Construction method and application of specific chromatogram of officinal magnolia flower standard decoction or traditional Chinese medicine formula granules

Abstract

The invention discloses a construction method of a specific chromatogram of a standard decoction or formula granule of flos magnoliae officinalis, which comprises the following steps: preparing a reference substance solution which comprises a reference substance solution of a reference medicinal material and a reference substance solution of a reference substance, the reference substance solution comprises a magnolin B reference substance, a magnolol reference substance and a honokiol reference substance; preparing the officinal magnolia flower standard decoction or formula granules into a test solution; a preset amount of the reference substance solution and the test solution are taken and injected into a liquid chromatograph, the liquid chromatograph takes octadecylsilane chemically bonded silica as a filler, acetonitrile as a mobile phase A and a phosphoric acid solution as a mobile phase B for gradient elution, and the characteristic chromatogram is established. The method can provide a data basis for quality control of standard decoction or formula granules and medicinal materials of flos magnoliae officinalis, and ensures the stability and controllability of product quality.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine quality analysis and detection, and in particular to a method for constructing a characteristic spectrum of a magnolia bark flower standard decoction or traditional Chinese medicine formula granules and an application thereof. Background Art

[0002] Magnolia officinalis flower is the dried flower bud of Magnolia officinalis Rehb.et Wils. or Magnolia officinalis Rehd.et Wils.var.biloba Rehb.et Wils. of the Magnoliaceae family. It has the effects of aromatic dehumidification, regulating qi and relieving fullness. In the 2020 edition of the Chinese Pharmacopoeia, the content of magnolol and honokiol is used as the quality evaluation index of Magnolia officinalis flower under the item of Magnolia officinalis flower medicinal materials. However, due to the diversity and complexity of the effective ingredients of traditional Chinese medicine, the content determination of a single ingredient is not enough to explain the intrinsic quality of traditional Chinese medicine, and it also lacks specificity. The fingerprint spectrum / characteristic spectrum of traditional Chinese medicine can provide more comprehensive and rich information, so it is often used in the quality control research of traditional Chinese medicine. At present, there are few research reports on the fingerprint / characteristic spectrum of Magnolia officinalis flower. Most of the existing studies focus on the research of fingerprint spectrum or characteristic spectrum of medicinal materials, and there is no research report on the use of fingerprint / characteristic spectrum in traditional Chinese medicine preparations with Magnolia officinalis flower as raw material.

[0003] Traditional Chinese medicine is mainly used in the form of decoctions. Decoctions are the material basis for Chinese medicine to exert its efficacy and have been clinically practiced 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 to reflect the clinical effectiveness of Chinese medicine and the spectrum-effect relationship. Chinese medicine standard decoctions are standardized preparations of traditional Chinese medicine decoctions. They refer to modern extraction methods and are prepared by standardized processes. 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 formulas. They are often used in clinical practice to replace traditional decoction pieces.

[0004] In summary, there are few research reports on the fingerprint or characteristic spectrum of Magnolia officinalis flower. The existing research reports only focus on the chemical composition of Magnolia officinalis flower medicinal material itself, and the research on chemical composition is mostly concentrated on fat-soluble components such as magnolol and honokiol. The water-soluble components in Magnolia officinalis flower and the changes in chemical composition during the decoction of Magnolia officinalis flower decoction are ignored. In the clinical use of Magnolia officinalis flower, whether in the form of decoction or formula granules, water is used as the solvent. After decoction or industrial extraction, the type, content or proportion of active ingredients may change to a certain extent, resulting in changes in the material basis for the clinical efficacy of traditional Chinese medicine. Therefore, studying the medicinal material itself alone is insufficient for evaluating the clinical effectiveness and monitoring the quality of the medicinal material. It is necessary to establish a quality control method for formula granules that is compatible with the effective substances of traditional Chinese medicine decoctions based on the effective substances 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 or Chinese medicine formula granules of Magnolia officinalis flower. The method has good reproducibility, accuracy and reliability, and can provide a data basis for mass production quality control of the standard decoction or formula granules of Magnolia officinalis flower, thereby ensuring the stability and controllability of the product quality of the standard decoction or formula granules of Magnolia officinalis flower.

[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 includes a reference medicinal material reference solution and a reference substance reference solution, the reference substance reference solution includes 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] Preset amounts of reference solution and test solution were taken and injected into a liquid chromatograph, which used 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 to establish a characteristic spectrum.

