Method for constructing trichosanthes kirilowii maxim medicinal material fingerprint spectrum and determining content of trichosanthes kirilowii maxim index components

Abstract

The invention relates to a method for constructing a trichosanthes kirilowii maxim medicinal material fingerprint spectrum and determining the content of trichosanthes kirilowii maxim index components. The method comprises the following steps: taking a trichosanthes kirilowii maxim medicinal material to prepare a test solution; taking rutin, chrysoeriol and cucurbitacine D to prepare a reference substance solution; respectively injecting the test solution and the reference solution into a liquid chromatograph-mass spectrometer, obtaining a fingerprint spectrum of the trichosanthes kirilowii maxim medicinal material according to the liquid chromatograph-mass spectrometer, establishing a UHPLC-QQQ-MS quantitative detection method, and determining the content of the index components in the trichosanthes kirilowii maxim medicinal material in an MRM mode. According to the method for constructing the snakegourd fruit medicinal material fingerprint spectrum and measuring the content, provided by the invention, the evaluation standard of snakegourd fruits is comprehensively improved, and the effectiveness of the snakegourd fruit medicinal material in clinical use is ensured.

Description

[0001] The present invention is a divisional application of the invention application with application number 202310314109.1, application date March 28, 2023, and invention name “A method for constructing a fingerprint of Trichosanthes kirilowii medicinal materials and determining the content of index components of Trichosanthes kirilowii”. Technical Field

[0002] The invention belongs to the technical field of traditional Chinese medicine quality analysis and detection, and particularly relates to a method for constructing a medicinal material fingerprint of Trichosanthes kirilowii and determining the content of index components of Trichosanthes kirilowii. Background Art

[0003] Trichosanthes kirilowii Maxim or Trichosanthes rosthornii Harms, a plant of the Cucurbitaceae family, is a dried and mature fruit. It has the effects of clearing away heat and removing phlegm, relieving chest tightness and dispersing stagnation, and moistening the intestines.

[0004] Trichosanthes kirilowii is mainly produced in Shandong, Hebei, Anhui and other places, and is distributed in most parts of the country. It is necessary to use effective technical means to evaluate the consistency of the quality of Trichosanthes kirilowii medicinal materials to ensure the safety, effectiveness and quality control of Trichosanthes kirilowii medicinal materials.

[0005] However, there is no control standard for the consistency evaluation of the quality of Trichosanthes kirilowii. The 2020 edition of the Chinese Pharmacopoeia lacks a method for determining the content of the index components of Trichosanthes kirilowii. It only stipulates the content requirements of 3,29-dibenzoyltriol in Trichosanthes kirilowii seeds and stir-fried Trichosanthes kirilowii seeds. The current research work is difficult to effectively control the overall quality of Trichosanthes kirilowii medicinal materials.

[0006] Therefore, it is urgent to develop a method for constructing the fingerprint of Trichosanthes kirilowii medicinal materials and determining its content so as to be applied to the quality evaluation of Trichosanthes kirilowii medicinal materials. Summary of the invention

[0007] The present invention aims to solve one of the technical problems existing in the prior art to at least a certain extent. To this end, the present invention provides a method for constructing a fingerprint of a medicinal material Trichosanthes kirilowii and determining the content of an index component of the medicinal material Trichosanthes kirilowii.

[0008] According to one aspect of the present invention, a method for constructing a fingerprint of a Trichosanthes kirilowii medicinal material is provided, comprising: preparing a test solution from the Trichosanthes kirilowii medicinal material; preparing a reference solution from rutin, cyperus rotundus and cucurbitacin D; respectively injecting the test solution and the reference solution into a liquid chromatography-mass spectrometer, and obtaining the fingerprint of the Trichosanthes kirilowii medicinal material according to a liquid chromatography-mass spectrometry method.

[0009] Preferably, the fingerprint of the Trichosanthes kirilowii Maxim. medicinal material contains 11 characteristic peaks, numbered from peak 1 to peak 11; peak 4 is rutin, peak 10 is chrysoeriol, and peak 11 is cucurbitacin D. The characteristic peaks in the LC-MS fingerprint and the identification of the characteristic peaks are as follows:

[0010]

[0011] Preferably, taking peak 10 as the S peak, the relative retention time ranges of peaks 1 to 9 and peak 11 relative to the S peak are respectively: 0.148 - 0.152, 0.101 - 0.103, 0.310 - 0.315, 0.361 - 0.368, 0.471 - 0.476, 0.574 - 0.575, 0.649 - 0.652, 0.761 - 0.766, 0.847 - 0.851, 1.020 - 1.025.

