CN101590060A - The composition and use thereof of ligustrazine and salvianolic acid B - Google Patents
The composition and use thereof of ligustrazine and salvianolic acid B Download PDFInfo
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Abstract
The invention provides a kind of is the pharmaceutical composition of active component with ligustrazine and salvianolic acid B, the purity of ligustrazine and salvianolic acid B is more than or equal to 80%, 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 and cancer etc.
Description
Technical field
The present invention relates to a kind of is the pharmaceutical composition of active component and at the pharmacology activity research for the treatment of cardiac and cerebral vascular diseases with ligustrazine 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.
Rhizoma Chuanxiong is a conventional Chinese medicine, and the beginning is stated from Shennong's Herbal, classifies middle product as.Chinese Pharmacopoeia (version in 2005) regulation Rhizoma Chuanxiong is the dry rhizome of samphire Rhizoma Chuanxiong Ligusticum chuanxiong Hort..Rhizoma Chuanxiong has blood-activating and qi-promoting, and functions such as wind-expelling pain-stopping are used for menoxenia, amenorrhea dysmenorrhea ， mass in the abdomen stomachache, the twinge of the breast side of body, tumbling and swelling, headache, rheumatic arthralgia.
GUANXINNING ZHUSHEYE is made up of Radix Salviae Miltiorrhizae and Rhizoma Chuanxiong, has functions such as blood circulation promoting and blood stasis dispelling, freeing vessels and nourishing heart, is used for angina pectoris [the Sanitation Ministry medicine standard, the 17 174 pages].
Salvianolic acid B is the main active ingredient in the Radix Salviae Miltiorrhizae, and ligustrazine is the main active in the Rhizoma Chuanxiong, and the two has many-sided pharmacological action, and identical pharmacological action is arranged, 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 may 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 salvianolic acid B
The chemical constitution of ligustrazine
With ligustrazine and salvianolic acid B be the pharmaceutical composition of active component 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 ligustrazine 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 ligustrazine and salvianolic acid B compositions is better than the pharmacologically active of ligustrazine 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, treats some and old and feeble relevant disease as prescription drugs in some countries of Europe, as 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 the protection of cell injury and anti-apoptotic or the like; the effective dose of salvianolic acid B is 0.007～0.7ug/ml; and EGb761 is 50～100ug/ml; differ 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 may 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 generation evolution 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, 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 by 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, may be relevant as the effect of tryptophan sample 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) test on the person 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 better than 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-prostaglandin F
1A (6-keto-PGF
1A), PGD
2(PGD
2) and the total growing amount of prostaglandin.Show that salvianolic acid B may be the main component of Radix Salviae Miltiorrhizae anti-liver injury, it improves the total growing amount of liver non-parenchymal cell prostaglandin (PGs) may 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, may play a role in the formation and development process of atherosclerosis (AS).Salvianolic acid B raises to the inductive endothelial permeability of VEGF 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, in the U937 of In vitro culture foam cell model, find, 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 has direct protective action to the rat capillary endothelium (CMEC) of tumor necrosis factor a (TNF-a) damage, its effect may 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 myocardial ischemia-reperfusion damage 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 may 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 may 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 whole blood viscosity, packed cell volume and aggregate index.
The pharmacological action of ligustrazine
Ligustrazine injection is used existing three more than ten years in China, be mainly used in ischemic heart desease such as coronary heart disease, angina pectoris, myocardial infarction, also be used for ischemic cerebrovascular, evident in efficacy, and non-evident effect, be subjected to doctors and patients' high praise deeply, become one of at present clinical Chinese medicine preparation the most commonly used.
One, cardiovascular pharmacological effect [Chinese microcirculation; 2003,7 (4): 257～259]
1, pharmaceutical research shows that ligustrazine has definite function of resisting myocardial ischemia.The myocardial ischemia-anoxemia that ligustrazine causes the rabbit injection of pituitrin has obvious resistant function, causes the dog experimental myocardial infarction to have to the ligation coronary artery to dwindle infarction size, alleviates lesion degree, reduces the effect of myocardial necrosis amount.Electron microscopic observation has the certain protection effect to the myocardial cell mitochondrion.Along with ischemic myocardiums such as open heart operations under thrombolytic therapy, coronary artery bypass, the extracorporeal circulation pour into the application of medical skill again, saved a large amount of myocardial ischemia patients' life, but perfusion itself also can cause myocardial damage and affect the treatment again.A large amount of pharmacological experiment proof ligustrazine can make ischemic myocardium avoid ischemical reperfusion injury.Ligustrazine can suppress ischemical reperfusion injury institute proarrhythmia, shorten the arrhythmia persistent period, reduce to quiver in the chamber and the incidence rate of chamber speed, reduce ST section behind the ischemical reperfusion injury raise, shorten hole rule recovery time, improve cardiac hemodynamic unusual, increase left ventricle intraventricular pressure peak value and maximum positive and negative rate of change thereof, suppress the anoxia _ reoxygenation damage the myocardial cell contracture, improve the damaging cells survival rate, suppress K in the myocardial cell
+/ Na
+The reduction of concentration ratio, alleviate the apoptosis that ischemical reperfusion injury causes.
