Mongolian medicine sword bean formula granules and establishment method of fingerprint spectrum of Mongolian medicine sword bean formula granules

Abstract

The invention belongs to the field of Mongolian medicine preparation technology and quality detection, and relates to Mongolian medicine sword bean formula granules and a fingerprint spectrum establishment method thereof. The preparation method of the Mongolian medicine sword bean formula granules comprises the following steps: mixing sword bean medicinal material fine powder with ethanol with any concentration in the range of 25%-35%, and granulating to obtain the sword bean formula granules. The spectrum establishment method comprises the following steps: preparing a mixed reference substance and a test solution; respectively detecting the obtained test sample and the reference substance solution by adopting ultra-high performance liquid chromatography to obtain corresponding chromatograms; comparing the chromatogram of the sample to be tested with the chromatogram of the reference substance, and identifying five components in the sample to be tested; 15 batches of sword bean formula granule chromatograms are imported into traditional Chinese medicine fingerprint similarity evaluation software, common peaks are marked, a fingerprint containing 11 common characteristic peaks is established, and a control spectrum is generated at the same time. According to the invention, the single sword bean medicinal material is prepared into the granules, and the establishment method of the fingerprint spectrum of the granules is established, so that a reference method is provided for quality control of the granules while the defects of traditional pill powder are made up.

Description

Technical Field

[0001] The invention belongs to the field of Mongolian medicine preparation technology and quality detection technology, and specifically relates to a Mongolian medicine sword bean formula granule and a method for establishing a fingerprint spectrum thereof. Background Art

[0002] The Mongolian name of sword bean is Boren-Shaosha. It comes from the dried mature seeds of the legume plant Canavalia gladiata (Jacq.) DC. It tastes sweet, is mild, greasy and soft. It has the effects of clearing kidney heat and tonifying the kidney. It is often used to treat kidney deficiency, kidney cold, kidney heat, low back pain, and can also be used for vomiting, cold hiccups and other diseases. Literature reports that sword bean contains chemical components such as phenols, flavonoids, terpenes, and amino acids. Modern pharmacological studies have shown that it has anti-tumor, anti-inflammatory, hypoglycemic, hypolipidemic, and antioxidant effects.

[0003] Mongolian medicine has a unique theoretical basis. In many preparations and prescriptions, Mongolian medicine is accustomed to using raw powder as medicine, which has the problems of large dosage, inconvenience in carrying, difficulty in swallowing, poor adaptability, etc., and this kind of medicinal powder is very easy to absorb moisture, has harsh storage conditions, poor fluidity, and cannot be adapted to modern intelligent dispensing machines. The present invention makes the single Mongolian medicine sword bean into sword bean formula granules, which can not only retain the characteristics of Mongolian medicine, but also solve the problems of Mongolian medicine traditional dosage forms not being easy to carry and poor patient compliance. While ensuring the efficacy, it has the advantages of convenient adjustment, good fluidity, weak hygroscopicity, unified specifications and standards, and adaptation to modern intelligent dispensing machines. However, due to the different physical properties of each medicinal material, the excipients, wetting agents and their proportions suitable for different medicinal materials are also different, which is not obvious to those skilled in the art, which also increases the difficulty of preparing Mongolian medicine formula granules.

[0004] The invention can analyze the overall chemical composition of the sword bean formula granules by establishing a fingerprint spectrum, scientifically and comprehensively evaluate the quality of the sword bean formula granules, and provide a scientific basis for their clinical application. Summary of the invention

[0005] In view of the above-mentioned technical problems, the present invention aims to propose a method for establishing a spectrum of Mongolian medicine sword bean formula granules and their fingerprints. Sword bean formula granules are single-flavor granules with original powder as medicine; the preparation method of sword bean formula granules is to mix sword bean medicinal material fine powder with a suitable wetting agent and then wet granulate them, which can better improve patient compliance and retain the characteristics of Mongolian medicine. The method for establishing the fingerprint spectrum of sword bean formula granules can comprehensively and scientifically reflect the intrinsic chemical characteristic information of sword bean formula granules, providing a basis for its quality detection.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] A Mongolian medicine sword bean formula granule comprises sword bean and a wetting agent; the wetting agent comprises ethanol of any concentration within the range of 25% to 35%; the ratio of the mass of sword bean to the volume of ethanol of any concentration is (8 to 12): (2.0 to 4.5); the mass fraction of sword bean in the Mongolian medicine sword bean formula granule is 100%.

[0008] Preferably, the ratio of sword bean to any concentration of wetting agent is (9-11):(2.5-4.0).

[0009] More preferably, the ratio of sword bean to any concentration of wetting agent is (9.5-10.5):(2.9-3.7).

[0010] Furthermore, the angle of repose of the sword bean formula particles is less than 35 degrees, the particle size is less than 10%, the water content is less than 8%, and the moisture absorption rate is less than 12%.

[0011] The present invention also provides a method for preparing the sword bean formula granules, comprising the following steps:

[0012] (1) crushing and sieving the sword bean medicinal material to obtain Mongolian medicine sword bean medicinal material fine powder;

[0013] (2) The fine powder of sword bean medicinal material is mixed with a wetting agent of any concentration, and after granulation and granulation, the sword bean formula granules are obtained.

[0014] Preferably, the mesh size of the sieve in step (1) is 70 to 130 meshes.

[0015] Preferably, the granulation method in step (2) is wet granulation; the mesh size of the sieve used in the wet granulation is 10 to 20 meshes; and the mixing time is 25 to 35 minutes.

