Mongolian medicine fried tribulus terrestris formula granules, construction method of fingerprint spectrum thereof and fingerprint spectrum

Abstract

The invention belongs to the technical field of Mongolian medicine preparations and quality detection, and particularly relates to a Mongolian medicine fried tribulus terrestris formula granule, a construction method of a fingerprint spectrum thereof and the fingerprint spectrum. The Mongolian medicine fried tribulus terrestris formula granules comprise fried tribulus terrestris fine powder and pharmaceutic adjuvants. The fingerprint spectrum construction method comprises the following steps: taking fried tribulus terrestris formula granules, extracting, filtering, determining by adopting ultra-high performance liquid chromatography, constructing a fingerprint spectrum, and carrying out compound identification on common peaks by adopting'double-standard one-quality '. The prepared Mongolian medicine fried tribulus terrestris formula granules are high in forming rate, good in fluidity, small in hygroscopicity and good in stability, the constructed fingerprint spectrum has the advantages of being good in stability, high in precision, good in repeatability and the like, the inherent quality of the fried tribulus terrestris formula granules can be comprehensively reflected, and compared with fingerprint spectrum construction completely through liquid chromatography, the operation cost is lower; compared with a common HPLC fingerprint spectrum, the analysis speed is higher, and the consumed mobile phase solution is less.

Description

Technical Field

[0001] The invention belongs to the technical field of Mongolian medicine preparation and quality detection, and particularly relates to a Mongolian medicine stir-fried tribulus terrestris formula granule and a construction method and fingerprint spectrum thereof. Background Art

[0002] Fried Tribulus terrestris (TRIBULI FRUCTUS) is the dried mature fruit of Tribulus terrestris L., a plant of the Tribulaceae family. In the Mongolian medical theory system, fried Tribulus terrestris is sweet, slightly bitter, warm, light, sharp and rare. It can dispel kidney cold, calm "He Yi", diuresis, reduce swelling and strengthen the body. It is used for frequent urination, urine retention, kidney cold, kidney "He Yi" disease, low back pain, wandering pain, spermatorrhea, impotence, edema. Modern research shows that fried Tribulus terrestris has the effects of lowering blood pressure, regulating immunity, detoxifying and relieving itching, and inhibiting the effect of acetylcholine. Traditional Mongolian medicine tends to grind the medicinal materials into powder and use them directly as medicine, but this method is poor in patient adaptability, swallowing is difficult, and the taste is not good; and this kind of medicinal powder is very easy to absorb moisture, has poor fluidity, and cannot be adapted to modern automatic dispensing machines. In response to the above problems, Mongolian medicine formula granules came into being. While ensuring the efficacy of the medicine, they have the advantages of good taste, convenient dosage adjustment, good fluidity, weak hygroscopicity, and unified specifications and standards. However, due to the different physical properties of each medicinal material, the excipients and their proportions suitable for different medicinal materials are also different, which is not obvious to technicians in this field, which also increases the difficulty of preparing Mongolian medicine formula granules. Fingerprint technology has the characteristics of integrity and fuzziness, and can more comprehensively reflect the intrinsic components of the drug. It has been widely used in the quality evaluation and authenticity identification of traditional Chinese medicine and ethnic medicine, and has also been widely recognized internationally, becoming a new generation of quality control methods for traditional medicines.

[0003] However, there are currently no patents or literature reports on ultra-high performance liquid phase fingerprints of stir-fried Tribulus terrestris medicinal materials and related preparations. The 2020 edition of the Chinese Pharmacopoeia still uses ultraviolet-visible spectrophotometry to determine the content of effective parts as the quality evaluation standard for Tribulus terrestris. How to improve the quality control method of stir-fried Tribulus terrestris has become an urgent problem to be solved. Although the fingerprint spectrum can fully reflect the intrinsic components of the drug, it is difficult to provide detailed information about specific compounds. Therefore, some scholars use liquid-mass chromatography to construct fingerprints, but the cost of using liquid-mass chromatography to construct fingerprints is too high, making it difficult to popularize. In addition, some scholars have tried to use liquid phase to construct fingerprints and use liquid-mass chromatography to qualitatively identify the chemical components in the fingerprint spectrum, but due to the different internal structures of liquid phase and liquid-mass instruments, even under the same chromatographic column and liquid phase conditions, the same compound still has a large difference in retention time, making it impossible for the chromatographic peaks on the liquid phase and liquid-mass chromatography to correspond. In addition, the components of traditional Chinese medicine are complex, and there may be some reference compounds that are not commercially available and cannot be obtained, making it impossible to identify the peaks of the fingerprint spectrum. There are currently no literature or patent reports on solutions to these problems. Summary of the invention

[0004] The present invention provides a Mongolian medicine stir-fried tribulus formula granule and a preparation method thereof, aiming to solve the problems of poor compliance, inconvenience in carrying, easy moisture absorption, poor fluidity, etc. of the traditional stir-fried tribulus administration method (i.e. stir-fried tribulus powder). At the same time, a construction method of stir-fried tribulus formula granule fingerprint spectrum is provided, which belongs to the "double standard one mass" method, and the "internal standard" is used to infer the retention time of the unknown chromatographic peak in the ultra-high performance liquid chromatogram on the ultra-high performance liquid phase-mass spectrometry, so that the ultra-high performance liquid phase corresponds to the chromatographic peak on the ultra-high performance liquid phase-mass spectrometry, which is a bridge connecting the two; the "external standard" is to use a reference substance to identify the peak of the liquid chromatogram, which is also a verification of the results of the ultra-high performance liquid phase-mass spectrometry qualitative compound; the "one mass" is to use the ultra-high performance liquid phase-mass spectrometry to qualitatively identify the compound, and the three are combined into "double standard one mass". On the one hand, it aims to make up for the defect that a single fingerprint spectrum can only vaguely evaluate the similarity of medicinal materials but cannot provide specific chemical composition information; on the other hand, it aims to provide a fast, accurate and low-cost quality evaluation method to ensure the quality controllability, safety and effectiveness of fried Tribulus terrestris formula granules, overcome the defect of the singleness of existing quality control methods, and provide a scientific basis for improving the quality standards of fried Tribulus terrestris and improving the quality inspection system of ethnic medicines.

[0005] In order to achieve the above-mentioned purpose of the present invention, the following technical scheme is adopted.

[0006] The invention provides a Mongolian medicine fried tribulus terrestris formula granule, the components of which include fried tribulus terrestris fine powder and medicinal excipients.

[0007] Furthermore, the stir-fried Tribulus terrestris fine powder is the Mongolian medicine stir-fried Tribulus terrestris medicinal material that can pass through a sieve of 80 to 120 meshes after being crushed, and the stir-fried Tribulus terrestris medicinal material is an authentic medicinal material.

[0008] Furthermore, the pharmaceutical excipients are commonly used excipients in pharmacy, including one or a combination of sodium carboxymethyl cellulose (CMC-NaPH7), hydroxypropyl methylcellulose (HPMC E5), and polyvinyl pyrrolidone (PVP K-25, PVP K-30).

[0009] Furthermore, the added amount of sodium carboxymethylcellulose (CMC-Na PH7) in the pharmaceutical excipient is 1wt% to 5wt%.

[0010] Furthermore, the added amount of hydroxypropyl methylcellulose (HPMC E5) in the pharmaceutical excipient is 3wt% to 7wt%.

[0011] Furthermore, the added amount of polyvinyl pyrrolidone (PVP K-25, PVP K-30) in the pharmaceutical excipient is 3wt% to 7wt%.

