CN111693618B - Construction method and detection method of UPLC characteristic spectrum of magnolia flower medicinal material - Google Patents

Abstract

The invention discloses a construction method of UPLC characteristic spectrum of officinal magnolia flower medicinal material, which comprises the following steps: (1) taking appropriate amount of magnolol and honokiol reference substance, adding methanol to obtain mixed solution containing magnolol and honokiol, and making into reference substance solution; (2) extracting flos Magnoliae with methanol to obtain sample solution; (3) respectively and precisely absorbing the reference substance solution and the test solution, injecting the reference substance solution and the test solution into a liquid chromatograph, and performing gradient elution by using octadecylsilane chemically bonded silica as a filling agent, acetonitrile as a mobile phase A and formic acid solution as a mobile phase B in the liquid chromatograph to establish a UPLC characteristic spectrum of the magnolia flower medicinal material. Correspondingly, the invention also provides a detection method for identifying the magnolia officinalis flower medicinal material and the pseudo magnolia delavayi. The method has good reproducibility, accuracy and reliability, can effectively control the quality of the magnolia officinalis flower medicinal material through the established UPLC characteristic spectrum, and realizes the identification of the magnolia officinalis flower medicinal material and the fake magnolia denudata.

Description

Construction method and detection method of UPLC characteristic spectrum of magnolia flower medicinal material

Technical Field

The invention relates to the technical field of traditional Chinese medicine quality analysis and detection, in particular to a construction method of UPLC characteristic spectrum of magnolia officinalis flower medicinal material and a detection method for identifying magnolia officinalis flower medicinal material and pseudo magnolia denudate thereof.

Background

The flos Magnoliae officinalis is dried flower bud of Magnolia officinalis of Magnoliaceae, Magnolia officinalis of Magnolia officinalis Rehd. It is bitter and slightly warm in nature. It enters spleen and stomach meridians. Fragrant, resolving dampness, regulating qi-flowing and relieving epigastric distention. The traditional Chinese medicine composition is mainly used for treating spleen and stomach damp and yin qi stagnation, chest and stomach stuffiness, fullness and distention, and no flavor of grain. The magnolia officinalis is used as a traditional material and a rare Chinese medicinal material tree species in China, belongs to the national II-level key protection wild plant, is a national second-level endangered protection plant, is a national second-level Chinese medicinal material, and is a special product in China.

According to the report of the literature, the main components of magnolia officinalis flowers are lignan compounds, sesquiterpene compounds and the like, wherein the quality control research mainly focuses on the content measurement of two index components, namely magnolol and honokiol, but the composition of the Chinese medicinal materials is greatly influenced by the basic source, the harvesting season, the production place processing mode and the like, so that the overall detection technology of the characteristic spectrum of the Chinese medicinal materials is established and popularized to improve the detection level capable of representing the effectiveness of the Chinese medicinal materials, and the problem to be solved urgently is solved.

Disclosure of Invention

The invention aims to solve the technical problem of providing a construction method of UPLC characteristic spectrum of magnolia officinalis flower medicinal material, which has good reproducibility, accuracy and reliability, can effectively control the quality of magnolia officinalis flower medicinal material through the established UPLC characteristic spectrum, and realizes the identification of magnolia officinalis flower medicinal material and pseudo magnolia delavayi.

The invention aims to solve the technical problem of providing a detection method for identifying the magnolia flower medicinal material and the pseudo magnolia denudata, wherein the detection method is simple, can quickly and conveniently identify the magnolia flower medicinal material and the pseudo magnolia denudata and has accurate result.

In order to achieve the technical effects, the characteristic spectrums of magnolia officinalis flowers in different producing areas are researched, the characteristic spectrum of a magnolia officinalis flower medicinal material is established, and the quality control level of the medicinal material is improved. The invention provides a construction method of UPLC characteristic spectrum of officinal magnolia flower medicinal material, which comprises the following steps:

(1) accurately weighing appropriate amount of magnolol and honokiol reference substance, and adding methanol to obtain mixed solution containing magnolol and honokiol to obtain reference substance solution;

(2) extracting magnolia officinalis flower medicinal material by using 65-75% methanol to prepare a test solution;

(3) respectively and precisely absorbing the reference substance solution and the test solution, injecting the reference substance solution and the test solution into a liquid chromatograph, and performing gradient elution by using octadecylsilane chemically bonded silica as a filling agent, acetonitrile as a mobile phase A and formic acid solution as a mobile phase B in the liquid chromatograph to establish a UPLC characteristic spectrum of the magnolia flower medicinal material.

Wherein the gradient elution is performed according to the following procedure:

0-3 min, wherein the mobile phase A is 8%, and the mobile phase B is 92%;

3-19 min, wherein the mobile phase A is 8% → 13%, and the mobile phase B is 92% → 87%;

19-22 min, wherein the mobile phase A is from 13% → 13.5%, and the mobile phase B is from 87% → 86.5%;

22-25 min, wherein the mobile phase A is 13.5% → 60%, and the mobile phase B is 86.5% → 40%;

25-32 min, wherein the mobile phase A is 60% and the mobile phase B is 40%;

32-33 min, wherein the mobile phase A is 60% → 8%, and the mobile phase B is 40% → 92%;

33-40 min, wherein the mobile phase A is 8% and the mobile phase B is 92%.

Preferably, the reference solution is prepared by the following method:

taking appropriate amount of magnolol and honokiol reference substance, precisely weighing, adding methanol to obtain mixed solution containing magnolol 60 μ g and honokiol 40 μ g per 1ml, and making into reference solution.

