CN113419000A - Method for identifying panax notoginseng with 25 heads and less than 80 heads based on non-targeted metabonomics - Google Patents
Method for identifying panax notoginseng with 25 heads and less than 80 heads based on non-targeted metabonomics Download PDFInfo
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Abstract
The invention provides a method for identifying panax notoginseng with 25 heads and less than 80 heads based on non-targeted metabonomics, belonging to the field of analysis and detection of traditional Chinese medicinal materials and comprising the following steps: s1, leaching the pseudo-ginseng metabolite by using a methanol solution as a solvent to obtain a test sample; s2, carrying out non-targeted component determination on the pseudo-ginseng metabolite extracting solution by adopting a UHPLC LTQ Oribitrap MS combined technology to obtain original data; s3, sequentially carrying out preprocessing and multivariate statistical analysis on the original data; s4, screening differential metabolites by taking the variable importance projection indexes as screening indexes; the method is based on the panax notoginseng integrity analysis, combines the UHPLC LTQ Oribitrap MS technology with non-targeted metabonomics, and combines the principal component analysis to screen the different metabolites of panax notoginseng with different specifications, can be used for accurately distinguishing panax notoginseng with different commodity specifications, and provides theoretical basis for the commodity specification standards of medicinal materials in the panax notoginseng market.
Description
Technical Field
The invention relates to the field of analysis and detection of traditional Chinese medicinal materials, in particular to a method for identifying panax notoginseng with 25 heads and panax notoginseng with less than 80 heads based on non-targeted metabonomics.
Background
Notoginseng is also named as radix linderae and radix stephaniae tetrandrae, is the dried root and rhizome of Panax notoginseng (Araliaceae) Panax notoginseng (Burk.) F.H.Chen, has very wide clinical application in Chinese medicine, and is also the traditional rare Chinese medicine and incised wound medicine in China. Pseudo-ginseng is sweet in nature and taste, slightly bitter and warm, enters liver and stomach meridians, and has the functions and main indications of dissipating blood stasis, stopping bleeding, diminishing swelling and relieving pain. Can be used for treating hemoptysis, hematemesis, epistaxis, hematochezia, metrorrhagia, traumatic hemorrhage, thoracico-abdominal pain, and traumatic injury with swelling and pain.
According to records of seventy-six medicinal material commodity specification standards issued by the ministry, the pseudo-ginseng specification grades can be graded according to the harvesting season, the medicinal parts and the sizes (weight) of the medicinal materials; in the market, the weight of the main root of pseudo-ginseng is the main way to distinguish different grades of pseudo-ginseng, and the price difference of pseudo-ginseng with different sizes (weights) is very large.
At present, the researches on the internal components of pseudo-ginseng with different sizes (weights) are less, the researches mainly comprise the steps of measuring the polysaccharide content of the pseudo-ginseng by using a phenol-sulfuric acid method, measuring volatile components by using GC-MS (gas chromatography-mass spectrometry), and the like, and the researches do not provide clear internal components of the pseudo-ginseng as internal indexes for distinguishing commercial medicinal materials, so that the practical significance is realized for exploring the difference metabolites of the pseudo-ginseng with different sizes and specifications and searching the internal component indexes of the pseudo-ginseng.
Disclosure of Invention
Aiming at the problems, the invention provides a method for identifying panax notoginseng with 25 heads and panax notoginseng below 80 heads based on non-targeted metabonomics, which aims to integrate ultra-high performance liquid chromatography-linear ion trap-electrostatic field orbit trap mass spectrum combined technology (UHPLC LTQ Oribitrap MS) and non-targeted metabonomics means to mine mass spectrum data of 25 heads of panax notoginseng and panax notoginseng below 80 heads, screen differential metabolites of the mass spectrum data and provide basis for standardizing the commodity specification standard of medicinal materials in the panax notoginseng market.
The purpose of the invention is realized by adopting the following technical scheme:
a method for identifying 25-80 pseudo-ginseng based on non-targeted metabonomics comprises the following steps:
s1, extracting metabolites of the pseudo-ginseng sample;
leaching the pseudo-ginseng metabolite by using a methanol solution as a solvent to obtain a test sample;
s2, measuring non-target components;
performing non-targeted component determination on the pseudo-ginseng metabolite extracting solution by adopting a UHPLC (ultra high performance liquid chromatography) LTQ Oribitrap MS (Mass Spectrometry) combined technology to obtain original data;
s3, processing and analyzing data
Sequentially carrying out pretreatment and multivariate statistical analysis on the original data;
s4, finding differential metabolites through data mining;
and (4) screening differential metabolites by taking the variable importance projection indexes as screening indexes.
