Detection method and application of a Chinese chive bulb preparation

Abstract

The present invention provides a detection method and its application for Allium macrostemon Bunge preparations. The detection method includes the following steps: (1) preparing the test Allium macrostemon Bunge preparation into an Allium macrostemon Bunge test sample solution; (2) performing high performance liquid chromatography (HPLC) detection on the Allium macrostemon Bunge test sample solution to obtain a characteristic chromatogram. The HPLC detection conditions are as follows: the detection wavelength is 250 - 258 nm, mobile phase A is acetonitrile, mobile phase B is water, gradient elution is performed, and the flow rate is 0.7 - 0.9 mL / min. The detection method of the present invention can detect Allium macrostemon Bunge preparations efficiently, accurately and stably, obtain multiple common characteristic peaks, with good separation degree and reproducibility of the characteristic peaks, and can effectively identify Allium macrostemon Bunge preparations with Allium macrostemon Bunge as the original plant and Allium macrostemon Bunge preparations with Allium chinense G. Don as the original plant.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a detection method and application of a macrostemon bunge preparation. Background Art

[0002] Macrostemon bunge (scientific name: Allium macrostemon Bunge) is a plant of the genus Allium in the Liliaceae family. Its roots are white and used medicinally, known as macrostemon bunge. The bulb is nearly spherical, and the outer skin of the bulb is black, papery or membranous, not broken, but in specimens, it often only remains the white inner skin due to shedding. There are 3-5 leaves, semi-cylindrical, or triangular semi-cylindrical due to the well-developed longitudinal ridges on the back, hollow, with grooves on the upper surface. The ovary is nearly spherical, with a concave nectary with a curtain at the base of the ventral suture. The style extends outside the perianth. The flowering and fruiting period is from May to July.

[0003] It is produced in all provinces and regions of China, growing on slopes, hills, valleys or grasslands below an altitude of 1500 meters. In a very few areas (Yunnan and Tibet), it can also be found on slopes at an altitude of 3000 meters. It is also distributed in Russia, North Korea and Japan. The bulb is used medicinally and can also be used as a vegetable, and has been cultivated in a few areas.

[0004] Macrostemon bunge is pungent, bitter, warm in nature and non-toxic, and has the effects of regulating qi, widening the chest, promoting yang and resolving stasis. Traditional Chinese medicine has long been used to treat diseases such as chest tightness and stabbing pain, tenesmus after diarrhea, and asthma.

[0005] In the Chinese Pharmacopoeia 2020 Edition, there are two origins for macrostemon bunge medicinal materials, namely the dried bulbs of the plants Allium macrostemon Bge. or Allium chinense G.Don of the Liliaceae family. The Chinese Pharmacopoeia only includes two origins for medicinal use. Through the description of the characteristics of the medicinal materials, Allium macrostemon Bge. is oval-shaped, and Allium chinense G.Don is long oval-shaped. This description can distinguish the two medicinal materials, but after being processed into formula granules, the original characteristics of the medicinal materials are lost and they can no longer be distinguished. For formula granules, the relevant "Technical Requirements" clearly stipulate that for varieties with multiple origins, the origins should be fixed, so it is particularly important to establish an effective method for identifying the origins.

[0006] CN113759057A discloses a characteristic chromatogram of an allium macrostemon bunge water extract and its preparation and a construction method thereof. The construction method includes the following steps: (1) preparation of an allium macrostemon bunge test solution; (2) taking the allium macrostemon bunge test solution and detecting it by high performance liquid chromatography. Using octadecylsilane chemically bonded silica gel as the filler and water-methanol as the mobile phase, the gradient elution program includes: 0→22 min, and the volume percentage of methanol in the mobile phase is: 5%→11%. A characteristic chromatogram is obtained, realizing the separation of common characteristic peaks including adenosine and thymidine. The elution program is simple, the obtained characteristic chromatogram has a stable baseline, less noise interference, good peak shapes of characteristic peaks and high resolution, and the peak positions of adenosine and thymidine can be accurately located, fully reflecting the integrity and characteristics of the allium macrostemon bunge water extract and its preparation, and providing a basis for the quality detection and control of the allium macrostemon bunge water extract and its preparation.

[0007] Although some detection methods of allium macrostemon bunge extracts and their characteristic chromatograms have been disclosed in the prior art, there are still problems such as a small number of common characteristic peaks, poor peak shapes of characteristic peaks, and low resolution. Moreover, the prior art is mainly applied to the quality standard control of allium macrostemon bunge extracts, and no research has been carried out on the distinction of different origins of allium macrostemon bunge extracts. Therefore, developing a detection method for allium macrostemon bunge preparations that can efficiently, accurately, and stably detect allium macrostemon bunge extracts, obtain multiple common characteristic peaks, have good resolution of characteristic peaks, and good reproducibility, and can be applied to distinguish allium macrostemon bunge extracts of different origins is the research focus in this field. Summary of the Invention

[0008] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a detection method and application of allium macrostemon bunge preparations, which can efficiently, accurately, and stably detect allium macrostemon bunge preparations, obtain multiple common characteristic peaks, have good resolution of characteristic peaks, and good reproducibility, and can be applied to distinguish allium macrostemon bunge formula granules of different origins.

