CN101428108A - Preparation method of Chinese medicine formulation - Google Patents

Preparation method of Chinese medicine formulation Download PDF

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CN101428108A
CN101428108A CNA2007101662894A CN200710166289A CN101428108A CN 101428108 A CN101428108 A CN 101428108A CN A2007101662894 A CNA2007101662894 A CN A2007101662894A CN 200710166289 A CN200710166289 A CN 200710166289A CN 101428108 A CN101428108 A CN 101428108A
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preparation
chinese medicine
ultrafiltration
decocting liquid
filter
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曾雄辉
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Abstract

The invention belongs to the technical field of traditional Chinese medicine, in particular to a preparation method of a traditional Chinese medicine preparation. In order to better reflect the essence of the traditional Chinese medicine, the invention proposes the guideline that the traditional Chinese medicine preparation is in line with the ancient way, medicine is extracted by water, and the preparation method combines the modern concentration and granulation processes, thereby summing up preparation process parameters and applicable excipients which are most matched with the traditional Chinese medicine preparation. The accumulated experiences of the traditional medicine are retained to the maximum extent, and the preparation method can also meet the needs of preparation forms which are applicable to the rapid pace of modern society, such as powder, dropping pills, granules, tablets, capsules, and the like.

Description

A kind of preparation method of Chinese medicine preparation
[technical field]
The invention belongs to technical field of Chinese medicines, be specifically related to a kind of preparation method of Chinese medicine preparation.
[background technology]
The present invention derives from shenqi pill, it is famous hospital, shenqi pill has another name called " Cui Shi eight flavor balls ", this side comes from Zhang Zhongjing " Medical Treasures of the Golden Chamber ", by Radix Rehmanniae, Rhizoma Dioscoreae, Fructus Corni, Rhizoma Alismatis, Cortex Moutan, Poria, Ramulus Cinnamomi, gun additioner eight herbal medicines are formed, be used for the treatment of the asthenia of deficiency of kidney-QI due to weak, quench one's thirst, phlegm retention, beriberi, diseases such as married woman's anuria during pregnancy. on this basis, ancient Chinese medicine doctor is through repeatedly practical exploration, constantly we's the clinical scope of application is expanded, make it one of become so far still in name side that tcm clinical practice is widely used. review over more than 10 year relevant shenqi pill clinical application research and report nearly hundred pieces, as can be seen, its therapeutic domain relates to nerve, endocrine, immunity, digestion, circulation, breathe, urinary system, a plurality of systems such as reproduction are in containing, outward, the woman, youngster, the oral cavity, eye, otorhinolaryngology, skin, numerous diseases of a plurality of subjects such as senile disease.
In recent decades, the production of Chinese herbal medicine has realized mechanization and semi-mechanization to a certain degree.Chinese medicine often is considered to that active constituent content is low, impurity is many, quality is unstable, so medication is based upon on the empirical basis more, can not integrate with modern medicine.For addressing this problem, Chinese medicine must be walked the road of extraction and purification.The extraction of Chinese medicine comprises many unit operationss such as leaching, clarification, filtration and evaporation.
[summary of the invention]
The preparation method that the purpose of this invention is to provide a kind of new Chinese medicine preparation.Basis of the present invention prescription derives from shenqi pill.
Chinese medicine preparation of the present invention, its prescription consists of: Radix Rehmanniae 5 weight portions, Radix Achyranthis Bidentatae, Fructus Corni, Rhizoma Dioscoreae, Semen Plantaginis, Rhizoma Alismatis, Poria, each 3 weight portion of Cortex Moutan, Cortex cinnamomi japonici (Ramulus Cinnamomi), each 1 weight portion of process of preparing Chinese medicine Radix Aconiti Lateralis Preparata end, preparation method is for to decoct with water Radix Rehmanniae, Radix Achyranthis Bidentatae, Fructus Corni, Rhizoma Dioscoreae, Semen Plantaginis, Rhizoma Alismatis, Poria, Cortex Moutan, Cortex cinnamomi japonici (Ramulus Cinnamomi) and the process of preparing Chinese medicine Radix Aconiti Lateralis Preparata end 2-4 time, each 0.5-3 hour, merge decocting liquid, filter the back concentrate fluid extract, add behind the adjuvant with behind the fluidized bed granulation useful in preparing drug formulations.