Preferably, the test solution is prepared by the following method:

precisely weighing 1.0-2.0g of coarse powder of magnolia flower, placing the coarse powder into a conical flask with a plug, precisely adding 10-20ml of 70% methanol, weighing the weight, carrying out ultrasonic treatment for 20-30 minutes, cooling, complementing the loss weight with 70% methanol, shaking up, filtering, and taking the subsequent filtrate.

Further preferably, the test solution is prepared by the following method:

1.0g of coarse powder of magnolia officinalis anther is precisely weighed, the coarse powder is placed in a conical flask with a plug, 10ml of 70% methanol is precisely added, the weight is weighed, ultrasonic treatment is carried out for 30 minutes, the power of the ultrasonic treatment is 300W, the frequency is 40kHz, the mixture is cooled, the loss weight is complemented with 70% methanol, the mixture is shaken uniformly, and the subsequent filtrate is filtered, thus obtaining the magnolia officinalis anther.

Preferably, the UPLC characteristic spectrum of the magnolia flower medicinal material is established by the following method:

precisely sucking 1 μ l of reference solution and sample solution respectively, and injecting into a liquid chromatograph, wherein the liquid chromatograph has octadecylsilane chemically bonded silica as filler, column length of 100mm, inner diameter of 2.1mm, and particle diameter of 1.8 μm; gradient elution is carried out by taking acetonitrile as a mobile phase A and 0.1% formic acid solution as a mobile phase B, the flow rate is 0.35ml per minute, the column temperature is 30 ℃, and the detection wavelength is 300 nm.

Preferably, the UPLC characteristic spectrum of the magnolia flower medicinal material includes 5 characteristic peaks, which are peak 1, peak 2, peak 3, peak 4 and peak 5, wherein peak 4 corresponding to the honokiol reference is an S peak, the relative retention time of peak 1, peak 2, peak 3 and the S peak should be within ± 10% of a specified value, and the specified value includes: peak 1 was 0.37, Peak 2 was 0.52, Peak 3 was 0.64.

Correspondingly, the invention also provides a detection method for identifying magnolia officinalis flowers and pseudo magnolia delavayi thereof, which comprises the following steps:

(1) accurately weighing appropriate amount of magnolol and honokiol reference substance, and adding methanol to obtain mixed solution containing magnolol and honokiol to obtain reference substance solution;

(2) extracting magnolia officinalis flower medicinal material by using 65-75% methanol to prepare a test solution;

(3) precisely absorbing a reference substance solution and a test solution respectively, injecting the reference substance solution and the test solution into a liquid chromatograph, and performing gradient elution by using octadecylsilane chemically bonded silica as a filling agent, acetonitrile as a mobile phase A and a formic acid solution as a mobile phase B in the liquid chromatograph to establish a UPLC characteristic spectrum of the magnolia flower medicinal material;

(4) injecting a preset amount of sample solution into a liquid chromatograph, wherein the liquid chromatograph uses octadecylsilane chemically bonded silica as a filler, acetonitrile as a mobile phase A and formic acid solution as a mobile phase B for gradient elution to obtain a sample chromatogram;

(5) comparing the UPLC characteristic spectrums of the chromatogram of the test sample and the Magnolia officinalis flower medicinal material, and judging the Magnolia officinalis flower medicinal material and the pseudo Magnolia officinalis flower thereof according to the relative retention time of each characteristic peak of the chromatogram of the test sample.

Preferably, the UPLC characteristic spectrum of the magnolia flower medicinal material comprises 5 characteristic peaks, namely peak 1, peak 2, peak 3, peak 4 and peak 5, wherein peak 4 corresponding to the honokiol reference substance is an S peak;

Calculating the relative retention time of the characteristic peak 1, peak 2, peak 3 and the S peak, if the relative retention time of the characteristic peak 1, peak 2, peak 3 and the S peak is within ± 10% of a specified value, the specified value comprising: judging the magnolia flower medicinal material if the peak 1 is 0.37, the peak 2 is 0.52 and the peak 3 is 0.64;

and if the corresponding retention time positions of the peak 4 and the peak 5 have no spectrum peak, determining that the pseudomagnolia delavayi is a pseudomagnolia delavayi.

Preferably, the gradient elution is performed according to the following procedure:

0-3 min, wherein the mobile phase A is 8% and the mobile phase B is 92%;

3-19 min, wherein the mobile phase A is 8% → 13%, and the mobile phase B is 92% → 87%;

19-22 min, wherein the mobile phase A is from 13% → 13.5%, and the mobile phase B is from 87% → 86.5%;

22-25 min, wherein the mobile phase A is 13.5% → 60%, and the mobile phase B is 86.5% → 40%;

25-32 min, wherein the mobile phase A is 60% and the mobile phase B is 40%;

32-33 min, wherein the mobile phase A is 60% → 8%, and the mobile phase B is 40% → 92%;

33-40 min, wherein the mobile phase A is 8% and the mobile phase B is 92%.

At the present stage, the identification of the magnolia flower medicinal material mainly comprises the aspects of character, content determination, thin-layer chromatography, ultraviolet spectrum and the like, and the aspects are not strong in characteristics and have small difference. The UPLC characteristic spectrum of the officinal magnolia flower medicinal material is established, and the invention has the following beneficial effects:

(1) The method utilizes 65-75% methanol to extract magnolia officinalis flower medicinal materials, and injects the magnolia officinalis flower medicinal materials into a liquid chromatograph, the liquid chromatograph performs gradient elution according to the specification by using octadecylsilane chemically bonded silica as a filling agent, acetonitrile as a mobile phase A and 0.1% formic acid solution as a mobile phase B, and a UPLC characteristic map of the magnolia officinalis flower medicinal materials is established. The UPLC characteristic spectrum of the magnolia flower medicinal material can fully display the characteristics of the chemical components of the magnolia flower medicinal material, the characteristic peak information content is rich, the method is good in reproducibility, accurate, reliable and good in stability, the established UPLC characteristic spectrum can be used for effectively identifying the magnolia flower medicinal material, the magnolia flower medicinal material and a fake magnolia delavayi can be distinguished, and the quality of the magnolia flower medicinal material can be effectively controlled.

