CN109030674A - A kind of radix bupleuri reference extract and its preparation method and application - Google Patents
A kind of radix bupleuri reference extract and its preparation method and application Download PDFInfo
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- CN109030674A CN109030674A CN201810728903.XA CN201810728903A CN109030674A CN 109030674 A CN109030674 A CN 109030674A CN 201810728903 A CN201810728903 A CN 201810728903A CN 109030674 A CN109030674 A CN 109030674A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
Abstract
The present invention provides a kind of radix bupleuri reference extracts, it is derived from: carrying out multiple low-temperature extraction, concentration, drying to Radix Bupleuri powder, the bupleurum extract of different batches is obtained, then the bupleurum extract of different batches is deployed, obtains radix bupleuri reference extract.Radix bupleuri reference extract of the invention ensure that the consistency of the radix bupleuri reference extract of different batches because being that the extract of different batches deploy；And its character is stable, uniform and easy to use.The radix bupleuri reference extract is upper in application, it is as control for radix bupleuri and containing in the control of the quality of radix bupleuri/radix bupleuri effective component medicinal material or Chinese materia medica preparation, in this quality control procedure, it is that radix bupleuri reference extract progress simple process can be used directly, it is easy to operate, especially in multicomponent assay, the preparation of reference extract solution is easier than the preparation of reference substance solution, not only Qualitive test can be carried out to medicinal material or Chinese materia medica preparation, but also can be also used for sxemiquantitative even quantitative analysis.
Description
Technical field
The present invention relates to quality control technologies for traditional Chinese medicine fields, more particularly to a kind of preparation side of radix bupleuri reference extract
Method.
Background technique
Existing traditional Chinese medicine quality control mode is substantially the development along Natural Medicine Chemistry, a certain or several to Chinese medicine
Effective component is the analysis method and the not only design of difinite quality but also the quality standard that can be quantified of target, referring to the matter of external botanical medicine
Amount control method is used for reference the mode of chemicals quality control, and is identified by document report foundation is simply physical and chemical accordingly, then
Develop to the identification based on spectrum, chromatography and the quality standard of assay.It is all multicomponent synthesis per Chinese medicine simply
Body also indicates that one or two using in medicinal material even several ingredients as medicinal material matter which dictates that its distinctive globality and ambiguity
There are significant limitations for the evaluation method of amount.1990 editions " Chinese Pharmacopoeias " increase the indentification by TLC of control medicinal material, make
The identification for obtaining Chinese medicine and Chinese patent drug has very big progress.Chinese medicine and external herbal medicine increasingly pay attention to multicomponent or multiple groups at present
The detection divided, such as the quality control of German ginkgo biloba p.e.The analysis method of finger-print, can be on the whole to medicinal material
Quality control is carried out, currently still there is very big researching value.There are two types of " right in terms of controlling quality of medicinal material for Chinese Pharmacopoeia at present
According to product ", chemical reference substance and Chinese medicine control medicinal material.Wherein chemical reference substance can be used for Qualitive test and the quantitative analysis of medicinal material,
Chinese medicine control medicinal material is then used for microscopical characters and thin layer identifies.However, chemical reference substance and Chinese medicine control medicinal material are in traditional Chinese medicine quality
There is its limitation in control.Firstly, chemical component diversification, single or several compounds can not reflect medicinal material in Chinese medicine
Overall picture, and existing standard often has many loopholes, and the phenomenon that adulterating happens occasionally.Chinese medicine control medicinal material is produced
The influence on ground, growing environment, it is difficult to ensure that per batch of uniform quality, and it is only used for Qualitive test, it can not reflect medicinal material
The height of component content.
Chinese medicine reference extract is the character and stable components prepared with Chinese medicine, can be used for qualitative or quantitative analysis
Extract, Mr. Xie Peishan proposes four basic demands (BSCS) to Chinese medicine reference extract and reference extract is answered
Have several primary conditions: Buthenticity, crude drug source is reliable, representative；Specificity, the detection used
Method specificity；Con-sistency, different batches reference extract should be consistent；StBbility, character is stable, uniformly,
It is easy to use.With the methods of high performance thin layer chromatography finger-print and high-efficiency liquid-phase fingerprint, it can be used for determining for medicinal material
Property identify, by the reference extract of external standard method mark, sxemiquantitative and quantitative analysis detection can be further utilized to.Chinese medicine control
Extract will have important meaning to traditional Chinese medicine quality control.
Summary of the invention
That the technical problem to be solved by the present invention is to provide a kind of batch consistency is good, character is stable, uniform and easy to use
Radix bupleuri reference extract, the present invention also provides the preparation method and application of the radix bupleuri reference extract, i.e., by the radix bupleuri
Reference extract is for radix bupleuri and containing in the control of the quality of radix bupleuri/radix bupleuri effective component medicinal material or Chinese materia medica preparation.
First aspect present invention provides a kind of radix bupleuri reference extract, derives from: carrying out to Radix Bupleuri powder more
The secondary bupleurum extract extract, be concentrated, being dried to obtain different batches, then deploys the bupleurum extract of different batches,
Obtain radix bupleuri reference extract.
Preferably, the sum of content of saikoside A and saikosaponin D is not less than 0.30% in the radix bupleuri reference extract
And it is not higher than 3.00%.
Preferably, the figure for using thin-layered chromatography and/or high performance liquid chromatography to obtain the radix bupleuri reference extract
It is consistent to compose corresponding raw medicinal material.
It is highly preferred that being measured to the radix bupleuri reference extract using thin-layered chromatography inspection and/or high performance liquid chromatography
To chromatogram in saikoside A it is consistent with the corresponding chemical reference substance of saikosaponin D or its corresponding raw medicinal material.
Radix Bupleuri powder repeatedly to be extracted, the concentration of the saikoside A and saikosaponin D that contain it improve,
To obtain the bupleurum extract of different batches.Described is repeatedly 1-7 times.
