CN111929373A - Identification method of medlar color-related metabolites - Google Patents
Identification method of medlar color-related metabolites Download PDFInfo
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- CN111929373A CN111929373A CN202010630232.0A CN202010630232A CN111929373A CN 111929373 A CN111929373 A CN 111929373A CN 202010630232 A CN202010630232 A CN 202010630232A CN 111929373 A CN111929373 A CN 111929373A
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- glucoside
- rutinoside
- anthocyanin
- petunia
- delphinidin
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- 235000017784 Mespilus germanica Nutrition 0.000 title claims abstract description 28
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- 238000000034 method Methods 0.000 title claims abstract description 23
- 240000002624 Mespilus germanica Species 0.000 title 1
- 150000004636 anthocyanins Chemical class 0.000 claims abstract description 42
- 229930002877 anthocyanin Natural products 0.000 claims abstract description 40
- 235000010208 anthocyanin Nutrition 0.000 claims abstract description 40
- 239000004410 anthocyanin Substances 0.000 claims abstract description 40
- 244000182216 Mimusops elengi Species 0.000 claims abstract description 27
- 235000013399 edible fruits Nutrition 0.000 claims abstract description 27
- 235000015459 Lycium barbarum Nutrition 0.000 claims abstract description 20
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- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 15
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- KKDMZZAUAIZTFK-DKJRWSDTSA-N 5-hydroxy-2-(4-hydroxy-3,5-dimethoxyphenyl)-3-[(2s,3r,4s,5s,6r)-3,4,5-trihydroxy-6-[[(1r,2r,3r,4s,5r)-2,3,4-trihydroxy-5-methylcyclohexyl]oxymethyl]oxan-2-yl]oxychromen-7-one Chemical compound COC1=C(O)C(OC)=CC(C2=C(C=C3C(O)=CC(=O)C=C3O2)O[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO[C@H]3[C@@H]([C@H](O)[C@@H](O)[C@H](C)C3)O)O2)O)=C1 KKDMZZAUAIZTFK-DKJRWSDTSA-N 0.000 claims description 4
- 238000010811 Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Methods 0.000 claims description 4
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- 239000003153 chemical reaction reagent Substances 0.000 claims description 4
- VEVZSMAEJFVWIL-UHFFFAOYSA-O cyanidin Natural products [O+]=1C2=CC(O)=CC(O)=C2C=C(O)C=1C1=CC=C(O)C(O)=C1 VEVZSMAEJFVWIL-UHFFFAOYSA-O 0.000 claims description 4
- 238000010828 elution Methods 0.000 claims description 4
- 125000003132 pyranosyl group Chemical group 0.000 claims description 4
- 150000003839 salts Chemical class 0.000 claims description 4
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- -1 cyanidin galactoside Chemical class 0.000 claims description 3
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 3
- 239000012498 ultrapure water Substances 0.000 claims description 3
- OIZFQAFWYYKPMR-PEVLUNPASA-N Delphinidin 3-O-glucoside Natural products O([C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1)C=1[C-](c2cc(O)c(O)c(O)c2)[O+]c2c(c(O)cc(O)c2)C=1 OIZFQAFWYYKPMR-PEVLUNPASA-N 0.000 claims description 2
- OVVGHDNPYGTYIT-ROUHPGRKSA-N alpha-L-Rhap-(1->6)-D-Glcp Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)C(O)O1 OVVGHDNPYGTYIT-ROUHPGRKSA-N 0.000 claims description 2
- XENHPQQLDPAYIJ-PEVLUNPASA-O delphinidin 3-O-beta-D-glucoside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC2=C(O)C=C(O)C=C2[O+]=C1C1=CC(O)=C(O)C(O)=C1 XENHPQQLDPAYIJ-PEVLUNPASA-O 0.000 claims description 2
- XCTGXGVGJYACEI-LCENJUANSA-O delphinidin 3-O-beta-D-glucoside-5-O-beta-D-glucoside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC(C(=[O+]C1=CC(O)=C2)C=3C=C(O)C(O)=C(O)C=3)=CC1=C2O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 XCTGXGVGJYACEI-LCENJUANSA-O 0.000 claims description 2
- 229930182478 glucoside Natural products 0.000 claims description 2
