Method for constructing characteristic chromatogram of bran-fried gordon euryale seed or preparation thereof and method for identifying bran-fried gordon euryale seed and crude gordon euryale seed

Abstract

The invention provides a construction method of a bran-fried gordon euryale seed or a preparation characteristic spectrum thereof. The invention also provides a method for identifying the bran-fried gordon euryale seed and the crude gordon euryale seed. The method for detecting the characteristic chromatogram of the bran-fried gordon euryale seed and the preparation thereof can be used for detecting the quality of bran-fried gordon euryale seed decoction pieces-standard decoction and formula granules and identifying crude gordon euryale seed products and bran-fried gordon euryale seed processed products, the result is reliable, and a new method is provided for controlling the quality of the bran-fried gordon euryale seeds.

Description

Technical Field

[0001] This invention relates to a method for constructing a characteristic spectrum of stir-fried fox nuts or its preparations, and a method for identifying stir-fried fox nuts and raw fox nuts. Background Technology

[0002] Euryale ferox Salisb., a plant in the Nymphaeaceae family, is the dried, mature seed kernel. It is listed in the 2020 edition of the Chinese Pharmacopoeia and has the effects of tonifying the kidneys and securing essence, strengthening the spleen and stopping diarrhea, and eliminating dampness and stopping leukorrhea. Clinically, it is mainly used for spermatorrhea, enuresis, frequent urination, chronic diarrhea due to spleen deficiency, leukorrhea, and vaginal discharge. Fried euryale ferox is a processed form of Euryale ferox. Fried euryale ferox differs from raw Euryale ferox in processing methods, medicinal properties, and suitable populations. Raw Euryale ferox is more "cooling and tonifying," suitable for those with kidney deficiency or damp-heat constitution, focusing on "strengthening the kidneys and eliminating dampness." Fried euryale ferox is more "warming and tonifying," suitable for those with chronic diarrhea due to spleen deficiency, focusing on "strengthening the spleen and astringing."

[0003] The main difficulty in distinguishing between raw and stir-fried fox nuts lies in their similarity in appearance and properties. Raw fox nuts are brownish-red or reddish-brown on the surface, with a white cross-section and a strong powdery texture. Stir-fried fox nuts are slightly yellow or pale yellow in color, with slight scorch marks on the surface after stir-frying, but the overall color change is not drastic, especially in low light or dry conditions, where the color difference from raw fox nuts is minimal, making misjudgment easy. Stir-fried fox nuts are slightly brittle, making a crisp sound when broken, and the cross-section is slightly scorched yellow. Raw fox nuts are hard, with a clean white cross-section, but both are broken granules, small in size (5–8 mm), with subtle differences in texture, making accurate differentiation by touch difficult for non-professionals. Stir-fried fox nuts should have a slight roasted wheat bran aroma, but this aroma is volatile, especially during long-term storage or when packaging is not properly sealed, causing the odor to fade and become similar to the "bland" taste of raw fox nuts, making it unreliable as a basis for identification. Especially after being prepared into formulation granules or preparations, it becomes even more difficult to distinguish between the two.

[0004] Application No. 202111348032.7, Invention Title: A Method for Identifying Gorgon Fruit and Fried Gorgon Fruit and Its Application, This invention provides a method for identifying gorgon fruit and fried gorgon fruit and its application. The identification method includes the following steps: (1) Mixing the sample to be tested with a solvent and refluxing to extract, collecting the filtrate and mixing it with an organic solution and letting it stand, then taking the supernatant to desolvent, dissolving the residue with an organic solution and filtering to obtain test solution A; (2) Performing liquid chromatography detection on test solution A obtained in step (1), comparing the detection results with the characteristic spectrum, and determining whether the sample to be tested is gorgon fruit or fried gorgon fruit.

[0005] Application No. 202310181336.1, entitled "Construction Method and Use of Characteristic Chromatography of Euryale Ferox and its Preparations," provides characteristic chromatograms of Euryale Ferox and its preparations, and a method for constructing them. In the method for constructing the characteristic chromatograms of Euryale Ferox and its preparations, octadecylsilane-bonded silica gel is used as the filler, and an aqueous solution of formic acid-acetonitrile is used as the mobile phase for gradient elution. It was found that only two elution cycles were needed to obtain nine common characteristic peaks, and these common characteristic peaks were well separated. Neither of the above two patents can distinguish between Euryale Ferox and stir-fried Euryale Ferox. Summary of the Invention

[0006] Based on this, the present invention establishes a characteristic chromatographic identification method for bran-fried foxnut and its preparations. The method has high stability and good repeatability, and can effectively and comprehensively reflect the condition of bran-fried foxnut products and their preparations, providing a basis for the subsequent formulation of quality standards.

