A method for constructing a characteristic spectrum of Dajianzhong Decoction and a method for determining the content of its effective components.

Abstract

This invention belongs to the field of pharmaceutical analysis technology, specifically relating to a method for constructing characteristic chromatograms of Dajianzhong Decoction and a method for determining the content of its effective components. The construction method includes the preparation of a test solution, and under specific conditions, characteristic chromatograms 1 and 2 are obtained using UPLC-UV-ELSD chromatography. Characteristic chromatogram 1, obtained under ultra-high performance liquid chromatography and a UV detector, is specific to dried ginger and roasted Sichuan pepper, with characteristic peaks mainly consisting of gingerol components in dried ginger and amide alkaloids in roasted Sichuan pepper. Characteristic chromatogram 2, obtained under ultra-high performance liquid chromatography and an evaporative light scattering detector, is specific to ginseng, consisting entirely of ginsenosides, thus solving the problem of large differences in detection wavelengths and mutual interference between the two major categories of components.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for constructing a characteristic fingerprint of Dajianzhong Decoction and a method for determining the content of active ingredients. Background Art

[0002] Dajianzhong Decoction comes from "Synopsis of Prescriptions of the Golden Chamber · Treatise on the Pulse, Syndrome, and Treatment of Abdominal Fullness, Cold Hernia, and Retained Food - Chapter 10", and the prescription consists of two heaping tablespoons of Sichuan pepper (remove the sweat), four liang of dried ginger, two liang of ginseng, and one sheng of gelatinized maltose. Its efficacy is to warm the middle - jiao and replenish deficiency, reduce adverse qi and relieve pain, and it is mainly used to treat the syndrome of weakness of middle - yang and internal exuberance of yin - cold. In the formula, Sichuan pepper is pungent, hot, and greatly warming, warming the middle - jiao to disperse cold, reducing adverse qi and relieving pain, and can also expel roundworms and kill insects, serving as the monarch drug; dried ginger is pungent, hot, and greatly warming, warming the middle - jiao to disperse cold, harmonizing the stomach and stopping vomiting, serving as the minister drug; due to the deficiency of middle - yang resulting in internal exuberance of yin - cold, ginseng is used, which is sweet and warm, tonifying the spleen and stomach, and supporting healthy qi, serving as the assistant drug; gelatinized maltose supplements the middle - jiao and replenishes deficiency, relieves spasm and pain, and also moderates the pungent and intense nature of Sichuan pepper and dried ginger, serving as the guiding drug. The four herbs are combined to form a formula for warming the middle - jiao and replenishing deficiency, reducing adverse qi and relieving pain.

[0003] The medicinal herbs of Sichuan pepper (stir - fried), dried ginger, and ginseng in Dajianzhong Decoction are all included in the first volume of the Chinese Pharmacopoeia (2020 Edition). In the current pharmacopoeia standard of dried ginger, there is a quantitative standard for the index component 6 - gingerol. In the current pharmacopoeia standard of ginseng, there are quantitative standards for ginsenosides Rg1, Re, and Rb1. There is no quantitative standard for index components in stir - fried Sichuan pepper. There are many relevant literatures on the quality research of each single medicinal herb, but there are few reports on the quality research of Dajianzhong Decoction or related prescriptions. In the prior art, in the quality evaluation of Dajianzhong Decoction formula granules based on traditional decoctions by Zhang Yijing et al., the detection effect was not good, the baseline was not stable, and the detection results did not match the actual situation. In the research on the quality standard of Dajianzhong Decoction granules by Li Xiang et al., the prescription they targeted did not match the key information of the current classic famous prescription, and gelatinized maltose was not added. After adding gelatinized maltose to the current prescription, the prepared sample was similar to an extract, and it was difficult to prepare the test sample for physical detection and analysis. Chinese patent document CN111505196 A discloses a quality control method for the substance reference of Dajianzhong Decoction, and this detection method has the defects of being cumbersome and time - consuming. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the defects of the existing detection methods for Dajianzhong Decoction, such as long detection period, poor detection effect (such as unstable baseline, inconsistent with the actual situation, cumbersome operation due to targeting single medicinal materials), and poor reproducibility. Thus, a method for constructing a characteristic fingerprint of Dajianzhong Decoction and a method for determining the content of active ingredients are provided.

[0005] For this purpose, the present invention provides the following technical solutions.

[0006] The first aspect of the present invention provides a method for constructing a characteristic fingerprint of Dajianzhong Decoction, comprising the following steps:

[0007] Preparation of test solution: Prepare a test solution from the test sample;

[0008] UPLC-UV-ELSD chromatography was used for detection. The chromatographic conditions of UPLC-UV-ELSD chromatography included: methanol as mobile phase A, formic acid aqueous solution as mobile phase B, and gradient elution. The gradient elution program included: 0-12 min, 50% mobile phase A, 50% mobile phase B; 12-15 min, 50%→65% mobile phase A, 50%→35% mobile phase B; 15-30 min, 65%→70% mobile phase A, 35%→30% mobile phase B; 30-35 min, 70%→80% mobile phase A, 30%→20% mobile phase B; 35-40 min, 80% mobile phase A, 20% mobile phase B.

[0009] Ultra-high performance liquid chromatography (UPLC)-ultraviolet (UV)-evaporative light scattering detection (ELSD).

[0010] The chromatographic conditions for the UPLC-UV-ELSD chromatography method described in the construction method further include:

[0011] Detector: The ultra-high performance liquid chromatograph is equipped with a UV detector and an evaporative light scattering detector in series; and / or,

[0012] The detection wavelength of the ultraviolet detector is 270 nm to 290 nm; and / or,

[0013] The carrier gas flow rate of the evaporative light scattering detector is 2.3–2.8 L / min; and / or,

[0014] The drift tube temperature is 90–100°C; and / or,

[0015] The column temperature of the ultra-high performance liquid chromatograph is 25–35 °C; and / or,

[0016] The flow rate is 0.35–0.45 ml / min; and / or,

[0017] The chromatographic column was an Agilent Infinity Lab Poroshell 120EC-C18, with specifications of an inner diameter of 3.0 mm, a column length of 150 mm, and a particle size of 2.7 μm; and / or,

[0018] Use 0.05–0.1% formic acid aqueous solution as mobile phase B; and / or,

[0019] The sample size is 2–4 μL.

[0020] The chromatographic conditions for the UPLC-UV-ELSD method described above are as follows: methanol as mobile phase A, 0.1% formic acid aqueous solution as mobile phase B; column temperature 30℃, flow rate 0.4 ml / min, sample volume 3 μL; UV detector detection wavelength 280 nm, theoretical plate number calculated based on 6-gingerol peak should not be less than 10000; evaporative light scattering detector carrier gas flow rate 2.5 L / min, drift tube temperature 95℃, theoretical plate number calculated based on ginsenoside Rb1 peak should not be less than 10000.

[0021] The construction method yields feature map 1 and / or feature map 2;

[0022] The characteristic spectrum 1 has 8 characteristic peaks. Taking peak 1 as the reference peak, the specified values ​​for the relative retention times of peaks 2, 3, 4, 5, 6, 7, and 8 are 1.138, 1.177, 1.219, 1.393, 1.467, 1.580, and 1.651, respectively; the relative retention times are within ±8% of the specified values.

[0023] The characteristic spectrum 2 has 8 characteristic peaks. Taking peak 5 as the reference peak, the specified values ​​for the relative retention times of peaks 3, 4, 6, 7 and 8 are 0.680, 0.928, 1.030, 1.129 and 1.253, respectively; the relative retention times are within ±8% of the specified values.

[0024] In the characteristic spectrum 1, peak 1 is 6-gingerol;

[0025] Preferably, in the characteristic spectrum 2, peak 1 is ginsenoside Re, peak 2 is ginsenoside Rg1, and peak 5 is ginsenoside Rb1.

[0026] The method for preparing the test sample solution includes: taking the test sample, diluting it, adding an extraction solvent, extracting it, and obtaining the test sample solution;

[0027] Preferably, the extraction solvent is water-saturated n-butanol;

[0028] Preferably, the extraction is performed 3-5 times;

[0029] Preferably, the dilution factor is 2-4 times.

[0030] The test sample was either a decoction or a paste of Dajianzhong Decoction.

[0031] The construction method further includes the step of preparing a reference solution;

[0032] Preferably, at least one of 6-gingerol, hydroxy-α-salicornin, hydroxy-β-salicornin, ginsenoside Rg1, ginsenoside Re and ginsenoside Rb1 is used as a reference standard.

[0033] Preferably, each 1 ml of the reference solution contains 0.15–0.25 mg of 6-gingerol, 0.25–0.35 mg of hydroxy-α-salicornin, 0.025–0.035 mg of hydroxy-β-salicornin, 0.15–0.25 mg of ginsenoside Rg1, 0.15–0.25 mg of ginsenoside Re, and 0.15–0.25 mg of ginsenoside Rb1.

[0034] The second aspect of the present invention provides a method for determining the content of effective components in Dajianzhong Decoction, which uses the above-mentioned construction method and employs the external standard method to determine the content of effective components.

[0035] The active ingredients include at least one of 6-gingerol, hydroxy-α-salicornin, hydroxy-β-salicornin, ginsenoside Rg1, ginsenoside Re, and ginsenoside Rb1.

[0036] The technical solution of this invention has the following advantages:

[0037] 1. The present invention provides a method for constructing the characteristic spectrum of Dajianzhong Decoction. This method uses a UV detector and an evaporative light scattering detector in series to obtain characteristic spectrum 1 and characteristic spectrum 2. Characteristic spectrum 1, obtained under ultra-high performance liquid chromatography and a UV detector, is specific to dried ginger and roasted Sichuan pepper, with characteristic peaks mainly consisting of gingerol components in dried ginger and amide alkaloid components in roasted Sichuan pepper. Characteristic spectrum 2, obtained under ultra-high performance liquid chromatography and an evaporative light scattering detector, is a characteristic spectrum specific to ginseng, consisting entirely of ginsenosides. This method solves the problem of large differences in detection wavelengths and mutual interference between the two categories of components.

