CN110715994B - Method for analyzing difference chemical components of spina date seed and spina date seed by using UHPLC-Q-Orbitrap MS - Google Patents
Method for analyzing difference chemical components of spina date seed and spina date seed by using UHPLC-Q-Orbitrap MS Download PDFInfo
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Abstract
The invention belongs to the technical field of traditional Chinese medicine analysis and traditional Chinese medicine quality control, and provides a method for analyzing different chemical components of spina date seed and spina date seed by using UHPLC-Q-Orbitrap MS, wherein medicinal powder of the spina date seed and the spina date seed is subjected to Soxhlet extraction and degreasing by petroleum ether, heated and refluxed by 70% ethanol, and filtered to obtain a sample solution; carrying out chemical component analysis on the sample solution by using UHPLC-Q-Orbitrap-MS, and carrying out peak matching, peak alignment, noise filtering and normalization on mass spectrum data by using Compound Discover software to obtain an Excel table containing the molecular weight, retention time and peak area of the Compound; and importing the compound peak area data matrix into Simca-p software for multivariate statistical analysis, and finding and identifying the different chemical components of the spina date seed and the spina date seed by combining an ROC curve. Discloses the material basis of the wild jujube kernel and the semen ziziphi spinosae, and provides reference for establishing a novel traditional Chinese medicine quality evaluation system.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine analysis and traditional Chinese medicine quality control, and particularly relates to a method for analyzing differential chemical components of spina date seed and spina date seed by using UHPLC-Q-Orbitrap MS, in particular to a method for analyzing differential chemical components of the spina date seed and the spina date seed by using a high-resolution mass spectrometry technology.
Background
The spina date seed is dry mature seed of Ziziphus jujuba Mill.var. spinosa Hu ex H.F.Chou in Ziziphus in Rhamnaceae, and the spina date seed is mature seed of Ziziphus mauritiana Lam in Ziziphus in the same family as the spina date seed, which is commonly called as "Ziziphus mauritianus seed", "Burkholda seed" or "imported spina date seed". Both of them have the traditional actions of tranquilizing mind and arresting sweating. The spina date seed recorded in Chinese pharmacopoeia of 2015 edition is sweet, sour and neutral; it enters liver, gallbladder and heart meridians. Nourish heart and tonify liver, calm heart and induce tranquilization, arrest sweating, promote fluid production. Can be used for treating vexation, insomnia, palpitation, dreaminess, asthenia, hyperhidrosis, body fluid deficiency, and thirst. The semen ziziphi spinosae is used as a traditional Chinese medicine in Yunnan province. The 2005 edition of the traditional Chinese medicine Standard of Yunnan province records that the spina date seed is sweet and flat; it enters heart and liver meridians. Has the effects of calming heart and tranquilizing mind, relieving restlessness and arresting sweating, and is used for treating vexation and insomnia, palpitation, dysphoria, sweating due to debility and the like. Along with the increase of life pressure, the number of people suffering from insomnia is continuously increased, and the clinical use frequency of the spina date seeds as a preferred traditional Chinese medicine for treating the insomnia is as high as 67.3%. In recent years, the market demand of the spina date seeds is continuously increased, wild resources are gradually shrunk, so that the price is continuously increased, and a large number of Yunnan and Burma natural jujube kernels are brought into the medicinal material market. Because the character characteristics of the wild jujube kernel and the Chinese date kernel are similar to the traditional efficacy, and the prices of the wild jujube kernel and the Chinese date kernel are different, the phenomenon that a large number of Chinese date kernels pretend to be the wild jujube kernel for sale is caused, and the wild jujube kernels and the Chinese date kernels are relatively disordered in the market and clinical application of traditional Chinese medicinal materials. For this reason, the clear chemical difference between the two will provide a solid experimental foundation for establishing scientific quality control standards.
Disclosure of Invention
The invention provides a method for analyzing different chemical components of wild jujube kernels and wild jujube kernels by using UHPLC-Q-Orbitrap MS in order to clarify the chemical difference between the wild jujube kernels and the wild jujube kernels.
The invention is realized by the following technical scheme: a method for analyzing difference chemical components of semen Ziziphi Spinosae and semen Ziziphi Spinosae by UHPLC-Q-Orbitrap MS comprises subjecting semen Ziziphi Spinosae and semen Ziziphi Spinosae medicinal powder to Soxhlet extraction with petroleum ether for defatting, extracting with 70% ethanol under heating and refluxing, and filtering to obtain sample solution; carrying out ultra-high performance liquid chromatography-quadrupole-electrostatic field orbit trap high resolution mass spectrum UHPLC-Q-Orbitrap-MS on sample solutions of spina date seed and spina date seed to analyze chemical components in the samples, and carrying out processing such as peak matching, peak alignment, noise filtering, normalization and the like on mass spectrum data through Compound Discover software to obtain an Excel table containing Compound molecular weight, retention time and peak area; and (3) introducing the data matrix containing the compound peak area into Simca-p software for multivariate statistical analysis, and finding and identifying the different chemical components of the spina date seed and the spina date seed by combining an ROC curve.
