CN105816472A - Pharmaceutical composition, medicine and purpose thereof - Google Patents
Pharmaceutical composition, medicine and purpose thereof Download PDFInfo
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Abstract
The invention discloses a pharmaceutical composition, a medicine and a purpose thereof. The pharmaceutical composition contains geniposide, ligustrazine and puerarin. The inventor was surprised to find that the pharmaceutical composition has anti-inflammatory effect, anticoagulation effect, and effects for preventing or treating inflammation and brain infarct injury after focal cerebral ischemia reperfusion injury as well as ischemic stroke.
Description
Technical field
The present invention relates to field of medicaments, especially traditional Chinese medicine effective ingredient and application thereof, especially relate to a kind of antiinflammatory, anticoagulation, prevent or treat the inflammation after focal brain ischemia-reperfusion injury and infarct lesion and the compositions of cerebral infarction.
Background technology
Ischemic cerebrovascular is the commonly encountered diseases and frequently-occurring disease that harm human life is healthy, has a strong impact on human survival quality.Focal cerebral ischemia often results in the delayed ischemic neurological deficits such as patient's sensory disturbance, transience dizziness, aphasia, and severe patient may result in hemiplegia, disturbance of consciousness or outbreak etc. of dampinging off；In general, in cerebral ischemia occurs latter 4.5 hours, thromboembolism treatment is effective, but ischemic tissue of brain is after recovering hemoperfusion, and brain tissue impairment increases the weight of further, referred to as cerebral ischemia reperfusion injury, and its mechanism is extremely complex not yet to be illustrated.In recent years research shows, the inflammatory reaction of cerebral ischemia re-pouring tissues following MCAO in rats local excessive is the one of the main reasons causing reperfusion injury, inflammatory cell gathers and inflammatory factor release constitutes the ischemia injury transformation to inflammatory injury, causes the more macrolesion of Reperfu-sion phase cerebral tissue.
Therefore, after effectively preventing brain tissue ischemia, the method for inflammatory reaction needs to be studied further.
Summary of the invention
It is contemplated that one of technical problem solved the most to a certain extent in correlation technique.To this end, the invention provides a kind of pharmaceutical composition, said composition contains jasminoidin, ligustrazine and puerarin.
It should be noted that the jasminoidin being previously mentioned in this article, ligustrazine and puerarin are commercially available.Jasminoidin, ligustrazine and puerarin are three kinds of important traditional Chinese medicine effective ingredient; it is surprisingly found by the inventors that the described pharmaceutical composition comprising these three traditional Chinese medicine effective ingredient has following remarkable effect: protect the cerebral tissue (the infarct area that cerebral ischemic reperfusion in rats causes in one embodiment of the invention, can be alleviated) of cerebral ischemia reperfusion injury；Suppress multiple inflammatory correlation factors, such as the synthesis such as nitric oxide, endothelin-1, thromboxane A2 and adhesion molecule or release, swelling that suppression telangiectasis causes, ooze out increase, many antiinflammatory actions such as suppression inflammation leukoplania in mid-term；Suppress synthesis and the release of up to 16 kinds of inflammatory correlation factors further, including 9 kinds of interleukin class factor IL-1 α, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12 (p70), IL-12 (p40), and monocyte chemoattractant protein-1 (MCP-1), eotaxin (Eotaxin), interferon-γ (IFN-γ), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony stimutaing factor (GM-CSF), the synthesis such as the factor (RANTES) of the normal T-cell excretion of modulated activation and the mice keratinocyte source sexual cell factor (KC) or release, there is significant antiinflammatory action；Meanwhile, described pharmaceutical composition can be obviously prolonged prothrombin time and thrombin time.The application acting as its inflammation after prevention or treatment focal brain ischemia-reperfusion injury and infarct lesion and cerebral infarction of the antiinflammatory of described pharmaceutical composition, anticoagulation and protection cerebral tissue provides new thinking and foundation.
According to embodiments of the invention, in described pharmaceutical composition, the preferred weight ratio of jasminoidin, ligustrazine and puerarin is (50～229): (15～200): (114～286).Most preferably, in described pharmaceutical composition, the weight ratio of jasminoidin, ligustrazine and puerarin is 1:4:3.Thus, it is possible to further enhance the antiinflammatory of prescription, blood coagulation resisting function, alleviate the infarct area that cerebral ischemic reperfusion in rats causes further, thus reach the effect of anti-cerebral ischemia reperfusion injury.
Further, according to another aspect of the invention, based on aforementioned pharmaceutical compositions, present invention also offers a kind of medicine, described medicine includes aforementioned pharmaceutical compositions.According to embodiments of the invention, said medicine can further include pharmaceutically acceptable excipient, carrier, adjuvant or their combination in any.Inventor finds, medicine according to embodiments of the present invention can also have following remarkable effect: protects the cerebral tissue (in one embodiment of the invention, can alleviate the infarct area that cerebral ischemic reperfusion in rats causes) of cerebral ischemia reperfusion injury；Suppress multiple inflammatory correlation factors, such as the synthesis such as nitric oxide, endothelin-1, thromboxane A2 and adhesion molecule or release, swelling that suppression telangiectasis causes, ooze out increase, many antiinflammatory actions such as suppression inflammation leukoplania in mid-term；Suppress synthesis and the release of up to 16 kinds of inflammatory correlation factors further, including 9 kinds of interleukin class factor IL-1 α, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12 (p70), IL-12 (p40), and monocyte chemoattractant protein-1 (MCP-1), eotaxin (Eotaxin), interferon-γ (IFN-γ), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony stimutaing factor (GM-CSF), the synthesis such as the factor (RANTES) of the normal T-cell excretion of modulated activation and the mice keratinocyte source sexual cell factor (KC) or release, there is significant antiinflammatory action；Meanwhile, described pharmaceutical composition can be obviously prolonged prothrombin time and thrombin time.The application acting as its inflammation after prevention or treatment focal brain ischemia-reperfusion injury and infarct lesion and cerebral infarction of the antiinflammatory of described pharmaceutical composition, anticoagulation and protection cerebral tissue provides new thinking and foundation.