[0012] Preferably, the chromatographic conditions of the liquid chromatography - mass spectrometry method include: stationary phase: a chromatographic column filled with octadecylsilyl - bonded silica gel; mobile phase: mobile phase A is water containing 0.1% (v / v) formic acid, and mobile phase B is an acetonitrile solution containing 0.1% (v / v) formic acid; gradient elution is adopted, and the flow rate is 0.3 mL / min.

[0013] Preferably, the chromatographic column is a CORTECS UPLC T3 Column (2.1×100 mm, 1.6 μm) chromatographic column.

[0014] Preferably, the gradient elution is carried out according to the following procedure: 0.0 - 1.0 min, mobile phase A changes from 85% → 84%, and mobile phase B changes from 15% → 16%; 1.0 - 5.5 min, mobile phase A remains at 84%, and mobile phase B remains at 16%; 5.5 - 6.0 min, mobile phase A changes from 84% → 77%, and mobile phase B changes from 16% → 23%; 6.0 - 16.0 min, mobile phase A changes from 77% → 66%, and mobile phase B changes from 23% → 34%.

[0015] Preferably, the mass spectrometry conditions of the liquid chromatography - mass spectrometry method include: ion source is ESI; ionization mode is negative ion mode; back - flush nitrogen temperature is 325 °C, back - flush nitrogen flow rate is 11.0 L / min, nebulizing gas flow rate is 45 psi, auxiliary nitrogen temperature is 350 °C, auxiliary nitrogen flow rate is 11.0 L / min, capillary voltage is 3500 V, in - source collision voltage is 175 V, cone - hole voltage is 65.0 V; scanning mode: full scan; scanning range: m / z 60 - 1700.

[0016] Preferably, the preparation method of the test solution includes: extracting and filtering the Trichosanthes kirilowii Maxim. medicinal material with a first solvent to obtain the test solution.

[0017] Preferably, the preparation method of the test sample solution comprises: adding 1 ml of the first solvent to 100 mg of the Trichosanthes kirilowii Maxim. medicinal material; soaking for 60 minutes and then performing ultrasonic extraction at 40 kHz and 30 °C for 30 minutes; centrifuging to obtain a first supernatant and a first residue; adding 1 ml of the first solvent to the first residue; performing ultrasonic extraction at 40 kHz and 30 °C for 30 minutes; centrifuging to obtain a second supernatant and a second residue; combining the first supernatant and the second supernatant to obtain the test sample solution, wherein the first solvent is 50%-95% ethanol.

[0018] According to another aspect of the present invention, there is provided a method for determining the content of the index components of the Trichosanthes kirilowii Maxim. medicinal material, comprising: preparing a test sample solution from the Trichosanthes kirilowii Maxim. medicinal material to be tested; preparing a reference substance solution from rutin, perlolyrine, chrysoeriol and cucurbitacin D; injecting the test sample solution and the reference substance solution into a liquid chromatography-mass spectrometry instrument respectively, and obtaining a test spectrum of the Trichosanthes kirilowii Maxim. medicinal material to be tested and a reference spectrum of the reference substance solution according to the liquid chromatography-mass spectrometry method; calculating the contents of the index components rutin, perlolyrine, chrysoeriol and cucurbitacin D in the Trichosanthes kirilowii Maxim. medicinal material to be tested according to the concentration of the reference substance solution, the peak area of the reference substance solution in the reference spectrum, and the peak area of the component corresponding to the Trichosanthes kirilowii Maxim. medicinal material to be tested and the reference substance solution in the test spectrum.

[0019] Preferably, the following relationship is satisfied between the concentration of the reference substance solution and the peak area of the reference substance solution in the reference spectrum: the first regression equation of rutin is y = 235579.05x + 1670.62 (r 2 = 0.9995, with a weight of 1 / x), the linear range is 0.025 - 2.5 μg / mL, and the detection limit is 0.005 μg / mL (S / N ≥ 3); the second regression equation of perlolyrine is y = 184480.64x + 13364.70 (r 2 = 0.9994, with a weight of 1 / x), the linear range is 0.05 - 5 μg / mL, and the detection limit is 0.01 μg / mL (S / N ≥ 3); the third regression equation of chrysoeriol is y = 493941.66x + 48.93 (r 2 = 0.9995, with a weight of 1 / x), the linear range is 0.025 - 2.5 μg / mL, and the detection limit is 0.005 μg / mL (S / N ≥ 3); the fourth regression equation of cucurbitacin D is y = 52814.15x - 340.56 (r 2= 0.9997, with a weight of 1 / x), the linear range is 0.025 - 5 μg / mL, and the detection limit is 0.005 μg / mL (S / N ≥ 3); among them, in the first regression equation, the second regression equation, the third regression equation and the fourth regression equation, the abscissa is the concentration of the reference solution, and the ordinate is the peak area in the reference chromatogram.

