Method for detecting specific chromatogram of granules for relieving depression and soothing nerves

Abstract

The invention discloses a method for detecting the characteristic chromatogram of granules for relieving depression and soothing nerves, which specifically comprises the following steps of: performing chromatographic condition and system applicability test: performing gradient elution by taking octadecylsilane chemically bonded silica as a filler, acetonitrile as a mobile phase A and 0.1% phosphoric acid as a mobile phase B, and detecting the wavelength of 210 nm; preparing a test solution; preparing a reference substance solution; respectively and precisely sucking the reference substance solution and the test solution and injecting the reference substance solution and the test solution into a high performance liquid chromatograph for determination, analysis and the like. The method disclosed by the invention is simple to operate, strong in specificity, short in detection time and accurate in detection result, can be used for quickly identifying the depression-resolving and nerve-soothing granules, can be used for comprehensively and quickly evaluating the quality of the depression-resolving and nerve-soothing granules, is beneficial to quality identification and quality control of the depression-resolving and nerve-soothing granules, and is suitable for popularization and application. Therefore, the safety and effectiveness of clinical medication are ensured.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine detection, and in particular to a method for detecting characteristic spectra of Jieyu Anshen granules. Background Art

[0002] Jieyu Anshen Granules are composed of 16 medicinal materials, including Bupleurum, jujube, Acorus calamus, Pinellia ternata, stir-fried Atractylodes macrocephala, floating wheat, processed Polygala tenuifolia, roasted licorice, stir-fried Gardenia jasminoides, lily, Arisaema cinerea, Curcuma aromatica, dragon teeth, stir-fried Ziziphus jujuba seeds, Poria cocos, and Angelica sinensis. It has the functions of soothing the liver and relieving depression, calming the mind and settling the spirit. It is used for mental stimulation such as emotional discomfort, liver depression and qi stagnation, anxiety, insomnia, forgetfulness, menopausal syndrome, neurosis, etc. Jieyu Anshen Granules are a compound preparation of traditional Chinese medicine, containing multiple traditional Chinese medicines and complex chemical components. Effective quality control is an important basis for ensuring its clinical efficacy. However, the current standard [Content Assay] Jieyu Anshen Granules only controls the gardenia glycoside in stir-fried Gardenia jasminoides, and cannot effectively and holistically evaluate the quality of Jieyu Anshen Granules, and it is difficult to effectively identify the quality of Jieyu Anshen Granules. Therefore, it is necessary to seek an effective method that can comprehensively control the quality of Jieyu Anshen Granules qualitatively or quantitatively, and a detection method that is conducive to promotion.

[0003] With the improvement of quality control technology of traditional Chinese medicine and compound preparations, the content determination of index components can no longer reflect the quality of traditional Chinese medicine and compound preparations. Characteristic spectrum is an effective method to show the overall quality of traditional Chinese medicine and compound preparations in modern analytical technology. The characteristic spectrum of traditional Chinese medicine comprehensively and holistically reflects the intrinsic quality of medicinal materials and can provide an effective reference method for the quality evaluation and control of traditional Chinese medicine. The 2020 edition of the "Chinese Pharmacopoeia" also adds the detection of characteristic spectrum for some traditional Chinese medicine varieties. Compared with traditional Chinese medicine detection methods, the characteristic spectrum method is of far-reaching significance in terms of accuracy. The drug detection process will not be limited by the form of the drug, and the stability, accuracy and reliability can be greatly guaranteed. Therefore, the establishment of the characteristic spectrum detection method of Jieyu Anshen Granules can not only make up for the shortcomings of the quality control of Jieyu Anshen Granules, but also facilitate the comprehensive evaluation and control of the quality of the preparations, thereby ensuring the safety and effectiveness of clinical medication. Summary of the invention

[0004] The purpose of the present invention is to provide a characteristic spectrum detection method for Jieyu Anshen Granules. The method has good specificity and short detection time. The method can realize rapid identification of Jieyu Anshen Granules and can evaluate the quality of Jieyu Anshen Granules more comprehensively, quickly and accurately, which is beneficial to the quality identification and quality control of Jieyu Anshen Granules, thereby ensuring the safety and effectiveness of clinical medication.

