Construction method and application of characteristic spectrum of Momordica cochinchinensis or Momordica cochinchinensis kernel

Abstract

The present invention belongs to the technical field of drug detection, and specifically relates to a construction method and application of a characteristic spectrum of Momordica cochinchinensis or Momordica cochinchinensis kernel. The construction method comprises constructing a characteristic spectrum of a test sample, and the construction method comprises the following steps: preparation of a test sample solution; wherein, the chromatographic conditions are: octadecylsilane bonded silica gel as a filler, formic acid as a mobile phase A, acetonitrile as a mobile phase B, and gradient elution. The construction method can be used to identify Momordica cochinchinensis kernel and Momordica cochinchinensis, and the separation effect of characteristic peaks in the obtained characteristic spectrum is good, and the effective chemical components in Momordica cochinchinensis or Momordica cochinchinensis kernel can be fully displayed, and 7 characteristic peaks can be determined, with more information, high accuracy, short detection time, and no interference between characteristic peaks. The characteristic spectrum obtained by the construction method has good reproducibility, and can be fully controlled from raw materials to particles. The method is fast, reliable, stable, and highly precise.

Description

Technical Field

[0001] The invention belongs to the technical field of drug detection, and particularly relates to a method for constructing a characteristic spectrum of Momordica cochinchinensis or Momordica cochinchinensis kernel and an application thereof. Background Art

[0002] Momordica cochinchinensis (Lour.) Spreng., also known as wood crab, earthen wood turtle and shell wood turtle, is the dried mature seed of the Cucurbitaceae plant Momordica cochinchinensis (Lour.) Spreng. The mature fruit is harvested in winter, split open, sun-dried until half dry, the pulp is removed, the seeds are taken out and dried; the shell is removed to obtain the Momordica cochinchinensis kernel, which was first recorded in Rihuazi. It has the effects of reducing swelling, dispersing nodules, detoxifying, chasing away wind and relieving pain. It tastes bitter, slightly sweet, warm in nature and toxic.

[0003] At present, the 2020 edition of the Chinese Pharmacopoeia only defines the properties, microscopic identification and content determination of Momordica cochinchinensis kernels. Momordica cochinchinensis kernel formula granules are obtained by extraction, concentration, drying, preparation and other steps of Momordica cochinchinensis kernels, which have lost the original appearance and characteristics of the medicinal material; in addition, there is also the problem of direct feeding of Momordica cochinchinensis medicinal material in the production process of Momordica cochinchinensis kernel formula granules, which makes it impossible to effectively distinguish Momordica cochinchinensis kernel formula granules from Momordica cochinchinensis formula granules.

[0004] The existing technology has the following problems when studying the chemical components of Momordica cochinchinensis: ① Chloroform-petroleum ether is used as the extraction solvent for the test solution, and concentrated sulfuric acid and other solvents need to be added during the extraction process, resulting in complicated extraction process steps for the test solution. The extraction solvent used is relatively harsh and unfriendly, and the characteristic peaks in the obtained characteristic spectrum are greatly interfered, which is not suitable for the identification of Momordica cochinchinensis and Momordica cochinchinensis kernels. ② Petroleum ether is used as the extraction solvent, and hydrochloric acid and other solvents need to be added during the extraction process. The preparation of the test solution is complicated, and the reproducibility of the obtained characteristic spectrum is poor. ③ The number of characteristic peaks in the obtained characteristic spectrum is small, and the effective components covered are small. Summary of the invention

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the defects of the prior art that the seeds of Momordica charantia and Momordica charantia kernel cannot be identified, and that the characteristic spectrum covers few effective ingredients when constructing liquid chromatography, thereby providing a method for constructing a characteristic spectrum of Momordica charantia or Momordica charantia kernel and its application.

[0006] To this end, the present invention provides the following technical solutions.

[0007] The present invention provides a method for constructing a characteristic spectrum of Momordica cochinchinensis or Momordica cochinchinensis kernel, comprising constructing a characteristic spectrum of a test sample, wherein the construction method comprises the following steps:

[0008] Preparation of test solution;

[0009] Chromatographic conditions: octadecylsilane bonded silica gel as filler, formic acid as mobile phase A, acetonitrile as mobile phase B, gradient elution;

[0010] The gradient elution procedure includes: 0-1min, the volume percentage of mobile phase A is 100%, and the volume percentage of mobile phase B is 0%; 1-2min, the volume percentage of mobile phase A is 100-98%, and the volume percentage of mobile phase B is 0-2%; 2-15min, the volume percentage of mobile phase A is 98-92%, and the volume percentage of mobile phase B is 2-8%; 15-25min, the volume percentage of mobile phase A is 92-85%, and the volume percentage of mobile phase B is 8-15%; 25-27min, the volume percentage of mobile phase A is 85-100%, and the volume percentage of mobile phase B is 15%-0; 25-30min, the volume percentage of mobile phase A is 100%, and the volume percentage of mobile phase B is 0;

[0011] Determination: Take the test solution, inject it into the ultra-high performance liquid chromatography instrument, and determine it.