[0038] 2. The method for constructing the characteristic spectrum of Dajianzhong Decoction provided by the present invention reduces the total detection time to 40 minutes, and the obtained characteristic spectrum has a stable baseline, good separation between characteristic peaks, and good reproducibility.

[0039] 3. The method for constructing the characteristic chromatogram of Dajianzhong Decoction provided by this invention is a multi-evaluation method that simultaneously measures the content of six active ingredients while determining the characteristic chromatogram, with no interference from negative samples. The method has passed methodological validation, and the results show good specificity, linearity, repeatability, accuracy, stability of the test solution, and robustness. When preparing the test solution, water-saturated n-butanol is used as the extraction solvent, and dilution before extraction ensures sufficient extraction of the active ingredients.

[0040] 4. The method for determining the content of effective components in Dajianzhong Decoction provided by this invention shortens the detection time, allows for multiple evaluations in one test, makes full use of the characteristics of different detectors, and analyzes different types of chemical components at the same time. The detection method is highly efficient and energy-saving. Attached Figure Description

[0041] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0042] Figure 1 This is the control characteristic spectrum 1 of the 15 batches of Dajianzhong Decoction reference samples in Experimental Example 1 of this invention;

[0043] Figure 2 This is the characteristic spectrum 1 obtained from 15 batches of Dajianzhong Decoction reference samples in Experiment Example 1 of this invention;

[0044] Figure 3 This is the chromatogram of the negative sample and the reference sample when establishing characteristic chromatogram 1 in Experimental Example 1 of the present invention;

[0045] Figure 4 This refers to the chromatogram and characteristic chromatogram 1 of the reference standard in Experimental Example 1 of the present invention when establishing characteristic chromatogram 1;

[0046] Figure 5 This is the comparative characteristic spectrum 2 of the 15 batches of Dajianzhong Decoction reference samples in Experiment Example 1 of this invention;

[0047] Figure 6 This is the characteristic spectrum 2 obtained from 15 batches of Dajianzhong Decoction reference samples in Experiment Example 1 of this invention;

[0048] Figure 7 This is the chromatogram of the negative sample and the reference sample when establishing characteristic chromatogram 2 in Experiment Example 1 of the present invention;

[0049] Figure 8 This is the chromatogram of the reference standard in Experiment Example 1 of the present invention when establishing characteristic chromatogram 2;

[0050] Figure 9 This is the feature map 2 of Experimental Example 1 of the present invention;

[0051] Figure 10 This refers to the characteristic chromatogram 1 obtained from different chromatographic columns in Section 1.2 of Experimental Example 2 of this invention;

[0052] Figure 11 This is the characteristic spectrum 1 obtained from different mobile phases in Section 1.3 of Experimental Example 2 of this invention;

[0053] Figure 12 This is the characteristic spectrum 1 obtained at different column temperatures in Section 1.4 of Experimental Example 2 of this invention;

[0054] Figure 13 This is the characteristic spectrum 1 obtained from different flow rates in section 1.5 of Experimental Example 2 of this invention;

[0055] Figure 14 This is the chromatogram of the blank experiment and the investigation of double retention time in Section 2.4 of Experimental Example 2 of the present invention;

[0056] Figure 15 These are the chromatograms obtained from different chromatographic columns in Section 1.1 of Experimental Example 3 of this invention;

[0057] Figure 16 These are chromatograms obtained from different mobile phases in Experimental Example 3 of this invention, section 1.2.

[0058] Figure 17 These are the chromatograms obtained at different column temperatures in Section 1.3 of Experimental Example 3 of this invention;

[0059] Figure 18 These are the chromatograms obtained at different flow rates in Section 1.3 of Experimental Example 3 of this invention.

[0060] Figure 19 This is the chromatogram obtained in Section 2.4 of Experimental Example 3 of this invention;

[0061] Figure 20 The spectrum is obtained from the specificity experiment in section 2.6 of Embodiment 2 of this invention. Detailed Implementation

[0062] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0063] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0064] instrument:

[0065] Ultra-high performance liquid chromatograph: Waters ACQUITY UPLC H-CLASS series, ALLTECH ELSD6000 evaporative light scattering detector; Ultrasonic cleaner: KQ-500DE CNC ultrasonic cleaner, power 500W, frequency 40kHz; Analytical balance: German Sartorius BT25S, BS2202S and BS124S.

[0066] Drug trials:

[0067] Acetonitrile was chromatographically pure (OCEANPAK, Tianjin Biaoshiqi Technology Development Co., Ltd.), methanol was chromatographically pure (Fisher Scientific), water was ultrapure water, anhydrous ethanol was analytically pure (Beijing Tongguang Fine Chemical Co., Ltd.), ethanol (95%) was analytically pure (Beijing Tongguang Fine Chemical Co., Ltd.), phosphoric acid was analytically pure (Beijing Tongguang Fine Chemical Co., Ltd.), anhydrous formic acid (98%) (Sinopharm Chemical Reagent Co., Ltd.), glacial acetic acid was analytically pure (Beijing Chemical Plant), and n-butanol was analytically pure (Beijing Tongguang Fine Chemical Co., Ltd.).

[0068] 6-Gingerol, batch number 111833-201806, purity 99.90%, China National Institutes for Food and Drug Control; Hydroxy-α-Sinopsin, batch number PS012248, purity >95%, Chengdu Pusi Biotechnology Co., Ltd.; Hydroxy-β-Sinopsin, batch number DSTDQ071601, purity ≥98%, Chengdu Dester Biotechnology Co., Ltd.; Ginsenoside Re, batch number 110754-202129, purity 96.0%, China National Institutes for Food and Drug Control; Ginsenoside Rg1, batch number 110703-202034, purity 94.0%, China National Institutes for Food and Drug Control; Ginsenoside Rb1, batch number 110704-202129, purity 94.3%, China National Institutes for Food and Drug Control; Ginsenoside Rb2, batch number 111 715-201203, purity 93.8%, China National Institutes for Food and Drug Control; Ginsenoside Rb3, batch number 111686-201504, purity 97.0%, China National Institutes for Food and Drug Control; Ginsenoside Rf, batch number MUST-16041002, purity 99.62%, Chengdu Manster Biotechnology Co., Ltd.; Ginsenoside Ro, batch number DST160910-031, purity ≥98%, Chengdu Dester Biotechnology Co., Ltd.; Ginsenoside Rd, batch number 130713, purity >99%, Shanghai Ronghe Pharmaceutical Technology Co., Ltd.; Ginsenoside Rg2, batch number 130622, purity >98%, Shanghai Ronghe Pharmaceutical Technology Co., Ltd.; Ginsenoside Rc, batch number 130724, purity >99%, Shanghai Ronghe Pharmaceutical Technology Co., Ltd.

[0069] The preparation method of the Da Jian Zhong Tang reference sample used in the following examples and experimental cases includes: 91.8g of stir-fried Sichuan pepper, dried ginger, and ginseng were weighed according to the prescription ratio, placed in a clay pot, and 800ml of water was added. The mixture was brought to a boil over high heat, then simmered over low heat until approximately 400ml remained. The mixture was filtered, the dregs were discarded, and 200ml of maltose was added to the decoction. The mixture was brought to a boil over high heat, then simmered over low heat until approximately 300ml remained, yielding the Da Jian Zhong Tang reference sample. Fifteen batches each of different batches of stir-fried Sichuan pepper, dried ginger, and ginseng were randomly combined to prepare a total of 15 batches of Da Jian Zhong Tang reference samples, with batch numbers corresponding to DJZT0901 to DJZT0915. The Da Jian Zhong Tang prescription, by weight, includes 9 parts stir-fried Sichuan pepper, 55.2 parts dried ginger, and 27.6 parts ginseng.

[0070] Example 1

[0071] This embodiment provides a method for constructing the feature map of Dajianzhong Decoction, including the following steps:

[0072] Preparation of the test solution: Take the Dajianzhong Decoction reference sample, shake well, take about 16g, weigh it, put it in a 50ml volumetric flask, add water to dilute to the mark (the volume calculated based on 16g of Dajianzhong Decoction reference sample is about 12.5ml, the dilution factor is about 4 times), shake well, accurately measure 25ml, put it in a separatory funnel, add water-saturated n-butanol and shake to extract 4 times, 25ml each time, combine the n-butanol extracts, recover the solvent to dryness, add an appropriate amount of 70% methanol to dissolve the residue, transfer it to a 5ml volumetric flask, add 70% methanol to the mark, shake well, filter, take the filtrate to obtain the test solution.

[0073] Preparation of reference solution: Take an appropriate amount of 6-gingerol reference standard, accurately weigh it, and add methanol to prepare a solution containing 0.2 mg of 6-gingerol reference standard per 1 ml, thus obtaining reference solution 1.

[0074] Take appropriate amounts of hydroxy-α-sanshool reference standard and hydroxy-β-sanshool reference standard, accurately weigh them, and add methanol to prepare a mixed solution containing 0.3 mg of hydroxy-α-sanshool reference standard and 0.03 mg of hydroxy-β-sanshool per 1 ml, thus obtaining reference solution 2.

[0075] Take appropriate amounts of ginsenoside Rg1 reference standard, ginsenoside Re reference standard, and ginsenoside Rb1 reference standard, accurately weigh them, and add 60% methanol to prepare a mixed solution containing 0.2 mg of each reference standard per 1 ml, thus obtaining reference solution 3.

[0076] Accurately weigh the ginsenosides Rg2, Rf, Ro, Rb2, Rb3, Rc, and Rd reference standards, and add 60% methanol to prepare a series of reference solutions containing 0.2 mg of each per ml, collectively referred to as reference solution 4.

