Construction methods and applications of characteristic chromatograms of Euryale ferox and its preparations

Abstract

The invention belongs to the technical field of traditional Chinese medicine detection, and specifically provides a characteristic spectrum of a gorgon fruit and a preparation thereof and a construction method thereof. In the construction method of the characteristic spectrum of the gorgon fruit and the preparation thereof, octadecylsilane bonded silica gel is used as a filler, an aqueous solution of formic acid-acetonitrile is used as a mobile phase, and gradient elution is performed. It is found that 9 common characteristic peaks can be obtained by using only 2 elution procedures, and good separation of these common characteristic peaks is achieved. The elution procedure is simple, the baseline of the obtained characteristic spectrum is stable, the noise interference is small, the characteristic peak shape is good, and the separation degree is high. The method can distinguish the gorgon fruit from its confusing products, provide a basis for the quality detection and control of the gorgon fruit and the preparation thereof, and achieve the characterization of the overall components of the gorgon fruit and the preparation thereof, with stable reproducibility.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a method for constructing a characteristic spectrum of gorgon fruit and a preparation thereof and uses thereof. Background Art

[0002] Gorgon fruit is the dried mature kernel of Euryale ferox Salisb., a plant of the Nymphaeaceae family. It is spherical, mostly broken, and the diameter of the intact one is 5 to 8 mm. The surface has a brownish red or reddish brown endocarp, one end is yellowish white, accounting for about 1 / 3 of the whole body, with a concave hilum mark, which is white after removing the endocarp. It is hard, white in cross section, and powdery. It has a slight odor and a light taste. It is distributed from Heilongjiang to Yunnan, Guangdong and other places. It has the effects of nourishing the kidney and consolidating essence, tonifying the spleen and stopping diarrhea, removing dampness and stopping leukorrhea. It is often used for spermatorrhea, enuresis, frequent urination, spleen deficiency and chronic diarrhea, leukorrhea, and leukorrhea.

[0003] The 2020 edition of the Chinese Pharmacopoeia only includes property identification and thin-layer chromatography identification. So far, there is no clear chemical composition index to evaluate Gorgon fruit medicinal materials. Simply measuring the amount of one or several effective ingredients can no longer fully reflect the quality of the medicinal materials. Chinese herbal formula granules are extracted from Chinese herbal medicine slices with water, and are produced by extraction, vacuum concentration, spray drying, granulation and other processes. Their clinical efficacy should be consistent with that of decoctions. Standard decoctions are the material benchmark for measuring whether Chinese herbal formula granules are basically consistent with decoctions. However, whether it is a standard decoction or a Chinese herbal granule, it has lost the microscopic and property identification characteristics of the original medicinal materials and decoction pieces, that is, it is impossible to inspect and identify the shape, size, color, surface, texture, etc. of the medicinal materials. Therefore, it is necessary to establish a method for rapid identification of the characteristic spectrum of Gorgon fruit and its preparations, so as to provide a basis for effective control and scientific evaluation of the quality of Gorgon fruit and its preparations. Summary of the invention

[0004] Therefore, the object of the present invention is to provide a method for constructing a characteristic spectrum of Gorgon fruit and its preparations and uses thereof. The method establishes a characteristic spectrum of the variety according to the characteristics of Gorgon fruit and its pharmaceutical preparations (standard decoctions or formula granules), realizes effective separation of each characteristic peak, increases the number of characteristic peaks, and shortens the detection and analysis time, improves the separation effect, and can quickly and effectively identify authentic Gorgon fruit pharmaceutical preparations and easily confused products, providing a scientific basis for comprehensively establishing quality control standards for Gorgon fruit pharmaceutical preparations.

[0005] The present invention provides a method for constructing a characteristic spectrum of Gorgon fruit and its preparation, comprising the following steps:

[0006] (1) Preparation of Euryale ferox test solution;

[0007] (2) A solution of a Euryale ferox test sample was detected by high performance liquid chromatography to obtain a characteristic spectrum of the test sample, using octadecylsilane bonded silica gel as a filler and an aqueous solution of formic acid-acetonitrile as a mobile phase for gradient elution. The gradient elution program included: 0→3 minutes→38 minutes, and the volume percentage of acetonitrile in the mobile phase was 1%→1%→20%-24%.

[0008] Furthermore, step (1) comprises:

[0009] 1) Take a Gorgon fruit sample, add a solvent to extract, and obtain an extract;

[0010] 2) Separate the solid and liquid of the extract and take the liquid, which is the test sample solution.

[0011] Furthermore, step (1) satisfies any one or more of the following AC:

[0012] A. In step 1), the extraction solvent of the Euryale ferox test solution is selected from a 30%-70% methanol aqueous solution, and 30-100 times the amount of the solvent is added;

[0013] B. In step 1), the extraction method is ultrasonic extraction or reflux extraction, and the extraction time is 30min-1h;

[0014] C. In step 2), the solid-liquid separation is independently selected from centrifugation or filtration.

[0015] Furthermore, the chromatographic conditions of the high performance liquid chromatography in step (2) also include: a detection wavelength of 308-312 nm, a flow rate of 0.25-0.32 ml / min, a column temperature of 23-27° C., and an injection volume of 2-5 μl;

[0016] Preferably, the volume percentage of formic acid in the aqueous solution of formic acid is 0.48-0.52%.

