A method for constructing HPLC characteristic chromatogram of pumpkin stem medicinal material, decoction piece, standard decoction and formula granules

Abstract

The application provides a pumpkin stem medicinal material, decoction piece, standard decoction and formula granule HPLC characteristic spectrum construction method, comprising: A) extracting pumpkin stem raw materials with a solvent to obtain a test solution; B) determining the test solution by high performance liquid chromatography to obtain the HPLC characteristic spectrum of the pumpkin stem raw material; the high performance liquid chromatography chromatographic conditions are that the chromatographic column is a C18 column; the mobile phase A is acetonitrile, the mobile phase B is 0.2% phosphoric acid, and gradient elution is adopted. The application adopts high performance liquid chromatography, selects acetonitrile-0.2% phosphoric acid as the mobile phase for gradient elution, establishes the HPLC characteristic spectrum of the pumpkin stem medicinal material, decoction piece, standard decoction and formula granule with vanillic acid as a reference, and has good repeatability and precision, and the method is stable and reliable, so that the quality of the pumpkin stem medicinal material, decoction piece, standard decoction and formula granule can be controlled.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of analytical detection technology, in particular to a HPLC characteristic chromatogram construction method of pumpkin pedicel medicinal materials, decoction pieces, standard decoction and formula granules. BACKGROUND

[0002] Pumpkin pedicel is the dried pedicel of Cucurbita moschata (Duch. ex Lam.) Duch. ex Poir. of Cucurbitaceae. After the melon is ripe, it is picked and dried. It has the effects of harmonizing the middle, tonifying qi, calming the fetus, detoxifying and astringing the sore. It is used for treating stomach qi reversal, fetal movement instability and sore. The standard decoction is a freeze-dried powder prepared by decocting the processed medicinal materials in water according to a fixed preparation process.

[0003] In order to ensure the uniformity and stability of the quality of pumpkin pedicel medicinal materials, decoction pieces and standard decoction, a new characteristic chromatogram method is established to control the quality. SUMMARY

[0004] Therefore, the present application provides a HPLC characteristic chromatogram construction method of pumpkin pedicel medicinal materials, decoction pieces, standard decoction and formula granules. The HPLC characteristic chromatogram construction method of pumpkin pedicel medicinal materials, decoction pieces, standard decoction and formula granules is stable and reliable, and can control the quality of pumpkin pedicel medicinal materials, decoction pieces, standard decoction and formula granules.

[0005] The present application provides a HPLC characteristic chromatogram construction method of pumpkin pedicel medicinal materials, decoction pieces, standard decoction and formula granules, comprising:

[0006] A) pumpkin pedicel raw materials are extracted with a solvent to obtain a test solution;

[0007] B) the test solution is determined by high performance liquid chromatography to obtain the HPLC characteristic chromatogram of the pumpkin pedicel raw materials;

[0008] The high performance liquid chromatography has the following chromatographic conditions: the chromatographic column is a C18 column; the mobile phase A is acetonitrile, the mobile phase B is 0.2% acetic acid, and gradient elution is adopted.

[0009] The HPLC characteristic chromatogram construction method of pumpkin pedicel medicinal materials, decoction pieces, standard decoction and formula granules provided by the present application first takes pumpkin pedicel raw materials, extracts the raw materials with a solvent to obtain a test solution. The solvent is preferably 70% ethanol.

[0010] According to the present application, the pumpkin pedicel raw materials are pumpkin pedicel medicinal materials, decoction pieces, standard decoction and formula granules.

[0011] According to the present application, the pumpkin pedicel raw materials are extracted with 70% ethanol by heating and refluxing or ultrasonic extraction for 30 minutes, cooled, shaken and filtered to obtain the test solution.

[0012] The sample preparation mode of the medicinal materials and decoction pieces is preferably reflux extraction, and the standard decoction and granules are preferably ultrasonic extraction.

[0013] The ratio of the mass of the pumpkin stem raw material g to the volume of the solvent mL is (0.5-2):20.

[0014] The above extraction solvent chromatographic peak information is large and has good effect.

[0015] The pumpkin stem raw material is pumpkin stem medicinal material, pumpkin stem decoction piece or decoction. The present application does not limit it, and the above raw materials can be quality controlled and qualitatively detected by the method of the present application.

[0016] The present application also includes the preparation of reference solution: taking vanillic acid control, using 70% ethanol to dissolve, obtaining control reference solution;

[0017] Taking pumpkin stem control medicinal material and 70% ethanol to heat reflux extraction, obtaining control medicinal material reference solution;

[0018] The control and control medicinal material reference solution are determined by high performance liquid chromatography, and the chromatograms of the control and control medicinal material reference are obtained; and the components of the HPLC characteristic map of the pumpkin stem medicinal material, decoction piece, standard decoction and its formula granule are qualitatively determined according to the chromatograms of the control and control medicinal material reference.

[0019] The mobile phase A is acetonitrile, the mobile phase B is 0.2% acetic acid solution, and the gradient elution is carried out.

[0020] The gradient elution is preferably specific as follows:

[0021] 0-30min, A phase: 7%, B phase: 93%;

[0022] 30-50min, A phase: 7%-13%, B phase: 93%-87%;

[0023] 50-58min, A phase: 13%, B phase: 87%.

[0024] The present application has good baseline separation under the above elution gradient, and the separation degree of each peak is good, and the baseline is stable.

[0025] C18 column, specification 5um, 4.6*250mm; column temperature 30℃.

[0026] The chromatographic column has symmetrical chromatographic peaks, good separation degree and moderate peak time under the above 30℃ condition.

[0027] The flow rate of the mobile phase is preferably 1.0ml / min.

[0028] The present application finds that each chromatographic peak is well separated, the peak shape is relatively symmetrical, the separation degree is moderate, and the flow rate is 1.0 ml / min, which is the most preferred scheme.

[0029] The detection wavelength of the present application is preferably 270 nm.

[0030] The present inventors find that the chromatographic information is rich at 270 nm, the chromatographic peak information is large, each component has good absorption, the response value is moderate, the separation degree of each peak is good, and the baseline is stable.

[0031] The injection amount of the present application is preferably 10 μL.

[0032] The present application has the beneficial effects that under one liquid chromatography condition, the material group of pumpkin peduncle medicinal materials, decoction pieces, standard decoction and formula granules is controlled by fingerprint, and vanillic acid is used for positioning the fingerprint; the cost of detection can be greatly reduced, and qualitative detection is realized.

[0033] The similarity of the HPLC characteristic spectrum of the pumpkin peduncle medicinal materials, decoction pieces, standard decoction and formula granules is evaluated by using the traditional Chinese medicine chromatographic fingerprint similarity evaluation system to obtain the HPLC standard characteristic spectrum of the pumpkin peduncle medicinal materials, decoction pieces, standard decoction and formula granules composed of 7 characteristic peaks, wherein peak 2 (S): vanillic acid peak.

[0034] In the standard decoction characteristic spectrum of the pumpkin peduncle, the vanillic acid peak is used as the reference peak S peak, the relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time is within ±10% of the specified value, and the specified value is 0.70 (peak 1), 1.13 (peak 3), 1.19 (peak 4), 1.48 (peak 5), 1.62 (peak 6), and 1.82 (peak 7).

[0035] Quality judgment standard: take the pumpkin peduncle medicinal materials, decoction pieces, standard decoction and formula granules samples, operate according to the above method to obtain the characteristic spectrum of the pumpkin peduncle medicinal materials, decoction pieces, standard decoction and formula granules, analyze the standard characteristic spectrum and sample characteristic spectrum of the pumpkin peduncle medicinal materials, decoction pieces, standard decoction and formula granules by using the 2012 edition of the national pharmacopoeia commission "traditional Chinese medicine chromatographic fingerprint similarity evaluation system", and the similarity is greater than 0.90.

[0036] The method provided by the present application can effectively monitor the quality of different batches of pumpkin peduncle medicinal materials, decoction pieces, standard decoction and formula granules, so that the quality is stable, the method has the characteristics of high precision and good reproducibility, and is conducive to the comprehensive monitoring of the quality of the product.