[0020] Preferably, the chromatographic conditions of the liquid chromatography - mass spectrometry method include: stationary phase: a chromatographic column filled with octadecylsilyl-bonded silica gel; mobile phase: mobile phase A is water containing 0.1% (v / v) formic acid, and mobile phase B is an acetonitrile solution containing 0.1% (v / v) formic acid; gradient elution is adopted, and the flow rate is 0.3 mL / min.

[0021] Preferably, the gradient elution is carried out according to the following procedure: from 0.0 - 3.0 min, mobile phase A changes from 80% → 70%, and mobile phase B changes from 20% → 30%; from 3.0 - 9.0 min, mobile phase A changes from 70% → 68.5%, and mobile phase B changes from 30% → 31.5%.

[0022] Preferably, the mass spectrometry conditions of the liquid chromatography - mass spectrometry method include: the ion source adopts the ESI positive and negative ion switching mode, the positive ion mode is from 0 to 4 min, and the negative ion mode is from 4 to 9 min; the scanning mode is the MRM scanning mode; the temperature of the backflush nitrogen is 325 °C, the flow rate of the backflush nitrogen is 10.0 L / min, the flow rate of the nebulizing gas is 40 psig, and the capillary voltage is 4000 V.

[0023] Preferably, the MRM scanning parameters of rutin, loline, chrysoeriol and cucurbitacin D in the index components are:

[0024]

[0025] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a method for constructing a fingerprint spectrum of Trichosanthes kirilowii Maxim. and determining the content of index components. By optimizing the liquid phase elution method, the method realizes the effective separation of different types of index components in Trichosanthes kirilowii Maxim., and uses the ion and fragment information listed by the mass spectrometry to identify and authenticate the chromatographic peaks in the fingerprint spectrum. At the same time, a quantitative analysis method for the index components in Trichosanthes kirilowii Maxim. is established. The present invention stably and comprehensively improves the evaluation standard of Trichosanthes kirilowii Maxim. medicinal materials, ensures the effectiveness of the medicinal materials in clinical use, and provides a reference for the overall control of the quality of Trichosanthes kirilowii Maxim. medicinal materials. Description of the Drawings

[0026] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of this application. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0027] Figure 1 Shows the fingerprint liquid chromatography elution method 01 chromatogram (a) and 02 chromatogram (b);

[0028] Figure 2 Shows the comparison between the reference solution control spectrum and the LC-MS fingerprint test solution spectrum of the test sample;

[0029] Figure 3 Shows the fingerprint spectra of 15 batches of Trichosanthes kirilowii Maxim. medicinal materials provided by the present invention, and the sample numbers are shown in Table 1;

[0030] Figure 4 Shows the reference fingerprint spectrum generated by the average method from the fingerprint spectra of 15 batches of Trichosanthes kirilowii Maxim. medicinal materials provided by the present invention;

[0031] Figure 5 Shows the content determination liquid chromatography elution method -1 chromatogram (a) and method -2 chromatogram (b); and

[0032] Figure 6 Shows the comparison between the reference solution control spectrum of the content determination mixed reference solution and the test spectrum of the test sample solution for LC-MS content determination. Detailed implementation manners

[0033] The following listed examples are to enable those skilled in the art to understand the present invention more clearly. It should be noted that the following examples do not limit the protection scope required by the present invention and are only illustrative examples. The raw materials, reagents or devices mentioned in the following examples can be obtained from commercial channels or by known existing methods without special instructions. The origin information of the Trichosanthes kirilowii Maxim. medicinal materials used in the present invention is shown in Table 1.

[0034] Table 1: Origin information of 15 batches of Trichosanthes kirilowii Maxim. medicinal materials

[0035]

[0036] Example 1

[0037] Method for constructing the fingerprint spectrum of Trichosanthes kirilowii Maxim. medicinal materials

[0038] This example provides a method for constructing the fingerprint spectrum of Trichosanthes kirilowii Maxim. medicinal materials, including: preparing the Trichosanthes kirilowii Maxim. medicinal materials into a test solution; preparing rutin, chrysoeriol and cucurbitacin D into a reference solution; injecting the test solution and the reference solution into a liquid chromatography - mass spectrometry instrument respectively, and obtaining the fingerprint spectrum of Trichosanthes kirilowii Maxim. medicinal materials according to the liquid chromatography - mass spectrometry method. The following provides the specific construction method.

[0039] 1. Preparation of the test solution

[0040] Accurately weigh 100.00 mg of Trichosanthes kirilowii Maxim. medicinal material powder (passed through No. 3 sieve) (error less than 0.05 mg), add 1 mL of 50%-95% ethanol, soak for 60 min, extract by ultrasound for 30 min (40 kHz, 30 °C), centrifuge, take the supernatant. Add 1 mL of 50%-95% ethanol to the residue, extract by ultrasound for 30 min (40 kHz, 30 °C), centrifuge, take the supernatant. Combine the supernatants of the two times to obtain the test solution for LC-MS fingerprint. Among them, ethanol is preferably 75%.

