish larvae were recorded. The results are as follows: Figure 7 As shown.

[0066] from Figure 7 It can be seen that, compared with the normal control group, the doses of 0.5 μg / ml, 1 μg / ml and 2 μg / ml of trichomoniasis penicillin had no significant effect on the number of tail flicks, body length, heart rate and mortality of zebrafish fry, indicating that trichomoniasis penicillin has no obvious toxicity.

[0067] 2. Cytotoxicity test of penicillin for fine wrinkles

[0068] BV2 microglia were co-incubated with fine-wrinkle penicillin (0.1 μg / ml, 1 μg / ml, 10 μg / ml) for 24 hours as the experimental group, with normal BV2 cells serving as the normal control group. Cell viability was assessed using a CCK-8 assay kit, and the results are as follows: Figure 8 As shown. From Figure 8 It can be seen that, at experimental doses of 0.1 μg / ml, 1 μg / ml and 10 μg / ml, penicillin had no significant effect on the viability of BV2 cells, indicating that penicillin has no obvious toxicity.

[0069] 3. Toxicity test of penicillin in mice

[0070] An experimental group was set up, in which C57BL / 6J mice were administered penicillin via gavage at a dosage of 20 mg / kg (20 mg penicillin per kg of mouse). Normal mice served as the control group. The daily behavior of the mice was observed, and their body weight was recorded continuously for 30 days. The results are as follows: Figure 9 As shown. Observations revealed no significant abnormalities in the daily behavior of the 20 experimental mice; on day 27, one mouse died. This mouse showed no obvious abnormalities before death, and autopsy revealed no abnormalities in the brain, heart, liver, spleen, lungs, or kidneys. This death was considered a random event. From Figure 9 It can be seen that at the experimental dose of 20 mg / kg, penicillin had no significant effect on the weight change of mice, indicating that penicillin has no obvious toxicity. Further conversion between mouse and human doses using the dosage conversion method in "Pharmacological Experimental Methodology" yields a human dose of 2.19 mg / kg for mice. Therefore, a human dose of penicillin at 2.19 mg / kg or lower is safe.

Claims

1. Use of penicillin rugosum in the preparation of a product for preventing or treating ischemic encephalopathy.

2. The use of the fine-creped penicillin according to claim 1 in the manufacture of a product for the prevention or treatment of ischemic encephalopathy, characterized in that, Penicillin rugosum can significantly improve the behavior score after ischemia, cerebral thrombosis and blood vessel diameter.

3. The use of the fine-creped penicillin according to claim 1 in the manufacture of a product for the prevention or treatment of ischemic encephalopathy, characterized in that, The product is a drug or food.

4. The use of the fine-creped penicillin according to claim 1 in the manufacture of a product for the prevention or treatment of ischemic encephalopathy, characterized in that, The ischemic encephalopathy includes cerebral thrombosis and middle artery embolism.

5. The use of the fine-creped penicillin according to claim 3 in the manufacture of a product for the prevention or treatment of ischemic encephalopathy, characterized in that, The dosage form of the drug includes but is not limited to injection, capsule, tablet, granule, gel, sustained-release preparation, oral liquid, dripping pill or nano preparation.

6. The use of the fine-creped penicillin according to claim 3 in the manufacture of a product for the prevention or treatment of ischemic encephalopathy, characterized in that, The drug includes pharmaceutically acceptable excipients, including fillers, disintegrants, lubricants, suspending agents, binders, sweeteners, flavorings, preservatives, bases and the like.

7. The use of the fine-creped penicillin according to claim 3 in the manufacture of a product for the prevention or treatment of ischemic encephalopathy, characterized in that, The food includes functional food or health products.

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