[0037] The characteristic spectrum of pumpkin stem medicinal materials, decoction pieces, standard decoction and formula granules is established, vanillic acid is used as a reference, the order of each characteristic peak and the correlation with medicinal materials and intermediate products are focused on, the overall quality appearance characteristics of the products can be comprehensively evaluated, and the method is scientific and reliable.

[0038] The characteristic spectrum method newly established in the application can detect the components with greater polarity in pumpkin stem and standard decoction thereof.

[0039] The application provides a HPLC characteristic spectrum construction method for pumpkin stem medicinal materials, decoction pieces, standard decoction and formula granules, which comprises the following steps: A) extracting pumpkin stem raw materials by using a solvent to obtain a to-be-tested solution; and B) determining the to-be-tested solution by using a high performance liquid chromatography (HPLC) method to obtain the HPLC characteristic spectrum of the pumpkin stem raw materials; the HPLC method has the following chromatographic conditions: a C18 column is used as the chromatographic column; acetonitrile is used as mobile phase A, and 0.2% acetic acid is used as mobile phase B; and gradient elution is performed. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 It is a chromatogram of pumpkin stem medicinal materials at different wavelengths according to Embodiment 1 of the application;

[0041] Figure 2 It is a column temperature investigation result diagram according to Embodiment 1 of the application;

[0042] Figure 3 It is a flow rate investigation result diagram according to Embodiment 1 of the application;

[0043] Figure 4 It is a delay investigation result diagram according to Embodiment 1 of the application;

[0044] Figure 5 Investigation results of extraction solvents;

[0045] Figure 6 Investigation of extraction methods;

[0046] Figure 7 Investigation of solvent addition amount;

[0047] Figure 8Extraction methods were examined;

[0048] Figure 9 This is a chromatographic peak identification result diagram;

[0049] Figure 10 Vanillic acid spectrum - reference standard;

[0050] Figure 11 Vanillic acid spectrum - medicinal material;

[0051] Figure 12 Figures showing the results of investigations using different instruments;

[0052] Figure 13 Investigations using different chromatographic columns;

[0053] Figure 14 Characteristic atlas of pumpkin stem medicinal materials;

[0054] Figure 15 A comparative characteristic spectrum of pumpkin stem medicinal material;

[0055] Figure 16 Characteristic spectrum of medicinal slices;

[0056] Figure 17 A reference image for the characteristic images of pumpkin stem slices;

[0057] Figure 18 Chromatograms of pumpkin stem-labeled soup at different wavelengths;

[0058] Figure 19 The results of the column temperature investigation are shown in the figure.

[0059] Figure 20 The results of the flow velocity study;

[0060] Figure 21 The graph shows the results of the delay investigation;

[0061] Figure 22 Results of solvent extraction investigation;

[0062] Figure 23 To examine the extraction method;

[0063] Figure 24 For the purpose of examining the extraction time;

[0064] Figure 25 To investigate the amount of solvent added;

[0065] Figure 26 A graph showing the results of chromatographic peak identification;

[0066] Figure 27 Vanillic acid spectrum - reference standard;

[0067] Figure 28Vanillic acid spectrum - standard decoction

[0068] Figure 29 Different instrument investigation results figure

[0069] Figure 30 Chromatographic column durability investigation

[0070] Figure 31 Pumpkin stem standard decoction characteristic spectrum

[0071] Figure 32 Pumpkin stem standard decoction characteristic spectrum control spectrum

[0072] Figure 33 Pumpkin stem granule different mobile phase chromatogram

[0073] Figure 34 Pumpkin stem formula granule different wavelength chromatogram

[0074] Figure 35 Pumpkin stem formula granule column temperature investigation chromatogram

[0075] Figure 36 Flow rate investigation

[0076] Figure 37 Delay investigation results figure

[0077] Figure 38 Extraction solvent investigation

[0078] Figure 39 Extraction method investigation

[0079] Figure 40 Extraction time investigation

[0080] Figure 41 Solvent addition amount investigation

[0081] Figure 42 Chromatographic peak identification

[0082] Figure 43 Vanillic acid spectrum - control

[0083] Figure 44 Vanillic acid spectrum - granule

[0084] Figure 45 Different instrument investigation

[0085] Figure 46 Different chromatographic column investigation

[0086] Figure 47 Pumpkin stem formula granule characteristic spectrum

[0087] Figure 48The control atlas of the characteristic atlas of the pumpkin stem formula granules;

[0088] Figure 49 It can be seen that the detection method of cucurbitacin components is not suitable for the detection of characteristic atlas of pumpkin stem medicinal materials and granules.

[0089] Figure 50 The characteristic spectrum of Comparative Example 2;

[0090] Figure 51 The characteristic spectrum of Comparative Example 3. DETAILED DESCRIPTION

[0091] The present application provides a kind of pumpkin stem medicinal materials, piece, standard decoction and its formula granule HPLC characteristic atlas construction method, and those skilled in the art can refer to the content of this paper, and appropriately improve process parameters to realize.In particular, it needs to be pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they all belong to the scope protected by the present application.The method and application of the present application have been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this paper without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0092] In order to further illustrate the present application, the following embodiments are described in detail.

[0093] High performance liquid chromatograph: chromatograph 1 (Waters e2695 type high performance liquid chromatograph), chromatograph 2 (Shimadzu LC-20AD type high performance liquid chromatograph), chromatograph 3 (Agilent 1260 type ultra high performance liquid chromatograph)

[0094] Electronic balance: ME204E / 02, MS205DM, XP26 (Mettler-Toledo Instruments Co., Ltd.);

[0095] Ultrapure water machine: cell type 1810A (Shanghai Moore Scientific Instruments Co., Ltd.);

[0096] Ultrasonic cleaner: KQ-600DB type (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0097] Chromatographic column: chromatographic column 1 (Kromasil 100-5-C18 4.6×250mm, 5μm), chromatographic column 2 (waters Symmetry @ C18 4.6×250mm, 5μm), chromatographic column 3 (Eliyte Hypersil ODS2 4.6×250mm, 5μm)

[0098] 2 reagents and reagents

[0099] Acetonitrile (Sigma-Aldrich, chromatographically pure); acetic acid (Chengdu Kolon Chemicals Co., Ltd., chromatographically pure); water was ultrapure water; other reagents were all of analytical purity.

[0100] Vanillic acid (China Institute for Food and Drug Control, batch number: 110776-201503, content 99.8%)

[0101] Pumpkin stem control medicinal materials (Chengdu Desit Biotechnology Co., Ltd., batch number: DSTYN003401)

[0102] Pumpkin stem medicinal materials batch number: A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11, A12, A13, A14, A15, A16, A17, A18, A19, A20, A21

[0103] Pumpkin stem decoction pieces (prepared by Sichuan Xinglvese Pharmaceutical Technology Development Co., Ltd., batch number: S1, S2, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12, S13, S14, S15, S16, S17, S18, S19, S20, S21).

[0104] Pumpkin stem standard decoction (prepared by Sichuan Xinglvese Pharmaceutical Technology Development Co., Ltd., batch number: B1, B2, B3, B4, B5, B6, B7, B8, B9, B10, B11, B12, B13, B14, B15, B16, B17, B18, B19, B20, B21).

[0105] Pumpkin stem formula granules batch number: K1, K2, K3.

[0106] Example 1 HPLC characteristic chromatogram of pumpkin stem medicinal materials

[0107] 1.1 Chromatographic conditions and system suitability test Octadecylsilane-bonded silica gel as filler (5 μm, 4.6 x 250 mm; column temperature 30 °C); acetonitrile as mobile phase A, 0.2% acetic acid solution as mobile phase B, gradient elution according to the following table; flow rate was 1.0 ml per minute; column temperature was 30 °C; detection wavelength was 270 nm. Theoretical plate number should not be less than 4000 calculated by vanillic acid peak.

[0108] Table 1

[0109]

[0110] Reference solution preparation: 2 g of pumpkin stem control medicinal material was placed in a conical flask with a stopper, 20 ml of 70% ethanol was added, weighed, refluxed for 30 minutes, cooled, weighed again, and the weight was made up with 70% ethanol, filtered, and used as the control medicinal material reference solution. An appropriate amount of vanillic acid control was precisely weighed, dissolved in 70% ethanol to prepare a solution containing 7 ug of vanillic acid per 1 ml, and used as the control reference solution.

