CN103245687B - A kind of test method of quality control based on component structure of danshen injections - Google Patents
A kind of test method of quality control based on component structure of danshen injections Download PDFInfo
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- CN103245687B CN103245687B CN201310164760.1A CN201310164760A CN103245687B CN 103245687 B CN103245687 B CN 103245687B CN 201310164760 A CN201310164760 A CN 201310164760A CN 103245687 B CN103245687 B CN 103245687B
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Abstract
The invention provides a kind of quality control new detecting method based on component structure of danshen injections.The present invention judges the quality of quality by characterizing the phenolic acid component and carbohydrate components of bulk property in danshen injections, and optimal standard is：Phenolic acid component：Carbohydrate components content is (1% 95%):(90%‑1%)；And danshensu in root of red-rooted salvia phenolic acid, protocatechualdehyde, caffeic acid, remove danshensu salvianolic acid E 1, go the composition of each composition of danshensu salvianolic acid E 2, salviandic acid A, tanshin polyphenolic acid B, salvianolic acid C, salvianolic acid D, Rosmarinic acid than being respectively (1% 95%):(5%‑95%):(1%‑20%):(1%‑20%):(5%‑20%):(1%‑50%):(5%‑20%):(1%‑10%):(1%‑20%):(1%‑20%)；The content summation of phenolic acid component and carbohydrate components is more than the 35 90% of total solid；The content of phenolic acid component is not less than the 40 80% of total solid.
Description
Technical field
The present invention relates to the quality control standard of compound Chinese medicinal preparation, it is especially a kind of for danshen injections based on group
The test method of quality control field of separation structure.
Background technology
The effective substance of Chinese medicine preparation has global feature according to certain composition by multiple components than what is constituted
Components group.Modern pharmacology and fitochemical studies result show, Chinese medicine preparation plays the material base of drug effect, and non-certain is single
Composition works, but is realized and acted synergistically by Mutiple Targets, multicomponent.At present, researcher gradually recognizes Chinese medicine preparation
Material base is studied and quality control can not walk traditional plant approach for seeking monomer, with certain active component or toxic component
Content formulate quality standard, but will the Overall View based on Chinese medicine, with " component " be point of penetration, illustrate the material base of Chinese medicine simultaneously
It is finally reached the purpose for the traditional Chinese medicine quality control for meeting overall feature.Material foundation of tcm " component structure is theoretical " thinks Chinese medicine system
Agent material base is an orderly entirety, with " three generating multi-dimension hierarchical structures ", is made up of various ingredients, and each component
Inside also by multiple compositions necessarily proportioning constitute, with all there is certain architectural feature in component between component, this with present with certain
Index content just controls the method for Chinese medicine preparation quality to compare, and both realizes controllability, the globality of Chinese medicine is embodied again.
Danshen injections is clinical conventional Chinese medicine preparation, has one's ideas straightened out with promoting blood circulation and removing blood stasis, qi-regulating, expands blood vessel and promote hat
The effect of shape blood flow volume, for prevention and treatment of diseases such as angina pectoris, myocardial infarctions.Danshen injections mainly contains water
Dissolubility salvianolic acid compound and carbohydrate, such as danshensu, protocatechualdehyde, caffeic acid, remove danshensu salvianolic acid E 1, remove danshensu
Salvianolic acid E 2, salviandic acid A, tanshin polyphenolic acid B, salvianolic acid C, salvianolic acid D, Rosmarinic acid etc..These compounds of studies have shown that all have
Certain cardiovascular and cerebrovascular protective effect.
The content of the invention
Goal of the invention：
The present invention is will be according to traditional Chinese medicine globality and systematic feature, and the quality control for solving danshen injections shows
Having deficiency, there is provided a kind of multidimensional structure method of quality control for representing component characteristicses.
