A method for constructing a characteristic spectrum of a sago seed preparation and its application

Abstract

The invention discloses a method for constructing a characteristic spectrum of a sago seed preparation and its application. The method adopts ultra-high performance liquid chromatography and reasonably controls the chromatographic method to form a full picture of the characteristic spectrum of the sago seed preparation. A comparative study is conducted on the preparations of fake European horse chestnut, Yunnan horse chestnut, and different-origin sago seed (Hippophae rhamnoides), sago seed (Zhejiang horse chestnut) and sago seed (Castoria truncatum) to more comprehensively reflect the characteristics of the sago seed preparation, provide a new analytical means for the intrinsic quality control of the sago seed preparation, and achieve the purpose of distinguishing between authenticity and different origins. The method of the present invention is simple, reproducible, accurate and reliable, easy to operate, short in time, low in solvent consumption, and has little environmental pollution.

Description

Technical Field

[0001] The invention belongs to the field of traditional Chinese medicine identification, and particularly relates to a method for constructing a characteristic spectrum of a Salacia fruit preparation and an application thereof. Background Art

[0002] The seeds of Aesculus chinensis were first recorded in the Compendium of Materia Medica. They have the effects of soothing the liver and regulating qi, harmonizing the stomach and relieving pain. They are mainly used to treat liver and stomach qi stagnation, chest and abdominal distension, and epigastric pain. Modern pharmacological studies have shown that the seeds of Aesculus chinensis have anti-inflammatory, anti-tumor, and gastrointestinal protective effects. There are about 30 species of Aesculus genus worldwide. The medicinal resources of Aesculus chinensis in my country are mainly wild, with about 10 species distributed in most parts of Central my country, East China, Northwest China, and Southwest China. There are many varieties, but the only authentic Aesculus chinensis included in the 2020 edition of the Chinese Pharmacopoeia are three species of Aesculus chinensis: Aesculus chinensis Bge., Aesculus chinensis Bge.var.chekiangensis (Huet Fang) Fang, or Aesculus wilsonii Rehd., all of which belong to the Aesculaceae family. However, due to the increasing clinical demand and the tight supply of wild medicinal resources, easily confused products such as European horse chestnut and Yunnan horse chestnut are often circulated in the medicinal material market as the medicinal material of Aesculus fragrans.

[0003] Existing technologies for testing the quality of sargassum seeds primarily rely on identifying the medicinal properties of the seeds and performing liquid phase analysis of certain components. For example, identifying medicinal properties to distinguish sargassum seeds from different sources is not suitable for quality control of sargassum seed preparations that have lost their medicinal properties. In the 2020 edition of the Chinese Pharmacopoeia, under the item of Sago asiatica, aescin A is used as a quality evaluation indicator for Sago asiatica medicinal materials. Nie Fang et al. used high performance liquid chromatography to determine the content of aescin A, B, C, and D in Sago asiatica medicinal materials (Nie Fang, Lv Weiqi, Tang Shengling. Simultaneous determination of four aescins in Sago asiatica from 14 origins by HPLC [J], Chinese Patent Medicine, 2018, 40(12): 2703-2708). Shi Zhaohua et al. used high performance liquid chromatography to establish a fingerprint method for Sago asiatica medicinal materials, detected the chemical components of Sago asiatica medicinal materials from different origins, and performed cluster analysis (Shi Zhaohua, Ye Lichun, Guan Xiaoyu, et al. Establishment of HPLC fingerprint and its application in medicinal material identification [J], Chinese Journal of Experimental Traditional Chinese Medicine, 2018, 24(14): 52-56). Sago asiatica contains rich chemical components, and the content of aescin alone cannot fully evaluate its quality. All of these methods used a 0.2% aqueous phosphoric acid solution as the mobile phase. Excessively low pH can cause irreversible damage to the chromatographic column, increasing testing costs. Gao Jing used high-performance liquid chromatography to analyze the fingerprints of S. truncatum seeds from different origins and employed LC-MS to infer the identification of the main chromatographic peaks (Gao Jing. Study on the Fingerprint of S. truncatum Seeds and the Extraction and Purification Process of the Active Component Aescin [D], Beijing University of Chinese Medicine, 2018). However, no method for identifying the authenticity of S. truncatum seeds or their different origins was established, and the quality of S. truncatum seed preparations could not be effectively controlled. Summary of the Invention

[0004] Purpose of the invention: The first purpose of the present invention is to provide a comprehensive, simple and fast method for constructing a characteristic map of Sauerkraut preparations; the second purpose of the present invention is to provide the application of this construction method in identifying the authenticity and different origins of Sauerkraut preparations.