[0012] The present invention also provides a method for preparing Mongolian medicine stir-fried Tribulus terrestris formula granules, which specifically comprises the following steps:

[0013] Step 1: grind the Mongolian medicine stir-fried Tribulus terrestris and sieve it to obtain stir-fried Tribulus terrestris fine powder;

[0014] Step 2: mixing the stir-fried fine powder of Tribulus terrestris obtained in step 1 with pharmaceutical excipients and purified water, and performing wet granulation;

[0015] Step 3: granulate the granules obtained in step 2 and dry them, and then granulate them after drying to obtain Mongolian medicine stir-fried Tribulus terrestris formula granules.

[0016] Preferably, the sieving in step 1 is through a 80-120 mesh sieve, and the sieved portion is collected as the stir-fried Tribulus terrestris fine powder.

[0017] Preferably, the pure water in step 2 is medicinal purified water, and the added amount is 60-80 mL / (100 g fried Tribulus terrestris powder).

[0018] Preferably, the mixing time in step 2 is 20 to 40 minutes.

[0019] Preferably, the mesh size of the sieve used for the wet granulation in step 2 is 10 to 20 meshes.

[0020] Preferably, the drying temperature in step 3 is 40° C. to 60° C., and the drying time is 3 h to 6 h.

[0021] Preferably, the mesh size of the sieve used for granulation in step 3 is 14 to 25 meshes.

[0022] Furthermore, the water content of the Mongolian medicine fried tribulus terrestris formula granules is less than 8%; the angle of repose of the Mongolian medicine fried tribulus terrestris formula granules is less than 40°, the moisture absorption rate is less than 15%, and the particle size is less than 10%.

[0023] The present invention also provides a method for constructing a fingerprint spectrum of Mongolian medicine stir-fried Tribulus terrestris formula particles, comprising the following steps:

[0024] Step 1: grind the stir-fried Tribulus terrestris formula granules into fine powder, accurately add the extraction solvent, perform ultrasonic extraction, filter, and take the filtrate to obtain the sample solution;

[0025] Step 2: The sample solution obtained in step 1 is tested by ultra-high performance liquid chromatography to obtain a sample chromatogram;

[0026] Step 3: import the sample chromatogram obtained in step 2 into the chromatographic evaluation software, mark the common peaks, establish the fingerprint spectrum, generate the reference spectrum, and calculate the similarity between the fingerprint spectrum of each batch of samples and the reference spectrum, and evaluate the quality of multiple batches of stir-fried Tribulus terrestris formula granules by the similarity;

[0027] Step 4: Select a suitable internal standard component, add methanol to mix, prepare an internal standard solution, mix the internal standard solution with the sample solution to prepare a test solution;

[0028] Step 5: Detecting the test solution by ultra-high performance liquid chromatography to obtain an ultra-high performance liquid chromatogram of the test solution;

[0029] Step 6: The test solution is tested by ultra-high performance liquid chromatography-mass spectrometry to obtain an ultra-high performance liquid chromatography-mass spectrometry ion chromatogram, primary and secondary mass spectrometry related information of the test sample;

[0030] Step 7: Select an unknown chromatographic peak with a retention time close to that of the internal standard component chromatographic peak on the ultra-high performance liquid chromatogram obtained in step 5, and infer its retention time on ultra-high performance liquid chromatography-mass spectrometry by calculating the relative retention time of the unknown chromatographic peak and the internal standard component chromatographic peak on the ultra-high performance liquid chromatogram. The specific calculation formula is T x2 =T r2 (T x1 / T r1 );(T x1 is the retention time of the unknown chromatographic peak on the ultra-high performance liquid chromatogram, T r1 is the retention time of the internal standard component chromatographic peak on the ultra-high performance liquid chromatogram, T x2 is the retention time of the unknown chromatographic peak on UPLC-MS, T r2 is the retention time of the chromatographic peak of the internal standard component on ultra-high performance liquid chromatography-mass spectrometry);

[0031] Step 8: Further analyze and qualitatively identify the chemical components corresponding to the unknown chromatographic peak according to the mass spectrum at the retention time of the unknown chromatographic peak inferred in step 7;

[0032] Step 9: Repeating steps 7 and 8 can achieve qualitative identification of all unknown chromatographic peaks in the ultra-high performance liquid chromatogram;

[0033] Step 10: According to the qualitative results, select the corresponding compound standard and perform injection analysis according to the liquid phase conditions of step 2, and confirm the ultra-high performance liquid chromatogram with the reference substance.

[0034] Preferably, in step 1, the extraction solvent is 100% methanol; the volume ratio of the stir-fried Tribulus terrestris formula granules to the extraction solvent is 1:100, such as 1 g:100 mL.

[0035] Preferably, in step 1, the power of the ultrasonic extraction is 160-200 W; the frequency of the ultrasound is 59 kHz; and the extraction time is 30 min.

[0036] Preferably, in step 2, the detection wavelength of the ultra-high performance liquid chromatography is 250nm~340nm, preferably 340nm; the chromatographic column used in the ultra-high performance liquid chromatography is a C18 chromatographic column (100×2.1mm, 1.8μm), and the flow rate of the ultra-high performance liquid chromatography is 0.2mL / min; the column temperature of the ultra-high performance liquid chromatography is 25~35°C, preferably 30°C; the injection volume of the ultra-high performance liquid chromatography is 1μL~5μL, preferably 2μL; the mobile phase of the ultra-high performance liquid chromatography is acetonitrile as mobile phase A and 0.1% formic acid water as mobile phase B, and gradient elution is performed.

[0037] Further, the gradient elution procedure comprises:

[0038] From 0 to 10 min, the volume fraction of mobile phase A changes from 13% to 17% to 16% to 20%;

[0039] At 10-11 min, the volume fraction of mobile phase A changed from 16%-20% to 23%-27%;

[0040] From 11 to 30 min, the volume fraction of mobile phase A changes from 23% to 27% to 33% to 37%;

[0041] At 30-35 min, the volume fraction of mobile phase A changed from 33%-37% to 58%-62%;

[0042] From 35 to 40 min, the volume fraction of mobile phase A changed from 58% to 62% to 73% to 77%.

[0043] The elution procedure is preferably:

[0044] From 0 to 10 min, the volume fraction of mobile phase A changed from 15% to 18%;

[0045] At 10-11 min, the volume fraction of mobile phase A changed from 18% to 25%;

[0046] From 11 to 30 min, the volume fraction of mobile phase A changed from 25% to 35%;

[0047] 30-35 min, the volume fraction of mobile phase A changed from 35% to 60%;

[0048] From 35 to 40 min, the volume fraction of mobile phase A changed from 60% to 75%.

[0049] Preferably, in step 3, the chromatographic evaluation software adopts the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)".

[0050] Preferably, in step 3, a total of 16 common peaks are labeled, with peak No. 9 being used as the reference peak, and the relative retention times and relative peak areas of the 16 peaks are as follows:

[0051] Peak 1: relative retention time is 0.231-0.234, relative peak area is 0.057-0.066;

[0052] Peak 2: relative retention time is 0.338-0.344, relative peak area is 0.090-0.104;

[0053] Peak 3: relative retention time is 0.369-0.376, relative peak area is 0.067-0.077;

[0054] Peak 4: relative retention time is 0.484-0.489, relative peak area is 0.123-0.146;

[0055] Peak 5: relative retention time is 0.510-0.517, relative peak area is 0.037-0.049;

[0056] Peak 6: relative retention time is 0.535-0.542, relative peak area is 0.052-0.062;

[0057] Peak 7: relative retention time is 0.787-0.795, relative peak area is 0.066-0.080;

[0058] Peak 8: relative retention time is 0.813-0.818, relative peak area is 0.107-0.124;

[0059] Peak 9: relative retention time is 1.000, relative peak area is 1.000;

[0060] Peak 10: relative retention time is 1.049-1.052, relative peak area is 0.067-0.087;

[0061] Peak 11: relative retention time is 1.347-1.350, relative peak area is 0.063-0.076;

[0062] Peak 12: relative retention time is 1.670-1.674, relative peak area is 0.063-0.067;

[0063] Peak 13: relative retention time is 1.803-1.808, relative peak area is 0.095-0.118;

[0064] Peak 14: relative retention time is 1.825-1.832, relative peak area is 0.057-0.079;

[0065] Peak 15: relative retention time is 2.045-2.053, relative peak area is 0.014-0.016;

[0066] Peak 16: relative retention time is 2.208~2.215, and relative peak area is 1.427~1.195.

