CN103665099A - Effective polypeptide parts in Polybia spp. insect venom, and cardio-cerebral thrombosis use thereof - Google Patents

Abstract

The invention relates to effective polypeptide parts in Polybia spp. insect venom, and a cardio-cerebral thrombosis use thereof. The effective parts are obtained through a step of dialyzing crude Polybia spp. insect venom through a 25KDa dialysis membrane, and a step of freeze-drying or reduced pressure concentration drying of the above obtained dialysis liquid. A series of pharmacodynamic experiments verify that the effective biotoxin polypeptide parts and transdermal absorption preparations thereof can substantially control the cerebral thrombus, can recover the mobility of cerebral palsy animals, can mitigate the ischemic brain injury, have stronger pharmacodynamic performances than first-line medicines comprising plavix, Xingnaojing and the like, and can be expected to be used for preparing innovative biochemical medicines for preventing and treating cardiovascular and cerebrovascular diseases in order to prevent and treat ischemic cardiocardiovascular diseases, such as myocardial infarction, cardiovascular thrombus, cerebral thrombus, cerebral infarction, acute/chronic cerebral apoplexy, and sequelae caused by cerebral infarction injury, like lateral hemiplegia, hemidysesthesia, hemiopia, facial and lingual paralysis, aphasia and general paralysis.

Description

Different abdomen Vespa insect bee venom polypeptide efficient part and anti-heart and brain thrombus purposes thereof

Technical field

The present invention relates to medical technical field, particularly, the present invention relates to preparation method and the medicinal use of polypeptide efficient part in a kind of different abdomen Vespa Polybia spp. insect.This efficient part is to be dialysed with the dialysis membrane of 25KDa by the thick bee venom of different abdomen Vespa wasp, and lye is dried and obtains through lyophilize or concentrating under reduced pressure.Inventor's pharmacology pharmacodynamic experiment inside and outside a series of animal bodies is found, this polypeptide efficient part has significantly prevents and treats the effect that cerebral thrombosis generated, recovered brain paralysis animal activity ability, alleviates ischemic brain injury, can expect and for the preparation of control cardiovascular and cerebrovascular diseases, comprise medicine, healthcare products, medicine equipment and the cosmetics of everyday use of ischemic cerebrovascular and ischemic cardiovascular.

Background technology

Cardiovascular and cerebrovascular diseases is in the world today, to threaten one of disease that the mankind are the most serious, and its M & M has surpassed tumour and leapt to the first in the world.At present, there are hyperpietic 600,000,000 people in the whole world, Prevalence of Hypertension approximately 10%.The first cause of death of the cardiovascular and cerebrovascular diseases Ye Shi China such as heart disease and stroke.Beijing Municipal Health Bureau discloses, and after the eighties in last century, the sickness rate of Beijing area cardiovascular diseases and cerebral apoplexy is all the trend of obvious rising, and by 2006, cardiovascular and cerebrovascular diseases accounted for 44% of the total cause of death of resident, had become first killer of Beijing resident.The data of ministry of Health of China announcement in 2005 shows: in the general mortality rate of Chinese population, mortality of cardio and cerebral vascular disease is high ranks first, and city resident's cerebrovascular mortality ratio reaches 21.2%, deaths from heart disease rate 17.9%; And urban residents' cerebrovascular mortality ratio also reaches 21.2%, deaths from heart disease rate 11.8%.China has 3,000,000 people nearly to die from cardiovascular disorder every year, has every day people more than 7000 to die from cardiovascular disorder, approximately falls down 1 every 12 seconds.Nearly 35～55 years old more than ten years male sex's myocardial infarction death gathers way the fastest.Along with the quickening of growth in the living standard and rhythm of life, cardiovascular and cerebrovascular diseases has now entered window phase, and Future Ten year is by eruption and prevalence.

Cardiovascular and cerebrovascular diseases is the general name of two large class diseases, and it can be divided into cardiovascular disorder and cerebrovascular disease.Cardiovascular disorder be take coronary heart disease as main, and coronary heart disease claims again coronary atherosclerotic heart disease, is because the coronary artery generation of supply myocardial blood is atherosis, and arteries is narrowed down, and deficiency myocardial blood supply causes; Because coronary artery pathological changes causes luminal stenosis or inaccessible clinical symptom, on time length, degree weight, be not quite similar, therefore can show as the various ways such as recessive heart trouble, stenocardia, myocardial infarction, myocardiosclerosis and sudden cardiac death.Cerebro-vascular diseases refers to the hemorrhage or thrombosis of rupture of blood vessel in brain, and what cause take one group of disease that brain hemorrhagic or ischemia injury symptom be main clinical manifestation, is commonly called as cerebral apoplexy.

Cardiovascular and cerebrovascular diseases has become " first killer " of harm people life and health, has the feature of " sickness rate is high, disability rate is high, mortality ratio is high, recurrence rate is high, complication many " it " four is high by more than one ".In Clinical types, be divided into (1) complete apoplexy: after morbidity, neurological deficit symptom is heavier more complete, and often (<6 hour) peaks within a few hours.(2) advancing stroke: the rear neurological deficit symptom of morbidity was made progress gradually or is staged and increases the weight of in 48 hours.(3) reversibility ischemia neurological deficit (RIND): after morbidity, neural disappearance symptom is lighter, continues more than 24 hours, but can recover in 3 weeks.

Cerebro-vascular diseases particularly, refer to the hemorrhage or thrombosis of rupture of blood vessel in brain, what cause take one group of disease that brain hemorrhagic or ischemia injury symptom be main clinical manifestation, claims again cerebrovascular accident or cerebral apoplexy, being commonly called as cerebral apoplexy, is to work as first three roughly one of dead disease.Clinical epidemiology data shows, China's incidence of stroke 1,500,000/year, dead 1,000,000/year.According to foreign statistic data, cerebro-vascular diseases be take ischemia as common, and cerebral infarction accounts for 59.2%～85%.And hematencephalon is except Japan, generally below 20%.China's new complete apoplexy 280 examples of rural survey in 1984, subarachnoid hemorrhage accounts for 3.9%, hematencephalon accounts for 44.6%, cerebral thrombosis accounts for 46.4%, cerebral embolism accounts for 2.5%, person accounts for 2.9% to be difficult to somatotype.By above data, can find out in ischemic cerebrovascular that cerebral thrombosis incidence probability is for the highest.

At present, in the treatment for cerebral thrombosis, main (1) super early stage thromboembolism treatment that adopts: object is thrombus, recovers rapidly infarcted region blood perfusion, alleviates neuronal damage; Thrombolysis should just likely be saved ischemic half blanking bar in the therapeutic time window in onset 6 hours.(2) anticoagulant therapy: object is anti-hemostasis suppository expansion and new thrombosis; Common drug has heparin, Low molecular heparin and warfarin etc.; During treatment, should monitor clotting time and prothrombin time, also must have the antagonists such as vitamin K, protamine sulfate, to process the hemorrhage complication of possibility.(3) Cerebral protection: can adopt calcium ion channel blocker, magnesium ion, anti-excitatory amino acid mediator, free-radical scavengers and mild hypothermia therapy.(4) fine treatment being fallen: by Fibrinogen in degraded blood, strengthens Fibrinolytic Activities, suppress thrombosis; Medicine has: fiber eliminating enzyme, batroxobin, ancrod and Lumbrukinase etc.(5) platelet aggregation-against treatment: fall ill in latter 48 hours and give acetylsalicylic acid 100～300 mg/day to the acute cerebral infarction patient without selecting, can reduce mortality ratio and recurrence rate, but not application simultaneously when carrying out thrombolysis and anticoagulant therapy, in order to avoid increase hemorrhage risk; Other anti-platelet aggregation agents also can be applied as ticlopidine, clopidogrel etc.(6) other drug treatment: vasodilator can cause stealing in brain blood and increase the weight of cerebral edema, should be cautious use of or need not; Neurocyte nutrition agent comprises three classes: affect energy metabolism class (acute phase should not be used), affect amino acid and polypeptide class, mediator and acceptor class affect the nerves.

Along with the raising day by day to environmental requirement, and the chemicals Side effect of developing is increasing, makes the world of medicine, medicine enterprise and patient have to eye to turn to " the green medicine " of wide spectrum, low toxicity, use safety.The biochemical new drug of plant and animal natural green resisting cardiovascular disease that constantly research and development make new advances is the declared policy that the populous nation of tens00000000 populations must carry out.

Because ischemic cerebrovascular pathogenesis is complicated, current also many to its medicine, in these medicines, there are plant amedica, animal drugs and mineral drug.And animal pharmaceuticals effect aspect treatment cerebrovascular disease is remarkable, always enjoy medical circle to pay close attention to, for the research of animal pharmaceuticals treatment cerebrovascular disease more have leech preparation, snake venom preparation, an earthworm preparation etc.

