Characteristic chromatogram of herba epimedii, characteristic chromatogram construction method and identification method

Abstract

The invention relates to the technical field of pharmaceutical analysis, and particularly provides a characteristic chromatogram of epimedium villosum, a characteristic chromatogram construction method and an identification method. According to the method, an epimedium villosum medicinal material is pretreated, and the characteristic chromatogram of the epimedium villosum is identified by means of a chromatographic analysis technology; the epimedium villosum and the planted epimedium villosum are distinguished through the relative peak area and the characteristic peak of the chromatographic peak, so that the technical problem that the wild epimedium villosum and the planted epimedium villosum cannot be distinguished by the existing methods such as appearance identification, thin-layer identification and DNA identification is solved, and the correct source of the wild epimedium villosum is effectively ensured; the method has the advantages of simple identification method and accurate result.

Description

Technical Field

[0001] The present invention relates to the technical field of drug analysis, and in particular to a characteristic spectrum of Epimedium pubescens, a characteristic spectrum construction method and an identification method. Background Art

[0002] There are many sources of Epimedium for medicinal use. The Flora of China published in 2001 recorded more than 40 species of Epimedium in China. At present, the 2020 edition of the Chinese Pharmacopoeia includes four sources of Epimedium, which are the dried leaves of Epimedium brevicomu Maxim., Epimedium sagittatum (Sieb. et Zucc.) Maxim., Epimedium pubescens Maxim. or Epimedium koreanum Nakai. They are harvested in summer and autumn when the stems and leaves are lush, and dried in the sun or in the shade.

[0003] The production areas of Epimedium are widely distributed, and the production areas of Epimedium of each origin are relatively fixed.

[0004] Epimedium can be propagated through two methods: rhizomes and seeds. Due to the low yield of artificial cultivation and the lack of scale, most of the original Epimedium medicinal materials currently circulating in the market come from wild resources. The content of icariin in Epimedium pubescens medicinal materials harvested at different times varies greatly.

[0005] There is a big difference in the yield between wild and cultivated Epimedium pubescens. The annual output of wild Epimedium pubescens is over 2,000 tons nationwide, while the annual output of cultivated Epimedium pubescens is around 200 tons. At present, wild Epimedium pubescens is one of the mainstream varieties of Epimedium medicinal materials, with a market share of 39%. If harvested properly, wild Epimedium pubescens can meet basic needs.

[0006] After more than a year of cultivation, the "Guiyi No. 1" high-yield epimedium cultivation test site in Shawan District, Leshan City, Sichuan Province has jointly cultivated high-quality epimedium varieties independently, which have the advantages of higher yield, shorter maturity cycle and higher content. It is in a leading position in the domestic epimedium industry. The current annual output of epimedium cultivation in the country is about 200 tons.

[0007] There are several problems at present: (1) The technical problem that the wild and cultivated Epimedium pubescens cannot be distinguished by appearance identification, thin layer identification and DNA identification; (2) There are great differences between wild and cultivated Epimedium pubescens in terms of yield, content and chromatographic performance; (3) From the perspective of market demand, the prospects for Chinese herbal medicine cultivation are very broad. However, Chinese herbal medicine cultivation also faces some challenges and problems. For example, the quality and safety of Chinese medicinal materials have always been one of the bottlenecks restricting its development. The traditional clinical medication habit is to use wild medicinal materials. Modern pharmacological research believes that the total flavonoids of wild Epimedium pubescens have the effects of promoting immune regulation, anti-osteoporosis, anti-myocardial hypoxia, improving cardiac blood rheology, promoting male reproductive system and reproductive endocrine function, anti-inflammatory, anti-tumor, and anti-aging. The safety, dosage and pharmacological effects of cultivated Epimedium pubescens have not been studied in depth. Therefore, a method for distinguishing Epimedium pubescens from cultivated Epimedium pubescens is very necessary when screening and using medicinal materials. Summary of the invention

[0008] In view of this, the present invention proposes a method for distinguishing wild Epimedium pubescens and cultivated Epimedium pubescens, in order to solve the problem of difficulty in distinguishing wild Epimedium pubescens from cultivated Epimedium pubescens.