[0067] Preferably, the 16 common peaks are peak 1 for caffeic acid, peak 2 for quercetin-3-gentiobioside, peak 3 for vanillin, peak 4 for ferulic acid, peak 5 for rutin, peak 6 for hyperoside, peak 7 for avenanthramide E, peak 8 for N-caffeoyltyramine, peak 9 for tribulamide, peak 10 for diferuloylputrescine, peak 11 for N-[4,5-dihydroxy-2-[[4-[hydroxy(phenyl)methyl]-5-oxo-3-phenyl-2H-furan-2-yl]oxy]-6-(methoxymethyl)oxa-3-yl]acetamide, peak 12 for apigenin-7-xyloside, peak 13 for crotonamide, peak 14 for cannabinoid F, peak 15 for chrysin-5-xyloside, and peak 16 for ethyl p-methoxycinnamate.

[0068] Preferably, in step 3, the similarity comparison result is that the similarity between the fingerprint of each batch of test sample and the fingerprint of the control is >0.90.

[0069] Furthermore, in step 4, the internal standard component is a common known Chinese medicine chemical component standard substance, and the selection principle is: it has good separation from other components in the sample to be tested on the ultra-high performance liquid chromatogram and has good ultraviolet absorption under the same chromatographic conditions.

[0070] Furthermore, the ultra-high performance liquid chromatography conditions in step 5 and the ultra-high performance liquid chromatography conditions in ultra-high performance liquid chromatography-mass spectrometry in step 6 should be consistent with the chromatographic conditions in step 2.

[0071] Furthermore, for the unknown chromatographic peak with a retention time close to the internal standard component chromatographic peak in step 7, the range of the similar retention time is recommended to be within ±5 min; if the retention time of the unknown chromatographic peak is outside ±5 min, the chromatographic peak with a retention time close to the internal standard component can be selected from the chromatographic peaks analyzed and qualitatively analyzed in step 8 as the chromatographic peak.

[0072] Furthermore, in step 7, the internal standard component chromatographic peak is the internal standard component added to the test solution in step 4 or the chemical component analyzed and qualitatively analyzed in step 8.

[0073] Preferably, in step 10, the compound standards are vanillin, ferulic acid, rutin, hyperoside, and ethyl p-methoxycinnamate, and the concentrations of the injection analysis are 6.92 μg / mL, 2.389 μg / mL, 1.780 μg / mL, 2.552 μg / mL, and 117.719 μg / mL, respectively.

[0074] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0075] 1. The present invention optimizes the types and dosages of pharmaceutical excipients, selects sodium carboxymethylcellulose (CMC-Na PH7), hydroxypropyl methylcellulose (HPMC E5), and polyvinyl pyrrolidone (PVP K-25, PVP K-30) from the commonly used pharmaceutical excipients, and further optimizes the addition amount of each pharmaceutical excipient. The optimized pharmaceutical excipients can avoid clogging the granulation machine, making the granulation process smooth and the granulation yield rate high.

[0076] 2. The present invention proposes and prepares Mongolian medicine stir-fried Tribulus terrestris formula granules, which only use a single medicinal excipient and purified water, with a simple formula, reduced cost, simplified production process, and minimized the effect of excipients on the efficacy of medicinal materials. Compared with traditional stir-fried Tribulus terrestris powder, the angle of repose decreases by 17.8° to 21.3°, the fluidity increases, which is conducive to clinical drug adjustment and can be adapted to modern automatic dispensing machines; the moisture absorption rate decreases by 4.1% to 7.8%, the hygroscopicity decreases, and the stability increases; the palatability is improved, and the patient compliance is improved. The improvement of the Mongolian medicine dosage form is achieved, and the problems of poor fluidity, easy moisture absorption, harsh storage conditions, inconvenience in taking, and difficulty in carrying in the traditional Mongolian medicine administration method are solved, and further contributions are made to the development of Mongolian medicine and other ethnic medicines.

[0077] 3. The present invention establishes a UPLC fingerprint determination method for Mongolian medicine stir-fried Tribulus terrestris formula granules, and optimizes the detection conditions of UPLC, including the composition of the mobile phase, the detection wavelength, the gradient elution program, the column temperature, the flow rate, the injection volume and other parameters. Using the optimized method and parameters, the constructed UPLC fingerprint has a stable baseline, good separation, high precision and good repeatability, which can fully reflect the intrinsic quality of Mongolian medicine stir-fried Tribulus terrestris, fill the gap of the domestic lack of stir-fried Tribulus terrestris related fingerprint construction method, and improve the existing quality standards. In addition, based on the "double standard one quality" technology, the complete compound information corresponding to the common peak is provided, and peak identification can also be performed for some reference compounds that are extremely expensive or not commercially available and unavailable on the market. Compared with the traditional HPLC fingerprint analysis speed is faster, and the mobile phase solution consumed is less; compared with the complete use of liquid mass to construct the fingerprint, this method only requires a few injections of liquid mass, and the operating cost is low, which provides a more scientific and reasonable basis for the quality control of stir-fried Tribulus terrestris. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 The present invention is a flow chart of the preparation process of the stir-fried Tribulus terrestris formula granules;

[0079] Figure 2 The fingerprints and the control fingerprints of 20 batches of stir-fried Tribulus terrestris formula granules in Example 5 of the present invention (S1-S20 are the sample fingerprints of 20 batches of stir-fried Tribulus terrestris formula granules, and R is the control fingerprint);

[0080] Figure 3 This is the ultra-high performance liquid chromatogram of the test sample in Example 5 of the present invention;

[0081] Figure 4 The secondary mass spectra of 16 compounds in Example 5 of the present invention are as follows (A is caffeic acid, B is quercetin-3-gentiobioside, C is vanillin, D is ferulic acid, E is rutin, F is hyperoside, G is avenanthramide E, H is N-caffeoyltyramine, I is tribulamide, J is diferuloylputrescine, K is N-[4,5-dihydroxy-2-[[4-[hydroxy(phenyl)methyl]-5-oxo-3-phenyl-2H-furan-2-yl]oxy]-6-(methoxymethyl)oxa-3-yl]acetamide, L is apigenin-7-xyloside, M is crosamide, N is cannabinoid F, O is chrysin-5-xyloside, and P is ethyl p-methoxycinnamate);

[0082] Figure 5 The main cleavage pathways of the 9 compounds in Example 5 of the present invention (A is caffeic acid, B is quercetin-3-gentiobioside, C is vanillin, D is ferulic acid, E is rutin, F is hyperoside, G is avenanthramide E, H is N-caffeoyltyramine, and I is tribulamide);

[0083] Figure 6 are the main cleavage pathways of the 7 compounds in Example 5 of the present invention (J is diferuloyl putrescine, K is N-[4,5-dihydroxy-2-[[4-[hydroxy(phenyl)methyl]-5-oxo-3-phenyl-2H-furan-2-yl]oxy]-6-(methoxymethyl)oxa-3-yl]acetamide, L is apigenin-7-xyloside, M is crosamide, N is cannabinoid F, O is chrysin-5-xyloside, and P is ethyl p-methoxycinnamate);

