CN115326981B - Method for measuring content of various components in garden balsam medicinal material - Google Patents
Method for measuring content of various components in garden balsam medicinal material Download PDFInfo
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- CN115326981B CN115326981B CN202211006013.0A CN202211006013A CN115326981B CN 115326981 B CN115326981 B CN 115326981B CN 202211006013 A CN202211006013 A CN 202211006013A CN 115326981 B CN115326981 B CN 115326981B
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G01N30/72—Mass spectrometers
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- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
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Abstract
The invention relates to the technical field of traditional Chinese medicine detection, in particular to a method for measuring the content of various components in a garden balsam medicinal material. The invention uses UPLC-MS/MS method, and uses the Acquisy I-Class UPLC BEH C18 chromatographic column (2.1 mm x 100mm, inner diameter 1.7 μm) for analysis. The chromatographic column and the autosampler were maintained at 40℃and 25℃respectively, the flow rate was 0.3mL/min, and the sample loading was 1. Mu.L. The mobile phase consisted of 0.1% formic acid in water a and 0.1% acetonitrile formic acid B. According to the method disclosed by the invention, protocatechuic acid, epicatechin, chlorogenic acid, quercetin, bai Zhushu glycoside and phyllanthin A in the garden balsam medicinal material can be rapidly and simultaneously separated, and the separation effect is good. Can be used for content measurement of protocatechuic acid, epicatechin, chlorogenic acid, quercetin, bai Zhushu glycoside and phyllanthin A in garden balsam medicinal material, and provides experimental basis for improving quality control level of garden balsam medicinal material.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicine detection, in particular to a method for measuring the content of various components in a garden balsam medicinal material.
Background
The garden balsam is a dry whole plant of the rhododendron phyllanthum Gaultheria yunnanensis (Franch.) of the Ericaceae family, is mainly distributed in the places such as Yunnan, sichuan, guangxi, guizhou and the like, can be used as a medicine, has flat property and pungent taste, has the effects of clearing heat and detoxicating, activating blood and removing stasis, dispelling wind and removing temperature, reducing qi and relieving asthma and the like, is used for treating dizziness, common cold due to wind-cold, cough and asthma, amenorrhea, traumatic swelling and pain, rheumatic arthralgia and the like, and is also described in Yunnan BenCao (1459) by Mingqianlan, guangxi plant resource, yunnan Chinese herbal medicine and other local books. The current standards of each province are not unified for the medicine application part of the garden balsam, and the plant name in the 'Yunnan province Chinese medicinal material standard (second album. Yi nationality medicine)', is the dry overground part of the Yunnan white fungus. The garden balsam recorded in Miao medicine roll of China herbal is the whole plant or root of Yunnan white pearl, and the medicinal part collected in Guizhou Chinese herbal medicine and national medicinal material quality standard (2003 edition) is the whole plant. The clinically common compound patent medicine containing the garden balsam comprises 'Yunnan white pearl syrup', 'compound tendon-stretching capsule', 'gout medicine' and the like, is a conventional medicinal material for treating rheumatoid diseases of Miao nationality, and has outstanding curative effect. The medical development value of the garden balsam medicinal material is higher, but the medical parts of the garden balsam medicinal material are different in standards and works in all places, so that the improvement of the quality control level of the garden balsam medicinal material and the deep development and utilization of medicinal material resources are limited.
The content of the components of different medicinal parts of the same medicinal material can be greatly different, for example, compared with the content of forsythoside A in different parts of fructus forsythiae, the content of forsythoside A in fructus forsythiae leaves is highest, the content of forsythoside A in green fructus forsythiae, continuous flower and fructus forsythiae is higher, and the content of forsythoside A in old fructus forsythiae is lower. At present, the quality control of the garden balsam is less studied, and except for the identification study of thin layer chromatography, only the content of single or one type of components such as salicylic acid, total flavone, quercetin, lignan glycoside, methyl salicylate and the like in the garden balsam is measured. Zeng detected Gaultheria yunnanensis and its formulations using the CE-ED method, including (+) -catechin, rutin, gentisic acid, vanillic acid, salicylic acid, quercetin and protocatechuic acid were identified and assayed. However, the amount of the sample was small, and the different portions thereof were not analyzed.
