CN101564388B

CN101564388B - Composition for Danshensu and danshinolic acid B and use thereof

Info

Publication number: CN101564388B
Application number: CN2009101192008A
Authority: CN
Inventors: 魏峰
Original assignee: BEIJING HUANJING HONGFANG BIOTECHNOLOGY Co Ltd
Current assignee: Harbin Pharmaceutical Group Sanjing Nuojie Prarmaceutical Co., Ltd.
Priority date: 2008-04-21
Filing date: 2009-03-09
Publication date: 2012-03-14
Anticipated expiration: 2029-03-09
Also published as: CN101564388A

Abstract

The invention provides a medicament composite using Danshensu and danshinolic acid B as active ingredient. Purification of Danshensu and danshinolic acid B or purification of Danshensu salt and danshinolic acid B salt is larger than or equal to 80%. The medicament composition can be used for treating coronary disease, angina, ischemic cerebral, atherosclerosis, myocardial ischemia or myocardial infarction, chronic heptic B fibrosis, fatty liver, senile dementia, cancer, or the like.

Description

The composition and use thereof of danshensu and salvianolic acid B

Technical field

The present invention relates to a kind of is the pharmaceutical composition of active component and at the pharmacology activity research of treating cardiac and cerebral vascular diseases with danshensu and salvianolic acid B.

Background technology

Radix Salviae Miltiorrhizae is a conventional Chinese medicine, and the beginning is stated from Shennong's Herbal, classifies as top grade.Chinese Pharmacopoeia (version in 2005) regulation Radix Salviae Miltiorrhizae is the dry root and rhizome of labiate Radix Salviae Miltiorrhizae Salvia miltiorrhiza Bge..Radix Salviae Miltiorrhizae has stasis-dispelling and pain-killing, promoting blood circulation to restore menstrual flow, and the functions such as relieving restlessness that clear away heart-fire are used for menoxenia, amenorrhea dysmenorrhea ， lumps in the chest and abdomen, breast ventral spine pain, pyretic arthralgia pain, skin infection swells and ache, dysphoria and insomnia, hepatosplenomegaly, angina pectoris.

Danshensu and salvianolic acid B are the main active ingredients in the Radix Salviae Miltiorrhizae, have many-sided pharmacological action, and the two has identical pharmacological action, and different pharmacological actions is also arranged.Because the two molecular structure differs greatly, so the difference of pharmacological action is inevitable; Even identical pharmacological action, its mechanism of action also possibly be different.The two is combined, can play mutually remedying, the effect of learning from other's strong points to offset one's weaknesses makes drug effect better.

The chemical constitution of danshensu

The chemical constitution of salvianolic acid B

With danshensu and salvianolic acid B be active component pharmaceutical composition and pharmacologically active thereof or the application aspect the treatment disease do not appear in the newspapers to.The invention discloses a kind of is the pharmaceutical composition of active component with danshensu and salvianolic acid B, and provides pharmacological evaluation to prove the pharmacologically active of this pharmaceutical composition; Pharmacological evaluation shows that the pharmacologically active of danshensu and salvianolic acid B compositions is better than the pharmacologically active of danshensu and the pharmacologically active of salvianolic acid B, and significant difference is arranged.

The pharmacological action of salvianolic acid B

Folium Ginkgo extract EGb761 has become one of best-selling health product on the American market, and some and old and feeble relevant disease are treated as prescription drugs by some countries in Europe, like AD (presenile dementia).Institute of Materia Medica,Chinese Academy of Medical Sciences professor Zhang Juntian compares the pharmacological action of salvianolic acid B and EGb761; No matter be antioxidation, remove free radical, a variety of causes and cause protection and anti-apoptotic of cell injury or the like; The effective dose of salvianolic acid B is 0.007～0.7ug/ml, and EGb761 is 50～100ug/ml, differs 100～1000 times; The two all shows treating cerebral ischemia at the MCAO model, and the effective dose of salvianolic acid B is lower tens times more than than EGb761.Salvianolic acid B is better than EGb761 to the inhibitory action of β-AP neurotoxicity (main diseases of AD because of), also is better than melatonin.Salvianolic acid B has so good activity possibly come from 2 points: strong anti-oxidation activity and calcium antagonism.The antioxidant activity of salvianolic acid B is vitamin E, mannitol and EGb761 100 times.The too high generation with free radical of intracellular calcium concentration too much is the key factor of neuronal damage, death; Salvianolic acid B is a powerful antioxidant, and can eliminate intracellular calcium overload, and it is developed to the treatment medicine for senile dementia, has important significance for theories and use value undoubtedly.Can think that salvianolic acid B is that after arteannuin, Radix Ginseng another has Chinese medicine [Chinese pharmacology communication, 2006 of international influence; 23 (4): the 12 national neuropsychopharmacology seminar plenary lecture.Acta Pharmaceutica Sinica, 2006; 41 (8): 706～711.The medicine Leader, 2007; 26 (2): 107～110].

One, protection cerebrovascular

(1) ferrum dependency lipid peroxidation (IDLPO) plays important role in the incidence and development process of brain injury.Salvianolic acid B is influential to rat cerebral even slurry ferrum dependency lipid peroxidation, can suppress the generation of rat cerebral even slurry malonaldehyde, is dose-effect relationship, suggesting effect mechanism relevant with the ferrum sequestration (Chinese gerontology magazine, June the 17th in 1997 rolled up).(2) glutamic acid (Glu) and amyloid-beta [A β _1-40] can cause that the release of neuron NO increases, cause neuronic damage, produce neurotoxicity; Cell viability when salvianolic acid B can significantly increase neural cell injury reduces lactic acid dehydrogenase (LDH) release rate, reduces the release of NO; Reach the effect of neuroprotective cell; Be expected to treat senile dementia [Acta Pharmaceutica Sinica, 2000,35 (12): 881～885].(3) mitochondrion is the main source of supply organism self-energy, and the caused energy battier of cerebral ischemia re-pouring is main relevant with mitochondrial function damage.Salvianolic acid B has good antioxidation, can dwindle stalk match area behind the focal cerebral ischemia.Salvianolic acid B can obviously suppress active decline of SOD and the decline of GSH content in the caused brain mitochondria of cerebral ischemia re-pouring, reduces mitochondrial injury simultaneously, the effect of performance protection brain function (Chinese pharmacology communication, 2003 the 20 volume first phases).(4) VEGF (VEGF) can cause that cerebrovascular permeability raises, and salvianolic acid B can significantly suppress the blood vessel permeation of VEGF, points out it that cerebrovascular is had certain protection effect (PLA's Acta Pharmaceutica Sinica, the 18th the 1st phase of volume of February in 2002).

Two, the liver protecting

(1) salvianolic acid B can promote hepatocyte growth; The collagenation that suppresses cell; The hypertrophy that can suppress In vitro culture HSC (hepatic stellate cell); This inhibitory action and antioxidation have certain relation, and the prompting salvianolic acid B has certain effect of anti hepatic fibrosis [Chinese hepatopathy magazine, the 4th the 4th phase of volume of December in 1996; World Chinese digests magazine, and 2002,10 (3): 317～319].(2) the part mechanism of salvianolic acid B anti-hepatic fibrosis is to have suppressed activation fat-storing cell propagation; And through suppress fat-storing cell cellulation epimatrix reduced collagen fiber in liver deposition and disturbed the formation of Di Shi gap basement membrane to have alleviated sinus hepaticus hair cell vascularization (Chinese hepatopathy magazine, the 4th the 2nd phase of volume of June in 1996).(3) salvianolic acid B has its PGI of promotion for the liver adherent cell ₂The effect that generates then is inhibitory action for the cyclo-oxygenase in the liver adherent cell homogenate, maybe be relevant as the effect of tryptophan appearance cofactor with salvianolic acid B.PGI ₂Microcirculating state to hepatic sinusoid has regulating action, and the inhibition of cyclo-oxygenase is relevant with anti-inflammatory effect, and the mechanism (CHINA JOURNAL OF CHINESE MATERIA MEDICA, 1994 the 19th the 2nd phases of volume) of salvianolic acid B anti-liver injury can partly be described.(4) the former primary cultures of rat of the external damage of carbon tetrachloride hepatocellular [ ³H] the thymidine incorporation significantly reduces, and the ALT activity significantly increases in the culture fluid, the salvianolic acid B this pathological change of inhibitory cell effectively (CHINA JOURNAL OF CHINESE MATERIA MEDICA, 1997 the 22nd the 5th phases of volume).(5) on the person, test and show that salvianolic acid B can effectively reverse chb hepatic fibrosis (histology's reversion rate reaches 36.67%), to the improvement of serum HA content, the comprehensive decline of 4 fibrosis serological index and B ultrasonic integration change and are superior to IFN-r.Salvianolic acid B and IFN-r all can improve chb patient symptom and sign and liver function; Do not find the clinical adverse (medical research communication, 2003 the 32nd the 2nd phases of volume) of salvianolic acid B.(6) salvianolic acid B Chinese People's Anti-Japanese Military and Political College Mus D-galactosamine hepatic injury can significantly reduce serum alanine aminotransferase (ALT), Aspartic Acid aminotransferase (AST) activity, alleviates hepatic necrosis.Improve liver non-parenchymal cell arachidonic acid metabolite 6-ketone group-PGF ₁A (6-keto-PGF ₁A), PGD ₂(PGD ₂) and the total growing amount of prostaglandin.Show that salvianolic acid B possibly be the main component of Radix Salviae Miltiorrhizae anti-liver injury, it improves the total growing amount of liver non-parenchymal cell prostaglandin (PGs) possibly be one of mechanism of its anti-liver injury (Chinese combination of Chinese and Western medicine magazine, 1993 the 13rd the 6th phases of volume).