[0007] This invention provides a method for constructing the characteristic spectrum of stir-fried fox nuts or its preparations, which includes the following steps:

[0008] a. Preparation of the test solution:

[0009] The medicinal material of Euryale ferox was dissolved and extracted using a solvent to obtain the test solution;

[0010] b. The test solution was analyzed by high performance liquid chromatography to obtain the characteristic chromatogram of Euryale ferox or its preparations;

[0011] The high-performance liquid chromatography (HPLC) conditions are as follows: a C18 column; methanol as mobile phase A; 0.2% formic acid solution as mobile phase B; gradient elution; and the elution conditions are as follows:

[0012]

[0013] The method for preparing the test solution is as follows:

[0014] Take the Euryale ferox herb, add water and decoct, filter, evaporate the filtrate to dryness, add 30% methanol to the residue, sonicate, cool, shake well, filter, and take the filtrate as the test solution.

[0015] The ultrasonic conditions are: power 600W, frequency 40kHz.

[0016] It also includes the preparation of reference solutions, wherein the references include stir-fried fox nuts as a reference herb, gallic acid as a reference standard, and 5-hydroxymethylfurfural as a reference standard;

[0017] The preparation method of the reference solution of the bran-fried foxnut is as follows: take the reference medicinal material, add water and decoct, filter, evaporate the filtrate to dryness, add 30% methanol to the residue, sonicate, cool, shake well, filter, and take the filtrate as the reference solution of the reference medicinal material.

[0018] The preparation method of the reference solution of gallic acid reference standard and 5-hydroxymethylfurfural reference standard is as follows: take appropriate amounts of gallic acid reference standard and 5-hydroxymethylfurfural reference standard, accurately weigh them, and add methanol to prepare the reference solution.

[0019] The chromatographic column is a C18 column with a length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm.

[0020] The mobile phase flow rate is 1.0 ml per minute; the column temperature is 30°C; the detection wavelength is 270 nm; and the theoretical plate number, calculated based on the gallic acid peak, should be no less than 5000.

[0021] The feature spectrum contains 6 feature peaks with relative retention times of: peak 1: 0.44, peak 2: 0.64, peak 3: 0.92, peak 4: 1.0, peak 5: 2.41, and peak 6: 2.46, with the relative retention times fluctuating within ±10%.

[0022] Peak 2 is gallic acid, and peak 4 is 5-hydroxymethylfurfural.

[0023] The bran-fried foxnut and its preparations are bran-fried foxnut slices, standard decoctions, or formula granules.

[0024] This invention also provides a method for identifying stir-fried fox nuts and raw fox nuts, which includes the following steps:

[0025] a. Take the sample to be tested;

[0026] b. Obtain the characteristic spectrum according to the method for constructing the characteristic spectrum of bran-fried foxnut or its preparation; if the relative retention times are completely consistent, it is bran-fried foxnut; if there is no peak 3, it is raw foxnut.

[0027] The method for detecting the characteristic chromatograms of bran-fried fox nuts and their preparations of the present invention can be used for the quality detection of bran-fried fox nut slices, standard decoctions, and formula granules, and can also distinguish between raw fox nuts and bran-fried fox nut products. The results are reliable, providing a new method for the quality control of bran-fried fox nuts. Attached Figure Description

[0028] Figure 1 Chromatogram of stir-fried fox nut slices - 230nm;

[0029] Figure 2 Chromatogram of stir-fried fox nut slices - 250nm;

[0030] Figure 3 Chromatogram of stir-fried fox nut slices - 270nm;

[0031] Figure 4 Chromatogram of stir-fried fox nut slices - 290nm;

[0032] Figure 5 Chromatogram of stir-fried fox nut slices - 310 nm;

[0033] Figure 6 Column temperature investigation;

[0034] Figure 7 Flow velocity study;

[0035] Figure 8 Delayed investigation;

[0036] Figure 9 Extraction solvent investigation;

[0037] Figure 10 Extraction time consideration;

[0038] Figure 11 Investigation of solvent addition amount;

[0039] Figure 12 Sampling quantity;

[0040] Figure 13 Chromatographic peak identification;

[0041] Figure 14 Different instruments;

[0042] Figure 15 Different chromatographic columns;

[0043] Figure 16 Characteristic chromatograms of 16 batches of stir-fried foxnut slices (S1-S16 are: FCQS-230701, FCQS-230802, FCQS-230803, FCQS-230804, FCQS-230805, FCQS-230806, FCQS-230807, FCQS-230808, FCQS-230809, FCQS-230810, FCQS-230811, FCQS-230812, FCQS-230813, FCQS-230814, FCQS-230815, FCQS-230816);

[0044] Figure 17 Comparative chromatogram of stir-fried foxnut slices (peak 2(S): gallic acid; peak 4: 5-hydroxymethylfurfural);

[0045] Figure 18Atlas of Euryale ferox medicinal materials (S1-S16 are: QS-YC-01, QS-YC-02, QS-YC-03, QS-YC-04, QS-YC-05, QS-YC-06, QS-YC-07, QS-YC-08, QS-YC-09, QS-YC-10, QS-YC-11, QS-YC-12, QS-YC-13, QS-YC-14, QS-YC-15, QS-YC-16 respectively);

