A Mongolian medicine stir-fried tribulus terrestris formula granule and its fingerprint construction method and fingerprint

Abstract

The present invention belongs to the technical field of Mongolian medicine preparation and quality inspection, and particularly relates to a Mongolian medicine stir-fried Tribulus terrestris formula granule and a method for constructing its fingerprint and a fingerprint. The Mongolian medicine stir-fried Tribulus terrestris formula granule comprises stir-fried Tribulus terrestris fine powder and pharmaceutical excipients; the fingerprint construction method comprises: extracting the stir-fried Tribulus terrestris formula granule, filtering it, measuring it using ultra-high performance liquid chromatography, constructing a fingerprint, and using "double label and one mass spectrometry" to identify compounds on common peaks. The Mongolian medicine stir-fried Tribulus terrestris formula granule prepared by the present invention has a high forming rate, good fluidity, low hygroscopicity, and good stability. The constructed fingerprint has the advantages of good stability, high precision, and good repeatability. It can fully reflect the intrinsic quality of the stir-fried Tribulus terrestris formula granule, has lower operating costs than completely using liquid chromatography to construct a fingerprint, is faster than ordinary HPLC fingerprint analysis, and consumes less mobile phase solution.

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] Stir-fried Tribulus terrestris (TRIBULI FRUCTUS) is the dried, mature fruit of Tribulus terrestris L., a plant of the Tribulaceae family. According to Mongolian medicine, stir-fried Tribulus terrestris is sweet, slightly bitter, warm, mild, sharp, and thin. It can dispel kidney cold, calm kidney disease, promote urination, reduce swelling, and strengthen the body. It is used to treat frequent urination, urinary retention, kidney cold, kidney disease, lower back and leg pain, wandering pain syndrome, nocturnal emission, impotence, and edema. Modern research indicates that stir-fried Tribulus terrestris has antihypertensive, immune-modulating, detoxifying, and antipruritic properties, as well as acetylcholine inhibition. Traditional Mongolian medicine prefers to grind the herb into a powder for direct use, but this method is difficult for patients to adapt to, resulting in difficulty swallowing and an unpleasant taste. Furthermore, this powder easily absorbs moisture, has poor flowability, and is unsuitable for modern automatic dispensing machines. To address these issues, Mongolian medicine granules emerged as the times require. While ensuring efficacy, they offer advantages such as a pleasant taste, easy dosage adjustment, good fluidity, low hygroscopicity, and uniform specifications and standards. However, due to the varying physical properties of each medicinal material, the appropriate excipients and their proportions for each medicinal material vary, which is not obvious to those skilled in the art. This complicates the preparation of Mongolian medicine granules. Fingerprint technology, characterized by its integrity and ambiguity, can more comprehensively reflect the intrinsic components of a drug. It is now widely used in the quality evaluation and authenticity verification of traditional Chinese medicine and ethnic medicines, and has gained widespread international recognition, 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 chromatography (HPLC) fingerprints for stir-fried Tribulus terrestris and related preparations. The 2020 edition of the Chinese Pharmacopoeia still uses UV-visible spectrophotometry to determine the content of the active ingredient as the quality evaluation standard for Tribulus terrestris. Improving the quality control methods for stir-fried Tribulus terrestris has become a pressing issue. While fingerprints can comprehensively reflect the intrinsic components of a drug, they struggle to provide detailed information about specific compounds. Therefore, some researchers have used liquid chromatography-mass spectrometry to construct fingerprints, but this approach is prohibitively expensive, making it difficult to popularize. Furthermore, some researchers have attempted to construct fingerprints using HPLC and qualitatively identify the chemical components in fingerprints. However, due to the different internal structures of HPLC and HPLC instruments, even under the same chromatographic column and HPLC conditions, the retention times of the same compound can vary significantly, making it impossible to match chromatographic peaks between HPLC and HPLC. Furthermore, the complex composition of traditional Chinese medicines may include some unavailable or unavailable reference compounds, making fingerprint peak identification impossible. Currently, no literature or patents have reported solutions to these problems. Summary of the Invention

