A method for constructing the characteristic chromatograms of stir-fried black sesame herbs, stir-fried black sesame decoction pieces, standard decoctions and their formula granules and its application

Abstract

The present invention provides a method for constructing characteristic chromatograms of stir-fried black sesame herbs, stir-fried black sesame decoction pieces, standard decoctions and their formula granules, and its application, comprising the following steps: A) Dissolving the stir-fried black sesame raw materials with 70% methanol, extracting to obtain a test solution; B) Measuring the test solution by high-performance liquid chromatography to obtain the characteristic chromatograms of stir-fried black sesame herbs, stir-fried black sesame decoction pieces, standard decoctions and their formula granules; the chromatographic conditions include: a C18 column, mobile phase A is acetonitrile, mobile phase B is 0.1% phosphoric acid solution, and gradient elution is performed. The present invention adopts high-performance liquid chromatography, selects acetonitrile-0.1% phosphoric acid solution as the mobile phase for gradient elution, and uses sesamin as the reference substance to establish characteristic chromatograms of stir-fried black sesame herbs, stir-fried black sesame decoction pieces, standard decoctions and their formula granules with good repeatability and precision, and the method is stable and reliable, and can control the quality of stir-fried black sesame herbs, stir-fried black sesame decoction pieces, standard decoctions and their formula granules.

Description

Technical Field

[0001] The present invention belongs to the technical field of analytical detection, and particularly relates to a method for constructing characteristic chromatograms of stir-fried black sesame herbs, stir-fried black sesame decoction pieces, standard decoctions and their formula granules, and their applications. Background Art

[0002] Black sesame is a variety that can be used both as medicine and food, and is cultivated throughout the country. It mainly has the effects of tonifying the liver and kidney, benefiting essence and blood, and moistening the intestines and relieving constipation. Clinically, it is mainly used for the treatment of diseases such as deficiency of essence and blood, dizziness and blurred vision, tinnitus and deafness, early whitening of hair and beard, hair loss after illness, and intestinal dryness and constipation. Black sesame is often used in clinical prescriptions and is the main raw material of traditional Chinese patent medicines such as Zhigancao Mixture, Shouwu Pills, and Wanling Jin'gu Ointment.

[0003] At present, Li Linyan et al. have conducted research on the medicinal components, functions and applications of black sesame herbs, but black sesame is often applied clinically in the form of preparations. There is no literature on the quality standard research of stir-fried black sesame formula granules. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a method for constructing characteristic chromatograms of stir-fried black sesame herbs, stir-fried black sesame decoction pieces, standard decoctions and their formula granules, and their applications. This method conducts research on single varieties of stir-fried black sesame formula granules, scientifically identifies stir-fried black sesame, has the characteristics of comprehensive detection and simple operation, can effectively identify stir-fried black sesame formula granules, and can ensure the uniformity and stability of the quality of stir-fried black sesame formula granules. This method is applicable to stir-fried black sesame herbs, stir-fried black sesame decoction pieces, standard decoctions and formula granules.

[0005] The present invention provides a method for constructing characteristic chromatograms of stir-fried black sesame herbs, stir-fried black sesame decoction pieces, standard decoctions and their formula granules, comprising the following steps:

[0006] A) Dissolve the stir-fried black sesame raw material with 70% methanol, extract to obtain a test solution;

[0007] B) Determine the test solution by high performance liquid chromatography to obtain characteristic chromatograms of stir-fried black sesame herbs, stir-fried black sesame decoction pieces, standard decoctions and their formula granules;

[0008] The chromatographic conditions of the high performance liquid chromatography include: a C18 column, mobile phase A is acetonitrile, mobile phase B is 0.1% phosphoric acid solution, and gradient elution.

[0009] In the present invention, both the 70% methanol and the 0.1% phosphoric acid solution are volume fractions; the 0.1% phosphoric acid solution is 0.1% phosphoric acid aqueous solution.

[0010] When 70% methanol is used as the extraction solvent in the present invention, the chromatographic peak information is abundant. In the present invention, if the raw material of stir-fried black sesame is the standard decoction or formula granules of stir-fried black sesame, the mass ratio of the raw material of stir-fried black sesame to the volume of 70% methanol is 0.1 g:(45 - 55) mL;

[0011] If the raw material of stir-fried black sesame is the medicinal material or cut crude drug of stir-fried black sesame, the mass ratio of the raw material of stir-fried black sesame to the volume of 70% methanol is 1 g:(45 - 55) mL.

[0012] When 70% methanol is used as the extraction solvent, the addition amount is appropriate, making the chromatographic peak area of the characteristic chromatogram moderate. In the specific embodiments of the present invention, if the raw material of stir-fried black sesame is the standard decoction or formula granules of stir-fried black sesame, the mass ratio of the raw material of stir-fried black sesame to the volume of 70% methanol is 0.1 g:50 mL; if the raw material of stir-fried black sesame is the medicinal material or cut crude drug of stir-fried black sesame, the mass ratio of the raw material of stir-fried black sesame to the volume of 70% methanol is 1 g:50 mL.

[0013] In the present invention, the extraction is ultrasonic extraction, which is simple and convenient; the power of the ultrasonic extraction is 550 - 650 W, the frequency of the ultrasonic extraction is 35 - 45 kHz, and the time of the ultrasonic extraction is 28 - 32 min. In the specific embodiments, the power of the ultrasonic extraction is 600 W, the frequency of the ultrasonic extraction is 40 kHz, and the time of the ultrasonic extraction is 30 min.