[0084] Figure 7 Peak identification of the ultra-high performance liquid chromatography of the test sample for the "external standard" reference substance of the present invention (A is the chromatogram of the test sample, and B is the chromatogram of the "external standard" reference substance);

[0085] Figure 8 It is the ion chromatogram of peaks 1 to 16 and peak R of the test sample in Example 5 of the present invention by ultra-high performance liquid chromatography-mass spectrometry;

[0086] Fig. 9 The chromatograms obtained under different mobile phase compositions in Experimental Example 1 of the present invention (S1 is acetonitrile-water, S2 is acetonitrile-0.1% formic acid water);

[0087] Fig.10 The chromatograms measured under different column temperatures in Experimental Example 1 of the present invention (S1 is a column temperature of 25°C, S2 is a column temperature of 30°C, and S3 is a column temperature of 35°C);

[0088] Fig.11 The chromatograms obtained under different detection wavelengths in Experimental Example 1 of the present invention (S1 is 250 nm, S2 is 300 nm, and S3 is 340 nm);

[0089] Fig.12 The chromatograms obtained under different injection volumes in Experimental Example 1 of the present invention (S1 is 1 μL, S2 is 2 μL);

[0090] Fig.13 The chromatograms obtained under different elution gradients in Experimental Example 1 of the present invention (S1 is gradient 3, S2 is gradient 2, and S3 is gradient 1);

[0091] Fig.14 This is the specific chromatogram in Experimental Example 2 of the present invention (A is the sample solution of the stir-fried Tribulus terrestris formula granules; B is the mixed reference solution; C is the blank solution (methanol)). DETAILED DESCRIPTION

[0092] The technical solution of the present invention is further described below through specific implementation methods. The embodiments are only used to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0093] 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 value and the maximum value) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, including the numerical range 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.

[0094] 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.

[0095] In the present invention, weight may be μg, mg, g, kg or other mass units known in the chemical industry.

[0096] In the specific implementation method, the verification of specificity, precision, repeatability, stability and other items is carried out in accordance with the relevant guiding principles in the appendix of the 2020 edition of the "Chinese Pharmacopoeia".

[0097] In the present invention, the water content of the Mongolian medicine stir-fried tribulus terrestris formula granules is preferably less than 8%; the angle of repose of the Mongolian medicine stir-fried tribulus terrestris formula granules is less than 40°; the particle size of the Mongolian medicine stir-fried tribulus terrestris formula granules is determined according to the particle size and particle size distribution method (General Rule 0982 Second Method) of the "Chinese Pharmacopoeia" (2020 Edition), and the particle size of the Mongolian medicine stir-fried tribulus terrestris formula granules is less than 10%. Compared with stir-fried tribulus terrestris powder, the prepared formula granules have a decreased angle of repose, increased fluidity, decreased hygroscopicity, and increased stability, which is convenient for packaging, transportation, and storage; the palatability is improved, and the patient compliance is improved.

[0098] The present invention has no special requirements on the source of the Mongolian medicine, and it can be purchased from a Mongolian medicine slice processing factory with GMP qualification, such as Inner Mongolia Qimeng Co., Ltd.

[0099] In the present invention, the granulation is preferably carried out in a granulator; the present invention does not specifically limit the specifications and parameters of the granulator. A conventional granulator in the art can be used. In the present invention, the inspection of the Mongolian medicine stir-fried Tribulus terrestris formula granules includes dosage form inspection and quality standard identification. The dosage form inspection includes particle size, moisture, filling difference and filling amount, all of which are inspected according to the method in the general rules 0104 of the fourth volume of the "Chinese Pharmacopoeia"; the medicinal material quality standard identification is inspected according to the provisions of the "Chinese Pharmacopoeia 2020 Edition" under the same name decoction pieces.

[0100] In the present invention, the above processes of pulverizing, screening, mixing, wet granulation, drying, granulation and subpackaging are preferably carried out in a Class D clean area.

[0101] The materials, instruments, reagents and other information used in the specific embodiments of the present invention can be as follows. Unless otherwise specified, they are all commercially available products.

[0102] Instruments and models: Agilent 1290 Infinity II analytical liquid chromatography purification system (Agilent Technologies, USA, including quaternary pump, DAD detector, Agilent OpenLab workstation), Vanquish ultra-high performance liquid chromatography and Q-Exactive Orbitrap mass spectrometer system (Thermo Fisher Scientific, USA, electrospray ionization source), MCA10.6S-2CCN-M one-millionth electronic balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.), SG3300H ultrasonic cleaner (Shanghai Guante Ultrasonic Instrument Co., Ltd.), UHPLC XB-C18 (2.1 mm × 100 mm, 1.8 μm) column.

[0103] Raw materials and test drugs: The fried Tribulus terrestris formula granules (batch numbers CJLPFKL-1, CJLPFKL-2, CJLPFKL-3, CJLPFKL-4, CJLPFKL-5, CJLPFKL-6, CJLPFKL-7, CJLPFKL-8, CJLPFKL-9, CJLPFKL-10, CJLPFKL-11, CJLPFKL-12, CJLPFKL-13, CJLPFKL-14, CJLPFKL-15, CJLPFKL-16, CJLPFKL-17, CJLPFKL-18, CJLPFKL-19, CJLPFKL-20,) were made by our laboratory, and the fried Tribulus terrestris raw materials were provided by Inner Mongolia Qimeng Co., Ltd.

[0104] Reference substances: vanillin (batch number: 110753-202119, purity: 98%); ferulic acid (batch number: 221102, purity: 98%) was purchased from Chengdu Zhibiao Huachun Biotechnology Co., Ltd.; rutin (batch number: MUST-22111214, purity: 98%) was purchased from Chengdu Munster Biotechnology Co., Ltd.; hyperoside (batch number: CRN0087, purity: 98%) was purchased from Hubei Cuiyuan Biotechnology Co., Ltd.; ethyl p-methoxycinnamate (batch number: E88920HI01, purity: 98%) was purchased from Tianjin Xiens Biochemical Technology Co., Ltd.; acetonitrile (Thermo Fisher Scientific, USA, chromatographic grade), methanol (Tianjin Komiou Chemical Reagent Co., Ltd., analytical grade), formic acid (Tianjin Damao Chemical Reagent Factory), and purified water (Hangzhou Wahaha Group Co., Ltd.).

[0105] Example 1.

[0106] This embodiment provides a method for preparing Mongolian medicine stir-fried Tribulus terrestris formula granules, comprising the following steps:

[0107] Step 1: grind the Mongolian medicine stir-fried Tribulus terrestris and pass it through a 100-mesh sieve to obtain stir-fried Tribulus terrestris fine powder;

[0108] Step 2: Mix the stir-fried Tribulus terrestris powder with 1.5 wt% sodium carboxymethylcellulose (CMC-Na PH7) for 30 minutes, then add 71 mL / (100 g stir-fried Tribulus terrestris powder) of purified water and stir evenly to obtain a soft material. The soft material is placed in a 10-mesh granulator for granulation.

[0109] Step 3: Dry the obtained granules at 60° C. for 4 h, and after drying, sieve the granules with a 14-mesh sieve to obtain Mongolian medicine stir-fried Tribulus terrestris formula granules.

[0110] The preparation process of the Mongolian medicine stir-fried Tribulus terrestris formula granules is shown in Figure 1 The test results of the Mongolian medicine stir-fried Tribulus terrestris formula granules and the performance comparison results with stir-fried Tribulus terrestris powder are shown in Table 1.

[0111] Table 1 Test results of Mongolian medicine stir-fried Tribulus terrestris formula granules and performance comparison results with stir-fried Tribulus terrestris powder.

[0112]

[0113] Example 2.

