CN109459515A - A kind of Herba Epimedii reference extract (arrow leaf) and its application - Google Patents
A kind of Herba Epimedii reference extract (arrow leaf) and its application Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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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
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Abstract
The present invention provides a kind of Herba Epimedii reference extract (arrow leaves), it is derived from: being carried out repeatedly extraction to epimedium herb (arrow leaf) powder and is obtained the Shorthorned Epimedium P.E of different batches, then the Shorthorned Epimedium P.E of different batches is deployed, obtains Herba Epimedii reference extract (arrow leaf).Herba Epimedii reference extract (arrow leaf) of the invention ensure that the consistency of the Herba Epimedii reference extract (arrow leaf) of different batches because being that the extract of different batches deploy；And its character is stable, uniform and easy to use.The Herba Epimedii reference extract (arrow leaf) is upper in application, it is as control for Herba Epimedii and containing in the control of the quality of Herba Epimedii/epimedium active constituent medicinal material or Chinese materia medica preparation, in this quality control procedure, it is that Herba Epimedii reference extract (arrow leaf) 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 Herba Epimedii reference extract (arrow leaf)
Preparation 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 (ASCS) to Chinese medicine reference extract and reference extract is answered
Have several primary conditions: Authenticity, crude drug source is reliable, representative；Specificity, the detection used
Method specificity；Con-sistency, different batches reference extract should be consistent；Stability, 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
Herba Epimedii reference extract (arrow leaf), the present invention also provides the applications of the Herba Epimedii reference extract (arrow leaf), i.e., by institute
Herba Epimedii reference extract (arrow leaf) is stated for Herba Epimedii and containing Herba Epimedii/epimedium active constituent medicinal material or Chinese materia medica preparation
In quality control.
Compared with prior art, the invention has the following advantages:
Herba Epimedii reference extract (arrow leaf) of the invention overcomes because being that the extract of different batches deploy
Because Chinese medicine control medicinal material is influenced by the place of production, growing environment, it is difficult to ensure that the defect per batch of uniform quality, to guarantee
The consistency of the Herba Epimedii reference extract of different batches (arrow leaf)；And its character is stable, uniform and easy to use.It is described
Herba Epimedii reference extract (arrow leaf) application it is upper, be by Herba Epimedii reference extract (arrow leaf) after quantitative analysis, as
Control for Herba Epimedii and containing the quality of Herba Epimedii/epimedium active constituent medicinal material or Chinese materia medica preparation control in, in this quality
It is that Herba Epimedii reference extract (arrow leaf) progress simple process can be used directly in control process, it is easy to operate, especially exist
When multicomponent assay, the preparation of reference extract solution is easier than the preparation of reference substance solution, not only can be to medicine
Material or Chinese materia medica preparation carry out Qualitive test, and can be also used for sxemiquantitative even quantitative analysis.
Detailed description of the invention
Fig. 1 is to obtain for using chemical reference substance as reference substance to Herba Epimedii raw medicinal material (arrow leaf) progress thin-layered chromatography
The thin-layer chromatogram arrived.Wherein the chromatographic band of label S1, S2 respectively corresponds as reference substance epimedin C, icariin, and 1-6 is successively
Correspond to Epimedium sagittatum raw medicinal material 1, Epimedium sagittatum raw medicinal material 2, Epimedium sagittatum raw medicinal material 3, Epimedium sagittatum
Raw medicinal material 4, Epimedium sagittatum raw medicinal material 5 and Epimedium sagittatum raw medicinal material 6.
Fig. 2 is to carry out high performance liquid chromatography to Herba Epimedii raw medicinal material (arrow leaf) for using chemical reference substance as reference substance
The HPLC finger-print stacking chart that method obtains, 1-6 respectively corresponds Epimedium sagittatum raw medicinal material 1, Epimedium sagittatum from top to bottom
Raw medicinal material 2, Epimedium sagittatum raw medicinal material 3, Epimedium sagittatum raw medicinal material 4, Epimedium sagittatum raw medicinal material 5 and arrow leaf are excessive
Sheep leaves of pulse plants raw medicinal material 6, S1, S2 correspond to chemical reference substance: epimedin C, icariin, R correspond to common pattern.
Fig. 3 is Herba Epimedii reference extract (arrow leaf) preparation flow figure.
Fig. 4 is Herba Epimedii reference extract (arrow leaf) and Epimedium sagittatum extraction raw medicinal material common pattern stacking chart, S1,
S2 corresponds to chemical reference substance, and ERS is corresponded to Herba Epimedii reference extract (arrow leaf), and R is Epimedium sagittatum raw medicinal material common pattern.
Fig. 5 is to carry out thin layer color to commercially available medicinal material for using chemical reference substance and Herba Epimedii reference extract as reference substance
The thin-layer chromatogram that spectrometry obtains.Wherein the chromatographic band of label S is respectively corresponded from the bottom up as reference substance epimedin C, Herba Epimedii
Glycosides, 5-14 are corresponding in turn to as Herba Epimedii 1, Herba Epimedii 2, Herba Epimedii 3, Herba Epimedii 4, Herba Epimedii 5, Herba Epimedii 6, Herba Epimedii 7, excessive sheep
The leaves of pulse plants 8, Herba Epimedii 9 and Herba Epimedii 10.1 corresponds to Herba Epimedii reference extract, and 2 correspond to main constituents in Yinyanghuo Granules, 3 correspond to it is excessive
Sheep leaves of pulse plants control medicinal material, 4 correspond to korean epimedium herb control medicinal material.