[0017] Furthermore, the construction method also includes the step of preparing a reference solution by using gallic acid and a solubilizing agent, and the step of detecting the reference solution by high performance liquid chromatography according to any of the construction methods described above to obtain a reference spectrum of the reference substance. Preferably, the solvent is selected from a 30%-70% methanol aqueous solution. Preferably, each 1 mL of the reference solution contains 10-100 μg of gallic acid.

[0018] Furthermore, the preparation method of the reference solution includes the following steps: the construction method also includes the steps of using the residue obtained after water extraction and drying of the Euryale ferox reference medicinal material and then obtaining a reference medicinal material reference solution according to any of the above-mentioned construction methods, and detecting the reference solution by high performance liquid chromatography according to any of the above-mentioned construction methods to obtain a reference medicinal material reference atlas.

[0019] Furthermore, the characteristic spectrum of the Gorgon fruit and its preparation has 9 characteristic peaks, peak 1 corresponds to the retention time of the gallic acid reference substance reference peak, the peak corresponding to the gallic acid reference substance reference peak is the S peak, the relative retention time of each characteristic peak and the S peak is within the range of ±10% of the specified value, and the specified value is: 1.26 (peak 2), 2.11 (peak 3), 3.21 (peak 4), 3.57 (peak 5), 4.81 (peak 6), 5.81 (peak 7), 7.41 (peak 8), 7.62 (peak 9), the relative peak area of ​​peak 7, peak 9 and S peak is within the specified value range, and the specified value is: not more than 0.68 (peak 7) and 0.45 (peak 9).

[0020] The present invention also provides the use of the method for constructing the characteristic spectrum of the Gorgon fruit and its preparation described in any one of the above items in the identification of Gorgon fruit products and counterfeits or in the quality detection of Gorgon fruit products.

[0021] The present invention also provides a method for distinguishing a Gorgon fruit product from a counterfeit product, comprising the step of comparing a characteristic spectrum of the product to be identified with a reference characteristic spectrum of the Gorgon fruit and its preparation; the characteristic spectrum of the product to be identified is obtained by using the Gorgon fruit product to be identified as a test sample according to any of the construction methods described above, and the reference characteristic spectrum of the Gorgon fruit and its preparation is selected from any one of the following (1)-(4):

[0022] (1) It has 9 common characteristic peaks, peak 1 corresponds to the retention time of the peak of the gallic acid reference substance, the peak corresponding to the peak of the gallic acid reference substance is the S peak, the relative retention time of each characteristic peak and the S peak is within the range of ±10% of the specified value, the specified values ​​of the relative retention time of peaks 2 to 9 are: 1.26, 2.11, 3.21, 3.57, 4.81, 5.81, 7.41, 7.62, respectively, the relative peak area of ​​peak 7 and S peak is not more than 0.68, preferably, the relative peak area of ​​peak 9 and S peak is not more than 0.45;

[0023] (2) using a single batch or multiple batches of Euryale ferox test samples to obtain a Euryale ferox characteristic spectrum according to any of the construction methods described above;

[0024] (3) Using multiple batches of Euryale ferox test samples and obtaining characteristic spectra according to any of the construction methods described above, a control characteristic spectrum is prepared by using the average value or median method.

[0025] The present invention also provides a quality detection method for Gorgon fruit and its preparation, characterized in that it comprises the step of comparing the characteristic spectrum of the Gorgon fruit product to be tested with the reference characteristic spectrum of the Gorgon fruit and its preparation; the characteristic spectrum of the Gorgon fruit product to be tested is obtained by using the Gorgon fruit product to be tested as a test sample according to any of the construction methods described above, and the reference characteristic spectrum of the Gorgon fruit and its preparation is selected from any one of the following (1)-(4):

[0026] (1) It has 9 common characteristic peaks, and the RSD of the retention time of each characteristic peak and the specified value of the retention time of the following characteristic peaks is less than 5.0%, and the specified values ​​of the retention time of each characteristic peak are 3.976min, 5.025min, 8.405min, 12.767min, 14.195min, 19.112min, 23.119min, 29.476min and 30.304min respectively;

[0027] (2) It has 9 common characteristic peaks, peak 1 corresponds to the retention time of the peak of the gallic acid reference substance, the peak corresponding to the peak of the gallic acid reference substance is the S peak, the relative retention time of each characteristic peak and the S peak is within the range of ±10% of the specified value, the specified values ​​of the relative retention time of peaks 2 to 9 are: 1.26, 2.11, 3.21, 3.57, 4.81, 5.81, 7.41, 7.62, respectively, the relative peak area of ​​peak 7 and S peak is not more than 0.68, preferably, the relative peak area of ​​peak 9 and S peak is not more than 0.45;

[0028] (3) using a single batch or multiple batches of Euryale ferox test samples to obtain a Euryale ferox characteristic spectrum according to any of the construction methods described above;

[0029] (4) Using multiple batches of Euryale ferox test samples and obtaining characteristic spectra according to any of the construction methods described above, a control characteristic spectrum is prepared by using the average value or median method.

