A method for constructing characteristic maps of Siegesbeckia sibiricum medicinal preparations of different origins

Abstract

The present invention belongs to the technical field of traditional Chinese medicine detection, and specifically relates to a method for constructing characteristic spectra of Siegesbeckia herba medicinal preparations of different origins. By adopting a specific test solution preparation method and matching chromatographic conditions, complete separation of 14 to 15 common characteristic peaks, including caffeic acid, 3,7-di-O-methylquercetin and isochlorogenic acid C, is achieved, with good peak shape, stable baseline, and short detection time. Characteristic spectra of Siegesbeckia herba medicinal preparations of different origins are constructed, fully demonstrating the chemical component characteristics of Siegesbeckia herba of different origins, providing a basis for identifying and distinguishing Siegesbeckia herba medicinal preparations of different origins. The method is stable, has high precision, good stability, good reproducibility, strong characteristicity, and rich chromatographic information.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a method for constructing characteristic spectra of Siegesbeckia sibiricum medicinal preparations of different origins. Background Art

[0002] Siegesbeckia orientalis L., Siegesbeckia pubescens Makino, or Siegesbeckia glabrescens Makino, all of the Asteraceae family. Siegesbeckia (Siegesbeckia) formula granules have anti-rheumatic, joint-tonifying, and detoxifying properties. They are commonly used clinically to treat rheumatic pain, weakness in the muscles and bones, soreness of the waist and knees, paralysis of the limbs, hemiplegia, and urticaria. The 2020 edition of the Chinese Pharmacopoeia specifies that the original plant of Siegesbeckia is Siegesbeckia orientalis L., Siegesbeckia pubescens Makino, or Siegesbeckia glabrescens Makino. The herbal materials of Siegesbeckia scoparia based on Siegesbeckia scoparia and Siegesbeckia glandularis have great differences in the content of active ingredients and efficacy. The properties of Siegesbeckia scoparia slices from different sources are relatively similar, and inexperienced personnel cannot accurately identify them. The 2020 edition of the Chinese Pharmacopoeia does not distinguish the sources in the identification description of Siegesbeckia scoparia, making it difficult to ensure the accuracy of the medicine. This method cannot be applied to the standard decoction of Siegesbeckia scoparia or the formula granules of Siegesbeckia scoparia based on Siegesbeckia scoparia and Siegesbeckia glandularis.

[0003] Chinese herbal formula granules and standard decoctions are common dosage forms of Herba Siegesbeckiae. Among them, Chinese herbal formula granules are extracted from Chinese herbal medicine slices with water and produced through extraction, drying, granulation and other processes. Their clinical efficacy should be consistent with that of the decoction. The standard decoction is a material benchmark for measuring whether Chinese herbal formula granules are basically consistent with the decoction. However, both standard decoctions and Chinese herbal granules have lost the identification characteristics of the original medicinal materials, that is, they cannot be inspected and identified from the shape, size, texture, etc. of the medicinal materials. At present, there are no reports on the characteristic spectrum of Herba Siegesbeckiae formula granules. Since Chinese herbal formula granules no longer have the characteristics of medicinal material identification, the identification method of the medicinal material is not suitable for the identification of preparations made from Herba Siegesbeckiae, such as Herba Siegesbeckiae formula granules. Summary of the Invention

[0004] Therefore, the present invention solves the problem that the methods in the prior art cannot accurately identify Siegesbeckia medicinal preparations based on Siegesbeckia, Siegesbeckia glandularis, and Siegesbeckia hairy stem. It provides a method for constructing characteristic spectra of Siegesbeckia medicinal preparations with different origins, establishes characteristic spectra of Siegesbeckia medicinal preparations with different origins, identifies and distinguishes Siegesbeckia medicinal preparations based on Siegesbeckia, Siegesbeckia glandularis, and Siegesbeckia hairy stem, and provides a rapid and reliable detection method for interspecies identification of Siegesbeckia medicinal preparations.

[0005] The present invention provides a method for constructing characteristic spectra of Siegesbeckia sibiricum medicinal preparations of different origins, comprising the following steps:

[0006] (1) Preparation of test solution: Weigh the test sample, extract with water to obtain an extract, separate the solid and liquid, take the liquid, extract with ethyl acetate, take the ethyl acetate solution, dry, add a solvent to dissolve, separate the solid and liquid, and take the liquid to obtain the test solution;

[0007] (2) The sample solution was detected by ultra-high performance liquid chromatography using octadecylsilane bonded silica gel as the filler, the mobile phase comprising acetonitrile and aqueous phosphoric acid solution, the gradient elution program comprising: 0 → 6 min → 11 min → 21 min → 41 min → 49 min → 55 min, and the volume percentage of acetonitrile in the mobile phase was: 5% → 8% → 11% → 15% → 24% → 49% → 83%.

[0008] Furthermore, step (2) also satisfies at least one of the following 1)-3):

[0009] 1) Flow rate: 0.29-0.31 mL / min, column temperature: 28-32°C;

[0010] 2) The volume percentage of phosphoric acid in the phosphoric acid aqueous solution is 0.08-0.12%;

[0011] 3) The detection wavelength is 325nm-335nm.

