CN109459515B - Herba epimedii control extract (arrow leaf) and application thereof - Google Patents

Abstract

The invention provides an epimedium control extract (arrow leaf), which is derived from the following components in percentage by weight: extracting herba Epimedii powder for multiple times to obtain herba Epimedii extracts of different batches, and blending herba Epimedii extracts of different batches to obtain herba Epimedii control extract (arrow leaf). The epimedium control extract (arrow leaf) is prepared by blending different batches of extracts, so that the consistency of the epimedium control extracts (arrow leaf) of different batches is ensured; and the character is stable, uniform and convenient to use. The epimedium control extract (arrow leaf) is used as a control in the quality control of epimedium and medicinal materials or Chinese medicinal preparations containing active ingredients of the epimedium/epimedium in the application, the epimedium control extract (arrow leaf) can be directly used by simple treatment in the quality control process, the operation is simple and convenient, particularly, when the multi-ingredient content is measured, the preparation of a control extract solution is simpler and more convenient than that of a control solution, the qualitative identification of the medicinal materials or the Chinese medicinal preparations can be carried out, and the epimedium control extract (arrow leaf) can also be used for semi-quantitative or even quantitative analysis.

Description

Herba epimedii control extract (arrow leaf) and application thereof

Technical Field

The invention relates to the technical field of traditional Chinese medicine quality control, in particular to a preparation method of an epimedium control extract (arrow leaf).

Background

The quality control mode of the traditional Chinese medicine is basically along the development of natural medicinal chemistry, an analysis method for taking one or more active ingredients of the traditional Chinese medicine as targets and the concept of qualitative and quantifiable quality standard, the quality control method of foreign plant medicines is referred, the mode of chemical medicine quality control is referred, corresponding simple physicochemical identification is established by means of literature reports, and the quality standard of identification and content determination mainly based on spectrum and chromatogram is developed. Each Chinese medicinal material is a multi-component complex, which determines the unique integrity and fuzziness of the Chinese medicinal material, and also shows that the evaluation method of taking one, two or even a plurality of components in the Chinese medicinal material as the quality of the Chinese medicinal material has great limitation. The 1990 edition of Chinese pharmacopoeia increases the thin-layer chromatography identification of reference medicinal materials, so that the identification of traditional Chinese medicines and Chinese patent medicines has great progress. At present, Chinese medicine and foreign herbal medicine pay more and more attention to the detection of multiple components or multiple components, such as the quality control of German ginkgo biloba extract. The analysis method of the fingerprint spectrum can carry out quality control on the medicinal materials on the whole, and still has great research value at present. At present, Chinese pharmacopoeia has two kinds of reference substances, namely a chemical reference substance and a traditional Chinese medicine reference medicinal material, in the aspect of controlling the quality of medicinal materials. The chemical reference substance can be used for qualitative identification and quantitative analysis of medicinal materials, and the traditional Chinese medicine reference medicinal material can be used for microscopic identification and thin-layer identification. However, both chemical reference substances and traditional Chinese medicine reference medicinal materials have limitations in traditional Chinese medicine quality control. Firstly, the chemical components in the traditional Chinese medicine are diversified, single or a plurality of compounds cannot reflect the whole appearance of the medicinal materials, and the existing standard often has a plurality of holes so as to have the phenomenon of being insufficient. The traditional Chinese medicine reference medicinal materials are influenced by the producing area and the growth environment, the quality consistency of each batch is difficult to ensure, and the traditional Chinese medicine reference medicinal materials can only be used for qualitative identification and cannot reflect the content of the medicinal material components.

The traditional Chinese medicine control extract is an extract which is prepared from traditional Chinese medicinal materials, has stable properties and components, can be used for qualitative or quantitative analysis, and has four basic requirements (ASCS) for the traditional Chinese medicine control extract by Mr. schehren, which also has several basic conditions: the herbal material source is reliable and representative; specificity, Specificity of the detection method used; con-sistence, the control extracts should remain consistent from batch to batch; stability, stable and uniform properties and convenient use. The method can be used for qualitative identification of medicinal materials by using high performance thin layer chromatography fingerprint and high performance liquid chromatography fingerprint, and the control extract standardized by external standard method can be further used for semi-quantitative and quantitative analysis and detection. The traditional Chinese medicine control extract has important significance for controlling the quality of the traditional Chinese medicine.

Disclosure of Invention

The invention aims to provide an epimedium control extract (arrow leaf) which has good batch consistency, stable and uniform properties and is convenient to use, and the invention also provides application of the epimedium control extract (arrow leaf), namely, the epimedium control extract (arrow leaf) is used for quality control of epimedium and medicinal materials or Chinese medicinal preparations containing active ingredients of the epimedium/epimedium.