[0009] The technical solution of the present invention is achieved as follows: The present invention provides a method for constructing a characteristic spectrum of Epimedium pubescens, comprising the following steps:

[0010] Step 1, preparation of reference solution: take wild Epimedium pubescens leaves, add water, heat and reflux, filter, evaporate the filtrate to dryness, add ethanol solution to dissolve the residue, filter, take the filtrate to obtain a medicinal material reference solution; take epimedin C, icariin, icariin-3-O-rhamnoside reference substances, add methanol to dissolve to obtain a reference substance solution;

[0011] Step 2, preparation of the test solution: take the leaves of Epimedium pubescens, crush and sieve, add water, heat and reflux, filter, evaporate the filtrate to dryness, add ethanol solution to dissolve the residue, filter, and take the filtrate to obtain the test solution;

[0012] Step 3, spectrum construction: inject the Chinese medicinal material reference solution and the reference substance solution in step 1 and the test solution in step 2 into a high performance liquid chromatograph for detection to construct a characteristic spectrum of Epimedium pubescens or its preparation.

[0013] In some embodiments, in step one, the method for preparing the medicinal material reference solution includes: taking 0.2 g of wild Epimedium pubescens, placing it in a stoppered conical flask, adding 20 ml of water, heating and refluxing for 60 min, shaking, filtering, evaporating the filtrate to dryness, adding 5 ml of dilute ethanol to dissolve the residue, shaking, filtering, and taking the filtrate as a control medicinal material reference solution.

[0014] In some embodiments, in step one, the method for preparing the reference substance solution includes: taking appropriate amounts of epimedin C, icariin, and icariin-3-O-rhamnoside reference substances, accurately weighing them, and adding methanol to prepare a solution containing 200 μg of epimedin C, 100 μg of icariin, and 50 μg of icariin-3-O-rhamnoside per 1 ml, respectively, as the reference substance solution.

[0015] In some embodiments, the method for preparing the test solution includes: taking Epimedium pubescentis leaves, crushing them through a No. 3 sieve, taking 0.2 g, accurately weighing them, placing them in a stoppered conical flask, adding 20 ml of water, heating and refluxing for 60 min, shaking well, filtering, evaporating the filtrate to dryness, adding 5 ml of dilute ethanol to the residue to dissolve it, shaking well, filtering, and taking the filtrate to obtain the solution.

[0016] In some embodiments, in step three, the chromatographic conditions of the high performance liquid chromatograph include: using octadecylsilane bonded silica gel as a filler; using acetonitrile as mobile phase A, water as mobile phase B, and gradient elution; and the detection wavelength is 270 nm.

[0017] In some embodiments, in step three, the gradient elution process is:

[0018] 0-20min, mobile phase A is 15-28%, mobile phase B is 85-72%;

[0019] 20-40min, mobile phase A is 28-29%, mobile phase B is 72-71%;

[0020] 40-80min, mobile phase A was 29-58%, mobile phase B was 71-42%.

[0021] In some embodiments, in the chromatographic analysis conditions, the theoretical plate number calculated based on the icariin peak should be no less than 6000.

[0022] In the second aspect, the present invention also provides a characteristic spectrum of Epimedium pubescens, which comprises 13 characteristic peaks, and the 13 characteristic peaks include: characteristic peaks of hyperoside, epimedin A, epimedin B, epimedin C, icariin, 6"'-deoxy-4"'-carbonyl-β-D-allosyl icariin, baouoside VII, icariin I, 2"-O-rhamnosyl icariin II, and baouoside I.

[0023] In a third aspect, the present invention further provides a method for identifying Epimedium pubescens, comprising the following steps:

[0024] Step 1, preparation of the test solution: take the leaves of Epimedium pubescens to be tested, crush and sieve, add water, heat and reflux, filter, evaporate the filtrate to dryness, add ethanol solution to dissolve the residue, filter, and take the filtrate to obtain the test solution;

[0025] Step 2: inject the test solution into a high performance liquid chromatograph for detection to obtain a corresponding spectrum to be identified;

[0026] Step 3: Compare the spectrum to be identified with the characteristic spectrum constructed by the method described in claim 1 to determine whether it is wild Epimedium pubescentis.

[0027] In some embodiments, the judgment method includes: if there are 13 characteristic peaks and the relative peak area of ​​epimedin C to icariin is between 1.0-2.5, it is wild Epimedium pubescens; if there are only 12 characteristic peaks and the relative peak area of ​​epimedin C to icariin is between 0.20-0.25, it is cultivated Epimedium pubescens.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The present invention uses chromatography analysis technology to distinguish between Epimedium pubescens and cultivated Epimedium pubescens through the relative peak area and characteristic peaks of the chromatographic peaks, thereby solving the technical problem that existing methods such as appearance identification, thin layer identification and DNA identification cannot distinguish between wild and cultivated Epimedium pubescens, effectively ensuring the correct source of wild Epimedium pubescens, and having the advantages of simple identification method and accurate result. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0031] Figure 1 This is the characteristic spectrum of the wild Epimedium pubescens of the present invention;