Technical scheme：
A kind of test method of quality control based on component of danshen injections, the feature of this method is to danshen injections
Constitute structure and component inner structure between the component of middle phenolic acid and carbohydrate components to be controlled, using before HPLC finger-prints, post spreading out
Biochemical HPLC and NMR technology determine the finite element for representing globality, so that entirety and systematically control danshen injections group
Point and composition in content ratio between composition.Weigh danshen injections quality optimality criterion be：
Phenolic acid component：The content control range of carbohydrate components is (1%-95%):(90%-1%)；And the red sage root in root of red-rooted salvia phenolic acid
Element, protocatechualdehyde, caffeic acid, go danshensu salvianolic acid E 1, go danshensu salvianolic acid E 2, salviandic acid A, tanshin polyphenolic acid B, salvianolic acid C,
The composition of each composition of salvianolic acid D, Rosmarinic acid is than being respectively (1%-95%):(5%-95%):(1%-20%):(1%-20%):(5%-
20%):(1%-50%):(5%-20%):(1%-10%):(1%-20%):(1%-20%)；The content of phenolic acid component and carbohydrate components is total
With the 35-90% more than total solid；The content of phenolic acid component is not less than the 40-80% of total solid.
Beneficial effect：
Traditional Chinese medicine preparation product is to pass through the performance effect that acts synergistically by numerous chemical compositions.The drug effect thing of traditional Chinese medicine preparation product
The a certain component that matter basis is made up of multicomponent or multicomponent is constituted, and the bioactivity between each component has significant difference,
Action target spot and mechanism are also not quite similar；Each compound structure is there is also difference in component, bioactivity there is also difference,
Therefore, these components and composition play optimum medicine efficacy material base constitute exist between certain uniqueness, i.e. component or
The composition architectural feature of uniqueness is there is in component between compound.Danshen injections mainly includes phenolic acid and carbohydrate components, this
A little components have certain protection Cardiovascular, and research shows that root of red-rooted salvia phenolic acid has nerve and Cerebral ischemia protection, protection cardiac muscle
The effect such as damage, antithrombotic and suppression platelet activation, improvement diabetic vascular complications, the propagation for suppressing vascular cell.Sugar
Class is also one of material base constituent component of its effect, and red sage root carbohydrate has antioxidation activity and improves mouse resist oxygen lack energy
Power, can also play protection H by raising prohibitin protein expressions2O2Cause the effect of myocardial cell injury.Therefore, to the red sage root
The quality control of parenteral solution, it is impossible to overall activity composition is only replaced with danshensu or protocatechualdehyde.Importantly, constituting drug effect
The composition architectural feature of material base is ignored.Therefore, it is theoretical based on component structure, illustrate the phenolic acid and carbohydrate of danshen injections
Architectural feature between component and in component, will realize the real control to danshen injections quality, it is ensured that its efficacy and saferry.
Instant invention overcomes the existing deficiency to danshen injections control of product quality detection method, a kind of injection of danshen is likely to become
The quality control new detecting method of liquid.
Brief description of the drawings
Fig. 1 danshen injections multidimensional structure quality control schematic diagrames
Liposoluble ingredient finger-print in Fig. 2 danshen injectionses, (wherein 1.5- hydroxymethylfurfurals；2. Sodium Danshensu;3. it is former
Catechu aldehyde;4. caffeic acid;5. remove danshensu salvianolic acid E 1;6. remove danshensu salvianolic acid E 2;7. salvianolic acid D;8. Rosmarinic acid;9.
Tanshin polyphenolic acid B;10. salviandic acid A；11, salvianolic acid C)
Fig. 3 250ml:16g specification danshen injections phenolic acid components HPLC finger-prints
Fig. 4 250ml:12g specification danshen injections phenolic acid components HPLC finger-prints
Fig. 5 danshen injectionses carbohydrate hydrolysis HPLC figures（1. the galactolipin of 2. glucose of mannose 3.)