[0016] Preferably, the drying temperature is 55-65°C.

[0017] Preferably, the obtained dry granules are sized, and the mesh size of the sieve used for sizing is 14 to 24 meshes.

[0018] The Mongolian medicine sword bean formula granules provided by the present invention are prepared by using traditional single-flavor sword bean medicinal materials as raw materials through the steps of crushing, sieving, adding wetting agents, mixing, making soft materials, granulating, drying, granulating, quality inspection, etc. The Mongolian medicine sword bean formula granules of the present invention have a drug loading of 100%, which is a new type of granules with unified specifications, unified dosages, and unified dosage forms (implementing granule standards: particle size, fluidity (angle of repose), water content, moisture absorption rate, original drug content, and microbial limit are all unified standards; the drug loading of the same sword bean formula granules is fixed and unified), which comprehensively improves the controllability and stability of the quality of Mongolian medicine pieces, can ensure the safety and effectiveness of Mongolian medicine, and is convenient for the clinical prescription of Mongolian medicine. The sword bean formula granules provided by the present invention improve the dosage of the pieces to make granules under the premise of fully retaining the traditional properties of Mongolian medicine and the clinical medication habits, which can not only more accurately control the dosage, greatly reduce the dosage of auxiliary materials, protect volatile components to a large extent, improve patient compliance, but also overcome the problems of inconvenience in carrying traditional Mongolian medicine preparations.

[0019] The fingerprint spectrum establishment method of the Mongolian medicine sword bean formula granules comprises the following steps:

[0020] Step 1, preparation of mixed reference solution;

[0021] Step 2, preparation of test solution;

[0022] Step 3, the test solution and the reference solution obtained in step 1 and step 2 are respectively detected by ultra-high performance liquid chromatography UPLC to obtain a chromatogram of the test sample and a chromatogram of the reference solution;

[0023] Step 4: Compare the chromatograms of the test sample obtained in step 3 with those of the reference sample, and identify the five chemical components in the test sample, gallic acid, L-tryptophan, methyl gallate, isoflurane, and p-methoxycinnamic acid; import the chromatograms of 15 batches of sword bean formula granules into the traditional Chinese medicine fingerprint similarity evaluation software, mark the common peaks, establish a fingerprint spectrum containing 11 common characteristic peaks, and generate a reference spectrum at the same time.

[0024] The detection conditions of the above UPLC method include: mobile phase A is 0.14% formic acid aqueous solution, mobile phase B is methanol; gradient elution is used; the procedure of the gradient elution includes:

[0025] From 0 min to 4 min, the volume fraction of mobile phase B changed from 9% to 11% to 18% to 20%;

[0026] From 4 to 12 minutes, the volume fraction of mobile phase B changed from 18% to 20% to 26% to 28%;

[0027] From 12 to 15 minutes, the volume fraction of mobile phase B changed from 26% to 28% to 32% to 34%;

[0028] From 15 to 23 minutes, the volume fraction of mobile phase B changed from 32% to 34% to 36% to 38%;

[0029] From 23 to 32 minutes, the volume fraction of mobile phase B changed from 36% to 38% to 63% to 65%.

[0030] Specifically, the above-mentioned gradient elution includes:

[0031] From 0 min to 4 min, the volume fraction of mobile phase B changed from 10% to 19%;

[0032] From 4 to 12 min, the volume fraction of mobile phase B changed from 19% to 27%;

[0033] From 12 to 15 minutes, the volume fraction of mobile phase B changed from 27% to 33%;

[0034] From 15 to 23 minutes, the volume fraction of mobile phase B changed from 33% to 37%;

[0035] From 23min to 32min, the volume fraction of mobile phase B changed from 37% to 64%.

[0036] Furthermore, the mobile phase B is methanol, and the volume fraction of formic acid in the formic acid aqueous solution is 0.1% to 0.2%.

[0037] Preferably, mobile phase B is a formic acid aqueous solution with a volume fraction of 0.14%.

[0038] Furthermore, the ultraviolet detection wavelength is 260nm to 290nm. For example, in different embodiments, the ultraviolet detection wavelength can be 265nm, 270nm, or 290nm, preferably 270nm.

[0039] Furthermore, the column temperature of the chromatographic column is 25-35°C. For example, in different embodiments, the column temperature of the chromatographic column can be 25°C, 30°C, or 35°C; preferably 30°C.

[0040] Furthermore, the injection volume is 2 μL to 5 μL. For example, in different embodiments, the injection volume may be 2 μL or 3 μL, preferably 2 μL.

[0041] Furthermore, the chromatographic column used in the UPLC method is a C18 chromatographic column, preferably UHPLC XB-C18 (2.1mm×100mm, 1.8μm).

[0042] Preferably, the detection conditions of the UPLC method include:

[0043] Mobile phase A was 0.14% formic acid in water, and mobile phase B was methanol;

[0044] The UV detection wavelength is 270nm;

[0045] The column temperature was 30°C;

[0046] The flow rate was 0.2 mL / min.

[0047] The injection volume was 2 μL;

[0048] The chromatographic column is UHPLC XB-C18 (2.1mm×100mm, 1.8μm);

[0049] Furthermore, the preparation method of the test solution comprises:

[0050] Take the sword bean formula granules, grind them into powder, use methanol solution as the extraction solvent to perform ultrasonic extraction on the sword bean formula granules, make up the lost weight with the extraction solvent, filter, and take the filtrate to obtain the sword bean formula granule test solution.

