CN106727605B - Application of cyclovirobuxine D in preparing medicine for preventing or treating cerebral arterial thrombosis - Google Patents
Application of cyclovirobuxine D in preparing medicine for preventing or treating cerebral arterial thrombosis Download PDFInfo
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- CN106727605B CN106727605B CN201710084332.6A CN201710084332A CN106727605B CN 106727605 B CN106727605 B CN 106727605B CN 201710084332 A CN201710084332 A CN 201710084332A CN 106727605 B CN106727605 B CN 106727605B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
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- Pharmacology & Pharmacy (AREA)
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Abstract
The invention discloses an application of cyclovirobuxine D in preparing a medicament for preventing or treating cerebral arterial thrombosis, wherein symptoms such as cerebral apoplexy, cerebral infarction, cerebral edema and the like can appear in animals or human under the condition of cerebral ischemia; the cerebral infarction range is narrowed, and cerebral edema is reduced; protecting nerve cell injury caused by apoplexy, improving acute global cerebral ischemia reperfusion injury and focal cerebral ischemia reperfusion injury, and treating ischemic apoplexy.
Description
Technical Field
The invention belongs to the field of pharmaceutical chemicals, and particularly relates to application of cyclovirobuxine D and pharmaceutically acceptable salts thereof in preparation of medicines for preventing or treating cerebral arterial thrombosis, which comprises the steps of improving nerve function damage caused by ischemia, protecting nerve cell damage caused by cerebral apoplexy, reducing cerebral infarction range, protecting brain damage, reducing cerebral edema, resisting oxidation, improving acute global cerebral ischemia reperfusion injury and focal cerebral ischemia reperfusion injury.
Background
The vitamin E Poncirus D is an alkaloid (structural formula shown in figure), belongs to steroid alkaloid, can be extracted from Buxus microphylla of Buxus and other plants of the same genus, and has effects of reducing myocardial oxygen consumption, enhancing myocardial contractility, increasing coronary blood flow, preventing arrhythmia, relieving angina pectoris, etc., and has definite therapeutic effect. At present, cyclovirobuxine D has been recorded and recorded by Chinese pharmacopoeia (2015 edition) according to medicinal plant oil and fat and extract, and oral tablets prepared from cyclovirobuxine D have been collected and carried by Chinese pharmacopoeia (2015 edition) according to prescription preparation and single preparation, have the effects of promoting qi circulation, activating blood circulation, dredging collaterals and relieving pain, and are used for treating thoracic obstruction, heart pain and intermittent pulse caused by qi stagnation and blood stasis; coronary heart disease and arrhythmia with the above syndromes.
The only Huangyangning tablets of the cyclovirobuxine D preparation on the market at home are approved by about ten manufacturers, the specifications are 0.5mg and 1mg per tablet, the daily dose is 2-6mg per day, and the main indications are coronary heart disease and arrhythmia. The Huangyangning tablet has the defects of slow oral effect, low bioavailability, cardiac toxicity accumulation and long peak reaching time of blood concentration, is mainly used for treating mild and moderate patients clinically and is not beneficial to treating severe patients and patients with dysphagia.
CN20111017742.6 discloses a natural medicine cyclovirobuxine D derivative, a preparation method and an application thereof, and pharmacodynamic test results show that the compounds and the derivatives thereof can effectively treat cardiovascular and cerebrovascular diseases, and are mainly used for treating angina, arrhythmia, hypertension or hyperlipidemia.
CN01133058.9 discloses a soluble inorganic salt of cyclovirobuxine D, its preparation and pharmaceutical preparations containing them. The invention relates to a preparation method of water-soluble inorganic salt of cyclovirobuxine D and application of injection thereof in treating coronary heart disease and arrhythmia.
CN02148452 discloses a cyclovirobuxine D salt preparation and its preparation method, which mainly uses cyclovirobuxine D as main drug, and mixes with hydrochloric acid or phosphoric acid, sodium dihydrogen phosphate and mannitol to make injection, in which the cyclovirobuxine D is converted into hydrochloride or phosphate. The preparation is administered intravenously for treating cardiovascular disease and cerebrovascular disease, has effects of activating qi-flowing, promoting blood circulation, dredging collaterals and relieving pain, and can be used for treating thoracic obstruction, cardialgia, coronary atherosclerotic heart disease, and angina pectoris.
At present, the clinical indications of cyclovirobuxine D are all used for treating angina pectoris, coronary heart disease, arrhythmia and other cardiac diseases, and the development of the therapeutic effect of cyclovirobuxine D on other diseases has great clinical therapeutic significance.