[0114] This embodiment provides a method for preparing Mongolian medicine stir-fried Tribulus terrestris formula granules, comprising the following steps:

[0115] Step 1: grind the stir-fried Tribulus terrestris medicinal material and pass it through a 100-mesh sieve to obtain stir-fried Tribulus terrestris fine powder;

[0116] Step 2: Mix the stir-fried Tribulus terrestris powder with 3 wt% sodium carboxymethylcellulose (CMC-Na PH7) for 30 minutes, then add 66 mL / (100 g stir-fried Tribulus terrestris powder) of purified water and stir evenly to obtain a soft material. The soft material is placed in a 10-mesh granulator for granulation.

[0117] Step 3: Dry the obtained granules at 60° C. for 4 h, and after drying, sieve the granules with a 14-mesh sieve to obtain Mongolian medicine stir-fried Tribulus terrestris formula granules.

[0118] The test results of the Mongolian medicine stir-fried Tribulus terrestris formula granules and the performance comparison results with the Mongolian medicine stir-fried Tribulus terrestris powder are shown in Table 2.

[0119] Table 2 Test results of Mongolian medicine stir-fried Tribulus terrestris formula granules and performance comparison results with stir-fried Tribulus terrestris powder.

[0120]

[0121]

[0122] Example 3.

[0123] This embodiment provides a method for preparing Mongolian medicine stir-fried Tribulus terrestris formula granules, comprising the following steps:

[0124] Step 1: grind the stir-fried Tribulus terrestris medicinal material and pass it through a 100-mesh sieve to obtain stir-fried Tribulus terrestris fine powder;

[0125] Step 2: Mix the stir-fried Tribulus terrestris powder with 5wt% hydroxypropyl methylcellulose (HPMC E5) for 30 minutes, then add 70mL / (100g stir-fried Tribulus terrestris powder) of purified water and stir evenly to obtain a soft material. Place the soft material in a 10-mesh granulator for granulation;

[0126] Step 3: Dry the obtained granules at 50° C. for 5 h, and after drying, sieve the granules with a 14-mesh sieve to obtain Mongolian medicine stir-fried Tribulus terrestris formula granules.

[0127] The test results of the Mongolian medicine stir-fried Tribulus terrestris formula granules and the performance comparison results with the Mongolian medicine stir-fried Tribulus terrestris powder are shown in Table 3.

[0128] Table 3 Test results of Mongolian medicine stir-fried Tribulus terrestris formula granules and performance comparison results with stir-fried Tribulus terrestris powder.

[0129]

[0130] Example 4.

[0131] This embodiment provides a method for preparing Mongolian medicine stir-fried Tribulus terrestris formula granules, comprising the following steps:

[0132] Step 1: grind the stir-fried Tribulus terrestris medicinal material and pass it through a 100-mesh sieve to obtain stir-fried Tribulus terrestris fine powder;

[0133] Step 2: Mix the stir-fried Tribulus terrestris powder with 6 wt% polyvinyl pyrrolidone (PVPK-25, PVPK-30) for 30 minutes, then add 65 mL / (100 g stir-fried Tribulus terrestris powder) of purified water and stir evenly to obtain a soft material. The soft material is placed in a 10-mesh granulator for granulation.

[0134] Step 3: Dry the obtained granules at 40° C. for 6 h, and after drying, sieve the granules with a 14-mesh sieve to obtain Mongolian medicine stir-fried Tribulus terrestris formula granules.

[0135] The test results of the Mongolian medicine stir-fried Tribulus terrestris formula granules and the performance comparison results with the Mongolian medicine stir-fried Tribulus terrestris powder are shown in Table 4.

[0136] Table 4 Test results of Mongolian medicine stir-fried Tribulus terrestris formula granules and performance comparison results with stir-fried Tribulus terrestris powder.

[0137]

[0138] Comparative example 1.

[0139] Step 1: grind the stir-fried Tribulus terrestris medicinal material and pass it through a 100-mesh sieve to obtain stir-fried Tribulus terrestris fine powder;

[0140] Step 2: The stir-fried Tribulus terrestris powder is mixed with the auxiliary materials in Table 5 and purified water respectively and stirred to obtain a soft material, and the soft material is placed in a 10-mesh granulator for granulation.

[0141] Step 3: Dry the obtained granules at 60° C. for 4 h, and after drying, sieve the granules with a 14-mesh sieve to obtain 10 combinations of Mongolian medicine stir-fried Tribulus terrestris formula granules (see Table 5).

[0142] The performance of the stir-fried Tribulus terrestris formula granules and stir-fried Tribulus terrestris powder prepared in Examples 1-4 was compared with the performance of the stir-fried Tribulus terrestris formula granules of 10 combinations obtained in the above step 3. The comparison results are shown in Table 6.

[0143] Table 5 Combinations of different excipients and purified water.

[0144]

[0145] Table 6 Comparison of performance of different stir-fried Tribulus terrestris formula granules and stir-fried Tribulus terrestris powder.

[0146]

[0147]

[0148] The results of the above examples and comparative examples show that compared with traditional fried tribulus powder, the angle of repose of the fried tribulus terrestris formula granules prepared by the method of this embodiment is reduced by 17.8° to 21.3°, the fluidity is increased, it is convenient for dosing, and it can be adapted to modern automatic dispensing machines; the moisture absorption rate is reduced by 4.1% to 7.8%, the hygroscopicity is reduced, and the stability is increased; the palatability is improved, and the patient compliance is improved. The present invention optimizes the types and addition amounts of pharmaceutical excipients. The three pharmaceutical excipients (sodium carboxymethyl cellulose (CMC-Na PH7), hydroxypropyl methylcellulose (HPMC E5), polyvinyl pyrrolidone (PVP K-25, PVP K-30)) compared with other excipients, the particle size of the prepared stir-fried tribulus formula granules decreased by 38.3%-40.0%, the angle of repose decreased by 7.5°-11.0°, the moisture absorption rate decreased by 2.0%-5.6%, the particle yield rate increased by 38.6%-40.0%, and the palatability improved; compared with other addition amounts, the addition amounts of the four medicinal excipients preferably selected in the present invention can make the soft material moderate in the preparation process of the stir-fried tribulus formula granules, easy to mix, avoid clogging the granulation machine, and the granulation process is smooth. The particle size of the prepared stir-fried tribulus formula granules decreased by 9.5%-24.6%, the angle of repose decreased by 4.3°-9.8°, the moisture absorption rate decreased by 1.3%-5.0%, the particle yield rate increased by 12.1%-26.7%, and the palatability improved.

[0149] Example 5.

[0150] This embodiment provides a method for constructing a fingerprint spectrum of Mongolian medicine stir-fried Tribulus terrestris formula granules, comprising the following steps:

[0151] Step 1: grind 20 batches of stir-fried Tribulus terrestris formula granules (1.5wt% sodium carboxymethyl cellulose, 98.5wt% stir-fried Tribulus terrestris fine powder), accurately weigh 1g of each, place in a 250mL stoppered conical flask, add 100mL of methanol, accurately weigh the weight, perform ultrasonic extraction (200W, frequency 59kHz) for 30min, cool, make up the lost weight with methanol solution, shake well, filter, and take the filtrate to obtain 20 batches of stir-fried Tribulus terrestris formula granule sample solutions;

[0152] Step 2: The 20 batches of stir-fried Tribulus terrestris formula granule sample solutions obtained in step 1 are tested by ultra-high performance liquid chromatography to obtain chromatograms of 20 batches of samples. The chromatographic conditions are as follows:

[0153] Chromatographic columns UHPLC XB-C18 (2.1 mm × 100 mm, 1.8 μm) chromatographic column; mobile phase: acetonitrile as mobile phase A, 0.1% formic acid aqueous solution as mobile phase B, gradient elution as specified in the following table (see Table 7); column temperature 30°C; flow rate: 0.2 ml / min; detection wavelength 340 nm; reference wavelength 400 nm; injection volume: 2 μL.

