CN104155399B - A kind of method of quality control of four rhizoma cyperi medicine materical crude slice processed - Google Patents
A kind of method of quality control of four rhizoma cyperi medicine materical crude slice processed Download PDFInfo
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Abstract
The invention discloses a kind of method of quality control of four rhizoma cyperi medicine materical crude slice processed, the present invention sets up four rhizoma cyperi HPLC finger-prints processed, with active component α-cyperolone for reference peaks, the content of α-cyperolone in Calculation Basis peak, the fingerprint peaks of other main active for reference, calculates each active component fingerprint peaks in the relative content of α-cyperolone with α-cyperolone.Method of the present invention has operability, only needs the relative content that can control other unknown principle active component with 1 known reference substance α-cyperolone component content, reaches a kind of scientific approach of control four rhizoma cyperi processed prepared slice quality.The method is simple, is easy to promote, and decreases the use amount of reference substance, and solve market monomeric substance reference substance deficiency and the problem of costliness, four rhizoma cyperi prepared slice qualities processed are stablized, and drug action strengthens, and contributes to promoting Chinese Medicine Industry fast-developing.
Description
Technical field
The present invention relates to quality control technologies for traditional Chinese medicine field, particularly relate to a kind of method of quality control of four rhizoma cyperi medicine materical crude slice processed.
Background technology
Rhizoma cyperi is the dry rhizome of sedge nutgrass flatsedge CyperusrotundusL..Begin to be loaded in " Mingyi Bielu ", having effect of dispersing stagnated hepatoqi, menstruction regulating and pain relieving, is the key medicine of Fuke Tiaojing pain relieving, have the laudatory title of " Directorate-General DG of gas disease; the chief commander of female section ", is clinically used for the treatment of the various gynecological diseases such as dysmenorrhoea, menstrual disorder, functional uterine bleeding.Due to rhizoma cyperi concoct before and after pharmacological action and chemical composition there is larger change, to be therefore clinically used as medicine mainly with processed product.
Chinese medicine preparation is an ancient pharmaceutical technology, existing many traditional processing procedures are still main based on experience inheriting, lack the further investigation of modern science and technology means, cause prepared slice quality to be difficult to control, cannot with to produce greatly and the development of modern traditional Chinese medicine adapts.In order to unified processing technique and modern Chinese herbal medicine concoct the paces developed, modern technology and classic method organically blend by the present invention, tradition is concocted theory and is complemented each other with modern science intension, and solid foundation is established in the modern times succession and the development that can be traditional school technique.
The process of preparing Chinese medicine object of rhizoma cyperi mainly reduces bitter taste and fragrant dry gas, improves curative effect, changes or strengthen pharmaceutically-active position and trend.Along with the development of Chinese medicine preparation, due to the difference of north and south medication custom, the rhizoma cyperi concocting method that ancient times are numerous or lost or further develop, result in rhizoma cyperi now and concoct the situation of " each method in various places; a medicine number method ", the concocting method continued to use according to the literature has more than more than 20 kinds such as vinegar system, wine system, four rhizoma cyperis processed, seven rhizoma cyperis processed, honey system, charcoal processing, ginger system, salt.In Jiangxi, Fujian, the surrounding area such as Taiwan, commonly use four method for makings and concoct rhizoma cyperi, processing procedure shows unique characteristics, enjoy a very good reputation at medicine circle, but processing technique is still main based on experience inheriting, lack the method for quality control of modern science and technology means, cannot accomplish " steady quality, technological specification, medicine materical crude slice are controlled ", which greatly limits the succession of its unique processing technique and apply.
For this reason; modern science and technology handy root canal preparation is adopted to carry out quality control to four rhizoma cyperi medicine materical crude slice processed; realize the large-scale production of four rhizoma cyperis processed; promotion Chinese Medicine Industry is fast-developing; this is the needs of Respect and development Chinese medicine tradition processing procedure, is also to ensure that the key point of " steady quality, technological specification, medicine materical crude slice are controlled " accomplished by Chinese medicine preparation medicine materical crude slice.