The method comprises the following specific steps:
(1) preparation of a sample: precisely weighing 2g of wild jujube seed sample powder of each batch, placing the wild jujube seed sample powder in Soxhlet extraction, adding petroleum ether for heating reflux, volatilizing a solvent from medicine residues, transferring the medicine residues to a round-bottom flask, adding 70% ethanol for heating reflux for 2 hours, filtering, washing the medicine residues with 70% ethanol, combining a washing solution and a filtrate, recovering the solvent until the solvent is dried, adding methanol to the residues for dissolving, transferring the residues to a 10mL volumetric flask, filtering through a 0.45 mu m filter membrane, and taking a subsequent filtrate to obtain a sample solution; respectively taking equal amount of solution from each sample solution, mixing uniformly, and placing in a liquid phase vial as quality control and QC sample;
(2) liquid chromatography-mass spectrometry conditions:
chromatographic conditions are as follows: thermo fisher U3000 ultra high performance liquid chromatograph with ACQUITY chromatographic column
An HSS T3 column with the thickness of 150mm multiplied by 2.1mm and the thickness of 1.8 mu m, a mobile phase of acetonitrile A and 0.1% formic acid water B, and a gradient elution program of 0-8 min and 5% → 17% A; 8-10 min, 17% A; 10-11 min, 17 → 18% A; 11-12 min, 18 → 20% A; 12-22 min, 20 → 33% A; 22-25 min, 33 → 100%; 25-27 min, 100 → 100% A; 27-30 min, 100 → 5% A; 30-35 min, 5% → 5%. The flow rate is 0.25 mL/min; the column temperature was 35 ℃; the sample injection amount is 3 mu L;
mass spectrum conditions: thermo ScientificTM Q ExactiveTMThe Orbitrap mass spectrometer Germany scans simultaneously with positive and negative ion switching using electrospray ionization source ESI. Spray voltage: 3.5KV (+), 2.5KV (-); sheath gas flow rate: 35 arb; auxiliary gas flow rate Aux gas flow rate: 10 arb; capillary temperature Capillary temperatureAnd (5) percent: 320 ℃; mass spectrometry scan mode: the scanning range of full scan mode full scan is 100-1500 m/z, the resolution is 70000, and the in-source induced collision cracking voltage is set to be 0 eV; MS/MS scanning mode: using data dependent scanning dd-ms2Resolution 17500, using NCE gradient energy;
(3) differential chemical composition analysis: calculating the detected raw of the spina date seed and the spina date seed in different batches by using Compound Discover software to obtain an Excel table of the metabolite peak area, and introducing Simca-p 13.0 to perform PCA analysis, PLS-DA analysis, OPLS-DA analysis and s-plot analysis; binding to a VIP value greater than 1, identifying 40 differential chemical components;
(4) performing clustering heat map analysis on the 40 differential chemical components;
(5) performing relative quantitative analysis on the 40 different chemical components;
(6) and (3) verifying the effectiveness of the differential chemical components by using an area AUC value under the ROC curve, and carrying out ROC curve analysis on 40 differential chemical components.
The petroleum ether added in the step (1) is in a boiling range of 60-90 ℃ and is soaked for 8 hours in 90 mL.
The mass spectrum scanning mode adopted in the step (2) is data dependent scanning DDA, and the NCE gradient energy is 15, 35 and 50.
Identifying the differential chemical components in the step (3), wherein the compound with the reference substance is identified according to the retention time, the parent ion and the secondary fragment information; and (3) comparing the parent ion and secondary fragment information of the Compound to identify the Compound without a reference substance, and referring to the matching result of the Compound Discover software.
In the step (6), the AUC values of the 40 differential chemical components are all larger than 0.92, and the AUC values are used as the marked differential chemical components for distinguishing the spina date seed from the spina date seed.
In the step (3), 40 different chemical components of the spina date seed and the spina date seed are identified, wherein the chemical components are 2 saponins, 19 flavones and 19 alkaloids.
