Method for constructing characteristic map of Fagopyrum serrata

Abstract

The present invention discloses a method for constructing a characteristic spectrum of golden buckwheat. The characteristic spectrum has gallic acid, protocatechuic acid, protocatechuic aldehyde, catechin, epicatechin, 4-coumaric acid, and procyanidin B2 as characteristic components. By using an efficient detection method, the common peaks of golden buckwheat samples of different origins are confirmed to construct a characteristic spectrum. The method can distinguish the counterfeit products of golden buckwheat, Smilax glabra and Smilax china, and with the flavonoids contained in golden buckwheat as representative components, it can more comprehensively reflect the product quality. The method is suitable for rapid and comprehensive quality control of golden buckwheat medicinal materials, decoction pieces, extracts, standard decoctions, formula granules, and benchmark samples, preparation intermediates, and finished products of golden buckwheat classic prescriptions. The method is efficient, easy to operate, and suitable for quality monitoring of continuous production.

Description

Technical Field

[0001] The present invention relates to a method for constructing a characteristic map of golden buckwheat, and in particular to a method for constructing a characteristic map of golden buckwheat that can identify counterfeits efficiently and reliably. Background Art

[0002] Fagopyrum dibotrys (D. Don) Hara is the dried rhizome of the Polygonaceae plant. It has a cool nature, a slightly pungent and astringent flavor, and enters the lung meridian. It clears heat and detoxifies, dispels pus, and removes blood stasis. It is used to treat lung abscesses with pus vomiting, lung-heat cough and asthma, and painful swelling of the pubic region. In recent years, there has been extensive research on its anti-inflammatory, analgesic, sedative, antihypertensive, immunosuppressive, and antiarrhythmic effects.

[0003] So far, there are few studies on fingerprints or characteristic spectra that can reveal its intrinsic quality. He Meishan et al. used HPLC to determine the fingerprint of golden buckwheat medicinal materials, but the detection time was long, 60 minutes, and the sample preparation was relatively complicated, and the separation of the characteristic peaks of the spectrum was poor (He Meishan, Qian Binghui, Wang Zhaolong, et al. Study on the HPLC fingerprint of golden buckwheat [J]. Chinese Pharmacist, 2005, (03): 217-219.). Lu Huan et al. used HPLC to determine the fingerprint of golden buckwheat slices, but the detection time was long, 90 minutes, and the sample preparation was relatively complicated, the baseline was not stable, and the separation of the characteristic peaks of the spectrum was poor (Lu Huan, Nie Peng, Zeng Wenxue, et al. Study on the HPLC fingerprint of golden buckwheat [J]. Journal of Jiangxi University of Traditional Chinese Medicine, 2012, 24 (03): 39-42.).

[0004] Both golden buckwheat and wild smilax are known as "Tu Fu Ling" (Smilax glabra), and both possess the properties of clearing heat and detoxifying. However, the names of these two medicinal herbs are often confused. Currently, literature reports only distinguish between golden buckwheat and wild smilax, regarding their origins, properties, and clinical applications, but lack characterization or thin-layer identification methods. Furthermore, "Tu Fu Ling" and "Smilax glabra" have been used and recorded in historical herbal texts, and their names are often confused across different regions. Therefore, distinguishing golden buckwheat, wild smilax, and "Smilax glabra" is crucial to ensure clinical drug safety and efficacy. Summary of the Invention

[0005] Purpose of the invention: In view of the shortcomings of existing quality control methods for Fagopyrum serrata, such as the inability to distinguish counterfeits, limited quality control ingredients, complicated operations, and low detection efficiency, the present invention aims to provide a method for constructing a characteristic map of Fagopyrum serrata that can identify counterfeits, is efficient, and reliable.

[0006] Technical solution: The method for constructing the characteristic spectrum of golden buckwheat of the present invention comprises the following steps:

[0007] (1) preparing a test solution and a reference solution: the reference solution includes a control medicinal material and a reference substance, wherein the reference substance is gallic acid and 4-coumaric acid;

[0008] (2) The test solution and the reference solution were determined by high performance liquid chromatography. The chromatographic conditions were as follows: chromatographic column: octadecylsilane bonded silica gel column, mobile phase: phase A is acetonitrile, phase B is 0.1% phosphoric acid solution; gradient elution program: 0 min → 8 min → 10 min → 15 min → 29 min, volume percentage of phase A: 1% → 6% → 7% → 7% → 8%; flow rate: 0.30-0.40 mL / min, column temperature: 25-35°C;

[0009] (3) Confirm the common peaks by comparing with the reference and construct the characteristic spectrum.

[0010] "Reference substances" and "reference medicinal materials" refer to standard substances used for identification, inspection, content determination and calibration of the performance of testing instruments.