[0111] Test solution preparation: 2 g of pumpkin stem powder (passed through a No. 3 sieve) was prepared into a test solution with the control medicinal material reference solution.

[0112] Determination method: 10 ul of the reference solution and the test solution were precisely taken and injected into the liquid chromatograph for determination.

[0113] 1.2 Selection of mobile phase

[0114] Reference pumpkin stem formula granule characteristic chromatogram method: acetonitrile-0.2% phosphoric acid, acetonitrile-0.2% acetic acid, and acetonitrile-0.2% formic acid were used as the mobile phase for investigation, and the determination mobile phase of the characteristic chromatogram of pumpkin stem medicinal material was determined to be acetonitrile-0.2% acetic acid.

[0115] 1.3 Wavelength selection

[0116] Based on the above proposed experimental conditions, the test solution was scanned by a diode array detector at full wavelength, and the chromatograms of the test solution at 254 nm, 270 nm, and 300 nm were extracted, respectively. The results are shown in Figure 1 , Figure 1 the different wavelength chromatograms of the pumpkin stem medicinal material of Example 1 of the present application.

[0117] 1.4 Column temperature investigation

[0118] Based on the above proposed experimental conditions, the column temperature was investigated at 25°C, 30°C, and 35°C, respectively. The results are shown in Figure 2 , Figure 2 the column temperature investigation results of Example 1 of the present application.

[0119] Table 2 Column temperature investigation-retention time

[0120]

[0121] Table 3 Column temperature investigation-relative retention time

[0122]

[0123] The results show that when the column temperature is 30°C, the chromatogram peak shape is relatively symmetrical, the separation degree is good, and the peak is complete. Therefore, the column temperature is determined to be 30°C.

[0124] 1.5 Flow rate investigation

[0125] On the basis of the above proposed experimental conditions, the flow rate of 0.8 ml / min, 1.0 ml / min, 1.2 ml / min were investigated respectively. The results are shown in the following table and the chromatogram of Example 1 of the present application. Figure 3 Figure 3 The flow rate of Example 1 of the present application is shown in the following table and the chromatogram.

[0126] Table 4 Retention time of flow rate investigation

[0127]

[0128]

[0129] Table 5 Relative retention time of flow rate investigation

[0130]

[0131] The results show that when the flow rate is 1.0 ml / min, the peak shape of the chromatogram is better and the separation degree is moderate. Therefore, the flow rate is determined to be 1.0 ml / min.

[0132] 1.6 Delayed investigation

[0133] On the basis of the above proposed experimental conditions, the delayed test was carried out.

[0134] The results are shown in the following table and the chromatogram of Example 1 of the present application. Figure 4 Figure 4 The results show that the sample has basically no chromatographic peak after 58 minutes, so the sample detection time is determined to be 58 minutes.

[0135] The results show that the sample has basically no chromatographic peak after 58 minutes, so the sample detection time is determined to be 58 minutes.

[0136] In summary, the chromatographic conditions and system suitability test of pumpkin stem medicinal material characteristic map are determined as follows: octadecylsilane bonded silica gel is used as the filler (the column length is 250 mm, the inner diameter is 4.6 mm, and the particle size is 5 μm); acetonitrile is used as the mobile phase A, and 0.2% acetic acid solution is used as the mobile phase B, which is gradient eluted according to the following table; the flow rate is 1.0 ml / min; the column temperature is 30℃; and the detection wavelength is 270 nm. The theoretical plate number calculated according to the peak of vanillic acid should not be less than 4000.

[0137]

[0138] 1.7 Preparation of test solution investigation

[0139] 1.7.1 Extraction solvent investigation

[0140] ​​Take pumpkin stem medicinal material powder (over three screen, batch number: A21) 2.0g, put in the conical flask with plug, respectively to test product extraction solvent is methanol, 30% ethanol, 50% ethanol, 70% ethanol, ethanol, water 20ml are investigated, tight plug, reflux 30 minutes, cool, shake, filter, take the filtrate, namely.

[0141] According to the results of "1.1 chromatographic conditions and system suitability test" sample determination, the results are shown in Figure 5 . Figure 5 Extraction solvent investigation results.

[0142] The results show that: when the extraction solvent is 70% ethanol, the peak shape of each characteristic peak is good, and the separation degree is moderate, and the extraction solvent is temporarily determined as 70% ethanol.

[0143] 1.7.2 extraction time investigation

[0144] Take pumpkin stem medicinal material powder (over three screen, batch number: A21) 2.0g, put in the conical flask with plug, add 70% ethanol 20ml, reflux, respectively to test product extraction time for 20 minutes, 30 minutes, 60 minutes are investigated, cool, shake, filter, take the filtrate, namely.

[0145] According to the results of "1.1 chromatographic conditions and system suitability test" sample determination, the results are shown in Figure 6 . Figure 6 Extraction method investigation.

[0146] As can be seen from the above figure, the results show that when the extraction time is 30 minutes, the chromatogram peak shape and separation degree are good. Therefore, the extraction time is determined as 30 minutes.

[0147] 1.7.3 solvent addition amount investigation

[0148] Take pumpkin stem medicinal material powder (over three screen, batch number: A21) 2.0g, put in the conical flask with plug, respectively add 70% methanol 15ml, 20ml, 50ml are investigated, tight plug, reflux 30 minutes, cool, shake, filter, take the filtrate, namely. The results are shown in Figure 7 . Figure 7 Solvent addition amount investigation.

[0149] The results show that when the extraction solvent addition amount is 20ml, the peak shape and separation degree of each chromatographic peak are good, so the solvent amount is selected as 20ml.

[0150] 1.7.4 extraction method investigation

[0151] Take pumpkin stem medicinal material powder (over three screen, batch number: A21) 2.0g, put in the conical flask with plug, add 70% ethanol 20ml, reflux for 30 minutes, cool, shake, filter, take the filtrate, as test solution; In addition, take the powder 2.0g, put in the conical flask with plug, add 70% ethanol 20ml, tightly plug, ultrasonic treatment (power 600W, frequency 40kHz) for 30 minutes, cool, shake, filter, take the filtrate, that is. See Figure 13 .

[0152] According to the results of "1.1 chromatographic conditions and system suitability test", the results are shown in Figure 8 . Figure 8 Extraction method investigation.

[0153] The results show that when the test sample is decocted and ultrasonic extracted, the peak shape is separated better, so the test sample extraction method is determined as reflux extraction.

[0154] In summary, the preparation method of pumpkin stem medicinal material characteristic map test solution is determined as follows: take pumpkin stem medicinal material powder (over three screen) 2.0g, put in the conical flask with plug, add 70% ethanol 20ml, heat reflux for 30 minutes, cool, shake, filter, take the filtrate, as test solution.

[0155] 1.8 Methodology investigation

[0156] 1.8.1 Chromatographic peak identification

[0157] Preparation of test solution: according to the experimental conditions determined in 1.1 above, prepare the test solution of pumpkin stem medicinal material.

[0158] Preparation of reference solution: take 2g of pumpkin stem control medicinal material, put in the conical flask with plug, add 70% ethanol 20ml, heat reflux for 30 minutes, cool, shake, filter, take the filtrate, as control medicinal material reference solution.

[0159] Vanillic acid control solution: appropriate amount of vanillic acid control solution was accurately weighed and dissolved in 70% ethanol to prepare a solution containing 7μg per 1ml as the reference solution of the control.

[0160] Preparation of negative control solution: according to the experimental conditions determined above, prepare the negative control solution of pumpkin stem medicinal material.

[0161] The peaks of pumpkin stem medicinal material characteristic map were located. The results are shown in Figure 9 , Figure 9 The chromatographic peak identification results are shown in the figure. Figure 10 Vanillic acid spectrum- control; Figure 11 Vanillic acid spectrum- medicinal material. The results show that peak 2 is vanillic acid. In the following methodology investigation, 7 characteristic peaks in the sample were investigated.

[0162] 1.8.2 Precision test

[0163] Take the pumpkin stem medicinal materials (batch number: A21) test solution, according to the proposed experimental method of 6 times, 10ul each time, calculate the relative retention time of each characteristic peak.

[0164] Table 6 Precision investigation - relative retention time

[0165]

[0166] The results showed that the relative retention time RSD of each characteristic peak of the sample was 0.03% to 0.13%, and the instrument precision was good.

