CN101829203B - A kind of Chinese medicine composition promotes the application in nitric oxide generating medicine in preparation - Google Patents
A kind of Chinese medicine composition promotes the application in nitric oxide generating medicine in preparation Download PDFInfo
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Abstract
The invention discloses the application of a kind of Chinese medicine composition in preparation promotion nitric oxide generating medicine.Chinese medicine composition of the present invention is made up of Radix Ginseng, Hirudo, Scolopendra, Scorpio, Eupolyphaga Seu Steleophaga, Periostracum Cicadae, Radix Paeoniae Rubra, Borneolum Syntheticum etc., has effect of benefiting QI for activating blood circulation, disperse blood stasis and dredge collateral.Prove that Chinese medicine composition of the present invention can increase the concentration of Plasma Nitric Oxide by test, alleviate myocardial ischemia reperfusion injury.
Description
Technical field
The present invention relates to a kind of novelty teabag of Chinese medicine composition, particularly, the present invention relates to the application of a kind of Chinese medicine composition in preparation promotion nitric oxide generating medicine, belong to application in TCM field.
Background technology
Acute myocardial infarction (Acute Myocardial Infarction, AMI) is fallen ill dangerous, and case fatality rate is high, and harm is large.Thus Coronary recanalization treatment (thrombolytic or emergency treatment calcification score) makes the Coronary recanalization of blocking as early as possible, recovers cardiac muscle effectively Reperfu-sion, has also become most important target and evaluation index.But still there is following problem after AMI Coronary recanalization and make cardiac muscle completely income: 1, impurity free vacancy disordering or Coronary slow flow phenomenon, though namely arteria coronaria is opened (mechanical blocking), cardiac muscle does not recover effective Reperfu-sion and shows as slow blood flow or no blood.Its result cardiac muscle thoroughly downright bad, infarction size expands, and the incidence rate of ventricular dilatation and reconstruct, Lower cardiac function and the severe complication such as heart failure and malignant arrhythmia increases, and hospital mortality increases 5-10 doubly; 2, myocardial ischemia reperfusion injury, namely ischemic myocardium is after recovery blood flow Reperfu-sion, increases the weight of its structural deterioration on the contrary, causes cell death, cause infarction size to expand, cause the further infringement of cardiac function, and affect the prognosis of AMI patient.
NO can regulate antiotasis, suppresses platelet and leukocytic gathering, adhesion, minimizing response to oxidative stress protect blood vessel structure integrity.No matter be that increase is exogenous or endogenous NO can both reduce ischemical reperfusion injury and reduce the adhesion of leukocyte and endotheliocyte after having there are some researches show By Reperfusion of Acute Myocardial Infarction.Some known endothelial functions of can protecting, as statins, endothelin-receptor antagonists and adiponectin etc., can reduce ischemical reperfusion injury and mechanism of action is all relevant with the generation of NO.
The present invention is the improvement invention carried out on the basis of No. 01131203.3 and No. 200410048292.2 patent, quotes in full the content of this two patent document record at this.Above-mentioned two patents this Chinese medicine composition unexposed promotes the application in nitric oxide generating medicine in preparation.
Summary of the invention
The object of the invention is to provide the application of a kind of Chinese medicine composition in preparation promotion nitric oxide generating medicine;
Preferably, the invention provides this Chinese medicine composition and prepare the application alleviated in the medicine of myocardial ischemia reperfusion injury.
The present invention applies described Chinese medicine composition containing Scorpio, Hirudo, Scolopendra, Eupolyphaga Seu Steleophaga, Periostracum Cicadae five kinds of insect medicines, add Radix Ginseng, Borneolum Syntheticum, Radix Paeoniae Rubra etc., there is QI invigorating, invigorate blood circulation, blood stasis dispelling, wind dispelling, the function such as dredging collateral, meridian qi and blood is run, passages through which vital energy circulates is moistened foster, and cold is dispelled, and healthy energy is recovered, can effectively promote that NO generates, alleviate myocardial ischemia reperfusion injury.