(2) The UPLC characteristic spectrum of the magnolia flower medicinal material is utilized, the relative retention time of each characteristic peak of the chromatogram of the test sample is calculated, the magnolia flower medicinal material and the pseudo magnolia delavayi can be effectively identified, the detection method is simple, the detection result can be quickly and conveniently obtained, and the result is accurate.

Drawings

FIG. 1 is a flow chart of a construction method of UPLC characteristic spectrum of Magnolia officinalis flower medicinal material.

FIG. 2 is a flow chart of the detection method for identifying Magnolia officinalis anther material and its counterfeit Magnolia officinalis of the present invention.

FIG. 3 is a reference characteristic spectrum of Magnolia officinalis flower medicinal material established in the present invention.

FIG. 4 is a control characteristic spectrum of Magnolia officinalis pseudovariety Magnolia denudata.

FIG. 5 shows absorption spectrum of cortex Lycii at the wavelength of flos Magnoliae officinalis.

FIG. 6 is a characteristic map of Magnolia officinalis flowers in different water phases.

FIG. 7 is a Magnolia officinalis flower profile for different chromatography columns.

FIG. 8 is a Magnolia flower profile at different flow rates.

FIG. 9 is a characteristic spectrum of Magnolia officinalis flowers at different column temperatures.

FIG. 10 is a characteristic map of Magnolia officinalis flowers with different extraction solvents.

FIG. 11 is a characteristic map of Magnolia officinalis flowers at different extraction times.

FIG. 12 is an overlay of the characteristic spectrum of the medicinal materials of 15 batches of Magnolia officinalis flowers in example 1 of the present invention.

FIG. 13 is an overlay of 3 batches of Magnolia officinalis pseudolites, Magnolia denudata, in example 2 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings.

As shown in figure 1, the invention provides a construction method of UPLC characteristic spectrum of Magnolia officinalis flower medicinal material by UPLC-UV technology, which comprises the following steps:

s101, precisely weighing a proper amount of magnolol and a proper amount of honokiol reference substances, and adding methanol to prepare a mixed solution containing magnolol and honokiol to prepare a reference substance solution;

Wherein the content ratio of magnolol to honokiol is (2-4): 2.

Preferably, the reference solution is prepared by the following method:

taking appropriate amount of magnolol and honokiol reference substance, precisely weighing, adding methanol to obtain mixed solution containing magnolol 60 μ g and honokiol 40 μ g per 1ml, and making into reference solution.

S102, taking a magnolia officinalis flower medicinal material, and extracting by using 65-75% methanol to prepare a test solution;

preferably, the test solution is prepared by the following method:

precisely weighing 1.0-2.0g of coarse powder of magnolia flower, placing the coarse powder into a conical flask with a plug, precisely adding 10-20ml of 70% methanol, weighing the weight, carrying out ultrasonic treatment for 20-30 minutes, cooling, complementing the loss weight with 70% methanol, shaking up, filtering, and taking the subsequent filtrate.

More preferably, the test solution is prepared by the following method:

1.0g of coarse powder of magnolia officinalis anther is precisely weighed, the coarse powder is placed in a conical flask with a plug, 10ml of 70% methanol is precisely added, the weight is weighed, ultrasonic treatment is carried out for 30 minutes, the power of the ultrasonic treatment is 300W, the frequency is 40kHz, the mixture is cooled, the loss weight is complemented with 70% methanol, the mixture is shaken uniformly, and the subsequent filtrate is filtered, thus obtaining the magnolia officinalis anther.

S103, precisely absorbing the reference substance solution and the test solution respectively, injecting the reference substance solution and the test solution into a liquid chromatograph, and performing gradient elution by using octadecylsilane chemically bonded silica as a filling agent, acetonitrile as a mobile phase A and formic acid solution as a mobile phase B in the liquid chromatograph to establish a UPLC characteristic spectrum of the magnolia flower medicinal material.

The invention takes acetonitrile as a mobile phase A, a formic acid solution as a mobile phase B, and the mobile phase B is preferably a 0.1% formic acid solution.

Preferably, the UPLC characteristic spectrum of the officinal magnolia flower medicinal material is established by the following method:

precisely sucking 1 μ l of reference solution and sample solution respectively, and injecting into a liquid chromatograph, wherein the liquid chromatograph has octadecylsilane chemically bonded silica as filler, column length of 100mm, inner diameter of 2.1mm, and particle diameter of 1.8 μm; gradient elution was performed according to Table 1 using acetonitrile as mobile phase A and 0.1% formic acid solution as mobile phase B at a flow rate of 0.35ml per minute, a column temperature of 30 ℃ and a detection wavelength of 300 nm. The number of theoretical plates should be not less than 10000 calculated according to honokiol peak.