[0005] To achieve the above object, the present invention adopts the following technical solution: A method for detecting the characteristic spectrum of Jieyu Anshen granules is carried out by high performance liquid chromatography, and specifically comprises the following steps: (1) Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as the filler; acetonitrile was used as the mobile phase A, 0.1% phosphoric acid was used as the mobile phase B, and gradient elution was performed according to the provisions in Table 1 below; the flow rate was 1 ml per minute; the detection wavelength was 210 nm;

[0006] (2) Preparation of test solution: Take the product under the item of difference in loading amount, grind it into powder, take about 2 g or 1 g (without sucrose), weigh it accurately, put it into a stoppered conical flask, add 20 ml of 70% methanol accurately, weigh it, treat it with ultrasound for 30 minutes (power 600 W, frequency 40 kHz), let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain the solution; (3) Preparation of reference solution: Take 0.12 g of licorice control medicinal material, 0.16 g of polygala control medicinal material, 0.16 g of gardenia control medicinal material, 0.4 g of lily control medicinal material, and 0.2 g of spiny jujube seed control medicinal material, and prepare the reference mixed control medicinal material solution according to the preparation method of the test solution; Accurately weigh appropriate amounts of gardenia glycoside reference substance, liquiritin reference substance, spinosin reference substance, and 3,6'-dicorinoyl sucrose reference substance, and add 70% methanol to prepare a mixed reference substance solution containing 100 μg gardenia glycoside, 20 μg liquiritin reference substance, 5 μg spinosin reference substance, and 5 μg 3,6'-dicorinoyl sucrose per 1 ml; (4) Determination method: Accurately pipette 10 µl of reference solution and test solution respectively, inject into high performance liquid chromatography, and determine; The characteristic spectrum of the test sample should present 11 main characteristic peaks corresponding to the retention times in the spectrum of the reference medicinal material, among which the chromatographic peaks 2, 5, 6, and 8 should correspond to the retention times of the chromatographic peaks of the gardenia glycoside reference substance, spinosin reference substance, liquiritin reference substance, and 3,6'-diesinapoylsucrose reference substance, respectively.

[0007] Furthermore, in the above-mentioned characteristic spectrum detection method of Jieyu Anshen Granules, in step (4), 10µl of the reference solution and the test solution are respectively accurately aspirated, injected into a high performance liquid chromatograph, and measured and compared with the corresponding spectra of the reference solution and the test solution, so as to construct a characteristic spectrum of Jieyu Anshen Granules consisting of 11 common characteristic peaks. Among the 11 common peaks of the characteristic spectrum of Jieyu Anshen Granules, peak 2 is gardenia glycoside, peak 5 is spinosin, peak 6 is liquiritin, and peak 8 is 3,6'-diesinapoylsucrose. During the detection, the characteristic chromatogram of the test sample to be identified is compared and analyzed with the corresponding characteristic spectrum constructed.

[0008] The beneficial effects of the present invention are: 1. The present invention discloses a method for detecting the characteristic spectrum of Jieyu Anshen Granules. By reasonably controlling the HPLC conditions, the characteristic peaks of the obtained spectrum have good separation effect and many characteristic peaks. By comparing the presence or absence of characteristic peaks in the spectrum, the Jieyu Anshen Granules can be quickly identified, and the quality of the Jieyu Anshen Granules can be evaluated more comprehensively, quickly and accurately. The present invention provides a more comprehensive and rapid detection and identification method for the Jieyu Anshen Granules.

[0009] 2. The characteristic spectrum detection method of the Jieyu Anshen Granules of the present invention can be used to quickly and accurately determine whether the Jieyu Anshen Granules contain the Chinese medicinal materials of processed Polygala tenuifolia, stir-fried Ziziphus jujuba seeds, roasted Licorice root, stir-fried Gardenia jasminoides and Lilium, providing a strong guarantee for the qualitative identification of the Jieyu Anshen Granules.

[0010] 3. The method of the present invention can be further used to construct a characteristic spectrum of Jieyu Anshen Granules consisting of 11 common characteristic peaks, with many characteristic peaks, good separation of each characteristic peak, and good peak shape. The quality of Jieyu Anshen Granules can be evaluated more comprehensively, quickly and accurately, and then the Jieyu Anshen Granules can be identified and the quality controlled.