Fig. 5-1 is excessive to extract (arrow leaf) and main constituents in Yinyanghuo Granules, Herba Epimedii control medicinal material, Korea for Herba Epimedii
Sheep leaves of pulse plants control medicinal material and epimedium herb carry out high performance thin layer chromatography figure digital scanning map.Band 1 is that Herba Epimedii control is extracted
Object (arrow leaf), band 2 are Herba Epimedii control medicinal material, and band 3 is korean epimedium herb control medicinal material, and band 4 is Herba Epimedii formula
Grain, band 5-14 are corresponding in turn to Herba Epimedii 1, Herba Epimedii 2, Herba Epimedii 3, Herba Epimedii 4, Herba Epimedii 5, Herba Epimedii 6, Herba Epimedii 7, excessive
The sheep leaves of pulse plants 8, Herba Epimedii 9 and Herba Epimedii 10.
Fig. 6 is to carry out efficient liquid to commercially available medicinal material for using chemical reference substance and Herba Epimedii reference extract as reference substance
The HPLC finger-print stacking chart that phase chromatography obtains, from top to bottom 4-15 be corresponding in turn to Herba Epimedii 1, Herba Epimedii 2, Herba Epimedii 3,
Herba Epimedii 4, Herba Epimedii 5, Herba Epimedii 6, Herba Epimedii 7, Herba Epimedii 8, Herba Epimedii 9 and Herba Epimedii 10.1 extracts for Herba Epimedii control
Object, 2 correspond to Herba Epimedii control medicinal material, and 3 correspond to korean epimedium herb control medicinal material, and 4 correspond to main constituents in Yinyanghuo Granules, S1,
S2 corresponds to chemical reference substance: epimedin C, icariin.
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:
Epimedium herb is purchased from the major medicinal material market in the whole nation, is accredited as Berberidaceae plant Epimedium sagittatum through professor Xie Peishan
The dried leaf of Epimedium sagittatum (Sieb.et Zucc.) Maxim.
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 ATS, thin-layer chromatography double flute
Expansion cylinder, Chromatogram Immersion Device III chromatography colour developing dipping tank and TLC visualizer thin layer
Chromatography video camera (Switzerland, CAMAG).
Agilent 1260series high performance liquid chromatograph is furnished with DAD detector (U.S., Agilent
Technologies)。
Methanol, ethyl alcohol, formic acid, acetic acid, phosphoric acid, butyl acetate, acetone are to analyze pure (Guangzhou Chemical Reagent Factory).
Acetonitrile is chromatographically pure (Merck KGaA)；
Methanol is chromatographically pure (Merck KGaA)；
Pure water；
Epimedin C indicates purity >=95.5%, (National Institute for Food and Drugs Control, lot number: 111780-
201503)；
Icariin (National Institute for Food and Drugs Control, lot number: 110737-200415)；
Herba Epimedii control medicinal material (National Institute for Food and Drugs Control, lot number: 121632-201502)
Korean epimedium herb control medicinal material (National Institute for Food and Drugs Control, lot number: 121032-201302)
Test medicinal material Herba Epimedii:
Epimedium sagittatum raw medicinal material 6 batches buyings are in the major medicinal material market in the whole nation, Epimedium sagittatum raw medicinal material 1-6；
Epimedium herb 10 batches buyings are in the major pharmacy in Guangzhou and medicinal material market, Herba Epimedii 1-10；
Main constituents in Yinyanghuo Granules is purchased in big pharmacy and medicinal material market；
Embodiment 1: the screening of Herba Epimedii raw medicinal material
Present embodiments provide the screening technique of the raw medicinal material of Herba Epimedii reference extract of the present invention (arrow leaf).
With thin-layered chromatography and high performance liquid chromatography respectively by reference substance of chemical reference substance to Herba Epimedii raw material
Medicinal material (arrow leaf) is analyzed and is screened.
Epimedium herb is purchased from the major medicinal material market in the whole nation, is accredited as Berberidaceae plant Epimedium sagittatum through professor Xie Peishan
The dried leaf of Epimedium sagittatum (Sieb.et Zucc.) Maxim.Identified 6 batches of epimedium herbs are that arrow leaf is excessive
The sheep leaves of pulse plants, complying with standard can be spare.
1 thin-layer chromatography
1.1 sample preparation
Chemical reference substance solution: precision weighs icariin and epimedin C chemical reference substance is appropriate, and respectively plus methanol is prepared
Solution at every 1mL containing about 0.5mg, as reference substance solution.
Medicinal material test solution: taking Herba Epimedii raw medicinal material powder 0.5g, accurately weighed, sets in 150ml stuffed conical flask,
It is added 50% ethyl alcohol 50mL, heating water bath heat flows back 80 DEG C of 30min, lets cool filtering, is transferred to evaporating dish and is evaporated, and residue is with 70%
Ethyl alcohol dissolution, is transferred in 5mL volumetric flask, is settled to scale, shake up, and the filter membrane for taking supernatant to cross 0.22 μm is to get medicinal material confession
Test sample solution.Medicinal material is bought from the major medicinal material market in the whole nation, respectively Epimedium sagittatum raw medicinal material 1, Epimedium sagittatum bulk pharmaceutical chemicals
Material 2, Epimedium sagittatum raw medicinal material 3, Epimedium sagittatum raw medicinal material 4, Epimedium sagittatum raw medicinal material 5 and Epimedium sagittatum are former
Expect medicinal material 6.
