Construction method and application of high performance liquid chromatography characteristic fingerprint of Lycopodiastrum casuarinoides

Abstract

The present invention provides a method for constructing a high-performance liquid chromatography (HPLC) characteristic fingerprint of Lycopodiastrum casuarinoides and its application, comprising the following steps: extracting a test sample with a solvent to obtain a test solution; determining the test solution by high-performance liquid chromatography to obtain the HPLC characteristic fingerprint of Lycopodiastrum casuarinoides; the chromatographic conditions of the high-performance liquid chromatography are as follows: the chromatographic column is a C18 column; mobile phase A is methanol, and mobile phase B is 0.1% acetic acid solution, with gradient elution. The present invention adopts high-performance liquid chromatography, selects methanol-0.1% acetic acid solution as the mobile phase for gradient elution, uses protocatechuic acid, 4-hydroxybenzoic acid, p-hydroxybenzaldehyde, and 4-coumaric acid as reference substances, and establishes the HPLC characteristic fingerprints of Lycopodiastrum casuarinoides herbs, cut pieces, extracts, and their formula granules. The method has good repeatability and precision, and is stable and reliable, and can control the quality of Lycopodiastrum casuarinoides cut pieces, herbs, extracts, and their formula granules.

Description

Technical Field

[0001] The present invention belongs to the technical field of detection, and particularly relates to a method for constructing a high-performance liquid chromatography characteristic fingerprint of Lycopodiastrum casuarinoides and its application. Background Art

[0002] Lycopodiastrum casuarinoides is a commonly used traditional Chinese medicine in Sichuan Province. It is the dried aerial part of Lycopodiastrum casuarinoides (Spring.) Holub. of the family Lycopodiaceae. Lycopodiastrum casuarinoides is sweet in taste and warm in nature, and belongs to the liver, spleen, and kidney meridians. It has the effects of dispelling wind and dampness, promoting blood circulation and relaxing tendons, and is commonly used to treat rheumatic numbness, traumatic sprains, bone and muscle pain, menstrual abdominal pain and other diseases. The chemical composition research of Lycopodiastrum casuarinoides mainly focuses on lycopodium alkaloids. In addition, it also contains aliphatic, flavonoid, coumarin, terpene and other components, but there are few reports in the existing literature. Modern pharmacological studies have shown that lycopodium alkaloids are the main active components of Lycopodiastrum casuarinoides, with cholinesterase inhibitory activity, anti-tumor, anti-inflammatory and other effects.

[0003] Lycopodiastrum casuarinoides is included in the "Sichuan Provincial Standards for Chinese Medicinal Materials" 2010 edition and the "Guangxi Provincial Standards for Chinese Medicinal Materials". The standards only specify its character, identification, inspection and extract. There are few literatures on the quality control method research of Lycopodiastrum casuarinoides, and only one article on the analysis of heavy metals and harmful elements in Lycopodiastrum casuarinoides has been published. The related research on the characteristic fingerprint of Lycopodiastrum casuarinoides is still blank. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a method for constructing a high-performance liquid chromatography characteristic fingerprint of Lycopodiastrum casuarinoides and its application. The method for constructing the high-performance liquid chromatography characteristic fingerprint of Lycopodiastrum casuarinoides provided by the present invention is stable and reliable, and can control the quality of Lycopodiastrum casuarinoides decoction pieces, medicinal materials, extracts and formula granules.

[0005] The present invention provides a method for constructing a high-performance liquid chromatography characteristic fingerprint of Lycopodiastrum casuarinoides, comprising the following steps:

[0006] Performing solvent extraction on the test sample to obtain a test solution;

[0007] Measuring the test solution by high-performance liquid chromatography to obtain a high-performance liquid chromatography characteristic fingerprint of Lycopodiastrum casuarinoides;

[0008] The chromatographic conditions of the high-performance liquid chromatography are as follows: the chromatographic column is a C18 column; mobile phase A is methanol, and mobile phase B is 0.1% acetic acid solution, with gradient elution.

[0009] The present invention has established a high-performance liquid chromatography characteristic fingerprint method, which is applicable to the detection of Lycopodiastrum casuarinoides medicinal materials, Lycopodiastrum casuarinoides decoction pieces, Lycopodiastrum casuarinoides extracts and Lycopodiastrum casuarinoides formula granules. The method disclosed by the present invention can be used as an important supplement to the existing quality control methods.

[0010] In the present invention, the 0.1% acetic acid solution is 0.1% aqueous acetic acid solution, and the 70% methanol is 70% aqueous methanol solution; both the 0.1% acetic acid solution and the 70% methanol are volume fractions.

[0011] In the present invention, the test sample is extracted with a solvent to obtain a test solution.

[0012] The present invention finds that when 70% methanol is used as the extraction solvent, the chromatographic peak information is large and the shapes of each chromatographic peak are better. Therefore, the solvent used for extraction is 70% methanol;

[0013] The extraction method is ultrasonic extraction; the extraction time is 15 - 25 min; the power of the ultrasonic extraction is 580 - 620 W, and the frequency of the ultrasonic extraction is 35 - 45 kHz. In a specific embodiment, the power of the ultrasonic extraction is 600 W, the frequency of the ultrasonic extraction is 40 kHz, and the extraction time is 20 min.

[0014] The mass ratio (g) of the test sample to the volume ratio (mL) of the extraction solvent is (0.4 - 0.6):20, preferably (0.45 - 0.55):20. In a specific embodiment, the mass ratio (g) of the test sample to the volume ratio (mL) of the extraction solvent is 0.5:20.

[0015] Specifically, the preparation process of the test sample is as follows: Take 0.5 g of the test sample, place it in a stoppered conical flask, add 20 ml of 70% methanol solution, ultrasonically treat (power 600 W, frequency 40 kHz) for 20 minutes, let it cool, shake well, filter, and take the subsequent filtrate to obtain the test solution.