Three, protection cardiovascular

1, atherosclerosis

(1) VEGF (VEGF) can strengthen the permeability of vascular endothelial cell, possibly in the formation and development process of atherosclerosis (AS), play a role.Salvianolic acid B raises to the inductive endothelial permeability of VEGF has significant inhibitory effect, and the prompting salvianolic acid B has therapeutical effect [Chinese J Pharmacol Toxicol, 2003,17 (1): 19～23] to AS.(2) foam cell is the characteristic pathological cells that occurs in atherosclerosis (AS) speckle; In AS speckle tissue, the expression of VEGF obviously raises, and in the U937 of In vitro culture foam cell model, finds; Salvianolic acid B can be the expression of the inhibition foam cell VEGF of dose dependent; For prevention and the treatment of salvianolic acid B clinical practice in AS provides further according to [Acta Pharmaceutica Sinica, 2002,37 (2): 86～89].(3) salvianolic acid B to tumor necrosis factor a (the rat CMEC (CMEC) of the damage of TNF-a) has direct protective action, its effect maybe with its stabilizing cell membrane, anti peroxidation of lipid relevant [the Tianjin traditional Chinese medical science, 2002; 19 (6): 38～39].

2, myocardial ischemia-reperfusion

(1) myocardial ischemia-reperfusion can produce major injury to myocardial cell, brings out arrhythmia, and severe patient can cause death.The damage of myocardial ischemia-reperfusion property can produce a large amount of oxygen-derived free radicals, and then causes the lipid peroxidation of myocardial cell membrane, and this peroxidation is one of major reason that causes the myocardial cell reperfusion injury.Salvianolic acid B has very strong antioxidation, can remove oxygen-derived free radicals, so myocardial ischemia reperfusion injury is had protective effect (Chinese Pharmaceutical Journal, 2003 the 38th the 8th phases of volume; Traditional Chinese Medicinal College of Liaoning's journal, 2004 the 6th the 1st phases of volume).(2) also have experiment to show that it is one of main reason that causes myocardial ischemia reperfusion injury that myocardial cell calcium overload and oxygen-derived free radicals increase, salvianolic acid B can suppress the Ca in the myocardial cell anoxia reoxygenation process ²⁺Overload is one of its mechanism of bringing into play the myocardial ischemia reperfusion injury myocardium protecting action [modern combination of Chinese and Western medicine magazine, 2004; 13 (1)].At first and the position of easy damaged, therefore, the protective effect of CMEC research had crucial meaning in the control myocardial ischemia disease when (3) heart microvascular endothelial cell (CMEC) was the heart ischemia anoxia.The salvianolic acid B pretreatment can activate CMEC endogenous delay protection mechanism, has brought into play antagonism myocardial ischemia-anoxemia damage effect [the Tianjin traditional Chinese medical science, 2002; 19 (1): 41～42].(4) salvianolic acid B can improve the endothelial cell damage that the anoxia reoxygenation causes, increases the release of endothelial cell nitric oxide.Salvianolic acid B improves the synthetic this mechanism of nitric oxide possibly improve relevant [the Acta Pharmacol Sin 2002 of endotheliocyte anoxia reoxygenation injury with it; 23 (10): 930～936].

3, myocardial infarction

(1) salvianolic acid B has therapeutical effect (Chinese patent medicine, 2004 the 26th the 3rd phases of volume) to dog myocardial infarction.(2) salvianolic acid B is influential to pathomorphology in the rat heart muscle infarction process; Can reduce myocardial infarction area, promote the growth of blood capillary, quicken the reparation of infarction kitchen range; What it reduced the myocardial infarction kitchen range possibly mechanism be to improve the content of SOD in the cell; Reduce the toxic action of radical pair myocardial cell, salvianolic acid B is a kind of effective anti-oxidants (Tianjin College of Traditional Chinese Medicine journal, 2004 the 23rd the 1st phase of volume).

4, hemorheology

Basis and clinical studies show, ischemic cardio cerebrovascular diseases blood samples of patients viscosity raises, and blood viscosity lowering is one of important means of control ischemic cardio cerebrovascular diseases.Salvianolic acid B has tangible reduction effect (microcirculation is learned magazine, 2003 01 phases) to rabbit WBV, packed cell volume and aggregate index.

The pharmacological action of danshensu

One, study of anti-atherogenic effect

(1) Radix Salviae Miltiorrhizae have the synthetic effect of cell endogenous cholesterol (Ch) that suppresses.It has reduction cholesterolemia (Ch), protection blood vessel barrier, prevents the effect that lipidosis and atherosclerosis (AS) speckle form.In addition, danshensu also has the effect of lipotropism protein oxidation, thereby can prevent damage [Chinese herbal medicine, 1991 of oxidation lipoprotein pair cell; 22 (1): 20～23].(2) in recent years, the oxidative modification lipoprotein theory of atherosclerosis generation receives countries in the world scholar's extensive concern.The danshensu that this experiment is found has the effect that suppresses the cellular oxidation modified LDL, for danshensu is further used for atherosclerotic control theoretical foundation [Nanjing Medical University's journal, 1994 is provided; 14 (4): 529～531].(3) danshensu can effectively protect body to avoid the nitrite anions damage, prevents atherosclerotic formation; Danshensu has the effect that suppresses the cellular oxidation modified LDL, and the amount of its oxidation resistance and danshensu is proportionate, and the prompting danshensu can be used for atherosclerotic control, and [contemporary Chinese is used pharmaceutical journal, 2002; 19 (2): 132～134].

Two, hepatoprotective effect

(1) hepatic fibrosis has tangible preventive and therapeutic effect to danshensu to rat immunity property.Main mechanism is that danshensu is to HSC (hepatic stellate cell) tangible inhibited proliferation [Chinese hepatopathy magazine, 2003; 11 (5): 288～290].(2) potassium cyanide causes hepatocyte injury and the free radical toxicity effect is closely related, and the effect that danshensu has stabilizing cell membrane, removes free radical is so have protective effect [combination of Chinese and Western medicine hepatopathy magazine, 1996 to experimental hepatocyte injury; 6 (3): 29～32].(3) danshensu can direct cell cultured supernatant be bred, and is dose-dependence; D-galactosamine is caused hepatocyte injury to danshensu and necrosis also has protective effect [Nanjing Medical University's journal, 1996; 16 (4): 346～348].(4) endotoxin can directly cause hepatocyte injury, and danshensu can pass through enhance SOD vigor, reduces the generation of NO and brings into play protective effect [combination of Chinese and Western medicine hepatopathy magazine, 1999 to the endotoxin liver damage; 9 (2): 30～32].(5) danshensu has anti-TAA (thioacetamide) hepatic injury and the effect that stimulates damage back liver cell regeneration, and its protective effect is higher than the piglets hepatocyte growth-promoting factors (PHGF) [combination of Chinese and Western medicine hepatopathy magazine, 1998,8 (3): 153～155] of market sale.(6) under the multiple hepatopathy, the particularly situation of serious hepatopathy, intestinal endotoxemia often appears.Danshensu improves Liver Microcirculation, thereby alleviates acute heavy hepatic injury through reducing intestinal source property level of endotoxin.Danshensu can alleviate the liver vessel contraction through reducing Endothelin (ET) content, improves the liver blood perfusion.Danshensu can produce through suppressing iNOS expression decreased toxic action NO; Protective effects such as microcirculation improvement, anticoagulant have been brought into play and promote eNOS to express the NO that induces; Reduce simultaneously the generation of ET again, regulate vaso-active substance ET, NO balance, thereby improve impaired liver microcirculation [Shanxi medical magazine; 2006,35 (2): 108～110; (6): 507～508].(7) insulin resistant is a key factor that causes non-alcoholic fatty liver disease, causes hepatocellular sugar to utilize obstacle; Danshensu can obviously improve the utilization of fatty liver cell to glucose, improves the reactivity to insulin, and the phenomenon of reduction insulin resistant [world Chinese digests magazine, and 2005; 13 (15): 1907～1909].(8) danshensu can significantly improve the hepatocyte function of nonalcoholic fatty liver model Mus, strengthens the resistance of liver to exogenous oxygen-derived free radicals.Though simvastatin can reduce serum and organize level of lipid; But liver inner lipid content obviously increases; ALT, more not medication of AST group significantly raise, and the inspection of hepatopathy reason finds that fatty apparition increases the weight of, though explain that its effect for reducing blood fat is obvious; But hyperlipemia-fatty liver is increased its fatty deposition [Jiangsu medicine, 2005 on the contrary; 31 (10): 787～789].