Animal pharmaceuticals protection ischemic brain injury many by reducing excitatory neurotransmitter and sour toxic action, suppress lipid peroxidation and nitration reaction, the approach such as reaction and inhibited apoptosis that reduce inflammation realize, and have the cerebral protection of many target spots, multi-level, too many levels.Zootoxin and extract thereof are in the therapeutic action of cerebral ischemia and Mechanism Study thereof, the more of research is (1) snake venom: the snake venom preparation that national Bureau of Drugs Supervision approval listing now is also used when in treatment cerebrovascular disease mainly comprises defibrase, agkistrodon shedaoensis embolism-resisting enzyme (SVATE I), Ahylysantinfarctase (SVATE), Jiangsu and Zhejiang Provinces Ahylysantinfarctase (SVATE II), Effect of Agkistrodon acutus Enzyme, Xiao Shuan ling, Thrombolytic enzyme, and by Japan push to Chinese market DF-521 (DF-521), be commonly called as batroxobin.(2) leech preparation: r-hirudin is a peptide species, modern pharmacology studies have shown that, r-hirudin has anti-freezing, antiplatelet gathers, improves the pharmacological actions such as microcirculation, and clinical trials show in a large number, and r-hirudin is than more effectively prevention of deep vein thrombosis formation of heparin.(3) earthworm preparation: the activeconstituents Lumbrukinase of earthworm can suppress thrombosis after cerebral ischemia, alleviate brain tissue impairment, prompting Lumbrukinase can be used as the control medicine of cerebrovascular disease.(4) scorpio: Scorpion Venom Fibrinolytic Active Peptide has provide protection to cerebral ischemia/reperfusion injury of rats, its mechanism may be relevant with inhibition expression of inflammatory cytokines.(5) gadfly: gadfly extracting solution has weak antithrombin effect, the polysaccharose substance containing in your knurl horsefly Hybomitra erberi (Brauer) gadfly can significantly be shown and extend mouse, rat clotting time, and can reduce the activity of the inside and outside source blood coagulation system factor, increase the vigor of fibrinolytic system, thus the formation of control thrombus.(6) Ground Beetle: 1 mg/kg of quiet note of Ground Beetle aqueous extract is after 10 minutes, and folder closes ventpipe and causes Animal Anoxia, finds that it can make rabbit anoxia tolerance function obviously strengthen, and Ground Beetle aqueous extract can obviously extend mouse hypoxia endurance time; Strengthen the activity of SOD in cerebral tissue, reduce NOS active, increase GSH content and the content that reduces NO, MDA.Illustrate that Ground Beetle extracting solution has certain provide protection to cerebral ischemia-reperfusion in mice; Its mechanism may be relevant with generation anti-oxidant and inhibition radicals NO.(7) spider: brave line spider venom-I(HWTX-I) subarachnoid space medication has certain provide protection to global cerebral ischemia/reperfusion; Its mechanism may be to play a role by suppressing the dead signal Signal Transduction Pathways activation of Fas molecule startup.(8) centipede: centipede has anti-freezing and thrombus dissolving effect, mechanism may with its venom in proteolytic enzyme, hemolytic factor and other blood system activation factor etc. closely related; Adopt line bolt method to set up arteria cerebri media Focal Cerebral Ischemia-Reperfusion in Rats model, prompting centipede extracting solution can be by reducing the content of Focal Cerebral Ischemia-Reperfusion in Rats plasma vWF and TPO, improve the damage that cerebral ischemia re-pouring causes, improve endothelial cell damage and platelet function, effectively suppress platelet adhesion reaction and gathering, prevent thrombosis, thereby alleviate the damage that cerebral ischemia re-pouring causes, can be used as the approach of control cardiovascular and cerebrovascular diseases.In addition also have the animal pharmaceuticals such as stiff silkworm, Cornu Bubali, Moschus also to can be applicable to the control of cerebral thrombosis disease.[Zhao Hairong, Wu Xiumei, Zhang Chenggui, Guo Yun glue, Yang Zizhong, Zhang Jingxin, Zhao Yu, etc.; Animal drugs on treatment of cerebral ischemia and its mechanism, CHAMC, 2014].In addition, the bat saliva of phyllostome, bee variety phallotoxins have also been reported thrombolysis composition.

Honeybee bee venom (bee venom) is present in the elements such as the venom in Bee Venom capsule, carbon containing, hydrogen, sulphur, phosphorus, calcium, chlorine, nitrogen, is transparent liquid, reacts acid.Honeybee bee venom has haemolysis and blood coagulation resisting function, when therapeutic dose, human body is seldom caused to hemolytic reaction, when larger dose, can make in vivo and in vitro blood coagulation time obviously extend, and shows that bee venom has therapeutic action promoting blood circulation and removing blood stasis.Also there is in addition the effect that reduces thromboxane, on the microcirculatory basis of improvement, work to alleviate joint symptom.

About all kinds of bee venom, all there is research both at home and abroad.(1) foreign study situation: the report that a piece of delivering of Austrian physician Phillip Terc in 1888 is entitled as " biting and rheumatismal particular associative about honeybee " has pulled open modern honeybee and treated the especially research prelude of melissotherapy (Bee venom therapy, BVT).The apiarist Ma Yezi charles (Charles Mraz) of Vermont ,Usa, through repeatedly practical application, be sure of that bee venom has the autoimmune disorder of alleviation as the effect of the symptom of multiple sclerosis and rheumatism and anti-inflammatory.Within 1967, first Habermanm isolates the melittin being comprised of 26 amino acid from honeybee bee venom, and has analyzed aminoacid sequence, proves in its structure without disulfide linkage.Hirai in 1979 etc. isolate the cationic polypeptide toxoid of a kind of l4 of containing amino-acid residue first from yellow wasp (Vespula lewisii) venom, are Mastopara(MP) one of wasp venom intoxicating, lethal Main Factors.Because of its function that there is induction mouse mast cell degranulation and discharge histamine, so be referred to as " the histamine release factor ".Nineteen ninety-five Reita etc. confirm sub-doses membrane attack complex can reduce complement to the damage base of cell on, further observed the impact of sub-doses melittin on complement damaging cells.Find that sub-doses melittin can obviously reduce the dissolution rate of complement to cell.Within 1997, Shamsher utilizes synthetic melittin to observe the impact on TypeⅡsecretoryphospholipaseA2; Result shows, melittin can suppress the activity of various sources TypeⅡsecretoryphospholipaseA2, to the inhibiting rate of Phospholipase A2 activity in patient with rheumatoid arthritis synovia, can reach 96%.(2) domestic research situation: within 1987 5, like cicada find after deliberation full bee venom and various component thereof in body or external zooperal pharmacological action different.More than 1996 Xiao Dong etc. are to apis mellifera poison component research discovery, the effect that bee venom has induced platelet to assemble.The researchs such as Xu Peng in 1996 find that bee venom has effect promoting blood circulation and removing blood stasis.Calendar year 2001 king General Guan Yu's Tomb etc. utilizes intestinal bacteria to study and express melittin precursor protein.The researchs such as Zhang Chen in 2004 show, melittin can disturb normally dividing a word with a hyphen at the end of a line of liver cancer cell cell cycle, and cell block, in the S phase, and is to dose-dependently, cause the accumulation of a large amount of S cells.It is little that Wang Bin in 2005 etc. find that mellitin is used separately the impact of NK killing activity.The enhancement of chimeric protein NK killing activity is the most remarkable, and kill rate is significantly higher than Normal group.The conspicuous synthetic that waits of red legend in 2009, containing the apamin gene of enteropeptidase cleavage site, has built the intestinal bacteria fusion expression vector containing apamin, has realized the amalgamation and expression of apamin and gst gene.Lee in 2009 likes that sword, Liang Feng find that melittin is inhibited to the propagation of gastric carcinoma cells BGC in vitro.Application prospect [the Heng Liu etc. as the insect medicine of new health industry about wasp aptoxin have been delivered in the medicinal extraordinary insect exploitation of the Dali College national local joint project research centre at inventor place, Utilization of Polislidae wasp venom as potential new insects drugs in the R & D wellness industry, International Journal of Biotechnology for Wellness Industry, 2012,1:241-249].

Different abdomen Vespa, Polybia spp., belong to Insecta Pterygota Hymenoptera (Hymenoptera) Clistogastra (Apocrita) Aculeata (Aculeata) Vespoidea (Vespoidea) Polybiidae (Polybiidae) insect, state, the Yunnan-Guizhou Plateau extensively distributes, belong to Social wasps, knot nest is gregarious.Because a lot of these unique genus insect resourceses concentrate on Yunnan-guizhou Area, domestic other provinces and external colleague are all less to its research and active report thereof.In the series of studies process of the pharmaceutical insects resisting cardiovascular class disease medicament in the medicinal extraordinary insect exploitation of the Dali College Dui Yunnan-Guizhou Plateau, national local joint project research centre at inventor place, this is belonged to various insects and carried out comprehensive comprehensively collection, kind evaluation; And further implemented the different abdomen wasp of variant kind bee venom collection, purifying, obtain different extract parts, the multiple pharmacodynamics that has compared its resisting cardiovascular class disease is active, has completed drug development previous research work.