[0016] The present invention determines the test solution by high performance liquid chromatography to obtain the high performance liquid chromatography characteristic fingerprint of Lycopodiastrum casuarinoides.

[0017] In the present invention, the chromatographic conditions of the high performance liquid chromatography are as follows: The chromatographic column is a C18 column; mobile phase A is methanol, mobile phase B is 0.1% acetic acid solution, and gradient elution is used. The present invention finds that the chromatogram baseline is relatively stable, there are many chromatographic peaks, and the peak shapes are better under the gradient elution conditions of methanol - 0.1% acetic acid solution. Therefore, the gradient elution of methanol - 0.1% acetic acid solution is used as the mobile phase for the determination method of the characteristic fingerprint of Lycopodiastrum casuarinoides medicinal materials.

[0018] The present invention finds that when the column temperature is 20°C, the chromatogram peak shape is symmetric and the resolution is better; when the flow rate is 0.25 mL / min, the chromatogram peak shape is better and the resolution is moderate.

[0019] The present invention finds that when the detection wavelength is 265 nm, the chromatographic peak information is larger and the chromatogram baseline is more stable. Therefore, the detection wavelength is 265 nm.

[0020] The injection volume is 1 μL, and the theoretical plate number calculated based on the 4 - hydroxybenzoic acid peak should be not less than 5000.

[0021] In the present invention, the gradient elution is specifically as follows:

[0022] From 0 to 2 min, phase A: 1%, phase B: 99%;

[0023] From 2 to 5 min, phase A: 1 - 6%, phase B: 99 - 94%;

[0024] From 5 to 10 min, phase A: 6 - 14%, phase B: 94 - 86%;

[0025] From 10 to 16 min, phase A: 14 - 20%, phase B: 86 - 80%;

[0026] From 16 to 28 min, phase A: 20 - 35%, phase B: 80 - 65%.

[0027] In the present invention, the test sample raw materials are one or more of Lycopodiastrum casuarinoides herbs, cut herbs, Lycopodiastrum casuarinoides extracts or Lycopodiastrum casuarinoides formula granules;

[0028] The present invention uses the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint to evaluate the similarity of the characteristic fingerprint of Lycopodiastrum casuarinoides, and obtains the HPLC standard characteristic fingerprint of Lycopodiastrum casuarinoides composed of 7 characteristic peaks. Among them, peak 3 is protocatechuic acid; peak 4 is 4-hydroxybenzoic acid; peak 6 is p-hydroxymethylbenzaldehyde; peak 7 is 4-coumaric acid.

[0029] The present invention uses the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint to evaluate the similarity of the HPLC characteristic fingerprint of Lycopodiastrum casuarinoides herbs, and obtains the HPLC standard characteristic fingerprint of Lycopodiastrum casuarinoides herbs composed of 10 characteristic peaks. Among them, the peak corresponding to the 4-hydroxybenzoic acid reference substance is the S peak; in the said standard characteristic fingerprint, calculate the relative retention time of each characteristic peak and the S peak, and its relative retention time should be within ±10% of the specified value; the specified values are: 0.29 (peak 1), 0.32 (peak 2), 0.78 (peak 3), 1.09 (peak 5), 1.16 (peak 6), 1.39 (peak 7).

[0030] The present invention uses the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint to evaluate the similarity of the HPLC characteristic fingerprint of Lycopodiastrum casuarinoides cut herbs, and obtains the HPLC standard characteristic fingerprint of Lycopodiastrum casuarinoides cut herbs composed of 10 characteristic peaks. Among them, the peak corresponding to the 4-hydroxybenzoic acid reference substance is the S peak; in the said standard characteristic fingerprint, calculate the relative retention time of each characteristic peak and the S peak, and its relative retention time should be within ±10% of the specified value; the specified values are: 0.30 (peak 1), 0.32 (peak 2), 0.78 (peak 3), 1.09 (peak 5), 1.16 (peak 6), 1.39 (peak 7).

[0031] The present invention uses a similarity evaluation system for traditional Chinese medicine chromatographic fingerprint to evaluate the similarity of the HPLC characteristic fingerprint of Shujincao formula granules, and obtains an HPLC standard characteristic fingerprint of Shujincao formula granules composed of 10 characteristic peaks. The peak corresponding to the 4-hydroxybenzoic acid reference substance is the S peak; in the said standard characteristic fingerprint, the relative retention times of each characteristic peak and the S peak are calculated, and the relative retention times should be within ±10% of the specified values; the specified values are: 0.29 (peak 1), 0.31 (peak 2), 0.78 (peak 3), 1.09 (peak 5), 1.16 (peak 6), 1.39 (peak 7).

[0032] The present invention uses a similarity evaluation system for traditional Chinese medicine chromatographic fingerprint to evaluate the similarity of the HPLC characteristic fingerprint of Shujincao extract, and obtains an HPLC standard characteristic fingerprint of Shujincao extract composed of 10 characteristic peaks. The peak corresponding to the 4-hydroxybenzoic acid reference substance is the S peak; in the said standard characteristic fingerprint, the relative retention times of each characteristic peak and the S peak are calculated, and the relative retention times should be within ±10% of the specified values; the specified values are: 0.31 (peak 1), 0.33 (peak 2), 0.78 (peak 3), 1.09 (peak 5), 1.16 (peak 6), 1.38 (peak 7).

[0033] The method provided by the present invention can effectively monitor the quality of Shujincao in different batches, making its quality stable. The method has the characteristics of high precision and good reproducibility, which is conducive to comprehensively monitoring the quality of products.