[0004] The present invention provides a Mongolian medicine stir-fried Tribulus terrestris formula granule and a preparation method thereof, aiming to solve the problems of poor compliance, inconvenience in carrying, easy moisture absorption, and poor fluidity associated with the traditional stir-fried Tribulus terrestris administration method (i.e., stir-fried Tribulus terrestris powder). A method for constructing a fingerprint of the stir-fried Tribulus terrestris formula granule is also provided. This construction method belongs to the "double-label-one-mass" method. The "internal standard" is used to infer the retention time of an unknown chromatographic peak in an ultra-high performance liquid chromatography (ULHPLC-MS) on an ULHPLC-MS, so that the chromatographic peaks on the ULHPLC-MS correspond to each other, serving as a bridge connecting the two. The "external standard" is used to identify peaks in the liquid chromatogram using a reference substance, which also verifies the results of the qualitative compound identification using the ULHPLC-MS. The "one-mass" method is used to qualitatively identify the compound using the ULHPLC-MS. The three methods are combined into a "double-label-one-mass" method. 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 testing system of ethnic medicine.

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

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

[0007] Furthermore, the stir-fried Tribulus terrestris fine powder is the product of Mongolian 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 carboxymethylcellulose (CMC-NaPH7), hydroxypropyl methylcellulose (HPMC E5), and polyvinylpyrrolidone (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 polyvinylpyrrolidone (PVP K-25, PVP K-30) in the pharmaceutical excipient is 3wt% to 7wt%.

[0012] The present invention also provides a preparation method of 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 Tribulus terrestris powder obtained in step 1 with pharmaceutical excipients and purified water, and performing wet granulation;

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

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

[0017] Preferably, the purified water in step 2 is medicinal purified water, and the added amount is 60 to 80 mL / (100 g of 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 in 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 granules, comprising the following steps:

[0024] Step 1: Grind the stir-fried Tribulus terrestris granules into powder, precisely add the extraction solvent, perform ultrasonic extraction, filter, and obtain 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 a fingerprint spectrum, generate a 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 based on the similarity;

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

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

[0029] Step 6: The test sample solution is detected by ultra-high performance liquid chromatography-mass spectrometry to obtain the 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 estimate 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 chromatography, T r1 T is the retention time of the internal standard component chromatographic peak on the ultra-high performance liquid chromatography. x2 is the retention time of the unknown chromatographic peak on ultra-high performance liquid chromatography-mass spectrometry, T r2 is the retention time of the internal standard component chromatographic peak on ultra-performance liquid chromatography-mass spectrometry);

[0031] Step 8: Based on the mass spectrum at the retention time of the unknown chromatographic peak inferred in step 7, further analyze and qualitatively identify the chemical component corresponding to the unknown chromatographic peak;

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

[0033] Step 10: Based on 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 chromatography with a 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 250 nm to 340 nm, preferably 340 nm; the chromatographic column used in the ultra-high performance liquid chromatography is a C18 chromatographic column (100×2.1 mm, 1.8 μm), and the flow rate of the ultra-high performance liquid chromatography is 0.2 mL / min; the column temperature of the ultra-high performance liquid chromatography is 25 to 35° C., preferably 30° C.; the injection volume of the ultra-high performance liquid chromatography is 1 μL to 5 μL, preferably 2 μL; 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] Furthermore, the gradient elution procedure includes:

[0038] From 0 to 10 min, the volume fraction of mobile phase A changed 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 changed 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 was changed from 15% to 18%;

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

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

[0047] From 30 to 35 min, the volume fraction of mobile phase A was 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 marked, with peak 9 being used as the reference peak. 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 greater than 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 chromatography chart 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 the ultra-high performance liquid chromatography-mass spectrometry in step 6 should be consistent with the chromatography 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 minutes; if the retention time of the unknown chromatographic peak is outside ±5 minutes, 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 of the internal standard component.