Medicine can be with the form being configured to any patient of being suitable for or object in need is administered according to an embodiment of the invention, such as according to embodiments of the invention, described medicine is injection dosage form, thus, it is possible to improve bioavailability and the curative effect of this medicine further.
According to a further aspect in the invention, present invention also offers aforementioned pharmaceutical compositions or the said medicine purposes in preparing medicine, described medicine is at least one following: antiinflammatory；Anticoagulation；Inflammation after prevention or treatment focal brain ischemia-reperfusion injury；Infarct lesion after prevention or treatment Focal Cerebral Ischemia Reperfusion；And prevent or treatment cerebral infarction.
Accompanying drawing explanation
Above-mentioned and/or the additional aspect of the present invention and advantage will be apparent from easy to understand, wherein from combining the accompanying drawings below description to embodiment:
Fig. 1 shows rat brain slice TTC dyeing according to an embodiment of the invention.
Detailed description of the invention
Embodiments of the invention are described below in detail, and the example of described embodiment is shown in the drawings.The embodiment described below with reference to accompanying drawing is exemplary, it is intended to is used for explaining the present invention, and is not considered as limiting the invention.
Embodiment 1: Chinese medicinal components compound recipe improves focal brain ischemia-reperfusion injury in rats model experiment
1. experimental technique:
1.1. laboratory animal packet and Pharmaceutical setting
Laboratory animal is grouped: 45 SPF level SD rats, male, body weight 250 ± 10g, after laboratory condition adaptability is fed 3 days, is randomly divided into following 3 groups: sham operated rats, model group and Chinese medicinal components compound recipe group.Chinese medicinal components compound recipe group gives Chinese medicinal components compound injection 0.6ml/100g continuously in operation injection in first three day, and sham operated rats and model group such as give at the dosage water for injection.The damage model of focal cerebral ischemia in rats 4h Reperfu-sion 20h is prepared with reference to following modeling method.
Chinese medicinal components compound injection configures: 500mg jasminoidin, 2000mg ligustrazine and 1500mg puerarin are dissolved in 1000mlPBS solution, and its concentration is 4mg/mL, and solution filters with 0.2 μm syringe type filter, is stored in the tapered centrifuge tube of 50mL standby.More than operation is all carried out in super-clean bench.
1.2 modeling methods: focal brain ischemia-reperfusion injury model is carried out with reference to after bolt line blocked method (MCAO) in the intraluminal middle cerebral artery occlusion in rats of ZeaLonga1 suitably improvement.In short, for model group and Chinese medicinal components compound recipe group, rat is after chloral hydrate (35mg/100g) general anaesthesia, and operation separates left common carotid, and appears common carotid artery crotch, internal carotid artery and external carotid artery.Free external carotid artery, inserts nylon wire in external carotid artery proximal part away from crotch 4mm otch, enters internal carotid artery under direct-view, and intubating length is about (18 ± 0.5) mm, to anterior cerebral artery near-end, blocks middle cerebral artery blood supply completely.During 4 hours Reperfu-sion of ischemia, soft pumpback nylon wire to external carotid artery crotch, recovers intraluminal middle cerebral artery occlusion in rats blood supply.Sham-operation comparison is simply not inserted into nylon fishing line, the same model group of remaining step.Postoperative giving suitable nursing, ADEQUATE DIET is fed, it is ensured that the temperature of feeding environment, humidity and illumination.Keep body temperature (37 ± 0.5) DEG C.Draw materials after 20h hour and carry out experiment detection.
The nervous symptoms scoring of 1.3 cerebral ischemia-reperfusion injury in rats models
Modelling is complete after animal is clear-headed, observes the behavior expression of animal, and the standards of grading with reference to ZeaLonga carry out 5 points of Neuroscore processed.Standards of grading are as follows.
0 point: impassivity functional impairment symptom (limb activity is normal)
1 point: slight focal function of nervous system symptom disappearance (can not full extension on the left of forelimb)
2 points: focal the helping of moderate can lack (rotating during walking) to hemiplegia side and right side
3 points: severe neurological afunction (is toppled over during autonomic movement) to the right
4 points: pole severe injury (can not spontaneous be walked, level of consciousness reduces)
1.4 rat cerebral infarction stereometries (TTC method)
1.4.1 experimental principle: TTC (2,3,5-triphenyltetrazolium chloride) is the proton acceptor of pyridine in respiratory chain-nucleotide structure enzyme system, take on a red color with the dehydrogenase reaction in normal structure, and dehydrogenase activity declines in ischemic tissue, it is impossible to reaction, therefore organize in pale asphyxia.TTC dyeing can distinguish normal structure and ischemic tissue.