The content of saponin components represented by the 2 saponins A and B in the spina date seeds is high;
of the 19 flavones: 4 flavonoid components 6 '(S) -O- (3-glc-indole-acetyl) spinosin, 6' (R) -O- (3-glc-indole-acetyl) -spinosin, nicotiflorin and camelliaside B are high in content in the spina date seeds; the remaining 15 flavone components, namely spinosin, 6 '-silallylspinosin, 6' -p-glycosylspinosin, 6 '- (3', 4', 5' -trimethoxyl) -cinnamylspinosin, isovitexin-2 '-O-beta-D-glucopyranoside, 6' -dihydrophaseylsporin, 6 '-phaseolyspinosin, 6' - (4 '-O-beta-D-glucopyranoside, 6' - (4 '-O-beta-D-glucopyranosyl) -benzoxystrobin, isoascosin-2' -O- (6-carboxylyl) -glucopyranoside, 6 '- (4' -O-beta-D-glucopyranoside) -vanillyloxystrobin, 6 '-p-hydroxysilyl,' -N '-3' -3 '-ethyl-2' ethyl-beta-D-glucopyranoside, 'beta-D-glucopyranosyl) -vanillyloxystrobin, 6' beta-3 'beta-D-3' -glucopyranoside, The content of epi-6 '- (N-beta-D-glucopyranosyl) -2', 3 '-dihydro-2' -oxo-3 '-yl-acetate spinosin, vicenin II and 6' -ferroylsporin in the jujube kernel is high;
of 19 alkaloids: the content of 7 alkaloid components, namely, magnoflorine, 3R-N-glc-3-hydroxy-indole acid, lotusine B, ramosine A, caaverine, asimilobine and noruciferine, in the spina date seed is high; the remaining 12 alkaloids, sanjoin A, lotusanine A, sanjoin B, sanjoin G2, C-11 epicer of sanjoin G1, sanjoin F, sanjoin G1, amphenidine D, mucronine J, norrisorbidine, nuciferine and n-metaminimobine, are high in the jujube kernel.
Compared with the prior art, the invention has the following advantages: 1. applying UHPLC-Q-Orbitrap-MS high-resolution mass spectrometry technology to provide accurate molecular mass of the compound and accurately identify chemical components in the spina date seed and the spina date seed; 2. by means of high-throughput sample analysis capability and multivariate statistical analysis of plant metabonomics technology, the difference chemical components of the spina date seed and the spina date seed are found out through qualitative and relative quantitative analysis; 3. and (4) verifying the effectiveness of the differential chemical components according to the AUC value of the area under the ROC curve. The invention discloses the two different chemical substance bases and provides reference for establishing a comprehensive and accurate traditional Chinese medicine quality evaluation system.
Drawings
FIG. 1 is a Base Peak Ion (BPI) graph of spine date kernels and spine date kernels;
FIG. 2 is a PCA plot of spine date seed, spine date seed and QC samples; in the figure: a positive ion mode; b, negative ion mode;
FIG. 3 is a plot of PLS-DA, Permutation, OPLS-DA and s-plot of spina date seed and spina date seed samples in positive ion mode;
FIG. 4 is a plot of PLS-DA, Permutation, OPLS-DA and s-plot of spina date seed and semen Ziziphi Spinosae samples in negative ion mode;
FIG. 5 clustering chart of the differential chemical composition of spine date seed and spine date seed
FIG. 6 is a graph showing the relative contents of different chemical components in Ziziphus jujuba Mill and Ziziphus jujuba Mill (white is saponin, gray is flavone, and black is alkaloid).
Detailed Description
The method for analyzing the chemical difference between the spina date seed and the spina date seed based on the UHPLC-Q-Orbitrap-MS technology is further described with reference to the following specific examples, but the scope of the invention is not limited thereto.
Example 1: the method for establishing UHPLC-Q-Orbitrap-MS analysis of the spina date seed and the spina date seed comprises the following steps:
and (3) collecting a sample in the step (1). The method comprises the following specific steps: the spina date seed used in the experiment is identified to be dry mature seed of Ziziphus jujuba Mill.var.spinosa (Bunge) Hu ex H.F.Chou of Rhamnaceae plant by assistant professor Duchenghui of Shanxi traditional Chinese medicine university; the semen Ziziphi Spinosae is dry mature seed of Ziziphus mauritiana Lam. of Rhamnaceae. A total of 16 batches of wild jujube kernel samples and 13 batches of structured jujube kernel samples are collected and stored in the modern research center of traditional Chinese medicine of Shanxi university, and the detailed information is shown in Table 1.