[0046] Figure 19 Characteristic chromatograms of standard decoction of stir-fried fox nuts (S1-S16 are: FCQS-BT-01, FCQS-BT-02, FCQS-BT-03, FCQS-BT-04, FCQS-BT-05, FCQS-BT-06, FCQS-BT-07, FCQS-BT-08, FCQS-BT-09, FCQS-BT-10, FCQS-BT-11, FCQS-BT-12, FCQS-BT-13, FCQS-BT-14, FCQS-BT-15, FCQS-BT-16);

[0047] Figure 20 Characteristic spectrum of stir-fried fox nuts (S1-S3 are FCQS-KL-01, FCQS-KL-02, and FCQS-KL-03 respectively);

[0048] Figure 21 Compare with the characteristic chromatogram (peak 2(S): gallic acid; peak 3: 5-hydroxymethylfurfural);

[0049] Figure 22 Comparison with characteristic chromatograms (peak 2(S): gallic acid; peak 3: 5-hydroxymethylfurfural; column: DiamonsilPlus, 4.6 mm × 250 mm, 5 μm);

[0050] Figure 23 Compare with the characteristic chromatogram (peak 2(S): gallic acid; peak 4: 5-hydroxymethylfurfural);

[0051] Figure 24 Characteristic spectrum of stir-fried foxnut formula granules (peak 2(S): gallic acid; peak 4: 5-hydroxymethylfurfural);

[0052] Figure 25 Comparison chart of Euryale ferox medicinal material, Euryale ferox slices stir-fried with wheat bran, Euryale ferox decoction stir-fried with wheat bran, and Euryale ferox granule formula. Detailed Implementation

[0053] Example 1: Detection of the characteristic chromatograms of stir-fried foxnut slices

[0054] 1. Instruments and Materials

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

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

[0057] Ultrapure water system: Cellular type 1810A (Shanghai Moler Scientific Instruments Co., Ltd.);

[0058] Ultrasonic cleaner: KQ-600DB model (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0059] Chromatographic columns: Diamonsil Plus 5μm 250×4.6mm Column; Diamonsil ODS 4.6×250mm 5μm; Agilent 5TC C18 4.6×250mm 5μm;

[0060] Methanol and phosphoric acid were of chromatographic grade, water was ultrapure water, and all other reagents were of analytical grade.

[0061] Gallic acid (China National Institutes for Food and Drug Control, batch number: 110831-201906, content: 91.5%);

[0062] 5-Hydroxymethylfurfural reference standard (China National Institutes for Food and Drug Control, batch number: 111626-202316, purity: 98.4%);

[0063] Reference material for Euryale ferox: (China National Institutes for Food and Drug Control, batch number: 121421-202105);

[0064] Gorgon medicinal materials: QS-YC-01, QS-YC-02, QS-YC-03, QS-YC-04, QS-YC-05, QS-YC-06, QS-YC-07, QS-YC- 08. QS-YC-09, QS-YC-10, QS-YC-11, QS-YC-12, QS-YC-13, QS-YC-14, QS-YC-15, QS-YC-16;

[0065] Batch number of braised gorgon fruit drink: FCQS-YP-01, FCQS-YP-02, FCQS-YP-03, FCQS-YP-04, FCQS-YP-05, FCQS-YP-06, FCQS-YP-07, FCQS-Y P-08, FCQS-YP-09, FCQS-YP-10, FCQS-YP-11, FCQS-YP-12, FCQS-YP-13, FCQS-YP-14, FCQS-YP-15, FCQS-YP-16;

[0066] Stir-fried glutinous rice bran soup batch number: FCQS-BT-01, FCQS-BT-02, FCQS-BT-03, FCQS-BT-04, FCQS-BT-05, FCQS-BT-06, FCQS-BT-07, FCQS-B T-08, FCQS-BT-09, FCQS-BT-10, FCQS-BT-11, FCQS-BT-12, FCQS-BT-13, FCQS-BT-14, FCQS-BT-15, FCQS-BT-16;

[0067] Batch numbers for stir-fried fox nuts: FCQS-KL-01, FCQS-KL-02, FCQS-KL-03.

[0068] 2 Feature Map Detection Method

[0069] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); methanol as mobile phase A and 0.2% formic acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 1.0 mL / min; column temperature 30 °C; detection wavelength 270 nm. The theoretical plate number, calculated based on the gallic acid peak, should be no less than 5000.

[0070]

[0071] Preparation of the reference solution: Take 1g of Euryale ferox reference material, add 25ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 25ml of 30% methanol to the residue, sonicate (power 600W, frequency 40kHz) for 20 minutes, cool, shake well, filter, and take the filtrate as the reference solution. Separately, take the reference solution from the [Content Determination] section as the reference solution.

[0072] Preparation of the test solution: Take 1g of stir-fried Euryale ferox granules (passed through a No. 2 sieve) and prepare the test solution together with the reference solution of the control medicinal material.