[0005] Technical solution: The method for constructing the characteristic spectrum of the Salacia fructus preparation of the present invention comprises the following steps:

[0006] (1) Take a sample of the Salacia chinensis preparation, add an organic solvent, extract, cool, filter, and the filtrate is the sample solution;

[0007] (2) Take epicatechin and add solvent to prepare a reference solution;

[0008] (3) Take the test solution and the reference solution, inject them into the ultra performance liquid chromatograph respectively, perform ultra performance liquid chromatography detection, and record the characteristic chromatogram; the characteristic spectrum has 8 characteristic peaks, peak 3 is epicatechin, and the relative retention time of the remaining characteristic peaks and peak 3 is calculated. The relative retention time is within the range of ±10% of the specified value. The specified values ​​of peaks 1 to 2 are: 0.54 and 0.91, respectively, and the specified values ​​of peaks 4 to 8 are: 1.27, 1.79, 1.93, 2.51, and 2.98, respectively.

[0009] Preferably, the preparation in step (1) includes a standard decoction or a formula granule; the organic solvent includes water, methanol, or a mixed solution of water and methanol; the extraction method includes ultrasound, shaking, or reflux; and the extraction time is 15 to 60 minutes.

[0010] Preferably, the solvent in step (2) is 10-50% methanol.

[0011] Preferably, the chromatographic column of the ultra-high performance liquid chromatography in step (3) is Eclipse Plus C18, CORTECS UPLC T3 or ZORBAX SB-C18 RRHD.

[0012] Preferably, the column temperature of the ultra-high performance liquid chromatograph in step (3) is 25-35° C.; the flow rate is 0.25-0.35 ml / min; the detection wavelength is 210-230 nm; acetonitrile is used as mobile phase A, and 0.01%-0.2% phosphoric acid solution is used as mobile phase B for gradient elution.

[0013] Preferably, the gradient elution is: 0-2 min, the volume fraction of mobile phase A is 6→8%, and the volume fraction of mobile phase B is 94→92%; 2-16 min, the volume fraction of mobile phase A is 8→9%, and the volume fraction of mobile phase B is 92→91%; 16-28 min, the volume fraction of mobile phase A is 9→12%, and the volume fraction of mobile phase B is 91→88%; 28-45 min, the volume fraction of mobile phase A is 12→14%, and the volume fraction of mobile phase B is 88→86%.

[0014] The invention discloses an application of the method for constructing the characteristic spectrum of the Sauerkraut fruit preparation in identifying the authenticity and different origins of the Sauerkraut fruit preparation.

[0015] The invention discloses an application of the method for constructing a characteristic spectrum of a sago seed preparation of the present invention in distinguishing Aesculus hippocastanum, Aesculus yunnanensis, Aesculus hippocastanum (Horse Chestnut), Aesculus zekyronicus (Zhejiang Aesculus), and Aesculus tianshiensis (Lavender).

[0016] The specific application method of the application of the present invention is:

[0017] (1) Take a sample of the Salacia chinensis preparation, add an organic solvent, extract, cool, filter, and the filtrate is the sample solution;

[0018] (2) Take epicatechin and add solvent to prepare a reference solution;

[0019] (3) Take the test solution and the reference solution, inject them into the ultra-high performance liquid chromatograph respectively, perform ultra-high performance liquid chromatography detection, and record the characteristic chromatogram; the characteristic spectrum has 8 characteristic peaks, peak 3 is epicatechin, and the relative retention time of the remaining characteristic peaks and peak 3 is calculated. The relative retention time is within the range of ±10% of the specified value. The specified values ​​of peaks 1 to 2 are: 0.54 and 0.91, respectively, and the specified values ​​of peaks 4 to 8 are: 1.27, 1.79, 1.93, 2.51, and 2.98, respectively;