The most each experimental group rat is put to death after 20 hours in 4 hours Reperfu-sion of ischemia according to experimental design, and broken end takes brain, food slicer of requiring mental skill by 2mm thickness, away from antinion 1,3,5, crown incision brain at 7mm.Brain section is placed in 2% tetrazole red (TTC), puts into 37 DEG C of incubator 130min, frequently stir brain sheet so that it is uniform contact is to dyeing liquor.Use Image-ProPlus6.0 image analysis system to calculate infarct size, each infarct size of brain sheet is added up with the product of thickness, it is thus achieved that Infarction volume.Observe medicine the brain protection of cerebral ischemia-reperfusion injury in rats is acted on.
2 experimental results
The left brain of rats in sham-operated group is showed no infarct, and the left brain of model group has obvious infarct, and the left brain cerebral infarction volume of Chinese medicinal components compound recipe group is significantly less than model group, and each group Rat Right brain is showed no infarct (Fig. 1, table 1).Chinese medicinal components compound recipe group rat symptom score is substantially better than model group (table 1).Show the reliability of this Chinese medicinal components compound treatment focal brain ischemia-reperfusion injury in rats, illustrate that this Chinese medicinal components compound recipe has good curative effect to cerebral infarction simultaneously.
Table 1 respectively organizes rat cerebral infarction area and neural behavior scoring compares
| Group | Infarct and brain volume ratio (%) | Neuroscore |
| Model group | 30.10±5.87 | 2.5±0.5 |
| Chinese medicinal components compound recipe group | 21.57±10.14* | 1.9±0.4* |
Remarks: Chinese medicinal components compound recipe group compares with model group, *: P ＜ 0.05；*: P ＜ 0.01.
The antiinflammatory action experimentation of embodiment 2 Chinese medicinal components compound recipe
Focal Ischemia-Reperfusion in Rats is postoperative, and body produces inflammatory reaction, discharges various inflammatory factor, causes reperfusion injury.Inflammatory cell gathers and inflammatory factor release constitutes the ischemia injury transformation to inflammatory injury, causes the more macrolesion of Reperfu-sion phase cerebral tissue.The antiinflammatory action of Chinese medicinal components compound recipe contributes to effectively preventing brain tissue ischemia reperfusion injury, reaches therapeutic purposes.Therefore, in the present embodiment, utilize the macrophage RAW264.7 inflammatory cell model that lipopolysaccharide (LPS) is induced, high throughput proteomics technology based on liquid phase protein chip technology, by measuring the impact on this modeled inflammation Cytokine expression profile of the Chinese medicinal components compound recipe difference compatibility, antiinflammatory action and mechanism thereof to Chinese medicinal components compound recipe have carried out the comprehensive research evaluation of system.Experimental result shows, the Chinese medicinal components compound recipe group (being specifically shown in Table 2) of 9 kinds of different compatibilities all has its effect expressed of suppression to inflammation cytokine, and Chinese medicinal components compound recipe group definite effect on antiinflammatory action of these different compatibilities is described.
1. experimental technique
1.1 Pharmaceutical setting
Jasminoidin, ligustrazine and puerarin and lipopolysaccharide are dissolved in DMEM in high glucose culture fluid respectively, and being each configured to concentration, to be the mother solution of 1mg/mL standby, and solution filters with 0.2 μm syringe type filter, and it is standby that subpackage is stored in the tapered centrifuge tube of 15mL.More than operation is all carried out in super-clean bench.
1.2 cells are cultivated
Mouse monokaryon macrophage RAW264.7 is with containing 10% hyclone, 100U/mL penicillin and the DMEM in high glucose culture fluid of 100 μ g/mL streptomycins, at 37 DEG C, 5%CO2Incubator in cultivate.Within every 2～3 days, changing liquid, the RAW264.7 cell that trophophase growth conditions of taking the logarithm is good is tested.
1.3 each component difference formulation manipulation in Chinese medicinal components compound recipe group
According to document and previous experiments, jasminoidin, three Chinese medicinal components of ligustrazine and puerarin are formed 9 kinds of different compatibility Chinese medicinal components compound recipe groups according to various dose level, after keeping 3 kinds of Chinese medicinal components compatibilities, the total concentration of final function cells is 400ug/ml, carries out antiinflammatory action experimentation.9 kinds of different compatibilities Chinese medicinal components compound recipe group (being specifically shown in Table 2).
Table 2 different compatibility Chinese medicinal components compound recipe group test dose design table
1.49 kinds of different compatibility Chinese medicinal components compound recipe groups cytotoxicity to RAW264.7 cell
Cell toxicant test philosophy: CCK-8 test kit is a kind of fast high-sensitive degree detection kit being widely used in cell proliferation and cytotoxicity analysis.Its ultimate principle is: in this reagent, containing WST 8, (its chemical name is: 2-(2-methoxyl group-4-nitrobenzophenone)-3-(4-nitrobenzophenone)-5-(2,4-disulfonic acid benzene)-2H-tetrazolium monosodium salt), it is reduced to yellow first product (Formazandye) with high water soluble under the effect of electron carrier 1-methoxyl group-5-toluphenazine dimethyl sulfate (1-MethoxyPMS) by the dehydrogenase in cell mitochondrial.Cytotoxicity is the biggest, then color is the most shallow.CCK-8 test kit is Japan's east core chemistry institute product.