[0034] The characteristic fingerprint of Shujincao established by the present invention uses protocatechuic acid, 4-hydroxybenzoic acid, p-hydroxybenzaldehyde, and 4-coumaric acid as reference substances, pays attention to the order of each characteristic peak and the correlation with the medicinal materials and intermediate products, can comprehensively evaluate the overall quality characteristics of the product, and the method is scientific and reliable.

[0035] The present invention provides a method for identifying Shujincao medicinal materials, cut pieces, extracts and their formula granules, and uses the method described in the above technical solution for detection and analyzes the detection results.

[0036] The present invention has established characteristic fingerprints of Shujincao medicinal materials, Shujincao cut pieces, Shujincao extracts and Shujincao formula granules, confirmed 7 common characteristic peaks, and studied their relative retention times, ensuring the stability of their chemical composition and the safety of use. Four of the index components were identified, overcoming the defect that it is difficult to reflect the overall content by measuring the content of a single component. It can control the internal quality of Shujincao and its related preparations from the overall and macroscopic aspects, ensure the curative effect of the drug, and make the quality control of its related preparations more regular. The method of the present invention has good stability, high precision, good reproducibility, is fast and convenient, and is easy to master and popularize.

[0037] The present invention provides a method for constructing a high-performance liquid chromatography (HPLC) characteristic fingerprint of Lycopodiastrum casuarinoides, comprising the following steps: subjecting a test sample to solvent extraction to obtain a test solution; measuring the test solution by high-performance liquid chromatography to obtain the HPLC characteristic fingerprint of Lycopodiastrum casuarinoides; the chromatographic conditions of the high-performance liquid chromatography are as follows: the chromatographic column is a C18 column; mobile phase A is methanol, and mobile phase B is 0.1% acetic acid solution, with gradient elution. The present invention uses high-performance liquid chromatography, selects methanol-0.1% acetic acid solution as the mobile phase for gradient elution, and uses protocatechuic acid, 4-hydroxybenzoic acid, p-hydroxybenzaldehyde, and 4-coumaric acid as reference substances to establish the HPLC characteristic fingerprints of Lycopodiastrum casuarinoides cut crude drugs, crude drugs, extracts, and their formula granules. The method has good repeatability and precision, and is stable and reliable, and can control the quality of Lycopodiastrum casuarinoides cut crude drugs, crude drugs, extracts, and their formula granules. Description of the Drawings

[0038] Figure 1 For the comparative characteristic fingerprints under different mobile phase conditions;

[0039] Figure 2 For the column temperature investigation;

[0040] Figure 3 For the flow rate investigation;

[0041] Figure 4 For the detection wavelength investigation;

[0042] Figure 5 For the extraction method investigation;

[0043] Figure 6 For the extraction solvent investigation;

[0044] Figure 7 For the extraction time investigation;

[0045] Figure 8 For the test sample weighing amount investigation;

[0046] Figure 9 For the specificity and characteristic peak identification diagram;

[0047] Figure 10 For the delay investigation chromatogram;

[0048] Figure 11 For the instrument durability investigation;

[0049] Figure 12 For the comparison diagram of different chromatographic columns;

[0050] Figure 13 For the control characteristic fingerprint of Lycopodiastrum casuarinoides extract;

[0051] Figure 14 For the control characteristic fingerprint of Lycopodiastrum casuarinoides formula granules;

[0052] Figure 15 It is a comparative characteristic spectrum of herbal medicine for relaxing muscles;

[0053] Figure 16 This is a comparative characteristic spectrum of Shujincao medicinal pieces. DETAILED DESCRIPTION

[0054] In order to further illustrate the present invention, the construction method of the high-performance liquid phase characteristic spectrum of Shujincao provided by the present invention and its application are described in detail below in combination with the embodiments, but they should not be understood as limiting the scope of protection of the present invention.

[0055] Example 1

[0056] 1. Instruments and test drugs

[0057] 1.1 Instrument

[0058] Liquid chromatograph: Waters H-Class Plus ultra-high performance liquid chromatograph, Agilent 1290 high performance liquid chromatograph;

[0059] Electronic balance: ME204E, XPE26 (Mettler-Toledo Instrument Co., Ltd.);

[0060] Ultrapure water machine: Cell type 1810A (Shanghai Moller Scientific Instrument Co., Ltd.);

[0061] Ultrasonic cleaner: KQ-600DB (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0062] 1.2 Reagents: Methanol and glacial acetic acid were of chromatographic grade, water was ultrapure water (prepared in the laboratory), and the other reagents were of analytical grade.

[0063] 1.3 Drug testing

[0064] Shujincao control medicinal material (Shanghai Hongyong Biotechnology Co., Ltd., batch number: 380003-202104);

[0065] Protocatechuic acid (China Food and Drug Inspection Institute, batch number 110809-201906, purity 97.7%);

[0066] 4-Hydroxybenzoic acid (China Food and Drug Inspection Institute, batch number 101149-202204, purity 100%);

[0067] p-Hydroxybenzaldehyde (Sichuan Weikeqi Biotechnology Co., Ltd., batch number wkq18010307, ​​purity 98%);

[0068] 4-Coumaric acid (China Food and Drug Control Institutes, batch number: 112037-202102, purity 99.7%);

[0069] Lot numbers of Lycopodiastrum casuarinoides herbs: SJC-YC-01, SJC-YC-01, SJC-YC-02, SJC-YC-03, SJC-YC-04, SJC-YC-05, SJC-YC-06, SJC-YC-07, SJC-YC-08, SJC-YC-09, SJC-YC-10, SJC-YC-11, SJC-YC-12, SJC-YC-13, SJC-YC-14, SJC-YC-15, SJC-YC-16, SJC-YC-17, SJC-YC-18, SJC-YC-19, SJC-YC-20;