Table 129 batch sample information table
| Numbering | Sample (I) | Producing area | Purchasing place | Time of |
| 1 | Wild jujube seed | Hebei river | Anguo medicinal material market | 2017.08 |
| 2 | Wild jujube seed | Hebei river | Anguo medicinal material market | 2017.08 |
| 3 | Wild jujube seed | Hebei river | Anguo medicinal material market | 2017.08 |
| 4 | Wild jujube seed | Shandong (mountain east) | Anguo medicinal material market | 2017.08 |
| 5 | Wild jujube seed | Shanxi province | Anguo medicinal material market | 2017.08 |
| 6 | Wild jujube seed | Shanxi province | Anguo medicinal material market | 2017.08 |
| 7 | Wild jujube seed | Shanxi province | Anguo medicinal material market | 2017.08 |
| 8 | Wild jujube seed | Shanxi province | Anguo medicinal material market | 2017.08 |
| 9 | Wild jujube seed | Gansu | Anguo medicinal material market | 2017.08 |
| 10 | Wild jujube seed | Hebei river | Anguo medicinal material market | 2017.08 |
| 11 | Wild jujube seed | Shandong (mountain east) | Anguo medicinal material market | 2017.08 |
| 12 | Wild jujube seed | Shandong (mountain east) | Anguo medicinal material market | 2017.08 |
| 13 | Wild jujube seed | Shanxi province | Medicine for promoting new country | 2017.10 |
| 14 | Wild jujube seed | Liaoning medicine | Zanhuang capsule | 2017.11 |
| 15 | Wild jujube seed | Hebei river | Yulin medicinal material market | 2017.04 |
| 16 | Wild jujube seed | Hebei river | Yulin medicinal material market | 2017.04 |
| 17 | Lizi kernel | Guizhou province | Guizhou province | 2016.10 |
| 18 | Lizi kernel | Guizhou province | Guizhou province | 2016.10 |
| 19 | Lizi kernel | Yunnan province | Chrysanthemum medicinal material market | 2016.11 |
| 20 | Lizi kernel | Yunnan province | Yulin medicinal material market | 2016.04 |
| 21 | Lizi kernel | Yunnan province | Kunming (a Chinese herbal medicine) | 2017.08 |
| 22 | Lizi kernel | Yunnan province | Kunming (a Chinese herbal medicine) | 2017.08 |
| 23 | Lizi kernel | Burma | Kunming (a Chinese herbal medicine) | 2017.08 |
| 24 | Lizi kernel | Burma | Kunming (a Chinese herbal medicine) | 2017.08 |
| 25 | Lizi kernel | Burma | Anguo medicinal material market | 2017.08 |
| 26 | Lizi kernel | Burma | Anguo medicinal material market | 2017.08 |
| 27 | Lizi kernel | Burma | Anguo medicinal material market | 2017.08 |
| 28 | Lizi kernel | Burma | Anguo medicinal material market | 2017.08 |
| 29 | Lizi kernel | Burma | Anguo medicinal material market | 2017.08 |
And (2) preparing a reference substance. 24 reference substances, namely, lindera aggregate, magnoflorine, veegum, spicinetin, swertisin, kaempferol-3-O-rutinoside, 6' -feruloyl spicinetin, spinasaponin A, spinasaponin B, betulinic acid, betulin, american tea acid, adenosine, vitexin, isovitexin, apigenin, naringin, quercetin, kaempferol, hesperetin, genkwanin, genistein, isorhamnetin and puerarin are purchased from Guanguan Biotechnology limited company, Virgine Inc., Sichuan province and Chengdui-Fengsi Biotechnology Limited company respectively.
And (3) preparing a reference substance and a sample. The method comprises the following specific steps: precisely weighing appropriate amount of each reference substance, adding methanol to obtain reference substance stock solution with corresponding concentration, mixing the reference substance stock solutions, and adding methanol to obtain corresponding mixed reference substance solution.
Precisely weighing about 2g of wild jujube seed sample powder (screened by a No. four sieve) of each batch, placing the wild jujube seed sample powder into a Soxhlet extractor, adding 90mL of petroleum ether (60-90 ℃), heating and refluxing for 4 hours, volatilizing the solvent in the dregs, transferring the dregs to a round-bottom flask, adding 40mL of 70% ethanol, heating and refluxing for 2 hours, filtering, washing the dregs with 5mL of 70% ethanol, combining the washing solution and the filtrate, recovering the solvent to be dry, dissolving the residue with methanol, transferring the residue to a 10mL volumetric flask, filtering with a 0.22 mu m filter membrane, and taking the subsequent filtrate to obtain a sample solution. Respectively taking equal amount of solution from each sample solution, mixing uniformly, placing in a liquid phase small bottle as a Quality Control (QC) sample, continuously performing QC sample investigation instrument precision for 6 times before analysis, and performing QC sample operation every 8 samples after sample injection.