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

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

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

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

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

[0015] 50min~68min, mobile phase A changes from 30%→85%, and mobile phase B changes from 70%→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-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, put it in a stoppered conical flask, add 20ml-30ml of 65%-75% methanol, ultrasonically treat it for 25min-35min, cool it, 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, magnolioside and honokiol reference substances, weigh them accurately, and add methanol to make solutions containing 20 μg 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 includes 10 common peaks, among which Peak 1 corresponds to 3-0-p-coumarylquinic acid, Peak 3 corresponds to magnolioside B, Peak 4 corresponds to magnolioside F, Peak 5 corresponds to magnolioside A, Peak 9 corresponds to magnolioside, and Peak 10 corresponds to magnolioside.

[0028] In one embodiment, in the characteristic spectrum of the standard decoction or formula granules of Magnolia officinalis flower, the magnolin B peak is used as the reference peak S1, and 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%;

[0029] Taking the magnolia officinalis 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%.

[0030] And, the use of the characteristic spectrum of the standard decoction or formula granules of Magnolia officinalis flower obtained by the method for constructing the characteristic spectrum of the standard decoction or formula granules of Magnolia officinalis flower in any one of (1) to (3):

[0031] (1) Authenticity identification and ingredient testing of Magnolia officinalis flower standard decoction;

[0032] (2) Authenticity identification and ingredient testing of Magnolia officinalis flower granules;

[0033] (3) Differentiation of the standard decoction of Magnolia officinalis flower from that of Magnolia grandiflora and Michelia scoparia.

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

[0035] The present invention is based on the material basis characteristics of the standard decoction of Magnolia officinalis flower, adopts high performance liquid chromatography (HPLC) to establish the characteristic spectrum standard of the standard decoction of Magnolia officinalis flower, and takes the material basis of the standard decoction as a reference to establish the characteristic spectrum standard of Magnolia officinalis flower formula granules, so as to achieve rapid and comprehensive quality monitoring of the standard decoction and formula granules of Magnolia officinalis flower, the method is stable, the precision is high, and the reproducibility is good, which is of great significance for the research on the clinical effectiveness of Magnolia officinalis flower medicinal materials and related preparations and the spectrum-effect relationship, and at the same time provides an important reference for the quality control of the standard decoction and formula granules of Magnolia officinalis flower. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 It is a characteristic spectrum of the standard decoction of Magnolia officinalis flower in Example 1 of the present invention - a chromatogram of different wavelengths;

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

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

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

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

[0044] Fig. 9 It 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] Fig.10 It is the characteristic spectrum of Magnolia officinalis flower formula granules in Example 1 of the present invention - the investigation spectrum of different extraction times;

[0046] Fig.11 It 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] Fig.12 It is the reference characteristic spectrum of the standard decoction of Magnolia officinalis flower in Example 1 of the present invention, wherein Peak 1: 3-0-p-coumarylquinic acid; Peak 3 (S1): Magnolia glycoside B; Peak 4: Magnolia glycoside F; Peak 5: Magnolia glycoside A; Peak 9 (S2): Honokiol; Peak 10: Magnolia glycoside;

[0048] Fig.13 It is the total ion flow diagram and ultraviolet absorption chromatogram of the test solution of Magnolia officinalis flower standard decoction in Example 1 of the present invention;

[0049] Fig.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] Fig.15It is the characteristic spectrum of 3 batches of Magnolia officinalis flower formula granules in Example 1 of the present invention;

[0051] Fig.16 It is a reference characteristic spectrum of Magnolia officinalis flower formula granules in Example 1 of the present invention, wherein Peak 1: 3-0-p-coumarylquinic acid; Peak 3 (S1): Magnolia glycoside B; Peak 5: Magnolia glycoside A; Peak 9 (S2): Honokiol; Peak 10: Magnokiol;

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

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

[0054] Fig.19 It is a superposition of characteristic spectra of three batches of Magnolia officinalis standard decoction in Example 4 of the present invention;

[0055] Fig. 20 It is a superposition of characteristic spectra of two batches of standard decoctions of Michelia scoparia in Example 4 of the present invention;

[0056] Fig.21 It is a superposition diagram of characteristic spectra of Magnolia officinalis flower and Magnolia grandiflora standard decoction and Michelia scoparia standard decoction in Example 4 of the present invention. DETAILED DESCRIPTION

