CN103251677A - Tongmai oral liquid raw material medicine extract and preparation method and application thereof - Google Patents
Tongmai oral liquid raw material medicine extract and preparation method and application thereof Download PDFInfo
- Publication number
- CN103251677A CN103251677A CN2013102109907A CN201310210990A CN103251677A CN 103251677 A CN103251677 A CN 103251677A CN 2013102109907 A CN2013102109907 A CN 2013102109907A CN 201310210990 A CN201310210990 A CN 201310210990A CN 103251677 A CN103251677 A CN 103251677A
- Authority
- CN
- China
- Prior art keywords
- extract
- crude drug
- tongmai
- raw material
- preparation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Medicines Containing Plant Substances (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Abstract
The invention provides a Tongmai oral liquid raw material medicine extract which is characterized in that the ratio of the Tongmai oral liquid raw material medicines of radix salviae miltiorrhizae, radix puerariae and rhizoma ligustici chuanxiong is 1:1:1. The content weight ratio of the active ingredients in the raw material medicine extract is as follows: tanshinol:puerarin:ferulic acid:tanshinone IIA is (1.0-1.2):(1.0-1.2):(1.0-1.2):1.0. According to the Tongmai oral liquid raw material medicine extract provided by the invention, the Tongmai oral liquid raw material medicines are extracted by use of 50-70% ethanol solvent, each active ingredient can be effectively extracted, the extraction effect is the best, the utilization rate of the traditional Chinese medicine raw materials is improved, and the waste of production cost is reduced. Meanwhile, the pharmacological activity of the raw material medicine extract prepared by the method is obviously superior to that of the original preparation technology, and a piece of good news is brought to patients suffering cardiovascular and cerebrovascular diseases.
Description
Technical field
The present invention relates to TONGMAI KOUFUYE crude drug extract and its production and use.
Background technology
TONGMAI KOUFUYE is made up of Radix Salviae Miltiorrhizae, Rhizoma Chuanxiong, Radix Puerariae three flavor medicines, and the function blood circulation and channel invigorating is used for ischemic cardio cerebrovascular diseases, arteriosclerosis, cerebral thrombosis, cerebral ischemia, coronary heart disease, anginal treatment is recorded in the 20 in the Sanitation Ministry medicine standard Chinese traditional patent formulation preparation.TONGMAI KOUFUYE prescription classics, flavour of a drug refine, dosage is abundant, curative effect is sure, yet numerous reason causes its market share not satisfactory, has hindered the paces of growing into the big kind of Chinese patent medicine.
The side of promoting blood circulation has the effect of blood circulation and channel invigorating, is mainly used in treating ischemic cardio cerebrovascular diseases, arteriosclerosis, coronary heart disease etc. clinically.According to the literature, the effective ingredient of TONGMAI KOUFUYE is mainly the water soluble ingredient that three kinds of medical materials contain, as danshensu, ferulic acid, puerarin etc.Yet modern study shows that the effective substance of Treated with Radix Salviae Miltiorrhizae cardiovascular and cerebrovascular disease is mainly fat-soluble phenanthrenequione constituents (tanshinone IIA, cryptotanshinone etc.) and water miscible liposoluble ingredient (danshensu, salvianolic acid B etc.).Tanshinone IIA can increase coronary flow, improves the myocardial metabolism disorder that causes after the anoxia, thereby improves the ability of myocardium anoxia enduring.Danshensu has remarkable coronary artery dilator, increases coronary flow, reduces the effect of myocardial oxygen consumption and increase myocardial contraction.In addition, the effective ingredient that Rhizoma Chuanxiong contains mainly is divided three classes: be the nitrogen-containing compound of representative with the ligustrazine, be the organic acid composition of representative based on the benzene peptide class of ligustilide with the ferulic acid, wherein ligustrazine and ligustilide are soluble in organic solvents such as ethanol, and ferulic acid is soluble in water.Ligustrazine has coronary artery dilator, increases blood flow volume, the pharmacological action of lax vascular smooth muscle, and ferulic acid energy anticoagulant, antithrombotic, releasing vascular smooth muscle spasm, and can improve myocardial ischemia.
Therefore, the inventor draws preliminary conclusion from the prior art deduction: there is certain defective in the existing preparation technology of TONGMAI KOUFUYE, and it has only emphasized the water soluble ingredient of three kinds of medical materials, and has ignored fat-soluble effective ingredient such as tanshinone IIA, ligustilide.In order to improve the utilization rate of Chinese material medicine resource, strengthen the drug activity of original preparation, demand a kind of new method for extracting of TONGMAI KOUFUYE crude drug urgently.
Summary of the invention
The object of the present invention is to provide the better TONGMAI KOUFUYE crude drug of a kind of drug activity extract and its production and use.
The invention provides a kind of TONGMAI KOUFUYE crude drug extract, wherein, TONGMAI KOUFUYE crude drug proportioning is: Radix Salviae Miltiorrhizae: Radix Puerariae: Rhizoma Chuanxiong=1:1:1; In the described crude drug extract, the active constituent content weight ratio is as follows: danshensu: puerarin: ferulic acid: tanshinone=(1.0～1.2): (1.0～1.2): (1.0～1.2): 1.0.
Further, in the described crude drug extract, contain the effective ingredient of following percentage by weight:
Danshensu 1.7～2.57%, puerarin 1.7～2.7%, ferulic acid 1.5～2.6%, tanshinone 1.5～2.6%.
A nearlyer step ground, described crude drug extract, after high performance liquid chromatography detected, chromatogram was as shown in Figure 1; Chromatographic condition is as follows:
Chromatographic column: octadecyl silane post;
Detect wavelength: 280nm;
Column temperature: 30 ° of C;
Flow velocity: 1.0ml/min;
Mobile phase: with acetonitrile-0.1% glacial acetic acid aqueous solution gradient elution, elution program is as follows:
The present invention also provides above-mentioned TONGMAI KOUFUYE crude drug preparation method of extract, and it comprises following operating procedure:
(1) weighting raw materials by weight ratio: Radix Salviae Miltiorrhizae, Rhizoma Chuanxiong, Radix Puerariae;
(2) with the crude drug mixing, with 50～70%v/v ethanol extraction, merge extractive liquid, behind the recovery solvent, namely gets TONGMAI KOUFUYE crude drug extract.
