CN103393708B - Ginsenoside-ophiopogonin D composition for treating cardiovascular and cerebrovascular diseases - Google Patents
Ginsenoside-ophiopogonin D composition for treating cardiovascular and cerebrovascular diseases Download PDFInfo
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Abstract
A ginsenoside-ophiopogonin D composition for treating cardiovascular and cerebrovascular diseases comprises the components by weight percent: 28-32% of ginsenoside Rb1, 18-22% of ginsenoside Rg1, 14-16% of ginsenoside Rb2, 14-16% of ginsenoside Rc, 8-12% of ginsenoside Re, and 14-16% of ophiopogonin D. The advantages are that: the provided ginsenoside-ophiopogonin D composition has fixed and scientific components, controllable and stable quality, substantial drug effect and higher security, same or slightly better drug effects on (myocardial, cerebral and lung) ischemia-reperfusion injury resistance, thrombosis resistance, arteriosclerosis resistance, arrhythmia resistance and the like than shenmai injection, and more safety on acute toxicity, abnormal toxicity, hemolysis, coacervation and the like than shenmai injection. The ginsenoside-ophiopogonin D composition has the advantages of clear and fixed components, more stable and controllable quality, substantial treatment effect and higher security.
Description
Technical field
The present invention relates to medical art, be specifically related to a kind of ginsenoside, ophiopogonin D compositions for the treatment of cardiovascular and cerebrovascular disease, specifically by ginsenoside Rb
1, ginsenoside Rg
1, ginsenoside Rb
2, Ginsenoside Rc, ginsenoside Re, ophiopogonin D composition.
Background technology
Cardiovascular and cerebrovascular disease is the primary disease threatening human health.According to the necrology display of the World Health Organization (WHO) about global various disease in 1996, whole world Patients with Cardiovascular/Cerebrovascular Diseases has 4.5 hundred million people at least, cause dead and maximum disease that disables, coronary heart disease, apoplexy and other deaths from heart disease number amount to 1,400 ten thousand, account for 28.8% of total death toll, and in the trend risen year by year, according to incompletely statistics, within 2000, die from that cardiovascular and cerebrovascular disease has accounted for total death toll 34%, account for the over half of disease death population.
Situation and the WHO of China add up roughly similar.The percentage ratio that cardiovascular and cerebrovascular disease in 1999 causes human mortality to account for total cause of the death rises to 39.4%, is also the No.1 disease and the main cause that cause human mortality.The patients with cerebral apoplexy of China existing more than 6,000,000, wherein 75% disability to some extent, 4% is heavy residual, and 1,500,000 new carbuncle in the occipital region apoplexy patient and 750,000 patients with coronary heart disease of also having an appointment every year, hypertension, hyperlipidaemic conditions large contingent especially, patient now exceedes hundred million.The family caused thus, medical treatment and financial burden have become the social problem that can not despise.Cardiovascular and cerebrovascular disease oneself become one of main public health problem in the whole world, development treatment cardiovascular and cerebrovascular disease newtype drug, become very urgent thing.
Radix Ginseng has strongly invigorating primordial QI, and multiple arteries and veins takes off admittedly, and invigorating the spleen to benefit the lung, promotes the production of body fluid and nourish blood, the effect of Fructus Alpiniae Oxyphyllae of calming the nerves.Ginsenoside is the topmost active component of Radix Ginseng, from Radix Ginseng, isolates GINSENOSIDE R0, Ra
1, Ra
2, Ra
3, Rb
1, Rb
2, Rb
3, Rc, Rd, Re, Rf, Rg
1, Rg
2, Rg
3, Rh
1, Rh
2, Rh
3, Rs
1, Rs
2monomer ginsenoside is planted Deng more than 40.Modern study shows, ginsenoside has infection, removing toxic substances, resisting fatigue, antitumor, antioxidation, vasodilator, promotion biosynthesis and suppresses the effects such as platelet aggregation.Wherein ginsenoside Rg
1, Re, Rb
1, Rb
2, Rc content in Radix Ginseng is the highest, and shows multiple pharmacologically active, major embodiment is as follows:
Ginsenoside Rb
lmain pharmacological have: 1, resist myocardial ischemia reperfusion injury; 2, anti-ischemia-reperfusion lung injury; 3, anti-cerebral ischemia damnification; 4, peripheral nerve-cell regeneration is promoted; 5, learning and memory of little mouse function is improved; 6, anti-aging effects.
Ginsenoside Rg
1main pharmacological have: 1, to the protective effect of myocardial ischemia reperfusion injury; 2, to arrhythmia and atherosclerotic therapeutical effect; 3, to the protective effect of cerebral ischemia reperfusion injury; 4, central nervous system's protective effect; 5, immunologic enhancement; 6, the pharmacological action such as defying age, nootropics.
Ginsenoside Rb
2main pharmacological has: 1, to lipometabolic effect, can prevent and reduce the generation of hyperlipidemia and atherosclerosis; There is disassimilation to cholesterol and promote Excretion; Suppress body to the absorption of fat.2, to the effect of cardiovascular system, there is calcium channel blocking action and anti-radical action; The myocardial cell oxidative damage that excessive xanthine one xanthine oxidase brings out can be resisted.3, the effect of DNA, RNA and protein synthesis is promoted.4, Tumor suppression growth and diffusion.5, to the effect of central nervous system, have the effect suppressing morphine tolerance and dependency to occur, can antagonism morphine paroxysmal pain effect and stiff live reaction, simultaneously can the effect of antagonism morphine elevate body temperature, weaken the antinociceptic effect that the administration of the morphine ventricles of the brain causes.6, to the glycometabolic effect of liver, suppress G-6-Pase to suppress the glyconeogenesis of diabetes rat, increase ATP content in tissue, reduce AMP content, thus change the metabolic patterns in diabetes rat body, blood glucose and liver glycogen content are obviously reduced.7, the effect of proliferation of human retinal pigment epithelium is suppressed.