- 150000008131 glucosides Chemical class 0.000 claims description 2
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- 238000004445 quantitative analysis Methods 0.000 claims description 2
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- 238000011160 research Methods 0.000 abstract description 19
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- KZMACGJDUUWFCH-UHFFFAOYSA-O malvidin Chemical compound COC1=C(O)C(OC)=CC(C=2C(=CC=3C(O)=CC(O)=CC=3[O+]=2)O)=C1 KZMACGJDUUWFCH-UHFFFAOYSA-O 0.000 description 2
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- 238000001946 ultra-performance liquid chromatography-mass spectrometry Methods 0.000 description 2
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- 240000001549 Ipomoea eriocarpa Species 0.000 description 1
- 235000005146 Ipomoea eriocarpa Nutrition 0.000 description 1
- YKRGDOXKVOZESV-WRJNSLSBSA-N Paeoniflorin Chemical compound C([C@]12[C@H]3O[C@]4(O)C[C@](O3)([C@]1(C[C@@H]42)O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)C)OC(=O)C1=CC=CC=C1 YKRGDOXKVOZESV-WRJNSLSBSA-N 0.000 description 1
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- 150000002215 flavonoids Chemical class 0.000 description 1
- 235000017173 flavonoids Nutrition 0.000 description 1
- NWKFECICNXDNOQ-UHFFFAOYSA-N flavylium Chemical compound C1=CC=CC=C1C1=CC=C(C=CC=C2)C2=[O+]1 NWKFECICNXDNOQ-UHFFFAOYSA-N 0.000 description 1
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- 210000004185 liver Anatomy 0.000 description 1
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- 235000009584 malvidin Nutrition 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
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- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- HKUHOPQRJKPJCJ-UHFFFAOYSA-N pelargonidin Natural products OC1=Cc2c(O)cc(O)cc2OC1c1ccc(O)cc1 HKUHOPQRJKPJCJ-UHFFFAOYSA-N 0.000 description 1
- 235000006251 pelargonidin Nutrition 0.000 description 1
- YPVZJXMTXCOTJN-UHFFFAOYSA-N pelargonidin chloride Chemical compound [Cl-].C1=CC(O)=CC=C1C(C(=C1)O)=[O+]C2=C1C(O)=CC(O)=C2 YPVZJXMTXCOTJN-UHFFFAOYSA-N 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- Life Sciences & Earth Sciences (AREA)
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- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
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Abstract
The invention provides an identification method of a medlar color-related metabolite, which comprises the following steps: after preprocessing medlar fruit samples of medlar varieties with different colors, extracting anthocyanin, and carrying out qualitative and quantitative detection on the anthocyanin; analyzing anthocyanin detection data, and positioning chromatographic ion peaks to obtain a wolfberry anthocyanin database; then quantitatively analyzing, and screening to obtain specific metabolites related to the color shade of the medlar fruits. The invention has the beneficial effects that: the method provides a foundation for the subsequent deeper research on specific substances with light and dark colors of the medlar, and simultaneously provides convenience for the research on subsequent metabolites and the breeding research; the accuracy of anthocyanin detection is improved by exploring the test conditions of ultra-high performance liquid chromatography-mass spectrometry; the method for identifying the metabolites related to the colors of the Chinese wolfberry fruits is provided, and an anthocyanin database is constructed, so that a material basis is provided for the future research, and a research idea is provided for the future identification of the metabolites related to the colors of the plants.
Description
Technical Field
The invention belongs to the field of analytical chemistry, and particularly relates to an identification method of a medlar color-related metabolite.