Disclosure of Invention
Aiming at the defects of the prior art, the invention discloses a new application of cyclovirobuxine D and pharmaceutically acceptable salts thereof in preparing medicines for preventing or treating cerebral arterial thrombosis, which comprises improving nerve function damage (including motor and sensory nerves) caused by ischemia; the cerebral infarction range is narrowed, and cerebral edema is reduced; protecting nerve cell injury caused by apoplexy, improving acute global cerebral ischemia reperfusion injury and focal cerebral ischemia reperfusion injury, and treating ischemic apoplexy.
The specific technical scheme of the invention is as follows:
the invention aims to disclose a new application of cyclovirobuxine D and pharmaceutically acceptable salts thereof in preparing medicines for preventing or treating cerebral arterial thrombosis. In order to improve the water solubility of cyclovirobuxine D, the pharmaceutically acceptable salt is preferably hydrochloride, sulfate or phosphate. The administration can be oral or injection, preferably intravenous drip or intravenous injection.
Cerebral apoplexy (also called apoplexy) and cerebrovascular accident (CVA) is an acute cerebrovascular disease, is a group of diseases of brain tissue damage caused by the sudden rupture of cerebral vessels or the blood failing to flow into the brain due to the blockage of blood vessels, including ischemic and hemorrhagic apoplexy, and has the characteristics of acute disease onset, critical disease condition, serious sequelae and high disability rate and multiple complications. Cerebral apoplexy is one of the main diseases affecting human health, and is a common disease and a frequently encountered disease of middle-aged and elderly people.
Edaravone is a brain protective agent (free radical scavenger) which can scavenge free radicals and inhibit lipid peroxidation, thereby inhibiting oxidative damage of brain cells, vascular endothelial cells and nerve cells. Ligustrazine hydrochloride is mainly used for treating ischemic cerebrovascular diseases such as cerebral insufficiency, cerebral thrombosis, cerebral embolism and other ischemic vascular diseases such as coronary atherosclerotic heart disease, angiitis, etc. The research of the invention carries out comparative research on the cyclovirobuxine D, the edaravone injection and the ligustrazine hydrochloride for injection, and finds that the effects of the invention on improving the nerve function damage (including motor and sensory nerves) caused by ischemia, reducing the cerebral infarction range, reducing the cerebral edema, improving the SOD activity of superoxide dismutase, resisting oxidation, protecting nerve cell damage caused by cerebral apoplexy and the like are obviously superior to the effects of the edaravone injection and the ligustrazine hydrochloride for injection, and the oral effect is inferior to the intravenous administration.
Vinpocetine can improve various symptoms induced by cerebral infarction sequelae, cerebral hemorrhage sequelae, cerebral arteriosclerosis and the like. The study of the invention compares the cyclovirobuxine D with edaravone injection and vinpocetine injection, and finds that the cyclovirobuxine D can significantly improve the nerve function injury caused by acute global cerebral ischemia reperfusion of rats and the focal cerebral ischemia reperfusion injury of rats.
Drawings
FIG. 1 shows the cerebral infarction range of rats of each administration group in the experiment of the focal cerebral ischemia-reperfusion injury of rats of example 6.
Detailed Description
Terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art, unless otherwise specified.
The present invention is described in further detail below with reference to specific examples and with reference to the data. It will be understood that this example is intended to illustrate the invention and not to limit the scope of the invention in any way.
In the following examples, various procedures and methods not described in detail are conventional methods well known in the art.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 Effect of Cyclovirobuxine D on the cranial nerve function in rats
1. Preparation of the model
SD rats (SPF grade, male, weight 298.4 + -10.1 (276-321) g) were anesthetized with isoflurane gas by VMR desktop small animal anesthesia machine (Matrx, USA) and fixed in lateral position. Cutting skin at the midpoint of a connecting line between an external auditory canal and an canthus, separating muscles layer by layer, exposing a zygomatic arch and a temporal bone scale, reaming the zygomatic arch, under an operating microscope, drilling a 2 mm-diameter small cranial window in a dental drill at a position about 2mm below the front connection position of the zygomatic arch and the temporal bone scale, exposing a middle cerebral artery, applying a small piece of filter paper to the middle artery, dripping 10 mu l of 50% FeCl3 on the filter paper, taking out the filter paper after 30min, cleaning the filter paper with physiological saline, adding 2-3 drops of penicillin solution (1.6 multiplied by 105U/mL) to prevent wound infection, suturing layer by layer, and returning to a cage for feeding. The sham group was operated in the same manner as the other animals except that FeCl3 was not added dropwise.