[0012] In the construction method, the chromatographic conditions are as follows: chromatographic column specifications: column length 150 mm, inner diameter 2.1 mm, particle size 1.7 μm; flow rate 0.28-0.32 ml / min, column temperature 32-35° C., detection wavelength 254 nm, 0.1% formic acid as mobile phase A, and the theoretical plate number calculated based on the p-hydroxybenzoic acid peak is not less than 3000.

[0013] The test sample is at least one of Momordica cochinchinensis decoction pieces, Momordica cochinchinensis formula granules, Momordica cochinchinensis standard decoction freeze-dried powder, Momordica cochinchinensis medicinal materials, Momordica cochinchinensis formula granules and Momordica cochinchinensis standard decoction freeze-dried powder.

[0014] The test solution is a water extract;

[0015] Preferably, when preparing the test solution, the ratio of the mass of the test sample to the volume of the extraction solvent is 0.008-0.014 g / ml.

[0016] The method for preparing the test solution comprises: taking the test sample, adding water, ultrasonic treatment, and filtering;

[0017] Preferably, the ultrasonic treatment has a power of 700 W, a frequency of 40 kHz, and a time of 30±min.

[0018] The construction method also includes the preparation of a reference solution;

[0019] Preferably, when preparing the reference substance solution, the ratio of the reference substance mass to the extraction solvent volume is 0.004-0.007 g / ml;

[0020] Preferably, the Momordica charantia control medicinal material is used as a reference.

[0021] The construction method also includes the preparation of a reference solution;

[0022] Preferably, the reference substance is 2-5 μg per 1 ml of the reference substance solution;

[0023] Preferably, p-hydroxybenzoic acid is used as a reference substance.

[0024] The characteristic spectrum obtained by the construction method includes 7 characteristic peaks;

[0025] Taking peak 5 as the reference peak, the specified values ​​of relative retention times of peaks 1, 2, 3, 4, 6 and 7 are 0.24, 0.32, 0.81, 0.93, 1.48 and 1.68 respectively;

[0026] Among them, the relative retention time of the characteristic peak is within ±10% of the specified value.

[0027] The present invention also provides a method for identifying Momordica cochinchinensis and Momordica cochinchinensis kernels, wherein the characteristic spectrum of the sample to be tested is obtained by adopting the above construction method, and the identification is performed based on the ratio of the peak areas of Peak 3 and Peak 5 in the characteristic spectrum of the sample to be tested.

[0028] Formula I was used for identification. When β1>0.3, the sample to be tested was Momordica cochinchinensis kernel;

[0029] When β1≤0.3, the sample to be tested is Momordica charantia;

[0030]

[0031] Among them, S3 is the peak area of ​​peak 3, and S5 is the peak area of ​​peak 5.

[0032] The technical solution of the present invention has the following advantages:

[0033] 1. The method for constructing the characteristic spectrum of Momordica cochinchinensis or Momordica cochinchinensis kernel provided by the present invention comprises constructing the characteristic spectrum of a test sample, and the construction method comprises the following steps: preparing a test sample solution; wherein the chromatographic conditions are: using octadecylsilane bonded silica gel as a filler, using formic acid as a mobile phase A, using acetonitrile as a mobile phase B, and gradient elution, and the gradient elution procedure comprises: 0-1min, the volume percentage of mobile phase A is 100%, and the volume percentage of mobile phase B is 0%; 1-2min, the volume percentage of mobile phase A is 100-98%, and the volume percentage of mobile phase B is 100-98%. The volume percentage of mobile phase B is 0-2%; 2-15min, the volume percentage of mobile phase A is 98-92%, and the volume percentage of mobile phase B is 2-8%; 15-25min, the volume percentage of mobile phase A is 92-85%, and the volume percentage of mobile phase B is 8-15%; 25-27min, the volume percentage of mobile phase A is 85-100%, and the volume percentage of mobile phase B is 15%-0; 25-30min, the volume percentage of mobile phase A is 100%, and the volume percentage of mobile phase B is 0. The construction method can be used to identify Momordica cochinchinensis kernel and Momordica cochinchinensis, and the separation effect of characteristic peaks in the obtained characteristic spectrum is good, and the effective chemical components in Momordica cochinchinensis or Momordica cochinchinensis kernel can be fully displayed, and 7 characteristic peaks can be determined, with more information, high accuracy, short detection time, and no interference between characteristic peaks. The characteristic spectrum obtained by the construction method has good reproducibility, and can be fully controlled from raw materials to particles. The method is fast, reliable, stable, and highly precise.

[0034] 2. The method for identifying Momordica cochinchinensis and Momordica cochinchinensis kernel provided by the present invention can quickly, reliably, comprehensively and accurately identify Momordica cochinchinensis and Momordica cochinchinensis kernel. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0036] Figure 1 This is the characteristic spectrum of different batches of Momordica charantia seed formula granules in Example 1 of the present invention;

[0037] Figure 2 This is a comparison chart of the Momordica charantia seed formula granules of Example 1 of the present invention;

[0038] Figure 3 This is the characteristic spectrum of different batches of Momordica cochinchinensis medicinal materials in Example 2 of the present invention;

[0039] Figure 4It is a comparison chart of different batches of Momordica cochinchinensis medicinal materials in Example 2 of the present invention;

[0040] Figure 5 It is the characteristic spectrum of different batches of Momordica charcoal slices in Example 3 of the present invention;