TABLE 1 gradient elution Table

The method takes acetonitrile as a mobile phase A and 0.1% formic acid solution as a mobile phase B, and adopts a specific gradient elution program, so that the characteristic components in the magnolia flowers can be well chromatographically separated, and the magnolia flowers can be efficiently identified. Furthermore, the peak area of each spectral peak at a wavelength of 300nm is large,

The UPLC characteristic spectrum of the magnolia flower medicinal material obtained by the present invention includes 5 characteristic peaks, as shown in fig. 3, which are peak 1, peak 2, peak 3, peak 4 and peak 5, respectively, wherein the retention time of peaks 4 and 5 is the same as that of honokiol and magnolol in a reference solution, respectively, and the retention time of peak 4 corresponding to the honokiol reference is the S peak, and the relative retention time of peak 1, peak 2, peak 3 and the S peak should be within ± 10% of a specified value, and the specified value includes: peak 1 is 0.37, Peak 2 is 0.52, Peak 3 is 0.64, so as to identify the officinal magnolia flower medicinal material.

As shown in FIG. 4, in the chromatogram of Magnolia officinalis pseudolite Magnolia denudata, there is no chromatographic peak at the corresponding retention time position of honokiol peak and magnolol peak. Therefore, the magnolia officinalis flower medicinal material and the pseudo magnolia delavayi can be distinguished.

Therefore, the UPLC characteristic spectrum of the magnolia flower medicinal material can fully display the characteristics of the chemical components of the magnolia flower medicinal material, the characteristic peak information content is rich, the method has good reproducibility, accuracy and reliability and good stability, the established UPLC characteristic spectrum can realize effective identification of the magnolia flower medicinal material, the magnolia flower medicinal material and the pseudo-magnolia denudata can be distinguished, and the quality of the magnolia flower medicinal material can be effectively controlled.

Correspondingly, as shown in fig. 2, the invention also provides a detection method for identifying magnolia officinalis anther material and pseudo magnolia delavayi thereof, which comprises the following steps:

S201, precisely weighing a proper amount of magnolol and a proper amount of honokiol reference substances, and adding methanol to prepare a mixed solution containing magnolol and honokiol to prepare a reference substance solution;

s202, taking a magnolia officinalis flower medicinal material, and extracting by using 65-75% methanol to prepare a test solution;

s203, precisely absorbing the reference substance solution and the test solution respectively, injecting the reference substance solution and the test solution into a liquid chromatograph, and performing gradient elution by using octadecylsilane chemically bonded silica as a filling agent, acetonitrile as a mobile phase A and formic acid solution as a mobile phase B in the liquid chromatograph to establish a UPLC characteristic map of the magnolia flower medicinal material;

s204, injecting a preset amount of a sample solution into a liquid chromatograph, and performing gradient elution on the liquid chromatograph by using octadecylsilane chemically bonded silica as a filler, acetonitrile as a mobile phase A and a formic acid solution as a mobile phase B to obtain a sample chromatogram;

s205, comparing the chromatogram of the test sample with the UPLC characteristic spectrum of the magnolia flower medicinal material, and judging the magnolia flower medicinal material and the pseudo magnolia denudate thereof according to the relative retention time of each characteristic peak of the chromatogram of the test sample.

The UPLC characteristic spectrum of the magnolia flower medicinal material comprises 5 characteristic peaks, namely a peak 1, a peak 2, a peak 3, a peak 4 and a peak 5, wherein the peak 4 corresponding to the honokiol reference substance is an S peak;

Calculating the relative retention time of the characteristic peak 1, peak 2, peak 3 and the S peak, if the relative retention time of the characteristic peak 1, peak 2, peak 3 and the S peak is within ± 10% of a specified value, the specified value comprising: judging the magnolia flower medicinal material if the peak 1 is 0.37, the peak 2 is 0.52 and the peak 3 is 0.64;

and if the corresponding retention time positions of the peak 4 and the peak 5 have no spectrum peak, determining that the pseudomagnolia delavayi is a pseudomagnolia delavayi.

The UPLC characteristic spectrum of the magnolia flower medicinal material is utilized, the relative retention time of each characteristic peak of the chromatogram of the test sample is calculated, the magnolia flower medicinal material and the pseudo magnolia delavayi can be effectively identified, the detection method is simple, the detection result can be quickly and conveniently obtained, and the result is accurate.

It should be noted that, in the detection method for identifying magnolia officinalis flower medicinal material and its counterfeit magnolia denudate, the method for establishing the UPLC characteristic spectrum of magnolia officinalis flower medicinal material is the same as that described above, and is not described herein again.

Further, the invention demonstrates the construction method of UPLC characteristic spectrum of Magnolia officinalis flower medicinal material by combining the following experimental data, which comprises the following steps:

1. instrument and reagent

1.1, instrument: ultra-high performance liquid chromatograph

2. Investigation of chromatographic conditions

2.1 selection of wavelength

294nm, 300nm and 310nm are selected as detection wavelengths of standard decoction samples of magnolia officinalis flowers (magnolia officinalis), full-wave scanning is carried out, absorption spectra of the samples in the range of 190-400 nm are recorded, and the result is shown in figure 5.

The result of FIG. 5 shows that the peak area of each spectral peak is larger at 300nm wavelength, therefore, 300nm is selected as the detection wavelength for establishing the characteristic spectrum of the standard decoction of Magnolia officinalis flowers (Magnolia officinalis).

2.2 examination of the aqueous phase

In the experiment, three mobile phases of 0.1% formic acid solution, 0.1% phosphoric acid solution and 0.1% acetic acid solution are selected for comparison, and the optimal water phase is screened.

In fig. 6, a chromatogram corresponding to a 0.1% formic acid solution is shown in curve 1, a chromatogram corresponding to a 0.1% phosphoric acid solution is shown in curve 2, and a chromatogram corresponding to a 0.1% acetic acid solution is shown in curve 3.

The results in fig. 6 show that by comparing the chromatograms of 3 different mobile phases, the information of the peaks is incomplete when 0.1% acetic acid solution is selected as the aqueous phase; when 0.1% phosphoric acid solution is selected as the water phase, the separation degree of partial peaks is poor, when 0.1% formic acid solution is selected, the information of the peaks is complete, and the separation degree of the peaks is good, so that 0.1% formic acid solution is selected as the water phase for establishing the characteristic spectrum of the magnolia flowers.