[0009] To achieve the purpose of this invention, the following technical solutions are adopted:

[0010] In the first aspect, the present invention provides a detection method for allium macrostemon bunge preparations. The detection method includes the following steps:

[0011] (1) Prepare the allium macrostemon bunge preparation to be tested into an allium macrostemon bunge test solution;

[0012] (2) Take the allium macrostemon bunge test solution and detect it by high performance liquid chromatography to obtain a characteristic chromatogram;

[0013] The conditions for detection by high performance liquid chromatography are as follows: the detection wavelength is 250 - 258 nm, for example, it can be 251 nm, 252 nm, 253 nm, 254 nm, 255 nm, 256 nm or 257 nm, etc., preferably 254 nm; mobile phase A is acetonitrile, mobile phase B is water, gradient elution is carried out, the flow rate is 0.7 - 0.9 mL / min, for example, it can be 0.75 mL / min, 0.8 mL / min or 0.85 mL / min, etc., preferably 0.8 mL / min.

[0014] A detection method for Allium macrostemon Bunge preparations provided by the present invention uses high performance liquid chromatography to detect Allium macrostemon Bunge preparations. Under the conditions that the detection wavelength is 250 - 258 nm (preferably 254 nm), the flow rate is 0.7 - 0.9 mL / min (preferably 0.8 mL / min), mobile phase A is acetonitrile, and mobile phase B is water, gradient elution is carried out, and the characteristic chromatogram of Allium macrostemon Bunge preparations can be obtained stably and accurately. There are multiple common characteristic peaks in the chromatogram, and there is good resolution between each characteristic peak. The characteristic chromatogram has good reproducibility. The test method provided by the present invention has high precision, can effectively identify two original plants of Allium macrostemon Bunge (small root garlic) formula granules and Allium macrostemon Bunge (Allium chinense G. Don) formula granules, effectively control the product quality, and ensure the clinical efficacy. Although high performance liquid chromatography is also used to detect Allium macrostemon Bunge preparations in the prior art, the identification method for the original plants of Allium macrostemon Bunge has not been reported. The detection method for Allium macrostemon Bunge preparations provided by the present invention can determine the content of the main active ingredients in Allium macrostemon Bunge preparations while detecting the characteristic chromatogram of Allium macrostemon Bunge preparations, and the obtained characteristic chromatogram can achieve the identification of the original plants, effectively saving the detection time and detection cost.

[0015] Preferably, the process of gradient elution is as follows: from 0 - 9 min, for example, it can be 2 min, 4 min, 6 min or 8 min, etc., mobile phase A: 0, mobile phase B: 100%; from 9 - 14 min, for example, it can be 10 min, 11 min, 12 min or 13 min, etc., mobile phase A: 0 - 3%, for example, it can be 1%, 2% or 2.5%, etc., mobile phase B: 100 - 97%, for example, it can be 99%, 98% or 97.5%, etc.; from 14 - 21 min, for example, it can be 15 min, 16 min, 18 min or 20 min, etc., mobile phase A: 3%, mobile phase B: 97%; from 21 - 35 min, for example, it can be 22 min, 24 min, 26 min, 28 min, 30 min, 32 min or 34 min, etc., mobile phase A: 3 - 10%, for example, it can be 4%, 6% or 8%, etc., mobile phase B: 97 - 90%, for example, it can be 96%, 94% or 92%, etc.

[0016] Preferably, the filler of the chromatographic column used for detection is octadecylsilane chemically bonded silica gel.

[0017] Preferably, the column temperature for the detection is 28 - 32°C, for example, it can be 29°C, 30°C, or 31°C, etc., and preferably it is 30°C.

[0018] Other specific point values within the above numerical ranges can be selected, and will not be elaborated one by one here.

[0019] Preferably, the Allium macrostemon Bunge preparation is Allium macrostemon Bunge formula granules.

[0020] Preferably, the Allium macrostemon Bunge preparation is selected from the original plant of Allium macrostemon Bunge or Allium chinense G. Don.

[0021] Preferably, the preparation method of the Allium macrostemon Bunge test solution is as follows: extracting the Allium macrostemon Bunge preparation with a solvent, and then performing solid-liquid separation, and the obtained liquid is the Allium macrostemon Bunge test solution.

[0022] Preferably, the solvent is selected from at least one of methanol aqueous solution, water, and ethanol aqueous solution, and preferably it is methanol aqueous solution.

[0023] Preferably, the material-liquid ratio for the extraction is 1:(15 - 25), for example, it can be 1:16, 1:18, 1:20, 1:22, or 1:24, etc., and preferably it is 1:20. Calculated based on 1 g of the Allium macrostemon Bunge preparation, the solvent is 15 - 25 mL.

[0024] Preferably, the extraction method is reflux or ultrasonic, and preferably it is ultrasonic.

[0025] Preferably, the extraction time is 20 - 40 min, for example, it can be 22 min, 24 min, 26 min, 28 min, 30 min, 32 min, 34 min, 36 min, or 38 min, etc., and preferably it is 30 min.

[0026] Preferably, the solid-liquid separation is selected from centrifugation or filtration.

[0027] Preferably, the detection method further includes the step of preparing a reference solution, and the step of detecting the reference solution to obtain a characteristic chromatogram by using the same detection method as the test solution.

[0028] Preferably, the reference substance in the reference solution includes any one of guanosine, adenosine, thymidine, guanosine, uracil, or tryptophan.

[0029] Preferably, the mass concentration of the reference substance in the reference solution is 0.01 - 0.1 mg / mL, for example, it can be 0.02 mg / mL, 0.04 mg / mL, 0.06 mg / mL, or 0.08 mg / mL, etc.

[0030] Preferably, the solvent in the reference solution is methanol aqueous solution.

[0031] Preferably, the mass percentage content of methanol in the aqueous methanol solution is 5-15%, for example, it can be 6%, 8%, 10%, 12% or 14%, etc., and preferably 10%.

