CN114487151A - Method for identifying phellodendron amurense and application thereof - Google Patents
Method for identifying phellodendron amurense and application thereof Download PDFInfo
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Abstract
The invention relates to a pharmaceutical analysis technology, and discloses an identification method of phellodendron amurense and application thereof. The identification method of the golden cypress comprises the following steps: crushing a sample to be tested, mixing the crushed sample with an extraction solvent, and performing extraction treatment and filtration to obtain a sample solution; preparing a first control solution, respectively carrying out liquid chromatography analysis on the sample solution and the first control solution to obtain a sample liquid chromatogram and a first control liquid chromatogram, introducing the sample liquid chromatogram and the first control liquid chromatogram into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system for matching to generate a control chromatogram of a sample to be tested, and then identifying a cortex phellodendri sample I through chromatogram data analysis; preparing a second control solution, respectively carrying out thin-layer chromatography on the sample solution and the second control solution, and identifying a cortex phellodendri sample II by observing a strip of the thin-layer chromatography under an ultraviolet lamp; and comparing and verifying the cortex phellodendri sample I and the cortex phellodendri sample II to identify the cortex phellodendri. The identification method can effectively promote the safety and effectiveness of clinical medication of the phellodendron.
Description
Technical Field
The invention relates to a pharmaceutical analysis technology, in particular to an identification method of phellodendron amurense and application thereof.
Background
Traditional Chinese medicine Cortex Phellodendri (Phellodendri Chinensis Cortex) is the dried bark of Phellodendri Phellodendron Chinensis schneid, a Phellodendri bark plant of the rutaceae family, widely distributed in the southwest region of china, and is also known as Phellodendri Cortex; cortex Phellodendri (Phellodendron Amurense Cortex) is dried bark of Phellodendron Amurense Rupr of Rutaceae; both of them are commonly used Chinese herbs, which have the actions of clearing heat and drying dampness, purging fire and removing heat, removing toxicity and curing sores, and are commonly used for treating damp-heat dysentery, jaundice and dark urine, pruritus vulvae, stranguria with heat, pain, beriberi, flaccid wall, bone steaming and internal heat, etc.
Since the beginning of the Chinese pharmacopoeia of 2005 edition, "Chuan Huang Bai" and "guan Huang Bai" were classified into two herbs. Although the Chinese pharmacopoeia provides that phellodendron amurense and phellodendron amurense have the same efficacy and flavor, with the progress of modern analysis technology, the components, especially the content, are still different, and the importance degree of the pharmacological and biological activities of the phellodendron amurense and phellodendron amurense is also different. The phellodendron amurense has better quality clinically, but the phenomenon of the mixture of phellodendron amurense and phellodendron amurense is still found in daily inspection. In order to ensure the safety and effectiveness of clinical medication, it is necessary to effectively identify phellodendron amurense or phellodendron amurense.
In the current literature report, the accuracy of the identification method of the golden cypress still needs to be improved, and a reliable and accurate identification method of the golden cypress is established, so that reference can be provided for perfecting the quality control of the traditional Chinese medicine golden cypress (including the golden cypress in Sichuan and the golden cypress in Guanguan provinces), and the safety and the effectiveness of clinical medication of the golden cypress are promoted.
Disclosure of Invention
The invention aims to overcome the problem that the accuracy of phellodendron bark identification needs to be improved in the prior art, and provides an identification method of phellodendron bark and application thereof.
In order to achieve the above object, a first aspect of the present invention provides a method for identifying phellodendron amurense, comprising the steps of:
(1) crushing a sample to be tested, mixing the crushed sample with an extraction solvent, carrying out extraction treatment to obtain a sample extracting solution, and filtering the sample extracting solution to obtain a sample solution;
(2) preparing a first control solution containing a first control substance, performing liquid chromatography analysis on the sample solution and the first control solution in the step (1) to obtain a sample liquid chromatogram and a first control liquid chromatogram, introducing the sample liquid chromatogram and the first control liquid chromatogram into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system for matching to generate a control chromatogram of a test sample, and then identifying a cortex phellodendri sample I from the test sample through chromatogram data analysis;
(3) preparing a second control solution containing a second control substance, respectively carrying out thin-layer chromatography on the sample solution and the second control solution in the step (1), observing a strip of the thin-layer chromatography under an ultraviolet lamp, and identifying a cortex phellodendri sample II from the test sample;
(4) and (3) comparing the phellodendron bark sample I obtained in the step (2) with the phellodendron bark sample II obtained in the step (3) to verify so as to identify phellodendron bark.
Preferably, the pulverizing process in step (1) comprises: and crushing the sample to be tested, and then sieving the crushed sample with a 65-mesh sieve to obtain sample particles with the particle size of less than 65 meshes.
