CN103623212B - A kind of preparation method and application consolidating ball - Google Patents
A kind of preparation method and application consolidating ball Download PDFInfo
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- CN103623212B CN103623212B CN201310633732.XA CN201310633732A CN103623212B CN 103623212 B CN103623212 B CN 103623212B CN 201310633732 A CN201310633732 A CN 201310633732A CN 103623212 B CN103623212 B CN 103623212B
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- radix rehmanniae
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- 238000002360 preparation method Methods 0.000 title claims abstract description 21
- 239000003814 drug Substances 0.000 claims abstract description 17
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 15
- 208000028591 pheochromocytoma Diseases 0.000 claims abstract description 15
- 229940079593 drug Drugs 0.000 claims abstract description 14
- 238000000194 supercritical-fluid extraction Methods 0.000 claims abstract description 13
- 241000756943 Codonopsis Species 0.000 claims abstract description 12
- 230000004663 cell proliferation Effects 0.000 claims abstract description 11
- 230000001629 suppression Effects 0.000 claims abstract description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 15
- 239000000843 powder Substances 0.000 claims description 10
- 238000000605 extraction Methods 0.000 claims description 9
- 239000012567 medical material Substances 0.000 claims description 9
- 239000006187 pill Substances 0.000 claims description 8
- 238000002156 mixing Methods 0.000 claims description 7
- 238000002474 experimental method Methods 0.000 description 8
- 238000000034 method Methods 0.000 description 7
- 229960004756 ethanol Drugs 0.000 description 5
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- 230000010261 cell growth Effects 0.000 description 3
- 239000012531 culture fluid Substances 0.000 description 3
- 229960000935 dehydrated alcohol Drugs 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- WEEMDRWIKYCTQM-UHFFFAOYSA-N 2,6-dimethoxybenzenecarbothioamide Chemical compound COC1=CC=CC(OC)=C1C(N)=S WEEMDRWIKYCTQM-UHFFFAOYSA-N 0.000 description 1
- QZKDXSOKZHLFHE-UHFFFAOYSA-N 4-(1,3-benzodioxol-5-yl)benzoic acid Chemical compound C1=CC(C(=O)O)=CC=C1C1=CC=C(OCO2)C2=C1 QZKDXSOKZHLFHE-UHFFFAOYSA-N 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 208000000616 Hemoptysis Diseases 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 229920002334 Spandex Polymers 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000010165 autogamy Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000010219 correlation analysis Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000012738 dissolution medium Substances 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000004900 laundering Methods 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- -1 mixing Substances 0.000 description 1
- 238000000465 moulding Methods 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 238000005498 polishing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 239000004759 spandex Substances 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 229960002385 streptomycin sulfate Drugs 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 230000035900 sweating Effects 0.000 description 1
- 235000019640 taste Nutrition 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
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- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention provides a kind of preparation method consolidating ball, by Radix Rehmanniae Preparata 100g, Radix Codonopsis 50g, Radix Rehmanniae 100g, Radix Asparagi 100g, Radix Ophiopogonis 100g as crude drug, employing supercritical extraction is prepared from, content is improved a lot, dose reduces, and present invention also offers and consolidates the application of ball in preparation suppression Clonal Rat Pheochromocytoma tumor PC-12 cell proliferation.
Description
Technical field
The present invention relates to technical field of traditional Chinese medicine preparation, be specifically related to a kind of preparation method and the application that consolidate ball.
Background technology
Consolidate ball and be recorded in Ministry of Public Health standard WS3-B-0238-90, prescription be Radix Rehmanniae Preparata 100g, Radix Codonopsis 50g, Radix Rehmanniae 100g, Radix Asparagi 100g, Radix Ophiopogonis 100g, the above five tastes, Radix Codonopsis, Radix Ophiopogonis powder are broken into fine powder, and Radix Rehmanniae Preparata, Radix Rehmanniae, Radix Asparagi decoct with water secondary, 3 hours first times, second time 2 hours, collecting decoction, filters, filtrate is concentrated into the clear paste that relative density is greater than 1.30 (20 DEG C), adds above-mentioned powder and appropriate amount of starch, mixing, pill, drying, polishing, to obtain final product.Energy YIN nourishing and QI supplementing, lung heat clearing pathogenic fire reducing.For deficiency of both QI and YIN, disease sees hectic fever, cough hemoptysis, and body is thin and weak, spontaneous sweating, weak or Tianjin wound etc. after being ill.
In prior art, not yet have and consolidate ball at the report extracting employing supercritical technology in preparation, and adopt the method for playing powder and decoction, technique is coarse, backward, and impurity is many, causes patient's consumption excessive, is inconvenient to take, and has had a strong impact on this product and has applied clinically.