Ginsenoside Rc's main pharmacological has: 1, antithrombotic and arteriosclerosis effect.2, antiarrhythmic effect.3, anti-overoxidation activity.4, calcium channel blocking action.5, inhibit activities is had to cAMP phosphodiesterase.6, promote that blood plasma secretion corticosterone is active.7, hepatoprotective effect.8, excitation is had to protein tyrosine kinase.
Ginsenoside Re's main pharmacological has: 1, ischemia resisting arrhythmia; 2, resist myocardial ischemia; 3, the dual regulation of cell membrane mobility; 4, stronger sedation and antidiuretic activity; 5, anti-parkinson effect; 6, anti-senescence function; 7, hypoglycemic activity; 8, proliferation of bone marrow cells effect is promoted.
Radix Ophiopogonis has Yin-nourishing and body fluid promoting, and lung moistening such as to clear away heart-fire at the effect, is the important composition flavour of a drug of clinical treatment cardiovascular disease.Its main component is steroidal saponin, polysaccharide and homoisoflavone compounds, pharmacological research shows: total saponins has resist myocardial ischemia damage and anti-arrhythmia effect, in river Radix Ophiopogonis, total saponins is mainly containing ophiopogonin D, ophiopogonin D main pharmacological has: significantly suppress the early stage peripheral blood neutrophil activation of venous thrombosis models rat, adhere to, alleviate thrombus weight, reduce serum sICAM-1 content, reduce inflammatory reaction, inhibition thrombosis.Stability line mitochondrial membrane potential, reduces flow of calcium ions, increases cell viability, has certain protective effect to human umbilical vein endothelial cell (HUVEC).In addition, ophiopogonin D can also have cough-relieving, relieving asthma, effect of antiinflammatory.
Application number be 201010225595.2 " comprise the pharmaceutical composition of red ginseng saponin extract and radix ophiopogonis saponin extract, Preparation Method And The Use ", application number is 200510063087.8 " a kind of pharmaceutical composition and its production and use ", application number is " ginseng freeze-drying powdery injection and the method for quality control thereof " of 200510108139.9, application number is " a kind of ginseng glucose injection " of 200510108140.1, application number be 200710121028.0 " a kind of be used for the treatment of cardiovascular disease compositions and and preparation method thereof " patent of invention in, relate to ginsenoside, ophiopogonin, be mixture, and the ratio of monomer saponin in all not clear and definite mixture, ratio is suitable just can play correct drug effect, ratio its drug effect improper may be contrary, thus cause the toxic and side effects to human body.This is doctor, patient and family members thereof are unwilling to see.
Ginsenoside, ophiopogonin are mainly present in SHENMAI ZHUSHEYE, and SHENMAI ZHUSHEYE has supplementing QI to prevent collapse, YIN nourishing and the production of body fluid promoting, the effect of raw arteries and veins.Be used for the treatment of the shock of type of deficiency of both QI and YIN, coronary heart disease, viral myocarditis, chronic cardiopulmonary disease, granulocytopenia.The immune function of tumour patient can be improved, when share with chemotherapeutics, have certain potentiation, and the toxicity caused by chemotherapeutics can be reduced.
But the chemical composition of SHENMAI ZHUSHEYE is very complicated, known at present and only have ginsenoside Rb as quality control index
1, ginsenoside Rg
1, ginsenoside Re, and ginsenoside Rb
2, Ginsenoside Rc, composition content in SHENMAI ZHUSHEYE such as ophiopogonin D is not low, but control when quality proportioning, and because of Radix Ginseng Rubra, Radix Ophiopogonis medical material individual variation and the difference of each producer preparation technology cause ginsenoside Rb in different batches SHENMAI ZHUSHEYE
1, ginsenoside Rg
1, ginsenoside Rb
2, Ginsenoside Rc, ginsenoside Re, ophiopogonin D content all has the difference of difference in various degree and ratio, exactly because ginsenoside Rb
1, ginsenoside Rg
1, ginsenoside Rb
2, Ginsenoside Rc, ginsenoside Re, the content of ophiopogonin D and the difference caused, its pharmacodynamic stability is not high, and when this adds increased Clinical practice, the danger of untoward reaction occurs patient, and adverse reaction rate is rising year by year.
In addition, human body is gradually obvious to the dependency of medicine, and when a certain individuality uses a kind of medicine, its use amount is constantly increasing and expanding, and in drug component, the ratio stability of each composition is just most important.How to determine stable component ratio parameter, thus the safety issue brought just seems more and more important.
Summary of the invention
The defect of the present invention just in order to solve the problem, provides a kind of ginsenoside, ophiopogonin D compositions for the treatment of cardiovascular and cerebrovascular disease.
The present invention adopts following technical scheme to realize.