[0167] 1.8.3 Reproducibility test

[0168] Take 6 portions of pumpkin stem medicinal materials (batch number: A21) test solution, according to the proposed experimental method for preparation and determination.

[0169] Table 7 Reproducibility test - relative retention time

[0170]

[0171] The results showed that the relative retention time RSD of 6 samples was 0.00% to 0.11%, indicating that the method was reproducible.

[0172] 1.8.4 Intermediate precision test

[0173] 1.8.4.1 Different instrument test

[0174] On the basis of the above proposed experimental conditions, two portions of pumpkin stem medicinal materials (batch number: A21) were weighed and test solution was prepared, and determination was carried out on chromatograph 1, chromatograph 2 and chromatograph 3 respectively.

[0175] The results are shown in Figure 12 The figure of different instrument test. Figure 12

[0176] Table 8 Instrument durability test - relative retention time

[0177]

[0178]

[0179] The results showed that when the test solution was detected by the above 3 instruments, the RSD of the relative retention time of each characteristic peak was 0.52% to 3.21%, indicating that the instrument durability was good.

[0180] 1.8.4.2 Different personnel and time test ​

[0181] Based on the experimental conditions in 1.1 above, two portions of pumpkin stem medicinal material (batch number: A21) were weighed by different personnel (A, B) at different times (T1, T2), respectively, to prepare the test samples for determination.

[0182] Table 9 Personnel and time investigation - relative retention time

[0183]

[0184] The results show that the RSD of the relative retention time of each characteristic peak is 0.05% to 0.56% when the same sample is determined by different personnel at different times, and the method has good stability.

[0185] 1.8.5 Investigation of column durability

[0186] Based on the experimental conditions above, column 1, column 2 and column 3 were used for analysis investigation, respectively. The results are shown in Table 10. Figure 13 . Figure 13 Investigation of different columns.

[0187] Table 10 Investigation of column durability - relative retention time

[0188]

[0189] The results show that the RSD of the relative retention time of each characteristic peak is 0.70% to 7.42% when the sample is detected by the above three columns, indicating that the column has good durability.

[0190] 1.8.6 Stability investigation

[0191] Based on the experimental conditions in 1.1 above, the same test sample solution was taken and determined at 0h, 3h, 6h, 9h, 15h and 24h, respectively.

[0192] Table 11 Stability investigation - retention time

[0193]

[0194] The results show that the RSD of the retention time of each characteristic peak is 0.07% to 0.21%, and the sample solution is stable within 24 hours.

[0195] In summary, the RSD of the relative retention time of each characteristic peak meets the requirements in the above investigations, and the method is good. The above 7 characteristic peaks are included in the subsequent investigation.

[0196] 1.8.7 Determination of characteristic peaks and establishment of control chromatogram

[0197] 1.6.7.1 Verification results of 21 batches of pumpkin stem medicinal materials

[0198] The test solution of 21 batches of medicinal materials was prepared according to the results of "1.1 Preparation of Test Solution of Test Sample", and was determined and analyzed by injection according to the results of "1.1 Chromatographic Conditions and System Suitability Test", and the relative retention time was calculated. The results are shown in Table 12. Figure 14 . Figure 14 The characteristic chromatogram of pumpkin stem medicinal materials.

[0199] (1-S21 are: A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11, A12, A13, A14, A15, A16, A17, A18, A19, A20, A21).

[0200] Table 12 Relative retention time of characteristic chromatogram of pumpkin stem medicinal materials

[0201]

[0202] According to the principle of stable relative retention time and detectable peaks in each batch of samples, a total of 7 peaks with good reproducibility were selected as characteristic peaks. The relative retention time RSD of 7 characteristic peaks of 21 batches of pumpkin stem medicinal materials was less than 10%.

[0203] Finally, it is stipulated that 7 characteristic peaks should appear in the test sample chromatogram, and should correspond to the retention time of 7 characteristic peaks in the reference chromatogram of the control medicinal material, and the peak corresponding to the vanillic acid reference peak is S peak. The relative retention time of each characteristic peak to S peak should be within ±10% of the specified value. The specified value is 0.70 (peak 1), 1.13 (peak 3), 1.19 (peak 4), 1.48 (peak 5), 1.62 (peak 6), 1.82 (peak 7).

[0204] The 21 batches of pumpkin stem medicinal materials were synthesized by using Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition), and the control characteristic chromatogram of the characteristic chromatogram of pumpkin stem medicinal materials was established, as shown in Figure 15 . Figure 15 The control characteristic chromatogram of the characteristic chromatogram of pumpkin stem medicinal materials is shown in Figure 1.

[0205] Example 2 HPLC characteristic chromatogram of pumpkin stem decoction pieces

[0206] Since the pumpkin stem decoction pieces are directly sliced from the pumpkin stem medicinal materials, only the decoction pieces are verified

[0207] 2.1 The 21 batches of pumpkin stem decoction pieces were determined by using the conditions 1.1 determined in Example 1. The results are shown in Table 13. Figure 16 , Figure 16The image shows the characteristic chromatogram of the medicinal slices; among them, peak 2 (S): vanillic acid (S1-S21 are respectively: 1, S2, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12, S13, S14, S15, S16, S17, S18, S19, S20, S21)).

[0208] Table 13 Relative retention time of characteristic chromatograms of medicinal slices

[0209]

[0210]

[0211] Based on the principles of stable relative retention times, detectability across all batches of samples, and relatively high peak values, seven peaks with good repeatability were selected as characteristic peaks. The relative retention times (RSDs) of the seven characteristic peaks from 21 batches of pumpkin stem slices were all less than 10.0%.

[0212] The final specification stipulates that the chromatogram of the test sample should show 7 characteristic peaks, and the retention times should correspond to the 7 characteristic peaks in the chromatogram of the reference medicinal material. The peak corresponding to the vanillic acid reference standard peak is designated as the S peak. The relative retention times of each characteristic peak and the S peak should be calculated. These relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.70 (peak 1), 1.13 (peak 3), 1.19 (peak 4), 1.48 (peak 5), 1.62 (peak 6), and 1.82 (peak 7).

[0213] The chromatographic fingerprint similarity evaluation system for traditional Chinese medicine (2012 version) was used to synthesize characteristic chromatograms of 21 batches of pumpkin stem slices, and a reference chromatogram for the characteristic chromatograms of pumpkin stem slices was established. The results are as follows: Figure 17 , Figure 17 The reference chromatogram is for the characteristic chromatogram of pumpkin stem slices. Peak 2 (S): vanillic acid; column 1.

[0214] Example 3: HPLC characteristic spectrum of pumpkin stem standard decoction

[0215] 3.1 Proposed chromatographic conditions

[0216] Using octadecylsilane-bonded silica gel as the packing material; acetonitrile as mobile phase A and 0.2% acetic acid solution as mobile phase B, gradient elution was performed according to the specifications in the table below; the flow rate was 1.0 ml per minute; the column temperature was 30℃; and the detection wavelength was 270 nm. The theoretical plate number, calculated based on the vanillic acid peak, should be no less than 4000.

[0217]

[0218] Reference solution preparation: Take 2 g of pumpkin stem control medicinal material, add 20 ml of 70% ethanol into a conical flask with a stopper, weigh, reflux for 30 minutes, cool, re-weigh, make up the weight with 70% ethanol, filter, and use as the control medicinal material reference solution. Take the vanillic acid control product, accurately weigh, add 70% ethanol solution to prepare a solution containing 7 ug of vanillic acid per 1 ml, and use as the control product reference solution.

[0219] Test solution preparation: Take 0.5 g of the product, add 20 ml of 70% ethanol into a conical flask with a stopper, weigh, ultrasonic treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, re-weigh, make up the lost weight with 70% ethanol, shake well, and obtain.

[0220] Determination method: accurately take 10 ul of the reference solution and the test solution respectively, inject into the liquid chromatograph, and determine.

[0221] 3.2 Chromatographic conditions and system suitability test

[0222] 3.2.1 Mobile phase selection

[0223] Reference pumpkin stem formula granule characteristic chromatogram method: using acetonitrile-0.2% phosphoric acid, acetonitrile-0.2% acetic acid, acetonitrile-0.2% formic acid as the mobile phase, the determination of the characteristic chromatogram of pumpkin stem medicinal material is determined as: acetonitrile-0.2% acetic acid.

