A method for establishing a Mongolian medicine formula granule of canavalia and its fingerprint spectrum

Abstract

The present application belongs to Mongolian medicine preparation technology and quality detection field, and relates to a kind of Mongolian medicine sword bean formula granules and the method for establishing fingerprint spectrum thereof.Preparation method of Mongolian medicine sword bean formula granules includes: sword bean medicinal material fine powder is mixed with any concentration of ethanol in the range of 25%~35%, after granulation whole grain, obtain sword bean formula granules.The method for establishing spectrum: prepare mixed reference substance and test solution;The obtained test and reference substance solution is detected by ultra-high performance liquid chromatography respectively, and the corresponding chromatogram is obtained;The test and reference substance chromatogram obtained is compared, and 5 components in test are identified;The chromatogram of 15 batches of sword bean formula granules is introduced into traditional Chinese medicine fingerprint spectrum similarity evaluation software, common peak is marked, and the fingerprint spectrum containing 11 common characteristic peaks is established, and the control spectrum is generated simultaneously.The present application prepares single sword bean medicinal material into granules, and establishes the method for establishing fingerprint spectrum thereof, which makes up the defects of traditional pill and powder, and provides a reference method for quality control.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of Mongolian medicine preparation and quality detection, and particularly relates to a Mongolian medicine Canavalia gladiata formula granule and a method for establishing a fingerprint spectrum thereof. BACKGROUND

[0002] The Mongolian name of Canavalia gladiata is Burin-shasha, which is derived from the dry mature seeds of Canavalia gladiata (Jacq.) DC. of the Leguminosae family, has a sweet taste, a flat nature, and is oily and soft; has the effects of clearing kidney heat and tonifying the kidney, and is commonly used for treating kidney deficiency, kidney cold, kidney heat, lumbago, and the like, and can also be used for treating vomiting, cold and hiccup. Literature reports that Canavalia gladiata contains phenolic compounds, flavonoids, terpenes, amino acids and other chemical components. Modern pharmacological studies show that it has the effects of anti-tumor, anti-inflammatory, blood glucose lowering, blood lipid lowering, and antioxidant.

[0003] The theoretical basis of Mongolian medicine is unique, and in many preparations and prescriptions, the Mongolian medicine habit is to use original powder as medicine, which has problems of large dosage, inconvenience to carry, difficulty in swallowing, poor adaptability, and the like, and the powder of such medicinal material is extremely easy to absorb moisture, has harsh storage conditions, and poor flowability, and cannot be adapted to modern intelligent dispensing machines. The present application prepares Canavalia gladiata formula granule from single Mongolian medicine Canavalia gladiata, which not only can retain the characteristics of Mongolian medicine, but also can solve the problems of poor portability and poor patient compliance of traditional Mongolian medicine dosage forms, and has the advantages of convenient adjustment, good flowability, weak moisture absorption, unified specifications, and adaptation to modern intelligent dispensing machines while ensuring the efficacy. However, due to the different physical properties of each medicinal material, the suitable excipients, wetting agents and their proportions for different medicinal materials are different, which is not obvious to those skilled in the art, and this also increases the difficulty of preparing Mongolian medicine formula granules.

[0004] The present application can analyze the overall chemical composition of the Canavalia gladiata formula granule by establishing the fingerprint spectrum of the Canavalia gladiata formula granule, and scientifically and comprehensively evaluate the quality thereof, thereby providing a scientific basis for clinical application. SUMMARY

[0005] In view of the above-mentioned technical problems, the present application aims to provide a Mongolian medicine Canavalia gladiata formula granule and a method for establishing a fingerprint spectrum thereof. The Canavalia gladiata formula granule is a single-ingredient granule prepared by using original powder as medicine; the preparation method of the Canavalia gladiata formula granule is to mix Canavalia gladiata medicinal material fine powder with a suitable wetting agent and then obtain the Canavalia gladiata formula granule by wet granulation, which can better improve the compliance of patients and retain the characteristics of Mongolian medicine. The method for establishing the fingerprint spectrum of the Canavalia gladiata formula granule can comprehensively and scientifically reflect the internal chemical characteristic information of the Canavalia gladiata formula granule, thereby providing a basis for quality detection.

[0006] To achieve the above-mentioned purposes, the present application adopts the following technical solutions:

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

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

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

[0010] Further, the rest angle of the formula granule of kidney bean is <35°, the particle size is <10%, the water content is <8%, and the wetting rate is <12%.

[0011] The application also provides a preparation method of the formula granule of kidney bean, comprising the following steps:

[0012] (1) crushing and sieving kidney bean medicinal materials to obtain Mongolian medicine kidney bean fine powder;

[0013] (2) mixing the kidney bean fine powder with any concentration of wetting agent, granulating and whole-graining to obtain the formula granule of kidney bean.