Treat the ginsenoside of cardiovascular and cerebrovascular disease, an ophiopogonin D compositions, the component of said composition comprises ginsenoside Rb
1, ginsenoside Rg
1, ginsenoside Rb
2, Ginsenoside Rc, ginsenoside Re, ophiopogonin D; The percentage by weight of component described in it is: ginsenoside Rb
128 ~ 32%, ginsenoside Rg
118 ~ 22%, ginsenoside Rb
214 ~ 16%, Ginsenoside Rc 14 ~ 16%, and ginsenoside Re 8 ~ 12%, ophiopogonin D 14 ~ 16%.
Compositions of the present invention contains pharmaceutically conventional carrier.
Ginsenoside Rb of the present invention
l, ginsenoside Rg
1, ginsenoside Rb
2, Ginsenoside Rc, ginsenoside Re, derive from crude drug Radix Ginseng Rubra, Radix Ginseng, Radix Panacis Quinquefolii, semisynthetic ginsenoside, ophiopogonin D derives from crude drug river Radix Ophiopogonis, semisynthetic ophiopogonin D, and can buy from the market, purity requirement is 85% ~ 99.9%.
A kind of ginsenoside, the ophiopogonin D compositions for the treatment of cardiovascular and cerebrovascular disease that the present invention relates to, can add pharmaceutically conventional carrier, be prepared into the preparation of various routine.Described preparation can be the preparations such as injection, powder ampoule agent for injection, tablet, powder, granule, capsule, drop pill.
The invention has the advantages that, instant invention overcomes the deficiencies in the prior art, provide that a kind of component is fixed, science, quality controllable, the stable and significant ginsenoside of drug effect, ophiopogonin D compositions.Monomer ginsenoside Rb of the present invention
1, ginsenoside Rg
1, ginsenoside Rb
2, Ginsenoside Rc, ginsenoside Re, ophiopogonin D special ratios combination after, in anti-(cardiac muscle, brain, lung) ischemical reperfusion injury, antithrombotic, arteriosclerosis, arrhythmia etc. drug effect and SHENMAI ZHUSHEYE equal to or slightly better, more safer than SHENMAI ZHUSHEYE in acute toxicity, undue toxicity, haemolysis and the safeties such as cohesion.Composition in existing SHENMAI ZHUSHEYE is unclear, and quality is wayward, and pharmacodynamic stability is not high, and one-tenth of the present invention is distinguished one from the other fixing, and quality is more stable, controlled, and this just improves its safety and effectiveness greatly.Moreover the application is when share with other chemotherapeutics, and potentiation comparatively prior art is more obvious, and patient can obtain medical treatment and rehabilitation in the short period of time.
Specific embodiments
Below in conjunction with detailed description of the invention, the present invention is further explained.
The preparation of ginsenoside, ophiopogonin D
Get Radix Ginseng Rubra thin slice (1 ~ 2mm), add 5 times of 75% alcohol reflux 3 times, each 2 hours, collect extracting solution, filter, get decompression filtrate recycling ethanol to without alcohol taste, add water and make the solution of every lml containing 0.5g crude drug, upper macroporous resin column (D class macroporous resin, the resin height of bed is about 20 with diameter ratio), respectively with water, 20%, 40%, 60%, the ethanol of 80% carries out gradient elution, differentiate through TLC, merge containing simple target compound (ginsenoside Rg respectively
1, ginsenoside Re, ginsenoside Rb
1, ginsenoside Rb
2, Ginsenoside Rc) how component, evaporate to dryness, add appropriate 95% dissolve with ethanol again, upper neutral alumina column (sample and alumina weight are than being 1:60), be that 95% ~ 50% ethanol carries out gradient elution by concentration respectively, collect eluent and carry out TLC discriminating, merge containing single blur portion, evaporate to dryness, obtains the ginsenoside Rg that purity is 85% ~ 99.9%
1, ginsenoside Re, ginsenoside Rb
1, ginsenoside Rb
2, Ginsenoside Rc.
Get Radix Ophiopogonis, add 5 times of 75% alcohol reflux 2 times, each 2 hours, collect extracting solution, filter, get decompression filtrate recycling ethanol extremely without alcohol taste, add water and make the solution of every lml containing 0.5g crude drug, upper macroporous resin column (D class macroporous resin, the resin height of bed is about 20 with diameter ratio), use water respectively, 30%, the ethanol of 80% carries out gradient elution, collect the component of the ethanol eluting of 30% ~ 80%, evaporate to dryness, add appropriate 95% dissolve with ethanol again, upper neutral alumina column (sample and alumina weight are than being 1:60), be that 95% ~ 60% ethanol carries out gradient elution by concentration respectively, collect eluent and carry out TLC discriminating, merge the part containing ophiopogonin D, evaporate to dryness.Be separated through reverse phase silica gel post, carry out gradient elution with methanol-water (20:80 ~ 50:50), collect eluent and carry out TLC discriminating, merge containing single blur portion, evaporate to dryness, obtains the ophiopogonin D that purity is 85% ~ 99.9%.
The detection of ginsenoside, ophiopogonin D
Ginsenoside measures according to high performance liquid chromatography (Chinese Pharmacopoeia version in 2010 annex VI D).
Chromatographic condition and system suitability immobile phase adopt Waters symmetry shield
tMrP18 chromatographic column (4.6mm × 250mm; 5.0 μm); Column temperature 30 DEG C, being mobile phase A with acetonitrile, take water as Mobile phase B, and according to the form below carries out gradient elution; Determined wavelength is 203nm.Number of theoretical plate is by ginsenoside Rb
1peak calculates should be not less than 1350000.