[0030] The technical solution of the present invention has the following advantages:

[0031] 1. The method for constructing the characteristic spectrum of Gorgon fruit and its preparation provided by the present invention uses octadecylsilane bonded silica gel as a filler and an aqueous solution of formic acid-acetonitrile as a mobile phase. It is found that 9 common characteristic peaks can be obtained by using only 2 elution programs, and these common characteristic peaks are well separated. The obtained characteristic spectrum has a stable baseline, good characteristic peak shape, high separation, and short detection time, which provides a basis for the quality detection and control of Gorgon fruit and its preparation, realizes the characterization of the overall components of Gorgon fruit and its preparation, and has stable reproducibility.

[0032] The above-mentioned construction method is not only applicable to the identification of Gorgon fruit medicinal materials, but also can be used to identify authentic and counterfeit Gorgon fruit pharmaceutical preparations, and can more comprehensively monitor the quality of Gorgon fruit formula granules.

[0033] 2. The method for constructing the characteristic spectrum of Gorgon fruit and its preparation provided by the present invention has good peak shape and separation degree of characteristic peaks and good repeatability of characteristic peaks, and has better separation efficiency, peak capacity and sensitivity; the method has high working efficiency, and has the advantages of simplicity, stability, high precision and good repeatability. It can be applied to different types of Gorgon fruit preparations, can control the consistency of the quality of Gorgon fruit preparations, and quickly realize the quality control of different types of Gorgon fruit preparations.

[0034] The detection method of the Gorgon fruit preparation provided by the present invention can better reflect and characterize the characteristic peaks of the types and numbers of the overall material components of the chemical components in the Gorgon fruit preparation by controlling the chromatographic conditions, thereby achieving quality control of the Gorgon fruit preparation according to the relative retention time of each characteristic peak. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0036] Figure 1 This is the gallic acid reference substance spectrum obtained in Example 4;

[0037] Figure 2 This is the sample spectrum of the Euryale ferox formula granules obtained in Example 4;

[0038] Figure 3 This is the characteristic spectrum of the Euryale ferox reference medicinal material obtained in Example 4;

[0039] Figure 4 is the chromatogram of gradient 1 of Experimental Example 1;

[0040] Figure 5 is the chromatogram of gradient 2 of Experimental Example 1;

[0041] Figure 6 is the chromatogram of gradient 3 of Experimental Example 1;

[0042] Figure 7 is the chromatogram of gradient 4 of Experimental Example 1;

[0043] Figure 8 This is the chromatogram with a flow rate of 0.25 ml / min;

[0044] Fig. 9 This is the chromatogram with a flow rate of 0.30 ml / min;

[0045] Fig.10 This is the chromatogram with a flow rate of 0.32 ml / min;

[0046] Fig.11 The chromatogram was investigated for a column temperature of 23°C;

[0047] Fig.12 The chromatogram was investigated at a column temperature of 25°C;

[0048] Fig.13 The chromatogram was investigated for a column temperature of 27°C;

[0049] Fig.14 The chromatogram was investigated for 0.48% formic acid concentration;

[0050] Fig.15 The chromatogram is 0.5% formic acid concentration;

[0051] Fig.16 The chromatogram is 0.52% formic acid concentration;

[0052] Fig.17 The characteristic spectra of three batches of Gorgon Fruit formula granules;

[0053] Fig.18 The characteristic spectra of 18 batches of Gorgon Fruit standard decoction freeze-dried powder;

[0054] Fig.19 This is the reference characteristic spectrum of Gorgon Fruit Formula Granules;

[0055] Fig. 20 is the instrument precision chromatogram;

[0056] Fig.21 is a repetitive chromatogram;

[0057] Fig. 22 is the intermediate precision chromatogram;

[0058] Fig.23 is the stability chromatogram;

[0059] Fig.24 Comparison of characteristic spectra of Gorgon fruit and lotus seeds, S1: Gorgon fruit; S2-S4: lotus seeds;

[0060] Fig.25 Comparison of characteristic spectra of Gorgon fruit, white sorghum and red sorghum, S1: Gorgon fruit; S2-S4: white sorghum; S5-S7: red sorghum. DETAILED DESCRIPTION

[0061] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0062] If the specific experimental steps or conditions are not specified in the embodiments, the operations or conditions of the conventional experimental steps described in the literature in the field can be followed. If the manufacturer of the reagents or instruments used is not specified, they are all conventional reagent products that can be obtained commercially. Gorgon fruit slices are prepared using Gorgon fruit medicinal materials according to the method of the "Chinese Pharmacopoeia". % formic acid solution or % formic acid water represents the volume percentage of formic acid in the formic acid aqueous solution. Gorgon fruit slice standard decoction freeze-dried powder and Gorgon fruit formula granules are prepared according to conventional methods in the field. For example, the Gorgon fruit slice standard decoction freeze-dried powder and Gorgon fruit formula granules in the present invention are prepared by the methods of the following Examples 1 and 2, respectively.

[0063] Example 1 Gorgon Fruit Decoction Standard Decoction Freeze-dried Powder

[0064] The present embodiment provides a method for preparing a freeze-dried powder of a standard decoction of Euryale ferox slices, comprising the following steps: taking Euryale ferox medicinal materials, removing impurities and crushing according to the Chinese Pharmacopoeia, decocting in a casserole twice, adding 8 times the weight of water to soak for 30 minutes for the first time, boiling over high heat first, then decocting over low heat for 30 minutes, filtering, adding 6 times the weight of water to boil over high heat for the second time, then decocting over low heat for 20 minutes, filtering, combining the filtrate, concentrating the filtrate (60° C.) to a concentrated extract with a relative density of 1.08 to 1.12 g / ml, placing it in a freeze dryer for freeze drying, and crushing it into powder to obtain the concentrate.