[0020] Using the formula Calculate the A value; use the formula Calculate the B value; use the formula Calculate the C value;

[0021] Wherein, S1 is the peak area of ​​characteristic peak 1, S4 is the peak area of ​​characteristic peak 4, S7 is the peak area of ​​characteristic peak 7, and S8 is the peak area of ​​characteristic peak 8;

[0022] When A≥20, it is Aesculus hippocastanum; when A<20 and B≥0.3, it is Aesculus chinensis (Tianshi chestnut); when A<20, B<0.3 and C≤0.2, it is Aesculus chinensis (Zhejiang horse chestnut); when A<20, B<0.3 and C≥0.4, it is Aesculus chinensis (Horse chestnut); when A<20, B<0.3 and 0.2<C<0.4, it is Aesculus yunnanensis.

[0023] The invention discloses an application of the method for constructing a characteristic spectrum of a Sauerkraut preparation in the quality inspection of Sauerkraut standard decoctions and formula granules.

[0024] The present invention establishes a quality control method for Salacia seed preparations, controls the intrinsic quality of Salacia seed preparation products, provides a new analytical means for the intrinsic quality control of Salacia seed preparation products, and achieves the purpose of distinguishing the authenticity and different origins of Salacia seed.

[0025] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The present invention adopts ultra-high performance liquid chromatography and reasonably controls the chromatographic method, uses acetonitrile as mobile phase A and 0.01%-0.2% phosphoric acid solution as mobile phase B for gradient elution, establishes 8 common peaks, and constitutes the full picture of the characteristic spectrum of the sago seed; 2. The method for constructing the characteristic spectrum of the sago seed of the present invention is simple, reproducible, accurate and reliable, easy to operate, with a stable baseline and good chromatographic peak separation; 3. The present invention conducts comparative studies on counterfeit European horse chestnut, Yunnan horse chestnut, and preparations of sago seed (Horse Chestnut) of different origins, sago seed (Zhejiang Horse Chestnut) and sago seed (Tianshi Li), through the construction of the sago seed characteristic spectrum, which can more comprehensively reflect the characteristics of the sago seed preparation, provide a new analytical means for the intrinsic quality control of the sago seed preparation, and achieve the purpose of distinguishing authenticity and different origins. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 UPLC chromatograms of Salacia seeds granules at different phosphoric acid concentrations;

[0027] Figure 2 This is the DAD-3D image of the granules of the salacia seeds;

[0028] Figure 3 UPLC chromatograms of Salacia chinensis formula granules at different detection wavelengths;

[0029] Figure 4 UPLC diagrams for different extraction solvents;

[0030] Figure 5 UPLC diagrams of different extraction methods;

[0031] Figure 6 UPLC diagrams for different extraction times;

[0032] Figure 7 To investigate the specificity of the granules of the salacia seeds;

[0033] Figure 8 is the separation effect of different chromatographic columns;

[0034] Figure 9 The characteristic spectra of Salacia chinensis using different column temperatures;

[0035] Figure 10 This is the characteristic spectrum of the salvia miltiorrhiza using different flow rates;

[0036] Figure 11 The characteristic spectra of Salacia chinensis using different brands of ultra-high performance liquid chromatography instruments are shown below;

[0037] Figure 12 This is the characteristic spectrum of multiple batches of standard decoction of Salacia chinensis;

[0038] Figure 13 UPLC chart of the salacia seeds granules and reference substance solution;

[0039] Figure 14 This is a comparison chart of the characteristics of the genuine and fake Sala seeds and the standard decoctions of different origins;

[0040] Figure 15 This is a flow chart for distinguishing the authenticity of Sala seeds and their different origins. DETAILED DESCRIPTION

[0041] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0042] Example 1

[0043] This example is used to confirm the optimal solution for the quality evaluation method of Salacia chinensis, specifically including the comparison of chromatographic conditions, wavelength selection, preparation of reference solution, and preparation of test solution, and the feasibility of the method is confirmed through methodological verification.