The applicant carried out chemical component analysis on the garden balsam, and selected 6 components of protocatechuic acid, epicatechin, chlorogenic acid, quercetin, bai Zhushu glycoside and phyllanthin A which are closely related to the pharmacological action of the garden balsam from 18 components identified as the mass.
Therefore, it is necessary to develop a method for simultaneously measuring 6 components of protocatechuic acid, epicatechin, chlorogenic acid, quercetin, bai Zhushu glycoside and phyllanthin A in a garden balsam medicinal material.
Disclosure of Invention
In order to solve the technical problems in the prior art, the invention provides a method for measuring the content of various components in a garden balsam medicinal material, which comprises the following steps:
A method for measuring the content of various components in herba speranskiae tuberculatae comprises measuring by UPLC-MS/MS method; analysis was performed using an Acquity I-Class UPLC BEH C18 column (2.1 mm. Times.100 mm, inner diameter 1.7 μm); the chromatographic column and the automatic sampler are respectively kept at 40 ℃ and 25 ℃, the flow rate is 0.3mL/min, and the sampling amount is 1 mu L; the mobile phase consists of 0.1% formic acid aqueous solution A and 0.1% acetonitrile formic acid B; the elution gradient was as follows: 0-0.5min, 10-10% B;0.5-2.5min;10% -40% of B;2.5-4 minutes, 40% -90% B;4-4.5 minutes, 90% -90% of B;4.5-6 minutes, 90% -10% of B.
Further, the UPLC-MS/MS method uses a UPLC-MS/MS system consisting of an ACQUITY I-Class UPLC system equipped with an electrospray ionization source and a XEVO TQS-triple quadrupole tandem mass spectrometer. Further, the UPLC-MS/MS system was further equipped with Waters VanGuard BEH C (2.1 mm. Times.5 mm,1.7 μm) column as a guard column to filter impurities.
Further, the UPLC-MS/MS method has the following mass spectrometer parameters: capillary voltage 3.0kV; capillary ionization voltage 3.0kV; the temperature of the ion source is 120 ℃; spraying gas and back-flushing gas, N2; the flow rate of the desolventizing agent is 650L/h; and desolvation gas temperature was 350 ℃, quantified using Multiple Reaction Monitoring (MRM) mode.
Further, parameters of the analyte in the multi-reaction monitoring mode are as follows:
Further, the sample solution is prepared by the following steps: precisely weighing herba speranskiae tuberculatae, precisely adding 25mL of 50% methanol, weighing, refluxing for 2h, cooling, adding 50% methanol for weight compensation, taking 200 μL of mixed solution of 50% methanol and 800 μL, centrifuging 12000rpm for 10min, and collecting supernatant.
Further, the reference substance solution is prepared by the following steps: precisely weighing a proper amount of reference substance, and diluting with methanol to 10mL to obtain stock solutions of chlorogenic acid (1.020 g/L), protocatechuic acid (1.035 g/L), epicatechin (1.018 g/L), quercetin (0.878 g/L), bai Zhushu glycoside (1.030 g/L) and phyllanthin A (1.116 g/L), respectively; precisely measuring the six reference substance stock solutions and diluting to final concentration of chlorogenic acid (0.012 mg/mL), protocatechuic acid (0.041 mg/mL), epicatechin (0.101 mg/mL), quercetin (0.137 mg/mL), bai Zhushu glycoside (0.077 mg/mL) and phyllanthin A (0.071 mgm/L); precisely measuring 100 μl of stock solutions of the final concentrations of the six reference substances, mixing to obtain mixed reference substance solution, sequentially diluting with 2 times dilution method, and storing in-20deg.C environment.
Further, the content measurement of the multiple components is to measure the content of protocatechuic acid, epicatechin, chlorogenic acid, quercetin, bai Zhushu glycoside and phyllanthin A in the garden balsam medicinal material at the same time.
Further, the data obtained for the UPLC-MS/MS method were all obtained using MasslynxTM V4.1.1 software (Waters corp., millford, MA, USA) and processed using QuanlynaTM V4.1 (Waters corp., millford, MA, USA) workstation.