Second aspect of the present invention provides the preparation method of radix bupleuri reference extract described in one kind, comprising the following steps:
Step 1: extracting: taking Radix Bupleuri powder to mix with ethyl alcohol, filtered after extraction, obtain filtrate 1 and filter residue 1, will filter
Slag 1 merges with filtrate 1 Ji Wei extracting solution in the same manner by extracting by the filtrate collected is extracted, and will extract
Liquid is concentrated to get radix bupleuri dry cream；
Step 2: preparing bupleurum extract: by gained radix bupleuri dry cream, being dissolved in ethyl alcohol, the ethyl alcohol for obtaining radix bupleuri dry cream is molten
Liquid, adds auxiliary material, and drying and screening obtains bupleurum extract；
Step 3: allotment: the bupleurum extract of different batches being deployed, radix bupleuri reference extract is obtained.
It preferably, further include with thin-layered chromatography and/or high performance liquid chromatography pair between the step 3 and step 4
The step of saikoside A and saikosaponin D of the bupleurum extract of different batches are detected.
Preferably, extraction of the step 1 into step 3, concentration and drying are carried out by the way of low-temperature treatment.
It is highly preferred that the low temperature is 20 DEG C~60 DEG C.
Preferably, filter residue 1 through in the step one in the same manner and being extracted, the number of extraction is N
It is secondary, wherein 6 >=N >=0, and N is integer.
It is highly preferred that the N is 3.
Preferably, the concentration of alcohol described in step 1 is 30%~95%.
It is highly preferred that concentration of alcohol is 70% in the step 1.
Preferably, the w/v of the Radix Bupleuri powder in the step 1 and ethyl alcohol is 1:5~1:45, weight
Measurement unit is g, and the measurement unit of volume is ml.
It is highly preferred that the w/v of Radix Bupleuri powder and ethyl alcohol in the step 1 is 1:10.
Preferably, the extracting mode in the step 1 are as follows: homogenate extraction.
Preferably, the homogenate extraction time in the step 1 is 1~5min.
It is highly preferred that the homogenate extraction time in the step 1 is 1min.
Preferably, the filtering in the step 1 is with Medium speed filter paper or 2000 mesh net filtrations.
It is highly preferred that the filtering in the step 1 is to use Medium speed filter paper.
Preferably, in the step 2 radix bupleuri dry cream and ethyl alcohol w/v: 1:2~1:10.
Preferably, auxiliary material used is superfine silica gel powder and/or medical starch in the step 2.
Preferably, the auxiliary material is superfine silica gel powder.
Preferably, the step 2 is 10%~50% micro mist that its weight is added in the ethanol solution of radix bupleuri dry cream
Silica gel.
Preferably, the step 2 is 40% superfine silica gel powder that its weight is added in the ethanol solution of radix bupleuri dry cream.
It preferably, was 90~200 mesh net filtrations after the step 2 low temperature drying.
It preferably, was 110 mesh net filtrations after the step 2 low temperature drying.
Preferably, the total of saikoside A and saikosaponin D in finally obtained reference extract is deployed in the step 3
Content must not be lower than 0.30% and not higher than 3.00%.
Preferably, the figure for using thin-layered chromatography and/or high performance liquid chromatography to obtain the radix bupleuri reference extract
It is consistent to compose corresponding raw medicinal material.
It is highly preferred that being measured to the radix bupleuri reference extract using thin-layered chromatography inspection and/or high performance liquid chromatography
To chromatogram in saikoside A it is consistent with the corresponding chemical reference substance of saikosaponin D or its corresponding raw medicinal material.
Third aspect present invention provides above-mentioned radix bupleuri reference extract answering in the identification of medicinal material or Chinese materia medica preparation
With.
Preferably, the identification is for radix bupleuri or containing radix bupleuri/medicinal material of radix bupleuri effective component or the quality of Chinese materia medica preparation
In control.
Preferably, the method that the described application uses is as follows: using the radix bupleuri reference extract of claim 1 as
Reference material, passes through thin-layered chromatography together with medicinal material or Chinese materia medica preparation and/or high performance liquid chromatography is detected, according to detection
Comparative result judgement.
Preferably, the thin-layered chromatography and/or high performance liquid chromatography are to radix bupleuri reference extract, medicinal material or Chinese medicine
Saikoside A and saikosaponin D in preparation are detected.
Preferably, affiliated thin-layered chromatography testing conditions are as follows:
Testing conditions are as follows:
Lamellae: TLC G60 prefabricated board (Merck)；
Point sample: 2 μ l, the long 8mm of ribbon point sample；
Solvent: methylene chloride: ethyl acetate: methanol: water=30:40:15:3；
Expansion mode: adding solvent 12ml in side in double flute expansion cylinder (20cm × 10cm), common to pre-equilibrate 15 minutes；
The drying of lamellae: heating 15 minutes after point sample under 50 DEG C of heating plates guarantees the drying of lamellae；
It inspects: after the residual solvents that go back are taken out in spray, spraying with -10% sulfuric acid ethyl alcohol color developing agent of 2% paradime thylaminobenzaldehyde,
1min is heated in 105 DEG C of heating plates, is placed in fluorescence viewing chromatogram image under ultraviolet lamp (366nm)；
Preferably, the high performance liquid chromatography testing conditions are as follows:
Chromatographic apparatus: Agilent 1260series high performance liquid chromatograph, equipped with DAD detector (U.S.,
AgilentTechnologies)
Chromatographic column: Agilent Zorbax SB-C18 (4.6mm × 250mm, 5 μm)；
Mobile phase: A- acetonitrile, B- water；
Gradient elution program: 0-15min:25%A → 35%A, 15-38min:35%A → 45%A, 38-60min:45%
A → 60%A, 60-70min:60%A → 100%A；
0-15min:75%B → 65%B, 15-38min:65%B → 55%B, 38-60min:55%B → 40%B, 60-
70min:40%B → 0%B；
Detection wavelength 203nm (DAD detector)；Flow velocity 1.0ml/min；10 μ l of sample volume；25 DEG C of column temperature, runing time:
70min。
Compared with prior art, the invention has the following advantages:
Radix bupleuri reference extract of the invention overcomes because being that the extract of different batches deploy because of Chinese medicine pair
It is influenced according to medicinal material by the place of production, growing environment, it is difficult to ensure that the defect per batch of uniform quality, to ensure that different batches
The consistency of secondary radix bupleuri reference extract；The low temperature control that ensure that preparation overall process, effectively prevents the change of effective component
Change, and its character is stable, uniform and easy to use.The radix bupleuri reference extract is upper in application, is to compare radix bupleuri to extract
Object is after quantitative analysis, as control for radix bupleuri and containing radix bupleuri/medicinal material of radix bupleuri effective component or the quality of Chinese materia medica preparation
It is that radix bupleuri reference extract progress simple process can be used directly in this quality control procedure in control, operation letter
Just, especially in multicomponent assay, the preparation of reference extract solution is easier than the preparation of reference substance solution, not only
Qualitive test can be carried out to medicinal material or Chinese materia medica preparation, and can be also used for sxemiquantitative even quantitative analysis.