[0044] 1. Instruments and samples

[0045] Waters ACQUIYT UPLC-H-Class ultra-high performance liquid chromatography system; Waters Quaternary Solvent-Manager quaternary pump; Sample-Manager-FIN autosampler; Waters UPLC-PDA detector; Empower-3 chromatography workstation; Agilent Technologies 1290 Infinity ultra-high performance liquid chromatograph; 1290 DAD diode array detector; 1290 MCT column oven; 1290 Vial sampler autosampler; 1290 Flexible pump quaternary pump; OpenLAB CDS 2.3 chromatography workstation; Thermo Vanquish ultra-high performance liquid chromatograph; Chromeleam 7.2SR4 workstation; METTLER TOLEDO XP6 one-millionth balance (Mettler-Toledo (Shanghai Instrument) Co., Ltd.); ME204E electronic analytical balance (Mettler-Toledo Instrument (Shanghai) Co., Ltd.); PL-J100 mechanical ultrasonic cleaning machine (Dongguan Kangshijie Ultrasonic Technology Co., Ltd.); HH-4 digital display constant temperature water bath (Changzhou Guohua Electric Co., Ltd.); Milli-Q pure water system (Millipore Corporation); TGL-16C centrifuge (Shanghai Anting Scientific Instrument Factory).

[0046] The sarocarp formula granules (batch numbers: S19(KL), S20(KL), S21(KL)) were provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0047] The different origins of Aesculus hippocastanum meet all the requirements under Aesculus hippocastanum in the 2020 edition of the Chinese Pharmacopoeia. The information on the fake European horse chestnut and Yunnan horse chestnut is shown in Table 1.

[0048] Table 1

[0049]

[0050]

[0051] 2. Source of reference substances

[0052] Epicatechin (batch number: 110878-201703) was purchased from the China Food and Drug Administration.

[0053] 3. Determine the chromatographic conditions

[0054] An Eclipse Plus C18 column (2.1 mm × 100 mm, 1.8 μm) was used; acetonitrile was used as mobile phase A, and 0.01% to 0.2% phosphoric acid solution was used as mobile phase B, with gradient elution as specified in Table 2; the flow rate was 0.3 ml / min; and the column temperature was 30°C. The theoretical plate number, calculated based on epicatechin, should be no less than 5000.

[0055] Table 2 Gradient elution program in Example 1

[0056]

[0057] Depend on Figure 1 It can be seen that when mobile phase B is 0.01% to 0.2% phosphoric acid solution, all eight chromatographic peaks in the characteristic spectrum of the sago palm fruit are stable, the baseline is relatively smooth, and the spectrum is beautiful. Different phosphoric acid concentrations have little effect on the characteristic spectrum of the sago palm fruit. Subsequent experiments will use 0.1% phosphoric acid solution for further investigation.

[0058] 4. Wavelength selection

[0059] Take an appropriate amount of Salacia sutchuenensis formula granules (batch number: S19 (KL)), grind it into powder, take about 0.5g, put it into a stoppered conical flask, add 25ml of 70% methanol, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, take it out, let it cool, shake it well, filter it, and take the filtrate to obtain the Salacia sutchuenensis formula granule solution as the test solution. Accurately aspirate the test solution separately and inject it into the liquid chromatograph (flow rate of 0.3ml per minute; column temperature of 30℃), use acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, perform gradient elution according to the provisions in Table 2, and record the absorption spectrum in the range of 190-400nm (see Figure 2 ).

[0060] Depend on Figure 3 It can be seen that at the detection wavelength of 210-230nm, the chromatographic peak information of the characteristic spectrum of the sago seed is relatively rich, the response value is high, and the baseline is relatively stable, which can be used as the detection wavelength of the characteristic spectrum of the sago seed. At the detection wavelength of 220nm, the chromatographic peak size is relatively consistent, so the detection wavelength can be preferably 220nm.