Further, concentration of alcohol is 60～70%v/v.
Preferably, concentration of alcohol is 70%v/v.
Wherein, the concrete technology of ethanol extraction is as follows: add 20～40 times of volume of ethanol of crude drug gross mass, reflux, extract, 3 times, each 1 hour.
Further, the ethanol volume is 30 times of crude drug gross mass.
Concentration of alcohol of the present invention is all represented with volume ratio, but, if in large-scale production process, for convenient configuration solvent, can weight ratio be unit.
The present invention also provides the purposes of above-mentioned TONGMAI KOUFUYE crude drug extract in the medicine of preparation prevention or treatment myocardial ischemia or cerebral ischemia.
Further, described medicine is the medicine that improves myocardial damage due to the myocardial ischemia.
Further, described medicine is the medicine that improves brain tissue impairment due to the cerebral ischemia.
Adopt 50～70% alcohol solvents that the TONGMAI KOUFUYE crude drug is extracted among the present invention, can effectively extract various active component, extraction effect is best, has improved the utilization rate of raw material of Chinese medicine, has reduced the waste of production cost.Simultaneously, the crude drug extract that this method prepares, drug activity obviously is better than former preparation process, and the patient has brought Gospel for cardiovascular and cerebrovascular disease.
Description of drawings
The HPLC-DAD chromatogram of Fig. 1 danshensu reference substance (A) and need testing solution (B)
The HPLC-DAD chromatogram of Fig. 2 puerarin reference substance (A) and need testing solution (B)
The HPLC-DAD chromatogram of Fig. 3 ferulic acid reference substance (A) and need testing solution (B)
The HPLC-DAD chromatogram of Fig. 4 ligustrazine hydrochloride reference substance (A) and need testing solution (B)
The HPLC-DAD chromatogram of Fig. 5 salvianolic acid B reference substance (A) and need testing solution (B)
The HPLC-DAD chromatogram of Fig. 6 tanshinone reference substance (A) and need testing solution (B)
The HPLC-DAD chromatogram of Fig. 7 mixing reference substance (A) and need testing solution (B)
The HPLC-DAD chromatogram of Fig. 8 Rhizoma Chuanxiong medical material (A) and need testing solution (B)
The HPLC-DAD chromatogram of Fig. 9 Radix Puerariae medical material (A) and need testing solution (B)
The HPLC-DAD chromatogram of Figure 10 red rooted salvia (A) and need testing solution (B)
Figure 11 extraction process 2(A) and extraction process 1(B) the HPLC-DAD chromatogram
Extraction process 2(B) and the HPLC-DAD chromatogram of FUFANG DANSHEN PIAN (C) Figure 12 extraction process 1(A),
Extraction process 2(B) and the HPLC-DAD chromatogram of the former technology of TONGMAI KOUFUYE (C) Figure 13 extraction process 1(A),
The chromatogram of Figure 14 the present invention 60% alcohol extraction process products therefrom
The specific embodiment
The preparation of embodiment 1 crude drug extract of the present invention
Get Radix Salviae Miltiorrhizae, Rhizoma Chuanxiong, each 10g(1:1:1 of Radix Puerariae medical material), mixing adds 60% ethanol 300ml, and reflux 1 hour repeats to extract three times, and extracting solution filters, and merges, and filtrate recycling ethanol also is concentrated into extractum, namely gets the crude drug extract.
The preparation of embodiment 2 crude drug extracts of the present invention
Get Radix Salviae Miltiorrhizae, Rhizoma Chuanxiong, each 100g(1:1:1 of Radix Puerariae medical material), mixing adds 55% ethanol 4000ml, and reflux 1 hour repeats to extract three times, and extracting solution filters, and merges, and filtrate recycling ethanol also is concentrated into extractum, namely gets the crude drug extract.
The preparation of embodiment 3 crude drug extracts of the present invention
Get Radix Salviae Miltiorrhizae, Rhizoma Chuanxiong, each 100g(1:1:1 of Radix Puerariae medical material), mixing adds 65% ethanol 200ml, and reflux 1 hour repeats to extract three times, and extracting solution filters, and merges, and filtrate recycling ethanol also is concentrated into extractum, namely gets the crude drug extract.
The preparation of embodiment 4 crude drug extracts of the present invention
Get Radix Salviae Miltiorrhizae, Rhizoma Chuanxiong, each 10g(1:1:1 of Radix Puerariae medical material), mixing adds 50% ethanol 200ml, and supersound extraction 0.5 hour repeats to extract three times, and extracting solution filters, and merges, and filtrate recycling ethanol also is concentrated into extractum, namely gets the crude drug extract.
The preparation of embodiment 5 crude drug extracts of the present invention
Get Radix Salviae Miltiorrhizae, Rhizoma Chuanxiong, each 10g(1:1:1 of Radix Puerariae medical material), mixing adds 70% ethanol 400ml, normal temperature dipping 5 hours, repercolation extracts, and extracting solution filters, and merges, and filtrate recycling ethanol also is concentrated into extractum, namely gets the crude drug extract.
The screening of embodiment 6 preparation methoies of the present invention
1. test apparatus and material
1.1 instrument, reagent
Agilent 1200 series of high efficiency chromatograph of liquid (U.S. Agilent company), configuration: quaternary pump (G1354A), online vacuum degassing machine (G1322A/G1379B), standard automatic sampler (G1329A), diode array detector (G1315B/C), instrument control and data handling system (Agilent Chemstation), the HT-340K column oven.
Sartorius BP121s electronic balance (Beijing Sai Duolisi scientific instrument company limited); AS10200 ultrasonic cleaner (Tianjin Ao Tesaisi Instr Ltd.); W201 thermostat water bath (Shen, Shanghai is along bio tech ltd); The excellent general ultrapure water machine of ULUP-I-10T (Chengdu Ultra Pure Science ﹠ Technology Co., Ltd);
Acetonitrile is chromatographically pure (Fisher), and water is ultra-pure water, and all the other reagent are analytical pure.
1.2 reagent
Rhizoma Chuanxiong, Radix Puerariae and red rooted salvia are all purchased the international commerce and trade city lotus pond Chinese crude drug specialized market in Chengdu.