[0224] 3.2.2 Wavelength selection

[0225] On the basis of "3.1 proposed chromatographic conditions", the test solution was scanned by diode array detector at full wave band, and the chromatograms of the test solution at 254 nm, 270 nm and 280 nm were extracted, respectively, as shown in Figure 18 . Figure 18 The chromatograms of pumpkin stem decoction at different wavelengths.

[0226] The results showed that when the detection wavelength was 270 nm, the amount of chromatographic peak information was larger, and the baseline of the chromatogram was more stable, so the detection wavelength was determined as 270 nm.

[0227] 3.2.3 Column temperature investigation

[0228] On the basis of "3.1 proposed chromatographic conditions", the column temperature was investigated at 25 ℃, 30 ℃ and 35 ℃, respectively. The results are shown in Figure 19 , wherein Figure 19 is the column temperature investigation result graph.

[0229] Table 14 Column temperature investigation-retention time

[0230]

[0231] Table 15 Column temperature investigation - relative retention time

[0232]

[0233] 3.2.4 Flow rate investigation

[0234] On the basis of "3.1 proposed chromatographic conditions", the flow rate was investigated at 0.8 ml / min, 1.0 ml / min and 1.2 ml / min, respectively.

[0235] The results are shown in Table 16. Figure 20 .Table 16 Flow rate investigation - relative retention time Figure 20

[0236] Table 16 Flow rate investigation - relative retention time

[0237]

[0238] The results show that when the flow rate is 1.0 ml / min, the chromatogram peak shape is good and the separation degree is moderate. Therefore, the flow rate is determined to be 1.0 ml / min.

[0239] 3.2.5 Delayed investigation

[0240] On the basis of the above 3.1 proposed experimental conditions, the delayed test was carried out. Figure 21 The results are shown in Figure 3.2.5. The results show that the sample has no chromatographic peak after 58 min, so the sample detection time is determined to be 58 min.

[0241] In summary, the chromatographic conditions and system suitability test of pumpkin stem standard decoction characteristic map are determined as follows: octadecylsilane bonded silica gel as the filler; acetonitrile as the mobile phase A, 0.2% acetic acid solution as the mobile phase B, gradient elution according to the following table; the flow rate is 1.0 ml / min; the column temperature is 30°C; the detection wavelength is 270 nm. The theoretical plate number calculated by vanillin acid peak should not be less than 4000.

[0242]

[0243] 3.3 Preparation of test solution investigation

[0244] 3.3.1 Extraction solvent investigation

[0245] Take 0.5 g of pumpkin stem standard decoction (batch number: B21) and place it in a conical flask with a plug. Investigate the extraction solvent for the test sample as follows: 20 ml of methanol, 30% ethanol, 50% ethanol, 70% ethanol, ethanol and water, respectively, tightly plug, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate, i.e. the test solution.

[0246] ​Determination by injection according to the results of "3.2 Chromatographic conditions and system suitability test", the results are shown in Table 3.2 Figure 22 . Figure 22 The results show that when the extraction solvent is 70% ethanol, the peak shape of each characteristic peak is good, and the separation degree is moderate. Therefore, the extraction solvent is temporarily determined as 70% ethanol.

[0247] 3.3.2 Extraction method investigation

[0248] Take 0.5 g of pumpkin stem standard decoction (batch number: B21) and place it in a conical flask with a stopper. Add 20 ml of 70% ethanol, tightly seal the flask, and investigate the extraction method of the test sample by reflux and ultrasonic extraction. The extraction time is 30 minutes. After cooling, shake well, filter, and take the filtrate, which is the test sample.

[0249] Determination by injection according to the results of "3.2 Chromatographic conditions and system suitability test", the results are shown in Table 3.2 Figure 23 . Figure 23 Extraction method investigation.

[0250] The results show that there is little difference in the effect of reflux and ultrasonic extraction of the test sample. The ultrasonic method is fast and simple, so the extraction method of the test sample is determined as ultrasonic extraction.

[0251] 3.3.3 Extraction time investigation

[0252] Take 0.5 g of pumpkin stem standard decoction (batch number: B21) and place it in a conical flask with a stopper. Add 20 ml of 70% ethanol, tightly seal the flask, and investigate the extraction method of the test sample by reflux and ultrasonic extraction. The extraction time is 30 minutes. After cooling, shake well, filter, and take the filtrate, which is the test sample. Figure 24 . Figure 24 Extraction time investigation. The results show that when the extraction time is 30 minutes, the chromatogram peak shape and separation degree are good. Therefore, the extraction time is determined as 30 minutes.

[0253] 3.3.4 Solvent addition amount investigation

[0254] Take 0.5 g of pumpkin stem standard decoction (batch number: B21) and place it in a conical flask with a stopper. Add 10 ml, 20 ml, and 50 ml of 70% ethanol, respectively, and investigate the extraction method of the test sample. Tightly seal the flask, ultrasonic treatment for 30 minutes, cool, shake well, filter, and take the filtrate, which is the test sample.

[0255] Determination by injection according to the results of "3.2 Chromatographic conditions and system suitability test", the results are shown in Table 3.2 Figure 25 . Figure 25The results showed that the addition amount of solvent was 20 ml, the peak shape and separation degree were good, so the solvent amount was selected as 20 ml. In summary, the preparation method of pumpkin stem decoction characteristic map test solution was determined as follows: taking 0.5 g of the product, placing it in a conical flask with a plug, adding 70% ethanol 20 ml, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, cooling, shaking, filtering, and taking the filtrate, i.e. the test solution.

[0256] 3.4 Methodology investigation

[0257] 3.4.1 Chromatographic peak identification

[0258] Preparation of test solution: according to the experimental conditions proposed in 3.2 above, prepare the test solution of pumpkin stem standard decoction.

[0259] Preparation of reference solution: take 2 g of pumpkin stem control drug, place it in a conical flask with a plug, add 70% ethanol 20 ml, weigh, reflux for 30 minutes, cool, reweigh, make up the weight with 70% ethanol, filter, and take as the reference solution of the control drug;

[0260] Take a suitable amount of vanillic acid reference substance, accurately weigh, add 70% ethanol solution to prepare a solution containing 7 ug of vanillic acid per 1 ml, and obtain it.

[0261] Preparation of negative control solution: according to the experimental conditions proposed in 3.2 above, prepare the negative control solution of pumpkin stem standard decoction.

[0262] Positioning of pumpkin stem standard decoction characteristic map peaks. The results are shown in Figure 26 , Figure 26 the chromatographic peak identification results chart; Figure 27 the vanillic acid spectrum chart - reference substance; Figure 28 the vanillic acid spectrum chart - standard decoction. The results showed that peak 2 was vanillic acid. In the following methodology investigation, 7 characteristic peaks in the sample were investigated.

[0263] 3.4.2 Precision test

[0264] Take the test solution of pumpkin stem standard decoction (batch number: B21), according to the proposed experimental method, continuously sample 6 times, 10 μl each time, and calculate the retention time of each characteristic peak.

[0265] Table 17 Precision investigation - retention time

[0266]

[0267] The results showed that the retention time RSD of each characteristic peak was 0.11% to 0.28%, indicating that the instrument precision was good.

[0268] 3.4.3 Reproducibility investigation

[0269] Precisely weigh 6 portions of pumpkin stem standard decoction (batch number: B21), prepare and determine according to the proposed experimental method, and calculate the relative retention time of each characteristic peak.

[0270] Table 18 Reproducibility investigation-relative retention time

[0271]

[0272]

[0273] The results show that the relative retention time RSD of the 6 samples is 0.09% to 0.11%, indicating that the method has good reproducibility.

[0274] 3.4.4 Intermediate precision investigation

[0275] 3.4.4.1 Different instrument investigation

[0276] Based on the experimental conditions proposed in 3.2 above, two portions of pumpkin stem standard decoction (batch number: B21) were precisely weighed, and the test sample solution was prepared, which was determined on chromatograph 1, chromatograph 2, and chromatograph 3, respectively, and the relative retention time of each characteristic peak was calculated. The results are shown in Figure 29 . Figure 29 The figure of the results of different instrument investigation is shown.