[0061] 5. Preparation of reference solution

[0062] Take an appropriate amount of epicatechin reference substance, accurately weigh it, and add 30% methanol to make a solution containing 40 μg per 1 ml, which is used as the reference substance solution.

[0063] 6. Preparation of test solution

[0064] (1) Investigation of different extraction solvents

[0065] Take an appropriate amount of Salacia sutchuenensis granules (batch number: S19 (KL)), grind them into powder, take about 0.5g, a total of 6 groups, accurately weigh, place in a stoppered conical flask, accurately add water, 10% methanol, 30% methanol, 50% methanol, 70% methanol, methanol 25ml each, plug, weigh, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, let cool, weigh again, make up the lost weight with the corresponding solvent, shake well, filter, take the filtrate, and obtain the test solution. Accurately aspirate 2μl of each test solution, inject into the liquid chromatograph, and measure according to the chromatographic conditions determined above. The results are shown in the figure. Figure 4 And Table 3 below.

[0066] Table 3 Comparison of extraction efficiency of different extraction solvents (peak area / sample weight)

[0067]

[0068] When using different solvents for extraction, the chromatographic peak shapes were all good, and the overall extraction efficiencies were relatively similar, making all suitable extraction solvents for preparing the sample solution for the characteristic spectrum of the saury seeds. Considering the sum of peak area / sample weight and the optimal chromatographic peak shape, 70% methanol solution was the preferred extraction solvent for the characteristic spectrum determination of the saury seeds formula granules.

[0069] (2) Investigation of different extraction methods

[0070] Take an appropriate amount of Salacia sutchuenensis granules (batch number: S19 (KL)), grind them into powder, take about 0.5g, a total of 3 groups, accurately weigh, place in a stoppered conical flask, accurately add 25ml of 70% methanol, seal, weigh, ultrasonically treat (power 600W, frequency 40kHz), shake to extract, heat and reflux for 30 minutes, let cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, take the filtrate, and obtain the test solution. Accurately draw 2μl of each test solution, inject into the liquid chromatograph, and measure according to the chromatographic conditions determined above. The results are shown in the figure. Figure 5 and Table 4.

[0071] Table 4 Comparison of extraction efficiency of different extraction methods (peak area / sample weight)

[0072]

[0073] When ultrasound, shaking, and reflux were used as extraction methods, the number of chromatographic peaks was consistent, the peak shapes were good, and the overall extraction efficiency was relatively close. All of these extraction methods can be used as extraction methods for preparing the sample solution with the characteristic spectrum of the seed of Sauerkraut. From the perspective of ease of operation, ultrasound extraction is the preferred extraction method for determining the characteristic spectrum of the seed of Sauerkraut granules.

[0074] (3) Investigation of different extraction times

[0075] Take an appropriate amount of Salacia sutchuenensis granules (batch number: S19 (KL)), grind them into powder, take about 0.5g, a total of 4 groups, accurately weigh, place in a stoppered conical flask, accurately add 25ml of 70% methanol, seal, weigh, ultrasonically treat (power 600W, frequency 40kHz) for 15 minutes, 30 minutes, 45 minutes and 60 minutes respectively, shake well, filter, and take the filtrate to obtain the test solution. Accurately take 2μl of each test solution, inject it into the liquid chromatograph, and measure it according to the chromatographic conditions determined above. The results are shown in the figure. Figure 6 And Table 5.

[0076] Table 5 Comparison of extraction efficiency at different extraction times (peak area / sample weight)

[0077]

[0078] When the ultrasonication time was 15 minutes, 30 minutes, 45 minutes, and 60 minutes, the number of chromatographic peaks was consistent, the peak shape was good, and the overall extraction efficiency was relatively close. All of these can be used as the extraction time for preparing the sample solution with the characteristic spectrum of S. truncatum. Considering the ease of operation and reduced energy consumption, the extraction time was preferably 30 minutes.

[0079] (4) Determination of the preparation method of the test solution

[0080] After investigation, the preferred method for preparing the test solution is: take an appropriate amount of the product, grind it into powder, take about 0.5 g, place it in a stoppered conical flask, accurately add 25 ml of 70% methanol, seal it tightly, and ultrasonically treat it (power 600 W, frequency 40 kHz) for 30 minutes. Let it cool, shake it well, filter it, and take the filtrate to obtain it.