Detailed description of the invention
Fig. 1 is to carry out thin layer color to radix bupleuri raw medicinal material for using chemical reference substance and radix bupleuri control medicinal material as reference substance
The thin-layer chromatogram that spectrometry obtains.Wherein the chromatographic band of label S1, S2 respectively corresponds as reference substance saikoside A and saikoside
D, 5 correspond to radix bupleuri control medicinal material, and 1-4 is corresponding in turn to radix bupleuri raw medicinal material 1, radix bupleuri raw medicinal material 2,3 and of radix bupleuri raw medicinal material
Radix bupleuri raw medicinal material 4.
Fig. 2 is to carry out efficient liquid phase for using chemical reference substance and radix bupleuri control medicinal material as reference substance radix bupleuri raw medicinal material
The HPLC finger-print stacking chart that chromatography obtains, 1-4 respectively corresponds radix bupleuri raw medicinal material 1, radix bupleuri raw medicinal material from top to bottom
2, radix bupleuri raw medicinal material 3 and radix bupleuri raw medicinal material 4,5 correspond to radix bupleuri control medicinal material, and S corresponds to chemical reference substance, and R is corresponding shared
Mode.
Fig. 3 is radix bupleuri reference extract preparation flow figure.
Fig. 4 is that radix bupleuri reference extract and radix bupleuri extract raw medicinal material common pattern stacking chart, and S corresponds to chemical reference substance,
ERS corresponds to radix bupleuri reference extract, and R is radix bupleuri raw medicinal material common pattern.
Fig. 5 is for using chemical reference substance, radix bupleuri control medicinal material and radix bupleuri reference extract being reference substance to commercially available medicine
Material carries out the thin-layer chromatogram that thin-layered chromatography obtains.Wherein the chromatographic band of label S1, S2 respectively corresponds as reference substance radix bupleuri soap
Glycosides A and saikosaponin D, 10 correspond to radix bupleuri control medicinal material, and 11 correspond to radix bupleuri reference extract, and 1-9 is corresponding in turn to radix bupleuri medicine
Material 1, Radix Bupleuri 2, Radix Bupleuri 3, Radix Bupleuri 4, RADIX BUPLEURI SCORZONERAEFOLII 5, RADIX BUPLEURI SCORZONERAEFOLII 6, Radix Bupleuri 5, Radix Bupleuri 6 and radix bupleuri medicine
Material 7.
Fig. 6 is Radix Bupleuri and reference extract high performance thin layer chromatography figure digital scanning map, and 10 correspond to radix bupleuri control
Medicinal material, 11 correspond to radix bupleuri reference extract, and 1-9 is corresponding in turn to Radix Bupleuri 1, Radix Bupleuri 2, Radix Bupleuri 3, Radix Bupleuri
4, RADIX BUPLEURI SCORZONERAEFOLII 5, RADIX BUPLEURI SCORZONERAEFOLII 6, Radix Bupleuri 5, Radix Bupleuri 6 and Radix Bupleuri 7.
Fig. 7 is for using chemical reference substance, radix bupleuri control medicinal material and radix bupleuri reference extract being reference substance to commercially available medicine
Material carries out the HPLC finger-print stacking chart that high performance liquid chromatography obtains, and 3-9 is corresponding in turn to Radix Bupleuri 1, bavin from top to bottom
Hu medicinal material 2, Radix Bupleuri 3, Radix Bupleuri 4, Radix Bupleuri 5, Radix Bupleuri 6, Radix Bupleuri 7, Radix Bupleuri 8.1 is radix bupleuri pair
Radix bupleuri control medicinal material is corresponded to according to extract, 2, S1, S2 respectively correspond chemical reference substance saikoside A and saikosaponin D, and R pairs
Answer common pattern.
Term is explained:
Accurately weighed: denotion takes one thousandth of the weight accurately to taken weight.
It is weighed: to weigh weight accurately to 1 the percent of taken weight.
Specific embodiment
The embodiment of the present invention is described below in detail.The embodiments described below is exemplary, and is only used for explaining this hair
It is bright, and be not considered as limiting the invention.Particular technique or condition are not specified in embodiment, according to text in the art
It offers described technology or conditions or is carried out according to product description.Reagents or instruments used without specified manufacturer,
For can be with conventional products that are commercially available.Term as used herein "and/or" includes one or more relevant listed
Any and all combinations of project.
The source of the instrument and material that use in following embodiment is as follows:
It is envisaged for preparing the medicinal material of reference extract:
Radix Bupleuri is purchased from the major medicinal material market in the whole nation, is accredited as umbelliferae bupleurum through professor Xie Peishan
The dry root of Bupleurum chinense DC.
The semi-automatic point sample instrument of 5 thin-layer chromatography of Linomat, the full-automatic point sample instrument of 4 thin-layer chromatography of BTS, thin-layer chromatography double flute
Expansion cylinder, Chromatogram Immersion Device III chromatography colour developing dipping tank and TLCvisualizer thin layer color
It composes video camera (Switzerland, CAMAG).