Preferably, the extraction solvent in step (1) is a methanol solution containing hydrochloric acid.
Preferably, the mass ratio of hydrochloric acid to methanol in the extraction solvent is 0.5-2: 100.
preferably, the extraction process in step (1) comprises: and carrying out ultrasonic treatment on the mixed solution obtained by mixing for 25-35min, and then carrying out solid-liquid separation.
Preferably, the filtration is membrane filtration, and the pore size of a filter membrane of the membrane filtration is 0.2-0.45 μm.
Preferably, in the step (2), the first control substance contains berberine hydrochloride, magnoflorine and palmatine hydrochloride.
Preferably, the concentration of berberine hydrochloride in the first control solution is 0.005-2mg/mL, the concentration of magnoflorine is 0.005-0.5mg/mL, and the concentration of palmatine hydrochloride is 0.005-0.5 mg/mL.
Preferably, the solvent of the first control solution is methanol.
Preferably, the conditions of the liquid chromatography in step (2) include: the chromatographic column adopts Waters Atlantis T3, acetonitrile as a mobile phase A and an aqueous solution containing phosphoric acid and triethylamine as a mobile phase B, and the volume ratio of the mobile phase A to the mobile phase B is 0.1-19: 1, performing gradient elution; the column temperature is 15-25 ℃, the flow rate is 0.6-1mL/min, the wavelength is 200-250nm, and the sample injection amount is 1-3 muL.
Preferably, the content of phosphoric acid in the mobile phase B is 0.2-0.5 volume percent, and the content of triethylamine in the mobile phase B is 0.2-0.5 volume percent.
Preferably, the process of profile data analysis in step (2) comprises: and processing the control map data of the test sample by adopting principal component analysis and/or orthogonal partial least squares discriminant analysis.
Preferably, in the step (3), the second control contains palmatine hydrochloride and/or berberine hydrochloride.
Preferably, the concentrations of palmatine hydrochloride and berberine hydrochloride in the second control solution are 0.1-1mg/mL respectively.
Preferably, the solvent of the second control solution is a methanol solution containing hydrochloric acid, and the mass ratio of the hydrochloric acid to the methanol is 0.5-2: 100.
preferably, the thin layer chromatography process in step (3) comprises: and (3) spotting the sample solution and the second control solution on a silica gel thin-layer plate respectively, developing, taking out and drying, and then carrying out heating color development by using a color developing agent.
Preferably, the spotting amount of the spotting is 0.5-2 μ L, the developing developer is n-butanol-acetic acid-water solution, and the developer is ethanol solution containing concentrated sulfuric acid.
Preferably, the volume ratio of n-butanol to acetic acid to water in the developing solvent is 3-4: 0.2-0.7: 1, the content of concentrated sulfuric acid in the color developing agent is 3-8 vol%.
The second aspect of the present invention provides the use of the above identification method in distinguishing phellodendron amurense and easily-confused products thereof, quality control of phellodendron amurense and identification of genuine or fake phellodendron amurense, wherein the phellodendron amurense is phellodendron amurense and/or phellodendron amurense.
Through the technical scheme, the invention has the beneficial effects that:
the method for identifying the golden cypress combines the process of liquid chromatography identification and the process of thin-layer chromatography identification, so that the two identification results can be mutually verified, the accuracy of identifying the golden cypress and/or the golden cypress in the Sichuan and/or the golden cypress in the Guanhuang provinces is effectively improved, a foundation is laid for promoting the modernization and the internationalization processes of the golden cypress and establishing an effective quality control standard, and the safety and the effectiveness of clinical medication of the golden cypress can be further promoted.
Furthermore, in the thin-layer chromatography identification process, the color developing agent is used for developing color, so that the strips of the thin-layer chromatography are clearer and more in number, more detected compounds are obtained, and the identification of the phellodendron amurense sample and other samples is simpler and more intuitive, and the accuracy is improved.