Summary of the invention
The object of the present invention is to provide a kind of method of quality control of four rhizoma cyperi medicine materical crude slice processed.By adopting number of chemical mode identification technology, in conjunction with pharmacodynamics effect research, verify spectrum-effect relationship and isolate effect components, obtain multiple monomeric compound, confirm that chief active effect components material has 5 kinds by pharmacological testing, its retention time is respectively: 16.1min, 18.5min, 24.9min, 29.5min and 30.5min, and with confirmed 5 main active for prepared slice quality Con trolling index, formulate four rhizoma cyperi prepared slice quality control methods processed, specification concocts critical technical parameter, be beneficial to suitability for industrialized production quality control, guarantee the validity of clinical application.The present invention is intended to carry forward traditional processing technique, advances the process of the standardization of four rhizoma cyperis processed, standardization and suitability for industrialized production, promotes that it is applied on a large scale.
The present invention adopts following technical scheme:
The method of quality control of four rhizoma cyperi medicine materical crude slice processed of the present invention is as follows:
(1) high effective liquid chromatography for measuring condition:
Chromatographic column: DiamonsilC18; Determined wavelength: 220-280nm; Flow velocity: 0.5-1.5mL/min; Sample size: 20 μ L; Column temperature: room temperature; Mobile phase: methyl alcohol and double distilled water; Elution program: gradient elution;
(2) preparation of reference substance solution:
Precision measures α-cyperolone reference substance 0.05mL, is placed in 10mL volumetric flask, is diluted to scale, shakes up with 95% ethanol, and obtaining concentration is 5ug/mL reference substance solution;
(3) foundation of standard equation:
Accurate reference substance solution of drawing is mixed with series concentration, injection liquid chromatography, by the chromatographic condition analysis of step (1), measure peak area, with reference substance amount for horizontal ordinate, peak area value is that ordinate obtains standard equation y=136.75x+3.5384, R=0.9996;
(4) preparation of need testing solution:
Get four rhizoma cyperi meal 4g processed, add 95% ethanol 32mL, heating and refluxing extraction 3 times, each 2h, merging filtrate is settled to 100mL, obtains four rhizoma cyperi medicinal material liquid processed, with the filtering with microporous membrane of 0.45 μm, obtains need testing solution;
(5) foundation of HPLC finger-print:
Stratographic analysis is carried out to test sample, by the chromatographic condition analysis of step (1), obtain 5 kinds of principle active component, set up HPLC finger-print, using α-cyperolone for reference peaks is as reference peak, relative content mensuration is carried out to explored principle active component fingerprint peaks, according to computing formula: fingerprint peaks relative content=(fingerprint peaks peak area/reference peaks peak area) × reference peaks content, obtain principle active component fingerprint peaks relative content, set up the quantivative approach of 5 kinds of effect components.The methodological study test findings of the method all shows: retention time and the peak area RSD of 5 effect components fingerprint peakses are all less than 5%, shows that the reappearance of instrument precision, detection method, sample solution stability in 24 hours etc. is all good.
In step (1), the specification of chromatographic column: DiamonsilC18 is 250 × 4.6mm, 5 μm.
In step (1), preferred detection wavelength: 250nm; Flow velocity: 1.0mL/min.
In step (1), mobile phase: the volume ratio of methyl alcohol (A) and double distilled water (B) gradient elution is: 0-10min, 30%-70%A and 70%-30%B; 10-45min, 70%-100%A and 30%-0%B.
In step (1), the method for gradient elution is as shown in the table:
In step (5), 5 kinds of principle active component retention times are respectively: 16.1min, 18.5min, 24.9min, 29.5min and 30.5min.
