Construction method of characteristic chromatogram of cattail medicinal material and its decoction pieces, standard decoction and traditional Chinese medicine dispensing granules

Abstract

The application discloses a kind of cattail medicinal materials and its decoction pieces, standard decoction, the construction method of characteristic chromatogram of traditional Chinese medicine formula granules, it includes: cattail medicinal materials or its decoction pieces, standard decoction, traditional Chinese medicine formula granules are extracted using methanol or ethanol, obtain test solution;Vanillin, 4-ferulic acid, 1-O-p-coumaroyl glycerol, ephedra phenol, dehydrogenated ephedra phenol are dissolved using methanol or ethanol, obtain control solution;Using liquid chromatograph determination, the stationary phase of chromatographic column of liquid chromatograph is octadecylsilane bonding silica gel, methanol is mobile phase A, 0.05vol%-0.2vol% phosphoric acid aqueous solution is mobile phase B and carries out gradient elution.This application constructs the information of characteristic chromatogram rich, can quickly, comprehensively reflect the quality of cattail medicinal materials or its decoction pieces, standard decoction, traditional Chinese medicine formula granules.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine identification technology, and in particular to a method for constructing characteristic spectra of Juncus effusus and its processed slices, standard decoctions, and traditional Chinese medicine formula granules. Background Technology

[0002] Juncus effusus L., a plant in the Juncaceae family, is the dried pith of its stem. It is sweet, bland, and slightly cold in nature; it enters the heart, lung, and small intestine meridians, and has the effects of clearing heart fire and promoting urination. It is used for insomnia due to heart palpitations, scanty and painful urination, and oral ulcers. After calcination, it has a hemostatic effect. Modern pharmacological studies have shown that Juncus effusus has anti-inflammatory, antioxidant, antihypertensive, anti-anxiety, and sedative effects. The 2020 edition of the Chinese Pharmacopoeia only includes tests for appearance, identification, examination, and extractives under the Juncus effusus herbal medicine section, lacking specific indicators such as content determination, indicating that the quality standards are still incomplete. Due to the diversity and complexity of effective components in traditional Chinese medicine, using a single component or measuring a few components is insufficient to explain the intrinsic quality of the medicinal material. Traditional Chinese medicine fingerprints / characteristic spectra can provide more comprehensive and richer information, and are therefore often used in quality control research of traditional Chinese medicinal materials. Traditional Chinese medicine primarily uses decoctions as its main form of administration. Decoctions are the material basis for the efficacy of Chinese medicine and have been practiced in clinical practice for thousands of years. Therefore, establishing a fingerprint or characteristic spectrum of standard Chinese medicine decoctions based on their effective components is of great significance for reflecting the clinical effectiveness of Chinese medicine and the spectrum-effect relationship.

[0003] Currently, there are few research reports on the fingerprint or characteristic spectrum of Juncus effusus. Existing research reports only focus on the medicinal material and some fat-soluble components, and there are few identifications of common fingerprint peaks. They ignore the water-soluble components in Juncus effusus decoction and the changes in chemical components during the decoction process. It is difficult to reflect the clinical effects of Juncus effusus, lacks a certain spectrum-effect relationship, and cannot reflect the material basis characteristics of clinical decoction. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for constructing characteristic spectra of Juncus effusus, its processed slices, standard decoctions, and traditional Chinese medicine granules, which are rich in characteristic peak information and can comprehensively reflect the quality characteristics of various forms of Juncus effusus.

[0005] To address the aforementioned technical problems, this invention provides a method for constructing characteristic spectra of Juncus effusus, its processed slices, standard decoctions, and traditional Chinese medicine granules, comprising:

[0006] The medicinal material of Juncus effusus or its processed slices, standard decoction, and traditional Chinese medicine formula granules were extracted using an extraction solvent to obtain the test solution;

[0007] Vanillin reference standard, 4-coumaric acid reference standard, 1-O-p-coumarylglycerol reference standard, erfol reference standard, and dehydroerfol reference standard were dissolved in an extraction solvent to obtain a reference standard solution;

[0008] The test solution and reference solution were analyzed by liquid chromatography to obtain characteristic chromatograms of Juncus effusus or its processed slices, standard decoctions and traditional Chinese medicine granules.

[0009] The extraction solvent is selected from 50 vol% to 100 vol% methanol or 50 vol% to 100 vol% ethanol;

[0010] The liquid chromatograph uses an octadecylsilane-bonded silica gel column as the stationary phase, methanol as mobile phase A, and a 0.05 vol%–0.2 vol% phosphoric acid aqueous solution as mobile phase B for gradient elution. The gradient elution curve is as follows:

[0011] From 0 min to 2 min, mobile phase A was 15% and mobile phase B was 85%.

[0012] Between 2 and 8 minutes, mobile phase A decreased from 15% to 18%, and mobile phase B decreased from 85% to 82%.

[0013] Between 8 and 16 minutes, mobile phase A decreased from 18% to 33%, and mobile phase B decreased from 82% to 67%.

[0014] Between 16 and 23 minutes, mobile phase A decreased from 33% to 50%, and mobile phase B decreased from 67% to 50%.

[0015] 23 min to 25 min, mobile phase A is 50%, mobile phase B is 50%;

[0016] Between 25 and 38 minutes, mobile phase A decreased from 50% to 65%, and mobile phase B decreased from 50% to 35%.