Drawings
FIG. 1 is an HPLC profile of sample 1, sample 28 and the first control solution of example 1, wherein A is sample 1, B is sample 28, and C is the first control solution; the peak 1 is phellodendrine hydrochloride, the peak 2 is magnoflorine, the peaks 3-7 are unknown compounds, the peak 8 is jateorhizine hydrochloride, the peak 9 is berberine hydrochloride, and the peak 10 is palmatine hydrochloride;
FIG. 2 is a comparison of the characteristic maps of all samples and the comparison maps of Phellodendri cortex and Phellodendri cortex in example 1, wherein A is the characteristic map of sample 1-sample 27, B is the characteristic map of sample 28-sample 33, and C is the comparison map of Phellodendri cortex and Phellodendri cortex; the peak 1 is phellodendrine hydrochloride, the peak 2 is magnoflorine, the peaks 3-7 are unknown compounds, the peak 8 is jateorhizine hydrochloride, the peak 9 is berberine hydrochloride, and the peak 10 is palmatine hydrochloride;
FIG. 3 is a score plot of the PCA analysis in example 1;
FIG. 4 is a load diagram of the PCA analysis in example 1, wherein peak 1 is phellodendrine hydrochloride, peak 2 is magnoflorine, peaks 3-7 are unknown compounds, peak 8 is jatrorrhizine hydrochloride, peak 9 is berberine hydrochloride, and peak 10 is palmatine hydrochloride;
FIG. 5 is a score plot of the OPLS-DA analysis in example 1;
FIG. 6 is a graph of the variable contribution of the OPLS-DA analysis in example 1, wherein peak 1 is phellodendrine hydrochloride, peak 2 is magnoflorine, peaks 3-7 are unknown compounds, peak 8 is jatrorrhizine hydrochloride, peak 9 is berberine hydrochloride, and peak 10 is palmatine hydrochloride;
FIG. 7 is a thin layer chromatogram of the second control solution, samples 1 to 3, and samples 28 to 30 in example 1, wherein 1 represents palmatine hydrochloride, 2 represents berberine hydrochloride, 3-5 represents samples 1 to 3, and 6-8 represents samples 28 to 30, respectively;
FIG. 8 is a thin layer chromatogram of the second control, samples 4 to 11, and samples 28 to 30 of example 1, wherein 1 represents palmatine hydrochloride, 2 represents berberine hydrochloride, 3-10 represents samples 4 to 11, and 11-13 represents samples 28 to 30, respectively;
FIG. 9 is a thin layer chromatogram of the second control, samples 12 to 19, and samples 31 to 33 in example 1, wherein 1 represents palmatine hydrochloride, 2 represents berberine hydrochloride, 14 to 16 represent samples 31 to 33, and 17 to 24 represent samples 12 to 19, respectively;
FIG. 10 is a thin layer chromatogram of the second control solution, samples 20 to 27, and samples 31 to 33 in example 1, wherein 1 represents palmatine hydrochloride, 2 represents berberine hydrochloride, 25 to 32 represent samples 20 to 27, and 33 to 35 represent samples 31 to 33, respectively;
FIG. 11 is a liquid chromatogram of sample 22 and sample 28 in example 1, wherein A represents the liquid chromatogram of sample 22 and B represents the liquid chromatogram of sample 28;
FIG. 12 is a liquid chromatogram of sample 14 in example 1.
Detailed Description
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.
In a first aspect, the present invention provides a method for identifying phellodendron bark, comprising the following steps:
(1) crushing a sample to be tested, mixing the crushed sample with an extraction solvent, carrying out extraction treatment to obtain a sample extracting solution, and filtering the sample extracting solution to obtain a sample solution;
(2) preparing a first control solution containing a first control substance, performing liquid chromatography analysis on the sample solution and the first control solution in the step (1) to obtain a sample liquid chromatogram and a first control liquid chromatogram, introducing the sample liquid chromatogram and the first control liquid chromatogram into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system for matching to generate a control chromatogram of a test sample, and then identifying a cortex phellodendri sample I from the test sample through chromatogram data analysis;
(3) preparing a second control solution containing a second control substance, respectively carrying out thin-layer chromatography on the sample solution and the second control solution in the step (1), observing a strip of the thin-layer chromatography under an ultraviolet lamp, and identifying a cortex phellodendri sample II from the test sample;
(4) and (3) comparing the phellodendron bark sample I obtained in the step (2) with the phellodendron bark sample II obtained in the step (3) to verify so as to identify phellodendron bark.
When phellodendron amurense is identified by using a thin-layer chromatography identification method of phellodendron amurense and phellodendron amurense recorded in Chinese pharmacopoeia, the problems of unclear chromatographic strips, low identification accuracy and the like exist. In the research and development process of quality control on the golden cypress, the inventor of the invention unexpectedly finds that after the golden cypress sample I is identified by combining the first contrast solution with the liquid chromatography, the golden cypress is further identified again by combining the second contrast solution with the thin-layer chromatography to obtain the golden cypress sample I, and the golden cypress is identified by comparing the identification result of the liquid chromatography, so that the accuracy of identifying the golden cypress and/or the golden cypress can be effectively improved, and the safety and the effectiveness of clinical medication of the golden cypress can be further promoted.
In the invention, the method for identifying phellodendron bark can be used for distinguishing phellodendron bark and easily confused products thereof, or distinguishing related phellodendron bark and easily confused products thereof, or performing quality control and quality grade division on phellodendron bark and/or related phellodendron bark, or identifying the authenticity of phellodendron bark and/or related phellodendron bark. In addition, the identification method of the phellodendron bark provided by the invention can also be used for distinguishing the phellodendron bark from the phellodendron bark.