2.3 inspection of the column

In this experiment, 3 kinds of chromatographic columns including Waters HSS T3(2.1 mm. times.100 mm, 1.8 μm), Waters BEH C18(2.1 mm. times.100 mm, 1.7 μm) and Agilent SB C18(2.1 mm. times.100 mm, 1.8 μm) were selected for comparison, and the best chromatographic column was selected.

In fig. 7, curve 1 is a chromatogram for Waters HSS T3, curve 2 is a chromatogram for Waters BEH C18, and curve 3 is a chromatogram for Agilent SB C18.

The results in fig. 7 show that by comparing the chromatograms of 3 different columns, when a Waters HSS T3 column is selected, the peak information is more complete and the peak separation degree of a Waters HSS T3 column is better, so that a Waters HSS T3 column (2.1mm × 100mm, 1.8 μm) is selected as the column for establishing the characteristic spectrum of magnolia flowers.

2.4 investigation of flow Rate

In the experiment, three flow rates of 0.25ml per minute, 0.30ml per minute and 0.35ml per minute are selected for comparison, and the optimal flow rate is screened.

In fig. 8, curve 1 is a chromatogram for a flow rate of 0.30ml per minute, curve 2 is a chromatogram for a flow rate of 0.25ml per minute, and curve 3 is a chromatogram for a flow rate of 0.35ml per minute.

The results of fig. 8 show that by comparing the chromatograms of 3 different flow rates, when 0.30 ml/min and 0.25 ml/min were selected as the flow rate, the peak information was more complete; when the flow rate is 0.35ml per minute, the peak information is complete, the separation effect is better than 0.30ml per minute and 0.25ml per minute, so 0.35ml per minute is selected as the flow rate for establishing the characteristic spectrum of the magnolia officinalis flowers.

2.5 examination of column temperature

In the experiment, three column temperatures of 25 ℃, 30 ℃ and 35 ℃ are selected for comparison, and the optimal column temperature is screened.

In fig. 9, a curve 1 is a chromatogram corresponding to a column temperature of 30 ℃, a curve 2 is a chromatogram corresponding to a column temperature of 35 ℃, and a curve 3 is a chromatogram corresponding to a column temperature of 25 ℃.

The results of fig. 9 show that: comparing 3 chromatograms with different column temperatures, selecting 25 ℃, 30 ℃ and 35 ℃ as column temperatures, when the column temperature is 30 ℃, the information of peaks is complete, and the separation effect is superior to 25 ℃ and 35 ℃, so that the column temperature of 30 ℃ is selected as the column temperature for establishing the characteristic spectrum of the magnolia flowers.

2.6 determination of chromatographic conditions

Chromatographic conditions are as follows: the column was Waters HSS T3(2.1 mm. times.100 mm, 1.8 μm); the sample amount is 1 mul; the flow rate is 0.35ml per minute, and the column temperature is 30 ℃; acetonitrile is used as a mobile phase A, 0.1% formic acid solution is used as a mobile phase B, gradient elution is carried out according to the specification in the following table, the flow rate is 0.35ml/min, and the detection wavelength is as follows: 300nm, column temperature: at 30 ℃.

TABLE 2-1 gradient elution Table

3. Preparation of reference solutions

Referring to the preparation method of a reference substance solution under the item of 'Chinese pharmacopoeia' in 2015, a proper amount of magnolol and honokiol reference substance is precisely weighed and added with methanol to prepare a mixed solution containing 40 mu g of honokiol and 60 mu g of magnolol per 1ml, thus obtaining the reference substance mixed reference substance solution of the reference substance.

4. Examination of pretreatment method of test solution

And inspecting the extraction solvent, extraction time and extraction mode of the magnolia officinalis flower medicinal material, and determining the sample pretreatment method of the magnolia officinalis flower medicinal material characteristic spectrum.

4.1 examination of extraction solvent

The experiment inspects the influence of different extraction solvents on the characteristic spectrum of the magnolia officinalis flower medicinal material, selects 70% methanol, 70% ethanol and ethanol as the extraction solvents, compares the influence of the different extraction solvents on the characteristic spectrum of the magnolia officinalis flower by observing the temporary peak shapes and separation effects of 4 characteristic peaks and calculating the total peak area/sample weighing amount of the 4 characteristic peaks, and selects the optimal extraction solvent.

Taking about 1.0G of coarse powder (G1806107) of magnolia officinalis anther, precisely weighing, paralleling 4 groups, placing 2 parts of each group into a conical flask with a plug, precisely adding methanol, 70% ethanol, 70% methanol and 10ml of ethanol respectively, weighing, ultrasonically treating (power 300W, frequency 40kHz) for 30 minutes, cooling, weighing again, supplementing the weight loss with corresponding solvent, shaking up, filtering, and taking the subsequent filtrate. Precisely sucking 1 μ l, injecting into liquid chromatograph, and measuring. The results of the experiment are shown in Table 4-1 below and FIG. 10.

TABLE 4-1 comparison of different solvents extracted from Magnolia officinalis flowers by their characteristic spectra

In fig. 10, a curve 1 is a chromatogram corresponding to an extraction solvent of 70% methanol, a curve 2 is a chromatogram corresponding to an extraction solvent of methanol, a curve 3 is a chromatogram corresponding to an extraction solvent of 70% ethanol, and a curve 4 is a chromatogram corresponding to an extraction solvent of ethanol.