[0032] Other specific point values within the above numerical ranges can be selected and will not be elaborated one by one here.

[0033] Preferably, the detection method includes the following steps:

[0034] (1) Weigh the Allium macrostemon Bunge formula granules to be tested, add 10-20 times the amount of solvent for extraction. The solvent is selected from at least one of aqueous methanol solution, water, and aqueous ethanol solution. The extraction is reflux extraction or ultrasonic extraction for 20-40 min, and then centrifugation or filtration is carried out for solid-liquid separation. Take the liquid to obtain the Allium macrostemon Bunge test solution.

[0035] (2) Take the Allium macrostemon Bunge test solution for high performance liquid chromatography detection to obtain a characteristic chromatogram. The detection wavelength is 250-258 nm. Mobile phase A is acetonitrile, and mobile phase B is water. Gradient elution is carried out at a flow rate of 0.7-0.9 mL / min.

[0036] The process of the gradient elution is as follows: 0-9 min, mobile phase A: 0, mobile phase B: 100%; 9-14 min, mobile phase A: 0-3%, mobile phase B: 100-97%; 14-21 min, mobile phase A: 3%, mobile phase B: 97%; 21-35 min, mobile phase A: 3-10%, mobile phase B: 97-90%.

[0037] In the second aspect, the present invention provides an application of the detection method as described in the first aspect in determining the content of guanosine and / or adenosine in the Allium macrostemon Bunge extract.

[0038] In the third aspect, the present invention provides an application of the detection method as described in the first aspect in identifying Allium macrostemon Bunge extracts with different original plants.

[0039] Preferably, the characteristic chromatograms of the Allium macrostemon Bunge extracts with different original plants are different;

[0040] Preferably, the differences include at least one of the number of characteristic peaks, the shape of characteristic peaks, the area of characteristic peaks, the retention time of characteristic peaks, or the half-peak width of characteristic peaks.

[0041] Preferably, the characteristic chromatogram of the Allium macrostemon Bunge preparation with the original plant of Allium macrostemon L. var. radicosum Regel includes 10 characteristic peaks, and the characteristic chromatogram of the Allium macrostemon Bunge preparation with the original plant of Allium chinense G. Don includes 5 characteristic peaks.

[0042] Preferably, the error of the relative retention time of the 10 characteristic peaks with respect to the reference peak does not exceed ±10%.

[0043] Preferably, the error of the relative retention time of the 5 characteristic peaks with respect to the reference peak does not exceed ±10%.

[0044] Preferably, taking the guanosine peak as the reference peak, according to the elution order, the relative retention times of the characteristic peaks are respectively: 0.28 - 0.29, for example, it can be 0.282, 0.284, 0.286 or 0.288, etc.; 0.39 - 0.40, for example, it can be 0.392, 0.394, 0.396 or 0.398, etc.; 0.45 - 0.46, for example, it can be 0.452, 0.454, 0.456 or 0.458, etc.; 0.57 - 0.58, for example, it can be 0.572, 0.574, 0.576 or 0.578, etc.; 0.92 - 0.93, for example, it can be 0.922, 0.924, 0.926 or 0.928, etc.; 1.00; 1.20 - 1.21, for example, it can be 1.202, 1.204, 1.206 or 1.208, etc.; 1.27 - 1.28, for example, it can be 1.272, 1.274, 1.276 or 1.278, etc.; 1.43 - 1.44, for example, it can be 1.432, 1.434, 1.436 or 1.438, etc.; 1.51 - 1.52, for example, it can be 1.512, 1.514, 1.516 or 1.518, etc.

[0045] Preferably, according to the elution order, the relative retention times of the characteristic peaks are respectively: 0.29, 0.40, 0.45, 0.57, 0.93, 1.00, 1.21, 1.28, 1.44, 1.51.

[0046] Preferably, taking the guanosine peak as the reference peak, according to the elution order, the relative retention times of the 5 characteristic peaks are respectively: 0.30 - 0.32, for example, it can be 0.30, 0.31 or 0.32, etc.; 0.60 - 0.62, for example, it can be 0.60, 0.61 or 0.62, etc.; 1.00; 1.30 - 1.30, for example, it can be 1.30, 1.31 or 1.32, etc.; 1.00.

[0047] Preferably, according to the elution order, the relative retention times of the characteristic peaks are respectively: 0.31, 0.61, 1.00, 1.31, 1.00.

[0048] Other specific point values within the above numerical ranges can be selected, and will not be elaborated one by one here.

[0049] Compared with the prior art, the present invention has the following beneficial effects:

[0050] A detection method for Allium macrostemon Bunge preparations provided by the present invention uses high performance liquid chromatography to detect Allium macrostemon Bunge preparations under specific conditions, and can stably and accurately detect the characteristic chromatogram of Allium macrostemon Bunge preparations. There are multiple common characteristic peaks in the chromatogram, and there is good resolution between each characteristic peak. The chromatogram has good reproducibility. The test method provided by the present invention has high precision, can effectively identify two original plants of Allium macrostemon Bunge (small root garlic) formula granules and Allium macrostemon Bunge (Allium chinense G. Don) formula granules, effectively control the product quality, ensure the clinical efficacy, and the detection method for Allium macrostemon Bunge preparations provided by the present invention can simultaneously determine the content of the main active ingredients in Allium macrostemon Bunge preparations while detecting the characteristic chromatogram of Allium macrostemon Bunge preparations, and the obtained characteristic chromatogram can achieve the identification of the original plant, effectively saving the detection time and detection cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 It is the characteristic chromatogram obtained by detecting in Example 1;