RAW264.7 cell contains 5 × 10 by every milliliter4Individual cell accesses 96 porocyte culture plate 0.2ml, cultivates 24 hours.9 kinds of different compatibility Chinese medicinal components compound recipe groups it are separately added into respectively in the hole of Tissue Culture Plate, the drug level making final function cells is as shown in table 2, each group includes 4 repeating holes, after cultivating 24 hours, every hole adds 10 μ lcck-8 solution of above-mentioned CCK-8 test kit configuration, hatch 1 hour, shake 1 minute, at 450nm wavelength, measure absorbance.The inhibitory action of medicine cell growth represents with suppression ratio.Suppression ratio=[(drug control hole absorbance-drug reaction hole absorbance)/(drug control hole absorbance-blank suction luminosity)] × 100%.Suppression ratio is the highest, represents that drug toxicity is the strongest.The results show these 9 kinds different compatibility Chinese medicinal components compound recipe group, to cytotoxic, can carry out follow-up test (being specifically shown in Table 3).
LPS induction RAW264.7 cell is expressed the effect of 23 kinds of inflammatory cytokines by 1.5 different compatibility Chinese medicinal components compound recipe groups
Cell is divided into Normal group, LPS model group (1 μ g/mLLPS), different compatibility Chinese medicinal components compound recipe group (being specifically shown in Table 2).The RAW264.7 cell that trophophase growth conditions of taking the logarithm is good is tested, and discards original culture fluid, adds appropriate DMEM in high glucose culture fluid, and piping and druming makes every milliliter containing 1 × 10 the most gently5The single cell suspension of individual cell.24 porocyte plate every hole inoculation 1mL cell suspension, cultivate 24 hours.In the hole of Tissue Culture Plate, 9 kinds of Chinese medicinal components compound recipe difference compatibility groups it are separately added into respectively according to experiment packet, the drug level making final function cells is as shown in table 2, lipopolysaccharide LPS is added after hatching 4 hours, make the final concentration of 1 μ g/mL (LPS of lipopolysaccharide in final solution, EscherichiaColiO111:B4, L2630, purchased from Sigma Co., USA).Each group of cell culture supernatant is collected after 20 hours, frozen standby in-80 DEG C of ultra cold storage freezers.
1.6.Bio-Plex200 liquid phase protein chip analyzes system 23 kinds of inflammatory cytokine secretion levels of detection
Bio-PlexProTMMouseCytokine23-plexAssay test kit is purchased from Bio-Rad company of the U.S..
Different compatibility Chinese medicinal components compound recipe group cell supernatant samples are at room temperature completely dissolved, and 10000r/min high-speed low temperature is centrifuged 10min, takes different compatibility Chinese medicinal components compound recipe group cell supernatant test kit and is equipped with buffer according to 1:10 dilution.Every hole adds 50 μ L anti-cytokine antibodies detection magnetic beads, washs 2 times, adds test kit outfit buffer, test kit outfit standard curve sample solution or different compatibility Chinese medicinal components compound recipe group cell supernatant to be detected sample to corresponding orifice plate and is respectively 50ul.Hatch 30min under room temperature on lucifuge 500r/min shaking table, wash 3 times.Being separately added in test kit Streptavidin-phycoerythrin (PE) fluorochrome 50 μ L in detection antibody 50 μ L and test kit successively, operation is ibid.nullEvery hole is equipped with the resuspended microballon of buffer with 125 μ L test kits，Put into Bio-Plex200 liquid phase protein chip and analyze system reading each hole fluorescence intensity,And calculate interleukin class factor IL-1 α in sample according to the fluorescence intensity of the standard substance of test kit outfit、IL-1β、IL-2、IL-3、IL-4、IL-5、IL-6、IL-9、IL-10、IL-12(p40)、IL-12(p70)、IL-13、IL-17，Tumor necrosis factor-alpha (TNF-α)、Eotaxin (Eotaxin)、Interferon-γ (IFN-γ)、Monocyte chemoattractant protein-1 (MCP-1)、Granulocyte colony-stimulating factor (G-CSF)、Granulocyte-macrophage colony stimutaing factor (GM-CSF)、Macrophage inflammatory protein-α (MIP-1 α)、Macrophage inflammatory protein-1 β (MIP-1 β)、The factor (RANTES) of the normal T-cell excretion of modulated activation、The mice keratinocyte source sexual cell factor (KC)，The concentration of above-mentioned 23 kinds of cytokines.According to experimental result, the uniform Design using LASSO mathematical modeling combines comprehensive weight method, obtain the comprehensive drug of different compatibility Chinese medicinal components compound recipe group anti-inflammatory activity, obtain the effective dose ratio range of the pharmaceutical composition suppression inflammation cytokine-expressing of these three active component composition.
1.7 statistical procedures
Experimental data mean ± standard deviationRepresenting, comparing employing single factor test variance detection method between group and carry out statistical procedures, P < 0.05 is expressed as having significant difference, and P < 0.01 is expressed as having pole significant difference.
2 experimental results
The 2.1 Chinese medicinal components compound recipe difference compatibility groups cytotoxicity to RAW264.7 cell
Cyto toxic experiment showed shows, the different compatibility Chinese medicinal components compound recipe groups of final concentration of 400 μ g/ml, the most free of toxic effects to RAW264.7 cell, the results are shown in Table 3.