[0073] The determination method involves precisely pipetting 10 μl each of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0074] 3. Chromatographic conditions and system suitability test

[0075] 3.1 Wavelength Selection

[0076] Based on the above-specified experimental conditions, a diode array detector was used to perform a full-band scan of the test solution, and chromatograms of the test solution at wavelengths of 230 nm, 250 nm, 270 nm, 290 nm, and 310 nm were extracted. See [link to chromatogram]. Figures 1-5 .

[0077] 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.

[0078] 3.2 Column Temperature Investigation

[0079] Based on the above-specified experimental conditions, investigations were conducted at column temperatures of 25℃, 30℃, and 35℃, respectively. (See attached figures.) Figure 6 .

[0080] The results showed that at a column temperature of 30℃, the chromatogram peaks were more symmetrical, the separation was better, and the peaks were more complete. Therefore, the column temperature was tentatively set at 30℃.

[0081] 3.3 Flow velocity investigation

[0082] Based on the above-specified experimental conditions, the flow rates of 0.8 ml / min, 1.0 ml / min, and 1.2 ml / min were investigated respectively. (See attached figures.) Figure 7 .

[0083] The results showed that at a flow rate of 1.0 ml / min, the chromatogram peak shape was good and the resolution was moderate. Therefore, the flow rate was tentatively set at 1.0 ml / min.

[0084] 3.4 Delayed Examination

[0085] Based on the above-specified experimental conditions, a delay test was conducted. The results are shown below. Figure 8 .

[0086] The results showed that the sample had virtually no chromatographic peaks after 50 minutes, so the sample detection time was set at 50 minutes.

[0087] In summary, the chromatographic conditions and system suitability test for the characteristic chromatograms 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); methanol as mobile phase A, and 0.2% formic acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 1.0 mL / min; column temperature 30 °C; detection wavelength 270 nm. The theoretical plate number, calculated based on the gallic acid peak, should be no less than 5000.

[0088]

[0089] 4 Preparation Investigation of Test Samples

[0090] 4.1 Investigation of Extraction Solvents

[0091] Take 1.5 g of the powder of stir-fried Euryale ferox seeds slices (sieved through No. 2 sieve) (batch number: FCQS - 230701), place it in a stoppered conical flask, add 25 ml of water, decoct for 30 minutes, cool, filter, evaporate the filtrate to dryness, and add 25 ml of water, 10% methanol, 30% methanol, 50% methanol, 70% methanol, methanol, and 50% ethanol respectively for investigation. Carry out ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the subsequent filtrate to obtain the sample. See Figure 9 .

[0092] The results show that when the extraction solvent is 30% methanol, the peak shapes of each characteristic peak are good and the resolution is moderate. The extraction solvent is tentatively determined to be 30% methanol.

[0093] 4.2 Investigation of Extraction Time

[0094] Take 1.5 g of the powder of stir-fried Euryale ferox seeds slices (sieved through No. 2 sieve) (batch number: FCQS - 230701), place it in a stoppered conical flask, add 25 ml of water, decoct for 30 minutes, cool, filter, evaporate the filtrate to dryness, add 25 ml of 30% methanol, and carry out ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes for investigation; take another 1.5 g of stir-fried Euryale ferox seeds slices, add 25 ml of 30% methanol, carry out ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes for investigation, cool, shake well, filter, and take the subsequent filtrate to obtain the sample. The results are shown in Figure 10 .

[0095] The results show that after decoction and then ultrasonic treatment, the peak shapes and resolution of the chromatogram are better and are similar to the characteristic peaks of the granules. Therefore, the extraction method is determined to be ultrasonic treatment after decoction.

[0096] 4.3 Investigation of Extraction Time

[0097] Take 1.5 g of the powder of stir-fried Euryale ferox seeds slices (sieved through No. 2 sieve) (batch number: FCQS - 230701), place it in a stoppered conical flask, add 25 ml of water, decoct for 30 minutes, cool, filter, evaporate the filtrate to dryness, add 25 ml of 30% methanol, and carry out ultrasonic treatment (power 600 W, frequency 40 kHz) for 20 minutes, 30 minutes, and 40 minutes respectively, cool, shake well, filter, and take the subsequent filtrate to obtain the sample. See Figure 11 .

[0098] The results show that when the extraction time is 20 minutes, complete extraction can be achieved. Therefore, the extraction time is selected as 20 minutes.

[0099] 4.4 Sampling Quantity Examination

[0100] Take 0.5g, 1.0g, and 1.5g of stir-fried foxnut slices (passed through a No. 2 sieve) (batch number: FCQS-230701) respectively, place them in a stoppered conical flask, add 25ml of water, decoct for 30 minutes, cool, filter, evaporate the filtrate to dryness, add 25ml of 30% methanol, sonicate (600W power, 40kHz frequency) for 20 minutes, cool, shake well, filter, and collect the filtrate to obtain the final product. See [link to product description]. Figure 12 .