The proportion of above-mentioned fluid extract is 1.15-1.30, preferred 1.15-1.20.
Above-mentioned decocting liquid can carry out centrifugation earlier, gets clarifying decocting liquid, and centrifugal rotation speed is 1500-20000 rev/min, and preferred 1500-10000 rev/min, more preferably 2000-8000 rev/min, most preferably 3000-5000 rev/min.
Above-mentioned decocting liquid can adopt the method for membrane filtration to filter.
Above-mentioned membrane filtration comprises one of filter method of employing ultrafiltration and nanofiltration or ultrafiltration and nanofiltration is used in combination.
Above-mentioned ultrafilter membrane is selected from cellulose diacetate film, three cellulose acetate membrane, cyanoethyl cellulose film, polysulfone membrane, sulfonated polysulfone membrane, poly (ether sulfone) film, sulfonated polyether sulfone film, polysulfonamides film, phenolphthalein side group polyarylsulfone (PAS) film, polyvinylidene fluoride film, polyacrylonitrile film, polyimide film, cellulose membrane, methyl methacrylate-acrylonitrile copolymer film, polyacrylonitrile-cellulose diacetate blend film, the dynamic ultrafilter membrane that forms, one of and the Modified Membrane of above-mentioned film, its molecular retention amount is below the 6000-10000.
The filter membrane aperture of above-mentioned ultrafiltration is: the 0.22-0.45 micron.
The membrane ultrafiltration device that above-mentioned ultrafiltration is adopted can be commercial doughnut or plate ultrafilter membrane separator.
Above-mentioned NF membrane molecular retention amount is 500-2000, preferred 500-1000, more preferably 700-1000.
The process of above-mentioned membrane filtration preferably with the process ultrafiltration earlier of decocting liquid, is carried out nanofiltration again.
Above-mentioned adjuvant is selected from microcrystalline Cellulose, powdery cellulose, mannitol, starch, lactose, gelatin, methylcellulose, dextrin, pregelatinized Starch, micropowder silica gel, hydroxypropyl methylcellulose, cross-linking sodium carboxymethyl cellulose, carboxymethyl starch sodium, Polyethylene Glycol, xylitol, lactose, glucose, glycine, mannitol, tartaric acid, silicon dioxide, one or more in calcium stearate and the magnesium stearate.
The said medicine preparation comprises granule, tablet, capsule, powder, drop pill.
The at present used extracting technique of Chinese medicine compound recipes that adopt certain density ethanol extraction Chinese medicine more, help reclaiming solvent like this and reduce impurity, but the method that adopts alcohol extraction has deviated from the experience accumulation of the treatment disease of Chinese medicine since 3000, has ignored for the purpose that can arrive " quality controllable " comparatively simply under the guiding theory of " modernization of Chinese medicine " " effectiveness " even " safety ".
In sum, the hysteresis of Chinese medicine extraction separating technology development becomes the bottleneck of traditional Chinese medicine development and existence, must be optimized, reform and strengthen original technology.The reinforcement technique of chemical separating and mass transfer will provide strong assurance for this reason, and realization Chinese medicine subject intersects with Chemical Engineering, will help realizing the modernization of Chinese medicine production equipment.With the Chinese medicine extraction separation process that induces one of notion, the theory of Chemical Engineering.Utilize available research achievements,,, technological process, production equipment, operating condition are done modernization overlay and careful groping, provided feasible scheme from basic influence factor's research staff in conjunction with the concrete condition that Chinese medicine is produced.
In order to return the marrow of traditional medicine treatment disease, the present invention proposes the guiding theory according to " abiding by Gu " with Chinese medicine preparation classics side, use the water extraction medicine, concentrated, granulating process in conjunction with modern have summed up the preparation process parameter and the suitable adjuvant that mate the most with Chinese medicine preparation.Kept the experience accumulation of traditional medicine to greatest extent, and it is allegro as dosage forms such as tablet, capsule, granule, powder, drop pills to adapt to modern society.