[0017] From 38 min to 42 min, mobile phase A was 65% and mobile phase B was 35%.

[0018] As an improvement to the above technical solution, the column length is 100mm to 150mm, the column diameter is 2mm to 3mm, the column temperature is 30℃ to 40℃, and the particle size of the stationary phase is 1.5μm to 2.1μm.

[0019] As an improvement to the above technical solution, the chromatographic column has a length of 100 mm, a diameter of 2.1 mm, a column temperature of 35 °C, and a stationary phase particle size of 1.6 μm.

[0020] As an improvement to the above technical solution, the mobile phase B is 0.1 vol% phosphoric acid.

[0021] As an improvement to the above technical solution, the flow rate of the liquid chromatograph is 0.25 mL / min to 0.35 mL / min, the injection volume of the test solution is 1 μL to 3 μL, and the injection volume of the reference solution is 1 μL to 3 μL.

[0022] The detection wavelength of the liquid chromatograph is 254nm to 282nm.

[0023] As an improvement to the above technical solution, the flow rate of the liquid chromatograph is 0.3 mL / min, the injection volume of the test solution is 2 μL, and the injection volume of the reference solution is 2 μL.

[0024] The detection wavelength of the liquid chromatograph is 282 nm.

[0025] As an improvement to the above technical solution, in the step of extracting the Juncus effusus herb or its processed slices, standard decoction, or traditional Chinese medicine formula granules with an extraction solvent to obtain a test solution, the extraction solvent is 100 vol% methanol, the extraction method is heating reflux extraction or ultrasonic extraction, and the extraction time is 15 min to 60 min.

[0026] As an improvement to the above technical solution, the method for preparing the test solution is as follows:

[0027] Accurately weigh 2g of Juncus effusus herb or its powdered form, place it in a stoppered conical flask, add 100mL of water, heat under reflux for 30 minutes, cool, filter, evaporate the filtrate to dryness, accurately add 25mL of methanol to the residue, weigh, and sonicate for 30 minutes using a power of 300W and a frequency of 40kHz, cool, weigh again, replenish the lost weight with methanol, shake well, filter, and collect the filtrate to obtain the final product.

[0028] As an improvement to the above technical solution, the method for preparing the test solution is as follows:

[0029] Take 0.1g of standard decoction of rush pith or rush pith Chinese medicine formula granules, weigh accurately, place in a stoppered conical flask, add 25mL of methanol accurately, weigh, and sonicate for 30 minutes at a power of 300W and a frequency of 40kHz. After cooling, weigh again, replenish the lost weight with methanol, shake well, filter, and collect the filtrate to obtain the final product.

[0030] As an improvement to the above technical solution, the characteristic spectrum includes 9 common peaks, of which peak 2 is vanillin peak, peak 3 is 4-coumaric acid peak, peak 4 is 1-O-p-coumarylglycerol peak, peak 6 is erfol peak, and peak 7 is dehydroerfol peak.

[0031] Using peak 3 as S1 peak, calculate the relative retention times of peaks 1 to 5; using peak 6 as S2 peak, calculate the relative retention times of peaks 7 to 9; the relative retention times of each peak are within ±10% of the specified values, and the specified values ​​for peaks 1 to 9 are: 0.47, 0.70, 1.00, 1.22–1.23, 1.45–1.47, 1.00, 1.03, 1.20, and 1.23, respectively.

[0032] Implementing this invention has the following beneficial effects:

[0033] Based on the analysis of the material basis of Juncus effusus, this invention constructs a characteristic spectrum of Juncus effusus, which can be used for the detection of medicinal materials, decoction pieces, standard decoctions, and Chinese medicine formula granules. It can quickly and comprehensively realize the quality monitoring of multiple characteristic components of Juncus effusus, which is of great significance for reflecting the clinical effectiveness of Chinese medicine and the spectrum-effect relationship. Attached Figure Description

[0034] Figure 1 These are characteristic chromatograms of Juncus effusus when different extraction solvents were used in Example 1;

[0035] Figure 2 These are characteristic chromatograms of the standard Juncus effusus decoction used in Example 1 with different extraction solvents;

[0036] Figure 3 These are characteristic chromatograms of Juncus effusus extracts obtained using different extraction methods in Example 1;

[0037] Figure 4 These are characteristic chromatograms of the standard decoction of Juncus effusus when using different extraction methods in Example 1;

[0038] Figure 5 These are characteristic chromatograms of Juncus effusus extracts obtained using different extraction times in Example 1;

[0039] Figure 6 These are characteristic chromatograms of the standard Juncus effusus decoction prepared using different extraction times in Example 1;

[0040] Figure 7 This is a characteristic spectrum (3D) of the standard Juncus effusus decoction under full wavelength scanning in Example 1;

[0041] Figure 8 These are characteristic spectra of the standard Juncus effusus decoction using different absorption wavelengths in Example 1;

[0042] Figure 9 These are characteristic chromatograms of the standard Juncus effusus decoction used in Example 1 with different mobile phase gradients;

[0043] Figure 10 These are characteristic chromatograms of the Juncus effusus standard decoction using different chromatographic columns in Example 1;

[0044] Figure 11 This is a diagram showing the specificity of the characteristic spectra of the standard Juncus effusus decoction in Example 1;

[0045] Figure 12 This is a superimposed image of the characteristic spectra of 16 batches of standard Juncus effusus decoction in Example 1;

[0046] Figure 13 This is a comparative characteristic spectrum of the standard decoction of Juncus effusus in Example 1;

[0047] Figure 14 These are the total ion chromatogram and ultraviolet absorption chromatogram of the test solution of the standard decoction of Juncus effusus; where A is the TIC chromatogram in negative ion mode; B is the TIC chromatogram in positive ion mode; and C is the chromatogram under UV 282nm.