2. Grouping and administration of drugs
80 animals which are successfully molded are selected and randomly divided into 8 groups by adopting a random number table method, and each group comprises 10 animals. Respectively comprising a model group, a tested drug 5 dose group, a positive drug edaravone injection (Yangzhou pharmaceutical Co., Ltd., specification 20 ml: 30mg) group, and a ligustrazine hydrochloride injection (Hefei Pingguang pharmaceutical Co., Ltd., 80mg per bottle, white powder) group. The tested drugs are divided into an intravenous infusion group and an oral administration group, the D4 doses of cyclovirobuxine are respectively 0.125, 0.25, 0.5 and 1mg/kg in the intravenous infusion group, the D1 doses (po) of cyclovirobuxine are respectively 0.5mg/kg in the oral administration group, the edaravone injection intravenous infusion group is 6.0mg/kg in dose, the ligustrazine hydrochloride intravenous infusion group is injected in dose of 12.4mg/kg, and in addition, 10 sham operation groups are arranged.
The reagents are all diluted to the required concentration by normal saline before use for intravenous administration. The administration volume was 4ml/kgBW and the infusion time was 30 min. Sham and model control groups were given equal volumes of saline.
3. Effect on neural function
Animals were scored for neurological impairment by blinding before and 24h after dosing according to the scoring criteria in table 1, with a total score of 16. Data are expressed as mean ± sd
Representing that student's t-test, P was used for comparison between two groups<A difference of 0.05 is statistically significant, and the results are shown in Table 2.
TABLE 1 scoring criteria for degree of neurological impairment in MCAO rats
TABLE 2 Effect of Cyclovirobuxine D on the degree of neurological impairment in rats with cerebral ischemia: (
n＝10)
Note: 1. the difference between the value in parentheses and the value before administration;
2. comparison with sham group:△△△P<0.001；
3. comparison with model control group:*P<0.05；
4. clinical equivalent dose of cyclovirobuxine D vein compared to oral: p >0.05.
5. Clinical equivalent dose cyclovirobuxine D was compared with edaravone: p >0.05.
6. Comparing the clinic equivalent dose of cyclovirobuxine D with ligustrazine hydrochloride: p >0.05.
The results show that the animals in the model group have obvious difference in score compared with the animals in the sham operation group. When cyclovirobuxine D is administered intravenously at 0.125, 0.25, 0.5, 1mg/kg, compared with model control group, its impaired neurological dysfunction score is reduced by 3.6% (P <0.05), 6.7% (P >0.05), 9.7% (P <0.05), 9.5% (P <0.05), and when cyclovirobuxine D is administered orally at 0.5mg/kg, its impaired neurological dysfunction score is reduced by 7.8% (P < 0.01). When edaravone is administered at 6.0mg/kg, the degree of nerve function damage score is reduced by 10.8% (P <0.05) compared with that of the model control group, and the degree of nerve function damage score is reduced by 3.5% (P >0.05) after the ligustrazine hydrochloride injection is administered for treatment.
The results show that FeCl3After the cyclovirobuxine D is intravenously administered to rats in a model of focal cerebral infarction injury caused by induced middle cerebral artery thrombosis, the degree of nerve function damage score is improved in different degrees, and the model has certain dose dependence, and intravenous administration is superior to oral administration. The treatment effect is equivalent to that of edaravone and is superior to that of ligustrazine hydrochloride.
Example 2 Effect of Cyclovirobuxine D on the brain death Range of rats
Example 1 after the experiment, the brain was taken out, frozen rapidly, and cut into 2mm thick slices along the coronal plane, and the above-mentioned half brain slices were placed into 2% tetrazolium oxide (TTC, product of Solarbio) dye solution at intervals to avoidIncubating at 37 ℃ for 10min for dyeing, storing the digital image in a computer by adopting a digital camera imaging system, measuring the infarct area and the whole brain area by using image analysis system v 4.0 software (CMIAS pathological image acquisition and analysis system, Beijing aerospace university image center product), and calculating the cerebral infarction range (the percentage of the infarct area to the whole brain area). Data are expressed as mean ± sd
Representing that student's t-test, P was used for comparison between two groups<A difference of 0.05 is statistically significant. The results are shown in Table 3.
TABLE 3 Effect of Cyclovirobuxine D on the extent of cerebral infarction in rats: (
n＝10)
Note: 1. comparison with sham group:△△△P<0.001；
2. comparison with model control group:*P<0.05，**P<0.01；
3. clinical equivalent dose of cyclovirobuxine D vein compared to oral: p >0.05.
4. Clinical equivalent dose cyclovirobuxine D was compared with edaravone: p >0.05.
5. Comparing the clinic equivalent dose of cyclovirobuxine D with ligustrazine hydrochloride: p >0.05.
The results show that the infarct size of the model animals is obviously increased compared with that of the sham operation animals. When cyclovirobuxine D is administered at 0.125, 0.25, 0.5 and 1.0mg/kg, the cerebral infarction range is respectively reduced by 17.3% (P >0.05), 28.6% (P <0.05), 35.7% (P <0.05) and 36.8% (P <0.01) compared with the model control group. The oral administration of cyclovirobuxine D is reduced by 23.8 percent (P is more than 0.05) in the cerebral infarction range of 0.5 mg/kg; the edaravone is administered at 6.0mg/kg, and compared with the model control group, the cerebral infarction range is reduced by 38.3 percent (P < 0.01); when ligustrazine hydrochloride is given at 12.4mg/kg, the cerebral infarction range is reduced by 23.2% (P is more than 0.05).