[0011] This method detects multiple batches of test sample solutions, selects common peaks, verifies the presence of each common peak in the control medicinal material by referring to the reference medicinal material spectrum, and confirms the composition of the effective ingredient in combination with the characteristic peaks of the reference material.

[0012] Wherein, the chromatographic conditions of the HPLC determination are as follows:

[0013] Chromatographic column: octadecylsilane bonded silica gel column, mobile phase: phase A is acetonitrile, phase B is 0.1% phosphoric acid solution; gradient elution program: 0 min→8 min→10 min→15 min→29 min, volume percentage of phase A: 1%→6%→7%→7%→8%; flow rate: 0.30-0.40 mL / min, column temperature: 25-35°C, detector: ultraviolet detector, evaporative light scattering detector or charged aerosol detector, detection wavelength: 270 nm-290 nm, preferably 280 nm.

[0014] Furthermore, the chromatographic conditions are as follows:

[0015] Chromatographic column length: 100 mm, inner diameter: 2.1 mm, filler particle size: 2.7 μm; mobile phase: phase A is acetonitrile, phase B is 0.1% phosphoric acid solution; flow rate: 0.30-0.40 mL / min, column temperature: 25-35° C., detector: UV detector, detection wavelength: 280 nm.

[0016] In the above construction method, the origin of golden buckwheat is golden buckwheat. The test sample has a total of 10 peaks, that is, the characteristic peaks of the characteristic spectrum are 10, among which peak 2 and peak 9 are the characteristic peaks of gallic acid and 4-coumaric acid, respectively; taking the characteristic peak of 4-coumaric acid as the reference peak S, the relative retention times of the remaining characteristic peaks are 0.10±10% (peak 1), 0.14±10% (peak 2), 0.19±10% (peak 3), 0.26±10% (peak 4), 0.32±10% (peak 5), 0.44±10% (peak 6), 0.56±10% (peak 7), 0.86±10% (peak 8), and 1.07±10% (peak 10).

[0017] Specifically, the above-mentioned test samples are the medicinal materials, decoction pieces, extracts, standard decoctions, formula granules of golden buckwheat, or benchmark samples, preparation intermediates or finished products of classic prescriptions containing golden buckwheat. Among them, "medicinal materials" or "raw drugs" refer to Chinese medicine raw materials that have not been processed or made into finished products. "Decoction pieces" refer to Chinese medicines for prescription use that have been processed according to needs, or Chinese medicines that can be directly used in traditional Chinese medicine clinical practice. "Extracts" refer to extracts that meet certain quality standards and are prepared according to standardized production processes, for example, alcohol (such as methanol, ethanol, etc.) extracts, water extracts, etc. "Standard decoctions" refer to single-flavor Chinese medicine decoction pieces water decoctions prepared by standardized processes based on traditional Chinese medicine theory and clinical application, with reference to modern extraction methods. "Formulated granules" are granules made from single-flavor Chinese medicine decoction pieces processed according to traditional standards and then extracted and concentrated for use in traditional Chinese medicine clinical prescriptions. "Classic prescription material standards" refer to traditional Chinese medicines prepared using the preparation methods of ancient classic prescriptions recorded in ancient medical texts. Aside from the molding process, all other preparation methods should be essentially consistent with those recorded in the ancient medical texts. "Classic prescription finished granules" or "classic prescription standard granules" refer to granules made from the extraction and concentration of ancient classic prescriptions recorded in ancient medical texts, intended for use in TCM clinical formulations.

[0018] About the test solution and the reference medicinal material solution:

[0019] The above test samples were extracted with 25-50 mL / 1 g of water or alcohol-water solution.

[0020] The preparation method of the test solution is as follows:

[0021] Take the golden buckwheat sample powder, accurately weigh it, place it in a container, accurately add the extraction solvent, seal it, weigh it, let it cool after extraction, and then extract it with ethyl acetate. Combine the ethyl acetate liquid, evaporate it to dryness, dissolve the residue in 10% methanol and transfer it to a 5ml volumetric flask, shake it well, filter it, and take the filtrate to obtain it.

[0022] The above extraction solvent is water, alcohol or alcohol-water solution.

[0023] Furthermore, the alcohol or alcohol-water solution is methanol or methanol-water solution, wherein the volume fraction of methanol in the methanol-water solution does not exceed 70%, and can specifically be 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%; in order to further improve the extraction efficiency and make the peak shape of the chromatographic peak better, the concentration of the extraction solvent is preferably 0%-50% by volume of methanol.

[0024] The mass-to-volume ratio of golden buckwheat sample powder to extraction solvent is 1g:(25-100mL), specifically 1g:25mL, 1g:50mL, 1g:75mL, or 1g:100mL. To further optimize extraction efficiency and material dosage, 1g:50mL is preferred. Too low a sample concentration is detrimental to chromatographic peak detection, while too high a concentration can overload the column and result in poor peak shape.