The table 3 Chinese medicinal components compound recipe difference compatibility group cytotoxicity to RAW264.7 cell
LPS induction RAW264.7 cell is expressed the impact of 23 kinds of inflammatory cytokines by 2.2 Chinese medicinal components compound recipe difference compatibility groups
In normal RAW264.7 cell, various cytokines all have low expression, and after LPS (1 μ g/mL) stimulates, RAW264.7 cell expresses substantial amounts of cytokine (P < 0.01).Wherein cell is expressed the horizontal exceeding Bio-Plex200 liquid phase protein chip of MIP-1 α and MIP-1 β and is analyzed the maximum that system can detect, it is impossible to obtains truthful data these data and cannot be carried out statistics.Compared with model group, positive drug 5 μ g/mL Dexamethasone group can significantly lower the expression (P < 0.01) of IL-6, IL-9 and IL-12 (p70), can substantially lower the expression (P < 0.05) of IL-12 (p40), GM-CSF and RANTES；The action effect of Chinese medicinal components compound recipe group is better than Dexamethasone group, the expression (P < 0.01) of IL-4, IL-6, IL-10, IL-12 (p70), IL-17, GM-CSF, KC and MCP-1 can be significantly lowered, the RANTES expression (P < 0.05) of IL-1 α, IL-1 β, IL-2, IL-5, IL-9, IL-13, Eotaxin, IFN-γ sum can be substantially lowered；The comprehensive suppression ratio of antiinflammatory of Chinese medicinal components compound recipe group is higher than Dexamethasone group.Experimental result is shown in Table 4-10.Individual cells factor suppression ratio=(model group cytokine concentrations-medicine group cytokine concentrations)/model group cytokine concentrations × 100%；Comprehensive cytokine suppression ratio sum/21, suppression ratio=21 kind.
Table 4 blank group, model group and the impact on the RAW264.7 cells express cell factor of the positive control drug group
Remarks: model group compares with blank group, #:P ＜ 0.05；##:P ＜ 0.01.Administration group compares with model group, *: P ＜ 0.05；*: P ＜ 0.01.
Table 5 formula 1 group, 2 groups of impacts on the RAW264.7 cells express cell factor of formula
| Sequence number | Cytokine | 1 group of cytokine concentrations/pg/mL of formula | Suppression ratio (%) | 2 groups of cytokine concentrations/pg/mL of formula | Suppression ratio (%) |
| 1 | IL-1α | 525.090±52.382 | -4.1 | 487.180±12.784 | 3.4 |
| 2 | IL-1β | 7475.505±485.309 | 5.2 | 7639.470±172.916 | 3.1 |
| 3 | IL-2 | 225.145±19.707 | 18.6 | 242.125±40.256 | 12.4 |
| 4 | IL-3 | 81.915±8.888 | 23.3 | 93.150±3.012 | 12.7 |
| 5 | IL-4 | 328.800±4.639* | 8.6 | 354.625±24.134 | 1.4 |
| 6 | IL-5 | 74.685±0.601* | 11.6 | 73.410±6.025 | 13.1 |
| 7 | IL-6 | 1160.075±86.274** | 70.7 | 1277.105±44.484** | 67.8 |
| 8 | IL-9 | 2468.065±75.569 | 8.1 | 2489.375±90.545 | 7.3 |
| 9 | IL-10 | 730.900±15.641** | 51.5 | 717.985±23.483** | 52.3 |
| 10 | IL-12(p40) | 141.320±2.135** | 40.4 | 150.380±2.135* | 36.6 |
| 11 | IL-12(p70) | 2540.140±35.369** | 10.3 | 2578.635±2.722** | 9.0 |
| 12 | IL-13 | 4714.650±202.671 | 0.5 | 4566.700±45.863 | 3.6 |
| 13 | IL-17 | 170.545±2.411 | 10.4 | 170.550±10.875 | 10.4 |
| 14 | Eotaxin | 2174.185±59.065 | 19.0 | 2206.035±76.233 | 17.9 |
| 15 | G-CSF | 310133.645±27692.105* | 75.9 | 666564.535±30519.768* | 48.2 |
| 16 | GM-CSF | 2795.225±22.012** | 55.1 | 2882.810±32.583** | 53.7 |
| 17 | IFN-γ | 338.455±21.503 | 13.4 | 356.810±3.182 | 8.7 |
| 18 | KC | 66.905±7.757 | 12.6 | 68.870±5.346 | 10.0 |
| 19 | MCP-1 | 36482.655±301.235 | 12.8 | 38223.985±5713.812 | 8.6 |
| 20 | RANTES | 8482.255±438.611** | 37.3 | 11330.175±1296.289 | 16.3 |
| 21 | TNF-α | 138415.535±3853.159** | 21.3 | 198114.345±38407.205* | 17.2 |
| Comprehensive suppression ratio | 23.9 | 19.7 |
Remarks: administration group compares with model group, *: P ＜ 0.05；*: P ＜ 0.01.