[0051] The following describes the preparation conditions of the sword bean formula granule test solution:

[0052] In actual operation, the extraction is performed using the sword bean formula particles ground into powder; the powdered sword bean formula particles are powders that have passed through a No. 5 sieve or a No. 6 sieve.

[0053] The extraction method is ultrasonic extraction, the ultrasonic power is 160-200W, and the ultrasonic frequency is 59kHz.

[0054] The solid-liquid ratio of the sword bean formula particles to the extraction solvent is 0.5g:10mL; and the extraction time is 30min.

[0055] The preparation method of the mixed reference solution includes: accurately weighing gallic acid, L-tryptophan, methyl gallate, isofraquinone and p-methoxycinnamic acid, and using methanol as a solvent to dissolve the reference substance; in the mixed reference solution, the concentrations of gallic acid, L-tryptophan, methyl gallate, isofraquinone and p-methoxycinnamic acid are 2.86 μg / mL, 1.89 μg / mL, 0.56 μg / mL, 3.16 μg / mL and 1.27 μg / mL, respectively.

[0056] Taking peak 2 as the reference peak, the relative retention times and relative peak areas of the 11 common characteristic peaks are as follows:

[0057] Peak 1: relative retention time is 0.473-0.482, relative peak area is 2.797-3.754;

[0058] Peak 2: relative retention time is 1.000, relative peak area is 1.000;

[0059] Peak 3: relative retention time is 1.299-1.325, relative peak area is 0.359-0.563;

[0060] Peak 4: relative retention time is 1.465-1.493, relative peak area is 1.333-1.714;

[0061] Peak 5: relative retention time is 2.077-2.125, relative peak area is 0.850-1.072;

[0062] Peak 6: relative retention time is 2.530-2.596, relative peak area is 0.413-0.560;

[0063] Peak 7: relative retention time is 2.589-2.659, relative peak area is 2.139-2.662;

[0064] Peak 8: relative retention time is 3.227-3.309, relative peak area is 0.992-1.263;

[0065] Peak 9: relative retention time is 3.497-3.585, relative peak area is 0.542-0.733;

[0066] Peak 10: relative retention time is 4.337-4.472, relative peak area is 1.198-1.440;

[0067] Peak 11: relative retention time is 4.414-4.553, relative peak area is 0.797-0.938;

[0068] The present invention compares 11 common characteristic peaks in the fingerprint with reference substances, and identifies the chemical components corresponding to 5 common peaks in the fingerprint.

[0069] In the fingerprint spectrum, peak 1 is gallic acid, peak 2 is L-tryptophan, peak 3 is methyl gallate, peak 6 is isoflurane, and peak 10 is p-methoxycinnamic acid.

[0070] The test solutions of 15 batches of Jack Bean formula granules were tested by the above-mentioned UPLC method to obtain the chromatograms of each batch of the Jack Bean formula granules, and the control chromatograms were obtained by processing with the traditional Chinese medicine fingerprint similarity evaluation software.

[0071] In actual operation, the chromatogram of the sample to be tested can be imported into the traditional Chinese medicine fingerprint similarity evaluation software to calculate the similarity with the control spectrum, and the quality of the formula particles can be controlled by the similarity.

[0072] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0073] (1) The Mongolian medicine sword bean formula granules provided by the present invention, under the premise of fully retaining the traditional properties of Mongolian medicine and clinical medication habits, improve the dosage of the decoction pieces into granules, which can not only more accurately control the dosage, greatly reduce the dosage of auxiliary materials, and protect the volatile components to a large extent, but also overcome the shortcomings of the lack of Mongolian medicine dosage forms and other dosage forms that are difficult to take and not easy to carry, and have outstanding contributions to the development of new Mongolian medicine dosage forms, which can further promote the development of Mongolian medicine preparations. In addition, the preparation process used in the present invention is simple and can also meet the requirements of large-scale production.

[0074] (2) The UPLC fingerprint determination method of the Mongolian medicine sword bean formula granules established by the present invention achieves good separation of chromatographic peaks; the fingerprint obtained by the present invention contains 11 common characteristic peaks, and 5 chromatographic peaks are identified, namely gallic acid, L-tryptophan, methyl gallate, isoflurane and p-methoxycinnamic acid, which can more comprehensively, specifically and scientifically reflect the chemical composition and relative content of the Mongolian medicine sword bean formula granules, and can also better control the quality of the Mongolian medicine sword bean formula granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 A chromatogram of a mixed reference solution provided in Example 3 of the present invention;

[0076] Figure 2 UPLC chromatogram of the test solution of sword bean formula granules provided in Example 3 of the present invention;

[0077] Figure 3 A graph showing the specific test results provided by the present invention;

[0078] Figure 4 A comparison of different mobile phase systems provided in Example 4 of the present invention;

[0079] Figure 5 This is a comparative graph of the gradient elution procedure under the conditions of Example 5 of the present invention;

[0080] Figure 6 A comparison chart of different detection wavelengths provided in Example 6 of the present invention;

[0081] Figure 7 A comparison chart of different column temperatures provided in Example 7 of the present invention;

[0082] Figure 8 The chromatograms are of 15 batches of sword bean formula granules and a control spectrum in Example 8 of the present invention;

[0083] Fig. 9 The present invention is a flow chart of the preparation process of sword bean formula granules. DETAILED DESCRIPTION

[0084] In order to facilitate the understanding of the present invention, the technical solution of the present invention will be fully described below in conjunction with the accompanying drawings and specific implementation methods, but the embodiments described below are part of the embodiments of the present invention, rather than all of the embodiments, and are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0085] In the present invention, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0086] The temperature parameters in the present invention, if not specifically limited, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the precision range controlled by the instrument. Fluctuations within the range of ±0.5°C, ±0.4°C, ±0.3°C, ±0.2°C, and ±0.1°C are allowed.