According to document, China mostly is compound for the application of " bee venom ", and more for the research of honeybee bee venom.For the research of wild wasp, the relevant report of the efficient part treatment cardiovascular and cerebrovascular that in especially extraordinary different abdomen Vespa insect bee venom, purifying obtains there is not yet.Published data has: (1) process for purification class: Zhang Wenli in 2007, Sun Jing unit disclosed " process for purification of bee venom " (publication number CN101088514A); Disclose and a kind of thick bee venom is dissolved in and in ethanol, removes the impurity such as propolis and obtain refining bee venom.The same year thereafter, two people disclose again a kind of " separating and purifying method of mellitin " (publication number CN101089017A), described by thick bee venom water embathe, ethanol precipitation, ammonium hydroxide and n-butanol extraction, the sequence of operations such as acetone precipitation, the final preparation method who obtains electrophoresis level mellitin melittin by the desalination of G-10 post, G-25 column purification and desalination freeze-drying.2009, Zhang Wenli has declared again " method of purification of mellitin " (publication number CN101455287A), has described the improvement to above-mentioned patent system Preparation Method, thereby retained the mellitin tetramer, is not trapped, processing step reduces, the preparation method that productive rate improves.1994, Liu Jinhui, Wang Yuancheng, Yang Feng disclosed and a kind ofly with filtration, ultrafiltration, dialysis, have removed macromole and micromolecular Bee Venom polypeptide preparation method." a kind of hornet anti-bacterial peptide and its preparation method and application " (grant number CN100547000C) that Liu Wei China, Li Ming in 2009 declare, has described and a kind ofly by gel filtration chromatography, ion-exchange chromatography and anti-phase high pressure liquid chromatography, has carried out separation and purification to obtain molecular weight be 1387.7 daltonian single chain polypeptides.(2) application aspect of mellitin: 2005, Zheng Jiang etc. disclose a kind of " mellitin and application thereof " (publication number CN1704431A), have described with the small-molecular peptides of the synthetic INLKAIAALAKKLL-NH2 of resin and for the preparation of kill bacteria and the pyemic medicine for the treatment of.Within 2009, bad ren, Xu Xueqing have applied for " hornet anti-bacterial peptide and its preparation method and application " (grant number CN100475840C), having described molecular weight is the hornet anti-bacterial peptide of 1316.6 daltonian NH2-IDWKGIAAMAKI-COOH, be mainly to obtain by centrifugal, gel filtration chromatography, ion-exchange chromatography, reversed-phase HPLC column purification, and tested its restraining effect to bacterium, fungi, virus, tumour cell.(3) biotechnology and application aspect: calendar year 2001, U.S. Pan Pacific Pharmaceuticals Inc. discloses " gene of the useful property of bee venom protein and this bee venom protein of coding " (publication number CN1313895A), describe the nucleic acid that novel protein, its antibody and coding separated from meltittin venom change albumen, be used for the treatment of rheumatic arthritis and inflammation.2002, Zhang Sufang, Shi Wanjun, Cheng Jiaan, Zhang Chuanxi disclosed " polypeptide of precursor gene of ink chest wasp aptoxin bematolysis peptide and coding thereof and preparation method " (publication number CN1385526A) and " polypeptide and the preparation method of volume spot yellow wasp hemolysis peptide precursor gene and coding " (grant number CN1385525A).2005, Zhang Sufang, Shi Wanjun, Cheng Jiaan, Zhang Chuanxi, Shen Lirong etc. successively disclosed again " polypeptide of large wasp sedative peptide precursor gene and coding thereof and preparation method " (grant number CN1194089C) and " polypeptide of apis cerana Vespa magnifiac (Sonan). hemolysis peptide precursor gene and coding and preparation method " (grant number CN1233830C).2006, Wang Bin etc. disclosed " melittin and gene expressing interleukin II chimera protein " (publication number CN1754960A), as the genetically engineered drug for the treatment of tumour, attempted.The bright group of face in 2007, Xu Tianmin disclose " fusion rotein and the preparation thereof of urokinase type fibrinolysin activator a chain and melittin " (publication number CN101337992A), have described a kind ofly by urokinase type plasminogen activator a chain, to be combined fusion rotein, its preparation method and the effect of this fusion rotein in oncotherapy forming with melittin.2008, " restructuring ceeA-mil heterozygous genes antibacterial peptide " (publication number CN101323644A) declared in Rui Pu Bioceuticals Inc., according to cecropin A and melittin, by gene engineering method, build a kind of recombinant protein of yeast expression, test its anti-microbial activity, attempted whereby a kind of recombinant antibacterial peptide.2009, the Hu Zongli of University Of Chongqing etc. disclosed " Melittin gene fission yeast engineering bacteria and construction process thereof and application " (grant number CN101591622B).The same year, the Shen Li of Zhejiang University honor etc. discloses a kind of " apis cerana royal jelly antibacterial peptide AccRoyalisin gene and coded polypeptide and application " (publication number CN101705231A), and has tested the Royalisin product anti-mycotic activity with the restructuring of fermentation engineering method.Agricultural University Of South China in 2011 thanks to green plum etc. and discloses a kind of " a kind of restructuring mellitin and application thereof " (publication number CN102229664A), by this gene recombination pharmacological agent chicken salpingo disease.In the same year, the respectful silver of Zhejiang University's leaf etc. discloses a kind of " Pteromalus puparum venom serine protease inhibitor Pp-PI polypeptide and application " (publication number CN102260348); Be intended for genetically modified crops or plant modification fungal component.(4) preparation aspect, Yang Mengjun calendar year 2001 discloses a kind of " nano Hufeng preparation medicine and preparation method thereof " (publication number CN1366926A), has described and take wasp polypide as raw material, after micronizing, through series of processes, the nano Hufeng preparation of preparing by supersonic jet technology.Cui Ford of Shenyang Pharmaceutical University, very little winter plum " biodegradable microsphere injection of bee venom and preparation method thereof " of declaring in 2003 has been described for the microball preparation formulation of Bee Venom and has attempted, but there is no pharmacology embodiment.That Cui Fude in 2009 etc. have declared is again a kind of " for Melittin complex nanometer granule of oral administration and preparation method thereof " (publication number CN101406691A); Described by nanotechnology Bee Venom has been made to Melittin complex nanometer granule, for oral.Above preparation is all without the wasp poison efficient part of describing after purifying, the transdermal absorption formulation preparation method who especially originates in the different abdomen Vespa insect bee venom efficient part of the special population in Yunnan; More without its physiologically active with for the relevant report of the active aspect of the pharmacodynamics of cardiovascular and cerebrovascular.

Obviously, above-mentioned disclosed every invention is not all to being under the jurisdiction of the different abdomen Vespa caste of special population and the activity of anti-cerebral thrombosis aspect is studied.In view of organic sphere is because geography, the different abundant species diversities that bring up of climatope are most important on the impact of physiologically active, between the different kind of insect, textural difference varies, and only amino acid whose arrangement difference just causes bioactive greatest differences.So the special different abdomen Vespa insect toxins for southwest species diversity Yunnan Province the highest, that national caste is maximum carries out system acquisition and biological activity determination still belongs to the first time, so but very necessary.For biometric safeguard resource resource security, take the lead in reporting the significant of control cardiovascular and cerebrovascular diseases activity that the different abdomen Vespa of Yunnan Vespoidea Medical insect resources was not studied in the past.

The different abdomen Vespa of Polybiidae (Polybiidae) Polybia spp wasp, is distributed in China Yun-Gui Plateau, and Yunnan Province is 10 kinds of nomenclator's less thaies, are Yunnan Province's superior resources.Whether the different abdomen Vespa insect toxins extensively distributing for medicine source, the clear and definite Yunnan-Guizhou Plateau has treatment ischemic cardio cerebrovascular diseases activity and medicinal use thereof, the strength ratio of the percutaneous dosing TTS preparation for treating ischemic cardio cerebrovascular diseases of different abdomen Vespa bee venom polypeptide efficient part and thus instructs and further understands fully the structure of matter basis of wherein playing main pharmacological, and from molecular pharmacology basis, illustrate the multiple bioactive mechanism of action of different abdomen Vespa insect toxins, the R&D team in the medicinal extraordinary insect exploitation of Dali College national local joint project research centre commonly uses different abdomen Vespa insect and has carried out synthetical collection and pharmacodynamics activity research being distributed in domestic among the people in Yunnan Province.

Percutaneous dosing is a kind of method that medicine passes through percutaneous drug delivery, medicinal application is after skin, with constant speed (or approaching constant speed), pass stratum corneum, diffuse through skin, by capillary vessel absorption, enter body and circulate, produce whole body or local therapeutic effects, conventionally on document, be called Transcutaneous Therapeutic System (transdermal therapeutic system, be called for short TTS) or transdermal delivery system (transdermal drug delivery system is called for short TDDS).The feature of percutaneous dosing: (1) can avoid first pass effect and the deactivation of medicine in gi tract of liver, and the absorption of medicine is not subject to the impact of gastrointestinal factors, the individual difference of minimizing medication.(2) can maintain constant effective Plasma Concentration or physiological effect, the Plasma Concentration peak valley phenomenon of avoiding oral administration to cause, reduces toxic side effects.(3) reduce administration number of times, improve therapeutic efficacy, extend action time, avoid multiple dose administration, make most patient be easy to accept.(4) easy to use, independently medication of patient, also can cancel medication at any time.

Just because of these advantages, the research and development of tts system are one of focuses of current pharmaceutical preparation research and development, and development rapidly, if nitro-dur has been one of best-selling 50 kinds of medicines in the world since coming out the eighties always.Tts system, by the universally recognized third generation pharmaceutical preparation novel form after oral and injection of domestic the world of medicine, is the new and high technology that contemporary pharmacy industry ought to be grasped, and has the meaning of milestone on pharmaceutics.As third generation novel pharmaceutical formulation, tts system is not only in the world one of drug research field with fastest developing speed, and has produced huge economic benefit in developed countries such as Europe, the United States.Since first TTS product Scopolamine paster in 1981 comes out, TTS has been subject to the welcome of extensive patients immediately.It is reported, at Nicotin-TTS(Nicotine patch in 1992) sales volume just reach 1,000,000,000 dollars, considerably beyond other smoking cessation products, by U.S.'s < < epoch > > magazine, be chosen as one of ten most popular large products of U.S. then; 1999 are only the Estraderm(FemPatcs that Voltaren company and Lamisil company produce) and Nitroderm(nitroglycerine patch) sales volume reach respectively 3.8 hundred million and 3.3 hundred million dollars.Having potent narcotic analgesic fentanyl paster started selling in 2002 and just reaches 15.9 hundred million dollars of annual sales amounts.A kind of high in technological content, the Novel external patch that scope is wide, easy to use in the TTSShi world today, DEVELOPMENT PROSPECT is wide, and potential economic benefit is huge.

The medicine that in TTS product, transdermal administration has been selected mainly contains following a few class: [sexual hormoue] – Progesterone, testosterone, Norethisterone, LNG, estradiol, prostaglandin(PG), vassopressin, estrogen and progestogen+Progesterone etc.; [cardiovascular drug] – nifedipine, nitrendipine, amlodipine, sorbitrate, pannonit, nicardipine, Proprasylyte etc.; [neural system medicine] – Physostigmine, morphine, Dihydroetorphine, ketorolac, fentanyl etc.; [anti-inflammatory analgesic] – INDOMETHACIN, Ketoprofen, flurbiprofen, Ibuprofen BP/EP, diclofenac etc.; [anti-asthmatic] – tulobuterol, terbutaline etc.At present, international percutaneous dosing market development is swift and violent, makes rapid progress, and according to the World Health Organization's prediction, by 2025, the existing medication that has 1/3 will be adopted to transdermal formulation, and the output value of tts system will reach tens billion of dollars.Therefore, the research and development of TDDS administration product are subject to extensive concern, will in drug use history, turn over a new page.