[0014] The present invention also includes the preparation of the reference solution:

[0015] Prepare a reference solution of the reference substance with sesamin and methanol at a concentration of 25 μg / mL;

[0016] Take the reference crude drug of black sesame, decoct it with water, filter, evaporate to dryness, add 70% methanol to the residue, perform ultrasonic treatment, cool it, shake well, filter, and take the filtrate to obtain the reference solution of the reference crude drug.

[0017] Determine the test solution by high performance liquid chromatography to obtain the characteristic chromatograms of the medicinal materials, cut crude drugs, standard decoctions and their formula granules of stir-fried black sesame. The chromatographic conditions of the high performance liquid chromatography include: a C18 column, mobile phase A is acetonitrile, mobile phase B is 0.1% phosphoric acid solution, and gradient elution.

[0018] In the present invention, the gradient elution is specifically as follows;

[0019] 0 - 6 min, phase A: 5%, phase B: 95%;

[0020] 6 - 13 min, phase A: 5 - 15%, phase B: 95 - 85%;

[0021] 13 - 25 min, phase A: 15 - 25%, phase B: 85 - 75%;

[0022] 25 - 35 min, Phase A: 25 - 30%, Phase B: 75 - 70%;

[0023] 35 - 60 min, Phase A: 30 - 70%, Phase B: 70 - 30%;

[0024] 60 - 65 min, Phase A: 70%, Phase B: 30%.

[0025] In the present invention, the flow rate of the mobile phase is 1.0 ml / min, the chromatogram peak shape is good, and the resolution is moderate.

[0026] In the present invention, the column temperature is 35°C; when the column temperature is 35°C, the chromatogram peak shape is symmetrical and the resolution is good. The specification of the C18 column is 4.6 mm × 250 mm, 5 μm; in specific embodiments, the C18 column is HSS T3 5μm 250×4.6mm Column; ZORBAX SB - Aq StableBond Analytical 4.6×250mm 5 - Micron; Kromasil 100 - 5 - C18 4.6×250mm.

[0027] In the present invention, when the detection wavelength is 210 nm, the chromatogram peak information is larger and the chromatogram baseline is more stable, so the detection wavelength is 210 nm.

[0028] The theoretical plate number calculated based on the sesamin peak should be not less than 5000.

[0029] The injection volume of the test solution is 9 - 11 μL; in specific embodiments, the addition amounts of the test solution and the reference solution are 10 μL.

[0030] In the present invention, by measuring the same sample with different instruments, different personnel at different times, the precision and stability of this method are verified. The present invention uses a C18 chromatographic column, the chromatogram peak separation is good, the peaks appear clearly, and the chromatographic column has good durability. The present invention measures the test solution for 0 - 24 h and finds that the RSD of the retention time of the corresponding characteristic peak is between 0.09% and 0.89%, indicating that the sample solution is stable within 24 h.

[0031] The present invention uses a similarity evaluation system for traditional Chinese medicine chromatographic fingerprints to evaluate the similarities of stir-fried black sesame herbs, decoction pieces, standard decoctions, and formula granules, and obtains HPLC standard characteristic fingerprints of stir-fried black sesame herbs, decoction pieces, standard decoctions, and formula granules composed of 6 characteristic peaks, corresponding to the 6 characteristic retention times in the chromatogram of the reference material of the control herb. Taking the peak corresponding to the sesamin reference peak as the S peak, in the said standard characteristic fingerprint, calculate the relative retention times of each characteristic peak and the S peak, and the relative retention times are within ±10% of the specified values, and the specified values are respectively: 0.09 (peak 1), 0.23 (peak 2), 0.29 (peak 3), 0.51 (peak 4), 1.04 (peak 6).

[0032] The present invention also provides a characteristic fingerprint of stir-fried black sesame herbs, stir-fried black sesame decoction pieces, standard decoctions, and their formula granules, and detects and analyzes the detection results by using the method described in the above technical solution.

[0033] The present invention provides a method for constructing a characteristic fingerprint of stir-fried black sesame herbs, stir-fried black sesame decoction pieces, standard decoctions, and their formula granules, including the following steps: A) Dissolve the stir-fried black sesame raw material with 70% methanol, extract to obtain a test solution; B) Determine the test solution by high performance liquid chromatography to obtain the characteristic fingerprints of stir-fried black sesame herbs, stir-fried black sesame decoction pieces, standard decoctions, and their formula granules; the chromatographic conditions of the high performance liquid chromatography include: a C18 column, mobile phase A is acetonitrile, mobile phase B is 0.1% phosphoric acid solution, and gradient elution is used. The present invention uses high performance liquid chromatography, selects acetonitrile - 0.1% phosphoric acid solution as the mobile phase for gradient elution, and uses sesamin as the reference substance to establish a characteristic fingerprint of stir-fried black sesame herbs, stir-fried black sesame decoction pieces, standard decoctions, and their formula granules with good repeatability and precision, and the method is stable and reliable, and can control the quality of stir-fried black sesame herbs, stir-fried black sesame decoction pieces, standard decoctions, and their formula granules. Description of the Drawings

[0034] Figure 1 It is the chromatogram of stir-fried black sesame granules;