[0065] Example 2 Gorgon Fruit Formula Granules

[0066] This embodiment provides a method for preparing Gorgon fruit formula granules, comprising the following steps:

[0067] Take Gorgon fruit medicinal material. According to the Chinese Pharmacopoeia, after removing impurities and crushing, heat reflux extraction twice. For the first time, add 8 times the weight of water to soak for 30 minutes, heat reflux extraction for 30 minutes, filter, and add 6 times the weight of water for the second time to extract for 20 minutes, filter, combine the filtrate, concentrate the filtrate to a relative density of 1.00 g / ml to 1.10 g / ml at 60°C, add appropriate amount of auxiliary materials, dry, add appropriate amount of auxiliary materials, granulate, and obtain.

[0068] Example 3

[0069] This embodiment provides a method for constructing a characteristic spectrum of a freeze-dried powder of a standard decoction of Euryale ferox slices, comprising the following steps:

[0070] (1) Preparation of test solution: Take about 0.5 g of the freeze-dried powder of the standard decoction of Euryale ferox prepared in Example 1, place it in a stoppered conical flask, add 20 ml of 50% methanol, and ultrasonically treat (power 250 W, frequency 40 kHz) for 45 minutes. Cool, shake well, filter, and take the filtrate to obtain the solution.

[0071] (2) Preparation of reference solution: Take 2g of Euryale ferox reference medicinal material, add 100ml of water, heat and reflux for 45 minutes, filter, evaporate the filtrate, add 20ml of 50% methanol to the residue, ultrasonically treat (power 250W, frequency 40kHz) for 45 minutes, cool, shake well, filter, and use as reference medicinal material reference solution. Take an appropriate amount of gallic acid reference substance, accurately weigh, add 50% methanol to make a solution containing 40μg of gallic acid per 1ml, and use as reference substance solution.

[0072] (3) Ultra-high performance liquid chromatography detection: Accurately pipette 3 μl of the test solution and reference solution respectively, use octadecylsilane bonded silica gel as the filler, and the chromatographic column: BEH C18 (column length 150 mm, inner diameter 2.1 mm, particle size 1.7 μm); acetonitrile as mobile phase A, 0.5% formic acid solution as mobile phase B, gradient elution as specified in the table below; flow rate 0.3 ml per minute, column temperature 25°C, detection wavelength 310 nm. The theoretical plate number calculated based on the gallic acid peak should be no less than 5000.

[0073] Table 1 Gradient elution conditions

[0074]

[0075] Example 4

[0076] This embodiment provides a method for constructing a characteristic spectrum of Gorgon fruit formula particles, comprising the following steps:

[0077] (1) Preparation of test solution: Take about 0.5 g of the Euryale ferox granules prepared in Example 2, place in a stoppered conical flask, add 20 ml of 50% methanol, ultrasonically treat (power 250 W, frequency 40 kHz) for 45 minutes, cool, shake well, filter, and take the filtrate to obtain the solution.

[0078] (2) Preparation of reference solution: the same as step (2) in Example 3.

[0079] (3) Ultra-high performance liquid chromatography detection: the same as step (3) of Example 3.

[0080] Results Figure 1-3 As shown, the characteristic spectrum of the Euryale ferox formula granules has 9 characteristic peaks, among which peak 1 corresponds to the retention time of the reference peak of the gallic acid reference substance. The peak corresponding to the reference peak of the gallic acid reference substance is the S peak, and the relative retention times of each characteristic peak and the S peak are: 1.27 (peak 2), 2.13 (peak 3), 3.24 (peak 4), 3.60 (peak 5), 4.85 (peak 6), 5.87 (peak 7), 7.48 (peak 8), 7.69 (peak 9). The relative peak areas of peaks 7 and 9 and S peak are calculated, and the relative peak areas are 0.26 (peak 7) and 0.16 (peak 9).

[0081] Table 2 System suitability parameters of Euryale ferox granules and reference solution

[0082]

[0083]

[0084] Experimental Example 1 Optimization of Chromatographic Conditions

[0085] 1. Instrument:

[0086] Chromatograph: Waters ACQUITY UPLC I-Class chromatography system, including quaternary solvent manager (ACQ-QSM), automatic sample injector (ACQ-FTN), original imported chromatography column oven (ACQ-CM), diode array ultraviolet detector (ACQ-TUV), Empower chromatography management system;

[0087] Electronic analytical balance: Mettler Toledo NewClassic MS 1 / 100,000, Jing Tian FA2044A.

[0088] Chromatographic column:

[0089] Chromatographic column: BEH C18 (column length 150 mm, inner diameter 2.1 mm, particle size 1.7 μm).

[0090] 2. Drug testing:

[0091] Gallic acid reference substance (batch number: 110831-201906, purchased from China Food and Drug Inspection Institutes);

[0092] Gorgon fruit control medicinal material: purchased from China Food and Drug Inspection Institute, batch number 121421-201804.