[0048] Figure 15 This is a superimposed image of the characteristic spectra of 16 batches of rush pith herbs in Example 2;

[0049] Figure 16 This is a comparative characteristic spectrum of the rush pith herb in Example 2;

[0050] Figure 17 This is a superimposed image of the characteristic spectra of 16 batches of Juncus effusus slices in Example 3;

[0051] Figure 18 This is a comparative characteristic spectrum of the rush pith slices in Example 3;

[0052] Figure 19 This is a superimposed image of the characteristic spectra of the three batches of Juncus effusus herbal formula granules in Example 4;

[0053] Figure 20 This is a comparative characteristic spectrum of the rush pith herbal formula granules in Example 4;

[0054] Among them: peak 2 is vanillin; peak 3 is 4-coumaric acid; peak 4 is 1-O-p-coumarylglycerol; peak 6 is erfol; peak 7 is dehydroerfol. Detailed Implementation

[0055] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0056] Example 1: Construction of a Characteristic Map of Juncus effusus

[0057] 1. Preparation of the test solution

[0058] Medicinal materials and processed slices: Take about 2g of the powder (passed through a No. 3 sieve), accurately weigh it, place it in a stoppered conical flask, add 100mL of water, heat under reflux for 30 minutes, cool, filter, evaporate the filtrate to dryness, accurately add 25mL of methanol to the residue, weigh it, sonicate (power 300W, frequency 40kHz) for 30 minutes, cool, weigh it again, make up the lost weight with methanol, shake well, filter, and collect the filtrate to obtain the product.

[0059] Chinese medicine formula granules and standard decoctions: Take an appropriate amount of this product, grind it into a fine powder, take about 0.1g, weigh it accurately, place it in a stoppered conical flask, add 25mL of methanol accurately, weigh it, sonicate it (power 300W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the weight loss with methanol, shake it well, filter it, and take the filtrate to obtain the product.

[0060] 2. Preparation of reference solution

[0061] Take appropriate amounts of vanillin reference standard, 4-coumaric acid reference standard, 1-O-p-coumarylglycerol reference standard, erfol reference standard, and dehydroerfol reference standard, accurately weigh them, and add methanol to prepare a mixed solution containing 50 μg of each in 1 mL, as the reference solution.

[0062] 3. Chromatographic conditions

[0063] Chromatographic column: Waters Cortecs T3 (2.1 mm × 100 mm, 1.6 μm); mobile phase: methanol (A) - 0.1% phosphoric acid aqueous solution (B); elution mode: gradient elution; flow rate: 0.3 mL / min, column temperature: 35 °C, detection wavelength: 282 nm. The gradient elution program of the mobile phase is shown in Table 1.

[0064] Table 1 Elution Procedure

[0065]

[0066] 4. Measurement

[0067] Accurately pipette 2 μL of the reference solution and 2 μL of the test solution into the liquid chromatograph and determine the result.

[0068] 5. Methodological Examination

[0069] (1) Investigation of extraction solvent

[0070] This study investigated the effect of different extraction solvents on the characteristic spectra of Juncus effusus during the preparation of the test sample solution.

[0071] Specifically, for the Juncus effusus sample: Take approximately 2g of Juncus effusus powder (passed through a No. 3 sieve), make 6 parallel groups, 2 portions per group, accurately weigh them, place them in a stoppered conical flask, add 100mL of water, heat under reflux for 30 minutes, cool, filter, evaporate the filtrate to dryness, accurately add 25mL each of 100% methanol, 70% methanol, 50% methanol, 100% ethanol, 70% ethanol, and 50% ethanol to the residue, weigh them, sonicate (300W power, 40kHz frequency) for 30 minutes, remove, cool, weigh again, make up the lost weight with the corresponding solvent, shake well, filter, take the filtrate, and inject and analyze according to the chromatographic conditions determined in section "3". The results are shown in [see section 3]. Figure 1 As shown in the figure. The results show that the above six extraction solvents have little effect on the peak shape and separation effect of each characteristic peak, so they can all be used as extraction solvents. Furthermore, when 100% methanol is used as the extraction solvent, the overall response of each characteristic peak is the highest, so 100% methanol is preferred as the extraction solvent.

[0072] For the standard decoction sample of Juncus effusus: Take an appropriate amount of the standard decoction of Juncus effusus, grind it into a fine powder, take about 0.1g, divide it into 6 parallel groups, 2 portions per group, accurately weigh them, place them in a stoppered conical flask, and accurately add 25mL of 100% methanol, 70% methanol, 50% methanol, 100% ethanol, 70% ethanol, and 50% ethanol respectively, weigh them, sonicate (power 300W, frequency 40kHz) for 30 minutes, remove, cool, weigh again, make up the weight loss with the corresponding solvent, shake well, filter, and take the filtrate to obtain the sample. Inject and analyze according to the chromatographic conditions determined in section "3", and the results are shown in [the table below]. Figure 2 As shown in the figure. The results show that the above six extraction solvents have little effect on the peak shape and separation effect of each characteristic peak, so they can all be used as extraction solvents. Furthermore, when 100% methanol is used as the extraction solvent, the overall response of each characteristic peak is the highest, so 100% methanol is preferred as the extraction solvent.