[0035] Figure 2 It is the investigation of different column temperatures of stir-fried black sesame formula granules;

[0036] Figure 3 It is the investigation of different flow rates of stir-fried black sesame formula granules;

[0037] Figure 4 It is the investigation of the delay of stir-fried black sesame formula granules;

[0038] Figure 5 It is the investigation of different extraction solvents of stir-fried black sesame formula granules;

[0039] Figure 6 It is the investigation of the extraction method of stir-fried black sesame formula granules;

[0040] Figure 7 For the investigation of the extraction time of roasted black sesame formula granules;

[0041] Figure 8 For the investigation of the solvent addition amount of roasted black sesame formula granules;

[0042] Figure 9 For the identification of chromatographic peaks;

[0043] Figure 10 For the investigation of different instruments;

[0044] Figure 11 For the investigation of different chromatographic columns;

[0045] Figure 12 For the verification diagram of the characteristic spectrograms of 3 batches of roasted black sesame formula granules;

[0046] Figure 13 For the characteristic spectrogram of black sesame medicinal materials;

[0047] Figure 14 For the characteristic spectrogram of roasted black sesame decoction pieces;

[0048] Figure 15 For the characteristic spectrogram of roasted black sesame standard decoction;

[0049] Figure 16 For the reference spectrogram of the characteristic spectrogram of roasted black sesame formula granules;

[0050] Figure 17 For the reference spectrogram of the characteristic spectrogram of black sesame medicinal materials;

[0051] Figure 18 For the reference spectrogram of the characteristic spectrogram of roasted black sesame decoction pieces;

[0052] Figure 19 For the reference spectrogram of the characteristic spectrogram of roasted black sesame standard decoction. Specific implementation manners

[0053] In order to further illustrate the present invention, the following describes in detail a method for constructing the characteristic spectrograms of a roasted black sesame medicinal material, roasted black sesame decoction pieces, standard decoction and its formula granules provided by the present invention in combination with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0054] 1. Experimental instruments and materials

[0055] High performance liquid chromatographs: Shimadzu LC-20AD type high performance liquid chromatograph, Agilent 1260 type high performance liquid chromatograph, Waters e2695 type high performance liquid chromatograph;

[0056] Electronic balance: ME204E / 02, XPE26 (Mettler Toledo Instruments Co., Ltd.);

[0057] Ultra-pure water machine: Cell type 1810A (Shanghai Moller Scientific Instruments Co., Ltd.);

[0058] Ultrasonic cleaner: Model KQ600DB (600W, 40KHz; Kunshan Ultrasonic Instruments Co., Ltd.);

[0059] Chromatographic column: HSS T3 5μm 250×4.6mm Column; ZORBAX SB-AqStableBond Analytical 4.6×250mm 5-Micron; Kromasil 100-5-C18 4.6×250mm;

[0060] Acetonitrile and phosphoric acid are of chromatographic purity, water is ultra-pure water, and the rest of the reagents are of analytical purity.

[0061] Sesamin reference substance (National Institutes for Food and Drug Control, batch number: 110836-201706, content calculated as 99.9%);

[0062] Control crude drug of black sesame ((National Institutes for Food and Drug Control, batch number: 121339-201803);

[0063] Methanol (SIGMA company, chromatographic purity); water is ultra-pure water, and other reagents are of analytical purity.

[0064] Stir-fried black sesame formula granules CHZM-01, stir-fried black sesame formula granules CHZM-02, CHZM-03, CHZM-04;

[0065] Stir-fried black sesame crude drugs: CHZM-YC-01, CHZM-YC-02, CHZM-YC-03, CHZM-YC-04, CHZM-YC-05, CHZM-YC-06, CHZM-YC-07, CHZM-YC-08, CHZM-YC-09, CHZM-YC-10, CHZM-YC-11, CHZM-YC-12, CHZM-YC-13, CHZM-YC-14, CHZM-YC-15, CHZM-YC-16, CHZM-YC-17, CHZM-YC-18;

[0066] Stir-fried black sesame decoction pieces: CHZM-YP-01, CHZM-YP-02, CHZM-YP-03, CHZM-YP-04, CHZM-YP-05, CHZM-YP-06, CHZM-YP-07, CHZM-YP-08, CHZM-YP-09, CHZM-YP-10, CHZM-YP-11, CHZM-YP-12, CHZM-YP-13, CHZM-YP-14, CHZM-YP-15, CHZM-YP-16, CHZM-YP-17, CHZM-YP-18;

[0067] Stir-fried black sesame standard decoction: CHZM-BT-01, CHZM-BT-02, CHZM-BT-03, CHZM-BT-04, CHZM-BT-05, CHZM-BT-06, CHZM-BT-07, CHZM-BT-08, CHZM-BT-09, CHZM-BT-10, CHZM-BT-11, CHZM-BT-12, CHZM-BT-13, CHZM-BT-14, CHZM-BT-15, CHZM-BT-16, CHZM-BT-17, CHZM-BT-18.

[0068] Chromatographic conditions and system suitability test Use octadecylsilane chemically bonded silica gel as the filler (column length is 250 mm, inner diameter is 4.6 mm, particle size is 5 μm); use acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, and perform gradient elution according to the regulations in Table a below; the flow rate is 1.0 ml per minute; the column temperature is 35 °C; the detection wavelength is 210 nm. The number of theoretical plates calculated based on the sesamin peak should be not less than 5000.