According to the present invention, the sample to be tested in step (1) may be pulverized by a conventional pulverization method, such as a grinder, a pulverizer, etc. Preferably, the pulverizing process comprises: and crushing the sample to be tested, and then sieving the crushed sample with a 65-mesh sieve to obtain sample particles with the particle size of less than 65 meshes. That is, after the test sample is crushed, the particles which can pass through the No. four sieve are screened out by utilizing the No. four sieve of the traditional Chinese medicine for extraction. The inventors found that, in the preferred embodiment, it is advantageous to improve the extraction efficiency of the substance component in the sample to be tested, thereby optimizing the discrimination effect of the chromatography.
According to the present invention, the extraction solvent in step (1) may employ a conventional organic solvent, such as methanol, ethanol, n-propanol, isopropanol, etc. Preferably, the extraction solvent is a methanol solution containing hydrochloric acid. Further preferably, the mass ratio of hydrochloric acid to methanol in the extraction solvent is 0.5-2: 100. the inventors have found that in this preferred embodiment it is advantageous to optimise the extraction of the substance component in the test sample.
According to the present invention, the extraction treatment in step (1) may be performed such that the active ingredient in the sample to be tested is sufficiently dissolved in the extraction solvent. Preferably, the extraction process in step (1) comprises: and carrying out ultrasonic treatment on the mixed solution obtained by mixing for 25-35min, and carrying out solid-liquid separation to obtain supernatant, wherein the supernatant is the sample extracting solution. In the present invention, the solid-liquid separation may be performed by a method conventional in the art, such as centrifugation, filtration, standing, etc.
According to the invention, preferably, the filtration is membrane filtration, and the pore size of a filter membrane of the membrane filtration is 0.2-0.45 μm. The inventor finds that the preferred embodiment is beneficial to improving the accuracy of liquid chromatography and thin-layer chromatography of the sample liquid and effectively avoiding the interference of impurities.
According to the present invention, the first control can be obtained by using the component substances isolated from phellodendron amurense as disclosed in the prior art. Preferably, in the step (2), the first control substance contains berberine hydrochloride, magnoflorine and palmatine hydrochloride.
According to the invention, preferably, the concentration of berberine hydrochloride in the first control solution is 0.005-2mg/mL, the concentration of magnoflorine is 0.005-0.5mg/mL, and the concentration of palmatine hydrochloride is 0.005-0.5 mg/mL. The inventor finds that under the preferred embodiment, the peak effect of each reference substance in the liquid chromatography analysis is ensured, and the effectiveness and the accuracy of the phellodendron amurense identification are further improved.
According to the present invention, the solvent of the first control solution can be a conventional organic solvent, such as methanol, ethanol, n-propanol, isopropanol, etc. Preferably, the solvent of the first control solution is methanol.
According to the present invention, preferably, the conditions of the liquid chromatography in step (2) include: the chromatographic column adopts Waters Atlantis T3, acetonitrile as mobile phase A and an aqueous solution containing phosphoric acid and triethylamine as mobile phasePhase B, wherein the volume ratio of the mobile phase A to the mobile phase B is 0.1-19: 1, performing gradient elution; the column temperature is 15-25 ℃, the flow rate is 0.6-1mL/min, the wavelength is 200-250nm, and the sample injection amount is 1-3 muL. Illustratively, the conditions of the liquid chromatography analysis may specifically be: the chromatographic column adopts Waters
T3 (Volter Co., 5 μm, 4.6X 250 mm); acetonitrile is taken as a mobile phase A, and an aqueous solution containing phosphoric acid and triethylamine is taken as a mobile phase B; during the gradient elution, the volume ratio of the mobile phase A to the mobile phase B is: 0-25min, 10-25% A; 25-40 min; 25-40% of A; 40-55min, 40-95% A; 55-65% of A, 95%; the column temperature is 20 ℃; the flow rate is 0.8 mL/min; the wavelength is 230 nm; the amount of sample was 2. mu.L. The inventors have found that in this preferred embodiment, it is advantageous to improve the sensitivity and accuracy of the liquid chromatography analysis.
According to the invention, preferably, the content of phosphoric acid in the mobile phase B is 0.2 to 0.5 volume% and the content of triethylamine is 0.2 to 0.5 volume%. Specifically, the content of phosphoric acid in the mobile phase B was 0.3 vol%, and the content of triethylamine was 0.3 vol%.
According to the invention, the traditional Chinese medicine chromatogram fingerprint similarity evaluation system in the step (2) adopts a traditional Chinese medicine chromatogram fingerprint similarity evaluation system 2004A edition to perform data import, multi-point correction and data matching of liquid chromatogram so as to obtain a reference chromatogram of the sample to be tested.