The results show that FeCl3After the cyclovirobuxine D is intravenously administered to rats in a model of focal cerebral infarction injury caused by induced middle cerebral artery thrombosis, the cerebral infarction range is improved in different degrees, and certain dose dependence exists, and the intravenous administration of the same dose is superior to the oral administration. The treatment effect is equivalent to that of edaravone and is superior to that of ligustrazine hydrochloride.
Example 3 Effect of Cyclovirobuxine D on cerebral edema in rats
In example 2, the other half of the brain slice is subjected to dry-wet-weight brain water content test, namely the other half of the brain slice is weighed and then placed in an oven at 50 ℃ to be baked to constant weight, the dry weight of the brain slice is weighed, the brain water content is calculated, the brain water content (%) is (wet weight-dry weight)/wet weight × 100% according to the following formula, and the data are expressed as average numbers +/-standard deviation
Representing that student's t-test, P was used for comparison between two groups<A difference of 0.05 is statistically significant. The results are shown in Table 4.
TABLE 4 Effect of Cyclovirobuxine D on cerebral edema in rats: (
n＝10)
Note: 1. comparison with sham group:△△△P<0.001；
2. comparison with model control group: p <0.05, P < 0.01;
3. clinical equivalent dose of cyclovirobuxine D vein compared to oral: p >0.05.
4. Clinical equivalent dose cyclovirobuxine D was compared with edaravone: p >0.05.
5. Comparing the clinic equivalent dose of cyclovirobuxine D with ligustrazine hydrochloride: p >0.05.
The results show that the degree of cerebral edema is reduced by 1.3% (P >0.05), 1.8% (P <0.05), 2.4% (P <0.05), 2.7% (P <0.01) when the cyclovirobuxine D is administered intravenously at 0.125, 0.25, 0.5 and 1.0mg/kg, respectively, and the degree of cerebral edema is reduced by 0.7% (P >0.05) when the cyclovirobuxine D is administered orally at 0.5mg/kg compared with the model control group; edaravone was administered at 6.0mg/kg, with a 2.9% reduction in brain edema compared to the model control group (P < 0.01); when ligustrazine hydrochloride is given at 12.4mg/kg, compared with a model control group, the degree of cerebral edema is reduced by 2.3 percent (P is less than 0.01).
The results show that significant cerebral edema appears after cerebral ischemia of rats. After the cyclovirobuxine D is intravenously administered, the cerebral edema degree is improved in different degrees and has certain dose dependence, and the intravenous administration of the same dose is superior to the oral administration. The treatment effect of the cerebral edema is equivalent to that of edaravone and ligustrazine hydrochloride.
Example 4 Effect of Cyclovirobuxine D on rat oxidative stress factor levels
Example 1 after completion of the experiment, blood was taken from the abdominal aorta, and serum was isolated, and serum MDA and serum SOD were measured by TBA and hydroxylamine methods, respectively. Data are expressed as mean ± sd
Representing that student's t-test, P was used for comparison between two groups<A difference of 0.05 is statistically significant. The results are shown in Table 5.
TABLE 5 Effect of Cyclovirobuxine D on the level of oxidative stress factors in rats with cerebral ischemia (S)
n＝10)
Note: 1. comparison with sham group:△△P<0.01，△△△P<0.001；
2. comparison with model control group: p<0.05，***P<0.01，***P<0.001；
3. Clinical equivalent dose of cyclovirobuxine D vein compared to oral:#P<0.05。
4. clinical equivalent dose cyclovirobuxine D was compared with edaravone: p >0.05.
5. Comparing the clinic equivalent dose of cyclovirobuxine D with ligustrazine hydrochloride:#P<0.05。
the results showed that after intravenous administration of cyclovirobuxine D0.125, 0.25, 0.5 and 1.0mg/kg, MDA was reduced by 1.7% (P >0.05), 19.4% (P <0.01), 27.2% (P <0.001), 28.1% (P <0.001), respectively, compared to the model control group; reduction of 10.9% (P >0.05) of 0.5mg/kg MDA of cyclovirobuxine D given orally; when 6.0mg/kg of edaravone and 12.4mg/kg of ligustrazine hydrochloride are administered, MDA is respectively reduced by 34.6% (P <0.001) and 7.2% (P > 0.05).