[0069] Table a Gradient elution program

[0070]

[0071]

[0072] Preparation of reference substance solution Weigh 1 g of the reference black sesame medicinal material, add 50 ml of water, decoct for 30 minutes, filter, evaporate to dryness, add 50 ml of 70% methanol to the residue, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, let it cool, shake well, filter, and take the consecutive filtrate as the reference medicinal material reference substance solution. Separately weigh an appropriate amount of sesamin reference substance, accurately weigh it, dissolve it in methanol to make a solution containing 25 μg per 1 ml as the reference substance solution of the reference substance.

[0073] Preparation of the test solution: Weigh an appropriate amount (about 0.1 g) of the stir-fried black sesame formula granule CHZM-01, place it in a stoppered conical flask, add 50 ml of 70% methanol, stopper tightly, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool to room temperature, shake well, filter, and take the subsequent filtrate, which is the test solution.

[0074] Determination method: Accurately pipette 10 μl each of the reference solution and the test solution, inject them into the liquid chromatograph, and perform the determination to obtain the results.

[0075] 2. Determination of the detection wavelength

[0076] Based on the above-determined experimental conditions, use a diode array detector to perform a full-wavelength scan on the test solution, and separately extract the chromatograms of the test solution at wavelengths of 200 nm, 210 nm, 220 nm, 240 nm, 260 nm, and 300 nm. See Figure 1 , Figure 1 From bottom to top in

[0077] are 210 nm, 300 nm, 260 nm, 240 nm, 220 nm, and 200 nm respectively; the results show that when the detection wavelength is 210 nm, the chromatographic peak information is larger and the chromatogram baseline is more stable. Therefore, the detection wavelength is determined to be 210 nm.

[0078] Based on the above-determined experimental conditions, investigate the column temperature at 25 °C, 30 °C, and 35 °C respectively. As shown in Figure 2 and Table 1:

[0079] Table 1 Investigation of column temperature - relative retention time ratio of characteristic peaks

[0080]

[0081]

[0082] The results of the column temperature investigation show that when the column temperature is 35 °C, the chromatogram peak shape is symmetric and the resolution is good. Therefore, the column temperature is finally determined to be 35 °C for the characteristic chromatogram method of the stir-fried black sesame standard decoction.

[0083] 4. Investigation of the flow rate

[0084] Based on the above-determined experimental conditions, investigate the flow rate at 0.8 mL / min, 1 mL / min, and 1.2 mL / min respectively. As shown in Figure 3 and Table 2. Figure 3 It shows that when the flow rate is 1.0 ml / min, the chromatogram peak shape is good and the resolution is moderate. Therefore, the flow rate is determined to be 1.0 ml / min.

[0085] Table 2 Flow rate - relative retention time

[0086]

[0087] 5. Delayed Test

[0088] Based on the above - established experimental conditions, extend the chromatogram acquisition time to 140 min. As Figure 4 shown, the results indicate that there are basically no chromatographic peaks after 65 min for the sample. Therefore, the sample detection time is set at 65 min.

[0089] 6. Investigation of Extraction Solvent

[0090] Take about 0.1 g of this product (CHZM - 01), place it in a stoppered conical flask, and add 50 ml of methanol, 30% methanol, 50% methanol, 70% methanol, water, and 70% ethanol respectively. Close the stopper tightly, perform ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, let it cool, shake well, filter, and take the subsequent filtrate to obtain the sample, as Figure 5 shown; the results show that when 70% methanol is used as the extraction solvent, there is a large amount of chromatographic peak information. Therefore, the extraction solvent for the test sample is determined to be 70% methanol.

[0091] 7. Investigation of Extraction Method

[0092] Take about 0.1 g of this product, place it in a stoppered conical flask, add 50 ml of 70% methanol, close the stopper tightly, and investigate the extraction methods of the test sample by reflux and ultrasonic (power 600 W, frequency 40 kHz) respectively. The extraction time is 30 min, let it cool, shake well, filter, and take the subsequent filtrate to obtain the sample, as Figure 6 shown; the results show that the effects are the same when the test sample is extracted by ultrasonic extraction and reflux extraction respectively. Since the ultrasonic extraction operation is more convenient, the extraction method for the test sample is determined to be ultrasonic extraction.

[0093] 8. Investigation of Extraction Time

[0094] Take about 0.1 g of this product, place it in a stoppered conical flask, add 50 ml of 70% methanol, close the stopper tightly, perform ultrasonic treatment (power 600 W, frequency 40 kHz), and investigate the test sample at extraction times of 15 minutes, 30 minutes, and 40 minutes respectively. Let it cool, shake well, filter, and take the subsequent filtrate to obtain the sample, as Figure 7 shown; the results show that sufficient extraction is achieved at an extraction time of 30 minutes. Therefore, the extraction time for the test sample is determined to be 30 minutes.

[0095] 9. Investigation of Solvent Addition Amount

[0096] Take about 0.1 g of this product, grind it finely, take about 0.1 g, put it into a stoppered conical flask, add 20 ml, 25 ml, and 50 ml of 70% methanol respectively, stopper tightly, weigh accurately, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, shake well, filter, and take the subsequent filtrate to obtain the solution. See Figure 8 ; The results showed that when the solvent addition amount was 50 ml, the chromatographic peak areas of the characteristic chromatogram were moderate. Therefore, the solvent addition amount of the test sample was determined to be 50 ml.