[0041] 2. Preparation of reference substance solution

[0042] Weigh reference substances of rutin, chrysoeriol and cucurbitacin D respectively, dissolve them with methanol, and prepare rutin and cucurbitacin D reference substance solutions at a concentration of 1 mg / mL and a chrysoeriol reference substance solution at a concentration of 0.25 mg / mL respectively. Using methanol as the dilution solvent, prepare a reference substance mixed solution with a concentration of 5 μg / mL for all.

[0043] 3. Optimization of liquid chromatography-mass spectrometry conditions

[0044] Take a preset amount of reference substance solution and test solution, inject them into the LC-MS liquid chromatography-mass spectrometry instrument. Using octadecylsilane chemically bonded silica as the filler, use an aqueous solution containing 0.1% (v / v) formic acid as mobile phase A and an acetonitrile solution containing 0.1% (v / v) formic acid as mobile phase B for gradient elution to construct the LC-MS fingerprint of Trichosanthes kirilowii Maxim. medicinal material.

[0045] The high-resolution mass spectrometry detection conditions are: the ion source is ESI; the ionization mode is negative ion mode; the temperature of the backflush nitrogen is 325 °C, the flow rate of the backflush nitrogen is 11.0 L / min, the flow rate of the nebulizing gas is 45 psi, the temperature of the auxiliary nitrogen is 350 °C, the flow rate of the auxiliary nitrogen is 11.0 L / min, the capillary voltage is 3500 V, the in-source collision voltage is 175 V, the cone voltage is 65.0 V; the scanning mode: full scan; the scanning range: m / z 60 - 1700.

[0046] 3.1 Optimization of liquid chromatography elution method

[0047] The preliminary attempt of liquid chromatography elution method 01 is: 0.0 - 1.0 min: 15 - 17% B, 1.0 - 5.0 min: 17% B, 5.0 - 6.0 min: 17 - 29% B, 6.0 - 9.5 min: 29% B, 9.5 - 15.0 min: 29 - 35% B; 15.0 - 16.0 min: 35% - 38.5% B. Under this elution condition, characteristic peaks 3 and 4 could not be baseline separated, and characteristic peak 8 was affected by small shoulder peaks and could not be clearly presented ( Figure 1a); Based on the experimental results, the elution method 02 was optimized, and the elution gradient was as follows: 0.0 - 1.0 min: 15 - 16% B, 1.0 - 5.5 min: 16% B, 5.5 - 6.0 min: 16 - 23% B, 6.0 - 16.0 min: 23 - 34% B. Under these elution conditions, the peak shapes and resolutions of the common peaks were both satisfactory. Figure 1 b).

[0048] Table 2: Gradient elution table of LC-MS fingerprint

[0049]

[0050] 3.2 Determination of liquid chromatography conditions

[0051] The chromatographic column was CORTECS UPLC T3 Column (2.1×100 mm, 1.6 μm), the flow rate was 0.3 mL / min, and the column temperature was 30 °C. The eluent was an aqueous solution containing 0.1% (v / v) formic acid (mobile phase A) and an acetonitrile solution containing 0.1% (v / v) formic acid (mobile phase B). The injection volume was 1 μL. Based on the optimal gradient elution program selected in 3.1, the control map of the trichosanthes root reference substance solution and the map of the test solution of LC-MS fingerprint are shown in Figure 2 :

[0052] 0.0 - 1.0 min, mobile phase A changed from 85% → 84%, mobile phase B changed from 15% → 16%;

[0053] 1.0 - 5.5 min, mobile phase A remained at 84%, mobile phase B remained at 16%;

[0054] 5.5 - 6.0 min, mobile phase A changed from 84% → 77%, mobile phase B changed from 16% → 23%;

[0055] 6.0 - 16.0 min, mobile phase A changed from 77% → 66%, mobile phase B changed from 23% → 34%;

[0056] 4. Identification of chemical components in LC-MS fingerprint

[0057] Using the LC-MS method, the reference substance solution and the test solution were detected. By comparing the retention times of the chromatographic peaks of the compounds, the exact molecular weights, and the fragmentation rules of the secondary spectra, a total of 3 compounds corresponding to the chromatographic peaks were determined, namely rutin (peak 4), chrysoeriol (peak 10), and cucurbitacin D (peak 11).

