CN110715994A - 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 differential metabolites 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 semen Ziziphi Spinosae is wild jujube of Zizyphus of RhamnaceaeZiziphus jujubaMill. var.spinosaDried mature seed of (Bunge) Hu ex H.F. Chou, the kernel of Zizyphus jujube is Zizyphus spinosus of the same family as Zizyphus vulgarisZiziphus mauritianaLam. mature seed, commonly known as "Yunnan date seed", "Burma date seed" or "imported wild jujube 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-OrbitrapMS (ultra high performance liquid chromatography-orbital-polymerase chain reaction) 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 a data matrix containing compound peak areas into Simca-p software for multivariate statistical analysis, and finding and identifying the differential metabolites 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 2 g 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 10 mL 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: 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 HSST3 column, 150 mm multiplied by 2.1 mm 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 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 is 35 ℃; the sample introduction amount is 3 muL;
mass spectrum conditions: thermo ScientificTMQ ExactiveTMThe Orbitrap mass spectrometer Germany scans simultaneously with positive and negative ion switching using electrospray ionization source ESI. Spray voltage: 3.5 KV (+), 2.5 KV (-); sheath gas flow rate: 35 arb; auxiliary gas flow rate Aux gas flow rate: 10 arb; capillary temperature Capillary temperature: 320 ℃ C; 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 metabolite 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 metabolites;
(4) performing clustering heat map analysis on 40 differential metabolites;
(5) performing relative quantitative analysis on 40 differential metabolites;
(6) and (3) verifying the effectiveness of the differential metabolites by using an area AUC value under the ROC curve, and carrying out ROC curve analysis on 40 differential metabolites.
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 metabolites in the step (3), wherein the compounds with the reference substances are identified according to the retention time, the parent ions 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 metabolites are all larger than 0.92, and the AUC values are used as the marker differential metabolites for distinguishing the spina date seed from the spina date seed.
In the step (3), 40 different metabolites of the spina date seed and the spina date seed are identified, wherein the metabolites 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: the content of 4 flavonoid components 6'' (S) -O- (3-glc-indole-acetyl) spinosin, 6'' (R) -O- (3-glc-indole-acetyl) -spinosin, nicotinoid 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, amphenib D, mucronine J, norrisorbidine, nuciferine and n-methylasimimobine, are high in the wild jujube seed.
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, differential metabolites 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 metabolite according to an area AUC value 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 heatmap of different metabolites of spine date seed and spine date seed
FIG. 6 is a relative metabolite content chart of semen Ziziphi Spinosae and semen Ziziphi Spinosae (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 experimental spina date seed is identified as the Rhamnaceae plant spina date seed by assistant professor of Shanxi university of traditional Chinese medicine and morning gloryZiziphus jujubaMill. var.spinosaDried mature seeds of (Bunge) Hu ex h.f.chou; the semen Ziziphi Spinosae is Rhamnaceae plant fructus ChoerospondiatisZiziphus mauritianaDried mature seed of Lam. 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
And (2) preparing a reference substance. Purchasing 24 reference substances respectively from Chenguan Biotechnology GmbH, Wei-ke-Qi Biotechnology GmbH, and Chengdu-Feng-Si Biotechnology GmbH of Baoji City, including lindera aggregate, magnoflorine, Weicaining, spilanthol, swertisin, kaempferol-3-O-rutinoside, 6' ' ' -feruloylscinolone, spina date seed saponin A, spina date seed saponin B, betulinic acid, betulin, american tea acid, adenosine, vitexin, isovitexin, apigenin, naringin, quercetin, kaempferol, hesperetin, genkwanin, genistein, isorhamnetin and puerarin.