Described Chinese medicine composition is made up of the crude drug of following weight portion:
Radix Ginseng 3-10 Hirudo 3-11 Eupolyphaga Seu Steleophaga 5-10 Olibanum (system) 1-5 Radix Paeoniae Rubra 3-9 Lignum Dalbergiae Odoriferae 1-5
Lignum Santali Albi 1-5 Scorpio 3-9 Periostracum Cicadae 3-12 Scolopendra 1-3 Borneolum Syntheticum 1-7 Semen Ziziphi Spinosae (stir-fry) 3-10;
Preferably, this Chinese medicine composition is made up of the crude drug of following weight portion:
Radix Ginseng 6 Hirudo 10 Eupolyphaga Seu Steleophaga 7 Olibanum (system) 2 Radix Paeoniae Rubra 5 Lignum Dalbergiae Odoriferae 2
Lignum Santali Albi 2 Scorpio 7 Periostracum Cicadae 7 Scolopendra 1 Borneolum Syntheticum 5 Semen Ziziphi Spinosae (stir-fry) 5;
Or:
Radix Ginseng 10 Hirudo 8 Eupolyphaga Seu Steleophaga 7 Olibanum (system) 2 Radix Paeoniae Rubra 5 Lignum Dalbergiae Odoriferae 2
Lignum Santali Albi 2 Scorpio 9 Periostracum Cicadae 7 Scolopendra 1 Borneolum Syntheticum 5 Semen Ziziphi Spinosae (stir-fry) 5;
Or:
Radix Ginseng 6 Hirudo 11 Eupolyphaga Seu Steleophaga 7 Olibanum (system) 2 Radix Paeoniae Rubra 5 Lignum Dalbergiae Odoriferae 2
Lignum Santali Albi 2 Scorpio 3 Periostracum Cicadae 7 Scolopendra 1 Borneolum Syntheticum 5 Semen Ziziphi Spinosae (stir-fry) 5;
The active component of above-mentioned Chinese medicine composition is made up of following ingredients:
A mean diameter be less than 100 μm Scorpio, Hirudo, Scolopendra, Eupolyphaga Seu Steleophaga, Periostracum Cicadae and Olibanum (processed) medicated powder;
B Borneolum Syntheticum medicated powder;
The volatile oil that c is extracted by Lignum Dalbergiae Odoriferae and Lignum Santali Albi;
The alcohol-extracted extract of alcohol extract after d Radix Ginseng ethanol extraction after concentrated;
The water extracted immersing paste that the Aqueous extracts of the Aqueous extracts after the Aqueous extracts of the Lignum Dalbergiae Odoriferae after e extract component c and Lignum Santali Albi medicinal residues, Radix Paeoniae Rubra and Semen Ziziphi Spinosae (parched) decoct with water and the medicine residues of Radix Ginseng after extract component d filters, be condensed into after mixing.
In Chinese medicine composition of the present invention, as the latin name of the crude drug of active component and processing method thereof from " Chinese medicine voluminous dictionary " (in July, 1977, the first edition, Shanghai science tech publishing house) and " Chinese Pharmacopoeia " (version in 2005, Chemical Industry Press).
The preparation formulation that the present invention's application also discloses above-mentioned Chinese medicine composition is capsule, tablet, granule, powder, oral liquid or pill.
For enabling above-mentioned dosage form realize, the acceptable adjuvant of pharmacy need be added when preparing these dosage forms, such as: filler, disintegrating agent, lubricant, suspending agent, binding agent, sweeting agent, correctives, antiseptic, substrate etc.Filler comprises: starch, pregelatinized Starch, lactose, mannitol, chitin, microcrystalline Cellulose, sucrose etc.; Disintegrating agent comprises: starch, pregelatinized Starch, microcrystalline Cellulose, carboxymethyl starch sodium, crospolyvinylpyrrolidone, low-substituted hydroxypropyl cellulose, cross-linking sodium carboxymethyl cellulose etc.; Lubricant comprises: magnesium stearate, sodium lauryl sulphate, Pulvis Talci, silicon dioxide etc.; Suspending agent comprises: polyvinylpyrrolidone, microcrystalline Cellulose, sucrose, agar, hydroxypropyl emthylcellulose etc.; Binding agent comprises, starch slurry, polyvinylpyrrolidone, hydroxypropyl emthylcellulose etc.; Sweeting agent comprises: saccharin sodium, Aspartane, sucrose, cyclamate, enoxolone etc.; Correctives comprises: sweeting agent and various essence; Antiseptic comprises: parabens, benzoic acid, sodium benzoate, sorbic acid and its esters, benzalkonium bromide, acetic acid chloroethene are fixed, Folium eucalypti globueli (Eucalyptus globulus Labill.) wet goods; Substrate comprises: PEG6000, PEG4000, insect wax etc.
The capsule that the present invention applies described Chinese medicine composition is made preferably by following preparation method: cleaned by the five kinds of Chinese medicine such as the Hirudo of said ratio, Scorpio, Periostracum Cicadae, Eupolyphaga Seu Steleophaga, Scolopendra, oven drying at low temperature, for subsequent use; Lignum Santali Albi, Lignum Dalbergiae Odoriferae extract volatile oil, medicinal residues and aqueous solution for subsequent use; Radix Ginseng 70% alcohol heating reflux extracts secondary, 3 hours first times, second time 2 hours, and merge extractive liquid, reclaims ethanol extremely without alcohol taste; The medicinal residues of medicine residues of Radix Ginseng and Lignum Santali Albi, Lignum Dalbergiae Odoriferae merge with aqueous solution, add Radix Paeoniae Rubra, Semen Ziziphi Spinosae (stir-fry), decoct with water secondary, 3 hours first times, second time 2 hours, collecting decoction, filter, filtrate is concentrated into the clear paste that relative density is 1.20-1.25 (60 DEG C), add above-mentioned panaxynol extract, mixing, cold drying, is ground into fine powder; The five tastes such as Olibanum (system) and Hirudo are ground into fine powder altogether; Borneolum Syntheticum porphyrize, respectively with above-mentioned fine powder facing-up, mixing, sprays into volatile oil, and mixing, incapsulates, to obtain final product.