According to the present invention, preferably, the process of profile data analysis in step (2) comprises: and processing the control map data of the test sample by adopting principal component analysis and/or orthogonal partial least squares discriminant analysis so as to identify the phellodendron bark sample I from the test sample. The principal component analysis and/or the orthogonal partial least squares discriminant analysis can be performed using software commonly used in the art, such as Simca-P14.1 software.
According to the present invention, the second control can be obtained by using the component substances isolated from phellodendron amurense as disclosed in the prior art. Preferably, in the step (3), the second control contains palmatine hydrochloride and/or berberine hydrochloride.
According to the invention, preferably, the concentration of palmatine hydrochloride and berberine hydrochloride in the second control solution is 0.1-1mg/mL respectively. The inventors have found that it is advantageous in this preferred embodiment to ensure the clarity of the corresponding bands in thin layer chromatography.
According to the present invention, the solvent of the second control solution can be a conventional organic solvent, such as methanol, ethanol, n-propanol, isopropanol, etc. Preferably, the solvent of the second control solution is a methanol solution containing hydrochloric acid. Further preferably, the mass ratio of hydrochloric acid to methanol in the solvent of the second control solution is 0.5-2: 100.
according to the present invention, preferably, the process of thin layer chromatography in step (3) comprises: and (3) spotting the sample solution and the second control solution on a silica gel thin-layer plate respectively, developing, taking out and drying, and then carrying out heating color development by using a color developing agent. Wherein, the drying can adopt the mode of airing. The inventor finds that in the preferred embodiment, the color development is carried out by using the color developing agent, so that the thin layer chromatography is clearer in strip and more in number, more compounds are detected, and further, the identification of the phellodendron amurense sample and other samples is simpler and more intuitive, and the accuracy is improved.
According to the invention, preferably, the spotting amount of the spotting is 0.5-2 μ L, the developing solvent is n-butanol-acetic acid-water solution, and the color developing agent is ethanol solution containing concentrated sulfuric acid. The inventors have found that in this preferred embodiment it is advantageous to further improve the definition of the strips.
According to the invention, the volume ratio of n-butanol, acetic acid and water in the developing solvent is preferably 3-4: 0.2-0.7: 1, the content of concentrated sulfuric acid in the color developing agent is 3-8 vol%.
Based on the identification method of the golden cypress, the invention provides the application of the identification method in distinguishing the golden cypress and the confusable products thereof, the quality control of the golden cypress and the authenticity identification of the golden cypress, wherein the golden cypress is the golden cypress in Sichuan and/or Guanhuang.
According to a particularly preferred embodiment of the present invention, the method for identifying phellodendron amurense includes the steps of:
(1) crushing a sample to be tested, sieving the crushed sample with a 65-mesh sieve to obtain sample particles with the particle size of less than 65 meshes, mixing the sample particles with a methanol solution containing hydrochloric acid (the mass ratio of the hydrochloric acid to the methanol is 0.5-2: 100) to obtain a mixed solution, carrying out ultrasonic treatment on the mixed solution for 25-35min, carrying out solid-liquid separation to obtain a sample extracting solution, and filtering the sample extracting solution through a membrane with the pore size of 0.2-0.45 mu m to obtain a sample solution;
(2) preparing a first control solution containing berberine hydrochloride (with a concentration of 0.005-2mg/mL), magnoflorine hydrochloride (with a concentration of 0.005-0.5mg/mL) and palmatine hydrochloride (with a concentration of 0.005-0.5mg/mL), and performing liquid chromatography analysis on the sample solution and the first control solution in the step (1) respectively to obtain a sample liquid chromatogram and a first control liquid chromatogram, wherein the conditions of the liquid chromatography analysis comprise: the chromatographic column adopts Waters Atlantis T3, acetonitrile as a mobile phase A, an aqueous solution containing phosphoric acid and triethylamine as a mobile phase B, and the volume ratio of the mobile phase A to the mobile phase B is 0.1-19: 1, performing gradient elution; the column temperature is 15-25 ℃, the flow rate is 0.6-1mL/min, the wavelength is 200-250nm, and the sample injection amount is 1-3 mu L; introducing the sample liquid chromatogram and the first control liquid chromatogram into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system for matching to generate a control chromatogram of a test sample, then processing the control chromatogram data of the test sample by adopting principal component analysis and/or orthogonal partial least squares discriminant analysis, and identifying a cortex phellodendri sample I from the test sample;
(3) preparing a second control solution containing palmatine hydrochloride and/or berberine hydrochloride (with the concentration of 0.1-1mg/mL respectively), and spotting the sample solution and the second control solution in the step (1) on a silica gel thin layer plate by 0.5-2 μ L respectively, wherein the volume ratio of the sample solution to the second control solution is 3-4: 0.2-0.7: 1, taking out and drying the sample, spraying an ethanol solution containing concentrated sulfuric acid (the content is 3-8 vol%) as a color developing agent to perform heating color development, observing a strip of thin layer chromatography under an ultraviolet lamp, and identifying a cortex phellodendri sample II from the sample;
(4) and (3) comparing and verifying the phellodendron sample I obtained in the step (2) with the phellodendron sample II obtained in the step (3) to identify phellodendron.