[0093] Gorgon fruit formula granules: prepared according to the method of Example 2, batch numbers: 2205001Y, 2205002Y, 2205003Y. Methanol (Tiandi), acetonitrile (Tiandi), formic acid (CNW), acetic acid (Chengdu Chron Chemical Co., Ltd), phosphoric acid (Chengdu Chron Chemical Co., Ltd).

[0094] 3. UPLC method optimization

[0095] (1) Selection of mobile phase gradient

[0096] Table 3 UPLC characteristic spectrum chromatographic gradient 1

[0097]

[0098] Table 4 UPLC characteristic spectrum chromatographic gradient 2

[0099]

[0100] Table 5 UPLC characteristic spectrum chromatographic gradient 3

[0101]

[0102] Table 6 UPLC characteristic spectrum chromatographic gradient 4

[0103]

[0104]

[0105] Results Figure 4-7 As shown, from gradient 1 to gradient 2, the separation effect of the chromatographic peaks at 10-13 minutes is better, and more chromatographic peaks can be separated; from gradient 2 to gradient 3, the separation of the chromatographic peaks at 24-28 minutes is greatly improved; from peak 3 to peak 4, the chromatographic peak of peak 4 is separated, and two chromatographic peaks with good separation are obtained between 12 minutes and 14 minutes. It can be seen that compared with gradient 1, gradient 2-4 can significantly increase the number of separated chromatographic peaks, especially the use of gradient 4 can achieve effective separation of more chromatographic peaks.

[0106] (2) Investigation of different flow rates

[0107] Take the same portion of the Euryale ferox formula granule test solution prepared in Example 4, and analyze it at flow rates of 0.25, 0.30, and 0.32 mL / min according to the chromatographic conditions of Example 4, record the chromatogram, and analyze the separation effect of each characteristic peak. The results are shown in the following table and Figure 8-10 As shown, when the flow rate changes, the separation degree of each characteristic peak is greater than 1.5, among which the separation effect of peaks 4, 5 and 9 is affected. Therefore, the preferred solution recommends a fixed flow rate of 0.3 ml per minute.

[0108] Table 7 Chromatogram parameters of standard decoction of Euryale ferox (lyophilized powder) at different flow rates

[0109]

[0110]

[0111] (3) Investigation of different column temperatures

[0112] Take the same sample solution of the Euryale ferox formula granules prepared in Example 4, and analyze it at different column temperatures according to the chromatographic conditions of Example 4, record the chromatogram, and analyze the separation effect of each characteristic peak. The results are shown in the following table and Figure 11-13 As shown in the figure, when the column temperature changes, the separation degree of each characteristic peak is greater than 1.5, among which the separation effect of peaks 4, 5, and 7 is affected. Therefore, it is recommended to fix the column temperature at 25°C in the preferred scheme.

[0113] Table 8 Chromatographic parameters of Gorgon Fruit Decoction Standard Decoction (lyophilized powder) at different column temperatures

[0114]

[0115] (4) Investigation of different formic acid concentrations

[0116] Take the same sample solution of the Euryale ferox formula granules prepared in Example 4, and analyze it under different formic acid concentrations according to the chromatographic conditions of Example 4, record the chromatogram, and analyze the separation effect of each characteristic peak. The results are shown in the following table and Figure 14-16 As shown, when the formic acid concentration changes, the separation effect of each characteristic peak is slightly affected, indicating that 0.48%-0.52% formic acid solution is feasible.

[0117] Table 9 Chromatogram parameters of standard decoction of Euryale ferox (lyophilized powder) at different formic acid concentrations

[0118]

[0119] Experimental Example 2 Establishment of characteristic spectrum of Euryale ferox formula granules

[0120] 1. Instruments and test drugs

[0121] Same as Experimental Example 1.

[0122] 2. Construction of multi-batch feature maps

[0123] Three batches of Euryale ferox formula granule samples (prepared according to the method of Example 2, batch numbers: 2205001Y, 2205002Y, 2205003Y) were taken and the characteristic spectra of the three batches of Euryale ferox formula granules were obtained according to the high performance liquid chromatography detection of Example 4, as shown in Fig.17 shown.

[0124] Take Gorgon fruit medicinal materials and 18 batches of Gorgon fruit slices standard decoction freeze-dried powder samples (prepared according to the method of Example 1, batch numbers: 2103001Y, 2103002Y, 2103003Y, 2103004Y, 2103005Y, 2103006Y, 2103007Y, 2103008Y, 2103009Y, 2103010Y, 2103011Y, 2103012Y, 2103013Y, 2103014Y, 2103015Y, 210801Y, 210802Y, 210803Y) and obtain 18 batches of Gorgon fruit slices standard decoction freeze-dried powder characteristic spectra and Gorgon fruit reference medicinal material characteristic spectra according to the ultra-high performance liquid chromatography of Example 3, see Fig.18 The characteristic spectrum similarity evaluation software "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Committee was used to generate the reference characteristic spectrum, see Fig.19 shown.

[0125] The measurement results show that the characteristic spectra of 18 batches of standard decoctions of Gorgon fruit slices (lyophilized powder) and 3 batches of Gorgon fruit formula granules have a total of 9 characteristic peaks, among which the peak corresponding to the gallic acid reference peak is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time of each characteristic peak is within ±10% of the specified value.