After intravenous administration of cyclovirobuxine D0.125, 0.25, 0.5 and 1.0mg/kg, SOD increased by 2.3% (P >0.05), 25.3% (P <0.05), 30.2% (P <0.05), 23.2% (P <0.05), respectively, compared with the model control group; the 0.5mg/kg SOD of the cyclovirobuxine D is increased by 16.7 percent (P < 05 >); when 6.0mg/kg of edaravone and 12.4mg/kg of ligustrazine hydrochloride are administered, SOD is increased by 34.5% (P <0.05) and 19.6% (P >0.05), respectively.
The results show that FeCl3After cerebral ischemia, the serum MDA level is obviously increased and the SOD content is obviously reduced in a model rat with focal cerebral infarction injury caused by cerebral artery thrombosis. The cyclovirobuxine D is treated by single intravenous infusion, has the effects of resisting oxidation, improving the activity of superoxide dismutase SOD, reducing peroxidation metabolite MDA, and protecting nerve cell damage caused by cerebral apoplexy, and the intravenous administration mode with the same dosage is superior to oral administration. Compared with 2 positive medicines on the market, the antioxidant effect is superior to ligustrazine hydrochloride and is inferior to edaravone.
Example 5 Cyclovirobuxine D ameliorates acute whole brain ischemia reperfusion injury in rats
1. Grouping animals
150 SD rats (SPF grade, male, 290-dose 310g) are randomly divided into 8 groups according to the weight, a pseudo-operation group, a model control group, an edaravone group (6.0mg/kg, Yangzhou pharmaceutical Co., Ltd., specification 20 ml: 30mg), a vinpocetine group (3.0mg/kg, Henan Hongchong pharmaceutical Co., Ltd., specification 2 ml: 20mg), a cyclovirobuxine D high dose group (1.0mg/kg), a cyclovirobuxine D medium dose group (0.5mg/kg), a cyclovirobuxine D low dose group (0.25mg/kg) and a cyclovirobuxine D intragastric group (0.5 mg/kg). The number of animals in each group was 20 except the number of animals in the sham group was 10.
2. Preparation of the model
The procedure for four artery occlusion using the modified Pulsinelli method (four-vessel occlusion,4-VO) was as follows:
after 3% chloral hydrate (300mg/kg, i.p.) anesthetizing rats, removing hair and exposing skin in front of and behind the neck, fixing the rats on an operation table in a prone position, and disinfecting the skin conventionally.
Secondly, a longitudinal incision with the length of about 2cm is made from the occipital tuberosity to the tail side of the rat from the middle lower knife at the back of the neck, tissues are separated layer by layer until wing-shaped holes at two sides of the first cervical vertebra are exposed, the first cervical nerve is separated after the wing-shaped holes are exposed, and the artery of the two vertebral sides is scalded and closed by inserting a 1411 high-frequency electrotome into the transverse process hole. The surface of the wound is washed by penicillin sodium solution, and the wound is sutured layer by layer.
And thirdly, changing the body position, taking the supine position and fixing the four limbs. Cutting a 2 cm-long longitudinal incision from the median line in front of the neck to the suprasternal fossa from the thyroid cartilage, fully dissociating and exposing the common carotid arteries on both sides, drawing the common carotid arteries on both sides with a long silk thread, tying a knot, adjusting the length of the suture without tensioning, and suturing the wound in layers on the deep surface of the neck muscle. Rats were placed in a single cage and irradiated with a sear lamp until awake.
And fourthly, after the operation is finished, the rats are raised in cages and are allowed to eat freely for 24 hours before ischemia reperfusion is carried out. Strict fasting without drinking was performed within 12h before ischemia-reperfusion. The rats after ether anesthesia were fixed on the operating table in the supine position. The suture is removed from the original incision, the bilateral common carotid arteries are exposed again, and the two drawn threads are cut off for separate drawing. Bilateral common carotid arteries were clamped closed with a arteriole clamp. If the model is successfully made, corneal reflex disappears within 30s after the bilateral common carotid arteries of the rat are blocked, the pupils on the two sides are scattered, righting reflex disappears, respiration is deepened more rapidly, eyeballs are pale, and the model has no response to peripheral non-noxious stimulation. The arteriole clamp is released 30min after clamping, and blood supply is recovered. The surface of the wound is washed by penicillin sodium solution and the skin is sutured.
Fifthly, after the operation, the rat is placed on an electric blanket to keep the body temperature and be beneficial to the recovery after the operation.
The rats in the sham operation group only separate the skin and the muscle of the head and neck, do not electrically congeal the vertebral arteries on both sides, and do not clamp the common carotid arteries on both sides, and the other operations are the same.