[0041] Figure 6 It is the characteristic spectrum of different batches of Momordica charantia formula granules in Example 4 of the present invention. DETAILED DESCRIPTION

[0042] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0043] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0044] The following instruments and reagents are used in the examples:

[0045] instrument:

[0046] Analytical balance (Sartorius, SQP QUINTIX224-1CN, Germany);

[0047] Waters high performance liquid chromatograph (equipped with TUV detector, temperature-controlled column oven, temperature-controlled autosampler, USA);

[0048] High-speed desktop centrifuge (SIGMA, 1-14, Germany);

[0049] Ultrasonic cleaning apparatus (Tianjin Aotesense, AS20500BT, China);

[0050] Waters ACQUITY UPLC BEH Shield RP18 (2.1 mm×150 mm, 1.7 μm), serial number: 01993207018396 (Waters Corporation).

[0051] Drug testing:

[0052] Momordica charantia reference drug (batch number: 121418-200501, China Food and Drug Administration);

[0053] p-Hydroxybenzoic acid (batch number: PCS-2169, purity: ≥98%) was purchased from Shanghai Standard Standard Technology Service Co., Ltd.;

[0054] The batch numbers and origins of Momordica cochinchinensis are:

[0055] K298YC01 Luchuan County, Yulin City, Guangxi Zhuang Autonomous Region

[0056] K298YC02 Rong County, Yulin City, Guangxi Zhuang Autonomous Region

[0057] K298YC03 Bobai County, Yulin City, Guangxi Zhuang Autonomous Region

[0058] K298YC04Fuyuan County, Qujing City, Yunnan Province

[0059] K298YC05 Luoping County, Qujing City, Yunnan Province

[0060] K298YC06 Malong District, Qujing City, Yunnan Province

[0061] K298YC07 Heshan City, Laibin City, Guangxi Zhuang Autonomous Region

[0062] K298YC08 Xingbin District, Laibin City, Guangxi Zhuang Autonomous Region

[0063] K298YC09 Jiajiang County, Leshan City, Sichuan Province

[0064] K298YC10 Muchuan County, Leshan City, Sichuan Province

[0065] K298YC11 Tongshan County, Xianning City, Hubei Province

[0066] K298YC12 Jiayu County, Xianning City, Hubei Province

[0067] K298YC13 Jingxi County, Baise City, Guangxi Zhuang Autonomous Region

[0068] K298YC14 Tianyang District, Baise City, Guangxi Zhuang Autonomous Region

[0069] K298YC15 Pingguo City, Baise City, Guangxi Zhuang Autonomous Region;

[0070] Batch number of Momordica cochinchinensis seed slices:

[0071] K298YP16 Luchuan County, Yulin City, Guangxi Zhuang Autonomous Region

[0072] K298YP17 Rong County, Yulin City, Guangxi Zhuang Autonomous Region

[0073] K298YP18 Bobai County, Yulin City, Guangxi Zhuang Autonomous Region

[0074] K298YP19 Fuyuan County, Qujing City, Yunnan Province

[0075] K298YP20 Luoping County, Qujing City, Yunnan Province

[0076] K298YP21 Malong District, Qujing City, Yunnan Province

[0077] K298YP22 Heshan City, Laibin City, Guangxi Zhuang Autonomous Region

[0078] K298YP23 Xingbin District, Laibin City, Guangxi Zhuang Autonomous Region

[0079] K298YP24 Jiajiang County, Leshan City, Sichuan Province

[0080] K298YP25 Muchuan County, Leshan City, Sichuan Province

[0081] K298YP26 Tongshan County, Xianning City, Hubei Province

[0082] K298YP27 Jiayu County, Xianning City, Hubei Province

[0083] K298YP28 Jingxi County, Baise City, Guangxi Zhuang Autonomous Region

[0084] K298YP29 Tianyang District, Baise City, Guangxi Zhuang Autonomous Region

[0085] K298YP30 Pingguo City, Baise City, Guangxi Zhuang Autonomous Region;

[0086] Batch numbers of Momordica charantia formula granules: K298KL01, K298KL02, K298KL03, K298KL04, K298KL05, K298KL06, K298KL07, K298KL08, K298KL09, K298KL10, K298KL11, K298KL12, K298KL13, K298KL14, K298KL15;

[0087] The batch numbers of the formula granules of Momordica charantia seed are: K298KL16, K298KL17, K298KL18, K298KL19, K298KL20, K298KL21, K298KL22, K298KL23, K298KL24, K298KL25, K298KL26, K298KL27, K298KL28, K298KL29, and K298KL30.

[0088] Acetonitrile (Tianjin Bohai Chemical Reagent Co., Ltd., 20210220, chromatographic grade);

[0089] Methanol (Tianjin Bohai Chemical Reagent Co., Ltd., 20210301, analytical grade);

[0090] Formic acid (88%, Fisher chemical, Laboratory Grade).

[0091] Example 1

[0092] This embodiment provides a method for constructing a characteristic spectrum of Momordica charantia seed formula granules, comprising the following steps:

[0093] Preparation of test solution: Grind the test sample Momordica charantia seed granules into powder, take about 0.2g, weigh accurately, put it in a stoppered conical flask, add 25ml of water accurately, stopper tightly, weigh the weight, and treat with ultrasound for 30min at a power of 700W and a frequency of 40kHz. After cooling, weigh again, make up the lost weight with water, shake well, filter, and take the filtrate to obtain the solution.