Compared with the prior art, the invention has the following beneficial effects:

the epimedium control extract (arrow leaves) is prepared from different batches of extracts, so that the defect that the quality of each batch is difficult to ensure to be consistent due to the influence of production areas and growth environments on traditional Chinese medicine control medicinal materials is overcome, and the consistency of the epimedium control extracts (arrow leaves) of different batches is ensured; and the character is stable, uniform and convenient to use. The epimedium control extract (arrow leaf) is applied to quality control of epimedium and medicinal materials or Chinese medicinal preparations containing active ingredients of epimedium/epimedium after quantitative analysis of the epimedium control extract (arrow leaf), in the quality control process, the epimedium control extract (arrow leaf) is simply processed and can be directly used, the operation is simple and convenient, particularly when the multi-component content is measured, the preparation of a control extract solution is simpler and more convenient than that of a control solution, and the epimedium control extract (arrow leaf) not only can be used for qualitative identification of the medicinal materials or the Chinese medicinal preparations, but also can be used for semi-quantitative or even quantitative analysis.

Drawings

FIG. 1 is a thin layer chromatogram obtained by performing thin layer chromatography on herba Epimedii raw material (folium Aristolochiae Setchuensis) with chemical reference substance as reference substance. Wherein, the color bands marked by the symbols S1 and S2 respectively correspond to contrast products epimedin C and icariin, and 1-6 sequentially correspond to arrow leaf epimedium raw material medicinal material 1, arrow leaf epimedium raw material medicinal material 2, arrow leaf epimedium raw material medicinal material 3, arrow leaf epimedium raw material medicinal material 4, arrow leaf epimedium raw material medicinal material 5 and arrow leaf epimedium raw material medicinal material 6.

FIG. 2 is a superimposed graph of HPLC fingerprint obtained by performing high performance liquid chromatography on herba Epimedii raw material (arrow leaf) with chemical reference substance as reference substance, wherein the high performance liquid chromatography is performed from top to bottom 1-6 for arrow leaf herba Epimedii raw material 1, arrow leaf herba Epimedii raw material 2, arrow leaf herba Epimedii raw material 3, arrow leaf herba Epimedii raw material 4, arrow leaf herba Epimedii raw material 5 and arrow leaf herba Epimedii raw material 6, S1 and S2 for chemical reference substances: epimedin C, icariin, R correspond to the common pattern.

FIG. 3 is a flow chart of the preparation of herba Epimedii control extract (arrow leaf).

FIG. 4 is a superposition diagram of common mode of herba Epimedii control extract (arrow leaf) and herba Epimedii extract raw material, S1 and S2 correspond to chemical reference, ERS corresponds to herba Epimedii control extract (arrow leaf), and R is common mode of herba Epimedii raw material.

FIG. 5 is a thin layer chromatogram obtained by thin layer chromatography of commercially available medicinal materials with chemical control and herba Epimedii control extract as reference substances. Wherein the color band of reference sign S is from bottom to top corresponding to control product epimedin C and icariin, 5-14 are sequentially corresponding 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 is herba Epimedii control extract, 2 is herba Epimedii granule, 3 is herba Epimedii control medicinal material, and 4 is Korean herba Epimedii control medicinal material.

FIG. 5-1 is a high performance thin layer chromatogram digital scanning spectrum of herba Epimedii paired extract (arrow leaf) and herba Epimedii granule, herba Epimedii reference medicinal material, Korean herba Epimedii reference medicinal material and herba Epimedii medicinal material. The strip 1 is herba Epimedii control extract (arrow leaf), the strip 2 is herba Epimedii control medicinal material, the strip 3 is Korean herba Epimedii control medicinal material, the strip 4 is herba Epimedii granule, and the strips 5-14 correspond 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 sequentially.

FIG. 6 is a superimposed graph of HPLC fingerprint chromatogram obtained by performing high performance liquid chromatography on commercially available medicinal materials with chemical reference substance and herba Epimedii reference extract as reference substance, and the HPLC fingerprint chromatogram is from top to bottom 4-15 corresponding 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 is herba Epimedii control extract, 2 is herba Epimedii control medicinal material, 3 is Korean herba Epimedii control medicinal material, 4 is herba Epimedii granule, and S1 and S2 are chemical control substances: epimedin C and icariin.

Detailed Description

The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The following examples use the following sources of instruments and materials:

the drugs intended for the preparation of the control extract:

herba Epimedii is purchased from various major medicinal material markets in China, and identified as dry leaf of Epimedium sagittatum (Sieb. et Zucc.) Maxim of berberidaceae by professor Shebei mountain.

A Linomat 5 thin-layer chromatography semi-automatic spotting instrument, an ATS 4 thin-layer chromatography full-automatic spotting instrument, a thin-layer chromatography double-groove developing cylinder, a Chromatogram imaging Device III chromatographic development dipping groove and a TLC visualizer thin-layer chromatography camera (CAMAG, Switzerland).

Agilent 1260series HPLC chromatograph equipped with DAD detector (Agilent Technologies, USA).

Methanol, ethanol, formic acid, acetic acid, phosphoric acid, butyl acetate, acetone were all analytically pure (Guangzhou chemical laboratories).