Summary of the invention
Goal of the invention: in order to solve the problem, the object of the present invention is to provide a kind of preparation method consolidating ball.
Another object of the present invention is that providing a kind of consolidates the application of ball in preparation suppression Clonal Rat Pheochromocytoma tumor PC-12 cell proliferation.
Technical scheme: the object of the invention is by following scheme realize:
A kind of preparation method consolidating ball, by Radix Rehmanniae Preparata 100g, Radix Codonopsis 50g, Radix Rehmanniae 100g, Radix Asparagi 100g, Radix Ophiopogonis 100g make as crude drug, described method is made up of the following step: get Radix Rehmanniae Preparata 100g, Radix Codonopsis 50g, Radix Rehmanniae 100g, Radix Asparagi 100g, Radix Ophiopogonis 100g, join in CO2 supercritical extraction device, ethanol is as entrainer, and the percent by volume that entrainer accounts for total extractant is 4-6%, extracting pressure 15-30MPa, temperature 30-50 DEG C, CO
2flow 1-3m1/g crude drug min, extraction time 150-180min, obtains supercritical extract, is ground into fine powder, sieves, and mixing, make pill 150g, every 12 balls are equivalent to total medical material 3g.
Above-mentioned a kind of preparation method consolidating ball, described CO
2the percent by volume that supercritical extraction entrainer accounts for total extractant is 5%.
Above-mentioned a kind of preparation method consolidating ball, described CO
2the extracting pressure 20MPa of supercritical extraction, temperature 40 DEG C, CO
2flow 2ml/g crude drug min, extraction time 160min.
Above-mentioned a kind of ball that consolidates is preparing the application suppressed in Clonal Rat Pheochromocytoma tumor PC-12 cell proliferation, consolidate ball by Radix Rehmanniae Preparata 100g, Radix Codonopsis 50g, Radix Rehmanniae 100g, Radix Asparagi 100g, Radix Ophiopogonis 100g make as crude drug, preparation method is made up of the following step: get Radix Rehmanniae Preparata 100g, Radix Codonopsis 50g, Radix Rehmanniae 100g, Radix Asparagi 100g, Radix Ophiopogonis 100g, join CO
2in supercritical extraction device, ethanol is as entrainer, and the percent by volume that entrainer accounts for total extractant is 4-6%, extracting pressure 15-30MPa, temperature 30-50 DEG C, CO
2flow 1-3m1/g crude drug min, extraction time 150-180min, obtains supercritical extract, is ground into fine powder, sieves, and mixing, make pill 150g, every 12 balls are equivalent to total medical material 3g.
The above-mentioned ball that consolidates, preparing the application suppressed in Clonal Rat Pheochromocytoma tumor PC-12 cell proliferation, consolidates CO described in ball preparation method
2the percent by volume that supercritical extraction entrainer accounts for total extractant is 5%.
The above-mentioned ball that consolidates, preparing the application suppressed in Clonal Rat Pheochromocytoma tumor PC-12 cell proliferation, consolidates CO described in ball preparation method
2the extracting pressure 20MPa of supercritical extraction, temperature 40 DEG C, CO
2flow 2ml/g crude drug min, extraction time 160min.
In prior art, consolidate ball by the former technique of above-mentioned prescription and obtain 300g pill, oral, 10 ~ 12 balls, 3 times on the one, every 12 balls are equivalent to total medical material 3g, and what adopt present invention process to be prepared into consolidates ball 150g, and every 12 balls are equivalent to total medical material 3g, but the medical material amount contained is original 2 times, therefore 5 ~ 6 balls, 3 times on the one, greatly reduce dose under the condition with more active component.
Detailed description of the invention
Form by the following examples, foregoing of the present invention is described in further detail again, but this should be interpreted as that the scope of the above-mentioned theme of the present invention is only limitted to following example, all technology realized based on foregoing of the present invention all belong to scope of the present invention.
Embodiment 1
Get Radix Rehmanniae Preparata 100g, Radix Codonopsis 50g, Radix Rehmanniae 100g, Radix Asparagi 100g, Radix Ophiopogonis 100g join CO
2in supercritical extraction device, ethanol is as entrainer, and the percent by volume that entrainer accounts for total extractant is 4%, extracting pressure 15MPa, temperature 30 DEG C, CO
2flow 1m1/g crude drug min, extraction time 150min, obtains supercritical extract, is ground into fine powder, sieves, and mixing, make pill 150g, every 12 balls are equivalent to total medical material 3g.