Three, antiinflammatory and enhancing human body immunity function

(1) danshensu ability activated mononuclear phagocyte TNF secretion-α, IL-1,4 kinds of inflammatory cytokines such as IL-6 and IL-8 can also suppress the secretion by these 4 kinds of factors of endotaxin induction simultaneously.Though danshensu ability activated mononuclear phagocyte secrete cytokines, the amount that is produced all significantly is lower than the amount that is produced by endotaxin induction.Therefore infer that danshensu possibly have the regulating action of two aspects, on the one hand can antiinflammatory, on the other hand can enhancing body's immunological function [Chinese Journal of Immunology, 1995; 11 (6): 370～372].(2) danshensu can significantly suppress rat abdominal cavity macrophage generation PGE ₂(prostaglandin) and TXB ₂(thromboxane), its antiinflammatory action possibly be to suppress stream [Pharmacology and Clinics of Chinese Materia Medica, 04 phase of nineteen ninety] in the calcium.(3) in the past research shows that drug for invigorating blood circulation and eliminating stasis has effects such as immunomodulating.Radix Salviae Miltiorrhizae is the drug for invigorating blood circulation and eliminating stasis of using always, and this research shows that danshensu has facilitation to the secretion of IL-2 and IFN-r, and the glucocorticoid cetacort is inhibited to the secretion of IL-2 and IFN-r simultaneously.Thereby the immunological enhancement of danshensu and the immunosuppressive action of glucocorticoid [the Tianjin traditional Chinese medical science, 1995 have further been confirmed; 12 (2): 23～24].

Four, to the effect of cardiac muscle

(1) danshensu has effect [Chinese patent medicine research, 1981 that dwindle the scheming infarction size and alleviate the state of an illness; (2): 35～37].(2) danshensu has protective effect [Chinese Journal of Pathophysiology, 1989 to the rat myocardial ischemia and reperfusion damage; 5 (2): 65～69].(3) " myocardial ischemia reperfusion injury " rolls up relevant with the myocardial cell calcium concentration; Think that at present " calcium overload " is a principal element of reperfusion injury; Intracellular calcium overload is one of important pathological change of myocardial ischemia reperfusion injury, is considered to the final common pathway of cell death.Calcium overload has certain preventive and therapeutic effect in the danshensu pair cell, and effect is superior to SOD [Chinese Journal of Pathophysiology, 1999; 15 (10): 877～879].(4) generation of reperfusion arrhythmia and control are one of research focuses of cardiovascular field; Pour into the oxygen-derived free radicals hypothesis of arrhythmia again, obtain many clinical and zooperal confirmations.Danshensu can reduce the incidence of arrhythmia of pouring into arrhythmia and exogenous free yl induction more significantly, and this can remove oxygen-derived free radicals and alleviate intracellular calcium overload relevant [Chinese Chinese medicine science and technology, 2000 with danshensu; 7 (3): 171～172].The lipid peroxidation damage that mitochondrion took place when (5) danshensu was to the rat heart muscle ischemia/reperfusion has protective effect; Adopt ESR spin trapping technique to find that danshensu has scavenging action to exogenous oxygen-derived free radicals; Thereby infer; Danshensu possibly avoided lipid peroxidation damage [Chinese Journal of Pathophysiology, 1990 that oxygen-derived free radicals causes as a kind of scavenger protection myocardial mitochondria of oxygen-derived free radicals; 6 (6): 420～423].(6) danshensu can produce myocardium protecting action through suppressing the inductive apoptosis of cardiac muscle of Angiotensin II (Ang II), has the loose effect of the myocardial cell of preventing, thus control ventricular hypertrophy [combination of Chinese and Western medicine cardiovascular and cerebrovascular disease magazine, 2006; 4 (6): 494～495].

Five, microcirculation improvement and anticoagulation

(1) danshensu has effect [Shanghai Medical Univ's journal, 1987 of microcirculation improvement obstacle and reduction blood plasma lactic acid content; 14 (1): 25～29].(2) danshensu has effect [Shanghai Second Emdical University's journal, 1990 of improving body anticoagulant and fibrinolytic; 10 (3): 208～211].(3) danshensu has tangible anticoagulation and suppresses effect [combination of Chinese and Western medicine magazine, 1983 that external thrombus forms; 3 (5): 297～299].(4) investigate the influence of danshensu to platelet, leukocyte and vascular endothelial cell express cell adhesion molecule; The result shows that danshensu can suppress vascular endothelial cell and granulocyte express cell adhesion molecule; This possibly be one of mechanism of danshensu performance anti-thrombosis function [Chinese Pharmacological circular, 2000; 16 (6): 682～685].

Six, to the effect of blood vessel

(1) the remarkable diastole pig of danshensu ability stripped coronary artery [Acta Pharmaceutica Sinica, 1982; 17 (3): 226～228].(2) danshensu has ability [combination of Chinese and Western medicine magazine, 1984 that prostaglandins angiogenic substances such as TXA2 are synthesized and discharged to the inhibition platelet; 4 (9): 532～565].(3) danshensu has the effect of expansion artery, and can suppress platelet release and shrink material; Radix Salviae Miltiorrhizae have antagonism development of hypoxic pulmonary vasoconstriction effect, shows that danshensu possibly be the treatment of pulmonary heart disease, adult respiratory distress syndrome critical illness such as (ARDS) offer help [Chinese anesthesiology magazine, 1994; 14 (3): 163～165].(4) operation such as limb transplantation and vascular anastomosis can cause ischemical reperfusion injury, in a lot of dabbling again tissues that exsomatize, can be observed a large amount of superoxide radicals and has formed.Danshensu can reduce ischemia-reperfusion blood vessel H ₂O ₂Amount, oxygen free radical injury is had protective effect [Shanghai Medical Univ's journal, 1998; 25 (2): 115～117].

Seven, to the effect of hemocyte

(1) danshensu can obviously suppress hematoblastic aggregation; And under the danshensu effect of anticoagulant dosage; Platelet membrane is mobile obviously to be increased; Show that danshensu treatment coronary heart disease is effective, maybe with the molecular defect of correcting the patients with coronary heart disease platelet membrane, flowability relevant [Pharmacology and Clinics of Chinese Materia Medica, 1990 of platelet increasing film; 6 (4): 31～34].(2) danshensu can reduce erythrocytic destruction, and its action pathway is to improve erythrocytic negative charge, make and be difficult for each other gathering, and activates the fibrinolysin system, increases mechanical strength [Shandong Medical University's journal, 1991 of erythrocyte membrane; 29 (3): 237～239].(3) danshensu has calcium antagonism, relies on passage through the voltage that suppresses on the erythrocyte membrane, suppresses stream in the erythrocytic calcium, and the intracellular Ca2+ level is reduced.[The Medical College of Changzhi, Shanxi's journal, 1997; 11 (2): 106～107].(4) danshensu can obviously suppress active by the inductive rat platelet aggregation in vitro of ADP; TFT behind the prolongation electricity irritation rat carotid artery; Reduce " blood stasis " rat WBV, plasma viscosity, packed cell volume, erythrocyte electrophoretic time, Kazon yield stress and erythrocyte aggregation index, improve the body blood rheological characteristic.Danshensu can obviously reduce blood " dense, glue, gather, coagulate " state, has good function of promoting blood circulation to disperse blood clots [Chinese patent medicine, 2003; 25 (8): 637～639].

Eight, protection cerebrovascular

(1) danshensu can improve the behavior disorder of rats with cerebral ischemia due to the middle cerebral artery thromboembolism, reduces cerebral ischemia district infarct size [Chinese herbal medicine, 2005; 36 (7): 1041～1043].(2) danshensu can suppress mitochondrial membrane potential and the active reduction due to anoxia/sugar deficiency injury; Thereby effect with stability line mitochondrial membrane potential; Suppress the generation of neuronal apoptosis, the neurocyte during to cerebral ischemic injury has protective effect [Chinese Chinese medicine magazine, 2006; 21 (6): 329～332].(3) exist neuronal apoptosis in the cerebral tissue ischemia injury process, Ca in the cell ²⁺Overload is one of pathogenesis of ischemic brain injury, is considered to the initiating agent of apoptosis.Danshensu can suppress Ca in the cell ²⁺Raise, can alleviate the degree of cell injury, the neurocyte of ischemia injury is had protective effect [Chinese herbal medicine, 2004; 35 (8): 921～924].