Acetonitrile is chromatographically pure (Merck KGaA);

methanol as chromatographically pure (Merck KGaA);

purified water;

epimedin C, mark purity is more than or equal to 95.5 percent (China institute for food and drug testing, lot number: 111780-201503);

icariin (China institute for testing food and drug; batch No. 110737-;

herba Epimedii control material (China institute for testing food and drug products: 121632-201502)

Korean epimedium control drug (China food and drug testing research institute, batch number: 121032-201302)

Testing medicinal materials, namely epimedium:

6 batches of arrow-leaf epimedium raw material medicines are purchased in various national large medicinal material markets, and 1-6 batches of arrow-leaf epimedium raw material medicines are purchased;

herba Epimedii 10 batches are purchased in large drug stores and drug markets of Guangzhou, herba Epimedii 1-10;

herba Epimedii granule is purchased in large drugstore and medicinal material market;

example 1: screening of epimedium herb raw medicinal materials

This example provides a method for screening the raw material herbs of the present invention's herba Epimedii control extract (folium sagittariae sagittifoliae).

Analyzing and screening herba Epimedii raw material (folium Aristolochiae Kaempferi) by thin layer chromatography and high performance liquid chromatography with chemical reference substance as reference substance.

Herba Epimedii is purchased from various major medicinal material markets in China, and identified as dry leaf of Epimedium sagittatum (Sieb. et Zucc.) Maxim of berberidaceae by professor Shebei mountain. 6 batches of epimedium medicinal materials are identified to be epimedium sagittifolia, and can be used for standby after meeting the standard.

1 thin layer chromatography

1.1 sample preparation

Chemical control solution: a proper amount of icariin and epimedin C chemical reference substances are precisely weighed, and methanol is respectively added to prepare a solution containing about 0.5mg per 1mL as a reference substance solution.

The test solution of the medicinal materials: taking 0.5g of epimedium raw material powder, precisely weighing, placing in a 150mL conical flask with a plug, adding 50mL of 50% ethanol, heating in a water bath, carrying out hot reflux for 30min at 80 ℃, cooling, filtering, transferring to an evaporating dish, evaporating, dissolving residues with 70% ethanol, transferring to a 5mL volumetric flask, fixing the volume to a scale, shaking up, taking supernatant, and filtering with a 0.22 mu m filter membrane to obtain a test solution of the medicinal material. The medicinal materials are bought from various large medicinal material markets in China and are respectively arrow-leaf epimedium herb raw material medicinal material 1, arrow-leaf epimedium herb raw material medicinal material 2, arrow-leaf epimedium herb raw material medicinal material 3, arrow-leaf epimedium herb raw material medicinal material 4, arrow-leaf epimedium herb raw material medicinal material 5 and arrow-leaf epimedium herb raw material medicinal material 6.

1.2 thin layer chromatography detection

The detection conditions were as follows:

thin-layer plate: TLC G60 precast slabs (Merck);

sample application: the test solution of the medicinal materials: 2. mu.l, the remaining sample 4. mu.l; the strip-shaped sample application length is 8 mm;

developing agent: butyl acetate: acetone: formic acid: water 11:2:10:10, (layering at 10 ℃, taking the upper layer solution);

the unfolding mode is as follows: adding a developing agent to one side of a double-groove developing cylinder, pre-balancing for 15 minutes, and developing and ascending for 9 cm;

and (6) inspection: after the development, 5% aluminum trichloride ethanol solution was uniformly sprayed, the thin layer plate was placed on a thin layer heating plate and heated at 105 ℃ for 2 minutes, and the plate was placed under an ultraviolet lamp (366nm) to examine a fluorescence chromatogram image.

And (3) detection results: as shown in FIG. 1, the thin layer chromatogram was examined under an ultraviolet lamp (366nm) (T: 25 ℃, RH: 70%). The chromatographic bands marked by the labels S1 and S2 are respectively reference substances epimedin C and icariin, 1-6 correspond to arrow-leaf epimedium raw material medicinal material 1, arrow-leaf epimedium raw material medicinal material 2, arrow-leaf epimedium raw material medicinal material 3, arrow-leaf epimedium raw material medicinal material 4, arrow-leaf epimedium raw material medicinal material 5 and arrow-leaf epimedium raw material medicinal material 6 in sequence; as can be seen from figure 1, the separation condition of epimedin C and icariin is good, each batch of Epimedium sagittatum medicinal materials can show the same spots at the same position, the similarity between the Epimedium sagittatum medicinal materials of each batch is high, and the Epimedium sagittatum medicinal materials are primarily screened as standard control extracts to extract raw medicinal materials.

2 high performance liquid chromatography

2.1 sample preparation

Chemical control solution: accurately weighing appropriate amount of chemical reference substances such as epimedin C and icariin, and respectively preparing into 100 μ g/ml solution with methanol.

The test solution of the medicinal materials: taking 0.1g of each medicinal material powder, precisely weighing, adding 80mL of 50% ethanol, performing hydrothermal reflux for 1h, taking out, performing suction filtration, evaporating the filtrate to dryness, dissolving the residue in 30% methanol, transferring to a 5mL volumetric flask, fixing the volume to the scale, shaking up, standing overnight in a refrigerator (4 ℃), taking the supernatant, and filtering through a 0.22 mu m filter membrane to obtain the traditional Chinese medicine composition. The medicinal materials are bought from various large medicinal material markets in China and are respectively arrow-leaf epimedium herb raw material medicinal material 1, arrow-leaf epimedium herb raw material medicinal material 2, arrow-leaf epimedium herb raw material medicinal material 3, arrow-leaf epimedium herb raw material medicinal material 4, arrow-leaf epimedium herb raw material medicinal material 5 and arrow-leaf epimedium herb raw material medicinal material 6.