The method of quality control of four rhizoma cyperi medicine materical crude slice processed of the present invention, get rhizoma cyperi crude drug, follow traditional processing procedure, " wine, vinegar, salt, ginger " four kinds of auxiliary materials are adopted to concoct, number of chemical mode identification technology is selected to verify spectrum-effect relationship in conjunction with research methods such as pharmacodynamics effects, and carry out being separated, confirming activity, with the relative content of the fingerprint peaks and reference substance reference peaks that confirm active principle active component for evaluation index, formulate four rhizoma cyperi prepared slice quality control methods processed.The foundation of the method is conducive to the control of the industrialization four rhizoma cyperi medicine materical crude slice processed quality of production, guarantees the validity of prepared slices of Chinese crude drugs clinical application.The present invention is intended to carry forward traditional processing technique, advances the process of the standardization of four rhizoma cyperis processed, standardization and suitability for industrialized production, promotes that it is applied on a large scale.Feature of the present invention is: set up four rhizoma cyperi HPLC finger-prints processed, with active component α-cyperolone for reference peaks, the content of α-cyperolone in Calculation Basis peak, the fingerprint peaks of other main active for reference, calculates each active component fingerprint peaks in the relative content of α-cyperolone with α-cyperolone.Method has operability, only needs the relative content that can control other unknown principle active component with 1 known reference substance α-cyperolone component content, reaches a kind of scientific approach of control four rhizoma cyperi processed prepared slice quality.The method is simple, be easy to promote, decrease the use amount of reference substance, solve market monomeric substance reference substance deficiency and the problem of costliness, four rhizoma cyperi prepared slice qualities processed are stablized, and drug action strengthens, and contributes to promoting Chinese Medicine Industry fast-developing, this is the needs of Respect and development Chinese medicine tradition processing technique, is also to ensure that the key point of " steady quality, technological specification, medicine materical crude slice are controlled " accomplished by Chinese medicine preparation medicine materical crude slice.
Good effect of the present invention is as follows:
1. break through previously About The Quality of Sliced Herbal Medicine to control to select the higher composition of some or multiple content to be the analytical approach of index in research, according to the chemical composition of four rhizoma cyperis processed, the systematic study results such as pharmacodynamics effect, with confirmed effect components for evaluation index, the relative content of other unknown effect components is only controlled with a kind of known α-cyperolone composition reference substance content, set up prepared slice quality control method, solve the in short supply and expensive problem of reference substance, explore a kind of reference substance carries out fixing quantity method feasibility to multiple effect components, test method has operability.
2. the optimization that the foundation of the method for quality control of four rhizoma cyperi medicine materical crude slice processed can be processing procedure provides foundation.After optimizing, the anti-dysmenorrhoea pharmacodynamics effect of gained four rhizoma cyperi processed medicine materical crude slice improves a lot.
Accompanying drawing explanation
Fig. 1 is reference substance α-cyperolone finger-print.
Fig. 2 is four rhizoma cyperi medicine materical crude slice finger-prints processed.
Embodiment
The following examples describe in further detail of the present invention.
Embodiment 1
(1) high effective liquid chromatography for measuring condition:
Chromatographic column: DiamonsilC18; Determined wavelength: 250nm; Flow velocity: 1.0mL/min; Sample size: 20 μ L; Column temperature: room temperature; Mobile phase: methyl alcohol and double distilled water; Elution program: gradient elution;
(2) preparation of reference substance solution:
Precision measures α-cyperolone reference substance 0.05mL, is placed in 10mL volumetric flask, is diluted to scale, shakes up with 95% ethanol, and obtaining concentration is 5ug/mL reference substance solution;
(3) foundation of standard equation:
Accurate reference substance solution of drawing is mixed with series concentration, injection liquid chromatography, by the chromatographic condition analysis of step (1), measure peak area, with reference substance amount for horizontal ordinate, peak area value is that ordinate obtains standard equation y=136.75x+3.5384, R=0.9996.