Agilent 1260series high performance liquid chromatograph, equipped with DAD detector (U.S.,
AgilentTechnologies)。
Methylene chloride, ethyl alcohol, ethyl acetate, methanol, phosphoric acid, strong ammonia solution are to analyze pure (Guangzhou Chemical Reagent Factory).
Acetonitrile is chromatographically pure (Merck KGaA).
Saikoside A indicates purity >=97.3%, (National Institute for Food and Drugs Control；Lot number: 110778-
201510)；
Saikosaponin D indicates purity >=93.2%, (National Institute for Food and Drugs Control；Lot number: 110777-
201510)；
Radix bupleuri control medicinal material: National Institute for Food and Drugs Control；Lot number: 120992-201509；
Radix bupleuri raw medicinal material: 4 batches of buyings are in the major pharmacy in Guangzhou and medicinal material market, radix bupleuri raw medicinal material 1~4；
Radix Bupleuri test sample: Radix Bupleuri 7 batches and RADIX BUPLEURI SCORZONERAEFOLII 2 batches buyings are in the major pharmacy in Guangzhou and medicinal material market, bavin
Hu medicinal material 1~7, RADIX BUPLEURI SCORZONERAEFOLII 1, RADIX BUPLEURI SCORZONERAEFOLII 2.
Embodiment 1: the screening of radix bupleuri raw medicinal material
Present embodiments provide the screening technique of the raw medicinal material of radix bupleuri reference extract of the present invention.
With thin-layered chromatography and high performance liquid chromatography respectively using chemical reference substance and radix bupleuri control medicinal material as object of reference
Confrontation radix bupleuri raw medicinal material is analyzed and is screened.
Radix bupleuri raw medicinal material is purchased from the major medicinal material market in the whole nation, teaches through Xie Peishan and reflects in method in 2020 editions Chinese Pharmacopoeias
It is set to the dry root of umbelliferae bupleurum Bupleurum chinense DC.Identified 4 batches of medicinal materials comply with standard can be in case of
With.
1 thin-layer chromatography
1.1 sample preparation
Chemical reference substance solution: precision weighs saikoside A and saikosaponin D, is configured to the molten of 0.5mg/ml with ethyl alcohol
Liquid.
The preparation of radix bupleuri raw medicinal material solution: raw medicinal material powder is crossed into No. four sieves (250 ± 9.965 mesh), is weighed respectively
Ethyl alcohol 10ml is added in 0.5g, is ultrasonically treated 30 minutes, centrifugation 3 minutes (revolving speed is 3200 turns per minute), takes supernatant mistake
0.22um filter membrane is up to radix bupleuri raw medicinal material solution.
Radix bupleuri control medicinal material sample solution preparation: using 1.1 radix bupleuri raw medicinal material solution manufacturing method systems in embodiment 1
It is standby.
The detection of 1.2 thin-layer chromatographys
Testing conditions are as follows:
Lamellae: TLC G60 prefabricated board (Merck)；
Point sample: 2 μ l, the long 8mm of ribbon point sample；
Solvent: methylene chloride: ethyl acetate: methanol: water=30:40:15:3；
Expansion mode: adding solvent 12ml in side in double flute expansion cylinder (20cm × 10cm), common to pre-equilibrate 15 minutes；
The drying of lamellae: heating 15 minutes after point sample under 50 DEG C of heating plates guarantees the drying of lamellae；
It inspects: after the residual solvents that go back are taken out in spray, spraying with -10% sulfuric acid ethyl alcohol color developing agent of 2% paradime thylaminobenzaldehyde,
1min is heated in 105 DEG C of heating plates, is placed in fluorescence viewing chromatogram image under ultraviolet lamp (366nm).
Testing result: as shown in Figure 1, to inspect thin-layer chromatogram under ultraviolet lamp (366nm).The chromatographic band of label S1, S2
Respectively reference substance saikoside A and saikosaponin D；Chromatographic bands 1-4 is corresponding in turn to radix bupleuri raw medicinal material 1, radix bupleuri bulk pharmaceutical chemicals
Material 2, radix bupleuri raw medicinal material 3, radix bupleuri raw medicinal material 4；Chromatographic bands 5 are radix bupleuri control medicinal material.As seen from Figure 1, saikoside A and
Saikosaponin D separation situation is good, and each batch Radix Bupleuri can show identical spot in same position with radix bupleuri control medicinal material
Point, similarity is high between each batch Radix Bupleuri and radix bupleuri control medicinal material, and preliminary screening is that standard control extract extracts bulk pharmaceutical chemicals
Material.
2 high performance liquid chromatography
2.1 sample preparation
Chemical reference substance solution: taking chemical reference substance saikoside A and appropriate saikosaponin D, accurately weighed, uses first respectively
The solution of 0.5mg/ml is made in alcohol to obtain the final product.
The preparation of radix bupleuri raw medicinal material solution: taking medicinal material or Chinese materia medica preparation to cross No. four sieves (250 ± 9.965 mesh), precision weighs afterwards
0.5g is set in 50ml stuffed conical flask, and the methanol solution 25ml of the strong ammonia solution containing 5%, weighed weight, at ultrasonic 500w is added
Manage 30min after, put to room temperature, supply weightlessness with the methanol solution of 5% strong ammonia solution, shake up, with Medium speed filter paper (aperture 30~
50 μm) filtration, merged with methanol 20ml points of 2 washing containers and the dregs of a decoction, washing lotion with filtrate, recycling design to dry, residue first
Alcohol dissolution, is transferred to 5ml volumetric flask, with methanol constant volume to scale 5ml, shakes up, and crosses 0.22 μm of filter membrane, takes filtrate up to radix bupleuri
Raw medicinal material solution.
Radix bupleuri control medicinal material solution preparation: with 2.1 radix bupleuri raw medicinal material solution manufacturing methods preparation in embodiment 1.