[0032] Figure 2 The characteristic spectrum of the present invention for planting Epimedium pubescens;

[0033] Figure 3 This is a comparison diagram of the characteristic spectra of the cultivated Epimedium pubescens of the present invention and the wild Epimedium pubescens;

[0034] Figure 4 This is a comparison chart of the characteristic spectra of 10 batches of cultivated Epimedium pubescens and wild Epimedium pubescens reference substances in Example 3;

[0035] Figure 5 This is a comparison chart of the characteristic spectra of 25 batches of wild Epimedium pubescens in Example 2;

[0036] Figure 6 This is a diagram of the characteristics of wild Epimedium pubescens;

[0037] Figure 7 To plant the characteristics of Epimedium pubescens;

[0038] Figure 8 This is a micrograph of wild Epimedium pubescens;

[0039] Fig. 9 This is a micrograph of a plant of Epimedium pubescens;

[0040] Fig.10 This is a DNA difference map between cultivated Epimedium pubescens (EP(C)) and wild Epimedium pubescens (EP(W)). DETAILED DESCRIPTION

[0041] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the embodiments of the present invention belong. If the definitions set forth in this section are contrary to or otherwise inconsistent with the definitions set forth in the patents, patent applications, published patent applications, and other publications incorporated herein by reference, the definitions listed in this section take precedence over the definitions incorporated herein by reference.

[0043] Unless otherwise specified, the methods used in the following examples are all conventional methods. Unless otherwise specified, the materials, reagents and instruments used are all conventional materials, reagents and instruments in the art, and can be obtained by those skilled in the art through commercial channels.

[0044] Example 1

[0045] This embodiment relates to the construction of a characteristic spectrum of Epimedium pubescens, which specifically includes the following steps:

[0046] Take 0.2g of wild Epimedium pubescens medicinal material, place it in a stoppered conical bottle, add 20ml of water, heat and reflux for 60 minutes, shake well, filter, evaporate the filtrate, dissolve the residue in 5ml of dilute ethanol, shake well, filter, and take the filtrate as the medicinal material reference solution. Separately take 20mg of epimedin C, 10mg of icariin, and 5mg of icariin-3-O-rhamnoside, accurately weigh them in 100ml, add methanol to dissolve and make up to volume as the reference solution.

[0047] Take the collected leaves of Epimedium pubescens, crush them through a No. 3 sieve, take out about 0.2g, weigh them accurately, put them in a stoppered conical flask, add 20ml of water, heat and reflux for 60 minutes, shake well, filter, evaporate the filtrate to dryness, dissolve the residue in 5ml of dilute ethanol, shake well, filter, and take the filtrate to obtain the test solution.

[0048] The reference solution and the epimedium solution were analyzed by HPLC respectively. The chromatographic analysis conditions were as follows: octadecylsilane bonded silica gel was used as the filler; acetonitrile-water was used as the mobile phase for gradient elution as specified, and the elution gradient was as shown in the table below; the column temperature was 30°C, and the detection wavelength was 270nm; at the same time, the theoretical plate number calculated based on the icariin peak should not be less than 6000.

[0049] Time (minutes) Acetonitrile (%) water(%) 0～20 15→28 85→72 20～40 28→29 72→71 40～80 29→58 71→42

[0050] Accurately pipette 10 μL of each of the medicinal material reference solution, reference substance reference solution and test solution, and inject them into the liquid chromatograph.

[0051] Using the medicinal material reference solution and the reference substance reference solution as reference spectra, common peaks were selected from the characteristic spectra of the test solution to construct a characteristic spectrum of Epimedium pubescens. The constructed characteristic spectrum of wild Epimedium pubescens has 13 characteristic peaks, such as Figure 1 As shown, peak 1 was identified as hyperoside, peak 2 as epimedin A, peak 3 as epimedin B, peak 4 as epimedin C, peak 7 as icariin, peak 8 as 6"'-deoxy-4"'-carbonyl-β-D-allosyl icariin, peak 9 as baouoside VII, peak 10 as icariin I, peak 11 as 2"-O-rhamnosyl icariin II, and peak 13 as baouoside I.

[0052] The characteristic map of Epimedium pubescens planted is constructed as follows Figure 2 As shown, there is one more characteristic peak compared to the characteristic spectrum of wild Epimedium pubescens, which is identified as the characteristic peak of icariin-3-O-rhamnoside.