The present invention will be described in detail below by way of examples.
In the following examples, the ultrasonic treatment was performed using a digital ultrasonic cleaner manufactured by Kunshan ultrasonic instruments Inc., the thin-layer plate was heated using a TH-II thin-layer heater manufactured by Shanghai science and biochemistry technology Inc., the thin-layer plate was photographed using a GoodLook-1000 thin-layer chromatography imaging system manufactured by Shanghai science and biochemistry technology Inc., the thin-layer plate was spotted using a SP-20E full-automatic spotting instrument manufactured by Shanghai science and biochemistry technology Inc., the high performance liquid chromatography was performed using Agilent 1260 (equipped with a diode array detector) manufactured by Agilent science and technology Inc., Waters
T3 column was purchased from Vortish and 5 μm, 4.6X 250 mm.
In the following examples, the specific sources and numbers of samples of phellodendron amurense and phellodendron amurense are shown in Table 1; berberine hydrochloride (batch ST03070120), palmatine hydrochloride (batch ST03040120), phellodendrine hydrochloride (batch ST08670120), jatrorrhizine hydrochloride (batch ST03080120) and magnoflorine (batch ST15050120) are purchased from Shanghai Shidande Standard technical service, Inc., and all reference substances have purity of more than 98%; chromatographic grade methanol and acetonitrile were purchased from Sigma-Aldrich, usa; the water is purified water (Huarun Yibao drink Co., Ltd.); other raw materials and reagents are conventional commercial products.
TABLE 1
Example 1
(1) Taking 33 samples shown in table 1 as test samples, respectively crushing the test samples, sieving with a 65-mesh sieve to obtain sample particles with particle sizes smaller than 65 meshes, respectively mixing the sample particles with a methanol solution containing hydrochloric acid (the mass ratio of hydrochloric acid to methanol is 1: 100) to obtain a mixed solution, performing ultrasonic treatment on the mixed solution for 30min, filtering to obtain a sample extracting solution, and filtering the sample extracting solution with a 0.45-micron microporous filter membrane to obtain a sample solution;
(2) preparing a first control solution containing berberine hydrochloride (with the concentration of 0.5mg/mL), palmatine hydrochloride (with the concentration of 0.2mg/mL), jatrorrhizine hydrochloride (with the concentration of 0.2mg/mL), magnoflorine (with the concentration of 0.2mg/mL) and phellodendrine hydrochloride (with the concentration of 0.2mg/mL), and performing liquid chromatography analysis on the sample solution and the first control solution in the step (1) respectively to obtain a sample liquid chromatogram and a first control liquid chromatogram (HPLC characteristic maps of the sample 1, the sample 28 and the first control solution are shown in figure 1), wherein the conditions of the liquid chromatography analysis are as follows: the chromatographic column adopts Waters
T3 (Volter Co., 5 μm, 4.6X 250 mm); acetonitrile is taken as a mobile phase A, and an aqueous solution containing phosphoric acid and triethylamine is taken as a mobile phase B; during the gradient elution, the volume ratio of the mobile phase A to the mobile phase B is: 0-25min, 10-25% A; 25-40 min; 25-40% of A; 40-55min, 40-95% A; 55-65% and 95% of A; the column temperature is 20 ℃; the flow rate is 0.8 mL/min; the wavelength is 230 nm; the sample injection amount is 2 mu L; introducing the sample liquid chromatogram and the first control liquid chromatogram into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system 2004A edition for matching to generate a control map of the sample to be tested (the characteristic maps of the sample 1 to the sample 27, the characteristic map of the sample 28 to the sample 33 and the control map of the phellodendron amurense and the phellodendron amurense are shown in figure 2), introducing the control map data of the sample to be tested into Simca-P14.1 for PCA analysis (principal component analysis) and OPLS-DA analysis (orthogonal partial least squares discriminant analysis), wherein the PCA analysis score map and the load map are shown in figures 3 and 4, and the OPLS-DA analysis score map and the variable contribution map are shown in figures 5 and 6. As can be seen from the PCA score chart and the OPLS-DA score chart (FIGS. 3 and 5), all samples can be roughly classified into two types, i.e., one type is phellodendron amurense, the other type is phellodendron amurense, and the PCA is loadedIn the lotus graph and the OPLS-DA variable contribution graph (fig. 4 and fig. 6), peaks 9, 2, 7 and 10 in the liquid chromatogram are key components for identifying the phellodendron amurense and the phellodendron amurense. By analyzing the profiles of the samples and combining the results of PCA and OPLS-DA, it can be concluded that: the content of the palmatine hydrochloride absorption peak (peak 10) and the berberine hydrochloride absorption peak (peak 9) in phellodendron amurense is obviously lower than that of phellodendron amurense, the strength of the palmatine hydrochloride absorption peak (peak 10) in a