[0126] Table 10 Relative retention time results of characteristic spectra of 18 batches of Gorgon Fruit decoction standard decoction (lyophilized powder) and 3 batches of granules

[0127]

[0128] Table 11 Relative peak area results of characteristic spectra of 18 batches of Gorgon Fruit decoction standard decoction (lyophilized powder) and 3 batches of granules

[0129]

[0130] 3. Specified value

[0131] According to the research results, it is determined that the characteristic spectrum of the Euryale ferox formula granules should show 9 characteristic peaks in the chromatogram of the test sample, and should correspond to the retention time of the 9 characteristic peaks in the chromatogram of the reference medicinal material, among which Peak 1 should correspond to the retention time of the peak of the gallic acid reference material. The relative retention time of each characteristic peak and the S peak is calculated, and its relative retention time should be within the range of ±10% of the specified value, and the specified value is: 1.26 (peak 2), 2.11 (peak 3), 3.21 (peak 4), 3.57 (peak 5), 4.81 (peak 6), 5.81 (peak 7), 7.41 (peak 8), 7.62 (peak 9). The relative retention time and relative peak area results of the Euryale ferox formula granules control characteristic spectrum and Euryale ferox reference medicinal material characteristic spectrum are shown in the following table.

[0132] The reference spectrum was fitted using Mark peak fitting.

[0133] Table 12 Relative retention time of the comparison spectrum of Euryale ferox formula granules

[0134]

[0135] Table 13 Relative peak area of ​​Euryale ferox formula granules comparison spectrum

[0136]

[0137] Table 14 Relative retention time of characteristic spectra of Euryale ferox reference medicinal materials

[0138]

[0139] Table 15 Relative peak areas of characteristic spectra of Euryale ferox reference medicinal materials

[0140]

[0141] Experimental Example 4 Methodology Verification

[0142] 1. Instrument precision

[0143] Take the same sample solution of Euryale ferox formula granules prepared in Example 4, repeat the injection 6 times according to the chromatographic conditions of Example 4, record the chromatogram, measure the relative retention time and relative peak area of ​​6 characteristic peaks, and analyze them. The results are shown in the following table and Fig. 20 As shown in the figure, the RSD of the relative retention time and relative peak area of ​​the characteristic peaks were small, indicating good precision.

[0144] Table 16 Precision relative retention time test results

[0145]

[0146] Table 17 Precision relative peak area test results

[0147]

[0148]

[0149] 2. Repeatability

[0150] Take the same portion of Euryale ferox formula granules, prepare the test solution according to the method of Example 4 and inject it for analysis, record the chromatogram, measure the relative retention time and relative peak area of ​​9 characteristic peaks, and analyze them. The results are shown in the following table and Fig.21 As shown in the figure, the RSD of the relative retention time and relative peak area of ​​the characteristic peaks were small, indicating good repeatability.

[0151] Table 18 Repeatability relative retention time test results

[0152]

[0153] Table 19 Repeatability relative peak area test results

[0154]

[0155]

[0156] 3. Intermediate precision (different operators)

[0157] Two inspectors used the same equipment at different times to measure the same batch of Euryale ferox formula granules according to the method and chromatographic conditions under Example 4, and measured the relative retention time and relative peak area of ​​9 common peaks, and analyzed them. The results are shown in Fig. 22 As shown in the table below, the RSDs of relative retention time and relative peak area are small, indicating that the intermediate precision of this method is good.

[0158] Table 20 Intermediate precision relative retention time test results

[0159]

[0160] Table 21 Intermediate precision relative peak area test results

[0161]

[0162] 4. Stability test

[0163] Take the same portion of the Euryale ferox formula granule test solution prepared in Example 4, and analyze it at 0, 4, 8, 12, 18, and 24 hours according to the chromatographic conditions of Example 4, record the chromatogram, measure the relative retention time and relative peak area of ​​9 characteristic peaks, and analyze them. The results are shown in Fig.23 As shown in the table below, the RSDs of the relative retention time and relative peak area of ​​each characteristic peak are small, indicating that this method has good stability and can meet the needs of the measurement.

[0164] Table 22 Stability relative retention time test results

[0165]

[0166] Table 23 Stability relative peak area test results

[0167]

[0168] Example 5 Application of the method for constructing characteristic spectra of Gorgon fruit and its preparations in detecting Gorgon fruit and its preparations

[0169] The present embodiment provides a quality detection method for Gorgon fruit and its preparation, wherein different batches of Gorgon fruit formula granules are determined by the method of Example 4, a characteristic spectrum of the Gorgon fruit formula granule product to be tested is constructed, and then compared with the control characteristic spectrum constructed in Experimental Example 3, the characteristic spectrum of the Gorgon fruit formula granule product to be tested has 9 common characteristic peaks, peak 1 corresponds to the retention time of the reference peak of the gallic acid reference substance, the peak corresponding to the reference peak of the gallic acid reference substance is the S peak, the relative retention time of each characteristic peak and the S peak is within the range of ±10% of the specified value, and the specified value is: 1.26 (peak 2), 2.11 (peak 3), 3.21 (peak 4), 3.57 (peak 5), 4.81 (peak 6), 5.81 (peak 7), 7.41 (peak 8), 7.62 (peak 9), the relative peak area of ​​peak 7, peak 9 and S peak is within the specified value range, and the specified value is: not more than 0.68 (peak 7) not more than, 0.45 (peak 9). Therefore, it can be judged that the quality of this batch of Gorgon fruit formula granules is qualified.