[0081] 7. Methodological Validation

[0082] (1) Specificity inspection

[0083] Take the above-prepared saroko seed formula granule test solution and negative solution (a solution prepared by the excipient preparation method of the test sample) and inject them into the liquid chromatograph respectively, and detect them under the chromatographic conditions determined in 3 of Example 1. Figure 7 .

[0084] The experimental results show that solvents and excipients have no interference with the characteristic peaks of the granule spectrum of the sarovar formula.

[0085] (2) Investigation of precision

[0086] Accurately aspirate the test solution and perform the test under the above-mentioned chromatographic conditions. Inject 6 times in succession, 2 μl each time. Take Peak 3 as the reference peak S, and calculate the relative retention time and relative peak area of ​​the remaining characteristic peaks relative to the S peak. The results are shown in Tables 6 and 7.

[0087] Table 6 Precision test results (relative retention time)

[0088]

[0089]

[0090] Table 7 Precision test results (relative peak area)

[0091]

[0092] The results showed that the RSDs of the relative retention time and relative peak area of ​​each characteristic peak were less than 1%, indicating that the instrument had good precision.

[0093] (3) Stability investigation

[0094] Accurately aspirate the test solution and perform the test under the above-mentioned chromatographic conditions. Inject the sample every 4 to 8 hours for a total of 24 hours. Inject 2 μl each time. Take Peak 3 as the reference peak S and calculate the relative retention time and relative peak area of ​​the remaining characteristic peaks to the S peak. The results are shown in Tables 8 and 9.

[0095] Table 8 Stability test results (relative retention time)

[0096]

[0097] Table 9 Stability test results (relative peak area)

[0098]

[0099] The results showed that the RSDs of the relative retention time and relative peak area of ​​each characteristic peak were less than 2%, and the test solution had good stability within 24 hours.

[0100] (4) Repeatability study

[0101] Take 0.5 g of Salacia chinensis formula granules (batch number: S19 (KL)), accurately weighed, and paralleled 6 times. The test solution was prepared according to the above test solution preparation method, and 2 μl was injected respectively. Peak 3 was used as the reference peak S, and the relative retention time and relative peak area of ​​the remaining characteristic peaks and the S peak were calculated. The results are shown in Tables 10 and 11.

[0102] Table 10 Repeatability test results (relative retention time)

[0103]

[0104] Table 11 Repeatability test results (relative peak area)

[0105]

[0106]

[0107] The results showed that the RSDs of the relative retention time and relative peak area of ​​each characteristic peak were less than 2%, indicating a good repeatability test.

[0108] (5) Durability test

[0109] 1) Chromatographic column inspection

[0110] In this study, the separation performance of three different brands of chromatographic columns, Eclipse Plus C18 (Agilent, 2.1×100mm, 1.8μm) (denoted as EP C18 in the figure), CORTECS UPLC T3 (Waters, 2.1×100mm, 1.6μm) (denoted as CORTECS T3 in the figure), and ZORBAX SB-C18 RRHD (Agilent, 2.1×100mm, 1.8μm) (denoted as SB C18 in the figure) was investigated for the sample solution. The results are shown in Figure 8 and Table 12.

[0111] Table 12 Effect of chromatographic column on separation effect (relative retention time)

[0112]

[0113] The results showed that the saffron seeds samples could be well separated on Eclipse Plus C18, CORTECS UPLC T3 and ZORBAX SB-C18RRHD columns, and the method had good durability on different chromatographic columns.

[0114] 2) Investigation of column temperature

[0115] The separation effect of the saro seeds granules was investigated using an Eclipse Plus C18 (Agilent, 2.1×100 mm, 1.8 μm) column at column temperatures of 25°C, 30°C, and 35°C. Figure 9 and Table 13.

[0116] Table 13 Effect of column temperature on separation effect (relative retention time)

[0117]

[0118]

[0119] The results showed that the analytical method had good robustness when the column temperature was between 25°C and 35°C. The relative retention times of the characteristic peaks were all within the specified range, and the small fluctuation in column temperature met the system suitability requirements.