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

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

[0016] Preferably, the drying temperature is 55 to 65℃.

[0017] Preferably, the obtained dry granules are whole-grained, and the mesh number of the sieve used in the whole-graining is 14 to 24.

[0018] The Mongolian medicine formula granules of Canavalia provide a traditional single Canavalia medicinal material as a raw material, are prepared through the steps of crushing, sieving, adding a wetting agent, mixing, preparing soft material, granulating, drying, whole granulating, and quality inspection. The Mongolian medicine formula granules of Canavalia provided in the application have a drug loading of 100%, are a new type of granules (the particle size, flowability (angle of repose), water content, moisture absorption rate, original drug content, and microbial limit are all unified standards; the drug loading of the same type of Canavalia formula granules is fixed and unified), have unified specifications, unified dosages, and unified dosage form standards, comprehensively improve the controllability and stability of the quality of Mongolian medicine decoction pieces, can ensure the safety and effectiveness of Mongolian medicine, and are convenient for Mongolian medicine clinical formula medication. The Canavalia formula granules provided in the application improve the dosage form of the decoction pieces into granules on the premise of completely retaining the traditional properties and clinical medication habits of Mongolian medicine, can more accurately control the drug dosage, greatly reduce the amount of auxiliary materials, greatly protect volatile components, improve patient compliance, and overcome the inconvenience of carrying traditional Mongolian medicine preparations.

[0019] The fingerprint spectrum establishment method of the above-mentioned Mongolian medicine formula granules of Canavalia includes the following steps:

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

[0021] Step 2, preparation of a test sample solution;

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

[0023] Step 4: comparing the test sample chromatogram and the reference chromatogram obtained in step 3, identifying 5 chemical components in the test sample, namely gallic acid, L-tryptophan, methyl gallate, isofraxidin, and p-methoxycinnamic acid; introducing the chromatograms of 15 batches of Canavalia formula granules into traditional Chinese medicine fingerprint spectrum similarity evaluation software, marking common peaks, establishing a fingerprint spectrum containing 11 common characteristic peaks, and generating a control spectrum.

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

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

[0026] 4 min~12 min, the volume fraction of mobile phase B changes from 18%~20% to 26%~28%;

[0027] 12 min~15 min, the volume fraction of mobile phase B changes from 26%~28% to 32%~34%.

[0028] 15min~23min, the volume fraction of mobile phase B is changed from 33% to 37%;

[0029] 23min~32min, the volume fraction of mobile phase B is changed from 37% to 64%.

[0030] Specifically, the above gradient elution includes:

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

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

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

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

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

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

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

[0038] Further, the ultraviolet detection wavelength is 260nm~290nm, such as 265nm, 270nm, 290nm in different embodiments; preferably 270nm.

[0039] Further, the column temperature of the chromatographic column is 25~35℃, such as 25℃, 30℃, 35℃ in different embodiments; preferably 30℃.

[0040] Further, the injection volume is 2μL~5μL, such as 2μL, 3μL in different embodiments; preferably 2μL.

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

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

[0043] The mobile phase A is 0.14% formic acid aqueous solution, and the mobile phase B is methanol;

[0044] The ultraviolet detection wavelength is 270 nm.

[0045] The column temperature is 30℃.

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

[0047] The injection volume is 2 μL.

[0048] The chromatographic column is Ultimate® UHPLC XB-C18 (2.1 mm x 100 mm, 1.8 μm).

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

[0050] The test solution of the Canavalia formula granules is prepared by taking the Canavalia formula granules, grinding them into powder, ultrasonic extraction with methanol solution as the extraction solvent, supplementing the lost weight with the extraction solvent, filtering, and taking the filtrate.

[0051] The preparation conditions of the test solution of the Canavalia formula granules are described as follows:

[0052] In actual operation, the Canavalia formula granules are ground into powder for extraction; the powder of the Canavalia formula granules is powder passing through a No. 5 or No. 6 sieve.

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

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

[0055] The preparation method of the mixed reference solution comprises: accurately weighing gallic acid, L-tryptophan, methyl gallate, isofraxidin and p-methoxycinnamic acid, and dissolving the reference substances in methanol as the solvent; in the mixed reference solution, the concentrations of gallic acid, L-tryptophan, methyl gallate, isofraxidin 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 the peak No. 2 as the reference peak, the relative retention times and relative peak areas of the 11 common characteristic peaks are as follows:

[0057] The peak No. 1: the relative retention time is 0.473-0.482, and the relative peak area is 2.797-3.754.

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

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

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

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

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

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

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

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

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

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

[0068] The present application compares 11 common characteristic peaks in the fingerprint spectrum with the control product, and identifies the chemical components corresponding to the 5 common peaks in the fingerprint spectrum.

