CN110531000B - Metabolism marker for distinguishing white bean Chinese yew and taxus yunnanensis and detection method thereof - Google Patents
Metabolism marker for distinguishing white bean Chinese yew and taxus yunnanensis and detection method thereof Download PDFInfo
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Abstract
The invention discloses a metabolic marker for distinguishing white bean yew and taxus yunnanensis and a detection method thereof. The paclitaxel C is used as a metabolic marker for identifying the white bean yew and the taxus yunnanensis. The weight content of paclitaxel C in the white bean yew is 3-5 times of that of the taxus yunnanensis. The invention can be used for samples collected in the field, does not need to be frozen for storage, and the collected samples can be used for extraction and detection of metabolic markers by simple drying and storage at room temperature. The biomass of a detection sample required by the invention is less and is only 25mg, and the damage to rare taxus chinensis seedlings to be identified is less.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a metabolic marker for distinguishing white bean yew and taxus yunnanensis and a detection method thereof.
Background
Taxus chinensis belongs to shallow root plants, has unobvious main roots and developed lateral roots, is a natural rare anti-cancer plant which is known to be endangered to extinct in the world, is an ancient wiggle tree species which is left over by the fourth glacier, and has a history of 250 ten thousand years on the earth. The white bean cedar is a single-species plant which is sent to China in the third period and has scientific value for researching phylogeny of plants in the oceanic and taxus families.
Taxus chinensis and Douglas fir have great economic value, and taxol and derivatives thereof extracted from plant bodies of the Taxus chinensis and the Douglas fir are one of the most widely applied antitumor drugs in the world at present. Paclitaxel was the first antitumor active ingredient isolated from the bark of Taxus brevifolia, and is a specific drug for treating ovarian cancer and breast cancer. With the progress of research, paclitaxel has also been found to be effective against other cancers, such as: lung cancer, esophagus cancer, etc. also have obvious curative effect. The whole plant of Taxus chinensis can be used as medicine for treating diabetes, menoxenia, hypertension and other urinary system diseases. In addition, the taxus chinensis and the white bean cedar have wide development prospects in the aspect of garden appreciation. By using the special red and white fruits of the taxus chinensis and the white bean cedar, the forest trees become landscape trees in high-grade districts, parks and roads at present.
The white bean yew (pseudoteaxus chienii) is Taxaceae, and can be 4 m high; the bark is gray brown, the leaves are strip-shaped and are arranged into two rows, the tips of the two rows are convex, the base parts are nearly circular, the short stems are arranged, the upper surface is bright green, the lower surface is provided with two white air hole belts, the seeds are oval, the upper parts are slightly flat, the top ends are provided with convex small tips, the fleshy cupped arillus is white when the seeds are mature, and the base parts are provided with stored bracts. The plants are distributed on the upper parts of high mountains such as Longquan hills and Sanyangshan in south Zhejiang province, Jiangxi Jinggang mountain, Jiannan Shishan mountain and northwest Cili Jianya, Guangdong northern mammary source, Guangxi Lingui Siming mountain, Qifen mountain and Daming mountain in Shang Lin county, and Hangzhou province. Taxus yunnanensis (Taxus mairei) is evergreen arbor, the bark of which is light gray, and the bark of which is longitudinally split into long slices, the color of which is different from that of a stomatal band, and the side bands are light green and obvious. The water-soluble fertilizer is distributed in provinces in the south of Yangtze river basin, Henan and Shaanxi. The national level emphasizes the protection of wild plants. At present, the research on the white bean yew and the taxus yunnanensis has been carried out primarily, but the seedlings are highly similar in morphology, contain a certain amount of paclitaxel, and the growth environments are partially overlapped, which brings certain difficulty to the distinguishing and identifying work of the white bean yew and the taxus yunnanensis. Accurate and rapid identification and differentiation are the precondition for popularizing high-efficiency main cultivars and developing the protection of wild taxaceae resources.
Non-targeted metabolomics is a recently developed technology for studying small molecule metabolites in organisms. The technology can comprehensively analyze all metabolites of a specific organism, a specific cell or a specific physiological condition for quantitative and qualitative analysis, and find out the metabolites with characteristic difference. As an emerging discipline for the study of small molecule metabolites, metabolomics technology is a discipline that has been developed following transcriptome and proteomics. Through multiple means such as chromatogram, mass spectrum and even nuclear magnetic resonance, the non-targeted metabonomics technology accurately detects the content difference of metabolites in the material, and is widely applied to the research fields such as disease diagnosis, medicine research and development, environmental detection and basic theories of botany. With modern metabolome technology, the discovery of metabolic markers between different biological samples is currently a focus of research. In the field of botany, there are not many studies for screening metabolic markers using metabolomics technology and for identification between plant species.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a metabolome marker-paclitaxel C for identifying white bean yew and taxus yunnanensis, the content of the metabolome marker-paclitaxel C in the white bean yew is obviously higher than that of the taxus yunnanensis, so that a good technical means is provided for solving the problem of tree species identification in the breeding and forestation processes of the taxus yunnanensis.