[0277] Table 19 Instrument durability investigation-relative retention time

[0278]

[0279] The results show that when the test sample is detected by the above 3 instruments, the RSD of the relative retention time of each characteristic peak is 0.22% to 3.59%, indicating that the instrument has good durability.

[0280] 3.4.4.2 Different personnel and time investigation

[0281] Based on the experimental conditions proposed above, two portions of pumpkin stem standard decoction (batch number: B21) were precisely weighed by different personnel (A, B) at different times (T1, T2), and the test sample was prepared, determined, and the relative retention time of each characteristic peak was calculated.

[0282] Table 20 Personnel and time investigation-relative retention time

[0283]

[0284] The results show that when the same sample is determined by different personnel at different times, the RSD of the relative retention time of each characteristic peak is 0.07% to 0.15%, indicating that the method has good stability.

[0285] 3.4.5 Durability of the chromatographic column

[0286] Based on the above experimental conditions, the relative retention times of the characteristic peaks were calculated by analyzing the chromatographic columns 1, 2 and 3, respectively. The results are shown in Table 21. Figure 30 、 Figure 30 Durability of the chromatographic column

[0287] Table 21 Durability of the chromatographic column - relative retention times

[0288]

[0289] The results show that the RSD of the relative retention times of the characteristic peaks is 0.65% to 7.34% when the sample is detected by the above three chromatographic columns, indicating that the chromatographic column has good durability.

[0290] 3.4.6 Stability

[0291] Based on the above experimental conditions in 3.2, the same test solution was taken and measured at 0 h, 3 h, 6 h, 9 h, 15 h and 24 h.

[0292] Table 22 Stability - retention times

[0293]

[0294] The results show that the RSD of the retention times of the characteristic peaks is 0.22% to 3.28%, and the sample solution is stable within 24 hours.

[0295] In summary, the RSD of the relative retention times of the characteristic peaks meets the requirements in the above investigations, and the method is good. The above seven characteristic peaks are included in the subsequent investigation.

[0296] 3.4.7 Determination of characteristic peaks and establishment of the control chromatogram

[0297] 3.4.7.1 Verification results of 21 batches of pumpkin stem standard decoction

[0298] The method in 3.2 was used to analyze the characteristic chromatograms of 21 batches of samples, and the relative retention times were calculated.

[0299] Results Figure 31 , Figure 31 Characteristic chromatograms of pumpkin stem standard decoction (s1-s21: B1, B2, B3, B4, B5, B6, B7, B8, B9, B10, B11, B12, B13, B14, B15, B16, B17, B18, B19, B20, B21)

[0300] Table 23 Relative retention times of pumpkin stem standard decoction

[0301]

[0302] According to the principle of relative retention time stability and each batch sample can be detected and the peak is relatively high, a total of 7 peaks with better durability are selected as characteristic peaks. According to the results of methodological investigation and 21 batches of standard decoction verification, the theoretical plate number is tentatively determined to be not less than 4000 according to the peak of vanillic acid.

[0303] 3.4.7.2 Establishment of relative retention time limit

[0304] Methodological investigation and verification results:

[0305] Table 24 Methodological results RSD% of each item Standard-Relative retention time / Retention time

[0306]

[0307]

[0308] The relative retention time of each characteristic peak is stable, and within the average value ± 10%, so the relative retention time of each peak is tentatively determined to be ± 10%.

[0309] The final provision is that the test sample chromatogram should present 7 characteristic peaks, which should correspond to the retention time of the 7 characteristic peaks in the reference material chromatogram of the control medicinal material, among which peak 3, peak 4, peak 6 and peak 7 should correspond to the retention time of the corresponding reference substance peaks, and the peak corresponding to the vanillic acid reference substance peak is S peak. The relative retention time of each characteristic peak to S peak should be within ± 10% of the specified value. The specified value is: 0.70 (peak 1), 1.13 (peak 3), 1.19 (peak 4), 1.48 (peak 5), 1.62 (peak 6), 1.82 (peak 7).

[0310] The 21 batches of pumpkin peduncle standard decoction were synthesized by using Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version), and the control chromatogram of pumpkin peduncle standard decoction characteristic chromatogram was established as shown in Figure 32 Peak 2 (S): Vanillic acid, chromatographic column 1.

[0311] Example 4 HPLC characteristic chromatogram of pumpkin peduncle formula granules

[0312] 4.1 Tentative chromatographic conditions

[0313] Chromatographic conditions and system suitability tests were conducted based on the characteristic chromatographic conditions of the pumpkin stem standard decoction. The chromatographic conditions for the pumpkin stem formulation granules were determined as follows: Octadecylsilane-bonded silica gel was used as the packing material; acetonitrile was used as mobile phase A, and 0.2% acetic acid solution was used as mobile phase B, with gradient elution as specified in the table below; the flow rate was 1.0 ml per minute; the column temperature was 30℃; and the detection wavelength was 270 nm. The theoretical plate number, calculated based on the vanillic acid peak, should not be less than 4000.

[0314]

[0315] Preparation of the reference solution: Take 2g of pumpkin stem reference material, place it in a stoppered conical flask, add 20ml of 70% ethanol, weigh, reflux for 30 minutes, cool, weigh again, add 70% ethanol to make up the weight, filter, and use as the reference solution for the reference material. Take an appropriate amount of vanillic acid reference standard, accurately weigh it, add 70% ethanol solution to prepare a solution containing 7ug of vanillic acid per 1ml, and use as the reference solution for the standard.

[0316] Preparation of the test solution: Take an appropriate amount of this product, grind it into a fine powder, accurately weigh about 0.5 g, place it in a stoppered conical flask, accurately add 20 ml of 70% ethanol, seal tightly, weigh, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, weigh again, replenish the lost weight with 70% ethanol, shake well, and the test solution is ready.

[0317] The determination method involves precisely pipetting 10 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0318] 4.2 Chromatographic conditions and system suitability test

[0319] 4.2.1 Selection of mobile phase

[0320] Based on the experimental conditions proposed in section 4.1 above, the effects of acetonitrile-0.2% phosphoric acid, acetonitrile-0.2% acetic acid, and acetonitrile-0.2% formic acid were investigated respectively. The results are as follows... Figure 33 The above, Figure 33 Chromatograms of pumpkin stem particles using different mobile phases.

[0321] The results showed that when the mobile phase was acetonitrile-0.2% acetic acid, the chromatographic peak information was large, the resolution was good, and the elution time was moderate. The mobile phase was tentatively set as acetonitrile-0.2% acetic acid.

[0322] 4.2.2 Wavelength Selection

[0323] Based on the proposed chromatographic conditions in section 4.1, a diode array detector was used to perform a full-spectrum scan of the test solution, and chromatograms of the test solution at wavelengths of 254 nm, 270 nm, and 280 nm were extracted. The results are as follows: Figure 34 .Figure 34 Chromatograms of pumpkin stem formulation granules at different wavelengths; the results showed that the chromatographic peak information was greater and the chromatographic baseline was more stable at a detection wavelength of 270 nm, so the detection wavelength was determined to be 270 nm.

[0324] 4.2.3 Column Temperature Investigation

[0325] Based on the proposed chromatographic conditions in section 4.1, the effects were investigated at column temperatures of 25℃, 30℃, and 35℃. The results are as follows: Figure 35 . Figure 35 Chromatogram of pumpkin stem formulation granules under column temperature investigation;

[0326] Table 25 Column Temperature Study - Retention Time

[0327]

[0328] Table 26 Column Temperature Study - Relative Retention Time

[0329]

[0330] The results showed that at a column temperature of 30℃, the chromatographic peaks were relatively symmetrical, the resolution was good, the elution time was moderate, and the peaks were relatively complete. Therefore, the column temperature was tentatively set at 30℃.

[0331] 4.2.4 Flow velocity investigation

[0332] Based on the proposed chromatographic conditions in section 4.1, the flow rates of 0.8 ml / min, 1.0 ml / min, and 1.2 ml / min were investigated, and the results are shown in [Figure 1]. Figure 36 . Figure 36 Flow velocity study;

[0333] Table 27 Flow velocity study - retention time

[0334]

[0335] Table 28 Flow velocity study - relative retention time

[0336]

[0337] The results showed that a flow rate of 1.0 ml / min resulted in a better peak shape and moderate resolution in the chromatogram. Therefore, the flow rate was determined to be 1.0 ml / min.