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

[0070] The test sample solution of 15 batches of canavalia formula granules is detected according to the above UPLC method, the chromatogram of each batch of canavalia formula granules is obtained, and the control chromatogram is obtained by processing with 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, the similarity with the control chromatogram is calculated, and the quality control of the formula granules is carried out through the similarity.

[0072] Compared with the prior art, the present application has the following advantages.

[0073] (1) The Mongolian medicine Canavalia formula granules provided by the present application retain the traditional properties and clinical medication habits of Mongolian medicine, and improve the dosage form of decoction pieces into granules. The granules can more accurately control the dosage, greatly reduce the amount of auxiliary materials, greatly protect volatile components, overcome the lack of Mongolian medicine dosage forms and other dosage forms, and are not easy to carry, and have outstanding contributions to the development of new dosage forms of Mongolian medicine, and can further promote the development of Mongolian medicine preparations. In addition, the preparation process used in the present application is simple, and can also meet the requirements of large-scale production.

[0074] (2) The UPLC fingerprint determination method of the Mongolian medicine Canavalia formula granules established by the present application realizes good separation of chromatographic peaks; the fingerprint obtained by the present application contains 11 common characteristic peaks, and 5 chromatographic peaks are identified, which are gallic acid, L-tryptophan, methyl gallate, isoflavonoid and p-methoxycinnamic acid. The chemical composition and relative content of the Mongolian medicine Canavalia formula granules can be more comprehensive, specific and scientific, and the quality of the Mongolian medicine Canavalia formula granules can be better controlled. BRIEF DESCRIPTION OF DRAWINGS

[0075] Figure 1 The mixed reference solution chromatogram provided for Example 3 of the present application.

[0076] Figure 2 The UPLC chromatogram of the Canavalia formula granule test solution provided for Example 3 of the present application.

[0077] Figure 3 The specificity test result graph provided by the present application.

[0078] Figure 4 The comparative atlas of different mobile phase systems provided for Example 4 of the present application.

[0079] Figure 5 The comparative atlas of gradient elution procedures under the conditions of Example 5 of the present application.

[0080] Figure 6 The comparative atlas of different detection wavelengths provided for Example 6 of the present application.

[0081] Figure 7 The comparative atlas of different column temperatures provided for Example 7 of the present application.

[0082] Figure 8 The chromatogram of 15 batches of Canavalia formula granules and the control atlas of Example 8 of the present application.

[0083] Figure 9 The preparation process flow chart of the Canavalia formula granules of the present application. DETAILED DESCRIPTION

[0084] In order to facilitate the understanding of the present application, the technical solutions of the present application will be comprehensively described below in combination with the drawings and specific embodiments. However, the following described embodiments are part of the embodiments of the present application, but not all the embodiments, and are only used to illustrate the present application, and should not be regarded as limiting the scope of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present application.

[0085] In the present application, when a numerical interval (i.e. a numerical range) is involved, the optional numerical distribution within the above numerical interval is regarded as continuous, and includes the two numerical end points (i.e. the minimum value and the maximum value) of the numerical range and each numerical value between the two numerical end points, unless otherwise specified. When the numerical interval only refers to integers within the numerical interval, the two end point integers of the numerical range and each integer between the two end points are included, unless otherwise specified. In addition, when multiple ranges are provided to describe a feature or a characteristic, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all sub-ranges included therein.

[0086] In the present application, the temperature parameter, unless otherwise specified, allows for constant temperature treatment and also allows for variation within a certain temperature interval. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuation within a range such as ±0.5℃, ±0.4℃, ±0.3℃, ±0.2℃, ±0.1℃ is allowed.

[0087] In the present application, the weight can be μg, mg, g, kg, etc. mass units known in the chemical industry.

[0088] In the specific embodiments, the verification of items such as specificity, precision, repeatability, stability, etc. is carried out according to the methodological validation in the current edition of the Appendix of the People's Republic of China Pharmacopoeia.

[0089] The present application does not have specific requirements for the source of Mongolian medicinal materials, and can be purchased from a Mongolian medicinal decoction piece processing factory with GMP qualification, such as Inner Mongolia Qimeng Co., Ltd., etc.

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

[0091] Preferably, in the application, 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 application, the flowability, formability, lubricity, and compressibility of the Mongolian medicine sword bean medicinal material fine powder are poor, and the compressibility and formability of the medicinal powder are improved by adding a suitable wetting agent and granulating the medicinal material fine powder by a wet method. Compared with the medicinal material fine powder, the rest angle of the Mongolian medicine sword bean formula granules prepared in the application is reduced, the hygroscopicity is reduced, the stability is increased, and the flowability is increased. In addition, the hardness and brittleness of the Mongolian medicine sword bean formula granules provided in the application are good, facilitating transportation and storage.