2.2 high performance liquid chromatography detection
High performance liquid chromatography testing conditions are as follows:
Chromatographic apparatus: Agilent 1260series high performance liquid chromatograph, equipped with DAD detector (U.S.,
AgilentTechnologies)
Chromatographic column: Agilent Zorbax SB-C18 (4.6mm × 250mm, 5 μm)；
Mobile phase: A- acetonitrile, B- water；
Gradient elution program:
0-15min:25%A → 35%A, 15-38min:35%A → 45%A, 38-60min:45%A → 60%A, 60-
70min:60%A → 100%A；
0-15min:75%B → 65%B, 15-38min:65%B → 55%B, 38-60min:55%B → 40%B, 60-
70min:40%B → 0%B；
Detection wavelength 203nm (DAD detector)；Flow velocity 1.0ml/min；10 μ l of sample volume；25 DEG C of column temperature, runing time:
70min。
High effective liquid chromatography for detecting: respectively it is accurate draw chemical reference substance solution, radix bupleuri control medicinal material solution and
Each 10 μ l of radix bupleuri raw medicinal material solution injects high performance liquid chromatograph.
High performance liquid chromatography testing result:
The HPLC finger-print stacking chart of testing result as shown in Figure 2,1-4 respectively corresponds radix bupleuri raw medicinal material from top to bottom
1, radix bupleuri raw medicinal material 2, radix bupleuri raw medicinal material 3 and radix bupleuri raw medicinal material 4, S1, S2 respectively correspond chemical reference substance saikoside A
And saikosaponin D, 5 correspond to radix bupleuri control medicinal material, and R corresponds to common pattern.
As seen from Figure 2, the finger-print of radix bupleuri control medicinal material and 1-radix bupleuri of radix bupleuri raw medicinal material raw medicinal material 4 has
There is high consistency, establishes radix bupleuri using 2017Pro editions softwares of ChemPattern (Beijing Ke Maien Science and Technology Ltd.)
The finger-print common pattern of medicinal material, and similarity analysis is carried out to all samples.
According to set Radix Bupleuri finger-print common pattern, using included angle cosine algorithm according to each sample component and its
Peak area carries out similarity evaluation to 4 batches of radix bupleuri raw medicinal materials and radix bupleuri control medicinal material, and the results are shown in Table 1.
Table 1
| Sample | Similarity |
| Radix bupleuri raw medicinal material 1 | 0.990 |
| Radix bupleuri raw medicinal material 2 | 0.988 |
| Radix bupleuri raw medicinal material 3 | 0.921 |
| Radix bupleuri raw medicinal material 4 | 0.936 |
| Radix bupleuri control medicinal material | 0.983 |
| Common pattern | 1 |
According to the above similarity analysis as a result, the similarity of 4 batches of radix bupleuri raw medicinal materials and radix bupleuri control medicinal material is all very high, by
This this 4 batches of radix bupleuri raw medicinal material selects the raw medicinal material extracted for radix bupleuri standard control extract.
Embodiment 2: the preparation of radix bupleuri reference extract
The preparation method of radix bupleuri reference extract of the present invention is present embodiments provided, method flow diagram is as shown in Figure 3.
One: extracting
Radix Bupleuri 500g is chosen, powder is made, 24 mesh screens is crossed, the ethyl alcohol of its 20 times of volume of quality is then added
(it is g that i.e. solid-to-liquid ratio, which is the unit of 1:20 (w/V) quality, and the unit of volume is ml), middling speed qualitative filter paper after homogenate extraction 1min
Filtering, collects filter residue 1 and filtrate 1, filter residue 1 are extracted three times again by same procedure, and will extract the filtrate collected three times again
Merge with filtrate 1 Ji Wei extracting solution, is the 150g that gets dry extract by 50 DEG C of low temperature concentrations of extracting solution；
Two: preparing bupleurum extract
With 200ml70% ethyl alcohol by dry cream dissolution completely after, then plus dry cream quality 30% superfine silica gel powder (mountains and rivers medicine
It is auxiliary), with 50 DEG C of low temperature dryings of Rotary Evaporators, it crushed 110 meshes, obtain bupleurum extract.
4 batches of bupleurum extracts are prepared, are surveyed by reference substance saikoside A and saikosaponin D using high performance liquid chromatography
Fixed, testing result is as shown in table 2:
Table 2
Three: allotment
The bupleurum extract of 4 batches of step 2 is mixed in 1:1 ratio and blends to obtain radix bupleuri reference extract, finally
It is about 1:8.8 (g/g) that product, which corresponds to crude drug ratio,.Radix bupleuri reference extract and reference substance pass through reference substance and utilize efficient liquid
Phase chromatography measurement, wherein the measurement result of the content of each ingredient is as shown in table 3.
Table 3
Allotment standard are as follows: the sum of content of saikoside A and saikosaponin D should be made in final radix bupleuri reference extract no
It obtains lower than 0.30% and is not higher than 3.00% (content is in terms of wt%).The range of the content of each ingredient in this allotment standard is also
By the optimum data that repeatedly the comprehensive holding stability of experiment, consistency and subsequent application etc. obtain repeatedly.
Four: detection
The radix bupleuri reference extract that step 3 is deployed into is referred to by reference substance using high effective liquid chromatography for measuring
Line map carries out similarity detection with raw medicinal material common pattern map is prepared, and such as Fig. 4, ERS correspond to radix bupleuri reference extract,
S1, S2 respectively correspond chemical reference substance saikoside A and saikosaponin D, and R is radix bupleuri raw medicinal material common pattern.It obtains similar
Degree is 0.956, it can be seen that the similarity of radix bupleuri reference extract and Radix Bupleuri finger-print common pattern is high, this illustrates this
The radix bupleuri reference extract of invention can represent Radix Bupleuri.
The character analysis of 3 radix bupleuri reference extract of embodiment
1. apparent state: the radix bupleuri reference extract that embodiment 2 obtains is buff powder.
2. determination of moisture: being carried out according to 2015 editions Chinese Pharmacopoeia annex IX G (hypobaric drying method).Testing result is radix bupleuri
Reference extract water content is 3.5%.