Preferably, the extraction method in step S1 comprises pulverizing radix Notoginseng, sieving with 40 mesh sieve, weighing sieved radix Notoginseng powder 0.600g, adding 20ml 70% methanol solution, ultrasonic extracting for 40min, separating extractive solution, and filtering the extractive solution with 0.22 μm microporous membrane to obtain the sample.
Preferably, the measuring method in step S2 is specifically:
chromatographic conditions are as follows: waters ACQUITY UPLC HSS T3 chromatographic column with specification of 2.1mm × 100mm, 1.8 μm; the mobile phase A is an aqueous solution containing 0.1 percent of formic acid, the mobile phase B is acetonitrile, the elution gradient is 0-8min, and the concentration of the mobile phase A is 98-65 percent; 8-19min, 65-60% A, 19-27min, 60-10% A; 27-30min, 10% A; the column temperature is 30 ℃, the flow rate is 0.3ml/min, and the sample injection amount is 3 mul; mass spectrum conditions: the ion source is an electrospray ionization source, the positive ion mode and the negative ion mode are respectively scanned, the temperature of the heater is 350 ℃, the temperature of the capillary tube is 350 ℃, the voltage of the capillary tube is 35V, the spraying voltage is 3.5kV, the flow rate of sheath gas is 40arb, and the flow rate of auxiliary gas is 10 arb; performing full scanning on the primary mass spectrum of the test solution and the reference solution in an FT mode, wherein the scanning range is 100-1200, and the secondary mass spectrum adopts data-dependent scanning; mass spectral data were collected and analyzed using Xcalibur software.
Preferably, the preprocessing in step S3 is to extract mass spectrum data of 25 and 80 or less notoginseng collected by UHPLC LTQ Oribitrap MS through Progenesis QI software, and perform preprocessing including peak alignment, data normalization and quality control on the data;
preferably, the multivariate statistical analysis comprises principal component analysis, specifically, the preprocessed data is imported into Ezinfo software, and the principal component analysis method is used for analyzing the category, similarity and difference of the samples to characterize the differences of the samples.
Preferably, the variable importance projection indexes in step S4 include p value, vip value and Fold Change value, components with p value less than 0.05, vip > 1 and Fold Change > 1.5 are screened, components with high and low crossover in 25-head groups and 80-head or lower-head groups are further eliminated, and finally, differential metabolites of 25-head and 80-head or lower-head pseudo-ginseng groups are established.
The invention has the beneficial effects that:
(1) the invention is based on the panax notoginseng integrity analysis, carries out non-target metabolism total component detection on panax notoginseng from the alcohol extract angle, can accurately distinguish panax notoginseng with different commodity specifications by searching the panax notoginseng internal component indexes, using the integral difference and combining with the main component analysis to screen the different metabolites of panax notoginseng with different specifications, and provides theoretical basis for the panax notoginseng market medicinal material commodity specification standard.
(2) The non-targeted metabonomics is a research method for carrying out systematic and comprehensive analysis on the whole metabonomic group, obtaining data of a large amount of metabolites and searching for differential metabolites, and can be used for describing the difference between different types of medicaments.
(3) Compared with the traditional liquid mass (TOF MS and the like) detection, the UHPLC LTQ Oribitrap MS technology is adopted for data acquisition, so that higher mass number resolution and mass precision can be provided, high-accuracy mass number determination is realized, the mass number precision of a typical sample injection analyte is lower than 1ppm, and the limit of accurate quantitative identification of small molecular mass numbers is removed.
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The invention is further illustrated by means of the attached drawings, but the embodiments in the drawings do not constitute any limitation to the invention, and for a person skilled in the art, other drawings can be obtained on the basis of the following drawings without inventive effort.
FIG. 1 is a diagram showing the principal component analysis of 25 and 80 panax notoginseng under positive ion mode;
FIG. 2 is a diagram showing the principal component analysis of 25 and 80 panax notoginseng under negative ion mode;
FIG. 3 is a graph of the orthorhombic partial least squares discriminant analysis of 25 and 80 Notoginseng radix in positive ion mode;
FIG. 4 is a diagram of the orthorhombic partial least squares discriminant analysis of 25 and 80 Notoginseng radix in negative ion mode;
FIG. 5 is a heat map of positive ion pattern differential components;
FIG. 6 is a heat map of the differential composition of negative ion profile.
Detailed Description
The invention is further described with reference to the following examples.