[0057] In order to make the purpose, 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 medicinal materials used in this experiment were collected by the staff of the Purchasing Management Department of Guangdong Yifang Pharmaceutical Co., Ltd. The medicinal materials and decoction pieces were qualified by the Quality Center of Guangdong Yifang Pharmaceutical Co., Ltd. and met the relevant provisions of the "Magnolia officinalis Flower" in the 2020 edition of the Chinese Pharmacopoeia. They were determined to be the dried flower buds of Magnolia officinalis Rehd.et Wils. of the Magnoliaceae family. The origin information of the medicinal materials is shown in Table 1.

[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 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, put them 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), keep it slightly boiling for 30 minutes over low heat (200W), filter it while hot through a 200-mesh sieve, and quickly cool the filtrate with cold water. Add 10 times the amount of water for the second decoction, boil over high heat (500W), keep it slightly boiling for 25 minutes over low heat (200W), filter it while hot through a 200-mesh sieve, and quickly cool the filtrate 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 at 60℃~80℃ under reduced pressure 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 of Magnolia officinalis flower standard decoction and formula granules

[0071] 1. Chromatographic conditions

[0072] 1.1 Wavelength selection

[0073] In this embodiment, 260nm, 300nm, and 350nm are selected as the detection wavelengths of the standard decoction sample of Magnolia officinalis flower, and full-wave scanning is performed to record the absorption spectrum of the sample in the range of 190nm to 400nm. The results are shown in 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 Table 2 and Table 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 (4.6mm×150mm, 2.7μm) chromatographic column; acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution according to the elution gradient 2 in Table 3; flow rate 0.9ml per minute; column temperature 30°C; detection wavelength 300nm; injection volume 5μl.

[0084] 2. Preparation of reference substance solution

[0085] Take appropriate amounts of magnolioside B, honokiol and magnolioside reference substances, weigh them accurately, and add methanol to prepare solutions containing 20 μg of magnolioside B, honokiol and magnolioside 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 solvents

[0089] Take an appropriate amount of Magnolia officinalis flower standard decoction (No. BT6), grind it into powder, take about 0.5g, weigh it accurately, and place it in 6 parallel groups, 2 portions in each group, put it in a stoppered conical bottle, and accurately add 25ml of methanol, 50% methanol, 70% methanol, ethanol, 50% ethanol and 70% ethanol respectively, weigh it, ultrasonically treat 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 and analyze it according to the chromatographic conditions determined under "1.4". The results are shown in 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, weigh it accurately, and place it in two parallel groups, 2 portions in each group, put it in a stoppered conical bottle, accurately add 25ml of 70% methanol, weigh it, and treat it with ultrasound (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 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

[0093] Take an appropriate amount of Magnolia officinalis flower standard decoction (No. BT6), grind it into powder, take about 0.5g, weigh it accurately, and place it in 3 parallel groups, 2 portions in each group, put it in a stoppered conical bottle, accurately add 25ml of 70% methanol, weigh it, and treat it with ultrasound (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 and analyze it according to the chromatographic conditions determined under "1.4". The results are shown in Figure 7 As shown, the results showed that different extraction times had 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, cool it, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain it.

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

[0097] (1) Investigation of extraction solvents

[0098] Take an appropriate amount of Magnolia officinalis flower formula granules (No. KL01), grind it into powder, take about 0.5g, accurately weigh it, and place it in 6 parallel groups, 2 portions in each group, put it in a stoppered conical flask, accurately add 25ml of methanol, 50% methanol, 70% methanol, ethanol, 50% ethanol and 70% ethanol respectively, weigh it, and treat it with ultrasound (power 300W, frequency 40kHz) for 30 minutes, take it out, let it cool, weigh it again, make up the lost weight with the corresponding solvent, shake it well, filter it, take the filtrate, and analyze it according to the chromatographic conditions determined under "1.4". The results are shown in 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 formula granules (No. KL01), grind it into powder, take about 0.5g, accurately weigh it, and place it in 2 parallel groups, 2 portions in each group, put it in a stoppered conical bottle, accurately add 25ml of 70% methanol, weigh it, and treat it with ultrasound (power 300W, frequency 40kHz) for 30 minutes, heat it under reflux for 30 minutes, take it out, cool it, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, take the filtrate, and analyze it according to the chromatographic conditions determined under "1.4". The results are shown in Fig. 9 As shown, the results show that 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