Background
The anthocyanin is a natural edible pigment, is safe and nontoxic, has rich resources, and has certain physiological health care functions, such as clearing free radicals in vivo, resisting tumors and cancers, protecting the liver, preventing diabetes and the like. Anthocyanins have been studied as derivatives of 2-phenylbenzopyran cationic structures, which are typical of flavonoids. The food mainly contains seed anthocyanin, i.e. cyanidin, morning glory pigment, delphinidin, peoniflorin, pelargonidin and malvidin.
At present, many researches on plant anthocyanin are carried out, researches show that anthocyanin is also a key metabolite influencing fruit color, different anthocyanin contents and types are reasons for color differences of white, red, black and the like of fruits, and no relevant researches are carried out at present on the reason for the shade of the color of medlar.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for identifying metabolites related to the color of medlar.
The specific technical scheme is as follows:
a method for identifying a color-related metabolite of Lycium barbarum, wherein the method for identifying a color-related metabolite of Lycium barbarum comprises:
step S1: after preprocessing medlar fruit samples of medlar varieties with different colors, extracting anthocyanin, and carrying out qualitative and quantitative detection on the anthocyanin;
step S2: analyzing anthocyanin detection data, determining various anthocyanin structures, and then performing chromatographic ion peak positioning to obtain a wolfberry anthocyanin database;
and step S3, carrying out quantitative analysis according to anthocyanidin of different varieties of wolfberry fruits, and screening to obtain specific metabolites related to the color shade of the wolfberry fruits.
Further, in the step S1, qualitative and quantitative detection of anthocyanin is performed by UPLC-MS/MS detection.
Further, in the step S1, after the pretreatment of the medlar fruit sample, 10-90% by volume of methanol is added as an extraction reagent, and the anthocyanin derivative is extracted under the conditions that the mass concentration ratio of the material liquid is 1 (10-50), the ultrasonic treatment is carried out for 5-60 min, and the ultrasonic treatment temperature is 20-60 ℃.
Further, in the step S1, after the medlar fruit sample is pretreated, 70% by volume of methanol is added as an extraction reagent, and the anthocyanin derivative is extracted under the conditions that the mass concentration ratio of the material liquid is 1:10, the ultrasonic treatment is performed for 10min, and the ultrasonic treatment temperature is 40 ℃.
Further, in step S1, the mass spectrometry parameters of the ultra performance liquid chromatography-mass spectrometry detection are:
| ion mode | Positive ion scanning | Air pressure of air curtain | 40psi |
| Voltage of ion spray | 5500V | Temperature of | 550℃ |
| Ion source gas 1 | 50psi | Ion source gas 2 | 60psi |
| Air for collision | Medium | Scanning mode | MRM |
| Inlet voltage | 10V | Outlet voltage of collision cell | 10V |
。
Further, in step S1, the chromatographic test parameters of the ultra performance liquid chromatography-mass spectrometry detection are:
a chromatographic column: waters acquisition UPLC HSS T3C 181.8 μm, 2.1mm 100 mm;
mobile phase: the phase A is an ultrapure water solution of 0.1 percent by mass of acetic acid, and the phase B is an acetonitrile solution of 0.1 percent by mass of acetic acid; the flow rate is 0.3 ml/min; the column temperature is 40 ℃; the sample volume is 2 mul;
the mobile phase step elution is as follows in volume percent:
further, in step S2, the anthocyanin types in the anthocyanin database include: petunia 3, 5-diglucoside, petunia-3-O-rutinoside-5-O-glucoside, petunia pigment, petunia 3-O- [6-O- (4-O-p-caffeoyl-alpha-L-rhamnosyl pyranosyl) -beta-D-glucopyranoside ] -5-O- [ beta-D-glucopyranoside, petunia 3-O-p-coumaroyl-rutinoside, petunia-3-O-p-coumaroyl-rutinoside-5-O-glucoside, petunia-3-O-rutinoside (feruloyl) -5-O-glucoside, and pharmaceutically acceptable salts thereof, Petunidin-3-p-coumaric acid-sophoroside-5-O-glucoside, petunidin-3-p-coumaric acid-sophoroside-7-O-glucoside, petunidin-3-p-coumaric acid-sophoroside-7-glucoside, delphinidin-hexoside, delphinidin 3-sophoroside-5-rhamnoside, delphinidin 3-O-p-coumaroyl-glucoside, delphinidin 3-O-rutinoside-5-O-glucoside, delphinidin 3-O-rutinoside, delphinidin-3-O- [6-O- (4-O-p-coumaroyl-alpha-L-rhamnosyl-pyranosyl) -beta-D-glucopyranoside ] -5-O- [ beta-D-glucopyranoside ], delphinidin-3-O-p-coumaroyl rutinoside, delphinidin-3-p-coumaric acid-O-glucoside, delphinidin-3-O-glucoside, delphinidin-diglucoside, malvidin-rutinoside, malvidin-3-O-p-coumaroyl rutinoside-5-O-glucoside, malvidin-3-O-rutinoside (feruloyl) -5-O-glucoside, malvidin-3-O-rutinoside (glucoside-cis-p-coumaroyl) -5-O-glucoside, malvidin-3-O-rutinoside-5-O-glucoside, malvidin-3-p-coumaric acid-sophoroside-7-O-glucoside, green delphinidin-3, 5-O-diglucoside, peoniflorin-3-O-p-coumaroyl-rutinoside-5-0 glucoside, cyanidin-3-p-coumaric acid-O-glucoside and cyanidin galactoside.
Further, in the step S3, various anthocyanidins are quantitatively analyzed, and substances satisfying FC not less than 2 or not more than 0.5 and VIP value not less than 1 are screened to obtain specific metabolites.
Further, in the step S3, the specific metabolite is a petunidin derivative.
Further, the petunidin derivative comprises: petunia 3, 5-diglucoside, petunia-3-O-rutinoside-5-O-glucoside, petunia pigment, petunia 3-O- [6-O- (4-O-p-caffeoyl-alpha-L-rhamnosyl pyranosyl) -beta-D-glucopyranoside ] -5-O- [ beta-D-glucopyranoside, petunia 3-O-p-coumaroyl-rutinoside, petunia-3-O-p-coumaroyl-rutinoside-5-O-glucoside, petunia-3-O-rutinoside (feruloyl) -5-O-glucoside, and pharmaceutically acceptable salts thereof, Petunidin-3-p-coumaric acid-sophoroside-5-O-glucoside, petunidin-3-p-coumaric acid-sophoroside-7-O-glucoside and petunidin-3-p-coumaric acid-sophoroside-7-glucoside.
Compared with the prior art, the invention has the beneficial effects that: (1) the method provides a foundation for the subsequent deeper research on specific substances with light and dark colors of the medlar, and simultaneously provides convenience for the research on subsequent metabolites and the breeding research; (2) the accuracy of anthocyanin detection is improved by exploring the test conditions of ultra-high performance liquid chromatography-mass spectrometry; (3) the method for identifying the metabolites related to the colors of the Chinese wolfberry fruits is provided, and an anthocyanin database is constructed, so that a material basis is provided for the future research, and a research idea is provided for the future identification of the metabolites related to the colors of the plants.
Drawings
FIG. 1 is a photograph of a dark color Lycium barbarum color;
fig. 2 is a picture of the color of light-colored lycium barbarum.
Detailed Description
The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.
The color difference of the lycium barbarum is an important index of the anthocyanin metabolite difference, and the current research of technicians in the field only stays in the difference of metabolite groups of various lycium barbarum, for example, in the previous research, more qualitative research and quantitative research are carried out on anthocyanin of lycium ruthenicum, the fact that the content of petunidin in the lycium ruthenicum is higher in the lycium ruthenicum is also found in the research process, and the petunidin is used as a specific substance of the lycium ruthenicum. However, the research of the invention finds that even other varieties can present dark color systems under the condition of high content of the petunidin, the differences are not on the varieties, and the influence of a single factor on a certain variety is considered to be possibly limited.