The embodiment of the invention relates to a method for identifying 25-80 panax notoginseng based on non-targeted metabonomics, which comprises the following steps:
s1, extracting Notoginseng radix 25 and Notoginseng radix 80 below with 70% methanol;
s2, carrying out non-target component determination on 25 panax notoginseng heads and panax notoginseng heads below 80 by using UHPLC LTQ Oribitrap MS technology;
s3, extracting and analyzing the component information by using data processing software, and performing principal component analysis on the component information;
s4, finding differential metabolites through data mining;
specifically, the method comprises the following steps:
s1, extracting metabolites of the pseudo-ginseng sample;
collecting 25 batches of radix Notoginseng with different producing areas and 5 batches of radix Notoginseng with less than 80 heads for experiment, wherein the collected radix Notoginseng is radix Notoginseng with three years of growth and 2016 year of planting, and the producing areas of the radix Notoginseng are different; pulverizing Notoginseng radix, sieving with 40 mesh sieve, weighing sieved Notoginseng radix powder 0.600g, adding 20ml 70% methanol solution, ultrasonic extracting for 40min, separating extractive solution, and sieving with 0.22 μm microporous membrane to obtain 10 batches of Notoginseng radix metabolite extractive solution;
s2, measuring non-target components;
adopting UHPLC LTQ Oribitrap MS combined technology to carry out non-target component determination on the pseudo-ginseng metabolite extract, wherein the chromatographic conditions are as follows: waters ACQUITY UPLC HSS T3 chromatographic column with specification of 2.1mm × 100mm, 1.8 μm; the mobile phase A is an aqueous solution containing 0.1 percent of formic acid, the mobile phase B is acetonitrile, the elution gradient is 0-8min, and the concentration of the mobile phase A is 98-65 percent; 8-19min, 65% -60% A; 19-27min, 60% -10% A; 27-30min, 10% A; the column temperature is 30 ℃, the flow rate is 0.3ml/min, and the sample injection amount is 3 mul; mass spectrum conditions: the ion source is electrospray ionization (ESI), the positive and negative ion modes are scanned respectively, the heater temperature is 350 deg.C, the capillary voltage is 35V, the spray voltage is 3.5kV, and the sheath gas (N)2) Flow rate 40arb, auxiliary gas (N)2) Flow rate 10 arb; performing full scanning (with the resolution of 30000 and the m/z scanning range of 100-; collecting and analyzing mass spectrum data by using Xcalibur software;
s3, processing and analyzing data;
extracting mass spectrum data of panax notoginseng with 25 heads and less than 80 heads acquired by UHPLC LTQ Oribitrap MS by Progenisis QI software, preprocessing the data (including peak alignment, data normalization, quality control and the like), introducing the obtained data into Ezinfo software, analyzing the sample category, similarity and difference by methods such as principal component analysis and the like, representing the sample difference, and further mining the difference metabolites;
a large amount of multi-dimensional data can be converted into a scatter diagram which can be visually observed through the principal component score diagram, and the similarity and difference degree of observation variables are reflected by the distance between different observation samples;
s4, finding differential metabolites through data mining;
mining mass spectrum data of pseudo-ginseng with 25 heads and less than 80 heads, firstly calculating p values and FC (Fold Change) values of components by Progenis QI software, calculating vip values by orthogonal partial least squares (OPLS-DA) analysis, screening components with p values less than 0.05, vip more than 1 and Fold Change more than 1.5, obtaining 59 differential components in a positive ion mode, obtaining 83 differential components in a negative ion mode, specifically referring to Table 1, screening the obtained differential components, mainly calculating possible molecular formulas (most ppm is less than 10) by accurate molecular weight m/z for the identification method of the components without secondary fragments, and then comparing and identifying the possible molecular formulas with the identified components of the pseudo-ginseng in the literature; further removing components with high and low cross in the 25-head group and the groups of panax notoginseng below 80, removing components with low content and difficult peak area extraction, and finally establishing the differential metabolites of 25 boundary groups of 25 and panax notoginseng below 80, wherein the 25 components have high content in the panax notoginseng below 80, which is shown in table 2.
The heat map is used for visually representing the differential components, and the content difference of the found differential components between the two groups is found to be obvious.
TABLE 1 differential composition in Positive and negative ion modes
Table 2 establishes differential metabolites for the cut-off of 25 and 80 Notoginseng radix
And (3) identifying and verifying the following 25 and 80 panax notoginseng:
sample preparation: AG-25-1(25 pieces, Hebei Anguo medicinal materials market collection), AG-180-1(180 pieces, Hebei Anguo medicinal materials market collection)
The method comprises the following steps: the 25 and 80 panax notoginseng heads were collected and verified according to the aforementioned ingredient determination method with the 25 differential metabolites identified in table 2 to be able to distinguish the 25 and 80 panax notoginseng heads, the verification results are shown in table 3.