[0102] Take an appropriate amount of Magnolia officinalis flower formula granules (No. KL01), grind it into powder, take about 0.5g, accurately weigh it, and place it in 3 parallel groups, 2 portions in each group, put it in a stoppered conical bottle, accurately add 25ml of 70% methanol, and ultrasonically treat it (power 300W, frequency 40kHz) for 15 minutes, 30 minutes, and 60 minutes respectively. Take it out, cool it, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, take the filtrate, and analyze it according to the chromatographic conditions determined under "1.4". The results are shown in Fig.10 As shown, the results showed that different extraction times had little effect on the characteristic spectra 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 0.5g, accurately weigh, put it in a stoppered conical flask, accurately add 25ml of 70% methanol, weigh the weight, ultrasonically treat (power 300W, frequency 40kHz) for 30 minutes, cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate to obtain.

[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 18 batches of Magnolia officinalis flower standard decoction, and select 10 common peaks with known components, good peak shape and separation, high purity and large peak area as the characteristic peaks of the characteristic spectrum of Magnolia officinalis flower standard decoction, such as Fig.11 As shown in Table 5, the relative retention time and relative peak area of ​​peaks 1 to 5 and S1 were calculated by taking the chromatographic peak of magnolidin B as the reference peak S1; the relative retention time and relative peak area of ​​peaks 6 to 10 and S2 were calculated by taking the chromatographic peak of magnolidinone as the reference peak S2. The results are shown in Tables 5 and 6. The characteristic spectra of 18 batches of standard decoction of Magnolia officinalis flower were matched using the "Similarity Evaluation System of Chromatographic Fingerprint of Traditional Chinese Medicine", and the reference spectra were generated by the average method to establish the reference characteristic spectra of standard decoction of Magnolia officinalis flower, as shown in Table 6. Fig.12 , this characteristic spectrum has 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 determination 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 peaks, respectively; the peak corresponding to the magnolin B 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, and 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 in item "1.4".

[0117] ②Mass spectrometry conditions. Mass spectrometry parameters are shown in Table 7.

[0118] Table 7 Mass spectrometry parameters

[0119]

[0120] ③ Preparation of test solution

[0121] Same as under “3.1”.

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

[0123] ⑤ Identification of compounds

[0124] The test solution was tested using the above-mentioned liquid chromatography and mass spectrometry analysis conditions. The peak 1 in the characteristic spectrum of the standard decoction of Magnolia officinalis flower was confirmed to be 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 by comparing the retention time of the reference liquid chromatography and the accurate molecular weight and fragment ion of the mass spectrometry. The total ion flow diagram and ultraviolet absorption chromatogram of the test solution are shown in Fig.13 , 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. Methodological Validation

[0128] (1) Specificity inspection

[0129] Accurately pipette 5 μl of the test solution, reference solution and blank solvent from Magnolia officinalis flower standard decoction (No. BT6), inject into liquid chromatograph, and measure according to the chromatographic conditions determined under "1.4". The results are shown in Fig.14 As shown, the results show that the test sample chromatogram has the same chromatographic peak at the corresponding retention time as the reference sample chromatogram, and there is no interference from the blank solvent, indicating that the method has good specificity.

[0130] (2) Precision inspection

[0131] Take an appropriate amount of Magnolia officinalis flower standard decoction (No.: BT6), grind it into powder, take about 0.5g, weigh it accurately, prepare the test solution according to the test solution preparation method determined under "3.1", repeat the injection 6 times according to the chromatographic conditions determined under "1.4", take the chromatographic peak of magnolin B as the reference peak S1, calculate the relative retention time and relative peak area RSD values ​​of peaks 1 to 5 with S1 peak, and then within the range of 0.21% to 2.87%, take the chromatographic peak of honokiol as the reference peak S2, calculate the relative retention time and relative peak area RSD values ​​of peaks 6 to 10 with S2 peak, and within the range of 0.65% to 2.24%, all of which are less than 3.0%, indicating that the instrument has good precision.