Guaranteed the stable content of paeonol among the present invention, for the standardization production of preparation is laid a good foundation.By realization of the present invention, can be under the prerequisite that the effective ingredient in the Chinese medicine preparation is extracted to greatest extent, reducing in the extract macromolecular substances such as impurity such as tannin, polyphenol, resin, protein, phlegmatic temperament and solid particle effectively removes, keep the active ingredient in the Chinese medicine preparation of the present invention, for the preparation of oral drug preparation is provided convenience.
[specific embodiment]
Following embodiment further describes the present invention, but described embodiment only is used to illustrate the present invention rather than restriction the present invention.Following examples Chinese crude drug feeds intake and is Radix Rehmanniae 50 grams, Radix Achyranthis Bidentatae, Fructus Corni, Rhizoma Dioscoreae, Semen Plantaginis, Rhizoma Alismatis, Poria, each 30 gram of Cortex Moutan, Cortex cinnamomi japonici (Ramulus Cinnamomi), each 10 gram of Radix Aconiti Lateralis Preparata end.
Embodiment 1
Take by weighing the pulverizing medicinal materials of recipe quantity, decoct 2 times with water extraction, each 2 hours, amount of water was 12 times of medical material, merge the water extract, filter decompression and solvent recovery, get fluid extract (proportion is 1.13-1.18), add adjuvant boiling granulating in fluid bed, promptly get granular preparation of the present invention.
Embodiment 2
Take by weighing the pulverizing medicinal materials of recipe quantity, decoct 3 times with water extraction, each 2 hours, amount of water was 8 times of medical material, merge the water extract, filter, decompression and solvent recovery gets fluid extract (proportion is 1.15-1.20), add adjuvant boiling granulating in fluid bed, encapsulated, promptly obtain capsule preparations of the present invention.
Embodiment 3
Take by weighing the pulverizing medicinal materials of recipe quantity, decoct 2 times with water extraction, each 2.5 hours, amount of water was 10 times of medical material, merge the water extract, filter, decompression and solvent recovery gets fluid extract (proportion is 1.19-1.25), add adjuvant boiling granulating in fluid bed, add 800 gram vegetable oil, mix homogeneously promptly obtains soft capsule of the present invention with being pressed into soft capsule after the colloid mill grinding.
Embodiment 4
Take by weighing the pulverizing medicinal materials of recipe quantity, decoct 2 times each 2 hours with water extraction, amount of water is 8 times of medical material, merges the water extract, centrifugal (rotating speed is 2000 rev/mins), filter, filtrate is 6000 three cellulose acetate membrane ultrafiltration with molecular cut off, filter type employing cross flow filter.The operating condition of ultrafiltration technology is: the inlet pressure of ultrafiltration is 0.1 MPa, and low 0.5 kPa of the liquid outlet pressure ratio inlet pressure of ultrafiltration progressively boosts during ultrafiltration.In ultra-filtration process, adopt the periodic pressure fluctuation, the pressure wave moment is 0.1 MPa.Ultrafiltration gained filtrate decompression reclaims solvent, gets fluid extract (proportion is 1.10-1.25), adds adjuvant boiling granulating in fluid bed, promptly gets granular preparation of the present invention.
Embodiment 5
Take by weighing the pulverizing medicinal materials of recipe quantity, decoct 2 times each 2 hours with water extraction, amount of water is 8 times of medical material, merges the water extract, centrifugal (rotating speed is 5000 rev/mins), filter, filtrate is 6000 polysulfone membrane ultrafiltration with molecular cut off, filter type employing cross flow filter.The operating condition of ultrafiltration technology is: the inlet pressure of ultrafiltration is 0.1 MPa, and low 0.5 kPa of the liquid outlet pressure ratio inlet pressure of ultrafiltration progressively boosts during ultrafiltration.In ultra-filtration process, adopt the periodic pressure fluctuation, the pressure wave moment is 0.1 MPa.Ultrafiltration gained filtrate decompression reclaims solvent, gets fluid extract (proportion is 1.10-1.20), adds adjuvant boiling granulating in fluid bed, adds the adjuvant tabletting, promptly obtains tablet of the present invention.