[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 present invention has the following beneficial effects.

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

[0076] 2. The present invention proposes and prepares a Mongolian medicine stir-fried Tribulus terrestris formula granule, which uses only a single pharmaceutical excipient and purified water. This simplifies the formula, reduces costs, simplifies the production process, and minimizes the impact of excipients on the efficacy of the medicinal material. Compared with traditional stir-fried Tribulus terrestris powder, the angle of repose decreases by 17.8° to 21.3°, increasing fluidity, facilitating clinical drug dispensing and adapting to modern automatic dispensing machines. The moisture absorption rate decreases by 4.1% to 7.8%, reducing hygroscopicity and increasing stability. Palatability is improved, and patient compliance is enhanced. This improves the Mongolian medicine dosage form, resolving issues such as poor fluidity, moisture absorption, harsh storage conditions, inconvenience in administration, and portability associated with traditional Mongolian medicine administration methods. It also further contributes to the development of Mongolian medicine and other ethnic medicines.

[0077] 3. The present invention establishes a UPLC fingerprint determination method for the Mongolian medicine stir-fried Tribulus terrestris formula granules, optimizing the UPLC detection conditions, including the composition of the mobile phase, detection wavelength, gradient elution program, column temperature, flow rate, injection volume, and other parameters. Using the optimized method and parameters, the constructed UPLC fingerprint has a stable baseline, good resolution, high precision, and good repeatability, which can comprehensively reflect the intrinsic quality of the Mongolian medicine stir-fried Tribulus terrestris, filling the gap in the lack of stir-fried Tribulus terrestris related fingerprint construction methods in China and improving existing quality standards. In addition, based on the "double label and single mass" technology, it provides complete compound information corresponding to the common peaks, and can also perform peak identification for some extremely expensive or commercially unavailable reference compounds. Compared with traditional HPLC fingerprint analysis, the analysis speed is faster and the consumption of mobile phase solution is less; compared with the complete use of liquid chromatography-mass spectrometry to construct fingerprints, this method only requires a few injections of liquid chromatography-mass spectrometry, with low operating costs, providing a more scientific and reasonable basis for the quality control of stir-fried Tribulus terrestris. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

[0081] Figure 4 The following are the secondary mass spectra of the 16 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, 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 crotonamide, N is cannabinoid F, O is chrysin-5-xyloside, and P is ethyl p-methoxycinnamate);

[0082] Figure 5 The main fragmentation pathways of the nine 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 The main cleavage pathways of the seven compounds in Example 5 of the present invention (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 crotonamide, 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 chromatogram of the test sample is performed for the "external standard" reference substance of the present invention (A is the chromatogram of the test sample, B is the chromatogram of the "external standard" reference substance);

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

[0086] Figure 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] Figure 10 The chromatograms obtained 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] Figure 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] Figure 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] Figure 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] Figure 14 This is the specific chromatogram in Experimental Example 2 of the present invention (A is the sample solution of 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 illustrated below by means of specific implementation methods. The embodiments are only for helping to understand the present invention and should not be regarded as specific limitations of the present invention.

[0093] In the present invention, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional values ​​within the numerical interval is considered continuous and includes the two numerical endpoints of the numerical range (i.e., the minimum and maximum values), as well as every value between these two numerical endpoints. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, these ranges can be combined when including the numerical range to describe a feature or characteristic. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges subsumed therein.

[0094] The temperature parameters in the present invention, unless otherwise specified, may be either constant temperature or fluctuating within a certain temperature range. It should be understood that the constant temperature treatment allows for temperature fluctuations within the precision range of instrument control. Fluctuations within the ranges of, for example, ±0.5°C, ±0.4°C, ±0.3°C, ±0.2°C, or ±0.1°C are permitted.