[0093] The application also provides a preparation method of the Mongolian medicine sword bean formula granules, comprising the following steps:

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

[0095] (2) mixing the sword bean medicinal material fine powder with 25%-35% of any concentration range of ethanol to granulate to obtain sword bean formula granules.

[0096] In the application, the crushed material is preferably passed through a 100 mesh sieve, and the undersize material is the sword bean medicinal material fine powder.

[0097] In the application, the mixing time of the sword bean medicinal material fine powder and the wetting agent is 25-35 min, more preferably 28-32 min, and most preferably 30 min. In the application, because the selected wetting agent is scientific and suitable, the powder is uniformly crushed, and other factors result in more accurate and reliable mixing time.

[0098] The mixed material is granulated in the application, and the granulation method is preferably wet granulation. In the application, the mesh number of the granulation is 10-20, preferably 14. After the granulation is completed, the material is sized in the application, and the mesh number of the sizing is preferably 14-24, preferably 20. The specification and parameters of the granulator are not specially limited in the application, and a conventional granulator in the art can be used.

[0099] After granulation, the application preferably further comprises the steps of packaging and quality inspection. In the application, the prepared sword bean formula granules are packaged in packaging materials; the quality inspection comprises outer packaging quality inspection and pharmaceutical preparation quality inspection; the outer packaging quality inspection is checked by artificial inspection whether the outer packaging is intact or damaged; in the application, the pharmaceutical preparation quality inspection is preferably carried out according to the pharmaceutical quality standard, and in the application, the specific reference is made to Chinese Pharmacopoeia (2020 edition four general rules 0104).

[0100] However, they cannot be understood as limiting the scope of protection of the application.

[0101] Example 1.

[0102] Preparation of Mongolian medicine Canavalia formula granules: Take the fine powder of Canavalia medicinal materials that have passed through a 100-mesh sieve and any concentration of ethanol in the range of 25% to 35% (add 330 ml of wetting agent per 1 kg of fine powder of Canavalia medicinal materials), mix for 30 min, and make the mixture into soft material. Place the soft material in a granulator to perform wet granulation. The mesh number of the granulation is 14, the drying temperature is 60°C, the drying time is 3 h, and the granulation is completed to obtain Canavalia formula granules.

[0103] According to the inspection method in the Chinese Pharmacopoeia (2020 edition, four-part general rules 0104), the results are shown in Table 1.

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

[0105]

[0106] Example 2.

[0107] Preparation of Mongolian medicine Canavalia formula granules: Take the fine powder of Canavalia medicinal materials that have passed through a 100-mesh sieve and any concentration of ethanol in the range of 25% to 35% (add 400 ml of wetting agent per 1 kg of fine powder of Canavalia medicinal materials), mix for 30 min, and make the mixture into soft material. Place the soft material in a granulator to perform wet granulation. The mesh number of the granulation is 14, the drying temperature is 60°C, the drying time is 3 h, and the granulation is completed to obtain Canavalia formula granules.

[0108] According to the inspection method in the Chinese Pharmacopoeia (2020 edition, four-part general rules 0104), the results are shown in Table 2.

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

[0110]

[0111] The above example results show that: compared with the fine powder of Canavalia medicinal materials, the Canavalia formula granules prepared by the method of the example have a decreased rest angle, indicating increased fluidity and facilitating dose division; the hygroscopicity is decreased by 3.52% to 4.39%, indicating decreased hygroscopicity and increased stability. In addition, due to the addition of wetting agents and different amounts of wetting agents, the properties of the Canavalia formula granules also differ.

[0112] Comparative Example 1.

[0113] Preparation of Mongolian medicine Canavalia formula granules: take the fine powder of Canavalia medicament that has passed through a 100-mesh sieve and any one of the following wetting agents: water, 20% ethanol, 40% ethanol (add 300 ml of wetting agent per 1.2 kg of fine powder of Canavalia medicament), mix for 30 min, and then make the mixture into soft material, place the soft material in a granulator to perform wet granulation, the mesh number of the granulation is 14, the drying temperature is 60 DEG C, the drying time is 3 h, and the granulation is performed to obtain the Canavalia formula granules.

[0114] The inspection was performed according to the Chinese Pharmacopoeia (2020 edition, four-part general rules 0104), and the results are shown in Table 3.

[0115] Table 3: Inspection results of Mongolian medicine Canavalia formula granules.

[0116]

[0117] Comparative Example 2.

[0118] Preparation of Mongolian medicine Canavalia formula granules: take the fine powder of Canavalia medicament that has passed through a 100-mesh sieve and any one of the following wetting agents: water, 20% ethanol, 40% ethanol (add 300 ml of wetting agent per 1.2 kg of fine powder of Canavalia medicament), mix for 30 min, and then make the mixture into soft material, place the soft material in a granulator to perform wet granulation, the mesh number of the granulation is 14, the drying temperature is 60 DEG C, the drying time is 3 h, and the granulation is performed to obtain the Canavalia formula granules.