[0073] (2) Examination of extraction methods

[0074] This study investigated the effects of different extraction methods in the preparation of the test sample solution on the characteristic chromatograms of Juncus effusus.

[0075] Specifically, for the Juncus effusus sample: Take approximately 2g of Juncus effusus powder, in two parallel groups (two portions per group), accurately weigh the powder, place it in a stoppered conical flask, add 100mL of water, heat under reflux for 30 minutes, cool, filter, evaporate the filtrate to dryness, accurately add 25mL of methanol to the residue, weigh, and then sonicate (300W, 40kHz) for 30 minutes and heat under reflux for 30 minutes respectively. Remove, cool, weigh again, replenish the lost weight with methanol, shake well, filter, and take the filtrate for analysis under the chromatographic conditions determined in section "3". The results are shown in [Figure 1]. Figure 3As shown, the results indicate that different extraction methods have little impact on the characteristic spectrum of Juncus effusus. Furthermore, considering ease of operation, ultrasonic processing was chosen.

[0076] For the standard decoction sample of Juncus effusus: Take an appropriate amount of the standard decoction of Juncus effusus, grind it into a fine powder, take about 0.1g, divide it into two parallel groups, two portions per group, accurately weigh them, place them in a stoppered conical flask, accurately add 25mL of methanol, weigh them, sonicate (power 300W, frequency 40kHz) for 30 minutes, heat under reflux for 30 minutes, remove, cool, weigh them again, make up the weight loss with methanol, shake well, filter, and take the filtrate as the sample. Inject and analyze according to the chromatographic conditions determined in section "3". The results are shown in the figure. Figure 4 As shown, different extraction methods have little impact on the characteristic chromatogram of the standard Juncus effusus decoction. Furthermore, considering ease of operation, ultrasonic treatment was chosen.

[0077] (3) Examination of extraction time

[0078] This study investigated the effect of different extraction times during the preparation of the test sample solution on the characteristic spectra of Juncus effusus.

[0079] Specifically, for the Juncus effusus sample: Take approximately 2g of Juncus effusus powder (passed through a No. 3 sieve), make 3 parallel groups, 2 portions per group, accurately weigh them, place them in a stoppered conical flask, add 100mL of water, heat under reflux for 30 minutes, cool, filter, evaporate the filtrate to dryness, accurately add 25mL of methanol to the residue, weigh it, and sonicate it (power 300W, frequency 40kHz) for 15 minutes, 30 minutes, and 60 minutes respectively, cool it, weigh it again, make up the weight loss with methanol, shake well, filter, and collect the filtrate. Inject and analyze according to the chromatographic conditions under section "3". The results are shown in [Figure 1]. Figure 5 As shown, different extraction times have little impact on the characteristic spectrum of Juncus effusus, and extraction times ranging from 15 min to 60 min are acceptable. Furthermore, considering extraction efficiency and completeness, ultrasonic extraction for 30 min was selected.

[0080] For the standard decoction sample of Juncus effusus: Take an appropriate amount of the standard decoction of Juncus effusus, grind it into a fine powder, take about 0.1g, divide it into 3 parallel groups, 2 portions per group, accurately weigh them, place them in a stoppered conical flask, accurately add 25mL of methanol, weigh them, and sonicate (power 300W, frequency 40kHz) for 15 minutes, 30 minutes, and 60 minutes. Remove them, cool them, weigh them again, make up the weight loss with methanol, shake well, filter, and take the filtrate. Inject and analyze according to the chromatographic conditions determined in section "3". The results are shown in [Figure 1]. Figure 6 As shown, different extraction times have little impact on the characteristic spectrum of the standard Juncus effusus decoction; extraction times ranging from 15 to 60 minutes are acceptable. Furthermore, considering extraction efficiency and completeness, ultrasonic extraction for 30 minutes was selected.

[0081] (4) Full wavelength scan and investigation of the optimal absorption wavelength

[0082] This study investigated the effect of different detection wavelengths on the characteristic chromatograms of Juncus effusus in ultra-high performance liquid chromatography.

[0083] Specifically, take an appropriate amount of the standard Juncus effusus decoction sample and prepare the test solution according to the method in section "1". Except for the detection wavelength, analyze the sample according to the chromatographic conditions determined in section "3". Specifically, record the 3D spectrum within the detection wavelength range of 200–400 nm, and test the characteristic spectra of Juncus effusus at 254 nm, 282 nm, and 325 nm respectively. The results are as follows: Figure 7 and Figure 8 As shown.

[0084] The results show that all characteristic peaks appear when the detection wavelength is between 254 nm and 282 nm, reflecting a wealth of information. Therefore, 254 nm to 282 nm can be selected as the detection wavelength. Furthermore, at a wavelength of 282 nm, the response values ​​of each characteristic peak are larger, the baseline is stable, and interference is smaller. Therefore, 282 nm is preferred as the detection wavelength for the characteristic spectrum.

[0085] (5) Investigation of mobile phase gradient

[0086] This study investigated the effects of different mobile phase gradients on the characteristic chromatograms of Juncus effusus in ultra-high performance liquid chromatography.