[0077] Accurately pipette 2 μL of reference solution 1 and reference solution 2, 4 μL of reference solution 3, 2 μL of reference solution 4 and 3 μL of test solution into the chromatograph and determine.

[0078] The UPLC-UV-ELSD method was used for detection. An ultra-high performance liquid chromatograph equipped with a UV detector and a series evaporative light scattering detector was used to acquire UPLC-UV and UPLC-ELSD spectra, respectively. UPLC-UV simultaneously determined 6-gingerol, hydroxy-α-sanshool, and hydroxy-β-sanshool reference standards, while UPLC-ELSD simultaneously determined ginsenoside Rg1, ginsenoside Re, and ginsenoside Rb1 reference standards. The chromatographic conditions included a column temperature of 30℃, a flow rate of 0.4 ml / min, and an Agilent Infinity Lab Poroshell column. 120EC-C18, specifications: inner diameter 3.0 mm, column length 150 mm, particle size 2.7 μm; using methanol as mobile phase A and 0.1% formic acid aqueous solution as mobile phase B; gradient elution, the gradient elution program includes: 0-12 min, 50% mobile phase A, 50% mobile phase B; 12-15 min, 50%→65% mobile phase A, 50%→35% mobile phase B; 15-30 min, 65%→70% mobile phase A, 35%→30% mobile phase B; 30-35 min, 70%→80% mobile phase A, 30%→20% mobile phase B; 35-40 min, 80% mobile phase A, 20% mobile phase B. The UV detector has a detection wavelength of 280 nm, and the theoretical plate number, calculated based on the 6-gingerol peak, should be no less than 10,000. The carrier gas flow rate of the evaporative light scattering detector is 2.5 L / min, the drift tube temperature is 95 °C, and the theoretical plate number calculated based on the ginsenoside Rb1 peak should be no less than 10,000.

[0079] Example 1: Establishment of Feature Map

[0080] 1. Establishment of Feature Map 1

[0081] Fifteen batches of Dajianzhong Decoction reference samples were taken and test solutions were prepared according to Example 1. UPLC-UV characteristic spectra were measured and analyzed. The relative retention times were calculated using 6-gingerol as a reference. The results are shown in [Figure 1]. Figure 1 , Figure 2 and the table below.

[0082] Table 1. Relative retention times of each characteristic peak in the characteristic spectrum of 15 batches of reference samples.

[0083] Sample Name Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 DJZT0901 1.138 1.176 1.218 1.392 1.466 1.579 1.649 DJZT0902 1.138 1.177 1.219 1.393 1.466 1.579 1.650 DJZT0903 1.137 1.176 1.218 1.392 1.466 1.578 1.649 DJZT0904 1.139 1.177 1.220 1.395 1.469 1.583 1.654 DJZT0905 1.139 1.177 1.220 1.395 1.469 1.582 1.653 DJZT0906 1.138 1.177 1.219 1.394 1.468 1.581 1.652 DJZT0907 1.138 1.176 1.218 1.392 1.465 1.579 1.649 DJZT0908 1.139 1.178 1.220 1.395 1.469 1.582 1.654 DJZT0909 1.138 1.177 1.219 1.394 1.468 1.581 1.652 DJZT0910 1.138 1.177 1.219 1.394 1.468 1.581 1.653 DJZT0911 1.137 1.175 1.217 1.391 1.464 1.576 1.646 DJZT0912 1.138 1.176 1.218 1.392 1.465 1.578 1.649 DJZT0913 1.138 1.177 1.219 1.393 1.467 1.580 1.652 DJZT0914 1.138 1.177 1.219 1.393 1.467 1.581 1.652 DJZT0915 1.138 1.177 1.219 1.394 1.468 1.581 1.653 average value 1.138 1.177 1.219 1.393 1.467 1.580 1.651 RSD 0.05 0.06 0.07 0.09 0.11 0.12 0.14

[0084] The characteristic spectra of 15 batches of Dajianzhongtang reference samples were analyzed. Taking peak 1 as the reference peak, the specified values ​​of the relative retention times of peaks 2, 3, 4, 5, 6, 7 and 8 were 1.138, 1.177, 1.219, 1.393, 1.467, 1.580 and 1.651, respectively, with the relative retention times within ±8% of the specified values. Among them, peak 3 is hydroxy-α-sanshool and peak 4 is hydroxy-β-sanshool.

[0085] Determination of reference peaks and attribution of characteristic peaks: The following samples were taken: Da Jian Zhong Tang reference sample (test sample), dried ginger negative sample (difference from Da Jian Zhong Tang reference sample: lacking dried ginger, preparation method same), stir-fried Sichuan pepper negative sample (difference from Da Jian Zhong Tang reference sample: lacking Sichuan pepper, preparation method same), ginseng negative sample (difference from Da Jian Zhong Tang reference sample: lacking ginseng, preparation method same), and 6-gingerol, hydroxy-α-salicornin, and hydroxy-β-salicornin reference solutions. These were detected according to the characteristic chromatographic detection method. The corresponding chromatograms are shown below. Figure 3-4 The results showed that 6-gingerol had a good peak shape and good separation from adjacent peaks, and its stability was better than that of sanshool. Therefore, 6-gingerol was selected as the reference peak. Through negative sample analysis, among the eight selected characteristic peaks, peaks 1, 5, and 6 were specific to dried ginger, while peaks 2, 3, 4, 7, and 8 were specific to roasted Sichuan pepper.

[0086] 2. Establishment of Feature Map 2

[0087] Fifteen batches of Dajianzhong Decoction reference samples were taken and test solutions were prepared according to Example 1. UPLC-ELSD characteristic spectra were determined and analyzed. Using ginsenoside Rb1(S3) as a reference, the relative retention time was calculated. Results are shown below. Figure 5-6 and the table below.

[0088] Table 2. Relative retention times of each characteristic peak in the characteristic spectrum obtained from 15 batches of reference samples.

[0089] Sample Name Peak 3 Peak 4 Peak 6 Peak 7 Peak 8 DJZT0901 0.680 0.928 1.030 1.128 1.254 DJZT0902 0.680 0.928 1.030 1.129 1.254 DJZT0903 0.680 0.927 1.030 1.128 1.254 DJZT0904 0.678 0.928 1.030 1.129 1.250 DJZT0905 0.679 0.928 1.030 1.129 1.251 DJZT0906 0.680 0.928 1.030 1.128 1.253 DJZT0907 0.680 0.928 1.031 1.129 1.255 DJZT0908 0.679 0.928 1.030 1.129 1.251 DJZT0909 0.679 0.927 1.030 1.129 1.252 DJZT0910 0.679 0.927 1.033 1.129 1.252 DJZT0911 0.681 0.929 1.030 1.128 1.256 DJZT0912 0.680 0.928 1.030 1.128 1.255 DJZT0913 0.679 0.927 1.030 1.129 1.252 DJZT0914 0.679 0.927 1.031 1.129 1.252 DJZT0915 0.679 0.927 1.030 1.129 1.252 average value 0.680 0.928 1.030 1.129 1.253 RSD 0.11 0.07 0.08 0.04 0.14

[0090] Analysis of the characteristic chromatograms of 15 batches was conducted. Using peak 5 as the reference peak, the specified relative retention times of peaks 3, 4, 6, 7, and 8 were 0.680, 0.928, 1.030, 1.129, and 1.253, respectively; the relative retention times were within ±8% of the specified values. Comparison with the reference standard revealed that peak 1 was ginsenoside Re, peak 2 was ginsenoside Rg1, peak 3 was ginsenoside Rf, peak 4 was ginsenoside Ro, peak 5 was Rb1, peak 6 was ginsenoside Rc, peak 7 was ginsenoside Rb2, and peak 8 was ginsenoside Rd.

[0091] Selection and assignment of reference peaks: The Da Jian Zhong Tang reference sample, dried ginger negative sample, stir-fried Sichuan pepper negative sample, and ginseng negative sample were used; ginsenosides Re, Rg1, Rg2, Rf, Ro, Rb1, Rb2, Rb3, Rc, and Rd were used as reference standards. Detection was performed according to the characteristic chromatographic detection method. The corresponding chromatograms are shown below. Figure 7-9 The results showed that the peak shape of ginsenoside Rb1 in the chromatogram was good, and the separation effect from adjacent chromatographic peaks was also good. Therefore, the ginsenoside Rb1 peak was selected as the reference peak for calculating the relative retention time of characteristic peaks. Through negative sample analysis, the selected 8 characteristic peaks were found to be specific to ginseng and were all ginsenoside components.

[0092] Experimental Example 2: UPLC-UV Feature Map 1

[0093] 1. Determination of chromatographic conditions

[0094] 1.1 Selection of detection wavelength

[0095] A diode array detector was used to perform spectral scanning on the Dajianzhong Decoction sample, and three-dimensional spectra were collected. The results showed that the chromatographic peak absorption of the test solution at a wavelength of 270-280 nm was strong. Considering that the detection of gingerol was weaker than that of sanshool, the maximum absorption of gingerol at 280 nm was selected as the detection wavelength for the UV characteristic spectrum.

[0096] 1.2 Selection of Chromatographic Column

[0097] Using the chromatographic column as the variable, the test solution was prepared and measured according to Example 1, and the results are shown in [Figure 1]. Figure 10 Columns 1-3 were Agilent InfinityLab Poroshell 120EC-C18 columns, differing only in batch number: SNUSCFW16967, SNUSCFW17959, and SNUSCFW19792, respectively; column 4 was a CAPCELL CORE C18 A34RB01105; all columns had dimensions of 3.0 × 150 mm and 2.7 μm. The results showed that the detection effects of different columns were basically consistent, with good reproducibility.

[0098] 1.3 Selection of mobile phase

[0099] The test solution was prepared according to Example 1. With the mobile phase as the variable, mobile phase systems (AB) 1-4 were methanol-water, methanol-0.05% formic acid solution, methanol-0.1% formic acid solution, and methanol-0.1% glacial acetic acid solution, respectively. The gradient elution program shown in the table below was used, and the results were determined according to Example 1. Figure 11 The results showed that adding acid to the mobile phase helps in the detection of ginsenoside Ro; therefore, methanol-0.1% formic acid is preferred as the mobile phase.