[0119] The inspection was performed according to the Chinese Pharmacopoeia (2020 edition, four-part general rules 0104), and the results are shown in Table 4.

[0120] Table 4: Inspection results of Mongolian medicine Canavalia formula granules.

[0121]

[0122] The above comparative example results show that only the fine powder of Canavalia medicament mixed with ethanol in a concentration range of 25% to 35% and the ratio of the mass of Canavalia to the volume of ethanol in any concentration within the range specified in the application can obtain Mongolian medicine Canavalia formula granules with good yield, particle size, rest angle, and moisture absorption rate. In addition, not all wetting agents can be used for the preparation of Mongolian medicine Canavalia formula granules, such as water, 20% ethanol, 40% ethanol, and the like.

[0123] Example 3.

[0124] The application also provides a method for constructing a fingerprint spectrum of Mongolian medicine Canavalia formula granules. In specific embodiments, the instruments, medicinal materials, and reagents used are as follows, and are all commercially available products unless otherwise specified. Among them,

[0125] Instruments and models: Y-2 swing granulator (Shanghai Tianhe Pharmaceutical Machinery Co., Ltd.), Agilent 1290 Infinity II ultra-high performance liquid chromatograph (equipped with a four-component pump, a DAD detector, an OpenLab CDS workstation, Agilent Technologies, USA), Ultimate® UHPLC XB-C18 (2.1 mm x 100 mm, 1.8 μm) chromatographic column, SG3300H ultrasonic cleaner (Shanghai Guantai Ultrasonic Instruments Co., Ltd.), MCA6.6S-2CCN-M millionth electronic balance (Beijing Sartorius Scientific Instrument Co., Ltd.).

[0126] Raw materials and reagents: 15 batches of Canavalia gladiata powder (provided by Qimeng Co., Ltd.); Canavalia gladiata formula granules (batch numbers 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 self-made by the laboratory; L-tryptophan (batch number: GLR8RERG, purity: 99%) was purchased from Anhui Zesheng Technology Co., Ltd.; methyl gallate (batch number: CRN0403, purity ≥98%), isofraxidin (batch number: CRN2641, purity ≥98%) were purchased from Hubei Caiyuan Biological Technology Co., Ltd.; gallic acid (batch number: 110831-201906, purity: 91.5%) was purchased from China Institute for Drug Control; p-methoxycinnamic acid (batch number: 20240108-1, purity: 99.1%) was purchased from Huifang Luzhe New Material Co., Ltd.; methanol (chromatographically pure, USA, Sigma-Aldrich); methanol (analytically pure, Tianjin Kemio Chemical Reagent Co., Ltd.); formic acid (chromatographically pure, Shandong Xinya Chemical Co., Ltd.); pure water (Hangzhou Wahaha Group Co., Ltd.).

[0127] A method for constructing a fingerprint spectrum of a Mongolian medicine Canavalia gladiata formula granule, comprising the following steps:

[0128] The test solution of the Canavalia gladiata formula granule was detected by UPLC;

[0129] The chromatographic conditions of the UPLC method include: using Ultimate UHPLC XB-C18 (2.1mm*100mm, 1.8um) as a chromatographic column, using methanol (B)-0.14% formic acid water (A) as a mobile phase, and performing gradient elution; the program of the gradient elution includes: 0min~4min, the volume fraction of the mobile phase B changes from 10% to 19%; 4min~12min, the volume fraction of the mobile phase B changes from 19% to 27%; 12min~15min, the volume fraction of the mobile phase B changes from 27% to 33%; 15min~23min, the volume fraction of the mobile phase B changes from 33% to 37%; 23min~32min, the volume fraction of the mobile phase B changes from 37% to 64%.

[0130] In the specific embodiment of the application, the ultraviolet detection wavelength is 260nm~290nm, preferably 270nm.

[0131] As in different embodiments, the ultraviolet detection wavelength can be 265nm, 270nm, 290nm.

[0132] In the specific embodiment of the application, the column temperature of the chromatographic column is 25~35℃, preferably 30℃.

[0133] As in different embodiments, the column temperature of the chromatographic column can be 25℃, 30℃, 35℃, etc.

[0134] In the specific embodiment of the application, the injection volume is 2μL~5μL, preferably 2μL.

[0135] As in different embodiments, the injection volume can be 2μL, 3μL, etc.

[0136] In the specific embodiment of the application, further comprising: using the UPLC method to detect a mixed control solution; the mixed control solution includes gallic acid, L-tryptophan, methyl gallate, isoflavone and p-methoxycinnamic acid.