[0120] 3) Investigation of flow rate

[0121] The separation effect of the saro seeds granules at flow rates of 0.25 ml / min, 0.30 ml / min, and 0.35 ml / min was investigated using an Eclipse Plus C18 (Agilent, 2.1×100 mm, 1.8 μm) column. Figure 10 and Table 14.

[0122] Table 14 Effect of flow rate on separation effect (relative retention time)

[0123]

[0124] The results showed that when the flow rate was in the range of 0.25ml / min to 0.35ml / min, the relative retention time of each characteristic peak was within the specified range. Small flow rate changes could meet the system applicability requirements, and the analytical method had good durability.

[0125] 4) Investigation of different instruments

[0126] The separation of the saffron seed granules was investigated using an Eclipse Plus C18 (Agilent, 2.1×100 mm, 1.8 μm) column with Agilent 1290, Waters H-Class, and Thermo Vanquish instruments. The results are shown in Table 1. Figure 11 and Table 15.

[0127] Table 15 Effect of instruments on separation effect (relative retention time)

[0128]

[0129] The results showed that when the instruments were Agilent 1290, Waters H-Class and Thermo Vanquish, the relative retention times of the characteristic peaks were all within the specified range, with good durability. Different instruments could meet the system suitability requirements.

[0130] Example 2

[0131] This example uses the optimal solution confirmed in Example 1 to construct an ultra-high performance liquid chromatography characteristic spectrum of the Shorea fruit preparation, and detects the Shorea fruit preparation to determine the characteristic peaks, reference peaks, and relative retention time values.

[0132] 1. Selection of common peaks

[0133] Using the chromatographic conditions, reference solution preparation method and test solution preparation method determined in Example 1, characteristic spectra of multiple batches of Salacia fructus preparations were measured respectively. The results are as follows: Figure 12 As shown, the common peak was determined.

[0134] 2. Identification of reference substances

[0135] Accurately pipette 2 μl of each reference solution and test solution, inject them into the liquid chromatograph, and measure them according to the chromatographic conditions determined in Example 1 to obtain (see Figure 13 ).

[0136] Based on practical feasibility and identification of reference substances, the final characteristic spectrum specifies 8 common peaks as characteristic peaks of Salacia chinensis for quality evaluation, with peak 3 being epicatechin, serving as reference peak S.

[0137] 3. Peak positioning - determination of relative retention time

[0138] The characteristic chromatogram of the Salacia fructus preparation should show eight characteristic peaks, and their retention times should correspond to the eight characteristic peaks in the chromatogram of the reference medicinal material. Peak 3 should correspond to the retention time of the corresponding reference peak. The peak corresponding to the epicatechin reference is the S peak. Calculate the relative retention times of Peaks 1, 2, 4, 5, 6, and 8 relative to the S peak. These relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.54 (Peak 1), 0.91 (Peak 2), 1.27 (Peak 4), 1.79 (Peak 5), 1.93 (Peak 6), 2.51 (Peak 7), and 2.98 (Peak 8).

[0139] Example 3

[0140] In this example, the optimal solution confirmed in Example 1 was used to construct the ultra-high performance liquid chromatography characteristic spectrum of the Shorea fruit preparation, and 33 batches of Shorea fruit preparations were tested. The authenticity and different origins of the Shorea fruit preparations were identified by the characteristic spectrum (see Figure 14 and Tables 16–21).