3. Animal administration
Except for the periwinkle D group animals, the animals are administrated by intragastric administration, and the other animals are administrated by tail vein injection. Rats were allowed to recover before behavioural scoring and dosing. The concentration of cyclovirobuxine D injected into high, medium and low dose groups is 0.25mg/ml, 0.125mg/ml, 0.0625mg/ml, the concentration of cyclovirobuxine D administered into stomach of cyclovirobuxine D group is 0.125mg/ml, the volume of edaravone injection injected into tail vein of edaravone group rat is 1.5mg/ml, the volume of vinpocetine group animal injected into tail vein is 0.75mg/ml, the volume of vein and stomach is 4.0ml/kg, and the volume of physiological saline injected into tail vein of pseudo-surgery group and model control group rat is equal volume. Behavioral scoring is carried out 2 hours after the administration every day, the continuous 5 days are carried out, 2% Evans blue solution is injected into tail vein immediately after the behavioral scoring on the 5 th day, the administration volume is 4.0ml/kg, 3% chloral hydrate is used for anesthetizing rats after 2 hours, and the brain is taken after the heart is perfused and broken.
4. Neurological scoring
The neurological scores were scored blind according to the rat 25 score scoring method (table 6) and were scored for a total of 25.
5. Rat cerebrovascular permeability assay:
firstly, preparing an Evans blue standard curve:
accurately weighing 10.0mg of Evans blue solid, adding normal saline-acetone mixed solution (3:7V/V) for dissolving, fixing the volume to 0.5ml, preparing 20mg/ml Evans blue solution, and diluting 20mg/ml Evans blue solution to 20, 10, 5, 2.5 and 1.25 (mu g/ml) respectively. The absorbance was measured at 620nm using a physiological saline-acetone mixture as a blank. And (4) plotting the concentration as an abscissa and the absorbance as an ordinate to obtain a regression equation.
② quantitative determination of Evans blue:
weighing brain, homogenizing with 10% (W/V) physiological saline-acetone mixture (3:7V/V), sealing, standing overnight, centrifuging at 2500rpm for 15min, collecting supernatant, and measuring absorbance at 620nm with physiological saline-acetone mixture as blank control. And (5) calculating the content of Evans blue in the brain tissue of each animal according to the standard curve.
6. Statistical treatment
Statistical analysis was performed using SPSS 20.0 software. The measurement data is expressed by Mean +/-standard deviation (Mean +/-SD), the comparison between groups is carried out by adopting one-factor analysis of variance, the comparison between the two groups adopts t test, and the significance of P <0.05 is realized.
7. Results of the experiment
TABLE 6 Total cerebral ischemia rat neurological function scoring criteria
| Symptoms and signs | Index/min of cerebral apoplexy |
| Fluffy and fluffy up | 1 |
| Slow or reduced motion | 1 |
| Enhanced ear contact response | 1 |
| Head tilting or bowing body | 3 |
| The eyes are not open or closed | 3 |
| Drooping eyelid | 1 |
| The hind limbs extend outwards and backwards | 3 |
| Rotating ring in situ | 3 |
| Twitching or clonus | 3 |
| Weakness of limbs | 6 |
| Total score | 25 |
Note: mild to moderate cerebral ischemia < 10 points; severe cerebral ischemia is more than or equal to 10 minutes.
TABLE 7 groups of rats nerve function score (Mean + -SD)
Note: compared with the sham operation group:ΔP＜0.05，ΔΔp is less than 0.01; compared with the model control group:*P＜0.05，**p is less than 0.01. D1, D2, D3, D4 and D5 each represented 1 to 5 days.
TABLE 8 brain tissue Evans blue content (Mean SD) of rats in each group
Note: compared with the sham operation group:ΔP＜0.05，ΔΔp is less than 0.01; compared to the model set:*P＜0.05，**P＜0.01。
TABLE 9 mortality in the groups of rats
Compared with a sham operation group, the nerve function score and the evans blue content of brain tissue of the rat in the model control group are both remarkably increased (P < 0.01). Compared with a model control group, rats in an edaravone (6.0mg/kg) group are remarkably reduced in neurological function score on day 3 (P <0.05), remarkably reduced in neurological function score on days 4 and 5 (P <0.01), and remarkably reduced in brain tissue evans blue content (P < 0.05); the nerve function score and the brain tissue evans blue content of rats in the vinpocetine group (3.0mg/kg) are not obviously reduced (P > 0.05).
Compared with a model control group, the nerve function score of rats in the high dose group of cyclovirobuxine D is extremely reduced (P <0.01) on the 4 th day after administration; rats with low, medium and high doses of cyclovirobuxine D and gavage group of cyclovirobuxine D had a significant or very significant decrease in neurological scores on day 5 of administration (P <0.05, P < 0.01). On day 5 of administration, the brain tissue content of rats in each dose group of cyclovirobuxine D was reduced compared to the model control group, but not statistically significant (P > 0.05).