Summary of the invention

1, the invention provides a kind of is the pharmaceutical composition of active component with danshensu and salvianolic acid B, and its weight ratio is 1: 3～3: 1, and preferred weight ratio is 1: 2～2: 1,1: 1.5～1.5: 1, and 1: 1.2～1.2: 1,1: 1.

2, in this pharmaceutical composition, danshensu and salvianolic acid B can also exist with the form of salt, are preferably sodium salt and magnesium salt.

3, in this pharmaceutical composition, the purity of the purity of danshensu and salvianolic acid B or danshensu salt and salvianolic acid B salt is more than or equal to 80%; The purity of preferred danshensu and salvianolic acid B or the purity of danshensu salt and salvianolic acid B salt are more than or equal to 90%; The purity of further preferred danshensu and salvianolic acid B or the purity of danshensu salt and salvianolic acid B salt are more than or equal to 98%.

4, this pharmaceutical composition can be used for treating coronary heart disease, angina pectoris, ischemic cerebral thrombosis, atherosclerosis, myocardial ischemia or myocardial infarction, chronic viral hepatitis B hepatic fibrosis, fatty liver, senile dementia, cancer etc.

5, this pharmaceutical composition can be made into injection, oral formulations and external preparation.Injection comprises aqueous injection, injectable powder and infusion solution; Oral formulations comprises tablet, capsule, soft capsule, drop pill, granule, oral liquid, soft gelatin capsule, the watered pill; External preparation comprises transdermal absorption formulation; Also can be made into controlled release preparation and slow releasing preparation.

6, danshensu of the present invention and salvianolic acid B and salt thereof can be used any method preparation, and method for preparing is had no restriction.

7, the present invention has carried out pharmacological testing, has proved with danshensu and salvianolic acid B to be that the pharmaceutical composition of active component is at the medical usage of treatment aspect the cardiovascular and cerebrovascular disease.

The specific embodiment

Following each embodiment only is used to the present invention is described but not limitation of the present invention

Embodiment one

The preparation of danshensu sodium: by the patented method preparation, danshensu sodium purity is more than or equal to 98%.

Number of patent application: 200710166154.8

Patent name: the method for preparing of salvianic acid A sodium pure product

Radix Salviae Miltiorrhizae (individual sub-goods) 3kg adds decocting in water 3 times, adds 7 times of amounts of water for the first time, boils 8 hours, and back secondary respectively adds 5 times of amounts of water, respectively boils 4 hours, filters, and merges filtrating three times; Be concentrated in right amount, be cooled to room temperature, add 95% ethanol, the limit edged stirs, and containing the alcohol amount to solution is 50%; After room temperature is placed 12 hours, to filter, filtrating is concentrated into an amount of, is cooled to room temperature; Add 95% ethanol, the limit edged stirs, and containing the alcohol amount to solution is 80%, after room temperature is placed 12 hours, filters; Filtrating is concentrated into an amount of, is cooled to room temperature, adds the water of 4 times of amounts, and the limit edged stirs, and places 1-2 hour, filters; (AB-8 800g), uses water elution to macroporous adsorptive resins on the filtrating, collects eluent, and (the 60-100 order 250g), is used water elution to polyamide column on the eluent, collects eluent; Concentrate eluant is mixed silica gel 1000g (100-200 order) to an amount of, to baking oven, dries (65 ℃).Silica gel dress post, use ethyl acetate: formic acid (50: 0.1) is washed silicagel column, and the collection eluent is recycled to driedly, gets dry extract.Get dried cream, add dissolve with methanol, transfer pH value to alkalescence with sodium bicarbonate aqueous solution, place, crystallize gets danshensu sodium crystallization (danshensu sodium content is 98.85%).

Embodiment two

The preparation of salvianolic acid B: by the patented method preparation, salvianolic acid B purity is more than or equal to 98%.

Number of patent application: 200710166155.2

Patent name: the method for preparing of salvianolic acid B pure product

A Radix Salviae Miltiorrhizae goods 3kg adds 20 times of amounts of aqueous hydrochloric acid solution (pH value is less than 1.5), floods 20 times, each 10 hours, filters; Polyamide on the filtrating (the 60-100 order, 1kg) post is behind upward intact each subacid water retting liquid, with the removal of impurity of 2-3% ethanol aqueous wash polyamide column, then with the washing chromatographic column; After alcohol cleaned, wash chromatographic column, collect eluent, make pH value less than 2 with hydrochloric acid accent eluent with 0.03% sodium bicarbonate aqueous solution; Last macroporous adsorbent resin (AB-8,2kg) post is behind the upward intact macroporous adsorptive resins, with the 10% ethanol aqueous wash resin column removal of impurity; Reuse 95% ethanol is washed resin column, collects 95% ethanol elution, be recycled to dried, dry powder A.Get dry powder A, add 15 times of amount dissolvings of acetone, filtration is reclaimed filtrating to doing, and gets dry powder B; Get dry powder B, add 15 times of amount dissolvings of ethyl acetate, filtration is reclaimed filtrating to doing, and gets dry powder C.Get dry powder C, add 50% methanol aqueous solution and dissolve in right amount, last Sephadex LH-20 post is washed chromatographic column with 50% methanol aqueous solution, merges eluent according to TLC, be recycled to dried, dry powder D (content of danshinolic acid B is 99.35%).

Embodiment three

Preparation is the freeze-dried powder of active component with danshensu and salvianolic acid B

Get danshensu 40g, salvianolic acid B 40g is dissolved in the 5000ml water for injection, adds 30g mannitol; Stirring and dissolving adds sodium hydroxide and transfers between pH value to 4～7, and ultrafiltration obtains apyrogenic clear liquor; Lyophilizing is processed every and is contained danshensu 40mg, the freeze-dried powder of salvianolic acid B 40mg.

Embodiment four

Preparation is the tablet of active component with danshensu and salvianolic acid B

Get danshensu 80g, salvianolic acid B 80g, starch 160g, dextrin (powder) 20g, mixing is processed granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, and sugar coating promptly gets.

Embodiment five

Get danshensu 13g, salvianolic acid B 65g is dissolved in the 5000ml water for injection, adds 30g mannitol; Stirring and dissolving adds sodium hydroxide and transfers between pH value to 4～7, and ultrafiltration obtains apyrogenic clear liquor; Lyophilizing is processed every and is contained danshensu 13mg, the freeze-dried powder of salvianolic acid B 65mg.

Embodiment six

Get danshensu 65g, salvianolic acid B 13g is dissolved in the 5000ml water for injection, adds 30g mannitol; Stirring and dissolving adds sodium hydroxide and transfers between pH value to 4～7, and ultrafiltration obtains apyrogenic clear liquor; Lyophilizing is processed every and is contained danshensu 65mg, the freeze-dried powder of salvianolic acid B 13mg.

Embodiment seven

Get danshensu 17g, salvianolic acid B 68g is dissolved in the 5000ml water for injection, adds 30g mannitol; Stirring and dissolving adds sodium hydroxide and transfers between pH value to 4～7, and ultrafiltration obtains apyrogenic clear liquor; Lyophilizing is processed every and is contained danshensu 17mg, the freeze-dried powder of salvianolic acid B 68mg.

Embodiment eight

Get danshensu 68g, salvianolic acid B 17g is dissolved in the 5000ml water for injection, adds 30g mannitol; Stirring and dissolving adds sodium hydroxide and transfers between pH value to 4～7, and ultrafiltration obtains apyrogenic clear liquor; Lyophilizing is processed every and is contained danshensu 68mg, the freeze-dried powder of salvianolic acid B 17mg.

Embodiment nine

Get danshensu 40g, salvianolic acid B 120g, starch 160g, dextrin (powder) 20g, mixing is processed granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, and sugar coating promptly gets.

Embodiment ten

Get danshensu 120g, salvianolic acid B 40g, starch 160g, dextrin (powder) 20g, mixing is processed granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, and sugar coating promptly gets.

Embodiment 11

Get danshensu 46g, salvianolic acid B 115g, starch 160g, dextrin (powder) 20g, mixing is processed granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, and sugar coating promptly gets.

Embodiment 12

Get danshensu 115g, salvianolic acid B 46g, starch 160g, dextrin (powder) 20g, mixing is processed granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, and sugar coating promptly gets.

Embodiment 13

Get danshensu 80g, salvianolic acid B 88g, starch 160g, dextrin (powder) 20g, mixing is processed granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, and sugar coating promptly gets.

Embodiment 14

Get danshensu 88g, salvianolic acid B 80g, starch 160g, dextrin (powder) 20g, mixing is processed granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, and sugar coating promptly gets.

Embodiment 15

Get danshensu 70g, salvianolic acid B 91g, starch 160g, dextrin (powder) 20g, mixing is processed granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, and sugar coating promptly gets.

Embodiment 16

Get danshensu 91g, salvianolic acid B 70g, starch 160g, dextrin (powder) 20g, mixing is processed granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, and sugar coating promptly gets.