[0100] Table 3 Gradient elution program

[0101] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0-5 54 46 5-25 54→75 46→25 25-30 75→80 25→20 30-35 80 20

[0102] 1.4 Column Temperature Selection

[0103] Using column temperature as a variable, the test solution was prepared and measured according to Example 1, and the results are shown in [Figure 1]. Figure 12 The column temperatures were 25℃, 30℃, and 35℃. The results showed that column temperature between 25-35℃ had no significant effect on the separation of chromatographic peaks, with 30℃ being the optimal column temperature.

[0104] 1.5 Selection of Flow Rate

[0105] Using flow rate as a variable, the test solution was prepared and measured according to Example 1, and the results are shown in [Figure 1]. Figure 13 The flow rates were 0.35 ml / min, 0.40 ml / min, and 0.45 ml / min, respectively. The results showed that the flow rate between 0.35 and 0.45 ml / min had no significant effect on the separation of chromatographic peaks, and the results were basically consistent. The optimal flow rate was 0.40 ml / min.

[0106] Based on the above, the optimal UPLC-UV chromatographic conditions are determined as follows: An Agilent Infinity LabPoroshell 120EC-C18 column (3.0 × 150 mm, 2.7 μm) is used; methanol is used as mobile phase A, and 0.1% formic acid solution is used as mobile phase B, with gradient elution performed according to the table below; the flow rate is 0.40 mL / min; the column temperature is 30℃; and the detection wavelength is 280 nm. The theoretical plate number, calculated based on the 6-gingerol peak, should be no less than 10,000.

[0107] Table 4 Gradient elution program

[0108] Time (min) Mobile phase A (%) Mobile phase B (%) 0～12 50 50 12～15 50→65 50→35 15～30 65→70 35→30 30～35 70→80 30→20 35～40 80 20

[0109] 2. Methodological Validation

[0110] 2.1 Precision Experiment

[0111] The test solution was prepared according to Example 1 and injected six times consecutively. Using peak 1 (6-gingerol) as a reference peak, the relative retention time and RSD of each characteristic peak were calculated. The RSD values ​​of peaks 2 through 8 were 0.04%, 0.03%, 0.03%, 0.03%, 0.06%, 0.07%, and 0.07%, respectively. The results indicate that the instrument has good precision.

[0112] 2.2 Stability Experiment

[0113] The test solution was prepared according to Example 1, and measurements were taken at 0 h, 1.5 h, 4 h, 9 h, 15 h, 20 h, and 36 h after completion. Using 6-gingerol as a reference peak, the relative retention time and RSD of each characteristic peak were calculated. The RSD values ​​of peaks 2 through 8 were 0.05%, 0.05%, 0.08%, 0.12%, 0.15%, 0.18%, and 0.21%, respectively. The results indicate that the test solution exhibits good stability within 36 h.

[0114] 2.3 Repeatability Experiment

[0115] Six test solutions were prepared and measured according to Example 1. Using 6-gingerol as a reference peak, the relative retention time and RSD value of each characteristic peak were calculated. The RSD values ​​of peaks 2 through 8 were 0.05%, 0.00%, 0.03%, 0.04%, 0.03%, 0.05%, and 0.03%, respectively. The results show that the method has good repeatability and meets the detection requirements.

[0116] 2.4 Investigation of blank experiment and double retention time

[0117] The test solution was prepared according to Example 1. The chromatogram was measured with the collection time extended to 80 min as in Example 1. Simultaneously, the chromatograms of the blank solvent (70% methanol) and the blank mobile phase were measured according to Example 1. (See attached image.) Figure 14 The results showed that no obvious chromatographic peaks appeared after 40 min, and there was no interference from the blank solvent 70% methanol and the mobile phase.

[0118] Experimental Example 3: UPLC-ELSD Feature Map 2

[0119] 1. Determination of chromatographic conditions

[0120] An Alltech ELSD6000 ultra-high performance liquid chromatography (UPLC) detector was used with the drift tube temperature set at 95°C and the carrier gas flow rate at 2.5 L / min to study the UPLC-ELSD characteristic spectra of ginsenosides.

[0121] 1.1 Selection of chromatographic column

[0122] Using the chromatographic column as the variable, the test solution was prepared and measured according to Example 1, and the results are shown in [Figure 1]. Figure 15Columns 1-3 were Agilent InfinityLab Poroshell 120EC-C18 columns, differing only in batch number: SNUSCFW16967, SNUSCFW17959, and SNUSCFW19792, respectively; column 4 was a CAPCELL CORE C18 A34RB01105; all columns had dimensions of 3.0 × 150 mm and 2.7 μm. The results showed that the detection effects of different columns were basically consistent, with good reproducibility.

[0123] 1.2 Selection of mobile phase

[0124] The test solution was prepared according to Example 1. The elution procedure is shown in the table below, with the mobile phase as the variable. Mobile phase systems (AB) 1-4 were methanol-water, methanol-0.05% formic acid solution, methanol-0.1% formic acid solution, and methanol-0.1% glacial acetic acid solution, respectively. The results were determined according to Example 1 and are shown below. Figure 16 The results showed that adding acid to the mobile phase helped in the detection of ginsenoside Ro, and formic acid had a better separation effect than glacial acetic acid. Therefore, methanol-0.1% formic acid was preferred as the mobile phase.

[0125] Table 5 Washing Procedure

[0126] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～5 54 46 5～25 54→75 46→25 25～30 75→80 25→20 30～35 80 20

[0127] 1.3 Selection of Column Temperature

[0128] Using column temperature as a variable, the results were measured according to Example 1, and the results are shown below. Figure 17 The column temperatures were 25℃, 30℃, and 35℃. The results showed that column temperature between 25-35℃ had no significant effect on the separation of chromatographic peaks, with 30℃ being the optimal column temperature.

[0129] 1.4 Selection of Flow Rate

[0130] Using flow rate as the variable, the measurements were performed according to Example 1, and the results are shown below. Figure 18 The flow rates were 0.35 ml / min, 0.40 ml / min, and 0.45 ml / min, respectively. The results showed that the flow rate in the range of 0.35-0.45 ml / min had no significant effect on the separation of chromatographic peaks, and the results were basically consistent. The optimal flow rate was 0.40 ml / min.

[0131] Based on the above, the optimal UPLC-ELSD chromatographic conditions are determined as follows: An Agilent Infinity LabPoroshell 120EC-C18 column (3.0 × 150 mm, 2.7 μm) is used; methanol is used as mobile phase A, and 0.1% formic acid solution is used as mobile phase B, with gradient elution according to the table below; the flow rate is 0.40 mL / min; the column temperature is 30℃; an Alltech ELSD6000 detector is used; the drift tube temperature is 95℃; and the carrier gas flow rate is 2.5 L / min. The theoretical plate number, calculated based on ginsenoside Rb1, should not be less than 10,000.

[0132] Table 6 Gradient elution program

[0133]

[0134]

[0135] 2. Methodological Validation

[0136] 2.1 Precision Experiment

[0137] The test solution was prepared according to Example 1, and injected six times consecutively. Using peak 5 (ginsenoside Rb1) as the reference peak, the relative retention times and RSD values ​​of peaks 3, 4, 6, 7, and 8 were calculated. The RSD values ​​of peaks 3, 4, and 6 through 8 were 0.08%, 0.06%, 0.00%, 0.05%, and 0.08%, respectively. The results indicate that the instrument has good precision.

[0138] 2.2 Stability Experiment

[0139] The test solution was prepared according to Example 1. Retention times and RSDs of peaks 3, 4, 6, 7, and 8 were measured at 0 h, 1.5 h, 4 h, 9 h, 15 h, 20 h, and 36 h after completion. Using ginsenoside Rb1 as a reference peak, the RSDs of peaks 3, 4, 6, 7, and 8 were calculated. The RSDs of peaks 3, 4, and 6 through 8 were 0.18%, 0.06%, 0.04%, 0.07%, and 0.29%, respectively. The results indicate that the test solution exhibits good stability within 36 h.

[0140] 2.3 Repeatability Experiment

[0141] Six test solutions were prepared and measured according to Example 1. Using ginsenoside Rb1 as a reference peak, the relative retention times and RSD values ​​of peaks 3, 4, 6, 7, and 8 were calculated. The RSD values ​​of peaks 3, 4, and 6 through 8 were 0.06%, 0.06%, 0.00%, 0.05%, and 0.06%, respectively. The results are shown in the table below. The results indicate that the method has good repeatability and meets the detection requirements.

[0142] 2.4 Investigation of blank experiment and double retention time

[0143] The test solution was prepared and measured according to Example 1. The spectra of the blank solvent (70% methanol), the blank mobile phase, and the test solution with an extended sampling time of 80 min were measured according to Example 1, respectively. (See attached figures.) Figure 19 The results showed that no obvious chromatographic peaks appeared after 40 min, and there was no interference from the blank solvent 70% methanol and the mobile phase.

[0144] Example 2

[0145] This embodiment provides a method for determining the content of 6-gingerol, hydroxy-α-sanshool, and hydroxy-β-sanshool in a Dajianzhong decoction reference sample, including the following steps:

[0146] Preparation of the test solution: Take the Dajianzhong Decoction reference sample, shake well, take about 16g, weigh it, put it in a 50ml volumetric flask, dilute with water to the mark, shake well, accurately measure 25ml, put it in a separatory funnel, add water-saturated n-butanol and shake to extract 4 times, 25ml each time, combine the n-butanol extracts, recover the solvent to dryness, add an appropriate amount of 70% methanol to dissolve the residue, transfer it to a 5ml volumetric flask, add 70% methanol to the mark, shake well, filter, and take the filtrate to obtain the test solution.