[0101] The results showed that the peak shape was better when the sample size was 1.0g, so the sample size of the test sample was determined to be 1.0g.

[0102] 4.5 Determine the method for preparing the test sample

[0103] The preparation method of the test solution for the chromatogram is as follows: Take 1.0 g of stir-fried Euryale ferox powder (passed through a No. 2 sieve), place it in a stoppered conical flask, add 25 ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 25 ml of 30% methanol, sonicate (power 600 W, frequency 40 kHz) for 20 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0104] 5. Methodological Investigation

[0105] 5.1 Chromatographic Peak Identification

[0106] Preparation of the test solution: Prepare the test solution of stir-fried foxnut slices according to the experimental conditions proposed above.

[0107] Preparation of reference solution: Take 1g of Euryale ferox reference material, add 25ml of water, decoct for 30 minutes, filter, evaporate to dryness, add 25ml of 30% methanol to the residue, sonicate (600W power, 40kHz frequency) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution. Separately, take appropriate amounts of gallic acid reference standard and 5-hydroxymethylfurfural reference standard, accurately weigh them, add methanol to prepare a solution containing 50μg per ml, as the reference solution.

[0108] Preparation of negative control solution: Prepare negative control solution of stir-fried foxnut slices without bran according to the experimental conditions proposed above.

[0109] The characteristic spectral peaks of *Euphorbia pekinensis* were located. (See...) Figure 13 .

[0110] As shown in the figure, peak 2 is gallic acid and peak 4 is 5-hydroxymethylfurfural. In the following methodological investigation, six characteristic peaks in the sample were examined, and no interference was observed in the negative solution, indicating good specificity of the method.

[0111] 5.2 Precision test

[0112] Take the test solution of stir-fried foxnut slices (batch number: FCQS-230701), inject 10 μl of the sample six times consecutively according to the proposed experimental method, and calculate the retention time of each characteristic peak. See Table 1.

[0113] Table 1 Precision test - retention time

[0114]

[0115] The results show that the retention time RSD of each characteristic peak is 0.02%-0.07%, indicating that the method has good precision.

[0116] 5.3 Repeatability Test

[0117] Take 6 portions of the test solution of stir-fried foxnut slices (batch number: FCQS-230701) and prepare and determine it according to the proposed experimental method. See Table 2.

[0118] Table 2 Repeatability Tests - Relative Retention Time Ratios

[0119]

[0120]

[0121] The results showed that the relative retention time RSD of the six samples was 0.02%–0.07%, indicating that the method had good reproducibility.

[0122] 5.4 Intermediate Precision Examination

[0123] Based on the above-planned experimental conditions, three portions of stir-fried foxnut slices (batch number: FCQS-230701) were weighed and used to prepare test solutions. These solutions were then analyzed using Agilent 1260, Thermo Fisher Scientific, and Waters e2695 high-performance liquid chromatographs, respectively. See [link / reference]. Figure 14 Table 3.

[0124] Table 3 Intermediate Precision-Relative Retention Time Ratio

[0125]

[0126] The results showed that when the test samples were detected using the above three instruments, the RSD range of the relative retention times of each characteristic peak was 0.48%-0.70%. The instruments demonstrated good durability.

[0127] 5.5 Investigations by different personnel and at different times

[0128] Based on the experimental conditions outlined above, two samples of stir-fried foxnut slices (batch number: FCQS-230701) were weighed by different personnel (A and B) at different times (T1 and T2) to prepare test samples for determination. See Table 4.

[0129] Table 4. Personnel and Time Assessment - Relative Retention Time

[0130]

[0131] The results showed that when different personnel measured the same sample at different times, the RSD of the relative retention time of each characteristic peak was 0.02% to 0.04%, indicating good intermediate precision of the method.

[0132] 5.6 Durability Test

[0133] Based on the above-established experimental conditions, the chromatographic columns were tested as follows: Diamonsil Plus 5μm 250×4.6mm Column; Diamonsil ODS 4.6×250mm 5μm Column; and Agilent 5TC C18 4.6×250mm 5μm Column. Results are shown below. Figure 15 Table 5.

[0134] Table 5 Durability Test - Relative Retention Time Ratio

[0135]

[0136] The results showed that the chromatographic column exhibited good durability when used to detect samples.

[0137] 5.7 Stability Test

[0138] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 2h, 4h, 8h, 16h, and 24h. See Table 6.

[0139] Table 6 Stability Study - Relative Retention Time

[0140]

[0141] The results showed that the RSD of the retention time of the characteristic peak was between 0.02% and 0.26%, and the sample solution was stable within 24 hours.

[0142] In summary, the RSD of the relative retention times of each characteristic peak meets the requirements in all the above tests, indicating that the method is effective. The aforementioned six characteristic peaks will be included in subsequent investigations.

[0143] 6. Determination of characteristic peaks and establishment of reference spectra

[0144] Using this method, characteristic spectral analysis was performed on 16 batches of samples, and the relative retention time and relative peak area ratio were calculated. See Figure 16 Table 7.