The detection of 1.2 thin-layer chromatographys
Testing conditions are as follows:
Lamellae: TLC G60 prefabricated board (Merck)；
Point sample: medicinal material test solution: 2 μ l, 4 μ l of remaining sample；The long 8mm of ribbon point sample；
Solvent: butyl acetate: acetone: formic acid: water=11:2:10:10, (10 DEG C of lower leafs, take upper solution)；
Expansion mode: add solvent in double flute expansion cylinder side, pre-equilibrate 15 minutes, uplink 9cm is unfolded；
It inspects: is uniformly sprayed after expansion with 5% alchlor ethanol solution, lamellae sets in thin layer heating plate 105 DEG C
Heating 2 minutes, is placed in fluorescence viewing chromatogram image under ultraviolet lamp (366nm).
Testing result: as shown in Figure 1, to inspect thin-layer chromatogram (T:25 DEG C, RH:70%) under ultraviolet lamp (366nm).
The chromatographic band of label S1, S2 are respectively reference substance epimedin C and icariin, and 1-6 is corresponding in turn to Epimedium sagittatum raw medicinal material
1, Epimedium sagittatum raw medicinal material 2, Epimedium sagittatum raw medicinal material 3, Epimedium sagittatum raw medicinal material 4, Epimedium sagittatum raw material
Medicinal material 5 and Epimedium sagittatum raw medicinal material 6；As shown in Figure 1, epimedin C and icariin separation situation are good, each batch arrow leaf
Epimedium herb can show identical spot in same position, and similarity is high between each batch Epimedium sagittatum medicinal material, tentatively
Screening is that standard control extract extracts raw medicinal material.
2 high performance liquid chromatography
2.1 sample preparation
Chemical reference substance solution: taking chemical reference substance epimedin C, appropriate icariin, accurately weighed, is matched respectively with methanol
The solution of 100 μ g/ml is made to obtain the final product.
Medicinal material test solution: taking each medicinal powder 0.1g, accurately weighed, and 50% ethyl alcohol 80ml, hydro-thermal reflux 1h is added to take
Out, it filtering, filtrate is evaporated, and residue adds 30% methanol to dissolve, and it is transferred in 5mL volumetric flask, is settled to scale, shake up, refrigerator (4
DEG C) stand overnight, take supernatant through 0.22 μm of membrane filtration to get.Medicinal material is bought from the major medicinal material market in the whole nation, respectively arrow leaf
Herba Epimedii raw medicinal material 1, Epimedium sagittatum raw medicinal material 2, Epimedium sagittatum raw medicinal material 3, Epimedium sagittatum raw medicinal material 4,
Epimedium sagittatum raw medicinal material 5 and Epimedium sagittatum raw medicinal material 6.
2.2 high performance liquid chromatography detection
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 (4.6 × 250mm, 5 μm)；
Mobile phase: A- methanol, B- acetonitrile, C-0.5% acetic acid；
Gradient elution program: 0-30min:0%A → 0%A, 30-45min:0%A → 11%A, 45-65min:11%A
→ 4%A, 65-85min:4%A → 4%A, 85-90min:4%A → 0%A, 90-100min:0%A → 0%A；
0-30min:12%B → 25%B, 30-45min:25%B → 23.5%B, 45-65min:23.5%B → 35%
B, 65-85min:35%B → 35%B, 85-90min:35%B → 50%B, 90-100min:50%B → 50%B；
0-30min:88%C → 75%C, 30-45min:75%C → 65.5%C, 45-65min:65.5%C → 61%
C, 65-85min:61%C → 61%C, 85-90min:61%C → 50%C, 90-100min:50%C → 50%C；
Detection wavelength 270nm (DAD detector)；Flow velocity 1.0ml/min；20 μ l of sample volume；20 DEG C of column temperature, runing time:
100min。
High effective liquid chromatography for detecting: accurate chemical reference substance solution and the Epimedium sagittatum medicinal material drawn is for examination respectively
Each 20 μ l of product solution injects liquid chromatograph.
High performance liquid chromatography testing result:
Measurement records chromatogram to get HPLC finger-print stacking chart shown in Fig. 2, and 1-6 respectively corresponds arrow from top to bottom
Leaf Herba Epimedii raw medicinal material 1, Epimedium sagittatum raw medicinal material 2, Epimedium sagittatum raw medicinal material 3, Epimedium sagittatum raw medicinal material
4, Epimedium sagittatum raw medicinal material 5 and Epimedium sagittatum raw medicinal material 6, S1, S2 correspond to chemical reference substance: epimedin C, excessive sheep
Leaves of pulse plants glycosides, R correspond to common pattern.
As seen from Figure 2, the finger-print of 6 raw medicinal material of Epimedium sagittatum 1- Epimedium sagittatum has the consistent of height
Property, the finger-print common pattern of Epimedium sagittatum medicinal material is thus established, and similarity analysis is carried out to all samples.