Ginsenoside Rb is got in the preparation of reference substance solution
1, ginsenoside Rg
1, ginsenoside Rb
2, Ginsenoside Rc and ginsenoside Re's reference substance be appropriate, accurately weighed, add 80% acetonitrile and make every 1ml respectively containing 0.15mg, 0.10mg, 0.05,0.06,0.07 and the mixed solution of 0.05mg, to obtain final product.
Algoscopy is accurate respectively draws reference substance solution and each 10 μ l of test sample, injection liquid chromatography, measures, to obtain final product.
Ophiopogonin D measures according to high performance liquid chromatography (Chinese Pharmacopoeia version in 2010 annex VI D).
Chromatographic condition and system suitability UItimate XB-C18 chromatographic column (4.6mm × 250mm; 5-Micron);
Column temperature 30 DEG C, flow velocity 1.0ml/min, sample size 20 μ l; Detector: detector (Altech ELSD2000ES), gas flow 2.5L/min, drift ease pipe temperature 95 DEG C; Mobile phase is water: acetonitrile (48:52).Number of theoretical plate calculates should be not less than 3000 by ophiopogonin D peak.
The preparation of reference substance solution respectively precision to take ophiopogonin D reference substance appropriate, add dissolve with methanol, making concentration is ophiopogonin D 0.5mg/ml reference substance solution.
Algoscopy is accurate respectively draws reference substance solution 10 μ l, 20 μ l and test sample 20 μ l, injection liquid chromatography, measures, to obtain final product.
Embodiment 1 prepares the injection of compositions of the present invention
By sample prepared by above-mentioned steps, then detect by above-mentioned method, purity is respectively ginsenoside Rb
199.6%, ginsenoside Rg
199.5%, ginsenoside Rb
299.0%, Ginsenoside Rc 99.2%, and ginsenoside Re 99.3%, ophiopogonin D 99.4%.
Ginsenoside Rb
132%, ginsenoside Rg
118%, ginsenoside Rb
214%, Ginsenoside Rc 14%, and ginsenoside Re 8%, ophiopogonin D 14%, and add appropriate water for injection and dissolve, ultrafiltration, embedding, makes injection.
Embodiment 2 prepares the injection of compositions of the present invention
By sample prepared by above-mentioned steps, then detect by above-mentioned method, purity is respectively ginsenoside Rb
191.3%, ginsenoside Rg
192.1%, ginsenoside Rb
290.0%, Ginsenoside Rc 91.5%, and ginsenoside Re 90.8%, ophiopogonin D 91.4%.
Ginsenoside Rb
128%, ginsenoside Rg
120%, ginsenoside Rb
214%, Ginsenoside Rc 14%, and ginsenoside Re 10%, ophiopogonin D 14%, and add appropriate water for injection and dissolve, then add proper amount of active carbon, filter, embedding, makes injection.
Embodiment 3 prepares the freeze-dried powder of compositions of the present invention
By sample prepared by above-mentioned steps, then detect by above-mentioned method, purity is respectively ginsenoside Rb
185.6%, ginsenoside Rg
186.2%, ginsenoside Rb
285.1%, Ginsenoside Rc 85.8%, and ginsenoside Re 85.3%, ophiopogonin D 86.0%.
Ginsenoside Rb
128%, ginsenoside Rg
122%, ginsenoside Rb
214%, Ginsenoside Rc 14%, and ginsenoside Re 8%, ophiopogonin D 14%, and add appropriate water for injection and dissolve, filter, embedding, makes freeze-dried powder.
Embodiment 4 prepares the tablet of compositions of the present invention
By sample prepared by above-mentioned steps, then detect by above-mentioned method, purity is respectively ginsenoside Rb
192.3%, ginsenoside Rg
192.8%, ginsenoside Rb
292.1%, Ginsenoside Rc 92.5%, and ginsenoside Re 92.9%, ophiopogonin D 92.4%.
Ginsenoside Rb
128%, ginsenoside Rg
118%, ginsenoside Rb
214%, Ginsenoside Rc 14%, and ginsenoside Re 12%, and ophiopogonin D 14%, is prepared into tablet according to a conventional method.
Embodiment 5 prepares the granule of compositions of the present invention
By sample prepared by above-mentioned steps, then detect by above-mentioned method, purity is respectively ginsenoside Rb
198.5%, ginsenoside Rg
198.4%, ginsenoside Rb
298.3%, Ginsenoside Rc 98.7%, and ginsenoside Re 98.2%, ophiopogonin D 98.1%.
Ginsenoside Rb
128%, ginsenoside Rg
118%, ginsenoside Rb
216%, Ginsenoside Rc 14%, and ginsenoside Re 8%, and ophiopogonin D 16%, is prepared into granule according to a conventional method.
Embodiment 6 prepares the capsule of compositions of the present invention
The sample that people prepares by above-mentioned steps, then detect by above-mentioned method, purity is respectively ginsenoside Rb
188.9%, ginsenoside Rg
188.7%, ginsenoside Rb
288.3%, Ginsenoside Rc 88.2%, and ginsenoside Re 88.6%, ophiopogonin D 88.5%.
Ginsenoside Rb
128%, ginsenoside Rg
118%, ginsenoside Rb
214%, Ginsenoside Rc 16%, and ginsenoside Re 8%, and ophiopogonin D 16%, is prepared into capsule according to a conventional method.
Embodiment 7 prepares the drop pill of compositions of the present invention
By sample prepared by above-mentioned steps, then detect by above-mentioned method, purity is respectively ginsenoside Rb
194.3%, ginsenoside Rg
194.8%, ginsenoside Rb
294.1%, Ginsenoside Rc 94.7%, and ginsenoside Re 94.5%, ophiopogonin D 94.2%.