The specific operation steps of the invention are as follows:
(1) Preparation of a control solution:
Precisely weighing a proper amount of reference substance, and diluting with methanol to 10mL to obtain stock solutions of chlorogenic acid (1.020 g/L), protocatechuic acid (1.035 g/L), epicatechin (1.018 g/L), quercetin (0.878 g/L), bai Zhushu glycoside (1.030 g/L) and phyllanthin A (1.116 g/L), respectively. The six control stock solutions were precisely measured and diluted to final concentrations of chlorogenic acid (0.012 mg/mL), protocatechuic acid (0.041 mg/mL), epicatechin (0.101 mg/mL), quercetin (0.137 mg/mL), bai Zhushu glycoside (0.077 mg/mL) and phyllanthin A (0.071 mgm/L). Precisely measuring 100 μl of stock solutions of the six reference substances with final concentration, mixing to obtain mixed reference substance solution, sequentially diluting with 2 times dilution method, and storing in refrigerator (-20deg.C).
(2) Preparation of test solution:
Precisely weighing about 0.2g of medicinal material, precisely adding 25mL of 50% methanol, refluxing for 2h after weighing, cooling, adding 50% methanol for weight compensation, taking 200 mu L of mixed solution of 800 mu L of 50% methanol, centrifuging 12000rpm for 10min, taking supernatant, and sampling with a sampling amount of 1 mu L.
(3) UPLC-MS/MS treatment:
taking a mixed reference substance solution and a test substance solution respectively, and carrying out UPLC-MS/MS determination according to the following conditions:
The UPLC-MS/MS system consisted of an ACQUITY I-Class UPLC system equipped with an electrospray ionization source and XEVO TQS-triple quadrupole tandem mass spectrometer. Analysis was performed using an Acquisy I-Class UPLC BEH C18 column (2.1 mm. Times.100 mm, inner diameter 1.7 μm). The system was also equipped with Waters VanGuard BEH C (2.1 mm. Times.5 mm,1.7 μm) chromatography columns. The chromatographic column and the autosampler were maintained at 40℃and 25℃respectively, the flow rate was 0.3mL/min, and the sample loading was 1. Mu.L. The mobile phase consisted of 0.1% formic acid in water a and 0.1% acetonitrile formic acid B. The elution gradient was as follows: 0-0.5min, 10-10% B;0.5-2.5min;10% -40% of B;2.5-4 minutes, 40% -90% B;4-4.5 minutes, 90% -90% of B;4.5-6 minutes, 90% -10% of B.
The mass spectrometer parameters were as follows: capillary voltage 3.0kV; capillary ionization voltage 3.0kV; the temperature of the ion source is 120 ℃; spraying gas and back-flushing gas, N2; the flow rate of the desolventizing agent is 650L/h; and the desolvation gas temperature was 350 ℃. Quantification was performed using the Multiple Reaction Monitoring (MRM) mode. The best parameters for the analytes in MRM mode are listed in Table 2. All data were obtained using MasslynxTM V4.1.1 software (Waters corp., millford, MA, USA) and processed using QuanlynaTM V4.1 (Waters corp., millford, MA, USA) workstation.
Compared with the prior art, the invention has the technical effects that:
The invention adopts UPLC-MS/MS method, and adopts UPLC-MS/MS system composed of ACQUITY I-Class UPLC system equipped with electrospray ionization source and XEVO TQS-triple quadrupole tandem mass spectrometer. Analysis was performed using an Acquisy I-Class UPLC BEH C18 column (2.1 mm. Times.100 mm, inner diameter 1.7 μm). The chromatographic column and the autosampler were maintained at 40℃and 25℃respectively, the flow rate was 0.3mL/min, and the sample loading was 1. Mu.L. The mobile phase consisted of 0.1% formic acid in water a and 0.1% acetonitrile formic acid B. The elution gradient was as follows: 0-0.5min, 10-10% B;0.5-2.5min;10% -40% of B;2.5-4 minutes, 40% -90% B;4-4.5 minutes, 90% -90% of B;4.5-6 minutes, 90% -10% of B. The mass spectrometer parameters were as follows: capillary voltage 3.0kV; capillary ionization voltage 3.0kV; the temperature of the ion source is 120 ℃; spraying gas and back-flushing gas, N2; the flow rate of the desolventizing agent is 650L/h; and the desolvation gas temperature was 350 ℃. Quantification was performed using the Multiple Reaction Monitoring (MRM) mode.