Or the present invention applies described capsule and makes preferably by following preparation method:
A) weight ratio of crude drug is: Radix Ginseng 3-10 part, Hirudo 3-11 part, Eupolyphaga Seu Steleophaga 5-10 part, Olibanum (processed) 1-5 part, Radix Paeoniae Rubra 3-9 part, Lignum Dalbergiae Odoriferae 1-5 part, Lignum Santali Albi 1-5 part, Scorpio 3-9 part, Periostracum Cicadae 3-12 part, Scolopendra 1-3 part, Borneolum Syntheticum 1-7 part, Semen Ziziphi Spinosae (parched) 3-10 part;
B) pulverizing medicinal materials technique:
By Scorpio, Hirudo, Scolopendra, Eupolyphaga Seu Steleophaga and Periostracum Cicadae five kinds of worm medicines after cleaning, washing process and clean the Olibanum (processed) after the process of preparing Chinese medicine and prepare burden by prescription, pulverized by pulverizer, medicated powder fineness reaches more than 80 orders; Medicated powder after coarse powder carries out micronizing through various superfine communication technique, makes medicated powder mean diameter be less than 100 μm; Medical material to be comminuted, after cleaning, drying sterilizing, is prepared burden;
C) concentrated and drying process is extracted:
Use water extraction again after Lignum Dalbergiae Odoriferae and the first extracting in water volatile oil of Lignum Santali Albi, Radix Paeoniae Rubra and Semen Ziziphi Spinosae decoct with water, after Aqueous extracts filters, and one-tenth extractum to be concentrated; After Radix Ginseng ethanol extraction, then use water extraction, alcohol extract is condensed into alcohol-extracted extract after reclaiming ethanol, and Aqueous extracts is condensed into the water extracted immersing paste after filtering and mixing with all Aqueous extracts;
D) preparation process:
Superfine powder flour is added, then by step c in Fluidbedgranulatingdrier) gained extracts extractum and sprays into granulation; By the granule made through granulate, add Borneolum Syntheticum fine powder, spray into the volatile oil extracted by Lignum Dalbergiae Odoriferae and Lignum Santali Albi, by capsule filler filling after mixing, make capsule.
Or the present invention applies described capsule and makes preferably by following preparation method:
A) weight ratio of crude drug is: Radix Ginseng 3-10 part, Hirudo 3-11 part, Eupolyphaga Seu Steleophaga 5-10 part, Olibanum (system) 1-5 part, Radix Paeoniae Rubra 3-9 part, Lignum Dalbergiae Odoriferae 1-5 part, Lignum Santali Albi 1-5 part, Scorpio 3-9 part, Periostracum Cicadae 3-12 part, Scolopendra 1-3 part, Borneolum Syntheticum 1-7 part, Semen Ziziphi Spinosae (parched) 3-10 part;
B) pulverizing medicinal materials technique:
By Scorpio, Hirudo, Scolopendra, Eupolyphaga Seu Steleophaga and Periostracum Cicadae five kinds of worm medicines after cleaning, washing process and clean the Olibanum (processed) after the process of preparing Chinese medicine and prepare burden by prescription, pulverized by pulverizer, medicated powder fineness reaches more than 80 orders; Medicated powder after coarse powder carries out micronizing through various superfine communication technique, makes medicated powder mean diameter be less than 100 μm; Medical material to be comminuted, after cleaning, drying sterilizing, is prepared burden;
C) concentrated and drying process is extracted:
Use water extraction again after Lignum Dalbergiae Odoriferae and the first extracting in water volatile oil of Lignum Santali Albi, Radix Paeoniae Rubra and Semen Ziziphi Spinosae decoct with water, after Aqueous extracts filters, and one-tenth extractum to be concentrated; After Radix Ginseng ethanol extraction, then use water extraction, alcohol extract is condensed into alcohol-extracted extract after reclaiming ethanol, and Aqueous extracts is condensed into the water extracted immersing paste after filtering and mixing with all Aqueous extracts, and extractum Direct spraying is dried to spray powder;
D) preparation process:
By superfine powder flour and step c) be added in Fluidbedgranulatingdrier together with gained spray drying powder, then spray solvent and make granule; By the granule made through granulate, add Borneolum Syntheticum fine powder, spray into the volatile oil extracted by Lignum Dalbergiae Odoriferae and Lignum Santali Albi, by capsule filler filling after mixing, make capsule.
The present invention applies the consumption of described Chinese medicine composition, is converted to the weight of raw medicinal material, is each 0.8-3 gram, daily 2-4 time, is preferably each 1.11-2.22 gram, daily three times.
Accompanying drawing explanation
Fig. 1: myocardial ischemia-reperfusion model is at different time points anterior descending coronary (LAD) mean blood flow: Chinese miniature pig is divided into sham operated rats (sham, n=7), placebo (placebo, n=7), medicine (tongxinluo of the present invention, and medication combined N-nitro-l-arginine (TXL+L-NNA, the n=6) processed group of the present invention n=7).Data represent with mean ± standard error.
*represent that comparing p < 0.05, # represents and compare p < 0.05 with placebo group with sham operated rats.