[0141] A value calculation formula:

[0142] B value calculation formula:

[0143] C value calculation formula:

[0144] Wherein, S1 is the peak area of ​​characteristic peak 1, S4 is the peak area of ​​characteristic peak 4, S7 is the peak area of ​​characteristic peak 7, and S8 is the peak area of ​​characteristic peak 8;

[0145] Table 1618 A value, B value, C value in the characteristic spectrum of the standard decoction of Psoralea corylifolia (Tianshi Li)

[0146]

[0147]

[0148] Table 173 A value, B value, C value in the characteristic spectrum of the granules of the formula of the seeds of the Chinese chestnut (Tianshi chestnut)

[0149]

[0150] Table 183 A value, B value, C value in the characteristic spectrum of the standard decoction of Aesculus hippocastanum (Horseidolia)

[0151]

[0152] Table 193 A value, B value, C value in the characteristic spectrum of the standard decoction of Aesculus hippocastanum (Zhejiang Aesculus)

[0153]

[0154] Table 203 A value, B value, C value in the characteristic spectrum of the standard decoction of Yunnan Aesculus hippocastanum

[0155]

[0156] Table 2. A value, B value, C value in the characteristic spectrum of 13 batches of standard decoction of Aesculus hippocastanum

[0157]

[0158] use Figure 15 Process identification.

[0159] Identification results: S1~S21 are Aesculus hippocastanum (Tianshi chestnut), S22~S24 are Aesculus hippocastanum (Horse Chestnut), S25~S27 are Aesculus hippocastanum (Zhejiang horse chestnut), S28~S30 are Aesculus yunnanensis, and S31~S33 are Aesculus hippocastanum.

Claims

1. A method for constructing a characteristic spectrum of a Salacia fruit preparation, characterized in that: The following steps are involved: (1) Take the test sample of the Salacia chinensis preparation, add the extraction solvent, extract, cool, filter, and the filtrate is the test sample solution; (2) Take epicatechin and add solvent to prepare a reference solution; (3) Take the test solution and the reference solution, inject them into the ultra-high performance liquid chromatograph respectively, perform ultra-high performance liquid chromatography detection, and record the characteristic chromatogram; The characteristic spectrum has 8 characteristic peaks, peak 3 is epicatechin, and the relative retention times of the remaining characteristic peaks and peak 3 are calculated. The relative retention times are all within the range of ±10% of the specified values. The specified values ​​of peaks 1 to 2 are 0.54 and 0.91, respectively, and the specified values ​​of peaks 4 to 8 are 1.27, 1.79, 1.93, 2.51, and 2.98, respectively. The preparation in step (1) includes a standard decoction or a formula granule; the extraction solvent is water, methanol or a methanol-water solution; The chromatographic column of the ultra-high performance liquid chromatograph in step (3) is Eclipse Plus C18, CORTECS UPLC T3 or ZORBAX SB-C18 RRHD; the Eclipse Plus C18 is Agilent, 2.1×100mm, 1.8μm; the CORTECS UPLC T3 is Waters, 2.1×100mm, 1.6μm; the ZORBAX SB-C18 RRHD is Agilent, 2.1×100mm, 1.8μm; Detection wavelength: 210-230 nm; acetonitrile was used as mobile phase A, and 0.01%-0.2% phosphoric acid solution was used as mobile phase B for gradient elution; the gradient elution was as follows: 0-2 min, the volume fraction of mobile phase A was 6→8%, and the volume fraction of mobile phase B was 94→92%; 2-16 min, the volume fraction of mobile phase A was 8→9%, and the volume fraction of mobile phase B was 92→91%; 16-28 min, the volume fraction of mobile phase A was 9→12%, and the volume fraction of mobile phase B was 91→88%; 28-45 min, the volume fraction of mobile phase A was 12→14%, and the volume fraction of mobile phase B was 88→86%.

2. The method for constructing the characteristic spectrum of the Salacia fructus preparation according to claim 1, wherein: The extraction method in step (1) includes ultrasound, shaking or reflux; the extraction time is 15 to 60 minutes.

3. The method for constructing the characteristic spectrum of the Salacia fructus preparation according to claim 1, wherein: The solvent in step (2) is 10-50% methanol.

4. The method for constructing the characteristic spectrum of the Salacia fructus preparation according to claim 1, wherein: The column temperature of the ultra-high performance liquid chromatography in step (3) is 25-35°C; the flow rate is 0.25-0.35 ml / min.

5. Application of a method for constructing a characteristic spectrum of a Sauerkraut preparation according to any one of claims 1 to 4 in quality inspection of Sauerkraut standard decoctions and formula granules.

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