[0094] Preparation of reference solution: Take 1.0 g Momordica cochinchinensis reference medicinal material, place it in a conical flask, add 25 ml of water, ultrasonically treat it for 30 minutes, cool it, shake it well, filter it, and take the filtrate to obtain the reference solution.

[0095] Preparation of reference solution: Take an appropriate amount of p-hydroxybenzoic acid reference substance and add methanol to prepare a solution containing 2 μg of p-hydroxybenzoic acid per 1 ml. This solution is used as the reference solution.

[0096] Chromatographic conditions: octadecylsilane bonded silica gel as filler (specifications: column length 150 mm, inner diameter 2.1 mm, particle size 1.7 μm); 0.1% formic acid as mobile phase A, acetonitrile as mobile phase B, gradient elution, gradient elution program see Table 1; flow rate 0.3 ml / min; column temperature 35°C; detection wavelength 254 nm; theoretical plate number calculated based on p-hydroxybenzoic acid peak should be no less than 3000;

[0097] Table 1 Gradient elution program

[0098] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～1 100 0 1～2 100→98 0→2 2～15 98→92 2→8 15～25 92→85 8→15 25～27 85→100 15→0 25～30 100 0

[0099] Determination: Accurately pipette 4 μL of reference solution, control solution and test solution respectively, inject into ultra-high performance liquid chromatography, determine, import the spectrum into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" for data matching, establish the characteristic spectrum of different batches of Momordica charantia formula granules, and obtain the control characteristic spectrum.

[0100] This example establishes characteristic spectra of three different batches of Momordica Coconut Seed Formula Granules, with batch numbers K298KL16, K298KL17, and K298KL18, respectively. The characteristic spectra of each batch of Momordica Coconut Seed Formula Granules are shown in Figure 1 , see the reference map Figure 2 .from Figure 1 and Figure 2It can be seen that the characteristic spectrum of the formula granules of Momordica charantia seed presents 7 characteristic peaks. Compared with the reference solution, peak 5 is p-hydroxybenzoic acid, peak 5 is taken as the S peak, and the relative retention time of the characteristic peaks is within ±10% of the specified value. The specified values ​​of the relative retention times of peaks 1, 2, 3, 4, 6 and 7 are 0.24, 0.32, 0.81, 0.93, 1.48 and 1.68, respectively.

[0101] Example 2

[0102] This embodiment provides a method for constructing a characteristic spectrum of Momordica cochinchinensis medicinal material, comprising the following steps:

[0103] Preparation of test solution: Grind the test sample Momordica charantia seed into powder, take about 0.2g, weigh accurately, put it in a stoppered conical flask, add 25ml of water accurately, stopper it, weigh it, and treat it ultrasonically for 30min at a power of 700W and a frequency of 40kHz. After cooling, weigh it again, make up the lost weight with water, shake well, filter, and take the filtrate to obtain the solution.

[0104] Preparation of reference solution: Take 1.0 g Momordica cochinchinensis reference medicinal material, place it in a conical flask, add 25 ml of water, ultrasonically treat it for 30 minutes, cool it, shake it well, filter it, and take the filtrate to obtain the reference solution.

[0105] Preparation of reference solution: Take an appropriate amount of p-hydroxybenzoic acid reference substance and add methanol to prepare a solution containing 2 μg of p-hydroxybenzoic acid per 1 ml. This solution is used as the reference solution.

[0106] Chromatographic conditions: octadecylsilane bonded silica gel as filler (specifications: column length 150 mm, inner diameter 2.1 mm, particle size 1.7 μm); 0.1% formic acid as mobile phase A, acetonitrile as mobile phase B, gradient elution, gradient elution program is the same as Table 1; flow rate 0.3 ml / min; column temperature 35°C; detection wavelength 254 nm; theoretical plate number calculated based on p-hydroxybenzoic acid peak should be no less than 3000;

[0107] Accurately pipette 4 μL of the reference solution, control solution and test solution respectively, inject into the ultra-high performance liquid chromatograph for measurement, import the spectrum into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" for data matching, establish the characteristic spectrum of different batches of Momordica charantia formula granules, and obtain the control characteristic spectrum.

[0108] This example establishes characteristic spectra of 15 different batches of Momordica cochinchinensis medicinal materials. The characteristic spectra of each batch of Momordica cochinchinensis medicinal materials are shown in Figure 3 , see the reference map Figure 4 .from Figure 3 and Figure 4It can be seen that the characteristic spectrum of Momordica cochinchinensis medicinal material presents 7 characteristic peaks. Compared with the reference solution, peak 5 is p-hydroxybenzoic acid, peak 5 is taken as the S peak, and the relative retention time of the characteristic peaks is within ±10% of the specified value. The specified values ​​of the relative retention times of peaks 1, 2, 3, 4, 6 and 7 are 0.24, 0.32, 0.81, 0.93, 1.48 and 1.68, respectively. Among them, the batch numbers of the 15 batches of Momordica charantia medicinal materials are: K298YC01, K298 YC02, K298 YC03, K298 YC04, K298 YC05, K298 YC06, K298YC07, K298YC08, K298YC09, K298YC10, K298YC11, K298YC12, K298YC13, K298YC14 and K298YC15.