Fig. 6 red sage root carbohydrate components nuclear-magnetism pulse sequence diagrams
Fig. 7 red sage root carbohydrate components chemical shift figures
Fig. 8 red sage root carbohydrate components area integral figures
Embodiment
With reference to quality control detection side based on component structure for danshen injections of the embodiment to the present invention
Method is described in detail：
Embodiment
Test method of quality control step one：
The salvianolic acid content in danshen injections is determined, qualitative, quantitative sign is carried out to each phenolic acid components
1. instrument：
Highly effective liquid phase chromatographic system：(configuration vacuum degassing machine, enters Agilent Technologies of the Agilent1100 U.S. automatically
Sample device, quaternary pump, VWD G1314A, DAD G1315B；Agilent ChemStation A10.02 chromatographic work stations)；
2. chromatographic condition
Chromatographic column：Shiseido Capcell pak MG C18 (4.6 × 250mm, 5 μm) chromatographic columns and Shiseido MG
C18 guard columns (4.6 × 12.5mm, 5 μm)；Mobile phase is the phosphoric acid water of acetonitrile (A) -0.075%, and elution program is：Acetonitrile (A) 0-
17min, 5%A-12%A；17-42min, 12%A-18%A；42-46min, 18%A-18%A；46-58min, 18%A-27%A；58-
65min, 27%A-27%A；65-75min, 27%A-50%A；75-85min, 50%A-80%A.Flow velocity is 1.0mL/min；Detect ripple
A length of 270nm；Column temperature is 30oC；Sampling volume is 10 μ L.
3. the preparation of reference substance solution:
Precision weighs danshensu, protocatechualdehyde, caffeic acid, removes danshensu salvianolic acid E 1, removes danshensu salvianolic acid E 2, red phenol
Sour A, tanshin polyphenolic acid B, salvianolic acid C, salvianolic acid D, Rosmarinic acid totally 10 liposoluble ingredient reference substances：
It is respectively placed in 10ml brown volumetric flasks, plus methanol dissolving, 10ml is settled to, each reference substance concentration is followed successively by
100.53μg/ml、95.60μg/ml、85.49μg/ml、74.26μg/ml、58.62μg/ml、120.36μg/ml、57.18μg/
ml、66.21μg/ml、59.78μg/ml、117.64μg/ml.1ml is respectively taken to mix to obtain mixed reference substance solution.
4. experimental method and result：
Take reference substance and danshen injections (250ml:16g and 250ml:12g specifications) sample solution, carried out by chromatographic condition
Analysis, determines danshensu, protocatechualdehyde, caffeic acid, removes danshensu salvianolic acid E 1, removes danshensu salvianolic acid E 2, salviandic acid A, pellet
Phenolic acid B, salvianolic acid C, salvianolic acid D, the peak area of Rosmarinic acid, calculate total phenolics content in danshen injections；Total phenolics and sugar
Class component, other components constitute ratio；Each composition constitutes ratio in total phenolics.Concrete outcome is shown in Fig. 2, Fig. 3 and Fig. 4, table 1, table 2, table
3。
Total phenolics content in the danshen injections of table 1（250ml：16g）
Total phenolics content in the danshen injections of table 2（250ml：12g）
The danshen injections total phenolics of table 3 and carbohydrate components, other components constitute ratio；Each composition constitutes ratio in total phenolics
Test method of quality control step 2：
Qualitative, quantitative sign is carried out to the carbohydrate content in danshen injections.HPLC chromatogram is shown in Fig. 5.
1. instrument：
Highly effective liquid phase chromatographic system：(configuration vacuum degassing machine, enters Agilent Technologies of the Agilent1100 U.S. automatically
Sample device, quaternary pump, VWD G1314A, DAD G1315B；Agilent ChemStation A10.02 chromatographic work stations)；
2. chromatographic condition
Chromatographic column：Agilent Zorbax SB-C18（4.6 × 250mm, 5 μm）Chromatographic column and SB-C18（4.6×10mm,
5μm）Guard column；Mobile phase is that acetonitrile (A) -0.05M potassium dihydrogen phosphate aqueous solutions carry out gradient elution:1-25min, 16.5%-
16.5%A；25-55min, 16.5%-24%A；55-60min, 24%-24%A；Flow velocity：1.0ml/min；Detection wavelength：245nm；Post
Temperature：45℃；Sample size：10μl.