(4) preparation of need testing solution:
Get four rhizoma cyperi meal 4g processed, add 95% ethanol 32mL, heating and refluxing extraction 3 times, each 2h, merging filtrate is settled to 100mL, obtains four rhizoma cyperi medicinal material liquid processed, with the filtering with microporous membrane of 0.45 μm, obtains need testing solution;
(5) foundation of HPLC finger-print:
Stratographic analysis is carried out to test sample, by the chromatographic condition analysis of step (1), obtain 5 kinds of principle active component, set up HPLC finger-print, using α-cyperolone for reference peaks is as reference peak, relative content mensuration is carried out to explored principle active component fingerprint peaks, according to computing formula: fingerprint peaks relative content=(fingerprint peaks peak area/reference peaks peak area) × reference peaks content, obtain principle active component fingerprint peaks relative content, set up the quantivative approach of 5 kinds of effect components.The methodological study test findings of the method all shows: retention time and the peak area RSD of 5 effect components fingerprint peakses are all less than 5%, shows that the reappearance of instrument precision, detection method, sample solution stability in 24 hours etc. is all good.
In step (1), the specification of chromatographic column: DiamonsilC18 is 250 × 4.6mm, 5 μm.
In step (1), mobile phase: methyl alcohol and double distilled water gradient elution.
In step (1), the method for gradient elution is as shown in the table:
Table 1 elution program
In step (5), 5 kinds of principle active component retention times are respectively: 16.1min, 18.5min, 24.9min, 29.5min and 30.5min.
In Fig. 2, the cubage method of 5 kinds of principle active component such as α-cyperolone:
The calculating of α-cyperolone reference peaks content
According to set up normal equation y=136.75x+3.5384, measure α-cyperolone finger-print peak area (i.e. α-cyperolone reference peaks peak area) by HPLC, substitute into equation and calculate its content (being α-cyperolone reference peaks content);
The relative content computing formula of other 4 kinds of effective constituents:
Effective constituent fingerprint peaks relative content=(effective constituent fingerprint peaks peak area/α-cyperolone reference peaks peak area) × α-cyperolone reference peaks content, obtains principle active component fingerprint peaks relative content.
The principal ingredient compound that retention time is respectively 2 content of 24.9min and 29.5min higher is respectively through NMR Structural Identification: cyperotundone and α-cyperone (α-cyperolone).Other 3 compositions need to confirm further.
Methodological study test findings
Select four rhizoma cyperi samples processed, instrument precision, stability of solution and experimental technique repeatability are investigated.Result shows: the retention time of 5 effect components fingerprint peakses and the RSD of peak area are all less than 5%.
Precision test
Get same need testing solution, by above-mentioned chromatographic condition, continuous sample introduction 6 times, record finger-print.With α-cyperolone for reference peak, calculate retention time and the peak area of 5 effective constituents, the accuracy of detecting instrument, result is as table 2.
Table 2 Precision test result
Stability test
Get same need testing solution, by above-mentioned chromatographic condition, measured respectively at 0,2,4,6,8,12 and 24 hour, record finger-print, with α-cyperolone for reference peak, calculate retention time and the peak area of each one-tenth swarming, detect the stability of need testing solution in 24 hours, test findings is as table 3.
Table 3 stability test result
Reappearance is tested
Prepare 6 part of four rhizoma cyperi need testing solution processed according to sample preparation methods parallel extraction, respectively sequentially determining, record finger-print, with α-cyperolone for reference peak, calculate retention time and the peak area of each effective constituent.Detect the reappearance of this test method, test findings is as table 4.
Table 4 reproducible test results
Four rhizoma cyperis processed affect test findings to dysmenorrhea model in mice
Table 5 four rhizoma cyperi processed affects test findings (x ± s) to dysmenorrhea model in mice
Note: compare with control group, " * " represents P<0.05, and " * * " represents P<0.O1
Although illustrate and describe embodiments of the invention, for the ordinary skill in the art, be appreciated that and can carry out multiple change, amendment, replacement and modification to these embodiments without departing from the principles and spirit of the present invention, scope of the present invention is by claims and equivalents thereof.