[0012] Furthermore, step (1) further satisfies any one or more of the following AG:

[0013] A. The ratio of the mass of the test sample to the volume of water is 0.5-2:30; the relationship between mass and volume is g / mL;

[0014] B. The extraction method is reflux extraction or ultrasonic extraction;

[0015] C. Extraction time is ≥20 min, preferably 30-120 min;

[0016] D. The solid-liquid separation is selected from centrifugation or membrane filtration;

[0017] E. Extract 1 to 3 times with ethyl acetate, with the ratio of the mass of the test sample to the volume of ethyl acetate each time being 0.5-2:45; the relationship between mass and volume is g / mL;

[0018] F. The ratio of the mass of the test sample to the volume of the solution after adding the solvent is 0.5-2:2; the relationship between mass and volume is g / mL;

[0019] G. The solvent is methanol or methanol-water solution.

[0020] Furthermore, the construction method further includes the step of preparing a reference solution using at least one of caffeic acid, 3,7-di-O-methylquercetin and isochlorogenic acid C as a solubilizer, and the step of detecting the reference solution by ultra-performance liquid chromatography according to any of the above construction methods to obtain a reference spectrum.

[0021] Furthermore, each 1 mL of the reference solution contains at least one of 1-100 μg of caffeic acid, 1-100 μg of 3,7-di-O-methylquercetin, and 1-100 μg of isochlorogenic acid C; and / or the solvent used in the preparation of the reference solution is selected from methanol or methanol-water solution.

[0022] Furthermore, the construction method also includes the steps of using the extract obtained by water extraction of the Herba Siegesbeckiae control medicinal material to prepare a control medicinal material reference solution according to the preparation method of the test solution in any of the above-mentioned construction methods, and detecting the control medicinal material reference solution by ultra-high performance liquid chromatography according to any of the above-mentioned construction methods to obtain a reference medicinal material reference atlas. Preferably, after the Herba Siegesbeckiae control medicinal material is extracted with water, it is filtered, the liquid is dried, and then the control medicinal material reference solution is prepared according to the preparation method of the test solution in the above-mentioned construction method.

[0023] Furthermore, the Siegesbeckia medicinal preparations of different origins are Siegesbeckia medicinal preparations of Siegesbeckia origin, Siegesbeckia glandularis origin, or Siegesbeckia hairy stem origin.

[0024] Furthermore, the characteristic spectrum of the Siegesbeckia herba medicinal preparation of the Siegesbeckia herba has 14 characteristic peaks, peak 2 corresponds to the peak of the caffeic acid reference substance, peak 2 is the S peak, and the relative retention times of peaks 1 to peak 14 and the S peak are within the range of ±10% of the specified values, and the specified values ​​of peaks 1 to peak 14 are: 0.60, 1.00, 1.51, 1.59, 1.87, 2.27, 2.49, 2.70, 3.72, 4.12, 4.17, 5.20, 5.72, and 6.96, respectively;

[0025] The characteristic spectrum of the Siegesbeckia herba medicinal preparation of the Siegesbeckia glandularis origin has 15 characteristic peaks, peak 2 corresponds to the peak of the caffeic acid reference substance, peak 2 is taken as the S peak, and the relative retention times of peaks 1 to 5, peak α, and peaks 6 to 14 and the S peak are within the range of ±10% of the specified values, and the specified values ​​of peaks 1 to 15 are: 0.60, 1.00, 1.51, 1.59, 1.87, 2.10, 2.27, 2.49, 2.70, 3.72, 4.12, 4.17, 5.20, 5.72, and 6.96, respectively;

[0026] The characteristic spectrum of the Siegesbeckia herba medicinal preparation derived from the hairy stem Siegesbeckia herba has 15 characteristic peaks, peak 2 corresponds to the peak of the caffeic acid reference substance, peak 2 is taken as the S peak, and the relative retention times of peak 1, peak β, peak 2-peak 14 and the S peak are within the range of ±10% of the specified value, and the specified values ​​of peaks 1-peak 15 are: 0.60, 0.93, 1.00, 1.51, 1.59, 1.87, 2.27, 2.49, 2.70, 3.72, 4.12, 4.17, 5.20, 5.72, and 6.96, respectively.

[0027] The present invention also provides a method for identifying Siegesbeckia medicinal preparations of different origins, comprising the steps of constructing a characteristic spectrum of the Siegesbeckia product according to any of the above-mentioned construction methods.

[0028] The present invention also provides a method for quality detection of Siegesbeckia medicinal preparations of different origins, comprising the steps of constructing a characteristic spectrum of the Siegesbeckia product according to any of the above-mentioned construction methods.