[0011] 4. The characteristic spectrum detection method of the Jieyu Anshen Granules of the present invention has the characteristics of simple operation, strong specificity, short detection time, accurate detection results, good separation, high precision, good repeatability and good stability. The detection method of the present invention can be used to evaluate and control the intrinsic quality of the Jieyu Anshen Granules more comprehensively, thereby ensuring the safety and effectiveness of clinical medication. The method of the present invention can provide a reliable basis for the authenticity identification and intrinsic quality of the Jieyu Anshen Granules, and provide a reference basis for further development and formulation of its quality standards. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Chromatogram of the reference medicinal material solution, in which, peak 2: gardenia glycoside, peak 5: spinosin, peak 6: liquiritin B, peak 8: 3,6'-diesinapoylsucrose; Figure 2 Chromatogram of mixed reference solution, in which, 2, gardenia glycoside, 5, spinosin, 6, liquiritin, 8, 3,6'-diesinapoylsucrose; Figure 3 Chromatograms of three batches of stir-fried Gardenia jasminoides and Gardenia jasminoides reference herbs (S1 is the reference herb); Figure 4 Chromatograms of 10 batches of stir-fried Ziziphus jujuba seeds and Ziziphus jujuba seeds as a reference medicinal material (S1 is the reference medicinal material); Figure 5 Chromatograms of 3 batches of lily medicinal materials and lily control medicinal materials (S1 is the control medicinal material); Figure 6 Chromatograms of 19 batches of roasted licorice herbs and licorice reference herbs (S1 is the reference herb); Figure 7 Chromatograms of 10 batches of processed Polygala tenuifolia medicinal materials and Polygala tenuifolia control medicinal materials (S1 is the control medicinal material); Figure 8 Chromatogram of Jieyu Anshen Granule sample; Fig. 9 HPLC graphs of gardenia glycosides, gardenia-deficient negative, gardenia control medicinal materials, and samples; Fig.10 HPLC graphs of spinosin, negative jujube seed, control jujube seed, and samples; Fig.11 Lack of lily negative, lily control medicinal materials, sample HPLC chart; Fig.12 HPLC graphs of liquiritin, licorice control medicinal materials, samples, and licorice-deficient negative; Fig.13 HPLC chart of 3,6-dicinapoylsucrose, lack of Polygala negative, Polygala control medicinal materials, and samples; Fig.14 Characteristic spectra (S1-S20) and control fingerprints (R) of 20 batches of Jieyu Anshen granules. DETAILED DESCRIPTION Example 1

[0013] 1. Instruments and test drugs Waters high performance liquid chromatograph; ML204 electronic analytical balance (Mettler-Toledo Instrument Shanghai Co., Ltd.). Gardenia glycoside (provided by China Food and Drug Inspection Institutes, for content determination, batch number: 110749-201919, content 97.1%); Liquorice glycoside (provided by China Food and Drug Inspection Institutes, for content determination, batch number: 111610-201908, content 95.0%); Spinosin (provided by China Food and Drug Inspection Institutes, for content determination, batch number: 111869-202005, content 94.8%); 3,6'-bisinacoylsucrose (provided by China Food and Drug Inspection Institutes, for content determination, batch number: 111848-202006, content 96.5%); Acetonitrile was chromatographically pure, and other reagents were analytically pure.

[0014] All 145 batches of Jieyu Anshen Granules are samples for the national drug random inspection in 2023.

[0015] Methods: The Jieyu Anshen Granules used in this study were produced by Jilin Province Luobang Pharmaceutical Co., Ltd., batch number: 2110001; the Jieyu Anshen Granules (without sucrose) were produced by Jilin Jierji Pharmaceutical Co., Ltd., batch number: 2206201.

[0016] 2. Investigation of experimental conditions and verification of methodology 2.1 Selection of mobile phase The effects of elution with methanol-water, acetonitrile-water, methanol-0.1% phosphoric acid solution, acetonitrile-0.1% phosphoric acid solution as mobile phases, as well as different gradients and flow rates were compared. After exploration, it was determined that acetonitrile was used as mobile phase A and 0.1% phosphoric acid was used as mobile phase B. The gradient was as shown in Table 1. Under this gradient, the separation effect was good and the peak shape was symmetrical.