[0087] Specifically, take an appropriate amount of the standard decoction sample of Juncus effusus and prepare the test solution according to the method in section "1". Except for the gradient elution conditions, inject and analyze according to the chromatographic conditions determined in section "3". Specifically, elute using four different mobile phase gradients as shown in Tables 2 to 5, and the results are as follows: Figure 9 As shown in the figure. The results show that when gradient 4 is selected, the response values ​​of each characteristic peak are larger, the baseline is stable, the interference is smaller, and the separation effect of each chromatographic peak is better. Therefore, considering all factors, gradient 4 is selected as the final elution gradient.

[0088] Table 2 Gradient 1

[0089]

[0090] Table 3 Gradient 2

[0091]

[0092] Table 4 Gradient 3

[0093]

[0094] Table 5 Gradient 4

[0095]

[0096] (6) Chromatographic column investigation

[0097] This study investigated the effects of different chromatographic columns on the characteristic chromatograms of Juncus effusus in ultra-high performance liquid chromatography.

[0098] Specifically, take an appropriate amount of the standard decoction sample of Juncus effusus and prepare the test solution according to the method in section "1". Except for the chromatographic column, analyze the sample according to the chromatographic conditions determined in section "3". Specifically, Waters Cortecs T3 (2.1mm × 100mm, 1.6μm), Agilent Eclipse Plus C18 (2.1mm × 100mm, 1.8μm), YMC Triart C18 (2.1mm × 100mm, 1.9μm), Waters BEH C18 (2.1mm × 100mm, 1.7μm), and Waters HSS T3 C18 (2.1mm × 100mm, 1.8μm) were used as chromatographic columns, and the results are as follows: Figure 10 As shown in the figure. The results show that when the Waters Cortecs T3 (2.1 mm × 100 mm, 1.6 μm) column is used as the chromatographic column, the response values ​​of each characteristic peak are large, the baseline is stable, the interference is small, and the separation effect of each chromatographic peak is good. Therefore, the Waters Cortecs T3 (2.1 mm × 100 mm, 1.6 μm) column is selected as the optimal chromatographic column.

[0099] 6. Methodological Validation

[0100] (1) Specificity examination

[0101] Accurately pipette 2 μL each of the test solution of the Juncus effusus standard decoction, the above-mentioned reference solution, and the blank solvent, and inject them into the liquid chromatograph. Determine the chromatographic conditions according to section "3". Results are shown below. Figure 11 The results showed that the chromatogram of the test sample had the same chromatographic peak at the corresponding retention time as that of the reference sample, and there was no interference from the blank solvent, indicating that the method has good specificity.

[0102] (2) Precision test

[0103] Take approximately 0.1 g of the standard decoction of Juncus effusus, accurately weigh it, and prepare the test solution according to the preparation method in section "1". Inject the sample 6 times repeatedly under the chromatographic conditions determined in section "3". Using the chromatographic peak of 4-coumaric acid as the reference peak S1, calculate the RSD values ​​of the relative retention time and relative peak area of ​​peaks 1-5 with peak S1. The values ​​should be within the range of 0.18% to 2.31%. Using the chromatographic peak of erofol as the reference peak S2, calculate the RSD values ​​of the relative retention time and relative peak area of ​​peaks 7-9 with peak S2. The values ​​should be within the range of 0.28% to 2.32%. All values ​​are less than 3.0%, indicating that the instrument precision is good.

[0104] (3) Stability test

[0105] Accurately weigh approximately 0.1 g of the standard decoction of Juncus effusus and prepare the test solution according to the preparation method in section "1". Analyze the sample at 0, 2, 4, 8, 15, 18, and 24 hours according to the chromatographic conditions determined in section "3". Using the 4-coumaric acid chromatographic peak as the reference peak S1, calculate the relative retention time and relative peak area RSD values ​​of peaks 1-5 with peak S1. The values ​​are within the range of 0.39% to 2.571%. Using the erofol chromatographic peak as the reference peak S2, calculate the relative retention time and relative peak area RSD values ​​of peaks 7-9 with peak S2. The values ​​are within the range of 0.48% to 2.62%, all less than 3.0%, indicating that the test solution has good stability within 24 hours.

[0106] (4) Repeated examination

[0107] Accurately weigh approximately 0.1 g of the standard decoction of Juncus effusus, prepare six parallel portions, and prepare the test solution according to the preparation method in section "1". Analyze the samples under the chromatographic conditions determined in section "3". Using the 4-coumaric acid peak as the reference peak S1, calculate the relative retention time and relative peak area (RSD) values ​​between peaks 1-5 and peak S1. The values ​​should be within the range of 0.62% to 2.96%. Using the erofol peak as the reference peak S2, calculate the relative retention time and relative peak area (RSD) values ​​between peaks 7-9 and peak S2. The values ​​should be within the range of 0.575% to 2.68%. All values ​​are less than 3.0%, indicating good repeatability of the method.

[0108] (5) Durability

[0109] (5.1) Column temperature

[0110] Accurately weigh approximately 0.1 g of the standard decoction of Juncus effusus and prepare the test solution according to the preparation method in section "1". Except for the column temperatures of 33℃, 35℃ and 37℃, the other chromatographic conditions are the same as those specified in section "3". Inject and analyze the samples separately. Using the 4-coumaric acid chromatographic peak as the reference peak S1, calculate the relative retention time and relative peak area RSD values ​​of peaks 1 to 5 with peak S1. The values ​​are within the range of 0.08% to 2.25%. Using the erofol chromatographic peak as the reference peak S2, calculate the relative retention time and relative peak area RSD values ​​of peaks 7 to 9 with peak S2. The values ​​are within the range of 0.66% to 2.44%. All values ​​are less than 3.0%, indicating that the method has good robustness to small temperature variations.