2.2 high Performance liquid chromatography detection

The detection conditions of the high performance liquid chromatography are as follows:

chromatography apparatus: agilent 1260series HPLC with DAD detector (Agilent Technologies, USA)

A chromatographic column: agilent ZORBAX SB-C18 (4.6X 250mm,5 μm);

mobile phase: a-methanol, B-acetonitrile, C-0.5% acetic acid;

gradient elution procedure: 0-30 min: 0% A → 0% A, 30-45 min: 0% A → 11% A, 45-65 min: 11% A → 4% A, 65-85 min: 4% A → 4% A, 85-90 min: 4% A → 0% A, 90-100 min: 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); the flow rate is 1.0 ml/min; the sample volume is 20 mul; column temperature 20 ℃, run time: for 100 min.

The detection method of the high performance liquid chromatography comprises the following steps: precisely sucking 20 μ l of chemical reference solution and 20 μ l of test solution of Epimedium sagittatum, respectively, and injecting into liquid chromatograph.

And (3) detecting results by high performance liquid chromatography:

measuring, recording chromatogram, and obtaining HPLC fingerprint superposition chart shown in figure 2, wherein 1-6 from top to bottom correspond to Epimedium sagittatum raw material medicinal material 1, Epimedium sagittatum raw material medicinal material 2, Epimedium sagittatum raw material medicinal material 3, Epimedium sagittatum raw material medicinal material 4, Epimedium sagittatum raw material medicinal material 5 and Epimedium sagittatum raw material medicinal material 6 respectively, and S1 and S2 correspond to chemical reference substances: epimedin C, icariin, R correspond to the consensus pattern.

As can be seen from fig. 2, the fingerprint spectrums of the raw medicinal materials Epimedium sagittatum L.1-Epimedium sagittatum L.6 have high consistency, so that a common fingerprint spectrum mode of Epimedium sagittatum L.is established, and similarity analysis is performed on all samples.

According to a set Epimedium sagittatum medicinal material fingerprint spectrum common mode, similarity evaluation is carried out on 6 batches of Epimedium sagittatum medicinal material samples by adopting an included angle cosine algorithm according to each sample component and the peak area thereof, and the result is shown in Table 1.

TABLE 1

Sample (I)	Degree of similarity
		Epimedium sagittatum (Maxim.) Maxim crude drug 1	1.00
Epimedium sagittatum raw material medicine 2	0.99
		Epimedium sagittatum (Maxim.) Maxim raw material medicine3	0.99
Epimedium sagittatum (Maxim.) Maxim crude drug 4	0.99
		Epimedium sagittatum (Maxim.) Maxim raw material 5	0.99
Epimedium sagittatum raw material medicine 6	0.99
		Common mode	1.00

According to the similarity analysis results, the similarity of the 6 batches of Epimedium sagittatum Maxim raw material medicinal materials is more than or equal to 0.99, the similarity is very high, and the Epimedium sagittatum Maxim standard control extract (Epimedium sagittatum Maxim) can be selected to be extracted to obtain the raw material medicinal materials.

Example 2: preparation of Epimedium control extract

This example provides a method for preparing herba Epimedii control extract (folium sagittariae sagittifoliae) of the present invention, and the flow chart of the method is shown in FIG. 3.

Firstly, the method comprises the following steps: extraction of

Selecting epimedium medicinal materials (arrow leaves), preparing the medicinal materials into powder, adding 50% ethanol (namely the solid-to-liquid ratio is 1: 25(w/V)) with the mass of 25 times of the weight of the medicinal materials, heating and refluxing for 30min, filtering the medicinal materials by medium-speed qualitative filter paper, collecting filter residue 1 and filter liquor 1, extracting the filter residue 1 again by the same method, combining the filter liquor collected by the extraction of the first time with the filter liquor 1 to obtain extracting solution, evaporating the solvent of the extracting solution until no ethanol smell exists to obtain epimedium crude extracts (arrow leaves), fixing the epimedium crude extracts (arrow leaves) by using a 1:20 macroporous resin column, adsorbing for 30-60 min, washing with pure water until no color, eluting with 70% ethanol, eluting until no color exists, combining alcohol eluent and recovering until the dry to obtain epimedium fine extracts (arrow leaves);

II, secondly: preparation of Epimedium extract (arrow leaf)

Dissolving herba Epimedii fine extract (folium sagittariae sagittifoliae) with 80% ethanol, adding 30% silica gel micropowder (assisted by Shanhe medicinal materials) of herba Epimedii fine extract (folium sagittifoliae), evaporating to dryness with rotary evaporator, pulverizing, and sieving with 110 mesh sieve to obtain herba Epimedii extract (folium sagittariae sagittifoliae).