[0070] Lot numbers of Lycopodiastrum casuarinoides cut crude drugs: SJC-YP-01, SJC-YP-02, SJC-YP-03, SJC-YP-04, SJC-YP-05, SJC-YP-06, SJC-YP-07, SJC-YP-08, SJC-YP-09, SJC-YP-10, SJC-YP-11, SJC-YP-12, SJC-YP-13, SJC-YP-14, SJC-YP-15, SJC-YP-16, SJC-YP-17, SJC-YP-18, SJC-YP-19, SJC-YP-20;

[0071] Lot numbers of Lycopodiastrum casuarinoides extracts: SJC-BT-01, SJC-BT-02, SJC-BT-03, SJC-BT-04, SJC-BT-05, SJC-BT-06, SJC-BT-07, SJC-BT-08, SJC-BT-09, SJC-BT-10, SJC-BT-11, SJC-BT-12, SJC-BT-13, SJC-BT-14, SJC-BT-15, SJC-BT-16, SJC-BT-17, SJC-BT-18, SJC-BT-19, SJC-BT-20;

[0072] Lot numbers of Lycopodiastrum casuarinoides formula granules: SJC-CP-01, SJC-CP-02, SJC-CP-03.

[0073] 2. Establishment of characteristic spectrum method

[0074] Chromatographic conditions and system suitability test Using octadecylsilane chemically bonded silica gel as the filler (column length: 150 mm, inner diameter: 2.1 mm, particle size: 1.9 μm); using methanol as mobile phase A and 0.1% acetic acid solution as mobile phase B, gradient elution was carried out according to the regulations in Table 1 below; the flow rate was 0.25 ml per minute; the column temperature was 20 °C; the detection wavelength was 265 nm. The number of theoretical plates calculated with respect to the 4-hydroxybenzoic acid peak should be not less than 5000.

[0075] Table 1 Gradient Elution Program

[0076]

[0077] Preparation of reference solution: Weigh accurately about 1 g of the reference crude drug of Lycopodiastrum casuarinoides, add 50 ml of water, decoct for 30 minutes, centrifuge, evaporate the supernatant to dryness, dissolve the residue in 20 ml of 70% methanol solution, ultrasonically treat (power 600 W, frequency 40 kHz) for 20 minutes, cool to room temperature, shake well, filter, and take the subsequent filtrate as the reference solution of the reference crude drug. Separately, weigh accurately an appropriate amount of 4-hydroxybenzoic acid reference substance, dissolve it in 70% ethanol to prepare a solution containing 10 μg of 4-hydroxybenzoic acid per 1 ml as the reference solution of the reference substance.

[0078] Preparation of test solution: Weigh accurately about 0.5 g of the powder of Lycopodiastrum casuarinoides extract, place it in a stoppered conical flask, add 20 ml of 70% methanol solution, ultrasonically treat (power 600 W, frequency 40 kHz) for 20 minutes, cool to room temperature, shake well, filter, and take the subsequent filtrate, which is the test solution.

[0079] Determination method: Accurately pipette 1 μl each of the reference solution and the test solution, inject them into the liquid chromatograph, and perform the determination to establish the high-performance liquid chromatography characteristic fingerprint.

[0080] 2.1 Investigation of chromatographic conditions

[0081] 2.1.1 Investigation of mobile phase

[0082] According to the above chromatographic conditions, the effects of methanol - 0.1% formic acid, methanol - 0.1% acetic acid, and methanol - 0.1% phosphoric acid on the separation effect of chromatographic peaks were investigated respectively. The results are as Figure 1 shown. The results show that under the gradient elution conditions, the baseline of the chromatogram of methanol - 0.1% acetic acid solution is relatively stable, there are many chromatographic peaks, and the peak shapes are good. Therefore, the gradient elution of methanol - 0.1% acetic acid solution is used as the mobile phase for the determination method of the characteristic fingerprint of Lycopodiastrum casuarinoides crude drug.

[0083] 2.1.2 Investigation of column temperature

[0084] On the basis of the above - determined experimental conditions, the column temperatures of 20 °C, 25 °C, and 30 °C were investigated respectively. See Figure 2 . The results of the column temperature investigation show that when the column temperature is 20 °C, the chromatogram peak shape is symmetric and the resolution is good. Therefore, 20 °C is finally determined as the column temperature for the characteristic fingerprint method of Lycopodiastrum casuarinoides extract.

[0085] 2.1.3 Investigation of flow rate

[0086] On the basis of the above - determined experimental conditions, the flow rates of 0.2 ml / min, 0.25 ml / min, and 0.3 ml / min were investigated respectively. See Figure 3. The results showed that when the flow rate was 0.25 ml / min, the chromatogram had a good peak shape and moderate resolution. Therefore, the flow rate was determined to be 0.25 ml / min.

[0087] 2.1.4 Detection Wavelength Investigation

[0088] Under the above chromatographic conditions, the detection was carried out at detection wavelengths of 240 nm, 265 nm, 280 nm, and 300 nm respectively. See Figure 4 , and the results showed that when the detection wavelength was 265 nm, the chromatographic peak had more information and the chromatogram baseline was more stable. Therefore, the detection wavelength was determined to be 265 nm.

[0089] 2.1.5 Summary

[0090] To sum up, the chromatographic conditions and system suitability test for the characteristic chromatogram of the extract of Lycopodiastrum casuarinoides were determined as follows: octadecylsilane chemically bonded silica gel was used as the filler (column length was 150 mm, inner diameter was 2.1 mm, particle size was

[0091] 1.9 μm); methanol was used as mobile phase A, and 0.1% acetic acid solution was used as mobile phase B, and gradient elution was carried out according to the regulations in Table 1 above; the flow rate was 0.25 ml per minute; the column temperature was 20 °C; the detection wavelength was 265 nm. The theoretical plate number calculated by the peak of 4-hydroxybenzoic acid should not be less than 5000.