2, the ligustrazine mechanism that resists myocardial ischemia has coronary artery dilating, protection arteria coronaria endothelium, microcirculation improvement, increase myocardial nutritional flow amount, antiplatelet aggregation and thrombosis, reduction blood viscosity, improves hemorheological property, reduces myocardial oxygen consumption, suppresses oxygen-derived free radicals, anti peroxidation of lipid, improves stream in the metabolism inhibitory state, retardance myocardial cell calcium of myocardial cell, regulates aspects such as outward potassium flow.(1) coronary artery dilating: ligustrazine expansion coronary vasodilator, increase coronary flow, improve myocardial oxygen delivery and protect heart.Ligustrazine shrinks dog arteria coronaria due to the endothelin-1 obvious antagonism.(2) protection arteria coronaria endothelium: vascular endothelial cell damage is not only atherosclerotic initiating agent, and the development of coronary heart disease is played an important role, and the protection vascular endothelial cell is the new way of this class disease of control.Ligustrazine improves the interior NO level of body and reduces ET level in the body by protection arteria coronaria endothelium, alleviates ischemia and reperfusion injury.(3) microcirculation improvement: ligustrazine can make microcirculation blood flow velocity and the open number of blood capillary increase.No matter the microcirculation disturbance that the Golden Hamster norepinephrine is caused all has clear improvement at aspects such as bore, flow velocity, flow and capillary vessel numbers, and is wherein the most obvious to the arteriole effect.(4) antiplatelet aggregation and thrombosis: ligustrazine has depolymerisation to the accumulative platelet of patients with coronary heart disease, and can reduce surface activity of blood platelet.Its mechanism of action is as follows: a: regulate TXA
2/ PGI
2System: TXA
2/ PGI
2Balance adjustment platelet function and thrombosis.Ligustrazine has the effect that suppresses the platelet arachidonic acid metabolic, can be by suppressing platelet T XA
2Produce, promote vascular endothelial cell to produce PGI simultaneously
2, keep TXA
2/ PGI
2Balance.B: " calcium antagonist " effect: ligustrazine can make platelet cAMP content raise nearly one times, and " calcium pump " on the activating cell film makes Ca in the platelet
2+Concentration reduces, blocking-up Ca
2+To platelet activation and prostaglandin metabolism.(5) reduce the blood viscosity: ligustrazine has the erythrocyte and the platelet surface electric charge of raising, improves erythrocyte deformability, promotes fibrinolytic, reduces blood viscosity, improves the hemorheological property effect.(6) reduce myocardial oxygen consumption: reduce the treatment that myocardial oxygen consumption helps ischemic coronary heart disease.Ligustrazine has negative frequency conversion effect, and the sinuatrial node perfusion that exsomatizes shows ligustrazine (1～2) * 10
-3G/ml concentration can make the pace-making frequency of knot Zhou Zilv fiber reduce rapidly, and is the negative frequency conversion effect of dose dependent.Ligustrazine has reduced myocardial oxygen consumption by negative chronotropic's property of this appropriateness.(7) suppress the oxygen-derived free radicals effect: the generation of ligustrazine by reducing oxygen-derived free radicals, directly remove oxygen-derived free radicals, improve that body is removed the oxygen-derived free radicals ability, the mechanism such as formation that reduce lipid peroxide have protective effect to ischemic myocardium.Confirmed all that in the clinical research of multiple zoopery and old myocardial infarction, open heart surgery with extracorporeal circulation ligustrazine can make that MDA obviously reduces in the ischemic myocardial tissue, and followed superoxide dismutase (SOD) vigor and glutathion peroxidase/lipid peroxide (GSH-Px/LPO) obviously to increase.(8) improve the metabolism inhibitory state of myocardial cell: ligustrazine can improve cultivates the myocardial cell anoxia when lacking sugar
3The H-leucine and
3The incorporation efficiency of H-uridine promotes protein, RNA to synthesize, and induces nitricoxide synthase mRNA to lack the expression of sugared myocardial cell in anoxia, thereby improves the metabolism inhibitory state of myocardial cell, strengthens the tolerance of cell to hypoxic-ischemic.(9) stream in the retardance calcium: block the expansible arteria coronaria of stream in the calcium, alleviate ischemic myocardium " calcium overload " infringement.Adopt full cell patch tongs technology to confirm that ligustrazine has the concentration dependent retarding effect to the flesh L-of guinea-pig ventricular type Calcium Current.Adult rat ventricular muscles L-type Calcium Current is also had the obvious suppression effect, and be concentration dependent and voltage-dependent, its mechanism may be brought into play effect for ligustrazine combines with the calcium channel of inactivated state with high-affinity.