TABLE 3 validation of differential metabolites
Analyzing the data in table 3, it can be seen that 24 of the 25 different components have a higher content in the AG-180-1 (below 80) panax notoginseng than in the AG-25-1 (25) panax notoginseng, which meets the found rule of high or low content of 25 panax notoginseng and below 80 panax notoginseng; only the content of 5.68-409.1829 m/z in AG-180-1 (below 80) pseudo-ginseng is lower than that of AG-25-1 (25) pseudo-ginseng, which does not accord with the content rule determined in the early stage, and the total correct rate of the differential components screened by the method of the invention can reach 96%. Therefore, the differential metabolites screened by the method can well distinguish 25 panax notoginseng and 180 panax notoginseng in content.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention, and not for limiting the protection scope of the present invention, although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (6)
1. A method for identifying 25-80 pseudo-ginseng based on non-targeted metabonomics is characterized by comprising the following steps:
s1, extracting metabolites of the pseudo-ginseng sample;
leaching the pseudo-ginseng metabolite by using a methanol solution as a solvent to obtain a test sample;
s2, measuring non-target components;
performing non-targeted component determination on the pseudo-ginseng metabolite extracting solution by adopting a UHPLC (ultra high performance liquid chromatography) LTQ Oribitrap MS (Mass Spectrometry) combined technology to obtain original data;
s3, processing and analyzing data;
sequentially carrying out pretreatment and multivariate statistical analysis on the original data;
s4, finding differential metabolites through data mining;
and (4) screening differential metabolites by taking the variable importance projection indexes as screening indexes.
2. The method for identifying panax notoginseng under 25 and 80 heads based on non-targeted metabonomics as claimed in claim 1, wherein the extraction method of step S1 comprises pulverizing panax notoginseng, sieving with a 40 mesh sieve, weighing 0.600g of sieved panax notoginseng powder, adding 20ml of 70% methanol solution, ultrasonically extracting for 40min, separating the extract, and filtering the extract with a 0.22 μm microporous membrane to obtain the test sample.
3. The method for identifying panax notoginseng 25 and 80 as follows based on non-targeted metabonomics according to claim 1, wherein the determination method of step S2 is specifically:
chromatographic conditions are as follows: waters ACQUITY UPLC HSS T3 chromatographic column with specification of 2.1mm × 100mm, 1.8 μm; the mobile phase A is an aqueous solution containing 0.1 percent of formic acid, the mobile phase B is acetonitrile, the elution gradient is 0-8min, and the concentration of the mobile phase A is 98-65 percent; 8-19min, 65-60% A, 19-27min, 60-10% A; 27-30min, 10% A; the column temperature is 30 ℃, the flow rate is 0.3ml/min, and the sample injection amount is 3 mul; mass spectrum conditions: the ion source is an electrospray ionization source, the positive ion mode and the negative ion mode are respectively scanned, the temperature of the heater is 350 ℃, the temperature of the capillary tube is 350 ℃, the voltage of the capillary tube is 35V, the spraying voltage is 3.5kV, the flow rate of sheath gas is 40arb, and the flow rate of auxiliary gas is 10 arb; performing full scanning on the primary mass spectrum of the test solution and the reference solution in an FT mode, wherein the scanning range is 100-1200, and the secondary mass spectrum adopts data-dependent scanning; mass spectral data were collected and analyzed using Xcalibur software.
4. The method of claim 1, wherein the preprocessing in step S3 is to extract mass spectrum data of 25 and 80 panax notoginseng collected by UHPLC LTQ Oribitrap MS through Progenesis QI software, and perform preprocessing on the data, including peak alignment, data normalization and quality control.
5. The method for identifying panax notoginseng under 25 and 80 based on non-targeted metabonomics according to claim 1, wherein the multivariate statistical analysis comprises principal component analysis, and specifically, the preprocessed data is introduced into Ezinfo software, and the categories, similarities and differences of the samples are analyzed by the principal component analysis method to characterize the differences of the samples.
6. The method of claim 1, wherein the variable importance projection indicators in step S4 comprise p-value, vip-value and Fold Change-value, the p-value and the Fold Change-value of the components are calculated using Progenesis QI software, the vip-value is calculated by orthogonal partial least squares discriminant analysis, the components with p-value < 0.05, vip > 1 and Fold Change > 1.5 are selected, the components with high and low crossover in the 25-head group and the 80-head-lower pseudo-ginseng group are further rejected, and the metabolites that differentiate the 25-head and 80-head-lower pseudo-ginseng are finally established.
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