[0025] The extraction is carried out by ultrasound, heating reflux or shaking. In order to simplify the extraction operation, ultrasonic treatment is preferably used. The power and frequency of the ultrasound have little effect on the present invention. In some preferred embodiments, the power of the ultrasonic treatment is 250W and the frequency is 40kHz.

[0026] The extraction treatment time is 5 to 60 minutes, specifically 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes or 60 minutes. In order to further optimize the extraction effect and treatment cycle, the extraction treatment time is preferably 10 to 60 minutes.

[0027] More specifically, the preparation method of the above-mentioned test solution is as follows:

[0028] Take golden buckwheat sample powder, accurately weigh it, put it in a stoppered conical flask, accurately add water or a methanol-water solution with a concentration of 10% to 70%, seal it, weigh it, and extract it by ultrasonic, heating or shaking for 10 to 60 minutes. Then cool it, shake it well, filter it, and extract the filtrate by shaking with ethyl acetate for 1 to 4 times. Combine the ethyl acetate solution each time, evaporate it to dryness, dissolve the residue in water or a methanol-water solution with a concentration of 10% to 70% and transfer it to a 5 to 20 ml volumetric flask, shake it well, filter it, and take the filtrate to obtain the filtrate. The mass volume ratio of the golden buckwheat sample powder to the water or the methanol-water solution with a concentration of 10% to 70% is 1g:(25 to 100 mL).

[0029] The preparation method of the above-mentioned control medicinal material reference solution is as follows:

[0030] Take the control medicinal material, accurately weigh it, place it in a container (such as a stoppered conical flask), accurately add the extraction solvent, seal it, weigh it, extract it (such as ultrasonic, heating reflux or shaking), let it cool, shake it well, filter it, and extract the filtrate by shaking with ethyl acetate. Combine the ethyl acetate solution each time, evaporate it to dryness, dissolve the residue in the extraction solvent and transfer it to a volumetric flask, shake it well, filter it, and take the filtrate to obtain it.

[0031] In order to simplify the practical steps, the extraction solvent used to prepare the control medicinal material reference solution can be the same as the extraction solvent used to prepare the test sample solution. The concentration of the extraction solvent is 0% to 70% by volume of methanol, specifically water or 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% methanol; preferably, the concentration of the extraction solvent is 0% to 50% by volume of methanol.

[0032] The mass volume ratio of the control medicinal material to water or a methanol-water solution with a concentration of 0% to 50% is 1 g: (25 to 100 mL), preferably 1 g: 50 mL.

[0033] More specifically, the preparation method of the control medicinal material reference solution is as follows:

[0034] Take 0.5 g of the control medicinal material, place it in a stoppered conical flask, add 25 mL of water, heat and reflux for 30 minutes, cool, shake, filter, and extract the filtrate by shaking with ethyl acetate twice, 25 mL each time. Combine the ethyl acetate and evaporate to dryness. Dissolve the residue in 10% methanol and transfer it to a 5 mL volumetric flask, add 10% methanol to the scale, shake, filter, and take the filtrate as the control medicinal material reference solution.

[0035] About reference substance solution:

[0036] The above-mentioned reference substances are dissolved in water, alcohol or alcohol-water solution.

[0037] The preparation method of the above-mentioned reference substance solution is as follows:

[0038] Take an appropriate amount of reference substance, weigh it accurately, add solvent, and mix until it dissolves clearly.

[0039] Furthermore, the above-mentioned alcohol or alcohol-water solution is methanol or methanol-water solution, wherein the methanol volume fraction of the methanol-water solution is not higher than 70%, and can specifically be 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%; preferably, the concentration of the extraction solvent is 5%-50% by volume of methanol.

[0040] Specifically, the concentration of the gallic acid reference solution is 50 μg / mL, and the concentration of the 4-coumaric acid reference solution is 50 μg / mL.

[0041] Preferably, in the above construction method, the chromatographic column is selected from a Poroshell 120CS C18 (2.1×100 mm, 2.7 μm) chromatographic column, and the flow rate is 0.30 to 0.40 mL / min.