[0017]

[0018] 2.2 Solution preparation 2.2.1 Preparation of test solution: Take the product under the item of difference in filling amount, grind it into powder, take about 2g or 1g (without sucrose), accurately weigh it, put it in a stoppered conical flask, accurately add 20ml of 70% methanol, weigh it, ultrasonicate for 30 minutes (power 600W, frequency 40kHz), cool it, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0019] 2.2.2 Preparation of reference solution 2.2.2.1 Preparation of reference drug solution According to the prescription, the dosage of each medicinal material was calculated when the sample sampling volume was 2g. Therefore, 0.12g of licorice control medicinal material, 0.16g of polygala control medicinal material, 0.16g of gardenia control medicinal material, 0.4g of lily control medicinal material, and 0.2g of spinach seed control medicinal material were taken. The mixed reference control medicinal material solution was prepared according to the preparation method of the test sample solution.

[0020] 2.2.2.2 Preparation of reference substance solution Accurately weigh 5.501 mg (content 97.1%) of gardenia glycoside reference substance and 5.121 mg (content 95.0%) of liquiritin reference substance and place them in 5 ml volumetric flasks respectively, and 5.538 mg (content 94.8%) of spinosin reference substance and 5.716 mg (content 96.5%) of 3,6'-dicorinoylsucrose reference substance and place them in 10 ml volumetric flasks respectively, add 70% methanol to dilute to the scale, shake well, and obtain (gardenia glycoside 1.0683 mg / ml, liquiritin reference substance 0.9730 mg / ml, spinosin reference substance 0.5250 mg / ml, 3,6'-dicorinoylsucrose 0.5516 mg / ml).

[0021] Accurately measure 1.00 ml of Gardenia glycoside reference stock solution, 200 μl of Licorice glycoside reference stock solution, 100 μl each of Spinosin reference stock solution and 3,6'-dicerinoylsucrose reference stock solution, place them in the same 5 ml volumetric flask, add 70% methanol to dilute to the scale as the mixed reference solution.

[0022] 2.2.3 Preparation of negative sample solution 2.2.3.1 Preparation of negative sample solution lacking stir-fried Gardenia, negative sample solution lacking stir-fried Ziziphus jujuba seeds, negative sample solution lacking roasted Licorice root, and negative sample solution lacking processed Polygala tenuifolia: According to the prescription and preparation method of the 2020 edition of the "Chinese Pharmacopoeia", the negative sample lacking stir-fried Gardenia, the negative sample lacking stir-fried Ziziphus jujuba seeds, the negative sample lacking roasted Licorice root, and the negative sample lacking processed Polygala tenuifolia were prepared respectively, and each negative sample solution was prepared according to the preparation of the test sample solution.

[0023] 2.2.3.2 Preparation of negative sample solution lacking lily: Weigh a certain amount of negative sample lacking lily according to the prescription ratio, and prepare the negative sample solution according to the preparation of the test solution.

[0024] 2.3 Characteristic peak confirmation This study investigated the characteristic peaks of five medicinal materials in Jieyu Anshen Granules, including stir-fried gardenia, stir-fried sour jujube seeds, lily, roasted licorice, and processed polygala. Among them, gardenia glycoside (peak 2) in stir-fried gardenia, spinosin (peak 5) in stir-fried sour jujube seeds, liquiritin (peak 6) in roasted licorice, and 3,6'-diesinapoylsucrose (peak 8) in processed polygala are all indicator components specified in the 2020 edition of the Chinese Pharmacopoeia.

[0025] Three batches of stir-fried Gardenia jasminoides, 10 batches of stir-fried Semen jujuba, 3 batches of Lilium, 19 batches of roasted Licorice, 10 batches of processed Polygala and their respective control materials were prepared into test solutions according to the above method and injected for analysis. The collected chromatograms were compared with the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition), see Figures 3 to 7 .

[0026] Combined with the chromatogram of Jieyu Anshen Granules, see Figure 8 It is clear that the characteristic peak No. 1 (unknown) in the sample comes from processed Gardenia jasminoides; the characteristic peak No. 3 (unknown) comes from Ziziphus jujuba seed; the characteristic peaks No. 4 and 7 (unknown) come from Lilium; the characteristic peak No. 9 (unknown) comes from Licorice; the characteristic peaks No. 10 and 11 (unknown) come from processed Polygala tenuifolia; the chromatographic peaks No. 2, 5, 6, and 8 should be compared with the Gardenia glycoside reference substance, Spinosin reference substance, Licorice glycoside reference substance, and 3,6'-diesinapoylsucrose reference substance, respectively.