[0338] 4.2.5 Delayedness Assessment

[0339] Based on the proposed chromatographic conditions in section 4.1, a delay test was conducted. The results are as follows: Figure 37 As shown, Figure 37The results show the effects of the delayed detection. The results indicate that the sample showed virtually no chromatographic peaks after 58 minutes; therefore, the sample detection time was set at 58 minutes.

[0340] In summary, the chromatographic conditions and system suitability test for the characteristic chromatogram of pumpkin stem formulation granules were determined as follows: Octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile as mobile phase A, and 0.2% acetic acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 1.0 mL / min; column temperature 30℃; detection wavelength 270 nm. The theoretical plate number, calculated based on the vanillic acid peak, should not be less than 4000.

[0341]

[0342] 4.3 Preparation of the test solution

[0343] 4.3.1 Investigation of Extraction Solvents

[0344] Take 0.5g of pumpkin stem formula granules (batch number: K1), grind them into a fine powder, and test them with 20ml each of 30% ethanol, 50% ethanol, 70% ethanol, ethanol, methanol, and water as extraction solvents. Sonicate the sample (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and collect the filtrate to obtain the final product.

[0345] The sample was injected and analyzed according to the results of "4.2 Chromatographic Conditions and System Suitability Test". The results are shown in [Figure Number]. Figure 38 . Figure 38 Extraction solvent investigation: The results showed that when the extraction solvent was 70% ethanol, the peak shapes of each characteristic peak were good and the separation was moderate. Therefore, the extraction solvent was determined to be 70% ethanol.

[0346] 4.3.2 Examination of Extraction Methods

[0347] Take 0.5g of pumpkin stem formula granules (batch number: K1), grind them into a fine powder, add 20ml of 70% ethanol, and investigate the extraction methods of the test sample when reflux and ultrasound (power 600W, frequency 40kHz) are used respectively. The extraction time is 30 minutes. After cooling, shake well, filter, and take the filtrate to obtain the sample.

[0348] The sample was then injected and analyzed according to the results of "4.2 Chromatographic Conditions and System Suitability Test". The results are shown in [the table below]. Figure 39 . Figure 39 Extraction methods were investigated; the results showed that there was little difference in the effectiveness of ultrasonic and reflux extraction of the test sample, and the peak shape and separation of each characteristic peak were good. Moreover, the ultrasonic method was fast and simple, so ultrasonic extraction was determined to be the extraction method for the test sample.

[0349] 4.3.3 Examination of extraction time

[0350] Take pumpkin stem formula granules (batch number: K1) 0.5 g, grind, add 70% ethanol 20 ml, ultrasonic (power 600 W, frequency 40 kHz) treatment, respectively, the test sample extraction time is 20 minutes, 30 minutes, 60 minutes, respectively, to investigate, cool, shake, filter, take the filtrate, that is.

[0351] According to the results of "4.2 chromatographic conditions and system suitability test", the results are shown in Figure 40 . Figure 40 Extraction time investigation; the results show that the extraction time is 30 minutes and 60 minutes, the extraction is more sufficient, so the extraction time is determined as 30 minutes.

[0352] 4.3.4 Solvent addition amount investigation

[0353] Take pumpkin stem formula granules (batch number: K1) 0.5 g, grind, respectively, add 70% ethanol 15 ml, 20 ml, 50 ml for investigation, ultrasonic (power 600 W, frequency 40 kHz) treatment 30 minutes, cool, shake, filter, take the filtrate, that is.

[0354] According to the results of "4.2 chromatographic conditions and system suitability test", the results are shown in Figure 41 . Figure 41 Solvent addition amount investigation;

[0355] The results show that the solvent addition amount is 20 ml, the peak shape and separation degree of each chromatographic peak is good, so the solvent addition amount is selected as 20 ml.

[0356] In summary, the preparation method of pumpkin stem formula granules characteristic map test solution is determined as follows: take 0.5 g of the product, place it in a conical flask with a plug, add 70% ethanol 20 ml, weigh, ultrasonic 30 minutes, cool, weigh again, make up the weight with 70% ethanol, filter, and get.

[0357] 4.4 Methodology

[0358] 4.4.1 Chromatographic peak identification

[0359] Preparation of test solution: according to the above proposed experimental conditions, prepare the test solution of pumpkin stem formula granules.

[0360] Preparation of reference solution: take 2 g of pumpkin stem control drug, place it in a conical flask with a plug, add 70% ethanol 20 ml, weigh, reflux 30 minutes, cool, weigh again, make up the weight with 70% ethanol, filter, and get the control drug reference solution

[0361] Take vanillic acid reference substance, accurately weigh, add 70% ethanol solution to prepare a solution containing 7 ug of vanillic acid per 1 ml, as the reference solution of the reference substance.

[0362] Preparation of negative control solution: The negative control solution of pumpkin stem formula granules was prepared according to the experimental conditions as above.

[0363] The characteristic peaks of pumpkin stem formula granules were located.

[0364] According to the results of "4.2 Chromatographic conditions and system suitability test", the results are shown in Table 2. Figure 42 . Figure 42 The chromatographic peaks were assigned. Figure 43 Vanillic acid spectrum- reference substance; Figure 44 Vanillic acid spectrum- granules.

[0365] The results showed that peak 2 was vanillic acid. In the following methodological study, 7 characteristic peaks in the sample were investigated.

[0366] 4.4.2 Precision test

[0367] The pumpkin stem formula granules (batch number: K1) test solution was taken, and the relative retention time of each characteristic peak was calculated according to the proposed experimental method for 6 times in succession.

[0368] Table 29 Precision investigation- relative retention time

[0369]

[0370] The results showed that the relative retention time RSD of each characteristic peak of the sample was in the range of 0-0.08%, and the precision of the instrument was good.

[0371] 4.4.3 Repeatability investigation

[0372] Six portions of pumpkin stem formula granules (batch number: K1) were accurately weighed, and prepared and determined according to the proposed experimental method.

[0373] Table 30 Repeatability investigation- relative retention time

[0374]

[0375]

[0376] The results showed that the relative retention time RSD of the 6 samples was in the range of 0.03%-0.06%, indicating that the method had good repeatability.

[0377] 4.4.4 Intermediate precision investigation

[0378] 4.4.4.1 Investigation of different instruments

[0379] On the basis of the above experimental conditions, pumpkin stem formula granules (batch number: K1) were precisely weighed to prepare the test solution, which was determined on chromatograph 1, chromatograph 2 and chromatograph 3, respectively.

[0380] Figure 45 The different instruments were investigated.

[0381] Table 31 Instrument durability investigation - relative retention time

[0382]

[0383] The results show that when the above three instruments are used to detect the test sample, the RSD of the relative retention time of each characteristic peak is 0.05% to 3.97%, and the instrument durability is good.

[0384] 4.4.4.2 Different personnel and time investigation

[0385] On the basis of the above experimental conditions, pumpkin stem formula granules (batch number: K1) were precisely weighed by different personnel (A, B) at different times (T1, T2) to prepare the test sample, which was determined.

[0386] Table 32 Personnel and time investigation - relative retention time

[0387]

[0388] The results show that when the same sample is determined by different personnel at different times, the RSD of the relative retention time of each characteristic peak is 0.12% to 0.22%, and the method is stable.

[0389] 4.5 Chromatographic column durability investigation

[0390] On the basis of the above experimental conditions of 4.1, chromatographic column 1, chromatographic column 2 and chromatographic column 3 were analyzed and investigated. The results are shown in Figure 46 , Figure 46 The results of the investigation of different chromatographic columns are shown in the figure.

[0391] Table 33 Chromatographic column durability investigation - relative retention time

[0392]

[0393] The results show that when the above three chromatographic columns are used to detect the sample, the RSD of the relative retention time of each characteristic peak is 0.68% to 7.28%, and three chromatographic columns can detect each characteristic peak.

[0394] 4.6 Stability

[0395] On the basis of the experimental conditions set out above in 4.1, the same test solution was taken and measured at 0 h, 3 h, 6 h, 9 h, 15 h and 24 h.