[0058] Using the LC-MS method, by comparing with the structures reported in the literature, retention times, and combining the fragmentation rules of tandem mass spectrometry, the compounds corresponding to another 8 chromatographic peaks were identified, namely 6,9-dihydroxy-4,7-megastigmadien-3-one-9-O-[α-L-apifuranosyl-(1→2)-β-D-glucopyranoside] or its isomers (peak 1), 2,7-dimethyl-2,4-dienedeca-α,ω-diacid-8-O-β-D-glu or its isomers (peaks 2, 3, 5), Chrysoeriol-7-O-β-D-glucoside or its isomers (peak 6), Propyl 2-(ethoxymethyl)-5-formyl-1H-pyrrole-1-carboxylate or its isomers (peak 7), 23,24-dihydrokhekadaengoside E or its isomers (peak 8), Opercurins A or its isomers (peak 9).

[0059] The fingerprint of Trichosanthes kirilowii Maxim. medicinal materials contains 11 characteristic peaks, numbered from peak 1 to peak 11. Peak 4 is rutin, peak 10 is chrysoeriol, and peak 11 is cucurbitacin D. The identification of characteristic peaks and characteristic peaks is shown in Table 3 for details.

[0060] Table 3: Identification of LC-MS characteristic peaks and characteristic peaks

[0061]

[0062] Taking peak 10 as the S peak, the relative retention time ranges of peaks 1 - 9 and peak 11 relative to the S peak are: 0.148 - 0.152, 0.101 - 0.103, 0.310 - 0.315, 0.361 - 0.368, 0.471 - 0.476, 0.574 - 0.575, 0.649 - 0.652, 0.761 - 0.766, 0.847 - 0.851, 1.020 - 1.025.

[0063] 5. Similarity evaluation of fingerprint

[0064] The LC-MS base peak map txt format of the test solution of 15 batches of Trichosanthes kirilowii Maxim. medicinal materials was imported into the "Chinese Chromatographic Fingerprint Similarity Evaluation System" (V2.0) software. Taking chrysoeriol (peak 10) as the reference peak, the fingerprint of 15 batches of Trichosanthes kirilowii Maxim. medicinal materials was established ( Figure 3 ), and the reference map was generated by the average method ( Figure 4) Calculate the similarity. The results show that the similarities of the fingerprint spectra of 15 Trichosanthis Fructus herbs are all above 0.900, indicating that the fingerprint spectrum can better evaluate the quality of Trichosanthis Fructus herbs. The specific similarity results are shown in Table 4, and the sample numbers can be seen in Table 1.

[0065] Table 4: Similarities of LC-MS Fingerprint Spectra of 15 Batches of Trichosanthis Fructus Herbs

[0066]

[0067] Example 2

[0068] This example provides a method for determining the contents of marker components in Trichosanthis Fructus herbs, including: preparing a test sample solution from the Trichosanthis Fructus herb to be tested; preparing a reference substance solution from rutin, loline, chrysoeriol, and cucurbitacin D; injecting the test sample solution and the reference substance solution into a liquid chromatography-mass spectrometry (LC-MS) instrument respectively, and obtaining the test spectrum of the Trichosanthis Fructus herb to be tested and the reference spectrum of the reference substance solution according to the LC-MS method; calculating the contents of the marker components rutin, loline, chrysoeriol, and cucurbitacin D in the Trichosanthis Fructus herb to be tested according to the concentration of the reference substance solution, the peak area of the reference substance solution in the reference spectrum, and the peak area of the component corresponding to the Trichosanthis Fructus herb to be tested and the reference substance solution in the test spectrum. The following provides the specific determination method.

[0069] 1. MRM Detection Parameters of Reference Substances

[0070] Based on 4 reference substances (including the marker components rutin, loline, chrysoeriol, and cucurbitacin D), establish the MRM quantitative method for 15 batches of Trichosanthis Fructus samples, and the MRM parameters are shown in Table 5.

[0071] Table 5: MRM Detection Parameters of 4 Reference Substances

[0072]

[0073] 2. Optimization of Experimental Conditions of Quantitative Method

[0074] The preliminary attempt of the liquid phase elution method -1 is: 0.0 - 0.5 min: 20% - 20% B, 0.5 - 3.0 min: 20% - 30% B, 3.0 - 7.0 min: 30% - 35% B, 7.0 - 9.0 min: 35% - 95% B. Under this elution condition, chrysoeriol and cucurbitacin D elute and peak simultaneously, and baseline separation cannot be achieved. Figure 5a); Based on the experimental results, the elution method - 2 was optimized, and the elution gradient was: 0.0 - 3.0 min: 20% - 30% B, 3.0 - 9.0 min: 30% - 31.5% B. The baseline separation of chrysoeriol and cucurbitacin D was achieved under a relatively gentle elution gradient. Figure 5 b).