In domestic patch market, traditional dog skin plaster (ancient percutaneous dosing mode) class Chinese medicine patch has occupied a greater part of above market share, and the market share of Chinese medicine and pharmaceutical chemicals transdermal patch is insignificant.Why smaller current domestic preparation capable of permeating skin market scale is, is mainly the restriction that is subject to domestic preparation technique level.China's tts system also exists the supporting level of obvious gap, particularly industrial production lower aspect research and development compared with developed countries, not yet can form scale production ability.The animal drugs percutaneous dosing building scientific research platform project that the medicinal extraordinary insect exploitation of Dali College national local joint project research centre is born has been done a large amount of preparations to this.Not only purchase world advanced person's a collection of TTS instrumentations such as bar cloth cream coating machine, also introduced energetically the Research Team of specializing in preparation capable of permeating skin.All kinds of bee venom TTS preparations in the present invention, the insect drug transdermal preparation GMP of Ji Shi Dali College Yunnan Province innovation team is prepared in workshop.

Prevent and treat the method for cerebral thrombosis mainly: thrombolysis, platelet aggregation-against, anti-freezing.But improve patient's survival rate and the difficult problem that quality of life is still cerebrovascular disease therapy.Even if survival, also has wherein approximately 50% patient can leave sequela in various degree.Therefore, improve the understanding of this disease, explore effective methods for the treatment of and medicine, the task of top priority of real genus, also has important theory value and clinical meaning undoubtedly.Inventor team from the thrombolytic effect of medicine, anticoagulating active, medicine on the impact of platelet function, copy cerebral thrombosis four aspects and start with, copying on the basis of cerebral thrombosis animal model, test corresponding neurophysiology and physiological and biochemical index, inquire into the impact of medicine on this model, to solve substantial problem for cerebrovascular disease.

In the research of the Vespoidea insect that the Yunnan-Guizhou Plateau is collected, we find that the thick bee venom collecting has blood coagulation resisting function, and test of many times finds that the efficient part that is less than 25KDa has the effect that antagonism Level In Rats With Focal Cerebral Ischemia damages then.The inventor also finds: the material medicine effect for the animal brains such as mouse, rat and nose is better than the route of administration such as other are oral, intramuscular injection, quiet note outward.Therefore, the inventor has designed with modern preparation technique and has prepared a series of transdermal absorption formulations, and by various these TTS formulation applications in resisting cardiovascular disease pharmacology and effect experiment, find: wasp poison polypeptide efficient part preparation capable of permeating skin has the advantage can not be substituted, and its onset time is fast, toxicity is low, action intensity is large, using dosage is little.The most important thing is, once find that subject has drug withdrawal at any time after malaise symptoms or toxic reaction, has guaranteed the patient's of laboratory animal and clinical use security.

The present invention uses different inside and outsides model, has tested different abdomen Vespa bee venom polypeptide efficient part that the thick bee venom of different abdomen Vespa prepares via dialysis membrane and transdermal absorption formulation thereof (take hydrogel, cataplasma, liniment the be example) pharmacologically active to mouse thrombus, cerebral infarction, platelet aggregation, decapitated mice breathing time etc.Test-results is found: different abdomen Vespa bee venom polypeptide efficient part and transdermal absorption formulation thereof have the effect of significant antithrombotic, anti-cerebral infarction, platelet aggregation-against, prolongation decapitated mice breathing time etc.There is the drug activity of part TTS pharmaceutical preparation over a line medication and the cookle class best-selling drugs of multiple cerebral ischemia diseases.Can be used as the further deeply exploitation of control cardiovascular and cerebrovascular diseases novelty natural drug, thereby form the present invention.

Summary of the invention

The object of this invention is to provide a kind of polypeptide efficient part extracting and preparation method thereof from different abdomen Vespa wasp, it is characterized in that: this efficient part is from the thick bee venom of different abdomen Vespa wasp, to take the dialysis membrane dialysis that molecular weight cut-off is 25KDa, lye is dried and obtains through lyophilize or concentrating under reduced pressure, and wherein content of peptides accounting is not less than 50%.

Another object of the present invention has been to provide the pharmaceutical use for the preparation of control cardiovascular and cerebrovascular diseases by different abdomen Vespa wasp polypeptide efficient part, it is characterized in that: described cardiovascular and cerebrovascular diseases refers to ischemic cerebrovascular, ischemic cardiovascular; Specifically refer to heart stalk, thrombus of heart blood vessel, cerebral thrombosis, cerebral infarction, urgency/chronic cerebral apoplexy, and the sequela of being brought by cerebral infarction injury is as lateral deviation paralysis, hemianesthesia, hemianopsia, face tongue paralysis, aphasia, total paralysis etc.This pharmaceutical use is embodied by pharmaceutical preparation, medicine equipment, cosmetics of everyday use form; The form of described pharmaceutical preparation, medicine equipment, daily chemical products is hydrogel, frothy gel, liniment, cataplasma, lyophilized powder, aqua, aerosol, suppository, externally-applied liniment, ointment.

A further object of the present invention has been to provide a kind of pharmaceutical composition with control cardiovascular and cerebrovascular diseases activity, it is characterized in that: this pharmaceutical composition contains the polypeptide efficient part, pharmaceutical excipient, pharmaceutical carrier or the dressing that extract from different abdomen Vespa wasp for the treatment of significant quantity.

Animal material medicine bee venom polypeptide in the present invention picks up from the different abdomen Vespa of Hymenoptera Vespoidea (Vespidae) Polybiidae (Polybiidae) (Polybia spp.) insect polypide.

The invention provides the efficient part that a kind of different abdomen Vespa bee venom is prepared through dialysis membrane, and take the transdermal absorption formulations such as hydrogel that its bulk drug prepares as main ingredient, cataplasma, liniment and controlled release or slow release formulation or nanometer formulation; And, the inventor first by above-mentioned formulation application in antithrombotic, anti-cerebral infarction, platelet aggregation-against, prolongation decapitated mice breathing time, reduce the medicinal use of cerebral edema etc.

Its bulk drug of polypeptide efficient part of preparing through dialysis membrane of the present invention or its pharmacologically acceptable salt are the hydrogel that main ingredient is prepared, cataplasma, the transdermal absorption formulations such as liniment and controlled release thereof or slow release formulation or its nanometer formulation can also be with the medicine of the Cardiovarscular class illness of now having gone on the market as fallen fibrinolytic suppository, anticoagulant, and brain protection preparation is as calcium ion channel blocker, magnesium ion, anti-excitatory amino acid mediator, free-radical scavengers, medicament for resisting platelet aggregation, vasodilator, the types of drugs such as nerve cell-protective agents are combined use or cross-reference, for example, with heparin, Low molecular heparin, warfarin, vitamin K, protamine sulfate, fiber eliminating enzyme, batroxobin, ancrod, defibrase, agkistrodon shedaoensis embolism-resisting enzyme (SVATE I), Ahylysantinfarctase (SVATE), Jiangsu and Zhejiang Provinces Ahylysantinfarctase (SVATE II), Effect of Agkistrodon acutus Enzyme, Xiao Shuan ling, Thrombolytic enzyme, acetylsalicylic acid, ticlopidine, clopidogrel, r-hirudin and leech preparation, snake venom preparation, Lumbrukinase and earthworm preparation, tiger line spider venom-I(HWTX-I), the combinations such as mellitin such as honeybee bee venom (bee venom) and melittin, or and XINGNAOJING, nimodipine, XUESAITONG, anxious effect is rescued heart pellet, Ligustrazine, the drug regimens such as Plavix, prepare composition or the compound preparation with treatment cardiovascular and cerebrovascular diseases effect, can expect and become the medicine for the treatment of cardiovascular and cerebrovascular diseases, healthcare products, cosmetics of everyday use and medicine equipment.Above-mentioned various kinds of drug composition or medicine, healthcare products, cosmetics of everyday use and medicine equipment can adopt several formulations, comprise the Transdermal absorption formulation medicines such as hydrogel, frothy gel, cataplasma, liniment, ointment, comprise the now conventional preparation of generally acknowledged pharmaceutics general knowledge of employing and various slowly-releasings, controlled release form or nanometer formulation.

Up to now, in previous literature and find no and the preparation technology of different abdomen Vespa insect bee venom polypeptide efficient part provided by the invention and the bibliographical information of method, the dialysis membrane of use special pore size distribution of the present invention is prepared different abdomen Vespa bee venom polypeptide efficient part method tool and is had an unexpected effect.And, up to now, Chinese and foreign documents there is no the different abdomen Vespa bee venom polypeptide efficient part collecting for Yunnan and carries out the report for ischemic cerebrovascular, ischemic cardiovascular activity research, does not more have this bee venom polypeptide efficient part to make TTS(transdermal absorption formulation) for preventing and treating the report of cardiovascular and cerebrovascular diseases.The research comprehensively by different abdomen Vespa bee venom polypeptide efficient part TTS preparation prevention and treatment ischemic cardio cerebrovascular diseases of the property of the present invention is directed to still belongs to the first time.In addition its bulk drug of efficient part that, different abdomen Vespa bee venom dialysis membrane is prepared and transdermal absorption formulation thereof all have the effect of significant antithrombotic, anti-cerebral infarction, platelet aggregation-against, prolongation decapitated mice breathing time, minimizing cerebral edema etc.; Also all belong to beyond thought result, thereby can expect and be developed further into medicine or the pharmaceutical composition into treatment ischemic cardio cerebrovascular diseases class.Completed thus the present invention.

Usefulness of the present invention is: using first molecular weight cut-off (MWCO) is that the dialysis membrane of 25 kilodaltons (25KDa) is prepared different abdomen Vespa insect bee venom polypeptide efficient part, and has found first the potentiality of this efficient part in the pharmaceutical field of exploitation control cardio-cerebralvascular diseases; For exploitation treatment ischemic cerebrovascular, ischemic cardiovascular, comprise heart stalk, thrombus of heart blood vessel, cerebral thrombosis, cerebral infarction, urgency/chronic cerebral apoplexy, and the sequela of being brought by cerebral infarction injury provides the basic substance of new animal pharmaceuticals as the original new drug of lateral deviation paralysis, hemianesthesia, hemianopsia, face tongue paralysis, aphasia, total paralysis etc.The another feature of the present invention is: the transdermal absorption formulation of making according to different abdomen Vespa insect bee venom polypeptide efficient part has rapid-action, active extensive, safe advantage, make the many TTS preparations of this efficient part activity be better than the clinical line medication of cardiovascular and cerebrovascular, there are quite wide market outlook.A present invention again feature is: this genus hymenopteran growth and breeding ability is vigorous, in the Yunnan-Guizhou Plateau, extensively distribute, be convenient to cultivation, every queen bee can hatch hundreds and thousands of filial generations every year, be conducive to become stable and Sustainable Exploitation bio-pharmaceutical resource, be also conducive to Poor Mountainous Area peasant simultaneously and cultivate this type of medicine source property animal and reach the object of shaking off poverty.There is potential great social profit and economic benefit.This medicine material derives from the Vespoidea insect of natural cultivation, and its bee venom source quantity is stable, quality controllable; Bee venom polypeptide efficient part preparation process is easy, green natural, and cost is low, pollutes littlely, is beneficial to scale operation.