[0042] The present invention provides a method for constructing a characteristic spectrum of golden buckwheat. The characteristic spectrum uses gallic acid, protocatechuic acid, protocatechuic aldehyde, catechin, epicatechin, 4-coumaric acid, and procyanidin B2 as characteristic components. Using an efficient detection method, the common peaks of golden buckwheat samples of different origins are identified to construct the characteristic spectrum. This method can distinguish the counterfeit golden buckwheat products, such as Smilax glabra and Smilax china, and, using the flavonoids contained in golden buckwheat as representative components, can more comprehensively reflect product quality. Compared with the prior art (Lu Huan, Nie Peng, Zeng Wenxue, et al. Research on HPLC fingerprint of golden buckwheat [J]. Journal of Jiangxi University of Traditional Chinese Medicine, 2012, 24(03): 39-42. Lu Huan, Nie Peng, Zeng Wenxue, et al. Research on HPLC fingerprint of golden buckwheat [J]. Journal of Jiangxi University of Traditional Chinese Medicine, 2012, 24(03): 39-42.), the characteristic spectrum of the present application has characteristic peaks (peak 3-peak 10) that are not found in the prior art. The reason is that the gradient elution procedure in the chromatographic conditions of the present application is different from that of the reference document; and ethyl acetate extraction is used in the sample solution preparation. Finally, the present application obtains a characteristic spectrum of golden buckwheat with good characteristic peak separation and strong reliability.

[0043] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0044] (1) The characteristic spectrum constructed by this method can distinguish samples of different counterfeits, and with the flavonoids and lignans contained in golden buckwheat as representative components, it can more comprehensively reflect the product quality;

[0045] (2) The method is efficient, easy to operate, suitable for quality monitoring of continuous production, and widely applicable. It can be used for quality control of golden buckwheat medicinal materials, decoction pieces, extracts, standard decoctions, formula granules, as well as benchmark samples, preparation intermediates and finished products of golden buckwheat classic prescriptions. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is the UPLC chromatogram comparing the reference herb Fagopyrum truncatum with the sample;

[0047] Figure 2 UPLC chromatograms of golden buckwheat samples under different extraction solvent conditions;

[0048] Figure 3This is the UPLC overlay diagram of the standard decoction sample of golden buckwheat;

[0049] Figure 4 This is the UPLC overlay diagram of the standard decoction sample of golden buckwheat;

[0050] Figure 5 UPLC chromatograms of golden buckwheat samples under different flow rate conditions;

[0051] Figure 6 UPLC chromatograms of golden buckwheat samples under different column temperature conditions;

[0052] Figure 7 UPLC chromatograms of golden buckwheat samples under different chromatographic column conditions;

[0053] Figure 8 This is the UPLC overlay image of the golden buckwheat medicinal material sample;

[0054] Figure 9 The UPLC chromatograms are compared between the medicinal material Fagopyrum scabra and the counterfeit medicinal materials Smilax china and Smilax glabra.

[0055] Figure 10 The UPLC chromatograms are compared between the standard decoction of golden buckwheat and the counterfeit standard decoction samples of Smilax china and Smilax glabra. DETAILED DESCRIPTION

[0056] The technical solution of the present invention will be further described below in conjunction with embodiments.

[0057] Instruments, reagents and samples

[0058] Waters Acquity UPLC ultra-high performance liquid chromatograph (Waters); Empower 3 workstation (Waters); Agilent Technologies 1290 Infinity ultra-high performance liquid chromatograph; 1290DAD diode array detector; 1290MCT column oven; 1290Vialsampler autosampler; 1290Fiexible pump quaternary pump; OpenLAB CDS2.3 chromatography workstation; KQ-250E ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); ME204E / 02 electronic analytical balance (Mettler-Toledo Instrument (Shanghai) Co., Ltd.); HY-4 oscillator (Jintan Kexing Instrument Factory); Milli-Q IQ water purification system (Millipore); AS165W centrifuge (Azov (Shanghai) Trading Co., Ltd.); GKC114 temperature-controlled water bath (Nantong Huatai Experimental Instrument Co., Ltd.). Acetonitrile (chromatographic grade, Thermo Fisher Scientific) Fisher Company); phosphoric acid (chromatographic grade, Aladdin Company); water was ultrapure water; other reagents were of analytical grade.

[0059] Gallic acid reference substance and 4-coumaric acid reference substance were purchased from China Food and Drug Inspection Institute with batch numbers 110831-201906 and 112037-202102, respectively.

[0060] The reference medicinal material Fagopyrum scabra was purchased from the China Food and Drug Inspection Institute with batch number 121114-201403.

[0061] The golden buckwheat medicinal material was collected and identified as golden buckwheat by Jiangyin Tianjiang Pharmaceutical Co., Ltd., and the golden buckwheat standard decoction was prepared by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0062] Example 1: Construction of characteristic spectrum of standard decoction of golden buckwheat

[0063] 1. Preparation of reference solution

[0064] Take 0.5g of the standard decoction of golden buckwheat and place it in a stoppered conical flask. Add 25ml of water and heat under reflux for 30 minutes. Let cool, shake well, and filter. The filtrate is extracted twice with ethyl acetate by shaking, 25ml each time. The ethyl acetate is combined and evaporated to dryness. The residue is dissolved in 10% methanol and transferred to a 5ml volumetric flask. 10% methanol is added to the mark, shake well, and filter. The filtrate is used as the reference solution for the reference medicinal material. An appropriate amount of gallic acid and 4-coumaric acid reference substances are accurately weighed and added with 10% methanol to make a solution containing 50μg per 1ml. This is used as the reference solution for the reference substance.