[0132] (3) Repeatability study

[0133] Take about 0.5 g of the same batch of Magnolia officinalis flower standard decoction (No.: BT6), accurately weigh it, and make 6 parallel portions. According to the test solution preparation method determined under "3.1", 6 test solutions were prepared. According to the chromatographic conditions determined under "1.4", the sample was injected and analyzed. The chromatographic peak of magnolin B was used as the reference peak S1, and the relative retention time and relative peak area RSD values ​​of peaks 1 to 5 and S1 were calculated to be in the range of 0.36% to 2.17%. The chromatographic peak of honokiol was used as the reference peak S2, and the relative retention time and relative peak area RSD values ​​of peaks 6 to 10 and S2 were calculated to be in the range of 0.67% to 2.94%, which were all less than 3.0%, indicating that the method had good repeatability.

[0134] 7. Establishment of the characteristic spectrum of Magnolia officinalis flower formula granules

[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 in "3.2", accurately pipette the above test solution, and inject and measure according to the chromatographic conditions in "1.4". The characteristic spectrum overlay of the 3 batches of samples is shown in Fig.15 As shown, the chromatographic peak of magnoside B was used as the reference peak S1, and the relative retention time and relative peak area of ​​peaks 1 to 5 with respect to peak 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 with respect to peak 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 by the average method to establish a reference characteristic spectrum of Magnolia officinalis flower formula granules, such as Fig.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: 10 characteristic peaks should be present in the chromatogram of the test sample, 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 magnolia glycoside reference substance is the S1 peak, and the relative retention times of peaks 1-2, 4-5 and S1 are calculated; the peak corresponding to the honokiol reference substance is the S2 peak, and the relative retention times of peaks 6-8 and S2 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).

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

[0143] Chromatographic conditions: octadecylsilane bonded silica gel as 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 per minute; column temperature: 30° C.; detection wavelength: 300 nm.

[0144] Preparation of reference solution: Preparation of reference solution: Take about 1g of Magnolia officinalis flower reference medicinal material, place it in a stoppered conical bottle, add 50ml of water, heat and reflux for 30 minutes, cool, filter, evaporate the filtrate, add 25ml of 70% methanol to the residue, ultrasonically treat (power 300W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference medicinal material reference solution. Take appropriate amounts of magnoside B, magnolol, and honokiol reference substances, accurately weigh them, and add methanol to make a solution containing 20μg of magnoside B, honokiol, and magnolol per 1ml.

[0145] Preparation of test solution: Take an appropriate amount of standard decoction of Magnolia officinalis flower, grind it into powder, take about 0.5g, weigh it accurately, put it in a stoppered conical flask, add 25ml of 70% methanol accurately, weigh it, treat it with ultrasound (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 the test solution.

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

[0147] Determination results: as shown in Table 11 and Fig.17 As shown in the figure, the characteristic spectrum of the standard decoction of Magnolia officinalis flower numbered BT5 has 10 characteristic peaks, of which peak 3, peak 9, and peak 10 correspond to the retention time of the corresponding reference substance peaks; the peak corresponding to the magnolol reference is the S1 peak, and the relative retention time of peaks 1-2, peaks 4-5 and S1 peak is calculated; the peak corresponding to the honokiol reference is the S2 peak, and the relative retention time of peaks 6-8 and S2 peak is calculated, and 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 characteristic spectrum of Magnolia officinalis flower formula granules (No.: KL02)

[0151] Chromatographic conditions: octadecylsilane bonded silica gel as 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 per minute; column temperature: 30° C.; detection wavelength: 300 nm.

[0152] Preparation of reference solution: Preparation of reference solution: Take about 1g of Magnolia officinalis flower reference medicinal material, place it in a stoppered conical bottle, add 50ml of water, heat and reflux for 30 minutes, cool, filter, evaporate the filtrate, add 25ml of 70% methanol to the residue, ultrasonically treat (power 300W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference medicinal material reference solution. Take appropriate amounts of magnoside B, magnolol, and honokiol reference substances, accurately weigh them, and add methanol to make a solution containing 20μg of magnoside B, honokiol, and magnolol per 1ml.