[0087] In the present invention, weight may be μg, mg, g, kg or other mass units known in the chemical industry.

[0088] The verification of specificity, precision, repeatability, stability and other items in the specific implementation method is carried out according to the relevant guiding principles in the appendix of the current edition of the "Pharmacopoeia of the People's Republic of China".

[0089] The present invention has no specific requirements on the source of Mongolian medicinal materials, and they can be purchased from Mongolian medicinal pieces processing factories with GMP qualifications, such as Inner Mongolia Qimeng Co., Ltd.

[0090] The Mongolian medicine sword bean formula granules provided by the invention are prepared from raw materials including sword bean medicinal material fine powder and a wetting agent, wherein the wetting agent includes ethanol in a concentration range of 25% to 35%; the ratio of the sword bean mass to the ethanol volume at any concentration is (8 to 12): (2.0 to 4.5); and the mass fraction of the sword bean in the Mongolian medicine sword bean formula granules is 100%.

[0091] Preferably, in the present invention, the ratio of sword bean to any concentration of wetting agent is (9-11):(2.5-4.0); more preferably (9.5-10.5):(2.9-3.7).

[0092] In the present invention, the fluidity, formability, lubricity and compressibility of the Mongolian medicine sword bean powder are relatively poor. The present invention improves the compressibility and formability of the powder by adding a suitable wetting agent and wet granulating the powder into formula granules. Compared with the fine powder of the medicinal material, the Mongolian medicine sword bean formula granules prepared have a decreased angle of repose, decreased hygroscopicity, increased stability and increased fluidity. In addition, the Mongolian medicine sword bean formula granules provided by the present invention have good hardness and brittleness, and are convenient for transportation and storage.

[0093] The present invention also provides a method for preparing Mongolian medicine sword bean formula granules, comprising the following steps:

[0094] (1) grinding the sword bean medicinal material through a 70-130 mesh sieve to obtain a sword bean medicinal material fine powder;

[0095] (2) The fine powder of the sword bean medicinal material is mixed with ethanol in any concentration range of 25% to 35% to obtain sword bean formula granules.

[0096] In the present invention, the crushed material is preferably passed through a 100-mesh sieve, and the sieved material obtained is the sword bean medicinal material fine powder.

[0097] In the present invention, the mixing time of the sword bean medicinal material powder and the wetting agent is 25 to 35 minutes, more preferably 28 to 32 minutes, and most preferably 30 minutes; in the present invention, the mixing time is more accurate and reliable because the selected wetting agent is scientific and appropriate, the medicinal powder is crushed evenly, and other factors.

[0098] The present invention granulates the mixed material, and the granulation method is preferably wet granulation. In the present invention, the mesh number of the granulation is 10 to 20 meshes, preferably 14 meshes. After the granulation is completed, the present invention performs granulation, and the mesh number of the granulation is preferably 14 to 24 meshes, preferably 20 meshes. The present invention does not specifically limit the specifications and parameters of the granulator, and a conventional granulator in the art can be used.

[0099] After granulation, the present invention preferably further includes the steps of subpackaging and quality inspection. In the present invention, the subpackaging preferably involves subpackaging the prepared sword bean formula granules into packaging materials; the quality inspection includes outer packaging quality inspection and drug preparation quality inspection; the outer packaging quality inspection is performed by manually checking whether the outer packaging is intact or damaged; in the present invention, the drug preparation quality inspection is preferably performed in accordance with drug quality standards, and in the present invention, it is specifically performed with reference to the Chinese Pharmacopoeia (2020 Edition Four General Rules 0104).

[0100] However, they should not be construed as limiting the scope of protection of the present invention.

[0101] Example 1.

[0102] Preparation of Mongolian medicine sword bean formula granules: respectively take sword bean medicinal material fine powder that has passed through a 100-mesh sieve and ethanol of any concentration in the range of 25% to 35% (add 330 ml of wetting agent for every 1 kg of sword bean medicinal material fine powder), mix for 30 minutes, make the mixed material into a soft material, place it in a granulator for wet granulation, the granulation mesh number is 14 mesh, the drying temperature is 60°C, the drying time is 3 hours, and the granules are granulated to obtain sword bean formula granules.

[0103] The test was carried out in accordance with the "Chinese Pharmacopoeia" (2020 Edition, Part IV, General Rules 0104), and the results are shown in Table 1.

[0104] Table 1 Test results of Mongolian medicine sword bean formula granules and comparison with sword bean powder.

[0105]

[0106] Example 2.

[0107] Preparation of Mongolian medicine sword bean formula granules: respectively take sword bean medicinal material fine powder that has passed through a 100-mesh sieve and ethanol of any concentration in the range of 25% to 35% (add 400 ml of wetting agent for every 1 kg of sword bean medicinal material fine powder), mix for 30 minutes, make the mixed material into a soft material, place it in a granulator for wet granulation, the granulation mesh number is 14 mesh, the drying temperature is 60°C, the drying time is 3 hours, and the granules are granulated to obtain sword bean formula granules.

[0108] The test was carried out in accordance with the "Chinese Pharmacopoeia" (2020 Edition, Part IV, General Rules 0104), and the results are shown in Table 2.

