Construction method and application of characteristic chromatogram of magnolia officinalis flower standard decoction or traditional Chinese medicine dispensing granules

Abstract

The application discloses a method for constructing a characteristic spectrum of a magnolia officinalis flower standard decoction or formula granule, which comprises the following steps: preparing a reference solution, wherein the reference solution comprises a control medicinal material reference solution and a control reference solution, the control reference solution comprises magnolianoside B control, magnolol control and honokiol control; preparing a test sample solution from the magnolia officinalis flower standard decoction or formula granule; injecting a preset amount of the reference solution and the test sample solution into a liquid chromatograph, wherein the liquid chromatograph adopts octadecylsilane bonded silica gel as a filler, acetonitrile as a mobile phase A and a phosphoric acid solution as a mobile phase B for gradient elution, and a characteristic spectrum is established. The application can provide a data basis for quality control of the magnolia officinalis flower standard decoction or formula granule and medicinal materials, and guarantee the stability and controllability of product quality.

Description

Technical Field

[0001] The present invention relates to the technical field of quality analysis and detection of traditional Chinese medicines, and in particular to a method for constructing a characteristic spectrum of a standard decoction of Magnolia officinalis flower or a traditional Chinese medicine formula granule and its application. Background Art

[0002] Magnolia officinalis flower is the dried flower bud of Magnolia officinalis (Magnolia officinalis Rehb. et Wils.) or Magnolia officinalis Rehd. et Wils. var. biloba Rehb. et Wils., both of the Magnoliaceae family. It has the effects of aromatic dehumidification, regulating qi and relieving fullness. The 2020 edition of the Chinese Pharmacopoeia uses the content of magnolol and honokiol as quality evaluation indicators for Magnolia officinalis flower under the item of Magnolia officinalis flower medicinal materials. However, due to the diversity and complexity of the active ingredients in traditional Chinese medicine, the content determination of a single component is not sufficient to illustrate the intrinsic quality of traditional Chinese medicine and lacks specificity. The fingerprint / characteristic spectrum of traditional Chinese medicine can provide more comprehensive and rich information and is therefore often used in quality control research of traditional Chinese medicine. Currently, there are few reports on the fingerprint / characteristic spectrum of Magnolia officinalis flower. Most of the existing studies focus on the study of fingerprints or characteristic spectra of medicinal materials, while there are no reports on the application of fingerprints / characteristic spectra to traditional Chinese medicine preparations using Magnolia officinalis flower as raw materials.

[0003] Traditional Chinese medicine is mainly used in the form of decoctions. Decoctions are the material basis for the efficacy of Chinese medicine and have been used in clinical practice for thousands of years. Therefore, establishing the fingerprint or characteristic spectrum of Chinese medicine decoctions based on the effective ingredients of Chinese medicine decoctions is of great significance for reflecting the clinical effectiveness and spectrum-effect relationship of Chinese medicine. Standard Chinese medicine decoctions are standardized preparations of traditional Chinese medicine decoctions. They are single-ingredient Chinese medicine decoctions prepared by standardized processes with reference to modern extraction methods. As a standard substance and standard system, they are used to standardize the quality of modern Chinese medicine preparations such as formula granules. Chinese medicine formula granules use water as a solvent and are made through modern industrial extraction, concentration, drying, and granulation. They are a new type of Chinese medicine preparation that can be added or subtracted according to symptoms and used in prescriptions. They are often used in clinical practice to replace traditional decoction pieces.

[0004] In summary, there are currently few reports on the fingerprint or characteristic spectrum of Magnolia officinalis flower. Existing studies have focused solely on the chemical composition of the Magnolia officinalis flower medicinal material itself, and most of these studies have concentrated on fat-soluble components such as magnolol and honokiol. Water-soluble components in Magnolia officinalis flower and changes in chemical composition during the decoction of Magnolia officinalis flower decoctions have been overlooked. Clinically, Magnolia officinalis flower, whether in the form of decoctions or formulated granules, uses water as a solvent. Decoction or industrial extraction may alter the type, content, or ratio of active ingredients, leading to changes in the material basis for the clinical efficacy of traditional Chinese medicine. Therefore, studying the medicinal material itself alone is insufficient for evaluating its clinical effectiveness and monitoring its quality. It is necessary to establish quality control methods for formulated granules that are compatible with the active ingredients of traditional Chinese medicine decoctions, based on the active ingredients of the decoction. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for constructing a characteristic spectrum of a standard decoction of Magnolia officinalis flower or a Chinese medicine formula granule. The method has good reproducibility, accuracy and reliability, and can provide a data basis for the mass production quality control of the standard decoction of Magnolia officinalis flower or the formula granule, thereby ensuring the stability and controllability of the product quality of the standard decoction of Magnolia officinalis flower or the formula granule.

[0006] In order to solve the above technical problems, the present invention provides a method for constructing a characteristic spectrum of a standard decoction or formula granules of Magnolia officinalis flower, comprising the following steps:

[0007] Prepare a reference solution, the reference solution comprising a control medicinal material reference solution and a reference substance reference solution, wherein the reference substance reference solution comprises a magnolol reference substance, a magnolol reference substance, and a honokiol reference substance;

[0008] Prepare the test solution by taking the standard decoction or formula granules of Magnolia officinalis flower;

[0009] Take a preset amount of reference solution and test solution and inject them into a liquid chromatograph. The liquid chromatograph uses octadecylsilane bonded silica gel as a filler, acetonitrile as mobile phase A, and phosphoric acid solution as mobile phase B for gradient elution to establish a characteristic spectrum.