[0170] Experimental Example 6 Application of the method for constructing characteristic spectra of Gorgon fruit and its preparations in identifying Gorgon fruit and its preparations

[0171] The main counterfeits of Gorgon fruit are red sorghum, white sorghum and lotus seeds, but their nature, taste and functions are different and they should not be mixed.

[0172] The present embodiment provides a method for identifying Gorgon Fruit and its preparation, comprising the following steps: using the red sorghum decoction standard decoction freeze-dried powder, white sorghum decoction standard decoction freeze-dried powder, and lotus seed decoction standard decoction freeze-dried powder to be identified as test samples, preparing the test sample solution according to the method of Example 3, and detecting under the chromatographic conditions of Example 3, and the results are shown in the following table. Among them, the red sorghum decoction standard decoction freeze-dried powder (red sorghum 1-3), the white sorghum decoction standard decoction freeze-dried powder (white sorghum 1-3), and the lotus seed decoction standard decoction freeze-dried powder (lotus seed 1-3) of different batches are respectively taken from three batches of red sorghum medicinal materials, white sorghum medicinal materials, and lotus seed medicinal materials, and then the impurities are removed and crushed according to the provisions of the pharmacopoeia, and then prepared according to the preparation method of Example 1.

[0173] Table 24 Relative retention time results of characteristic spectra of lotus seed, white sorghum and red sorghum decoction standard decoction

[0174]

[0175] Table 25 Results of relative peak area determination of characteristic spectrum of standard decoction of lotus seeds, white sorghum and red sorghum pieces

[0176]

[0177]

[0178] The test results were analyzed by using the above method to construct the characteristic spectrum and comparing it with the control characteristic spectrum constructed in Experimental Example 3. Figure 24-25 As shown in the figure, the standard decoction of lotus seed slices has larger chromatographic peaks at 12 minutes and 18-19 minutes. At the same time, the characteristic peak area of ​​gallic acid peak in the standard decoction of lotus seed slices is significantly lower than that of Gorgon fruit. Therefore, the relative peak areas of each characteristic peak and gallic acid peak are larger, which can be clearly distinguished from Gorgon fruit.

[0179] The white sorghum standard decoction and the red sorghum standard decoction all showed large chromatographic peaks at 17-18 minutes, 20-21 minutes, and around 25 minutes. The characteristic peak area of ​​the gallic acid peak was significantly lower than that of Gorgon fruit, so the relative peak areas of each characteristic peak and the gallic acid peak were all large. Peak 7 was higher in white sorghum and red sorghum, and Peak 9 was not detected in white sorghum, but was detected at a higher level in red sorghum.

[0180] In combination with the above distinctions, the relative peak area of ​​Peak 7 is used to distinguish Euryale gorgoniensis from lotus seeds, red sorghum, and white sorghum, and the relative peak area of ​​Peak 9 is used to distinguish Euryale gorgoniensis from lotus seeds and red sorghum. The results are shown in the table below.

[0181] Table 26 Relative peak areas of peak 7 and peak 9

[0182] Relative peak area Gorgon Fruit Lotus seeds Red Sorghum White sorghum Peak 7 0.111-0.525 45.093-89.284 3.364-4.224 9.867-25.575 Peak 9 0.117-0.348 4.910-13.191 4.346-11.302 /

[0183] From the above data, it can be seen that the relative peak areas of Peak 7, Peak 9 and S peak (gallic acid) of Gorgon fruit are quite different from those of the counterfeit products. It is stipulated that the relative peak area of ​​Peak 7 shall not be higher than 0.68 (0.525×1.3=0.682), and the relative peak area of ​​Peak 9 shall not be higher than 0.45 (0.348×1.3=0.452).

[0184] This confirms that this method can be used to distinguish red sorghum, white sorghum, lotus seeds and water chestnuts.

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

Claims

1. A method for constructing a characteristic spectrum of Gorgon fruit and its preparation, characterized in that: The following steps are included: (1) Preparation of Euryale ferox test solution; (2) A solution of a Euryale ferox test sample was tested by high performance liquid chromatography to obtain a characteristic spectrum of the test sample. Octadecylsilane bonded silica gel was used as a filler and an aqueous solution of formic acid-acetonitrile was used as a mobile phase for gradient elution. The gradient elution program included: 0→3 minutes→38 minutes. The volume percentage of acetonitrile in the mobile phase was 1%→1%→20%-24%. Step (1) included: 1) Take the Euryale ferox test sample, add solvent to extract, and obtain the extract; 2) Separate the solid and liquid of the extract and take the liquid, which is the test solution; In step 1), the extraction solvent of the Euryale ferox test solution is selected from a 30%-70% methanol aqueous solution, and 30-100 times the amount of the solvent is added; In step 1), the extraction method is ultrasonic extraction or reflux extraction, and the volume percentage of formic acid in the aqueous solution of formic acid is 0.48-0.52%.

2. The construction method according to claim 1, characterized in that: Step (1) satisfies any one or more of the following AB: A. Extraction time is 30min-1h; B. In step 2), the solid-liquid separation is independently selected from centrifugation or filtration.