[0145] Table 7. Relative retention times of 16 batches of stir-fried fox nut slices

[0146]

[0147]

[0148] Based on the principles of stable relative retention times, detectability across all batches of samples, and relatively high peak values, six peaks with good repeatability were selected as characteristic peaks. The relative retention times (RSDs) of the six characteristic peaks from 16 batches of stir-fried foxnut slices were all less than 3.0%.

[0149] The final specification stipulates that the chromatogram of the test sample should show 6 characteristic peaks. Except for peak 3, the retention times should correspond to the 5 characteristic peaks in the chromatogram of the reference medicinal material. Peak 4 should correspond to the retention time of the corresponding reference standard peak. The peak corresponding to the 5-hydroxymethylfurfural reference standard is designated as the S peak. The relative retention times of each characteristic peak and the S peak should be calculated and should be within ±10% of the specified values. The specified values ​​are: 0.44 (peak 1), 0.64 (peak 2), 0.92 (peak 3), 2.41 (peak 5), and 2.46 (peak 6).

[0150] The chromatographic fingerprint similarity evaluation system for traditional Chinese medicine (2012 version) was used to synthesize the characteristic chromatograms of 16 batches of stir-fried foxnut slices, and a reference characteristic chromatogram for stir-fried foxnut slices was established. See [link / reference]. Figure 17 .

[0151] Verification results of 16 batches of Euryale ferox medicinal materials

[0152] The proposed method was used to determine the characteristic spectra of 16 batches of this product, and the relative retention times were calculated. For example... Figure 18 As shown in Table 8.

[0153] Table 8. Relative retention time of characteristic chromatograms of Euryale ferox medicinal materials

[0154]

[0155]

[0156] Verification results of 16 batches of bran-fried foxnut standard soup

[0157] The proposed method was used to determine the characteristic spectra of 16 batches of this product, and the relative retention times were calculated. For example... Figure 19 As shown in Table 9.

[0158] Table 9. Relative retention time of 16 batches of standard decoction of stir-fried fox nuts

[0159]

[0160]

[0161] Verification Results of 3 Batches of Stir-Fried Gorgon Fruit Granules

[0162] The characteristic fingerprints of 3 batches of samples of this product were determined by the proposed method, and the relative retention times were calculated. As Figure 20 shown in Table 10

[0163] Table 10 Relative Retention Times of 3 Batches of Stir-Fried Gorgon Fruit Formula Granules

[0164]

[0165] Example 2 Characteristic Fingerprint of Stir-Fried Gorgon Fruit Slices of the Present Invention

[0166] Chromatographic Conditions and System Suitability Test Octadecylsilyl silica gel was used as the filler (column length was 250 mm, inner diameter was 4.6 mm, particle size was 5 μm); methanol was used as mobile phase A, and 0.2% formic acid solution was used as mobile phase B, and gradient elution was carried out according to the regulations in the following table; the flow rate was 1.0 ml per minute; the column temperature was 30 °C; the detection wavelength was 270 nm. The number of theoretical plates calculated by the gallic acid peak should not be less than 5000

[0167]

[0168] Preparation of Reference Solution Weigh 1 g of Gorgon fruit reference medicinal material, add 25 ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 25 ml of 30% methanol, ultrasonically treat (power 600 W, frequency 40 kHz) for 20 minutes, cool, shake well, filter, and take the subsequent filtrate as the reference medicinal material reference solution. Separately weigh an appropriate amount of gallic acid reference substance, accurately weigh it, dissolve it in 50% methanol to prepare a solution containing 25 μg per 1 ml, and that is the reference substance reference solution

[0169] Preparation of Test Solution Weigh 1 g of stir-fried Gorgon fruit powder (sieved through No. 2 sieve), and prepare the test solution in the same way as the reference medicinal material reference solution

[0170] Determination Method: Accurately pipette 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and determine to obtain the results

[0171] The chromatogram of the test sample should show 6 characteristic peaks. Except for peak 3, the retention times should correspond to the 5 characteristic peaks in the chromatogram of the reference medicinal material. Peak 4 should correspond to the retention time of the corresponding reference standard peak. The peak corresponding to the 5-hydroxymethylfurfural reference is the S peak. Calculate the relative retention times of each characteristic peak and the S peak; the relative retention times should be within ±10% of the specified values. Specified values ​​are: 0.44 (peak 1), 0.64 (peak 2), 0.92 (peak 3), 2.41 (peak 5), and 2.46 (peak 6). (See...) Figure 21 )

[0172] Example 3: Characteristic Atlas of Euryale Ferox (Gorgon Fruit)

[0173] Chromatographic conditions and system suitability test

[0174] The column was packed with octadecylsilane-bonded silica gel (250 mm column length, 4.6 mm inner diameter, 5 μm particle size); methanol was used as mobile phase A, and 0.2% formic acid solution was used as mobile phase B, with gradient elution performed according to the specifications in the table below; the flow rate was 1.0 mL / min; the column temperature was 30 °C; and the detection wavelength was 270 nm. The theoretical plate number, calculated based on the gallic acid peak, should be no less than 5000.