[0052] Figure 2 It is the confirmation diagram of each characteristic peak in the characteristic chromatogram of Example 1;

[0053] Figure 3 It is the 3D full scan diagram of the test solution of Allium macrostemon Bunge prepared in Example 1 at 254 nm;

[0054] Figure 4 It is the characteristic chromatogram obtained by detecting 18 batches of Allium macrostemon Bunge formula granules in Examples 35 - 52;

[0055] Figure 5 It is the characteristic chromatogram obtained by detecting in Examples 53 - 58;

[0056] Figure 6 It is the characteristic chromatogram obtained by detecting in Example 59. DETAILED DESCRIPTION OF THE INVENTION

[0057] The technical solutions of the present invention will be further described below through specific embodiments. Those skilled in the art should understand that the embodiments are only for helping to understand the present invention and should not be regarded as specific limitations on the present invention.

[0058] As used herein, the terms "comprising", "including", "having", "containing" or any other variation thereof are intended to cover non - exclusive inclusion. For example, a composition, step, method, article or device containing the listed elements is not necessarily limited to those elements, but may also include other elements not explicitly listed or elements inherent to such composition, step, method, article or device.

[0059] "Optional" or "any one" means that the subsequent described matter or event may or may not occur, and this description includes the case where the event occurs and the case where the event does not occur.

[0060] The indefinite articles "a" and "an" before the elements or components of the present invention do not limit the quantity requirements (i.e., the number of occurrences) of the elements or components. Therefore, "a" or "an" should be construed as including one or at least one, and the singular form of the element or component also includes the plural form, unless the quantity clearly refers only to the singular form.

[0061] The descriptions of the terms "one embodiment", "some embodiments", "exemplarily", "specific examples", or "some examples" described in the present invention mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this document, the schematic expressions of the above terms are not necessarily directed to the same embodiment or example.

[0062] In the following examples, the sources of the instruments, reagents, and test drugs are as follows:

[0063] Instruments:

[0064] High-performance liquid chromatograph (Shimadzu LC-2010A, TUV detector, Labsolutions operating system);

[0065] High-performance liquid chromatograph (Shimadzu LC-2010A, Waters e2695, UV detector);

[0066] ME104E electronic balance (Mettler Toledo);

[0067] Chromatographic column (Waters C18, column length 250 mm, column inner diameter 4.6 mm, particle size 5 μm).

[0068] Test drugs:

[0069] Scallion Bulb formula granules:

[0070] The original plant is Allium macrostemon Bunge: 200618-725800-01, 200529-724400-02, 200610-713800-03, 200703-725800-04, 200703-725800-05, 200703-043100-06, 200703-043100-07, 200703-725800-08, 200703-043100-09, 200703-725800-10, 200703-043100-11, 200703-727000-12, 200703-710000-13, 200703-710038-14, 200703-712000-15, 200703-714100-17, 200703-724400-18, 202201, 202202, 202203;

[0071] The original plant is Allium chinense G. Don: 202204, 202205, 202206, 202207;

[0072] Adenosine reference substance (batch number: 110879-201703, purity 99.7%, National Institutes for Food and Drug Control);

[0073] Thymidine reference substance (batch number: 110879-201703, National Institutes for Food and Drug Control);

[0074] Uracil reference substance (batch number: 100469-201302, purity 99.6%, National Institutes for Food and Drug Control);

[0075] Guanosine reference substance (batch number: 111977-201501, purity 93.6%, National Institutes for Food and Drug Control);

[0076] Tryptophan reference substance (batch number: 140686-201904, purity 99.9%, National Institutes for Food and Drug Control);

[0077] Uridine reference substance (batch number: 110887-2018-03, purity 99.5%, National Institutes for Food and Drug Control).

[0078] Reagents:

[0079] Acetonitrile: chromatographic grade (Merk);

[0080] Water is distilled water (Watsons);

[0081] Other reagents are all of analytical grade.

[0082] Example 1

[0083] This embodiment provides a detection method for Allium macrostemon Bunge preparations, and the detection method is as follows:

[0084] (1) Weigh 0.5 g of the formula granules of the test Allium macrostemon Bunge (Allium macrostemon Bunge) into a stoppered conical flask, add 10 mL of 10% methanol aqueous solution, and then extract by ultrasonic wave (power 250 W, frequency 40 kHz) for 30 min. Let it cool to room temperature, weigh it, make up the lost weight with 10% methanol aqueous solution, shake well, filter, take the liquid to obtain the test solution of Allium macrostemon Bunge.

[0085] (2) Precisely pipette 10 μL of the test solution of Allium macrostemon Bunge and inject it into a high-performance liquid chromatograph. Use a C18 chromatographic column, the packing material is octadecylsilyl-bonded silica gel, the detection wavelength is 254 nm, the column temperature is 30 °C, the mobile phase A is acetonitrile, the mobile phase B is water, and gradient elution is carried out. The flow rate is 0.8 mL / min, and the theoretical plate number calculated by the guanyosine peak should be not less than 50,000. The gradient elution process is shown in Table 1:

[0086] Table 1

[0087] Time (min) Mobile Phase A (%) Mobile Phase B (%) 0-9 0 100 9-14 0→3 100→97 14-21 3 97 21-35 3→10 97→90

[0088] The characteristic chromatogram obtained by the detection in Example 1 is as Figure 1 shown; the detection results of the retention time for 6 repeated injections are shown in Table 2; the detection results of the peak area for 6 repeated injections are shown in Table 3:

[0089] Table 2

[0090]

[0091] Table 3

[0092]

[0093]

[0094] According to the test results in Table 2 and Table 3, it can be known that the relative retention time RSD of each characteristic peak in the 6 repeated experiment results is in the range of 0.03 - 0.17%, and the RSD of the relative peak area is in the range of 0.10 - 3.42%, indicating that the detection method provided by the present invention has good precision.