[0095] In the present invention, weight can be μg, mg, g, kg and 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 Mongolian medicine stir-fried Tribulus terrestris formula granules preferably have a moisture content of less than 8%; the angle of repose of the Mongolian medicine stir-fried Tribulus terrestris formula granules is less than 40°; and the particle size of the Mongolian medicine stir-fried Tribulus terrestris formula granules is less than 10% as measured by the particle size and particle size distribution method (General Chapter 0982, Method 2) of the "Chinese Pharmacopoeia" (2020 edition). Compared with stir-fried Tribulus terrestris powder, the prepared formula granules have a reduced angle of repose, increased fluidity, decreased hygroscopicity, and increased stability, making them easier to package, transport, and store; and they also have improved palatability and enhanced patient compliance.

[0098] The present invention has no special requirements on the source of the above-mentioned Mongolian medicine, and it can be purchased from a Mongolian medicine 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 this field 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 in accordance with the method in the general rules 0104 of the fourth volume of the "Chinese Pharmacopoeia"; the medicinal material quality standard identification is inspected in accordance with 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, and reagents used in the specific embodiments of the present invention are 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 spectrometry system (Thermo Fisher Scientific, USA, electrospray ionization source), MCA10.6S-2CCN-M one-millionth electronic balance (Sartorius Scientific Instrument (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: Stir-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 in our laboratory, and the stir-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%) were 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 preparation method of 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 pH 7) 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 then 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 preparation method of 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 pH 7) 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 then 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 preparation method of 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 5 wt% hydroxypropyl methylcellulose (HPMC E5) for 30 minutes, then add 70 mL / (100 g 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 then 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 preparation method of 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% polyvinylpyrrolidone (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 then 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 was mixed with the auxiliary materials in Table 5 and purified water respectively, and stirred to obtain a soft material, which was then placed in a 10-mesh granulator for granulation.

[0141] Step 3: The obtained granules were dried at 60° C. for 4 h, and after drying, sieved using 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 10 combinations of stir-fried Tribulus terrestris formula granules obtained in step 3 above. 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 the 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 preferred by the present invention (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 terrestris formula granules decreased by 38.3% to 40.0%, the angle of repose decreased by 7.5° to 11.0°, the moisture absorption rate decreased by 2.0% to 5.6%, the granule yield rate increased by 38.6% to 40.0%, and the palatability improved; compared with other addition amounts of the four preferred medicinal excipients of the present invention, the soft material is moderate in the preparation process of the stir-fried tribulus terrestris formula granules, mixing is easy, clogging of the granulation machine can be avoided, the granulation process is smooth, the particle size of the prepared stir-fried tribulus terrestris formula granules decreased by 9.5% to 24.6%, the angle of repose decreased by 4.3° to 9.8°, the moisture absorption rate decreased by 1.3% to 5.0%, the granule yield rate increased by 12.1% to 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 carboxymethylcellulose, 98.5wt% stir-fried Tribulus terrestris fine powder) and accurately weigh 1g of each. Place the mixture in a 250mL stoppered conical flask, add 100mL of methanol, accurately weigh the weight, and perform ultrasonic extraction (200W, frequency 59kHz) for 30min. Cool the mixture, 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 were tested using ultra-performance liquid chromatography to obtain chromatograms of 20 batches of samples. The chromatographic conditions were as follows:

[0153] Chromatographic columns UHPLC XB-C18 column (2.1 mm × 100 mm, 1.8 μm); mobile phase: acetonitrile as mobile phase A, 0.1% formic acid aqueous solution as mobile phase B, gradient elution as specified in the table below (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 20 batches of sample chromatograms obtained in step 2 into the 2012 edition of the "Chinese Herbal Chromatographic Fingerprint Similarity Evaluation System" and use the median method to generate superimposed fingerprints and reference fingerprints (see Figure 2 Using the generated control fingerprint R as a reference, the similarity of the fingerprints of each batch of stir-fried Tribulus terrestris granules was evaluated. The results showed that the similarity of the fingerprints of all 20 batches of stir-fried Tribulus terrestris granules was above 0.90 (see Table 8). The good similarity indicates that the overall quality of the stir-fried Tribulus terrestris 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 column (2.1 mm × 100 mm, 1.8 μm); 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 ultra-high performance liquid chromatogram of the test sample was obtained (as shown in FIG. Figure 3 ).