[0175]

[0176] Preparation of the reference solution: Take 1g of Euryale ferox reference material, add 25ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 25ml of 30% methanol to the residue, sonicate (power 600W, frequency 40kHz) for 20 minutes, cool, shake well, filter, and take the filtrate as the reference solution. Separately, take an appropriate amount of gallic acid reference standard, accurately weigh it, add 50% methanol to prepare a solution containing 25μg per ml, which is the reference solution.

[0177] Preparation of the test solution: Take 1.0 g of Euryale ferox (passed through a No. 2 sieve) and prepare the test solution together with the reference solution of the control medicinal material.

[0178] The determination method involves precisely pipetting 10 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0179] The chromatogram of the test sample should show five characteristic peaks, and the retention times should correspond to the five characteristic peaks in the chromatogram of the reference medicinal material. Peak 3 should correspond to the retention time of the reference standard peak. The peak corresponding to the gallic acid reference standard peak is the S peak. Calculate the relative retention times of each characteristic peak and the S peak; the relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.44 (peak 1), 0.64 (peak 2), 2.41 (peak 4), and 2.46 (peak 5). (See...) Figure 22 )

[0180] Example 4: Characteristic chromatogram of the standard decoction of stir-fried fox nuts of the present invention

[0181] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); methanol as mobile phase A and 0.2% formic acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 1.0 mL / min; column temperature 30 °C; detection wavelength 270 nm. The theoretical plate number, calculated based on the gallic acid peak, should be no less than 5000.

[0182]

[0183] Preparation of the reference solution: Take 1g of Euryale ferox reference material, add 25ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 25ml of 30% methanol to the residue, sonicate (power 600W, frequency 40kHz) for 20 minutes, cool, shake well, filter, and take the filtrate as the reference solution. Separately, take an appropriate amount of gallic acid reference standard, accurately weigh it, add 50% methanol to prepare a solution containing 25μg per ml, which is the reference solution.

[0184] Preparation of the test solution: Take 0.2g of wheat bran-fried foxnut powder, place it in a stoppered conical flask, add 25ml of 30% methanol, seal tightly, sonicate (power 600W, frequency 40kHz) for 20 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0185] The determination method involves precisely pipetting 10 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0186] The chromatogram of the test sample should show 6 characteristic peaks. Except for peak 3, the retention times should correspond to the 5 characteristic peaks in the chromatogram of the reference medicinal material. Peak 4 should correspond to the retention time of the corresponding reference standard peak. The peak corresponding to the 5-hydroxymethylfurfural reference is the S peak. Calculate the relative retention times of each characteristic peak and the S peak; the relative retention times should be within ±10% of the specified values. Specified values ​​are: 0.44 (peak 1), 0.64 (peak 2), 0.92 (peak 3), 2.41 (peak 5), and 2.46 (peak 6). (See...) Figure 23 )

[0187] Example 5: Characteristic chromatogram of the bran-fried foxnut formula of the present invention, and identification method of raw foxnut.

[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 methanol as mobile phase A and 0.2% formic acid solution as mobile phase B, and perform gradient elution according to the regulations in the following table; the flow rate is 1.0 ml per minute; the column temperature is 30 °C; the detection wavelength is 270 nm. The number of theoretical plates calculated based on the gallic acid peak should be not less than 5000.

[0189]

[0190] Preparation of reference substance solution: Take 1 g of the control crude drug of Euryale ferox Salisb., add 25 ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 25 ml of 30% methanol, perform ultrasonic treatment (power 600 W, frequency 40 kHz) for 20 minutes, cool, shake well, filter, and take the subsequent filtrate as the reference substance solution of the control crude drug. Separately weigh an appropriate amount of gallic acid reference substance accurately, dissolve it in 50% methanol to prepare a solution containing 25 μg per 1 ml, and that is the reference substance solution of the reference substance.

[0191] Preparation of test solution: Take an appropriate amount of stir-fried Euryale ferox Salisb. granules, grind them finely, take 0.2 g, place it in a stoppered conical flask, add 25 ml of 30% methanol, stopper tightly, perform ultrasonic treatment (power 600 W, frequency 40 kHz) for 20 minutes, cool, shake well, filter, and take the subsequent filtrate, and that is it.

[0192] Assay method: Accurately pipette 10 μl each of the reference substance solution and the test solution, inject them into the liquid chromatograph, and determine, and that is it.