[0029] The technical solution of the present invention has the following advantages:

[0030] 1. The method for constructing characteristic profiles of Siegesbeckia sibiricum medicinal preparations of different origins provided by the present invention is used in combination with a specific test solution preparation method and chromatographic conditions. Specifically, the test solution is prepared by the following method: preparation of the test solution: weighing the test sample, adding water to extract to obtain an extract, solid-liquid separation, taking the liquid, extracting with ethyl acetate, taking the ethyl acetate solution, drying, adding methanol to dissolve, solid-liquid separation, taking the liquid, and obtaining the test solution; and the following chromatographic conditions: using octadecylsilane bonded silica as a filler, the mobile phase includes acetonitrile and phosphoric acid aqueous solution, and the gradient elution program includes: 0→6min→11min→21mi n→41min→49min→55min, the volume percentage of acetonitrile in the mobile phase was: 5%→8%→11%→15%→24%→49%→83%, achieving complete separation of 14-15 common characteristic peaks including caffeic acid, 3,7-di-O-methylquercetin and isochlorogenic acid C, with good peak shape, stable baseline and short detection time. Characteristic profiles of Siegesbeckia medicinal preparations of different origins were constructed, fully demonstrating the chemical composition characteristics of Siegesbeckia medicinal preparations of different origins, providing a basis for the identification and differentiation of Siegesbeckia medicinal preparations of different origins. The method is stable, with high precision, good stability, good reproducibility, strong characteristicity and rich chromatographic information.

[0031] 2. The identification method of different Siegesbeckia medicinal preparations provided by the present invention can be used to identify and distinguish two formula granules with different Siegesbeckia origins, providing a rapid and reliable detection method for the identification of the origin of Siegesbeckia (Siegesbeckia) formula granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0033] Figure 1 The characteristic spectra and control characteristic spectra of 18 batches of Siegesbeckia sibiricum (Siegesbeckia sibiricum) formula granules in Example 1, wherein S1(14)-S18(14) are the characteristic spectra of 18 batches of Siegesbeckia sibiricum (Siegesbeckia sibiricum) formula granules, and R(14) is the control characteristic spectra;

[0034] Figure 2 This is the reference characteristic spectrum of Siegesbeckia sibiricum (Siegesbeckia sibiricum) formula granules;

[0035] Figure 3 Characteristic spectra of three batches of Siegesbeckia sibiricum (Schizonepeta adenophora) granules;

[0036] Figure 4 This is the reference characteristic spectrum of Siegesbeckia sibiricum (Schizonepeta adenophora) formula granules;

[0037] Figure 5 This is a comparison of the characteristic spectra of three types of Siegesbeckia sibiricum formula granules;

[0038] Figure 6 The chromatograms obtained under gradient conditions 1 and 2 in Experimental Example 1 are shown in order from bottom to top: gradient conditions 1 and 2;

[0039] Figure 7 This is the chromatogram obtained under gradient condition 3 in Experimental Example 1;

[0040] Figure 8 is the PDA spectrum in Experimental Example 1;

[0041] Figure 9 This is the chromatogram of the test solution and mixed reference solution of Siegesbeckia sibiricum (Siegesbeckia sibiricum) formula granules in Experimental Example 1. From top to bottom, it is the test solution and mixed reference solution of Siegesbeckia sibiricum (Siegesbeckia sibiricum) formula granules;

[0042] Figure 10 Characteristic spectra of the Herba Siegesbeckiae (Siegesbeckia) formula granules and Herba Siegesbeckiae (Schizonepeta adenophorae) formula granules obtained in Comparative Example 1;

[0043] Figure 11 This is the characteristic spectrum of the Siegesbeckia sibiricum (Siegesbeckia sibiricum) formula granules and Siegesbeckia sibiricum (Siegesbeckia sibiricum) formula granules obtained in Comparative Example 2. DETAILED DESCRIPTION

[0044] The following examples are provided to better further understand the present invention, but are not limited to the best mode of implementation, and do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the enlightenment of the present invention or by combining the features of the present invention with other prior arts falls within the scope of protection of the present invention. If the specific experimental steps or conditions are not specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional reagent products that can be obtained commercially. Siegesbeckia (Siegesbeckia) represents the Siegesbeckia of the Siegesbeckia origin, and the same applies to the other Siegesbeckia (Glandular Siegesbeckia) and Siegesbeckia (Hairy Stem Siegesbeckia). The base is indicated in brackets.

[0045] The pharmaceutical preparation of Siegesbeckia serrata described in the present invention is prepared by the following method: taking Siegesbeckia serrata, heating and refluxing extraction at least once, adding 6 to 12 times the weight of water each time for at least 0.5 hours, filtering, combining the filtrates, concentrating the filtrates to a relative density of 1.05-1.10 g / mL at 60°C, adding conventional excipients, and preparing clinically acceptable tablets, capsules, pills, granules, honey-refined pills, sustained-release preparations, immediate-release preparations, controlled-release preparations, oral liquid preparations or injection preparations according to conventional processes. The pharmaceutically acceptable excipients include fillers, disintegrants, lubricants, suspending agents, binders, sweeteners, flavoring agents, preservatives, bases, etc. Fillers include: starch, pregelatinized starch, lactose, mannitol, chitin, microcrystalline cellulose, sucrose, etc.; disintegrants include: starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone, low-substituted hydroxypropyl cellulose, cross-linked sodium carboxymethyl cellulose, etc.; lubricants include: magnesium stearate, sodium lauryl sulfate, talc, silicon dioxide, etc.; suspending agents include: polyvinyl pyrrolidone, microcrystalline cellulose, sucrose, agar, hydroxypropyl methylcellulose, etc.; binders include: starch slurry, polyvinyl pyrrolidone, hydroxypropyl methylcellulose, etc.; sweeteners include: sodium saccharin, aspartame, sucrose, cyclamate, glycyrrhetinic acid, etc.; flavorings include: sweeteners and various flavors; preservatives include: parabens, benzoic acid, sodium benzoate, sorbic acid and its salts, benzalkonium bromide, chloroethidine acetate, eucalyptus oil, etc.; matrices include: PEG6000, PEG4000, insect wax, etc.