Fig. 2: myocardial ischemia, impurity free vacancy disordering and infarct size measurement result: dangerous area (AAR) is defined as the percentage ratio that ischemic region myocardial Mass Measured accounts for left ventricular mass; The percentage ratio that myocardial infarction weight accounts for hazardous area myocardial Mass Measured is Infarct area (IR); The percentage ratio that cardiac muscle impurity free vacancy disordering district weight accounts for hazardous area myocardial Mass Measured is impurity free vacancy disordering district area (NRR).China miniature pig is divided into sham operated rats (sham, N=7), placebo (placebo, N=7), medicine (tongxinluo of the present invention,) and the medication combined N-nitro-l-arginine of the present invention (tongxinluo plus L-NNA, N=6) processed group N=7).Data represent with mean ± standard error.
*p < 0.05 is for there being significant difference.
Fig. 3: pig myocardium organizes eNOS (eNOS) and beta-actin (β-actin) protein immunization imprinting to detect figure: respectively from the impurity free vacancy disordering district the (the 1st from the present invention medication combined N-nitro-l-arginine (1-3 road), drug treating group of the present invention (4-6 road) and placebo group (7-9 road), 4 and 7 roads), the cardiac muscular tissue in Zai Liu district (the 2nd, 5 and 8 road) and non-ischemic region (the 3rd, 6 and 9 road).Often organize 3 animals, every animal repeats 3 row protein immunoblot inspections
Fig. 4: pig myocardium tissue blood vessel endothelium calcium conglutination element (VE-vadherin) and beta-actin (β-actin) protein immunization imprinting detect figure: respectively from from the impurity free vacancy disordering district (the 1st, 4 and 7 road) of the present invention medication combined N-nitro-l-arginine (1-3 road), drug treating group of the present invention (4-6 road) and placebo group (7-9 road), the cardiac muscular tissue in Zai Liu district (the 2nd, 5 and 8 road) and non-ischemic region (the 3rd, 6 and 9 road).Often organize 3 animals, every animal is repeated 3 protein immunoblots and detects.
Fig. 5: pig impurity free vacancy disordering district (NRR), Zai Liu district (RR) and non-ischemic region (NON) myocardium myeloperoxidase (MPO) (MPO) activity: Chinese miniature pig is divided into sham operated rats (sham, n=7), placebo (placebo, n=7), medicine (tongxinluo of the present invention,) and the medication combined N-nitro-l-arginine of the present invention (tongxinluo plus L-NNA, n=6) n=7).Data represent with mean ± standard error.
*represent p < 0.05.
Detailed description of the invention
Embodiment 1:
A) crude drug formula is:
Radix Ginseng 39.6g Hirudo 72.6g Eupolyphaga Seu Steleophaga 46.2g Olibanum (system) 13.2g
Radix Paeoniae Rubra 33g Lignum Dalbergiae Odoriferae 13.2g Lignum Santali Albi 13.2g Scorpio 19.8g
Periostracum Cicadae 46.2g Scolopendra 6.6g Borneolum Syntheticum 33g Semen Ziziphi Spinosae (stir-fry) 33g;
B) pulverizing medicinal materials technique:
By Scorpio, Hirudo, Scolopendra, Eupolyphaga Seu Steleophaga and Periostracum Cicadae five kinds of worm medicines after cleaning, washing process and clean the Olibanum (processed) after the process of preparing Chinese medicine and prepare burden by prescription, pulverized by pulverizer, medicated powder fineness reaches more than 80 orders; Medicated powder after coarse powder carries out micronizing through various superfine communication technique, makes medicated powder mean diameter be less than 30-40 μm; Medical material to be comminuted, after cleaning, drying sterilizing, is prepared burden;
C) concentrated and drying process is extracted:
Use water extraction again after Lignum Dalbergiae Odoriferae and the first extracting in water volatile oil of Lignum Santali Albi, Radix Paeoniae Rubra and Semen Ziziphi Spinosae add suitable quantity of water and decoct secondary, each 3 hours, merge Aqueous extracts, after filtration, and one-tenth extractum to be concentrated; Radix Ginseng with appropriate 70% ethanol extraction secondary, each 3 hours, merge extractive liquid, reclaim ethanol extremely without alcohol taste, use water extraction again, it is 0.9 ~ 1.1 alcohol-extracted extract that alcohol extract is condensed into 60 DEG C of mensuration relative densities, and Aqueous extracts is concentrated into 60 DEG C after filtering and mixing with above-mentioned all Aqueous extracts and measures the clear paste that relative density is 0.9 ~ 1.1, for subsequent use;
D) preparation process:
Superfine powder flour is added, then by step c in Fluidbedgranulatingdrier) gained extracts extractum and sprays into granulation; By the granule made through granulate, add Borneolum Syntheticum fine powder, spray into the volatile oil extracted by Lignum Dalbergiae Odoriferae and Lignum Santali Albi, by capsule filler filling after mixing, make 1000 capsules.
The consumption of medicine of the present invention is each 2-4 grain, daily three times.