Embodiment 17

Get danshensu 65g, salvianolic acid B 91g, starch 160g, dextrin (powder) 20g, mixing is processed granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, and sugar coating promptly gets.

Embodiment 18

Get danshensu 91g, salvianolic acid B 65g, starch 160g, dextrin (powder) 20g, mixing is processed granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, and sugar coating promptly gets.

Embodiment 19

Get danshensu 60g, salvianolic acid B 96g, starch 160g, dextrin (powder) 20g, mixing is processed granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, and sugar coating promptly gets.

Embodiment 20

Get danshensu 96g, salvianolic acid B 60g, starch 160g, dextrin (powder) 20g, mixing is processed granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, and sugar coating promptly gets.

Embodiment 21

Get danshensu 60g, salvianolic acid B 102g, starch 160g, dextrin (powder) 20g, mixing is processed granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, and sugar coating promptly gets.

Embodiment 22

Get danshensu 102g, salvianolic acid B 60g, starch 160g, dextrin (powder) 20g, mixing is processed granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, and sugar coating promptly gets.

Embodiment 23

Get danshensu 55g, salvianolic acid B 99g, starch 160g, dextrin (powder) 20g, mixing is processed granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, and sugar coating promptly gets.

Embodiment 24

Get danshensu 99g, salvianolic acid B 55g, starch 160g, dextrin (powder) 20g, mixing is processed granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, and sugar coating promptly gets.

Embodiment 25

Get danshensu 60g, salvianolic acid B 114g, starch 160g, dextrin (powder) 20g, mixing is processed granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, and sugar coating promptly gets.

Embodiment 26

Get danshensu 114g, salvianolic acid B 60g, starch 160g, dextrin (powder) 20g, mixing is processed granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, and sugar coating promptly gets.

Embodiment 27

Get danshensu 50g, salvianolic acid B 105g, starch 160g, dextrin (powder) 20g, mixing is processed granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, and sugar coating promptly gets.

Embodiment 28

Get danshensu 105g, salvianolic acid B 50g, starch 160g, dextrin (powder) 20g, mixing is processed granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, and sugar coating promptly gets.

Embodiment 29

Get danshensu 50g, salvianolic acid B 110g, starch 160g, dextrin (powder) 20g, mixing is processed granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, and sugar coating promptly gets.

Embodiment 30

Get danshensu 110g, salvianolic acid B 50g, starch 160g, dextrin (powder) 20g, mixing is processed granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, and sugar coating promptly gets.

The embodiment hentriaconta-

Get danshensu 50g, salvianolic acid B 115g, starch 160g, dextrin (powder) 20g, mixing is processed granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, and sugar coating promptly gets.

Embodiment 32

Get danshensu 115g, salvianolic acid B 50g, starch 160g, dextrin (powder) 20g, mixing is processed granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, and sugar coating promptly gets.

Embodiment 33

Get danshensu 45g, salvianolic acid B 108g, starch 160g, dextrin (powder) 20g, mixing is processed granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, and sugar coating promptly gets.

Embodiment 34

Get danshensu 108g, salvianolic acid B 45g, starch 160g, dextrin (powder) 20g, mixing is processed granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, and sugar coating promptly gets.

Embodiment 35

Get danshensu 45g, salvianolic acid B 117g, starch 160g, dextrin (powder) 20g, mixing is processed granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, and sugar coating promptly gets.

Embodiment 36

Get danshensu 117g, salvianolic acid B 45g, starch 160g, dextrin (powder) 20g, mixing is processed granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, and sugar coating promptly gets.

Embodiment 37

Get danshensu 40g, salvianolic acid B 108g, starch 160g, dextrin (powder) 20g, mixing is processed granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, and sugar coating promptly gets.

Embodiment 38

Get danshensu 108g, salvianolic acid B 40g, starch 160g, dextrin (powder) 20g, mixing is processed granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, and sugar coating promptly gets.

Embodiment 39

Get danshensu 40g, salvianolic acid B 112g, starch 160g, dextrin (powder) 20g, mixing is processed granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, and sugar coating promptly gets.

Embodiment 40

Get danshensu 112g, salvianolic acid B 40g, starch 160g, dextrin (powder) 20g, mixing is processed granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, and sugar coating promptly gets.

Embodiment 41

Get danshensu 40g, salvianolic acid B 116g, starch 160g, dextrin (powder) 20g, mixing is processed granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, and sugar coating promptly gets.

Embodiment 42

Get danshensu 116g, salvianolic acid B 40g, starch 160g, dextrin (powder) 20g, mixing is processed granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, and sugar coating promptly gets.

Embodiment 43

The preparation of danshensu sodium:

Radix Salviae Miltiorrhizae 3kg adds decocting in water 3 times, adds 7 times of amounts of water for the first time, boils 8 hours, and back secondary respectively adds 5 times of amounts of water, respectively boils 4 hours, filters, and merges filtrating three times; Be concentrated in right amount, be cooled to room temperature, add 95% ethanol, the limit edged stirs, and containing the alcohol amount to solution is 50%; After room temperature is placed 12 hours, to filter, filtrating is concentrated into an amount of, is cooled to room temperature; Add 95% ethanol, the limit edged stirs, and containing the alcohol amount to solution is 80%, after room temperature is placed 12 hours, filters; Filtrating is concentrated into an amount of, is cooled to room temperature, adds the water of 4 times of amounts, and the limit edged stirs, and places 1-2 hour, filters; (AB-8 800g), uses water elution to macroporous adsorptive resins on the filtrating, collects eluent, and (the 60-100 order 250g), is used water elution to polyamide column on the eluent, collects eluent; Concentrate eluant is mixed silica gel 1000g (100-200 order) to an amount of, to baking oven, dries (65 ℃).Silica gel dress post, use ethyl acetate: formic acid (50: 0.1) is washed silicagel column, collects eluent, be recycled to dried, must dry powder, danshensu sodium content is 92.27%.

Embodiment 44

The preparation of danshensu sodium:

Radix Salviae Miltiorrhizae 3kg adds decocting in water 3 times, adds 7 times of amounts of water for the first time, boils 8 hours, and back secondary respectively adds 5 times of amounts of water, respectively boils 4 hours, filters, and merges filtrating three times; Be concentrated in right amount, be cooled to room temperature, add 95% ethanol, the limit edged stirs, and containing the alcohol amount to solution is 50%; After room temperature is placed 12 hours, to filter, filtrating is concentrated into an amount of, is cooled to room temperature; Add 95% ethanol, the limit edged stirs, and containing the alcohol amount to solution is 80%, after room temperature is placed 12 hours, filters; Filtrating is concentrated into an amount of, is cooled to room temperature, adds the water of 4 times of amounts, and the limit edged stirs, and places 1-2 hour, filters; (AB-8 800g), uses water elution to macroporous adsorptive resins on the filtrating, collects eluent, and (the 60-100 order 250g), is used water elution to polyamide column on the eluent, collects eluent; Concentrate eluant gets dry powder to doing, and danshensu sodium content is 81.86%.

Embodiment 45

The preparation of salvianolic acid B:

Radix Salviae Miltiorrhizae 3kg adds 20 times of amounts of aqueous hydrochloric acid solution (pH value is less than 1.5), floods 20 times, each 10 hours, filters; Polyamide on the filtrating (the 60-100 order, 1kg) post is behind upward intact each subacid water retting liquid, with the removal of impurity of 2-3% ethanol aqueous wash polyamide column, then with the washing chromatographic column; After alcohol cleaned, wash chromatographic column, collect eluent, make pH value less than 2 with hydrochloric acid accent eluent with 0.03% sodium bicarbonate aqueous solution; Last macroporous adsorbent resin (AB-8,2kg) post is behind the upward intact macroporous adsorptive resins, with the 10% ethanol aqueous wash resin column removal of impurity; Reuse 50% ethanol is washed resin column, collects 50% ethanol elution, be recycled to dried, dry powder A.Get dry powder A, add 15 times of amount dissolvings of acetone, filtration is reclaimed filtrating to doing, and gets dry powder B; Get dry powder B, add 15 times of amount dissolvings of ethyl acetate, filtration is reclaimed filtrating to doing, and gets dry powder C, and content of danshinolic acid B is 91.16%.

Embodiment 46

The preparation of salvianolic acid B:

Radix Salviae Miltiorrhizae 3kg adds 20 times of amounts of aqueous hydrochloric acid solution (pH value is less than 1.5), floods each 10 hours 20 times; Filter, and polyamide on the filtrating (the 60-100 order, 1kg) post is behind upward intact each subacid water retting liquid; With the removal of impurity of 2-3% ethanol aqueous wash polyamide column, use the washing chromatographic column then, after alcohol is cleaned, wash chromatographic column with 0.03% sodium bicarbonate aqueous solution; Collect eluent, transfer eluent to make pH value with hydrochloric acid, last macroporous adsorbent resin (AB-8,2kg) post less than 2; After having gone up macroporous adsorptive resins, with the 10% ethanol aqueous wash resin column removal of impurity, reuse 70% ethanol is washed resin column, collects 70% ethanol elution; Be recycled to dried, dry powder A, content of danshinolic acid B is 82.95%.