Ginsenoside Rb
130%, ginsenoside Rg
118%, ginsenoside Rb
214%, Ginsenoside Rc 14%, and ginsenoside Re 8%, and ophiopogonin D 16%, is prepared into drop pill according to a conventional method.
Embodiment 8 prepares the tablet of compositions of the present invention
By sample prepared by above-mentioned steps, then detect by above-mentioned method, purity is respectively ginsenoside Rb
198.7%, ginsenoside Rg
198.4%, ginsenoside Rb
296.4%, Ginsenoside Rc 97.6%, and ginsenoside Re 95.5%, ophiopogonin D 94.8%.
Ginsenoside Rb
128%, ginsenoside Rg
118%, ginsenoside Rb
215%, Ginsenoside Rc 15%, and ginsenoside Re 8%, and ophiopogonin D 16%, is prepared into drop pill according to a conventional method.
Embodiment 9 prepares the capsule of compositions of the present invention
By sample prepared by above-mentioned steps, then detect by above-mentioned method, purity is respectively ginsenoside Rb
199.4%, ginsenoside Rg
195.8%, ginsenoside Rb
290.6%, Ginsenoside Rc 96.8%, and ginsenoside Re 94.4%, ophiopogonin D 94.6%.
Ginsenoside Rb
128%, ginsenoside Rg
118%, ginsenoside Rb
215%, Ginsenoside Rc 16%, and ginsenoside Re 8%, and ophiopogonin D 15%, is prepared into drop pill according to a conventional method.
Embodiment 10 prepares the drop pill of compositions of the present invention
By sample prepared by above-mentioned steps, then detect by above-mentioned method, purity is respectively ginsenoside Rb
193.8%, ginsenoside Rg
196.8%, ginsenoside Rb
293.6%, Ginsenoside Rc 94.8%, and ginsenoside Re 95.5%, ophiopogonin D 94.6%.
Ginsenoside Rb
128%, ginsenoside Rg
118%, ginsenoside Rb
216%, Ginsenoside Rc 15%, and ginsenoside Re 8%, and ophiopogonin D 15%, is prepared into drop pill according to a conventional method.
Embodiment 11 prepares the injection of compositions of the present invention
By sample prepared by above-mentioned steps, then detect by above-mentioned method, purity is respectively ginsenoside Rb
196.6%, ginsenoside Rg
194.5%, ginsenoside Rb
293.0%, Ginsenoside Rc 91.2%, and ginsenoside Re 95.3%, ophiopogonin D 94.4%.
Ginsenoside Rb
129%, ginsenoside Rg
121%, ginsenoside Rb
214%, Ginsenoside Rc 14%, and ginsenoside Re 8%, ophiopogonin D 14%, and add appropriate water for injection and dissolve, ultrafiltration, embedding, makes injection.
Embodiment 12 prepares the freeze-dried powder of compositions of the present invention
By sample prepared by above-mentioned steps, then detect by above-mentioned method, purity is respectively ginsenoside Rb
193.6%, ginsenoside Rg
197.5%, ginsenoside Rb
294.0%, Ginsenoside Rc 93.2%, and ginsenoside Re 96.3%, ophiopogonin D 97.4%.
Ginsenoside Rb
131%, ginsenoside Rg
119%, ginsenoside Rb
214%, Ginsenoside Rc 14%, and ginsenoside Re 8%, ophiopogonin D 14%, and add appropriate water for injection and dissolve, ultrafiltration, embedding, makes injection.
Compositions of the present invention is on myocardial ischemia-reperfusion injury impact experiment
1, test material
1.1 medicines: injection prepared by the embodiment of the present invention 1, injection prepared by embodiment 2, injectable powder prepared by embodiment 3, SHENMAI ZHUSHEYE, 10ml/ props up, and is produced by Dali Pharmaceutical Co., Ltd., lot number: 1203242.
1.2 experimental animals: SD rat, regular grade, laboratory animal room of unming Medical College provides (occupancy permit number: SYXK(Yunnan) 2010-0008).
1.3 main agents: chlorination nitroxyl chloride NBT (N-BT) reagent, Shanghai advance chemical reagent work; SOD, MOD test kit, Bioengineering Research Institute is built up in Nanjing; LDH, CK test kit, Zhongsheng Beikong Biological Science & Technology Co., Ltd.; TNF-a, ICAM-1 enzyme linked immunological kit, company of Beijing astronomical phenomena one Bang Ding medical science association.
1.4 key instruments: PowerVision6001 detection system, PowerVision Products; Electric pulmotor (MODEL CS-3), Shanghai Medical Equipment Factory; Microplate reader (RT-6000), Lei Du company of the U.S.; Multi-media color Pathologic image analysis system (HPIAS-1000 type), Beijing Kong Hai company; Ultraviolet spectrophotometer (TU-1800SPC), Beijing Puxi General Instrument Co., Ltd; Full automatic biochemical apparatus (BIOSYSTEMS A25), Chengdu bass reaches Instrument Ltd.; Analytical balance (CPA225D), Sai Duolisi.
2, experimental technique
2.1 rats are at the foundation of body Ischemia-Reperfusion Injury Model, grouping, dosage
Healthy SD rat (180 ~ 240g), 10% urethane anesthesia, open breast, tracheal intubation, connect electric pulmotor (inspiratory/expiratory 1:2, respiratory frequency 30 times/min, airway pressure 3kPa), ramus descendens anterior arteriae coronariae sinistrae root wears ligature, balances after 10 minutes, except Normal group, following coronary artery occlusion causes ischemia, duodenal administration at once after ligation.Ligation 40min forms ischemia model, decontrols ligature afterwards and fills with 2h formation reperfusion injury model again.