Two, cerebrovascular pharmacological action [modern combination of Chinese and Western medicine magazine; 2006,15 (10): 1398～1399]
Ischemic cerebrovascular accounts for 70%～80% of whole cerebrovascular clinically, and ligustrazine is to the protective effect of cranial nerve tissue, and except vasodilator, inhibition thrombosis, also comprise: (1) improves erythrocyte deformability, reduces blood viscosity.(2) generation of lipid peroxide (LPO) in the minimizing rat ischemia cerebral tissue increases the content of superoxide dismutase (SOD) and glutathion peroxidase (GSH-Px), thereby alleviates radical damage.(3) suppress the c-jun protein expression, downward modulation cerebral ischemia-expression of c-fos when pouring into again, the expression of rise bcl-2 has neuroprotective.And ligustrazine energy and Ca
2+Channel receptor is combination reversibly, regulates Ca
2+Cell in flow, keep the mitochondrial integrity of neuron.Ligustrazine and bcl-2 may all act on the neuron linear plastochondria, the protection cerebral tissue, and the brain injury when alleviating cerebral ischemia re-pouring improves the toleration of neuron to hypoxic-ischemic.(4) can suppress Ca
2+Overload, ligustrazine blocking-up cell Ca
2+Interior stream, the spasm of releasing blood vessel reduces neuron and is poisoned to death, the cytoclasis that the blocking-up free radical causes.Ligustrazine also can be by suppressing Ca
2+Overload, synergism takes place with bcl-2 in the generation of minimizing free radical etc., also can bring into play anti-apoptotic effects by suppressing short apoptogene such as c-fos, p53 etc.(5) interleukin 8 (IL-8) level raises during cerebral ischemia re-pouring, and ligustrazine can suppress the synthetic of IL-8 and discharge, thus vicious cycle and chain reaction between blocking-up IL-8 and inflammatory reaction and the oxygen-derived free radicals.(6) quicken ischemia neurocyte HSP70 gene in the expression of transcribing with translation skill, and then make that HSP70 is synthetic to be increased, make neurocyte produce tolerance, thereby play protecting neuron from acute to ischemia.
Summary of the invention
1, the invention provides a kind of is the pharmaceutical composition of active component with ligustrazine and salvianolic acid B, and its weight ratio is 1: 10～10: 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, ligustrazine can be ligustrazine hydrochloride and ligustrazine phosphate, and salvianolic acid B can exist with the form of salt, is preferably sodium salt and magnesium salt.
3, in this pharmaceutical composition, the purity of ligustrazine and salvianolic acid B is more than or equal to 80%; The purity of preferred ligustrazine and salvianolic acid B is more than or equal to 90%; The purity of further preferred ligustrazine and salvianolic acid B is 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, ligustrazine of the present invention and salvianolic acid B can be with the preparations of any method, to preparation method without any restriction.
7, the present invention has carried out pharmacological testing, has proved with ligustrazine 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
Ligustrazine phosphate (composite): purchase in Beijing, Beijing pharmaceutcal corporation, Ltd; Batch number: 070707;
Quality standard: meet " Chinese pharmacopoeia version in 2005.
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 preparation method 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, filter (the 60-100 order of polyamide on the filtrate, 1kg) post, after having gone up each subacid water retting liquid,, wash chromatographic column then with water with the removal of impurity of 2-3% ethanol aqueous wash polyamide column, after alcohol cleaned, wash chromatographic column with 0.03% sodium bicarbonate aqueous solution, collect eluent, transfer eluent to make pH value less than 2 with hydrochloric acid, 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, collect 95% ethanol elution, be recycled to dried, dry powder A.Get dry powder A, add 15 times of amounts of acetone and dissolve, filter, reclaim filtrate, get dry powder B to doing; Get dry powder B, add 15 times of amounts of ethyl acetate and dissolve, filter, reclaim filtrate, get dry powder C to doing.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 ligustrazine and salvianolic acid B
Get ligustrazine 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 made every and is contained ligustrazine 40mg, the freeze-dried powder of salvianolic acid B 40mg.
Embodiment four
Preparation is the tablet of active component with ligustrazine and salvianolic acid B
Get ligustrazine 80g, salvianolic acid B 80g, starch 160g, dextrin (powder) 20g, mixing is made granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, sugar coating, promptly.
Embodiment five
Preparation is the freeze-dried powder of active component with ligustrazine and salvianolic acid B
Get ligustrazine 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 made every and is contained ligustrazine 13mg, the freeze-dried powder of salvianolic acid B 65mg.
Embodiment six
Preparation is the freeze-dried powder of active component with ligustrazine and salvianolic acid B
Get ligustrazine 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 made every and is contained ligustrazine 65mg, the freeze-dried powder of salvianolic acid B 13mg.
Embodiment seven
Preparation is the freeze-dried powder of active component with ligustrazine and salvianolic acid B
Get ligustrazine 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 made every and is contained ligustrazine 17mg, the freeze-dried powder of salvianolic acid B 68mg.
Embodiment eight
Preparation is the freeze-dried powder of active component with ligustrazine and salvianolic acid B
Get ligustrazine 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 made every and is contained ligustrazine 68mg, the freeze-dried powder of salvianolic acid B 17mg.
Embodiment nine
Preparation is the tablet of active component with ligustrazine and salvianolic acid B
Get ligustrazine 40g, salvianolic acid B 120g, starch 160g, dextrin (powder) 20g, mixing is made granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, sugar coating, promptly.