[0027] 2.4 Investigation of test solution preparation 2.4.1 Selection of extraction method and extraction time 2.4.1.1 Compare different ultrasonic extraction times and heating reflux extraction.

[0028] Jieyu Anshen Granules Ultrasonic extraction: Take the product under the item of difference in loading amount, grind it into powder, take about 2g, weigh it accurately, put it in a stoppered conical flask, accurately add 20ml of 70% methanol, weigh it, ultrasonicate for 15 minutes, 30 minutes, and 60 minutes (power 600W, frequency 40kHz), cool it, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0029] Heating reflux extraction: Take the product under the item of difference in loading amount, grind it into powder, take about 2g, weigh it accurately, put it in a stoppered conical flask, accurately add 20ml of 70% methanol, weigh it, heat and reflux for 30 minutes, cool, weigh it again, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate to obtain it.

[0030] Jieyu Anshen Granules (Sugar-free) Ultrasonic extraction: Take the product under the item of difference in loading amount, grind it into powder, take about 1g, weigh it accurately, put it in a stoppered conical flask, accurately add 20ml of 70% methanol, weigh it, ultrasonicate for 15 minutes, 30 minutes, and 60 minutes (power 600W, frequency 40kHz), cool it, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0031] Heating reflux extraction: Take the product under the item of difference in loading amount, grind it into powder, take about 1g, accurately weigh it, put it in a stoppered conical flask, accurately add 20ml of 70% methanol, weigh the weight, heat and reflux for 30 minutes, cool, weigh it again, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate to obtain it.

[0032] The results showed that the contents of the tested components in ultrasonic extraction and heating reflux extraction of sucrose-containing and sucrose-free granules were similar, and ultrasonic extraction for 30 minutes was relatively complete, so ultrasonic extraction for 30 minutes was selected. See Tables 2 and 3 for details.

[0033]

[0034]

[0035] 2.4.2 Selection of extraction solvent type The extraction effects of six different solvents, including methanol, 70% methanol, 50% methanol, 20 ml of 50% ethanol, 20 ml of 70% ethanol, and 20 ml of water, were investigated respectively.

[0036] Take the contents of this product under the item of filling quantity difference, grind it finely, take about 2g or 1g (without sucrose), weigh it accurately, put it in a stoppered conical flask, accurately add 20ml of each of the above 6 solvents, weigh it, ultrasonicate for 30 minutes (power 600W, frequency 40kHz), cool it, weigh it again, make up the lost weight with the above solvents respectively, shake it well, filter it, and take the filtrate to obtain it.

[0037] The test results show that the extraction contents of alcohol solutions of different concentrations are relatively close, all higher than the content of water extraction. The content of the tested components extracted by 70% methanol is slightly higher than that of the other five solvents, so 70% methanol is selected as the extraction solvent, see Tables 4 and 5 for details.

[0038]

[0039]

[0040] 2.4.3 Selection of the amount of extraction solvent added The extraction effects of three different addition volumes of 70% methanol, 10ml, 20ml and 40ml, were investigated respectively.

[0041] Take the contents of this product under the item of filling amount difference, grind it finely, take about 2g or 1g (without sucrose), weigh it accurately, put it in a stoppered conical flask, accurately add 10ml, 20ml, and 40ml of 70% methanol respectively, weigh it, ultrasonicate for 30 minutes (power 600W, frequency 40kHz), cool it, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0042] The test results show that when the extraction solvent volume is 20ml, the components to be tested can be completely extracted. Therefore, 20ml of 70% methanol is selected as the extraction solvent, see Table 6-7 for details.

[0043]

[0044]

[0045] Based on the above research, the preparation method of the test solution is proposed as follows: Take the product under the item of filling amount difference, grind it into powder, take about 2g or 1g (without sucrose), weigh it accurately, put it in a stoppered conical flask, add 20ml of 70% methanol accurately, weigh it, ultrasonicate for 30 minutes (power 600W, frequency 40kHz), cool it, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0046] 2.5 Exclusivity According to the prescription and preparation method of the 2020 edition of the Chinese Pharmacopoeia, negative samples lacking stir-fried Gardenia, lacking stir-fried Ziziphus jujuba seeds, lacking Lilium bulgurii seeds, lacking roasted Licorice root, lacking processed Polygala tenuifolia, and lacking Acorus tatarinowii were prepared, and the respective negative sample solutions were prepared according to the preparation of the test solution. The chromatograms of the negative sample solutions did not show the same peaks as the respective control medicinal material reference solutions.