[0053] The comparison of the characteristic spectrum of cultivated Epimedium pubescens and wild Epimedium pubescens is shown in the figure below. Figure 3 shown.

[0054] The map construction method in the above embodiments can also be applied to other preparations of Epimedium pubescens.

[0055] Example 2

[0056] This embodiment relates to the identification of wild Epimedium pubescens, and the specific steps are as follows:

[0057] Take the leaves of Epimedium pubescentis to be tested, crush them through a No. 3 sieve, take out about 0.2g, weigh them accurately, put them in a stoppered conical flask, add 20ml of water, heat and reflux for 60 minutes, shake well, filter, evaporate the filtrate to dryness, dissolve the residue in 5ml of dilute ethanol, shake well, filter, and take the filtrate to obtain the test solution.

[0058] The test solution was subjected to high performance liquid chromatography analysis under the same analysis conditions as in Example 1.

[0059] The leaves of Epimedium pubescens to be tested were taken from 25 batches of different origins, as shown in the following table:

[0060]

[0061]

[0062] The relative peak areas of epimedin C and icariin in the above different batches of Epimedium pubescens were between 1.0-2.5, and there were 13 characteristic peaks, so 25 batches of Epimedium pubescens were determined to be wild Epimedium pubescens.

[0063] The detection profiles of the above 25 batches of Epimedium pubescens are as follows: Figure 5 shown.

[0064] Example 3

[0065] This embodiment relates to the identification of planting Epimedium pubescens, and the specific steps are as follows:

[0066] Take the leaves of Epimedium pubescentis to be tested, crush them through a No. 3 sieve, take out about 0.2g, weigh them accurately, put them in a stoppered conical flask, add 20ml of water, heat and reflux for 60 minutes, shake well, filter, evaporate the filtrate to dryness, dissolve the residue in 5ml of dilute ethanol, shake well, filter, and take the filtrate to obtain the test solution.

[0067] The test solution was subjected to high performance liquid chromatography analysis under the same analysis conditions as in Example 1.

[0068] The leaves of Epimedium pubescens to be tested were taken from 10 batches of artificially grown products from different origins, as shown in the following table:

[0069]

[0070]

[0071] The relative peak areas of epimedin C and icariin in 10 batches of Epimedium pubescens were between 0.20 and 0.25, and there were 12 characteristic peaks, and one more characteristic peak, icariin-3-O-rhamnoside, was found. The 10 batches of Epimedium pubescens were identified as cultivated Epimedium pubescens. Figure 4 shown.

[0072] Comparative Example 1

[0073] This comparative example adopts property identification:

[0074] Epimedium pubescens has a three-part compound leaf; the lower surface of the leaf and the petiole are densely covered with velvety soft hairs. Figure 6 As shown in the figure, the characteristics of the medicinal material of Epimedium pubescens are as follows Figure 7 As shown, it is impossible to distinguish between Epimedium pubescens and cultivated Epimedium pubescens.

[0075] Comparative Example 2

[0076] Microscopic identification, the surface of the leaves of Epimedium pubescens: the stomata of the lower epidermis are sparse, with many slender non-glandular hairs. The characteristics of wild Epimedium pubescens are shown in the figure below. Figure 8 As shown in the figure, the characteristics of the medicinal material of Epimedium pubescens are as follows Fig. 9 As shown, it is impossible to distinguish between Epimedium pubescens and cultivated Epimedium pubescens.

[0077] Comparative Example 3

[0078] DNA identification showed that the difference sites of chloroplast SSR (Simple Sequence Repeats) and SNP (Single Nucleotide Polymorphisms) of Epimedium pubescens were very few and had no obvious pattern. Fig.10 As shown, it is impossible to distinguish between Epimedium pubescens and cultivated Epimedium pubescens.

[0079] It can be seen from the embodiments and comparative experiments that, with the help of chromatographic analysis technology, the relative peak area and characteristic peaks of the chromatographic peaks are used to distinguish between Epimedium pubescens and cultivated Epimedium pubescens, which solves the technical problem that the existing appearance identification, thin layer identification and DNA identification methods cannot distinguish between wild and cultivated Epimedium pubescens, effectively ensures the correct source of wild Epimedium pubescens, and has the advantages of simple identification method and accurate results.