Embodiment 2
A) crude drug formula is:
Radix Ginseng 66g Hirudo 52.8g Eupolyphaga Seu Steleophaga 46.2g Olibanum (system) 13.2g
Radix Paeoniae Rubra 33g Lignum Dalbergiae Odoriferae 13.2g Lignum Santali Albi 13.2g Scorpio 59.4g
Periostracum Cicadae 46.2g Scolopendra 6.6g Borneolum Syntheticum 33g Semen Ziziphi Spinosae (stir-fry) 33g
B) pulverizing medicinal materials technique:
By Scorpio, Hirudo, Scolopendra, Eupolyphaga Seu Steleophaga and Periostracum Cicadae five kinds of worm medicines after cleaning, washing process and clean the Olibanum (processed) after the process of preparing Chinese medicine and prepare burden by prescription, pulverized by pulverizer, medicated powder fineness reaches more than 80 orders; Medicated powder after coarse powder carries out micronizing through various superfine communication technique, makes medicated powder mean diameter be less than 70-90 μm; Medical material to be comminuted, after cleaning, drying sterilizing, is prepared burden;
C) concentrated and drying process is extracted:
Use water extraction again after Lignum Dalbergiae Odoriferae and the first extracting in water volatile oil of Lignum Santali Albi, Radix Paeoniae Rubra and Semen Ziziphi Spinosae add suitable quantity of water and decoct secondary, each 3 hours, merge Aqueous extracts, after filtration, and one-tenth extractum to be concentrated; Radix Ginseng with appropriate 70% ethanol extraction secondary, each 3 hours, merge extractive liquid, reclaim ethanol extremely without alcohol taste, use water extraction again, alcohol extract is condensed into relative density and is determined as 1.0 ~ 1.05 alcohol-extracted extracts at 60 DEG C, and Aqueous extracts is concentrated into 60 DEG C after filtering and mixing with above-mentioned all Aqueous extracts and measures the clear paste that relative density is 1.0 ~ 1.1, for subsequent use;
D) preparation process:
Superfine powder flour is added, then by step c in Fluidbedgranulatingdrier) gained extracts extractum and sprays into granulation; By the granule made through granulate, add Borneolum Syntheticum fine powder, spray into the volatile oil extracted by Lignum Dalbergiae Odoriferae and Lignum Santali Albi, preparation process is pressed into 1000 routinely.
The consumption of medicine of the present invention is each 2-4 sheet, daily three times.
Embodiment 3: the preparation of pill
A) crude drug formula is:
Radix Ginseng 39.6g Hirudo 66g Eupolyphaga Seu Steleophaga 46.2g Olibanum (system) 13.2g
Radix Paeoniae Rubra 33g Lignum Dalbergiae Odoriferae 13.2g Lignum Santali Albi 13.2g Scorpio 46.2g
Periostracum Cicadae 46.2g Scolopendra 6.6g Borneolum Syntheticum 33g Semen Ziziphi Spinosae (stir-fry) 33g
B) pulverizing medicinal materials technique:
By Scorpio, Hirudo, Scolopendra, Eupolyphaga Seu Steleophaga and Periostracum Cicadae five kinds of worm medicines after cleaning, washing process and clean the Olibanum (processed) after the process of preparing Chinese medicine and prepare burden by prescription, pulverized by pulverizer, medicated powder fineness reaches more than 80 orders; Medicated powder after coarse powder carries out micronizing through various superfine communication technique, makes medicated powder mean diameter 10-20 μm; Medical material to be comminuted, after cleaning, drying sterilizing, is prepared burden;
C) concentrated and drying process is extracted:
Use water extraction again after Lignum Dalbergiae Odoriferae and the first extracting in water volatile oil of Lignum Santali Albi, Radix Paeoniae Rubra and Semen Ziziphi Spinosae add suitable quantity of water and decoct secondary, each 3 hours, merge Aqueous extracts, after filtration, and one-tenth extractum to be concentrated; Radix Ginseng with appropriate 70% ethanol extraction secondary, each 3 hours, merge extractive liquid, reclaim ethanol extremely without alcohol taste, use water extraction again, it is 0.9 ~ 1.0 alcohol-extracted extract that alcohol extract is condensed into 60 DEG C of mensuration relative densities, and Aqueous extracts is concentrated into 60 DEG C after filtering and mixing with above-mentioned all Aqueous extracts and measures the clear paste that relative density is 1.0 ~ 1.1, for subsequent use;
D) preparation process:
Preparation process routinely, makes 1000 pills.
The consumption of medicine of the present invention is each 2-4 grain, daily three times.
Experimental example:
Promote that nitric oxide generates for illustrating Chinese medicine composition of the present invention, alleviate the application of myocardial ischemia reperfusion injury, carried out following test to prove its curative effect with the capsule (hereinafter referred to as medicine of the present invention) obtained by above-described embodiment 1 method, but it can not form any restriction to scope of the present invention.
1 materials and methods
1.1 experiment groupings
Select Chinese miniature pig 28 (being purchased from Beijing Agricultural University), male and female are not limit, body weight 25 ± 5 kilograms, be divided into sham operated rats, placebo group, medicine group of the present invention and the present invention's medication combined nitric oxide synthase inhibitors N-nitro-L-arginine (L-NNA) processed group at random, often organize 7.