[0193] Six characteristic peaks should appear in the chromatogram of the test sample. Except for peak 3, the retention times of the other 5 characteristic peaks should correspond to those in the reference substance chromatogram of the control crude drug. Among them, the retention time of peak 4 should correspond to that of the corresponding reference substance peak of the reference substance. The peak corresponding to the 5-hydroxymethylfurfural 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 ±10% of the specified values. The specified values are: 0.44 (peak 1), 0.64 (peak 2), 0.92 (peak 3), 2.41 (peak 5), 2.46 (peak 6). (See Figure 24 )

[0194] Using the Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints (2012 Edition), synthesize the characteristic fingerprints of 16 batches of Euryale ferox Salisb. crude drugs into a control fingerprint of Euryale ferox Salisb. crude drugs, synthesize the characteristic fingerprints of 16 batches of stir-fried Euryale ferox Salisb. cut pieces into a control fingerprint of cut pieces, synthesize the characteristic fingerprints of 16 batches of stir-fried Euryale ferox Salisb. standard decoctions into a control fingerprint of standard decoctions, and synthesize the characteristic fingerprints of 3 batches of stir-fried Euryale ferox Salisb. formula granules into a control fingerprint of formula granules. Compare the above control fingerprints of Euryale ferox Salisb. crude drugs, stir-fried Euryale ferox Salisb. cut pieces, stir-fried Euryale ferox Salisb. standard decoctions, and stir-fried Euryale ferox Salisb. formula granules, see Figure 25 .

[0195] Depend on Figure 25 It can be seen that the presence or absence of peak 3 can distinguish between raw Euryale ferox and processed Euryale ferox products and their preparations. This method can identify raw Euryale ferox products and processed Euryale ferox products and their preparations.

Claims

1. A method for constructing a characteristic spectrum of stir-fried fox nuts or its formulations, characterized in that: It includes the following steps: a. Preparation of the test solution: The medicinal material of Euryale ferox was dissolved and extracted using a solvent to obtain the test solution; b. The test solution was analyzed by high performance liquid chromatography to obtain the characteristic chromatogram of Euryale ferox or its preparations; The high-performance liquid chromatography (HPLC) conditions are as follows: a C18 column; methanol as mobile phase A; 0.2% formic acid solution as mobile phase B; gradient elution; and the elution conditions are as follows: 。 2. The method for constructing the characteristic spectrum of stir-fried fox nuts or their preparations according to claim 1, characterized in that: The method for preparing the test solution is as follows: Take the Euryale ferox herb, add water and decoct, filter, evaporate the filtrate to dryness, add 30% methanol to the residue, sonicate, cool, shake well, filter, and take the filtrate as the test solution.

3. The method for constructing the characteristic spectrum of stir-fried fox nuts or their preparations according to claim 2, characterized in that: The ultrasonic conditions were: power 600W, frequency 40kHz.

4. The method for constructing the characteristic spectrum of stir-fried fox nuts or their preparations according to claim 1, characterized in that: It also includes the preparation of reference solutions, wherein the references include stir-fried fox nuts as a reference herb, gallic acid as a reference standard, and 5-hydroxymethylfurfural as a reference standard; The preparation method of the reference solution of the bran-fried foxnut is as follows: take the reference medicinal material, add water and decoct, filter, evaporate the filtrate to dryness, add 30% methanol to the residue, sonicate, cool, shake well, filter, and take the filtrate as the reference solution of the reference medicinal material. The preparation method of the reference solution of gallic acid reference standard and 5-hydroxymethylfurfural reference standard is as follows: take appropriate amounts of gallic acid reference standard and 5-hydroxymethylfurfural reference standard, accurately weigh them, and add methanol to prepare the reference solution.

5. The method for constructing the characteristic spectrum of Euryale ferox or its preparations according to claim 1, characterized in that: The chromatographic column is a C18 column with a length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm.

6. The method for constructing the characteristic spectrum of Euryale ferox or its preparations according to claim 1, characterized in that: The mobile phase flow rate is 1.0 ml per minute; the column temperature is 30°C; the detection wavelength is 270 nm; and the theoretical plate number, calculated based on the gallic acid peak, should be no less than 5000.

7. The method for constructing the characteristic spectrum of stir-fried foxnut or its preparation according to any one of claims 1-6, characterized in that: The characteristic spectrum contains 6 characteristic peaks with relative retention times of: peak 1: 0.44, peak 2: 0.64, peak 3: 0.92, peak 4: 1.0, peak 5: 2.41, and peak 6: 2.46, with the relative retention times fluctuating within ±10%.

8. The method for constructing the characteristic spectrum of stir-fried foxnut or its preparation according to claim 7, characterized in that: Peak 2 is gallic acid, and peak 4 is 5-hydroxymethylfurfural.

9. The method for constructing the characteristic spectrum of stir-fried foxnut or its preparation according to any one of claims 1-8, characterized in that: The bran-fried foxnut and its preparations are bran-fried foxnut slices, standard decoctions, or formula granules.

10. A method for distinguishing between stir-fried fox nuts and raw fox nuts, characterized in that: It includes the following steps: a. Take the sample to be tested; b. The characteristic spectrum is obtained by the method of constructing the characteristic spectrum of stir-fried foxnut or its preparation according to any one of claims 1-9; if the relative retention times are completely consistent, it is stir-fried foxnut; if there is no peak 3, it is raw foxnut.

Images