[0046] The test sample in this example is Siegesbeckia sibiricum granules. The specific preparation method of the Siegesbeckia sibiricum granules is as follows: 5000 g of Siegesbeckia sibiricum slices are decocted with water, filtered, and the filtrate is concentrated into a clear paste (the dry extract yield is 13% to 20%). An appropriate amount of auxiliary materials is added, dried, and an appropriate amount of auxiliary materials is added again, mixed, and granulated to obtain 1000 g. Twenty-one batches of Siegesbeckia sibiricum granules of different origins were prepared in the present invention. The production areas are shown in the table below.

[0047] Table 1 21 batches of Siegesbeckia scoparia granules with different origins

[0048]

[0049]

[0050] Example 1

[0051] This embodiment provides a method for constructing a UPLC characteristic spectrum of Siegesbeckia sibiricum formula granules and a method for identifying the same, comprising the following steps:

[0052] Preparation of reference solution: Take 2.0 g of Siegesbeckia sibiricum (Siegesbeckia sibiricum) as a control medicinal material, add 30 mL of water, boil for 30 minutes, filter, and prepare the reference medicinal material solution in the same manner as the test sample. Take appropriate amounts of caffeic acid, isochlorogenic acid C, and 3,7-di-0-methylquercetin reference substances and add methanol to prepare a mixed control solution containing 45 μg of caffeic acid, 40 μg of isochlorogenic acid C, and 60 μg of 3,7-di-0-methylquercetin per 1 mL. This will serve as the reference solution.

[0053] Preparation of test solution: Take an appropriate amount of the test sample, grind it into powder, take about 1.0 g, place it in a stoppered conical flask, add 30 mL of water, ultrasonically treat for 30 minutes, cool, filter, and extract twice with ethyl acetate, 45 mL each time, combine the ethyl acetate, evaporate to dryness, dissolve the residue in methanol and make up to 2 mL, shake well, filter, and take the filtrate.

[0054] Determination method: Accurately pipette 1 μl of each reference solution and test solution into an ultra-high performance liquid chromatograph for determination. Chromatographic conditions are as follows: octadecylsilane bonded silica gel as the filler (column length, 100 mm, inner diameter, 2.1 mm, particle size, 1.6 μm); acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution as specified in the table below; flow rate, 0.3 ml / min; detection wavelength, 330 nm; column temperature, 30°C; theoretical plate number, calculated based on the caffeic acid peak, should be no less than 49,000.

[0055]

[0056] Example 2

[0057] 18 batches of Herba Siegesbeckiae (Siegesbeckia) granules were used as test samples. Characteristic spectra of 18 Herba Siegesbeckiae (Siegesbeckia) granules were constructed according to the method of Example 1. The spectra were imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" for data matching, and a control characteristic spectrum was established (such as Figure 1-2 shown).

[0058] The control characteristic spectrum of the formula granule sample of Siegesbeckia scoparia (Siegesbeckia) includes 14 common characteristic peaks, among which peak 2 corresponds to caffeic acid; the peak corresponding to the caffeic acid reference peak is the S peak, and the relative retention times of peaks 1 to 14 and the S peak are: 0.60, 1.00, 1.51, 1.59, 1.87, 2.27, 2.49, 2.70, 3.72, 4.12, 4.17, 5.20, 5.72, and 6.96, respectively.

[0059] The characteristic spectra of 18 samples of Siegesbeckia scoparia (Siegesbeckia) formula granules all have 14 characteristic peaks, peak 2 corresponds to the peak of the caffeic acid reference substance, peak 2 is the S peak, and the relative retention times of peaks 1 to 14 and the S peak are within the range of ±10% of the specified values. The specified values ​​of peaks 1 to 14 are: 0.60, 1.00, 1.51, 1.59, 1.87, 2.27, 2.49, 2.70, 3.72, 4.12, 4.17, 5.20, 5.72, and 6.96, respectively.

[0060] Example 3

[0061] Three batches of Herba Siegesbeckiae (Glandular Stem Siegesbeckiae) granules were used as test samples. The characteristic spectra of three Herba Siegesbeckiae (Glandular Stem Siegesbeckiae) granules were constructed according to the method of Example 1. The spectra were imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" for data matching, and a control characteristic spectrum was established (such as Figure 3-4 shown).

[0062] The control characteristic spectrum of the formula granules of Siegesbeckia sibiricum (Siegelia aegypti) includes 15 common characteristic peaks, which are numbered in sequence according to retention time. There is a peak α between peak 5 and peak 6, and peak 2 corresponds to caffeic acid. The peak corresponding to the caffeic acid reference peak is the S peak. The relative retention times of peaks 1 to 5, peak α, and peak 6 to 14 and the S peak are within the range of ±10% of the specified values. The specified values ​​of peaks 1 to 15 are: 0.60, 1.00, 1.51, 1.59, 1.87, 2.10, 2.27, 2.49, 2.70, 3.72, 4.12, 4.17, 5.20, 5.72, and 6.96, respectively.