[0161] Step 6: The test solution was detected by ultra-high performance liquid chromatography-mass spectrometry. The chromatographic conditions were as follows: chromatographic column UHPLC XB-C18 column (2.1 mm × 100 mm, 1.8 μm); 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. Mass spectrometry conditions were as follows: electrospray ionization (ESI) source, spray voltages: 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; scan mode: Full MS / dd-MS2. Ultra-high performance liquid chromatography-mass spectrometry ion chromatograms, primary and secondary mass spectra were obtained for the sample.

[0162] Step 7: Select peak 10 on the ultra-high performance liquid chromatogram obtained in step 5, and calculate the relative retention time of peak 10 and the quercetin chromatographic peak (R peak) on the ultra-high performance liquid chromatogram to estimate its retention time on ultra-high performance liquid chromatography-mass spectrometry. 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 ultra-high performance liquid chromatography (19.87 min), T x2 is the retention time of peak 11 on ultra-high performance liquid chromatography-mass spectrometry, 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 was 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 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, peak 10 is benzoic acid, peak 11 is benzoic acid, peak 12 is benzoic acid, peak 13 is benzoic acid, peak 14 is benzoic acid, peak 15 is benzoic acid, peak 16 is benzoic acid, peak 17 is benzoic acid, peak 18 is benzoic acid, peak 19 is benzoic acid, peak 20 is benzoic acid, peak 21 is benzoic acid, peak 22 is benzoic acid, peak 23 is benzoic acid, peak 24 is benzoic acid, peak 25 is benzoic acid, peak 26 is benzoic acid, peak 27 is benzoic acid, peak 28 is benzoic acid, peak 29 is benzoic acid, peak 30 is benzoic acid, peak 31 is benzoic acid, peak 32 is benzoic acid, peak 33 is benzoic acid, peak 34 is benzoic acid, peak 35 is benzoic acid, peak 36 is benzoic acid, peak 37 is benzoic acid, peak 38 is benzoic acid, peak 39 is benzoic acid, peak 40 is benzoic acid, peak 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. Figure 7 The concentrations of the five chemical components were vanillin 6.92 μg / mL, ferulic acid 2.389 μg / mL, rutin 1.780 μg / mL, hyperoside 2.552 μg / mL, and ethyl p-methoxycinnamate 117.719 μg / mL.

[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 errors between the retention times of the compounds corresponding to the 16 common peaks inferred by this invention and the measured values ​​on ultra-high performance liquid chromatography-mass spectrometry are shown in Table 9.

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

[0168]

[0169]

[0170] The results of the above examples show that the retention times of the chemical components estimated using the method of this example are within 0.3 minutes of the actual measured values ​​by ULCP-MS. ULCP-MS correlation can be achieved, providing detailed chemical composition information for 16 common peaks in the fingerprint of the stir-fried Tribulus terrestris granules (see Table 10), of which 5 compounds have been confirmed using reference substances.

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

[0172]

[0173] Note: The items marked with “*” have been confirmed by the reference materials.

[0174] Experimental example.

[0175] Investigation on the liquid phase conditions and methodology of the fingerprint spectrum of stir-fried Tribulus terrestris formula granules was carried out 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 stir-fried Tribulus terrestris formula granules, prepare sample solution according to the method described in step 1 under 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. Figure 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 under 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 is best and the separation is best when the column temperature is 30°C. Figure 10 .