[0092] Example 2 Investigation on the Preparation of Test Samples

[0093] 2.2.1 Investigation on Extraction Method

[0094] Take about 0.5 g of the powder of the extract of Lycopodiastrum casuarinoides (batch number: SJC-BT-01), accurately weigh it, place it in a stoppered conical flask, add 20 ml of 70% methanol, and ultrasonically extract (power 600 W, frequency 40 kHz) for 20 minutes and reflux extract for 20 minutes respectively. Let it cool, shake well, filter, and take the subsequent filtrate to obtain the sample. The results are shown in Figure 5 .

[0095] The results showed that there was no significant difference in the chromatograms of the test samples by ultrasonic extraction and reflux extraction, and the ultrasonic method was rapid and simple. Therefore, the extraction method of the test sample was determined to be ultrasonic extraction.

[0096] 2.2.2 Investigation on Extraction Solvent

[0097] Take about 0.5 g of the powder of the extract of Lycopodiastrum casuarinoides (batch number: SJC-BT-01), accurately weigh it, place it in a stoppered conical flask, and add 20 ml of water, 30% methanol, 70% methanol, methanol, 30% ethanol, 70% ethanol, and ethanol respectively. Ultrasonically treat (power 600 W, frequency 40 kHz) for 20 minutes, let it cool, shake well, filter, and take the subsequent filtrate to obtain the sample. The results are shown in Figure 6 . From Figure 6It can be seen that when 70% methanol is used as the extraction solvent, the chromatographic peak information is large and the peak shapes of each chromatographic peak are better. Therefore, the extraction solvent is determined to be 70% methanol.

[0098] 2.2.3 Investigation of extraction time

[0099] Take about 0.5 g of the powder of the extract of Lycopodiastrum casuarinoides (batch number: SJC-BT-01), accurately weigh it, place it in a stoppered conical flask, add 20 ml of 70% methanol, and ultrasonically extract (power 600 W, frequency 40 kHz) for 10 minutes, 20 minutes, and 30 minutes respectively. Let it cool, shake well, filter, and take the continuous filtrate to obtain the sample solution. The results are shown in Figure 7 . From Figure 7 It can be seen that when the extraction time is 20 minutes, the extraction can be sufficient. Therefore, the extraction time of the test sample is determined to be 20 minutes.

[0100] 2.2.4 Investigation of the weighing amount of the test sample

[0101] Take about 0.3 g, 0.5 g, and 1.0 g of the powder of this product (batch number: SJC-BT-01) respectively, accurately weigh them, place them in a stoppered conical flask, accurately add 20 ml of 70% methanol, ultrasonically treat (power 600 W, frequency 40 kHz) for 20 minutes, let it cool, shake well, filter, and take the continuous filtrate to obtain the sample solution. The results are shown in Figure 8 . From Figure 8 It can be seen that when the weighing amount of the test sample is 0.5 g, the peak height and peak area of the characteristic chromatogram are more appropriate and the baseline is relatively stable. Therefore, the weighing amount of the test sample is determined to be 0.5 g.

[0102] 2.2.5 Summary

[0103] According to the above results, the preparation method of the test sample is determined as follows: Take about 0.5 g of the powder of this product, accurately weigh it, place it in a stoppered conical flask, accurately add 20 ml of 70% methanol, ultrasonically treat (power 600 W, frequency 40 kHz) for 20 minutes, let it cool, shake well, filter, and take the continuous filtrate to obtain the sample solution.

[0104] 2.3 Methodology investigation

[0105] 2.3.1 Specificity test and chromatographic peak identification

[0106] Preparation of the test sample solution: Take about 0.5 g of the powder of this product, place it in a stoppered conical flask, add 20 ml of 70% methanol solution, ultrasonically treat (power 600 W, frequency 40 kHz) for 20 minutes, let it cool, shake well, filter, and take the continuous filtrate to obtain the sample solution.

[0107] Preparation of reference solution of control crude drug: Take 1 g of the control crude drug of Lycopodiastrum casuarinoides, add 50 ml of water, decoct for 30 minutes, centrifuge, take the supernatant and evaporate to dryness, dissolve the residue in 20 ml of 70% methanol solution, and ultrasonically treat (power 600 W, frequency 40 kHz) for 20 minutes. Let it cool, shake well, filter, and take the consecutive filtrate as the reference solution of control crude drug.

[0108] Preparation of reference solution for peak location: Take appropriate amounts of reference substances of protocatechuic acid, 4-hydroxybenzoic acid, p-hydroxybenzaldehyde, and 4-coumaric acid, and separately prepare solutions containing 10 μg per 1 ml with 70% methanol as the reference solutions for peak location.

[0109] Preparation of negative control solution: Prepare the negative control solution without the extract of Lycopodiastrum casuarinoides according to the experimental conditions determined above.

[0110] Inject and detect according to the chromatographic conditions determined above, and the results are shown in Figure 9 : The negative sample does not interfere with the detection of the chromatographic peaks of the test sample, indicating good specificity of this method. Among the 7 characteristic peaks of the test sample, peak 3 is protocatechuic acid, peak 4 is 4-hydroxymethylbenzoic acid, peak 6 is p-hydroxymethylbenzaldehyde, and peak 7 is 4-coumaric acid. Taking the peak corresponding to 4-hydroxybenzoic acid as the S peak, in the subsequent methodological investigation, the 7 peaks in the chromatogram of the test sample are investigated.

[0111] 2.3.2 Delayedness investigation

[0112] Take the powder of the test sample, and the retention time of the chromatographic conditions is twice that of the method for constructing the foregoing characteristic chromatogram. Inject 10 μl, and the results are as shown in Figure 10 .