[0063] The characteristic spectra of the three samples of Siegesbeckia scoparia (Siegelia aegypti) formula granules all have 15 characteristic peaks, which are numbered in sequence according to the retention time. There is a peak α between peak 5 and peak 6, peak 2 corresponds to the peak of the caffeic acid reference substance, and peak 2 is the S peak. The relative retention times of peaks 1 to 5, peak α, and peak 6 to 14 and the S peak are within the range of ±10% of the specified values. The specified values ​​of peaks 1 to 15 are 0.60, 1.00, 1.51, 1.59, 1.87, 2.10, 2.27, 2.49, 2.70, 3.72, 4.12, 4.17, 5.20, 5.72, and 6.96, respectively.

[0064] Example 4

[0065] The characteristic spectrum of the Herba Siegesbeckiae (Hairy Stem Siegesbeckiae) formula granules was constructed according to the method of Example 1. Figure 5 As shown, the characteristic spectrum of the formula granules of Siegesbeckia scoparia (hairy stem Siegesbeckia scoparia) has 15 characteristic peaks, which are numbered in sequence according to the retention time. There is a peak β between peak 1 and peak 2, peak 2 corresponds to the peak of the caffeic acid reference substance, and peak 2 is the S peak. The relative retention times of peak 1, peak β, peak 2-peak 14 and the S peak are within the range of ±10% of the specified value, and the specified values ​​of peak 1-peak 15 are: 0.60, 0.93, 1.00, 1.51, 1.59, 1.87, 2.27, 2.49, 2.70, 3.72, 4.12, 4.17, 5.20, 5.72, and 6.96, respectively.

[0066] Example 5

[0067] Comparison of the characteristic spectra of the three different original formula granule samples constructed in Examples 2-4 revealed that:

[0068] ① Identification is performed based on whether there is an obvious characteristic peak between peak 1 and peak 2 or whether peak β exists in the characteristic spectrum of the granule sample of Siegesbeckia sibiricum (Siegesbeckia sibiricum).

[0069] There is an obvious exclusive peak (peak β) between peak 1 and peak 2 of Siegesbeckia hirsuta, which is different from that of Siegesbeckia protothecoides and Siegesbeckia glandularis. Therefore, whether there is an obvious exclusive peak between peak 1 and peak 2 can identify Siegesbeckia hirsuta. That is, if there is a characteristic peak or peak β between peak 1 and peak 2, it is Siegesbeckia hirsuta, otherwise it is not.

[0070] ② The relative peak areas of peaks 9 and 11 among the 14 characteristic peaks in the characteristic spectrum of the granule sample of Siegesbeckia sibiricum (Siegesbeckia sibiricum) were used as indicators for identification.

[0071] Compared to Siegesbeckia sibiricum, the characteristic peaks of Siegesbeckia sibiricum and Siegesbeckia tricholoma were significantly higher, especially Peaks 9 and 11. The relative peak areas of Peaks 9 and 11 are as follows.

[0072] Table 2 Relative peak area results of peak 9 and peak 11 of 18 batches of Siegesbeckia sibiricum granules

[0073]

[0074]

[0075] After discarding the higher discrete values, the relative peak areas of the identification peaks between batches of Siegesbeckia serrata (Siegesbeckia serrata) were calculated, and the RSD% range was 24.4%-55.3%. Among them, the RSD value of peak 11 was less than 25%, which was relatively stable, and the peak area was larger, making it more accurate as an identification peak.

[0076] For the identification peak of Siegesbeckia serrata (peak 11), peak 9 is the S peak. The measured relative peak area range is 0.651-2.049, with a mean of 1.51 and a mean ± 30% range of 1.05-1.96. The mean ± 3SD range is 0.41-2.62. Peak 9 is the S peak, and the relative peak area of ​​peak 11 must not be less than 0.65 (rounded to the measured lower limit).

[0077] Table 3 Relative peak area results of peak 9 and peak 11 of glandular peduncle base and hairy peduncle base of Siegesbeckia serrata samples

[0078]

[0079] The data showed that the relative peak areas of Peak 11 and Peak 9 for the Adenophora stalk-based Herba Siegesbeckiae Granules and their control, and the Hairy Stem-based Herba Siegesbeckiae Granules and their control ranged from 0.150 to 0.432. That is, if the relative peak area of ​​Peak 11 to Peak 9 is above 0.65, it is Siegesbeckia stalk-based Herba Siegesbeckiae, otherwise it is not.

[0080] ③ Identification was performed based on whether there was an obvious characteristic peak between peak 5 and peak 6 in the characteristic spectrum of the granule sample of Siegesbeckia sibiricum (Siegesbeckia sibiricum).