[0109] Table 2 Test results of Mongolian medicine sword bean formula granules and comparison with sword bean powder.

[0110]

[0111]

[0112] The results of the above examples show that compared with the fine powder of sword bean medicinal material, the angle of repose of the sword bean formula granules prepared by the method of this example is reduced, indicating that the fluidity is increased and it is convenient to divide the dosage; the moisture absorption rate is reduced by 3.52% to 4.39%, indicating that the hygroscopicity is reduced and the stability is increased. In addition, due to the different wetting agents and the amount of wetting agents added, the properties of the sword bean formula granules are also different.

[0113] Comparative example 1.

[0114] Preparation of Mongolian medicine sword bean formula granules: respectively take sword bean medicinal material fine powder that has passed through a 100-mesh sieve and ethanol of any concentration in the range of 25% to 35% (add 150 ml of wetting agent for every 1.2 kg of sword bean medicinal material fine powder), mix for 30 minutes, make the mixed material into a soft material, place it in a granulator for wet granulation, the granulation mesh number is 14 mesh, the drying temperature is 60°C, the drying time is 3 hours, and the granules are granulated to obtain sword bean formula granules.

[0115] The test was carried out in accordance with the "Chinese Pharmacopoeia" (2020 Edition, Part IV, General Rules 0104), and the results are shown in Table 3.

[0116] Table 3 Test results of Mongolian medicine sword bean formula granules.

[0117]

[0118] Comparative example 2.

[0119] Preparation of Mongolian medicine sword bean formula granules: take sword bean medicinal material fine powder that has passed through a 100-mesh sieve and any wetting agent: water, 20% ethanol, 40% ethanol (add 300 ml of wetting agent for every 1.2 kg of sword bean medicinal material fine powder), mix for 30 minutes, make the mixed material into a soft material, place it in a granulator for wet granulation, the granulation mesh number is 14 mesh, the drying temperature is 60°C, the drying time is 3 hours, and the granules are granulated to obtain sword bean formula granules.

[0120] The test was carried out in accordance with the "Chinese Pharmacopoeia" (2020 Edition, Part IV, General Rules 0104), and the results are shown in Table 4.

[0121] Table 4 Test results of Mongolian medicine sword bean formula granules.

[0122]

[0123] The above comparative example results show that: only the sword bean medicinal material fine powder is mixed with ethanol in the concentration range of 25% to 35%, and the ratio of sword bean mass to any concentration ethanol volume can obtain Mongolian medicine sword bean formula particles with good yield, particle size, angle of repose and moisture absorption rate within the scope specified by the present invention. In addition, not all wetting agents can be used for the preparation of sword bean Mongolian medicine sword bean formula particles, such as wetting agents such as water, 20% ethanol, 40% ethanol, etc., which are not suitable for preparing Mongolian medicine sword bean formula particles.

[0124] Example 3.

[0125] The present invention also provides a method for constructing a fingerprint spectrum of Mongolian medicine sword bean formula particles. The information of the instruments, medicinal materials and reagents used in the specific embodiments is as follows. Unless otherwise specified, they are all commercially available products.

[0126] Instruments and models: Y-2 swing granulator (Shanghai Tianhe Pharmaceutical Machinery Co., Ltd.), Agilent 1290 Infinity II ultra-high performance liquid chromatograph (equipped with quaternary pump, DAD detector, OpenLab CDS workstation, Agilent Technologies, USA), UHPLC XB-C18 (2.1 mm × 100 mm, 1.8 μm) chromatographic column, SG3300H ultrasonic cleaner (Shanghai Guante Ultrasonic Instrument Co., Ltd.), MCA6.6S-2CCN-M one-millionth electronic balance (Beijing Sartorius Scientific Instrument Co., Ltd.).

[0127] Raw materials and test drugs: 15 batches of Jack Bean powder (provided by Qimeng Co., Ltd.); Jack Bean granules (batch number DDPFKL-1, DDPFKL-2, DDPFKL-3, DDPFKL-4, DDPFKL-5, DDPFKL-6, DDPFKL-7, DDPFKL-8, DDPFKL-9, DDPFKL-10, DDPFKL-11, DDPFKL-12, DDPFKL-13, DDPFKL-14, DDPFKL-15) were made by our laboratory; L-tryptophan (batch number: GLR8RERG, purity: 99%) was purchased from Anhui Zesheng Technology Co., Ltd.; gallic acid Methyl ester (batch number: CRN0403, purity ≥98%) and isoflurane (batch number: CRN2641, purity ≥98%) were purchased from Hubei Cuiyuan Biotechnology Co., Ltd.; gallic acid (batch number: 110831-201906, purity: 91.5%) was purchased from China Food and Drug Inspection Institute; p-methoxycinnamic acid (batch number: 20240108-1, purity: 99.1%) was purchased from Huaifang Luze New Materials Co., Ltd.; methanol (chromatographic grade, Thermo Fisher Scientific, USA), methanol (analytical grade, Tianjin Comeo Chemical Reagent Co., Ltd.), formic acid (chromatographic grade, Shandong Xiya Chemical Co., Ltd.), and purified water (Hangzhou Wahaha Group Co., Ltd.).