Claims (3)
1. a method of quality control for four rhizoma cyperi medicine materical crude slice processed, is characterized in that: described method is as follows:
(1) high effective liquid chromatography for measuring condition:
Chromatographic column: DiamonsilC18; Determined wavelength: 220-280nm; Flow velocity: 0.5-1.5mL/min; Sample size: 20 μ L; Column temperature: room temperature; Mobile phase: methyl alcohol and double distilled water; Gradient elution, mobile phase: the volume ratio of methyl alcohol and double distilled water gradient elution is: 0-10min, 30%-70% methyl alcohol and 70%-30% double distilled water; 10-45min, 70%-100% methyl alcohol and 30%-0% double distilled water;
(2) preparation of reference substance solution:
Precision measures α-cyperolone reference substance 0.05mL, is placed in 10mL volumetric flask, is diluted to scale, shakes up with 95% ethanol, and obtaining concentration is 5 μ g/mL reference substance solution;
(3) foundation of standard equation:
Accurate reference substance solution of drawing is mixed with series concentration, injection liquid chromatography, by the chromatographic condition analysis of step (1), measure peak area, with reference substance amount for horizontal ordinate, peak area value is that ordinate obtains standard equation y=136.75x+3.5384, R=0.9996;
(4) preparation of need testing solution:
Get four rhizoma cyperi meal 4g processed, add 95% ethanol 32mL, heating and refluxing extraction 3 times, each 2h, merging filtrate is settled to 100mL, obtains four rhizoma cyperi medicinal material liquid processed, with the filtering with microporous membrane of 0.45 μm, obtains need testing solution;
(5) foundation of HPLC finger-print:
Stratographic analysis is carried out to test sample, by the chromatographic condition analysis of step (1), obtain 5 kinds of principle active component, 5 kinds of principle active component retention times are respectively: 16.1min, 18.5min, 24.9min, 29.5min and 30.5min, set up HPLC finger-print, using α-cyperolone for reference peaks is as reference peak, relative content mensuration is carried out to explored principle active component fingerprint peaks, according to computing formula: fingerprint peaks relative content=(fingerprint peaks peak area/reference peaks peak area) × reference peaks content, obtain principle active component fingerprint peaks relative content, set up the quantivative approach of 5 kinds of effect components.
2. the method for quality control of four rhizoma cyperi medicine materical crude slice processed as claimed in claim 1, is characterized in that: in step (1), and the specification of chromatographic column: DiamonsilC18 is 250mm × 4.6mm, 5 μm.
3. the method for quality control of four rhizoma cyperi medicine materical crude slice processed as claimed in claim 1, is characterized in that: in step (1), determined wavelength: 250nm; Flow velocity: 1.0mL/min.
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| CN108426963B (en) * | 2018-05-31 | 2020-12-25 | 山东中医药大学 | Method for constructing HPLC fingerprint of vinegar rhizoma cyperi |
| CN112666277B (en) * | 2020-11-30 | 2022-07-08 | 四川新绿色药业科技发展有限公司 | HPLC (high Performance liquid chromatography) characteristic spectrum construction and detection method for rhizoma cyperi medicinal materials, decoction pieces, standard decoction and formula granules |
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Effective date of registration: 20170306 Address after: 332000 Jiangxi province Jiujiang Xunyang District Chengdong Industrial Park No. 3 base Patentee after: JIANGXI GUANGGUI CHINESE HERBAL MEDICINE CO.,LTD. Address before: 330004 Jiangxi city of Nanchang province Wanli District Hing Wan Road No. 818 Patentee before: JIANGXI UNIVERSITY OF TRADITIONAL CHINESE MEDICINE |
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Address after: 332000 Park 3, Chengdong Industrial Base, Xunyang District, Jiujiang City, Jiangxi Province Patentee after: Jiangxi Longkaihe Traditional Chinese Medicine Slices Co.,Ltd. Address before: 332000 Park 3, Chengdong Industrial Base, Xunyang District, Jiujiang City, Jiangxi Province Patentee before: JIANGXI GUANGGUI CHINESE HERBAL MEDICINE CO.,LTD. |