[0010] In one embodiment, the gradient elution is performed according to the following procedure:

[0011] 0-10 min, mobile phase A is 10%, mobile phase B is 90%;

[0012] 10-25 min, mobile phase A from 10% to 14%, mobile phase B from 90% to 86%;

[0013] 25-39 min, mobile phase A from 14% to 20%, mobile phase B from 86% to 80%;

[0014] 39-50 min, mobile phase A from 20% to 30%, mobile phase B from 80% to 70%;

[0015] From 50 to 68 minutes, the concentration of mobile phase A increased from 30% to 85%, and that of mobile phase B increased from 70% to 15%.

[0016] In one embodiment, the concentration of the phosphoric acid solution is 0.05% to 0.15%.

[0017] In one embodiment, the preset amount of the reference solution and the test solution is 4.5 μL to 5.5 μL;

[0018] The liquid chromatograph uses octadecylsilane bonded silica gel as a filler, with a column length of 150 mm, an inner diameter of 4.6 mm, a particle size of 2.7 μm, and a column temperature of 29°C to 31°C;

[0019] When performing gradient elution, the flow rate of the mobile phase is 0.8-1.0 ml / min;

[0020] The ultraviolet detection wavelength of the liquid chromatograph is 290nm to 310nm.

[0021] In one embodiment, the test solution is prepared by the following method:

[0022] Take the standard decoction or formula granules of Magnolia officinalis flower, grind it into powder, weigh 0.45g~0.55g, place it in a stoppered conical flask, add 20ml~30ml of 65%~75% methanol, ultrasonically treat it for 25min~35min, let it cool, weigh it again, make up the lost weight with 65%~75% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0023] In one embodiment, the reference substance solution is prepared according to the following method:

[0024] Take appropriate amounts of magnolioside B, magnolol, and honokiol reference substances, weigh them accurately, and add methanol to prepare solutions containing 20 μg each of magnolioside B, honokiol, and honokiol per 1 ml to obtain reference substance solutions.

[0025] In one embodiment, the control medicinal material reference solution is prepared by the following method:

[0026] Take 0.9g~1.1g of Magnolia officinalis flower reference medicinal material, place it in a stoppered conical flask, add 45ml~55ml of water, heat and reflux for 25min~35min, cool, filter, evaporate the filtrate to dryness, add 20ml~30ml of 65%~75% methanol to the residue, ultrasonically treat for 25min~35min, cool, shake well, filter, and take the filtrate as the reference solution of the control medicinal material.

[0027] In one embodiment, the characteristic spectrum comprises 10 common peaks, wherein the peak No. 1 corresponds to 3-0-p-coumaroylquinic acid, the peak No. 3 corresponds to magnoliavinse B, the peak No. 4 corresponds to magnoliavinse F, the peak No. 5 corresponds to magnoliavinse A, the peak No. 9 corresponds to honokiol, and the peak No. 10 corresponds to magnolol.

[0028] In one embodiment, in the characteristic spectrum of the Magnolia officinalis Rehd. et Wils. standard decoction or formula granules, taking the peak of magnoliavinse B as the reference peak S1, the relative retention time of the peak No. 1 to the peak No. 2, the peak No. 4 to the peak No. 5 meets the following conditions: the peak No. 1 is 0.84, the peak No. 2 is 0.93, the peak No. 4 is 1.32, and the peak No. 5 is 1.37, and the relative standard deviation is within ±10%.

[0029] Taking the peak of honokiol as the reference peak S2, the relative retention time of the peak No. 6 to the peak No. 8 meets the following conditions: the peak No. 6 is 0.61, the peak No. 7 is 0.64, and the peak No. 8 is 0.66, and the relative standard deviation is within ±10%.

[0030] and the application of the characteristic spectrum of the Magnolia officinalis Rehd. et Wils. standard decoction or formula granules obtained by the method for constructing the characteristic spectrum of the Magnolia officinalis Rehd. et Wils. standard decoction or formula granules in any one of (1) to (3):

[0031] (1) the authenticity identification and component detection of the Magnolia officinalis Rehd. et Wils. standard decoction;

[0032] (2) the authenticity identification and component detection of the Magnolia officinalis Rehd. et Wils. formula granules;

[0033] (3) the differentiation of the Magnolia officinalis Rehd. et Wils. standard decoction from the Magnolia sprengeri standard decoction and the Magnolia officinalis Rehd. et Wils. var. biloba standard decoction.

[0034] The implementation of the present application has the following beneficial effects:

[0035] Based on the material basis characteristics of the Magnolia officinalis Rehd. et Wils. standard decoction, the present application establishes the characteristic spectrum standard of the Magnolia officinalis Rehd. et Wils. standard decoction by using high performance liquid chromatography (HPLC), and establishes the characteristic spectrum standard of the Magnolia officinalis Rehd. et Wils. formula granules by taking the material basis of the standard decoction as a reference, so as to realize the rapid and comprehensive quality monitoring of the Magnolia officinalis Rehd. et Wils. standard decoction and formula granules, and the method is stable, has high precision, and good reproducibility, which has very important significance for reflecting the clinical effectiveness of the Magnolia officinalis Rehd. et Wils. medicinal material and related preparations and the research on the spectrum-effect relationship, and simultaneously provides an important reference for the quality control of the Magnolia officinalis Rehd. et Wils. standard decoction and formula granules. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 is the chromatogram of the characteristic spectrum of the Magnolia officinalis Rehd. et Wils. standard decoction at different wavelengths in Example 1 of the present application;

[0037] Figure 2 This is a characteristic spectrum of the standard decoction of Magnolia officinalis flower in Example 1 of the present invention - elution gradient 1 chromatogram;

[0038] Figure 3 This is the characteristic spectrum of the standard decoction of Magnolia officinalis flower in Example 1 of the present invention - elution gradient 2 chromatogram;