Experiment is 10 groups: normal group, model group, injectable powder, the SHENMAI ZHUSHEYE high low dose group of injection prepared by the embodiment of the present invention 1, the injection of embodiment 2 preparation, embodiment 3 preparation, (5 for SOD, MDA assay in N-BT dyeing and serum often to organize 15; 5 measure for Serum LDH, CK; 5 for TNF-a, ICAM-1 assay in serum.Duodenal administration, normal and model group to normal saline, the embodiment of the present invention 1 prepare injection, execute injection prepared by example 4, injectable powder prepared by embodiment 3 (add normal saline dilution become every ml to be respectively 0.1 containing ginsenoside Rb1, ginsenoside Rg1, ginsenoside Rb2, Ginsenoside Rc, ginsenoside Re, ophiopogonin D, 0.35,0.3,0.03,0.2,0.02mg), SHENMAI ZHUSHEYE group high and low dose is respectively to 12ml/kg, 6ml/kg.
The mensuration of 2.2 myocardial ischemia infarction sizes
Rat ischemia 40min, wins heart after filling with 2h again, and normal saline flushing is clean, is evenly cut into 5, weighs, and N-BT dyes, image analyzer calculating myocardium infarct size, myocardial infarction weight, infarct/ventricle (heart) area percentage.
The assay of SOD, MDA, LDH, CK in 2.3 serum
Fill with abdominal aortic blood after 2 hours again, natural separation serum ,-20 DEG C of preservations.Concrete step is poly-is undertaken by test kit description, and visible spectrophotometer detects the OD value of sample SOD, MDA, calculated activity and content; Full automatic biochemical apparatus detects CK, LDH.
TNF-a, ICAM-1 assay in 2.4 serum
Double antibody sandwich-ELISA (ELISA) detects TNF-a, ICAM-1 content in serum.Get the quantitative EIA test kit of TNF-a, ICAM-1, if gauge orifice 8 hole, every hole adds sample diluting liquid 100ul, and the first hole adds standard substance 100ul, with sample injector sucking-off 100ul after mixing, moves to the second hole.So repeatedly oppose and be doubly diluted to the 7th hole, and from the 7th hole and go out 100ul and discard, octal is blank control wells.Treat that in gaging hole, every hole adds blood serum sample 100ul, Sptting plate is fully mixed rearmounted 37 DEG C, 120min, cleaning mixture fully washs 4-6 time, filter paper print is dry, first antibody working solution 50ul is added in every hole, mix rearmounted 37 DEG C, 60min washes plate, every hole enzyme-added mark working solution 100ul, 37 DEG C, 60min washes plate, every hole adds nitrite ion 100ul, 37 DEG C, 5-10min, every hole adds 1 stop buffer mixing, microplate reader 492nm place surveys OD value, with standard substance 1000, 500, 250, 125, 62.5, 32, 16, the OD value of 0PG/ml is mapped on semilogarithmic paper, draw standard curve.OD value is asked on the graph and is calculated TNF-a, ICAM-1 concentration in sample per sample.
2.5 myocardial pathology are observed
Win heart after filling with 2 hours again, ice normal saline washes down blood, and under ligature, the thick cardiac muscle of 2mm is got at 3mm place, liquid nitrogen freezing ,-20 DEG C of frozen sections, thick 8 μm of sheet, and each sample standard deviation does HE dyeing, observes myocardial histopathology and changes.
3, statistical disposition
Numerical value employing average scholar standard deviation represents (
), SPSS11.5 statistical software makes noe-way ANOVA and analyzes.
4, result and conclusion
4.1 impacts on Myocardial Ischemia-reperfusion Injury infarction size
After N-BT dyeing, normal myocardium is skipper, and infarct cardiac muscle is not painted.Ischemia-reperfusion injury model group infraction is serious, each administration group can obviously reduce infarct size, alleviates infarcted region weight, reduce infarcted region/ventricle (heart) percentage rate, difference tool statistical significance (p < 0.05), the results are shown in Table 1 compared with model group.
An impact (mistake of table 1 Myocardial Ischemia-reperfusion Injury infarction size! Undefined bookmark.
n=5)
Note: * P < 0.05, * * P < 0.01 compared with model group
After ischemia-reperfusion, myocardial infarction area, infarcted region weight increase, and infarcted region/ventricle (heart) percentage rate increases; Injection prepared by the embodiment of the present invention 1, the injection of embodiment 2 preparation, the injectable powder of embodiment 3 preparation, SHENMAI ZHUSHEYE high low dose group can obviously reduce infarct size, alleviate infarcted region weight, reduce infarcted region/ventricle (heart) percentage rate, difference tool statistical significance (p < 0.05) compared with model group.
4.2 impacts on SOD, MDA in serum
In normal group serum, SOD activity higher (459.6 soil 14.0) U/ml, MDA (2.9 scholar 0.8) nmol/ml content is low, and after Ischemia Reperfusion, SOD activity is significantly reduced to (369.2 scholar 25.1) U/ml, MDA and is increased to (6.4 soil 1.2) nmol/ml.