3. uniformity test: 3 batch radix bupleuri reference extracts are prepared according to the method for embodiment 2, after measured between each batch
The thin-layer chromatography testing result difference very little for saikoside A and saikosaponin D, saikoside A and saikosaponin D it is glimmering
Hot spot point has apparent display, and is distributed very consistent；Through its ferulaic acid content of high performance liquid chromatography detection in allotment standard model
Within enclosing.Therefore the radix bupleuri reference extract consistency being prepared using the preparation method of radix bupleuri reference extract of the invention
It is very good.
4. stability test:
The test sample for the radix bupleuri reference extract for taking the method according to embodiment 2 of 3 different batches to prepare, according to country
The relevant regulations of " requirement of national drug standards substance Development Techniques " that pharmacopoeia commission works out, inspection target include character and molten
Xie Xing.
Test sample opening is placed in suitable clean container, is placed 10 days at a temperature of 60 DEG C, is taken in the 5th day and the 10th day
Sample is detected by stability high spot reviews project.
The results show that before and after hot test test sample character without significant change, before and after hot test thin-layer chromatogram also without
Significant change, dissolution rate measurement result carry out Independent samples t-test with SPSS, and calculated result P > 0.05 illustrates no conspicuousness
Difference.
Comparative example 3
Difference with embodiment 3 is: (can suitably provide comparative example)
Embodiment 4
Present embodiments provide the application for the radix bupleuri reference extract that embodiment 2 is prepared.
With thin-layered chromatography and high performance liquid chromatography respectively with the control of chemical reference substance, radix bupleuri control medicinal material and radix bupleuri
Extract is that reference substance analyzes commercially available medicinal material.
1 thin-layer chromatography
1.1 sample preparation
The preparation of chemical reference substance solution: precision weighs saikoside A and saikosaponin D, is configured to 0.5mg/ml with ethyl alcohol
Solution.
The preparation of Radix Bupleuri test solution: each 0.5g of medicinal powder (crossing 24 mesh screens) is taken, ethyl alcohol 10ml is added, surpasses
Sonication 30 minutes, centrifugation 3 minutes (revolving speed is 3200 turns per minute), supernatant is taken to cross 0.22um filter membrane up to medicinal material test sample
Solution.Medicinal material is bought from Guangzhou, respectively Radix Bupleuri 1, Radix Bupleuri 2, Radix Bupleuri 3, Radix Bupleuri 4, RADIX BUPLEURI SCORZONERAEFOLII 1, south
Radix bupleuri 2, Radix Bupleuri 5, bavin medicinal material Hu 6 and Radix Bupleuri 7.
Radix bupleuri sample solution preparation: using the identical method preparation of 1.1 Radix Bupleuri test solutions in embodiment 4.
The preparation of radix bupleuri reference extract solution: taking radix bupleuri ERS appropriate, accurately weighed, and methanol is added to dissolve, ultrasonic treatment
30min is made into the concentration of 15mg/ml, crosses 0.22 μm of filter membrane up to bupleurum extract reference substance solution.
The detection of 1.2 thin-layer chromatographys
Testing conditions are as follows:
Lamellae: TLC G60 prefabricated board (Merck)；
Point sample: 2 μ l, the long 8mm of ribbon point sample；
Solvent: methylene chloride: ethyl acetate: methanol: water=30:40:15:3；
Expansion mode: adding solvent 12ml in side in double flute expansion cylinder (20cm × 10cm), common to pre-equilibrate 15 minutes；
The drying of lamellae: heating 15 minutes after point sample under 50 DEG C of heating plates guarantees the drying of lamellae；
It inspects: after the residual solvents that go back are taken out in spray, spraying with -10% sulfuric acid ethyl alcohol color developing agent of 2% paradime thylaminobenzaldehyde,
1min is heated in 105 DEG C of heating plates, is placed in fluorescence viewing chromatogram image under ultraviolet lamp (366nm).
Testing result: as shown in figure 5, to inspect thin-layer chromatogram under ultraviolet lamp (366nm).The chromatographic band of label S1, S2
Respectively reference substance saikoside A and saikosaponin D, chromatographic bands 1-9 are corresponding in turn to Radix Bupleuri 1, Radix Bupleuri 2, radix bupleuri
Medicinal material 3, Radix Bupleuri 4, RADIX BUPLEURI SCORZONERAEFOLII 5, RADIX BUPLEURI SCORZONERAEFOLII 6, Radix Bupleuri 5, Radix Bupleuri 6 and Radix Bupleuri 7.10 correspond to radix bupleuri pair
Radix bupleuri reference extract is corresponded to according to medicinal material, 11.
Interpretation of result: as shown in Figure 5, saikoside A and saikosaponin D separation situation it is preferable, radix bupleuri reference extract and
Radix Bupleuri can show identical spot, RADIX BUPLEURI SCORZONERAEFOLII medicinal material (band 5, band 6) distribution situation and northern bavin in same position
(band 1-4) is significantly different recklessly, can be good at distinguishing by the method.
Fig. 6: Radix Bupleuri and reference extract high performance thin layer chromatography figure digital scanning map.
2 high performance liquid chromatography
2.1 sample preparation
The preparation of chemical reference substance solution: taking chemical reference substance saikoside A and appropriate saikosaponin D, accurately weighed, respectively
The solution of 0.5mg/ml is made with methanol to obtain the final product.
Radix bupleuri reference extract solution: it is appropriate that precision weighs radix bupleuri reference extract, adds methanol ultrasonic dissolution 30 minutes, matches
Be made the solution of the 1ml solvent reference extract of radix bupleuri containing 15mg to get.
The preparation of Radix Bupleuri test solution: precision weighs 0.5g after taking medicinal material or Chinese materia medica preparation to cross No. four sieves, sets 50ml
In stuffed conical flask, it is added the methanol solution 25ml of the strong ammonia solution containing 5%, weighed weight, after ultrasonic 500w processing 30min,
It lets cool, supplies weightlessness, shake up, with Medium speed filter paper (30~50 μm of aperture) filtration, with methanol 20ml points of 2 washing containers and medicines
Slag, washing lotion merge with filtrate, and to doing, residue is dissolved recycling design with methanol, are transferred to 5ml volumetric flask, with methanol constant volume to quarter
Degree, shakes up, and crosses 0.22 μm of filter membrane, takes filtrate to obtain the final product.