Example 1
Pretreatment experiment of medlar
Optimizing an extraction solvent, optimizing a material-liquid ratio, optimizing extraction time and the like, extracting more medlar anthocyanin by comparing different pretreatment methods, and carrying out a cross experiment in the following variables to select an optimal extraction condition.
TABLE 1 different volume percentages of methanol solvent
TABLE 2 different feed-to-liquid ratios
| Different feed-liquid ratios (g/ml) | 1:10 | 1:20 | 1:30 | 1:40 | 1:50 |
TABLE 3 different ultrasound times
| Different ultrasonic time (min) | 5 | 10 | 20 | 30 | 60 |
TABLE 4 different ultrasound temperatures
| Different ultrasound temperatures (. degree. C.) | 20 | 30 | 40 | 50 | 60 |
The optimization steps are as follows:
1.1, taking methanol as an extracting agent, and investigating the extracting effect of different methanol volume percentages (10-90%) on the petunidin compounds. And simultaneously fixing the mass concentration ratio (1:10g/mL) and the ultrasonic time (10min) of the feed liquid, carrying out ultrasonic treatment at room temperature, and carrying out UPLC-MS/MS detection after extraction. The results are shown in Table 5:
TABLE 5 different methanol volume percent ion chromatogram peak areas
1.2 according to the results, the volume percent of the methanol is selected to be 70%, and the material-liquid ratio, the ultrasonic time and the ultrasonic temperature are optimized in the same way. By optimizing the conditions, the optimal extraction result of the substances can be obtained when the methanol concentration is 70%, the feed liquid mass concentration ratio is 1:10g/mL, the ultrasonic treatment is carried out for 10min and the ultrasonic treatment temperature is 40 ℃, and the detection result of the UPLC-MS/MS is shown in Table 6:
TABLE 6 ion chromatogram peak area at 70% by volume methanol test conditions
Example 2
Liquid phase and mass spectrum conditions of fructus Lycii anthocyanidin
The data acquisition instrument system mainly comprises Ultra Performance Liquid Chromatography (UPLC) and Tandem mass spectrometry (MS/MS).
Chromatographic conditions are as follows:
(1) a chromatographic column: waters acquisition UPLC HSS T3C 181.8 μm, 2.1mm 100 mm;
(2) mobile phase: a is ultrapure water, B is acetonitrile, 0.1% (volume percent) acetic acid is added;
(3) the flow rate is 0.3 ml/min; the column temperature is 40 ℃; the sample volume was 2. mu.l.
The mobile phase step elution is as follows in volume percent:
TABLE 7 elution gradient
Mass spectrum conditions:
TABLE 8 Mass Spectrometry parameters
| Ion mode | Positive ion scanning | Air pressure of air curtain | 40psi |
| Voltage of ion spray | 5500V | Temperature of | 550℃ |
| Ion source gas 1 | 50psi | Ion source gas 2 | 60psi |
| Air for collision | Medium | Scanning mode | MRM |
| Inlet voltage | 10V | Outlet voltage of collision cell | 10V |
Example 3
17 medlar germplasm with different fruit colors are selected, wherein the medlar germplasm comprises 8 medlar with dark fruit color and 9 medlar with light fruit color.
Qualitative and quantitative detection is carried out on the anthocyanin content of the medlar fruits by utilizing the embodiment 1 and the embodiment 2, standard qualification is carried out on inquired anthocyanins, manual spectrum decomposition is carried out on unknown anthocyanins and ion chromatographic peaks are positioned, and 32 anthocyanins metabolites are identified in total, as shown in a table 9; subsequently, samples were numbered 1-17, respectively, as shown in Table 10; and obtaining retention time and peak area of the substances through peak identification, peak alignment, peak matching, normalization and the like, calculating a VIP value by using simca 14.0 software, and calculating the difference multiple of the metabolite content by using excel.