Embodiment ten
Preparation is the tablet of active component with ligustrazine and salvianolic acid B
Get ligustrazine 120g, salvianolic acid B 40g, starch 160g, dextrin (powder) 20g, mixing is made granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, sugar coating, promptly.
Embodiment 11
Preparation is the tablet of active component with ligustrazine and salvianolic acid B
Get ligustrazine 46g, salvianolic acid B 115g, starch 160g, dextrin (powder) 20g, mixing is made granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, sugar coating, promptly.
Embodiment 12
Preparation is the tablet of active component with ligustrazine and salvianolic acid B
Get ligustrazine 115g, salvianolic acid B 46g, starch 160g, dextrin (powder) 20g, mixing is made granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, sugar coating, promptly.
Embodiment 13
Preparation is the tablet of active component with ligustrazine and salvianolic acid B
Get ligustrazine 80g, salvianolic acid B 88g, starch 160g, dextrin (powder) 20g, mixing is made granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, sugar coating, promptly.
Embodiment 14
Preparation is the tablet of active component with ligustrazine and salvianolic acid B
Get ligustrazine 88g, salvianolic acid B 80g, starch 160g, dextrin (powder) 20g, mixing is made granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, sugar coating, promptly.
Embodiment 15
Preparation is the tablet of active component with ligustrazine and salvianolic acid B
Get ligustrazine 70g, salvianolic acid B 91g, starch 160g, dextrin (powder) 20g, mixing is made granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, sugar coating, promptly.
Embodiment 16
Preparation is the tablet of active component with ligustrazine and salvianolic acid B
Get ligustrazine 91g, salvianolic acid B 70g, starch 160g, dextrin (powder) 20g, mixing is made granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, sugar coating, promptly.
Embodiment 17
Preparation is the tablet of active component with ligustrazine and salvianolic acid B
Get ligustrazine 65g, salvianolic acid B 91g, starch 160g, dextrin (powder) 20g, mixing is made granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, sugar coating, promptly.
Embodiment 18
Preparation is the tablet of active component with ligustrazine and salvianolic acid B
Get ligustrazine 91g, salvianolic acid B 65g, starch 160g, dextrin (powder) 20g, mixing is made granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, sugar coating, promptly.
Embodiment 19
Preparation is the tablet of active component with ligustrazine and salvianolic acid B
Get ligustrazine 60g, salvianolic acid B 96g, starch 160g, dextrin (powder) 20g, mixing is made granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, sugar coating, promptly.
Embodiment 20
Preparation is the tablet of active component with ligustrazine and salvianolic acid B
Get ligustrazine 96g, salvianolic acid B 60g, starch 160g, dextrin (powder) 20g, mixing is made granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, sugar coating, promptly.
Embodiment 21
Preparation is the tablet of active component with ligustrazine and salvianolic acid B
Get ligustrazine 60g, salvianolic acid B 102g, starch 160g, dextrin (powder) 20g, mixing is made granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, sugar coating, promptly.
Embodiment 22
Preparation is the tablet of active component with ligustrazine and salvianolic acid B
Get ligustrazine 102g, salvianolic acid B 60g, starch 160g, dextrin (powder) 20g, mixing is made granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, sugar coating, promptly.
Embodiment 23
Preparation is the tablet of active component with ligustrazine and salvianolic acid B
Get ligustrazine 55g, salvianolic acid B 99g, starch 160g, dextrin (powder) 20g, mixing is made granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, sugar coating, promptly.
Embodiment 24
Preparation is the tablet of active component with ligustrazine and salvianolic acid B
Get ligustrazine 99g, salvianolic acid B 55g, starch 160g, dextrin (powder) 20g, mixing is made granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, sugar coating, promptly.
Embodiment 25
Preparation is the tablet of active component with ligustrazine and salvianolic acid B
Get ligustrazine 60g, salvianolic acid B 114g, starch 160g, dextrin (powder) 20g, mixing is made granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, sugar coating, promptly.
Embodiment 26
Preparation is the tablet of active component with ligustrazine and salvianolic acid B
Get ligustrazine 114g, salvianolic acid B 60g, starch 160g, dextrin (powder) 20g, mixing is made granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, sugar coating, promptly.
Embodiment 27
Preparation is the tablet of active component with ligustrazine and salvianolic acid B
Get ligustrazine 50g, salvianolic acid B 105g, starch 160g, dextrin (powder) 20g, mixing is made granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, sugar coating, promptly.
Embodiment 28
Preparation is the tablet of active component with ligustrazine and salvianolic acid B
Get ligustrazine 105g, salvianolic acid B 50g, starch 160g, dextrin (powder) 20g, mixing is made granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, sugar coating, promptly.
Embodiment 29
Preparation is the tablet of active component with ligustrazine and salvianolic acid B
Get ligustrazine 50g, salvianolic acid B 110g, starch 160g, dextrin (powder) 20g, mixing is made granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, sugar coating, promptly.
Embodiment 30
Preparation is the tablet of active component with ligustrazine and salvianolic acid B
Get ligustrazine 110g, salvianolic acid B 50g, starch 160g, dextrin (powder) 20g, mixing is made granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, sugar coating, promptly.