[0097] 10. Determine the preparation method of the test sample

[0098] Take an appropriate amount of this product (batch number: CHZM - 01), grind it finely, take 0.1 g, weigh accurately, put it into a stoppered conical flask, add 50 mL of 70% methanol, stopper tightly, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool to room temperature, shake well, filter, and take the subsequent filtrate to obtain the solution.

[0099] 11. Methodology investigation

[0100] 11.1 Chromatographic peak identification

[0101] Preparation of the test sample solution: Prepare the test sample solution of the stir - fried black sesame formula granules according to the above - determined experimental conditions.

[0102] Preparation of the reference substance solution: Take an appropriate amount of sesamin reference substance, weigh accurately, and dissolve it in methanol to make a solution containing 25 μg per 1 mL to obtain the solution.

[0103] Preparation of the control medicinal material solution: Take 1 g of the black sesame control medicinal material, add 50 ml of water, decoct for 30 minutes, filter, evaporate to dryness, dissolve the residue in 50 ml of 70% methanol, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool to room temperature, shake well, filter, and take the subsequent filtrate as the control medicinal material reference substance solution.

[0104] Preparation of the negative control solution: Prepare the negative control solution without the stir - fried black sesame formula granules according to the above - determined experimental conditions.

[0105] The present invention locates the characteristic chromatogram peaks of the stir - fried black sesame formula granules. As Figure 9 shown.

[0106] 11.2 Precision test

[0107] Take the test sample solution of the stir - fried black sesame formula granules (batch number: CHZM - 01), inject 10 μL each time continuously for 6 times according to the determined experimental method, and calculate the retention time and peak area of each characteristic peak. As shown in Table 3 and Table 4.

[0108] Table 3 Precision investigation - retention time

[0109]

[0110]

[0111] The results showed that the RSD of the retention times of the characteristic peaks for precision was 0.04% - 0.39%. The method had good precision.

[0112] 11.3 Repeatability investigation

[0113] Accurately weigh 6 portions of the roasted black sesame formula granules (batch number: CHZM - 01), and prepare and determine them according to the proposed experimental method. As shown in Table 4.

[0114] Table 4 Repeatability investigation - relative retention time

[0115]

[0116] The results showed that the RSD of the relative retention times of the characteristic peaks for repeatability was 0.01% - 0.57%. The method had good repeatability.

[0117] 11.4 Intermediate precision investigation

[0118] 11.4.1 Investigation with different instruments

[0119] On the basis of the above - proposed experimental conditions, accurately weigh two portions of the roasted black sesame formula granules (batch number: CHZM - 01) respectively, prepare the test solution, and determine them on Agilent 1260, Shimadzu LC - 20AD, and Waters 2695 high - performance liquid chromatographs respectively. As Figure 10 , shown in Table 5.

[0120] Table 5 Instrument durability investigation - relative retention time ratio

[0121]

[0122]

[0123] 11.4.2 Investigation with different personnel and time

[0124] On the basis of the above - proposed experimental conditions, different personnel (A, B) accurately weigh two portions of the roasted black sesame formula granules (batch number: CHZM - 01) respectively at different times (T1, T2), prepare the test samples, and conduct the determination. As shown in Table 6.

[0125] Table 6 Personnel and time investigation - relative retention time

[0126]

[0127] The results showed that when the same sample was determined by different personnel at different times, the method had good stability.

[0128] 11.5 Durability Investigation

[0129] 11.5.1 Chromatographic Column Durability Investigation

[0130] Based on the above - determined experimental conditions, the following chromatographic columns were investigated respectively: HSS T3 5μm 250×4.6mm Column; ZORBAX SB - Aq StableBond Analytical 4.6×250mm 5 - Micron; Kromasil 100 - 5 - C18 4.6×250mm. As shown in Figure 11 , Table 7. It can be seen from Figure 11 that the peak separation of the chromatograms of each chromatographic column is good and the peaks appear clearly, indicating good durability of the chromatographic columns.

[0131] Table 7 Chromatographic Column Durability Investigation - Relative Retention Time

[0132]

[0133] 11.5.2 Stability Investigation

[0134] Based on the above - determined experimental conditions, the same test sample solution was taken and measured at 0h, 4h, 8h, 12h, 16h, and 24h respectively. As shown in Table 8.

[0135] Table 8 Stability Investigation - Retention Time

[0136]

[0137] The results show that the RSD of the retention time of the corresponding characteristic peaks is between 0.09% and 0.89%, indicating that the sample solution is stable within 24 hours.

[0138] 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 6 characteristic peaks were included in the subsequent investigations.

[0139] Example 1 Verification Results of 3 Batches of Stir - Fried Black Sesame Formula Granules

[0140] The characteristic fingerprints of 3 batches of this product were determined by the proposed method, and the relative retention time was calculated. As shown in Figure 12 , Table 9.

[0141] Table 9 Relative Retention Time of 3 Batches of Stir - Fried Black Sesame Formula Granules

[0142]

[0143] According to the principle of stable relative retention time, detectable in each batch of samples, and relatively high peaks, a total of 6 peaks with good repeatability were selected as characteristic peaks. The results showed that when Peak 5 was used as the S peak, the relative standard deviation (RSD) of the relative retention time of the characteristic peaks in 3 batches of stir-fried black sesame formula granules was less than 1%.