[0179] 1.3 Investigation of different detection wavelengths: In the early exploration of experimental conditions, the DAD detector was used to perform ultraviolet full wavelength scanning on the stir-fried Tribulus terrestris formula granules. It was found that the number of chromatographic peaks at a wavelength of 340nm was the largest, and all had good ultraviolet absorption. Stir-fried Tribulus terrestris formula granules were taken, and the sample solution was prepared according to the method described in step 1 under Example 1. The analysis was performed according to the liquid phase conditions described in step 2. The chromatograms at wavelengths of 250nm, 300nm, and 340nm were compared. The results showed that the baseline in the 250nm chromatogram was unstable, and the response value of some common peaks in the 300nm chromatogram was too large, which put a lot of pressure on the equipment. The 340nm chromatogram had the best peak shape, appropriate response value, and the best separation, as shown in Figure 2. Figure 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 under Example 1, analyze according to the liquid phase conditions described in step 2, and compare the chromatograms of injection volumes of 1 μL and 2 μL. The results show that the chromatogram of 2 μL has the best peak shape, appropriate response value, and the best separation, see Figure 12 .

[0181] 1.5 Investigation of different elution gradients: Take stir-fried Tribulus terrestris granules and prepare sample solution according to the method described in step 1 of Example 1. Use acetonitrile (A)-0.1% formic acid aqueous solution (B) as mobile phase and perform elution according to the gradients shown in Tables 11-13. Column temperature 30°C; 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. Figure 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: Investigation of the methodology of ultra-high performance liquid chromatography.

[0190] 2.1 Precision test: Take the stir-fried Tribulus terrestris formula granules, prepare a 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, and use peak No. 9 as the reference peak S to calculate the relative retention time and relative peak area of ​​each chromatographic peak.

[0191] The results of the precision test showed (see Tables 14 and 15) that the relative retention time RSD values ​​of each chromatographic peak of the stir-fried Tribulus terrestris formula granules were all less than 0.2%, and the relative peak area RSD values ​​were all less than 2.00%, indicating that the instrument had 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 the sample 6 times continuously, record the chromatogram, and use peak No. 9 as the reference peak S to calculate the relative retention time and relative peak area of ​​each chromatographic peak.

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

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

[0200]

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

[0202]

[0203]

[0204] 2.3 Stability test: Take the stir-fried Tribulus terrestris formula granules, prepare a sample solution according to the method described in step 1 under Example 1, and according to the liquid phase conditions described in step 2, inject and detect at 0h, 2h, 4h, 8h, 16h, and 24h, record the chromatogram, and use peak No. 9 as the reference peak S to calculate the relative retention time and relative peak area of ​​each chromatographic peak.

[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 sample was stable within 24 hours.

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

[0207]

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

[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 solution containing 6.92μg, 2.389μg, 1.780μg, 2.552μg, and 117.719μg per 1mL. Take the stir-fried Tribulus terrestris formula granules, and prepare 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 FIG. Figure 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 merely express the embodiments of the present invention, and the description thereof is relatively specific and detailed, but it should not be understood as limiting the scope of the invention patent. Those skilled in the art should understand that, without departing from the concept of the present invention, several variations and improvements can be made. It is not necessary and impossible to enumerate all the embodiments here, and the obvious changes or modifications derived therefrom are still within the scope of protection of the present invention. The Mongolian medicine stir-fried Tribulus terrestris formula granules, the preparation method thereof, and the fingerprint spectrum construction method in the present invention achieve excellent results only when applied to the Mongolian medicine stir-fried Tribulus terrestris of the present invention, and are not applicable to other Mongolian medicines. Therefore, the technical solution required to be protected by the claims has outstanding substantive features and significant progress.