According to the method disclosed by the invention, protocatechuic acid, epicatechin, chlorogenic acid, quercetin, bai Zhushu glycoside and phyllanthin A in the garden balsam medicinal material can be rapidly and simultaneously separated, and the separation effect is good. The sample recovery rate of the method is 94.93-105.4%, and RSD is less than or equal to 4.12%. The method is simple, rapid, accurate and repeatable, can be used for content measurement of protocatechuic acid, epicatechin, chlorogenic acid, quercetin, bai Zhushu glycoside and phyllanthin A in the garden balsam medicinal material, and provides experimental basis for improving quality control level of the garden balsam medicinal material.
Drawings
FIG. 1 is a total ion flow diagram of the positive ion mode of the Gaultheria Yunnanensis mass spectrum HPLC/Q Exactive Plus MS (a. Scopolamine; b. Eugenol; c. Kaempferol; d. Ursolic acid).
Fig. 2 is a total ion flow chart of the diatom oophora root HPLC/Q Exactive Plus MS mass spectrum negative ion mode (a. Protocatechuic acid; B. Procyanidin A2; c. Chlorogenic acid; d. Procyanidin B2; e. Vanillic acid; f. Epicatechin; g. Bai Zhushu glycoside; h. Salicylic acid; i. Benzoic acid; j. Quercetin; k. Phyllanthin a; l. Quercetin; m. Oleanolic acid; n. Palmitic acid).
FIG. 3 is a graph of the specificity of six components (from left to right: mix standard, sample, blank solvent) (T1. Protocatechuic acid; T2. Quercetin; T3. Bai Zhushu glycoside; T4. Epicatechin; T5. Dianthrin A; T6. Chlorogenic acid).
FIG. 4 shows the chemical formulas of 6 content-measuring components (T1. Protocatechuic acid; T2. Quercetin; T3. Bai Zhushu glycoside; T4. Epicatechin; T5. Dianthrin A; T6 chlorogenic acid).
FIG. 5 is a hierarchical cluster analysis dendrogram.
Fig. 6 is a graph showing the analysis of the principal components of the diathesis, and the score of the load graph (b).
Detailed Description
The technical scheme of the present invention is further defined below in conjunction with the specific embodiments, but the scope of the claimed invention is not limited to the description.
Examples:
Instrument and material:
Instrument:
UHPLC/Q Exactive Plus MS high resolution mass spectrometry instrument (Thermo FISHER SCIENTIFIC, U.S.); ACQUITYUPLC H-Class ultra performance liquid chromatography (Waters, USA); allegra 30R centrifuges low temperature high speed centrifuges (Beckman Coulter, U.S.A.); multitube vortex oscillator (VX-III, beijing pedal science and technology Co., ltd.); EL204 electronic balance (meltler-tolido instruments limited); micropipettes (Eppendorf, germany); laboratory-specific ultra-pure water machine (WP-UP-III-20, sichuan Wo Teer technology development Co., ltd.); xex TQMS triple quadrupole mass spectrometer (Woltzian, america, including autosampler, binary gradient pump, column oven, vacuum degasser, masslynx4.1 mass spectrometry workstation)
Reagent:
Samples of the bone penetrating incense were collected from different areas of the Guizhou province of China and were supplied by Guizhou Babrio pharmaceutical Co. Chlorogenic acid (110753-201817), protocatechuic acid (111538-201606) and quercetin (111538-201606) reference substances are purchased from Chinese food and drug verification institute. Epicatechin control (AF 8030805) was purchased from Chengdu Biotechnology Inc. Bai Zhushu glycoside and Dian Bai Zhu glycoside A control were prepared by laboratory and had a lot number of 20190223. The purity of all the control products is more than 98 percent. The other reagents were all analytically pure.
The method comprises the following steps:
Preparation of a control solution:
Precisely weighing a proper amount of reference substance, and diluting with methanol to 10mL to obtain stock solutions of chlorogenic acid (1.020 g/L), protocatechuic acid (1.035 g/L), epicatechin (1.018 g/L), quercetin (0.878 g/L), bai Zhushu glycoside (1.030 g/L) and phyllanthin A (1.116 g/L), respectively. The six control stock solutions were precisely measured and diluted to final concentrations of chlorogenic acid (0.012 mg/mL), protocatechuic acid (0.041 mg/mL), epicatechin (0.101 mg/mL), quercetin (0.137 mg/mL), bai Zhushu glycoside (0.077 mg/mL) and phyllanthin A (0.071 mgm/L). Precisely measuring 100 μl of stock solutions of the six reference substances with final concentration, mixing to obtain mixed reference substance solution, sequentially diluting with 2 times dilution method, and storing in refrigerator (-20deg.C).