[0065] 2. Preparation of test solution

[0066] Take 0.5g of crude powder of this product, place it in a stoppered conical flask, add 25ml of water, heat and reflux for 1 hour, cool, shake well, filter, and extract the filtrate by shaking with ethyl acetate twice, 25ml each time. Combine the ethyl acetate solutions, evaporate to dryness, dissolve the residue in 10% methanol and transfer it to a 5ml volumetric flask, add 10% methanol to the scale, shake well, filter, and take the filtrate to obtain.

[0067] 3. Construction of feature maps

[0068] (1) Chromatographic conditions

[0069] A Poroshell 120CS C18 column (2.1 mm × 100 mm, 2.7 μm) was used with acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, using gradient elution as specified in the table below; the flow rate was 0.35 mL / min; the column temperature was 30°C; the detection wavelength was 280 nm, and the number of theoretical plates calculated based on the 4-coumaric acid peak should be no less than 3000.

[0070]

[0071] (2) Testing and results

[0072] Accurately pipette 2 μL of each reference solution and test solution into the liquid chromatograph. Figure 1 .

[0073] Conclusion: The results showed that the detection baseline was stable, the target components were well separated, and the analysis could be completed within 30 minutes.

[0074] Example 2: Investigation of extraction solvents for sample pretreatment

[0075] 1. Preparation of reference solution

[0076] The relevant method is the same as Example 1.

[0077] 2. Preparation of test solution

[0078] Take about 0.5g of the powder of this product, divide it into 5 parallel portions, accurately weigh them, place them in a stoppered conical flask, accurately add 25ml each of water, 30% methanol, 50% methanol, 70% methanol and methanol, stopper it, weigh the weight, heat and reflux for 1 hour, cool, weigh again, make up the lost weight with the corresponding solvent, shake well, filter, and extract twice with ethyl acetate by shaking, 25ml each time, combine the ethyl acetate, evaporate to dryness, dissolve the residue in 10% methanol and transfer it to a 5ml volumetric flask, add 10% methanol to the scale, shake well, filter, and take the filtrate.

[0079] 3. Construction of feature maps

[0080] (1) Chromatographic conditions

[0081] The relevant method is the same as Example 1.

[0082] (2) Testing and results

[0083] Accurately pipette 2 μL of each test solution and reference solution, inject into the liquid chromatograph, and calculate the ratio of the total peak area of ​​the test sample to the sample weight. The results are shown in Table 1. Figure 2 .

[0084] Table 1 Comparison of different extraction solvents

[0085]

[0086] Conclusion: The chromatographic peak information of the sample extracted with water is rich and the peak area is large, while the chromatographic peak information of the sample extracted with other solvents is incomplete, so water is selected as the extraction solvent.

[0087] Example 3: Investigation of characteristic spectra of multiple batches of standard decoctions of golden buckwheat

[0088] 1. Preparation of reference solution

[0089] The relevant method is the same as Example 1.

[0090] 2. Preparation of test solution

[0091] The relevant method is the same as Example 1.

[0092] 3. Construction of feature maps

[0093] (1) Chromatographic conditions

[0094] The relevant method is the same as Example 1.

[0095] (2) Testing and results

[0096] 2 μL of each test solution and reference solution were accurately aspirated and injected into the liquid chromatograph. The spectral data were then imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" recommended by the National Pharmacopoeia Committee for result analysis. Peak No. 9 (identified as 4-coumaric acid by the reference substance) was taken as the S peak. The relative retention time of each peak and the S peak was calculated to establish the characteristic spectrum of the standard decoction of golden buckwheat. The results are shown in Table 2. Figure 3 .

[0097] Table 2 Determination results of different batches of standard decoction of golden buckwheat (relative retention time)

[0098]

[0099] Conclusion: Peaks 1 to 10 were consistently present in all batches of standard decoctions of Golden Fagopyrum truncatum. Peaks 2, 4, 5, 7, 8, 9, and 10 correspond to gallic acid, protocatechuic acid, protocatechualdehyde, catechin, epicatechin, 4-coumaric acid, and procyanidin B2, respectively. In addition to identifying common flavonoids such as protocatechuic acid, catechin, and procyanidin B2, this method also adds 4-coumaric acid, a lignan, indicating that this method further enriches the quality evaluation of Golden Fagopyrum truncatum.