Table 6 formula 3 groups, 4 groups of impacts on the RAW264.7 cells express cell factor of formula
| Sequence number | Cytokine | 3 groups of cytokine concentrations/pg/mL of formula | Suppression ratio (%) | 4 groups of cytokine concentrations/pg/mL of formula | Suppression ratio (%) |
| 1 | IL-1α | 458.400±10.649 | 9.1 | 471.470±19.318 | 6.5 |
| 2 | IL-1β | 7881.485±804.299 | 0.0 | 7464.095±52.008 | 5.3 |
| 3 | IL-2 | 254.080±35.171 | 8.1 | 237.265±49.490 | 14.2 |
| 4 | IL-3 | 88.235±9.963 | 17.4 | 87.590±16.914 | 18.0 |
| 5 | IL-4 | 345.800±11.653 | 3.8 | 344.700±8.542 | 4.1 |
| 6 | IL-5 | 72.130±4.214 | 14.6 | 72.985±5.424 | 13.6 |
| 7 | IL-6 | 1068.555±137.681** | 73.0 | 1214.435±33.609** | 69.4 |
| 8 | IL-9 | 2542.215±180.432 | 5.3 | 2561.365±3.755 | 4.6 |
| 9 | IL-10 | 759.440±29.925** | 49.6 | 747.445±54.695* | 50.4 |
| 10 | IL-12(p40) | 142.830±4.271* | 39.7 | 151.135±1.068* | 36.2 |
| 11 | IL-12(p70) | 2619.585±196.597 | 7.5 | 2573.200±174.387 | 9.2 |
| 12 | IL-13 | 4991.610±32.555 | -5.4 | 4654.210±353.355 | 1.7 |
| 13 | IL-17 | 176.110±11.498 | 7.4 | 182.530±3.635 | 4.1 |
| 14 | Eotaxin | 2400.570±40.927 | 10.6 | 2305.185±223.410 | 14.2 |
| 15 | G-CSF | 494730.265±922.895* | 61.6 | 959183.185±163789.711 | 25.5 |
| 16 | GM-CSF | 2854.370±83.622** | 54.1 | 2826.285±14.602** | 54.6 |
| 17 | IFN-γ | 367.625±17.840 | 5.9 | 357.710±3.182 | 8.4 |
| 18 | KC | 67.445±1.110 | 11.9 | 70.180±0.184 | 8.3 |
| 19 | MCP-1 | 34473.485±3816.503 | 17.6 | 29393.320±419.399* | 29.7 |
| 20 | RANTES | 8720.815±1553.068* | 35.6 | 10589.130±524.334* | 21.8 |
| 21 | TNF-α | 192264.405±39457.895* | 17.6 | 179207.815±13062.094** | 18.5 |
| Comprehensive suppression ratio | 21.2 | 19.9 |
Remarks: administration group compares with model group, *: P ＜ 0.05；*: P ＜ 0.01.
Table 7 formula 5 groups, 6 groups of impacts on the RAW264.7 cells express cell factor of formula
| Sequence number | Cytokine | 5 groups of cytokine concentrations/pg/mL of formula | Suppression ratio (%) | 6 groups of cytokine concentrations/pg/mL of formula | Suppression ratio (%) |
| 1 | IL-1α | 447.735±27.386 | 11.2 | 410.590±14.581* | 18.6 |
| 2 | IL-1β | 7536.935±432.926 | 4.4 | 7566.170±34.620 | 4.0 |
| 3 | IL-2 | 250.540±56.767 | 9.4 | 273.515±1.775 | 1.1 |
| 4 | IL-3 | 79.965±21.659 | 25.1 | 72.540±5.360 | 32.1 |
| 5 | IL-4 | 331.555±17.812 | 7.8 | 340.875±20.174 | 5.2 |
| 6 | IL-5 | 66.590±8.443 | 21.2 | 69.575±5.424 | 17.6 |
| 7 | IL-6 | 1421.535±147.538** | 64.1 | 1197.450±283.352** | 69.8 |
| 8 | IL-9 | 2281.565±218.503 | 15.0 | 2311.325±221.812 | 13.9 |
| 9 | IL-10 | 738.185±75.583* | 51.0 | 706.905±28.730** | 53.1 |
| 10 | IL-12(p40) | 138.650±16.589 | 41.5 | 155.660±10.663 | 34.3 |
| 11 | IL-12(p70) | 2450.080±130.065 | 13.5 | 2517.090±57.078* | 11.1 |
| 12 | IL-13 | 4700.890±104.624 | 0.8 | 4367.160±65.733 | 7.8 |
| 13 | IL-17 | 168.835±3.627 | 11.3 | 172.685±4.236 | 9.2 |
| 14 | Eotaxin | 2211.835±280.587 | 17.6 | 2212.365±232.065 | 17.6 |
| 15 | G-CSF | 914210.700±250724.933 | 29.0 | 560856.395±248394.047 | 56.5 |
| 16 | GM-CSF | 2813.055±91.464** | 54.8 | 2805.610±0.001** | 54.9 |
| 17 | IFN-γ | 346.290±19.332 | 11.4 | 341.110±2.531* | 12.7 |
| 18 | KC | 66.395±1.110 | 13.3 | 67.440±2.588* | 11.9 |
| 19 | MCP-1 | 28868.310±378.995* | 31.0 | 38920.530±3786.910 | 7.0 |
| 20 | RANTES | 10573.215±206.115** | 21.9 | 10661.350±1048.201 | 21.2 |
| 21 | TNF-α | 199123.6±15417.643** | 17.1 | 206953.535±21906.472** | 16.5 |
| Comprehensive suppression ratio | 22.5 | 22.7 |
Remarks: administration group compares with model group, *: P ＜ 0.05；*: P ＜ 0.01.