Embodiment 6
Take by weighing the pulverizing medicinal materials of recipe quantity, decoct 3 times with water extraction, each 2 hours, amount of water was 6 times of medical material, merged the water extract, filtered, and filtrate is 10000 polysulfone membrane ultrafiltration with molecular cut off, filter type employing cross flow filter.The operating condition of ultrafiltration technology is: the inlet pressure of ultrafiltration is 0.1 MPa, and low 0.5 kPa of the liquid outlet pressure ratio inlet pressure of ultrafiltration progressively boosts during ultrafiltration.In ultra-filtration process, adopt the periodic pressure fluctuation, the pressure wave moment is 0.1 MPa.It is to handle in 500 the rolling nanofiltration device that ultrafiltration gained filtrate is selected molecular cut off for use through system, wherein the nanofiltration amount of water can be controlled in about 0.75 times of former ultrafiltrate, get fluid extract (proportion is 1.28-1.30), add adjuvant boiling granulating in fluid bed, add the adjuvant tabletting, promptly obtain tablet of the present invention.
Embodiment 7
Take by weighing the pulverizing medicinal materials of recipe quantity, decoct 2 times with water extraction, each 2 hours, amount of water was 6 times of medical material, merged the water extract, filtered, and filtrate is 8000 three cellulose acetate membrane ultrafiltration with molecular cut off, filter type employing cross flow filter.The operating condition of ultrafiltration technology is: the inlet pressure of ultrafiltration is 0.1 MPa, and low 0.5 kPa of the liquid outlet pressure ratio inlet pressure of ultrafiltration progressively boosts during ultrafiltration.In ultra-filtration process, adopt the periodic pressure fluctuation, the pressure wave moment is 0.1 MPa.It is to handle in 500 the rolling nanofiltration device that ultrafiltration gained filtrate is selected molecular cut off for use through system, wherein the nanofiltration amount of water can be controlled in about 0.85 times of former ultrafiltrate, get fluid extract (proportion is 1.24-1.30), add adjuvant boiling granulating in fluid bed, promptly get granular preparation of the present invention.
Embodiment 8
Take by weighing the pulverizing medicinal materials of recipe quantity, decoct 3 times with water extraction, each 2 hours, amount of water was 8 times of medical material, merged the water extract, filtered, and filtrate is 6000 cyanoethyl cellulose membrane ultrafiltration with molecular cut off, filter type employing cross flow filter.The operating condition of ultrafiltration technology is: the inlet pressure of ultrafiltration is 0.1 MPa, and low 0.5 kPa of the liquid outlet pressure ratio inlet pressure of ultrafiltration progressively boosts during ultrafiltration.In ultra-filtration process, adopt the periodic pressure fluctuation, the pressure wave moment is 0.1 MPa.It is to handle in 500 the rolling nanofiltration device that ultrafiltration gained filtrate is selected molecular cut off for use through system, wherein the nanofiltration amount of water can be controlled in about 0.8 times of former ultrafiltrate, get fluid extract (proportion is 1.25-1.30), add adjuvant boiling granulating in fluid bed, encapsulated, promptly obtain capsule preparations of the present invention.
Determining of the amount of experimental example 1-dry extract of the present invention
Embodiment 1-8 fluid extract with among the present invention does not add adjuvant, and directly lyophilization obtains dry extract, data such as following table 1:
The data of the amount of table 1 dry extract of the present invention
Figure A200710166289D00071
By realization of the present invention, can be under the prerequisite that the effective ingredient in the Chinese medicine preparation of the present invention is extracted to greatest extent, reducing in the extract macromolecular substances such as impurity such as tannin, polyphenol, resin, protein, phlegmatic temperament and solid particle effectively removes, the active ingredient that keeps medicine is for the preparation of oral drug preparation is provided convenience.
Experimental example 2-carries out the liquid phase analysis research of Chinese medicine preparation with high performance liquid chromatograph
One, materials and methods
(1), experimental drug
The standard substance paeonol that this research is used is available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute.
(2), instrument and equipment and experiment condition
1. instrument and equipment
Liquid phase pump: Applied Biosystems (ABI) 400 * 2
Injection valve: Rheodyne Type 7125 (10 μ L loop)
The automatic gradient controller of mobile phase controller: ABI
Detector: ABI 1000s photodiode array detector
2. analysis condition
Pre-column: μ-Bondapak TMC18 (Millipore, Milford, MA, USA)
Chromatographic column: Cosmosil 5C18-MS, 5 μ m, 25cm * 4.6mm (Nacalai Tesque, Kyoto, Japan)
Mobile phase: (A) 0-50 mM/rise KH2PO4 is adjusted to pH2.5-6.5 with 5% H3PO4
(B)H2O/CH3CN:30/70-10/90(V/V)
Flow velocity: 1.0 ml/min
Detect wavelength: 230nm
Gradient elution program: as table 1
(3), experimental technique
1. the discussion of the mobile phase of different salt densities
With optimal chromatographic column, in mobile phase (A), use respectively 0,10,20,30,40 and 50 mMs/liter potassium dihydrogen phosphate, analyze with the gradient elution program of table 1, each group repeats biphasic injection.