Give injection prepared by the embodiment of the present invention 1, injectable powder prepared by injection prepared by embodiment 2, embodiment 3, after SHENMAI ZHUSHEYE height low dosage, in serum, SOD, MDA change, difference tool statistical significance (p < 0.05), the results are shown in Table 2 compared with model group.
Table 2 rat myocardial ischemia and reperfusion on the impact of SOD in serum and MDA (
n=5)
Note: * p < 0.05, * * p < 0.01, * * * p < 0.001 compared with model group
In Reperfusion injury wound model serum, the reduction of SOD activity, MAD content raise; Injection prepared by the embodiment of the present invention 1, the injection of embodiment 2 preparation, the injectable powder of embodiment 3 preparation, SHENMAI ZHUSHEYE high low dose group can increase SOD activity in serum, reduce MDA content, difference tool statistical significance (p < 0.05) compared with model group.
4.3 impacts on LDH, CK in serum
In normal group serum, LDH, CK value is (546.4 scholar 151.7) U/L, (638.4 scholar 139.5) U/L, is increased to (3831.3 scholar 1496.0) U/L, (6754.3 scholar 2086.2) U/L after Ischemia Reperfusion.After each medicine process, in serum, LHD, KC content reduces, injectable powder, SHENMAI ZHUSHEYE high low dose group difference tool meaning (p < 0.05) compared with model group of injection prepared by the embodiment of the present invention 1, the injection of embodiment 2 preparation, embodiment 3 preparation, the results are shown in Table 3.
Table 3 rat myocardial ischemia and reperfusion on the impact of Serum LDH and CK (
n=5)
Note: * p < 0.05, * * p < 0.01, * * * p < 0.001 compared with model group
In Reperfusion injury wound model serum, KC, LHD serum increases; Injection prepared by the embodiment of the present invention 1, the injection of embodiment 2 preparation, the injectable powder of embodiment 3 preparation, SHENMAI ZHUSHEYE high low dose group can suppress the release of KC and LHD, difference tool statistical significance (p < 0.05) compared with model group.
4.4 impacts on TNF-a, cIAM-1 content in serum
After ischemical reperfusion injury, serum TNF-a, ICAM-1 are elevated to (70.022 scholar 3.088) PG/ml, (50.280 scholar 1.035) PG/ml by normal group (62.810 scholar 1.890) GP/ml, (46.901 scholar 0.418) PG/ml respectively, difference tool statistical significance (P<0.05); Injection prepared by the embodiment of the present invention 1, the injection of embodiment 2 preparation, the injectable powder of embodiment 3 preparation, SHENMAI ZHUSHEYE high low dose group significantly can suppress the excessive secretion of TNF-a, CIAM-1, difference has statistical significance (P<0.05) compared with model group, the results are shown in Table 4.
Table 4 is on the impact (mistake of myocardial ischemia reperfusion injury serum TNF-a, IAcM-1 content! Undefined bookmark.
n=5)
Note: * p < 0.05, * * p < 0.01, * * * p < 0.001 compared with model group
In Reperfusion injury wound model serum, TNF-a and CIAM-1 raises; Injection prepared by the embodiment of the present invention 1, the injection of embodiment 2 preparation, the injectable powder of embodiment 3 preparation, SHENMAI ZHUSHEYE high low dose group significantly can suppress TNF-a, the excessive secretion of IcAM-1, difference has statistical significance (p < 0.05) compared with model group.
4.5 impacts that myocardial histopathology is changed
HE colored light sem observation, normal myocardial cells marshalling, uniform coloring, after birth is complete, without degeneration necrosis, myocardial cell arrangement disorder after ischemical reperfusion injury, painted inequality, part myocardial cell cloudy swelling, cardiac muscle fiber unclear transverse striation of muscle fiber or disappearance, karyorhexis disappears; After the process of SSTG side, myocardial cell arranges that comparatively model group is neat, cardiac muscle fiber mould stricture of vagina is comparatively clear, and painted relatively uniform, edema alleviates, and degeneration necrosis scope is less.
HE colored light sem observation, each group rat infarcted region myocardial cell arrangement disorder after modeling, painted uneven, subregion myocardial cell cloudy swelling, cardiac muscle fiber unclear transverse striation of muscle fiber or disappearance, karyorhexis disappears, after SSTG process, infarcted region myocardial cell degeneration changes lighter with necrosis, painted relatively uniform, hydropic degeneration alleviates, and Myocardial necrosis size is less.
Medicine of the present invention and the comparative study of injection containing ginseng extract formulations toxic
Carry out toxicity test according to (Chinese Pharmacopoeia version in 2010 annex XVIII B abnormal toxicity tests method) and reference acute toxicity test method to the present invention and SHENMAI ZHUSHEJI to compare.
Table 5 the acute toxicity tests (LD50)
Contrast two result of the tests above, find that product of the present invention is in toxicity and undue toxicity two, are unexpectedly improved, and the safety of product is significantly ensured, goal of the invention is achieved.
Medicine of the present invention and the comparative study of injection containing ginseng extract hemolytic test
Test method according to (Chinese Pharmacopoeia version in 2010 annex XVIII B haemolysis with cohesion inspection technique) carries out hemolytic test, observes medicine of the present invention and the haemolysis of SHENMAI ZHUSHEYE under variable concentrations and the situation of cohesion.
1, test material
1.1 medicines: injection prepared by the embodiment of the present invention 1, injection prepared by embodiment 2, the injectable powder of embodiment 3 preparation, SHENMAI ZHUSHEYE 10ml/ props up, and is produced by Dali Pharmaceutical Co., Ltd., lot number: 1203242.