Embodiment 2
Get Radix Rehmanniae Preparata 100g, Radix Codonopsis 50g, Radix Rehmanniae 100g, Radix Asparagi 100g, Radix Ophiopogonis 100g join CO
2in supercritical extraction device, ethanol is as entrainer, and the percent by volume that entrainer accounts for total extractant is 6%, extracting pressure 30MPa, temperature 50 C, CO
2flow 3m1/g crude drug min, extraction time 180min, obtains supercritical extract, is ground into fine powder, sieves, and mixing, make pill 150g, every 12 balls are equivalent to total medical material 3g.
Embodiment 3
Get Radix Rehmanniae Preparata 100g, Radix Codonopsis 50g, Radix Rehmanniae 100g, Radix Asparagi 100g, Radix Ophiopogonis 100g join CO
2in supercritical extraction device, ethanol is as entrainer, and the percent by volume that entrainer accounts for total extractant is 5%, extracting pressure 20MPa, temperature 40 DEG C, CO
2flow 2m1/g crude drug min, extraction time 160min, obtains supercritical extract, is ground into fine powder, sieves, and mixing, make pill 150g, every 12 balls are equivalent to total medical material 3g.
Embodiment 4: consolidate the experimentation data that ball suppresses Clonal Rat Pheochromocytoma tumor PC-12 cell proliferation
1 experiment material
1.1 experiment cell strains
Clonal Rat Pheochromocytoma tumor PC-12 cell, Nanjing Medical University's laboratory cell storehouse, DMEM+10%FBS cellar culture.
1.2 Experimental agents
Drugs: the present invention consolidates ball: prepare by embodiment 3 method.
Medicinal liquid liquid storage: take 100mg and consolidate ball, is dissolved in 5ml dehydrated alcohol, 0.2 μm of frit, 500 μ ldoff pipe subpackages ,-20 DEG C of storages, and simultaneously 0.2 μm of frit dehydrated alcohol is in order to the use of matched group.
1.3 experiment reagent
DMEM(GIBCO company Cat.No.12100-061Lot.No.758137); Hyclone (Tian Hang bio tech ltd, Zhejiang Lot.No.100419); NaHCO3(Shanghai hundred million chemical reagent company limited Cat.No.11810-033Lot.No.1088387 of a specified duration); Trypsin(AMRESCO company lot number: 2010/04); EDTA(AMRESCO company lot number: 2009/10); PenicillinGSodiumSalt(AMRESCO company lot number: 2010242); StreptomycinSulfate(AMRESCO company lot number: 2010382); Dehydrated alcohol (Nanjing Chemistry Reagent Co., Ltd.'s lot number: 080310182); MTT (Biosharp lot number: 0793); PBS(laboratory autogamy); 1.4 experiment equipment
Lycra inverted microscope (German Leica model: DM1L); Visible-ultraviolet light microwell plate detector (MD company of U.S. model: SPECTRAMAX190); CO2 incubator (FORMA model: 3111); Super-clean bench (safe and sound Inc. of Su Jing group moulding number: SW-CJ-ZFD); Pure water instrument (Spring company of U.S. model: S/N020579); Accurate pipettor (French Gilson Inc model: P2); Electronic balance (German Sai Duolisi company limited model: BT323S); Full-automatic high-pressure autoclave (Japanese SANYO company model: MLS-3020); Table electrothermal air dry oven (the accurate experimental facilities company in Shanghai model: DHG9123A); Refrigerator (Siemens Company's model: KG18V21TI); Liquid nitrogen container (CBS model: 2001); Low speed centrifuge (Anting Scientific Instrument Factory, Shanghai's model: KA-1000); 0.2 μm of filter (MILLIPORE model: SLGP033RB); 10cm culture dish (NEST company), 96 well culture plates (NEST company); Cell counting count board; Centrifuge tube, pipet, Tips are some.
2 experimental techniques
1) Clonal Rat Pheochromocytoma tumor PC-12 cell DMEM+10%FBS in 37 DEG C, 5%CO2 carries out cellar culture (10cm culture dish), when Growth of Cells is to logarithmic (log) phase, collecting cell, discards culture fluid, PBS fine laundering 3 times, add 3ml0.25% trypsin-0.04%EDTA, after 37 DEG C of digestion 2min, add 5ml complete medium neutralization reaction wherein, proceeded in centrifuge tube after piping and druming cell, the centrifugal 5min of 1000rpm, adjustment concentration of cell suspension 3 × 104/ml.
2) enter in 96 well culture plates by cell kind, every hole adds cell suspension 180 μ l, and culture plate puts into cell culture incubator (37 DEG C, 5%CO2) cellar culture.
3) according to cell growth status, generally grow to 50%-70%, add and consolidate ball solution, continue to cultivate 24h.
4) add 20 μ lMTT solution (5mg/ml, i.e. 0.5%MTT) after 24h, continue to cultivate 4h.