[0095] Test Example 1

[0096] Precisely weigh the reference substances of adenosine, guanosine, uracil, thymidine, uridine, and tryptophan, add 70% methanol to prepare a 20 μg / mL solution, and obtain the reference substance solutions of adenosine, guanosine, uracil, thymidine, uridine, and tryptophan. Accurately pipette 10 μL each of the test sample solution of Allium macrostemon Bunge prepared in Example 1, the reference substance solution of adenosine, the reference substance solution of thymidine, the reference substance solution of uracil, the reference substance solution of uridine, the reference substance solution of tryptophan, and the reference substance solution of guanosine, inject them into a high-performance liquid chromatograph, and perform the determination according to the detection method in step (2) of Example 1. Confirm each characteristic peak. The confirmation diagrams of each characteristic peak are as Figure 2 shown. As can be seen from the figure, by comparing with the characteristic chromatogram of the reference substance, it is determined that peak 1 is uracil, peak 4 is uridine, peak 6 is guanosine, peak 7 is thymidine, peak 8 is tryptophan, and peak 9 is adenosine.

[0097] Examples 2 - 3

[0098] This example provides a detection method for Allium macrostemon Bunge preparations, and the detection method is as follows:

[0099] The difference from Example 1 is only that the flow rates in step (2) are 0.7 mL / min and 0.9 mL / min respectively, and other materials, steps, and parameters are the same as those in Example 1.

[0100] The retention time results obtained by the methods described in Examples 1 - 3 are shown in Table 4; the peak area results obtained by the methods described in Examples 1 - 3 are shown in Table 5:

[0101] Table 4

[0102]

[0103] Table 5

[0104]

[0105] According to the detection results in Table 4 and Table 5, it can be seen that the RSD values of the relative retention time and relative peak area exceed the range of 5% under different flow rates, indicating that this method has relatively strict requirements for the flow rate. In Example 1, that is, when the flow rate is 0.8 mL / min, the relative retention time and relative peak area are relatively more stable and closer to the average value. Therefore, the preferred flow rate is 0.8 mL / min.

[0106] Examples 4 - 5

[0107] This example provides a detection method for Allium macrostemon Bunge preparations, and the detection method is as follows:

[0108] The difference from Example 1 is only that the column temperatures in step (2) are 28°C and 32°C respectively, and other materials, steps, and parameters are the same as those in Example 1.

[0109] The retention time results detected by the methods described in Examples 1, 4, and 5 are shown in Table 6; the peak area results detected by the methods described in Examples 1, 4, and 5 are shown in Table 7:

[0110] Table 6

[0111]

[0112] Table 7

[0113]

[0114]

[0115] According to the detection results in Table 6 and Table 7, under different column temperature conditions, the relative retention time and the RSD value of the relative peak area are both within 5%, indicating that the detection method has good durability for column temperature.

[0116] Examples 6 - 7

[0117] This example provides a detection method for Allium macrostemon Bunge preparations, and the detection method is as follows:

[0118] The difference from Example 1 is only that the detection wavelengths in step (2) are 252 nm and 256 nm, and other materials, steps, and parameters are the same as those in Example 1.

[0119] The retention time results detected by the methods described in Examples 1, 6, and 7 are shown in Table 8; the peak area results detected by the methods described in Examples 1, 6, and 7 are shown in Table 9:

[0120] Table 8

[0121]

[0122] Table 9

[0123]

[0124]

[0125] According to the detection results in Table 8 and Table 9, under different detection wavelength conditions, the relative retention time and the RSD value of the relative peak area are both within 5%, indicating that the detection method has good durability for the detection wavelength. Perform a 3D full scan on the test solution, and the test result spectrum is as Figure 3 shown. The results show that the characteristic spectrum information is relatively rich near 254 nm. Therefore, the detection wavelength is preferably 254 nm.

[0126] Example 8

[0127] This embodiment provides a method for detecting Allium macrostemon Bunge preparations, and the detection method is as follows:

[0128] The difference from Example 1 is only that step (1) is different. Step (1) of this embodiment is: Weigh 0.5 g of the test Allium macrostemon Bunge (Allium macrostemon Bunge) formula granules into a stoppered conical flask, add 10 mL of 10% methanol aqueous solution, reflux and extract for 30 min, cool to room temperature, weigh, make up the lost weight with 10% methanol aqueous solution, shake well, filter, take the liquid to obtain the Allium macrostemon Bunge test solution. Other materials, steps and parameters are the same as those in Example 1.

[0129] The retention time results detected by the methods described in Examples 1 and 8 are shown in Table 10; the peak area results detected by the methods described in Examples 1 and 8 are shown in Table 11:

[0130] Table 10

[0131]

[0132]

[0133] Table 11

[0134]

[0135] According to the detection results in Table 10 and Table 11, it can be seen that under different extraction conditions, the relative retention time and relative peak area in the characteristic chromatogram have small differences. Therefore, both ultrasonic extraction and reflux extraction can be used, and among them, the ultrasonic treatment operation is more convenient.

[0136] Examples 9 - 11

[0137] This embodiment provides a method for detecting Allium macrostemon Bunge preparations, and the detection method is as follows:

[0138] The difference from Example 1 is only that the extraction agents in step (1) are 30% methanol aqueous solution, 10% ethanol aqueous solution, and 30% ethanol aqueous solution respectively, and other materials, steps and parameters are the same as those in Example 1.