The application of the paclitaxel C as a metabolic marker for identifying the white bean yew and the taxus yunnanensis has the following chemical characteristics: the molecular formula is C46H57NO14Molecular weight is 847.9, and the chemical structural formula is as follows:
the weight content of paclitaxel C in the white bean yew is 3-5 times of that of the taxus yunnanensis.
Preferably, the method is used for efficiently distinguishing the white bean Chinese yew seedlings from the taxus yunnanensis seedlings.
The invention also aims to provide a detection method of the white bean yew and the taxus yunnanensis, and particularly relates to a detection method of the white bean yew and the taxus yunnanensis
1) The metabolite of the ground white-bean yew or Yunnan yew branch sample is extracted by 45-55% methanol buffer solution.
Adding 120 μ L of 50% methanol precooled to-20 deg.C by volume into 25mg Taxus chinensis sample, shaking gently for 1min, incubating at room temperature for 10min, standing at-20 deg.C overnight, centrifuging at 4000 Xg for 20min, and storing the supernatant at-80 deg.C or drying;
2) performing LC-MS analysis on the supernatant of the extractive solution, and judging according to the mass content of paclitaxel C, wherein the extract is white Douglas fir if the mass content of paclitaxel C is higher than 0.05mg/g, and is Taxus yunnanensis if the mass content of paclitaxel C is lower than 0.01 mg/g;
the conditions of the high performance gas chromatography were as follows:
the hue system is: SCIEX UPLC system;
the chromatographic column temperature was: 35 ℃;
the chromatographic column is as follows: agilent acid acrylamide columns (100mm x 2.1mm, 1.7 μm, Waters, UK);
mobile phase: mobile phase A is 25mM ammonium acetate +25mM ammonia water, and mobile phase B is 9:1 IPA of ACN +0.1% formic acid;
the mobile phase gradient was: 95% of mobile phase B in 0-0.5min, 95% to 65% of mobile phase B in 0.5-9.5min, 65% to 40% of mobile phase B in 9.5-10.5min, 40% to 95% of mobile phase B in 10.5-12min, and 95% of mobile phase B in 12.2-15 min;
the mass spectrometry conditions were as follows:
high resolution mass spectrometer: TripleTOF type 5600 plus;
the shielding Gas is set to be 30PSI, the ion source Gas1 is 60PSI, and the ion source Gas2 is 60 PSI;
the heating temperature is 650 ℃;
for the positive ion mode, the ion spray voltage is 5000V respectively, and for the negative ion mode, the ion spray voltage floating is-4500V respectively;
the mass spectrum data acquisition adopts an IDA mode, and the TOF mass range is 60-1200 Da.
The invention also aims to provide application of the metabolic marker in a kit for identifying the white bean yew and the taxus yunnanensis. The kit comprises a standard substance of the paclitaxel C and/or a detection reagent for detecting the paclitaxel C.
The invention has the following beneficial effects:
1. the invention has the advantages of high precision, programmed operation, high sensitivity and accurate and reliable result.
2. The invention can be used for samples collected in the field, does not need to be frozen for storage, and the collected samples can be used for extraction and detection of metabolic markers by simple drying and storage at room temperature. The biomass of a detection sample required by the invention is less, and is only 25mg, so that the method has little damage to rare Chinese yew seedlings to be identified.
3. The invention utilizes a method of combining high performance chromatography and mass spectrometry to obtain non-target metabolic spectra of the white bean Chinese yew and the Yunnan Chinese yew, identifies a series of metabolites in different tree species, and obtains the differential metabolites between the white bean Chinese yew and the Yunnan Chinese yew through quantitative analysis. And screening to obtain the metabolites with obvious differences by a P value significance analysis method.
Drawings
Fig. 1 is an annotated picture of metabolites of white bean yew and taxus yunnanensis.
FIG. 2 shows the PCA analysis results of the metabolites of Douglas fir and Taxus yunnanensis.
FIG. 3 is a standard mass spectrum of paclitaxel C.
FIG. 4 shows the results of paclitaxel C content in BD1-10 (Douglas fir) and YN1-10 (Taxus yunnanensis).
Detailed Description
In order to make the objects, techniques and features of the present patent more apparent, the following detailed description of the present patent will proceed with reference being made to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present patent and are not limiting upon the present patent.