[0137] More specifically, the embodiment also provides a method for constructing a fingerprint of Mongolian medicine kidney bean formula granules, including the following steps:

[0138] (1) Preparation of the control solution

[0139] Accurately weigh appropriate amount of gallic acid, L-tryptophan, methyl gallate, isofraxidin and p-methoxycinnamic acid reference substances, respectively, into 10 ml volumetric flasks, dissolve with methanol solution to prepare reference substance stock solutions of gallic acid, L-tryptophan, methyl gallate, isofraxidin and p-methoxycinnamic acid with mass concentrations of 96.67 μg / ml, 63.67 μg / ml, 19 μg / ml, 106.67 μg / ml and 43 μg / ml, respectively. Accurately transfer appropriate amount of each reference substance stock solution into a volumetric flask in turn, dilute to the mark with methanol solution, shake well to prepare a mixed reference substance solution containing 2.86 μg, 1.89 μg, 0.56 μg, 3.16 μg and 1.27 μg per 1 ml.

[0140] (2) Preparation of test sample solution

[0141] Take appropriate amount of mucuna formula granules, grind finely, accurately weigh 0.500 g, and place it in a 10 ml volumetric flask. Add appropriate amount of methanol solution, ultrasonic extraction (power 200 W, frequency 59 kHz) for 30 min, cool down, dilute to the mark with methanol solution, shake well, filter, and take the filtrate to obtain the test sample solution of mucuna formula granules.

[0142] (3) Ultra-high performance liquid chromatography conditions

[0143] Chromatographic column: Ultimate® UHPLC XB-C18 (2.1 mm x 100 mm, 1.8 μm); mobile phase: methanol (B)-0.14% formic acid water (A), gradient elution according to the following table (see Table 5); detection wavelength 270 nm; column temperature 30°C; flow rate: 0.2 ml / min; injection volume: 2 μL.

[0144] Table 5 Gradient elution program (volume fraction).

[0145]

[0146] (4) Detection steps

[0147] Accurately pipette 2 μL of the mixed reference substance solution of step (1) and the test sample solution of step (2) into the ultra-high performance liquid chromatograph, respectively, for determination, and record the chromatogram as shown in Figure 1 , Figure 2 By comparison with gallic acid, L-tryptophan, methyl gallate, isofraxidin and p-methoxycinnamic acid reference substances, the results show that peak No. 1 is gallic acid, peak No. 2 is L-tryptophan, peak No. 3 is methyl gallate, peak No. 6 is isofraxidin, and peak No. 10 is p-methoxycinnamic acid.

[0148] Example 4.

[0149] The present example refers to the detection method of Example 3, the difference is that: step (3) in the ultra performance liquid chromatography conditions, the mobile phase composition is different; the mobile phase composition specific information is shown in Table 6, and the corresponding obtained chromatogram is shown in Figure 4 .

[0150] Table 6: Mobile phase composition and corresponding number.

[0151]

[0152] Example 5.

[0153] The present example refers to the detection method of Example 3, the difference is that: step (3) in the ultra performance liquid chromatography conditions, the mobile phase composition is different; the mobile phase composition specific information is shown in Table 6, and the corresponding obtained chromatogram is shown in Figure 5 .

[0154] Table 7: Gradient elution program (volume fraction).

[0155]

[0156] Example 6.

[0157] The present example refers to the detection method of Example 3, the difference is that: step (3) in the ultra performance liquid chromatography conditions, the detection wavelength is different. The detection wavelength specific information is shown in Table 8, and the corresponding obtained chromatogram is shown in Figure 6 .

[0158] Table 8: Detection wavelength and corresponding number.

[0159]

[0160] Example 7.

[0161] The present example refers to the detection method of Example 3, the difference is that: step (3) in the ultra performance liquid chromatography conditions, the column temperature is different. The column temperature specific information is shown in Table 9, and the corresponding obtained chromatogram is shown in Figure 7 .

[0162] Table 9: Column temperature and corresponding number.

[0163]

[0164] Example 8.

[0165] The present example provides a method for establishing 15 batches of jack bean formula granules and control chromatographic peaks, including the following steps:

[0166] Take 15 batches of sword bean formula granules for testing, respectively according to the method of step (2) in Example 3 to prepare the test solution, and according to the chromatographic conditions of step (3) to inject and measure, and record the chromatogram. The obtained chromatogram is introduced into "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition", and the median method is used to generate 15 batches of sword bean formula granule fingerprint (S1-S15) and control chromatogram (R), see Figure 8 .

[0167] Take L-tryptophan (peak 2) as the reference peak, calculate the RSD values of the relative retention time and relative peak area of each common peak of 15 batches of sword bean formula granules.

