CN104034900B - Novel method for determination of cytoprotection biological activity in traditional Chinese medicinal materials or traditional Chinese medicine preparations - Google Patents
Novel method for determination of cytoprotection biological activity in traditional Chinese medicinal materials or traditional Chinese medicine preparations Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
- G01N33/561—Immunoelectrophoresis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
- G01N33/5041—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects involving analysis of members of signalling pathways
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/91—Transferases (2.)
- G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- G01N2333/91205—Phosphotransferases in general
- G01N2333/9121—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases
Abstract
The invention discloses a method for determination of cytoprotection biological activity in traditional Chinese medicinal materials like leech and earthworm or traditional Chinese medicine preparations composed of leech and earthworm. In-vitro experiments prove that the extract of leech or earthworm can realize dose-related activation of a signal transduction pathway where the kinase ERK is located. Thus, cytoprotection activity in medicinal preparations containing the medicinal component leech or earthworm can be reflected by determining activation of kinases like ERK through protein immunoblotting, and the method provided by the invention has good repeatability.
Description
Technical field
The newfound a kind of biological activity that the present invention relates to Chinese medicine preparation or the quality determining method developed according to this activity, specifically, the present invention relates to newfound biological activity and the assay method thereof of a kind of Chinese medicine preparation containing Hirudo or Pheretima medicinal ingredient, the method can be used for the quality control of product or the determination of effective ingredient.
Background technology
In recent years, the blocking effect of mitogen activated protein kinases family (mitogen activated protein kinases, MAPKs) effect in adaptive cytoprotection is increasingly subject to pay attention to.MAPK enzyme belongs to tyrosine-kinase enzyme system, is that signal forwards intracellular transmitter to from extracellular.It can induce the quick expression of some irritability albumen; such as heat shock protein, superoxide dismutase, nitricoxide synthase, ATP sensitive potassium channel, Antioxidative Factors etc., and the increase that these endogenous effector molecules are expressed is the material base of delayed preconditioning protection.
Extracellular signal-regulated kinase (Extracellular signal-regulated kinases, ERKs) is one of member of MAPK family, is divided into ERK1 (p44) and two kinds of isoforms of ERK2 (p42).Extracellular different stimulated such as mitogen, somatomedin, angiotensin ò and oxidative stress etc. all can activate ERKs.The ERKs of activation is primarily involved in cell growth, differentiation and Anti-G value.(Guyton KZ, Liu Y, Gorospe M, Xu Q, the Holbrook NJ.Activation of mitogen-activated protein kinase by H such as Guyton202.Role in cell survival following oxidantinjury.J Biol Chem.1996Feb23；271 (8): 4138-42.) report, antioxidant stress injury can be improved cell survival rate by ERK.nullNeurotransmitter norepinephrine can strengthen (the Troadec JD of the protective effect to dopaminergic neuron by activating ERK1/2，Marien M，Mourlevat S，eT a1.Activation of the mitogen-activated protein kinase(ERK(1/2))signaling pathway by cyclic AMP potentiates the neuroprotective effect of the neurotransmitter noradrenaline on dopaminergic neurons.Mol Pharmacol.2002Nov；62 (5): 1043-52.).The activation of ERK also can promote growth (the Hansen Tv of PC12 cell, Rehfeld JF, Nielsen FC.KCl potentiates forskolin-induced PC12cell neurite outgrowth via protein kinase A and extracellular signal-regulated kinase signaling pathways.Neurosci Lett.2003Aug14；347 (1): 57-61.).Increasing research shows, ERK plays an important role in the cytoprotective that multiple pretreatment causes.Also it is reported that; ERK1/2 take part in medicine or Cardioprotection (the Fryer RM of Ischemic reperfusion induction; Hsu AK, Gross GJ.ERK and p38MAP kinase activation are components of opioid-induced delayed cardioprotection.Basic Res Cardiol.2001Apr；96 (2): 136-42.).Therefore, the cytoprotection of ERK is many.
