CN110455964A - The analysis of Coumarins ingredient and identification method in decomposite leaf Radix Angelicae Pubescentis alcohol extract - Google Patents
The analysis of Coumarins ingredient and identification method in decomposite leaf Radix Angelicae Pubescentis alcohol extract Download PDFInfo
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Abstract
The present invention provides the analysis of Coumarins ingredient and identification method in decomposite leaf Radix Angelicae Pubescentis alcohol extract, belongs to chemical composition analysis technical field.Step is S1: extracting the effective component in decomposite leaf Radix Angelicae Pubescentis medicinal material using the method for ethyl alcohol heating ultrasonic extraction, decomposite leaf Radix Angelicae Pubescentis test solution is made；Coumarins standard items are weighed, standard solution is configured to；S2: test solution and standard solution are detected using ultra performance liquid chromatography-quadrupole time-of-flight mass spec-trometry method；S3: analyzing mass spectrographic data using PeakView1.2 software, by database rapid screening and second mass analysis, from a variety of Coumarins ingredients of decomposite leaf Radix Angelicae Pubescentis alcohol extract Rapid identification.The present invention isolates and identifies 32 coumarin kind compounds from decomposite leaf Radix Angelicae Pubescentis, realizes more comprehensive and system the analysis of decomposite leaf Radix Angelicae Pubescentis coumarin, provides basis for the qualitative analysis of Coumarins ingredient and decomposite leaf Radix Angelicae Pubescentis pharmacological research.
Description
Technical field
The present invention relates to chemical composition analysis technical fields, and in particular to Coumarins ingredient in decomposite leaf Radix Angelicae Pubescentis alcohol extract
Analysis and identification method.
Background technique
Cumarin is the secondary metabolites being distributed widely in plant kingdom, often the cinnamic acid close with source of students, flavonoids,
The associations such as lignanoid, are distributed widely in higher plant, especially Umbelliferae, Rutaceae, composite family, pulse family, orchid family, Solanaceae, auspicious
In the plants such as fragrant section, Saxifragaceae and Oleaceae and microbial metabolic products.Cumarin has coordinate plant growth, antibacterial disease-resistant
Poison, relaxing smooth muscle, absorbs ultraviolet light and the multiple biological activities such as anti-radiation at anticoagulation.So far it is planted from Rutaceae both citrus
A variety of coumarin kind compounds are isolated in object.Umbelliferae Radix Angelicae Pubescentis platymiscium decomposite leaf Radix Angelicae Pubescentis Heracleum millefolium
Diels, the entitled Ba Mubao of Tibetan medicine, and have alias Chiba Radix Angelicae Pubescentis, Tibetan Angelica, it is distributed mainly on China Tibet, Qinghai, Gansu, four
River, Yunnan are a kind of common Tibetan medicines.Decomposite leaf Radix Angelicae Pubescentis nature and flavor are pungent, bitter, tepor, for treating wind-cold-dampness arthralgia, lumbocrural pain, Shao Yin
Lie prostrate wind headache, lumbocrural pain, the diseases such as headache dentalgia.Wherein, main active constituent is therefore cumarin is built in decomposite leaf Radix Angelicae Pubescentis
Stand it is a kind of efficiently quickly extracting and developing purifying and identification decomposite leaf Radix Angelicae Pubescentis in cumarin method for deepen decomposite leaf Radix Angelicae Pubescentis medicine
It manages Effect study and improves quality control system and all have important meaning.
It is relatively more to the research of Radix Angelicae Pubescentis both at home and abroad at present, it is relatively fewer to the research of decomposite leaf Radix Angelicae Pubescentis.Rich Kaohsiung etc. passes through column
Chromatography and infrared spectrophotometer are separated from decomposite leaf Radix Angelicae Pubescentis ethanol extract identifies 11 compounds, only identifies 6 kinds of perfume (or spice)
Legumin class chemical component still lacks grind to the system research and pharmacological action of Coumarins chemical component in decomposite leaf Radix Angelicae Pubescentis at present
Study carefully.Ultra performance liquid chromatography-quadrupole time-of-flight mass spec-trometry combination (UHPLC-Q-TOF/MS) technology has high-resolution, height
Sensitive qualitative analysis ability and powerful structural characterization ability, extensive utilization to chemical composition of Chinese materia medica analysis quick separating with
In identification.But at present in the analysis and identification method of Coumarins ingredient in decomposite leaf angelol extract, there are no apply the skill
How the relevant report of art sets chromatography and mass spectrographic condition, while efficiently separating a variety of Coumarins ingredients, and carry out to it
Identification, is current urgent problem to be solved.
Summary of the invention
Goal of the invention of the invention is: in view of the above problems, providing fragrant in a kind of decomposite leaf Radix Angelicae Pubescentis alcohol extract
The analysis of legumin constituents and identification method isolate and identify 32 coumarin kind compounds from decomposite leaf Radix Angelicae Pubescentis, realize
Decomposite leaf Radix Angelicae Pubescentis coumarin more comprehensively and the analysis of system, for the qualitative analysis of Coumarins ingredient and decomposite leaf Radix Angelicae Pubescentis pharmacological research
Basis is provided.