And (4) performing liquid chromatography-mass spectrometry analysis. The method comprises the following specific steps:
instruments and reagents: thermo fisher U3000 ultra-high performance liquid chromatographAn on-line degasser, a quaternary gradient pump, a column oven, an ultraviolet detector, and an autosampler (Thermo Fisher Scientific Co., USA) are providedTM Q ExactiveTMOrbitrap mass spectrometer (Germany); one hundred thousand analytical balances model CPA225D, Sartorius beijing instrument systems ltd, germany; vacuum centrifugal concentrator, Eppendorf, germany; xcalibur software (version 3.0) software; compound Discover (3.0) software. Acetonitrile and formic acid were both obtained from Fisher, and methanol was obtained from merck. Other reagents were analytically pure.
Chromatographic conditions are as follows: the chromatographic column is
HSS T3 column (150mm × 2.1mm, 1.8 μm), mobile phase acetonitrile (A), 0.1% formic acid (B), gradient elution program of 0-8 min, 5% → 17% A; 8-10 min, 17% A; 10-11 min, 17 → 18% A; 11-12 min, 18 → 20% A; 12-22 min, 20 → 33% A; 22-27 min, 33 → 100%; 27-29 min, 100 → 100% A; 29-32 min, 100 → 5% A; 32-35 min, 5% → 5%. The flow rate is 0.25 mL/min; the column temperature was 35 ℃; the amount of sample was 3. mu.L.
Mass spectrum conditions: the positive and negative ions are switched and scanned simultaneously using an electrospray ionization source (ESI). Spray voltage (Spray voltage): 3.5KV (+), 2.5KV (-); sheath gas flow rate (Sheath gas flow rate): 35 arb; auxiliary gas flow rate (Aux gas flow rate): 10 arb; capillary temperature (Capillary temperature): 320 ℃; mass spectrometry scan mode: the scanning range of a full scan mode (full scan) is 100-1500 m/z, the resolution is 70000, and the induced collision cracking voltage in a source is set to be 0 eV; MS/MS scanning mode: using data dependent scanning (dd-ms)2) Resolution 17500, NCE energy set to 15eV, 35eV, and 50eV, respectively.
And (6) processing data of the Compound Discover software. The method comprises the following specific steps: introducing raw data (raw files) of 16 batches of spina date seeds, 13 batches of jujube kernels, 10 QC samples and blank methanol into Compound Discover software, deconvoluting chromatographic peaks, denoising, smoothing, correcting baselines and aligning the peaks. And compounds are matched by databases of Mass Band, Nature Chemistry, Nist, Plant Metabolic Network, Planta Pilot de Quimica Fina, plantaCyc, PubMed, Royal Society of Chemistry, RSC least Chemistry Wiki, Springer Nature, Web of science, Wikipedia. And respectively calculating the original data in a positive and negative ion mode to obtain an Excel data table containing the molecular formula, retention time and peak area of the compound.
Example 2: the method for analyzing the wild jujube kernel and the wild jujube kernel by adopting a chemometric method comprises the following steps:
and (3) inspecting the precision of the instrument in the step (1). The method comprises the following specific steps: example 1 an Excel table was obtained in step (6), and a peak area matrix of 16 spine date seeds, 13 spine date kernels and 10 QC samples in the table was introduced into Simca-p (13.0) software to perform Principal Component Analysis (PCA). In both positive and negative ion mode, QC samples were tightly packed together, as shown in fig. 2, indicating that the instrument was well-defined during the experiment.
And (2) PCA analysis. The method comprises the following specific steps: the peak area matrix of 16 spine date kernels and 13 spine date kernel samples in the Excel table in the step (6) of example 1 was introduced into Simca-p (13.0) software for principal component analysis. The results show that R of the PCA model2X＝0.86，Q2The result is 0.95, which shows that the model is stable and has strong prediction capability. As shown in FIGS. 3A and 4A, the spina date seed and the spina date seed can be obviously separated in the positive and negative ion mode, and can be self-polymerized into one type, which shows that the chemical difference between the spina date seed and the spina date seed is obvious.