Preparation of test solution:
Precisely weighing about 0.2g of medicinal material, precisely adding 25mL of 50% methanol, refluxing for 2h after weighing, cooling, adding 50% methanol for weight compensation, taking 200 mu L of mixed solution of 800 mu L of 50% methanol, centrifuging 12000rpm for 10min, taking supernatant, and sampling with a sampling amount of 1 mu L.
High resolution mass spectrometry instrument conditions:
LC system: vanquish horizon; chromatographic column: hypersil gold aQ (2.1X100 mm,1.9 μm); mobile phase: 0.04% acetic acid water (a) -0.04% acetic acid acetonitrile (B); flow rate: 0.35mL/min; sample injection amount: 2. Mu.L, elution gradient is shown in Table 1. In Q Exactive Plus MS systems, electrospray ion sources are used to detect in positive and negative ion scan modes, respectively, under the following conditions: spray voltage: 3.8kV (+)/3.2 kV (-); evaporating temperature: 350 ℃; protective gas: 40arb; auxiliary gas: 10arb; capillary temperature: 320 ℃; s-lens: 60; the general method comprises the following steps: fullms-ddms; scanning range: m/z is 120-1500; resolution (MS 1): 70,000; MS/MS resolution: 17,500; step NCE: 20. 40, 60.
TABLE 1 chromatographic elution gradient
| Time (min) | A％ | B％ |
| 0.0 | 98 | 2 |
| 0.5 | 98 | 2 |
| 18.5 | 2 | 98 |
| 21.5 | 2 | 98 |
| 21.6 | 2 | 98 |
| 24 | 98 | 2 |
UPLC-MS/MS instrument conditions:
The UPLC-MS/MS system consisted of an ACQUITY I-Class UPLC system equipped with an electrospray ionization source and XEVO TQS-triple quadrupole tandem mass spectrometer. Analysis was performed using an Acquisy I-Class UPLC BEH C18 column (2.1 mm. Times.100 mm, inner diameter 1.7 μm). The system was also equipped with Waters VanGuard BEH C (2.1 mm. Times.5 mm,1.7 μm) chromatography columns. The chromatographic column and the autosampler were maintained at 40℃and 25℃respectively, the flow rate was 0.3mL/min, and the sample loading was 1. Mu.L. The mobile phase consisted of 0.1% formic acid in water a and 0.1% acetonitrile formic acid B. The elution gradient was as follows: 0-0.5min, 10-10% B;0.5-2.5min;10% -40% of B;2.5-4 minutes, 40% -90% B;4-4.5 minutes, 90% -90% of B;4.5-6 minutes, 90% -10% of B.
The mass spectrometer parameters were as follows: capillary voltage 3.0kV; capillary ionization voltage 3.0kV; the temperature of the ion source is 120 ℃; spraying gas and back-flushing gas, N2; the flow rate of the desolventizing agent is 650L/h; and the desolvation gas temperature was 350 ℃. Quantification was performed using the Multiple Reaction Monitoring (MRM) mode. The best parameters for the analytes in MRM mode are listed in Table 2. All data were obtained using MasslynxTM V4.1.1 software (Waters corp., millford, MA, USA) and processed using QuanlynaTM V4.1 (Waters corp., millford, MA, USA) workstation.
TABLE 2 optimal parameters for 6 Components in MRM mode
| Composition of the components | Molecular formula | Quantitative ion pairs | Taper hole voltage | Collision energy |
| Protocatechuic acid | C7H6O4 | 152.9-109 | 30 | 15 |
| Quercetin | C21H20O11 | 447.1-300.8 | 30 | 15 |
| Bai Zhushu glycoside | C19H26O12 | 491-293 | 30 | 15 |
| Epicatechin | C15H14O6 | 289.1-244.6 | 35 | 15 |
| Dian Baizhu glycoside A | C27H36O12 | 551-419.1 | 30 | 15 |
| Chlorogenic acid | C16H18O9 | 353-191 | 30 | 15 |
Results:
quick identification of chemical components by high-resolution liquid chromatography-mass spectrometry:
The UHPLC/Q Exactive Plus MS liquid chromatography-mass spectrometry system is used for detecting main chemical components in the garden balsam sample, and according to multi-stage mass spectrum information, the compound is automatically identified by multi-library matching through Compound Discoverer 3.1.1 software (Thermo FISHER SCIENTIFIC, MA, USA), and 18 chemical components in the positive (figure 1) mode and the negative (figure 2) mode are respectively determined. The mass spectrum information obtained by the detection is shown in table 3.