TONGMAI KOUFUYE purchases in the big pharmacy of Chengdu Bao Yuantang that (manufacturer:, FUFANG DANSHEN PIAN is purchased in the happy big pharmacy of Chengdu Jinniu District Rong (manufacturer: Guangzhou Baiyunshan Heji Huangpu Chinese Medicine Co., Ltd.) Hunan Central South Kelun Medicine Co., Ltd.).
Danshensu reference substance (lot number: D-014-120706); Salvianolic acid B reference substance (lot number: D-012-120629) available from Chengdu Rui Fensi bio tech ltd.
Ferulic acid reference substance (lot number: 0773-9708, confession assay usefulness), puerarin reference substance (lot number: 0752-9806, confession assay usefulness), tanshinone reference substance (lot number: 10766-200314, for assay with) and ligustrazine hydrochloride reference substance (lot number: 110817-200305, for assay with) all available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute;
2. method and result
2.1 sample preparation
(1) former technology:
Get Radix Salviae Miltiorrhizae, Radix Puerariae and each 10g(1:1:1 of Rhizoma Chuanxiong medical material), more than three flavors, decoct with water secondary, 1.5 hours for the first time, 1 hour for the second time, collecting decoction filtered, filtrate is concentrated into relative density 1.18～1.22 (50 ℃), puts coldly, adds ethanol and makes and contain alcohol amount and reach 65%.Cold preservation 48 hours filters, and filtrate recycling ethanol is not to there being the alcohol flavor, and to be concentrated into relative density be 1.10～1.14 (50 ℃) (extract A of promoting blood circulation).
(2) extraction process 1:
New technology is 1.: get Radix Salviae Miltiorrhizae, Radix Puerariae and each 10g(1:1:1 of Rhizoma Chuanxiong medical material), mixing adds ethanol 300mL, reflux 1h, extracting solution filters, and filtrate recycling ethanol also is concentrated into an amount of.Medicinal residues add 40% ethanol 300mL, reflux 1 hour, and extracting solution filters, and filtrate recycling ethanol also is concentrated into an amount of.Medicinal residues add water 300mL again, decoct 2 hours, and decocting liquid filters, and evaporate to dryness merges three kinds of extractum, and is standby.(extract B 1 of promoting blood circulation).
New technology is 1.: get Radix Salviae Miltiorrhizae, Radix Puerariae and each 10g(1:1:1 of Rhizoma Chuanxiong medical material), mixing adds ethanol 300mL, reflux 1h, extracting solution filters, and filtrate recycling ethanol also is concentrated into an amount of.Medicinal residues add 50% ethanol 300mL, reflux 1 hour, and extracting solution filters, and filtrate recycling ethanol also is concentrated into an amount of.Medicinal residues add water 300mL again, decoct 2 hours, and decocting liquid filters, and evaporate to dryness merges three kinds of extractum, and is standby.(extract B 2 of promoting blood circulation).
New technology is 1.: get Radix Salviae Miltiorrhizae, Radix Puerariae and each 10g(1:1:1 of Rhizoma Chuanxiong medical material), mixing adds ethanol 300mL, reflux 1h, extracting solution filters, and filtrate recycling ethanol also is concentrated into an amount of.Medicinal residues add 60% ethanol 300mL, reflux 1 hour, and extracting solution filters, and filtrate recycling ethanol also is concentrated into an amount of.Medicinal residues add water 300mL again, decoct 2 hours, and decocting liquid filters, and evaporate to dryness closes three kinds of extractum, and is standby.(extract B 3 of promoting blood circulation).
(3) extraction process 2:
New technology is 2.: get Radix Salviae Miltiorrhizae, Rhizoma Chuanxiong, each 10g(1:1:1 of Radix Puerariae medical material), mixing adds 50% ethanol 300mL, and reflux 1 hour repeats to extract three times, and extracting solution filters, and merges, and filtrate recycling ethanol also is concentrated into extractum (extract C 1 of promoting blood circulation).
New technology is 2.: get Radix Salviae Miltiorrhizae, Rhizoma Chuanxiong, each 10g(1:1:1 of Radix Puerariae medical material), mixing adds 60% ethanol 300mL, and reflux 1 hour repeats to extract three times, and extracting solution filters, and merges, and filtrate recycling ethanol also is concentrated into extractum (extract C 2 of promoting blood circulation).
New technology is 2.: get Radix Salviae Miltiorrhizae, Rhizoma Chuanxiong, each 10g(1:1:1 of Radix Puerariae medical material), mixing adds 70% ethanol 300mL, and reflux 1 hour repeats to extract three times, and extracting solution filters, and merges, and filtrate recycling ethanol also is concentrated into extractum (extract C 3 of promoting blood circulation).
2.2 the investigation of chromatographic condition
2.2.1 the selection of mobile phase
Flow phase system such as acetonitrile-water, methanol-0.1% glacial acetic acid, acetonitrile-0.1% glacial acetic acid have mainly been examined or check.The result shows that acetonitrile-0.1% glacial acetic acid system is best, and the separating degree of main chromatographic peak is improved, and peak shape is good, so finally determine flow phase system is: A pump: acetonitrile; B pump: 0.1% glacial acetic acid aqueous solution.2.2.2 the selection of chromatographic column
Investigated Welchrom C
18Chromatographic column (4.6mm * 250mm, 5 μ m) and Xtimate
TMC
18Chromatographic column (4.6mm * 250mm, 5 μ m) uses two kinds of chromatographic columns that same sample is analyzed by the chromatographic condition of drafting, and according to concrete separation case, finally selects Xtimate
TMC
18Chromatographic column.
2.2.3 the optimization of condition of gradient elution
Behind the composition of having determined mobile phase, investigated the gradient elution program.Being initial ratio with 10% acetonitrile, is the end ratio with 75% acetonitrile.Separating degree with main chromatographic peak is evaluation criterion, has finally determined best condition of gradient elution.