And carrying out content analysis on the differential metabolites, wherein the screening standard is as follows: FC is more than or equal to 2 or less than or equal to 0.5, and VIP value is more than or equal to 1.
Meanwhile, correlation analysis is carried out on the metabolite content and the fruit color classification, and metabolites with correlation more than or equal to 0.4 are selected. By combining the two standards, the fact that the content of the petunidin metabolite is obviously related to the dark color of the medlar fruits is finally found, and the medlar fruits with the metabolite with high petunidin content often have dark color phenotypes.
32 anthocyanin metabolites in wolfberry fruit identified in Table 9
TABLE 1017 changes in petunidin content in different medlar fruit color samples
| Sample numbering | Sample name | Fruit colour | Color grouping | Area of petunidin peak |
| 1 | Lycium ruthenicum Murr | Black color | Deep colour | 1.57*106 |
| 2 | QH-13-08-06 | Black color | Deep colour | 8.00*105 |
| 3 | QH-13-08 | Dark red color | Deep colour | 6.06*105 |
| 4 | Zh-13-08-02 | Dark red color | Deep colour | 4.59*105 |
| 5 | QH-13-08-05 | Dark purple | Deep colour | 3.86*105 |
| 6 | HZ-13-01 | Purple color | Deep colour | 2.87*105 |
| 7 | Line 7 | Dark red color | Deep colour | 2.21*105 |
| 8 | Change of yellow fruit | Dark red color | Deep colour | 1.43*105 |
| 9 | West 2-161 | Orange colour | Light color | 1.31*104 |
| 10 | West 2-82 | Orange colour | Light color | 1.25*104 |
| 11 | West 11-147 | Orange colour | Light color | 1.24*104 |
| 12 | West 2-166 | Orange colour | Light color | 1.08*104 |
| 13 | West 2-143 | Orange colour | Light color | 9.28*103 |
| 14 | East 6-169 | Orange colour | Light color | 9.01*103 |
| 15 | West 2-45 | Orange colour | Light color | 7.66*103 |
| 16 | East 1-166 | Orange red | Light color | 3.96*103 |
| 17 | East 8-168 | Orange colour | Light color | 2.91*103 |
The number 2-17 is selected from wild or self-cultivated varieties, and as can be seen from the table above, the content of the petunidin derivative is high, the color of the medlar is dark, and otherwise, the color is light.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. A method for identifying a color-related metabolite of Lycium barbarum comprises the following steps:
step S1: after preprocessing medlar fruit samples of medlar varieties with different colors, extracting anthocyanin, and carrying out qualitative and quantitative detection on the anthocyanin;
step S2: analyzing anthocyanin detection data, determining various anthocyanin structures, and then performing chromatographic ion peak positioning to obtain a wolfberry anthocyanin database;
and step S3, carrying out quantitative analysis according to anthocyanidin of different varieties of wolfberry fruits, and screening to obtain specific metabolites related to the color shade of the wolfberry fruits.
2. The method of claim 1, wherein in step S1, qualitative and quantitative detection of anthocyanin is performed by UPLC-MS/MS detection.
3. The method for identifying color-related metabolites of Lycium barbarum as claimed in claim 1, wherein in step S1, the method comprises the steps of pre-treating a sample of Lycium barbarum fruit, adding 10-90% by volume of methanol as an extraction reagent, and extracting the anthocyanin derivative under conditions of a feed liquid mass concentration ratio of 1 (10-50), ultrasonic treatment for 5-60 min, and an ultrasonic temperature of 20-60 ℃.
4. The method for identifying color-related metabolites of Lycium barbarum as claimed in claim 1 or 3, wherein in step S1, the sample of Lycium barbarum fruit is pretreated and then methanol with a volume percentage of 70% is added as an extraction reagent, and the anthocyanin derivative is extracted under conditions of a feed liquid mass concentration ratio of 1:10, ultrasonic treatment for 10min and an ultrasonic temperature of 40 ℃.