TABLE 3 identification component Mass Spectrometry information in the Permeability to bone incense UHPLC/Q Exactive Plus MS Positive and negative ion mode
Specialization:
and (3) respectively taking the mixed standard solution, the sample and the blank solvent, injecting according to the UPLC-MS/MS instrument conditions, and recording a TIC chromatogram, wherein the TIC chromatogram is shown in figure 3. The results show that the method has good specificity.
Linear relationship:
And precisely sucking the mixed reference substance solution, and performing linear regression by taking a peak area integral value as an ordinate (y) and a reference quality concentration as an abscissa (x) according to the UPLC-MS/MS instrument condition described above to obtain regression equations and linear ranges of protocatechuic acid, epicatechin, chlorogenic acid, quercetin, bai Zhushu glycoside and phyllanthin A, wherein the regression equations and linear ranges are shown in Table 4, and the good linear relationship is shown.
TABLE 4 six component standard curves, LOD and LOQ
Precision:
The daily and diurnal precision of each analyte was studied by taking test solutions under the UPLC-MS/MS instrument conditions described above and determining six analytes in six repeated determinations over one day and three consecutive days, and the variation of peak area was used as precision in calculating RSD, and RSD values of protocatechuic acid, epicatechin, chlorogenic acid, quercetin, bai Zhushu glycoside, and phyllanthin a were all within acceptable ranges, and the results are shown in table 5, indicating that it has good precision.
Repeatability:
6 parts of the same batch of garden balsam medicinal materials are taken, a sample solution is prepared according to the method, and sample injection analysis is carried out according to a UPLC-MS/MS instrument, wherein the results are shown in Table 5, and the mass fractions RSD of protocatechuic acid, epicatechin, chlorogenic acid, quercetin, bai Zhushu glycoside and phyllanthin A are respectively 1.4%, 2.1%, 1.2%, 1.6%, 2.3% and 2.1%, so that the sample solution has good repeatability.
Stability:
The same sample solutions were taken and measured at 0, 2, 4, 8, 12 and 24 hours, peak areas were recorded, and the results are shown in Table 5, and the RSD of protocatechuic acid, epicatechin, chlorogenic acid, quercetin, bai Zhushu glycoside and phyllanthin A were 1.8%, 2.6%, 2.1%, 1.8%, 1.7% and 2.8%, respectively, and the results indicate that the sample solutions have good stability.
Table 5 precision, stability and repeatability of 6 components in the diathesis (n=6)
Recovery rate:
Recovery of analyte was determined on the same day using standard addition, three different concentrations of mixed standard solutions (50%, 100% and 150% of the known amount in the sample) were added to the sample, and the results were calculated by comparing the difference between the standard and non-standard samples analyzed under the same conditions by injection into a LC/MS under the term "3.4", respectively.
Table 6 recovery of 6 components in diatom ooze (n=9)
Determination of UPLC-ESI-MS/MS content of 6 components in 38 batches of garden balsam medicinal materials:
The UPLC-MS/MS is adopted to simultaneously carry out content measurement on 6 components of the garden balsam medicinal materials in different batches in 38 different areas, the chemical formulas of the components are shown in figure 4, and the content measurement results are shown in table 9.