2.2.4 detect the selection of wavelength
Adopt diode array detector (DAD) to do full wavelength scanner, investigate the finger printing under the different absorbing wavelength.With reference to corresponding document, high spot reviews the chromatogram characteristic at 254nm, 280nm and 320nm place.The result shows 254nm place part chromatographic peak (as ferulic acid) disappearance, and there is disappearance at 320nm place back segment chromatographic peak (as tanshinone).And have uv absorption preferably at each composition of 280nm place, and chromatograph information is the abundantest, and therefore selecting 280nm is the detection wavelength of finger printing.
2.2.5 the final chromatographic condition that adopts
According to above-mentioned result of study, determine that optimum chromatogram condition is: Xtimate
TMC
18Chromatographic column (4.6mm * 250mm, 5 μ m); Detect wavelength: 280nm; Column temperature: 30 ° of C; Flow velocity: 1.0mL/min; Sample size: 10 μ L; Sampling time: 60min; Mobile phase: A is acetonitrile mutually, and B is 0.1% glacial acetic acid aqueous solution mutually, carries out the binary gradient elution, and elution program sees Table 1.
Table 1 gradient elution program
2.3 the preparation of need testing solution
(1) extraction process 1:
Get the extract B 1～3 of promoting blood circulation, the accurate title, decide, and places conical flask, the accurate methanol solution 50mL that adds, close plug claims to decide weight, supersound extraction 30min takes out, and puts cold, claim again to decide weight, supply the weight that subtracts mistake with methanol, shake up, filter, get subsequent filtrate, cross 0.45 μ m microporous filter membrane, namely get need testing solution.
(2) extraction process 2:
Get the extract C 1～3 of promoting blood circulation, the accurate title, decide, and places conical flask, the accurate methanol solution 50mL that adds, close plug claims to decide weight, supersound extraction 30min takes out, and puts cold, claim again to decide weight, supply the weight that subtracts mistake with methanol, shake up, filter, get subsequent filtrate, cross 0.45 μ m microporous filter membrane, namely get need testing solution.
(3) TONGMAI KOUFUYE:
Ginseng is got the extract A of promoting blood circulation of former technology preparation,
Place conical flask, the accurate methanol solution 50mL that adds, close plug claims decide weight, supersound extraction 30min takes out, and puts coldly, claims decide weight again, supplies the weight that subtracts mistake with methanol, shakes up, subsequent filtrate is got in filtration, 0.45 μ m microporous filter membrane, namely get need testing solution.
3. the qualitative identification of chromatographic peak and signal belong to
3.1 carrying out signal, the reference substance contrast differentiates
Need testing solution with " extraction process 1 " is object of study.
The preparation of reference substance solution: it is an amount of to get danshensu, puerarin, salvianolic acid B, ligustrazine hydrochloride, ferulic acid, tanshinone reference substance, adds methanol respectively and makes certain density solution, namely gets each reference substance solution.Carry out the qualitative identification of need testing solution chromatographic peak under above-mentioned chromatographic condition, sample size is 10 μ L.The qualification result difference is Fig. 1-Fig. 7 of face as follows.
(1) danshensu
The result: the 5.780min peak of need testing solution (extraction process 1) HPLC collection of illustrative plates is qualitative to be danshensu.(2) puerarin
The result: the 11.807min peak of need testing solution (extraction process 1) HPLC collection of illustrative plates is qualitative to be puerarin.(3) ferulic acid
The result: the 19.673min peak of need testing solution (extraction process 1) HPLC collection of illustrative plates is qualitative to be ferulic acid.(4) ligustrazine hydrochloride
The result: do not detect ligustrazine hydrochloride in the need testing solution (extraction process 1), this may be because the too low detectability that do not reach of this composition content in need testing solution.
(5) salvianolic acid B
The result: the peak shape of salvianolic acid B is short and stout, asymmetric in the need testing solution (extraction process 1).Consult pertinent literature, find that this is relevant with the acid that mobile phase adds.Therefore, in order to take into account other four compositions of qualitative, quantitative, salvianolic acid B is given up in this research, does not do analysis.
(6) tanshinone
The result: the 51.942min peak of need testing solution (extraction process 1) HPLC collection of illustrative plates is qualitative to be tanshinone.
(7) mix reference substance
The result: the 5.780min peak of need testing solution (extraction process 1) HPLC collection of illustrative plates is qualitative, and to be that danshensu, 11.807min peak are qualitative be tanshinone for ferulic acid, 51.942min peak are qualitative for puerarin, 19.673min peak are qualitative.
3.2 the signal ownership is carried out in the contrast of single medical material
The preparation of Rhizoma Chuanxiong medical material need testing solution: get Rhizoma Chuanxiong medicinal powder 1.0g, the accurate title, decide, and adds 70% methanol solution 50mL, and supersound extraction 30min under the room temperature filters, and gets subsequent filtrate, crosses 0.45 μ m microporous filter membrane, namely gets Rhizoma Chuanxiong medical material need testing solution.
The preparation of Radix Puerariae medical material need testing solution: get Radix Puerariae medicinal powder 1.0g, the accurate title, decide, and adds 70% methanol solution 50mL, and supersound extraction 30min under the room temperature filters, and gets subsequent filtrate, crosses 0.45 μ m microporous filter membrane, namely gets Radix Puerariae medical material need testing solution.
The preparation of red rooted salvia need testing solution: get red rooted salvia powder 1.0g, the accurate title, decide, and adds 70% methanol solution 50mL, and supersound extraction 30min under the room temperature filters, and gets subsequent filtrate, crosses 0.45 μ m microporous filter membrane, namely gets the red rooted salvia need testing solution.
(1) need testing solution (extraction process 1) and the contrast of Rhizoma Chuanxiong medical material
Result: mainly contain 5 peaks in need testing solution (extraction process 1) the HPLC collection of illustrative plates and come from the Rhizoma Chuanxiong medical material, comprise the benzene peptide constituents (see figure 8) of low polarity such as ferulic acid and ligustilide.
(2) need testing solution (extraction process 1) and the contrast of Radix Puerariae medical material
Result: mainly contain 4 peaks in need testing solution (extraction process 1) the HPLC collection of illustrative plates and come from the Radix Puerariae medical material, as the puerarin (see figure 9).
(3) need testing solution (extraction process 1) and red rooted salvia contrast
Result: mainly contain 5 class peaks in need testing solution (extraction process 1) the HPLC collection of illustrative plates and come from red rooted salvia, comprise danshensu, salvianolic acid constituents, tanshinone and several liposoluble constituent (see figure 10).