Embodiment 47

Preparation is the freeze-dried powder of active component with danshensu (danshensu sodium content is 92.27%) and salvianolic acid B (content of danshinolic acid B is 91.16%)

Get danshensu 40g, salvianolic acid B 40g is dissolved in the 5000ml water for injection, adds 30g mannitol, and stirring and dissolving adds sodium hydroxide and transfers between pH value to 4～7, and ultrafiltration obtains apyrogenic clear liquor, and freeze-dried powder is processed in lyophilizing.

Embodiment 48

Preparation is the tablet of active component with danshensu (danshensu sodium content is 92.27%) and salvianolic acid B (content of danshinolic acid B is 91.16%)

Embodiment 49

Preparation is the freeze-dried powder of active component with danshensu (danshensu sodium content is 81.86%) and salvianolic acid B (content of danshinolic acid B is 82.95%)

Get danshensu 13g, salvianolic acid B 65g is dissolved in the 5000ml water for injection, adds 30g mannitol, and stirring and dissolving adds sodium hydroxide and transfers between pH value to 4～7, and ultrafiltration obtains apyrogenic clear liquor, and freeze-dried powder is processed in lyophilizing.

Embodiment 50

Preparation is the freeze-dried powder of active component with danshensu (danshensu sodium content is 81.86%) and salvianolic acid B (content of danshinolic acid B is 91.16%)

Get danshensu 65g, salvianolic acid B 13g is dissolved in the 5000ml water for injection, adds 30g mannitol, and stirring and dissolving adds sodium hydroxide and transfers between pH value to 4～7, and ultrafiltration obtains apyrogenic clear liquor, and freeze-dried powder is processed in lyophilizing.

Embodiment 51

Preparation is the freeze-dried powder of active component with danshensu (danshensu sodium content is 92.27%) and salvianolic acid B (content of danshinolic acid B is 82.95%)

Get danshensu 17g, salvianolic acid B 68g is dissolved in the 5000ml water for injection, adds 30g mannitol, and stirring and dissolving adds sodium hydroxide and transfers between pH value to 4～7, and ultrafiltration obtains apyrogenic clear liquor, and freeze-dried powder is processed in lyophilizing.

Embodiment 52

Get danshensu 68g, salvianolic acid B 17g is dissolved in the 5000ml water for injection, adds 30g mannitol, and stirring and dissolving adds sodium hydroxide and transfers between pH value to 4～7, and ultrafiltration obtains apyrogenic clear liquor, and freeze-dried powder is processed in lyophilizing.

Embodiment 53

Preparation is the tablet of active component with danshensu (danshensu sodium content is 81.86%) and salvianolic acid B (content of danshinolic acid B is 82.95%)

Embodiment 54

Preparation is the tablet of active component with danshensu (danshensu sodium content is 92.27%) and salvianolic acid B (content of danshinolic acid B is 82.95%)

Embodiment 55

Preparation is the tablet of active component with danshensu (danshensu sodium content is 81.86%) and salvianolic acid B (content of danshinolic acid B is 91.16%)

Pharmacological testing: medicine A is a danshensu sodium, and purity is 98.85%; Medicine B is a salvianolic acid B, and purity is 99.35%; Medicine A, B are provided by Beijing Keshilan Pharmaceutical Technology Co., Ltd..

Test Example one

The different proportionings of medicine A, B are to the influence of isolated rat heart coronary flow

1. experiment material

1.1 experimental apparatus and reagent

Powerlab/8sp Langendorff isolated heart perfusion system, ADInstrument Pty Ltd (Australia).Krebs-Henseleit liquid, it consists of (mmol/L): NaCl 118, and KCl 4.7, MgSO ₄7H ₂O1.2, CaCl ₂2.0, NaHCO ₃20, KH ₂PO ₄1.2 Glucose 11.1, pH value 7.2-7.4.

1.2 receive reagent thing and preparation

Two kinds of medicines are numbered A, B; Lot number 20071103 is provided by Beijing Keshilan Pharmaceutical Technology Co., Ltd..

Medicine A, B are made into A with Krebs-Henseleit liquid before the experiment: B is respectively 10mg: the medicinal liquid of 10mg/ml, 6.66mg: 13.33mg/ml, 5mg: 15mg/ml, 13.33mg: 6.66mg/ml, 15mg: 5mg/ml, 20mg: 0mg/ml, 0mg: 20mg/ml.

By A: B is respectively 2mg: 2mg, 1.33mg: 2.66mg, 1mg: 3mg, 2.66mg: 1.33mg, 3mg: 1mg, 4mg: 0mg, 0mg: the administration of 4mg/ heart, administration volume are 0.2ml.

1.3 laboratory animal

Healthy Wistar rat, male, body weight 350g ± 30g, Beijing Vital River Experimental Animals Technology Co., Ltd. provides.

2. experimental technique

2.1 group technology

Experiment is divided into medicine A group, medicine B group and A+B drug group, and the A+B drug group comprises 1: 1 proportioning group, 1: 2 proportioning group, 1: 3 proportioning group, 2: 1 proportioning groups, 3: 1 proportioning groups.Establish 7 groups altogether, every group of 14 rats.Group technology, drug dose and administration concentration are seen table 1.

Table 1 experiment group technology

2.2 experimental implementation method

The rat sacrificed by decapitation is taken out heart rapidly, places 4 ℃ of K-H liquid, discharges remained blood gently, and heart is connected to perfusion device and fixing, the beginning perfusion, and perfusate is kept 37 ℃, and feeds 95% oxygen.After treating that electrocardio is steady, perfusion pressure is adjusted in 60-70mmHg carries out the voltage stabilizing perfusion, coronary flow before the record administration of stable back slowly injects the medicinal liquid that the 0.2ml/ heart prepares through dosing holes, and the above-mentioned each item index of immediate record after administration finishes continues to observe 6 minutes.

2.3 detection index

Injection pressure (mmHg); Heart rate (inferior/minute); Before the administration and after the administration at once, 1,2,3,4,5 minute coronary flow (ml/min), coronary flow rate of change (%).

2.4 statistical procedures

(x ± s) expression adopts the SPSS10.0 statistical software to data, and rate of change relatively adopts one-way ANOVA to analyze between group, and the front and back measured value adopts paired t-test, and P＜0.05 is for there being significant difference with mean ± standard deviation.

3. result

3.1 respectively organize the influence of medicine to the isolated rat heart heart rate

Before the experiment perfusion pressure is adjusted in 60-70mmHg and carries out the voltage stabilizing perfusion, medicine does not make significant difference to the isolated rat heart heart rate, and the result sees table 2.

Respectively organize isolated rat heart injection pressure, heart rate before and after table 2 administration

3.2 respectively organize the influence of medicine to coronary flow

Coronary flow there was no significant difference (P＞0.05) between each group before the administration;

Respectively organize the isolated rat heart coronary flow after the administration rising is in various degree all arranged; With compare before the administration; Difference has statistical significance (P＜0.05 or P＜0.01); After the administration at once, 1,2,3,4 minute the time, each proportioning group has been compared significant difference (P＜0.05 or P＜0.01) with single medicament A, B; 1: 1 proportioning group of A+B medicine has the trend that is superior to other group; The result sees table 3.

4. conclusion

Aspect rising isolated rat heart coronary flow, in 5 proportionings (1: 1,1: 2,1: 3,2: 1,3: 1) group of forming by A, two medicines of B, be excellent with 1: 1 proportioning group.

Test Example two

The different proportionings with the B medicine of A are to the influence of mice normal pressure anoxia enduring

1. experiment material

1.1 animal

The KM mice, male and female half and half, 20-22g.Quality certification SCXK (capital) 2005-0013 is provided by Institute of Experimental Animals, Chinese Academy of Medical Sciences.

1.2 medicine

Two kinds of medicines are numbered A, B.Lot number 20071103 is provided by Beijing Keshilan Pharmaceutical Technology Co., Ltd..

Propranolol (10mg/ sheet), Tianjin Lisheng Pharmaceutical Co., Ltd. produces, lot number 0411015.

1.3 reagent

Vaseline, Tianjin petrochemistry trial (demonstration) plant, lot number 071019.

Sodica calx, Shanghai City are received brightness dried reagent factory, lot number 20040724.