Table 9 content of 6 ingredients of Gaultheria yunnanensis (mg/g, n=3)
Systematic cluster analysis (HCA) and Principal Component Analysis (PCA):
In order to analyze the quality stability and the variation of the content of each component among the different parts of the garden balsam medicinal materials as a whole, the study performed HCA and PCA on 38 batches of garden balsam medicinal materials by using SPSS 20 software (IBM corp., NY, USA). HCA was performed using Ward's method, selecting euclidean distance as a metric, and normalizing with the variable Z value score, and the results show that 38 samples were divided into two major clusters (I and II) according to the content variation of these 6 compounds, as shown in fig. 5. Cluster ii included most of the aerial parts, while cluster i included all of the roots, indicating a large difference in the composition of the compounds between the roots and the aerial parts. PCA analysis with these 6 compounds as variables resulted in load and score plots seen in fig. 6 (a) and (b). As shown in FIG. 6 (a), epicatechin, bai Zhushu glycoside and protocatechuic acid were distributed in exactly three different directions and were each located closer to the origin, indicating that there was no correlation between the contents of these three components, and it was presumed that there were three components that might be important factors for causing the quality change of the diathesis. As shown in fig. 6 (b), root medicinal materials in different regions are mainly shifted longitudinally in the component 2 (PC 2) axis direction, while aerial medicinal materials in different regions are mainly shifted toward the first quadrant. The shift may be related to the variables in these directions, consistent with the analysis of FIG. 6 (a)
Conclusion and discussion:
The research shows that the ethyl acetate active part of the speranskia herb root has strong analgesic and anti-inflammatory activity, and 10 chemical components including quercetin, chlorogenic acid, protocatechuic acid and the like are identified. The phyllanthin A has anti-arthritis pharmacological activity, and is effective component of herba speranskiae tuberculatae. Zhang B et al showed that Bai Zhushu glycosides (200 mg/kg) were able to significantly inhibit acetic acid-induced abdominal contractions in mice, and to inhibit swelling of the auricles in mice caused by balm oil, similar to the inhibition of equimolar amounts of aspirin production. Chlorogenic acid also showed good anti-inflammatory and analgesic activity in carrageenan-induced inflammation experiments and 10% formaldehyde pain experiments. Chen Yingkang and other experimental researches show that 25-100 mug/mL catechin can obviously improve local swelling and pain of joint and polyarthritis of rats with rheumatoid arthritis, and relieve symptoms of the rheumatoid arthritis, and is one of active ingredients of the diathesis. Therefore, the experiment selects 6 components of protocatechuic acid, epicatechin, chlorogenic acid, quercetin, bai Zhushu glycoside and phyllanthin A which are closely related to the pharmacological action of the garden balsam from 18 identified components for quality control study.
The load diagram is used for describing the image characteristics, the relation among the variable attribute characteristics can be known, and the differentiating diagram can obtain the differentiating condition among the samples. It can be seen from the calculation method of the principal component that the larger the absolute value of the load, the larger the influence on the principal component. As shown in fig. 6 (a), in the direction of component 1 (PC 1), the content of the phyllanthin a and the content of the quercetin are two poles, which may mean that the higher the content of the quercetin in the medicinal material is, the correspondingly lower the content of the phyllanthin a is, and vice versa, and the variable with obvious correlation obviously divides the parent medicinal material of the root and the overground parts into two parts. For epicatechin, bai Zhushu glycoside and protocatechuic acid, the respective contents of epicatechin and protocatechuic acid contribute to the contents of the components of the diatom-bone incense in different areas almost, one component always occupies the dominant position of the contents, and the variable with insignificant correlation forms the quality difference of medicinal materials in different areas to a large extent independently of the other two components.
In this assay result, bai Zhushu glycoside was significantly higher in aerial parts than in roots and had significant differences (aerial parts 2.127 ±1.275&1.318±1.075 roots, P < 0.05). And in the overground part of the deviated batch, the content of the component is obviously higher than that of other batches, the batches are mainly from the Pichia area, the Pichia is located in the western area of Guizhou, the topography is 1000 m higher than that of the middle area such as Guiyang, the annual average air temperature is lower than that of the middle area, and the environment with lower air temperature is presumed to be more favorable for the accumulation of Bai Zhushu glycoside in the overground part. Protocatechuic acid and epicatechin are the main factors for the shift in root score. Under the factors of epicatechin, the areas are affected most greatly, the epicatechin content is higher than that of other areas, and the epicatechin is located at lower latitude in the south of Guizhou, and is warmer and more moist than that of other areas, so that the condition of warmer climate can be presumed to be more suitable for the accumulation of epicatechin in roots. The total content of protocatechuic acid in plants is low, and under the influence of the protocatechuic acid, the scoring is deviated mainly from the roots of Guiyang areas. The research of the application provides experimental basis for further improving the quality control standard of different drug administration positions of the garden balsam and standardizing the drug administration positions.