According to set Epimedium sagittatum medicinal materials fingerprint common pattern, using included angle cosine algorithm according to each sample group
Divide and its peak area carries out similarity evaluations to 6 batches of Epimedium sagittatum medicinal material samples, the results are shown in Table 1.
Table 1
| Sample | Similarity |
| Epimedium sagittatum raw medicinal material 1 | 1.00 |
| Epimedium sagittatum raw medicinal material 2 | 0.99 |
| Epimedium sagittatum raw medicinal material 3 | 0.99 |
| Epimedium sagittatum raw medicinal material 4 | 0.99 |
| Epimedium sagittatum raw medicinal material 5 | 0.99 |
| Epimedium sagittatum raw medicinal material 6 | 0.99 |
| Common pattern | 1.00 |
According to the above similarity analysis as a result, similarity >=0.99 of 6 batches of Epimedium sagittatum raw medicinal materials, similarity is very
Height can be selected and extract to obtain raw medicinal material for Herba Epimedii standard control extract (arrow leaf).
Embodiment 2: the preparation of Herba Epimedii reference extract
The preparation method of Herba Epimedii reference extract of the present invention (arrow leaf) is present embodiments provided, method flow diagram is as schemed
Shown in 3.
One: extracting
It chooses epimedium herb (arrow leaf), powder is made, 50% ethyl alcohol that 25 times of volumes of its quality are then added is (i.e. solid
Liquor ratio is 1:25 (w/V)), and middling speed qualitative filter paper filters after heating and refluxing extraction 30min, collects filter residue 1 and filtrate 1, filter residue 1 are pressed
Same procedure is extracted once again, and is merged the filtrate once collected is extracted again with filtrate 1 Ji Wei extracting solution, by extracting solution
Solvent evaporated, up to Herba Epimedii crude extract (arrow leaf), fixes Herba Epimedii crude extract (arrow to no ethanol flavor with 1:20 macroporous resin column
Leaf), 30~60min is adsorbed, pure water is washed till no color, with 70% ethanol elution, is eluted to no color, merges alcohol eluen recycling
To dry up to Herba Epimedii extract (arrow leaf)；
Two: preparing Shorthorned Epimedium P.E (arrow leaf)
After smart (arrow leaf) the extract dissolution completely of appropriate 80% ethyl alcohol Herba Epimedii, then plus Herba Epimedii extract (arrow leaf)
30% superfine silica gel powder (mountains and rivers medicine is auxiliary), is evaporated with Rotary Evaporators, crushed 110 meshes, obtain Shorthorned Epimedium P.E (arrow
Leaf).
6 batches of Shorthorned Epimedium P.Es (arrow leaf) is prepared, high effective liquid chromatography for measuring is utilized by reference substance, testing result is such as
Shown in table 3:
Table 3
Three: allotment
The Shorthorned Epimedium P.E (arrow leaf) of 6 batches of step 2 is mixed in 1:1 ratio and blends to obtain Herba Epimedii control
Extract (arrow leaf), it is about 1:7 (g/g) that final products, which correspond to crude drug ratio, utilizes high performance liquid chromatography by reference substance
Method measurement, wherein the measurement result of the content of each ingredient is as shown in table 4.
Table 4
Allotment standard are as follows: should make epimedin C in final Herba Epimedii reference extract (arrow leaf) must not be lower than 13.00%
And it must not be lower than 3.00% with the content of icariin not higher than 17.00% and be not higher than 5.00% (content is in terms of wt%).This
The range of the content of each ingredient in allotment standard be also by repeatedly repeatedly experiment it is comprehensive keep stability, consistency and after
The optimum data that the application in face etc. obtains.
Four: detection
The Herba Epimedii reference extract (arrow leaf) that step 3 is deployed into is surveyed by reference substance using high performance liquid chromatography
Surely finger-print is obtained, carries out similarity detection with raw medicinal material common pattern map is prepared, such as Fig. 4, ERS correspond to Herba Epimedii
Reference extract (arrow leaf), R are Epimedium sagittatum raw medicinal material common pattern, and S1, S2 correspond to chemical reference substance: epimedin C,
Icariin.Obtaining similarity is 1.00, and Herba Epimedii reference extract (arrow leaf) and Epimedium sagittatum medicinal materials fingerprint are shared
The similarity of mode is very high, this illustrates that Herba Epimedii reference extract of the invention (arrow leaf) can represent Epimedium sagittatum medicinal material.
The character analysis of 3 Herba Epimedii reference extract of embodiment (arrow leaf)
1. apparent state: the Herba Epimedii reference extract (arrow leaf) that embodiment 2 obtains is light gray purple powder.
2. determination of moisture: being carried out according to 2015 editions Chinese Pharmacopoeia annex IX G (hypobaric drying method).Testing result is excessive sheep
Leaves of pulse plants reference extract (arrow leaf) water content is 0.50%.
3. uniformity test: preparing 3 batch Herba Epimedii reference extracts (arrow leaf) according to the method for embodiment 2, after measured
The thin-layer chromatography testing result difference very little for epimedin C and icariin between each batch, epimedin C and icariin
Fluorescence spot has apparent display, and is distributed very consistent；Through its epimedin C of high performance liquid chromatography detection and Icariin content
Within allotment critical field.Therefore it is prepared using the preparation method of Herba Epimedii reference extract (arrow leaf) of the invention
Herba Epimedii reference extract (arrow leaf) consistency is very good.