5. The method according to claim 3, wherein in step S1, the mass spectrometric parameters of the HPLC-MS are:
6. the method according to claim 3, wherein in step S1, the chromatographic test parameters of the HPLC-MS are as follows:
a chromatographic column: c181.8 μm, 2.1mm x 100 mm;
mobile phase: the phase A is an ultrapure water solution of 0.1 percent by mass of acetic acid, and the phase B is an acetonitrile solution of 0.1 percent by mass of acetic acid; the flow rate is 0.3 ml/min; the column temperature is 40 ℃; the sample volume is 2 mul;
the mobile phase step elution is as follows in volume percent:
7. the method of claim 1, wherein in step S2, the anthocyanin species in the database include: petunia 3, 5-diglucoside, petunia-3-O-rutinoside-5-O-glucoside, petunia pigment, petunia 3-O- [6-O- (4-O-p-caffeoyl-alpha-L-rhamnosyl pyranosyl) -beta-D-glucopyranoside ] -5-O- [ beta-D-glucopyranoside, petunia 3-O-p-coumaroyl-rutinoside, petunia-3-O-p-coumaroyl-rutinoside-5-O-glucoside, petunia-3-O-rutinoside (feruloyl) -5-O-glucoside, and pharmaceutically acceptable salts thereof, Petunidin-3-p-coumaric acid-sophoroside-5-O-glucoside, petunidin-3-p-coumaric acid-sophoroside-7-O-glucoside, petunidin-3-p-coumaric acid-sophoroside-7-glucoside, delphinidin-hexoside, delphinidin 3-sophoroside-5-rhamnoside, delphinidin 3-O-p-coumaroyl-glucoside, delphinidin 3-O-rutinoside-5-O-glucoside, delphinidin 3-O-rutinoside, delphinidin-3-O- [6-O- (4-O-p-coumaroyl-alpha-L-rhamnosyl-pyranosyl) -beta-D-glucopyranoside ] -5-O- [ beta-D-glucopyranoside ], delphinidin-3-O-p-coumaroyl rutinoside, delphinidin-3-p-coumaric acid-O-glucoside, delphinidin-3-O-glucoside, delphinidin-diglucoside, malvidin-rutinoside, malvidin-3-O-p-coumaroyl rutinoside-5-O-glucoside, malvidin-3-O-rutinoside (feruloyl) -5-O-glucoside, malvidin-3-O-rutinoside (glucoside-cis-p-coumaroyl) -5-O-glucoside, malvidin-3-O-rutinoside-5-O-glucoside, malvidin-3-p-coumaric acid-sophoroside-7-O-glucoside, green delphinidin-3, 5-O-diglucoside, peoniflorin-3-O-p-coumaroyl-rutinoside-5-0 glucoside, cyanidin-3-p-coumaric acid-O-glucoside and cyanidin galactoside.
8. The method for identifying color-related metabolites of Lycium barbarum as claimed in claim 1, wherein in step S3, each anthocyanin is quantitatively analyzed, and substances satisfying FC > 2 or < 0.5 and VIP > 1 are screened to obtain specific metabolites.
9. The method of claim 1, wherein in step S3, the specific metabolite is a petunidin derivative.
10. The method of claim 9, wherein the petunidin derivative comprises: petunia 3, 5-diglucoside, petunia-3-O-rutinoside-5-O-glucoside, petunia pigment, petunia 3-O- [6-O- (4-O-p-caffeoyl-alpha-L-rhamnosyl pyranosyl) -beta-D-glucopyranoside ] -5-O- [ beta-D-glucopyranoside, petunia 3-O-p-coumaroyl-rutinoside, petunia-3-O-p-coumaroyl-rutinoside-5-O-glucoside, petunia-3-O-rutinoside (feruloyl) -5-O-glucoside, and pharmaceutically acceptable salts thereof, Petunidin-3-p-coumaric acid-sophoroside-5-O-glucoside, petunidin-3-p-coumaric acid-sophoroside-7-O-glucoside and petunidin-3-p-coumaric acid-sophoroside-7-glucoside.
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