[0147] Accurately pipette 3 μL of the test solution and inject it into the chromatograph for determination. UPLC-UV-ELSD chromatography was used for detection. The ultra-high performance liquid chromatograph was equipped with a UV detector and a series evaporative light scattering detector. UPLC-UV and UPLC-ELSD spectra were acquired separately. Chromatographic conditions included: column temperature 30℃, flow rate 0.4 mL / min, and an Agilent Infinity Lab Poroshell column. 120EC-C18, specifications: inner diameter 3.0 mm, column length 150 mm, particle size 2.7 μm; using methanol as mobile phase A and 0.1% formic acid aqueous solution as mobile phase B; gradient elution, the gradient elution program includes: 0-12 min, 50% mobile phase A, 50% mobile phase B; 12-15 min, 50%→65% mobile phase A, 50%→35% mobile phase B; 15-30 min, 65%→70% mobile phase A, 35%→30% mobile phase B; 30-35 min, 70%→80% mobile phase A, 30%→20% mobile phase B; 35-40 min, 80% mobile phase A, 20% mobile phase B. The UV detector has a detection wavelength of 280 nm, and the theoretical plate number, calculated based on the 6-gingerol peak, should be no less than 10,000. The carrier gas flow rate of the evaporative light scattering detector is 2.5 L / min, the drift tube temperature is 95 °C, and the theoretical plate number calculated based on the ginsenoside Rb1 peak should be no less than 10,000.

[0148] 6-Gingerol and hydroxy-α-piperazine (C) were calculated using the external standard method. 16 H 25 NO2) and hydroxy-β-sanshool (C 16 H 25 The content of NO2.

[0149] Determination of the preparation of the test solution

[0150] Determination of extraction method

[0151] Pretreatment of Dajianzhong Decoction reference sample: Weigh an appropriate amount of Dajianzhong Decoction reference sample, dilute with water (dilution factor approximately 2), shake well to obtain diluted solution, and set aside.

[0152] Method 1: Accurately measure 10 ml of the above diluted solution and place it into a pretreated resin column (resin types: AB-8, HPD100, D101, glass column inner diameter 1.5 cm, resin amount 10 g). First, elute with 150 ml of water and discard it, then add 100 ml of ethanol to elute. Collect the ethanol eluent, evaporate to dryness under reduced pressure, dissolve the residue in 70% methanol and make up to 5 ml, shake well, filter, and obtain 3 test solutions.

[0153] Method 2: Prepare the test solution by varying the number of extractions (3, 4, 5, and 6). Accurately measure 10 ml of the diluted solution into a separatory funnel, add 10 ml of water-saturated n-butanol for extraction each time, combine the n-butanol layers, evaporate to dryness under reduced pressure, dissolve the residue in 70% methanol and bring the volume to 5 ml, shake well, filter, and obtain 4 test solutions.

[0154] The contents of 6-gingerol, hydroxy-α-sanshool and hydroxy-β-sanshool were determined according to the above method. The results are shown in the table below. Each group has 2 parallel experiments. mg / g represents the content of active ingredients in 1g of Dajianzhong Decoction reference sample.

[0155] Table 7. Content of active ingredients in test solution prepared by different methods

[0156]

[0157] During the experiment, Method 1, i.e., the macroporous adsorption resin method, resulted in a slow liquid flow rate and a long preparation time for the test solution. Considering factors such as efficiency and stability, Method 2, i.e., the extraction method, was preferred for preparing the test solution, with 3-5 extraction cycles. The determination of the dilution factor and the amount of extraction solvent was also discussed.

[0158] Diluent A: Weigh 80.4g of Dajianzhong Decoction reference sample into a 250ml volumetric flask, add water to the mark, shake well, and set aside to obtain a dilution with a dilution factor of 4.

[0159] Diluent B: Weigh 64.1g of Dajianzhong Decoction reference sample into a 100ml volumetric flask, add water to the mark, shake well, and set aside to obtain a dilution with a dilution factor of 2.

[0160] Diluent C: Weigh 40.2g of Dajianzhong Decoction reference sample into a 250ml volumetric flask, add water to the mark, shake well, and set aside to obtain a dilution with a dilution factor of 8.

[0161] Experiment 1-1: Accurately measure 25 ml of diluent A, add an equal volume of water-saturated n-butanol for extraction 4 times, combine the n-butanol layers, evaporate to dryness under reduced pressure, dissolve the residue in 70% methanol and make up to 5 ml; wherein, the volume ratio of diluent A to water-saturated n-butanol is 1:1.

[0162] Experiment 1-2: Accurately measure 25 ml of diluent A, add 50 ml of water-saturated n-butanol and extract 4 times, combine the n-butanol layers, evaporate to dryness under reduced pressure, dissolve the residue in 70% methanol and make up to 5 ml; wherein, the volume ratio of diluent A to water-saturated n-butanol is 1:2.

[0163] Experiment 2-1: Accurately measure 10 ml of diluent B, add an equal volume of water-saturated n-butanol for extraction 4 times, combine the n-butanol layers, evaporate to dryness under reduced pressure, dissolve the residue in 70% methanol and make up to 5 ml; wherein, the volume ratio of diluent B to water-saturated n-butanol is 1:1.

[0164] Experiment 2-2: Accurately measure 10 ml of diluent B, add 20 ml of water-saturated n-butanol and extract 4 times. Combine the n-butanol layers, evaporate to dryness under reduced pressure, dissolve the residue in 70% methanol and make up to 5 ml. The volume ratio of diluent B to water-saturated n-butanol is 1:2.

[0165] Experiment 3-1: Accurately measure 50 ml of diluent C, add 50 ml of water-saturated n-butanol and extract 4 times, combine the n-butanol layers, evaporate to dryness under reduced pressure, dissolve the residue in 70% methanol and make up to 5 ml; wherein, the volume ratio of diluent C to water-saturated n-butanol is 1:1.

[0166] The contents of 6-gingerol, hydroxy-α-sanshool and hydroxy-β-sanshool were determined according to the above method, and the results are shown in the table below. Two parallel experiments were conducted for each group, and the average value was taken.

[0167] Table 8. Content of active ingredients in test solutions obtained at different dilution ratios and extraction solvent volumes.

[0168]

[0169] The above results indicate that: ① The content of active ingredients obtained by diluting the sample with water-saturated n-butanol at a volume ratio of 1:1 and 1:2 is basically consistent, and extraction with a volume ratio of 1:1 for the diluent and water-saturated n-butanol is sufficient. ② The content of active ingredients in the test sample obtained by diluting the reference sample by 2 times and extracting it 4 times is slightly lower than that obtained by diluting it by 4 times and 8 times. However, the content of active ingredients in the test samples obtained by diluting the reference sample by 4 times and extracting it 4 times after dilution by 4 times and 8 times is similar. Therefore, diluting the reference sample with water by 4 times before extraction is sufficient.

[0170] In summary, the optimal method for preparing the test solution is as follows: Weigh 16.0 g (approximately 12.5 ml) of the Dajianzhong Decoction reference sample accurately, place it in a 50 ml volumetric flask, dilute with water to the mark, shake well, accurately measure 25 ml and place it in a separatory funnel, extract four times with water-saturated n-butanol in equal proportions, collect the n-butanol layer, evaporate the solvent under reduced pressure, dissolve the residue in 70% methanol and make up to 5 ml, shake well, filter, and collect the filtrate to obtain the test solution.

[0171] Chromatographic conditions and system suitability for determination of active ingredient content

[0172] 1.1 Selection of chromatographic column

[0173] Using the chromatographic column as a variable, the test solution was prepared according to Example 2, and the contents of 6-gingerol, hydroxy-α-sanshool, and hydroxy-β-sanshool were determined. The results are shown in the table below. Columns 1-3 were Agilent InfinityLab Poroshell 120EC-C18 columns, differing only in batch number: SNUSCFW16967, SNUSCFW17959, and SNUSCFW19792, respectively. Column 4 was a CAPCELL CORE C18 A34RB 01105; all columns had dimensions of 3.0 × 150 mm and 2.7 μm. The results showed that the detection effects of different columns were basically consistent, with good reproducibility.

[0174] Table 9 Results of investigation using different chromatographic columns

[0175] chromatographic column 6-Gingerol (mg / g) Hydroxy-α-sanshool (mg / g) Hydroxy-β-sanshool (mg / g) 1 0.0852 0.1260 0.0154 2 0.0872 0.1285 0.0157 3 0.0838 0.1266 0.0155 4 0.0846 0.1268 0.0153 RSD (%) 1.70 0.84 1.10

[0176] 1.2 Selection of Column Temperature

[0177] The test solution was prepared and measured according to Example 2, with column temperature as the variable. The results are shown in the table below. The column temperatures were 25℃, 30℃, and 35℃. The results show that the column temperature between 25-35℃ has no effect on the determination of the content of the active ingredient. The preferred column temperature in this invention is 30℃.

[0178] Table 10 Results of the investigation at different column temperatures

[0179] Column temperature 6-Gingerol (mg / g) Hydroxy-α-sanshool (mg / g) Hydroxy-β-sanshool (mg / g) 25 0.0850 0.1271 0.0157 30 0.0865 0.1290 0.0159 35 0.0850 0.1274 0.0157 RSD (%) 1.01 0.80 0.73

[0180] 1.3 Selection of Flow Rate

[0181] The test solution was prepared and measured according to Example 2, with flow rate as the variable. The results are shown in the table below. The flow rates were 0.35 ml / min, 0.40 ml / min, and 0.45 ml / min. The results show that the flow rate in the range of 0.35-0.45 ml / min has no effect on the determination of the content of the index component, and the preferred flow rate is 0.40 ml / min.