Table 8 formula 7 groups, 8 groups of impacts on the RAW264.7 cells express cell factor of formula
| Sequence number | Cytokine | 7 groups of cytokine concentrations/pg/mL of formula | Suppression ratio (%) | 8 groups of cytokine concentrations/pg/mL of formula | Suppression ratio (%) |
| 1 | IL-1α | 406.570±4.045* | 19.4 | 421.810±28.836 | 16.4 |
| 2 | IL-1β | 6675.660±900.995 | 15.3 | 6879.375±705.516 | 12.7 |
| 3 | IL-2 | 214.065±4.038** | 22.6 | 243.930±56.625 | 11.8 |
| 4 | IL-3 | 71.530±9.730 | 33.0 | 83.775±20.287 | 21.5 |
| 5 | IL-4 | 288.775±30.342* | 19.7 | 313.235±13.499* | 12.9 |
| 6 | IL-5 | 60.620±9.645 | 28.2 | 68.295±6.032 | 19.2 |
| 7 | IL-6 | 934.305±511.740* | 76.4 | 1158.175±312.025** | 70.8 |
| 8 | IL-9 | 2159.800±85.192* | 19.6 | 2431.895±284.207 | 9.4 |
| 9 | IL-10 | 662.395±86.458* | 56.0 | 661.515±69.431* | 56.1 |
| 10 | IL-12(p40) | 118.150±22.062 | 50.1 | 137.140±17.664 | 42.1 |
| 11 | IL-12(p70) | 2153.535±230.001 | 24.0 | 2335.515±161.751* | 17.5 |
| 12 | IL-13 | 4422.490±328.366 | 6.6 | 4436.775±137.879 | 6.3 |
| 13 | IL-17 | 142.445±19.226 | 25.1 | 164.565±4.830 | 13.5 |
| 14 | Eotaxin | 2015.675±102.212** | 24.9 | 2188.990±17.352 | 18.5 |
| 15 | G-CSF | 286725.755±200926.718* | 77.7 | 505252.600±136869.950* | 60.8 |
| 16 | GM-CSF | 2430.455±255.329** | 61.0 | 2669.975±144.073** | 57.1 |
| 17 | IFN-γ | 285.235±31.148* | 27.0 | 329.280±58.662 | 15.7 |
| 18 | KC | 56.375±10.840* | 26.4 | 64.955±1.294 | 15.1 |
| 19 | MCP-1 | 16927.830±11642.952 | 59.5 | 22401.245±3814.806* | 46.5 |
| 20 | RANTES | 6826.555±4308.041 | 49.6 | 8619.070±2418.263 | 36.3 |
| 21 | TNF-α | 107931.785±95531.844 | 23.4 | 168137.21±56621.858 | 19.2 |
| Comprehensive suppression ratio | 35.5 | 27.6 |
Remarks: administration group compares with model group, *: P ＜ 0.05；*: P ＜ 0.01.
The 9 groups of impacts on the RAW264.7 cells express cell factor of table 9 formula
| Sequence number | Cytokine | 9 groups of cytokine concentrations/pg/mL of formula | Suppression ratio (%) |
| 1 | IL-1α | 461.365±36.084 | 8.5 |
| 2 | IL-1β | 6900.835±536.220 | 12.5 |
| 3 | IL-2 | 271.845±1.775 | 1.7 |
| 4 | IL-3 | 90.315±0.997 | 15.4 |
| 5 | IL-4 | 336.485±18.604 | 6.4 |
| 6 | IL-5 | 70.852±0.001* | 16.1 |
| 7 | IL-6 | 1474.375±67.338** | 62.8 |
| 8 | IL-9 | 2508.185±11.307 | 6.6 |
| 9 | IL-10 | 725.370±7.821** | 51.8 |
| 10 | IL-12(p40) | 137.530±6.421* | 42.0 |
| 11 | IL-12(p70) | 2553.745±114.346 | 9.8 |
| 12 | IL-13 | 4548.145±85.199 | 4.0 |
| 13 | IL-17 | 166.700±4.228 | 12.4 |
| 14 | Eotaxin | 2237.945±31.106 | 16.7 |
| 15 | G-CSF | 550435.595±203998.786 | 57.3 |
| 16 | GM-CSF | 2745.765±3.698** | 55.9 |
| 17 | IFN-γ | 359.735±0.955 | 7.9 |
| 18 | KC | 68.490±0.368 | 10.5 |
| 19 | MCP-1 | 25727.555±1077.030* | 38.5 |
| 20 | RANTES | 8487.640±1632.497* | 37.3 |
| 21 | TNF-α | 184007.490±29398.841* | 18.1 |
| Comprehensive suppression ratio | 23.4 |
Remarks: administration group compares with model group, *: P ＜ 0.05；*: P ＜ 0.01.
The table 10 Chinese medicinal components compound recipe difference compatibility group comprehensive inhibitory action to inflammation cytokine
| Different compatibility Chinese medicinal components compound recipe groups | Comprehensive suppression ratio (%) |
| Compatibility 1 | 23.9 |
| Compatibility 2 | 19.7 |
| Compatibility 3 | 21.2 |
| Compatibility 4 | 19.9 |
| Compatibility 5 | 22.5 |
| Compatibility 6 | 22.7 |
| Compatibility 7 | 35.5 |
| Compatibility 8 | 27.6 |
| Compatibility 9 | 23.4 |
The blood coagulation resisting function research of embodiment 3 Chinese medicinal components compound recipe
1. experimental technique
1.1 Pharmaceutical setting
Jasminoidin, ligustrazine and puerarin are dissolved in PBS respectively, it is each configured to the mother solution that concentration is 2mg/mL, 6mg/mL and 1.5mg/mL, solution filters with 0.2 μm syringe type filter, be sub-packed in the tapered centrifuge tube of 15mL be stored in-20 DEG C standby, the mixed liquor of final concentration of 2000 μ g/ml it is configured to before using, wherein jasminoidin, ligustrazine and puerarin concentration are respectively 250 μ g/mL, 750 μ g/mL and 1000 μ g/mL, and above operation is all carried out in super-clean bench.Positive drug heparin sodium tests final concentration of 100U/ml.