Claims

1. A method for constructing a fingerprint of Mongolian medicine stir-fried Tribulus terrestris formula granules, characterized in that: The steps include: Step 1: Grind the stir-fried Tribulus terrestris granules into powder, precisely add the extraction solvent, perform ultrasonic extraction, filter, and obtain 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 a fingerprint spectrum, generate a 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 based on the similarity; Step 4: Select a suitable internal standard component, add methanol to mix, and prepare an internal standard solution. Mix the internal standard solution with the sample solution to prepare the test solution. Step 5: The test solution is tested by ultra-high performance liquid chromatography to obtain an ultra-high performance liquid chromatogram of the test sample; Step 6: The test solution was detected by ultra-high performance liquid chromatography-mass spectrometry. The chromatographic conditions were as follows: chromatographic column: Ultimate® UHPLC XB-C18 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; mass spectrometry conditions were as follows: electrospray ionization source ESI, spray voltage: 3.8 kV positive ion mode, -3.5 kV Negative ion mode; capillary temperature: 320°C, auxiliary heater temperature: 300°C; sheath gas pressure: 35arb, auxiliary gas pressure: 10arb; scan mode: Full MS / dd-MS2; obtain the test sample's ULHPLC-MS ion chromatogram, primary and secondary mass spectrometry related information; 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 ultra-high performance liquid chromatography-mass spectrometry, and Tr2 is the retention time of the internal standard component chromatographic peak on ultra-high performance liquid chromatography-mass spectrometry; Step 8: Based on the mass spectrum at the retention time of the unknown chromatographic peak inferred in step 7, further analyze and qualitatively identify the chemical component corresponding to the unknown chromatographic peak; Step 9: Repeat steps 7 and 8 to achieve qualitative identification of all unknown chromatographic peaks in the ultra-high performance liquid chromatogram; Step 10: Based on 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 chromatography with a reference substance; In steps 2 and 5, the detection wavelength of the ultra-high performance liquid chromatography was 340 nm; the chromatographic column used was a C18 column, the flow rate was 0.2 mL / min, the reference wavelength was 400 nm, the column temperature was 30° C., the injection volume was 2 μL, and gradient elution was performed using acetonitrile as mobile phase A and 0.1% formic acid water as mobile phase B. The gradient elution procedure includes: 0-10 min, volume fraction of mobile phase A 15%-18%; 10-11 min, volume fraction of mobile phase A 18%-25%; 11-30 min, volume fraction of mobile phase A 25%-35%; 30-35 min, volume fraction of mobile phase A 35%-60%; 35-40 min, volume fraction of mobile phase A 60%-75%; The Mongolian medicine stir-fried tribulus terrestris formula granules are composed of stir-fried tribulus terrestris fine 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 mesh after being crushed, and the stir-fried Tribulus terrestris powder is an authentic medicinal material. The pharmaceutical excipient is one or a combination of sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose, and polyvinyl pyrrolidone; The added amount of the sodium carboxymethyl cellulose is 1 wt% to 5 wt%; the added amount of the hydroxypropyl methyl cellulose is 3 wt% to 7 wt%; and the added amount of the polyvinyl pyrrolidone is 3 wt% to 7 wt%.

2. The method for constructing the fingerprint of the Mongolian medicine stir-fried Tribulus terrestris formula granules according to claim 1, wherein: In step 3, a total of 16 common peaks were marked, with peak 9 as the reference peak. The relative retention times and relative peak areas of the 16 peaks were 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.

3. The method for constructing an ultra-high performance liquid chromatography fingerprint of a stir-fried Tribulus terrestris formula granule according to claim 1, 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.

4. The method for constructing the fingerprint of the Mongolian medicine stir-fried Tribulus terrestris formula granules according to claim 1, wherein: In step 9, the qualitative identification of all the unknown chromatographic peaks is 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 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.

5. The method for constructing the fingerprint of the Mongolian medicine stir-fried Tribulus terrestris formula granules according to claim 1, 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.

6. The method for constructing the fingerprint of the Mongolian medicine stir-fried Tribulus terrestris formula granules according to claim 1, characterized in that: The preparation method of Mongolian medicine stir-fried tribulus terrestris formula granules specifically comprises the following steps: 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 Tribulus terrestris powder obtained in step 1 with pharmaceutical excipients and purified water, and performing wet granulation; Step 3: granulating and drying the granules obtained in step 2, and then re-granulating the granules after drying to obtain Mongolian medicine stir-fried Tribulus terrestris formula granules.

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

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