[0182] Table 11 Results of the investigation at different flow velocities

[0183]

[0184]

[0185] In summary, the preferred chromatographic conditions for determining the contents of 6-gingerol, hydroxy-α-salicornin, and hydroxy-β-salicornin are as follows: Octadecylsilane-bonded silica gel as the stationary phase (Agilent InfinityLab Poroshell 120EC-C18 3.0×150mm 2.7μm); methanol as mobile phase A and 0.1% formic acid solution as mobile phase B, with gradient elution as specified in the table below; column temperature of 30℃; flow rate of 0.40 ml / min; and detection wavelength of 280 nm. The theoretical plate number, calculated based on the 6-gingerol peak, should be no less than 10,000.

[0186] Table 12 Gradient elution program

[0187] Time (min) Mobile phase A (%) Mobile phase B (%) 0～12 50 50 12～15 50→65 50→35 15～30 65→70 35→30 30～35 70→80 30→20 35～40 80 20

[0188] 2. Methodological Validation

[0189] 2.1 Linear Examination

[0190] (1) Accurately weigh 20.11 mg of 6-gingerol reference standard (purity 99.9%), place it in a 10 ml volumetric flask, dissolve it in methanol, dilute to the mark, shake well to obtain the reference standard stock solution, and then dilute it to concentrations of 10.04, 50.22, 100.45, 200.90, 502.25, 1004.49, and 2008.99 μg / ml to obtain a series of standard solutions. Take 2 μl of each of the above standard solutions and determine them by high performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0512). Plot the standard curve with peak area as the ordinate and concentration as the abscissa. The linear equation is shown in the table below.

[0191] (2) Accurately weigh 11.95 mg of hydroxy-α-sanshool reference standard, place it in a 10 ml volumetric flask, dissolve it in methanol, dilute to the mark, shake well, and obtain the reference standard stock solution. Then dilute to concentrations of 5.84, 29.22, 58.45, 292.24, 584.47, and 1168.95 μg / ml to obtain a series of standard solutions. Take 2 μL of each solution and determine it by high performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0512). Plot the standard curve with peak area as the ordinate and concentration as the abscissa. The linear equation is shown in the table below.

[0192] (3) Accurately weigh 3.03 mg of hydroxy-β-sanshool reference standard, place it in a 10 ml volumetric flask, dissolve it in methanol, dilute it to the mark, shake well, and obtain the reference standard stock solution. Then dilute it to concentrations of 0.59, 2.97, 5.94, 29.69, 59.39, and 118.78 μg / ml to obtain a series of standard solutions. Take 2 μL of each solution and determine it by high performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0512). Plot the standard curve with peak area as the ordinate and concentration as the abscissa. The linear equation is shown in the table below.

[0193] Table 13 Standard curves and linear ranges of 6-gingerol, hydroxy-α-sanshool, and hydroxy-β-sanshool

[0194] Reference Standard curve <![CDATA[R 2 ]]> Linear range (μg / ml) 6-Gingerol Y = 3082.0454x - 17933.8436 0.9999 10.04～2008.99 Hydroxy-α-sanshool Y = 15918.5070x + 133679.1909 0.9993 5.84～1168.95 Hydroxy-β-piperidine Y = 30125.6027x + 1865.2662 1.0000 0.59～118.78

[0195] The results showed that the standard curves of 6-gingerol, hydroxy-α-salicornin and hydroxy-β-salicornin exhibited good linearity.

[0196] 2.2 Precision Experiment

[0197] A standard sample of Dajianzhong Decoction was accurately weighed and prepared into a test solution according to Example 2. The solution was injected six times consecutively, and the peak area was recorded. The RSD values ​​of each active ingredient after six consecutive injections were calculated. The RSDs for 6-gingerol, hydroxy-α-salicornin, and hydroxy-β-salicornin were 1.10%, 0.09%, and 0.14%, respectively. The results indicate that the method has good precision.

[0198] 2.3 Repeatability Experiment

[0199] 16g of the Dajianzhong Decoction reference sample was accurately weighed and used to prepare six test solutions according to Example 2. The content of the active ingredients was determined, and the RSD values ​​of each active ingredient in the six test solutions were calculated. The RSDs of 6-gingerol, hydroxy-α-sanshool, and hydroxy-β-sanshool were 2.28%, 2.44%, and 0.77%, respectively. The results show that the method has good repeatability.

[0200] 2.4 Stability Test

[0201] 16g of the Dajianzhong Decoction reference sample was accurately weighed and the test solution was prepared according to Example 2. Peak areas were measured at 0h, 1.5h, 4h, 9h, 15h, 20h, and 36h after preparation, and the RSD values ​​of the peak areas of each active ingredient at different times were calculated. The RSDs of 6-gingerol, hydroxy-α-sanshool, and hydroxy-β-sanshool were 1.27%, 0.68%, and 0.91%, respectively. The results indicate that the test solution has good stability within 36h.

[0202] 2.5 Accuracy Experiment

[0203] Take about 8.0g of Dajianzhong Decoction reference sample into a 50ml volumetric flask, dilute with water to the mark, shake well, and obtain a spare Dajianzhong Decoction reference sample. Make 6 copies of the sample. The contents of 6-gingerol in the spare Dajianzhong Decoction reference sample (recorded as weighed amount) are 0.0915mg / g, hydroxy-α-salicornin content is 0.1482mg / g, and hydroxy-β-salicornin content is 0.0158mg / g.

[0204] According to the 1:1 ratio of index component concentration, accurately transfer 180 μL of 6-gingerol reference solution (concentration 2.009 mg / ml), 500 μL of hydroxy-α-sanshool reference solution (concentration 1.1689 mg / ml), and 500 μL of hydroxy-β-sanshool reference solution (concentration 0.1188 mg / ml) into ground-glass stoppered Erlenmeyer flasks to prepare 6 portions respectively. After rapidly evaporating the solvent under reduced pressure, accurately add 25 ml of the prepared Dajianzhongtang reference sample solution to each portion, sonicate for 10 min, and transfer all to a separatory funnel. Prepare the test solution according to Example 2 and determine the content of the index component. Calculate the recovery rate. The results are as follows. The results show that this method meets the requirements of quantitative analysis. The spiking amount in Table 14 is the amount of active ingredient in the reference solution added to the prepared Dajianzhongtang reference sample; the 25 ml sample volume is the content of active ingredient in 25 ml of the prepared Dajianzhongtang reference sample solution; the detection value is the detection content of active ingredient in the test solution prepared according to Example 2.

[0205] Table 14 Accuracy Results

[0206]

[0207] 2.6 Specificity Experiment

[0208] Negative sample 1 (lacking dried ginger) and negative sample 2 (lacking roasted Sichuan pepper) were prepared separately. Test solutions were prepared according to Example 2 and measured. Results are shown below. Figure 20The preparation processes for negative samples 1 and 2 were the same as those for the Da Jian Zhong Tang reference sample, except that the dried ginger was removed from the prescription for negative sample 1, and the roasted Sichuan peppercorns were removed from the prescription for negative sample 2. The results showed that the negative samples had virtually no interference with the determination of the indicator component content, indicating that this method has good specificity.

[0209] 2.7 Durability Test

[0210] (1) Flow rate: With flow rate as the variable, the flow rates were 0.35 ml / min, 0.40 ml / min and 0.45 ml / min, respectively. The test solution was prepared according to Example 2 and the content of the index components was determined. The RSDs of 6-gingerol, hydroxy-α-salicornin and hydroxy-β-salicornin were 1.53%, 1.30% and 1.27%, respectively, indicating that the flow rate durability was good.

[0211] (2) Column temperature: The column temperature was used as a variable. The column temperatures were 25℃, 30℃ and 35℃. The test solution was prepared according to Example 2 and the content of the index components was determined. The RSDs of 6-gingerol, hydroxy-α-salicornin and hydroxy-β-salicornin were 1.01%, 0.80% and 0.73%, respectively, indicating that the column temperature durability was good.

[0212] (3) Chromatographic columns: Using the chromatographic columns as variables, columns 1-3 were three batches of Agilent InfinityLab Poroshell 120EC-C18 columns: SNUSCFW16967, SNUSCFW17959, and SNUSCFW19792; column 4 was a CAPCELL CORE C18A34RB 01105. The test solution was prepared according to Example 2, and the content of the index components was determined. The RSDs of 6-gingerol, hydroxy-α-sanshool, and hydroxy-β-sanshool were 1.70%, 0.84%, and 1.10%, respectively, indicating good robustness to the same column specifications.

[0213] Example 3

[0214] This embodiment provides a method for determining the content of ginsenosides Re, Rg1, and Rb1 in a reference sample of Dajianzhong Decoction, including the following steps:

[0215] Preparation of the test solution: Take the Dajianzhong Decoction reference sample, shake well, take about 16g, weigh it, put it in a 50ml volumetric flask, dilute with water to the mark, shake well, accurately measure 25ml, put it in a separatory funnel, add water-saturated n-butanol and shake to extract 4 times, 25ml each time, combine the n-butanol extracts, recover the solvent to dryness, add an appropriate amount of 70% methanol to dissolve the residue, transfer it to a 5ml volumetric flask, add 70% methanol to the mark, shake well, filter, and take the filtrate to obtain the test solution.

[0216] Accurately pipette 3 μL of the test solution and inject it into the chromatograph for determination. UPLC-UV-ELSD chromatography was used for detection. The ultra-high performance liquid chromatograph was equipped with a UV detector and a series evaporative light scattering detector. UPLC-UV and UPLC-ELSD spectra were acquired separately. Chromatographic conditions included: column temperature 30℃, flow rate 0.4 mL / min, and an Agilent Infinity Lab Poroshell column. 120EC-C18, specifications: inner diameter 3.0 mm, column length 150 mm, particle size 2.7 μm; using methanol as mobile phase A and 0.1% formic acid aqueous solution as mobile phase B; gradient elution, the gradient elution program includes: 0-12 min, 50% mobile phase A, 50% mobile phase B; 12-15 min, 50%→65% mobile phase A, 50%→35% mobile phase B; 15-30 min, 65%→70% mobile phase A, 35%→30% mobile phase B; 30-35 min, 70%→80% mobile phase A, 30%→20% mobile phase B; 35-40 min, 80% mobile phase A, 20% mobile phase B. The UV detector has a detection wavelength of 280 nm, and the theoretical plate number, calculated based on the 6-gingerol peak, should be no less than 10,000. The carrier gas flow rate of the evaporative light scattering detector is 2.5 L / min, the drift tube temperature is 95 °C, and the theoretical plate number calculated based on the ginsenoside Rb1 peak should be no less than 10,000.