[0139] The retention time results detected by the methods described in Examples 1 and 9 - 11 are shown in Table 12; the peak area results detected by the methods described in Examples 1 and 9 - 11 are shown in Table 13:

[0140] Table 12

[0141]

[0142]

[0143] Table 13

[0144]

[0145] According to the detection results in Table 12 and Table 13, it can be seen that different extractants have a great influence on the peak shape in the characteristic spectrum of the detection results. When extracted with 30% ethanol aqueous solution, the overall peak shape of the spectrum is poor and the resolution is low. When extracted with 30% methanol aqueous solution and 10% ethanol aqueous solution, a peak wrapping phenomenon appears at peak 2 and it cannot be integrated. Therefore, the extractant is preferably 10% methanol aqueous solution.

[0146] Examples 12 - 13

[0147] This example provides a detection method for Allium macrostemon Bunge preparations, and the detection method is as follows:

[0148] The difference from Example 1 is only that in step (1), the masses of the test Allium macrostemon Bunge (small root garlic) formula granules weighed are 0.2 g and 0.8 g respectively, and other materials, steps and parameters are the same as those in Example 1.

[0149] The retention time results obtained by the methods described in Examples 1, 12, and 13 are shown in Table 14; the peak area results obtained by the methods described in Examples 1, 12, and 13 are shown in Table 15:

[0150] Table 14

[0151]

[0152] Table 15

[0153]

[0154] According to the detection results in Table 14 and Table 15, it can be seen that in the spectra obtained by detecting different concentrations of the test sample solutions, the relative retention time and relative peak area have small differences. Combining the response values of each characteristic peak in the spectrum, in the examples, that is, when the concentration is 0.5 g / mL, the response values of each characteristic peak are moderate. Therefore, the concentration of the test sample solution in the present invention is preferably 0.5 g / mL.

[0155] Examples 14 - 15

[0156] This example provides a detection method for Allium macrostemon Bunge preparations, and the detection method is as follows:

[0157] The difference from Example 1 is only that in step (1), the extraction times are 20 min and 40 min respectively, and other materials, steps and parameters are the same as those in Example 1.

[0158] The retention time results obtained by the methods described in Examples 1, 14, and 15 are shown in Table 16; the peak area results obtained by the methods described in Examples 1, 14, and 15 are shown in Table 17:

[0159] Table 16

[0160]

[0161] Table 17

[0162]

[0163] According to the detection results in Table 16 and Table 17, it can be seen that different extraction times have little effect on the relative retention time and relative peak area in the detection result chromatogram, indicating that the extraction is complete at 20 min. To ensure complete extraction of Allium macrostemon Bunge formula granules with different batches and different origins, 30 min is preferably selected.

[0164] Examples 16 - 22

[0165] This example provides a detection method for Allium macrostemon Bunge preparations, and the detection method is as follows:

[0166] The difference from Example 1 is only that after step (1) is completed, the test solution is placed for 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, and 24 h respectively, and then step (2) is carried out. Other materials, steps, and parameters are the same as those in Example 1.

[0167] The retention time results detected by the methods described in Examples 1, 16 - 22 are shown in Table 18; the peak area results detected by the methods described in Examples 1, 16 - 22 are shown in Table 19:

[0168] Table 18

[0169]

[0170]

[0171] Table 19

[0172]

[0173] According to the detection results in Table 18 and Table 19, it can be seen that the relative retention time RSD of each characteristic peak within 24 h is in the range of 0.07 - 0.77%, and the relative peak area is in the range of 0.26 - 4.51%, indicating that the test solution prepared by the present invention has good stability within 24 h.

[0174] Examples 23 - 28

[0175] This example provides a detection method for Allium macrostemon Bunge preparations, and the detection method is as follows:

[0176] The difference from Example 1 is only that in step (1), 6 portions of the same batch of test Allium macrostemon Bunge (Allium macrostemon Bunge) formula granules with equal mass are weighed respectively. Other materials, steps, and parameters are the same as those in Example 1.

[0177] The retention time results obtained by the methods described in Examples 23-28 are shown in Table 20; the peak area results obtained by the methods described in Examples 23-28 are shown in Table 21:

[0178] Table 20

[0179]

[0180]

[0181] Table 21

[0182]

[0183] According to the test results in Table 20 and Table 21, it can be seen that the relative retention time RSD of each characteristic peak in the detection chromatogram is in the range of 1.77-2.02%, and the relative peak area RSD is in the range of 0.47-2.73%, indicating that the characteristic chromatogram obtained by the detection method of the present invention has good repeatability.

[0184] Examples 29-34

[0185] This example provides a detection method for Allium macrostemon Bunge preparations, and the detection method is as follows:

[0186] The difference from Examples 23-28 is only that in step (2), a Waters e2695 high performance liquid chromatograph and a UV detector are used, and other materials, steps and parameters are the same as those in Example 1.

[0187] The retention time results obtained by the methods described in Examples 29-34 are shown in Table 22:

[0188] Table 22

[0189]

[0190] According to the test results in Table 22, it can be seen that the relative retention time RSD of each characteristic peak in the chromatograms of different detection instruments is in the range of 1.35-2.51%, and the detection results meet the requirements of intermediate precision analysis.