4. stability test:
The test sample for the Herba Epimedii reference extract (arrow leaf) for taking the method according to embodiment 2 of 3 different batches to prepare,
According to the relevant regulations of " requirement of national drug standards substance Development Techniques " that Chinese Pharmacopoeia Commission works out, inspection target includes
Character and dissolubility.
Test sample opening is set in suitable clean container, is placed 10 days at a temperature of 60 DEG C, is sampled in the 5th day and the 10th day,
It 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.
Embodiment 4
Present embodiments provide the application for the Herba Epimedii reference extract (arrow leaf) that embodiment 2 is prepared.
It is excessive with chemical reference substance, Herba Epimedii control medicinal material, Korea respectively with thin-layered chromatography and high performance liquid chromatography
Sheep leaves of pulse plants control medicinal material and Herba Epimedii reference extract (arrow leaf) are that reference substance analyzes commercially available medicinal material.
1 thin-layer chromatography
1.1 sample preparation
Chemical reference substance solution: precision weighs icariin and epimedin C chemical reference substance is appropriate, and respectively plus methanol is prepared
Solution at every 1mL containing about 0.5mg, as reference substance solution.
Herba Epimedii reference extract (arrow leaf) solution: precision weighs the Herba Epimedii reference extract that embodiment 2 is prepared
(arrow leaf) 15mg, sets in 5mL volumetric flask, adds methanol 4mL, is ultrasonically treated 15 minutes, lets cool, add methanol to scale, shake up, mistake
0.22 μm of filter membrane to obtain the final product.
Main constituents in Yinyanghuo Granules: taking the granule for being equivalent to medicinal material 0.5g, and 70% ethyl alcohol dissolves constant volume to 5mL capacity
Bottle in, be settled to scale, shake up, the filter membrane for taking supernatant to cross 0.22 μm to get；
Medicinal material test solution: taking epimedium herb powder 0.5g, accurately weighed, sets in 150ml stuffed conical flask, is added
50% ethyl alcohol 50mL, heating water bath heat 80 DEG C of 30min of reflux, lets cool filtering, is transferred to evaporating dish and is evaporated, 70% ethyl alcohol of residue
Dissolution, is transferred in 5mL volumetric flask, is settled to scale, shake up, the filter membrane for taking supernatant to cross 0.22 μm is to get medicinal material test sample
Solution.Medicinal material is bought from the major pharmacy in Guangzhou, respectively Herba Epimedii 1, Herba Epimedii 2, Herba Epimedii 3, Herba Epimedii 4, Herba Epimedii 5, excessive sheep
The leaves of pulse plants 6, Herba Epimedii 7, Herba Epimedii 8, Herba Epimedii 9 and Herba Epimedii 10；Herba Epimedii control medicinal material solution, korean epimedium herb pair are prepared with method
According to medicinal material solution.
The detection of 1.2 thin-layer chromatographys
Testing conditions are as follows:
Lamellae: TLC G60 prefabricated board (Merck)；
Point sample: medicinal material test solution: 2 μ l, 4 μ l of remaining sample；The long 8mm of ribbon point sample；
Solvent: butyl acetate: acetone: formic acid: water=11:2:10:10, (10 DEG C of lower leafs, take upper solution)；
Expansion mode: add solvent in double flute expansion cylinder side, pre-equilibrate 15 minutes, uplink 9cm is unfolded；
It inspects: is uniformly sprayed after expansion with 5% alchlor ethanol solution, lamellae sets in thin layer heating plate 105 DEG C
Heating 2 minutes, is placed in fluorescence viewing chromatogram image under ultraviolet lamp (366nm)；
Testing result: as shown in figure 5, to inspect thin-layer chromatogram (T:25 DEG C, RH:70%) under ultraviolet lamp (366nm).
The chromatographic band of label S is respectively reference substance epimedin C and icariin from the bottom up, and 5-14 is corresponding in turn to Herba Epimedii 1, excessive sheep
The leaves of pulse plants 2, Herba Epimedii 3, Herba Epimedii 4, Herba Epimedii 5, Herba Epimedii 6, Herba Epimedii 7, Herba Epimedii 8, Herba Epimedii 9 and Herba Epimedii 10.1 corresponds to
Herba Epimedii reference extract, 2 correspond to main constituents in Yinyanghuo Granules, and 3 correspond to Herba Epimedii control medicinal material, and 4 correspond to the excessive sheep of Korea
Leaves of pulse plants control medicinal material.
Interpretation of result: by Fig. 5-1 it is found that epimedin C and icariin separation situation are preferable, Herba Epimedii reference extract
(arrow leaf) and main constituents in Yinyanghuo Granules, Herba Epimedii control medicinal material, korean epimedium herb control medicinal material and epimedium herb (Herba Epimedii
1-10) major spot (epimedin C and icariin) position consistency.From map find out Herba Epimedii reference extract (arrow leaf) with it is excessive
The sheep leaves of pulse plants 1 can show identical spot in same position, should be Epimedium sagittatum；Herba Epimedii reference extract (arrow leaf) with it is excessive
Sheep leaves of pulse plants granule, Herba Epimedii control medicinal material, korean epimedium herb control medicinal material and epimedium herb (Herba Epimedii 2-10) are slightly
Difference；It can learn that the medicinal material in pharmacy currently on the market is mostly korean epimedium herb and other a small amount of kinds from map
Epimedium herb, few Epimedium sagittatum medicinal material.