[0081] Among the 14 characteristic peaks in the characteristic spectra of 18 batches of Siegesbeckia sibiricum (Siegesbeckia sibiricum) formula granule samples and the characteristic spectra of the hairy pedunculate Siegesbeckia sibiricum formula granule samples, there is no obvious characteristic peak between peak 5 and peak 6, while the glandular pedunculate Siegesbeckia sibiricum formula granule samples have characteristic peaks. The specific results of the relative peak area between peak 5 and peak 6 of the glandular pedunculate Siegesbeckia sibiricum formula granule and peak 2 are as follows:

[0082] Table 4 Relative peak areas of characteristic peaks between peaks 5 and 6 of three batches of Adenophora stalk-based Herba Siegesbeckiae Granules

[0083]

[0084] The relative peak area of ​​peak α and peak 2 of the formula granules of the glandular peduncle of Siegesbeckia herba is in the range of 0.061 to 0.161. If the relative peak area of ​​peak α and peak 2 between peak 5 and peak 6 is greater than or equal to 0.061, it is the glandular peduncle, otherwise it is Siegesbeckia herba or hairy peduncle Siegesbeckia herba.

[0085] Experimental Example 1 Investigation of Construction Method

[0086] 1. Instruments, reagents and test drugs

[0087] Instrument: Waters ACQUITY H-Class ultra-high performance liquid chromatograph; PDA Detector; TUV Detector; Empower 3 chromatography workstation; XP26 (Mettler-Toledo), BSA124S electronic balance (Sartorius Scientific Instrument (Beijing) Co., Ltd.), BT25S electronic balance (Sartorius Scientific Instrument (Beijing) Co., Ltd.), KQ-500DB ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.);

[0088] Column: ACQUITY BEH C18 (2.1×100 mm, 1.7 μm);

[0089] C18 (2.1×100mm, 1.6μm);

[0090] ACQUITY BEH C18 (2.1×100mm, 1.7um).

[0091] Trial drug:

[0092] Caffeic acid reference substance (batch number: 110885-201703, purity, 99.7%, China Food and Drug Administration);

[0093] 3,7-di-O-methylquercetin reference substance (batch number: MUST-19031508, purity, 98.0%, Chengdu Munster Biotechnology Co., Ltd.);

[0094] Isochlorogenic acid C (batch number: AF20121801, purity, 98.0%, Chengdu Aifa Biotechnology Co., Ltd.);

[0095] Siegesbeckia sibiricum (Siegesbeckia sibiricum) as a control medicinal material (Batch No. 121572-201202, China Food and Drug Administration);

[0096] Siegesbeckia sibiricum (Siegesbeckia glandulosa) as a control medicinal material (Batch No. 121472-201102, China Food and Drug Administration);

[0097] Reagents: Acetonitrile (Merck, JA078530) and phosphoric acid (Fisher Scientific, 171289) were of chromatographic grade; water was distilled water (Watsons); and other reagents were of analytical grade.

[0098] 2. Preparation of test solution

[0099] Accurately weigh Siegesbeckia sibiricum granules and grind them finely. Take approximately 1.0 g and place in a stoppered conical flask. Add 30 mL of water and sonicate for 30 minutes. Allow to cool, filter, and extract twice with ethyl acetate, shaking each time using 45 mL. Combine the ethyl acetate extracts and evaporate to dryness. Dissolve the residue in methanol and dilute to 2 mL. Shake well, filter, and collect the filtrate to obtain the product.

[0100] 3. Optimization of chromatographic conditions

[0101] (1) Mobile phase gradient optimization experiment

[0102] The test solution prepared according to item 2 of this experimental example was detected by ultra-performance liquid chromatography, using octadecylsilane bonded silica gel as the filler (column length 100 mm, inner diameter 2.1 mm, particle size 1.6 μm); acetonitrile as mobile phase A, and 0.1% phosphoric acid solution as mobile phase B, respectively, with gradient elution according to the following gradient conditions; flow rate 0.3 ml per minute; detection wavelength 330 nm; column temperature 30°C; theoretical plate number calculated based on the caffeic acid peak should be not less than 49,000.

[0103] Table 5 Gradient conditions 1

[0104]

[0105] Table 6 Gradient Condition 2

[0106]

[0107] Table 7 Gradient Condition 3

[0108]

[0109] See Figure 6 and 7 As shown, the results show that compared with the gradient conditions, the baseline of the characteristic peaks of the chromatogram obtained under gradient condition 1 is stable, the separation effect of the characteristic peaks is significantly improved, and the peak area is more uniform.

[0110] (2) Investigation of detection wavelength

[0111] The Herba Siegesbeckiae Formula Granules were prepared according to the method in item 2 of this experimental example to prepare the Herba Siegesbeckiae Formula Granules test solution. Figure 8 ), the characteristic peak information at a wavelength of 328nm is richer than that at a wavelength of 330nm, so the wavelength is selected as 328nm.

[0112] 4. Identification of characteristic peaks

[0113] Take appropriate amounts of caffeic acid, isochlorogenic acid C and 3,7-di-O-methylquercetin reference substances, add methanol to prepare mixed control solutions containing 45 μg, 35 μg, 40 μg and 60 μg per 1 ml, as reference substance solutions.

[0114] The test solution of Herba Siegesbeckiae Granules was prepared according to the preparation method of Example 1. The reference solution and the Herba Siegesbeckiae Granules test solution were tested by ultra-high performance liquid chromatography according to the method of Example 1 and then compared. The results were as follows: Figure 9 ,

[0115] Summary: The retention times of peaks 2, 9 and 14 in the chromatogram of the test sample of Siegesbeckia serrata formula granules are consistent with those of the chromatographic peaks of caffeic acid, isochlorogenic acid C and 3,7-di-O-methylquercetin reference substances, respectively. It can be confirmed that peak 2 is caffeic acid, peak 9 is isochlorogenic acid C and peak 14 is 3,7-di-O-methylquercetin.