[0396] Table 34 Stability Investigation - Relative Retention Time

[0397]

[0398] The results show that the RSD of the relative retention time of the characteristic peaks is 0.05% to 0.09%, and the sample solution is stable within 24 hours.

[0399] In summary, the RSD of the relative retention time of each characteristic peak meets the requirements in the above investigations, and the method is good. The above 7 characteristic peaks are included in the subsequent investigation.

[0400] 4.5 Determination of Characteristic Peaks and Establishment of Control Chromatogram

[0401] 4.5.1 Verification Results of 3 Batches of Pumpkin Stem Formula Granules

[0402] 3 batches of pumpkin stem formula granules were verified, and the relative retention time was calculated by using this method to analyze the characteristic chromatograms of 3 batches of samples. The results are as follows Figure 47 Figure 47 The characteristic chromatogram of pumpkin stem formula granules is as follows

[0403] Table 35 Relative Retention Time of Pumpkin Stem Formula Granules

[0404]

[0405] According to the principles of stability of relative retention time and detection of each batch of samples with relatively high peaks, a total of 7 peaks with good reproducibility were selected as characteristic peaks. According to the results of the methodological investigation and the verification results of 3 batches of granules, the theoretical plate number was tentatively determined to be not less than 4000 calculated by the peak of vanillic acid.

[0406] 7.2 Establishment of Relative Retention Time Limit

[0407] Methodological investigation items and verification results:

[0408] Table 36 Summary of RSD% of Results of Each Item of Methodology - Relative Retention Time

[0409]

[0410] The relative retention time of each characteristic peak is stable and within the range of average value ± 10%, so the relative retention time limit of each peak is tentatively set to ± 10%.

[0411] ​The final specification stipulates that the chromatogram of the test sample should show 7 characteristic peaks, and the retention times should correspond to the 7 characteristic peaks in the chromatogram of the reference medicinal material. The peak corresponding to the vanillic acid reference standard peak is designated as the S peak. The relative retention times of each characteristic peak and the S peak should be calculated. These relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.70 (peak 1), 1.13 (peak 3), 1.19 (peak 4), 1.48 (peak 5), 1.62 (peak 6), and 1.82 (peak 7).

[0412] The similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine (2012 version) was used to synthesize three batches of pumpkin stem formulation granules, and a similarity model was established. Figure 48 The comparison spectrum of the characteristic spectrum of the pumpkin stem formula granules.

[0413] Comparative Example 1: Chromatographic conditions and system suitability test. An octadecylsilane-bonded silica gel column (Diamonsil C18 column (250 mm × 4.6 mm, 5 μm) was used as the packing material. Acetonitrile was used as mobile phase A, and 0.1% acetic acid solution was used as mobile phase B. Gradient elution was performed according to the specifications in Table 37. The flow rate was 1.0 mL per minute; the column temperature was 30 °C; and the detection wavelength was 234 nm.

[0414] Table 37

[0415]

[0416] Chromatogram as shown Figure 49 As shown, Figure 49 It can be seen that the detection method for cucurbitacin components is not applicable to the detection of characteristic spectra of pumpkin stem medicinal materials and granules.

[0417] Comparative Example 2 used octadecylsilane-bonded silica gel as the packing material (Diamonsil C18 column (250 mm × 4.6 mm, 5 μm), acetonitrile as mobile phase A, and 0.1% acetic acid solution as mobile phase B, and performed gradient elution according to the specifications in the table below; the flow rate was 1.0 mL per minute; the column temperature was 30 °C; and the detection wavelength was 234 nm. (Diamonsil C18 column (250 mm × 4.6 mm, 5 μm)).

[0418] Table 38

[0419]

[0420] Chromatogram as shown Figure 50 As shown, Figure 50It can be seen that the same mobile phase, using the same gradient of different S peak for comparative example 1 and comparative example 2 two determination, the results show that using this mobile phase gradient, pumpkin stem medicinal materials and finished products are not detected cucurbitacin B and vanillic acid components, pumpkin stem medicinal materials and finished products in each peak separation and peak type difference, so cucurbitaceae cucurbitacin class of components of the method is not suitable for the detection of pumpkin stem characteristic chromatogram.

[0421] Comparative example 3

[0422] Using the same mobile phase gradient, the mobile phase is changed to acetonitrile-0.2% acetic acid, acetonitrile-0.2% phosphoric acid, methanol-0.2% acetic acid test.

[0423] In the case of no change in the mobile phase gradient, select different mobile phase for investigation, the results show that with acetonitrile-0.2 acetic acid as mobile phase, the separation degree and peak type of each peak in pumpkin stem granules are good, which can effectively detect the fingerprint of pumpkin stem granules. As shown in Figure 51

[0424] The above only is the preferred embodiment of the present application, it should be pointed out that, for those skilled in the art, without departing from the principles of the present application, can also make a number of improvements and refinements, these improvements and refinements should also be considered as the protection scope of the present application.​

Claims

1. A method for constructing an HPLC characteristic chromatogram of pumpkin pedicel medicinal material, decoction piece, standard decoction and its formula granules, comprising: A) extracting pumpkin pedicel raw material with a solvent to obtain a test solution; and heating and refluxing or ultrasonic extracting pumpkin pedicel raw material with 70% ethanol; B) determining the test solution by high performance liquid chromatography to obtain an HPLC characteristic chromatogram of pumpkin pedicel raw material; The high performance liquid chromatography has the following chromatographic conditions: a C18 column; a mobile phase A of acetonitrile and a mobile phase B of 0.2% acetic acid; a detection wavelength of 270 nm; gradient elution; and the gradient elution is as follows: 0-30 min, A phase: 7%, B phase: 93%; 30-50 min, A phase: 7%-13%, B phase: 93%-87%; 50-58 min, A phase: 13%, B phase: 87%.

2. The method of claim 1, wherein, The method further comprises preparing a reference solution of a control and a control medicinal material: dissolving vanillic acid control in 70% ethanol to obtain a reference solution of the control; extracting pumpkin pedicel control medicinal material with 70% ethanol by heating and refluxing to obtain a reference solution of the control medicinal material; determining the reference solutions of the control and the control medicinal material by high performance liquid chromatography to obtain chromatograms of the reference solutions of the control and the control medicinal material, respectively; and qualitatively determining the components of the HPLC characteristic chromatogram of pumpkin pedicel medicinal material, decoction piece, standard decoction and its formula granules according to the chromatograms of the reference solutions of the control and the control medicinal material.

3. The method of claim 1, wherein, The chromatographic column is a C18 column with a specification of 5 μm and 4.6*250 mm, and the column temperature is 30°C.

4. The method of claim 3, wherein, The flow rate of the mobile phase is 1.0 mL / min, and the injection volume is 10 μL.

5. The method of claim 1, wherein, The similarity of the HPLC characteristic chromatogram of pumpkin pedicel medicinal material, decoction piece, standard decoction and its formula granules is evaluated by a traditional Chinese medicine chromatographic fingerprint similarity evaluation system to obtain an HPLC standard characteristic chromatogram of pumpkin pedicel medicinal material, decoction piece, standard decoction and its formula granules composed of seven characteristic peaks, wherein peak 2S is a vanillic acid peak.

6. The method of claim 5, wherein, In the standard decoction characteristic chromatogram of pumpkin pedicel, the vanillic acid peak is taken as a reference peak S peak, the relative retention times of the characteristic peaks to the S peak are calculated, the relative retention times are within ±10% of the specified values, and the specified values are 0.70 (peak 1), 1.13 (peak 3), 1.19 (peak 4), 1.48 (peak 5), 1.62 (peak 6) and 1.82 (peak 7), respectively.

7. The method of claim 1, wherein, In step A), the pumpkin pedicel raw material is pumpkin pedicel medicinal material, decoction piece, standard decoction and its formula granules.

8. The method of claim 1, wherein, Step A) is specifically as follows: heating and refluxing or ultrasonic extracting pumpkin pedicel raw material with 70% ethanol for 30 min, cooling, shaking and filtering to obtain the test solution.

9. The method of claim 8, wherein, The ratio of the mass of the pumpkin pedicel raw material (g) to the volume of the solvent (mL) is (0.5-2):20.

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