The results of Table 4-1 and FIG. 10 show that the ethanol solvent has a poor symmetry of characteristic peaks and a low response, as can be seen by comparing the characteristic patterns of the 4 extraction solvents. When the extraction solvent is 70% methanol and 70% ethanol, the total characteristic peak area/sample volume of the extract is not different and is higher than that of the methanol solvent, so that the 70% methanol is selected as the extraction solvent of the characteristic spectrum of the magnolia officinalis flower (magnolia officinalis).

4.2 examination of extraction methods

The experiment respectively inspects the influence of different extraction modes on the characteristic spectrum of the officinal magnolia flower medicinal material, selects ultrasonic and reflux as the extraction modes, and compares the characteristic spectrum results of the different extraction modes through temporarily determined 5 chromatographic peaks of total peak area/sample weighing amount and chromatogram.

Taking about 1.0G of coarse powder (G1806107) of magnolia officinalis anther, precisely weighing, paralleling 2 groups, placing 2 parts of each group into a conical flask with a plug, precisely adding 10ml of 70% methanol, weighing, respectively carrying out ultrasonic treatment (power 250W, frequency 40kHz) for 30 minutes, heating and refluxing for 30 minutes, cooling, weighing again, complementing the weight loss with 70% methanol, shaking uniformly, filtering, and taking the subsequent filtrate. Precisely sucking 1 μ l, injecting into liquid chromatograph, and measuring. The results of the experiment are shown in Table 4-2 below.

TABLE 4-2 comparison of different extraction methods of Magnolia officinalis flower medicinal material characteristic spectrum

The results in Table 4-2 show that the different extraction methods have little effect on the characteristic spectrum of the magnolia flower medicinal material by comparing ultrasonic extraction with reflux extraction, and ultrasonic treatment is selected as the extraction method in order to be consistent with the standard decoction.

4.3 extraction time inspection

The experiment respectively inspects the influence of different extraction time on the characteristic spectrum of the magnolia officinalis anther material, selects and inspects three different extraction time of 20 minutes, 30 minutes and 40 minutes, and compares the characteristic spectrum results of different extraction time through temporarily determined 5 chromatographic peaks, namely total peak area/sample weighing amount and a chromatogram.

Taking about 1.0G of coarse powder (G1806107) of magnolia officinalis anther material, precisely weighing, paralleling 3 groups, placing 2 parts of each group into a conical flask with a plug, precisely adding 10ml of 70% methanol, weighing, respectively carrying out ultrasonic treatment (power 300W, frequency 40kHz) for 20 minutes, 30 minutes and 40 minutes, cooling, weighing again, complementing the lost weight with 70% methanol, shaking up, filtering, and taking the subsequent filtrate. Precisely sucking 1 μ l, injecting into liquid chromatograph, and measuring.

TABLE 4-3 comparison of different extraction times of characteristic spectra of Magnolia officinalis flowers

In fig. 11, a curve 1 is a chromatogram corresponding to 20min of ultrasound, a curve 2 is a chromatogram corresponding to 30min of ultrasound, and a curve 3 is a chromatogram corresponding to 40min of ultrasound.

The results of tables 4-3 and FIG. 11 show that: the influence of different extraction times on the characteristic spectrum of the magnolia officinalis flower medicinal material is discovered by comparing the different extraction times, the influence of the different extraction times on the characteristic spectrum of the magnolia officinalis flower medicinal material is not large, and ultrasonic treatment is selected for 30 minutes to ensure complete extraction.

4.4 determination of preparation method of test solution

According to the experimental result, the pretreatment method of the magnolia officinalis anther material characteristic spectrum sample can be determined as follows:

precisely weighing 1.0g of coarse powder of flos Magnoliae, placing in a conical flask with a plug, precisely adding 10ml of 70% methanol, weighing, ultrasonically treating (power 300W and frequency 40kHz) for 30 min, cooling, supplementing the loss weight with 70% methanol, shaking, filtering, and collecting the filtrate.

5. Measurement method

Precisely sucking 1 μ l of reference solution and sample solution respectively, injecting into liquid chromatograph, and measuring.

The invention is further illustrated by the following examples

Example 1:

(1) sample information table

TABLE 515 batch Magnolia officinalis medicinal material information sheet

The 15 batches of magnolia flower medicinal materials all meet the regulation of the magnolia flower medicinal material item in the 2015 edition of Chinese pharmacopoeia.

(2) The characteristic spectrum method comprises the following steps:

chromatographic conditions chromatographic column: waters HSS T3 column (2.1 × 100mm, 1.8 μm) with acetonitrile as mobile phase a and 0.1% formic acid solution as mobile phase B, gradient elution was performed as specified in table 1, flow rate 0.35ml/min, detection wavelength: 300nm, column temperature: at 30 ℃.

Preparation of reference solution A proper amount of magnolol and honokiol reference substance is precisely weighed, and methanol is added to prepare a mixed solution containing 40 μ g of honokiol and 60 μ g of honokiol per 1 ml.

Preparing a test solution, namely taking about 1.0g of crude powder of magnolia officinalis flower (magnolia officinalis) medicinal material, precisely weighing, placing in a conical flask with a plug, precisely adding 10ml of 70% methanol, weighing, carrying out ultrasonic treatment (power is 300W, frequency is 40kHz) for 30 minutes, cooling, complementing the loss weight with 70% methanol, shaking up, filtering, and taking subsequent filtrate to obtain the magnolia officinalis flower (magnolia officinalis) medicinal material.

The determination method comprises precisely sucking reference solution and sample solution 1.0 μ l respectively, injecting into liquid chromatograph, and determining.