[0144] Formulation of the specified limit of relative retention time

[0145] The summary of each investigation item and verification result of the methodology is shown in Table 10:

[0146] Table 10 Summary of RSD% of the results of each item of the methodology - relative retention time

[0147]

[0148]

[0149] Therefore, the specified value of the relative retention time of each peak was tentatively set at ±10%.

[0150] Final regulation: The RSD of the relative retention time of each characteristic peak met the requirements in all the above investigations, and the method was good. Six characteristic peaks should be presented in the characteristic spectrum of the test sample, and the peak corresponding to the sesamin reference substance was the S peak.

[0151] Example 2 Verification results of 18 batches of stir-fried black sesame medicinal materials

[0152] The characteristic spectrum of 18 batches of samples of this product was determined by the proposed method, and the relative retention time was calculated. As shown in Figure 13 Table 11.

[0153] Table 11 Relative retention time of 18 batches of stir-fried black sesame medicinal materials

[0154]

[0155]

[0156] Example 3 Verification results of 18 batches of stir-fried black sesame decoction pieces

[0157] The characteristic spectrum of 18 batches of samples of this product was determined by the proposed method, and the relative retention time was calculated. As shown in Figure 14 Table 12.

[0158] Table 12 Relative retention time of 18 batches of stir-fried black sesame decoction pieces

[0159]

[0160]

[0161] Example 4 Verification results of 18 batches of standard decoctions of stir-fried black sesame

[0162] The characteristic fingerprints of 18 batches of this product were determined by the proposed method, and the relative retention times were calculated. As Figure 15 shown in Table 13.

[0163] Table 13 Relative Retention Times of 18 Batches of Stir-Fried Black Sesame Standard Decoctions

[0164]

[0165] Example 6 Formula Granules:

[0166] Chromatographic conditions and system suitability test Use octadecylsilane chemically bonded silica gel as the filler (column length is 250 mm, inner diameter is 4.6 mm, particle size is 5 μm); use acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, and perform gradient elution according to the regulations in Table a above; the flow rate is 1.0 ml per minute; the column temperature is 35 °C; the detection wavelength is 210 nm. The number of theoretical plates calculated based on the sesamin peak should be not less than 5000.

[0167] Preparation of reference substance solution Weigh 1 g of black sesame reference medicinal material, add 50 ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 50 ml of 70% methanol, sonicate (power 600 W, frequency 40 kHz) for 30 minutes, cool to room temperature, shake well, filter, and take the subsequent filtrate as the reference medicinal material reference substance solution. Separately, weigh an appropriate amount of sesamin reference substance accurately, dissolve it in methanol to prepare a solution containing 25 μg per 1 ml as the reference substance solution of the reference substance.

[0168] Preparation of test solution for formula granules Take an appropriate amount of this product, grind it finely, take about 0.1 g, place it in a stoppered conical flask, accurately add 50 ml of 70% methanol, sonicate (power 600 W, frequency 40 kHz) for 30 minutes, cool to room temperature, shake well, filter, and take the subsequent filtrate, that is obtained.

[0169] Determination method for formula granules Accurately pipette 10 μl each of the reference substance solution and the test solution, inject them into the liquid chromatograph, and perform the determination to obtain.

[0170] Six characteristic peaks should appear in the chromatogram of the test sample, and their retention times should correspond to those of the six characteristic peaks in the chromatogram of the reference medicinal material reference substance. The peak corresponding to the sesamin reference substance peak of the reference substance is the S peak. Calculate the relative retention times of each characteristic peak and the S peak, and the relative retention times should be within the range of ±10% of the specified values. The specified values are: 0.09 (peak 1), 0.23 (peak 2), 0.29 (peak 3), 0.51 (peak 4), 1.04 (peak 6).

[0171] Three batches of stir-fried black sesame formula granules were synthesized using the Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines (2012 Edition) to establish a reference chromatogram for the characteristic fingerprints of stir-fried black sesame formula granules. AsFigure 16 As shown, among which, peak 5 (S): sesamin; chromatographic column: HSS T3, 4.6 mm × 250 mm, 5 μm.

[0172] Example 7 Medicinal materials:

[0173] Chromatographic conditions and system suitability test Use octadecylsilane chemically bonded silica gel as the filler (column length is 250 mm, inner diameter is 4.6 mm, particle size is 5 μm); use acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, and perform gradient elution according to the regulations in Table a above; the flow rate is 1.0 ml per minute; the column temperature is 35 °C; the detection wavelength is 210 nm. The number of theoretical plates calculated based on the sesamin peak should be not less than 5000.

[0174] Preparation of reference substance solution Weigh accurately 1 g of the black sesame reference medicinal material, add 50 ml of water, decoct for 30 minutes, filter, evaporate to dryness, dissolve the residue in 50 ml of 70% methanol, perform ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, cool to room temperature, shake well, filter, and take the subsequent filtrate as the reference medicinal material reference substance solution. Separately weigh an appropriate amount of sesamin reference substance accurately, dissolve it in methanol to prepare a solution containing 25 μg per 1 ml as the reference substance reference substance solution.