[0080] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for constructing a characteristic spectrum of Epimedium pubescens, characterized in that: The steps include: Step 1, preparation of reference solution: take wild Epimedium pubescens leaves, add water, heat and reflux, filter, evaporate the filtrate to dryness, add ethanol solution to dissolve the residue, filter, take the filtrate to obtain a medicinal material reference solution; take epimedin C, icariin, icariin-3-O-rhamnoside reference substances, add methanol to dissolve to obtain a reference substance solution; Step 2, preparation of the test solution: take the leaves of Epimedium pubescens, crush and sieve, add water, heat and reflux, filter, evaporate the filtrate to dryness, add ethanol solution to dissolve the residue, filter, and take the filtrate to obtain the test solution; Step 3, spectrum construction: inject the Chinese medicinal material reference solution and the reference substance solution in step 1 and the test solution in step 2 into a high performance liquid chromatograph for detection to construct a characteristic spectrum of Epimedium pubescens or its preparation.

2. The method for constructing a characteristic spectrum of Epimedium pubescens according to claim 1, characterized in that: In step 1, the preparation method of the medicinal material reference solution includes: taking 0.2 g of wild Epimedium pubescens, placing it in a stoppered conical flask, adding 20 ml of water, heating and refluxing for 60 minutes, shaking, filtering, evaporating the filtrate to dryness, adding 5 ml of dilute ethanol to dissolve the residue, shaking, filtering, and taking the filtrate as the control medicinal material reference solution.

3. The method for constructing a characteristic spectrum of Epimedium pubescens according to claim 1, characterized in that: In step 1, the preparation method of the reference substance solution includes: taking appropriate amounts of epimedin C, icariin, and icariin-3-O-rhamnoside reference substances, accurately weighing them, and adding methanol to prepare a solution containing 200 μg of epimedin C, 100 μg of icariin, and 50 μg of icariin-3-O-rhamnoside per 1 ml, respectively, as the reference substance solution.

4. The method for constructing a characteristic spectrum of Epimedium pubescens according to claim 1, characterized in that: The preparation method of the test solution comprises: taking leaves of Epimedium pubescens, crushing them through a No. 3 sieve, taking 0.2 g, accurately weighing them, placing them in a stoppered conical flask, adding 20 ml of water, heating and refluxing for 60 minutes, shaking them well, filtering them, evaporating the filtrate to dryness, dissolving the residue in 5 ml of dilute ethanol, shaking them well, filtering them, and taking the filtrate to obtain the solution.

5. The method for constructing a characteristic spectrum of Epimedium pubescens according to claim 1, characterized in that: In step three, the chromatographic conditions of the high performance liquid chromatograph include: using octadecylsilane bonded silica gel as a filler; using acetonitrile as mobile phase A, water as mobile phase B, and gradient elution; and the detection wavelength is 270 nm.

6. The method for constructing a characteristic spectrum of Epimedium pubescens according to claim 4, characterized in that: In step 3, the gradient elution process is: 0-20min, mobile phase A is 15-28%, mobile phase B is 85-72%; 20-40min, mobile phase A is 28-29%, mobile phase B is 72-71%; 40-80min, mobile phase A was 29-58%, mobile phase B was 71-42%.

7. The method for constructing a characteristic spectrum of Epimedium pubescens according to claim 5, characterized in that: In the chromatographic analysis conditions, the theoretical plate number calculated based on the icariin peak should be no less than 6000.

8. The characteristic spectrum of Epimedium pubescens constructed by the construction method described in any one of claims 1 to 7, characterized in that: The characteristic spectrum includes 13 characteristic peaks, and the 13 characteristic peaks include: characteristic peaks of hyperoside, epimedin A, epimedin B, epimedin C, icariin, 6"'-deoxy-4"'-carbonyl-β-D-allosyl icariin, baohuoside VII, icariin I, 2"-O-rhamnosyl icariin II, and baohuoside I.

9. A method for identifying Epimedium pubescens, characterized in that: The following steps are involved: Step 1, preparation of the test solution: take the leaves of Epimedium pubescens to be tested, crush and sieve, add water, heat and reflux, filter, evaporate the filtrate to dryness, add ethanol solution to dissolve the residue, filter, and take the filtrate to obtain the test solution; Step 2: inject the test solution into a high performance liquid chromatograph for detection to obtain a corresponding spectrum to be identified; Step 3: Compare the spectrum to be identified with the characteristic spectrum constructed by the method described in claim 1 to determine whether it is wild Epimedium pubescentis.

10. The method for identifying Epimedium pubescens according to claim 9, characterized in that: In step three, the judgment method includes: if there are 13 characteristic peaks and the relative peak area of ​​epimedin C and icariin is between 1.0-2.5, it is wild Epimedium pubescens; if there are only 12 characteristic peaks and the relative peak area of ​​epimedin C and icariin is between 0.20-0.25, it is cultivated Epimedium pubescens.

Images