[0039] Figure 4 This is a characteristic spectrum of the standard decoction of Magnolia officinalis flower in Example 1 of the present invention - chromatograms of different chromatographic columns;

[0040] Figure 5 This is a chromatogram of the characteristic spectrum of the standard decoction of Magnolia officinalis flower in Example 1 of the present invention - different extraction solvents;

[0041] Figure 6 This is a chromatogram of the characteristic spectrum of the standard decoction of Magnolia officinalis flower in Example 1 of the present invention - different extraction methods;

[0042] Figure 7 This is a chromatogram of the characteristic spectrum of the standard decoction of Magnolia officinalis flower in Example 1 of the present invention at different extraction times;

[0043] Figure 8 This is a characteristic spectrum of Magnolia officinalis flower formula granules in Example 1 of the present invention - an investigation spectrum of different extraction solvents;

[0044] Figure 9 This is a characteristic spectrum of Magnolia officinalis flower formula granules in Example 1 of the present invention - an investigation spectrum of different extraction methods;

[0045] Figure 10 This is a characteristic spectrum of Magnolia officinalis flower formula granules in Example 1 of the present invention - an investigation spectrum of different extraction times;

[0046] Figure 11 This is a common pattern diagram of the characteristic spectra of 18 batches of Magnolia officinalis flower standard decoction in Example 1 of the present invention;

[0047] Figure 12 This is the control characteristic spectrum of the standard decoction of Magnolia officinalis flower in Example 1 of the present invention, wherein, Peak 1: 3-O-p-coumarylquinic acid; Peak 3 (S1): Magnoside B; Peak 4: Magnoside F; Peak 5: Magnoside A; Peak 9 (S2): Honokiol; Peak 10: Magnokiol;

[0048] Figure 13 The total ion current graph and ultraviolet absorption chromatogram of the test solution of Magnolia officinalis flower standard decoction in Example 1 of the present invention are shown;

[0049] Figure 14 This is a chromatogram showing the specificity of the characteristic spectrum of the standard decoction of Magnolia officinalis flower in Example 1 of the present invention;

[0050] Figure 15This is the characteristic spectrum of three batches of Magnolia officinalis flower formula granules in Example 1 of the present invention;

[0051] Figure 16 This is a reference characteristic spectrum of the Magnolia officinalis flower formula granules in Example 1 of the present invention, wherein Peak 1: 3-O-p-coumarylquinic acid; Peak 3 (S1): Magnolin B; Peak 5: Magnolin A; Peak 9 (S2): Honokiol; Peak 10: Magnolin;

[0052] Figure 17 This is the characteristic spectrum of the standard decoction of Magnolia officinalis flower in Example 2 of the present invention (number: BT5);

[0053] Figure 18 This is the characteristic spectrum of Magnolia officinalis flower formula granules in Example 3 of the present invention (No.: KL02);

[0054] Figure 19 This is a superimposed graph of the characteristic spectra of three batches of Magnolia officinalis standard decoction in Example 4 of the present invention;

[0055] Figure 20 This is an overlay of characteristic spectra of two batches of standard decoctions of Michelia sempervirens in Example 4 of the present invention;

[0056] Figure 21 This is a superimposed graph of the characteristic spectra of the standard decoctions of Magnolia officinalis flower, Magnolia grandiflora, and Michelia serrata flower in Example 4 of the present invention. DETAILED DESCRIPTION

[0057] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings and specific implementation methods.

[0058] Example 1: Construction of UPLC characteristic profiles of Magnolia officinalis flower standard decoction and formula granules

[0059] 1. Sources of Magnolia officinalis Flower

[0060] The Magnolia officinalis flower used in this experiment was harvested by staff from the Purchasing Management Department of Guangdong Yifang Pharmaceutical Co., Ltd. The herbal medicine and herbal pieces were certified by the Quality Center of Guangdong Yifang Pharmaceutical Co., Ltd. and meet all relevant requirements for "Magnolia officinalis flower" in Part I of the 2020 edition of the Chinese Pharmacopoeia. They are identified as the dried flower buds of Magnolia officinalis Rehd. et Wils., a plant of the Magnoliaceae family. See Table 1 for the origin of the herbal medicine.

[0061] Table 1 Origin information of 18 batches of Magnolia officinalis flower medicinal materials

[0062]

[0063]

[0064] 2. Preparation of Magnolia officinalis Flower Pieces

[0065] Referring to the provisions under Magnolia officinalis flower in the first volume of the 2020 edition of the Chinese Pharmacopoeia: There is no decoction piece item under Magnolia officinalis flower, so we simply "remove impurities" and prepare 18 batches of Magnolia officinalis flower decoction pieces.

[0066] 3. Preparation of Magnolia officinalis Flower Standard Decoction

[0067] Take 100g of Magnolia officinalis flower slices, place in an electric ceramic pot, add water and decoct twice, add 12 times the amount of water for the first decoction, soak for 30 minutes, boil over high heat (500W), then keep it slightly boiling over low heat (200W) for 30 minutes, filter through a 200-mesh sieve while hot, and cool the filtrate quickly with cold water. Add 10 times the amount of water for the second decoction, boil over high heat (500W), then keep it slightly boiling over low heat (200W) for 25 minutes, filter through a 200-mesh sieve while hot, and cool the filtrate quickly with cold water. Combine the two decoctions, concentrate under reduced pressure, and freeze-dry to obtain a standard decoction freeze-dried powder.