The embodiment hentriaconta-
Preparation is the tablet of active component with ligustrazine and salvianolic acid B
Get ligustrazine 50g, salvianolic acid B 115g, starch 160g, dextrin (powder) 20g, mixing is made granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, sugar coating, promptly.
Embodiment 32
Preparation is the tablet of active component with ligustrazine and salvianolic acid B
Get ligustrazine 115g, salvianolic acid B 50g, starch 160g, dextrin (powder) 20g, mixing is made granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, sugar coating, promptly.
Embodiment 33
Preparation is the tablet of active component with ligustrazine and salvianolic acid B
Get ligustrazine 45g, salvianolic acid B 108g, starch 160g, dextrin (powder) 20g, mixing is made granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, sugar coating, promptly.
Embodiment 34
Preparation is the tablet of active component with ligustrazine and salvianolic acid B
Get ligustrazine 108g, salvianolic acid B 45g, starch 160g, dextrin (powder) 20g, mixing is made granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, sugar coating, promptly.
Embodiment 35
Preparation is the tablet of active component with ligustrazine and salvianolic acid B
Get ligustrazine 45g, salvianolic acid B 117g, starch 160g, dextrin (powder) 20g, mixing is made granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, sugar coating, promptly.
Embodiment 36
Preparation is the tablet of active component with ligustrazine and salvianolic acid B
Get ligustrazine 117g, salvianolic acid B 45g, starch 160g, dextrin (powder) 20g, mixing is made granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, sugar coating, promptly.
Embodiment 37
Preparation is the tablet of active component with ligustrazine and salvianolic acid B
Get ligustrazine 40g, salvianolic acid B 108g, starch 160g, dextrin (powder) 20g, mixing is made granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, sugar coating, promptly.
Embodiment 38
Preparation is the tablet of active component with ligustrazine and salvianolic acid B
Get ligustrazine 108g, salvianolic acid B 40g, starch 160g, dextrin (powder) 20g, mixing is made granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, sugar coating, promptly.
Embodiment 39
Preparation is the tablet of active component with ligustrazine and salvianolic acid B
Get ligustrazine 40g, salvianolic acid B 112g, starch 160g, dextrin (powder) 20g, mixing is made granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, sugar coating, promptly.
Embodiment 40
Preparation is the tablet of active component with ligustrazine and salvianolic acid B
Get ligustrazine 112g, salvianolic acid B 40g, starch 160g, dextrin (powder) 20g, mixing is made granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, sugar coating, promptly.
Embodiment 41
Preparation is the tablet of active component with ligustrazine and salvianolic acid B
Get ligustrazine 40g, salvianolic acid B 116g, starch 160g, dextrin (powder) 20g, mixing is made granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, sugar coating, promptly.
Embodiment 42
Preparation is the tablet of active component with ligustrazine and salvianolic acid B
Get ligustrazine 116g, salvianolic acid B 40g, starch 160g, dextrin (powder) 20g, mixing is made granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, sugar coating, promptly.
Embodiment 43
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, filter (the 60-100 order of polyamide on the filtrate, 1kg) post, after having gone up each subacid water retting liquid,, wash chromatographic column then with water with the removal of impurity of 2-3% ethanol aqueous wash polyamide column, after alcohol cleaned, wash chromatographic column with 0.03% sodium bicarbonate aqueous solution, collect eluent, transfer eluent to make pH value less than 2 with hydrochloric acid, 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, collect 50% ethanol elution, be recycled to dried, dry powder A.Get dry powder A, add 15 times of amounts of acetone and dissolve, filter, reclaim filtrate, get dry powder B to doing; Get dry powder B, add 15 times of amounts of ethyl acetate and dissolve, filter, reclaim filtrate to doing, get dry powder C, content of danshinolic acid B is 91.16%.
Embodiment 44
The preparation of salvianolic acid B:
Radix Salviae Miltiorrhizae 3kg, add 20 times of amounts of aqueous hydrochloric acid solution (pH value is less than 1.5), flood 20 times, each 10 hours, filter, (the 60-100 order of polyamide on the filtrate, 1kg) post is behind upward intact each subacid water retting liquid, with the removal of impurity of 2-3% ethanol aqueous wash polyamide column, wash chromatographic column then with water, after alcohol cleaned, wash chromatographic column with 0.03% sodium bicarbonate aqueous solution, the collection eluent, transfer eluent to make pH value with hydrochloric acid less than 2, 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 70% ethanol is washed resin column, collects 70% ethanol elution, is recycled to dried, get dry powder A, content of danshinolic acid B is 82.95%.
Embodiment 45
Preparation is the tablet of active component with ligustrazine and salvianolic acid B (content of danshinolic acid B is 82.95%)
Get ligustrazine 80g, salvianolic acid B 80g, starch 160g, dextrin (powder) 20g, mixing is made granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, sugar coating, promptly.
Embodiment 46
Preparation is the freeze-dried powder of active component with ligustrazine and salvianolic acid B (content of danshinolic acid B is 91.16%)
Get ligustrazine 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 made in lyophilizing.