[0175] Preparation of test solution Take an appropriate amount of stir-fried black sesame medicinal material (passing through No. 2 sieve), weigh about 1 g, add 50 ml of water, decoct for 30 minutes, filter, evaporate to dryness, dissolve the residue in 50 ml of 70% methanol, perform ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, cool to room temperature, shake well, filter, and take the subsequent filtrate, that is obtained.

[0176] Determination method Accurately pipette 10 μl each of the reference substance solution and the test solution, inject them into the liquid chromatograph, and perform the determination, then it can be obtained.

[0177] Six characteristic peaks should appear in the characteristic chromatogram of the test solution, and should correspond to the retention times of the six characteristic peaks in the reference chromatogram of the reference medicinal material. Among them, the peak corresponding to the sesamin reference substance is the S peak. Calculate the relative retention times of each characteristic peak to the S peak, and the relative retention times should be within ±10% of the specified values. The specified values are: 0.10 (peak 1), 0.23 (peak 2), 0.28 (peak 3), 0.51 (peak 4), 1.04 (peak 6).

[0178] Using the Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints (2012 Edition), 19 batches of stir-fried black sesame medicinal materials were synthesized to establish a reference chromatogram for the characteristic chromatogram of stir-fried black sesame medicinal materials. As Figure 17 shown, among which, peak 5 (S): sesamin, the chromatographic column used: HSS T3, 4.6 mm × 250 mm, 5 μm.

[0179] Example 8 Sliced Chinese Herbal Medicine:

[0180] Chromatographic Conditions and System Suitability Test Use octadecylsilane chemically bonded silica gel as the filler (column length is 250 mm, inner diameter is 4.6 mm, particle size is 5 μm); use acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, and perform gradient elution according to the regulations in Table a above; the flow rate is 1.0 ml per minute; the column temperature is 35 °C; the detection wavelength is 210 nm. The number of theoretical plates calculated based on the sesamin peak should be not less than 5000.

[0181] Preparation of Reference Solution Take 1 g of black sesame reference medicinal material, add 50 ml of water, decoct for 30 minutes, filter, evaporate to dryness, add 50 ml of 70% methanol to the residue, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the subsequent filtrate as the reference medicinal material reference solution.

[0182] Separately take an appropriate amount of sesamin reference substance, accurately weigh it, and make a solution containing 25 μg per 1 ml with methanol as the reference substance reference solution.

[0183] Preparation of Test Solution Take an appropriate amount of stir-fried black sesame sliced Chinese herbal medicine (sieved through No. 2 sieve), take about 1 g, add 50 ml of water, decoct for 30 minutes, filter, evaporate to dryness, add 50 ml of 70% methanol to the residue, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the subsequent filtrate to obtain the test solution.

[0184] Determination Method Accurately pipette 10 μl each of the reference solution and the test solution, inject them into the liquid chromatograph for determination to obtain the results.

[0185] Six characteristic peaks should be presented in the characteristic chromatogram of the test sample, and they should correspond to the retention times of the six characteristic peaks in the chromatogram of the reference medicinal material reference. Among them, the peak corresponding to the sesamin reference is the S peak. Calculate the relative retention times of each characteristic peak and the S peak, and the relative retention times should be within ±10% of the specified values. The specified values are: 0.10 (peak 1), 0.23 (peak 2), 0.28 (peak 3), 0.51 (peak 4), 1.04 (peak 6).

[0186] Using the Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints (2012 Edition), 19 batches of stir-fried black sesame sliced Chinese herbal medicine were synthesized to establish a reference chromatogram for the characteristic chromatogram of stir-fried black sesame sliced Chinese herbal medicine. As Figure 18 shown, among them, peak 5 (S): sesamin, and the chromatographic column used: HSS T3, 4.6 mm × 250 mm, 5 μm.

[0187] Example 9 Standard Decoction:

[0188] Chromatographic conditions and system suitability test: Use octadecylsilane chemically bonded silica gel as the filler (column length is 250 mm, inner diameter is 4.6 mm, particle size is 5 μm); use acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, and perform gradient elution according to the regulations in Table a above; the flow rate is 1.0 ml per minute; the column temperature is 35 °C; the detection wavelength is 210 nm. The number of theoretical plates calculated based on the sesamin peak should be not less than 5000.

[0189] Preparation of reference solution: Take 1 g of black sesame reference crude drug, add 50 ml of water, decoct for 30 minutes, filter, evaporate to dryness, add 50 ml of 70% methanol to the residue, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, let it cool, shake well, filter, and take the subsequent filtrate as the reference crude drug reference solution. Separately weigh an appropriate amount of sesamin reference substance accurately, dissolve it in methanol to prepare a solution containing 25 μg per 1 ml as the reference substance reference solution.

[0190] Preparation of test solution: Take an appropriate amount of stir-fried black sesame standard decoction, about 0.1 g, place it in a stoppered conical flask, add 50 ml of 70% methanol, tightly stopper, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, let it cool, shake well, filter, and take the subsequent filtrate, which is the test solution.

[0191] Determination method: Accurately pipette 10 μl each of the reference solution and the test solution, inject them into the liquid chromatograph for determination, and that's it.

[0192] In the chromatogram of the test sample, 6 characteristic peaks should appear and should correspond to the retention times of the 6 characteristic peaks in the chromatogram of the reference crude drug reference solution. Among them, the peak corresponding to the sesamin reference substance is the S peak. Calculate the relative retention times of each characteristic peak and the S peak, and the relative retention times should be within the range of ±10% of the specified values. The specified values are 0.09 (peak 1), 0.23 (peak 2), 0.28 (peak 3), 0.51 (peak 4), 1.04 (peak 6).