[0068] 4. Preparation of Magnolia Officinalis Flower Formula Granules

[0069] Take Magnolia officinalis flower medicinal material, select and remove impurities, and crush it with a crusher equipped with a 2.0 cm aperture screen to obtain "Magnolia officinalis flower slices". Take 4500g of Magnolia officinalis flower slices, add 13 times the amount of water, decoct for 1.0 hour, filter the decoction, and concentrate the filtrate under reduced pressure at 60℃~80℃ to a clear paste with a relative density of 1.07~1.09 (80℃), filter, spray dry, add appropriate amount of silicon dioxide and maltodextrin, mix well, dry granulate, make 1000g, and package. Prepare 3 batches of Magnolia officinalis flower formula granules, numbered KL01, KL02, and KL03 respectively.

[0070] 5. Establishment of characteristic profiles for standard decoction and formula granules of Magnolia officinalis flower

[0071] 1. Chromatographic conditions

[0072] 1.1 Wavelength selection

[0073] In this embodiment, 260nm, 300nm, and 350nm were selected as the detection wavelengths of the standard decoction sample of Magnolia officinalis flower, and full-wave scanning was performed to record the absorption spectrum of the sample in the range of 190nm to 400nm. The results are shown in FIG. Figure 1 The results showed that at a wavelength of 300 nm, the chromatographic peaks were more abundant and the peak area was larger. Therefore, 300 nm was selected as the detection wavelength for establishing the characteristic spectrum of the standard decoction of Magnolia officinalis flower.

[0074] 1.2 Optimization of elution gradient

[0075] Agilent poroshell 120EC C 18(4.6mm×150mm, 2.7μm) chromatographic column; acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution according to the provisions of Tables 2 and 3; flow rate 0.3ml per minute, column temperature 30℃; injection volume 2μl, results are shown in Figure 2 and Figure 3 As shown, the results show that when elution gradient 2 is used, the separation of each characteristic peak is better and the baseline is more stable, so gradient elution is performed according to elution gradient 2.

[0076] Table 2 Elution gradient 1

[0077]

[0078] Table 3 Elution gradient 2

[0079]

[0080] 1.3 Investigation of chromatographic columns

[0081] Compare Waters BEH C 18 Chromatographic column (3.0 mm × 150 mm, 2.5 μm), Agilent SB C 18 Chromatographic column (4.6m×150mm, 5μm), Poroshell 120EC-C 18 The effects of three chromatographic columns (4.6m×150mm, 2.7μm) on the characteristic spectrum of Magnolia officinalis flower standard decoction are shown in the following table. Figure 4 As shown in the figure, the results show that when the Agilent Poroshell 120EC-C18 column is selected, the characteristic peak information is complete and the separation is good.

[0082] 1.4 Determination of chromatographic conditions

[0083] Agilent poroshell 120EC C 18 (4.6 mm × 150 mm, 2.7 μm) chromatographic column; acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution according to elution gradient 2 in Table 3; flow rate, 0.9 ml / min; column temperature, 30°C; detection wavelength, 300 nm; injection volume, 5 μl.

[0084] 2. Preparation of reference substance solution

[0085] Take appropriate amounts of magnoside B, magnolol, and honokiol reference substances, weigh them accurately, and add methanol to prepare solutions containing 20 μg each of magnoside B, honokiol, and magnolol per 1 ml.

[0086] 3. Preparation of test solution

[0087] 3.1 Preparation of Magnolia officinalis flower standard decoction test solution

[0088] (1) Investigation of extraction solvent

[0089] Take an appropriate amount of Magnolia officinalis flower standard decoction (No. BT6), grind it into powder, take about 0.5g, accurately weigh it, and place it in 6 parallel groups, 2 portions in each group, in a stoppered conical flask, accurately add 25ml of methanol, 50% methanol, 70% methanol, ethanol, 50% ethanol and 70% ethanol respectively, weigh it, ultrasonicate it (300W, frequency 40kHz) for 30 minutes, let it cool, weigh it again, and then make up the lost weight with the corresponding solvent, shake it well, filter it, and take the filtrate. Inject it according to the chromatographic conditions determined under "1.4" and analyze the results. Figure 5 As shown, the results show that when 70% methanol is selected as the extraction solvent, the response values ​​of each characteristic peak are generally higher. Therefore, 70% methanol is selected as the extraction solvent.

[0090] (2) Investigation of extraction methods

[0091] Take an appropriate amount of Magnolia officinalis flower standard decoction (No. BT6), grind it into powder, take about 0.5g, accurately weigh it, and place it in two parallel groups, 2 portions in each group, place it in a stoppered conical flask, accurately add 25ml of 70% methanol, weigh it, and ultrasonically treat it (power 300W, frequency 40kHz) for 30 minutes, heat it under reflux for 30 minutes, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate. Inject it and analyze it according to the chromatographic conditions determined under "1.4". The results are shown in Figure 6 As shown in the results, different extraction methods have little effect on the characteristic spectrum of the standard decoction of Magnolia officinalis flower. Considering the convenience of operation, ultrasonic treatment was selected.

[0092] (3) Extraction time investigation

[0093] Take an appropriate amount of Magnolia officinalis flower standard decoction (No. BT6), grind it into powder, take about 0.5g, accurately weigh it, and place it in 3 parallel groups, 2 portions in each group, place it in a stoppered conical flask, accurately add 25ml of 70% methanol, weigh it, and ultrasonically treat it (power 300W, frequency 40kHz) for 15 minutes, 30 minutes, and 60 minutes respectively. Let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate. Inject it according to the chromatographic conditions determined under "1.4" and analyze the results. Figure 7 As shown in the figure, the results show that different extraction times have little effect on the characteristic spectrum of the standard decoction of Magnolia officinalis flower. In order to ensure complete extraction, ultrasonic extraction for 30 minutes was selected.