Example 1: extraction of metabolites of white bean Chinese yew and Yunnan Chinese yew and preparation of control sample
Treatment of experimental samples:
the small branches (twig) of Taxus chinensis are accurately weighed, and each 25mg is used for extracting metabolites.
And adding 120 mu L of precooled 45% -50% methanol extract into the ground taxus chinensis sample, and mixing in a centrifuge tube. Shaking gently for 1min, incubating at room temperature for 10min, standing at-20 deg.C overnight, centrifuging at 4000 Xg for 20min, and storing the supernatant at-80 deg.C or drying;
treatment of control samples:
to better analyze the quantitative differences between the two groups of samples from taxus yunnanensis and taxus yunnanensis, a group of control samples was prepared. The control sample was prepared as follows: taking 10 independent white bean yew experimental samples and 10 independent Yunnan yew experimental samples, and mixing the 20 samples in equal volume. The mixture was divided into 10 equal portions to prepare control samples.
Example 2: annotation of metabolites of Douglas fir and Taxus yunnanensis
In the implementation process of the invention, a large amount of mass spectrum data including peak (peak), retention time (retention time) and annotation information are detected. The obtained mass spectrum original data is converted into an XML format through software such as XCMS, CAMERA and metaX. The obtained data of different ions are identified by retention time and nuclear-to-proton ratio. The generated ion information was compared to its exact molecular weight and annotated by KEGG and HMDB online databases.
The present invention yielded a total of 7167 valid ion signatures, and thus 5871 successfully annotated metabolites. Annotation of the results indicates that most of the identified metabolites can be assigned to at least one metabolic pathway. The annotated metabolites are mainly classified into metabolic pathways such as amino acid metabolism, carbohydrate metabolism, terpenoid metabolism, lipidation metabolism, nucleotide metabolism, energy metabolism, and the like, and the specific results are shown in fig. 1.
Example 3: screening of marked differential metabolites of white bean Chinese yew and Yunnan Chinese yew
In order to screen the marked differential metabolites of the white bean yew and the taxus yunnanensis, the invention detects two main quality control parameters including Coefficient of Variation (Coefficient of Variation) and Principal Component Analysis (PCA). Among them, the coefficient of variation value was less than 30%, showing higher reproducibility. The main component analysis results show that the variance values of PC1 and PC2 are 73.85% and 2.60%, respectively, which indicates that the discrimination of the two taxus species is high, and the specific PCA analysis results are shown in FIG. 2. Statistical results show that a total screening results in 311 metabolites with significantly different accumulation, 150 of which are significantly accumulated in white bean yew, and another 161 of which are significantly accumulated in taxus yunnanensis. The above differential metabolites can be classified into a plurality of main metabolite classes including alkaloids, amino acids, flavones, hormones, lipids, terpenes, phenylpropanoids, saccharides, and the like. The main metabolites are greatly different between the white bean yew and the taxus yunnanensis. According to the invention, by analyzing the metabolites which are obviously differentially accumulated, the taxol C is identified to be the marked differential metabolite of the white bean yew and the taxus yunnanensis.
Example 4: paclitaxel C (Taxuyunnannin C) standard substance and liquid-mass spectrum thereof
A paclitaxel C (Taxuyunnannin C) standard is purchased from a chemical source on the internet, and is characterized as follows: CAS number 153415-45-3; the molecular weight is 847.9; molecular formula C46H57NO14(ii) a The density is 1.3 +/-0.1 g/cm3The boiling point was 932.3 + -65.0 deg.C (760 mmHg). The standard mass spectrum is shown in FIG. 3. And accurately measuring the content of the taxol C in the taxus chinensis sample by using the standard substance.
Example 5: reliability verification example for distinguishing marked differential metabolites of white bean cedar and taxus yunnanensis
The five-year-old white Douglas fir cultivation seedlings and the five-year-old Yunnan taxus chinensis cultivation seedlings are planted in the Hangzhou Master university experiment nursery. The collected samples are respectively numbered as: BD1-10 (Douglas fir) and YN1-10 (Taxus yunnanensis).
Fully drying collected Taxus chinensis twigs in a 40 ℃ oven, filtering through a sieve with the aperture of 0.42mm, accurately weighing 0.5g of dry powder for each sample, adding 15mL of 100% methanol, and uniformly mixing. The mixture was treated with 150W, 40kHz, ultrasonic waves for 30 min. After the lysate was centrifuged at 4000 Xg for 20min, the supernatant was transferred to a new centrifuge tube. After vacuum drying, the mixture was dissolved in 50% methanol again for use. And diluting the obtained sample by 10 times by volume, and carrying out UPLC-MS/MS quantitative detection. The detection result shows that the average content of paclitaxel C in the white bean yew is obviously higher than that of the taxus yunnanensis, and the specific result is shown in fig. 4. The above results demonstrate the reliability of paclitaxel C as a marker metabolite that distinguishes white bean yew from taxus yunnanensis. The invention screens and obtains a marker metabolite for distinguishing the white bean yew and the taxus yunnanensis based on a metabonomics technology, the required sample is only 25mg, and the accuracy rate is 100%.