Radix bupleuri sample solution preparation: method preparation identical with 2.1 Radix Bupleuri test solutions in embodiment 4.
2.2 high performance liquid chromatography detection
Prepared solution, testing conditions are as follows in high performance liquid chromatography detection 2.1:
Chromatographic apparatus: Agilent 1260series high performance liquid chromatograph, equipped with DAD detector (U.S.,
AgilentTechnologies)
Chromatographic column: Agilent Zorbax SB-C18 (4.6mm × 250mm, 5 μm)；
Mobile phase: A- acetonitrile, B- water；
Gradient elution program: 0-15min:25%A → 35%A, 15-38min:35%A → 45%A, 38-60min:45%
A → 60%A, 60-70min:60%A → 100%A；
0-15min:75%B → 65%B, 15-38min:65%B → 55%B, 38-60min:55%B → 40%B, 60-
70min:40%B → 0%B；
Detection wavelength 203nm (DAD detector)；Flow velocity 1.0ml/min；10 μ l of sample volume；25 DEG C of column temperature, runing time:
70min。
High effective liquid chromatography for detecting: accurate respectively to draw chemical reference substance solution, radix bupleuri control medicinal material solution；Bavin
Hu reference extract solution and each 10 μ l of medicinal material test solution inject liquid chromatograph.
High performance liquid chromatography testing result:
The HPLC finger-print stacking chart of testing result as shown in Figure 7,3-9 is corresponding in turn to Radix Bupleuri 1, bavin from top to bottom
Hu medicinal material 2, Radix Bupleuri 3, Radix Bupleuri 4, Radix Bupleuri 5, Radix Bupleuri 6 and Radix Bupleuri 7.1 is radix bupleuri reference extract,
2 be radix bupleuri control medicinal material, and S1, S2 respectively correspond chemical reference substance saikoside A and saikosaponin D, and R corresponds to common pattern.
As seen from Figure 7, the finger-print of Radix Bupleuri 1- Radix Bupleuri 7 and radix bupleuri control medicinal material has the one of height
Cause property.According to radix bupleuri reference extract finger-print, using included angle cosine algorithm according to each sample component and its peak area to 7 batches
Radix Bupleuri and radix bupleuri control medicinal material finger-print carry out similarity evaluation, and the results are shown in Table 4.
Table 4
| Sample | Similarity |
| Radix bupleuri 1 | 0.961 |
| Radix bupleuri 2 | 0.957 |
| Radix bupleuri 3 | 0.915 |
| Radix bupleuri 4 | 0.914 |
| Radix bupleuri 5 | 0.652 |
| Radix bupleuri 6 | 0.939 |
| Radix bupleuri 7 | 0.971 |
| Radix bupleuri control medicinal material | 0.914 |
| Common pattern | 0.93 |
| Radix bupleuri ERS | 1 |
According to the above similarity analysis as a result, 7 batches of Radix Bupleuris remaining sample and radix bupleuri control in addition to radix bupleuri radix bupleuri 5 are extracted
Similarity >=0.900 of object, similarity are very high.Show that the consistency of radix bupleuri reference extract and medicinal material of the invention is good, answers
For medicinal material or the sxemiquantitative discriminatory analysis of Chinese materia medica preparation.
According to the result of TLC and HPLC finger-print it is found that being examined to the radix bupleuri reference extract using thin-layered chromatography
The corresponding medicinal material of the chromatogram measured is consistent, more precisely, uses thin-layer chromatography to the radix bupleuri reference extract
Fluorescence spot at saikoside A and saikosaponin D position in the chromatogram that method detects and use high performance liquid chromatography
Chromatographic peak of the chromatogram that method detects at saikoside A and saikosaponin D position distinguishes corresponding chemical reference
Product or the consistent therefore of the invention radix bupleuri reference extract of its corresponding raw medicinal material can be applied to determining for medicinal material or Chinese materia medica preparation
Property identify, such as qualitative analysis is carried out to the saikoside A and saikosaponin D ingredient of medicinal material or Chinese materia medica preparation.
According to HPLC measurement result and statistical analysis, the bupleurum extract finger-print common pattern and radix bupleuri medicine of foundation
The similarity of material is high, and the similarity pole of radix bupleuri reference extract and Radix Bupleuri finger-print common pattern of the invention
Height, this explanation carry out saikoside A and saikosaponin D to medicinal material or Chinese materia medica preparation using radix bupleuri reference extract of the invention
Assay reliability is high, therefore radix bupleuri reference extract of the invention can be applied to the sxemiquantitative mirror of medicinal material or Chinese materia medica preparation
It does not analyze, can also be applied to radix bupleuri and containing in the control of the quality of radix bupleuri/radix bupleuri effective component medicinal material or Chinese materia medica preparation, such as it is right
The saikoside A and saikosaponin D ingredient of medicinal material or Chinese materia medica preparation carry out sxemiquantitative discriminatory analysis, or to radix bupleuri and contain bavin
Recklessly/radix bupleuri effective component medicinal material or Chinese materia medica preparation carry out half-quantitative detection and then control its quality.
Claims (10)
1. a kind of radix bupleuri reference extract, which is characterized in that it is derived from: Radix Bupleuri powder is repeatedly extracted, is concentrated,
It is dried to obtain the bupleurum extract of different batches, then the bupleurum extract of different batches is deployed, obtains radix bupleuri control
Extract；
Optional, the sum of content of saikoside A and saikosaponin D is not less than 0.30% and in the radix bupleuri reference extract
Higher than 3.00%；
It is optional, the map that the radix bupleuri reference extract is obtained using thin-layered chromatography and/or high performance liquid chromatography with
Its corresponding raw medicinal material is consistent；
Optional, the color that the radix bupleuri reference extract is measured using thin-layered chromatography inspection and/or high performance liquid chromatography
Saikoside A in spectrogram is consistent with the corresponding chemical reference substance of saikosaponin D or its corresponding raw medicinal material.