From the above, the method can rapidly and simultaneously separate protocatechuic acid, epicatechin, chlorogenic acid, quercetin, bai Zhushu glycoside and phyllanthin A in the garden balsam medicinal material, and has good separation effect. The sample recovery rate of the method is 94.93-105.4%, and RSD is less than or equal to 4.12%. The method is simple, convenient, quick, accurate and good in repeatability, can be used for measuring the content of protocatechuic acid, epicatechin, chlorogenic acid, quercetin, bai Zhushu glycoside and phyllanthin A in the garden balsam medicinal material, provides an experimental basis for improving the quality control level of the garden balsam medicinal material, and also provides an experimental basis for improving the quality control standard and the standard administration position of different administration positions of the garden balsam.
Finally, it should be noted that the above embodiments are merely representative examples of the present invention. Obviously, the technical solution of the invention is not limited to the above-described embodiments, but many variations are possible. All modifications directly derived or suggested to one skilled in the art from the present disclosure should be considered as being within the scope of the present invention.
Claims (4)
1. A method for measuring the content of various components in a garden balsam medicinal material is characterized in that a UPLC-MS/MS method is adopted for measuring; analysis was performed using an acquisition I-Class UPLC BEH C18 column, with the following specifications: 2.1 mm×100mm, inner diameter 1.7 μm; the chromatographic column and the automatic sampler are respectively kept at 40 ℃ and 25 ℃, the flow rate is 0.3 mL/min, and the sample injection amount is 1 mu L; the mobile phase consists of 0.1% formic acid aqueous solution A and 0.1% acetonitrile formic acid B; the elution gradient was as follows: 0-0.5 min, 10-10% B, 0.5-2.5 min; 10-40% of B, 2.5-4 minutes, 40-90% of B; 4-4.5 minutes, 90% -90% of B;4.5-6 minutes, 90% -10% of B;
The content measurement of the components is to measure the content of protocatechuic acid, epicatechin, chlorogenic acid, quercetin, bai Zhushu glycoside and phyllanthin A in the garden balsam medicinal material at the same time;
The UPLC-MS/MS method comprises an ACQUITY I-Class UPLC system provided with an electrospray ionization source and a XEVO TQS-triple quadrupole tandem mass spectrometer;
The UPLC-MS/MS system is also provided with Waters VanGuard BEH C chromatographic columns, and the specification of the UPLC-MS/MS system is as follows: 2.1 mm×5mm, 1.7 μm;
The mass spectrometer parameters of the UPLC-MS/MS method are as follows: capillary voltage 3.0kV; capillary ionization voltage 3.0kV; the temperature of the ion source is 120 ℃; spraying gas and back-flushing gas, N2; the flow rate of the desolventizing agent is 650L/h; and desolvation gas temperature was 350 ℃, quantified using a multiple reaction monitoring mode;
The parameters of the analytes in the multi-reaction monitoring mode are as follows:
。
2. the method for measuring the content of various components in the garden balsam medicinal material according to claim 1, wherein the sample solution is prepared by the following steps: precisely weighing the garden balsam medicinal material, precisely adding 50% methanol 25 mL, refluxing 2h after weighing, cooling, adding 50% methanol for weight compensation, taking 200 mu L of the mixture, adding 50% methanol 800 mu L of vortex mixing, centrifuging 12000 rpm for 10 min, and taking supernatant.
3. The method for measuring the content of various components in the garden balsam medicinal material according to claim 1, wherein the reference substance solution is prepared by the following steps: precisely weighing a proper amount of reference substances, and diluting to 10mL by using methanol to obtain stock solutions of 1.020 g/L chlorogenic acid, 1.035 g/L protocatechuic acid, 1.018 g/L epicatechin, 0.878 g/L quercetin, 1.030 g/L Bai Zhushu glycoside and 1.116 g/L Yunnan Bai Zhugan A respectively; precisely measuring the six reference substance stock solutions and diluting to the final concentration of 0.012 mg/mL, 0.041/mg/mL protocatechuic acid, 0.101/mg/mL epicatechin, 0.137/mg/mL quercetin, 0.077/mg/mL Bai Zhushu glycoside and 0.071 mgm/L Yunnan Bai Zhugan A; precisely measuring 100 mu L of final concentration stock solution of the six reference substances, mixing to obtain mixed reference substance solution, sequentially diluting with a 2-fold dilution method, and storing in an environment of-20 ℃ for later use.
4. The method for determining the content of various components in the garden balsam medicinal material according to claim 1, wherein the UPLC-MS/MS method is adopted, and the obtained data are obtained by using MasslynxTM V4.1 software and processed by using QuanlynaTM V4.1 workstation.
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