phellodendron amurense sample is very low, and the palmatine hydrochloride absorption peak (peak 10) cannot be detected in part of samples; in addition, the absorption intensity of the 7 th peak in phellodendron amurense is greater than that of phellodendron amurense, while the absorption intensity of the magnoflorine absorption peak (peak 2) is relatively small. Identifying cortex phellodendri amurense sample I as a sample 1-sample 21 and a sample 23-sample 27 from the test sample by analyzing the map characteristics, and identifying cortex phellodendri amurense sample I as a sample 28-sample 33; wherein, the liquid chromatogram of sample 22 is shown in fig. 11, the absorption peak (peak 10) of palmatine hydrochloride is more obvious, which indicates that the palmatine hydrochloride content is higher and is similar to the spectrum characteristic of phellodendron amurense rupr, but is not completely consistent with the spectrum characteristic of phellodendron amurense rupr, the overall characteristic is similar to that of phellodendron amurense rupr, in the PCA and OPLS-DA score maps, although the distribution of sample 22 is closer to that of phellodendron amurense rupr, there is a certain distance between the sample 22 and phellodendron amurense rupr, so the result is difficult to judge the specific category of sample 22;
(3) respectively preparing a solution containing palmatine hydrochloride (with the concentration of 0.5mg/mL) and a solution containing berberine hydrochloride (with the concentration of 0.5mg/mL) as second control solutions, respectively taking 1 mu L of the sample solution and the second control solution in the step (1) to be spotted on a silica gel G thin-layer plate (each silica gel G thin-layer plate contains two spotting of the second control solutions as controls), and carrying out the following steps of: 1: 2 as developing agent, then taking out, airing, spraying ethanol solution containing 5 vol% of concentrated sulfuric acid as color developing agent, heating until the spots are clear, observing the thin layer chromatography strip under 365nm ultraviolet light, and the thin layer chromatograms of the samples 1-33 are shown in figures 7-10 (in order to better display the color developing strip, shown in color pictures). According to the HPLC analysis research result, the fact that the amur corktree bark has a very obvious palmatine hydrochloride band can be found, and the amur corktree bark has no obvious palmatine hydrochloride band. Therefore, the phellodendron amurense sample II is identified as sample 1-sample 13 and sample 15-sample 27 from the test samples, and the phellodendron amurense sample II is identified as sample 28-sample 33, wherein the spots of the sample 14 at the palmatine hydrochloride are clearer (as shown in figure 9 by number 19) and the similarity with the phellodendron amurense sample is relatively higher compared with other identified phellodendron amurense sample II, so that the specific type of the sample 14 is difficult to judge by the result of the thin-layer chromatography;
(4) comparing and verifying the phellodendron amurense sample I obtained in the step (2) with the phellodendron amurense sample II obtained in the step (3), wherein the identification results of the sample 1-sample 13, the sample 15-sample 21 and the sample 23-sample 27 in the step (2) and the step (3) are consistent, so that the phellodendron amurense is determined; sample 28-sample 33 identified consistently in step (2) and step (3) to be determined as golden cypress;
the liquid chromatogram of the sample 14 in the step (2) is shown in fig. 12, the map characteristics are consistent with the cortex phellodendri contrast map, and the sample 14 can be accurately judged to be cortex phellodendri by integrating the experimental results of the step (2) and the step (3);
in the thin-layer chromatography of the sample 22 in the step (3), the palmatine hydrochloride spots are not obvious (as shown by the number 27 in fig. 10), the thin-layer spectrum characteristics of the palmatine hydrochloride spots are consistent with those of phellodendron amurense, and the sample 22 can be accurately judged to be phellodendron amurense by integrating the experimental results in the step (2) and the step (3).
In example 1, when the samples 25 and 26 were purchased, the manufacturer introduced and preliminarily identified the golden cypress, and analyzed in step (2), the map features of the golden cypress were consistent with those of golden cypress, and the golden cypress was identified, and further verified in step (3) to be the golden cypress. The sample 30 is introduced by a merchant when purchased and is preliminarily identified as phellodendron amurense, the spectral characteristics of the sample are consistent with those of the phellodendron amurense through the analysis of the step (2), the phellodendron amurense is determined as the phellodendron amurense, and the phellodendron amurense is further determined as the phellodendron amurense through the verification of the step (3). Therefore, the step (2) and the step (3) can accurately identify whether the sample sold in the market is phellodendron amurense or phellodendron amurense.