[0116] Example 2 Methodology Verification

[0117] 1. Repeatability

[0118] Six portions of Siegesbeckia sibiricum (Siegesbeckia sibiricum) granules were analyzed according to the method in Example 1 to obtain a characteristic spectrum. Peak 2 was used as the reference peak, and the relative peak area and relative retention time were calculated. The RSD was also calculated. The results are shown in the table below.

[0119] Table 8 Retention time and relative retention time of characteristic spectrum repeatability

[0120]

[0121] Table 9 Peak area and relative peak area of ​​characteristic spectrum repeatability

[0122]

[0123]

[0124] Summary: According to the repeatability investigation results, the RSD of the relative retention time of each characteristic peak is 0% to 1.3%, and the RSD of the relative peak area is in the range of 0% to 20.3%, indicating that the repeatability of the characteristic spectrum is good.

[0125] 2. Intermediate precision

[0126] Using a separate TUV detector, 6 portions of Siegesbeckia sibiricum (Siegesbeckia sibiricum) granules were analyzed according to the method in Example 1 to obtain a characteristic spectrum. Peak 2 was used as the reference peak, and the relative peak area and relative retention time were calculated. The RSD was also calculated. The results are shown in the table below.

[0127] Table 10 Retention time and relative retention time table of intermediate precision investigation of characteristic spectrum

[0128]

[0129] Table 11 Peak area and relative peak area of ​​intermediate precision investigation of characteristic spectrum

[0130]

[0131]

[0132] Summary: Using a different instrument, the TUV detector, for characteristic spectra, the relative retention time RSDs for each characteristic peak ranged from 0 to 0.8%, and the relative peak area RSDs ranged from 0 to 11.7%. The relative retention time RSDs for each instrument ranged from 0 to 9.2%, and the relative peak area RSDs ranged from 0 to 23.6%, indicating that different instrumental analyses had some influence on the characteristic spectra.

[0133] 3. Solution stability investigation

[0134] An appropriate amount of Siegesbeckia sibiricum (Siegesbeckia sibiricum) granules were taken and a test solution was prepared according to the method in Example 1. The solution was measured at 0 h, 3 h, 6 h, 9 h, 12 h, and 24 h using the method in Example 1 to obtain a characteristic spectrum. Using Peak 2 as the reference peak, the relative peak area and relative retention time were calculated. The RSD was also calculated. The results are shown in the table below.

[0135] Table 12 Stability relative retention time table

[0136]

[0137] Table 13 Stability relative peak area table

[0138]

[0139] Summary: The results of the stability experiment show that the chemical components in the test solution are stable within 24 hours, the RSD of the relative retention time of each characteristic peak is within 0-1.3%, and the RSD of the relative peak area is within the range of 0-10.7%, indicating that the analytical method is stable and reliable.

[0140] 4. Durability test

[0141] (1) Investigation of different column temperatures

[0142] Take an appropriate amount of Siegesbeckia sibiricum (Siegesbeckia sibiricum) granules, prepare a test solution according to the preparation method of the test solution in Example 1, and perform the assay according to the chromatographic conditions in Example 1. This experiment examined column temperatures of 28°C, 30°C, and 32°C to investigate the robustness of the chromatographic method at different column temperatures.

[0143] Table 14 Relative retention time table at different column temperatures

[0144]

[0145] Summary: From the above data, it can be seen that the RSD of the relative retention time of the characteristic spectra at different column temperatures is within 2.3%, and the relative retention times of the 14 peaks of the characteristic spectra are almost the same. This method has good tolerance to column temperature.

[0146] (2) Investigation of different acid concentrations

[0147] Take an appropriate amount of Siegesbeckia sibiricum (Siegesbeckia sibiricum) granules, prepare a test solution according to the preparation method of the test solution in Example 1, and perform the assay according to the chromatographic conditions in Example 1. This experiment examined formic acid solution concentrations of 0.08%, 0.10%, and 0.12% to examine the robustness of the chromatographic method for different formic acid solution concentrations.

[0148] Table 15 Relative retention time table at different acid concentrations

[0149]

[0150] Summary: From the above data, it can be seen that the relative retention time RSDs of the 12 peaks under different acid concentrations are within 2.3%, with small differences. The separation of the chromatographic peaks under different acid concentrations is good, which shows that slight changes in the concentration of phosphoric acid solution have little effect on this method.

[0151] Comparative Example 1

[0152] This comparative example provides a method for identifying Siegesbeckia sibiricum formula granules of different origins, comprising the following steps:

[0153] Preparation of test solution: Take 1.0 g of Siegesbeckia sibiricum (Siegesbeckia sibiricum) granules and 1.0 g of Siegesbeckia sibiricum (Siegesbeckia sibiricum) granules respectively, add 25 mL of water, boil for 60 minutes, filter, and obtain the solution.