[0193] Using the Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints (2012 Edition), 18 batches of stir-fried black sesame standard decoctions were synthesized to establish a reference chromatogram of the characteristic chromatogram of stir-fried black sesame standard decoction. As Figure 19 shown, among them, peak 5 (S): sesamin, and the chromatographic column used: HSS T3, 4.6 mm × 250 mm, 5 μm.

[0194] As can be seen from the above embodiments, the present invention provides a method for constructing characteristic chromatograms of stir-fried black sesame herbs, stir-fried black sesame decoction pieces, standard decoctions and their formula granules, comprising the following steps: A) Dissolve the stir-fried black sesame raw materials with 70% methanol, extract to obtain a test solution; B) Measure the test solution by high performance liquid chromatography to obtain the characteristic chromatograms of stir-fried black sesame herbs, stir-fried black sesame decoction pieces, standard decoctions and their formula granules; The chromatographic conditions of high performance liquid chromatography include: a C18 column, mobile phase A is acetonitrile, mobile phase B is 0.1% phosphoric acid solution, and gradient elution is used. The present invention adopts high performance liquid chromatography, selects acetonitrile-0.1% phosphoric acid solution as the mobile phase for gradient elution, uses sesamin as the reference substance, and establishes characteristic chromatograms of stir-fried black sesame herbs, stir-fried black sesame decoction pieces, standard decoctions and their formula granules with good repeatability and precision. The method is stable and reliable, and can control the quality of stir-fried black sesame herbs, stir-fried black sesame decoction pieces, standard decoctions and their formula granules.

[0195] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for constructing characteristic chromatograms of stir-fried black sesame herbs, stir-fried black sesame decoction pieces, standard decoctions and their formula granules, comprising the following steps: A) Dissolve the stir-fried black sesame raw material with 70% methanol and perform ultrasonic extraction to obtain a test solution; If the stir-fried black sesame raw material is a stir-fried black sesame standard decoction or formula granule, the mass ratio of the stir-fried black sesame raw material to the volume of 70% methanol is 0.1 g:(45 - 55) mL; If the stir-fried black sesame raw material is stir-fried black sesame herbs or decoction pieces, the mass ratio of the stir-fried black sesame raw material to the volume of 70% methanol is 1 g:(45 - 55) mL; The power of the ultrasonic extraction is 550 - 650 W, the frequency of the ultrasonic extraction is 35 - 45 kHz, and the time of the ultrasonic extraction is 28 - 32 min; B) Determine the test solution by high performance liquid chromatography to obtain characteristic chromatograms of stir-fried black sesame herbs, stir-fried black sesame decoction pieces, standard decoctions and their formula granules; The chromatographic conditions of high performance liquid chromatography include: a C18 column, mobile phase A is acetonitrile, mobile phase B is 0.1% phosphoric acid solution, and gradient elution; The specific gradient elution is as follows; 0 - 6 min, phase A: 5%, phase B: 95%; 6 - 13 min, phase A: 5 - 15%, phase B: 95 - 85%; 13 - 25 min, phase A: 15 - 25%, phase B: 85 - 75%; 25 - 35 min, phase A: 25 - 30%, phase B: 75 - 70%; 35 - 60 min, phase A: 30 - 70%, phase B: 70 - 30%; 60 - 65 min, phase A: 70%, phase B: 30%; The flow rate of the mobile phase is 1.0 ml / min; the detection wavelength is 210 nm, and the theoretical plate number calculated by the sesamin peak should be not less than 5000; The specification of the C18 column is 4.6 mm×250 mm, 5 μm; the column temperature is 35°C; It also includes the preparation of a reference substance solution: Prepare a reference substance solution of 25 μg / mL by mixing sesamin reference substance and methanol; Take the reference black sesame herbs, decoct with water, filter, evaporate to dryness, add 70% methanol to the residue, perform ultrasonic treatment, cool, shake well, filter, and take the filtrate to obtain a reference substance solution of the reference herbs; 2. The construction method according to claim 1, wherein The injection volume of the test solution is 9 - 11 μL.

3. The construction method according to claim 1, characterized in that, Use the similarity evaluation system for traditional Chinese medicine chromatographic fingerprints to evaluate the similarity of stir-fried black sesame herbs, decoction pieces, standard decoctions, and formula granules, and obtain an HPLC standard characteristic chromatogram of stir-fried black sesame herbs, decoction pieces, standard decoctions, and formula granules composed of 6 characteristic peaks, corresponding to the 6 characteristic retention times in the reference substance chromatogram of the reference herbs. Take the peak corresponding to the sesamin reference substance peak as the S peak. In the standard characteristic chromatogram, calculate the relative retention time of each characteristic peak to the S peak, and the relative retention time is within ±10% of the specified value. The specified values are: 0.09 (peak 1), 0.23 (peak 2), 0.29 (peak 3), 0.51 (peak 4), 1.04 (peak 6).

4. A method for identifying stir-fried black sesame herbs, stir-fried black sesame decoction pieces, standard decoctions and their formula granules, characterized in that, Perform detection using the method described in any one of claims 1 - 3 and analyze the detection results.

Images