[0128] A method for constructing a fingerprint spectrum of Mongolian medicine sword bean formula particles comprises the following steps:

[0129] The UPLC method was used to detect the test solution of the sword bean formula granules;

[0130] The chromatographic conditions of the UPLC method include: UHPLC XB-C18 (2.1 mm×100 mm, 1.8 μm) is used as a chromatographic column, and methanol (B)-0.14% formic acid water (A) is used as a mobile phase for gradient elution; the gradient elution program includes: from 0 min to 4 min, the volume fraction of the mobile phase B changes from 10% to 19%; from 4 min to 12 min, the volume fraction of the mobile phase B changes from 19% to 27%; from 12 min to 15 min, the volume fraction of the mobile phase B changes from 27% to 33%; from 15 min to 23 min, the volume fraction of the mobile phase B changes from 33% to 37%; from 23 min to 32 min, the volume fraction of the mobile phase B changes from 37% to 64%.

[0131] In a specific embodiment of the present invention, the ultraviolet detection wavelength is 260nm to 290nm, preferably 270nm.

[0132] For example, in different embodiments, the ultraviolet detection wavelength may be 265 nm, 270 nm, or 290 nm.

[0133] In a specific embodiment of the present invention, the column temperature of the chromatographic column is 25-35°C, preferably 30°C.

[0134] For example, in different embodiments, the column temperature of the chromatographic column can be 25°C, 30°C, 35°C, etc.

[0135] In a specific embodiment of the present invention, the injection volume is 2 μL to 5 μL, preferably 2 μL.

[0136] For example, in different embodiments, the injection volume can be 2 μL, 3 μL, etc.

[0137] In a specific embodiment of the present invention, it also includes: using the UPLC method to detect a mixed reference solution; the mixed reference includes gallic acid, L-tryptophan, methyl gallate, isoflurane and p-methoxycinnamic acid.

[0138] More specifically, this embodiment also provides a method for constructing a fingerprint spectrum of Mongolian medicine sword bean formula particles, comprising the following steps:

[0139] (1) Preparation of reference solution

[0140] Weigh gallic acid, L-tryptophan, methyl gallate, isofraquinone and p-methoxycinnamic acid reference substances accurately, dissolve them in methanol solution in a 10ml volumetric flask to prepare reference substance stock solutions with mass concentrations of 96.67μg / ml, 63.67μg / ml, 19μg / ml, 106.67μg / ml and 43μg / ml, respectively. Accurately pipette appropriate amounts of each reference substance stock solution into a volumetric flask, dilute to the mark with methanol solution, shake well, and prepare mixed reference substance solutions containing 2.86μg, 1.89μg, 0.56μg, 3.16μg and 1.27μg per 1ml.

[0141] (2) Preparation of test solution

[0142] Take an appropriate amount of sword bean formula granules, grind them into powder, accurately weigh 0.500g, put them in a 10ml volumetric flask, add an appropriate amount of methanol solution, ultrasonically extract (power 200W, frequency 59kHz) for 30min, cool, make up to the scale with methanol solution, shake well, filter, and take the filtrate to obtain the sword bean formula granule test solution.

[0143] (3) UPLC conditions

[0144] Chromatographic column: UHPLC XB-C18 (2.1 mm × 100 mm, 1.8 μm); mobile phase: methanol (B)-0.14% formic acid water (A), gradient elution as specified in the following table (see Table 5); detection wavelength 270 nm; column temperature 30 ° C; flow rate: 0.2 ml / min; injection volume: 2 μL.

[0145] Table 5 Gradient elution program (volume fraction).

[0146]

[0147] 4) Detection steps

[0148] Accurately pipette 2 μL of the mixed reference solution of step (1) and the test solution of step (2), inject them into the ultra-high performance liquid chromatograph, measure, and record the chromatogram. Figure 1 , Figure 2 ; After comparison with gallic acid, L-tryptophan, methyl gallate, isofraquinone and p-methoxycinnamic acid reference substances, the results showed that peak 1 was gallic acid, peak 2 was L-tryptophan, peak 3 was methyl gallate, peak 6 was isofraquinone, and peak 10 was p-methoxycinnamic acid.

[0149] Example 4.

[0150] This embodiment refers to the detection method of Example 3, except that: in the ultra-high performance liquid chromatography conditions of step (3), the mobile phase composition is different; the specific information of the mobile phase composition is shown in Table 6, and the corresponding chromatogram is shown in Figure 4 .

[0151] Table 6 Mobile phase composition and corresponding numbers.

[0152]

[0153] Example 5.

[0154] This comparative example is based on the detection method of Example 3, except that: in the ultra-high performance liquid chromatography conditions of step (3), the mobile phase elution gradient is different. The elution gradient of this comparative example is shown in Table 7, and the corresponding chromatogram is shown in Figure 5 .

[0155] Table 7 Gradient elution program (volume fraction).

[0156]

[0157] Example 6.

[0158] This example refers to the detection method of Example 3, except that: in the ultra-high performance liquid chromatography conditions of step (3), the detection wavelength is different. The specific information of the detection wavelength is shown in Table 8, and the corresponding chromatogram is shown in Figure 6 .

[0159] Table 8 Detection wavelengths and corresponding numbers.

[0160]

[0161] Example 7.

[0162] This embodiment refers to the detection method of Example 3, except that: in the ultra-high performance liquid chromatography conditions of step (3), the column temperature is different. The specific information of the column temperature is shown in Table 9, and the corresponding chromatogram is shown in Figure 7 .

[0163] Table 9 Column temperatures and corresponding numbers.

[0164]

[0165] Example 8.