[0154] Ultra-high performance liquid chromatography: 1 μl of each test solution of Herba Siegesbeckiae (Siegesbeckia) formula granules and Herba Siegesbeckiae (Siegesbeckia) formula granules were accurately aspirated and injected into an ultra-high performance liquid chromatograph for determination to obtain a characteristic graph. The chromatographic conditions were the same as those in Example 1.

[0155] See the results Figure 10 As shown, this method cannot effectively distinguish and identify Siegesbeckia sibiricum formula granules of different origins.

[0156] Comparative Example 2

[0157] This comparative example provides a method for identifying Siegesbeckia sibiricum formula granules of different origins, comprising the following steps:

[0158] Preparation of test solution: Take 1.0 g of Siegesbeckia sibiricum (Siegesbeckia sibiricum) granules and 1.0 g of Siegesbeckia sibiricum (Siegesbeckia sibiricum) granules respectively, add 25 mL of methanol, sonicate for 30 minutes, and filter to obtain the solution.

[0159] Ultra-high performance liquid chromatography: 1 μl of each test solution of Herba Siegesbeckiae (Siegesbeckia) formula granules and Herba Siegesbeckiae (Siegesbeckia) formula granules were accurately aspirated and injected into an ultra-high performance liquid chromatograph for determination to obtain a characteristic graph. The chromatographic conditions were the same as those in Example 1.

[0160] See the results Figure 11 As shown, this method cannot effectively distinguish and identify Siegesbeckia sibiricum formula granules of different origins.

[0161] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A method for constructing characteristic spectra of Siegesbeckia sibiricum medicinal preparations of different origins, characterized in that: The following steps are included: (1) Preparation of test solution: weigh the test sample, extract with water to obtain the extract, separate the solid and liquid, take the liquid, extract with ethyl acetate, take the ethyl acetate solution, dry, add solvent to dissolve, separate the solid and liquid, take the liquid to obtain the test solution; The invention also includes the steps of preparing a reference solution by using caffeic acid, 3,7-di-O-methylquercetin and isochlorogenic acid C and a solubilizing agent; (2) The test solution and the reference solution were detected by ultra-high performance liquid chromatography, using octadecylsilane bonded silica gel as the filler, the mobile phase comprising acetonitrile and phosphoric acid aqueous solution, the gradient elution program comprising: 0 → 6 min → 11 min → 21 min → 41 min → 49 min → 55 min, and the volume percentage of acetonitrile in the mobile phase being: 5% → 8% → 11% → 15% → 24% → 49% → 83%; The volume percentage of phosphoric acid in the phosphoric acid aqueous solution is 0.08-0.12%; The detection wavelength is 325nm-335nm; The chromatographic column used had a column length of 100 mm, an inner diameter of 2.1 mm, and a particle size of 1.6 μm; The Siegesbeckia medicinal preparations of different origins are Siegesbeckia medicinal preparations of Siegesbeckia origin, Siegesbeckia glandularis origin, or Siegesbeckia hairy stem origin; The Siegesbeckia sibiricum medicinal preparation is Siegesbeckia sibiricum formula granules.

2. The construction method according to claim 1, characterized in that Step (2) also satisfies a flow rate of 0.29-0.31 mL / min and a column temperature of 28-32°C.

3. The construction method according to claim 1, characterized in that The step (1) also satisfies any one or more of the following AG: A. The ratio of the mass of the test sample to the volume of water is 0.5-2:30; the relationship between mass and volume is g / mL; B. The extraction method is reflux extraction or ultrasonic extraction; C. Extraction time is ≥20 min; D. The solid-liquid separation is selected from centrifugation or membrane filtration; E. Extract 1 to 3 times with ethyl acetate, with the ratio of the mass of the test sample to the volume of ethyl acetate each time being 0.5-2:45; the relationship between mass and volume is g / mL; F. The ratio of the mass of the test sample to the volume of the solution after adding the solvent is 0.5-2:2; the relationship between mass and volume is g / mL; G. The solvent is methanol or methanol aqueous solution.

4. The construction method according to claim 3, characterized in that The extraction time is 30-120 min.

5. The construction method according to claim 1, characterized in that Each 1 mL of the reference solution contains 1-100 µg of caffeic acid, 1-100 µg of 3,7-di-O-methylquercetin, and 1-100 µg of isochlorogenic acid C; and / or, the solvent used in preparing the reference solution is selected from methanol or a methanol-water solution.

6. The construction method according to any one of claims 1 to 5, characterized in that: The construction method also includes the steps of using the extract obtained by extracting the control medicinal material Siegesbeckia scoparia with water to prepare a control medicinal material reference solution according to the preparation method of the test solution, and detecting the control medicinal material reference solution according to ultra-performance liquid chromatography to obtain a reference medicinal material reference atlas.

7. The construction method according to claim 6, characterized in that: The control medicinal material of Siegesbeckia scoparia was extracted with water, filtered, and the liquid was dried. Then, the control medicinal material reference solution was prepared according to the preparation method of the test solution.

8. A method for detecting the quality of Siegesbeckia sibiricum medicinal preparations of different origins, characterized in that: The method comprises the steps of obtaining a characteristic spectrum of the Siegesbeckia medicinal preparation according to the construction method described in any one of claims 1 to 7.

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