2 high performance liquid chromatography
2.1 sample preparation
Chemical reference substance solution: taking chemical reference substance epimedin C, appropriate icariin, accurately weighed, is matched respectively with methanol
The solution of 100 μ g/ml is made to obtain the final product.
Herba Epimedii reference extract (arrow leaf) solution: precision weighs Herba Epimedii ERS15mg, is placed in 5ml volumetric flask, adds
Water 4ml is ultrasonically treated 15 minutes.It lets cool, adds methanol to scale, shake up, solution takes subsequent filtrate, i.e., through 0.22 μm of membrane filtration
Obtain Herba Epimedii reference extract (arrow leaf) solution；
Main constituents in Yinyanghuo Granules: taking the granule for being equivalent to medicinal material 0.1g, and 30% ethyl alcohol dissolves constant volume to 5mL capacity
Bottle in, be settled to scale, shake up, refrigerator (4 DEG C) is stood overnight, take supernatant through 0.22 μm of membrane filtration to get；
Medicinal material test solution: taking each medicinal powder 0.1g, accurately weighed, and 50% ethyl alcohol 80ml, hydro-thermal reflux 1h is added to take
Out, it filtering, filtrate is evaporated, and residue adds 30% methanol to dissolve, and it is transferred in 5mL volumetric flask, is settled to scale, shake up, refrigerator (4
DEG C) stand overnight, take supernatant through 0.22 μm of membrane filtration to get.Pharmacy's medicinal material is bought from Guangzhou, be respectively as follows: Herba Epimedii 1,
Herba Epimedii 2, Herba Epimedii 3, Herba Epimedii 4, Herba Epimedii 5, Herba Epimedii 6, Herba Epimedii 7, Herba Epimedii 8, Herba Epimedii 9 and Herba Epimedii 10；Together
Method prepares Herba Epimedii control medicinal material solution, korean epimedium herb control medicinal material solution.
2.2 high performance liquid chromatography detection
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 (4.6 × 250mm, 5 μm)；
Mobile phase: A- methanol, B- acetonitrile, C-0.5% acetic acid；
Gradient elution program: 0-30min:0%A → 0%A, 30-45min:0%A → 11%A, 45-65min:11%A
→ 4%A, 65-85min:4%A → 4%A, 85-90min:4%A → 0%A, 90-100min:0%A → 0%A；
0-30min:12%B → 25%B, 30-45min:25%B → 23.5%B, 45-65min:23.5%B → 35%
B, 65-85min:35%B → 35%B, 85-90min:35%B → 50%B, 90-100min:50%B → 50%B；
0-30min:88%C → 75%C, 30-45min:75%C → 65.5%C, 45-65min:65.5%C → 61%
C, 65-85min:61%C → 61%C, 85-90min:61%C → 50%C, 90-100min:50%C → 50%C；
Detection wavelength 270nm (DAD detector)；Flow velocity 1.0ml/min；20 μ l of sample volume；20 DEG C of column temperature, runing time:
100min。
High effective liquid chromatography for detecting: respectively it is accurate draw chemical reference substance solution, Herba Epimedii control medicinal material solution,
Korean epimedium herb control medicinal material solution, Herba Epimedii reference extract (arrow leaf) solution and each 20 μ l of medicinal material test solution, injection
Liquid chromatograph.
High performance liquid chromatography testing result:
Measurement records chromatogram to get HPLC finger-print stacking chart shown in fig. 6, and 5-14 is corresponding in turn to from top to bottom
Herba Epimedii 1, Herba Epimedii 2, Herba Epimedii 3, Herba Epimedii 4, Herba Epimedii 5, Herba Epimedii 6, Herba Epimedii 7, Herba Epimedii 8, Herba Epimedii 9 and excessive sheep
The leaves of pulse plants 10.1 corresponds to Herba Epimedii reference extract (arrow leaf), and 2 correspond to Herba Epimedii control medicinal material, and 3 correspond to korean epimedium herb pair
Main constituents in Yinyanghuo Granules is corresponded to according to medicinal material, 4；S1, S2 correspond to chemical reference substance: epimedin C, icariin.
As seen from Figure 6, the finger-print difference of 10 medicinal material of Herba Epimedii 1- Herba Epimedii is larger.It is mentioned according to Herba Epimedii control
Object finger-print is taken, using included angle cosine algorithm according to each sample component and its peak area to 10 batches of epimedium herb finger-prints
Similarity evaluation is carried out, the results are shown in Table 5.