[0111] (5.2) Flow velocity

[0112] Accurately weigh approximately 0.1 g of the standard decoction of Juncus effusus and prepare the test solution according to the preparation method in section "1". Except for the flow rates of 0.28 mL / min, 0.30 mL / min, and 0.32 mL / min, the other chromatographic conditions are the same as those specified in section "3". Inject and analyze the samples separately. Using the 4-coumaric acid chromatographic peak as the reference peak S1, calculate the relative retention time and relative peak area RSD values ​​of peaks 1 to 5 with peak S1. The values ​​are within the range of 0.36% to 2.72%. Using the erofol chromatographic peak as the reference peak S2, calculate the relative retention time and relative peak area RSD values ​​of peaks 7 to 9 with peak S2. The values ​​are within the range of 0.12% to 2.09%. All values ​​are less than 3.0%, indicating that the method has good robustness to small variations in flow rate.

[0113] 7. Establishment of Feature Maps

[0114] According to the method in this embodiment, appropriate amounts of 16 batches of Juncus effusus standard decoction were taken, finely ground, and 16 test solutions were prepared according to the method determined in section "1". These solutions were then injected and analyzed under the chromatographic conditions in section "3". The characteristic chromatograms of the 16 batches of Juncus effusus standard decoction samples were determined, and the common peaks of the characteristic chromatograms of the 16 batches of Juncus effusus standard decoction were identified using the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software". Nine common peaks with known components, good peak shape and resolution, high purity, and large peak area were selected as the characteristic peaks of the Juncus effusus standard decoction. Figure 12 As shown, the 4-coumaric acid chromatographic peak, with a relatively moderate retention time and readily available reference standard, was used as reference peak S1. The relative retention times and relative peak areas of peaks 1-5 with peak S1 were calculated. Similarly, the erofol chromatographic peak was used as reference peak S2. The relative retention times and relative peak areas of peaks 7-9 with peak S2 were calculated. The results are shown in Tables 6 and 7. The UPLC characteristic chromatograms of 16 batches of Juncus effusus standard decoction were matched using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine." A reference chromatogram was generated using the average method, establishing a reference characteristic chromatogram for Juncus effusus standard decoction. Figure 13 As shown.

[0115] Table 6. Characteristic chromatograms (relative retention times) of 16 batches of standard Juncus effusus decoction

[0116]

[0117] Table 7. Characteristic chromatograms (relative peak areas) of 16 batches of standard Juncus effusus decoction

[0118]

[0119] Based on the test results of 16 batches of Juncus effusus standard decoction samples, the characteristic chromatogram standard for Juncus effusus standard decoction was determined as follows: the test sample chromatogram should show 9 characteristic peaks, among which peaks 3 and 6 should correspond to the retention times of the corresponding reference peaks; the peak corresponding to the 4-coumaric acid reference peak is peak S1, and the relative retention times of peaks 1, 2, 4, and 5 with peak S1 should be calculated. Their relative retention times should be within ±10% of the specified values, which are: 0.47 (peak 1), 0.70 (peak 2), 1.22 (peak 4), and 1.45 (peak 5); the peak corresponding to the erofol reference peak is peak S2, and the relative retention times of peaks 7-9 with peak S2 should be calculated. Their relative retention times should be within ±10% of the specified values, which are: 1.03 (peak 7), 1.20 (peak 8), and 1.23 (peak 9).

[0120] 8. Common Peak Identification

[0121] The common peaks in the standard decoction of Juncus effusus were identified using ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS). The UHPLC conditions are shown in section "3" (except for mobile phase B, which was 0.1 vol% formic acid). The preparation method of the test solution is shown in section "1," and the preparation method of the reference standard is shown in section "2." The mass spectrometry conditions are shown in Table 8.

[0122] Table 8 Mass Spectrometry Parameters

[0123]

[0124] The test solution was analyzed using the aforementioned ultra-high performance liquid chromatography (UHPLC) and mass spectrometry (MS / MS) conditions. By comparing the retention times of the reference standard in HPLC and analyzing the precise molecular weight and fragment ions in MS / MS, five components were identified in the characteristic chromatogram of the Juncus effusus standard decoction: vanillin (peak 2), 4-coumaric acid (peak 3), 1-O-p-coumarylglycerol (peak 4), erfol (peak 6), and dehydroerfol (peak 7). The total ion chromatogram and UV absorption chromatogram of the test solution are shown below. Figure 14 The specific identification results are shown in Table 9.

[0125] Table 9. Mass spectrometry identification results of compounds in the standard decoction of Juncus effusus.