[0100] Based on the determination results of multiple batches of standard decoction of golden buckwheat, it is stipulated that 10 characteristic peaks should be present in the chromatogram of the standard decoction of golden buckwheat, of which peaks 2 and 9 should correspond to the retention times of the gallic acid reference substance and 4-coumaric acid reference substance, respectively. Taking peak 9 as the reference peak, the relative retention times of the other characteristic peaks are 0.10±10% (peak 1), 0.14±10% (peak 2), 0.19±10% (peak 3), 0.26±10% (peak 4), 0.32±10% (peak 5), 0.44±10% (peak 6), 0.56±10% (peak 7), 0.86±10% (peak 8), and 1.07±10% (peak 10).

[0101] Example 4: Investigation of the stability of the test product

[0102] 1. Preparation of reference solution

[0103] The relevant method is the same as Example 1.

[0104] 2. Preparation of test solution

[0105] The relevant method is the same as Example 1.

[0106] 3. Construction of feature maps

[0107] (1) Chromatographic conditions

[0108] The relevant method is the same as Example 1.

[0109] (2) Testing and results

[0110] Every 4 hours, 2 μL of each reference solution and test solution was accurately aspirated and injected into the liquid chromatograph. The peak corresponding to Peak 9 (4-coumaric acid) was used as the reference peak to calculate the relative retention time and relative peak area of ​​each common peak. The results are shown in Table 3.

[0111] Table 3 Stability test results (relative retention time)

[0112]

[0113] Conclusion: The test solution has good stability within 24 hours (RSD%<2.0%).

[0114] Example 5: Specificity test

[0115] 1. Preparation of reference solution

[0116] The relevant method is the same as Example 1.

[0117] 2. Preparation of test solution

[0118] The relevant method is the same as Example 1.

[0119] 3. Construction of feature maps

[0120] (1) Chromatographic conditions

[0121] The relevant method is the same as Example 1.

[0122] (2) Testing and results

[0123] Accurately draw 2 μL of blank solvent and test solution respectively and inject them into liquid chromatograph. Figure 4 .

[0124] Conclusion: The results show that the blank solvent has no interference with the determination of the characteristic spectrum of the standard decoction of Fagopyrum serrata, and the method has strong specificity.

[0125] Example 6: Investigation of mobile phase flow rate

[0126] 1. Preparation of reference solution

[0127] The relevant method is the same as Example 1.

[0128] 2. Preparation of test solution

[0129] The relevant method is the same as Example 1.

[0130] 3. Construction of feature maps

[0131] (1) Chromatographic conditions

[0132] The flow rates of the mobile phase were set to 0.30 mL / min, 0.35 mL / min, and 0.40 mL / min, respectively. The rest of the relevant methods were the same as in Example 1.

[0133] (2) Testing and results

[0134] 2 μL of each reference solution and test solution were accurately drawn, and the mobile phase flow rates were set to 0.30 mL / min, 0.35 mL / min, and 0.40 mL / min, respectively. The samples were injected into the liquid chromatograph and the relative retention time of each characteristic peak was calculated. The results are shown in Table 4. Figure 5 .

[0135] Table 4 Effect of flow rate on separation effect (relative retention time)

[0136]

[0137] Conclusion: When the flow rate is between 0.25mL / min and 0.35mL / min, the separation effect of each characteristic peak is good, and the relative retention times are basically consistent, and the durability is good.

[0138] Example 7: Investigation of chromatographic column temperature

[0139] 1. Preparation of reference solution

[0140] The relevant method is the same as Example 1.

[0141] 2. Preparation of test solution

[0142] The relevant method is the same as Example 1.

[0143] 3. Construction of feature maps

[0144] (1) Chromatographic conditions

[0145] The column temperatures were set to 25°C, 30°C, and 35°C, respectively, and the rest of the relevant procedures were the same as in Example 1.

[0146] (2) Testing and results

[0147] 2 μL of each reference solution and test solution were accurately aspirated, and the column temperature was set to 25°C, 30°C, and 35°C, respectively. The samples were injected into the liquid chromatograph and the relative retention time of each characteristic peak was calculated. The results are shown in Table 5. Figure 6 .

[0148] Table 5 Effect of column temperature on separation effect (relative retention time)

[0149]

[0150] Conclusion: When the column temperature is between 25℃ and 35℃, the separation effect of each characteristic peak is better, the relative retention times are basically consistent, and the durability is good.

[0151] Example 8: Investigation of chromatographic column type

[0152] 1. Preparation of reference solution

[0153] The relevant method is the same as Example 1.

[0154] 2. Preparation of test solution

[0155] The relevant method is the same as Example 1.