(3) Results

As shown in fig. 12, fig. 12 is a feature map overlay of 15 batches of magnolia officinalis anther, and relative retention time and relative peak area of each remaining feature peak are calculated with reference to peak 4 and magnolol, and the experimental results are shown in tables 6 and 7 below.

Relative retention time of each characteristic peak of characteristic spectrum of cortex magnoliae officinalis flower medicinal materials in table 615 batches

Relative peak area of each characteristic peak of characteristic spectrum of Magnolia officinalis flower medicinal materials in Table 715 batches

As can be seen from tables 6 and 7, the characteristic spectra of 15 batches of magnolia officinalis anther materials all present 5 characteristic peaks, wherein the retention time of peaks 4 and 5 is the same as that of honokiol and magnolol in a reference solution, the peak 4 and the magnolol peak are taken as S peaks, and the RSD value of the relative retention time of the 5 characteristic peaks is 0.02-0.29%; the relative peak area RSD value is 47.07% -117.02%, and the result shows that the content of the corresponding components of the characteristic peak of the magnolia flower medicinal materials in different producing areas has certain difference. The relative peak area of the peak 1 is 0.149-0.821, the relative peak area of the peak 2 is 0.035-3.937, the relative peak area of the peak 3 is 0.074-1.665, and the relative peak area of the peak 5 is 0.275-1.433.

Similarity of Magnolia officinalis flowers in Table 815 batches

Similarity evaluation is carried out on the characteristic spectrums of the magnolia officinalis flowers in 15 batches, the similarity of the characteristic spectrums of the magnolia officinalis flowers in each production place is good, and the fact that the composition of the medicinal material has no obvious production place specificity is shown. The similarity value of the medicinal materials in individual batches is less than 0.9, and certain difference exists. Presumably, the reason is that the composition ratio of each component is affected due to differences in the harvesting time or the production location processing mode, so that the similarity of the characteristic map of part of the batches to the control is poor.

Example 2

(1) Sample information table

TABLE 915 batch Magnoliae officinalis flower medicinal material information sheet

(2) The characteristic spectrum method comprises the following steps:

chromatographic conditions chromatographic column: waters HSS T3 column (2.1 × 100mm, 1.8 μm) with acetonitrile as mobile phase a and 0.1% formic acid solution as mobile phase B, gradient elution was performed as specified in table 1, flow rate 0.35ml/min, detection wavelength: 300nm, column temperature: at 30 ℃.

Preparation of reference solution A proper amount of magnolol and honokiol reference substance is precisely weighed, and methanol is added to prepare a mixed solution containing 40 μ g of honokiol and 60 μ g of honokiol per 1 ml.

Preparing a test solution, namely taking about 1.0g of magnolia officinalis pseudolite magnolia delavayi coarse powder, precisely weighing, placing in a conical flask with a plug, precisely adding 10ml of 70% methanol, weighing, ultrasonically treating (with the power of 300W and the frequency of 40kHz) for 30 minutes, cooling, supplementing the reduced weight with 70% methanol, shaking uniformly, filtering, and taking a subsequent filtrate to obtain the magnolia officinalis pseudolite magnolia delavayi coarse powder.

The determination method comprises precisely sucking reference solution and sample solution 1.0 μ l each, injecting into liquid chromatograph, and determining.

(3) As a result, the

As shown in FIG. 13, FIG. 13 shows the overlay of the characteristic spectrum of Magnolia officinalis in 3 batches of the pseudomorpha of Magnolia officinalis. In the chromatogram of Magnolia officinalis pseudobulbifera, there is no chromatographic peak at the corresponding retention time position of honokiol peak and magnolol peak.

Therefore, according to the experimental results, the characteristic maps of the magnolia officinalis anther materials are determined, the similarity of the characteristic maps of the magnolia officinalis anther materials in each production place is good, and the fact that the composition of the medicinal material substance has no obvious production place specificity is shown; meanwhile, the method can effectively identify the authenticity of the magnolia officinalis flowers, and the corresponding retention time positions of the peak 4 and the peak 5 have no chromatographic peak, so that the magnolia officinalis flowers and the pseudo magnolia delavayi can be identified, and the quality evaluation system of the magnolia officinalis flowers is perfected.

While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.

Claims (8)

1. A construction method of UPLC characteristic spectrum of officinal magnolia flower medicinal material is characterized by comprising the following steps:

(1) Accurately weighing appropriate amount of magnolol and honokiol reference substance, adding methanol to obtain mixed solution containing magnolol and honokiol, and making into reference substance solution;

(2) extracting magnolia officinalis flower medicinal material by using 65-75% methanol to prepare a test solution;

(3) precisely absorbing a reference substance solution and a test solution respectively, injecting the reference substance solution and the test solution into a liquid chromatograph, and performing gradient elution on a chromatographic column of the liquid chromatograph by using octadecylsilane chemically bonded silica as a filler, acetonitrile as a mobile phase A and a formic acid solution as a mobile phase B to establish a UPLC characteristic spectrum of the magnolia flower medicinal material; the gradient elution was performed according to the following procedure:

0-3 min, wherein the mobile phase A is 8% and the mobile phase B is 92%;

3-19 min, wherein the mobile phase A is 8% → 13%, and the mobile phase B is 92% → 87%;

19-22 min, wherein the mobile phase A is from 13% → 13.5%, and the mobile phase B is from 87% → 86.5%;

22-25 min, wherein the mobile phase A is 13.5% → 60%, and the mobile phase B is 86.5% → 40%;

25-32 min, wherein the mobile phase A is 60% and the mobile phase B is 40%;

32-33 min, wherein the mobile phase A is 60% → 8%, and the mobile phase B is 40% → 92%;

33-40 min, wherein the mobile phase A is 8% and the mobile phase B is 92%.