6 batches of epimedium extracts (arrow leaves) were prepared and measured by high performance liquid chromatography using controls, with the test results shown in Table 3:

TABLE 3

Thirdly, the method comprises the following steps: blending

And (3) mixing and blending the epimedium extracts (arrow leaves) of the 6 batches in the step two according to the proportion of 1:1 to obtain epimedium control extracts (arrow leaves), wherein the proportion of the corresponding raw medicinal materials of the final product is about 1: 7(g/g), measured by high performance liquid chromatography using a control, wherein the measurement results of the contents of the respective components are shown in Table 4.

TABLE 4

The blending standard is as follows: the final herba Epimedii control extract (folium Aristolochiae Semtschatae) should have epimedin C content of no less than 13.00% and no more than 17.00% and icariin content of no less than 3.00% and no more than 5.00% (content in wt%). The content range of each component in the blending standard is also the best data obtained by comprehensively maintaining stability, consistency and later application through repeated experiments.

Fourthly, the method comprises the following steps: detection of

Determining herba Epimedii control extract (folium Epimedii) prepared in step three by high performance liquid chromatography to obtain fingerprint, and detecting similarity with common mode spectrum of raw material medicinal materials, as shown in FIG. 4, ERS corresponds to herba Epimedii control extract (folium Artemisiae Argyi), R is common mode of folium Epimedii, S1, S2 corresponds to chemical control: epimedin C and icariin. The obtained similarity is 1.00, and the similarity of common patterns of the epimedium control extract (arrow leaves) and the arrow leaf epimedium medicinal material fingerprint is very high, which indicates that the epimedium control extract (arrow leaves) can represent the arrow leaf epimedium medicinal material.

Example 3 analysis of the Properties of control extract of Epimedium (arrow leaf)

1. Apparent state: the epimedium control extract (arrow leaf) obtained in example 2 was a pale grayish purple powder.

2. And (3) moisture determination: according to 2015 version of appendix IX G of the Chinese pharmacopoeia (reduced pressure drying). The detection result shows that the water content of the herba Epimedii control extract (folium Aristolochiae Kaempferi) is 0.50%.

3. And (3) testing consistency: 3 batches of epimedium control extracts (arrow leaves) are prepared according to the method of example 2, and the thin-layer chromatography detection results of epimedin C and icariin measured among the batches have small difference, and fluorescent spots of the epimedin C and the icariin are obviously displayed and are distributed very uniformly; the contents of epimedin C and icariin are detected to be within the range of the preparation standard by high performance liquid chromatography. Therefore, the consistency of the epimedium control extract (arrow leaf) prepared by the preparation method of the epimedium control extract (arrow leaf) is very good.

4. And (3) stability testing:

taking 3 different batches of test samples of herba Epimedii reference extract (folium Aristolochiae Kaempferi) prepared by the method of example 2, and examining indexes including properties and solubility according to the relevant regulation of 'national drug standard substance development technical requirement' formulated by the Commission of the national pharmacopoeia.

The sample is placed in a clean container with an opening at 60 deg.C for 10 days, sampled on the 5 th and 10 th days, and tested according to the stability focus examination item.

The result shows that the characters of the test sample do not obviously change before and after the high-temperature test, the thin-layer chromatogram does not obviously change before and after the high-temperature test, the determination result of the dissolution rate is subjected to independent sample t-test by SPSS, the calculation result P is more than 0.05, and no significant difference exists.

Example 4

This example provides the use of the epimedium control extract (arrow leaf) prepared in example 2.

Analyzing commercially available medicinal materials by thin layer chromatography and high performance liquid chromatography with chemical reference substance, herba Epimedii reference substance, Korean herba Epimedii reference substance and herba Epimedii reference extract (folium Arisaemati) as reference substance.

1 thin layer chromatography

1.1 sample preparation

Chemical control solution: a proper amount of icariin and epimedin C chemical reference substances are precisely weighed, and methanol is respectively added to prepare a solution containing about 0.5mg per 1mL as a reference substance solution.

Epimedium control extract (arrow leaf) solution: precisely weighing 15mg of the epimedium control extract (arrow leaf) prepared in the example 2, putting the epimedium control extract (arrow leaf) into a 5mL volumetric flask, adding 4mL of methanol, carrying out ultrasonic treatment for 15 minutes, cooling, adding the methanol to the scale, shaking up, and filtering through a 0.22 mu m filter membrane to obtain the epimedium compound.

Herba epimedii formula granules: taking formula granules equivalent to 0.5g of medicinal materials, dissolving with 70% ethanol, fixing the volume to a 5mL volumetric flask, fixing the volume to a scale, shaking up, and filtering supernate with a 0.22 μm filter membrane to obtain the product;

the test solution of the medicinal materials: taking 0.5g of epimedium medicinal material powder, precisely weighing, placing in a 150mL conical flask with a plug, adding 50mL of 50% ethanol, heating in a water bath, carrying out hot reflux for 30min at 80 ℃, cooling, filtering, transferring to an evaporating dish, evaporating, dissolving residues with 70% ethanol, transferring to a 5mL volumetric flask, fixing the volume to a scale, shaking up, taking supernatant, and filtering with a 0.22 mu m filter membrane to obtain the medicinal material sample solution. The medicinal materials are purchased from large drugstores in Guangzhou, and are respectively 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; preparing herba Epimedii reference solution and Korean herba Epimedii reference solution by the same method.