And (3) analyzing the OPLS-DA and s-plot. The method comprises the following specific steps: in order to screen out the different components of the spina date seed and the spina date seed, supervised PLS-DA and OPLS-DA are used for analyzing the data. First, PLS-DA analysis was performed, and in order to further verify the reliability of the model, the corresponding PLS-DA model in FIGS. 3B and 4B was verified by a Permutation experiment (Permutation). The accumulated contribution rate R is obtained by randomly changing the arrangement sequence of the category variables Y for 200 times2And predictive power Q2. In which R of the experimental model is arranged in positive ion mode2Regression line and Q2The intercepts of the regression line and the vertical axis are respectively 0.474 and-0.483, and the experimental model is arranged in a negative ion modeR2Regression line and Q2The intercepts of the regression line and the vertical axis are respectively 0.474 and-0.483, and the rightmost end identifies the original Q of the model2Q of value greater than any of the left Y variables of the random permutation model2And the values (3B and 4B) show that the constructed PLS-DA discrimination model has no overfitting phenomenon, has good prediction capability and is reliable in display model. Further performing OPLS-DA analysis on the data, as shown in FIGS. 3C and 4C, the spina date seed and the semen ziziphi spinosae are clearly separated in the positive and negative ion mode, suggesting that the chemical components of the spina date seed and the semen ziziphi spinosae are significantly different. To find out these differential components, the overall differences of the two were further analyzed, and scatter plot analysis was performed, making s-plot plots (see FIGS. 3D and 4D). Each point on the S-curve in the figure represents a compound, and the components at the two ends of the curve are the components with larger difference. The contribution degree of the variables is described by a common variable load evaluation parameter (VIP) value, the variable with VIP & gt 1 is used as a characteristic variable, the different chemical components of the spine date seed and the spine date seed are found, and 431 difference variables are found.
Example 3: the identification of the differential chemical components comprises the following steps:
the method comprises the following specific steps: identifying the compound with the reference substance according to the retention time, the parent ion and the secondary fragment information; the Compound without the reference substance is firstly identified by comparing the parent ion and the secondary fragment information of the Compound according to the literature, and then the matching result of Compound Discover software is referred to. Excel tables including molecular weight, retention time, peak area and online database matched compounds were obtained using Compound Discover software. A total of 40 differential chemical components, 2 saponins, 19 flavonoids and 19 alkaloid components, were identified from 431 differential variables according to this method, see table 2.
Example 4: the differential metabolism relative content analysis comprises the following steps:
step (1) clustering heat map analysis of the differential chemical components. The method comprises the following specific steps: and (3) introducing the 40 differential chemical components identified in the step (4) into a MetabioAnalyst website for clustering heat map analysis (figure 5). Each column in the figure represents a sample and each pixel represents a metabolite. The color of each pixel point is transited from blue to red, wherein the color represents the relative content of the metabolites. Red indicates a higher level and blue indicates a lower level. Hierarchical clustering analysis shows that all spina date seeds are gathered into one group, and all spina date seeds are gathered into one group, so that the remarkable difference exists between the spina date seeds and the spina date seeds, and the screened remarkable different chemical components can be used as markers to obviously separate the two groups. FIG. 5 shows that the content of saponin components represented by jujuubide A and jujujuubide B in wild jujube seeds is high; 4 of the 19 flavone components have higher relative content in the spina date seeds, and 15 flavone components have higher relative content in the semen ziziphi spinosae; 7 of the 19 alkaloid components have relatively high content in the spina date seed, and 12 of the 19 alkaloid components have relatively high content in the semen ziziphi spinosae. This shows that the saponin component has a relatively high content in the semen Ziziphi Spinosae, and most of the flavone and alkaloid components have a relatively high content in the semen Ziziphi Spinosae.
And (2) carrying out relative quantitative analysis on the differential chemical components. The method comprises the following specific steps: the relative content analysis was performed by taking the normalized peak areas of 40 different chemical components of the spina date seed and the spina date seed in the step (6) of example 1 as a histogram (fig. 6). Figure 6 shows that both flavone and alkaloid content are significantly higher than saponin content. jujudioside A and jujujudioside B are contained in wild jujube seeds at high content. 4 flavonoid components 6' (S) -O- (3-glc-indole-acetyl) spinosin, 6' (R) -O- (3-glc-indole-acetyl) spinosin, nicotiflorin and camelliside B are contained in wild jujube, and the other 15 flavonoid components spinosin, 6' -silaspinosin, 6' -p-coumaryl spinosin, 6' - (3', 4', 5' -trimethoxyl) -cinnamylspinosin, isovitexin-2' -O-beta-D-glucopyranoside, 6' -dihydrosecoglycosylsin, 6' - (-) -phaseolyspinosin, 6' -O-beta-D-glucopyranoside, 6' -dihydrosecoglycosylsaponin, 6' - (4' -O-beta-D-glucopyranosyl) -spinosin, isoeugenyl-2 ' -6' -beta-D-glucopyranoside, 2' -beta-D-glucopyranoside-4 ' -glucopyranosin, and 6' -glucopyranoside-beta-4 ' -O-beta-D-glucopyranoside, 6 '-p-hydroxybenzoxysporin, 6' - (N-beta-D-glucopyranosyl) -2', 3' -dihydro-2 '-oxo-3' -yl-acetate spinosin, epi-6 '- (N-beta-D-glucopyranosyl) -2', 3 '-dihydro-2' -oxo-3 '-yl-acetate spinosin, vicenin II and 6' -ferroxystrobin are all contained in the jujube kernel in higher content. The content of 7 alkaloid components, namely, magnoflorine, 3R-N-glc-3-hydroxy-indenoceacetic acid, lotusine B, ramosine A, caaverine, asimilobine and norluciferine, in spina date seed is higher, and the content of the rest 12 alkaloids, namely, sanjoine A, lotusaine A, sanjoine B, sanjoine G2, C-11 injector of sanjoine G1, sanjoine F, sanjoine G1, amphenib D, mucronine J, norrisorbidine, nuciferine and N-methyisimobinine, in spina date seed is higher.