[0113] The results show that this characteristic chromatogram method can fully display the complete chromatographic information.

[0114] 2.3.3 Precision test

[0115] Prepare 1 portion of the test sample solution according to the determined experimental method, inject consecutively 6 times, and calculate the retention time of each characteristic peak. See Table 2.

[0116] Table 2 Precision investigation - Retention time

[0117]

[0118] It can be seen from the above that the precision of this instrument is good.

[0119] 2.3.4 Repeatability investigation

[0120] Prepare 6 portions of the test sample solution according to the determined experimental method, measure, and calculate the relative retention time of each characteristic peak. See Table 3.

[0121] Table 3 Repeatability investigation - Relative retention time

[0122]

[0123] As can be seen from the above, the method has good repeatability.

[0124] 2.3.5 Intermediate precision investigation

[0125] Six test solution samples were prepared by Operator A1 at time T1 according to the proposed experimental method and determined on Instrument C1; six test solution samples were prepared by Operator A2 at time T2 according to the proposed experimental method and determined on Instrument C2. The relative retention times of each characteristic peak were calculated, as shown in Figure 11 , as shown in Table 4.

[0126] Table 4 Investigation of operators and time - ratio of relative retention times

[0127]

[0128]

[0129] As can be seen from the above, the method has good intermediate precision.

[0130] 2.3.6 Column durability

[0131] On the basis of the above - proposed experimental conditions, three different types of chromatographic columns were used for investigation (Column 1: Shim - pack Scepter C18 - 120, 2.1 mm×150 mm, 1.9 μm; Column 2: Luna Omega PS C18 100A, 2.1 mm×150 mm, 1.6 μm; Column 3: ACQUITY UPLC HSS T3, 2.1 mm×150 mm, 1.8 μm). See Table 5, Figure 12 .

[0132] Table 5 Investigation of column durability - relative retention time

[0133]

[0134] From Figure 12 it can be seen that seven common peaks appeared in the three batches of chromatographic columns, and the separation effects were quite the same. The method has good column durability.

[0135] 2.3.7 Stability investigation

[0136] On the basis of the above - proposed experimental conditions, the same test solution sample was taken and determined at 0 h, 3 h, 7 h, 12 h, and 18 h respectively. See Table 6.

[0137] Table 6 Investigation of stability - retention time

[0138]

[0139] As can be seen from the above, the RSD of the retention time of the characteristic peaks is 0.54 - 1.47%, and the sample solution is relatively stable within 18 hours.

[0140] Verification and Method Establishment of the Characteristic Chromatogram of the Sample in Example 3

[0141] 3.1 Extract

[0142] Using the method for constructing the characteristic chromatogram described above, the characteristic chromatograms of 20 batches of Shujincao extracts were determined by the constructed characteristic chromatogram method and the test sample preparation method described above, and the relative retention time was calculated. The retention times of each characteristic peak were specified to establish a reference characteristic chromatogram. The verification results are shown in Table 7 below.

[0143] Table 7 Relative Retention Time of the Characteristic Peaks of the Verification Batches of 20 Batches of Shujincao Extracts

[0144]

[0145] According to the principle of stable relative retention time, and that all batches of samples can be detected and the peaks are relatively high, a total of 7 peaks with good repeatability were selected as characteristic peaks. The results show that when Peak 4 is used as the S peak, the RSD of the relative retention time of the characteristic peaks of the test samples of 20 batches of Shujincao extracts with salt is 0.00% - 1.19%, and they are included in the standard.

[0146] Therefore, it is finally specified that: there should be 7 characteristic peaks in the chromatogram of the test sample, and they should correspond to the retention times of the 7 characteristic peaks in the chromatogram of the reference medicinal material reference substance. The peak corresponding to the reference substance peak of 4-hydroxybenzoic acid reference substance is the S peak, and the relative retention time of each characteristic peak and the S peak is calculated. Its relative retention time should be within the range of ±10% of the specified value, and the specified values are: 0.31 (Peak 1), 0.33 (Peak 2), 0.78 (Peak 3), 1.09 (Peak 5), 1.16 (Peak 6), 1.38 (Peak 7).

[0147] Using the Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints (2012 Edition), the 20 batches of Shujincao extracts were synthesized to establish a reference chromatogram of Shujincao extracts. See Figure 13 , Figure 13 as the reference characteristic chromatogram of Shujincao extracts; among them, Peak 3: protocatechuic acid; Peak 4 (S): 4-hydroxybenzoic acid; Peak 6: p-hydroxybenzaldehyde; Peak 7: 4-coumaric acid.

[0148] 3.2 Formula Granules

[0149] In the present invention, the characteristic chromatograms of 3 batches of Shujincao formula granules with salt were determined by the method for constructing the characteristic chromatogram and the test sample preparation method described above, and the relative retention time was calculated. The verification results are shown in Table 8 below.

[0150] Table 8 Relative retention times of three batches of Shujincao formula granules

[0151]

[0152] According to the principle of stable relative retention time, all batches of samples can be detected and the peaks are relatively high, a total of 7 peaks with good repeatability were selected as characteristic peaks. The results showed that when peak 4 was used as the S peak, the relative retention time RSD of the 7 characteristic peaks of three batches of Shujincao formula granules was 0.00% - 1.20%, and they were included in the standard.

[0153] Finally, it is stipulated that: seven characteristic peaks should be presented in the chromatogram of the test sample, and the retention times of the seven characteristic peaks should correspond to those in the chromatogram of the reference medicinal material reference substance. The peak corresponding to the reference substance peak of 4-hydroxybenzoic acid reference substance is the S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time should be within the range of ±10% of the specified value. The specified values are: 0.29 (peak 1), 0.31 (peak 2), 0.78 (peak 3), 1.09 (peak 5), 1.16 (peak 6), 1.39 (peak 7).