[0109] Example 3

[0110] This embodiment provides a method for constructing a characteristic spectrum of Momordica charantia seeds, comprising the following steps:

[0111] Preparation of test solution: Grind the test sample Momordica charantia seed slices, take about 0.2g, weigh accurately, put it in a stoppered conical flask, add 25ml of water accurately, stopper tightly, weigh the weight, ultrasonically treat for 30min, power 700W, frequency 40kHz, weigh again after cooling, make up the lost weight with water, shake well, filter, and take the filtrate.

[0112] Preparation of reference solution: Take 1.0 g Momordica cochinchinensis reference medicinal material, place it in a conical flask, add 25 ml of water, ultrasonically treat it for 30 minutes, cool it, shake it well, filter it, and take the filtrate to obtain the reference solution.

[0113] Preparation of reference solution: Take an appropriate amount of p-hydroxybenzoic acid reference substance and add methanol to prepare a solution containing 2 μg of p-hydroxybenzoic acid per 1 ml. This solution is used as the reference solution.

[0114] Chromatographic conditions: octadecylsilane bonded silica gel as filler (specifications: column length 150 mm, inner diameter 2.1 mm, particle size 1.7 μm); 0.1% formic acid as mobile phase A, acetonitrile as mobile phase B, gradient elution, gradient elution program is the same as Table 1; flow rate 0.3 ml / min; column temperature 35°C; detection wavelength 254 nm; theoretical plate number calculated based on p-hydroxybenzoic acid peak should be no less than 3000;

[0115] Accurately pipette 4 μL of reference solution, control solution and test solution respectively, inject into ultra-high performance liquid chromatography and measure.

[0116] The characteristic spectra of 15 batches of Momordica cochinchinensis seeds are shown in Figure 5There are 7 characteristic peaks in total. Peak 5 is p-hydroxybenzoic acid. Peak 5 is taken as S peak. The relative retention time of the characteristic peaks is within ±10% of the specified value. The specified values ​​of the relative retention time of peaks 1, 2, 3, 4, 6 and 7 are 0.24, 0.32, 0.81, 0.93, 1.48 and 1.68 respectively. The batch numbers of the 15 batches of Momordica cochinchinensis decoction pieces are: K 298YP16, K298YP17, K298YP18, K298YP19, K298YP20, K298YP21, K298YP22, K298Y P23, K298YP24, K298YP25, K298YP26, K298YP27, K298YP28, K298YP29, K298YP30.

[0117] Example 4

[0118] This embodiment provides a method for constructing a characteristic spectrum of Momordica charantia formula granules, comprising the following steps:

[0119] Preparation of test solution: Grind the test sample Momordica charantia formula granules into powder, take about 0.2g, weigh accurately, put it in a stoppered conical flask, add 25ml of water accurately, stopper tightly, weigh the weight, ultrasonically treat for 30min, power 700W, frequency 40kHz, weigh again after cooling, make up the lost weight with water, shake well, filter, and take the filtrate to obtain.

[0120] Preparation of reference solution: Take 1.0 g Momordica cochinchinensis reference medicinal material, place it in a conical flask, add 25 ml of water, ultrasonically treat it for 30 minutes, cool it, shake it well, filter it, and take the filtrate to obtain the reference solution.

[0121] Preparation of reference solution: Take an appropriate amount of p-hydroxybenzoic acid reference substance and add methanol to make a solution containing 2 μg per 1 ml. This solution is used as the reference solution.

[0122] Chromatographic conditions: octadecylsilane bonded silica gel as filler (specifications: column length 150 mm, inner diameter 2.1 mm, particle size 1.7 μm); 0.1% formic acid as mobile phase A, acetonitrile as mobile phase B, gradient elution, gradient elution program is the same as Table 1; flow rate 0.3 ml / min; column temperature 35°C; detection wavelength 254 nm; theoretical plate number calculated based on p-hydroxybenzoic acid peak should be no less than 3000;

[0123] Accurately pipette 4 μL of reference solution, control solution and test solution respectively, inject into ultra-high performance liquid chromatography and measure.

[0124] The characteristic spectra of 3 batches of Momordica charantia granules are shown in Figure 6There are 7 characteristic peaks in total. Peak 5 is p-hydroxybenzoic acid. Peak 5 is taken as the S peak. The relative retention time of the characteristic peaks is within ±10% of the specified value. The specified values ​​of the relative retention time of Peak 1, Peak 2, Peak 3, Peak 4, Peak 6 and Peak 7 are 0.24, 0.32, 0.81, 0.93, 1.48 and 1.68, respectively. The batch numbers of the three batches of Momordica charantia formula granules are K298KL01, K298KL02 and K298KL03, respectively.

[0125] Experimental Example 5

[0126] This example provides a method for identifying Momordica cochinchinensis and Momordica cochinchinensis kernels. A characteristic spectrum of the sample to be tested is constructed according to the method in Example 1 to obtain a characteristic spectrum of the sample to be tested. The peak area ratio of Peak 3 and Peak 5 in the characteristic spectrum is then used for identification, as follows:

[0127] Preparation of the sample solution: Grind the sample to be tested, take about 0.2g, accurately weigh, place in a stoppered conical flask, accurately add 25ml of water, seal, weigh, and ultrasonically treat for 30min at a power of 700W and a frequency of 40kHz. After cooling, weigh again, make up the lost weight with water, shake well, filter, and take the filtrate.