[0126]

[0127]

[0128] Example 2: Characteristic Atlas of Juncus effusus

[0129] Sixteen batches of Juncus effusus were taken and injected for analysis according to the chromatographic conditions under "3" and the test solution preparation method determined under "1" in Example 1. The common peaks of the 16 batches of Juncus effusus were identified using the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System." Nine common peaks with retention times consistent with the standard decoction of Juncus effusus were selected as characteristic peaks of the characteristic chromatogram of Juncus effusus. Figure 15 As shown in Tables 10 and 11. Using the 4-coumaric acid chromatographic peak as reference peak S1, the relative retention times and relative peak areas of peaks 1-5 with peak S1 were calculated; using the erofol chromatographic peak as reference peak S2, the relative retention times and relative peak areas of peaks 7-9 with peak S2 were calculated, and the RSD values ​​were also calculated. The results showed that the characteristic chromatograms of 16 batches of Juncus effusus had 9 common peaks, consistent with the common peaks in the characteristic chromatograms of the Juncus effusus standard decoction. The UPLC characteristic chromatograms of the 16 batches of Juncus effusus were matched using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine," and a control chromatogram was generated using the average method to establish a control characteristic chromatogram for Juncus effusus, as shown in Tables 10 and 11. Figure 16 As shown.

[0130] Table 10. Characteristic images of 16 batches of Juncus effusus medicinal materials (relative retention time)

[0131]

[0132] Table 11. Characteristic chromatograms (relative peak areas) of 16 batches of Juncus effusus medicinal materials

[0133]

[0134]

[0135] Based on the test results of 16 batches of Juncus effusus samples, the characteristic chromatographic standard of Juncus effusus was determined as follows: the test sample chromatogram should show 9 characteristic peaks, among which peaks 3 and 6 should correspond to the retention times of the corresponding reference peaks; the peak corresponding to the 4-coumaric acid reference peak is peak S1, and the relative retention times of peaks 1, 2, 4, and 5 with peak S1 should be calculated. Their relative retention times should be within ±10% of the specified values, which are: 0.47 (peak 1), 0.70 (peak 2), 1.23 (peak 4), and 1.47 (peak 5); the peak corresponding to the erofol reference peak is peak S2, and the relative retention times of peaks 7-9 with peak S2 should be calculated. Their relative retention times should be within ±10% of the specified values, which are: 1.03 (peak 7), 1.20 (peak 8), and 1.23 (peak 9).

[0136] Example 3: Characteristic chromatogram of Juncus effusus slices

[0137] Sixteen batches of Juncus effusus decoction pieces were taken and injected for analysis according to the chromatographic conditions under "3" and the test solution preparation method determined under "1" in Example 1. The common peaks of the 16 batches of Juncus effusus decoction pieces were identified using the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System." Nine common peaks with retention times consistent with the standard decoction of Juncus effusus were selected as characteristic peaks of the characteristic chromatogram of the Juncus effusus decoction pieces. Figure 17 As shown. Using the 4-coumaric acid chromatographic peak as reference peak S1, the relative retention times and relative peak areas of peaks 1-5 with peak S were calculated; using the erofol chromatographic peak as reference peak S2, the relative retention times and relative peak areas of peaks 7-9 with peak S2 were calculated, and the RSD values ​​were calculated. The experimental results are shown in Tables 12 and 13. The UPLC characteristic chromatograms of 16 batches of Juncus effusus decoction pieces were matched using the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System," and a control chromatogram was generated using the average method to establish a control characteristic chromatogram for Juncus effusus decoction pieces, as shown below. Figure 18 As shown.

[0138] Table 12. Characteristic chromatograms of 16 batches of Juncus effusus slices (relative retention time)

[0139]

[0140] Table 13. Characteristic chromatograms (relative peak areas) of 16 batches of Juncus effusus slices.

[0141]

[0142] Example 4: Characteristic spectrum of Juncus effusus granules in traditional Chinese medicine formula

[0143] Three batches of Juncus effusus granules were taken and analyzed according to the chromatographic conditions under "3" and the test solution preparation method determined under "1" in Example 1. The characteristic chromatograms of the three batches of Juncus effusus granules were recorded. Figure 19 As shown. Using the 4-coumaric acid chromatographic peak as reference peak S1, the relative retention times and relative peak areas of peaks 1-5 with peak S were calculated; using the erofol chromatographic peak as reference peak S2, the relative retention times and relative peak areas of peaks 7-9 with peak S2 were calculated, and the RSD values ​​were calculated. The experimental results are shown in Tables 14 and 15. The UPLC characteristic chromatograms of three batches of Juncus effusus traditional Chinese medicine formula granules were matched using the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System," and a control chromatogram was generated using the average method to establish a control characteristic chromatogram for Juncus effusus traditional Chinese medicine formula granules, as shown below. Figure 20 As shown.

[0144] Table 14 Characteristic Spectra of Juncus effusus Granules in Traditional Chinese Medicine Formulas (Relative Retention Time)

[0145]

[0146]

[0147] Table 15 Characteristic Spectrum of Juncus effusus Granules in Traditional Chinese Medicine Formulas (Relative Peak Area)

[0148]

[0149] The above description is a preferred embodiment of the invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the invention, and these improvements and modifications are also considered to be within the scope of protection of the invention.