[0154] Table 7 Mobile phase gradient elution conditions.

[0155]

[0156] Step 3: Import the chromatograms of the 20 batches of samples obtained in step 2 into the 2012 edition of the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines" and use the median method to generate superimposed fingerprints and reference fingerprints (see Figure 2 ). The generated control fingerprint R was used as a reference to evaluate the similarity of the fingerprints of each batch of stir-fried Tribulus terrestris formula granules. The results showed that the similarity of the fingerprints of the 20 batches of stir-fried Tribulus terrestris formula granules was above 0.90 (see Table 8). The good similarity indicates that the overall quality of the stir-fried Tribulus terrestris formula granules is stable.

[0157] Table 8 Similarity evaluation results of fingerprint spectra of 20 batches of stir-fried Tribulus terrestris granules.

[0158]

[0159] Step 4: Select quercetin as the internal standard component, add methanol to mix, and prepare an internal standard solution. Mix the internal standard solution with the stir-fried Tribulus terrestris formula granule sample solution obtained in step 1 to prepare a test solution;

[0160] Step 5: The test solution is tested by ultra-high performance liquid chromatography. The chromatographic conditions are as follows: chromatographic column UHPLC XB-C18 (2.1mm×100mm, 1.8μm) chromatographic column; mobile phase: acetonitrile as mobile phase A, 0.1% formic acid aqueous solution as mobile phase B, gradient elution (1-10min, 15%-18% A; 10-11min, 18%-25% A; 11-30min, 25%-35% A; 30-35min, 35%-60% A; 35-40min, 60%-75% A); column temperature 30℃; flow rate: 0.2ml / min; detection wavelength 340nm; reference wavelength 400nm; injection volume: 2μL. The ultra-high performance liquid chromatogram of the test sample was obtained (such as Figure 3 ).

[0161] Step 6: The sample solution was tested by ultra-high performance liquid chromatography-mass spectrometry. The chromatographic conditions were as follows: chromatographic column UHPLC XB-C18 (2.1 mm × 100 mm, 1.8 μm) chromatographic column; mobile phase: acetonitrile as mobile phase A, 0.1% formic acid aqueous solution as mobile phase B, gradient elution (1-10 min, 15%-18% A; 10-11 min, 18%-25% A; 11-30 min, 25%-35% A; 30-35 min, 35%-60% A; 35-40 min, 60%-75% A); column temperature 30°C; flow rate: 0.2 ml / min; detection wavelength 340 nm; reference wavelength 400 nm; injection volume: 2 μL. The mass spectrometry conditions were as follows: electrospray ion source (ESI), spray voltage: 3.8 kV (ESI+), -3.5 kV (ESI-); capillary temperature: 320°C, auxiliary heater temperature: 300°C; sheath gas pressure: 35 arb, auxiliary gas pressure: 10 arb; scanning mode: Full MS / dd-MS2. The ion chromatogram, primary and secondary mass spectra of the sample were obtained.

[0162] Step 7: Select peak 10 on the ultra-high performance liquid chromatogram obtained in step 5, and infer its retention time on ultra-high performance liquid chromatography-mass spectrometry by calculating the relative retention time of peak 10 and the quercetin chromatogram peak (R peak) on the ultra-high performance liquid chromatogram. The specific calculation formula is T x2 =T r2 (T x1 / T r1 ). (T x1 is the retention time of peak 10 on the ultra-high performance liquid chromatography (18.47 min), T r1 is the retention time of quercetin peak on UPLC (19.87min), T x2 is the retention time of peak 11 on UPLC-MS, T r2 is the retention time of quercetin peak on ultra-high performance liquid chromatography-mass spectrometry (18.44 min), and the retention time of peak 10 on ultra-high performance liquid chromatography-mass spectrometry is calculated to be 17.14 min;

[0163] Step 8: Extract the mass spectrum at the retention time of 17.14±0.3min, and further analyze it by comparing with the database and literature. The chemical component corresponding to peak 10 is identified as diferuloylputrescine. Its secondary mass spectrum is as follows: Figure 4 -J, the main cleavage pathways are as follows Figure 6 -J shown;

[0164] Step 9: Repeat steps 7 and 8 to qualitatively identify the chemical components corresponding to peaks 1 to 9 and peaks 11 to 15 in the ultra-high performance liquid chromatogram of the test sample as follows: peak 1 is caffeic acid, peak 2 is quercetin-3-gentiobioside, peak 3 is vanillin, peak 4 is ferulic acid, peak 5 is rutin, peak 6 is hyperoside, peak 7 is aventuramide E, peak 8 is N-caffeoyltyramine, peak 9 is tribulamide, and peak 10 is quinoline. Peak 1 is N-[4,5-dihydroxy-2-[[4-[hydroxy(phenyl)methyl]-5-oxo-3-phenyl-2H-furan-2-yl]oxy]-6-(methoxymethyl)oxa-3-yl]acetamide, peak 12 is apigenin-7-xyloside, peak 13 is crotonamide, peak 14 is cannabinoid F, peak 15 is chrysin-5-xyloside, and peak 16 is ethyl p-methoxycinnamate. Its secondary mass spectrum is shown as follows Figure 4 As shown, the main cleavage pathways are Figure 5 , 6 shown.

[0165] Step 10: Select vanillin, ferulic acid, rutin, hyperoside, and ethyl p-methoxycinnamate standards and perform sample analysis according to the liquid phase conditions of step 2 in the embodiment, and use "external standard" to identify the peaks of the ultra-high performance liquid chromatogram, see Figure 7 The concentrations of the five chemical components were 6.92 μg / mL of vanillin, 2.389 μg / mL of ferulic acid, 1.780 μg / mL of rutin, 2.552 μg / mL of hyperoside, and 117.719 μg / mL of ethyl p-methoxycinnamate.

[0166] The total ion current of peaks 1 to 16 and peak R by ultra-high performance liquid chromatography-mass spectrometry is shown in the figure Figure 8 The error between the retention time of the compounds corresponding to the 16 common peaks inferred by this invention and the measured value on ultra-high performance liquid chromatography-mass spectrometry is shown in Table 9

[0167] Table 9 Comparison results of retention time estimation and measured values.

[0168]

[0169]

[0170] The results of the above examples show that the retention time of each chemical component inferred by the method of this example is less than 0.3min compared with the actual measured value. The correlation between ultra-high performance liquid chromatography and ultra-high performance liquid chromatography-mass spectrometry can be achieved, providing specific chemical component information of 16 common peaks for the fingerprint of stir-fried Tribulus terrestris formula granules (see Table 10), of which 5 compounds have been confirmed by reference substances.

[0171] Table 10 Compounds corresponding to the 16 common peaks.

[0172]

[0173] Note: Those marked with "*" have been confirmed by reference materials

[0174] Experimental example.

[0175] The liquid phase conditions and methodology of the fingerprint spectrum of stir-fried Tribulus terrestris formula granules were investigated and determined in accordance with General Chapter 0512 of the "Chinese Pharmacopoeia 2020 Edition".

[0176] Experimental Example 1 Investigation of ultra-high performance liquid chromatography conditions.

[0177] 1.1 Investigation of different mobile phase compositions: Take the stir-fried Tribulus terrestris formula granules, prepare the sample solution according to the method described in step 1 of Example 1, analyze according to the liquid phase conditions described in step 2, and compare the chromatograms of acetonitrile-0.1% formic acid water and acetonitrile-water as mobile phases. The results show that the chromatogram of the mobile phase composed of acetonitrile-0.1% formic acid aqueous solution has low baseline noise, the best peak shape, high response value, and good separation. Fig. 9 .