[0191] Examples 35-52

[0192] This example provides a detection method for Allium macrostemon Bunge preparations, and the detection method is as follows:

[0193] The difference from Example 1 is only that in step (1), the Allium macrostemon Bunge (small root garlic) formula granules are from 18 batches of Allium macrostemon Bunge formula granules, and other materials, steps and parameters are the same as those in Example 1.

[0194] The characteristic chromatograms obtained by Examples 35-52 are asFigure 4 As shown; the retention time results detected by the methods described in Examples 35 - 52 are shown in Table 23; the peak area results detected by the methods described in Examples 35 - 52 are shown in Table 24; the similarity between the characteristic chromatograms detected by the methods described in Examples 35 - 52 and the control characteristic chromatogram is shown in Table 25:

[0195] Table 23

[0196]

[0197]

[0198] Table 24

[0199]

[0200]

[0201] It can be seen from the detection results in Table 23 and Table 24 that among the characteristic chromatograms of 18 batches of Allium macrostemon Bunge formula granules, the relative retention times of each characteristic peak have small differences, and the RSD is within the range of ±5%, meeting the quality control requirements. The average values of the relative retention times can be selected as the measured values, which are 0.29 (peak 1), 0.40 (peak 2), 0.45 (peak 3), 0.57 (peak 4), 0.93 (peak 5), 1.00 (peak 6), 1.21 (peak 7), 1.28 (8), 1.44 (peak 9), 1.51 (peak 10) respectively, with an allowable error of ±10%; the differences in the relative peak areas of each characteristic peak are large, and the RSD is within the range of 13.31 - 176.58%, not meeting the quality control standards. Therefore, the relative peak area is not specified.

[0202] Table 25

[0203] Batch Number Similarity with the Control Characteristic Chromatogram Example 35 200618-725800-01 0.985 Example 36 200529-724400-02 0.994 Example 37 200610-713800-03 0.994 Example 38 200703-725800-04 0.995 Example 39 200703-725800-05 0.996 Example 40 200703-043100-06 0.996 Example 41 200703-043100-07 0.996 Example 42 200703-725800-08 0.995 Example 43 200703-043100-09 0.972 Example 44 200703-725800-10 0.996 Example 45 200703-043100-11 0.972 Example 46 200703-727000-12 0.996 Example 47 200703-710000-13 0.995 Example 48 200703-710038-14 0.993 Example 49 200703-712000-15 0.972 Example 50 200703-710000-16 0.996 Example 51 200703-714100-17 0.995 Example 52 200703-724400-18 0.994 — R 1

[0204] It can be seen from the detection results in Table 25 that the similarities between the characteristic chromatograms obtained by detecting 18 batches of Allium macrostemon Bunge formula granules and the control characteristic chromatogram are 0.972 - 0.996, all higher than 0.95, indicating that the detection method provided by the present invention has small differences in the characteristic chromatograms obtained when detecting different batches of Allium macrostemon Bunge formula granules and has good applicability.

[0205] Examples 53 - 58

[0206] This example provides a detection method for Allium macrostemon Bunge preparations, and the detection method is as follows:

[0207] The difference from Example 1 is only that the Allium macrostemon Bunge (small root garlic) formula granules in step (1) are respectively replaced with: Allium macrostemon Bunge (small root garlic) formula granules 202201, Allium macrostemon Bunge (small root garlic) formula granules 202202, Allium macrostemon Bunge (small root garlic) formula granules 202203, Allium macrostemon Bunge (Chinese leek) formula granules 202204, Allium macrostemon Bunge (Chinese leek) formula granules 202205, Allium macrostemon Bunge (Chinese leek) formula granules 202206, and other materials, steps and parameters are the same as those in Example 1.

[0208] The characteristic chromatograms obtained by detecting Examples 53 - 58 are as Figure 5 shown. As can be seen from the figure, compared with the Allium macrostemon Bunge (small root garlic) formula granules, in the chromatogram of the Allium macrostemon Bunge (Chinese leek) formula granules, peaks 1, 2, 3, 7, and 10 are significantly missing, and the peak shape of peak 5 is quite different. By specifying 10 characteristic peaks in the characteristic chromatogram of the Allium macrostemon Bunge (small root garlic) formula granules, Allium macrostemon Bunge with different original plants can be quickly distinguished.

[0209] Example 59

[0210] This example provides a detection method for Allium macrostemon Bunge preparations, and the detection method is as follows:

[0211] The difference from Example 1 is only that the Allium macrostemon Bunge (small root garlic) formula granules in step (1) are replaced with Allium macrostemon Bunge (Chinese leek) formula granules 202207, and other materials, steps and parameters are the same as those in Example 1.

[0212] The characteristic chromatogram obtained by detecting Example 59 is as Figure 6 shown.

[0213] The applicant declares that the present invention uses the above examples to illustrate the process method of the present invention, but the present invention is not limited to the above process steps, that is, it does not mean that the present invention must rely on the above process steps to be implemented. Those skilled in the art should understand that any improvement to the present invention, the equivalent replacement of the raw materials selected by the present invention, the addition of auxiliary components, the selection of specific methods, etc. all fall within the protection scope and the disclosure scope of the present invention.