Example 5: ROC curve analysis comprising the steps of:
and (4) verifying the effectiveness of the differential chemical components by using an area AUC value under the ROC curve. The method comprises the following specific steps: receiver operating characteristic curve (ROC curve) is widely used in medicine as a statistical tool for describing diagnostic accuracy. In metabolomics data analysis, ROC curves are often used to assess the effectiveness of metabolites as a means of identifying different sets of samples. The area under the ROC curve (AUC) value is typically used as an indicator of prediction accuracy. It is generally believed that the closer the AUC is to 1, the higher the diagnostic accuracy. When AUC is more than 0.75, the judgment method is high in accuracy. When AUC is less than or equal to 0.5, this diagnosis is of no value. The research adopts the area under the ROC curve to verify the effectiveness of the difference chemical components of the spina date seeds and the spina date seeds.
As shown in table 2, the areas under the ROC curves of 40 different chemical components of the spina date seed and the spina date seed are both greater than 0.92, which indicates that the 40 different chemical components can be used as the marked different chemical components for distinguishing the spina date seed from the spina date seed.
TABLE 2 Compound information identified based on UHPLC-Q-Orbitrap-MS technique
Note: AUC is the area under the ROC curve AUC value.
Claims (3)
1. A method for analyzing the difference chemical components of wild jujube kernel and wild jujube kernel by UHPLC-Q-Orbitrap MS is characterized in that: carrying out Soxhlet extraction and degreasing on the wild jujube kernel and the wild jujube kernel medicinal material powder by using petroleum ether, carrying out heating reflux extraction by using 70% ethanol, and filtering to obtain a sample solution; carrying out ultra-high performance liquid chromatography-quadrupole-electrostatic field orbit trap high resolution mass spectrum UHPLC-Q-Orbitrap-MS on sample solutions of spina date seed and spina date seed to analyze chemical components in the samples, and carrying out peak matching, peak alignment, noise filtering and normalization on mass spectrum data through Compound Discover software to obtain an Excel table containing Compound molecular weight, retention time and peak area; importing a data matrix containing compound peak areas into Simca-p software for multivariate statistical analysis, and finding and identifying the different chemical components of the spina date seed and the spina date seed by combining an ROC curve;
the method comprises the following specific steps:
(1) preparation of a sample: precisely weighing 2g of wild jujube seed sample powder of each batch, placing the wild jujube seed sample powder in Soxhlet extraction, adding petroleum ether for heating reflux, volatilizing a solvent from medicine residues, transferring the medicine residues to a round-bottom flask, adding 70% ethanol for heating reflux for 2 hours, filtering, washing the medicine residues with 70% ethanol, combining a washing solution and a filtrate, recovering the solvent until the solvent is dried, adding methanol to the residues for dissolving, transferring the residues to a 10mL volumetric flask, filtering through a 0.45 mu m filter membrane, and taking a subsequent filtrate to obtain a sample solution; respectively taking equal amount of solution from each sample solution, mixing uniformly, and placing in a liquid phase vial as a quality control QC sample; adding 90mL of petroleum ether with a boiling range of 60-90 ℃ for soaking for 8 hours;
(2) liquid chromatography-mass spectrometry conditions:
chromatographic conditions are as follows: the method comprises the following steps of (1) performing a Thermo fisher U3000 ultra-high performance liquid chromatograph, wherein a chromatographic column is an ACQUITY UPLC HSS T3 column, the thickness of the chromatographic column is 150mm multiplied by 2.1mm and 1.8 mu m, a mobile phase is acetonitrile A and 0.1% formic acid water B, a gradient elution program is 0-8 min, and the concentration of 5% → 17% A; 8-10 min, 17% A; 10-11 min, 17 → 18% A; 11-12 min, 18 → 20% A; 12-22 min, 20 → 33% A; 22-25 min, 33 → 100%; 25-27 min, 100 → 100% A; 27-30 min, 100 → 5% A; 30-35 min, 5% → 5%;