[0075] 3. Determination of the experimental conditions for the quantification method

[0076] The UHPLC - QQQ - MS conditions were as follows: The chromatographic column was a CORTECS UPLC T3 Column (2.1×100 mm, 1.6 μm), the flow rate was 0.3 mL / min, and the column temperature was 30°C. The eluent was an aqueous solution containing 0.1% (v / v) formic acid (mobile phase A) and an acetonitrile solution containing 0.1% (v / v) formic acid (mobile phase B). The injection volume was 2 μL, and gradient elution was carried out according to the following procedure. For the comparison between the reference chromatogram of the mixed reference standard solution for content determination and the LC - MS chromatogram of the sample solution to be measured for content determination, see Figure 6 .

[0077] 0.0 - 3.0 min, mobile phase A changed from 80% → 70%, mobile phase B changed from 20% → 30%;

[0078] 3.0 - 9.0 min, mobile phase A changed from 70% → 68.5%, mobile phase B changed from 30% → 31.5%.

[0079] The mass spectrometry detection was carried out in the positive and negative ion switching mode; the temperature of the back - blown nitrogen was 325°C, the flow rate of the back - blown nitrogen was 10.0 L / min, the flow rate of the nebulizing gas was 40 psi, the temperature of the auxiliary nitrogen was 350°C, the flow rate of the auxiliary nitrogen was 11.0 L / min, and the capillary voltage was 4000 V. The analytes were determined by LC - QQQ - MS in the MRM mode.

[0080] 4. Preparation of the sample solution to be measured

[0081] Accurately weigh 100.00 mg of the Trichosanthes kirilowii Maxim. sample powder (passed through No. 3 sieve), add 1 mL of 50 - 95% ethanol, soak for 1 h, extract ultrasonically for 30 min (40 kHz, 30°C), centrifuge, take the supernatant. Add 1 mL of 50 - 95% ethanol to the residue, extract ultrasonically for 30 min (40 kHz, 30°C), centrifuge, take the supernatant, and combine the two supernatants to obtain the test solution of the Trichosanthes kirilowii Maxim. medicinal material. Among them, ethanol is preferably 75%.

[0082] 5. Methodological verification

[0083] The methodological verification items include detection limit, linear range, precision, accuracy, recovery rate, matrix effect, and stability.

[0084] 5.1 Detection limit, linear range

[0085] Preparation of standard solution: Weigh an appropriate amount of rutin, loline, chrysoeriol, and cucurbitacin D reference substances accurately, dissolve them in methanol. Except for chrysoeriol which is made into a 0.25 mg / mL solution, the other three are respectively made into 1 mg / mL reference substance stock solutions. Take an appropriate amount of the reference substance stock solutions and mix them to make a solution containing 10 μg of loline, 5 μg of rutin, 5 μg of chrysoeriol, and 5 μg of cucurbitacin D in every 1 mL, that is, the mixed reference substance solution is obtained.

[0086] Gradiently dilute the mixed reference substance solution to prepare a series of linear working solutions. Dilute the stock solution to three concentrations of low, medium, and high as the quality control samples of the mixed reference substance. Among them, the low concentration is 0.25 μg of loline, 0.125 μg of chrysoeriol, 0.125 μg of rutin, and 0.125 μg of cucurbitacin D in every 1 mL; the medium concentration is 1 μg of loline, 0.5 μg of chrysoeriol, 0.5 μg of rutin, and 0.5 μg of cucurbitacin D in every 1 mL; the high concentration is 5 μg of loline, 2.5 μg of chrysoeriol, 2.5 μg of rutin, and 2.5 μg of cucurbitacin D in every 1 mL. The standard curve graph uses the concentration as the abscissa and the peak area as the ordinate, conducts linear regression, and obtains the standard curve.

[0087] The standard curve of rutin is y = 235579.05x + 1670.62 (r 2 = 0.9995, the weight is 1 / x), the linear range is 0.025 - 2.5 μg / mL, and the detection limit is 0.005 μg / mL (S / N≥3).

[0088] The standard curve of loline is y = 184480.64x + 13364.70 (r 2 = 0.9994, the weight is 1 / x), the linear range is 0.05 - 5 μg / mL, and the detection limit is 0.01 μg / mL (S / N≥3).

[0089] The standard curve of chrysoeriol is y = 493941.66x + 48.93 (r 2 = 0.9995, the weight is 1 / x), the linear range is 0.025 - 2.5 μg / mL, and the detection limit is 0.005 μg / mL (S / N≥3).

[0090] The standard curve of cucurbitacin D is y = 52814.15x - 340.56 (r 2 = 0.9997, the weight is 1 / x), the linear range is 0.025 - 5 μg / mL, and the detection limit is 0.005 μg / mL (S / N≥3).