1.2 reagent and instrument
Thioflavin S, azovan blue purchased from American Sigma company; Red tetrazolium is purchased from Beijing geneva Chemical Company; MPO test kit is that Bioengineering Research Institute's product is built up in Nanjing; Rabbit anti-human NOS3, β-actin polyclonal antibody, goat-anti people VE-cadherin polyclonal antibody equal purchased from American Santa Cruze company; Mouse-anti rabbit, mouse-anti sheep two resist purchased from middle mountain gold bridge biotech firm; Ultrasonic blood flow instrument and 1.5mm probe are produced by Transonic Systems company; Siemens 900c respirator; U.S. Biopac SystemMP-150 polygraph.
1.3 medicines and usage
Medicine of the present invention (being produced by Shijiazhuang Yiling Pharmaceutical Co., Ltd).Medicine 0.4g/kg of the present invention and 30ml drinking water are blended in first 3 hours of coronary ligation through Injection by stomach duct; The L-NNA of 10mg/kg is dissolved in 200ml normal saline, within first 50 minutes, is injected by femoral vein with 7ml/min at coronary ligation.Placebo group first 3 hours of coronary ligation through Injection by stomach duct 30ml drinking water.
1.4 experimental technique
Ketamine and stable mix preparation (70: 3) 730mg intramuscular injection induced anesthesia, then with 2mg/kg/h speed Continuous infusion.All animals equal per os circulation of qi promoting cannula, respirator controls ventilation, opens thoracic cavity, longitudinal incision pericardium along median sternotomy, expose heart, and pericardium to be sutured in thoracic wall is hanging basket shape.At left anterior descending coronary artery (LAD) near-end 1/3 place, free also threading, penetrate the silica gel tube intracavity ligation 1.5h that is about 3 ~ 4cm, then the 3h that loosens sets up acute myocardial infarction and re-perfusion model along ligature.A threading under sham operated rats arteria coronaria, not ligation, without acute myocardial infarction, also without Reperfu-sion.Blood pressure in surface electrocardiogram, femoral artery is monitored in art.Before coronary ligation, loosen at once, Reperfu-sion 180 minutes measures intraventricular pressure.Before coronary ligation, Reperfu-sion 5,30,60,120,180 minutes time measure LAD mean blood flow.All data are all presented at polygraph and use 3.8.1 version Acqknowledge software analysis.
The judgement of 1.5 impurity free vacancy disordering areas and method of drawing material
This research, with reference to the past experimental technique, at the end of experiment, injects the fluorescent dye thioflavin S of 4% of 1ml/kg simultaneously from left atrium, room, region aobvious fluorescence under 365nm wavelength fluorescent lamp that it arrives with blood flow, is Zai Liu district; The not aobvious fluorescence in impurity free vacancy disordering district; Again in original position again ligation LAD, the 2% azovan blue dyestuff of the 1ml/kg that reinjects from left atrium, azovan blue can not arrive ischemic region with blood flow, thus ischemic region not blueness; Non-ischemic region blueness.Take out heart immediately after putting to death animal, wipe out left and right auricle and right ventricle, swing in ice normal saline and wash away except residual blood; From direction, the apex of the heart centripetal end, be parallel to coronary sulcus and in order 6-7 sheet be cut in left room, take weight respectively and take pictures.Leave and take normal district, Zai Liu district and impurity free vacancy disordering district part Heart preservation in liquid nitrogen.Each cardiac muscle is put into 37 DEG C of 1% red tetrazolium phosphate buffer 15 minutes and takes pictures, the region of white is necrotic area cardiac muscle.Use LECA-QWIN software, calculate ischemia hazardous area respectively by the area method of quadrature to account for left ventricular mass percentage ratio and be expressed as dangerous area (AAR), infarcted region, impurity free vacancy disordering district account for ischemic region percentage by weight and are then expressed as Infarct area (IR) and impurity free vacancy disordering district area (NRR) respectively.
1.6 cardiac muscular tissue's myeloperoxidase (MPO) (MPO) determinations of activity
From liquid nitrogen, take out cardiac muscular tissue be about 100mg, smash with duroplasts parcel, remaining concrete operations are undertaken by test kit description.Distilled water returns to zero, and uses spectrophotometric measurement OD value at 460nm place.The H decomposing 1umol in the reaction system of 37 DEG C is organized in every gram
2o
2it is 1 enzyme activity unit.
1.7 immunoblottings survey protein content
Muscular tissue of coring from liquid nitrogen is about 100mg, mixes, pulverize, centrifugal extraction supernatant under ultrasonic disintegrator with the ice RIPA lysate of 1ml and 1mmol phenylmethyl sulfonylfluoride (PMSF), measures total protein concentration by BCA method.Adopt the discontinuous buffer system of sodium lauryl sulphate one denaturing polyacrylamide gel, successively join the separation gel of 8% and the concentrated glue of 5%; Get protein sample, loading 25ul (about containing protein 60 ug), successively with 80 and 120V electrophoresis, after about 100min, carries out electrotransfer in cellulose acetate membrane in transfering buffering liquid; Put into 5% defatted milk powder confining liquid room temperature and close 60min; Put into primary antibodie (1: 500 dilution) 4 DEG C to spend the night; Put into two anti-(1: 2000 dilution) room temperature again and hatch 1h; Develop with the super quick luminescent solution of ECL and expose on film.