Hirudo is the most traditional Chinese medicine, begins to be loaded in Shennong's Herbal, and the traditional Chinese medical science thinks that it has removing blood stasis, removing blood stasis, the effect stimulated the menstrual flow.Always it is used for treating lump in the abdomen, mass in the abdomen, blood stasis amenorrhea, traumatic injury.Modern medicine finds that Hirudo master, containing protein, contains 17 kinds of aminoacid, in addition including 8 kinds of aminoacid of needed by human, possibly together with 14 kinds of elements such as Zn, Mn, Fe, Co, Cr, Se, Mo, Ni.There is anticoagulant, reduce platelet aggregation rate, reduction blood fat, antiinflammatory action etc..Hirudin in Hirudo is to act on the strongest thrombin inhibitor, and it can not only stop fibrinogenic solidification, it is possible to stop the further blood stasis reaction of catalyzed by thrombin, such as the platelet response etc. of blood coagulation induction.Owing to hirudin can suppress free and on sludged blood thrombin effectively, therefore may be used to prevent formation and the extension of all kinds of thrombosis.
Pheretima is also Chinese medicine simply, is classified as low-grades at Shennong's Herbal, have heat clearing away arresting convulsion, dredging collateral, relieving asthma, effect of diuresis.For diseases such as high heat, coma, infantile convulsion tic, arthralgia, dyspnea and cough due to lung-heat, oliguria edema, hypertension after often parch.Li Shizhen (1518-1593 A.D.) referred to as has dredge the meridian passage, blood circulation promoting and blood stasis dispelling, prophylactic treatment cardiovascular and cerebrovascular disease effect in Compendium of Material Medica, has more than 40 kind of pharmacologic action in Compendium of Material Medica worm portion.Modern study shows, Pheretima contains abundant enzyme, and wherein fibrinoclase, Lumbrukinase, earthworm colloid enzyme have wide influence to internal blood coagulation and fibrinolytic system.Clinic such as Chinese medicine preparation containing Pheretima composition such as FUFANG DILONG JIAONANG, NAOXINTONG JIAONANG etc. is widely used in the preventing and treating of the diseases such as apoplexy.
Chinese medicine preparation containing Hirudo or Pheretima medical material has many-sided physiologically active such as anticoagulant, thrombolytic more, is used for the preventing and treating of cardiovascular and cerebrovascular vessel relevant disease clinically.In recent years, due to cytoprotective apply clinically increasingly extensive, the research to the cytoprotection of Chinese medicine also gets more and more.The a series of document report plurality of Chinese preparation containing Hirudo or Pheretima has obvious cell protection activity.By Hirudo and the Chinese medicine SHUXUETONG ZHUSHEYE of Pheretima two taste medical material prescription, research shows to suppress the neuronal apoptosis (Zhang Xuan of Focal Cerebral Ischemia (MCAO) rat model, the impact [J] on acute cerebral infarction in rats neuronal apoptosis and related gene expression of the Hu Changlin SHUXUETONG ZHUSHEYE. Jiangxi College of Traditional Chinese Medicine journal, 2005, (1): 58-60；); alleviate free radical mediated ischemic rat brain damage (cerebral protection [J] of Zhang Xuan, Wu Suning, Zhang Linting Shuxuetong injection on focal cerebral ischemia radical damage. practical cardio-cerebral-pulmo angiopathy magazine; 2007, (2): 94-95).Prescription contains the compound Chinese medicinal preparation NAOXINTONG JIAONANG of Pheretima; Brain Microvascular Endothelial simulation cerebral ischemia reperfusion injury is had significant protective effect (Liu Zhenquan, Xu Qiuping, Zhang Wensheng etc. [J]. Chinese Pharmaceutical Journal; 2007, (10): 733-736).But, due to the complexity of Chinese medicine ingredients, for the cell protection activity of this kind of Chinese medicine preparation, majority can only confirm activity, but can not specify its target spot or mechanism of action, does not the most well measure the extracorporeal biology method of this kind of activity.