4. the comparison and analysis of TONGMAI KOUFUYE HPLC collection of illustrative plates under the different extraction process conditions
4.1 intuitive analysis
(1) comparison of extraction process 1 and extraction process 2
The result: the chromatogram profile of extraction process 1 and technology 2 is closely similar.Therefore extraction process 1 has namely kept the water soluble ingredient of three flavor medical materials with ethanol-40～60% alcohol-water reflux, extract, respectively, has extracted liposoluble constituent again, and extraction process 2 is with 50～70% alcohol reflux three times.From the result of Figure 11, extraction process 1 and extraction process 2 difference are little, have all obtained good extraction effect.(2) extraction process 1, extraction process 2 and FUFANG DANSHEN PIAN are relatively
As can be seen from Figure 12, extraction process 1, extraction process 2 have similarity with the chromatogram of FUFANG DANSHEN PIAN, all contain the effective ingredient that contains in the red rooted salvias such as danshensu, salvianolic acid, tanshinone.Certainly, because the prescription difference of FUFANG DANSHEN PIAN, all the other chromatographic peaks have tangible difference.But by three's comparative study, can find that the extraction effect of extraction process 1, technology 2 and FUFANG DANSHEN PIAN is the same, all can obtain the fat-soluble and water soluble ingredient in the red rooted salvia.
(3) comparison of extraction process 1, extraction process 2 and TONGMAI KOUFUYE
As can be seen from Figure 13, the chromatogram of TONGMAI KOUFUYE and extraction process 1 and extraction process 2 has evident difference.Danshensu and content of puerarin and other two groups of difference are little in the TONGMAI KOUFUYE, but ferulaic acid content is obviously lower.In addition, the second half section chromatographic peak of TONGMAI KOUFUYE lacks substantially, shows liposoluble constituents such as not containing tanshinone, cryptotanshinone, ligustilide.Obviously, the preparation technology of this and TONGMAI KOUFUYE is closely bound up.TONGMAI KOUFUYE mainly adopts decocting to boil technology, has therefore extracted the water soluble ingredient in Radix Salviae Miltiorrhizae, Radix Puerariae and the Rhizoma Chuanxiong medical material, and does not extract the liposoluble constituent of three kinds of medical materials.
The chromatographic peak of extraction process 1 and extraction process 2 is the most comprehensive, has comprised water soluble ingredient and the liposoluble constituent of three kinds of medical materials.Wherein liposoluble constituent mainly comes from Rhizoma Chuanxiong and red rooted salvia, the effective ingredient such as tanshinone that benzene peptide effective constituents such as ligustilide that contains as the Rhizoma Chuanxiong medical material and red rooted salvia contain.
4.2 four kinds of active constituent content comparative study under the different extraction process conditions
It is an amount of that precision takes by weighing danshensu, puerarin, ferulic acid and tanshinone reference substance respectively, place different measuring bottles, with dissolve with methanol solution and be diluted to scale, shake up, get each reference substance storing solution (concentration be followed successively by 0.38,1.25,0.45,0.61mg/mL).Accurate above-mentioned danshensu storing solution 5mL, puerarin storing solution 5mL, ferulic acid storing solution 8mL, the tanshinone storing solution 6mL of drawing adds methanol solution and is settled to scale in the 50mL measuring bottle respectively, as mixing reference substance solution.
The accurate absorption mixed reference substance solution and each 10 μ L of need testing solution, and sample introduction is measured under above-mentioned chromatographic condition, and the record peak area calculates the content of danshensu, puerarin, ferulic acid and tanshinone in the different samples by external standard method, the results are shown in Table 2.
Four kinds of content of effective of table 2 are (mg/g) relatively
The result shows that Radix Salviae Miltiorrhizae, Radix Puerariae and Rhizoma Chuanxiong medical material (mixed proportion 1:1:1) are under different preparation process conditions, and the content of its effective ingredient (danshensu, puerarin, ferulic acid and tanshinone) has evident difference.The danshensu average content: new technology is 2.〉new technology 1. former technology; The puerarin average content: new technology is 2.〉new technology 1. former technology; The ferulic acid average content: new technology is 2.〉new technology 1. former technology; The tanshinone average content: new technology is 2.〉new technology 1. former technology.
Wherein have two point values to pay special attention to:
(1) TONGMAI KOUFUYE does not contain fat-soluble tanshinone composition; And water miscible ferulic acid component content is also lower.
(2) in the technology 1, though adopt alcohol, the water of variable concentrations to extract, active constituent content is lower than technology 2 in its extract.Above-mentioned experiment shows, adopts 50～70% alcohol solvents that the TONGMAI KOUFUYE crude drug is extracted, and can effectively extract various active component, and extraction effect is best, has improved the utilization rate of raw material of Chinese medicine, has reduced the waste of production cost; Wherein again with 60～70% extraction effect the bests.
Below example specifies beneficial effect of the present invention by experiment.
The comparative study of test example 1 treatment ischemic heart desease
1.1 experiment material, instrument
1.1.1 be subjected to the reagent thing
The extract A of promoting blood circulation.
The extract C 1 of promoting blood circulation.
The extract C 2 of promoting blood circulation.
The extract C 3 of promoting blood circulation.
FUFANG DANSHEN PIAN: lot number D2A025, manufacturer: Guangzhou Baiyunshan Heji Huangpu Chinese Medicine Co., Ltd. produces.Specification: every is equivalent to primary crude drug 0.6g; Usage and dosage: oral.One time 3,3 times on the one.
1.2 laboratory animal
The KM mice, the SPF level, ♀/♂ half and half, body weight 18.5～22.5g, reaching large bio tech ltd by Chengdu provides, the laboratory animal certification of fitness number: scxkc (111) 2008-24.
The SD rat, the SPF level, ♀/♂ half and half, body weight 180～220g, reaching large bio tech ltd by Chengdu provides, the laboratory animal certification of fitness number: scxkc (111) 2008-24.
1.3 experimental apparatus
▲ Thermo global function microplate reader, BIS-2113, U.S. Thermo Fisher Scientific Instr Ltd. produces.