[0217] The contents of ginsenosides Re, Rg1, and Rb1 were calculated using the external standard two-point method and logarithmic equation.

[0218] Determination of the preparation of the test solution

[0219] Determination of extraction method

[0220] Pretreatment of Dajianzhong Decoction reference sample: Weigh an appropriate amount of Dajianzhong Decoction reference sample, dilute with water (dilution factor approximately 2), shake well to obtain diluted solution, and set aside.

[0221] Method 1: Accurately measure 10 ml of the above diluted solution and place it into a pretreated resin column (resin types: AB-8, HPD100, D101, glass column inner diameter 1.5 cm, resin amount 10 g). First, elute with 150 ml of water and discard it, then add 100 ml of ethanol to elute. Collect the ethanol eluent, evaporate to dryness under reduced pressure, dissolve the residue in 70% methanol and make up to 5 ml, shake well, filter, and obtain 3 test solutions.

[0222] Method 2: Prepare the test solution by varying the number of extractions (3, 4, 5, and 6). Accurately measure 10 ml of the diluted solution into a separatory funnel, add 10 ml of water-saturated n-butanol for extraction each time, combine the n-butanol layers, evaporate to dryness under reduced pressure, dissolve the residue in 70% methanol and bring the volume to 5 ml, shake well, filter, and obtain 4 test solutions.

[0223] The contents of ginsenosides Re, Rg1, and Rb1 were determined according to the above method. The results are shown in the table below. Each group had two parallel experiments.

[0224] Table 15 Content of active ingredients in test solution prepared by different methods

[0225]

[0226]

[0227] In the experiment, the preparation of the test solution using method 1 was time-consuming. The difference between the two methods in the measured content of saponin components was not significant. Considering factors such as simplifying the steps, the preferred number of extractions was 3-5.

[0228] Determination of dilution ratio and extraction solvent volume

[0229] Diluent A: Weigh 80.4g of Dajianzhong Decoction reference sample into a 250ml volumetric flask, add water to the mark, shake well, and set aside to obtain a dilution with a dilution factor of 4.

[0230] Diluent B: Weigh 64.1g of Dajianzhong Decoction reference sample into a 100ml volumetric flask, add water to the mark, shake well, and set aside to obtain a dilution with a dilution factor of 2.

[0231] Diluent C: Weigh 40.2g of Dajianzhong Decoction reference sample into a 250ml volumetric flask, add water to the mark, shake well, and set aside to obtain a dilution with a dilution factor of 8.

[0232] Experiment 1-1: Accurately measure 25 ml of diluent A, add an equal volume of water-saturated n-butanol for extraction 4 times, combine the n-butanol layers, evaporate to dryness under reduced pressure, dissolve the residue in 70% methanol and make up to 5 ml; wherein, the volume ratio of diluent A to water-saturated n-butanol is 1:1.

[0233] Experiment 1-2: Accurately measure 25 ml of diluent A, add 50 ml of water-saturated n-butanol and extract 4 times, combine the n-butanol layers, evaporate to dryness under reduced pressure, dissolve the residue in 70% methanol and make up to 5 ml; wherein, the volume ratio of diluent A to water-saturated n-butanol is 1:2.

[0234] Experiment 2-1: Accurately measure 10 ml of diluent B, add an equal volume of water-saturated n-butanol for extraction 4 times, combine the n-butanol layers, evaporate to dryness under reduced pressure, dissolve the residue in 70% methanol and make up to 5 ml; wherein, the volume ratio of diluent B to water-saturated n-butanol is 1:1.

[0235] Experiment 2-2: Accurately measure 10 ml of diluent B, add 20 ml of water-saturated n-butanol and extract 4 times. Combine the n-butanol layers, evaporate to dryness under reduced pressure, dissolve the residue in 70% methanol and make up to 5 ml. The volume ratio of diluent B to water-saturated n-butanol is 1:2.

[0236] Experiment 3-1: Accurately measure 50 ml of diluent C, add 50 ml of water-saturated n-butanol and extract 4 times, combine the n-butanol layers, evaporate to dryness under reduced pressure, dissolve the residue in 70% methanol and make up to 5 ml; wherein, the volume ratio of diluent C to water-saturated n-butanol is 1:1.

[0237] The contents of ginsenosides Re, Rg1, and Rb1 were determined according to the above method, as shown in the table below. Each group had two parallel experiments, and the average value was taken.

[0238] Table 16. Content of active ingredients in test samples obtained at different dilution ratios and extraction solvent volumes.

[0239]

[0240] The above results indicate that the amount of n-butanol used for extraction and the dilution factor have little effect on the content of saponins.

[0241] Chromatographic conditions and system suitability for determination of active ingredient content

[0242] 1.1 Selection of chromatographic column

[0243] Chromatographic columns 1-4, all with dimensions of 3.0 × 150 mm and 2.7 μm, were selected. Columns 1-3 were Agilent InfinityLab Poroshell 120EC-C18 columns, differing only in batch number: SNUSCFW16967, SNUSCFW17959, and SNUSCFW19792, respectively. Column 4 was a CAPCELL CORE C18 A34RB01105. The results showed that the detection effects of different columns were basically consistent, with good reproducibility.

[0244] Table 17 Results of the investigation of different chromatographic columns

[0245]

[0246] 1.2 Selection of Column Temperature

[0247] The test solution was prepared and measured according to Example 3, with column temperature as the variable. The results are shown in the table below. The column temperatures were 25℃, 30℃, and 35℃. The results show that the column temperature between 25-35℃ has no effect on the determination of the content of the active ingredient. The preferred column temperature in this invention is 30℃.

[0248] Table 18 Results of investigation at different column temperatures

[0249]

[0250] 1.3 Selection of Flow Rate

[0251] The test solution was prepared and measured according to Example 3, with flow rate as the variable. The results are shown in the table below. The flow rates were 0.35 ml / min, 0.40 ml / min, and 0.45 ml / min. The results show that the flow rate in the range of 0.35-0.45 ml / min has no effect on the determination of the content of the index component, and the preferred flow rate is 0.40 ml / min.

[0252] Table 19 Results of the investigation at different flow velocities

[0253]

[0254] In summary, the optimal chromatographic conditions for determining ginsenosides Re, Rg1, and Rb1 are as follows: Octadecylsilane-bonded silica gel as the stationary phase (Agilent InfinityLab Poroshell 120EC-C18 3.0×150mm 2.7μm); methanol as mobile phase A and 0.1% formic acid solution as mobile phase B, with gradient elution as specified in the table below; column temperature 30℃; flow rate 0.40 ml / min; Alltech ELSD6000 detector; drift tube temperature 95℃; carrier gas flow rate 2.5 L / min. The theoretical plate number should be no less than 10,000 for the ginsenoside Rb1 peak.

[0255] Table 20 Gradient Elution Procedure

[0256] Time (min) Mobile phase A (%) Mobile phase B (%) 0～12 50 50 12～15 50→65 50→35 15～30 65→70 35→30 30～35 70→80 30→20 35～40 80 20

[0257] 2. Methodological Validation

[0258] 2.1 Linear Examination

[0259] (1) Preparation of reference stock solutions: Accurately weigh 10.78 mg of ginsenoside Re reference standard (purity 96.0%), place it in a 10 ml volumetric flask, dissolve it in methanol, dilute to the mark, and shake well to obtain ginsenoside Re reference stock solution; accurately weigh 10.79 mg of ginsenoside Rg1 reference standard (purity 98.50%), place it in a 10 ml volumetric flask, dissolve it in methanol, dilute to the mark, and shake well to obtain ginsenoside Rg1 reference stock solution; accurately weigh 11.20 mg of ginsenoside Rb1 reference standard (purity 94.3%), place it in a 10 ml volumetric flask, dissolve it in methanol, dilute to the mark, and shake well to obtain ginsenoside Rb1 reference stock solution.

[0260] (2) Preparation of mixed reference solution: Accurately measure 2 ml of each of the stock solutions of ginsenoside Re, Rg1 and Rb1 into the same 10 ml volumetric flask, dilute with water to the mark, mix well to obtain mixed reference solution 1; then accurately measure 1 ml of mixed reference solution 1 into the 10 ml volumetric flask, add 60% methanol to the mark, mix well to obtain mixed reference solution 2.

[0261] (3) Determination method: Accurately pipette 5 μl of mixed reference standard dilution 2 and 1, 2, 4, 6, 8 and 10 μl of mixed reference standard dilution 1, and determine according to high performance liquid chromatography (Chinese Pharmacopoeia 2020 edition, Part IV, General Chapter 0512). Plot a standard curve with the logarithm of peak area (Ln peak area) as the ordinate Y and the logarithm of injection volume (μg) (Ln injection volume) as the abscissa x.

[0262] Table 21 Results of ginsenoside content, peak area, etc. in different reference solutions

[0263]

[0264] Table 22 Standard curves and linear ranges of ginsenosides Re, Rg1, and Rb1

[0265] Reference Standard curve <![CDATA[R 2 ]]> Linear range (μg) Ginsenoside Re Y = 1.7856x + 1.7437 0.9998 103.50～2070.00 Ginsenoside Rg1 Y = 1.8077x + 1.6121 0.9995 106.30～2126.00 Ginsenoside Rb1 Y = 1.8438x + 1.3603 0.9990 105.60～2112.00

[0266] The results showed that the standard curves of ginsenosides Re, Rg1, and Rb1 exhibited good linearity.