Inventor have collected containing Hirudo, some Chinese medicine preparation of Pheretima medical material prescription, and is studied it.Find that this kind of Chinese medicine preparation has the kinase whose activity of active cell ERK; signal path associated protein or kinase target point to ERK kinases place are studied further; finding that EGFR, Src, MAPK, PKC, Akt, P13K equimolecular also can be activated, this is consistent with the cytoprotective associated signal paths described in accompanying drawing 1.
Summary of the invention
It is an object of the invention to overcome the deficiency of the existing Chinese medicine preparation cytoprotective biological activity determination method containing Hirudo or Pheretima medical material, it is provided that a kind of new Bioactivity detection method, to control the quality of product or can separate further and purification active substance.
What the present invention provided measures containing Hirudo or the Chinese medicine preparation cytoprotective biological activity determination method of Pheretima medical material, mainly comprises the steps that
(1) formulated need testing solution: for injection, can directly take injection as need testing solution, powder ampoule agent for injection can use physiological saline solution dissolve after as need testing solution, after solid preparation can use proper method to extract active component, with aseptic filtration after water, normal saline or buffer solution as need testing solution.
(2) cell is cultivated: use Tissue Culture Dish or cell bottle to cultivate cell, when reach 90-95% laminating spend time, Secondary Culture.
(3) test sample activates and prepares cell pyrolysis liquid: after passage cell reaches the laminating degree of 80-90%, uses serum-free medium to cultivate.After using the serum-free medium active cell 5～20min containing test sample, washing cell lysis.By cell pyrolysis liquid centrifuging and taking supernatant, carry out protein quantification mensuration.
(4) protein electrophoresis-immunoblotting assay: operate routinely, use ECL reagent detection signal, and quantitatively.
In described step (1), the test sample compound method of solid preparation is that precision weighs solid preparation 0.1～10g, water, normal saline or methanol, ethanol solution is used to extract, extracting mode can use immersion, heating extraction or supersound extraction, extracting solution is evaporated to be dried, with after water, normal saline or Tris buffer solution as need testing solution.
In described step (2), cell culture vessel uses the Tissue Culture Dish of 10cm diameter, it would however also be possible to employ Tissue Culture Flask.Trypsinization can be used according to cell type difference, it is also possible to need not when passing on.Cell cultivation refers to ATCC relevant cultivation explanation with passing on actual conditions.
The method that in described step (4), ECL test kit detection signal is quantitative can use and the signal on cellulose membrane carries out light-sensitive emulsion film developing, and then the gray scale to colour developing band is scanned integration acquisition.The equipment such as gel image analyser can also be used to be integrated quantitatively.With without supplying the solvent control gained signal value of reagent product as V1, the signal value of test sample gained is V2, then cytoprotective biological activity can represent with associated kinase activation rise rate:
Cell protection activity
Accompanying drawing explanation
Fig. 1: cytoprotective correlation molecule signal path schematic diagram during ischemical reperfusion injury
Fig. 2: SHUXUETONG ZHUSHEYE activates ERK kinase protein electrophoresis-western blot figure
Fig. 3: SHUXUETONG ZHUSHEYE activates Src kinase protein electrophoresis-western blot figure
Fig. 4: SHUXUETONG ZHUSHEYE activates Akt kinase protein electrophoresis-western blot figure
Fig. 5: SHUXUETONG ZHUSHEYE activates EGFR kinase protein electrophoresis-western blot figure
Fig. 6: NAOXINTONG activates ERK kinase protein electrophoresis-western blot figure
Detailed description of the invention
Following example are in order to illustrate the present invention, but are not limited to the scope of the present invention.SHUXUETONG ZHUSHEYE (main component Hirudo, Pheretima) in following example is provided by Mudanjiang Youbo Pharmaceutical Co., Ltd., and NAOXINTONG (main component Hirudo, Pheretima, Flos Carthami etc.) is provided by Shaanxi Buchang Pharmaceuticals Co., Ltd..If no special instructions, reagent is analytical pure.