The above embodiments are not intended to limit the present invention, and the present invention is not limited to the above embodiments, and all embodiments are within the scope of the present invention as long as they meet the requirements of the present invention.
Claims (5)
1. The application of paclitaxel C as a metabolic marker for identifying white bean yew and taxus yunnanensis is characterized in that the molecular formula is C46H57NO14Molecular weight 847.9, chemical formula as follows:
2. the use as claimed in claim 1, wherein the paclitaxel C content in white Douglas fir is 3-5 times that of Taxus yunnanensis.
3. Use according to any one of claims 1 to 2, for distinguishing young plants of Douglas fir from young plants of Taxus yunnanensis.
4. A detection method for distinguishing white bean yew and Yunnan yew is characterized by comprising the following steps:
1) extracting the metabolite from the ground white-bean yew or Yunnan yew branch sample by using a methanol solution;
adding 120 μ L of 50% methanol precooled to-20 deg.C by volume into 25mg Taxus chinensis sample, shaking gently for 1min, incubating at room temperature for 10min, standing at-20 deg.C overnight, centrifuging at 4000 Xg for 20min, and storing the supernatant at-80 deg.C or drying;
2) performing LC-MS analysis on the supernatant of the extractive solution, and judging according to the mass content of paclitaxel C, wherein the extract is white Douglas fir if the mass content of paclitaxel C is higher than 0.05mg/g, and is Taxus yunnanensis if the mass content of paclitaxel C is lower than 0.01 mg/g;
the conditions of the high performance gas chromatography were as follows:
the hue system is: SCIEX UPLC system;
the chromatographic column temperature was: 35 ℃;
the chromatographic column is as follows: an Agilent acidic acrylamide column;
mobile phase: the mobile phase A is 25mM ammonium acetate and 25mM ammonia water, and the mobile phase B is 9:1 IPA, ACN and 0.1% formic acid;
the mobile phase gradient was: 95% of mobile phase B in 0-0.5min, 95% to 65% of mobile phase B in 0.5-9.5min, 65% to 40% of mobile phase B in 9.5-10.5min, 40% to 95% of mobile phase B in 10.5-12min, and 95% of mobile phase B in 12.2-15 min;
the mass spectrometry conditions were as follows:
high resolution mass spectrometer: TripleTOF5600plus type;
the shielding Gas is set to be 30PSI, the ion source Gas1 is 60PSI, and the ion source Gas2 is 60 PSI;
the heating temperature is 650 ℃;
for the positive ion mode, the ion spray voltage was 5000V, for the negative ion mode, the ion spray voltage was-4500V;
the mass spectrum data acquisition adopts an IDA mode, and the TOF mass range is 60-1200 Da.
5. The application of paclitaxel C in a kit for identifying white bean yew and taxus yunnanensis is provided.
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| JP2005272565A (en) * | 2004-03-24 | 2005-10-06 | Soda Aromatic Co Ltd | Antioxidant |
| CN1791590A (en) * | 2003-03-17 | 2006-06-21 | 天然医药品公司 | Purification of taxanes and taxane mixtures using polyethyleneimine-bonded resins |
| CN103808851A (en) * | 2012-11-15 | 2014-05-21 | 刘胜远 | Thin-layer chromatography detection method of xylosyl-10-deacetyltaxol in taxus chinensis |
| CN109324127A (en) * | 2018-09-30 | 2019-02-12 | 东阳市杰迩威生物科技有限公司 | The detection method of taxol in Chinese yew extract |
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| CN1791590A (en) * | 2003-03-17 | 2006-06-21 | 天然医药品公司 | Purification of taxanes and taxane mixtures using polyethyleneimine-bonded resins |
| JP2005272565A (en) * | 2004-03-24 | 2005-10-06 | Soda Aromatic Co Ltd | Antioxidant |
| CN103808851A (en) * | 2012-11-15 | 2014-05-21 | 刘胜远 | Thin-layer chromatography detection method of xylosyl-10-deacetyltaxol in taxus chinensis |
| CN109324127A (en) * | 2018-09-30 | 2019-02-12 | 东阳市杰迩威生物科技有限公司 | The detection method of taxol in Chinese yew extract |
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