[0091] 5.2 Precision, accuracy, stability, recovery rate, matrix effect

[0092] Low, medium, and high concentration quality control samples were taken, and the UHPLC-QQQ-MS conditions were used to continuously inject samples for determination 3 times respectively, and continuously determined for 3 days. The relative standard deviation (RSD) values of the peak areas of 4 analytes to be detected were calculated to obtain the within-day precision and between-day precision; the accuracy was evaluated by the percentage ratio of the detected concentration to the actual concentration of the quality control sample. The results showed that the within-day and between-day precisions of the low, medium, and high concentration quality control samples were within ±15%, and the within-day and between-day accuracies were between 85% and 115%, meeting the requirements.

[0093] The stability was calculated from the peak areas of the same low, medium, and high concentration quality control samples, which were injected for detection within 48 h at 4°C according to the UHPLC-QQQ-MS conditions under item "3" of Example 2. The results showed that the RSD of the stability determination of the low, medium, and high concentration quality control samples was within ±15%, meeting the requirements.

[0094] According to the following formula, a mixed standard solution with a determined concentration was added to the sample to conduct the recovery experiment and matrix effect experiment. The results showed that both the recovery rate and the matrix effect were between 85% and 115%, meeting the requirements.

[0095] Recovery rate = (peak area of the sample after spiking - peak area of the sample) / peak area of the mixed standard solution with a determined concentration)

[0096] Matrix effect = (peak area of the sample after spiking after extraction - peak area of the sample) / peak area of the mixed standard solution with a determined concentration)

[0097] The above results (Tables 6 and 7) indicate that the established quantitative method can accurately, precisely, and stably quantify the index components in the Trichosanthis Kirilowii Maxim. medicinal material samples.

[0098] Table 6: Results of precision and accuracy experiments of related reference substances of Trichosanthis Kirilowii Maxim. medicinal materials

[0099]

[0100] Table 7: Results of stability, recovery rate, and matrix effect experiments of related reference substances of Trichosanthis Kirilowii Maxim. medicinal materials

[0101]

[0102] 6.15 Determination of the content of index components in Trichosanthis Kirilowii Maxim. medicinal materials

[0103] The contents of four components in 15 batches of Trichosanthes kirilowii Maxim. herbs were determined. The preparation method of Trichosanthes kirilowii Maxim. herbs in different batches was referred to the preparation of the test solution under item "4" of Example 2. The LC-MS detection parameters were referred to the selection of experimental conditions for the quantitative method under item "3" of Example 2. According to the LC-MS detection results, the components and contents of Trichosanthes kirilowii Maxim. herbs in different batches were analyzed. The content of loline in 15 batches of Trichosanthes kirilowii Maxim. herb samples was 0.00075% - 0.00455%, the content of chrysoeriol was 0.00059% - 0.00381%, the content of rutin was 0.00010% - 0.00342%, and the content of cucurbitacin D was 0.00007% - 0.00105%. The specific determination results are shown in Table 8.

[0104] Table 8: Quantitative results of index components in 15 batches of Trichosanthes kirilowii Maxim. herbs

[0105]

[0106] * The content calculation formula is: the content in Trichosanthes kirilowii Maxim. sample = m / M × 100% (where m is the content of the compound to be detected in the sample detected by linearity, and M is the initial mass of the Trichosanthes kirilowii Maxim. herb powder)

[0107] To sum up, the present invention provides a method for constructing the fingerprint of Trichosanthes kirilowii Maxim. herbs and determining the content of index components. This method makes up for the problem of poor specificity of the previous fingerprint of Trichosanthes kirilowii Maxim. herbs, optimizes the liquid phase elution method, effectively separates the compound peaks of index components, and on this basis, establishes an LC-MS fingerprint. The similarity of 15 batches of Trichosanthes kirilowii Maxim. herbs is above 0.900, indicating that this fingerprint can better evaluate the quality of Trichosanthes kirilowii Maxim. herbs. Based on the liquid phase conditions for the separation of index components, a UHPLC-QQQ-MS quantitative detection method is established to determine the contents of index components rutin, loline, chrysoeriol and cucurbitacin D in Trichosanthes kirilowii Maxim. The results show that the content of loline in Trichosanthes kirilowii Maxim. herb samples from different origins is 0.00075% - 0.00455%, the content of chrysoeriol is 0.00059% - 0.00381%, the content of rutin is 0.00010% - 0.00342%, and the content of cucurbitacin D is 0.00007% - 0.00105%. This method improves the evaluation standard of Trichosanthes kirilowii Maxim. and provides a reference for the overall and comprehensive quality control of Trichosanthes kirilowii Maxim.