[0156] 3. Construction of feature maps

[0157] (1) Chromatographic conditions

[0158] The chromatographic columns used were Aq-C18 (2.1×100 mm, 2.7 μm), CS C18 (2.1×100 mm, 2.7 μm), and Aq-C18 (2.1×150 mm, 2.7 μm), respectively. The rest of the relevant methods were the same as in Example 1.

[0159] (2) Testing and results

[0160] 2 μL of each reference solution and test solution were accurately aspirated, and the column temperatures were Aq-C18 (2.1×100mm, 2.7μm), CS C18 (2.1×100mm, 2.7μm), and Aq-C18 (2.1×150mm, 2.7μm), respectively. The samples were injected into the liquid chromatograph and the relative retention time of each characteristic peak was calculated. The results are shown in Table 6. Figure 7 .

[0161] Table 6 Effect of column type on separation effect (relative retention time)

[0162]

[0163]

[0164] Conclusion: Golden Fagopyrum samples were well separated on all three chromatographic columns, with the peak elution times being consistent on the Aq-C18 and CSC18 columns. Subsequent studies will use a Poroshell 120CS C18 (Agilent, 2.1 mm × 100 mm, 2.7 μm) column.

[0165] Example 8: Investigation of characteristic spectra of multiple batches of golden buckwheat medicinal materials

[0166] 1. Preparation of reference solution

[0167] The relevant method is the same as Example 1.

[0168] 2. Preparation of test solution

[0169] Take about 2.0 g of golden buckwheat medicinal material, and the rest of the relevant methods are the same as Example 1.

[0170] 3. Construction of feature maps

[0171] (1) Chromatographic conditions

[0172] The relevant method is the same as Example 1.

[0173] (2) Testing and results

[0174] 2 μL of each test solution and reference solution was accurately aspirated and injected into the liquid chromatograph. The spectral data were then imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" recommended by the National Pharmacopoeia Committee for result analysis. Peak No. 9 (identified as 4-coumaric acid by the reference substance) was taken as the S peak. The relative retention time of each peak and the S peak was calculated to establish the characteristic spectrum of golden buckwheat medicinal material. The results are shown in Table 7. Figure 8 .

[0175] Table 7 Sample determination results (relative retention time)

[0176]

[0177]

[0178] Conclusion: The results show that peaks 1 to 10 are consistently present in different batches of golden buckwheat. Peaks 2, 4, 5, 7, 8, 9, and 10 correspond to gallic acid, protocatechuic acid, protocatechualdehyde, catechin, epicatechin, 4-coumaric acid, and procyanidin B2, respectively. In addition to identifying common flavonoids such as protocatechuic acid, catechin, and procyanidin B2, this method also adds 4-coumaric acid, a lignan, indicating that this method further enriches the quality evaluation of golden buckwheat.

[0179] Based on the determination results of multiple batches of Fagopyrum truncatum, it is stipulated that 10 characteristic peaks should be present in the chromatogram of Fagopyrum truncatum, of which Peak 2 and Peak 9 should correspond to the retention times of the gallic acid reference substance and 4-coumaric acid reference substance, respectively. Taking Peak 9 as the reference peak, the relative retention times of the remaining characteristic peaks are 0.10±10% (Peak 1), 0.14±10% (Peak 2), 0.19±10% (Peak 3), 0.26±10% (Peak 4), 0.32±10% (Peak 5), 0.44±10% (Peak 6), 0.56±10% (Peak 7), 0.86±10% (Peak 8), and 1.07±10% (Peak 10).

[0180] Example 9: Comparison of characteristic spectra of golden buckwheat and counterfeit Chinese smilax and Chinese yam

[0181] 1. Preparation of reference solution

[0182] The relevant method is the same as Example 1.

[0183] 2. Preparation of test solution

[0184] Take about 2.0 g of golden buckwheat medicinal materials, smilax china medicinal materials, and smilax glabra medicinal materials respectively, and the rest of the relevant methods are the same as those in Example 1.

[0185] 3. Construction of feature maps

[0186] (1) Chromatographic conditions

[0187] The relevant method is the same as Example 1.

[0188] (2) Testing and results

[0189] Accurately pipette 2 μL of each test solution and reference solution into the liquid chromatograph, and compare the characteristic spectra. Figure 9 .

[0190] Conclusion: The characteristic spectrum of Fagopyrum truncatum sample contains 10 common peaks, of which peaks 2 and 9 should correspond to the retention times of gallic acid reference and 4-coumaric acid reference, respectively. Taking peak 9 as the reference peak, the relative retention times of the other characteristic peaks are 0.10±10% (peak 1), 0.14±10% (peak 2), 0.19±10% (peak 3), 0.26±10% (peak 4), 0.32±10% (peak 5), 0.44±10% (peak 6), 0.56±10% (peak 7), 0.86±10% (peak 8), and 1.07±10% (peak 10).