[0094] (4) Determination of the preparation method of the test solution of Magnolia officinalis flower standard decoction

[0095] According to the above experimental results, the sample pretreatment method of the characteristic spectrum of the standard decoction of Magnolia officinalis flower can be determined as follows: take an appropriate amount of the standard decoction of Magnolia officinalis flower, grind it into powder, take about 0.5 g, accurately weigh it, put it in a stoppered conical flask, accurately add 25 ml of 70% methanol, weigh it, ultrasonically treat it (power 300 W, frequency 40 kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it evenly, filter it, and take the filtrate to obtain it.

[0096] 3.2 Preparation of Magnolia officinalis flower formula granules test solution

[0097] (1) Investigation of extraction solvent

[0098] Take an appropriate amount of Magnolia officinalis flower granules (No. KL01), grind them into powder, take about 0.5g, accurately weigh, and divide into 6 parallel groups, 2 portions each, place in a stoppered conical flask, accurately add 25ml of methanol, 50% methanol, 70% methanol, ethanol, 50% ethanol and 70% ethanol respectively, weigh the weight, and ultrasonicate (power 300W, frequency 40kHz) for 30 minutes, remove, cool, weigh again, make up the lost weight with the corresponding solvent, shake well, filter, take the filtrate, and analyze according to the chromatographic conditions determined under "1.4". The results are shown in Figure 2. Figure 8 As shown, the results show that when 70% methanol is selected as the extraction solvent, the response values ​​of each characteristic peak are generally higher. Therefore, 70% methanol is selected as the extraction solvent.

[0099] (2) Investigation of extraction methods

[0100] Take an appropriate amount of Magnolia officinalis flower granules (No. KL01), grind them into powder, take about 0.5g, accurately weigh, and divide into two parallel groups, 2 portions each, place in a stoppered conical flask, accurately add 25ml of 70% methanol, weigh the weight, ultrasonically treat (power 300W, frequency 40kHz) for 30 minutes, heat under reflux for 30 minutes, remove, cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, take the filtrate, and analyze according to the chromatographic conditions determined under "1.4". The results are shown in Figure 2. Figure 9 As shown in the results, different extraction methods have little effect on the characteristic spectrum of Magnolia officinalis flower formula granules. Considering the convenience of operation, ultrasonic treatment was selected.

[0101] (3) Extraction time investigation

[0102] Take an appropriate amount of Magnolia officinalis flower granules (No. KL01), grind them into powder, take about 0.5g, accurately weigh, and divide into 3 parallel groups, 2 portions each, place in a stoppered conical flask, accurately add 25ml of 70% methanol, and ultrasonically treat (power 300W, frequency 40kHz) for 15 minutes, 30 minutes, and 60 minutes respectively. Remove, cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, take the filtrate, and analyze according to the chromatographic conditions determined under "1.4". The results are shown in Figure 2. Figure 10 As shown in the figure, the results show that different extraction times have little effect on the characteristic spectrum of Magnolia officinalis flower formula granules. In order to ensure complete extraction, ultrasonic extraction for 30 minutes was selected.

[0103] (4) Determination of the preparation method of the test solution of Magnolia officinalis flower formula granules

[0104] According to the above experimental results, the sample pretreatment method of the characteristic spectrum of Magnolia officinalis flower formula granules can be determined as follows: take an appropriate amount of Magnolia officinalis flower formula granules, grind them into powder, take about 0.5 g, accurately weigh, place in a stoppered conical flask, accurately add 25 ml of 70% methanol, weigh the weight, ultrasonically treat (power 300 W, frequency 40 kHz) for 30 minutes, let cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate.

[0105] 4. Establishment of the characteristic spectrum of Magnolia officinalis flower standard decoction

[0106] Take 18 batches of Magnolia officinalis flower standard decoction samples, prepare the test solution according to the test solution preparation method determined under "3.1", and perform sample injection determination according to the chromatographic conditions determined under "1.4". Use the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software" to identify the common peaks of the characteristic spectra of the 18 batches of Magnolia officinalis flower standard decoction, and select 10 common peaks with known ingredients, good peak shape and separation, high purity and large peak area as the characteristic peaks of the characteristic spectra of Magnolia officinalis flower standard decoction, such as Figure 11 As shown in Table 5, the relative retention time and relative peak area of ​​peaks 1 to 5 and S1 were calculated using the chromatographic peak of magnolol as the reference peak S1; the relative retention time and relative peak area of ​​peaks 6 to 10 and S2 were calculated using the chromatographic peak of magnolol as the reference peak S2. The results are shown in Tables 5 and 6. The characteristic spectra of 18 batches of Magnolia officinalis flower standard decoction were matched using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System", and a reference spectrum was generated by the average method to establish a reference characteristic spectrum of Magnolia officinalis flower standard decoction, as shown in Table 5. Figure 12 , this characteristic spectrum has a total of 10 characteristic peaks.

[0107] Table 5 Characteristic spectra of 18 batches of Magnolia officinalis flower standard decoction (relative retention time)

[0108]

[0109]

[0110] Table 6 Characteristic spectra of 18 batches of Magnolia officinalis flower standard decoction (relative peak area)

[0111]

[0112]

[0113] According to the test results of 18 batches of Magnolia officinalis flower standard decoction samples, the characteristic spectrum standard of Magnolia officinalis flower standard decoction was determined as follows: 10 characteristic peaks should be present in the chromatogram of the test sample, among which peak 3, peak 9, and peak 10 should correspond to the retention time of the corresponding reference substance peak, respectively. The peak corresponding to the magnolol reference substance is the S1 peak, and the relative retention times of peaks 1-2, peaks 4-5 and the S1 peak are calculated; the peak corresponding to the honokiol reference substance is the S2 peak, and the relative retention times of peaks 6-8 and the S2 peak are calculated. The relative retention times should be within ±10% of the specified values, and the specified values ​​are 0.84 (peak 1), 0.93 (peak 2), 1.32 (peak 4), 1.37 (peak 5), 0.61 (peak 6), 0.64 (peak 7), and 0.66 (peak 8).