1.8 statistical analysis
All variablees represent with mean ± standard error, compare and check with non-ginseng Kruskal-Wallis between many groups, and meaningful rear row Dunnett one factor analysis of variance compares group difference.Hemodynamic index is compared with duplicate test.Statistical significance is had with P < 0.05.Statistical analysis is carried out with SPSS 13.0 software.
2 results
2.1 animal dead situations
The medication combined L-NNA processed group of the present invention has a pig repeatedly to occur in myocardial ischemia process, and room is fast, quiver in room, rejects because rescuing failure.
2.2 hemodynamic results
The rate-pressure product (RPP) that different time points measures before ischemia, after Reperfu-sion, intraventricular pressure rate of change peak value (+dp/dt), left ventricular end diastolic presssure (LVDEP) and anterior descending branch arteria coronaria mean blood flow (CBF), specifically in table 1.
Table 1 hemodynamic index (mean ± standard error)
Note: RPP, rate pressure; CBF, anterior descending branch mean blood flow; LVEDP, left ventricular end diastolic presssure;
+ dp/dt, left ventricular pressure power rate of change peak value; Sham, sham operated rats; Placebo, placebo group; TXL, drug treating group of the present invention; TXL+L-NNA, the medication combined nitric oxide synthase inhibitors N-nitro-L-arginine processed group of the present invention
Before ischemia, the index RPP of reflecting myocardium oxygen consumption degree in the present invention medication combined L-NNA processed group apparently higher than sham-operation, placebo and medicine group of the present invention (P < 0.05).But during Reperfu-sion, RPP no significant difference between each group; The ventricular function represented with+dp/dt and LVDEP obviously declines (P < 0.05) during Reperfu-sion.But each time point during Reperfu-sion, compares no difference of science of statistics between three ischemia-reperfusion group; Before ischemia, coronary flow is zero difference between each group; Reperfu-sion is the increase of ischemia-reperfusion processed group coronary flow after 5 minutes, reduces gradually subsequently; Compare with sham-operation and drug treating group of the present invention, Reperfu-sion 120 minutes and 180 minutes time placebo group and the present invention's medication combined L-NNA processed group coronary flow obviously reduce (P < 0.05), as shown in Figure 1.
2.3 myocardial ischemia, impurity free vacancy disordering and infarct size measurement result
Placebo group, medicine group of the present invention and the medication combined L-NNA processed group AAR of the present invention are 31 ± 3%, 32 ± 4% and 28 ± 3% respectively, no significant difference between each group.With placebo group (90 ± 3%, 70 ± 4%) and the medication combined L-NNA processed group (95 ± 2% of the present invention, 63 ± 3%) compare, drug treating group (78 ± 3% of the present invention, 47 ± 5%) IR, NRR (P < 0.05) is considerably reduced, as shown in Figure 2.2.4 protein immunization mark notations measure impurity free vacancy disordering district, Zai Liu district and non-ischemic region cardiac muscular tissue eNOS (eNOS) and blood vessel endothelium calcium conglutination element (VE-vadherin) content
Cardiac muscular tissue eNOS content in Ge Zu impurity free vacancy disordering district is obviously less than Zai Liu district and non-ischemic region, but compares without obviously difference between each ischemia-reperfusion group, as shown in Figure 3; Impurity free vacancy disordering district VE-cadherin content Ye compare Zai Liu district, non-ischemic region obviously reduce; Compare with placebo group and the medication combined L-NNA processed group of the present invention, drug treating of the present invention significantly increases the content of impurity free vacancy disordering district VE-cadherin, as shown in Figure 4.
2.5 myocardium MPO determination of activity results
The MPO activity of placebo, medicine of the present invention and the present invention medication combined L-NNA processed group non-ischemic region cardiac muscle compares zero difference with sham operated rats.And MPO activity in impurity free vacancy disordering district is greater than Zai Liu district (P < 0.05), MPO activity in Zai Liu district is greater than non-ischemic region (P < 0.05).Compare with placebo and the medication combined L-NNA processed group of the present invention, drug treating of the present invention considerably reduces impurity free vacancy disordering district and Zai Liu district MPO activity (P < 0.05), as shown in Figure 5.
3 conclusions
Experimental result shows the medication combined L-NNA processed group of the present invention RPP before ischemia and obviously raises; prompting L-NNA process likely adds the oxygen consumption of cardiac muscle during myocardial ischemia; although but previously research confirms that application L-NNA can increase RPP separately; but can not have an impact to infarct size and regional myocardial blood flow; think in this experiment based on this; L-NNA drug treating itself does not increase myocardial ischemia reperfusion injury; but eliminate the myocardium protecting action of medicine of the present invention, show that the myocardium protecting action of medicine of the present invention generates relevant with NO.