Animal mortality: the total number of dead animals in the sham operation group and the model control group is 0; the total number of dead animals in the edaravone group is 1; the total number of dead animals in the vinpocetine group is 1; the total number of the animals killed in the cyclovirobuxine D high-dose group is 1; the total number of dead animals in the dose group in the cyclovirobuxine D is 1; the total number of the animals died in the low dose group of cyclovirobuxine D is 0; the number of the animals died in the group of the gavage D of the cyclovirobuxine is 1. The experimental animal deaths are shown in Table 9.
8. Conclusion
The cyclovirobuxine D with the dosage of 0.25-1.0mg/kg can obviously improve the acute global cerebral ischemia reperfusion nerve function damage of rats, but has no obvious improvement effect on the cerebral vascular permeability of rats.
Example 6 Cyclovirobuxine D ameliorates focal cerebral ischemia reperfusion injury in rats
1. Preparation of model animals
SD rats (SPF grade, 250-270g, male) 136 were taken, and a Middle Cerebral Artery Occlusion (MCAO) model of the rats was prepared by an internal carotid artery embolization method: rats were anesthetized with 3% chloral hydrate (300mg/kg, i.p.), on a supine operating table, the neck hair was cut, sterilized with iodophor, the skin was incised from the center, the tissue was separated, the right common carotid artery was exposed, the digastric and sternocleidomastoid muscles were pulled outward, sequentially free from the bifurcation of the common carotid artery to the head, the branches of the external carotid artery were ligated and cut: the inferior occipital artery and the superior thyroid artery. Ligation is carried out at the far end of the external carotid artery, and the external carotid artery is cut off to free the main trunk for standby. Then the internal carotid artery is separated, and a loose button is formed at the root of the common carotid artery by silk threads to clamp the common carotid artery and the internal carotid artery. The fishing line (length 50mm, diameter 0.26mm) is slowly pushed towards the intracranial carotid artery by the incision of the external carotid artery main trunk, and the artery clamp on the internal carotid artery is loosened after the fishing line enters the internal carotid artery. The bifurcation of the common carotid artery is used as a mark, resistance is felt when the carotid artery is pushed for about 18mm, namely, the carotid artery reaches the inside of a thinner anterior cerebral artery, all blood supply sources of a middle artery are blocked, the root of an external carotid artery is fastened to fix a fishing line, the common carotid artery is unfastened, and the skin is sutured. After 2 hours of ischemia, the fishing line was slowly drawn out, completing the MCAO-induced focal cerebral ischemia-reperfusion model. After anesthesia, rats in the sham operation group were exposed only to the bifurcation of the internal and external carotid arteries, without occluding the middle artery.
Preparing a fishing line: one end of a fishing line 50mm long and 0.26mm in diameter was marked with a marker, and a further marker was marked 18mm from the marked end and placed in physiological saline for later use.
2. Grouping and administration of drugs
136 rats were randomly divided into 8 groups by weight, a sham operation group, a model control group, an edaravone group (6.0mg/kg, Yangzhou pharmaceutical Co., Ltd., specification: 20 ml: 30mg), a vinpocetine group (3.0mg/kg, Henan hong pharmaceutical Co., Ltd., specification: 2 ml: 20mg), a cyclovirobuxine D high dose group (1.0mg/kg), a cyclovirobuxine D medium dose group (0.5mg/kg), a cyclovirobuxine D low dose group (0.25mg/kg) and a cyclovirobuxine D intragastric group (0.5 mg/kg). The number of animals in each group was 18 except the sham group, which was 10. Except for the periwinkle D group animals, the animals are administrated by intragastric administration, and the other animals are administrated by tail vein injection. Locomotion and mental function scoring and dosing were performed after cerebral ischemia reperfusion. Model animals with a neurological score of > 8 points were selected for subsequent experiments. The cyclovirobuxine D high, medium and low dose groups are injected with cyclovirobuxine D solution with concentration of 0.25mg/ml, 0.125mg/ml and 0.0625mg/ml through tail vein respectively, the gavage administration of cyclovirobuxine D solution with concentration of 0.125mg/ml in the gavage group, the rat of the edaravone group is injected with edaravone injection (1.5mg/ml) through tail vein, the animal of the vinpocetine group is injected with vinpocetine solution with 0.75mg/ml through tail vein, the intravenous and gavage administration volume is 4.0ml/kg, and the rat of the pseudo operation group and the model control group is injected with physiological saline with equal volume through tail vein. Nerve function scoring was performed 2 hours after daily administration, and rats were sacrificed 5 consecutive days, and on day 5, brains were removed, and cerebral infarction rate and brain water content were measured.
3. Degree of neurological impairment score
According to the scoring standard of the degree of the nerve function damage of the rat (see table 1), the degree of the nerve function damage is scored by a blind method, and the total score is 16. The results are shown in Table 10.