Embodiment 1: SHUXUETONG ZHUSHEYE activation kinase whose to ERK
(1) cell is cultivated:
Ren sus domestica epithelial cell (LLC-PK1) is purchased from ATCC, uses DMEM culture medium and adds 10% hyclone, 100units/ml penicillin, 100 μ g/ml streptomycins, and 5%CO2 incubator is cultivated.LLC-PK1 cell is cultivated in the Tissue Culture Dish of 10cm diameter.When reach 90-95% laminating spend time, use trypsin digestion cell, and in 1:7 ratio Secondary Culture.
(2) Shu Xue-tong activates and prepares cell pyrolysis liquid:
SHUXUETONG ZHUSHEYE is provided by Mudanjiang Youbo Pharmaceutical Co., Ltd., lot number 11112512.After LLC-PK1 cell tryptase enzymic digestion, in 1: 7 ratio Secondary Culture in 6 orifice plates of 3.5cm.After cell reaches the laminating degree of 80-90%, use serum-free medium overnight incubation.The DMEM culture medium of serum-free is changed in the next morning.After 20min, change the DMEM serum-free medium (Shu Xue-tong presses 1: 10 dilution proportion) containing Shu Xue-tong.After 37 DEG C of active cell 10min of Shu Xue-tong, use ice-cold phosphate buffer wash cell.Then with ice-cold RIPA buffer (containing 1%NP-40,1% sodium deoxycholate, 150mMNaCl, 1mM EDTA, 1mM PMSF, 1mM vanadic acid sodium, 1mM NaF, 10g/ml aprotinin, 10g/ml leupeptin, 50mM Tris-HCl, pH7.4) cell lysis.For the hole of 3.5cm a diameter of in 6 orifice plates, every hole adds RIPA buffer 100 μ l.From Tissue Culture Dish, scrape cell and be collected in 1.5ml centrifuge tube.Centrifuge tube 15min is slowly rocked with cell lysis in Cool Room 4 DEG C.Then by cell pyrolysis liquid 14000rpm low-temperature centrifugation 15min.Transfer supernatant, in another clean centrifuge tube, carries out Lowry method protein quantification and measures.In each sample, average protein concentration is about 2-3mg/ml.
(3) protein electrophoresis-immunoblotting assay:
Preparation Tris/ glycine gels, containing 10%Acry/Bis in the separation gel of bottom.After cell pyrolysis liquid that protein content is 50 μ g and the mixing of 10 μ l5X sample-loading buffers, use RIPA buffer polishing to cumulative volume 50 μ l.Boiling water boils 5min.Sample uses SDS-PAGE to separate, and uses cellulose membrane transferring film.Film uses TBST buffer (containing 10mM Tris-HCl, 150mM NaCl, 0.1%Tween20, the pH8.0) room temperature containing 1%BSA, 1% skim milk to block 1 hour.Film uses 4 DEG C of overnight incubation of pERK (P-p44/42Thr202/Tyr204) anti-(Cell Signaling Technology company).TBST buffer washes film 3 times, each 10 minutes.Film uses goat anti-rabbit igg-HRP two anti-(Santa Cruze company) incubated at room 1h.TBST buffer washes film 3 times, each 10 minutes.Use ECL test kit detection pERK signal, and quantitatively.
For detecting the expression of ERK albumen, carry out film stripping.The IgG-HRP of the antibody of ERK (Santa Cruze company) and goat-anti rabbit (Santa Cruze company, article No. Cat#SC-2004) it is configured in the TBST buffer containing 3% defatted milk powder, using above-mentioned one, two antibody is the total ERK of probe in detecting.
Protein electrophoresis-western blot figure that ERK activates is shown in accompanying drawing 2.From accompanying drawing 2, after SHUXUETONG ZHUSHEYE 10 times dilution, ERK activating more than 3 times for reference solvent, SHUXUETONG ZHUSHEYE 50 times dilution activity is the most obvious.