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 2 g 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 90 mL 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 40 mL of 70% ethanol, heating and refluxing for 2 hours, filtering, washing the dregs with 5 mL 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 10 mL 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: a Thermo Fisher U3000 ultra-high performance liquid chromatograph which is provided with an online degasser, a quaternary gradient pump, a column incubator, an ultraviolet detector and an automatic sample injector (Thermo Fisher Scientific company, USA), and Thermo ScientificTMQ ExactiveTMOrbitrap mass spectrometer (Germany); cOne hundred thousand analytical balances model PA225D, Sartorius Beijing Instrument systems, Inc. of 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 an ACQUITY UPLC HST 3 column (150 mm multiplied by 2.1 mm, 1.8 mu m), the mobile phase is acetonitrile (A) and 0.1% formic acid water (B), and the gradient elution program is 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; 22-27 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 sample size is 3 muL.
Mass spectrum conditions: the positive and negative ions are switched and scanned simultaneously using an electrospray ionization source (ESI). Spray voltage (Sprayvoltage): 3.5 KV (+), 2.5 KV (-); 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 in-source induced collision cracking voltage is set to be 0 eV; MS/MS scanning mode: using data dependent scanning (dd-ms)2) Resolution 17500, NCE energy set to 15 eV, 35 eV, and 50 eV, 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 MassBand, Nature Chemistry, Nist, Plant Metabolic Network, Planta Pilot de Quinica 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. Results show that of the PCA modelR 2 X=0.86，Q 2 And =0.95, the model is stable and has strong prediction capability. As shown in fig. 3A and 4A, the zizyphus jujube kernel and the zizyphus jujube kernel can be clearly separated in the positive and negative ion mode, and can self-aggregate into one type, which shows that the chemical difference between the two 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). By randomly changing the class variable 200 timesYObtaining the cumulative contribution rateR 2 And predictive capabilityQ 2 . In which experimental models are arranged in positive ion modeR 2 Regression line andQ 2 the 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 modeR 2 Regression line andQ 2 the intercepts of the regression line and the vertical axis are respectively 0.474 and-0.483, and the rightmost end identifies the original of the modelQ 2 With values greater than any of the left-hand random-arranged models of the Y variablesQ 2 And 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 a variable is described by a common variable load evaluation parameter (VIP) value, a variable with VIP & gt 1 is used as a characteristic variable, differential metabolites of spina date seed and spina date seed are found, and 431 differential variables are found in total.
Example 3: identification of differential metabolites comprising the steps of:
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 metabolites, 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) differential metabolite clustering heatmap analysis. The method comprises the following specific steps: introducing the 40 differential metabolites identified in step (4) into the MetabioAnalyst website for clustering heatmap analysis (FIG. 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 class, and all spina date seeds are gathered into one class, so that the spina date seeds and the spina date seeds have obvious difference, and screened obvious difference metabolites 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 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 metabolites. The method comprises the following specific steps: the relative content analysis was performed by taking the normalized peak areas of 40 different metabolites of spine date seed and spine date seed in 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' ' ' -sinapoxysporin, 6' ' ' -p-coumalic spinosin, 6' ' ' - (3' ' ',4' ' ',5' ' ' -trimethoxyl) -cindamycin, isovitexin-2' ' -O-beta-D-glucopyranoside, 6' ' ' -dihydrosecospinosin, 6' - (phaseosylvin, 6' -O-beta-D-glucopyranoside, 6' ' ' -dihydrosecoglycosylsaponin, 6' -4' -O-beta-D-glucopyranosin-2-glucopyranosyl-2-glucopyranosin-6 ' ' -pyrido-glycoside-6 ' ' -beta-glucopyranosin, 6' -pyridoxystrobin-2 ' ' -beta-D-glucopyranoside, 6' -beta-glucopyranosin-2-beta-D-glucopyranoside, 6' ' -beta-D-glucopyranosin, 6' ' -beta-2-D-glucopyranoside, 6' ' -beta-D-2-D-glucopyranoside, 6' -beta-D, 6'' '- (4' '' -O- β -D-glucopyranosyl) -vanillyl spinosin, 6'' '-p-hydroxybenzoyl spinosin, 6' '' - (N- β -D-glucopyranosyl) -2'' ',3' '' -dihydro-2'' '' '-oxo-3' '' -yl-acetate spinosin, epi-6'' '- (N- β -D-glucopyranosyl) -2' '',3'' '-dihydro-2' '' -oxo-3'' '-yl-acetate spinosin, vicenin II and 6' '' -ferroxystrobin are each present in the kernel at a relatively high level. The content of 7 alkaloid components, namely, magnoflorine, 3R-N-glc-3-hydroxy-indenoceacetic acid, lotusine B, ramosine A, caaverine, asimilobine and norlucucine, in the spina date seed is higher, and the content of the other 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-methyisimilorine, in the spina date seed is higher.