[0128] According to the "chromatographic conditions" in Example 1, the injection volume was 4 μL, and a characteristic spectrum of the sample to be tested was obtained; the peak area ratio of Peak 3 and Peak 5 in the characteristic spectrum of the sample to be tested was calculated according to Formula I, when β1>0.3, the sample to be tested was Momordica cochinchinensis kernel, and when β1≤0.3, the sample to be tested was Momordica cochinchinensis seed.

[0129]

[0130] Among them, S3 is the peak area of ​​peak 3, and S5 is the peak area of ​​peak 5.

[0131] Experimental Example 1 Methodological Verification

[0132] (1) Repeatability

[0133] 0.2 g of the same batch of Momordica Cocos seed formula granules (batch number K298KL17) were taken and prepared into test solutions according to the method under "Preparation of test solution" in Example 1, with a total of 6 portions, numbered 1-6 respectively, and then measured according to "Chromatographic conditions" and "Determination" in Example 1 to obtain characteristic spectra, and the relative retention time and relative peak area were calculated. The results are shown in Tables 2 and 3.

[0134] Table 2 Relative retention time of characteristic spectrum repeatability of Momordica cochinchinensis seed formula granules

[0135] Chromatographic peak number 1 2 3 4 5(S) 6 7 1 0.241 0.324 0.812 0.930 1.000 1.479 1.681 2 0.241 0.324 0.813 0.930 1.000 1.479 1.681 3 0.241 0.324 0.813 0.931 1.000 1.477 1.680 4 0.241 0.324 0.811 0.929 1.000 1.479 1.682 5 0.241 0.324 0.811 0.929 1.000 1.479 1.683 6 0.240 0.324 0.811 0.929 1.000 1.479 1.683 RSD / % 0.09 0.05 0.11 0.07 0.00 0.05 0.07

[0136] Table 3 Peak area and relative peak area of ​​characteristic spectrum repeatability of formula granules of Momordica cochinchinensis seeds

[0137] Chromatographic peak number 1 2 3 4 5(S) 6 7 1 0.096 0.085 0.103 0.026 1.000 0.029 0.174 2 0.096 0.084 0.102 0.027 1.000 0.029 0.174 3 0.097 0.085 0.102 0.025 1.000 0.030 0.171 4 0.098 0.086 0.104 0.027 1.000 0.030 0.173 5 0.098 0.086 0.108 0.027 1.000 0.032 0.173 6 0.100 0.088 0.108 0.027 1.000 0.031 0.172 RSD / % 1.5 1.4 2.9 2.9 0.0 3.2 0.7

[0138] From the results in Table 2 and Table 3, the relative retention time RSD of each characteristic peak of the characteristic spectrum obtained by the present invention is 0.05%-0.11%, and the RSD of the relative peak area is in the range of 0.7%-3.2%, indicating that the repeatability of the characteristic spectrum is good.

[0139] (2) Intermediate precision

[0140] 0.2 g of the same batch of Momordica cochinchinensis seed formula granules (batch number K298KL17) were taken and prepared into test solutions according to the method under "Preparation of test solution" in Example 1. There were 6 samples in total, numbered 1-6 respectively. An intermediate precision experiment was carried out using an Agilent ultra high performance liquid chromatograph different from that in Example 1. The characteristic spectra of the 6 samples were obtained according to the "Chromatographic conditions" and "Determination" of Example 1. The relative retention time and relative peak area were calculated. The results are shown in Tables 4 and 5.

[0141] Table 4 Retention time and relative retention time table of intermediate precision investigation of characteristic spectra

[0142]

[0143] Table 5 Peak area and relative peak area of ​​intermediate precision test of characteristic spectrum

[0144]

[0145]

[0146] From Tables 4 and 5, in the characteristic spectra obtained by Waters UPLC H-Class, TUV detector, the relative retention time RSD of each characteristic peak is in the range of 0.02%-0.09%, indicating that the intermediate precision of the characteristic spectra is good; compared with the repeatability sample, the relative retention time RSD of the two is in the range of 1.6%-3.5%, indicating that the relative retention time precision of the characteristic spectra is good; and the relative peak area RSD is 1.2%-1.9%, compared with the repeatability sample, the relative peak area RSD of the two is in the range of 1.0%-4.6%, indicating that different instrument analyses have little effect on the characteristic spectra.

[0147] (3) Stability investigation

[0148] 0.2 g of the same batch of Momordica Cocos seed formula granules (batch number K298KL17) were taken and prepared into test solutions according to the method under "Preparation of test solution" in Example 1, with a total of 7 portions. Then, the solutions were placed at room temperature for 0, 2, 4, 6, 9, 11, and 24 h, respectively, and then measured according to the "Chromatographic conditions" and "Determination" in Example 1 to obtain characteristic spectra, and the relative retention time and relative peak area were calculated. The results are shown in Tables 6 and 7.