[0114] 5. Identification of characteristic peaks

[0115] ①Liquid chromatography conditions

[0116] Except that mobile phase B is "0.1% acetic acid solution", the other chromatographic conditions are the same as those under "1.4".

[0117] ② Mass spectrometry conditions. See Table 7 for mass spectrometry parameters.

[0118] Table 7 Mass spectrometry parameters

[0119]

[0120] ③ Preparation of test solution

[0121] Same as under “3.1”.

[0122] ④Determination method: Accurately aspirate 1 μl of the test solution, inject it into liquid chromatography-mass spectrometry instrument, and determine the result.

[0123] ⑤ Identification of compounds

[0124] The test solution was tested using the above-mentioned liquid chromatography and mass spectrometry analysis conditions. By comparing the reference liquid chromatography retention time and the mass spectrometry accurate molecular weight and fragment ion comparative analysis, it was confirmed that peak 1 in the characteristic spectrum of the standard decoction of Magnolia officinalis flower was 3-0-p-coumarylquinic acid, peak 3 was magnoside B, peak 4 was magnoside F, peak 5 was magnoside A, peak 9 was honokiol, and peak 10 was magnolol. The total ion current and ultraviolet absorption chromatogram of the test solution are shown in FIG. Figure 13 The compound information is shown in Table 8.

[0125] Table 8 Mass spectrometry identification results of compounds in Magnolia officinalis flower standard decoction

[0126]

[0127] 6. Methodology verification

[0128] (1) Specificity investigation

[0129] 5 μl of each of the test sample solution, the reference substance solution of the control substance, and the blank solvent under Magnolia officinalis flower standard decoction (No. BT6) was precisely taken and injected into the liquid chromatograph, and determination was performed according to the chromatographic conditions determined in item “1.4”. The results are shown in Table 1, which shows that the test sample chromatogram has the same chromatographic peak at the retention time corresponding to the control substance chromatogram, and the blank solvent does not interfere, indicating that the method has good specificity. Figure 14

[0130] (2) Precision investigation

[0131] About 0.5 g of Magnolia officinalis flower standard decoction (No. BT6) was precisely weighed, and a test sample solution was prepared according to the test sample solution preparation method determined in item “3.1”. The sample was injected 6 times repeatedly according to the chromatographic conditions determined in item “1.4”. With magnoliobioside B chromatographic peak as the reference peak S1, the relative retention time and relative peak area RSD values of peaks 1-5 to S1 peak were calculated, which were in the range of 0.21%-2.87%. With honokiol chromatographic peak as the reference peak S2, the relative retention time and relative peak area RSD values of peaks 6-10 to S2 peak were calculated, which were in the range of 0.65%-2.24%, both less than 3.0%, indicating that the instrument precision was good.

[0132] (3) Reproducibility investigation

[0133] About 0.5 g of the same batch of Magnolia officinalis flower standard decoction (No. BT6) was precisely weighed, and 6 test sample solutions were prepared in parallel according to the test sample solution preparation method determined in item “3.1”. The sample was injected and analyzed according to the chromatographic conditions determined in item “1.4”. With magnoliobioside B chromatographic peak as the reference peak S1, the relative retention time and relative peak area RSD values of peaks 1-5 to S1 peak were calculated, which were in the range of 0.36%-2.17%. With honokiol chromatographic peak as the reference peak S2, the relative retention time and relative peak area RSD values of peaks 6-10 to S2 peak were calculated, which were in the range of 0.67%-2.94%, both less than 3.0%, indicating that the method had good reproducibility.

[0134] 7. Establishment of Magnolia officinalis flower formula granule characteristic chromatogram

[0135] ​Take 3 batches of Magnolia officinalis flower granules (No.: KL01, KL02 and KL03), prepare the test solution according to the test solution preparation method determined under "3.2", accurately pipette the above test solution, and inject the sample according to the chromatographic conditions under "1.4". The characteristic spectrum overlay of the 3 batches of samples is shown in Figure 15 As shown, the chromatographic peak of magnolol B was used as the reference peak S1, and the relative retention time and relative peak area of ​​peaks 1 to 5 and S1 were calculated. The chromatographic peak of magnolol was used as the reference peak S2, and the relative retention time and relative peak area of ​​peaks 6 to 10 and S2 were calculated. The results are shown in Tables 9 and 10.

[0136] Table 9 Characteristic Spectrum Sample Determination of Magnolia Officinalis Flower Formula Granules (Relative Retention Time)

[0137]

[0138]

[0139] Table 10 Characteristic spectrum of Magnolia officinalis flower formula granules sample determination (relative peak area)

[0140]

[0141] The characteristic spectra of the three batches of Magnolia officinalis flower formula granules were matched using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System", and a reference spectrum was generated using the average method to establish a reference characteristic spectrum of Magnolia officinalis flower formula granules, such as Figure 16 Referring to the research results of the characteristic spectrum of the standard decoction, the characteristic spectrum standard of Magnolia officinalis flower formula granules was determined as follows: the test sample chromatogram should present 10 characteristic peaks, of which peaks 3, 9, and 10 should correspond to the retention times of the corresponding reference substance peaks, respectively. The peak corresponding to the magnolol reference substance is the S1 peak, and the relative retention times of peaks 1-2, 4-5, and the S1 peak are calculated; the peak corresponding to the honokiol reference substance is the S2 peak, and the relative retention times of peaks 6-8 and the S2 peak are calculated. The relative retention times should be within ±10% of the specified values, which are 0.84 (peak 1), 0.93 (peak 2), 1.32 (peak 4), 1.37 (peak 5), 0.61 (peak 6), 0.64 (peak 7), and 0.66 (peak 8).