Table 1 is analyzed the gradient elution program of Chinese medicine preparation
Figure A200710166289D00081
2. the discussion of the mobile phase of different pH-values
With the biphosphate potassium concn of optimization, in mobile phase (A), add not commensurability 5% phosphoric acid or ammonia, the buffer solution of preparation different pH values, pH value is respectively 2.5,3.5,4.5,5.5 and 6.5, and each organizes repetitive operation twice.
3. the discussion of the mobile phase of different acetonitrile ratios
Because the adding of salt, mobile phase (B) must contain an amount of water, separates out to prevent salt, and obtains more smooth baseline, therefore prepares the mobile phase of different water/acetonitrile ratio, is respectively 30/70,25/75,20/80,15/85 and 10/90.Select the buffer solution condition of above-mentioned optimization to analyze, twice of every group of repetitive operation.
4. prepare standard solution and production standard curve
(1) gets 10.0 milligrams BPB (n-butyl p-hydroxy benzoate) and be dissolved in 10 milliliters 70% methanol aqueous solution, as internal standard product (IS) solution.Take by weighing 10.0 milligrams paeonol respectively, be made into 20 milliliters of standard substance mother solutions with 70% methanol aqueous solution.Every kind of standard substance mother solution got 5,2,1,0.5,0.1 and 0.05 milliliters again and placed measuring bottle then, adds 1 milliliter of internal standard product solution separately, is made into 10 milliliters of standard solutions with 70% methanol aqueous solution at last.With above-mentioned optimization condition, each concentration repeats double injection, injects 10 microlitres at every turn, gets its meansigma methods production standard curve.
(2) precision takes by weighing the Chinese medicine preparation powder of 1.0 grams, with 20 milliliters of 70% methanol aqueous solutions as extractant, ultrasound concussion 30 minutes, centrifugal filtration, triplicate, combining extraction liquid is concentrated into 10 milliliters.Get in 5 milliliters of concentrated solution to 10 milliliter measuring bottles, add 1 milliliter of internal standard product solution, be diluted to 10 milliliters with 70% methanol aqueous solution.Use 0.45 μ m filter to filter,, repeat double injection, inject 10 microlitres at every turn, average as analysis result as quantitative inspection liquid.
(4), the suitability of analysis condition assessment
1. repeatability
Get 2.0 milliliters of standard substance mother solutions to measuring bottle, add 1.0 milliliters of internal standard product solution, be diluted to 10.0 milliliters, as inspection liquid with 70% methanol aqueous solution.Repeat six injections on the same day, do not amount on the same day and repeat six injections, inject 10 microlitres at every turn.
2. the response rate
Precision takes by weighing 1.0 gram Chinese medicine preparation powder, with 20 milliliters of 70% methanol aqueous solutions, and as extractant, ultrasound concussion 30 minutes, centrifugal filtration, triplicate, combining extraction liquid is concentrated into 10 milliliters.Get 5.0 milliliters of concentrated solution to 10 milliliter measuring bottles, add 1.0 milliliters of standard substance mother solutions, add 1.0 milliliters of internal standard product solution again, be diluted to 10.0 milliliters with 70% methanol aqueous solution, after 0.45 μ m filter filters as quantitative inspection liquid.Repeat double injection, inject 10 microlitres at every turn, get the average result of double injection.
3. detectability
Dilution standard product solution progressively injects chromatographic column and detects, up to S/N than (signal to noise ratio) less than 3, calculate injection rate and concentration thereof.