Embodiment 47
Preparation is the tablet of active component with ligustrazine and salvianolic acid B (content of danshinolic acid B is 82.95%)
Get ligustrazine 80g, salvianolic acid B 100g, starch 160g, dextrin (powder) 20g, mixing is made granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, sugar coating, promptly.
Embodiment 48
Preparation is the freeze-dried powder of active component with ligustrazine and salvianolic acid B (content of danshinolic acid B is 91.16%)
Get ligustrazine 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 made in lyophilizing.
Embodiment 49
Preparation is the tablet of active component with ligustrazine and salvianolic acid B (content of danshinolic acid B is 91.16%)
Get ligustrazine 40g, salvianolic acid B 108g, starch 160g, dextrin (powder) 20g, mixing is made granule, and is dry below 60 ℃, adds magnesium stearate 6g, is pressed into 1000, sugar coating, promptly.
Embodiment 50
Preparation is the freeze-dried powder of active component with ligustrazine and salvianolic acid B (content of danshinolic acid B is 82.95%)
Get ligustrazine 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 made in lyophilizing.
Pharmacological testing: medicine B is a salvianolic acid B, and purity is 99.35%, is provided by Beijing Keshilan Pharmaceutical Technology Co., Ltd.; Medicine C is a ligustrazine phosphate, and purity is 100.0%, purchases in Beijing, Beijing pharmaceutcal corporation, Ltd.
Test example one
The different proportionings with B of medicine C 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 be subjected to reagent thing and preparation
Medicine ligustrazine (representing with C), salvianolic acid B (representing with B) are provided by Beijing Keshilan Pharmaceutical Technology Co., Ltd..
Medicine C, B are made into C 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 C: 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 C group, medicine B group and C+B medicine group, and C+B medicine 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 see 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 every 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, the results are shown in 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 organizing the isolated rat heart coronary flow after the administration all has in various degree rising, and compares before the administration, and difference has statistical significance (P＜0.05 or P＜0.01); Each proportioning group has been compared significant difference (P＜0.05 or P＜0.01) with single with medicine C, B after the administration, and 1: 1 proportioning group of C+B medicine has the trend that is better than other group; The results are shown in 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 two medicines of C, B, be excellent with 1: 1 proportioning group.
Test example two
The different proportionings with B of medicine C are to the influence of mice normal pressure anoxia enduring
Summary: purpose: the normal pressure resisting oxygen lack of observing ligustrazine, salvianolic acid B.Method: adopt mice normal pressure anoxia enduring model.The result: ligustrazine compares with the blank group under 72mg/kg dosage, and the effect of obvious prolongation mice normal pressure hypoxia endurance time is arranged.In the experiment of two medicine drug combinations, compare with the blank group, 72mg/kg dosage group in 72mg/kg, 36mg/kg dosage group and 2: 1 proportionings of 1: 1 proportioning all prolongs mice normal pressure hypoxia endurance time in various degree, and rate elongation is respectively 35.96%, 22.0%, 19.0%.And, obviously be better than the effect (P＜0.05) of single 72mg/kg dosage group with ligustrazine by the effect of the 72mg/kg group of 1: 1 proportioning.Conclusion: ligustrazine has tangible resisting oxygen lack, and effect is more excellent during in 1: 1 ratio drug combination with salvianolic acid B.
1. experiment material
1.1 animal
The KM mice, male and female half and half, 22-24g.Quality certification SCXK (capital) 2005-0013 is provided by Institute of Experimental Animals, Chinese Academy of Medical Sciences.
1.2 medicine
Ligustrazine phosphate (representing with C), salvianolic acid B (representing with B) are provided by Beijing Keshilan Pharmaceutical Technology Co., Ltd..
Propranolol (10mg/ sheet), Tianjin Lisheng Pharmaceutical Co., Ltd. produces.
1.3 reagent
Vaseline, Tianjin petrochemistry trial (demonstration) plant.
Sodica calx, Shanghai City are received brightness dried reagent factory.
2. experimental technique
The influence of administration 2.1 C, B prescription are distinguished the flavor of to mice normal pressure anoxia enduring
120 of mices, female, hero half and half is divided into 10 groups at random by body weight, sex, 12 every group.Establish the blank group respectively, C, B medicine single medicinal material administration group, dosage is divided into 72mg/kg, 36mg/kg, 18mg/kg, 9mg/kg group, positive control drug propranolol 50mg/kg group.C, 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]:
2.2 the different proportionings of C, B medicine to the influence of mice normal pressure anoxia enduring and with single comparison with the C medicine
180 of mices, female, hero half and half is divided into 15 groups at random by body weight, sex, 12 every group.Establish blank group, C+B administration group, positive control drug propranolol 50mg/kg group and single respectively with C medicine 72mg/kg group.Administration group dosage is provided with: the C+B medicine is by 3 proportioning administrations, and each proportioning is respectively established four dosage 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.C medicine group sodium chloride with 0.9% before experiment is mixed with solution, and concentration is 3.6mg/ml.The C+B medicine is mixed with the solution of respective concentration with 0.9% sodium chloride before experiment, four dosage group 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
The influence of administration 3.1 C, B prescription are distinguished the flavor of to mice normal pressure anoxia enduring
C medicine high dose group, single intraperitoneal administration 7 days compares with the blank group, can obviously prolong mice normal pressure hypoxia endurance time, and rate elongation is 21.35% (P＜0.05).The results are shown in Table 1.