The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.
Claims (10)
1. The identification method of the golden cypress is characterized by comprising the following steps:
(1) crushing a sample to be tested, mixing the crushed sample with an extraction solvent, carrying out extraction treatment to obtain a sample extracting solution, and filtering the sample extracting solution to obtain a sample solution;
(2) preparing a first control solution containing a first control substance, performing liquid chromatography analysis on the sample solution and the first control solution in the step (1) to obtain a sample liquid chromatogram and a first control liquid chromatogram, introducing the sample liquid chromatogram and the first control liquid chromatogram into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system for matching to generate a control chromatogram of a test sample, and then identifying a cortex phellodendri sample I from the test sample through chromatogram data analysis;
(3) preparing a second control solution containing a second control substance, respectively carrying out thin-layer chromatography on the sample solution and the second control solution in the step (1), observing a strip of the thin-layer chromatography under an ultraviolet lamp, and identifying a cortex phellodendri sample II from the test sample;
(4) and (3) comparing and verifying the phellodendron sample I obtained in the step (2) with the phellodendron sample II obtained in the step (3) to identify phellodendron.
2. The authentication method as claimed in claim 1, wherein the pulverization in the step (1) comprises: and crushing the sample to be tested, and then sieving the crushed sample with a 65-mesh sieve to obtain sample particles with the particle size of less than 65 meshes.
3. The identification method according to claim 1, wherein the extraction solvent in step (1) is a methanol solution containing hydrochloric acid;
preferably, the mass ratio of hydrochloric acid to methanol in the extraction solvent is 0.5-2: 100.
4. the authentication method according to claim 1, wherein the extraction process in step (1) comprises: carrying out ultrasonic treatment on the mixed solution obtained by mixing for 25-35min, and then carrying out solid-liquid separation;
preferably, the filtration is membrane filtration, and the pore size of a filter membrane of the membrane filtration is 0.2-0.45 μm.
5. The identification method according to any one of claims 1 to 4, wherein in the step (2), the first control substance comprises berberine hydrochloride, magnoflorine and palmatine hydrochloride;
preferably, the concentration of berberine hydrochloride in the first control solution is 0.005-2mg/mL, the concentration of magnoflorine is 0.005-0.5mg/mL, and the concentration of palmatine hydrochloride is 0.005-0.5 mg/mL;
preferably, the solvent of the first control solution is methanol.
6. The identification method according to any one of claims 1 to 4, wherein the conditions of the liquid chromatography in step (2) include: the chromatographic column adopts Waters Atlantis T3, acetonitrile as a mobile phase A and an aqueous solution containing phosphoric acid and triethylamine as a mobile phase B, and the volume ratio of the mobile phase A to the mobile phase B is 0.1-19: 1, performing gradient elution; the column temperature is 15-25 ℃, the flow rate is 0.6-1mL/min, the wavelength is 200-250nm, and the sample injection amount is 1-3 mu L;
preferably, the content of phosphoric acid in the mobile phase B is 0.2-0.5 volume percent, and the content of triethylamine in the mobile phase B is 0.2-0.5 volume percent.
7. The identification method according to any one of claims 1 to 4, wherein the process of profile data analysis in step (2) comprises: and processing the control map data of the test sample by adopting principal component analysis and/or orthogonal partial least squares discriminant analysis.
8. The identification method according to any one of claims 1 to 4, wherein in the step (3), the second control contains palmatine hydrochloride and/or berberine hydrochloride;
preferably, the concentrations of palmatine hydrochloride and berberine hydrochloride in the second control solution are respectively 0.1-1 mg/mL;
preferably, the solvent of the second control solution is a methanol solution containing hydrochloric acid, and the mass ratio of the hydrochloric acid to the methanol is 0.5-2: 100.
9. the identification method according to any one of claims 1 to 4, wherein the thin layer chromatography process in step (3) comprises: respectively spotting the sample solution and the second control solution on a silica gel thin-layer plate, developing, taking out and drying, and then heating and developing by using a developer;
preferably, the spotting amount of the spotting is 0.5-2 μ L, the developing developer is n-butanol-acetic acid-water solution, and the color developing agent is ethanol solution containing concentrated sulfuric acid;
preferably, the volume ratio of n-butanol to acetic acid to water in the developing solvent is 3-4: 0.2-0.7: 1, the content of concentrated sulfuric acid in the color developing agent is 3-8 vol%.
10. Use of the method according to any one of claims 1 to 9 for distinguishing between phellodendron amurense and confusable products thereof, quality control of phellodendron amurense, and authenticity identification of phellodendron amurense, wherein the phellodendron amurense is phellodendron amurense and/or phellodendron amurense.
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