2. experimental technique

2.1A, the administration of B prescription flavor is to the influence of mice normal pressure anoxia enduring

100 of mices, female, hero half and half is divided into 10 groups by body weight, sex, 10 every group at random.Establish the blank group respectively, A, B medicine single medicinal material administration group dosage are divided into 72mg/kg, 36mg/kg, 18mg/kg, 9mg/kg group, positive control drug propranolol 50mg/kg group.A, B medicine group are mixed with the solution of respective concentration with 0.9% sodium chloride before experiment, concentration is respectively 3.6mg/ml, 1.8mg/ml, 0.9mg/ml, 0.45mg/ml, and propranolol concentration is 2.5mg/ml.By body weight 0.2ml/10g intraperitoneal administration, once a day, successive administration 7 days, the blank group gives isopyknic 0.9% sodium chloride.0.5h after the last administration gets and respectively organizes each one of the close mice of body weight, as detecting animal with criticizing.Mice is put into the 250ml wide-mouth port grinding bottle that fills the 15g sodica calx rapidly, every bottle one (sealing), manual time-keeping immediately, the record mice is because of the anoxia death time.Breathing to dehisce for the last time is the death time, observes the time-to-live of mice, calculates the prolonged survival period percentage rate ^[1]

2.2A, the different proportionings of B medicine are to the influence of mice normal pressure anoxia enduring

140 of mices, female, hero half and half is divided into 14 groups by body weight, sex, 10 every group at random.Establish blank group, A+B administration group, positive control drug propranolol 50mg/kg group respectively.Administration group dosage is provided with: A, B medicine are by 3 proportioning administrations, and each proportioning is respectively established four dose groups, 1: 1 proportioning, and dosage is 72 (36+36) mg/kg, 36 (18+18) mg/kg, 18 (9+9) mg/kg, 9 (4.5+4.5) mg/kg.1: 2 proportioning, dosage are 72 (24+48) mg/kg, 36 (12+24) mg/kg, 18 (6+12) mg/kg, 9 (3+6) mg/kg.2: 1 proportionings, dosage are 72 (48+24) mg/kg, 36 (24+12) mg/kg, 18 (12+6) mg/kg, 9 (6+3) mg/kg group.A, B medicine are mixed with the solution of respective concentration with 0.9% sodium chloride before experiment, three proportioning administration concentration are respectively 3.6mg/ml, 1.8mg/ml, 0.9mg/ml, 0.45mg/ml, and propranolol concentration is 2.5mg/ml.By body weight 0.2ml/10g intraperitoneal administration, once a day, successive administration 7 days, the blank group gives isopyknic 0.9% sodium chloride (detection method is the same).

2.3 statistical method

(x ± s) expression relatively adopts the t check to experimental data between group, significant level is a standard with 0.05 and 0.01 with mean ± standard deviation.

3. experimental result

3.1A, the administration of B prescription flavor is to the influence of mice normal pressure anoxia enduring

A, B prescription flavor intraperitoneal administration 7 days compare with the blank group, all do not have obviously to prolong mice normal pressure hypoxia endurance time effect (P＞0.05).The result sees table 1.

The administration of table 1A.B prescription flavor is to influence (x ± s) (n=10) of mice normal pressure anoxia enduring

Annotate: compare with the blank group, ^*P＜0.01.

3.2A, the different proportionings of B medicine are to the influence of mice normal pressure anoxia enduring

A+B medicine intraperitoneal administration 7 days, with the blank group relatively, 1: 1 proportioning, wherein 72 (36+36) mg/kg, 36 (18+18) mg/kg group all prolong mice normal pressure hypoxia endurance time in various degree, rate elongation is respectively 21.4%, 30.9% (P＜0.05-0.01).2: 1 proportionings, wherein 72 (48+24) m/kg, 9 (6+3) mg/kg organize, and rate elongation is respectively 24.4%, 16.3% (P＜0.05).Propranolol can obviously prolong mice normal pressure hypoxia endurance time, and rate elongation is 71.4% (P＜0.01).The result sees table 2.

The different proportionings of table 2A, B medicine are to influence (x ± s) (n=10) of mice normal pressure anoxia enduring

Annotate: compare with the blank group, ^*P＜0.05, ^*P＜0.01.

4, conclusion

The A medicine of Isodose and the flavor administration of B prescription and blank group are relatively; The mice normal pressure anoxia enduring time-to-live there is not obvious influence; And A, two kinds of medicines of B are by after 1: 1 and the proportioning administration in 2: 1; All demonstrating prolongation mice anoxia enduring time-to-live effect in various degree, is excellent with 1: 1 proportioning wherein.

List of references

1. Qi Chen edits: herbal pharmacology research methodology, People's Health Publisher, 1996; 783-784. Test Example three

1: 1 proportioning of A&B medicine is to the experimentation that influences of rat heart muscle ischemia

Summary: purpose: observe of the influence of 1: 1 proportioning group of A&B medicine to the rat heart muscle ischemia.Method: adopt ligation rat coronary artery anterior descending branch to cause myocardial infarction and ischemia model, administration 7 days.Result: A: B (1: 1 ratio) medicine 72mg/kg dose groups can alleviate degree of myocardial ischemia, with model group comparing difference remarkable (P＜0.05).Conclusion: 1: 1 proportioning of A&B medicine can reduce the myocardial infarction degree at 72mg/kg dosage, has certain function of resisting myocardial ischemia.

1. purpose: observe of the influence of 1: 1 proportioning group of A&B medicine to the rat heart muscle ischemia

In jar stream heart experiment of exsomatizing, it is remarkable to obtain 1: 1 ratio effect of A&B medicine; To in the mice anoxia enduring experiment, obtain 1: 1 ratio of A&B medicine at 18mg/kg: the anoxia enduring death time prolongs (P＜0.01) under the 18mg/kg dosage at the A&B medicine.Therefore, this experiment has been carried out the influence experimentation of 1: 1 proportioning of A&B medicine to the rat heart muscle ischemia on above-mentioned two experiment basis.

2. materials and methods

2.1 animal

Laboratory animal: adult healthy SD rat, male, body weight (250 ± 20) g.Raise in the room ventilation cabinet, constant temperature (22 ± 2) ℃, naturally round the clock, free drinking water.Animal quality certification numbering SCXK (capital) 2004-0001.

2.2 divide into groups and administration

Rat divides into groups by body weight, is divided into 6 groups at random, 10 every group.

1) normal control group; 2) sham operated rats; 3) model group;

4) receive reagent A&B high dose group A: B=36mg/kg: 36mg/kg

5) receive dose groups A: B=18mg/kg: 18mg/kg among the reagent A&B

6) receive reagent A&B low dose group A: B=9mg/kg: 9mg/kg

Medication: overnight fasting before the art, freely drink water.Postoperative administration 7 days, 1h draws materials after the last administration.

2.3 animal model preparation

Animal faces upward the position and fixes with pentobarbital intraperitoneal injection of anesthesia (30mg/kg), leads with standard I I and monitors the animal electrocardiogram; Tracheostomize inserts tracheal intubation, meets animal artificial respirator pedestrian worker and breathes (90 times/minute, breathed ratio 1.5: 1); Left side the 4th～5 intercostal is opened breast, breaks pericardium, exposes heart, in ADC root threading (3/8 arc band pin stitching thread, 0/3 nylon wire), is equipped with ligation and uses; Stablized behind the threading 10 minutes, thoracic wall is sewed up in ligation (no ST section and T ripple changer eliminate), and restore nature is breathed.

2.4 key instrument

16 road physiology monitors (MP-150, U.S. BIOPAC); AE200 type electronic analytical balance (Shanghai, prunus mume (sieb.) sieb.et zucc. Teller-Tuo benefit Instr Ltd.).

2.5 statistical procedures

Carry out statistical analysis with the SPSS10.0 software kit, data are with (x ± s) expression relatively uses the t check between group, P＜0.05 is a significant difference, and P＜0.01 is a significant differences.

2.6 index detects

The mensuration of rat myocardium block area: 1h draws materials after the last administration, wins heart, takes out after placing-20 ℃ of refrigerator 30min, along 6 of the following crosscuts of heart ligature, puts the 10-15min (37 ℃ of water-baths) that dyes in 1% red tetrazolium (TTC) phosphate buffer.Perusal, infarcted region is canescence, and infarcted region does not take on a red color.Along the slough edge separation infarcted myocardium tissue and infarcted myocardium are not organized respectively and to be weighed, the percentage ratio that accounts for whole cardiac weight with infarcted myocardium is represented infarction size.

3. result

A: B (1: 1 ratio) medicine is to the influence of rat myocardium block area:

Myocardial ischemia is obvious behind the rat ligation ADC; Model group rat heart muscle necrotic area weight account for respectively left ventricle and whole-heartedly dirty weight 18.49%; 13.96%; Give A: B (1: 1 ratio) medicine after 7 days the myocardial ischemia situation improvement is in various degree all arranged, mainly with A: B (1: 1 ratio) medicine 72mg/kg dose effect is remarkable, with model group P＜0.05 relatively.See table 1, table 2.

Table 1 A: B (1: 1 ratio) medicine to the influence of rat myocardium block area (x ± s, N=10)

Table 2 A: B (1: 1 ratio) medicine to the influence of rat myocardium block area (x ± s, N=10)

Compare with model group ^*P＜0.05.