3.2C, the different proportionings of B medicine to the influence of mice normal pressure anoxia enduring and with single comparison with the C medicine
With the blank group relatively, all prolong mice normal pressure hypoxia endurance time in various degree in the 72mg/kg dosage group of 72mg/kg, 36mg/kg dosage group and 2: 1 proportionings of 1: 1 proportioning, rate elongation is respectively 35.96%, 22.0%, 19.0%.And, obviously be better than the effect (▲ P＜0.05) of single 72mg/kg dosage group with ligustrazine by the effect of the 72mg/kg group of 1: 1 proportioning.Propranolol can obviously prolong mice normal pressure hypoxia endurance time, and rate elongation is 65.5%.The results are shown in Table 2.
The administration of table 1C, B prescription flavor is to influence (x ± s) (n=12) of mice normal pressure anoxia enduring
Annotate: compare * P＜0.05, * * P＜0.01 with the blank group
The different proportionings of table 2C, B medicine are to influence (x ± s) (n=12) of mice normal pressure anoxia enduring
Annotate: compare * P＜0.05, * * P＜0.01 with the blank group; Compare ▲ P＜0.05 with the C group.
4, conclusion
During the administration of ligustrazine single, under the high dose mice normal pressure anoxia enduring time-to-live there is certain prolongation effect; And with salvianolic acid B in behind 1: 1 ratio administering drug combinations, the interaction energy that prolongs the mice anoxia enduring time-to-live obviously strengthens, and points out two medicines to have synergism, may be because due to two medicine effect links or the target spot difference.
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 C and B medicine is to the influence of rat heart muscle ischemia
Summary: purpose: observe medicine ligustrazine and salvianolic acid B when 1: 1 proportioning drug combination to the influence of rat heart muscle ischemia.Method: adopt ligation rat coronary artery anterior descending branch to cause myocardial infarction and ischemia model, administration 7 days.Result: 72mg/kg, 36mg/kg dosage group can alleviate degree of myocardial ischemia, with model group comparing difference remarkable (P＜0.01, P＜0.05).Conclusion: ligustrazine and salvianolic acid B drug combination can reduce the myocardial infarction degree, have tangible function of resisting myocardial ischemia.
1. purpose: observe of the influence of 1: 1 proportioning group of C and B medicine to the rat heart muscle ischemia
In the experiment of a stripped jar of stream heart and to the results suggest in the experiment of mice anoxia enduring, ligustrazine and salvianolic acid B effect when 1: 1 ratio is remarkable.Therefore, this experiment on above-mentioned two experiment basis, observed ligustrazine and salvianolic acid B two medicines when 1: 1 proportioning drug combination to the protective effect of rat heart muscle ischemia.
2. materials and methods
2.1 animal
Laboratory animal: adult healthy SD rat, male, body weight (260 ± 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 grouping and administration
Two kinds of medicines are respectively ligustrazine (representing with C) and salvianolic acid B (representing with B), are provided by Beijing Keshilan Pharmaceutical Technology Co., Ltd..
Rat is divided into 6 groups, 10 every group at random by the body weight grouping.
1) normal control group; 2) sham operated rats; 3) model group;
4) be subjected to reagent high dose group C+B=36mg/kg+36mg/kg
5) be subjected to dosage group C+B=18mg/kg+18mg/kg in the reagent
6) be subjected to reagent low dose group C+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 anterior descending coronary 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), recovers general breathing.
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
Influence to the rat myocardium block area:
Myocardial ischemia is obvious after the rat ligation anterior descending coronary, model group rat heart muscle necrotic area weight account for respectively left ventricle and whole-heartedly dirty weight 18.82%, 14.10%, be subjected to the reagent thing after 7 days, compare with model group, the myocardial ischemia situation of high, middle dosage group all has (P＜0.01 of improving in various degree; P＜0.05).See Table 1, table 2.
Table 1 be subjected to the reagent thing to the influence of rat myocardium block area (x ± s, N=10)
Table 2 be subjected to the reagent thing to the influence of rat myocardium block area (x ± s, N=10)
Annotate: compare * P＜0.05 with model group; * P＜0.01.
4. brief summary
Adopt ligation rat coronary artery anterior descending branch to cause myocardial infarction and ischemia model, irritate stomach and be subjected to reagent thing (1: 1 proportioning) 7 days (according to 72mg/kg, 36mg/kg, 18mg/kg dosage).The result shows: medicine can both alleviate degree of myocardial ischemia under 72mg/kg, 36mg/kg dosage, with remarkable (P＜0.01 of model group comparing difference; P＜0.05).
5. conclusion
Ligustrazine and salvianolic acid B be drug combination under 1: 1 proportioning, can alleviate the experimental mouse myocardial infarction area, has the Mus myocardial ischemia effect of the tangible Chinese People's Anti-Japanese Military and Political College.