[0142] Example 2 Determination of the characteristic spectrum of Magnolia officinalis flower standard decoction (No.: BT5)

[0143] Chromatographic conditions: octadecylsilane bonded silica gel as the filler (column length: 150 mm, inner diameter: 4.6 mm, particle size: 2.7 μm); acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution as specified in Table 3; flow rate: 0.9 ml / min; column temperature: 30°C; detection wavelength: 300 nm.

[0144] Preparation of Reference Solution: Prepare the reference solution by taking approximately 1 g of Magnolia officinalis flower as a control medicinal material and placing it in a stoppered conical flask. Add 50 ml of water and heat under reflux for 30 minutes. Allow to cool, filter, and evaporate the filtrate to dryness. Add 25 ml of 70% methanol to the residue and sonicate (power 300 W, frequency 40 kHz) for 30 minutes. Allow to cool, shake well, filter, and use the filtrate as the reference medicinal material solution. Accurately weigh appropriate amounts of magnolol, magnolol, and honokiol reference substances and add methanol to prepare solutions containing 20 μg each of magnolol, honokiol, and magnolol per 1 ml.

[0145] Preparation of test solution: Take an appropriate amount of standard decoction of Magnolia officinalis flower, grind it into powder, take about 0.5g, accurately weigh it, put it into a stoppered conical flask, accurately add 25ml of 70% methanol, weigh it, and ultrasonically treat it (power 300W, frequency 40kHz) for 30 minutes. Let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0146] Determination method: Accurately aspirate 5 μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0147] Test results: as shown in Table 11 and Figure 17 As shown, the characteristic spectrum of the standard decoction of Magnolia officinalis flower numbered BT5 has a total of 10 characteristic peaks, of which peaks 3, 9, and 10 correspond to the retention times of the corresponding reference substances; the peak corresponding to the magnolol reference substance is peak S1, and the relative retention times of peaks 1-2, 4-5 and S1 are calculated; the peak corresponding to the honokiol reference substance is peak S2, and the relative retention times of peaks 6-8 and S2 are calculated. Their relative retention times are: 0.84 (peak 1), 0.93 (peak 2), 1.33 (peak 4), 1.37 (peak 5), 0.61 (peak 6), 0.64 (peak 7), and 0.66 (peak 8).

[0148] Table 11 Calculation results of relative retention time of each characteristic peak of the characteristic spectrum of Magnolia officinalis flower standard decoction (No.: BT5)

[0149]

[0150] Example 3 Determination of the characteristic spectrum of Magnolia officinalis flower formula granules (No.: KL02)

[0151] Chromatographic conditions: octadecylsilane bonded silica gel as the filler (column length: 150 mm, inner diameter: 4.6 mm, particle size: 2.7 μm); acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution as specified in Table 3; flow rate: 0.9 ml / min; column temperature: 30°C; detection wavelength: 300 nm.

[0152] Preparation of Reference Solution: Prepare the reference solution by taking approximately 1 g of Magnolia officinalis flower as a control medicinal material and placing it in a stoppered conical flask. Add 50 ml of water and heat under reflux for 30 minutes. Allow to cool, filter, and evaporate the filtrate to dryness. Add 25 ml of 70% methanol to the residue and sonicate (power 300 W, frequency 40 kHz) for 30 minutes. Allow to cool, shake well, filter, and use the filtrate as the reference medicinal material solution. Accurately weigh appropriate amounts of magnolol, magnolol, and honokiol reference substances and add methanol to prepare solutions containing 20 μg each of magnolol, honokiol, and magnolol per 1 ml.

[0153] Preparation of test solution: Take an appropriate amount of this product, grind it into powder, take about 0.5g, accurately weigh it, put it into a stoppered conical flask, accurately add 25ml of 70% methanol, weigh it, and treat it ultrasonically (power 300W, frequency 40kHz) for 30 minutes. Let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate.

[0154] Determination method: Accurately aspirate 5 μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0155] Determination results; as shown in Table 12 and Figure 18 As shown, the characteristic spectrum of Magnolia officinalis flower formula granules numbered KL02 has a total of 10 characteristic peaks, of which peaks 3, 9, and 10 correspond to the retention times of the corresponding reference substances; the peak corresponding to the magnolol reference substance is peak S1, and the relative retention times of peaks 1-2, 4-5 and S1 are calculated; the peak corresponding to the honokiol reference substance is peak S2, and the relative retention times of peaks 6-8 and S2 are calculated. Their relative retention times are: 0.85 (peak 1), 0.93 (peak 2), 1.33 (peak 4), 1.37 (peak 5), 0.61 (peak 6), 0.64 (peak 7), and 0.66 (peak 8).

[0156] Table 12 Calculation results of relative retention time of each characteristic peak of the characteristic spectrum of Magnolia officinalis flower formula granules (No.: KL02)

[0157]

[0158]

[0159] Example 4 Distinction between Magnolia officinalis flower standard decoction, Magnolia grandiflora standard decoction and Michelia sempervirens flower standard decoction

[0160] Take 3 batches of standard decoction of Magnolia grandiflora (No.: GT1810111, GT1810112 and GT1810113) and 2 batches of standard decoction of Michelia serrata (No.: GT2303128, GT2303158) to make the test solution. Accurately aspirate the above test solution and inject the sample according to the chromatographic conditions under "1.4". The characteristic spectrum overlay of the 3 batches of standard decoction of Magnolia grandiflora is shown in Figure 19 As shown, the characteristic spectrum superposition of two batches of standard decoction of Michelia sempervirens is shown in Figure 20 As shown in the figure, the characteristic spectrum superposition of Magnolia officinalis flower standard decoction, Magnolia officinalis flower standard decoction and Michelia sempervirens flower standard decoction is shown in Figure 21 shown.