[0154] Three batches of Shujincao formula granules were synthesized using the Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines (2012 Edition) to establish a reference chromatogram of the characteristic chromatogram of Shujincao formula granules. See Figure 14 , Figure 14 as the reference characteristic chromatogram of Shujincao formula granules; among them, peak 3: protocatechuic acid; peak 4 (S): 4-hydroxybenzoic acid; peak 6: p-hydroxybenzaldehyde; peak 7: 4-coumaric acid.

[0155] 3.3 Medicinal materials

[0156] The characteristic chromatograms of 20 batches of Shujincao medicinal materials were determined using the aforementioned characteristic chromatogram construction method and test solution preparation method, and the relative retention time was calculated. The verification results are shown in Table 9 below.

[0157] Table 9 Relative retention times of characteristic peaks of 20 batches of Shujincao medicinal materials

[0158]

[0159] According to the principle of stable relative retention time, all batches of samples can be detected and the peaks are relatively high, a total of 7 peaks with good repeatability were selected as characteristic peaks. The results showed that when peak 4 was used as the S peak, the relative retention time RSD of the 7 characteristic peaks of 20 batches of Shujincao formula granules was 0.00% - 1.16%, and they were included in the standard.

[0160] Final requirement: Seven characteristic peaks should be presented in the chromatogram of the test sample, and the retention times of the seven characteristic peaks should correspond to those in the reference chromatogram of the control crude drug. The peak corresponding to the reference peak of 4-hydroxybenzoic acid reference substance is the S peak, and the relative retention times of each characteristic peak to the S peak are calculated. The relative retention times are within the range of the specified value ±10%, and the specified values are: 0.29 (peak 1), 0.32 (peak 2), 0.78 (peak 3), 1.09 (peak 5), 1.16 (peak 6), 1.39 (peak 7).

[0161] The similarity evaluation system for traditional Chinese medicine chromatographic fingerprints (2012 edition) was used to synthesize 20 batches of Lycopodiastrum casuarinoides herbs, and the reference chromatogram of the characteristic chromatograms of 20 batches of Lycopodiastrum casuarinoides herbs was established. See Figure 15 , Figure 15 as the reference characteristic chromatogram of Lycopodiastrum casuarinoides herbs; among them, peak 3: protocatechuic acid; peak 4 (S): 4-hydroxybenzoic acid; peak 6: p-hydroxybenzaldehyde; peak 7: 4-coumaric acid.

[0162] 3.4 Decoction Pieces

[0163] The characteristic chromatograms of 20 batches of Lycopodiastrum casuarinoides decoction pieces were determined by using the aforementioned characteristic chromatogram construction method and test solution preparation method, and the relative retention times were calculated. The verification results are shown in Table 10 below.

[0164] Table 10 Relative retention times of characteristic peaks of 20 batches of Lycopodiastrum casuarinoides decoction pieces

[0165]

[0166] According to the principle of stable relative retention time, all batches of samples can be detected and the peaks are relatively high, a total of 7 peaks with good repeatability were selected as characteristic peaks. The results showed that when peak 4 was used as the S peak, the RSD of the relative retention times of the 7 characteristic peaks of 20 batches of Lycopodiastrum casuarinoides formula granules was 0.00 - 0.88%, and they were included in the standard.

[0167] Final requirement: Seven characteristic peaks should be presented in the chromatogram of the test sample, and the retention times of the seven characteristic peaks should correspond to those in the reference chromatogram of the control crude drug. The peak corresponding to the reference peak of 4-hydroxybenzoic acid reference substance is the S peak, and the relative retention times of each characteristic peak to the S peak are calculated. The relative retention times are within the range of the specified value ±10%, and the specified values are: 0.30 (peak 1), 0.32 (peak 2), 0.78 (peak 3), 1.09 (peak 5), 1.16 (peak 6), 1.39 (peak 7).

[0168] The similarity evaluation system for traditional Chinese medicine chromatographic fingerprints (2012 edition) was used to synthesize 20 batches of Lycopodiastrum casuarinoides decoction pieces, and the reference chromatogram of the characteristic chromatograms of 20 batches of Lycopodiastrum casuarinoides decoction pieces was established. See Figure 16 , Figure 16This is the reference characteristic spectrum of Shujincao medicinal pieces; among them, Peak 3: protocatechuic acid; Peak 4 (S): 4-hydroxybenzoic acid; Peak 6: p-hydroxybenzaldehyde; Peak 7: 4-coumaric acid.

[0169] As can be seen from the above embodiments, the present invention discloses the construction and application of a high-performance liquid phase characteristic spectrum detection method for muscle-soothing herbal materials, decoction pieces, extracts, and muscle-soothing herbal granules. The method is applied to muscle-soothing herbal materials, decoction pieces, extracts, muscle-soothing herbal granules and related preparations, identifies 4 index components, confirms 7 common characteristic peaks, and studies their relative retention times, stipulates their relative retention times, establishes a control characteristic spectrum, fully displays the chemical composition characteristics of muscle-soothing herbal materials, and comprehensively reflects the quality information of muscle-soothing herbal materials, decoction pieces, extracts, and formula granules and related preparations, thereby being able to achieve comprehensive and effective control of the quality of muscle-soothing herbal materials, decoction pieces, extracts, formula granules and related preparations, and ensuring their chemical composition stability and safety in use. In addition to being applicable to the identification of the origin and related detection of muscle-soothing herbal materials and decoction pieces, the present invention has more advantages in the quality control of muscle-soothing herbal extracts, muscle-soothing herbal granules and related preparations.