[0047] In summary, this method has good specificity. Figures 9 to 13 , characteristic peaks 1 (unknown) and 2 (gardenoside) come from processed Gardenia jasminoides; characteristic peaks 3 (unknown) and 5 (spinosyl) come from Semen Ziziphi Spinosae; characteristic peaks 4 and 7 (unknown) come from Lilium bulgurica; characteristic peaks 6 (liquiritigenin) and 9 (unknown) come from Licorice; characteristic peaks 8 (3,6'-diesinapoylsucrose), 10 (unknown), and 11 (unknown) come from processed Polygala tenuifolia.

[0048] 2.6 Instrument precision The Jieyu Anshen granules test solution was injected 6 times continuously according to the proposed chromatographic conditions, and the characteristic spectrum was recorded. The retention time of the characteristic peak was calculated. The retention time RSD of each characteristic peak was <3.0%, and the results are shown in Tables 8 and 9, indicating that the instrument precision is good. The reference control medicinal material solution is abbreviated as control (the same below).

[0049]

[0050]

[0051] 2.7 Repeatability Take the Jieyu Anshen Granules test sample, and prepare 6 test sample solutions in parallel according to the proposed method. According to the proposed chromatographic conditions, the solution was injected into the HPLC, the characteristic spectrum was recorded, and the retention time of the characteristic peak was calculated. The results showed that the RSD of the retention time of each characteristic peak was less than 3.0%. The results are shown in Tables 10 and 11, indicating that the method has good repeatability.

[0052]

[0053]

[0054] 2.8 Stability The test solution of Jieyu Anshen Granules was taken and measured at 0h, 4h, 8h, 12h, 18h and 24h according to the proposed chromatographic conditions. The characteristic spectrum was recorded and the retention time of the characteristic peaks was calculated. The results showed that the RSD of the retention time of each characteristic peak was <3.0%. The results are shown in Tables 12 and 13, indicating that the test solution was stable within 24 hours.

[0055]

[0056]

[0057] 3. HPLC characteristic spectra of 20 batches of Jieyu Anshen granules samples Take 2 batches of Jieyu Anshen Granules from each manufacturer, a total of 20 batches (batch numbers: S1-S20), prepare the test solution according to the method under "2.2", and inject and record the chromatogram according to the chromatographic conditions under "2.1", that is, according to the chromatographic conditions specified in the method of the present invention. Use the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 Edition) for common peak matching, select 12 chromatographic peaks with good repeatability and high separation that can be attributed to the prescription medicinal flavors as common peaks, and generate HPLC characteristic spectra of 20 batches of samples. The characteristic spectra (S1-S20) and the control fingerprint (R) of 20 batches of Jieyu Anshen Granules are shown in Fig.14 . Example 2

[0058] A method for detecting the characteristic spectrum of Jieyu Anshen granules is carried out by high performance liquid chromatography, and specifically comprises the following steps: (1) Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as the filler; acetonitrile was used as the mobile phase A, 0.1% phosphoric acid was used as the mobile phase B, and gradient elution was performed according to the provisions in Table 1 below; the flow rate was 1 ml per minute; the detection wavelength was 210 nm;

[0059] (2) Preparation of test solution: Take the product under the item of difference in loading amount, grind it into powder, take about 2 g or 1 g (without sucrose), weigh it accurately, put it into a stoppered conical flask, add 20 ml of 70% methanol accurately, weigh it, treat it with ultrasound for 30 minutes (power 600 W, frequency 40 kHz), let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain the solution; (3) Preparation of reference solution: Take 0.12 g of licorice control medicinal material, 0.16 g of polygala control medicinal material, 0.16 g of gardenia control medicinal material, 0.4 g of lily control medicinal material, and 0.2 g of spiny jujube seed control medicinal material, and prepare the reference mixed control medicinal material solution according to the preparation method of the test solution; Accurately weigh appropriate amounts of gardenia glycoside reference substance, liquiritin reference substance, spinosin reference substance, and 3,6'-dicorinoyl sucrose reference substance, and add 70% methanol to prepare a mixed reference substance solution containing 100 μg gardenia glycoside, 20 μg liquiritin reference substance, 5 μg spinosin reference substance, and 5 μg 3,6'-dicorinoyl sucrose per 1 ml; (4) Determination method: Accurately pipette 10 µl of reference solution and test solution respectively, inject into high performance liquid chromatography, and determine; The characteristic spectrum of the test sample should present 11 main characteristic peaks corresponding to the retention times in the spectrum of the reference medicinal material, among which the chromatographic peaks 2, 5, 6, and 8 should correspond to the retention times of the chromatographic peaks of the gardenia glycoside reference substance, spinosin reference substance, liquiritin reference substance, and 3,6'-diesinapoylsucrose reference substance, respectively.