Measuring after myocardial ischemia that MPO is active can the degree that infiltrates in cardiac muscular tissue of quantitative response neutrophilic granulocyte.Impurity free vacancy disordering district MPO is active in Zai Liu district and non-ischemic region in experimental result display, this with observe by the method such as Electronic Speculum, SABC the result drawn and conform to.Compare with placebo group, drug treating group of the present invention considerably reduces Zai Liu district and impurity free vacancy disordering district MPO is active, points out medical preconditioning of the present invention to decrease the degree of neutrophilic granulocyte in tissue infiltration.Existing research display can reduce impurity free vacancy disordering area with lacking leukocytic perfusate perfusion ischemic myocardium, and after ischemia-reperfusion, NO can reduce granulocytic rolling, adhesion with endotheliocyte by regulating the generation of cell adhesion molecule.More than experiment shows L-NNA and the medication combined processed group Zai Liu district of the present invention and impurity free vacancy disordering district MPO activity obviously increases, and shows that medicine of the present invention reduces neutrophilic granulocyte infiltration in the tissue by the generation of change NO.
VE-cadherin is a kind of attachment proteins of endothelial-cell specific, has the effect of reusing to maintenance blood vessel endothelium structural intergrity.Adhering to connection protein function is the major reason causing cardiac interstitium edema and inflammatory cell infiltration extremely.VE-cadherin is very responsive to proteolytic enzyme, and the proteolytic enzyme of especially inflammatory cell activation release after myocardial ischemia-reperfusion, can directly cause VE-cadherin to degrade.More than after research display myocardial ischemia-reperfusion, impurity free vacancy disordering district cardiac muscle VE-cadherin obviously reduces, and this is consistent with result of study previously.Medical preconditioning of the present invention obviously increases impurity free vacancy disordering district VE-cadherin content, and with L-N NA coupling after VE-cadherin content reduce, show that medicine of the present invention increases VE-cadherin content and generates relevant with NO.
In sum; medicine of the present invention obviously can reduce myocardial ischemia reperfusion injury; reduce the infiltration of neutrophilic granulocyte in ischemic region, increase impurity free vacancy disordering district VE-cadherin content, NOS competitive type inhibitor L-NNA has then fully phased out these myocardium protecting actions of medicine of the present invention.Show that medicine of the present invention can increase the concentration of Plasma Nitric Oxide, effectively alleviate myocardial ischemia reperfusion injury.
Claims (1)
1. a Traditional Chinese medicine composition and N-nitro-L-arginine are preparing in myocardial ischemia-reperfusion the application raised in left ventricular end diastolic presssure medicine; The ratio of described Chinese medicine composition and N-nitro-L-arginine is 0.4g: 10mg; Described N-nitro-L-arginine uses with injection form; Described Chinese medicine composition is made up of following steps:
A) crude drug formula is:
Radix Ginseng 39.6g, Hirudo 72.6g, Eupolyphaga Seu Steleophaga 46.2g, Olibanum (processed) 13.2g, Radix Paeoniae Rubra 33g, Lignum Dalbergiae Odoriferae 13.2g, Lignum Santali Albi 13.2g, Scorpio 19.8g, Periostracum Cicadae 46.2g, Scolopendra 6.6g, Borneolum Syntheticum 33g, Semen Ziziphi Spinosae (parched) 33g;
B) pulverizing medicinal materials technique: by Scorpio, Hirudo, Scolopendra, Eupolyphaga Seu Steleophaga and Periostracum Cicadae five kinds of worm medicines after cleaning, washing process and clean the Olibanum (processed) after the process of preparing Chinese medicine and prepare burden by prescription, pulverized by pulverizer, medicated powder fineness reaches more than 80 orders; Medicated powder after coarse powder carries out micronizing through various superfine communication technique, makes medicated powder mean diameter be less than 30-40 μm; Medical material to be comminuted, after cleaning, drying sterilizing, is prepared burden;
C) concentrated and drying process is extracted: use water extraction again after Lignum Dalbergiae Odoriferae and the first extracting in water volatile oil of Lignum Santali Albi, Radix Paeoniae Rubra and Semen Ziziphi Spinosae add suitable quantity of water and decoct secondary, each 3 hours, merge Aqueous extracts, after filtration, one-tenth extractum to be concentrated; Radix Ginseng with appropriate 70% ethanol extraction secondary, each 3 hours, merge extractive liquid, reclaim ethanol extremely without alcohol taste, use water extraction again, it is 0.9 ~ 1.1 alcohol-extracted extract that alcohol extract is condensed into 60 DEG C of mensuration relative densities, and Aqueous extracts is concentrated into 60 DEG C after filtering and mixing with above-mentioned all Aqueous extracts and measures the clear paste that relative density is 0.9 ~ 1.1, for subsequent use;
D) preparation process: add superfine powder flour in Fluidbedgranulatingdrier, then by step c) gained extracts extractum and sprays into granulation; By the granule made through granulate, add Borneolum Syntheticum fine powder, spray into the volatile oil extracted by Lignum Dalbergiae Odoriferae and Lignum Santali Albi, by capsule filler filling after mixing, make 1000 capsules.
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