Table 5
| Sample | Similarity |
| Herba Epimedii 1 | 0.96 |
| Herba Epimedii 2 | 0.63 |
| Herba Epimedii 3 | 0.45 |
| Herba Epimedii 4 | 0.40 |
| Herba Epimedii 5 | 0.40 |
| Herba Epimedii 6 | 0.43 |
| Herba Epimedii 7 | 0.93 |
| Herba Epimedii 8 | 0.60 |
| Herba Epimedii 9 | 0.55 |
| Herba Epimedii 10 | 0.46 |
| Herba Epimedii control medicinal material | 0.51 |
| Korean epimedium herb control medicinal material | 0.39 |
| Main constituents in Yinyanghuo Granules | 0.67 |
| Herba Epimedii ERS | 1.00 |
According to the above similarity analysis as a result, 10 batches of medicinal material Herba Epimedii 1, Herba Epimedii 7 and Herba Epimedii reference extract (arrow
Leaf) similarity be respectively 0.98 and 0.95, similarity is very high, remaining commodity herb and Herba Epimedii reference extract similarity are very
It is low, this is because Herba Epimedii commodity herb is many kinds of, less Epimedium sagittatum in the market.
According to the result of TLC and HPLC finger-print it is found that using thin layer to the Herba Epimedii reference extract (arrow leaf)
The corresponding raw medicinal material of the chromatogram that chromatography detects is consistent, more precisely, compares and extracts to the Herba Epimedii
The epimedin C in chromatogram and the fluorescence spot at icariin position and use high that object is detected using thin-layered chromatography
Chromatographic peak of the chromatogram that effect liquid phase chromatogram method detects at epimedin C and icariin position distinguishes corresponding change
It learns reference substance or the consistent therefore of the invention Herba Epimedii reference extract (arrow leaf) of its corresponding raw medicinal material can be applied to medicinal material
Or the Qualitive test of Chinese materia medica preparation, such as qualitative analysis is carried out to the epimedin C and icariin ingredient of medicinal material or Chinese materia medica preparation
According to HPLC measurement result and statistical analysis, the Epimedium sagittatum of foundation extracts medicinal materials fingerprint common pattern
It is high with the similarity of Epimedium sagittatum medicinal material, and Herba Epimedii reference extract (arrow leaf) of the invention and Epimedium sagittatum mention
Take the similarity of medicinal materials fingerprint common pattern again high, this explanation uses Herba Epimedii reference extract (arrow leaf) of the invention
The assay reliability for carrying out epimedin C and icariin to epimedium herb or Chinese materia medica preparation is high therefore of the invention
Herba Epimedii reference extract (arrow leaf) can be applied to the sxemiquantitative discriminatory analysis of epimedium herb or Chinese materia medica preparation, can also be applied to
Herba Epimedii and containing in the control of the quality of Herba Epimedii/epimedium active constituent medicinal material or Chinese materia medica preparation, such as to medicinal material or Chinese medicine
The epimedin C and icariin ingredient of preparation carry out sxemiquantitative discriminatory analysis, or to Epimedium sagittatum and contain Herba Epimedii/excessive sheep
The medicinal material or Chinese materia medica preparation of leaves of pulse plants effective component carry out half-quantitative detection and then control its quality.
Claims (10)
1. a kind of Herba Epimedii reference extract (arrow leaf), which is characterized in that it is derived from: to epimedium herb (arrow leaf) powder into
Row repeatedly extracts and obtains the Shorthorned Epimedium P.E of different batches, then deploys, obtains to the Shorthorned Epimedium P.E of different batches
To Herba Epimedii reference extract (arrow leaf)；
Optional, it is described that epimedium herb (arrow leaf) powder is repeatedly extracted, it is the epimedin C and Herba Epimedii for containing it
The concentration of glycosides improves, to obtain the Shorthorned Epimedium P.E (arrow leaf) of different batches.
Optional, epimedin C must not be lower than 13.00% and not higher than 17.00% in the Herba Epimedii reference extract (arrow leaf)
Content with icariin must not be lower than 3.00% and not higher than 5.00%；
Optional, the Herba Epimedii reference extract (arrow leaf) is obtained using thin-layered chromatography and/or high performance liquid chromatography
Map it is corresponding raw medicinal material it is consistent；
It is optional, in the chromatogram detected to the Herba Epimedii reference extract (arrow leaf) using thin-layered chromatography towards the leaves of pulse plants
The fluorescence spot at C and icariin and the chromatogram detected using high performance liquid chromatography are determined in epimedin C and excessive sheep
It is consistent that chromatographic peak at leaves of pulse plants glycosides position should distinguish corresponding chemical reference substance or its corresponding raw medicinal material.
2. Herba Epimedii reference extract (arrow leaf) according to claim 1, which is characterized in that the Herba Epimedii reference extract
(arrow leaf) is prepared using following steps:
Step 1: extracting: taking epimedium herb (arrow leaf) powder to mix with ethyl alcohol, filtered after being heated to reflux, obtain filtrate 1 and filter
Slag 1, and filtrate 1 is recycled to no ethanol flavor and obtains Herba Epimedii crude extract (arrow leaf)；
Step 2: crossing column: Herba Epimedii crude extract (arrow leaf) is taken, is fixed with macroreticular resin, adsorbs, is washed till no color with pure water, then
With ethanol elution to no color, merge ethanol eluate, recycling design to dry Herba Epimedii extract (arrow leaf)；
Step 3: preparing Shorthorned Epimedium P.E (arrow leaf): gained Herba Epimedii essence being extracted (arrow leaf), is dissolved, is obtained excessive with ethyl alcohol
The alcoholic solution of sheep leaves of pulse plants extract (arrow leaf), adds auxiliary material, is evaporated sieving and obtains Shorthorned Epimedium P.E (arrow leaf)；
Step 4: allotment: the Shorthorned Epimedium P.E (arrow leaf) of different batches being deployed, Herba Epimedii reference extract is obtained
(arrow leaf).