Claims

1. A method for constructing characteristic chromatograms of Juncus effusus, its processed slices, standard decoctions, and traditional Chinese medicine granules, characterized in that, include: The medicinal material of Juncus effusus or its processed slices, standard decoction, and traditional Chinese medicine formula granules were extracted using an extraction solvent to obtain the test solution; Vanillin reference standard, 4-coumaric acid reference standard, 1- O - The coumaroyl glycerol reference standard, erfol reference standard, and dehydroerfol reference standard were dissolved in an extraction solvent to obtain a reference standard solution; The test solution and reference solution were analyzed by liquid chromatography to obtain characteristic chromatograms of Juncus effusus or its processed slices, standard decoctions and traditional Chinese medicine granules. The extraction solvent is selected from 50 vol% to 100 vol% methanol or 50 vol% to 100 vol% ethanol; The liquid chromatograph uses a Waters Cortecs T3 column with a length of 100 mm, a diameter of 2.1 mm, and a stationary phase particle size of 1.6 μm. The liquid chromatograph uses methanol as mobile phase A and a 0.05 vol%–0.2 vol% phosphoric acid aqueous solution as mobile phase B for gradient elution. The gradient elution curve is as follows: From 0 min to 2 min, mobile phase A was 15% and mobile phase B was 85%. Between 2 and 8 minutes, mobile phase A decreased from 15% to 18%, and mobile phase B decreased from 85% to 82%. From 8 min to 16 min, mobile phase A decreased from 18% to 33%, and mobile phase B decreased from 82% to 67%. From 16 min to 23 min, mobile phase A decreased from 33% to 50%, and mobile phase B decreased from 67% to 50%. 23-25 ​​min, mobile phase A is 50%, mobile phase B is 50%; Between 25 and 38 minutes, mobile phase A decreased from 50% to 65%, and mobile phase B decreased from 50% to 35%. From 38 min to 42 min, mobile phase A was 65% and mobile phase B was 35%. The detection wavelength of the liquid chromatograph is 254nm~282nm.

2. The method for constructing the characteristic spectrum of Juncus effusus herb and its processed slices, standard decoctions, and traditional Chinese medicine granules as described in claim 1, characterized in that, The column temperature of the chromatographic column is 30℃~40℃.

3. The method for constructing the characteristic spectrum of Juncus effusus herb and its processed slices, standard decoctions, and traditional Chinese medicine granules as described in claim 1 or 2, characterized in that, The column temperature of the chromatographic column is 35℃.

4. The method for constructing the characteristic spectrum of Juncus effusus herb and its processed slices, standard decoctions, and traditional Chinese medicine granules as described in claim 1, characterized in that, The mobile phase B is 0.1 vol% phosphoric acid.

5. The method for constructing the characteristic spectrum of Juncus effusus herb and its processed slices, standard decoctions, and traditional Chinese medicine granules as described in claim 1, characterized in that, The flow rate of the liquid chromatograph is 0.25 mL / min to 0.35 mL / min, the injection volume of the test solution is 1 μL to 3 μL, and the injection volume of the reference solution is 1 μL to 3 μL.

6. The method for constructing the characteristic chromatograms of Juncus effusus and its processed slices, standard decoctions, and traditional Chinese medicine granules as described in claim 1 or 5, characterized in that, The flow rate of the liquid chromatograph is 0.3 mL / min, the injection volume of the test solution is 2 μL, and the injection volume of the reference solution is 2 μL. The detection wavelength of the liquid chromatograph is 282 nm.

7. The method for constructing the characteristic spectrum of Juncus effusus herb and its processed slices, standard decoctions, and traditional Chinese medicine granules as described in claim 1, characterized in that, In the step of extracting the medicinal material of Juncus effusus or its processed slices, standard decoction, or traditional Chinese medicine formula granules with an extraction solvent to obtain the test solution, the extraction solvent is 100 vol% methanol, the extraction method is heating reflux extraction or ultrasonic extraction, and the extraction time is 15 min to 60 min.

8. The method for constructing the characteristic spectrum of Juncus effusus herb and its processed slices, standard decoctions, and traditional Chinese medicine granules as described in claim 1, characterized in that, The method for preparing the test solution is as follows: Accurately weigh 2g of Juncus effusus herb or its powdered form, place it in a stoppered conical flask, add 100mL of water, heat under reflux for 30 minutes, cool, filter, evaporate the filtrate to dryness, accurately add 25mL of methanol to the residue, weigh, and sonicate for 30 minutes using a power of 300W and a frequency of 40kHz, cool, weigh again, replenish the lost weight with methanol, shake well, filter, and collect the filtrate to obtain the final product.

9. The method for constructing the characteristic spectrum of Juncus effusus herb and its processed slices, standard decoctions, and traditional Chinese medicine granules as described in claim 1, characterized in that, The method for preparing the test solution is as follows: Take 0.1g of standard decoction of rush pith or rush pith Chinese medicine formula granules, weigh accurately, place in a stoppered conical flask, add 25mL of methanol accurately, weigh, and sonicate for 30 minutes at a power of 300W and a frequency of 40kHz. After cooling, weigh again, replenish the lost weight with methanol, shake well, filter, and collect the filtrate to obtain the final product.

10. The method for constructing the characteristic spectrum of Juncus effusus herb and its processed slices, standard decoctions, and traditional Chinese medicine granules as described in claim 1, characterized in that, The characteristic spectrum includes 9 common peaks, of which peak 2 is the vanillin peak, peak 3 is the 4-coumaric acid peak, and peak 4 is the 1- O - For coumaroylglycerol peak, peak 6 is the erfol peak, and peak 7 is the dehydroerfol peak; Using peak 3 as S1 peak, calculate the relative retention times of peaks 1 to 5; using peak 6 as S2 peak, calculate the relative retention times of peaks 7 to 9; the relative retention times of each peak are within ±10% of the specified values, and the specified values ​​for peaks 1 to 9 are: 0.47, 0.70, 1.00, 1.22~1.23, 1.45~1.47, 1.00, 1.03, 1.20, and 1.23, respectively.

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