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

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

[0155] Determination results; as shown in Table 12 and Fig.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 magnoliin B reference is the S1 peak, and the relative retention times of peaks 1-2, 4-5 and S1 are calculated; the peak corresponding to the honokiol reference is the S2 peak, 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 sempervivum standard decoction

[0160] Take 3 batches of Magnolia decoction standard decoction (No.: GT1810111, GT1810112 and GT1810113) and 2 batches of Michelia serrata standard decoction (No.: GT2303128, GT2303158) to make test solution, accurately pipette the above test solution, and inject and measure according to the chromatographic conditions under "1.4". The characteristic spectrum superposition of the 3 batches of Magnolia decoction standard decoction is shown in Fig.19 As shown, the characteristic spectrum superposition of two batches of standard decoction of Michelia sempervirens is shown in Fig. 20 As shown in the figure, the characteristic spectrum superposition of Magnolia officinalis flower standard decoction, Magnolia officinalis flower standard decoction and Michelia sempervivum flower standard decoction is shown in Fig.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 officinalis 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), and the standard decoction of Michelia officinalis 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 obviously 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 officinalis flower under the proposed chromatographic conditions, and provides a quality basis for the large-scale production of standard decoction or formula granules of Magnolia officinalis flower (Magnolia officinalis).

[0162] The above is a preferred embodiment of the invention. It should be pointed out that a person skilled in the art can make several improvements and modifications without departing from the principle of the invention. These improvements and modifications are also considered to be within the scope of protection of the invention.

Claims

1. A method for constructing a characteristic spectrum of a standard decoction or formula granules of Magnolia officinalis flower, characterized in that: The following steps are involved: Prepare a reference solution, the reference solution includes a reference medicinal material reference solution and a reference substance reference solution, the reference substance reference solution includes a magnolol reference substance, a magnolol reference substance, and a honokiol reference substance; Prepare the test solution by taking the standard decoction or formula granules of Magnolia officinalis flower; Preset amounts of reference solution and test solution were taken and injected into a liquid chromatograph, which used 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 to establish a characteristic spectrum.

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 gradient elution was carried out according to the following procedure: 0-10min, mobile phase A is 10%, mobile phase B is 90%; 10min-25min, mobile phase A from 10% to 14%, mobile phase B from 90% to 86%; 25min~39min, mobile phase A from 14%→20%, mobile phase B from 86%→80%; 39min~50min, mobile phase A from 20%→30%, mobile phase B from 80%→70%; 50min~68min, mobile phase A changes from 30%→85%, and mobile phase B changes from 70%→15%.

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 concentration of the phosphoric acid solution is 0.05% to 0.15%.

4. 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 to 5.5 μL; 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.; When performing gradient elution, the flow rate of the mobile phase is 0.8-1.0 ml / min; The ultraviolet detection wavelength of the liquid chromatograph is 290nm-310nm.

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: The test solution was prepared by the following method: Take the standard decoction or formula granules of Magnolia officinalis flower, grind it into powder, weigh 0.45g-0.55g, put it in a stoppered conical flask, add 20ml-30ml of 65%-75% methanol, ultrasonically treat it for 25min-35min, cool it, weigh it again, make up the lost weight with 65%-75% methanol, shake it well, filter it, and take the filtrate to obtain it.

6. 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 magnolioside B, magnolioside and honokiol reference substances, weigh them accurately, and add methanol to make solutions containing 20 μg of magnolioside B, honokiol and honokiol per 1 ml to obtain reference substance solutions.

7. 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 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.

8. 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 characteristic spectrum includes 10 common peaks, among which Peak 1 corresponds to 3-0-p-coumarylquinic acid, Peak 3 corresponds to magnolioside B, Peak 4 corresponds to magnolioside F, Peak 5 corresponds to magnolioside A, Peak 9 corresponds to magnolioside, and Peak 10 corresponds to magnolioside.

9. The method for constructing the characteristic spectrum of the standard decoction or formula granules of Magnolia officinalis flower according to claim 8, characterized in that: In the characteristic spectrum of the standard decoction or formula granules of Magnolia officinalis flower, the peak of magnolioside B is used as the reference peak S1, and 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 magnolia officinalis 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%.

10. Use of the characteristic spectrum of the standard decoction or formula granules of Magnolia officinalis Flower obtained by the method for constructing the characteristic spectrum of the standard decoction or formula granules of Magnolia officinalis Flower according to any one of claims 1 to 9 in any one of (1) to (3): (1) Authenticity identification and ingredient testing of Magnolia officinalis flower standard decoction; (2) Authenticity identification and ingredient testing of Magnolia officinalis flower granules; (3) Differentiation of the standard decoction of Magnolia officinalis flower from that of Magnolia grandiflora and Michelia scoparia.

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