3. Herba Epimedii reference extract (arrow leaf) according to claim 2, which is characterized in that described described in step 1
Filter residue 1 is extracted according to the extracting method of step 1 by n times, and n times are extracted the filtrate collected and are merged with filtrate 1 Ji Wei extraction
Extracting solution is recycled to no gas ethanol flavor and obtains Herba Epimedii crude extract (arrow leaf) by liquid；Wherein 6 >=N >=0, and N is integer；
Optional, the N is 2.
4. Herba Epimedii reference extract (arrow leaf) according to claim 2, which is characterized in that the extraction in the step 1
Solution ethanol concentration is 30%~95%；
Optional, it is 50% that solution ethanol concentration is extracted in the step 1.
5. Herba Epimedii reference extract (arrow leaf) according to claim 2, which is characterized in that the excessive sheep in the step 1
The w/v of leaves of pulse plants medicinal material (arrow leaf) powder and ethyl alcohol is 1:5~1:45；
Optional, the w/v of epimedium herb (arrow leaf) powder and ethyl alcohol in the step 1 is 1:25；
Optional, the heating and refluxing extraction time in the step 1 is 1~120min；
Optional, the heating and refluxing extraction time in the step 1 is 30min；
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. Herba Epimedii reference extract (arrow leaf) according to claim 2, which is characterized in that Herba Epimedii in the step 2
Crude extract and macroreticular resin fixed proportion are as follows: 1:10~1:100；
Optional, Herba Epimedii crude extract (arrow leaf) and macroreticular resin fixed proportion in the step 2 are as follows: 1:20；
Optional, Herba Epimedii crude extract (arrow leaf) is in the macroporous resin adsorption time in the step 2 are as follows: 1~100min；
Optional, Herba Epimedii crude extract (arrow leaf) is in the macroporous resin adsorption time in the step 2 are as follows: 50min；
Optional, Herba Epimedii crude extract (arrow leaf) elutes concentration of alcohol in macroreticular resin in the step 2 are as follows: 30%~95%；
Optional, Herba Epimedii crude extract (arrow leaf) elutes concentration of alcohol in macroreticular resin in the step 2 are as follows: 70%.
7. Herba Epimedii reference extract (arrow leaf) according to claim 2, which is characterized in that Herba Epimedii in the step 3
The w/v of extract (arrow leaf) and ethyl alcohol: 1:2~1:20；
Optional, dissolution concentration of alcohol used is 80% ethyl alcohol in the step 3.
Optional, auxiliary material used is superfine silica gel powder and/or medical starch in the step 3.
Optional, the auxiliary material is superfine silica gel powder；
Optional, the step 3 is to be added the 10%~50% of its weight in the aqueous solution of Herba Epimedii extract (arrow leaf)
Superfine silica gel powder；
Optional, the step 3 is the 30% micro mist silicon that its weight is added in the aqueous solution of Herba Epimedii extract (arrow leaf)
Glue；
It is optional, the step 3 be evaporated after be 90~200 mesh net filtrations；
It is optional, the step 3 be evaporated after be 110 mesh net filtrations.
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 epimedin C and icariin of the Shorthorned Epimedium P.E of batch are detected.
8. Herba Epimedii reference extract (arrow leaf) according to claim 2, which is characterized in that deployed in the step 4
Standard are as follows: should make epimedin C in finally obtained reference extract must not be lower than 13.00% and not higher than 17.00% and excessive sheep
The content of leaves of pulse plants glycosides must not be lower than 3.00% and not higher than 5.00%；
Optional, the Herba Epimedii reference extract (arrow leaf) is obtained using thin-layered chromatography and/or high performance liquid chromatography
Map it is corresponding raw medicinal material it is consistent；
It is optional, in the chromatogram detected to the Herba Epimedii reference extract (arrow leaf) using thin-layered chromatography towards the leaves of pulse plants
Determine the fluorescence spot at C and icariin position and the chromatogram that is detected using high performance liquid chromatography in epimedin C and
It is consistent that chromatographic peak at icariin position should distinguish corresponding chemical reference substance or its corresponding raw medicinal material.
9. the described in any item Herba Epimedii reference extracts of claim 1 to 8 (arrow leaf) are in the identification of medicinal material or Chinese materia medica preparation, or
Herba Epimedii or containing the quality of Herba Epimedii/epimedium active constituent medicinal material or Chinese materia medica preparation control in application.
10. application according to claim 8, which is characterized in that the identification of the medicinal material or Chinese materia medica preparation or Herba Epimedii and containing excessive
The method of the quality control of the sheep leaves of pulse plants/epimedium active constituent Chinese materia medica preparation are as follows: any one of claim 1-8 is prepared
Herba Epimedii reference extract (arrow leaf), medicinal material or Chinese materia medica preparation are detected with thin-layered chromatography and/or high performance liquid chromatography,
Comparison differentiates；
It is optional, the thin-layered chromatography and/or high performance liquid chromatography be to Herba Epimedii reference extract (arrow leaf), medicinal material or
Epimedin C and icariin in Chinese materia medica preparation are detected.
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