3. the preparation of standard solution
Take monose reference substance (the mannose 4.97mg/ml, glucose 5.04mg/ml, galactolipin 5.16mg/ml newly prepared）
Each 100 μ l are mixed to prepare mixing monose reference substance, add the 0.15M μ l of NaOH solution 300, and mixing takes 100 μ l to add 0.1M's
1-phenyl-3-methyl-5-pyrazolones ketone（PMP）The μ l of methanol solution 100, room temperature is cooled to after reacting 30min in 70 DEG C of water-baths, plus
The μ l of hydrochloric acid 100 for entering 0.075M are neutralized, and are stood after adding the chloroform dissolving of 0.7ml water and 1ml, vortex 2min, layering
Carefully extract lower floor chloroform layer out with syringe afterwards, remaining water layer adds the mixing of 1ml chloroforms vortex oscillation, repeat with
Upper process 3 times.Most water layer is standby with after 0.45 μm of membrane filtration at last.
4. the preparation of sample solution
0.5ml danshen injectionses are taken, 0.5ml water is added, mixed, 0.15M NaOH solution 1ml is added, mixes, takes 100 μ
L adds 0.2M 1-phenyl-3-methyl-5-pyrazolones ketone（PMP）The μ l of methanol solution 100, react cold after 30min in 70 DEG C of water-baths
But to room temperature, the μ l of hydrochloric acid 100 for adding 0.075M are neutralized, and add the chloroform dissolving of 0.7ml water and 1ml, vortex 2min
After stand, carefully extract with syringe lower floor's chloroform layer after layering out, remaining water layer adds 1ml chloroform vortex oscillations
Mixing, repeats above procedure 3 times.Most water layer is standby with after 0.45 μm of membrane filtration at last.
5. experimental method and result：
Reference substance and danshen injections sample solution are taken, is analyzed by chromatographic condition, each monose in determination sample solution
Peak area, with mannose, glucose, galactolipin reference substance is compared, calculate danshen injections in carbohydrate components content.
It the results are shown in Table 4.
The carbohydrate content assay result of table 4
Test method of quality control step 3：
Using measure of the magnetic resonance NMR technology to carbohydrate components in danshen injections solids
1. the preparation of sample
Buffer solution：1.5M PBS, 0.05%TSP（g/100ml）；
Need testing solution：The red sage root extracts solid 0.7948g and adds 2ml distilled waters, is vortexed, centrifugation（Without precipitation）, take supernatant
500ml adds 50ml buffer solutions, and mixing is produced.
2. instrument parameter is set
Nuclear-magnetism pulse-gFlipsy（Fig. 6）
Parameter setting is as follows：
3. calculation formula
MTSP:TSP molecular weight CTSPTSP molar concentration in nuclear magnetic tube
4. compound belongs to and content
It is shown in Table 5, Fig. 7, Fig. 8.
The carbohydrate content magnetic resonance NMR measurement results of table 5
Inventor has found by many experiments, when the phenolic acid component in danshen injections：The content control model of carbohydrate components
Enclose for (1%-95%):(90%-1%)；And danshensu in root of red-rooted salvia phenolic acid, protocatechualdehyde, caffeic acid, go danshensu salvianolic acid E 1,
The composition ratio for removing each composition of danshensu salvianolic acid E 2, salviandic acid A, tanshin polyphenolic acid B, salvianolic acid C, salvianolic acid D, Rosmarinic acid is respectively
(1%-95%):(5%-95%):(1%-20%):(1%-20%):(5%-20%):(1%-50%):(5%-20%):(1%-10%):(1%-
20%):(1%-20%)；The content summation of phenolic acid component and carbohydrate components is more than the 35-90% of total solid；The content of phenolic acid component
When being not less than the 40-80% of total solid, the quality of the danshen injections is optimal.
Above example is the Overall Steps of the test method of quality control of the danshen injections of the present invention, and at present to pellet
Join the quality control of parenteral solution only using composition danshensu and protocatechualdehyde as index, it is evident that this with it is contained in danshen injections
Many active components play drug effect mass action feature disagree, be also unscientific Quality Control detection method.Therefore, it is of the invention
Use what composition structure in the multicomponent and component of danshen injections drew for the test method of quality control of danshen injections
Optimum detection standard is more effective, scientific and reasonable.It is all the Patents to danshen injections in existing open source literature report
It is to be directed to extraction process or preparation process mostly, yet there are no the quality control whole detection side for component structure feature of the present invention
Method.