[0178] 1.2 Investigation of different column temperatures: Take the stir-fried Tribulus terrestris formula granules, prepare the sample solution according to the method described in step 1 of Example 1, analyze according to the liquid phase conditions described in step 2, and compare the chromatograms at column temperatures of 25°C, 30°C, and 35°C. The results show that the chromatogram peak shape and separation degree are the best when the column temperature is 30°C. Fig.10 .

[0179] 1.3 Investigation of different detection wavelengths: In the preliminary exploration of experimental conditions, the DAD detector was used to perform ultraviolet full-wavelength scanning on the stir-fried Tribulus terrestris formula granules, and it was found that the number of chromatographic peaks at a wavelength of 340nm was the largest, and all had good ultraviolet absorption. Take the stir-fried Tribulus terrestris formula granules, prepare the sample solution according to the method described in step 1 of Example 1, analyze it according to the liquid phase conditions described in step 2, and compare the chromatograms at wavelengths of 250nm, 300nm, and 340nm. The results show that the baseline in the 250nm chromatogram is unstable, and the response value of some common peaks in the 300nm chromatogram is too large, which puts a lot of pressure on the equipment. The 340nm chromatogram has the best peak shape, appropriate response value, and the best separation. Fig.11 .

[0180] 1.4 Investigation of different injection volumes: Take the stir-fried Tribulus terrestris formula granules, prepare the sample solution according to the method described in step 1 of Example 1, analyze according to the liquid phase conditions described in step 2, and compare the chromatograms with injection volumes of 1 μL and 2 μL. The results show that the chromatogram with 2 μL has the best peak shape, appropriate response value, and the best separation, see Fig.12 .

[0181] 1.5 Investigation of different elution gradients: Take the stir-fried Tribulus terrestris formula granules, prepare the sample solution according to the method described in step 1 of Example 1, use acetonitrile (A)-0.1% formic acid aqueous solution (B) as the mobile phase, and perform elution according to the gradients of Tables 11-13 respectively. Column temperature 30℃; detection wavelength 340nm; flow rate 0.2mL / min, injection volume 2μL. The results show that the chromatogram obtained under gradient 3 has a stable baseline, good separation, and good peak shape. Fig.13 .

[0182] Table 11 Gradient elution program 1.

[0183]

[0184] Table 12 Gradient elution program 2.

[0185]

[0186] Table 13 Gradient elution program 3.

[0187]

[0188]

[0189] Experimental Example 2: Methodological investigation of ultra-high performance liquid chromatography.

[0190] 2.1 Precision test: Take the stir-fried Tribulus terrestris formula granules, prepare the sample solution according to the method described in step 1 under Example 1, analyze according to the liquid phase conditions described in step 2, inject the sample 6 times continuously, record the chromatogram, take peak No. 9 as the reference peak S, and calculate the relative retention time and relative peak area of ​​each chromatographic peak.

[0191] The results of the precision test show (see Tables 14 and 15) that the relative retention time RSD values ​​of each chromatographic peak of the stir-fried Tribulus terrestris formula granules are all less than 0.2%, and the relative peak area RSD values ​​are all less than 2.00%, indicating that the instrument has good precision.

[0192] Table 14 Precision test - relative retention time.

[0193]

[0194] Table 15 Precision - relative peak area.

[0195]

[0196]

[0197] 2.2 Repeatability test: Take the stir-fried Tribulus terrestris formula granules, prepare 6 sample solutions in parallel according to the method described in step 1 under Example 1, analyze according to the liquid phase conditions described in step 2, inject 6 times continuously, record the chromatogram, take peak No. 9 as the reference peak S, and calculate the relative retention time and relative peak area of ​​each chromatographic peak.

[0198] The results of the repeatability test show (see Tables 16 and 17) that the RSDs of the relative retention times of the chromatographic peaks of the stir-fried Tribulus terrestris formula granules are all less than 0.40%, and the RSDs of the relative peak areas are all less than 3.00%. The experiment shows that the method has good repeatability.

[0199] Table 16 Repeatability test - relative retention time.

[0200]

[0201] Table 17 Repeatability test - relative peak areas.

[0202]

[0203]

[0204] 2.3 Stability test: Take the stir-fried Tribulus terrestris formula granules, prepare the sample solution according to the method described in step 1 under Example 1, and according to the liquid phase conditions described in step 2, sample injection and detection are performed at 0h, 2h, 4h, 8h, 16h, and 24h, respectively, and the chromatogram is recorded. Peak 9 is used as the reference peak S, and the relative retention time and relative peak area of ​​each chromatographic peak are calculated.

[0205] The results of the stability test showed (see Tables 18 and 19) that the relative retention time RSDs of the chromatographic peaks of the stir-fried Tribulus terrestris formula granules were all less than 0.70%, and the relative peak area RSDs were all less than 3.00%, indicating that the samples were stable within 24 hours.

[0206] Table 18 Stability test - relative retention time.

[0207]

[0208] Table 19 Stability test - relative peak areas.

[0209]

[0210] 2.4 Specificity test: Preparation of reference solution: Take appropriate amount of vanillin, ferulic acid, rutin, hyperoside, and ethyl p-methoxycinnamate reference substances respectively, weigh accurately, and add methanol to prepare mixed reference solutions containing 6.92μg, 2.389μg, 1.780μg, 2.552μg, and 117.719μg per 1mL. Take the stir-fried Tribulus terrestris formula granules, and prepare the sample solution and blank solution according to the method described in step 1 under Example 1. The above mixed reference solution, sample solution, and blank solution are analyzed in turn according to the liquid phase conditions described in step 2, and the chromatograms are recorded. The results are shown in Table 1. Fig.14 The blank solution (methanol) had no obvious effect on the chromatographic peaks of the sample solution and the reference solution.

[0211] The applicant declares that the above-mentioned embodiments only express the implementation mode of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. The technicians in the relevant technical field should understand that, without departing from the concept of the present invention, several deformations and improvements can be made. It is not necessary and impossible to enumerate all the implementation modes here, and the obvious changes or changes derived therefrom are still within the protection scope of the present invention. The Mongolian medicine stir-fried Tribulus terrestris formula granules and the preparation method and fingerprint spectrum construction method of the present invention are applied to the Mongolian medicine stir-fried Tribulus terrestris of the present invention to achieve excellent results, and are not applicable to other Mongolian medicines. Therefore, the technical solution required to be protected by the claims has outstanding substantive characteristics and significant progress.

Claims

1. A Mongolian medicine stir-fried Tribulus terrestris formula granule, characterized in that: The components include stir-fried Tribulus terrestris powder and medicinal excipients; The stir-fried Tribulus terrestris powder is a Mongolian medicine stir-fried Tribulus terrestris powder that can pass through a sieve of 80-120 meshes after being crushed, and the stir-fried Tribulus terrestris powder is a genuine Mongolian medicine. The pharmaceutical excipient is one or a combination of sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose and polyvinyl pyrrolidone.

2. The Mongolian medicine stir-fried Tribulus terrestris formula granules according to claim 1, characterized in that: The added amount of the sodium carboxymethyl cellulose is 1wt%~5wt%; the added amount of hydroxypropyl methyl cellulose is 3wt%~7wt%; and the added amount of polyvinyl pyrrolidone is 3wt%~7wt%.

3. A method for preparing Mongolian medicine stir-fried Tribulus terrestris formula granules, characterized in that: The specific steps include: Step 1: grind the Mongolian medicine stir-fried Tribulus terrestris and sieve it to obtain stir-fried Tribulus terrestris fine powder; Step 2: mixing the stir-fried fine powder of Tribulus terrestris obtained in step 1 with pharmaceutical excipients and purified water, and performing wet granulation; Step 3: granulate the granules obtained in step 2 and dry them, and then granulate them after drying to obtain Mongolian medicine stir-fried Tribulus terrestris formula granules.