[0108] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for constructing a fingerprint of Trichosanthes kirilowii Maxim. medicinal materials, characterized in that, Comprising: Preparing the Fructus Trichosanthis medicinal material into a test solution; Preparing rutin, chrysoeriol and cucurbitacin D into a reference solution; Injecting the test solution and the reference solution into a liquid chromatography-mass spectrometry instrument respectively, and obtaining the fingerprint of the Fructus Trichosanthis medicinal material according to the liquid chromatography-mass spectrometry method.

2. The construction method according to claim 1, characterized in that, In the fingerprint of the Fructus Trichosanthis medicinal material, 11 characteristic peaks are included. Peak 4 is rutin, peak 10 is chrysoeriol, and peak 11 is cucurbitacin D. The numbers of the characteristic peaks are peak 1 to peak 11; The identification of the characteristic peaks and the characteristic peaks is as follows:

3. The construction method according to claim 2, characterized in that, Taking peak 10 as the S peak, the relative retention time ranges of peak 1 to peak 9 and peak 11 relative to the S peak are respectively: 0.148～0.152、0.101～0.103、0.310～0.315、0.361～0.368、0.471～0.476、0.574～0.575、0.649～0.652、0.761～0.766、0.847～0.851、1.020～1.025。 4. The construction method according to claim 1, characterized in that, The chromatographic conditions of the liquid chromatography-mass spectrometry method include: Stationary phase: a chromatographic column filled with octadecylsilyl-bonded silica gel; Mobile phase: mobile phase A is water containing 0.1% (v / v) formic acid, and mobile phase B is acetonitrile solution containing 0.1% (v / v) formic acid; Gradient elution is adopted, and the flow rate is 0.3 mL / min.

5. The construction method according to claim 4, characterized in that The chromatographic column is a CORTECS UPLC T3 Column (2.1×100 mm, 1.6 μm) chromatographic column.

6. The construction method according to claim 4, wherein The gradient elution is carried out according to the following procedure: 0.0 - 1.0 min, mobile phase A changes from 85% → 84%, and mobile phase B changes from 15% → 16%; 1.0 - 5.5 min, mobile phase A remains at 84%, and mobile phase B remains at 16%; 5.5 - 6.0 min, mobile phase A changes from 84% → 77%, and mobile phase B changes from 16% → 23%; 6.0 - 16.0 min, mobile phase A changes from 77% → 66%, and mobile phase B changes from 23% → 34%.

7. The construction method according to claim 1, characterized in that The mass spectrometry conditions of the liquid chromatography-mass spectrometry method include: The ion source is ESI, and the ionization mode is negative ion mode; The temperature of the backflush nitrogen is 325 °C, the flow rate of the backflush nitrogen is 11.0 L / min, the flow rate of the nebulizing gas is 45 psi, the temperature of the auxiliary nitrogen is 350 °C, the flow rate of the auxiliary nitrogen is 11.0 L / min, the capillary voltage is 3500 V, the in-source collision voltage is 175 V, and the cone voltage is 65.0 V; Scanning mode: full scan; Scanning range: m / z 60 - 1700.

8. The construction method according to any one of claims 1 to 7, characterized in that The preparation method of the test solution includes: Extracting and filtering the Fructus Trichosanthis medicinal material with a first solvent to obtain the test solution.

9. The construction method according to claim 8, characterized in that The preparation method of the test solution includes: Adding 1 ml of the first solvent to 100 mg of the Fructus Trichosanthis medicinal material; After soaking for 60 minutes, ultrasonic extraction is carried out at 40 kHz and 30 °C for 30 minutes; Centrifuging to obtain a first supernatant and a first residue; Continuing to add 1 ml of the first solvent to the first residue; Carrying out ultrasonic extraction at 40 kHz and 30 °C for 30 minutes; Centrifuging to obtain a second supernatant and a second residue; Combining the first supernatant and the second supernatant to obtain the test solution, wherein the first solvent is 50% - 95% ethanol.

10. A method for determining the content of index components in Trichosanthes kirilowii Maxim. medicinal materials, characterized in that, Comprising: Preparing the to-be-detected Fructus Trichosanthis medicinal material into a to-be-detected sample solution; Preparing rutin, loline, chrysoeriol and cucurbitacin D into a reference solution; Inject the sample solution to be tested and the reference solution into a liquid chromatography-mass spectrometry (LC-MS) instrument respectively. According to the LC-MS method, obtain the test chromatogram of the Trichosanthes kirilowii Maxim. medicinal material to be tested and the reference chromatogram of the reference solution. Calculate the contents of the index components rutin, loliolide, chrysoeriol, and cucurbitacin D in the Trichosanthes kirilowii Maxim. medicinal material to be tested based on the concentration of the reference solution, the peak area of the reference solution in the reference chromatogram, and the peak area of the components corresponding to the Trichosanthes kirilowii Maxim. medicinal material to be tested and the reference solution in the test chromatogram.

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