the flow rate is 0.25 mL/min; the column temperature was 35 ℃; the sample introduction amount is 3 muL;
mass spectrum conditions: thermo ScientificTM Q ExactiveTMAn Orbitrap mass spectrometer, using an electrospray ionization source ESI, scanning positive and negative ions simultaneously;
spraying voltage: +3.5 KV, -2.5 KV; flow rate of sheath gas: 35 arb; flow rate of auxiliary gas: 10 arb; capillary temperature: 320 ℃; mass spectrometry scan mode: the scanning range of the full scanning mode is 100-1500 m/z, the resolution is 70000, and the in-source induced collision cracking voltage is set to be 0 eV; MS/MS scanning mode: using data dependent scanning dd-ms2Resolution 17500, using NCE gradient energy; the adopted mass spectrum scanning mode is data dependent scanning DDA, and NCE gradient energy is 15, 35 and 50;
(3) differential chemical composition analysis: calculating the detected raw of the spina date seed and the spina date seed in different batches by using Compound Discover software to obtain an Excel table of the peak area of the Compound, and introducing Simca-p 13.0 to perform PCA analysis, PLS-DA analysis, OPLS-DA analysis and s-plot analysis; binding to a VIP value greater than 1, identifying 40 differential chemical components; identifying the differential chemical components, namely identifying the compound with a reference substance according to retention time, parent ions and secondary fragment information; the Compound without the reference substance is characterized by firstly comparing parent ions and secondary fragment information of the Compound to identify the Compound and then referring to the matching result of Compound Discover software;
(4) performing clustering heat map analysis on the 40 differential chemical components;
(5) performing relative quantitative analysis on the 40 different chemical components;
(6) verifying the effectiveness of the differential chemical components by using an area AUC value under an ROC curve, and carrying out ROC curve analysis on 40 differential chemical components; AUC values of 40 differential chemical components are all larger than 0.92, and the AUC values are used as the marked differential chemical components for distinguishing the spina date seed and the spina date seed.
2. The method of claim 1, wherein the UHPLC-Q-Orbitrap MS is used to analyze the differential chemical composition of spine date kernels and spine date kernels, wherein: identifying 40 different chemical components of the spina date seed and the spina date seed in the step (3), wherein the different chemical components comprise 2 saponins, 19 flavones and 19 alkaloids;
the 2 saponins are jujujuboside A and jujujuboside B, and the content of the saponins in the spina date seeds is high;
of the 19 flavones: the content of 4 flavonoid components 6'' (S) -O- (3-glc-indole-acetyl) spinosin, 6'' (R) -O- (3-glc-indole-acetyl) spinosin, nicotiflorin and camelliaside B in the spina date seed is high; the remaining 15 flavone components, spinosin, 6'' '-sinapoylsporin, 6' '' -p-glycosylspinosin, 6'' '- (3' '',4'' ',5' '' -trimethoxyl) -cinmannosylspinosin, isovitexin-2'' -O-beta-D-glucopyranoside, 6'' '-dihydrophaseylsporin, 6' '' -phaseylsporinin, 6'' '- (4' '' -O-beta-D-glucopyranosyl) -xylospinosin, isoascogenin-2 '' -O- (6 '' -carboxyloyl) -glucopyranoside, 6'' '- (4' '' -O-beta-D-glucopyranosyl) -vanillyl-glucopyranoside, 6'' '-O-beta-D-glucopyranosyl-glucopyranosin, 6' '-hydroxysuccinimide, 6' '-beta-D-glucopyranosyl-glucopyranosin, 6' '-beta-D-glucopyranosyl-glucopyranosin, 6' '-beta-2' -glucopyranosin, 6'' -beta-D-glucopyranosyl-2-beta-D-glucopyranosin, 3' ' ' -dihydro-2' ' ' ' -oxo-3' ' ' -yl-acetate saponin, epi-6' ' ' - (N-beta-D-glucopyranosyl) -2' ' ',3' ' ' -dihydro-2' ' ' ' -oxo-3' ' ' -yl-acetate saponin, vicenin II and 6' ' ' -fructoylsaccharin are high in the jujube kernel;
of 19 alkaloids: the content of 7 alkaloid components, namely, magnoflorine, 3R-N-glc-3-hydroxy-indole acid, lotusine B, ramosine A, caaverine, asimilobine and noruciferine, in the spina date seed is high; the remaining 12 alkaloids, sanjoin A, lotusanine A, sanjoin B, sanjoin G2, C-11 epicer of sanjoin G1, sanjoin F, sanjoin G1, amphenidine D, mucronine J, norrisorbidine, nuciferine and n-metaminimobine, are high in the jujube kernel.
3. Use of the method of claim 1 or 2 for quality control of spine date seeds.
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