[0161] The experimental results show that the standard decoction of Magnolia officinalis flower is significantly different from the standard decoction of Magnolia officinalis flower and the standard decoction of Michelia sutchuenensis flower in characteristic spectra. After retention time comparison and spectral analysis, the standard decoction of Magnolia officinalis flower lacks peaks 2, 6, 8, 9 (honokiol) and 10 (magnokiol), while the standard decoction of Michelia sutchuenensis flower lacks peaks 1 to 5, 8, 9 (honokiol) and 10 (magnokiol), indicating that the chemical components contained in the three are quite different and the number of characteristic peaks is significantly different. Therefore, the present invention can distinguish Magnolia officinalis flower (Magnolia officinalis) from the standard decoction of Magnolia officinalis flower and the standard decoction of Michelia sutchuenensis flower under the proposed chromatographic conditions, providing a quality basis for the large-scale production of standard decoction of Magnolia officinalis flower (Magnolia officinalis) or formula granules.

[0162] The above is a preferred embodiment of the 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 principles of the invention. These improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. A method for constructing a characteristic spectrum of a standard decoction or formula granule of Magnolia officinalis flower, characterized in that: The following steps are involved: Prepare a reference solution, the reference solution comprising a control medicinal material reference solution and a reference substance reference solution, wherein the reference substance reference solution comprises a magnolol reference substance, a magnolol reference substance, and a honokiol reference substance; A test solution is prepared by taking a standard decoction or formula granules of Magnolia officinalis flower; the test solution is prepared by the following method: take a standard decoction or formula granules of Magnolia officinalis flower, grind it into powder, weigh 0.45g~0.55g, place it in a stoppered conical flask, add 20ml~30ml of 65%~75% methanol, ultrasonically treat it for 25min~35min, let it cool, weigh it again, make up the lost weight with 65%~75% methanol, shake it well, filter it, and take the filtrate to obtain the product; Take a preset amount of reference solution and test solution and inject them into a liquid chromatograph with an ultraviolet detection wavelength of 290nm~310nm. The liquid chromatograph uses an Agilent poroshell 120EC C 18 The chromatographic column has a length of 150 mm, an inner diameter of 4.6 mm, and a filler particle size of 2.7 μm. Gradient elution is performed using acetonitrile as mobile phase A and phosphoric acid solution as mobile phase B to establish a characteristic spectrum. Wherein, the gradient elution is carried out according to the following procedure: 0-10 min, mobile phase A was 10%, mobile phase B was 90%; 10 min to 25 min, mobile phase A from 10% to 14%, mobile phase B from 90% to 86%; 25min~39min, mobile phase A from 14% to 20%, mobile phase B from 86% to 80%; 39-50 min, mobile phase A from 20% to 30%, mobile phase B from 80% to 70%; 50-68 min, mobile phase A from 30% to 85%, mobile phase B from 70% to 15%; The concentration of the phosphoric acid solution is 0.05% to 0.15%; The characteristic spectrum includes 10 common peaks, among which peak 1 corresponds to 3-0-p-coumarylquinic acid, peak 3 corresponds to magnoside B, peak 4 corresponds to magnoside F, peak 5 corresponds to magnoside A, peak 9 corresponds to magnolol, and peak 10 corresponds to magnolol.

2. The method for constructing the characteristic spectrum of the standard decoction or formula granules of Magnolia officinalis flower according to claim 1, characterized in that: The preset volume of the reference solution and the test solution is 4.5 μL~5.5 μL; The column temperature of the liquid chromatograph is 29°C to 31°C; When performing the gradient elution, the flow rate of the mobile phase is 0.8-1.0 ml / min.

3. The method for constructing the characteristic spectrum of the standard decoction or formula granules of Magnolia officinalis flower according to claim 1, characterized in that: The reference substance solution was prepared according to the following method: Take appropriate amounts of magnolol B, magnolol, and honokiol reference substances, weigh them accurately, and add methanol to make solutions containing 20µg each of magnolol B, honokiol, and honokiol per 1ml to obtain reference substance solutions.

4. The method for constructing a characteristic spectrum of a standard decoction or formula granule of Magnolia officinalis flower according to claim 1, characterized in that: The control medicinal material reference solution is prepared by the following method: Take 0.9g~1.1g of Magnolia officinalis flower reference medicinal material, place it in a stoppered conical flask, add 45ml~55ml of water, heat and reflux for 25min~35min, cool, filter, evaporate the filtrate to dryness, add 20ml~30ml of 65%~75% methanol to the residue, ultrasonically treat for 25min~35min, cool, shake well, filter, and take the filtrate as the reference solution of the control medicinal material.

5. The method for constructing the characteristic spectrum of the standard decoction or formula granules of Magnolia officinalis flower according to claim 1, characterized in that: In the characteristic spectrum of the standard decoction or formula granules of Magnolia officinalis flower, with the magnololamine B peak as the reference peak S1, the relative retention times of peaks 1 to 2, 4 to 5, and the reference peak S1 are consistent with: peak 1 0.84, peak 2 0.93, peak 4 1.32, peak 5 1.37, and the relative standard deviation is within ±10%; Taking the magnolol peak as the reference peak S2, the relative retention times of peaks 6 to 8 and the reference peak S2 are consistent with: peak 6 0.61, peak 7 0.64, peak 8 0.66, and the relative standard deviation is within ±10%.

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