5) after 4h, buckle method removes supernatant, pats dry gently with absorbent paper, and every hole adds 200 μ l dimethyl sulfoxide, puts low-speed oscillation 10min on shaking table, crystal is fully dissolved.The light absorption value in each hole is measured at enzyme-linked immunosorbent assay instrument 490nm place.
6) background (do not add cell, only add culture fluid) is set, control wells (the medicine dissolution medium of cell, same concentrations, culture fluid, MTT, dimethyl sulfoxide) simultaneously, often organizes the multiple hole of setting 6.
7) result represents with the suppression ratio of medicine to cell:
Cell proliferation suppression ratio (%)=(control wells OD value-dosing holes OD value)/control wells OD value × 100%.Experiment repetition 3 times.
3 statistical dispositions
Adopt the correlation analysis in MicrosoftExcel2003 software and Studentt inspection, data represent with mean ± S.D..
4 experimental results
Statistical result showed after mtt assay experiment, compare with matched group, when dosage reaches 5mg/ml, to Clonal Rat Pheochromocytoma tumor PC-12 cell inhibitory effect variant (P<0.05), dosage this difference when 10mg/ml has significance (P<0.01), has pole significant difference (P<0.001) when dosage reaches 15-20mg/ml.
Table 1 consolidates ball to Clonal Rat Pheochromocytoma tumor PC-12 cell inhibitory effect influence research (X ± SD)
Note: compare with matched group, * P<0.01; * P<0.001
5 experiment conclusion
Consolidate ball and can suppress Clonal Rat Pheochromocytoma tumor PC-12 cell proliferation, reduce the Growth of Cells number of Clonal Rat Pheochromocytoma tumor PC-12 cell, this effect is dose dependent.
Claims (3)
1. consolidate the application of ball in preparation suppression Clonal Rat Pheochromocytoma tumor PC-12 cell proliferation, it is characterized in that consolidating ball by Radix Rehmanniae Preparata 100g, Radix Codonopsis 50g, Radix Rehmanniae 100g, Radix Asparagi 100g, Radix Ophiopogonis 100g make as crude drug, its preparation method is made up of the following step: get Radix Rehmanniae Preparata 100g, Radix Codonopsis 50g, Radix Rehmanniae 100g, Radix Asparagi 100g, Radix Ophiopogonis 100g, join CO
2in supercritical extraction device, ethanol is as entrainer, and the percent by volume that entrainer accounts for total extractant is 4-6%, extracting pressure 15-30MPa, temperature 30-50 DEG C, CO
2flow 1-3m1/g crude drug min, extraction time 150-180min, obtains supercritical extract, is ground into fine powder, sieves, and mixing, often make pill 150g, every 12 balls are equivalent to total medical material 3g.
2. consolidate the application of ball in preparation suppression Clonal Rat Pheochromocytoma tumor PC-12 cell proliferation according to claim 1, it is characterized in that consolidating CO described in ball preparation method
2the percent by volume that supercritical extraction entrainer accounts for total extractant is 5%.
3. consolidate the application of ball in preparation suppression Clonal Rat Pheochromocytoma tumor PC-12 cell proliferation according to claim 1, it is characterized in that consolidating CO described in ball preparation method
2the extracting pressure 20MPa of supercritical extraction, temperature 40 DEG C, CO
2flow 2ml/g crude drug min, extraction time 160min.
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| CN201310633732.XA CN103623212B (en) | 2013-12-02 | 2013-12-02 | A kind of preparation method and application consolidating ball |
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| CN201310633732.XA CN103623212B (en) | 2013-12-02 | 2013-12-02 | A kind of preparation method and application consolidating ball |
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| CN103623212A CN103623212A (en) | 2014-03-12 |
| CN103623212B true CN103623212B (en) | 2016-01-20 |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102988577A (en) * | 2012-10-15 | 2013-03-27 | 李正梅 | Preparation method and application of brain-soothing hypertension pill |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102988577A (en) * | 2012-10-15 | 2013-03-27 | 李正梅 | Preparation method and application of brain-soothing hypertension pill |
Non-Patent Citations (1)
| Title |
|---|
| 槲皮素对大鼠肾上腺嗜铬细胞瘤PC12细胞的诱导分化作用;张俊等;《中国微侵袭神经外科杂志》;20110420;第16卷(第4期);180-183 * |
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Effective date of registration: 20170605 Address after: Three No. 88 blue industrial park, 515325 Nanshan street, Guangdong, Puning Patentee after: GUANGDONG SANLAN PHARMACEUTICAL CO., LTD. Address before: 266000 Shandong province Qingdao City Red Road No. 31 North Building 201 Patentee before: Li Qiang |