Test example four
Ligustrazine and salvianolic acid B compositions are to the experimentation of focal cerebral ischemia in rats influence
Summary
Adopt the ferric chloride part to stick intraluminal middle cerebral artery occlusion in rats and cause the focal cerebral ischemia injury model,, inquire into and be subjected to the protective effect of reagent rat model by mensuration to rat model nervous symptoms and cerebral infarction scope.The result shows, the nervous symptoms of ligustrazine 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: ligustrazine and salvianolic acid B compositions can alleviate focal cerebral ischemia injury by suppressing cerebral thrombosis, and its curative effect is better than ligustrazine group and salvianolic acid B group.
Experiment purpose
Observe the influence of ligustrazine 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
Be subjected to reagent: C is a ligustrazine, and B is a salvianolic acid B, C: B=1 in the C+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.
Three, instrument: XTT stero microscope, Yunnan Optical Instruments Factory's product; AEG-220 type electronic analytical balance, Japanese Shimadzu company product; 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 4 groups at random by body weight, i.e. normal saline group, C group, B group, C+B group.Each treated animal is all in postoperative sublingual vein administration in 30 minutes, postoperative 2 hours and 23 hours intraperitoneal injection secondaries.Injected dose 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); right arm reclining is fixed; make 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, at the bone window that cheekbone and the close oral-lateral 1mm place of temporo squamosum joint make a diameter 2.5mm, clear up residue 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 ℃.
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 as 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.As bilateral resistance equity and strong, be designated as 0 fen; As 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, cerebellum 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
2HPO
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, account for the percentage ratio of full brain weight and Ipsilateral brain weight as the cerebral infarction scope with blocking tissue's weight.
Five, result
Above-mentioned experimental result represents with x ± s, and paired comparison t check between statistical test employing group the results are shown in Table 1 and table 2.
Table 1 is subjected to reagent to the influence of MCAT rat nervous symptoms (x ± s)
Table 2 is subjected to reagent to the influence of MCAT rat cerebral infarction scope (x ± s)
Annotate: compare with the normal saline group,
*P＜0.05,
*P＜0.01
Brief summary
As can be seen from the above table, the nervous symptoms of C+B, C, 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; And C+B, C, 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: C+B, C, B all can alleviate focal cerebral ischemia injury by suppressing cerebral thrombosis, and the curative effect of C+B group is better than C group and B group.
Claims (12)
1, a kind of is the pharmaceutical composition of active component with ligustrazine and salvianolic acid B, it is characterized in that weight ratio is 1: 10～10: 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, ligustrazine can be ligustrazine hydrochloride and ligustrazine phosphate, and salvianolic acid B can exist with the form of salt.
7, compositions according to claim 1 is characterized in that, salvianolic acid B salt is preferably sodium salt and magnesium salt.
8, compositions according to claim 1 is characterized in that, the purity of ligustrazine and salvianolic acid B is more than or equal to 80%.
9, compositions according to claim 1 is characterized in that, the purity of ligustrazine and salvianolic acid B is more than or equal to 90%.
10, compositions according to claim 1 is characterized in that, the purity of ligustrazine and salvianolic acid B is more than or equal to 98%.
11, compositions according to claim 1 is characterized in that, 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 and cancer.
12, compositions according to claim 1 is characterized in that, can be made into that said any dosage form can be made into injection, oral formulations and external preparation on the pharmaceutics; Injection comprises aqueous injection, injectable powder and infusion solution, and 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.
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| CNA2009101191999A CN101590060A (en) | 2008-05-26 | 2009-03-09 | The composition and use thereof of ligustrazine and salvianolic acid B |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104490883A (en) * | 2014-12-31 | 2015-04-08 | 四川川大华西药业股份有限公司 | Pharmaceutical composition used for treating cardiovascular and cerebrovascular diseases as well as preparation method and application of pharmaceutical composition |
| CN109157571A (en) * | 2018-11-20 | 2019-01-08 | 陈新忠 | A kind of drug and preparation method thereof for treating coronary atherosclerosis |
| CN110755440A (en) * | 2019-11-20 | 2020-02-07 | 中国海洋大学 | Application of ligustrazine and mannuronic acid composition in preparing medicine for treating thrombotic diseases and nervous system diseases |
-
2009
- 2009-03-09 CN CNA2009101191999A patent/CN101590060A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104490883A (en) * | 2014-12-31 | 2015-04-08 | 四川川大华西药业股份有限公司 | Pharmaceutical composition used for treating cardiovascular and cerebrovascular diseases as well as preparation method and application of pharmaceutical composition |
| CN104490883B (en) * | 2014-12-31 | 2017-06-16 | 四川川大华西药业股份有限公司 | It is a kind of to treat pharmaceutical composition of cardiovascular and cerebrovascular disease and its production and use |
| CN109157571A (en) * | 2018-11-20 | 2019-01-08 | 陈新忠 | A kind of drug and preparation method thereof for treating coronary atherosclerosis |
| CN110755440A (en) * | 2019-11-20 | 2020-02-07 | 中国海洋大学 | Application of ligustrazine and mannuronic acid composition in preparing medicine for treating thrombotic diseases and nervous system diseases |
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