1.2 Chinese medicinal components compound recipe In Vitro Anti Blood clottinies
5 SPF level SD rats, male, body weight 250 ± 10g, after laboratory condition adaptability is fed 3 days, with 10% chloral hydrate solution anesthesia, dosage is 0.3ml/100g, takes blood in coeliac artery.Mix according to blood consumption and 3.8% sodium citrate liquid by volume 1:9, with 3000r/min after mixing, centrifugal 10 minutes.Take supernatant, isolate platelet poor plasma, tested in two hours.Prediction Chinese medicinal components compound recipe, PBS solution group and heparin sodium group (100U/ml) after by volume 1:4 mix, use activated partial thromboplastin time (APTT), thrombin time (TT) and the prothrombin time (PT) of each group of semi-automatic Blood coagulation instrument mensuration with platelet poor plasma respectively.Partial thromboplastin time (APTT), thrombin time (TT) and prothrombin time (PT) test kit are purchased from Beijing Steellex Scientific Instrument Company.
1.2.1 activated partial thromboplastin time (APTT) measures
Open manual or semi-automatic coagulo meter, according to rule of operation setting program.APTT reagent equilibrates to room temperature, the reverse mixing of jog.By CaCl2Solution is preheating to 37 DEG C.Detect according to after table 11 order sample-adding.
Table 11APTT tests Loading sequence
| 1 | APTT reagent | 50μl |
| 2 | Medicine to be measured and platelet poor plasma (p p p) mixture | 50μl |
| 3 | Mix latter 37 DEG C and hatch 3 minutes | |
| 4 | The CaCl of preheating2Solution | 50μl |
| 5 | Mixing immediately, timing, record PCT |
1.2.2 thrombin time (TT) measures
Thrombin is dissolved and is configured to suitable concn solution, 37 DEG C of water-baths 10 minutes.Detect according to after table 12 order sample-adding.
Table 12TT tests Loading sequence
| 1 | Thrombin solution | 100μl |
| 2 | Medicine to be measured and platelet poor plasma (ppp) mixture | 100μl |
| 3 | Mixing immediately, timing, record PCT |
1.2.3. prothrombin time (PT) measures
Thromboplastin solution is dissolved and is configured to suitable concn solution, 37 DEG C of water-baths 10 minutes.Detect according to after table 13 order sample-adding.
Table 13TT tests Loading sequence
| 1 | Thromboplastin solution | 100μl |
| 2 | Medicine to be measured and platelet poor plasma (ppp) mixture | 50μl |
| 3 | Mixing immediately, timing, record PCT |
2 experimental results
Compared with PBS solution group, the average prothrombin time (PT) of Chinese medicinal components compound recipe group and average thrombin time (TT) have had and have been obviously prolonged, difference statistically significant (P < 0.05)；And average activation partial thromboplastin time (APTT) no significant difference (P ＞ 0.05) (table 14).
The impact on external thrombin time of blood plasma of the table 14 Chinese medicinal components compound recipe group
Remarks: administration group compares with PBS group, *: P ＜ 0.05；*: P ＜ 0.01.
In the description of this specification, the description of reference term " embodiment ", " some embodiments ", " example ", " concrete example " or " some examples " etc. means that the specific features, structure, material or the feature that combine this embodiment or example description are contained at least one embodiment or the example of the present invention.In this manual, the schematic representation of above-mentioned term is necessarily directed to identical embodiment or example.And, the specific features of description, structure, material or feature can be to combine in one or more embodiments in office or example in an appropriate manner.Additionally, in the case of the most conflicting, the feature of the different embodiments described in this specification or example and different embodiment or example can be combined and combine by those skilled in the art.
Although above it has been shown and described that embodiments of the invention, it is understandable that, above-described embodiment is exemplary, it is impossible to be interpreted as limitation of the present invention, and above-described embodiment can be changed, revises, replace and modification by those of ordinary skill in the art within the scope of the invention.
Claims (7)
1. a pharmaceutical composition, it is characterised in that contain:
Jasminoidin；
Ligustrazine；And
Puerarin.
Pharmaceutical composition the most according to claim 1, it is characterised in that the weight ratio of described jasminoidin, ligustrazine and puerarin is (50～229): (15～200): (114～286).
Pharmaceutical composition the most according to claim 2, it is characterised in that the weight ratio of described jasminoidin, ligustrazine and puerarin is 1:4:3.
4. a medicine, it is characterised in that including:
Pharmaceutical composition described in any one of claims 1 to 3.
Medicine the most according to claim 4, it is characterised in that farther include pharmaceutically acceptable excipient, carrier, adjuvant or their combination in any.
6. according to the medicine described in claim 4 or 5, it is characterised in that described medicine is injection dosage form.
7. the purposes in preparing medicine of the pharmaceutical composition described in any one of claims 1 to 3, described medicine is at least one following:
Antiinflammatory；
Anticoagulation；
Inflammation after prevention or treatment focal brain ischemia-reperfusion injury；
Infarct lesion after prevention or treatment Focal Cerebral Ischemia Reperfusion；And
Prevention or treatment cerebral infarction.
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| CN107397753A (en) * | 2017-08-10 | 2017-11-28 | 湖南文理学院 | A kind of medicine based on cape jasmine glycoside derivates and preparation method thereof |
| CN109813879A (en) * | 2017-11-21 | 2019-05-28 | 石家庄以岭药业股份有限公司 | A kind of biological detecting method of Chinese medicine composition |
| WO2019161568A1 (en) * | 2018-02-26 | 2019-08-29 | 钱彐群 | Drug and preparation method therefor |
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