[0166] This embodiment provides a method for establishing 15 batches of sword bean formula particles and control chromatographic peaks, comprising the following steps:

[0167] Take 15 batches of sword bean formula granules as test samples, prepare test sample solutions according to the method under step (2) in Example 3, inject and measure according to the chromatographic conditions under step (3), and record the chromatogram. The obtained chromatogram is imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition", and the median method is used to generate 15 batches of sword bean formula granules fingerprints (S1-S15) and control spectrum (R), see Figure 8 .

[0168] Taking L-tryptophan (peak 2) as the reference peak, the RSD values ​​of the relative retention time and relative peak area of ​​each common peak of 15 batches of sword bean formula granules were calculated.

[0169] The results showed (see Tables 10 and 11) that the relative retention time RSD values ​​of the common peaks of 15 batches of sword bean formula granules were 0.52% to 0.91%, indicating that the peak times of the common peaks of the established fingerprint spectra were relatively stable; the relative peak area RSD values ​​were 4.34% to 12.59%, indicating that there were certain differences in the chemical composition content of different batches of sword bean formula granules.

[0170] Using the generated control spectrum R as the reference spectrum, the similarity of the fingerprint spectra of 15 batches of Jack Bean formula granules was evaluated. The results showed that the similarity of the fingerprint spectra of 15 batches of Jack Bean formula granules was ≥0.90, see Table 12.

[0171] Table 10 Fingerprints of 15 batches of sword bean formula granules - relative retention time.

[0172]

[0173] Table 11 Fingerprints of 15 batches of sword bean formula granules - relative peak areas.

[0174]

[0175]

[0176] Table 12 Similarity evaluation results of fingerprint spectra of 15 batches of sword bean formula granules.

[0177]

[0178] Methodological investigation

[0179] 1) Specificity test

[0180] Take 1 portion of the test sample of the sword bean formula granules, prepare the test solution according to the method under step (2) in Example 3, accurately pipette 2 μL of the test solution, the mixed reference solution and the blank solution (methanol solution), respectively, and measure according to the chromatographic conditions under step (3), record the chromatogram, and the results are shown in Figure 3 .

[0181] 2) Precision test

[0182] Take 1 portion of the sword bean formula granules as a test sample, prepare a test solution according to the method under step (2) in Example 3, take the same portion of the test solution, and continuously inject 6 times according to the chromatographic conditions under step (3), record the chromatogram, and use L-tryptophan (peak 2) as the reference peak to calculate the RSD values ​​of the relative retention time and relative peak area of ​​the 11 common peaks.

[0183] The results of the precision test show (see Tables 13 and 14) that the relative retention time RSD values ​​of the common peaks of the sword bean formula granules are all less than 0.01%, and the relative peak area RSD values ​​are all less than 1.46%, indicating that the instrument has good precision.

[0184] Table 13 Precision test - relative retention time.

[0185]

[0186] Table 14 Precision test - relative peak area.

[0187]

[0188] 3) Repeatability test

[0189] Take 6 portions of the sword bean formula granules as a test sample, prepare a test solution according to the method under step (2) in Example 3, inject and measure according to the chromatographic conditions under step (3), record the chromatogram, use L-tryptophan (peak 2) as the reference peak, and calculate the RSD values ​​of the relative retention time and relative peak area of ​​11 common peaks.

[0190] The results of the repeatability test show (see Tables 15 and 16) that the relative retention time RSD values ​​of the common peaks of the sword bean formula granules are all less than 0.23%, and the relative peak area RSD values ​​are all less than 3.47%, indicating that the method has good repeatability.

[0191] Table 15 Repeatability test - relative retention times.

[0192]

[0193]

[0194] Table 16 Repeatability test - relative peak areas.

[0195]

[0196] 4) Stability test

[0197] Take 1 portion of the sword bean formula granule test sample, prepare a test solution according to the method under step (2) in Example 3, and according to the chromatographic conditions under step (3), inject and measure at 0h, 2h, 4h, 8h, 16h, and 24h, record the chromatogram, and use L-tryptophan (peak 2) as the reference peak to calculate the RSD values ​​of the relative retention time and relative peak area of ​​the 11 common peaks.

[0198] The results of the stability test show (see Tables 17 and 18) that the relative retention time RSD values ​​of the common peaks of the sword bean formula granules are all less than 0.97%, and the relative peak area RSD values ​​are all less than 3.72%, indicating that the test solution of the sword bean formula granules is stable within 24 hours.

[0199] Table 17 Stability test - relative retention time.

[0200]

[0201] Table 18 Stability test - relative peak areas.

[0202]

[0203]

[0204] The method of the present invention has high sensitivity and good reproducibility, and the obtained fingerprint spectrum has rich information about common characteristic peaks, which meets the integrity requirements of the fingerprint spectrum.

[0205] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some or all of the technical features may be replaced by equivalents. These modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention. The prescription process and analysis conditions of the Mongolian medicine formula granules created by the present invention are only applicable to sword bean medicinal materials, and are not applicable to other Mongolian medicinal materials. The prescription process of the Mongolian medicine sword bean formula granules established thereby is not obvious to those skilled in the art.