Claims (4)
1. a kind of test method of quality control based on component of danshen injections, specific method is to total phenol in danshen injections
Two aspects of structure and each component inner structure carry out detection control between the component of acid constituents and carbohydrate components, using following specific step
Suddenly：
Determine in danshen injections each phenolic acid in salvianolic acid specifically constitute, ratio and total phenolics content；
Determine carbohydrate composition and content in danshen injections；
Determine carbohydrate components and content in danshen injections solids；
It is characterized in that, the phenolic acid component in the parenteral solution:The content control range of carbohydrate components is (1%-95%):(90%-
1%)；And danshensu in root of red-rooted salvia phenolic acid, protocatechualdehyde, caffeic acid, go danshensu salvianolic acid E 1, go danshensu salvianolic acid E 2,
The composition of each composition of salviandic acid A, tanshin polyphenolic acid B, salvianolic acid C, salvianolic acid D, Rosmarinic acid is than being respectively (1%-95%):(5%-
95%):(1%-20%):(1%-20%):(5%-20%):(1%-50%):(5%-20%):(1%-10%):(1%-
20%):(1%-20%)；The content summation of phenolic acid component and carbohydrate components is more than the 35-90% of total solid；Phenolic acid component
The quality that content is not less than the 40-80% of the total solid parenteral solutions is optimal.
2. a kind of test method of quality control based on component of danshen injections according to claim 1, its feature exists
In determined with HPLC chromatogram method each phenolic acid in total phenolics specifically constitute, ratio and total phenolics content.
3. a kind of test method of quality control based on component of danshen injections according to claim 1, its feature exists
In using mannose, glucose, galactolipin is reference substance, and carbohydrate composition and content are determined with HPLC chromatogram method.
4. a kind of test method of quality control based on component of danshen injections according to claim 1, its feature exists
In composition and content that carbohydrate in solids in parenteral solution is determined using magnetic resonance NMR technology.
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| CN103713004B (en) * | 2013-11-08 | 2016-06-08 | 山东丹红制药有限公司 | A kind of magnetic resonance detection method of Chinese medicine injection |
| CN104931519B (en) * | 2015-04-16 | 2017-04-05 | 天津中医药大学 | A kind of detection method of content of salvia root polyphenol acid injection |
| CN107014853A (en) * | 2017-02-22 | 2017-08-04 | 天津中医药大学 | A kind of detection method of content of sodium tashinone II A sulfonate injection |
| CN115403653A (en) * | 2022-05-19 | 2022-11-29 | 首都医科大学 | D (+) -beta- (3, 4-dihydroxyphenyl) -lactyl-Pro-Ala-Lys, synthesis and application thereof |
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| JPS59136643A (en) * | 1983-01-26 | 1984-08-06 | Gifu Daigaku | Method for non-destructive screening of fruit |
| CN100450501C (en) * | 2004-03-17 | 2009-01-14 | 天津天士力制药股份有限公司 | Chinese medicine preparation for treating cardiovascular and cerebrovascular diseases and preparing process thereof |
| CN100522191C (en) * | 2005-08-12 | 2009-08-05 | 上海绿谷制药有限公司 | Fingerprint atlas detection method for tanshin polyphenolic acid salts |
| CN101036748B (en) * | 2007-04-18 | 2011-09-28 | 贵州信邦制药股份有限公司 | Detection method of the Yixinshu Chinese traditional medicine for supplementing qi and for promoting blood circulation by removing blood stasis |
| CN101129463B (en) * | 2007-08-20 | 2011-09-14 | 正大青春宝药业有限公司 | Method of preparing red sage root injection |
| CN102749348B (en) * | 2012-06-18 | 2014-06-11 | 河南省科高植物天然产物开发工程技术有限公司 | Method for identifying active components in medicinal plant |
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