Claims

1. A detection method for Allium macrostemon Bunge preparations, characterized in that, The detection method includes the following steps: (1) Prepare a test Allium macrostemon Bunge preparation into an Allium macrostemon Bunge test sample solution; The preparation method of the Allium macrostemon Bunge test sample solution is as follows: Extract the Allium macrostemon Bunge preparation with an aqueous methanol solution, and then perform solid-liquid separation. The resulting liquid is the Allium macrostemon Bunge test sample solution; The extraction method is reflux or ultrasonic; (2) Take the Allium macrostemon Bunge test sample solution and perform high performance liquid chromatography (HPLC) detection to obtain a characteristic chromatogram; The conditions for the HPLC detection are as follows: The detection wavelength is 250 - 258 nm, mobile phase A is acetonitrile, mobile phase B is water, gradient elution is performed, and the flow rate is 0.7 - 0.9 mL / min; The process of the gradient elution is as follows: 0 - 9 min, mobile phase A: 0, mobile phase B: 100%; 9 - 14 min, mobile phase A: 0 - 3%, mobile phase B: 100 - 97%; 14 - 21 min, mobile phase A: 3%, mobile phase B: 97%; 21 - 35 min, mobile phase A: 3 - 10%, mobile phase B: 97 - 90%; The filler of the chromatographic column used in the detection is octadecylsilyl bonded silica gel; The detection method further includes the steps of preparing a reference solution, and using the same detection method as the Allium macrostemon Bunge test sample solution to detect the reference solution to obtain a characteristic chromatogram; The reference substances in the reference solution include guanosine, adenosine, thymidine, uridine, uracil, and tryptophan.

2. The detection method according to claim 1, wherein The column temperature for the detection is 28 - 32 °C.

3. The detection method according to claim 2, characterized in that The column temperature for the detection is 30 °C.

4. The detection method according to claim 1, wherein The Allium macrostemon Bunge preparation is Allium macrostemon Bunge formula granules.

5. The detection method according to claim 1, wherein The Allium macrostemon Bunge preparation is selected from the original plant of Allium macrostemon Bunge or Allium chinense G. Don.

6. The detection method according to claim 1, wherein, The material-liquid ratio for the extraction is 1:(10 - 20).

7. The detection method according to claim 1, wherein The extraction time is 20 - 40 min.

8. The detection method according to claim 1, characterized in that The solid-liquid separation is selected from centrifugation or filtration.

9. The detection method according to claim 1, wherein The mass concentration of the reference substances in the reference solution is 0.01 - 0.1 mg / mL.

10. The detection method according to claim 1, wherein, The solvent in the reference solution is an aqueous methanol solution.

11. The detection method according to claim 10, wherein The mass percentage content of methanol in the aqueous methanol solution is 5 - 15%.

12. The detection method according to claim 1, wherein The detection method includes the following steps: (1) Weigh the test Allium macrostemon Bunge formula granules, add 10 - 20 times the amount of aqueous methanol solution for extraction. The extraction is reflux extraction or ultrasonic extraction, the time is 20 - 40 min, then perform centrifugation or filtration for solid-liquid separation, and take the liquid to obtain the Allium macrostemon Bunge test sample solution; (2) Take the Allium macrostemon Bunge test sample solution and perform high performance liquid chromatography (HPLC) detection to obtain a characteristic chromatogram. The detection wavelength is 250 - 258 nm, mobile phase A is acetonitrile, mobile phase B is water, gradient elution is performed, and the flow rate is 0.7 - 0.9 mL / min; The process of the gradient elution is as follows: 0 - 9 min, mobile phase A: 0, mobile phase B: 100%; 9 - 14 min, mobile phase A: 0 - 3%, mobile phase B: 100 - 97%; 14 - 21 min, mobile phase A: 3%, mobile phase B: 97%; 21 - 35 min, mobile phase A: 3 - 10%, mobile phase B: 97 - 90%.

13. Use of the detection method according to any one of claims 1 - 12 in determining the content of guanosine and / or adenosine in Allium macrostemon Bunge extract.

14. Use of the detection method according to any one of claims 1-12 in identifying Allium macrostemon Bunge extracts of different origins, characterized in that, The original sources include the original source of Allium macrostemon Bunge or the original source of Allium chinense G. Don.

15. The application according to claim 14, characterized in that, The characteristic fingerprints of the extracts of Allium macrostemon Bunge with different original sources are different.

16. The application according to claim 15, wherein The differences include at least one of the number of characteristic peaks, the shape of characteristic peaks, the area of characteristic peaks, the retention time of characteristic peaks, or the half-peak width of characteristic peaks.

17. The application according to claim 14, characterized in that, The characteristic fingerprint of the extract of Allium macrostemon Bunge with the original source of Allium macrostemon Bunge includes 10 characteristic peaks, and the characteristic fingerprint of the extract of Allium macrostemon Bunge with the original source of Allium chinense G. Don includes 5 characteristic peaks.

18. The application according to claim 17, wherein Taking the guanosine peak as the reference peak, according to the elution order, the relative retention times of the 10 characteristic peaks are respectively: 0.28 - 0.29, 0.39 - 0.40, 0.45 - 0.46, 0.57 - 0.58, 0.92 - 0.93, 1.00, 1.20 - 1.21, 1.27 - 1.28, 1.43 - 1.44, 1.51 - 1.

52.

19. The application according to claim 18, wherein Taking the guanosine peak as the reference peak, according to the elution order, the relative retention times of the 10 characteristic peaks are respectively: 0.29, 0.40, 0.45, 0.57, 0.93, 1.00, 1.21, 1.28, 1.44, 1.

51.

20. The application according to claim 17, wherein Taking the guanosine peak as the reference peak, according to the elution order, the relative retention times of the 5 characteristic peaks are respectively: 0.30 - 0.32, 0.60 - 0.62, 1.00, 1.30 - 1.30, 1.

00.

21. The application according to claim 20, characterized in that Taking the guanosine peak as the reference peak, according to the elution order, the relative retention times of the 5 characteristic peaks are respectively: 0.31, 0.61, 1.00, 1.31, 1.00.

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