[0168] The results show (see Tables 10 and 11) that the RSD values of the relative retention time of each common peak of 15 batches of sword bean formula granules are 0.52%~0.91%, indicating that the peak time of each common peak of the established fingerprint is relatively stable; the RSD values of the relative peak area are 4.34%~12.59%, indicating that there are certain differences in the content of chemical components of different batches of sword bean formula granules.

[0169] Take the generated control chromatogram R as the reference chromatogram to evaluate the similarity of the 15 batches of sword bean formula granule fingerprint, and the results show that the similarity of the 15 batches of sword bean formula granule fingerprint is ≥0.90, see Table 12.

[0170] Table 10 Fingerprint of 15 batches of sword bean formula granules-Relative retention time.

[0171]

[0172] Table 11 Fingerprint of 15 batches of sword bean formula granules-Relative peak area.

[0173]

[0174] Table 12 Fingerprint similarity evaluation results of 15 batches of sword bean formula granules.

[0175]

[0176] Methodology

[0177] 1) Specificity test

[0178] Take 1 part of sword bean formula granules for testing, prepare the test solution according to the method of step (2) in Example 3, precisely take 2 μL of the test solution, mixed control solution and blank solution (methanol solution), and measure according to the chromatographic conditions of step (3), and record the chromatogram, see Figure 3 .

[0179] 2) Precision test

[0180] Take 1 part of the test sample of Canavalia formula granules, and prepare the test sample solution according to the method in step (2) in Example 3. Take the same part of the test sample solution, and continuously inject 6 times under the chromatographic conditions in step (3), and record the chromatogram. Take L-tryptophan (peak 2) as the reference peak, and calculate the RSD values of the relative retention time and the relative peak area of 11 common peaks.

[0181] The precision test results show (see Tables 13 and 14) that the RSD values of the relative retention time of each common peak of the Canavalia formula granules are less than 0.01%, and the RSD values of the relative peak area are less than 1.46%, indicating that the instrument precision is good.

[0182] Table 13 Precision test-relative retention time.

[0183]

[0184] Table 14 Precision test-relative peak area.

[0185]

[0186] 3) Reproducibility test

[0187] Take 6 parts of the test sample of Canavalia formula granules, and prepare the test sample solution according to the method in step (2) in Example 3. Inject and determine under the chromatographic conditions in step (3), and record the chromatogram. Take L-tryptophan (peak 2) as the reference peak, and calculate the RSD values of the relative retention time and the relative peak area of 11 common peaks.

[0188] The reproducibility test results show (see Tables 15 and 16) that the RSD values of the relative retention time of each common peak of the Canavalia formula granules are less than 0.23%, and the RSD values of the relative peak area are less than 3.47%, indicating that the method has good reproducibility.

[0189] Table 15 Reproducibility test-relative retention time.

[0190]

[0191] Table 16 Reproducibility test-relative peak area.

[0192]

[0193] 4) Stability test

[0194] Take 1 part of the test sample of Canavalia formula granules, and prepare the test sample solution according to the method in step (2) in Example 3. Inject and determine under the chromatographic conditions in step (3) at 0 h, 2 h, 4 h, 8 h, 16 h, and 24 h, respectively, and record the chromatogram. Take L-tryptophan (peak 2) as the reference peak, and calculate the RSD values of the relative retention time and the relative peak area of 11 common peaks.

[0195] The stability test results show (see Tables 17 and 18) that the relative retention time RSD values of each common peak of the canavalia formula granules are less than 0.97%, and the relative peak area RSD values are less than 3.72%, indicating that the test sample solution of the canavalia formula granules is stable within 24 hours.

[0196] Table 17 Stability test - relative retention time.

[0197]

[0198] Table 18 Stability test - relative peak area.

[0199]

[0200] The method of the present application has high sensitivity and good reproducibility, and the obtained fingerprint spectrum has abundant information of common characteristic peaks, meeting the overall requirements of the fingerprint spectrum.

[0201] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application; the prescription process and analysis conditions of the Mongolian medicine formula granules of the present application are only suitable for canavalia medicinal materials, and are not suitable for other Mongolian medicinal materials, and the prescription process of the Mongolian medicine canavalia formula granules established thereby is not obvious to those skilled in the art.