Specific embodiments

In order to understand better essence of the present invention, with embodiment and pharmacology embodiment, illustrate to use respectively below and take the hydrogel that polypeptide efficient part that the thick bee venom of different abdomen Vespa prepares through dialysis membrane makes as main ingredient, cataplasma, the preparation method of the transdermal absorption formulations such as liniment, and the impact that collagen and adrenalin mixture inducing mouse thrombus in vivo is formed (treatment administration and prevention administration), on the cerebral protection of cerebral anoxia mouse (medicine on decapitated mice pant the impact of time), the pharmacological results such as antiplatelet aggregative activity, preparation and the new purposes in control cardiovascular and cerebrovascular biochemical drug development field thereof of this polypeptide efficient part are described.

Unless otherwise noted, per-cent of the present invention refers to weight percent.Mandatory declaration, embodiments of the invention are for the present invention rather than limitation of the present invention are described.The simple modifications that essence according to the present invention is carried out the present invention all belongs to the scope of protection of present invention.

embodiment 1: the preparation of east different abdomen wasp aptoxin polypeptide efficient part and liniment

The collection of the thick bee venom of the 1.1 different abdomen wasp in east:

It is domestic that the different abdomen wasp in east is picked up from Puer City, Yunnan Province, and honeybee body sample is accredited as the different abdomen wasp in Polybia orientalis Saussure(east according to the brave professor of medicinal extraordinary insect exploitation national local joint project research centre untrimmed book of Dali of Yunnan institute).Gather wasp poison device: use the pair lid formula collector of bee venom through the repacking of this Engineering Research Center, wooden frame, long 80 centimeters, wide 50 centimeters; 20 centimeters/wide 33 centimeters of embedded 4 lengths of a film are got malicious glass; Input voltage 9V, output automatic intermittent ripple voltage; Gather wasp poison method: the device of gathering venom is placed in before honeycomb, according to the routine rules of gathering venom, this nest east different abdomen wasp bee colony worker bee is gathered venom.Open venon extractor power switch, knock ground and honeycomb, attract the different abdomen wasp in east out to attack collector of bee venom.Gather venom after end, powered-down, sweeps the no longer different abdomen wasp in east of toxin expelling, collects the bee venom on sheet glass, packs in special freezing bottle plum Teller 100,000/scales/electronic balance weighing into.Merge the thick bee venom of the different abdomen wasp in east that collection obtains for several times and amount to 293 milligrams.

1.2 see through the purifying that dialysis membrane carries out thick poison:

According to conventional dialysis film, hold back macromole method, use the dialysis tubing of U.S.'s spectrum medical science (spectrum) brand, instant cellulose ester membrane, molecular weight cut-off (MWCO) is 25000 dalton (25KDa).Use pre-treatment and operation all to carry out to specifications.Add 9 ml distilled waters and dissolve the different abdomen wasp aptoxin in east, then carefully pour in dialysis tubing, tighten suitable for reading.The dialysis tubing installing is put into the beaker that fills distilled water, 4 ℃ of temperature environment shaking table concussion dialysed overnight, and constantly stirred.After 19 hours, get peritoneal effluent lyophilize, after the desalination of G-10 post, then freeze-drying, be the bulk drug of next step preparation use.Method obtains 211 milligrams of east different abdomen wasp aptoxin polypeptide efficient parts according to this.Through Forint phenol method inspection, in this efficient part, content of peptides is 81.1%.

The preparation of 1.3 east different abdomen wasp aptoxin polypeptide efficient part linimentes:

Liniment method processed: get 0.25 gram of glycerine, the 0.1 gram of different abdomen wasp aptoxin of tween-80 and east polypeptide efficient part is dissolved in 5 ml distilled waters, micro-ly add thermal shocking and make it to dissolve.Then with 20 milliliters of 2%CMC-Na rubber cement stirring and evenly mixings, 40 ℃ of insulations, treat bubble elimination, just applicable to the experimentation on animals in pharmacology embodiment.Use is during to mouse, and the liniment final concentration being mixed with is 0.75 milligram/30 microlitres.

embodiment 2. the preparation of the different abdomen wasp aptoxin of mole polypeptide efficient part and hydrogel adhesive

The collection of the thick poison of the different abdomen wasp of 2.1 mole:

It is domestic that the different abdomen wasp of mole is picked up from Ruili City, Yunnan Province, and honeybee body sample is accredited as Polybia stigma Smith according to the medicinal extraordinary insect exploitation national local joint project research centre Guo Yun glue professor of Dali of Yunnan institute.Gather wasp poison device: use the pair lid formula collector of bee venom through the repacking of this Engineering Research Center, wooden frame, long 80 centimeters, wide 50 centimeters; 20 centimeters/wide 33 centimeters of embedded 4 lengths of a film are got malicious glass; Input voltage 12V, output automatic intermittent ripple voltage; Gather wasp poison method: the device of gathering venom of uncoated is placed in before honeycomb, according to the routine rules of gathering venom, the different abdomen wasp of this nest mole bee colony worker bee is gathered venom.Open venon extractor power switch, knock ground and honeycomb mouth, attract the different abdomen wasp of mole out to attack collector of bee venom.Gather venom after end, powered-down, sweeps the no longer different abdomen wasp of mole of toxin expelling, collects the bee venom on sheet glass, packs in special freezing bottle plum Teller 100,000/scales/electronic balance weighing into.Merge the thick bee venom of the different abdomen wasp of mole that collection obtains for several times and amount to 283 milligrams.

2.2 see through the purifying that dialysis membrane carries out thick poison:

According to conventional dialysis film, hold back macromole method, use the dialysis tubing of U.S.'s spectrum medical science (spectrum), instant cellulose ester membrane, molecular weight cut-off is 25KDa.Use pre-treatment and operation all to carry out to specifications.Add 9 ml distilled waters and dissolve the thick bee venom of the different abdomen wasp of mole, then carefully pour in dialysis tubing, tighten suitable for reading.The dialysis tubing installing is put into the beaker that fills distilled water, 4 ℃ of temperature environment shaking table concussion dialysed overnight, and constantly stirred.After 18 o'clock, get peritoneal effluent lyophilize, after the desalination of G-10 post, then freeze-drying, be the bulk drug of next step preparation use.Obtain 207 milligrams of the different abdomen wasp aptoxin of mole polypeptide efficient parts.Through Forint phenol method inspection, in this efficient part, content of peptides is 87.2%.

The preparation of the different abdomen wasp aptoxin of 2.3 mole polypeptide efficient part hydrogel adhesive:

Hydrogel adhesive method processed: take 150 grams of glycerine, put into 500 ml beakers, take successively each 1 gram of dihydroxyaluminum aminoacetate, EDTA, be successively dissolved in glycerine, stir, then add 25 grams of NP-700, mix, obtain A liquid; 5.0 grams of carbomers are dissolved 4 hours, add 200 grams of distilled waters, obtain B liquid; A, B are mixed to get to C liquid.Get 1.0 grams, tartrate, add 117.5 grams of distilled waters and mix and obtain D liquid.The gradation of D liquid is poured in C, and stir, obtain hydrogel matrix.The hydrogel adhesive concentration needing according to pharmacology embodiment adds the different abdomen wasp aptoxin of mole polypeptide efficient part in hydrogel adhesive, stirs.Can be used for the experimentation on animals in pharmacology embodiment.Use is when mouse brain or nose, and the hydrogel adhesive final concentration being mixed with is 0.75 milligram/30 microlitres.

embodiment 3. the preparation of Sumatera different abdomen wasp aptoxin polypeptide efficient part cataplasma

The collection of the thick poison of the 3.1 different abdomen wasps in Sumatera:

It is domestic that the different abdomen wasp in Sumatera is picked up from Yunnan Province's Menhai, and honeybee body sample is accredited as Polybia sumatrensis Saussure according to national local joint project research centre professor Yang Zizhong of medicinal extraordinary insect exploitation of Dali of Yunnan institute.The basic device of gathering venom is used the pair lid formula collector of bee venom through the repacking of this Engineering Research Center, and wooden frame is long 80 centimeters, wide 50 centimeters; 20 centimeters/wide 33 centimeters of embedded 4 lengths of a film are got malicious glass; Input voltage 9V, output automatic intermittent ripple voltage; Wooden frame is painted redness with red paint in advance; On wooden frame, smearing in advance 20 ml concns is honeycomb and the honeybee body extract volatile solvent soln (preparation method: 100 grams of honeycombs and the homogenate of 80 worker bee honeybee bodies are ground of the different abdomen wasp in other a brood of Sumatera of 15 mg/ml, add 20 ml distilled waters, with ethyl acetate extraction 2 times, each 20 milliliters; After extraction liquid evaporated under reduced pressure solvent, with ether dissolution, be mixed with the extract volatile solvent soln of 15 mg/ml); Gathering the different abdomen wasp aptoxin in Sumatera smears the most effective in first 20 minutes.This venon extractor is positioned over to the ground that is covered with large red plastic cloth, slightly low dip 15 degree, form the intense stimulus signal to honeycomb bee colony.Then open venon extractor power switch, knock ground and honeycomb mouth, attract the different abdomen wasp in Sumatera out to attack collector of bee venom.Gather venom after end, powered-down, sweeps the no longer different abdomen wasp in Sumatera of toxin expelling, collects the bee venom on sheet glass, packs in special freezing bottle plum Teller 100,000/scales/electronic balance weighing into.232 milligrams of the merging thick bee venom of the different abdomen wasp in Sumatera that collection obtains for several times.