Example 5: ROC curve analysis comprising the steps of:
and (4) verifying the effectiveness of the differential metabolites 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 effectiveness of the different metabolites of the spina date seeds and the spina date seeds is verified by the area under the ROC curve.
As shown in Table 2, the areas under the ROC curves of the 40 differential metabolites of the spina date seed and the spina date seed are both greater than 0.92, which indicates that the 40 differential metabolites can be used as the marker differential metabolites 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 ROC curve.
Claims (9)
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, normalization and other treatment on mass spectrum data through CompundDiscover software to obtain an Excel table containing compound molecular weight, retention time and peak area; and (3) introducing a data matrix containing compound peak areas into Simca-p software for multivariate statistical analysis, and finding and identifying the differential metabolites of the spina date seed and the spina date seed by combining an ROC curve.
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: the method comprises the following specific steps:
(1) preparation of a sample: precisely weighing 2 g 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 10 mL 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: 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 HSST3 column, 150 mm multiplied by 2.1 mm 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 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 is 35 ℃; the sample introduction amount is 3 muL;
mass spectrum conditions: thermo ScientificTMQ ExactiveTMThe Orbitrap mass spectrometer Germany, using electrospray ionization source ESI, scanning simultaneously with positive and negative ion switching;
spray voltage: 3.5 KV (+), 2.5 KV (-); sheath gas flow rate: 35 arb; auxiliary gas flow rate Aux gas flow rate: 10 arb; capillary temperature Capillary temperature: 320 ℃ C; 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 metabolite 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 metabolites;
(4) performing clustering heat map analysis on 40 differential metabolites;
(5) performing relative quantitative analysis on 40 differential metabolites;
(6) and (3) verifying the effectiveness of the differential metabolites by using an area AUC value under the ROC curve, and carrying out ROC curve analysis on 40 differential metabolites.
3. The method of claim 2, wherein the UHPLC-Q-Orbitrap MS is used to analyze the differential chemical composition of spine date kernels and spine date kernels, wherein: 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.
4. The method of claim 2, wherein the UHPLC-Q-Orbitrap MS is used to analyze the differential chemical composition of spine date kernels and spine date kernels, wherein: 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.
5. The method of claim 2, wherein the UHPLC-Q-Orbitrap MS is used to analyze the differential chemical composition of spine date kernels and spine date kernels, wherein: identifying the differential metabolites in the step (3), wherein the compounds with the reference substances are identified according to the retention time, the parent ions 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.
6. The method of claim 2, wherein the UHPLC-Q-Orbitrap MS is used to analyze the differential chemical composition of spine date kernels and spine date kernels, wherein: in the step (6), the AUC values of the 40 differential metabolites are all larger than 0.92, and the AUC values are used as the marker differential metabolites for distinguishing the spina date seed from the spina date seed.
7. The method of claim 2, wherein the UHPLC-Q-Orbitrap MS is used to analyze the differential chemical composition of spine date kernels and spine date kernels, wherein: in the step (3), 40 different metabolites of the spina date seed and the spina date seed are identified, wherein the metabolites are 2 saponins, 19 flavones and 19 alkaloids.
8. The method of claim 7, wherein the UHPLC-Q-Orbitrap MS is used to analyze the differential chemical composition of spine date kernels and spine date kernels, wherein: the content of saponin components represented by the 2 saponins A and B 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, amphenib D, mucronine J, norrisorbidine, nuciferine and n-methylasimimobine, are high in the wild jujube seed.
9. Use of the method of claim 1 or 2 for quality control of spine date seeds.
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