Embodiment 2: SHUXUETONG ZHUSHEYE activation kinase whose to Src
Experimentation with embodiment 1, detects Src kinase whose activation situation after function cells 10min after Shu Xue-tong 10 times, 30 times of dilutions substantially, and it is the antibody for Src that immunoblotting uses.Protein electrophoresis-western blot figure that Src activates is shown in accompanying drawing 3.From accompanying drawing 3, after SHUXUETONG ZHUSHEYE 10 times dilution, obvious to Src Activation Activity, for 1.3 times of control solvent.
Embodiment 3: SHUXUETONG ZHUSHEYE activation kinase whose to Akt
Experimentation with embodiment 1, detects Akt kinase whose activation situation after function cells 10min after Shu Xue-tong 10 times dilution substantially, and it is the antibody for Akt that immunoblotting uses.Protein electrophoresis-western blot figure that Akt activates is shown in accompanying drawing 4.From accompanying drawing 4, act on 10 minutes after SHUXUETONG ZHUSHEYE 10 times dilution, obvious to Akt Activation Activity, for 1.7 times of control solvent.Embodiment 4: SHUXUETONG ZHUSHEYE activation kinase whose to EGFR
Experimentation with embodiment 1, detects Akt kinase whose activation situation after function cells 10min after Shu Xue-tong 10 times dilution substantially, and immunoblotting uses the antibody for EGFR.Protein electrophoresis-western blot figure that EGFR activates is shown in accompanying drawing 5.From accompanying drawing 5, act on 10 minutes after SHUXUETONG ZHUSHEYE 10 times dilution, obvious to EGFR Activation Activity, for 2.3 times of control solvent.
Embodiment 5: NAOXINTONG activation kinase whose to ERK
(1) cell is cultivated:
Ren sus domestica epithelial cell (LLC-PK1) is purchased from ATCC, uses DMEM culture medium and adds 10% hyclone, 100units/ml penicillin, 100 μ g/ml streptomycins, and 5%CO2 incubator is cultivated.LLC-PK1 cell is cultivated in the Tissue Culture Dish of 10cm diameter.When reach 90-95% laminating spend time, use trypsin digestion cell, and in 1: 7 ratio Secondary Culture.
(2) NAOXINTONG activates and prepares cell pyrolysis liquid:
NAOXINTONG JIAONANG is provided by Shaanxi Buchang Pharmaceuticals Co., Ltd., lot number 10094.Test liquid preparation method is as follows: takes capsule 20, pours out content and weigh.Taking content 2g, with 10% ethanol 30ml supersound extraction 3 times, each 10min, united extraction liquid, 40 DEG C of drying under reduced pressure, residue 5ml physiological saline solution is NAOXINTONG JIAONANG test liquid.After LLC-PKl cell tryptase enzymic digestion, in 1: 7 ratio Secondary Culture in 6 orifice plates of 3.Scm.After cell reaches the laminating degree of 80-90%, use serum-free medium overnight incubation.The DMEM culture medium of serum-free is changed in the next morning.After 20min, change the DMEM serum-free medium (NAOXINTONG presses 1: 10 dilution proportion) containing NAOXINTONG.After 37 DEG C of active cell 10min of NAOXINTONG, use ice-cold phosphate buffer wash cell.Then with ice-cold RIPA buffer (containing 1%NP-40,1% sodium deoxycholate, 150mM NaCl, 1mM EDTA, 1mM PMSF, 1mM vanadic acid sodium, 1mM NaF, 10g/ml aprotinin, 10g/ml leupeptin, 50mM Tris-HCl, pH7.4) cell lysis.For the hole of 3.5cm a diameter of in 6 orifice plates, every hole adds RIPA buffer 100 μ l.From Tissue Culture Dish, scrape cell and be collected in 1.5ml centrifuge tube.Centrifuge tube 15min is slowly rocked with cell lysis in Cool Room 4 DEG C.Then by cell pyrolysis liquid 14000rpm low-temperature centrifugation 15min.Transfer supernatant, in another clean centrifuge tube, carries out Lowry method protein quantification and measures.In each sample, average protein concentration is about 2-3mg/ml.(3) protein electrophoresis-immunoblotting assay:
Preparation Tris/ glycine gels, containing 10%Acry/Bis in the separation gel of bottom.After cell pyrolysis liquid that protein content is 50 μ g and the mixing of 10 μ l5X sample-loading buffers, use RIPA buffer polishing to cumulative volume 50 μ l.Boiling water boils 5min.Sample uses SDS-PAGE to separate, and uses cellulose membrane transferring film.Film uses TBST buffer (containing 10mM Tris-HCl, 150mM NaCl, 0.1%Tween20, the pH8.0) room temperature containing 1%BSA, 1% skim milk to block 1 hour.Film uses 4 DEG C of overnight incubation of pERK (P-p44/42Thr202/Tyr204) anti-(Cell Signaling Technology company).TBST buffer washes film 3 times, each 10 minutes.Film uses goat anti-rabbit igg-HRP two anti-(Santa Cruze company) incubated at room 1h.TBST buffer washes film 3 times, each 10 minutes.Use ECL test kit detection pERK signal, and quantitatively.For detecting the expression of ERK albumen, carry out film stripping.The IgG-HRP of the antibody of ERK (Santa Cruze company) and goat-anti rabbit (Santa Cruze company, article No. Cat#SC-2004) it is configured in the TBST buffer containing 3% defatted milk powder, using above-mentioned one, two antibody is the total ERK of probe in detecting.
Protein electrophoresis-western blot figure that ERK activates is shown in accompanying drawing 6.From accompanying drawing 6, after NAOXINTONG test liquid 10 times dilution, obvious to ERK Activation Activity, 5～15min reach to activate peak.
Claims (3)
1. the biological activity assay method of the Chinese medicine preparation containing Hirudo or Pheretima composition, it is characterised in that by detection ERK
The cytoprotective biological activity activating this Chinese medicine preparation of reflection of kinases place signal transduction pathway, the protein molecular detected includes
EGFR and/or Src and/or Akt, described detection method comprises the steps:
(1) preparation Chinese medicine preparation need testing solution: for injection, directly take injection as need testing solution, powder ampoule agent for injection
As need testing solution after using physiological saline solution to dissolve；For solid preparation, after using proper method to extract active component,
With aseptic filtration after water, normal saline or buffer solution as need testing solution；
(2) cell is cultivated: use Tissue Culture Dish or cell bottle to cultivate cell, when reach 90-95% laminating spend time, Secondary Culture；
(3) test sample activates and prepares cell pyrolysis liquid: after passage cell reaches the laminating degree of 80-90%, uses serum-free medium training
Support, after using the serum-free medium active cell 5～20min containing need testing solution, washing cell lysis, cell is cracked
Liquid centrifuging and taking supernatant, carries out protein quantification mensuration；
(4) protein electrophoresis-immunoblotting assay: operate routinely, use ECL reagent detection signal, and quantitatively.
Biological activity assay method the most according to claim 1, it is characterised in that tissue that cytoprotection relates to or
Organ has heart, kidney, liver, lung, cranial nerve and cerebrovascular.
Biological activity assay method the most according to claim 1, it is characterised in that the signal conduction of detection ERK kinases place is logical
The cell type used that activates on road is mammalian cell.
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| CN102507792A (en) * | 2011-11-17 | 2012-06-20 | 牡丹江友搏药业有限责任公司 | Quality detection method for Shuxuetong preparation |
| CN103604766A (en) * | 2013-11-25 | 2014-02-26 | 牡丹江友搏药业股份有限公司 | Biological quality control method of Shuxuetong injection |
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