3.2 see through the purifying that dialysis membrane carries out thick poison:

According to conventional dialysis film, hold back macromole method, use the dialysis tubing of U.S.'s spectrum medical science (spectrum), instant cellulose ester membrane (MWCO:25KDa).Use pre-treatment and operation all to carry out to specifications.Add 9 ml distilled waters and dissolve the different abdomen wasp aptoxin in Sumatera, then carefully pour in dialysis tubing, tighten suitable for reading.The dialysis tubing installing is put into the beaker that fills distilled water, 4 ℃ of temperature environment shaking table concussion dialysed overnight, and constantly stirred.After 16 hours, get peritoneal effluent lyophilize, after the desalination of G-10 post, then freeze-drying, be the bulk drug of next step preparation use.Obtain 187 milligrams of Sumatera different abdomen wasp efficient parts.Through Forint phenol method inspection, in this efficient part, content of peptides is 76.4%.

The preparation of 3.3 Sumatera different abdomen wasp aptoxin polypeptide efficient part cataplasmas:

Cataplasma method processed: 5 grams of carbomers are dissolved 4 hours, add 200 grams of distilled waters, obtain A liquid; Take 150 grams of glycerine, put into 500 ml beakers, take successively each 1 gram of dihydroxyaluminum aminoacetate and EDTA, be successively dissolved in glycerine, stir, then add 25 grams of NP-700, mix, obtain B liquid; A, B are mixed to get to C liquid.Get 1 gram, tartrate, add 117.5 grams of the distilled waters even D of the obtaining liquid that is mixed.The gradation of D liquid is poured in C, and stir, obtain matrix E.In the matrix of appropriate amount, add the Sumatera of preparing in 3.2 different abdomen wasp aptoxin polypeptide efficient part to pharmacology embodiment requirement, stir.Make the final concentration (the cataplasma content that guarantees 1.0 centimetres of 1.0 cm x during mouse experiment is 0.75 milligram) that the needed unit surface of pharmacological evaluation requires.In the medicinal extraordinary insect exploitation of Dali College national local joint project research centre, GMP pilot plant is coated with into the cataplasma of 7 centimetres of 5 cm x with cataplasma machine.During use, be cut into 1.0 centimetres of big or small bulks of 1.0 cm x and be affixed on mouse brain or nose, and good with immobilization with adhesive tape.

pharmacology embodiment 1. the provide protection (prevention administration) that east different abdomen wasp aptoxin polypeptide efficient part liniment forms collagen and adrenalin mixture inducing mouse thrombus in vivo

1.1 experiment purposes:

By observing the provide protection of medicine to the mouse thrombus of collagen protein-suprarenin induction, its anti thrombotic action of preliminary assessment.After the quiet note collagen and adrenalin of mouse mixture mixing inductor, owing to adopting homogenate technical finesse collagen protein inductor, make collagen protein easily by pulmonary circulation, menses flow to the cerebrovascular and form thrombus, can there is rapidly the symptom of half side hemiplegia of limb in animal, death generally occurred in 5 minutes, after quiet injecting medical liquid, record respectively the time that mouse hemiplegia forms, time and the hemiplegia of mouse survival do not recovered number, the mouse and the dead mouse that surpass 15 minutes were all calculated with 15 minutes.

1.2 experiment materials:

1.2.1 laboratory animal: male mice in kunming, body weight: 18～22 grams.

1.2.2 experiment equipment: disposable syringe (specification: 1 milliliter), 100 microlitre liquid-transfering guns, gavage pin each with before boiling water scalding), stopwatch, 5 milliliters of homogenizers etc.

1.2.3 main agents:

1.2.3.1 positive drug:

Acetylsalicylic acid (enteric coated tablet, Bayer HealthCare Co, lot number: BJ10402);

Chlorine pyrroles thunder, another name Plavix (Sai Nuofei, lot number: 2A627);

Ligustrazine (injection, Tri-Lion Pharmaceutical Co., Ltd., Harbin, lot number: 121101A3);

Collagen protein (professional biochemical reagents supplier).

1.2.3.2 black shield wasp aptoxin polypeptide efficient part: by the national local joint project research 1.2.3.2 east of the medicinal extraordinary insect exploitation of Dali College different abdomen wasp aptoxin polypeptide efficient part: developing national local joint project research centre by the medicinal extraordinary insect of Dali College provides, and preparation method is shown in embodiment 1.

1.2.4 the configuration of main agents: the preparation of collagen protein-suprarenin mixture, reference ([1] Zhu Hong, Wu Qiang, Xu Ming, Wu Yingying, Hu Xiuping, Zou Yuhong, Yang Yan, the experimental study [J] of Radix Astragali total extract inside and outside anti thrombotic action, Chinese Clinical pharmacology and therapeutics, 2005,08:17-920; [2] Wang Yanqiu, Liu Xianli, Liu Dongying, Wu Rongjie, Zou Xiangyang, Hou Lin, the research of Codiumfragile(sur.) Hariot. polysaccharide anticoagulation and anti thrombotic action [J], Anhui medicine, 2011, the method in 07:804-806) completes.Facing the used time takes 45 milligrams of collagen proteins, be soaked in 3～4 ml physiological salines more than 2 hours, homogenate, 2000 revs/min centrifugal 10 minutes, get supernatant liquor; 1.8 milliliters, suprarenin separately getting 1 grams per liter, adds in supernatant liquor, adds physiological saline to 40 milliliter, makes 1.125 mg/ml collagen proteins and 0.045 mg/ml epinephrine solution.With normal saline dilution multiple proportional, be 1.5 ﹕ 1, inductor is the collagen protein 20.02 7 mg/ml epinephrine solutions of 0.67 mg/ml.Dosage after dilution is collagen protein 268 micrograms/only and suprarenin 10 micrograms, that is 0.4 milliliter/of tail vein injection.

1.2.5 the preparation of positive drug: convert according to human-animal's dosage, calculate the mouse dosage of positive drug, and the day before yesterday gastric infusion person is prepared to 4 ℃ of preservations in experiment with physiological saline.

1.2.6 the preparation of the different abdomen wasp aptoxin in east polypeptide efficient part liniment: liniment preparation method is referring to 1.3 parts in embodiment 1.Mouse dosage is 0.75 milligram/20 grams, each 30 microlitres, and the concentration that liniment need to be prepared is 0.75 milligram/30 microlitres.

1.2.7 the preparation of positive drug liniment: according to the same process described in this pharmacology embodiment 1.2.6, preparation positive drug acetylsalicylic acid liniment, Plavix liniment, Ligustrazine pin liniment.Wherein, acetylsalicylic acid liniment compound concentration is 1.6 milligrams/30 microlitres; Plavix liniment compound concentration is 0.16 milligram/30 microlitres; Ligustrazine pin liniment compound concentration is 1.1 milligrams/30 microlitres.

1.3 experiment groupings:

60 mouse (male Kunming kind) are divided into 10 groups, 6 every group at random.

1.3.1 general route of administration positive drug group (3 groups) (acetylsalicylic acid gastric infusion group, Plavix gastric infusion group, Ligustrazine intravenous administration group);

1.3.2 three kinds of positive drug liniment groups (3 groups) (acetylsalicylic acid liniment group, Plavix liniment group, Ligustrazine liniment group);

1.3.3 (matrix group, smears the blank matrix of liniment that does not add any medicine to negative control group (2 groups); Physiological saline group);

1.3.4 administration group: east different abdomen wasp aptoxin polypeptide efficient part liniment group (2 groups) (the imitative temple of brain film administration group, nose film administration group).

1.4 experimental techniques:

Matrix group, nose are filmed the imitative temple administration group of group, brain quantitatively according to 30 microlitres/only smear, and gavage and intravenous injection, by 0.2 milliliter/10 grams, according to the drug administration preparing above, give seven days continuously, every one day, claim a body weight, mouse normally drinks water, diet.After last administration, 1 hour each group is from tail vein injection platelet aggregation collagen protein-suprarenin mixing solutions (0.4 milliliter /).Observe in 5 minutes dead mouse number and in 15 minutes mouse hemiplegia do not recover number, calculate the time of recovery of medicine to the protection ratio of mouse brain thrombus and mouse hemiplegia.

1.5 experimental results:

The impact experiment that the present embodiment Chinese traditional medicine forms collagen and adrenalin mixture inducing mouse thrombus in vivo belongs to prevention administration, after mouse tail vein injection inductor, all there is accelerated breathing, restless, ophthalmoptosis, turn white in physiological saline, matrix group, rapidly dead subsequently, mortality ratio is for very; Administration group, positive drug group, be short of breath after mouse induction mild or rapid to dead.With physiological saline group, matrix group comparison; east 2 liniment groups of different abdomen wasp aptoxin polypeptide efficient part all obviously reduce mouse death rate, and film administration group and the imitative temple administration group of brain of the prompting different abdomen in east black shield wasp aptoxin polypeptide efficient part nose is formed with significant protective effect to mouse thrombus.Experimental result is in Table 1.

The effect (prevention administration) of each experimental group of table 1 to collagen and adrenalin mixture induction thrombus dead mouse and recovery

1.6 experimental result explanations:

The quiet note collagen and adrenalin of mouse mixture mixing inductor makes collagen protein by pulmonary circulation; menses flow to the cerebrovascular and form thrombus; there is rapidly the symptom of half side hemiplegia of limb in animal; by observing the provide protection of medicine to the mouse thrombus of collagen protein-suprarenin induction, can its anti thrombotic action of preliminary assessment.In this pharmacology embodiment, east different abdomen wasp aptoxin polypeptide efficient part nose is filmed and is imitated temple administration group administration group and the imitative temple administration group of filming of filming, and the anti-mouse brain thrombus effect showing is better than Plavix liniment administration group, acetylsalicylic acid liniment administration group, Ligustrazine liniment administration group; It is suitable with a line medication Plavix gastric infusion group to cerebral thrombosis mouse hemiplegia restitution.The present embodiment has illustrated that east different abdomen wasp aptoxin polypeptide efficient part and transdermal absorption formulation thereof have quite potent anti-thrombosis ability, can play splendid protection and prophylactic effect to the formation of cardiovascular and cerebrovascular thrombus.