The preparation of 2% red blood cell suspension: get Sanguis Leporis seu oryctolagi 20 ~ 30ml, removing Fibrinogen, wash with sodium chloride injection about 10 times amount, the centrifugal 15min of 1500r/min, abandons supernatant, repeats 2 ~ 3 times, till the not aobvious redness of supernatant, abandon supernatant, gained erythrocyte sodium chloride injection is prepared 2% red blood cell suspension, is for experiment.
The preparation of test medicine: precision takes injectable powder prepared by the embodiment of the present invention 3, adds normal saline dilution and becomes every ml containing ginsenoside Rb
1, ginsenoside Rg
1, ginsenoside Rb
2, Ginsenoside Rc, ginsenoside Re, ophiopogonin D be respectively 0.1,0.35,0.3,0.03,0.2,0.02mg.
2, the method for inspection:
Get 7, test tube respectively, wherein 1 ~ No. 5 pipe is test medicine pipe, and No. 6 pipes are negative control pipe (only having 2% red blood cell suspension and sodium chloride injection), and No. 7 pipes are positive control pipe (only having 2% red blood cell suspension and distilled water).Add 2% red cell suspension, sodium chloride injection, distilled water shown according to the form below, the thermostatic water tank being placed in 37 DEG C ± 0.5 DEG C after mixing immediately carries out incubation.Start to observe once every 15 minutes, after 1h, interval is observed once for 1 hour, continuous 24 hours.
Table 7 medicine of the present invention and SHENMAI ZHUSHEYE sample haemolysis and cohesion viewing test
If the solution in test be clear and bright redness, acellular residual or have a small amount of erythrocyte residual at the bottom of pipe, show have haemolysis to occur; As erythrocyte all sinks, supernatant achromatism and clarity, shows to occur without haemolysis.
If have brownish red or rufous flocculent deposit in solution, do not disperse after jolting, show have red blood cell condensation to occur.If any the phenomenon of red blood cell condensation, cohesion or pseudo agglutination can be judged by the following method further.If condensation product can be uniformly dispersed again after test tube vibration, or condensation product is placed on microscope slide, drip 2 sodium chloride injections at microscope slide edge, put basis of microscopic observation, cohesion erythrocyte can be pseudo agglutination by the person of breaking up, if condensation product do not shaken loose or on slide not by the person of breaking up for condensing.
3, result judges:
Negative control pipe occurs without haemolysis and cohesion, when positive control pipe has haemolysis to occur, if the solution that test medicine the 3rd is managed in (0.3ml), in 3 hours, haemolysis and cohesion does not occur, judges that test medicine conforms with the regulations; If the solution that test medicine the 3rd is managed in (0.3ml), in 3 hours, haemolysis and cohesion occurs, judge that test medicine is against regulation.
4, result of the test:
Haemolysis and cohesion is there is not in the preparation of the embodiment of the present invention 1, embodiment 2, embodiment 3 preparation and the solution of SHENMAI ZHUSHEYE in the 1st pipe (0.3ml) in 3 hours, 2nd pipe (0.4ml) starts hemolytic reaction occurs for 24 hours, and the 3rd pipe (0.5ml) starts hemolytic reaction occurs for 20 hours; SHENMAI ZHUSHEYE the 2nd is managed (0.4ml) and is started hemolytic reaction occurs for 20 hours, and the 3rd pipe (0.5ml) starts hemolytic reaction occurs for 15 hours.
From hemolysis index, Chinese medicine haemolysis problem is also very important problem, and especially the haemolysis of saponin is the main cause that a lot of saponins can not be developed as injection always.Product of the present invention is compared with SHENMAI ZHUSHEYE, and hemolysis obviously reduces, thus more can improve the safety of product.
More than just for further illustrating the present invention, instead of be used for limiting the scope of the invention.Every detailed description of the invention made in scope and the various variations of applying, all belong to protection scope of the present invention.
Claims (3)
1. treat the ginsenoside of cardiovascular and cerebrovascular disease, an ophiopogonin D compositions, it is characterized in that, the component of said composition comprises ginsenoside Rb
1, ginsenoside Rg
1, ginsenoside Rb
2, Ginsenoside Rc, ginsenoside Re, ophiopogonin D; The percentage by weight of component described in it is: ginsenoside Rb
128 ~ 32%, ginsenoside Rg
118 ~ 22%, ginsenoside Rb
214 ~ 16%, Ginsenoside Rc 14 ~ 16%, and ginsenoside Re 8 ~ 12%, ophiopogonin D 14 ~ 16%.
2. a kind of ginsenoside, ophiopogonin D compositions for the treatment of cardiovascular and cerebrovascular disease according to claim 1, is characterised in that described in it: in the component of said composition, ginsenoside Rb
1, ginsenoside Rg
1, ginsenoside Rb
2, Ginsenoside Rc, ginsenoside Re, the purity of ophiopogonin D is 85% ~ 99.9%.
3. a kind of ginsenoside, the ophiopogonin D compositions for the treatment of cardiovascular and cerebrovascular disease according to claim 1 and 2, is characterized in that, said composition contains pharmaceutically conventional carrier.
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| CN102028700A (en) * | 2009-09-24 | 2011-04-27 | 昆明制药集团股份有限公司 | Medicinal composition and preparation method thereof |
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| CN102028700A (en) * | 2009-09-24 | 2011-04-27 | 昆明制药集团股份有限公司 | Medicinal composition and preparation method thereof |
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