2. a kind of preparation method of radix bupleuri reference extract described in claim 1, comprising the following steps:
Step 1: extracting: taking Radix Bupleuri powder to mix with ethyl alcohol, filtered after extraction, obtain filtrate 1 and filter residue 1, by filtrate 1
It closes liquid and is concentrated to get radix bupleuri dry cream；
Step 2: preparing bupleurum extract: by gained radix bupleuri dry cream, it is dissolved in ethyl alcohol, obtains the ethanol solution of radix bupleuri dry cream, then
Auxiliary material is added, drying and screening obtains bupleurum extract；
Step 3: allotment: the bupleurum extract of different batches being deployed, radix bupleuri reference extract is obtained；
Optional, it further include with thin-layered chromatography and/or high performance liquid chromatography between the step 3 and step 4 to difference
The step of saikoside A and saikosaponin D of the bupleurum extract of batch are detected；
Optional, extraction of the step 1 into step 3, concentration and drying are carried out by the way of low-temperature treatment；
Optional, the low temperature is 20 DEG C~60 DEG C.
3. preparation method according to claim 2, which is characterized in that filter residue 1 is according to step 1 in the step one
Extracting method is extracted by n times, and the filtrate that n times are extracted is merged with filtrate 1 Ji Wei extracting solution, and extracting solution is concentrated to get
Hu dry cream；Wherein 6 >=N >=0, and N is integer；
Optional, the N is 3.
4. preparation method according to claim 2, which is characterized in that the concentration of alcohol described in step 1 is 30%
~95%；
Optional, concentration of alcohol is 70% in the step 1.
5. preparation method according to claim 3, which is characterized in that Radix Bupleuri powder and ethyl alcohol in the step 1
W/v be 1:5~1:45；
Optional, the w/v of Radix Bupleuri powder and ethyl alcohol in the step 1 is 1:10；
Optional, the extracting mode in the step 1 are as follows: homogenate extraction；
Optional, the homogenate extraction time in the step 1 is 1~5min；
Optional, the homogenate extraction time in the step 1 is 1min；
Optional, the filtering in the step 1 is with Medium speed filter paper or 2000 mesh net filtrations；
Optional, the filtering in the step 1 is to use Medium speed filter paper.
6. preparation method according to claim 3, which is characterized in that the weight of radix bupleuri dry cream and ethyl alcohol in the step 2
Volume ratio: 1:2~1:10；
Optional, auxiliary material used is superfine silica gel powder and/or medical starch in the step 2.
Optional, the auxiliary material is superfine silica gel powder；
Optional, the step 2 is 10%~50% micro mist silicon that its weight is added in the ethanol solution of radix bupleuri dry cream
Glue；
Optional, the step 2 is 40% superfine silica gel powder that its weight is added in the ethanol solution of radix bupleuri dry cream；
Optional, it was 90~200 mesh net filtrations after the step 2 low temperature drying；
Optional, it was 110 mesh net filtrations after the step 2 low temperature drying.
7. preparation method according to claim 2, which is characterized in that allotment makes finally obtained control in the step 3
The total content of saikoside A and saikosaponin D must not be lower than 0.30% and not higher than 3.00% in extract；
It is optional, the map that the radix bupleuri reference extract is obtained using thin-layered chromatography and/or high performance liquid chromatography with
Its corresponding raw medicinal material is consistent；
Optional, the color that the radix bupleuri reference extract is measured using thin-layered chromatography inspection and/or high performance liquid chromatography
Saikoside A in spectrogram is consistent with the corresponding chemical reference substance of saikosaponin D or its corresponding raw medicinal material.
8. application of the radix bupleuri reference extract described in claim 1 in the identification of medicinal material or Chinese materia medica preparation.
9. application according to claim 8, it is characterized in that, the identification for radix bupleuri or containing radix bupleuri/radix bupleuri effectively at
In the quality control of the medicinal material or Chinese materia medica preparation that divide.
10. application according to claim 8 or claim 9, which is characterized in that the method that application uses is as follows: by claim 1-
The radix bupleuri reference extract passes through thin-layered chromatography and/or efficient liquid as reference material together with medicinal material or Chinese materia medica preparation
Phase chromatography is detected, according to testing result comparison judgement；
Optional, the thin-layered chromatography and/or high performance liquid chromatography are to radix bupleuri reference extract, medicinal material or Chinese materia medica preparation
In saikoside A and saikosaponin D detected；
Optional, affiliated thin-layered chromatography testing conditions are as follows:
Testing conditions are as follows:
Lamellae: TLC G60 prefabricated board (Merck)；
Point sample: 2 μ l, the long 8mm of ribbon point sample；
Solvent: methylene chloride: ethyl acetate: methanol: water=30:40:15:3；
Expansion mode: adding solvent 12ml in side in double flute expansion cylinder (20cm × 10cm), common to pre-equilibrate 15 minutes；Thin layer
The drying of plate: heating 15 minutes after point sample under 50 DEG C of heating plates guarantees the drying of lamellae；
Inspect: spray, which is taken out, goes back after residual solvents, spray with -10% sulfuric acid ethyl alcohol color developing agent of 2% paradime thylaminobenzaldehyde,
1min is heated in 105 DEG C of heating plates, is placed in fluorescence viewing chromatogram image under ultraviolet lamp (366nm)；
Optional, described high performance liquid chromatography testing conditions are as follows:
Chromatographic apparatus: Agilent 1260series high performance liquid chromatograph is furnished with DAD detector (U.S., Agilent
Technologies)
Chromatographic column: Agilent Zorbax SB-C18 (4.6mm × 250mm, 5 μm)；
Mobile phase: A- acetonitrile, B- water；
Gradient elution program: 0-15min:25%A → 35%A, 15-38min:35%A → 45%A, 38-60min:45%A →
60%A, 60-70min:60%A → 100%A；
0-15min:75%B → 65%B, 15-38min:65%B → 55%B, 38-60min:55%B → 40%B, 60-
70min:40%B → 0%B；
Detection wavelength 203nm (DAD detector)；Flow velocity 1.0ml/min；10 μ l of sample volume；25 DEG C of column temperature, runing time:
70min。
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