To the quiet note collagen and adrenalin of mouse mixture mixing inductor, thrombosed provide protection experiment is the important evaluation index of the universally recognized evaluation medicine of pharmacology educational circles to cerebral thrombosis disease preventive and therapeutic effect to medicine.By above-mentioned east different abdomen wasp aptoxin polypeptide efficient part and transdermal absorption formulation experimental result thereof, can be found out, different abdomen Vespa bee venom and relevant transdermal absorption formulation thereof are expected to become the medicine of new anti-cerebral thrombosis disease.

pharmacology embodiment 2: the provide protection (treatment administration) that east different abdomen wasp aptoxin polypeptide efficient part liniment forms collagen and adrenalin mixture inducing mouse thrombus in vivo

2.1 experiment purposes:

After inductor inducing mouse thrombus in vivo being formed by observation, treat again the experimental result of administration, observe the provide protection of medicine to the mouse thrombus of collagen protein-suprarenin induction, further evaluate its anti thrombotic action.Experiment mechanism is with pharmacology embodiment 1.1 parts.

2.2 experiment materials:

2.2.1 laboratory animal: male mouse of kunming, body weight: 18～22 grams.

2.2.2 experiment equipment: disposable syringe (specification: 1 milliliter), 100 microlitre liquid-transfering guns, gavage pin each with before boiling water scalding), stopwatch, 5 milliliters of homogenizers etc.

2.2.3 main agents:

2.2.3.1 positive drug:

Acetylsalicylic acid (enteric coated tablet, Bayer HealthCare Co, lot number: BJ10402);

Chlorine pyrroles thunder, another name Plavix (Sai Nuofei, lot number: 2A627);

Ligustrazine (injection, Tri-Lion Pharmaceutical Co., Ltd., Harbin, lot number: 121101A3);

Collagen protein (professional biochemical reagents supplier).

2.2.3.2 the different abdomen wasp aptoxin in east polypeptide efficient part (provided by the medicinal extraordinary insect exploitation of Dali College national local joint project research centre, preparation method is shown in embodiment 1).

2.2.4 the configuration of main agents: the preparation of collagen protein-suprarenin mixture, with pharmacology embodiment 1.

2.2.5 the preparation of positive drug: with pharmacology embodiment 1.According to human-animal's dosage, convert, calculate the mouse dosage of positive drug, and in experiment, the day before yesterday gastric infusion person is prepared to 4 ℃ of preservations with physiological saline.

2.2.6 the preparation of the different abdomen wasp aptoxin in east polypeptide efficient part liniment: with pharmacology embodiment 1.Liniment preparation method is referring to 1.3 parts in embodiment 1.Mouse dosage is 0.75 milligram/20 grams, each 30 microlitres, and the concentration that liniment need to be prepared is 0.75 milligram/30 microlitres.

2.3 experiment groupings:

48 mouse (hero) are divided into 8 groups at random, 6 every group.

2.3.1 general route of administration positive drug group (3 groups) (acetylsalicylic acid gastric infusion group, Plavix gastric infusion group, Ligustrazine intravenous administration group);

2.3.2 the results suggest of pharmacological evaluation 1, three kinds of positive drug linimentes (acetylsalicylic acid liniment, Plavix liniment, Ligustrazine liniment) do not have remarkable therapeutic action, because of revision test no longer in pharmacology the present embodiment.

2.3.3 (matrix group, smears the blank matrix of liniment that does not add any medicine for model group and 2 groups of negative control group; Physiological saline group);

2.3.4 administration group: east different abdomen wasp aptoxin polypeptide efficient part liniment group (2 groups) (the imitative temple of brain film administration group, nose film administration group).

2.4 experimental techniques:

First from mouse tail vein injectable collagen albumen and the adrenergic inductor system of mixing, 0.4 milliliter/only.After injection, mouse produces the cerebral thrombosis symptom of hemiplegia of limb immediately.Inject latter 1 minute, administration immediately, observe in 5 minutes dead mouse number and in 15 minutes mouse hemiplegia do not recover number.Calculate the protection ratio of medicine to mouse brain thrombus.For confirming whether success of model, the toxicity of the blank matrix of judgement liniment to mouse, for the mouse of physiological saline group and matrix group, give 0.4 milliliter of tail vein injection saline/only, inject and give respectively physiological saline and the blank matrix of liniment for latter 1 minute.Model group induction thrombosis, refuses administration.

2.5 experimental results: the impact that medicine forms collagen and adrenalin mixture inducing mouse thrombus in vivo

The impact that the present embodiment Chinese traditional medicine forms collagen and adrenalin mixture inducing mouse thrombus in vivo belongs to treatment administration.Hemiplegia occurs in very short time after mouse tail vein injection inductor, and expiratory dyspnea, ophthalmoptosis turn white, and mouse is dead rapidly subsequently.With model group comparison, the different abdomen wasp aptoxin polypeptide efficient part nose imitative temple administration group of administration group and brain of filming in east increases significantly to the survival rate of thrombus mouse.Experimental result is in Table 2.

The effect (treatment administration) of each experimental group of table 2 to collagen and adrenalin mixture induction thrombus dead mouse and recovery

2.6 experimental result explanations:

To the quiet note collagen and adrenalin of mouse mixture mixing inductor, thrombosed provide protection experiment is the important evaluation index of the universally recognized evaluation medicine of pharmacology educational circles to cerebral thrombosis disease preventive and therapeutic effect to medicine.After modeling formation hemiplegia, the treatment experiment (survival rate) of trial drug to mouse again, can more effectively evaluate tested medicine for the treatment ability of clinical common acute cerebral thrombosis.As can be seen from Table 2, the imitative temple of administration group and the brain administration of filming of filming of the different abdomen wasp aptoxin in east polypeptide efficient part nose shows the effect of unexpected mouse brain thrombus injury protection.The action intensity of the death that the administration group treatment chmice acute cerebral thrombosis of filming its nose causes is better than a line medication Plavix, acetylsalicylic acid and Ligustrazine.Demonstrate east different abdomen wasp aptoxin polypeptide efficient part and transdermal absorption formulation thereof (brain is imitated temple liniment) and there is quite potent anti-thrombosis ability, can play splendid protection, preventive and therapeutic action to the formation of cardiovascular and cerebrovascular thrombus.

Experimental result by above-mentioned east different abdomen wasp aptoxin polypeptide efficient part and transdermal absorption formulation thereof is found out again, the medicine that different abdomen Vespa bee venom and relevant transdermal absorption formulation thereof are expected to become new anti-cerebral thrombosis disease and prevent and treat cardiovascular and cerebrovascular major disease.

pharmacology embodiment 3: the different abdomen wasp aptoxin of mole polypeptide efficient part hydrogel adhesive is on the brain Protection of cerebral anoxia mouse (medicine on decapitated mice pant the impact of time)

3.1 experiment purposes:

By observing the breathing prolongation effect of medicine to decapitated mice, its protection of brain to cerebral anoxia animal effect of preliminary assessment.

3.2 experiment materials:

3.2.1 laboratory animal: Kunming mouse, male, body weight: 18～22 grams.

3.2.2 experiment equipment: 8 of instruments, stopwatch etc.

3.2.3 main agents:

3.2.3.1 positive drug:

Acetylsalicylic acid (enteric coated tablet, Bayer HealthCare Co, lot number: BJ10402);

Ligustrazine (injection, Tri-Lion Pharmaceutical Co., Ltd., Harbin, lot number: 121101A3).

3.2.3.2 the different abdomen wasp aptoxin of mole polypeptide efficient part hydrogel adhesive, provides (preparation method is shown in embodiment 2) by the medicinal extraordinary insect exploitation of Dali College national local joint project research centre.

3.2.3.3 the preparation of positive drug: with pharmacology embodiment 1.

3.2.2.4 the preparation of the different abdomen wasp aptoxin of mole polypeptide efficient part hydrogel adhesive: hydrogel adhesive preparation method is referring to 2.3 parts in embodiment 2.The hydrogel adhesive final concentration being mixed with is 0.75 milligram/30 microlitres.

3.3 experiment groupings:

30 mouse (hero) are divided into 6 groups at random, 5 every group.

3.3.1 general route of administration positive drug group (2 groups) (acetylsalicylic acid gastric infusion group, Ligustrazine intravenous administration group);

3.3.2 (hydrogel matrix group, does not add the blank matrix of the making aqueogel of any medicine to negative control group (2 groups); Physiological saline group);

3.3.3 administration group: the different abdomen wasp aptoxin of mole polypeptide efficient part hydrogel adhesive group (2 groups) (brain is imitated temple hydrogel administration group, nose hydrogel administration group).Use is when mouse brain or nose, and mouse dosage is 0.75 milligram/20 grams, each 30 microlitres.

3.4 experimental techniques:

After last administration 1 hour, from mouse ear back root part, break end fast, record the time s(second of panting after mouse broken end) and mouth breathing frequency n (inferior).

3.5 experimental results: in Table 3.

The brain Protection of the different abdomen wasp aptoxin of table 3 mole polypeptide efficient part hydrogel adhesive to cerebral anoxia mouse

Note: with the comparison of physiological saline group, * * P<0.01, * P<0.05

3.6 experimental result explanations:

As seen from Table 3, with the comparison of physiological saline group, two positive drug groups, the two groups of different abdomen wasp aptoxin of mole polypeptide efficient part hydrogel adhesive groups (brain is imitated temple hydrogel administration group, nose hydrogel administration group) all have significant difference (* P<0.05, * P<0.01), illustrate that the different abdomen wasp aptoxin of pharmacologic nevus polypeptide efficient part hydrogel adhesive can increase mouse broken end frequency of respiration (during cerebral anoxia, mouse has certain resistivity).With matrix group comparison, the two groups of different abdomen wasp aptoxin of mole polypeptide efficient part hydrogel adhesive groups can effectively extend decapitated mice pants the time, illustrates that administration group mouse has certain hypoxia-bearing capability.Experimental result shows that different abdomen Vespa bee venom polypeptide efficient part has certain provide protection for ischemic-hypoxic brain injury.

pharmacology embodiment 4: the experiment of Sumatera different abdomen wasp aptoxin polypeptide efficient part cataplasma platelet aggregation-against

4.1 experiment purposes:

Inspection Sumatera different abdomen wasp aptoxin polypeptide efficient part, as the potential quality of anti-heart and brain thrombus medicine, is developed anti-heart and brain thrombus medicine TTS preparation for next step scientific basis is provided.