2. The method for constructing UPLC profile of Magnolia bark flower medicinal material of claim 1, wherein the reference solution is prepared by the following method:

Taking appropriate amount of magnolol and honokiol reference substance, precisely weighing, adding methanol to obtain mixed solution containing magnolol 60 μ g and honokiol 40 μ g per 1ml, and making into reference solution.

3. The method for constructing UPLC characteristic spectrum of Magnolia officinalis flower medicinal material of claim 1, wherein the sample solution is prepared by the following method:

precisely weighing 1.0-2.0g of coarse powder of magnolia flower, placing the coarse powder into a conical flask with a plug, precisely adding 10-20ml of 70% methanol, weighing the weight, carrying out ultrasonic treatment for 20-30 minutes, cooling, complementing the loss weight with 70% methanol, shaking up, filtering, and taking the subsequent filtrate.

4. The method for constructing UPLC characteristic spectrum of Magnolia officinalis flower medicinal material of claim 3, wherein the sample solution is prepared by the following method:

1.0g of coarse powder of magnolia officinalis anther is precisely weighed, the coarse powder is placed in a conical flask with a plug, 10ml of 70% methanol is precisely added, the weight is weighed, ultrasonic treatment is carried out for 30 minutes, the power of the ultrasonic treatment is 300W, the frequency is 40kHz, the mixture is cooled, the loss weight is complemented with 70% methanol, the mixture is shaken uniformly, and the subsequent filtrate is filtered, thus obtaining the magnolia officinalis anther.

5. The method for constructing UPLC feature map of magnolia officinalis flower medicinal material as claimed in claim 1, wherein the UPLC feature map of magnolia officinalis flower medicinal material is established by the following method:

Precisely sucking 1 μ l of reference solution and sample solution respectively, and injecting into a liquid chromatograph with octadecylsilane chemically bonded silica as filler, column length of 100mm, inner diameter of 2.1mm, and particle diameter of 1.8 μm; and performing gradient elution by using acetonitrile as a mobile phase A and 0.1% formic acid solution as a mobile phase B at the flow rate of 0.35ml per minute, the column temperature of 30 ℃ and the detection wavelength of 300 nm.

6. The method for constructing UPLC feature map of magnolia flower medicinal material according to claim 1, wherein the UPLC feature map of magnolia flower medicinal material comprises 5 feature peaks, which are peak 1, peak 2, peak 3, peak 4 and peak 5, respectively, wherein peak 4 corresponding to honokiol reference is S peak, the relative retention time of peak 1, peak 2, peak 3 and S peak should be within ± 10% of the specified value, and the specified value comprises: peak 1 was 0.37, Peak 2 was 0.52, Peak 3 was 0.64.

7. A detection method for identifying magnolia officinalis flowers and pseudo magnolia delavayi is characterized by comprising the following steps:

(1) accurately weighing appropriate amount of magnolol and honokiol reference substance, and adding methanol to obtain mixed solution containing magnolol and honokiol to obtain reference substance solution;

(2) Extracting magnolia officinalis flower medicinal material by using 65-75% methanol to prepare a test solution;

(3) precisely absorbing a reference substance solution and a test solution respectively, injecting the reference substance solution and the test solution into a liquid chromatograph, and performing gradient elution by using octadecylsilane chemically bonded silica as a filling agent, acetonitrile as a mobile phase A and a formic acid solution as a mobile phase B in the liquid chromatograph to establish a UPLC characteristic spectrum of the magnolia flower medicinal material;

(4) injecting a preset amount of sample solution into a liquid chromatograph, wherein the liquid chromatograph uses octadecylsilane chemically bonded silica as a filler, acetonitrile as a mobile phase A and formic acid solution as a mobile phase B for gradient elution to obtain a sample chromatogram;

(5) comparing the sample chromatogram with UPLC characteristic spectrum of Magnolia officinalis flower medicinal material, and determining Magnolia officinalis flower medicinal material and its pseudo Magnolia officinalis flower by the relative retention time of each characteristic peak of the sample chromatogram;

the gradient elution was performed according to the following procedure:

0-3 min, wherein the mobile phase A is 8% and the mobile phase B is 92%;

3-19 min, wherein the mobile phase A is 8% → 13%, and the mobile phase B is 92% → 87%;

19-22 min, wherein the mobile phase A is from 13% → 13.5%, and the mobile phase B is from 87% → 86.5%;

22-25 min, wherein the mobile phase A is 13.5% → 60%, and the mobile phase B is 86.5% → 40%;

25-32 min, wherein the mobile phase A is 60% and the mobile phase B is 40%;

32-33 min, wherein the mobile phase A is 60% → 8%, and the mobile phase B is 40% → 92%;

33-40 min, wherein the mobile phase A is 8% and the mobile phase B is 92%.

8. The detection method for identifying magnolia officinalis flowers and magnolia officinalis counterfeit product, magnolia officinalis, as claimed in claim 7, wherein the UPLC signature of magnolia officinalis flowers comprises 5 signature peaks, peak 1, peak 2, peak 3, peak 4, and peak 5, wherein peak 4 corresponding to honokiol reference is S peak;

calculating the relative retention time of the characteristic peak 1, peak 2, peak 3 and the S peak, if the relative retention time of the characteristic peak 1, peak 2, peak 3 and the S peak is within ± 10% of a specified value, the specified value comprising: judging the magnolia flower medicinal material if the peak 1 is 0.37, the peak 2 is 0.52 and the peak 3 is 0.64;

and if the corresponding retention time positions of the peak 4 and the peak 5 have no spectrum peak, determining that the pseudomagnolia delavayi is a pseudomagnolia delavayi.

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