4. brief summary

Adopt ligation rat coronary artery anterior descending branch to cause myocardial infarction and ischemia model, irritate stomach and give 1: 1 proportioning 7 days (according to 72mg/kg, 36mg/kg, 18mg/kg dosage) of A&B medicine.The result shows: A: B (1: 1 ratio) medicine 72mg/kg dose groups can alleviate degree of myocardial ischemia, with model group comparing difference remarkable (P＜0.05);

5. conclusion

1: 1 proportioning of A&B medicine can alleviate myocardial infarction area at 72mg/kg dosage, A: B (1: 1 ratio) has certain Chinese People's Anti-Japanese Military and Political College's Mus myocardial ischemia effect at 72mg/kg dosage.

Test Example four

Danshensu and salvianolic acid B compositions are to the experimentation of focal cerebral ischemia in rats influence

Summary

Adopt the ferric chloride part to stick middle cerebral artery and cause the focal cerebral ischemia injury model,, inquire into and receive the protective effect of reagent rat model through mensuration to rat model nervous symptoms and cerebral infarction scope.The result shows; The nervous symptoms of danshensu and salvianolic acid B compositions 6 hours and 24 hours after postoperative intravenous injection in 30 minutes, postoperative 2 hours and 23 hours lumbar injections can obviously improve modeling obviously dwindles 24 hours cerebral infarction scope of animal pattern cerebral thrombosis (P＜0.01).Conclusion: danshensu and salvianolic acid B compositions can alleviate focal cerebral ischemia injury through suppressing cerebral thrombosis, and its curative effect is better than danshensu group and salvianolic acid B group.

Experiment purpose

Observe the influence of danshensu and the salvianolic acid B compositions focal cerebral ischemia in rats that blocking-up causes to intraluminal middle cerebral artery occlusion in rats.

Experiment material

One, animal: the SD rat, male, body weight 200-220g, the quality certification number: SCXK (capital) 2004-0001 is provided by Beijing Vital River Experimental Animals Technology Co., Ltd..

Two, medicine and reagent

Receive reagent: A is a danshensu, and purity is 98.85%; B is a salvianolic acid B, and purity is 99.35%; A: B=1 in the A+B group: 1.Provided by Beijing Keshilan Pharmaceutical Technology Co., Ltd. by reagent.Reagent: red tetrazolium (TTC) is pale yellow powder, Beijing Ma Shi fine chemicals company limited product, lot number: 011102.

Three, instrument: XTT stero microscope, Yunnan Optical Instruments Factory's product; AEG-220 type electronic analytical balance, Japanese Shimadzu Company products; The desk-top dentistry car of 307-6, Shanghai Dental Medical Apparatus and Instrument Factory's product; HZQ-C air bath agitator, Dongming, Harbin Medical Instruments factory product.

Experimental technique and result

One, grouping and administration

Laboratory animal is divided into five groups at random by body weight, i.e. sham operated rats, model control group, A group, B group, A+B group.Each treated animal is all in postoperative sublingual vein administration in 30 minutes, postoperative 2 hours and 23 hours intraperitoneal injection secondaries.Sham operated rats and model control group give isometric normal saline.ID adopts the 0.4ml/100g body weight.

Two, the middle cerebral artery thrombus model is made

The anesthesia of rats by intraperitoneal injection 10% chloral hydrate solution (350mg/kg), RAR is fixed, and makes a curved incision at paropia and external auditory meatus line mid point; Be about 1.5cm, pinch off temporalis and excision expose temporal bone; Under stero microscope, make the bone window of a diameter 2.5mm, cleaning residue at cheekbone and temporo squamosum joint near oral-lateral 1mm place with dental burr; Expose middle cerebral artery (between tractus olfactorius and inferior cerebral vein), put small pieces plastic sheeting protection blood vessel surrounding tissue.Have the small pieces quantitative filter paper of 50% ferric chloride solution, 10 μ L to apply on this section middle cerebral artery suction, take off filter paper behind the 30min, use the normal saline flushing local organization, layer-by-layer suture steams again and raises.Room temperature is controlled at 24 ℃.Sham operated rats is not except that dripping the ferric chloride solution, and all the other operating procedures shine with model.

Three, nervous symptoms standards of grading

To experimental animal 24h after surgery, carry out behavior and detect.Standards of grading: 1. carry the Mus tail and observe forelimb flexing situation, protract, be designated as 0 fen like two forelimb symmetries; As the offside forelimb of performing the operation shoulder flexing, elbow flexing, shoulder inward turning occur or has concurrently, is designated as 1 fen.2. animal is placed on the plane, push away both shoulders respectively, check resistance to side shifting.Like bilateral resistance equity and strong, be designated as 0 fen; Like operation collateral resistance is descended, be designated as 1 fen.3. animal two forelimbs are put on the wire netting, observed muscular tension.Bilateral muscular tension equity and be 0 minute effectively; Operation offside muscle of anterior limb tension force descends and is designated as 1 fen.4. carry the Mus tail, animal has ceaselessly to operation offside revolver, is designated as 1 fen.According to above standard scoring, full marks are 4 minutes, and mark is high more, and the behavior disorder of animal is serious more.

Four, the mensuration of cerebral infarction scope

Behind the animal via behavior scoring, broken end is got brain.The remainder that removes behind olfactory bulb, XIAONAO and the low brain stem is cut into 5 crown below 4 ℃, and (every 5ml dye liquor contains 4%TTC 1.5ml, 1M K rapidly the brain sheet to be placed the TTC dye liquor ₂HP0 ₄0.1ml all the other adding distil waters are to scale), 37 ℃ of lucifuge temperature were incubated 30 minutes, took out to be placed on the 24h that keeps in Dark Place in 10% formalin.The non-ischemic region in dyed back is a rose, and infarct is a white.White organized carefully to dig down weigh, the percentage ratio that accounts for full brain weight and Ipsilateral brain weight with blocking tissue's weight is as the cerebral infarction scope.

Five, result

Above-mentioned experimental result is represented with x ± s, paired comparison t check between statistical test employing group, and the result sees table 1 and table 2.

Table 1 receives reagent to the influence of MCAT rat nerves symptom (x ± s)

Table 2 receives reagent to the influence of MCAT rat cerebral infarction scope (x ± s)

Annotate: compare with model control group, ^*P＜0.05, ^*P＜0.01

Brief summary

Can find out the nervous symptoms of A+B, A, B 6 hours and 24 hours after postoperative intravenous injection in 30 minutes, postoperative 2 hours and 23 hours lumbar injections all can obviously improve modeling from last table; And A+B, A, B can also obviously dwindle 24 hours cerebral infarction scope of animal pattern cerebral thrombosis (P＜0.01, P＜0.05, P＜0.05).Conclusion: A+B, A, B all can alleviate focal cerebral ischemia injury through suppressing cerebral thrombosis, and the curative effect of A+B group is better than A group and B group.

Claims

1. one kind is the pharmaceutical composition of active component with danshensu and salvianolic acid B, it is characterized in that, weight ratio is 1: 3～3: 1.

2. compositions according to claim 1 is characterized in that, preferred weight ratio is 1: 2～2: 1.

3. compositions according to claim 1 is characterized in that, preferred weight ratio is 1: 1.5～1.5: 1.

4. compositions according to claim 1 is characterized in that, preferred weight ratio is 1: 1.2～1.2: 1.

5. compositions according to claim 1 is characterized in that, preferred weight ratio is 1: 1.

6. compositions according to claim 1 is characterized in that danshensu and salvianolic acid B exist with the form of salt.

7. compositions according to claim 6 is characterized in that, danshensu salt is tanshinol sodium, and salvianolic acid B salt is salvianolic acid B magnesium.

8. compositions according to claim 1 is characterized in that the purity of danshensu and salvianolic acid B is more than or equal to 80%.

9. compositions according to claim 6 is characterized in that, the purity of danshensu salt and salvianolic acid B salt is more than or equal to 80%.

10. compositions according to claim 1 is characterized in that the purity of danshensu and salvianolic acid B is more than or equal to 90%.

11. compositions according to claim 6 is characterized in that, the purity of danshensu salt and salvianolic acid B salt is more than or equal to 90%.

12. compositions according to claim 1 is characterized in that, the purity of danshensu and salvianolic acid B is more than or equal to 98%.

13. compositions according to claim 6 is characterized in that, the purity of danshensu salt and salvianolic acid B salt is more than or equal to 98%.

14. compositions according to claim 1 is characterized in that, is used to treat coronary heart disease, angina pectoris, ischemic cerebral thrombosis, atherosclerosis, myocardial ischemia or myocardial infarction, chronic viral hepatitis B hepatic fibrosis, fatty liver, senile dementia and cancer.

15. compositions according to claim 1 is characterized in that, processes aqueous injection, injectable powder, infusion solution, tablet, capsule, drop pill, granule, oral liquid, soft gelatin capsule, the watered pill, transdermal absorption formulation, controlled release preparation and slow releasing preparation.