▲ constant temperature shaking table: QYC-200 type, Shanghai Fu Ma Instr Ltd..
▲ refrigerated centrifuge: TG2-16C type, Anting Scientific Instrument Factory, Shanghai.
▲ electric-heated thermostatic water bath: model SK12-6, Ningbo of Zhejiang medical apparatus and instruments factory.
▲ AS10200 ultrasonic cleaner (Tianjin Ao Tesaisi Instr Ltd.).
▲ W201 thermostat water bath (Shen, Shanghai is along bio tech ltd).
The excellent general ultrapure water machine of ▲ ULUP-I-10T (Chengdu Ultra Pure Science ﹠ Technology Co., Ltd).
1.4 test kit
Mice ATP Elisa test kit: lot number: 121028, purchase in Shanghai biotechnology company limited.
Rats Glu Elisa test kit, lot number: 121015, purchase in Shanghai biotechnology company limited.
Rats Asp Elisa test kit, lot number: 121026, purchase in Shanghai biotechnology company limited.
Rats GABA Elisa test kit, lot number: 121020, purchase in Shanghai biotechnology company limited.
1.5 statistical method
Carry out statistical analysis with SPSS17.0 software.Data are with mean ± standard deviation
One factor analysis of variance is adopted in expression between group, carry out the LSD check between the neat person's group of variance, and the heterogeneity of variance person carries out Tamhane ' s T2 check.
1.6 experiment content and method
1.6.1 experiment grouping and administration
The KM mice is by the body weight random packet, and 10 every group, conduct respectively: 1. model control group: equal-volume pure water; 2. the extract A of promoting blood circulation group: 7.5gkg
-13. 1 group of the extract C of promoting blood circulation: 7.5gkg
-14. the extracting solution C2 that promotes blood circulation group: 7.5gkg
-15. 3 groups of the extract C of promoting blood circulation: 7.5gkg
-16. the positive is organized (FUFANG DANSHEN PIAN): 3.6gkg
-1Take by weighing 10 normal mouses at random as the normal control group 7. by body weight in addition: the equal-volume pure water.Every group of mice irritated stomach 1 time, continuous 7 days every day.
1.6.2 mouse cardiac muscle ischemia model clone method
Test 6d and begin, except the blank group, all the other respectively organize mice by 3mg/kg dosage lumbar injection isoproterenol, continuous two days.The injection of blank group mouse peritoneal gives the Isodose normal saline.
1.1.3 detection index
1. myocardial metabolism index determining: blood, 4000rmin are gathered in the mouse heart puncture
-1Centrifugal 10min collects supernatant, and the Elisa method is measured ATP content in the blood plasma.
2. myocardial tissue damage index: each microscopic examination result of experimental mice cardiac muscular tissue is carried out grade scoring, and as the quantizating index of curative effects such as Drug therapy myocardial ischemia, the result is in the myocardial tissue damage index.
1.2 experimental result
1.2.1 the influence to mice plasma ATP content
The results are shown in Table 3.
ATP content in each experimental mice blood plasma of table 3
Annotate: compare with model group:
*P＜0.01;
*P＜0.05; Compare with the extract A group:
△ △P＜0.01,
△P＜0.05.
As shown in Table 3, compare the extract C of promoting blood circulation 1, C2 and the significantly rising (P＜0.05) of C3 group with model group; Compare extract C 1, C2 and C3 group mice plasma ATP content not statistically significant with the extract A group of promoting blood circulation.
1.2.2 the influence to mouse cardiac muscle tissue injury index
The results are shown in Table 4.
Cardiac muscular tissue's microscopy standards of grading: 0 minute: no gangrenosum acne damage or inflammation; 1 minute: blood vessel was slightly expanded, edema; 2 minutes: vasodilation, organize inflammatory infiltration, slight downright bad; 3 minutes: serious downright bad, blood vessel was seriously expanded, lymph foilicie hyperplasia.
Each experimental mice myocardial tissue damage index of table 4
Annotate: compare with model group:
*P＜0.01;
*P＜0.05; Compare with the extract A group:
△ △P＜0.01,
△P＜0.05.
As shown in Table 4, with model group relatively, the extract C of promoting blood circulation 1, C2 and C3 group mouse cardiac muscle pathology damage index all have significantly and reduce (P＜0.05); With promote blood circulation extract A relatively, 3 groups of mouse cardiac muscle pathology damage of extract C index has significantly and reduces (P＜0.05), extract C 1 and C2 group not statistically significant.
The influence of 2 pairs of rat ischemia encephalopathy (HIE)s
2.1 experiment content and method
2.1.1 experiment grouping and administration
The SD rat is by body weight random packet, 10 every group, conduct respectively: model control group: equal-volume pure water; The extract A of promoting blood circulation group: 4.5gkg
-11 group of the extract C of promoting blood circulation: 4.5gkg
-1The extracting solution C2 that promotes blood circulation group: 4.5gkg
-13 groups of the extract C of promoting blood circulation: 4.5gkg
-1Positive group (FUFANG DANSHEN PIAN): 2.16gkg
-1In addition take by weighing 10 normal mouses at random as the normal control group by body weight: the equal-volume pure water.Every group of mice irritated stomach 1 time, continuous 7 days every day.
2.2.2 rat cerebral ischemia model copy method
Test 1h after the administration in the 7th day, each experimental group rats by intraperitoneal injection 10% chloral hydrate (0.35g/kg) anesthesia separates right carotid, except the blank group, all the other respectively organize rat with No. 5 syringe needles to cephalad direction inserting needle injection arachidonic acid 0.5mgkg
-1, blank group injection Isodose normal saline.Behind the 10min, inject the blue 100g body weight of 0.2% ivens 0.5mL, behind 5min, by right common carotid artery intubate blood sampling 3mL, add the 2%EDTA-Na that 0.3m is housed
2Plastic test tube, mixing at once is in 4 ℃ of preservations.Break end rapidly then, it is standby to get brain.
2.2.3 detection index
1. neurotransmitter: getting cerebral tissue, is the homogenate medium with the normal saline, 4000rmin
-1Centrifugal 15min collects supernatant, and the Elisa method is measured glutamic acid (Glu), aspartic acid (Asp), γ-An Jidingsuan (GABA) content in the blood plasma.