Claims

1. A method for establishing a fingerprint of a Mongolian medicine formula granule of Can Dou, characterized in that, The method comprises the following steps: Step 1, preparation of a mixed reference solution: accurately weigh gallic acid, L-tryptophan, methyl gallate, isofraxidin and p-methoxycinnamic acid, and dissolve the reference substances in methanol as a solvent; Step 2, preparation of a test solution: take the Canavalia formula granules, grind them, and then ultrasonically extract the ground Canavalia formula granules with methanol as an extraction solvent; after the extraction, the lost weight is made up with the extraction solvent, and then the filtrate is obtained, which is the test solution of the Canavalia formula granules; Step 3, the test solution and the reference solution obtained in steps 1 and 2 are respectively detected by UPLC to obtain the test chromatogram and the reference chromatogram; Step 4: compare the test chromatogram with the reference chromatogram, identify the five chemical components in the test solution, i.e. gallic acid, L-tryptophan, methyl gallate, isofraxidin and p-methoxycinnamic acid; mark the common peaks in the chromatograms of 15 batches of Canavalia formula granules, establish a fingerprint spectrum containing 11 common characteristic peaks, and generate a control 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 260 nm-290 nm; the column temperature of the chromatographic column is 25-35℃; the injection volume is 2 μL-5 μL; the chromatographic column used in the UPLC method is an Ultimate® UHPLC XB-C18, 2.1 mm×100 mm, 1.8 μm chromatographic column; gradient elution is adopted, and the gradient elution program includes: 0 min-4 min, the volume fraction of mobile phase B is 10%-19%; 4 min-12 min, the volume fraction of mobile phase B is 19%-27%; 12 min-15 min, the volume fraction of mobile phase B is 27%-33%; 15 min-23 min, the volume fraction of mobile phase B is 33%-37%; 23 min-32 min, the volume fraction of mobile phase B is 37%-64%; The Mongolian medicine Canavalia formula granules comprise Canavalia and a humectant; the humectant comprises ethanol with any concentration in the range of 25%-35%; the mass ratio of the Canavalia to the volume of ethanol with any concentration is 8-12:2.0-4.5, kg / L.

2. The establishment method according to claim 1, characterized by, In step 1, the concentrations of gallic acid, L-tryptophan, methyl gallate, isofraxidin and p-methoxycinnamic acid in the mixed reference solution 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 of claim 1, wherein, The ground Canavalia formula granules are powders passing through a No. 5 or No. 6 sieve; the ultrasonic power is 160-200 W; the solid-liquid ratio of the ground Canavalia formula granules to the extraction solvent is 0.5 g:10 mL; the volume fraction of methanol in the methanol aqueous solution is 100%; and the extraction time is 30 min.

4. A method of establishing according to any of claims 1-3, characterized in that, In the constructed fingerprint of the Mongolian medicine formula granules of Canavalia, peak No. 1 is gallic acid, peak No. 2 is L-tryptophan, peak No. 3 is methyl gallate, peak No. 6 is isofagomine, and peak No. 10 is p-methoxycinnamic acid; with peak No. 2 as the reference peak, the relative retention times and relative peak areas of the 11 common peaks are as follows: Peak No. 1: relative retention time is 0.473~0.482, and relative peak area is 2.797~3.754; Peak No. 2: relative retention time is 1.000, and relative peak area is 1.000; Peak No. 3: relative retention time is 1.299~1.325, and relative peak area is 0.359~0.563; Peak No. 4: relative retention time is 1.465~1.493, and relative peak area is 1.333~1.714; Peak No. 5: relative retention time is 2.077~2.125, and relative peak area is 0.850~1.072; Peak No. 6: relative retention time is 2.530~2.596, and relative peak area is 0.413~0.560; Peak No. 7: relative retention time is 2.589~2.659, and relative peak area is 2.139~2.662; Peak No. 8: relative retention time is 3.227~3.309, and relative peak area is 0.992~1.263; Peak No. 9: relative retention time is 3.497~3.585, and relative peak area is 0.542~0.733; Peak No. 10: relative retention time is 4.337~4.472, and relative peak area is 1.198~1.440; Peak No. 11: relative retention time is 4.414~4.553, and relative peak area is 0.797~0.

938.

5. A method of establishing according to any of claims 1-3, characterized by, The test solution of 15 batches of the formula granules of Canavalia is detected by the UPLC method, the chromatograms of the formula granules of Canavalia of each batch are obtained, and the control chromatogram is obtained by processing with the traditional Chinese medicine fingerprint similarity evaluation software.

6. The establishment method of claim 1, wherein, The preparation method of the formula granules of Canavalia comprises the following steps: (1) crushing and sieving the Canavalia medicinal material to obtain Mongolian medicine Canavalia fine powder; (2) mixing the Canavalia fine powder with any concentration of wetting agent, granulating, and obtaining the formula granules of Canavalia after whole-graining.

7. The establishment method according to claim 6, characterized by, In the step (1), the mesh number of the sieve is 70~130.

8. The establishment method according to claim 6, wherein, In the step (2), the granulation method is wet granulation, the mesh number of the sieve used in the wet granulation is 10~20, the mixing time is 25~35 min, the drying temperature is 55~65℃, and the mesh number of the sieve used in the whole-graining is 14~24.

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