[0060] Furthermore, in the above-mentioned characteristic spectrum detection method of Jieyu Anshen Granules, in step (4), 10µl of the reference solution and the test solution are respectively accurately aspirated, injected into a high performance liquid chromatograph, and the corresponding spectra of the reference solution and the test solution are measured and compared, so that a characteristic (fingerprint) spectrum of Jieyu Anshen Granules consisting of 11 common characteristic peaks can be constructed. Among the 11 common peaks of the characteristic (fingerprint) spectrum of Jieyu Anshen Granules, peak 2 is gardenia glycoside, peak 5 is spinosin, peak 6 is liquiritin, and peak 8 is 3,6'-diesinapoylsucrose. During the detection, the characteristic chromatogram of the test sample to be identified is compared and analyzed with the corresponding characteristic (fingerprint) spectrum constructed.

Claims

1. A method for detecting characteristic spectrum of Jieyu Anshen granules, characterized in that: The detection is carried out by high performance liquid chromatography, which specifically includes the following steps: (1) Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as the filler; acetonitrile was used as the mobile phase A, 0.1% phosphoric acid was used as the mobile phase B, and gradient elution was performed according to the provisions in Table 1 below; the flow rate was 1 ml per minute; the detection wavelength was 210 nm; (2) Preparation of test solution: Take the product under the item of difference in loading amount, grind it into powder, take about 2g or 1g (without sucrose), weigh it accurately, put it into a stoppered conical flask, add 20ml of 70% methanol accurately, weigh it, and treat it ultrasonically for 30 minutes at a power of 600W and a frequency of 40kHz; let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain the solution; (3) Preparation of reference solution: Take 0.12 g of licorice control medicinal material, 0.16 g of polygala control medicinal material, 0.16 g of gardenia control medicinal material, 0.4 g of lily control medicinal material, and 0.2 g of spiny jujube seed control medicinal material, and prepare the reference mixed control medicinal material solution according to the preparation method of the test solution; Accurately weigh appropriate amounts of gardenia glycoside reference substance, liquiritin reference substance, spinosin reference substance, and 3,6'-dicorinoyl sucrose reference substance, and add 70% methanol to prepare a mixed reference substance solution containing 100 μg gardenia glycoside, 20 μg liquiritin reference substance, 5 μg spinosin reference substance, and 5 μg 3,6'-dicorinoyl sucrose per 1 ml; (4) Determination method: Accurately pipette 10 µl of reference solution and test solution respectively, inject into high performance liquid chromatography, and determine; The characteristic spectrum of the test sample should present 11 main characteristic peaks corresponding to the retention times in the spectrum of the reference medicinal material, among which the chromatographic peaks 2, 5, 6, and 8 should correspond to the retention times of the chromatographic peaks of the gardenia glycoside reference substance, spinosin reference substance, liquiritin reference substance, and 3,6'-diesinapoylsucrose reference substance, respectively.

2. The characteristic spectrum detection method of Jieyu Anshen granules according to claim 1, characterized in that: In step (4), 10 µl of the reference solution and the test solution are respectively accurately aspirated and injected into a high performance liquid chromatograph to measure and compare the spectra corresponding to the reference solution and the test solution, thereby constructing a characteristic spectrum of the Jieyu Anshen Granules consisting of 11 common characteristic peaks. Among the 11 common peaks of the characteristic spectrum of the Jieyu Anshen Granules, peak 2 is gardenia glycoside, peak 5 is spinosin, peak 6 is liquiritin, and peak 8 is 3,6'-dicerinoylsucrose.

3. The characteristic spectrum detection method of Jieyu Anshen granules according to claim 2 is characterized in that: During the detection, the characteristic chromatogram of the sample to be identified is compared and analyzed with the corresponding characteristic spectrum constructed.

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