4.2 experiment materials:

4.2.1 laboratory animal: healthy Male Kunming strain mice, purchased from Dali College Experimental Animal Center, 18～22 grams of body weight.

4.2.2 experiment equipment: stopwatch, trace blood kapillary (internal diameter is 1 millimeter, and length is 10 centimetres), mouse stomach syringe needle, 1 milliliter of syringe, EP pipe, 200 microlitre micropipets, rifle are first-class.

4.2.3 experiment reagent:

4.2.3.1 the different abdomen wasp aptoxin in Sumatera polypeptide efficient part cataplasma: provide (preparing according to embodiment 3) by the national local United Engineering Center of the medicinal extraordinary insect exploitation of Dali College;

4.2.3.2 physiological saline (Kelun Pharm Ind Co., Ltd., Sichuan, lot number: N12102201-2);

4.2.3.3 Aspirin Enteric-coated Tablets (Bayer HealthCare Co, lot number: BJ10402);

4.2.3.4 urokinase injection (Biochemistry Tianjin Pharmaceutical Co, lot number: 20120403);

4.2.3.5 XUESAITONG PIAN (the Kunming Jin Tai get of pharmacy group medicine company limited-liability company, lot number: 130202).

4.3 experimental techniques:

4.3.1 reagent preparation

4.3.1.1 the preparation of positive drug etc.: Aspirin Enteric-coated Tablets (80 mgs/kg) and XUESAITONG PIAN (90 mgs/kg) are prepared with physiological saline in experiment the day before yesterday, 4 ℃ of preservations; Urokinase injection (100000U/ kilogram), experiment is made into 10000U/ milliliter with physiological saline, 4 ℃ of preservations the day before yesterday; Get before use 20 normal saline dilution to 200 microlitres for microlitre mother liquor; 4.3.1.2 the preparation of the different abdomen wasp aptoxin in Sumatera polypeptide efficient part cataplasma: in experiment fresh preparation the day before yesterday, 4 ℃ save backup.According to embodiment 2(2.4 part) and embodiment 4(4.3 part) described method makes a golden yellow tiger honeybee and ink chest wasp aptoxin polypeptide efficient part cataplasma respectively the final concentration (the cataplasma content that every 1.0 cm x is 1.0 centimetres is 0.75 milligram) that the needed unit surface of pharmacological evaluation requires.The bulk that is cut into 1.0 centimetres of 1.0 cm x during use is affixed on mouse brain or nose transdermal administration, and good with immobilization with adhesive tape.

4.3.2 administration and measuring method

4.3.2.1 grouping and administration: 42 male mices are divided into 7 groups, every group 6 at random, is respectively Sumatera different abdomen wasp aptoxin polypeptide efficient part cataplasma nose and pastes administration group (M1), Sumatera different abdomen wasp aptoxin polypeptide efficient part cataplasma temple administration groups (M2), Aspirin Enteric-coated Tablets group (sun 1), urokinase group (sun 2), XUESAITONG PIAN group (sun 3), catablasm base material control group (to 1), solvent control group (to 2).The next day of mouse, weigh once, and the amount of keeping on a diet.M1 group, M2 group is all 0.75 milligram of the cataplasma of 1.0 centimetres of every 1.0 cm x to cataplasma content of dispersion, and the bulk of 1.0 centimetres of 1.0 cm x shearing is affixed on to mouse brain or nose transdermal administration, and good with immobilization with adhesive tape.1 group, positive 3 groups of sun, all take gastric infusion to 2 groups, by 0.2 milliliter/10 grams administrations.To 1, give the catablasm base material (not pastille) of 1.0 centimetres of 1.0 cm x.Take above method successive administration seven days.Within the 8th day, with capillary tube technique, survey each treated animal clotting time.

4.3.2.2 capillary tube technique is measured the impact of medicine on the clotting time outside normal Mice Body: with glass capillary, in mouse intraocular corner of the eyes ball rear vein beard, get blood, autoblood flows into beginning timing in kapillary, blood lies against on desktop after filling with kapillary, one section of kapillary fractureed every 20 seconds, and slowly to the left and right both sides pull open, whether the observation place of fractureing has the solidifying silk of blood, observes to till having the solidifying silk of blood to occur.The blood of usining flows in glass capillary to occurring the clotting time of the interval time of the solidifying silk of blood as mouse.

4.4 experimental results:

As can be seen from Table 3, the result recording with capillary tube technique is: compare with solvent control group with matrix control group, positive drug group, Sumatera different abdomen wasp aptoxin polypeptide efficient part cataplasma nose paste administration and all there is significant difference (* P<0.05, * * P<0.05) in the measured external clotting time of cataplasma temple administration; Compare with three positive drug groups, Sumatera different abdomen wasp aptoxin polypeptide efficient part cataplasma nose pastes administration and the measured external clotting time of cataplasma temple administration is also significantly greater than positive drug (P<0.05).Can find out, the blood coagulation resisting function that different abdomen Vespa Sumatera different abdomen wasp aptoxin polypeptide efficient part cataplasma nose pastes administration and the administration of cataplasma temple is all better than an existing line medication greatly; Can expect that this genus wasp aptoxin polypeptide efficient part is developed to anticoagulation, antithrombotic reagent and transdermal absorption formulation, for preventing and treating cardiovascular and cerebrovascular ischemic disease.

Table 4 Sumatera different abdomen wasp aptoxin polypeptide efficient part cataplasma and control group are on the Mice Body impact in outer clotting time

(* * P<0.01, * P<0.05; With model group comparison) ( ^△p<0.05; With positive drug comparison)

4.5 presentation of results:

The change of lesion vessels structure is thrombotic prerequisite, thrombocyte and white corpuscle in its activation or attraction blood flow, make it to adhere to, be gathered in lesion, these cells that adhere to, assemble can discharge various active material, further increase the weight of vascular lesion and raise more cell, causing thrombosis.That is to say, in thrombosis, there is the mutual promoting action of reciprocal causation in endotheliocyte, thrombocyte, white corpuscle, forms vicious cycle.Platelet activation gathering and thrombosis and development are closely related, and platelet activation can cause thrombosis after assembling, and vasospasm, affects blood circulation, produce ischemic or block, thereby promoting cardiovascular and generation cerebrovascular disease.Therefore the impact that drugs is assembled platelet activation, for preventing that thrombotic diseases and exploitation control cardio-cerebralvascular diseases original new drug are most important.

In this experiment, according to the result recording, we find that Sumatera different abdomen wasp aptoxin polypeptide efficient part cataplasma nose pastes the external clotting time and positive and negative control group comparison of administration and temple administration, all has significant difference.Accordingly, the different abdomen wasp aptoxin in medicine to be measured Sumatera polypeptide efficient part has extremely significant platelet aggregation-against and anti-thrombosis function.Illustrate that different abdomen Vespa bee venom and efficient part thereof are worth further researching and developing into the novel biochemical medicine of anti-platelet aggregation, anti-thrombus of heart blood vessel, resisting cerebrovascular thrombus, control ischemic cardio cerebrovascular diseases very much.

When above-mentioned specification sheets elaboration is of the present invention, the object that embodiment and pharmacology embodiment are provided is simultaneously to illustrate actual mechanical process of the present invention and meaning of the present invention.In the time of within the scope of entering the claims in the present invention and its equivalent, practical application of the present invention comprises all general variations, cooperation, or improves.

Claims (6)

1. a bee venom polypeptide efficient part, is characterized in that: this efficient part is to obtain through dialysis membrane dialysis from the thick bee venom of the different abdomen Vespa of Hymenoptera Vespoidea insect; Wherein the molecular weight cut-off of dialysis membrane is 25KDa.

2. a preparation method who prepares the bee venom polypeptide efficient part described in right 1, it is characterized in that: dissolve the thick bee venom of wasp of collecting, pour in dialysis tubing, tighten suitable for readingly, the dialysis tubing installing is put into the beaker that fills distilled water, shaking table concussion dialysis, stir, after 12-24 hour, get peritoneal effluent dry, desalination, then be drying to obtain; Wherein dialysis tubing refers to that molecular weight cut-off is the dialysis tubing of 25KDa, is dried and refers to that lyophilize or concentrating under reduced pressure are dried, and the thick bee venom of wasp refers to the bee venom in the different abdomen Vespa of Hymenoptera Vespoidea insect body.

3. the arbitrary described bee venom polypeptide efficient part of claim 1-2, for the preparation of the purposes of control cardiovascular and cerebrovascular diseases medicament, is characterized in that: described cardiovascular and cerebrovascular diseases refers to ischemic cerebrovascular, ischemic cardiovascular.

4. according to the purposes of claim 3, it is characterized in that described cardiovascular and cerebrovascular diseases refers to heart stalk, thrombus of heart blood vessel, cerebral thrombosis, cerebral infarction, urgency/chronic cerebral apoplexy and/or cerebral infarction injury sequela; Wherein cerebral infarction injury sequela refers to lateral deviation paralysis, hemianesthesia, hemianopsia, face tongue paralysis, aphasia, total paralysis.

5. according to the purposes of claim 3, it is characterized in that: described purposes is embodied by pharmaceutical preparation, medicine equipment, cosmetics of everyday use form; The form of described pharmaceutical preparation, medicine equipment, daily chemical products is hydrogel, frothy gel, liniment, cataplasma, lyophilized powder, aqua, aerosol, suppository, externally-applied liniment, ointment.

6. a pharmaceutical composition with control cardiovascular and cerebrovascular diseases effect, is characterized in that: this pharmaceutical composition contain treatment significant quantity according to bee venom polypeptide efficient part, pharmaceutical excipient, pharmaceutical carrier or the dressing of claim 1.