4. The method for preparing the Mongolian medicine stir-fried Tribulus terrestris formula granules according to claim 3, characterized in that: The sieving in step 1 is sieving through a 80-120 mesh sieve; the purified water in step 2 is medicinal purified water, and the added amount is 60-80 mL / 100 g of fried Tribulus terrestris powder; the drying temperature in step 3 is 40° C.-60° C., and the drying time is 3 h-6 h.

5. A method for constructing a fingerprint spectrum of Mongolian medicine stir-fried Tribulus terrestris formula granules, characterized in that: The steps include: Step 1: grind the stir-fried Tribulus terrestris formula granules into fine powder, accurately add the extraction solvent, perform ultrasonic extraction, filter, and take the filtrate to obtain the sample solution; Step 2: The sample solution obtained in step 1 is tested by ultra-high performance liquid chromatography to obtain a sample chromatogram; Step 3: import the sample chromatogram obtained in step 2 into the chromatographic evaluation software, mark the common peaks, establish the fingerprint spectrum, generate the reference spectrum, and calculate the similarity between the fingerprint spectrum of each batch of samples and the reference spectrum, and evaluate the quality of multiple batches of stir-fried Tribulus terrestris formula granules by the similarity; Step 4: Select a suitable internal standard component, add methanol to mix, prepare an internal standard solution, mix the internal standard solution with the sample solution to prepare a test solution; Step 5: Detecting the test solution by ultra-high performance liquid chromatography to obtain an ultra-high performance liquid chromatogram of the test solution; Step 6: The test solution is tested by ultra-high performance liquid chromatography-mass spectrometry to obtain an ultra-high performance liquid chromatography-mass spectrometry ion chromatogram, primary and secondary mass spectrometry related information of the test sample; Step 7: Select an unknown chromatographic peak with a retention time close to that of the internal standard component chromatographic peak on the ultra-high performance liquid chromatogram obtained in step 5, and infer its retention time on ultra-high performance liquid chromatography-mass spectrometry by calculating the relative retention time of the unknown chromatographic peak and the internal standard component chromatographic peak on the ultra-high performance liquid chromatogram; the specific calculation formula is Tx2=Tr2 (Tx1 / Tr1), Tx1 is the retention time of the unknown chromatographic peak on the ultra-high performance liquid chromatogram, Tr1 is the retention time of the internal standard component chromatographic peak on the ultra-high performance liquid chromatogram, Tx2 is the retention time of the unknown chromatographic peak on the ultra-high performance liquid chromatography-mass spectrometry, and Tr2 is the retention time of the internal standard component chromatographic peak on the ultra-high performance liquid chromatography-mass spectrometry; Step 8: Further analyze and qualitatively identify the chemical components corresponding to the unknown chromatographic peak according to the mass spectrum at the retention time of the unknown chromatographic peak inferred in step 7; Step 9: Repeating steps 7 and 8 can achieve qualitative identification of all unknown chromatographic peaks in the ultra-high performance liquid chromatogram; Step 10: According to the qualitative results, select the corresponding compound standard and perform injection analysis according to the liquid phase conditions of step 2, and perform reference confirmation on the ultra-high performance liquid chromatogram; In the step 2, the detection wavelength of the ultra-high performance liquid chromatography is 250nm~340nm; the chromatographic column used is a C18 chromatographic column, the flow rate is 0.2mL / min; the column temperature is 25~35°C; the injection volume is 1μL~5μL; acetonitrile is used as mobile phase A, 0.1% formic acid water is used as mobile phase B, and gradient elution is performed; The gradient elution procedure includes: From 0 to 10 min, the volume fraction of mobile phase A changed from 13% to 17% to 16% to 20%; 10-11 min, the volume fraction of mobile phase A changed from 16%-20% to 23%-27%; From 11 to 30 min, the volume fraction of mobile phase A changed from 23% to 27% to 33% to 37%; From 30 to 35 min, the volume fraction of mobile phase A changed from 33% to 37% to 58% to 62%; From 35 to 40 min, the volume fraction of mobile phase A changed from 58% to 62% to 73% to 77%.

6. The method for constructing the fingerprint spectrum of the Mongolian medicine stir-fried Tribulus terrestris formula granules according to claim 5 is characterized in that: In step 3, 16 common peaks are labeled, with peak 9 as the reference peak. The relative retention times and relative peak areas of the 16 peaks are as follows: Peak 1: relative retention time is 0.231-0.234, relative peak area is 0.057-0.066; Peak 2: relative retention time is 0.338~0.344, relative peak area is 0.090~0.104; Peak 3: relative retention time is 0.369~0.376, relative peak area is 0.067~0.077; Peak 4: relative retention time is 0.484-0.489, relative peak area is 0.123-0.146; Peak 5: relative retention time is 0.510~0.517, relative peak area is 0.037~0.049; Peak 6: relative retention time is 0.535~0.542, relative peak area is 0.052~0.062; Peak 7: relative retention time is 0.787~0.795, relative peak area is 0.066~0.080; Peak 8: relative retention time is 0.813-0.818, relative peak area is 0.107-0.124; Peak 9: relative retention time is 1.000, relative peak area is 1.000; Peak 10: relative retention time is 1.049~1.052, relative peak area is 0.067~0.087; Peak 11: relative retention time is 1.347~1.350, relative peak area is 0.063~0.076; Peak 12: relative retention time is 1.670-1.674, relative peak area is 0.063-0.067; Peak 13: relative retention time is 1.803~1.808, relative peak area is 0.095~0.118; Peak 14: relative retention time is 1.825-1.832, relative peak area is 0.057-0.079; Peak 15: relative retention time is 2.045-2.053, relative peak area is 0.014-0.016; Peak 16: relative retention time is 2.208~2.215, and relative peak area is 1.427~1.

195.

7. The method for constructing the ultra-high performance liquid phase fingerprint of the stir-fried Tribulus terrestris formula granules according to claim 5, characterized in that: The internal standard component chromatographic peak in step 7 is the internal standard component added to the test solution in step 4, or a known chemical component in the sample to be tested.

8. The method for constructing the fingerprint spectrum of the Mongolian medicine stir-fried Tribulus terrestris formula granules according to claim 5 is characterized in that: In step 9, the qualitative identification of all the unknown chromatographic peaks is that peak 1 is caffeic acid, peak 2 is quercetin-3-gentiobioside, peak 3 is vanillin, peak 4 is ferulic acid, peak 5 is rutin, peak 6 is hyperoside, peak 7 is avenanthramide E, peak 8 is N-caffeoyltyramine, peak 9 is tribulamide, peak 10 is diferuloylputrescine, peak 11 is N-[ 4,5-dihydroxy-2-[[4-[hydroxy(phenyl)methyl]-5-oxo-3-phenyl-2H-furan-2-yl]oxy]-6-(methoxymethyl)oxa-3-yl]acetamide, peak 12 is apigenin-7-xyloside, peak 13 is crotonamide, peak 14 is cannabinoid F, peak 15 is chrysin-5-xyloside, and peak 16 is ethyl p-methoxycinnamate.

9. The method for constructing the fingerprint spectrum of the Mongolian medicine stir-fried Tribulus terrestris formula granules according to claim 5, characterized in that: In step 10, the compound standards are vanillin, ferulic acid, rutin, hyperoside, and ethyl p-methoxycinnamate, and the concentrations of the injection analysis are 6.92 μg / mL, 2.389 μg / mL, 1.780 μg / mL, 2.552 μg / mL, and 117.719 μg / mL, respectively.

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