[0267] 2.2 Precision Experiment

[0268] 16g of Dajianzhong Decoction reference sample was accurately weighed and the test solution was prepared according to Example 3. The sample was injected and measured 6 times consecutively, the peak area was recorded, and the RSD value of the active ingredients was calculated. The RSD values ​​of ginsenoside Re, ginsenoside Rg1 and ginsenoside Rb1 were 0.92%, 2.58% and 0.89% respectively, indicating that the method has good precision.

[0269] 2.3 Repeatability Experiment

[0270] Six portions of 16g ​​of Dajianzhong Decoction reference sample were accurately weighed and prepared according to Example 3. The peak area was measured and the RSD value of the active ingredients was calculated. The RSD values ​​of ginsenoside Re, ginsenoside Rg1 and ginsenoside Rb1 were 0.83%, 1.12% and 0.96%, respectively, indicating that the method has good repeatability.

[0271] 2.4 Stability Test

[0272] 16g of the Dajianzhong Decoction reference sample was accurately weighed and the test solution was prepared according to Example 3. The content of active ingredients was measured at 0h, 1.5h, 4h, 9h, 15h, 20h, and 36h after preparation. The peak area was recorded, and the RSD value of the active ingredients was calculated. The RSD values ​​of ginsenoside Re, ginsenoside Rg1, and ginsenoside Rb1 were 1.73%, 4.06%, and 1.64%, respectively. The results showed that the test solution was stable within 36 hours.

[0273] 2.5 Accuracy Experiment

[0274] Take about 8g of Dajianzhong Decoction reference sample into a 50ml volumetric flask, dilute with water to the mark, shake well, and obtain a spare Dajianzhong Decoction reference sample. Make 6 copies of the sample. The content of ginsenoside Re in the spare Dajianzhong Decoction reference sample (recorded as weighed amount) is 0.0835mg / g, the content of ginsenoside Rg1 is 0.0606mg / g, and the content of ginsenoside Rb1 is 0.0976mg / g.

[0275] According to the 1:1 concentration ratio of the index components, 300 μl of ginsenoside Re reference standard (concentration of 1.03488 mg / ml), 230 μl of ginsenoside Rg1 reference standard (concentration of 1.062815 mg / ml), and 370 μl of ginsenoside Rb1 reference standard (concentration of 1.0562 mg / ml) were accurately transferred to ground-glass stoppered Erlenmeyer flasks to prepare 6 portions of each. After rapidly evaporating the solvent under reduced pressure, 25 ml of each portion was accurately added to the prepared Dajianzhong Decoction reference sample solution. The solution was dissolved by sonication for 10 min, and all portions were transferred to a separatory funnel. The test solution was prepared according to Example 3, and the content of the index components was determined. The recovery rate was calculated, and the results are as follows. The results show that the method meets the requirements of quantitative analysis. In the table, the added amount is the content of the effective component in the reference solution; the 25 ml sample volume is the content of the effective component in 25 ml of the prepared Dajianzhong Decoction reference sample; the detection value is the detection content of the index component after preparing the test solution according to Example 3.

[0276] Table 23 Accuracy Results of Ginsenosides

[0277]

[0278] 2.6 Specificity Experiment

[0279] A negative sample lacking ginseng was prepared, and the negative sample was analyzed according to Example 3. The results showed that all components could be separated, the negative sample was free of interference, and the method had good specificity.

[0280] 2.7 Durability Test

[0281] (1) Flow rate: The flow rate was used as a variable. The flow rates were 0.35 ml / min, 0.40 ml / min and 0.45 ml / min. The test solution was prepared according to Example 3 and the content of the index components was determined. The RSDs of ginsenoside Re, Rg1 and ginsenoside Rb1 were 1.87%, 1.26% and 1.22%, respectively.

[0282] (2) Column temperature: The column temperature was used as a variable. The column temperatures were 25℃, 30℃ and 35℃. The test solution was prepared according to Example 3 and the content of the index components was determined. The RSDs of ginsenoside Re, Rg1 and ginsenoside Rb1 were 0.47%, 1.66% and 2.87%, respectively.

[0283] (3) Chromatographic columns: Using the chromatographic columns as variables, columns 1-3 were Agilent InfinityLab Poroshell 120EC-C18 columns, with three batch numbers: SNUSCFW16967, SNUSCFW17959, and SNUSCFW19792; column 4 was a CAPCELL CORE C18A34RB 01105 column. The test solution was prepared according to Example 3, and the content of the index components was determined. The RSDs of ginsenosides Re, Rg1, and Rb1 were 3.23%, 1.35%, and 0.34%, respectively.

[0284] Example 4

[0285] This embodiment provides a method for determining the content of active ingredients in 15 batches of Dajianzhong Decoction reference samples. The content of active ingredients in 15 batches of Dajianzhong Decoction was determined according to Examples 2 and 3, and the results are as follows:

[0286] Table 24 Content of effective ingredients in 15 batches of Dajianzhong Decoction reference samples

[0287]

[0288] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for constructing a characteristic map of Dajianzhong Decoction, characterized in that, Includes the following steps: Preparation of test solution: Prepare a test solution from the test sample; The preparation method of the test sample solution includes: taking the test sample, diluting it with water 2-4 times, adding water-saturated n-butanol, and extracting it 3-5 times to obtain the test sample solution; UPLC-UV-ELSD chromatography was used for detection. The chromatographic conditions of UPLC-UV-ELSD chromatography included: methanol as mobile phase A, formic acid aqueous solution as mobile phase B, and gradient elution. The gradient elution program was as follows: 0-12 min, 50% mobile phase A, 50% mobile phase B; 12-15 min, 50%→65% mobile phase A, 50%→35% mobile phase B; 15-30 min, 65%→70% mobile phase A, 35%→30% mobile phase B; 30-35 min, 70%→80% mobile phase A, 30%→20% mobile phase B; 35-40 min, 80% mobile phase A, 20% mobile phase B. The chromatographic column was an Agilent Infinity Lab Poroshell 120 EC-C18. Characteristic spectrum 1, obtained under ultra-high performance liquid chromatography and ultraviolet detection, is specific to dried ginger and stir-fried Sichuan pepper. In characteristic spectrum 1, peak 1 is 6-gingerol as a reference peak. Characteristic spectrum 2, obtained under ultra-high performance liquid chromatography and evaporative light scattering detection, is specific to ginseng. Peak 5 is ginsenoside Rb1 as a reference peak. The detection wavelength of the ultraviolet detector is 270 nm to 290 nm. The carrier gas flow rate of the evaporative light scattering detector is 2.3 to 2.8 L / min. The drift tube temperature is 90 to 100 °C.

2. The construction method according to claim 1, characterized in that, The chromatographic conditions for UPLC-UV-ELSD chromatography also include: Detector: The ultra-high performance liquid chromatograph is equipped with a UV detector and an evaporative light scattering detector in series; and / or, The column temperature of the ultra-high performance liquid chromatograph is 25~35℃; and / or, The flow rate is 0.35~0.45 ml / min; and / or, Column specifications: inner diameter 3.0 mm, column length 150 mm, particle size 2.7 µm; and / or, Use 0.05~0.1% formic acid aqueous solution as mobile phase B; and / or, The sample size is 2~4μL.

3. The construction method according to claim 1 or 2, characterized in that, Chromatographic conditions for UPLC-UV-ELSD: Methanol as mobile phase A, 0.1% formic acid aqueous solution as mobile phase B; column temperature 30℃, flow rate 0.4 ml / min, sample volume 3 μL; UV detector detection wavelength 280 nm, theoretical plate number calculated based on 6-gingerol peak should not be less than 10000; evaporative light scattering detector carrier gas flow rate 2.5 L / min, drift tube temperature 95℃, theoretical plate number calculated based on ginsenoside Rb1 peak should not be less than 10000.

4. The construction method according to claim 1, characterized in that, The construction method yields feature map 1 and feature map 2; The characteristic spectrum 1 has 8 characteristic peaks. Taking peak 1 as the reference peak, the specified values ​​for the relative retention times of peaks 2, 3, 4, 5, 6, 7, and 8 are 1.138, 1.177, 1.219, 1.393, 1.467, 1.580, and 1.651, respectively. The relative retention time is within ±8% of the specified value; The characteristic spectrum 2 has 8 characteristic peaks. Taking peak 5 as the reference peak, the specified values ​​for the relative retention times of peaks 3, 4, 6, 7 and 8 are 0.680, 0.928, 1.030, 1.129 and 1.253, respectively. The relative retention time is within ±8% of the specified value.

5. The construction method according to claim 4, characterized in that, In the characteristic spectrum 2, peak 1 is ginsenoside Re, and peak 2 is ginsenoside Rg1.

6. The construction method according to claim 1, characterized in that, The test sample was either a decoction or a paste of Dajianzhong Decoction.

7. The construction method according to claim 1, characterized in that, It also includes the step of preparing a reference solution; At least one of 6-gingerol, hydroxy-α-salicornin, hydroxy-β-salicornin, ginsenoside Rg1, ginsenoside Re and ginsenoside Rb1 was used as a reference standard. Each 1 ml of the reference solution contains 0.15–0.25 mg of 6-gingerol, 0.25–0.35 mg of hydroxy-α-salicornin, 0.025–0.035 mg of hydroxy-β-salicornin, 0.15–0.25 mg of ginsenoside Rg1, 0.15–0.25 mg of ginsenoside Re, and 0.15–0.25 mg of ginsenoside Rb1.

8. A method for determining the content of effective components in Dajianzhong Decoction, characterized in that, The content of active ingredients was determined by external standard method using the construction method described in any one of claims 1-7; the active ingredients include 6-gingerol, hydroxy-α-salicornin, hydroxy-β-salicornin, ginsenoside Rg1, ginsenoside Re and ginsenoside Rb1.

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