2. cerebral tissue damage index: each microscopic examination result of experimental group rat cerebral tissue is carried out grade scoring, and as the quantizating index of curative effects such as Drug therapy cerebral ischemia, the result is in the brain tissue impairment index.
2.2 experimental result
2.2.1 the influence to rat ischemia cerebral tissue Glu content
The results are shown in Table 5.
Each Glu of experimental group rat cerebral tissue content of table 5
Annotate: compare with model group:
*P＜0.01;
*P＜0.05; Compare with the extract A group:
△ △P＜0.01,
△P＜0.05.
As shown in Table 5, compare with model group, C1, C2 and C3 organize to have significantly and reduce (P＜0.05); Extract A group not statistically significant; With promote blood circulation extract A relatively, the extract C of promoting blood circulation 2 and the C3 group Glu of rat cerebral tissue content have significantly and reduce.
2.2.2 the influence to rat ischemia cerebral tissue Asp content
The results are shown in Table 6.
Each Asp of experimental group rat cerebral tissue content of table 6
Annotate: compare with model group:
*P＜0.01;
*P＜0.05; Compare with the extract A group:
△ △P＜0.01,
△P＜0.05.
As shown in Table 6, compare with model group, C1, C2 and C3 organize to have significantly and reduce (P＜0.05); Extract A group not statistically significant; With promote blood circulation extract A relatively, extract C 1, C2 and the C3 group Asp of rat cerebral tissue content have significantly and reduce (P＜0.05).
2.2.3 the influence to rat ischemia cerebral tissue GABA content
The results are shown in Table 7.
Each GABA of experimental group rat cerebral tissue content of table 7
Annotate: compare with model group:
*P＜0.01;
*P＜0.05; Compare with the extract A group:
△ △P＜0.01,
△P＜0.05.
As shown in Table 7, compare with model group, the extract C of promoting blood circulation 1, C2 and the C3 group GABA of rat cerebral tissue content enlarge markedly (P＜0.05); Extract A group not statistically significant; Compare with the extract A of promoting blood circulation, extract C 1, C2 and the C3 group GABA of rat cerebral tissue content enlarge markedly (P＜0.05).
2.2.5 the influence to rat cerebral tissue's damage index
The results are shown in Table 10.
Cerebral tissue microscopy standards of grading: 0 minute: no gangrenosum acne damage, edema or inflammation; 1 minute: downright bad, edema that cerebral tissue is slight; 2 minutes: the necrosis of cerebral tissue moderate, edema and organize inflammatory infiltration; 3 minutes: cerebral tissue was seriously downright bad, cell infiltration, lymph foilicie hyperplasia.
Each experimental group rat cerebral tissue damage index of table 10
Annotate: compare with model group:
*P＜0.01;
*P＜0.05; Compare with the extract A group:
△ △P＜0.01,
△P＜0.05.
As shown in Table 7, with model group relatively, the extract C of promoting blood circulation 1, C2 and C3 rat cerebral tissue pathology damage index have significantly and reduce (P＜0.05); Group extract A group not statistically significant; With promote blood circulation extract A relatively, the extract C of promoting blood circulation 1, C2 and C3 group rat cerebral tissue pathology damage index all have significantly and reduce (P＜0.05).
2.5 experiment brief summary
This experiment causes the rat cerebral ischemia model by common carotid artery injection arachidonic acid the pharmacological action of the extracting solution of promoting blood circulation of two kinds of obstructed technology preparations is verified that the result shows:
(1) TONGMAI KOUFUYE does not contain fat-soluble tanshinone composition, and water miscible ferulic acid component content is also lower.The present invention's 50～70% alcohol extraction processes can obviously keep fat-soluble tanshinone, and increase content of ferulic acid.
(2) in the extract of promoting blood circulation of the present invention's 50～70% alcohol extraction processes preparations danshensu, puerarin, ferulic acid and four kinds of effective ingredient average contents of tanshinone all more former technology is higher.
(3) for the treatment of mice ischemic heart desease: the present invention's 50～70% alcohol extraction processes more can improve ischemic myocardial cell ATP content than former technology, more can improve the mice IMN.
(4) for the treatment of rat cerebral ischemia: the present invention's 50～70% alcohol extraction processes more can reduce the content of ischemic rat brain Glu, Asp than former technology, and the GABA content that more can raise also more can improve the cerebral tissue ischemic necrosis.
In sum, adopt 50～70% alcohol solvents that the TONGMAI KOUFUYE crude drug is extracted among the present invention, can effectively extract various active component, extraction effect is best, has improved the utilization rate of raw material of Chinese medicine, has reduced the waste of production cost.Simultaneously, the crude drug extract that this method prepares, drug activity obviously is better than former preparation process, and the patient has brought Gospel for cardiovascular and cerebrovascular disease.
Claims (10)
1. TONGMAI KOUFUYE crude drug extract, it is characterized in that: TONGMAI KOUFUYE crude drug proportioning is: Radix Salviae Miltiorrhizae: Radix Puerariae: Rhizoma Chuanxiong=1:1:1; In the described crude drug extract, the active constituent content weight ratio is as follows: danshensu: puerarin: ferulic acid: tanshinone=(1.0～1.2): (1.0～1.2): (1.0～1.2): 1.0.
2. TONGMAI KOUFUYE crude drug extract according to claim 1 is characterized in that: in the described crude drug extract, contain the effective ingredient of following percentage by weight:
Danshensu 1.7～2.57%, puerarin 1.7～2.7%, ferulic acid 1.5～2.6%, tanshinone 1.5～2.6%.
3. TONGMAI KOUFUYE crude drug extract according to claim 1 and 2 is characterized in that: described crude drug extract, and after high performance liquid chromatography detected, chromatogram was as shown in figure 14; Chromatographic condition is as follows:
Chromatographic column: octadecyl silane post;
Detect wavelength: 280nm;
Column temperature: 30 ° of C;
Flow velocity: 1.0ml/min;
Mobile phase: with acetonitrile-0.1% glacial acetic acid aqueous solution gradient elution, elution program is as follows:
4. any described TONGMAI KOUFUYE crude drug preparation method of extract of claim 1-3, it is characterized in that: it comprises following operating procedure:
(1) weighting raw materials by weight ratio: Radix Salviae Miltiorrhizae, Rhizoma Chuanxiong, Radix Puerariae;
(2) with the crude drug mixing, with 50～70%v/v ethanol extraction, merge extractive liquid, behind the recovery solvent, namely gets TONGMAI KOUFUYE crude drug extract.