1.2 thin layer chromatography detection

The detection conditions were as follows:

thin-layer plate: TLC G60 precast slabs (Merck);

sample application: the test solution of the medicinal materials: 2. mu.l, the remaining sample 4. mu.l; the strip-shaped sample application length is 8 mm;

developing agent: butyl acetate: acetone: formic acid: water 11:2:10:10, (layering at 10 ℃, taking the upper layer solution);

the unfolding mode is as follows: adding a developing agent to one side of a double-groove developing cylinder, pre-balancing for 15 minutes, and developing and ascending for 9 cm;

and (6) inspection: after the development, 5% aluminum trichloride ethanol solution is uniformly sprayed, the thin layer plate is placed on a thin layer heating plate and heated for 2 minutes at 105 ℃, and a fluorescence chromatographic image is inspected under an ultraviolet lamp (366 nm);

and (3) detection results: as shown in FIG. 5, the thin layer chromatogram was examined under an ultraviolet lamp (366nm) (T: 25 ℃, RH: 70%). The color band of reference sign S is respectively reference substance epimedin C and icariin from bottom to top, 5-14 are respectively corresponding to epimedium 1, epimedium 2, epimedium 3, epimedium 4, epimedium 5, epimedium 6, epimedium 7, epimedium 8, epimedium 9 and epimedium 10. 1 is herba Epimedii control extract, 2 is herba Epimedii granule, 3 is herba Epimedii control medicinal material, and 4 is Korean herba Epimedii control medicinal material.

And (4) analyzing results: as can be seen from FIG. 5-1, the separation of epimedin C and icariin is better, the positions of the epimedium control extract (arrow leaf) and the epimedium prescription granule, the epimedium control medicinal material and the epimedium medicinal material (epimedium 1-10) main spots (epimedin C and icariin) are consistent. The map shows that the epimedium control extract (arrow leaf) and the epimedium 1 can display the same spot at the same position and are supposed to be arrow leaf epimedium; the herba Epimedii reference extract (folium sagittariae sagittifoliae) is slightly different from herba Epimedii granule, herba Epimedii reference medicinal material, Korean herba Epimedii reference medicinal material and herba Epimedii medicinal material (herba Epimedii 2-10); from the map, the medicinal materials in the pharmacy on the market are mostly Korean epimedium and a small amount of other varieties of epimedium medicinal materials, and the epimedium medicinal materials with few arrow leaves are known.

2 high performance liquid chromatography

2.1 sample preparation

Chemical control solution: accurately weighing appropriate amount of chemical reference substances such as epimedin C and icariin, and respectively preparing into 100 μ g/ml solution with methanol.

Epimedium control extract (arrow leaf) solution: precisely weighing herba Epimedii ERS15mg, placing in a 5ml volumetric flask, adding 4ml water, and performing ultrasonic treatment for 15 min. Cooling, adding methanol to scale, shaking, filtering with 0.22 μm filter membrane, and collecting filtrate to obtain herba Epimedii control extract (folium Aristolochiae Kaempferi) solution;

herba epimedii formula granules: taking formula granules equivalent to 0.1g of medicinal materials, dissolving with 30% ethanol, fixing the volume to a 5mL volumetric flask, fixing the volume to scale, shaking up, standing overnight in a refrigerator (4 ℃), and filtering supernate with a 0.22-micron filter membrane to obtain the traditional Chinese medicine composition;

the test solution of the medicinal materials: taking 0.1g of each medicinal material powder, precisely weighing, adding 80mL of 50% ethanol, performing hydrothermal reflux for 1h, taking out, performing suction filtration, evaporating the filtrate to dryness, dissolving the residue in 30% methanol, transferring to a 5mL volumetric flask, fixing the volume to the scale, shaking up, standing overnight in a refrigerator (4 ℃), taking the supernatant, and filtering through a 0.22 mu m filter membrane to obtain the traditional Chinese medicine composition. The medicinal materials of the pharmacy are purchased from Guangzhou and respectively: epimedium 1, epimedium 2, epimedium 3, epimedium 4, epimedium 5, epimedium 6, epimedium 7, epimedium 8, epimedium 9 and epimedium 10; preparing herba Epimedii reference solution and Korean herba Epimedii reference solution by the same method.

2.2 high Performance liquid chromatography detection

The detection conditions of the high performance liquid chromatography are as follows:

chromatography apparatus: agilent 1260series HPLC with DAD detector (Agilent Technologies, USA)

A chromatographic column: agilent ZORBAX SB-C18 (4.6X 250mm,5 μm);

mobile phase: a-methanol, B-acetonitrile, C-0.5% acetic acid;

gradient elution procedure: 0-30 min: 0% A → 0% A, 30-45 min: 0% A → 11% A, 45-65 min: 11% A → 4% A, 65-85 min: 4% A → 4% A, 85-90 min: 4% A → 0% A, 90-100 min: 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); the flow rate is 1.0 ml/min; the sample volume is 20 mul; column temperature 20 ℃, run time: for 100 min.