4. Determination of cerebral infarction Rate
Taking brain, rapidly freezing, cutting into 2mm thick slices along coronal plane, placing brain slices into 2% TTC staining solution, incubating at 37 deg.C in dark for 10min, and imaging with digital camera. The pale zone (infarct zone) and the non-pale zone (normal zone) were then separated and the cerebral infarction rate was calculated according to the following formula:
the cerebral infarction rate is pale area weight/(pale area weight + non-pale area weight) × 100%. The results are shown in Table 11.
5. Determination of brain Water content
Weighing brain slices, drying in a 50 ℃ oven to constant weight, weighing the dry weight, calculating the brain water content, and calculating the percentage of the brain water content according to the following formula:
percent brain water content ═ wet weight-dry weight/wet weight x 100%. The results are shown in Table 11.
6. Statistical treatment
Statistical analysis was performed using SPSS 17.0 software. The measurement data is expressed by Mean +/-standard deviation (Mean +/-SD), the comparison between groups is carried out by adopting one-factor analysis of variance, the comparison between the two groups adopts t test, and the significance of P <0.05 is realized.
7. Results of the experiment
TABLE 10 degree of nerve function impairment scores (Mean + -SD) for groups of rats
Note: compared with the sham operation group:△P＜0.05，△△p is less than 0.01; compared with the model control group:*P＜0.05，**p is less than 0.01. D1, D2, D3, D4 and D5 each represented 1 to 5 days.
TABLE 11 cerebral infarction Rate and brain Water content (Mean + -SD) of the groups of rats
Note: compared with the sham operation group:△P＜0.05，△△p is less than 0.01; compared with the model control group:*P＜0.05，**P＜0.01。
TABLE 12 number of dead rats in each group
Compared with a sham operation group, the nerve function score, the cerebral infarction rate and the brain water content of the rats in the model control group are all remarkably increased (P is less than 0.01). Compared with a model control group, rats in an edaravone (6.0mg/kg) group have extremely obviously reduced neurological function scores on days 4 and 5 after administration (P <0.01), and have obviously reduced cerebral infarction rate and brain water content (P < 0.05); the brain water content of rats in the vinpocetine group (3.0mg/kg) is obviously reduced (P <0.05), but the neurological score and the cerebral infarction rate are not obviously reduced (P > 0.05).
Compared with a model control group, rats with cyclovirobuxine D high dose and medium dose on days 4 and 5 are remarkably reduced in neurological function score (P <0.01) and rats with low dose (P < 0.05); the neurofunctional score of the mouse in the perivirobuxine D gavage group at 5 days is obviously reduced (P <0.05), but the neurofunctional score at 4 days is not obviously reduced (P > 0.05).
On the 5 th day of administration, compared with the model control group, the cerebral infarction rate of the rats of the cyclovirobuxine D high-dose group is remarkably reduced (P <0.05), and the brain water content is remarkably reduced (P < 0.01); the cerebral infarction rate and the brain water content of rats in the dose group of cyclovirobuxine D are both obviously reduced (P is less than 0.05); the cerebral infarction rate of the low-dose group of the cyclovirobuxine D is remarkably reduced (P <0.05), and the brain water content is remarkably reduced (P < 0.01); the brain water content of the rats in the cyclovirobuxine D gavage group is remarkably reduced (P <0.05), but the cerebral infarction rate is not remarkably reduced (P > 0.05).
Animal mortality: the total number of dead animals in the sham group was 0; the total number of dead animals in the model control group is 6; the total number of dead animals in the edaravone group was 8; the total number of dead animals in the vinpocetine group is 5; the total number of the animals died in the cyclovirobuxine D high-dose group is 4; the total number of dead animals in the dose group in the cyclovirobuxine D is 3; the total number of the animals died in the low dose group of cyclovirobuxine D is 6; the number of the animals died in the group of the gavage D of the cyclovirobuxine is 4. See table 12 for details.
8. Conclusion
The cyclovirobuxine D intravenous administration with 0.25-1.0mg/kg dose can obviously improve the focal cerebral ischemia reperfusion (I/R) injury of rats.
Claims (3)
1. The application of the cyclovirobuxine D and the pharmaceutically acceptable salt thereof as the only active ingredients in the preparation of the medicine for preventing or treating acute global cerebral ischemia-reperfusion nerve function injury is characterized in that the medicine is administrated by intravenous drip or intravenous injection, and the administration dose is 0.25-1 mg/kg.
2. The use according to claim 1, characterized in that said pharmaceutically acceptable salt of cyclovirobuxine D is selected from the hydrochloride, sulphate or phosphate.
3. The use according to claim 1, characterized in that said acute global cerebral ischemia-reperfusion nerve function injury is caused by ischemic stroke.
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| CN1679702A (en) * | 2005-01-28 | 2005-10-12 | 北京阜康仁生物制药科技有限公司 | Medicinal preparation containing notoginseng and flatstem milkvetch seed for cardio-cerebral blood vessel diseases and its preparing method |
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