[0170] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for constructing a high performance liquid chromatography characteristic fingerprint of Lycopodiastrum casuarinoides, comprising the following steps: The test sample is extracted with a solvent to obtain a test solution; The test solution is determined by high performance liquid chromatography to obtain the high performance liquid chromatography characteristic fingerprint of Lycopodiastrum casuarinoides; The chromatographic conditions of the high performance liquid chromatography are as follows: the chromatographic column is a C18 column; mobile phase A is methanol, and mobile phase B is 0.1% acetic acid solution, with gradient elution; The specific gradient elution is as follows: 0 - 2 min, phase A: 1%, phase B: 99%; 2 - 5 min, phase A: 1 - 6%, phase B: 99 - 94%; 5 - 10 min, phase A: 6 - 14%, phase B: 94 - 86%; 10 - 16 min, phase A: 14 - 20%, phase B: 86 - 80%; 16 - 28 min, phase A: 20 - 35%, phase B: 80 - 65%; The column temperature is 20 °C, the flow rate is 0.25 mL / min, the detection wavelength is 265 nm, the injection volume is 1 μL, and the theoretical plate number calculated based on the 4-hydroxybenzoic acid peak should be not less than 5000; It also includes the preparation of a reference substance solution: Take reference substances of protocatechuic acid, 4-hydroxybenzoic acid, p-hydroxybenzaldehyde, and 4-coumaric acid, dissolve them with 70% methanol to obtain a reference substance solution; The reference substance solution is determined by high performance liquid chromatography to obtain the chromatogram of the reference substance; and the components of the HPLC characteristic fingerprint of Lycopodiastrum casuarinoides are qualitatively determined according to the chromatogram of the reference substance.

2. The construction method according to claim 1, wherein The solvent used for extraction is 70% methanol; the extraction method is ultrasonic extraction; the extraction time is 15 - 25 min; The power of the ultrasonic extraction is 580 - 620 W, and the frequency of the ultrasonic extraction is 35 - 45 kHz; The mass ratio (g) of the test sample to the volume ratio (mL) of the extraction solvent is (0.4 - 0.6):

20.

3. The construction method according to claim 1, wherein The test sample raw materials are one or several of Lycopodiastrum casuarinoides herbs, cut herbs, Lycopodiastrum casuarinoides extracts, or Lycopodiastrum casuarinoides formula granules; The similarity of the Lycopodiastrum casuarinoides characteristic fingerprint is evaluated using the similarity evaluation system for traditional Chinese medicine chromatographic fingerprints to obtain the HPLC standard characteristic fingerprint of Lycopodiastrum casuarinoides composed of 7 characteristic peaks, where peak 3 is protocatechuic acid; peak 4 is 4-hydroxybenzoic acid; peak 6 is p-hydroxymethylbenzaldehyde; peak 7 is 4-coumaric acid.

4. The construction method according to claim 3, wherein The similarity of the HPLC characteristic fingerprint of Lycopodiastrum casuarinoides herbs is evaluated using the similarity evaluation system for traditional Chinese medicine chromatographic fingerprints to obtain the HPLC standard characteristic fingerprint of Lycopodiastrum casuarinoides herbs composed of 10 characteristic peaks, where the peak corresponding to the 4-hydroxybenzoic acid reference substance is the S peak; in the said standard characteristic fingerprint, calculate the relative retention time of each characteristic peak to the S peak, and its relative retention time should be within ±10% of the specified value; the specified values are: 0.29 (peak 1), 0.32 (peak 2), 0.78 (peak 3), 1.09 (peak 5), 1.16 (peak 6), 1.39 (peak 7).

5. The construction method according to claim 3, wherein The similarity of the HPLC characteristic fingerprint of Shujincao decoction pieces was evaluated using the similarity evaluation system for traditional Chinese medicine chromatographic fingerprints, and the HPLC standard characteristic fingerprint composed of 10 characteristic peaks was obtained. The peak corresponding to the reference substance 4-hydroxybenzoic acid was defined as the S peak. In the said standard characteristic fingerprint, the relative retention times of each characteristic peak and the S peak were calculated, and the relative retention times should be within ±10% of the specified values. The specified values were: 0.30 (peak 1), 0.32 (peak 2), 0.78 (peak 3), 1.09 (peak 5), 1.16 (peak 6), 1.39 (peak 7).

6. The construction method according to claim 3, wherein The similarity of the HPLC characteristic fingerprint of Shujincao formula granules was evaluated using the similarity evaluation system for traditional Chinese medicine chromatographic fingerprints, and the HPLC standard characteristic fingerprint composed of 10 characteristic peaks was obtained. The peak corresponding to the reference substance 4-hydroxybenzoic acid was defined as the S peak. In the said standard characteristic fingerprint, the relative retention times of each characteristic peak and the S peak were calculated, and the relative retention times should be within ±10% of the specified values. The specified values were: 0.29 (peak 1), 0.31 (peak 2), 0.78 (peak 3), 1.09 (peak 5), 1.16 (peak 6), 1.39 (peak 7). The similarity of the HPLC characteristic fingerprint of Shujincao extract was evaluated using the similarity evaluation system for traditional Chinese medicine chromatographic fingerprints, and the HPLC standard characteristic fingerprint composed of 10 characteristic peaks was obtained. The peak corresponding to the reference substance 4-hydroxybenzoic acid was defined as the S peak. In the said standard characteristic fingerprint, the relative retention times of each characteristic peak and the S peak were calculated, and the relative retention times should be within ±10% of the specified values. The specified values were: 0.31 (peak 1), 0.33 (peak 2), 0.78 (peak 3), 1.09 (peak 5), 1.16 (peak 6), 1.38 (peak 7).

7. A method for identifying Lycopodiastrum casuarinoides medicinal materials, cut pieces, extracts and their formula granules, characterized in that The method according to any one of claims 1 to 6 was used for detection, and the detection results were analyzed.

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