The detection method of the high performance liquid chromatography comprises the following steps: precisely sucking 20 μ l of chemical reference solution, herba Epimedii reference solution, Korean herba Epimedii reference solution, herba Epimedii reference extract (folium Epimedii) solution and medicinal material sample solution respectively, and injecting into liquid chromatograph.

And (3) detecting results by high performance liquid chromatography:

measuring, recording chromatogram to obtain HPLC fingerprint chromatogram stack diagram shown in FIG. 6, which sequentially corresponds 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 from top to bottom 5-14. 1 is herba Epimedii control extract (arrow leaf), 2 is herba Epimedii control medicinal material, 3 is Korean herba Epimedii control medicinal material, and 4 is herba Epimedii granule; s1, S2 correspond to chemical controls: epimedin C and icariin.

As can be seen from FIG. 6, the difference of fingerprint spectra of the medicinal materials Epimedium 1-Epimedium 10 is large. Similarity evaluation is carried out on the fingerprints of 10 batches of epimedium medicinal materials according to the fingerprints of the epimedium control extracts and the component and the peak area of each sample by adopting an included angle cosine algorithm, and the result is shown in table 5.

TABLE 5

Sample (I)	Degree of similarity
		Epimedium
1	0.96
		Epimedium 2	0.63
Epimedium 3	0.45
		Epimedium 4	0.40
Epimedium 5	0.40
		Epimedium 6	0.43
Epimedium 7	0.93
		Epimedium 8	0.60
Epimedium 9	0.55
		Epimedium 10	0.46
Herba Epimedii reference medicinal material	0.51
		Korean herba Epimedii control medicinal material	0.39
Epimedium formula particle	0.67
		Epimedium ERS	1.00

According to the similarity analysis results, the similarity of 10 batches of medicinal materials, namely the epimedium 1, the epimedium 7 and the epimedium control extract (arrow leaves) is 0.98 and 0.95 respectively, the similarity is very high, and the similarity of other commercial medicinal materials and the epimedium control extract is very low, because the epimedium commercial medicinal materials are various in types and few arrow leaf epimedium is in the market.

According to the results of TLC and HPLC finger-prints, the chromatogram obtained by detecting the herba Epimedii control extract (folium Epimeredis Indicae) by thin layer chromatography is consistent with the corresponding raw material, more precisely, the fluorescence spots at the positions of epimedin C and icariin in the chromatogram obtained by detecting the herba Epimedii control extract by thin layer chromatography and the chromatographic peaks at the positions of epimedin C and icariin in the chromatogram obtained by detecting the herba Epimedii control extract by high performance liquid chromatography are respectively consistent with the corresponding chemical reference or the corresponding raw material, therefore, the herba Epimedii control extract (folium Epimeredis Indicae) of the invention can be applied to the qualitative identification of medicinal materials or Chinese medicinal preparations, for example, the qualitative analysis of the components of epimedin C and icariin of medicinal materials or Chinese medicinal preparations

According to HPLC determination results and statistical analysis, the similarity between the established common fingerprint pattern of Epimedium sagittatum extract medicinal materials and Epimedium sagittatum medicinal materials is extremely high, and the similarity between the common fingerprint pattern of Epimedium sagittatum extract (Epimedium sagittatum) and Epimedium sagittatum extract medicinal materials is extremely high, which shows that the content determination reliability of epimedin C and icariin on Epimedium medicinal materials or Chinese medicinal preparations is extremely high by using the Epimedium sagittatum control extract (Epimedium sagittatum) of the invention, therefore, the Epimedium sagittatum control extract (Epimedium sagittatum) of the invention can be applied to semi-quantitative identification analysis of the Epimedium sagittatum medicinal materials or Chinese medicinal preparations, and also can be applied to quality control of the Epimedium sagittatum and the medicinal materials or Chinese medicinal preparations containing effective components, such as the semi-quantitative identification analysis of the epimedin C and the icariin medicinal materials or Chinese medicinal preparations, or semi-quantitatively detecting herba Epimedii and medicinal materials or Chinese medicinal preparation containing herba Epimedii/herba Epimedii effective components to control their quality.

Claims (1)

1. A thin-layer chromatography detection method for Epimedium sagittatum control extract solution or medicinal materials containing Epimedium sagittatum/Epimedium sagittatum active ingredients is characterized by comprising the following steps:

(1) preparation of Epimedium control extract

The epimedium control extract is derived from: extracting epimedium herb powder for multiple times to obtain epimedium extracts of different batches, and blending the epimedium extracts of different batches to obtain epimedium herb contrast extracts of sagittaria sagittifolia;

the epimedium sagittifolium medicinal material powder is extracted for multiple times, so that the concentration of epimedin C and icariin contained in the epimedium sagittifolium medicinal material powder is increased, and different batches of epimedium sagittifolium extracts are obtained;

the epimedium sagittatum control extract contains epimedin C not less than 13.00% and not more than 17.00% and icariin not less than 3.00% and not more than 5.00%;

the map obtained by adopting thin-layer chromatography and high performance liquid chromatography to the epimedium sagittifolia control extract is consistent with the corresponding raw medicinal materials;

fluorescence spots at the epimedin C and the icariin in a chromatogram obtained by detecting the epimedium sagittatum control extract by adopting thin-layer chromatography and chromatographic peaks at the epimedin C and the icariin positions of the chromatogram obtained by detecting the epimedium sagittatum control extract by adopting high performance liquid chromatography are respectively consistent with the corresponding chemical reference substances or the corresponding raw material medicines;

the epimedium sagittifolia control extract is prepared by the following steps:

step one, extraction: mixing epimedium sagittifolia medicinal material powder with ethanol, heating, refluxing, filtering to obtain a filtrate 1 and a filter residue 1, and recovering the filtrate 1 until ethanol smell does not exist to obtain an epimedium sagittifolia crude extract;

step two, column passing: fixing the crude extract of Epimedium sagittatum with macroporous resin, adsorbing, washing with pure water until no color exists, eluting with ethanol until no color exists, mixing the ethanol eluates, and recovering solvent to obtain dry Epimedium sagittatum extract;

step three, preparing the epimedium sagittifolium extract: dissolving the obtained Epimedium sagittatum fine extract with ethanol to obtain an alcoholic solution of the Epimedium sagittatum fine extract, adding auxiliary materials, drying by distillation and sieving to obtain Epimedium sagittatum extract;

step four, blending: blending epimedium sagittifolium extracts of different batches to obtain epimedium sagittifolium control extracts;

extracting the filter residue 1 in the step one for N times according to the extraction method in the step one, combining the filtrate collected by the extraction for N times with the filtrate 1 to obtain an extracting solution, and recovering the extracting solution until no ethanol smell exists to obtain an epimedium herb crude extract;

the N is 2;

the ethanol concentration of the extraction solution in the first step is 50 percent;

the weight volume ratio of epimedium sagittifolia medicinal material powder to ethanol in the step one is 1: 25;

the heating reflux extraction time in the step one is 30 min;

the filtration in the first step is to use medium-speed filter paper;

in the second step, the fixing ratio of the arrowleaf epimedium crude extract to the macroporous resin is as follows: 1: 20;

in the second step, the adsorption time of the epimedium herb crude extract in the macroporous resin is as follows: 50 min;

in the second step, the elution ethanol concentration of the arrowleaf epimedium crude extract in the macroporous resin is as follows: 70 percent;

the weight volume ratio of the epimedium brevicornum fine extract to the ethanol in the third step is as follows: 1: 2-1: 20;

the concentration of the dissolved ethanol used in the third step is 80 percent ethanol;

the auxiliary material is micro silica gel powder;

adding 30% of micro silica gel powder by weight into the aqueous solution of the epimedium arrow leaf refined extract;

drying the mixture in the third step to dryness, and filtering the mixture through a 110-mesh screen;

the step three and the step four also comprise a step of detecting epimedin C and icariin of different batches of epimedium extracts by thin-layer chromatography and high performance liquid chromatography;

the blending standard in the fourth step is as follows: the content of epimedin C in the finally obtained control extract is not less than 13.00% and not more than 17.00% and the content of icariin is not less than 3.00% and not more than 5.00%;

the map obtained by adopting thin-layer chromatography and high performance liquid chromatography to the epimedium sagittifolia control extract is consistent with the corresponding raw medicinal materials;

fluorescence spots at the positions of epimedin C and icariin in a chromatogram obtained by detecting the epimedium sagittatum comparison extract by adopting thin-layer chromatography and chromatographic peaks at the positions of epimedin C and icariin in the chromatogram obtained by detecting the epimedium sagittatum comparison extract by adopting high performance liquid chromatography are respectively consistent with a corresponding chemical reference substance or a corresponding raw material medicine thereof;

(2) detection by thin layer chromatography

Precisely weighing 15mg of the prepared epimedium sagittifolia control extract, placing the 15mg in a 5mL volumetric flask, adding 4mL of methanol, carrying out ultrasonic treatment for 15 minutes, cooling, adding methanol to a scale, shaking up, and filtering through a 0.22 mu m filter membrane to obtain epimedium sagittifolia control extract solution;

the thin-layer chromatography detection conditions are as follows:

thin-layer plate: TLC G60 precast slab Merck;

sample application: the test solution of the medicinal materials: 2. mu.l, the remaining sample 4. mu.l; the strip-shaped sample application length is 8 mm;

developing agent: butyl acetate: acetone: formic acid: layering with water =11:2:10:10 at 10 ℃, and taking the upper solution;

the unfolding mode is as follows: adding a developing agent to one side of a double-groove developing cylinder, pre-balancing for 15 minutes, and developing and ascending for 9 cm;

and (6) inspection: after the development, 5% aluminum trichloride ethanol solution is uniformly sprayed, the thin layer plate is placed on a thin layer heating plate and heated for 2 minutes at 105 ℃, and a fluorescence chromatographic image is inspected under an ultraviolet lamp 366 nm.

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