5. preparation method according to claim 4, it is characterized in that: concentration of alcohol is 60～70%v/v.
6. preparation method according to claim 5, it is characterized in that: concentration of alcohol is 70%v/v.
7. preparation method according to claim 4, it is characterized in that: the concrete technology of ethanol extraction is as follows: add 20～40 times of volume of ethanol of crude drug gross mass, reflux, extract, 3 times, each 1 hour.
8. the purposes of any described TONGMAI KOUFUYE crude drug extract of claim 1-3 in the medicine of preparation prevention or treatment myocardial ischemia or cerebral ischemia.
9. purposes according to claim 8 is characterized in that: described medicine is the medicine that improves myocardial damage due to the myocardial ischemia.
10. purposes according to claim 8 is characterized in that: described medicine is the medicine that improves brain tissue impairment due to the cerebral ischemia.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2013102109907A CN103251677A (en) | 2013-05-31 | 2013-05-31 | Tongmai oral liquid raw material medicine extract and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2013102109907A CN103251677A (en) | 2013-05-31 | 2013-05-31 | Tongmai oral liquid raw material medicine extract and preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103251677A true CN103251677A (en) | 2013-08-21 |
Family
ID=48956110
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2013102109907A Pending CN103251677A (en) | 2013-05-31 | 2013-05-31 | Tongmai oral liquid raw material medicine extract and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103251677A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103675117A (en) * | 2013-11-06 | 2014-03-26 | 大连天宇制药有限公司 | Establishment method and application of Zhengxintai tablet fingerprint |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1660202A (en) * | 2004-12-15 | 2005-08-31 | 武汉大学 | Nano medical preparation for promoting blood circulation and preparation method |
| CN1923227A (en) * | 2005-09-02 | 2007-03-07 | 天津药物研究院 | Traditional Chinese medicinal formulation for treating ischemic cardiovascular and cerebrovascular diseases and preparation method thereof |
-
2013
- 2013-05-31 CN CN2013102109907A patent/CN103251677A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1660202A (en) * | 2004-12-15 | 2005-08-31 | 武汉大学 | Nano medical preparation for promoting blood circulation and preparation method |
| CN1923227A (en) * | 2005-09-02 | 2007-03-07 | 天津药物研究院 | Traditional Chinese medicinal formulation for treating ischemic cardiovascular and cerebrovascular diseases and preparation method thereof |
Non-Patent Citations (4)
| Title |
|---|
| 余建清等: "正交试验优选通脉颗粒中葛根素的提取工艺", 《中国中医药信息杂志》 * |
| 曾俊芬等: "HPLC同时测定通脉口服液中5种有效成分的含量", 《中国药学杂志》 * |
| 郑雅楠: "通脉滴丸的制备工艺和质量标准研究", 《中国优秀硕士学位论文全文数据库工程科技I辑》 * |
| 雷嘉川等: "高效液相色谱法测定通脉颗粒中丹参酮ⅡA的含量", 《中国现代应用药学杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103675117A (en) * | 2013-11-06 | 2014-03-26 | 大连天宇制药有限公司 | Establishment method and application of Zhengxintai tablet fingerprint |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103207255B (en) | A kind of detection method of content of NAOXINTONG JIAONANG | |
| CN101721488B (en) | Pharmaceutical composition for treating liver diseases and prepration method thereof | |
| CN102488863A (en) | Chinese herbal medicine compound with anticancer effect, preparation method and detection method thereof | |
| CN104758515A (en) | Traditional Chinese medicinal composition for treating nephropathy as well as preparation method and detection method thereof | |
| CN104288245B (en) | Anti-aging and constitutional pharmaceutical composition and preparation method thereof and detection method | |
| Wu et al. | Study on major antitumor components in Yinchenhao decoction in vitro and in vivo based on hollow fiber cell fishing coupled with high performance liquid chromatography | |
| CN103239506B (en) | Tongmai oral liquid raw material extract | |
| CN103565866A (en) | Preparation method of panax notoginseng saponins | |
| CN102539599B (en) | Method for detecting liver-enhancing medicine | |
| CN112526014B (en) | Jinyinliang oral liquid fingerprint spectrum and establishing method thereof | |
| CN105911192A (en) | Semiliquidambar cathayensis blood-circulation-promoting and stasis-removing active part extracting method and fingerprint spectrum detecting method | |
| CN103784761A (en) | Drug for treatment of vertigo and preparation method thereof | |
| CN102920964B (en) | Traditional Chinese medicine preparation for curing cough | |
| CN101601700A (en) | Valeriana amurensis effective part extract and method of quality control thereof and medical usage | |
| CN100381813C (en) | Quality control of compound Danshen root drops | |
| CN102590423B (en) | Fingerprint spectrum determination method of ligusticum wallichii tea-blending granular preparation | |
| CN103251677A (en) | Tongmai oral liquid raw material medicine extract and preparation method and application thereof | |
| CN102100737A (en) | Medicinal composition containing general ginsenoside and total salvianolic acid and preparation method thereof | |
| CN102240320B (en) | Motherwort and astragalus capsule used for treating prostatic hyperplasia | |
| CN101642492B (en) | Drug for treating chronic pelvic inflammatory disease and preparation method thereof | |
| CN108226325A (en) | Roripa montana gives birth to the method for building up of arteries and veins oral liquid composition finger-print | |
| CN106361811A (en) | Tongmai pharmaceutical composition and preparation method thereof | |
| CN102038856A (en) | Content measuring method for four components in Chinese medicinal composition | |
| Zhang et al. | Improved effect of fresh ginseng paste (radix ginseng-ziziphus jujube) on hyperuricemia based on network pharmacology and molecular docking | |
| CN104297360A (en) | Detection method for compound Xueshuantong (thrombus clearing) preparation fingerprint chromatogram |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20130821 |