4. the quantitative analysis of preparation composition
Precision takes by weighing 1.0 gram Chinese medicine preparation granules (embodiment 7), with 20 milliliters of 70% methanol aqueous solutions as extractant, ultrasound concussion 30 minutes, centrifugal filtration, triplicate, combining extraction liquid is concentrated into 10 milliliters.Get in 5 milliliters of concentrated solution to 10 milliliter measuring bottles, add 1 milliliter of internal standard product solution, be diluted to 10 milliliters, filter through 0.45 μ m filter, as quantitative inspection liquid with 70% methanol aqueous solution.Repeat double injection, inject 10 microlitres at every turn, get its meansigma methods.
Experimental example 3-preparation index substance of the present invention assay
With embodiment 1-8 fluid extract, do not add adjuvant, directly lyophilization obtains dry extract according to above test condition detection level, and the gained paeonol content is as follows:
The effective ingredient paeonol is investigated in table 1 sample
Conclusion: guaranteed the stable content of paeonol among the present invention, for the standardization production of preparation is laid a good foundation.By realization of the present invention, can be under the prerequisite that the effective ingredient in the Chinese medicine preparation is extracted to greatest extent, reducing in the extract macromolecular substances such as impurity such as tannin, polyphenol, resin, protein, phlegmatic temperament and solid particle effectively removes, keep the active ingredient in the Chinese medicine preparation of the present invention, for the preparation of oral drug preparation is provided convenience.

Claims (7)

1. the preparation method of a Chinese medicine preparation, its prescription consists of: Radix Rehmanniae 5 weight portions, Radix Achyranthis Bidentatae, Fructus Corni, Rhizoma Dioscoreae, Semen Plantaginis, Rhizoma Alismatis, Poria, each 3 weight portion of Cortex Moutan, Cortex cinnamomi japonici (Ramulus Cinnamomi), each 1 weight portion of process of preparing Chinese medicine Radix Aconiti Lateralis Preparata end, it is characterized in that Radix Rehmanniae, Radix Achyranthis Bidentatae, Fructus Corni, Rhizoma Dioscoreae, Semen Plantaginis, Rhizoma Alismatis, Poria, Cortex Moutan, Cortex cinnamomi japonici (Ramulus Cinnamomi) and process of preparing Chinese medicine Radix Aconiti Lateralis Preparata end are decocted with water 2-4 time, each 0.5-3 hour, merge decocting liquid, filter the back concentrate fluid extract, add behind the adjuvant with behind the fluidized bed granulation useful in preparing drug formulations.
2. preparation method according to claim 1, the proportion that it is characterized in that described fluid extract is 1.15-1.30
3. preparation method according to claim 2, the proportion that it is characterized in that described fluid extract is 1.15-1.20.
4.. preparation method according to claim 1 is characterized in that described decocting liquid can carry out centrifugation earlier, gets clarifying decocting liquid, centrifugal rotation speed is 1500-20000 rev/min.
5. according to the arbitrary described preparation method of claim 1-4, it is characterized in that described decocting liquid can adopt the method for membrane filtration to filter.
6. preparation method according to claim 5 is characterized in that described membrane filtration comprises ultrafiltration and nanofiltration.
7. preparation method according to claim 6, the process that it is characterized in that described membrane filtration is carried out nanofiltration again for the process ultrafiltration earlier of decocting liquid.
CNA2007101662894A 2007-11-09 2007-11-09 Preparation method of Chinese medicine formulation Pending CN101428108A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102145102B (en) * 2010-02-04 2012-10-17 天津中新药业集团股份有限公司达仁堂制药厂 Traditional Chinese medicinal composition for warming and tonifying kidney yang and strengthening qi and promoting diuresis and preparation method thereof
CN102145103B (en) * 2010-02-04 2012-10-17 天津中新药业集团股份有限公司乐仁堂制药厂 Traditional Chinese medicine composition for warming and invigorating kidney yang and replenishing qi to promote diuresis and preparation method thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102145102B (en) * 2010-02-04 2012-10-17 天津中新药业集团股份有限公司达仁堂制药厂 Traditional Chinese medicinal composition for warming and tonifying kidney yang and strengthening qi and promoting diuresis and preparation method thereof
CN102145103B (en) * 2010-02-04 2012-10-17 天津中新药业集团股份有限公司乐仁堂制药厂 Traditional Chinese medicine composition for warming and invigorating kidney yang and replenishing qi to promote diuresis and preparation method thereof

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