[0149] Table 6 Retention time and relative retention time table of characteristic spectrum stability investigation

[0150] Chromatographic peak number 1 2 3 4 5(S) 6 7 0h 0.242 0.324 0.812 0.928 1.000 1.479 1.682 2h 0.240 0.324 0.813 0.930 1.000 1.479 1.682 4h 0.240 0.324 0.813 0.930 1.000 1.479 1.683 6h 0.240 0.323 0.812 0.930 1.000 1.479 1.684 9h 0.239 0.324 0.818 0.936 1.000 1.488 1.694 11h 0.241 0.327 0.815 0.931 1.000 1.473 1.676 24h 0.238 0.324 0.809 0.924 1.000 1.478 1.686 RSD / % 0.49 0.37 0.34 0.38 0.00 0.29 0.32

[0151] Table 7 Peak area and relative peak area of ​​characteristic spectrum stability test

[0152]

[0153]

[0154] According to the results in Tables 6 and 7, the chemical components in the test solution were stable within 24 h, the relative retention time RSD of each characteristic peak was in the range of 0.29%-0.49%, and the relative peak area RSD was in the range of 2.35%-3.02%, indicating that the method of the present invention is stable and reliable.

[0155] Experimental Example 2 Investigation of column temperature

[0156] The same batch of Momordica cochinchinensis seed formula granules (batch number K298KL17) were taken, ground, and prepared into test solutions according to the method under the item "Preparation of test solution" in Example 1, a total of 3 portions, and measured according to the items "Chromatographic conditions" and "Measurement" in Example 1 to obtain characteristic spectra. The difference between these 3 test solutions when obtaining characteristic spectra is that the column temperatures are different, which are 32°C, 35°C and 37°C, respectively. The results are shown in Table 8.

[0157] Table 8 Retention time and relative retention time table at different column temperatures

[0158]

[0159] Table 8 shows that at different column temperatures, the relative retention times of the seven characteristic peaks are within the range of ±10% of the specified value. The results show that the relative retention time of this method has good durability to column temperature.

[0160] Experimental Example 3 Investigation of flow rate

[0161] The same batch of Momordica cochinchinensis seed formula granules (batch number K298KL17) were taken, ground, and prepared into test solutions according to the method under the item "Preparation of test solution" in Example 1, a total of 3 portions, and measured according to the items "Chromatographic conditions" and "Measurement" in Example 1 to obtain characteristic spectra. The difference between these 3 test solutions when obtaining characteristic spectra is that the flow rates are different, which are 0.28 ml / min, 0.30 ml / min, and 0.32 ml / min, respectively. The results are shown in Table 9.

[0162] Table 9 Retention time and relative retention time table at different flow rates

[0163]

[0164] Table 9 shows that at different flow rates, the relative retention times of the seven characteristic peaks are within the range of ±10% of the specified value. The results show that the relative retention time of this method has good durability to flow rate.

[0165] Experimental Example 4 Investigation of Chromatographic Column

[0166] The same batch of Momordica cochinchinensis seed granules (batch number K298KL17) were taken and ground into powder. The test solution was prepared according to the method under the item "Preparation of the test solution" in Example 1. There were 3 parts in total. The characteristic spectra were obtained according to the items "Chromatographic conditions" and "Determination" in Example 1. The difference between the 3 test solutions in obtaining the characteristic spectra was that the chromatographic columns were different. The results are shown in Table 10. The chromatographic columns are as follows:

[0167] Column 1: ACQUITY HSS T3 (2.1×150 mm, 1.8 μm);

[0168] Column 2: ACQUITY UPLC BEH Shield RP18 (2.1 mm × 150 mm, 1.7 μm);

[0169] Column 3: ACQUITY T3 (2.1×150 mm, 1.6 μm)

[0170] Table 10 Retention time and relative retention time table at different flow rates

[0171]

[0172] The relative retention times of the seven characteristic peaks of the three different types of chromatographic columns do not completely conform to the specified value within the range of ±10%, and the peak areas vary greatly, indicating that the present invention is only applicable to the ACQUITY UPLC BEH Shield RP18 chromatographic column.

[0173] Experimental Example 5 Investigation of β1

[0174] (1) Fifteen batches of Momordica cochinchinensis seed granules were taken and test solutions were prepared according to the method under "Preparation of test solution" in Example 1. The characteristic spectrum was obtained according to the "Chromatographic conditions" and "Determination" items in Example 1, and the peak areas of Peak 3 and Peak 5 and their ratio were calculated. The results and the batch numbers of the Momordica cochinchinensis seed granules are shown in Table 11.

[0175] Table 11 Peak area and ratio of peak 3 and peak 5 in the characteristic spectrum of Momordica cochinchinensis formula granules

[0176]

[0177] (2) Fifteen batches of Momordica cochinchinensis granules were taken and test solutions were prepared according to the method under "Preparation of test solution" in Example 1. The test was performed according to the "Chromatographic conditions" and "Determination" items in Example 1 to obtain a characteristic spectrum, and the peak areas of Peak 3 and Peak 5 and their ratio were calculated. The results and the batch numbers of the Momordica cochinchinensis granules are shown in Table 12.

[0178] Table 12 Peak area and ratio of peak 3 and peak 5 in the characteristic spectrum of Momordica charantia formula granules

[0179]

[0180]

[0181] The comparison results of Momordica cochinchinensis formula granules and Momordica cochinchinensis kernel formula granules β1 are shown in Table 13.