3. The construction method according to claim 1, characterized in that: The chromatographic conditions of the high performance liquid chromatography in step (2) also include: a detection wavelength of 308-312 nm, a flow rate of 0.25-0.32 ml / min, a column temperature of 23-27° C., and an injection volume of 2-5 μl.

4. The construction method according to any one of claims 1 to 3, characterized in that: The construction method also includes the step of preparing a reference solution by using gallic acid as a solubilizer, and the step of detecting the reference solution by high performance liquid chromatography according to the construction method described in any one of claims 1 to 3 to obtain a reference spectrum of the reference.

5. The construction method according to claim 4, characterized in that: The solvent is selected from a 30%-70% methanol aqueous solution.

6. The construction method according to claim 4, characterized in that: Each 1 mL of the reference solution contains 10-100 μg of gallic acid.

7. The construction method according to any one of claims 1 to 3, characterized in that: The construction method also includes the step of using the residue obtained after water extraction and drying of the Euryale ferox reference medicinal material and then obtaining a reference medicinal material reference atlas according to the construction method described in any one of claims 1 to 3.

8. The construction method according to any one of claims 1 to 3, characterized in that: The characteristic spectrum of the Euryale ferox and its preparation has 9 characteristic peaks, peak 1 corresponds to the retention time of the reference peak of the gallic acid reference substance, the peak corresponding to the reference peak of the gallic acid reference substance is the S peak, the relative retention time of each characteristic peak and the S peak is within ±10% of the specified value, the specified values ​​of the relative retention time of peaks 2 to 9 are: 1.26, 2.11, 3.21, 3.57, 4.81, 5.81, 7.41, 7.62, respectively, and the relative peak areas of peaks 7 and 9 to the S peak are: not more than 0.68, not more than 0.45, respectively.

9. Use of the method for constructing characteristic maps of Gorgon fruit and its preparations according to any one of claims 1 to 8 in distinguishing Gorgon fruit products from counterfeits or in quality inspection of Gorgon fruit products.

10. A method for distinguishing Gorgon fruit products from counterfeit products, characterized in that: The method comprises the steps of comparing the characteristic spectrum of the product to be identified with the reference characteristic spectrum of Gorgon fruit and its preparation; the characteristic spectrum of the product to be identified is obtained by using the Gorgon fruit product to be identified as a test sample according to the construction method described in any one of claims 1 to 8, and the reference characteristic spectrum of Gorgon fruit and its preparation is selected from any one of the following (1) to (3): (1) It has 9 common characteristic peaks, peak 1 corresponds to the retention time of the gallic acid reference peak, the peak corresponding to the gallic acid reference peak is the S peak, the relative retention time of each characteristic peak and the S peak is within the range of ±10% of the specified value, the specified values ​​of the relative retention time of peaks 2 to 9 are: 1.26, 2.11, 3.21, 3.57, 4.81, 5.81, 7.41, 7.62, respectively, and the relative peak area of ​​peak 7 and S peak is not greater than 0.68; (2) A characteristic spectrum of Gorgon fruit obtained by the construction method described in any one of claims 1 to 8 using a single batch or multiple batches of Gorgon fruit test samples; (3) Using multiple batches of Euryale ferox test samples and the characteristic spectra obtained by the construction method described in any one of claims 1 to 8, a control characteristic spectrum is prepared by the average value or median method.

11. The method for distinguishing Gorgon fruit products from counterfeit products according to claim 10, characterized in that: The relative peak area between Peak 9 and Peak S is no more than 0.

45.

12. A quality detection method for Gorgon Fruit and its preparation, characterized in that: The method comprises the steps of comparing the characteristic spectrum of the Gorgon fruit product to be tested with the reference characteristic spectrum of Gorgon fruit and its preparation; the characteristic spectrum of the Gorgon fruit product to be tested is obtained by using the Gorgon fruit product to be tested as a test sample according to the construction method described in any one of claims 1 to 8, and the reference characteristic spectrum of Gorgon fruit and its preparation is selected from any one of the following (1) to (4): (1) It has 9 common characteristic peaks, and the RSD of the retention time of each characteristic peak and the specified value of the retention time of the following characteristic peaks is less than 5.0%. The specified values ​​of the retention time of each characteristic peak are 3.976min, 5.025min, 8.405min, 12.767min, 14.195min, 19.112min, 23.119min, 29.476min and 30.304min respectively; (2) It has 9 common characteristic peaks, peak 1 corresponds to the retention time of the gallic acid reference peak, the peak corresponding to the gallic acid reference peak is the S peak, the relative retention time of each characteristic peak and the S peak is within the range of ±10% of the specified value, the specified values ​​of the relative retention time of peaks 2 to 9 are: 1.26, 2.11, 3.21, 3.57, 4.81, 5.81, 7.41, 7.62, respectively, and the relative peak area of ​​peak 7 and S peak is not greater than 0.68; (3) A characteristic spectrum of Gorgon fruit obtained by the construction method described in any one of claims 1 to 8 using a single batch or multiple batches of Gorgon fruit test samples; (4) Using multiple batches of Euryale ferox test samples and the characteristic spectra obtained by the construction method described in any one of claims 1 to 8, a control characteristic spectrum is prepared by the average value or median method.

13. The quality detection method of Gorgon Fruit and its preparation according to claim 12, characterized in that: The relative peak area between Peak 9 and Peak S is no more than 0.45.

Images