[0191] Compared with the characteristic spectrum of Fagopyrum truncatum, Peaks 4, 9, and 10 were not detected in Smilax glabra. The characteristic peaks of the two are quite different, so this characteristic spectrum can effectively distinguish Fagopyrum truncatum from Smilax glabra. Compared with the characteristic spectrum of Fagopyrum truncatum, Peak 2 has a higher response value in Fagopyrum truncatum but was not detected in Smilax glabra. Therefore, this characteristic spectrum can effectively distinguish Fagopyrum truncatum from Smilax glabra.

[0192] Example 10: Comparison of characteristic spectra of standard decoctions of golden buckwheat and counterfeit products of smilax china and smilax glabra

[0193] 1. Preparation of reference solution

[0194] The relevant method is the same as Example 1.

[0195] 2. Preparation of test solution

[0196] Take about 0.5 g of standard decoction of golden buckwheat, standard decoction of sarsaparilla, and standard decoction of smilax glabra respectively, and the rest of the relevant methods are the same as those in Example 1.

[0197] 3. Construction of feature maps

[0198] (1) Chromatographic conditions

[0199] The relevant method is the same as Example 1.

[0200] (2) Testing and results

[0201] Accurately pipette 2 μL of each test solution and reference solution into the liquid chromatograph, and compare the characteristic spectra. Figure 10 .

[0202] Conclusion: The characteristic spectrum of Fagopyrum truncatum sample contains 10 common peaks, of which peaks 2 and 9 should correspond to the retention times of gallic acid reference and 4-coumaric acid reference, respectively. Taking peak 9 as the reference peak, the relative retention times of the other characteristic peaks are 0.10±10% (peak 1), 0.14±10% (peak 2), 0.19±10% (peak 3), 0.26±10% (peak 4), 0.32±10% (peak 5), 0.44±10% (peak 6), 0.56±10% (peak 7), 0.86±10% (peak 8), and 1.07±10% (peak 10).

[0203] Compared with the characteristic spectrum of the standard decoction of Fagopyrum scutellariae, peaks 4, 9, and 10 were not detected in the standard decoction of Smilax glabrae. The characteristic peaks of the two differed significantly, thus effectively distinguishing Fagopyrum scutellariae from Smilax glabrae. Compared with the characteristic spectrum of the standard decoction of Fagopyrum scutellariae, peak 2 had a higher response value in the standard decoction of Fagopyrum scutellariae but was not detected in the standard decoction of Smilax glabrae. Therefore, this characteristic spectrum effectively distinguished Fagopyrum scutellariae from the standard decoction of Smilax glabrae.

Claims

1. A method for constructing a characteristic map of Fagopyrum serrata, characterized in that: The construction method comprises the following steps: (1) Prepare a test solution and a reference solution: the reference includes a control medicinal material and a reference substance, wherein the reference substance is gallic acid and 4-coumaric acid; the test sample is a medicinal material, a decoction piece, a standard decoction or a formula granule of Fagopyrum truncatum; the test sample or the reference medicinal material is extracted with water and then extracted with ethyl acetate to obtain a test solution and a reference medicinal material solution; (2) The test solution and the reference solution were determined by high performance liquid chromatography. The chromatographic conditions were as follows: chromatographic column: octadecylsilane bonded silica gel column, mobile phase: phase A is acetonitrile, phase B is 0.1% phosphoric acid solution; gradient elution program: 0 min → 8 min → 10 min → 15 min → 29 min, volume percentage of phase A: 1% → 6% → 7% → 7% → 8%; flow rate: 0.30~0.40 mL / min, column temperature: 25~35℃, detector: UV detector, detection wavelength: 270nm~290nm; column length: 100 mm, inner diameter: 2.1 mm, filler particle size: 2.7 μm; (3) Confirm the common peaks by comparing with the reference material and construct a characteristic spectrum; the characteristic spectrum includes the characteristic peaks of gallic acid, protocatechuic acid, protocatechuic aldehyde, catechin, epicatechin, 4-coumaric acid and proanthocyanidin B2.

2. The method for constructing the characteristic spectrum of Fagopyrum serrata according to claim 1, wherein: The characteristic spectrum has 10 characteristic peaks, among which peak 2 and peak 9 are characteristic peaks of gallic acid and 4-coumaric acid, respectively.

3. The method for constructing the characteristic spectrum of Fagopyrum serrata according to claim 1, characterized in that: The chromatographic column is selected from a Poroshell 120 CS C18 chromatographic column, with a flow rate of 0.30-0.40 mL / min.

4. An application of a characteristic spectrum of Fagopyrum serratum constructed according to the construction method according to any one of claims 1 to 3 in the detection of Fagopyrum serratum.

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