[0182] Table 13 Comparison results of Momordica cochinchinensis formula granules and Momordica cochinchinensis kernel formula granules β1

[0183]

[0184] It can be seen from the results in Table 13 that the ratios of the peak areas of Peak 3 and Peak 5 of the Momordica chinensis formula granules and the Momordica chinensis kernel formula granules are quite different. The measured values ​​of the Momordica chinensis kernel formula granules are 0.38-0.77, with an average of 0.63, and the measured values ​​of the Momordica chinensis formula granules are 0.10-0.17, with an average of 0.12, indicating that the peak area ratios of 15 batches of Momordica chinensis kernel formula granules are greater than 0.3, and the peak area ratios of 15 batches of Momordica chinensis formula granules are less than 0.3. Therefore, when β1>0.3, it is a Momordica chinensis kernel formula granule sample, and when β1≤0.3, it is a Momordica chinensis formula granule sample; the method provided by the present invention is suitable for identifying Momordica chinensis and Momordica chinensis kernel.

[0185] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the scope of protection of the invention.

Claims

1. A method for constructing a characteristic spectrum of Momordica charantia or Momordica charantia kernel, characterized in that: The method comprises constructing a characteristic spectrum of a test sample, wherein the construction method comprises the following steps: The method for preparing the test solution includes: taking the test sample, adding water, ultrasonic treatment, and filtering; using p-hydroxybenzoic acid as a reference substance; Chromatographic conditions: octadecylsilane bonded silica as filler, 0.1% formic acid as mobile phase A, acetonitrile as mobile phase B, gradient elution; chromatographic column specifications: column length 150 mm, inner diameter 2.1 mm, particle size 1.7 μm; The gradient elution procedure includes: 0-1min, the volume percentage of mobile phase A is 100%, and the volume percentage of mobile phase B is 0%; 1-2min, the volume percentage of mobile phase A is 100-98%, and the volume percentage of mobile phase B is 0-2%; 2-15min, the volume percentage of mobile phase A is 98-92%, and the volume percentage of mobile phase B is 2-8%; 15-25min, the volume percentage of mobile phase A is 92-85%, and the volume percentage of mobile phase B is 8-15%; 25-27min, the volume percentage of mobile phase A is 85-100%, and the volume percentage of mobile phase B is 15%-0; 25-30min, the volume percentage of mobile phase A is 100%, and the volume percentage of mobile phase B is 0; Determination: Take the test solution, inject it into the ultra-high performance liquid chromatography instrument, and determine it.

2. The construction method according to claim 1, characterized in that: Chromatographic conditions: flow rate 0.28-0.32 ml / min, column temperature 32-35°C, detection wavelength 254 nm, theoretical plate number calculated based on the p-hydroxybenzoic acid peak is not less than 3000.

3. The construction method according to claim 1 or 2, characterized in that: The test sample is at least one of Momordica cochinchinensis decoction pieces, Momordica cochinchinensis formula granules, Momordica cochinchinensis standard decoction freeze-dried powder, Momordica cochinchinensis medicinal materials, Momordica cochinchinensis formula granules and Momordica cochinchinensis standard decoction freeze-dried powder.

4. The construction method according to claim 1 or 2, characterized in that: The test solution is a water extract.

5. The construction method according to claim 4, characterized in that: When preparing the test solution, the ratio of the mass of the test sample to the volume of the extraction solvent is 0.008-0.014 g / ml.

6. The construction method according to claim 1 or 2, characterized in that: The power of the ultrasonic treatment is 700 W, the frequency is 40 kHz, and the time is 30±min.

7. The construction method according to claim 1 or 2, characterized in that: The construction method also includes the preparation of a reference solution.

8. The construction method according to claim 7, characterized in that: When preparing the reference substance solution, the ratio of the reference substance mass to the extraction solvent volume is 0.004-0.007 g / ml.

9. The construction method according to claim 8, characterized in that: The Momordica charantia control medicinal material was used as a reference.

10. The construction method according to claim 1 or 2, characterized in that: The construction method also includes preparing the reference substance into a reference substance solution; The reference substance content in each 1 ml of the reference substance solution is 2-5 μg.

11. The construction method according to claim 1, characterized in that: The characteristic spectrum obtained by the construction method includes 7 characteristic peaks; Taking peak 5 as the reference peak, the specified values ​​of relative retention times of peaks 1, 2, 3, 4, 6 and 7 are 0.24, 0.32, 0.81, 0.93, 1.48 and 1.68 respectively; Among them, the relative retention time of the characteristic peak is within ±10% of the specified value.

12. A method for identifying Momordica cochinchinensis and Momordica cochinchinensis kernels, characterized in that: The characteristic spectrum of the sample to be tested is obtained by the construction method described in any one of claims 1 to 11, and identification is performed based on the ratio of the peak areas of Peak 3 and Peak 5 in the characteristic spectrum of the sample to be tested.

13. The identification method according to claim 12, characterized in that: Formula I was used for identification. When β1>0.3, the sample to be tested was Momordica cochinchinensis kernel; When β1≤0.3, the sample to be tested is Momordica charantia; Formula I; Among them, S3 is the peak area of ​​peak 3, and S5 is the peak area of ​​peak 5.

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