Claims

1. A method for establishing a fingerprint spectrum of Mongolian medicine sword bean formula particles, characterized in that: The steps include: Step 1, preparation of mixed reference solution; Step 2, preparation of test solution; Step 3, the test solution and the reference solution obtained in step 1 and step 2 are respectively detected by ultra-high performance liquid chromatography to obtain a chromatogram of the test sample and a chromatogram of the reference solution; Step 4: Compare the chromatograms of the test sample obtained in step 3 with those of the reference sample, and identify the five chemical components gallic acid, L-tryptophan, methyl gallate, isofraquinone, and p-methoxycinnamic acid in the test sample; import the chromatograms of 15 batches of sword bean formula granules into the similarity evaluation software for Chinese medicine chromatographic fingerprints, mark the common peaks, establish a fingerprint containing 11 common characteristic peaks, and generate a reference spectrum at the same time; The detection conditions of the UPLC method include: mobile phase A is 0.14% formic acid aqueous solution; mobile phase B is methanol, and the volume fraction of formic acid is 0.1%~0.2%; the ultraviolet detection wavelength is 260nm~290nm; the column temperature of the chromatographic column is 25~35℃; the injection volume is 2μL~5μL; the chromatographic column used in the UPLC method is a C18 chromatographic column; gradient elution is used, and the gradient elution procedure includes: From 0min to 4min, the volume fraction of mobile phase B changed from 9% to 11% to 18% to 20%; From 4 to 12 min, the volume fraction of mobile phase B changed from 18% to 20% to 26% to 28%; From 12 to 15 minutes, the volume fraction of mobile phase B changed from 26% to 28% to 32% to 34%; From 15min to 23min, the volume fraction of mobile phase B changed from 32% to 34% to 36% to 38%; From 23min to 32min, the volume fraction of mobile phase B changed from 36%~38% to 63%~65%.

2. The establishment method according to claim 1, characterized in that: In the step 1, the preparation of the mixed reference solution is as follows: accurately weighing gallic acid, L-tryptophan, methyl gallate, isofraquinone and p-methoxycinnamic acid, and using methanol as a solvent to dissolve the reference substance; in the mixed reference solution, the concentrations of gallic acid, L-tryptophan, methyl gallate, isofraquinone and p-methoxycinnamic acid are 2.86 μg / mL, 1.89 μg / mL, 0.56 μg / mL, 3.16 μg / mL and 1.27 μg / mL, respectively.

3. The establishment method according to claim 1, characterized in that: In the step 2, the preparation of the test solution includes: taking the sword bean formula granules, grinding them, ultrasonically extracting the ground sword bean formula granules with methanol as an extraction solvent, supplementing the lost weight with the extraction solvent, filtering, and taking the filtrate to obtain the sword bean formula granule test solution.

4. The establishment method according to claim 3, characterized in that: The ground sword bean formula particles are powders that pass through a No. 5 sieve or a No. 6 sieve; the ultrasonic power is 160-200W; the solid-liquid ratio of the ground sword bean formula particles to the extraction solvent is 0.5g:10mL; the volume fraction of methanol in the methanol aqueous solution is 100%; and the extraction time is 30min.

5. The construction method according to any one of claims 1 to 4, characterized in that: In the constructed fingerprint of Mongolian medicine sword bean formula granules, peak 1 is gallic acid, peak 2 is L-tryptophan, peak 3 is methyl gallate, peak 6 is isofraquinone, and peak 10 is p-methoxycinnamic acid; taking peak 2 as the reference peak, the relative retention times and relative peak areas of the 11 common peaks are as follows: Peak 1: relative retention time is 0.473~0.482, relative peak area is 2.797~3.754; Peak 2: relative retention time is 1.000, relative peak area is 1.000; Peak 3: relative retention time is 1.299~1.325, relative peak area is 0.359~0.563; Peak 4: relative retention time is 1.465-1.493, relative peak area is 1.333-1.714; Peak 5: relative retention time is 2.077~2.125, relative peak area is 0.850~1.072; Peak 6: relative retention time is 2.530~2.596, relative peak area is 0.413~0.560; Peak 7: relative retention time is 2.589~2.659, relative peak area is 2.139~2.662; Peak 8: relative retention time is 3.227~3.309, relative peak area is 0.992~1.263; Peak 9: relative retention time is 3.497~3.585, relative peak area is 0.542~0.733; Peak 10: relative retention time is 4.337-4.472, relative peak area is 1.198-1.440; Peak 11: relative retention time is 4.414~4.553, and relative peak area is 0.797~0.

938.

6. The construction method according to any one of claims 1 to 4, characterized in that The test solutions of 15 batches of Jack Bean formula granules were tested by the UPLC method to obtain the chromatograms of each batch of the Jack Bean formula granules, which were processed by traditional Chinese medicine fingerprint similarity evaluation software to obtain a control spectrum.

7. The Mongolian medicine sword bean formula granules in the construction method according to claim 1, characterized in that: The Mongolian medicine sword bean formula granules include sword beans and a wetting agent; the wetting agent includes ethanol at any concentration within the range of 25% to 35%; the ratio of the mass of the sword beans to the volume of ethanol at any concentration is (8 to 12): (2.0 to 4.5); the mass fraction of the sword beans in the Mongolian medicine sword bean formula granules is 100%.

8. The method for preparing sword bean formula granules according to claim 7, characterized in that: The following steps are involved: (1) crushing and sieving the sword bean medicinal material to obtain Mongolian medicine sword bean medicinal material fine powder; (2) The fine powder of sword bean medicinal material is mixed with a wetting agent of any concentration, and after granulation and granulation, the sword bean formula granules are obtained.

9. The preparation method according to claim 8, characterized in that: In the step (1), the mesh size of the sieve is 70-130 meshes.

10. The preparation method according to claim 8, characterized in that: In the step (2), the granulation method is wet granulation, the mesh size of the sieve used in the wet granulation is 10-20 mesh, the mixing time is 25-35 minutes, and the drying temperature is 55-65°C; the mesh size of the sieve used for granulation is 14-24 mesh.

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