To achieve the goals above, The technical solution adopted by the invention is as follows:
The analysis of Coumarins ingredient and identification method in decomposite leaf Radix Angelicae Pubescentis alcohol extract, comprising the following steps:
S1: the effective component in decomposite leaf Radix Angelicae Pubescentis medicinal material is extracted using the method for ethyl alcohol heating ultrasonic extraction, decomposite leaf Radix Angelicae Pubescentis is made and supplies
Test sample solution；Coumarins standard items are weighed, standard solution is configured to；
S2: test solution and standard solution are carried out using ultra performance liquid chromatography-quadrupole time-of-flight mass spec-trometry method
Detection；
S3: analyzing mass spectrographic data using PeakView1.2 software, passes through database rapid screening and second order ms point
Analysis, from a variety of Coumarins ingredients of decomposite leaf Radix Angelicae Pubescentis alcohol extract Rapid identification.
In the present invention, it is preferable that the specific practice in the step S1 are as follows:
The preparation of S1.1 test sample stock solution: decomposite leaf Radix Angelicae Pubescentis pulverizing medicinal materials are crossed into 80-120 mesh, precision weighs 5g, according to solid-liquid
Than the alcohol solution dipping 20min-60min for being 70% with volumetric concentration for 1:10-12, it is then heated to 45-50 DEG C, using super
Sound extracts 1-2h, then filters to take filtrate；Filter residue is repeated into aforesaid operations, merges 2 filtrates, is concentrated into no alcohol taste, obtains medicinal extract；
Medicinal extract is settled to 25mL with volumetric concentration for 50% methanol solution, obtains test sample stock solution, 4 DEG C of storages are spare；S1.2 standard items
The preparation of stock solution: before precision weighs psoralen, scopolactone, bergapten, Imperatorin, Osthole, oxidation
Seven kinds of Hu Su, angeloyloxy-columbianetin each 1mg of standard items are separately added into the methanol that 5mL volumetric concentration is 50% and dissolve,
It is 200 μ g/mL standard items stock solutions that mass concentration, which is made, and 4 DEG C of storages are spare；
The preparation of S1.3 test solution and standard solution: taking above-mentioned test sample stock solution and each 1mL of standard items stock solution, point
It is not transferred in 2mL centrifuge tube, is put into low-temperature and high-speed centrifuge with 13000rmin-1It is centrifuged 8-15min, is taken respectively after centrifugation
0.5mL supernatant in the sample injection bottle of 1.5mL to get decomposite leaf Radix Angelicae Pubescentis test solution and standard solution.
In the present invention, it is preferable that in the step S2, using ultra performance liquid chromatography-quadrupole time-of-flight mass spec-trometry side
Method respectively detects decomposite leaf Radix Angelicae Pubescentis test solution and standard solution, and determination condition is as follows:
Chromatographic condition: chromatographic column specification be 2.1 × 100mm, 1.7 μm；40 DEG C of column temperature, sample volume 4 μ L, flow velocity 0.25mL/min；
Using 0.1% aqueous formic acid as mobile phase A, gradient elution, the program of elution are carried out by Mobile phase B of acetonitrile are as follows: 0~10min,
5%~15%B；10~20min, 15%~25%B；20~30min, 25%~45%B；30~32min, 45%~47%B；32~
35min, 47%B；35~40min, 47%~50%B；40~45min, 50%~55%B；45~50min, 55%~95%B；50~
52min, 95%B；52~52.1min, 95%~5%B；52.1~55min, 5%B；
Mass Spectrometry Conditions: ESI electric spray ion source, positive ion mode detection are used；Mass scan range m/z100~1000, it is spraying
Voltage 5.5kV, removes 100 eV of cluster voltage, collision energy 45eV by 500 DEG C of ion source temperature, and collision energy is superimposed 15eV, gas curtain gas
Pressure 40psi, atomization gas and assist gas pressure power are 50psi, data acquisition time 55min, using TOF-MS-IDA-MS/MS
Mode acquires data, and IDA setting response is more than that 6 tops of 100cps carry out second order ms scanning, and daughter ion scans model
M/z:50~1000 are enclosed, the preferential carry out second level scanning of the condition is met, open dynamic background deduction；
Obtain total ion current figure, first mass spectrometric information and the second order ms information of compound.
In the present invention, it is preferable that in the step S3, by database rapid screening and second mass analysis, from decomposite leaf
The a variety of Coumarins ingredients of Radix Angelicae Pubescentis alcohol extract Rapid identification method particularly includes: using PeakView1.2 software to mass spectrographic
Data are analyzed；According to domestic and international specialized database Pubmed, ChemSpider, Metlin, Chinese Academy of Sciences's specialty chemical data
It is broken to collect coumarin kind compound title, molecular formula and second level for library and domestic and international decomposite leaf Radix Angelicae Pubescentis and cumarin research papers
Piece information tentatively establishes decomposite leaf Radix Angelicae Pubescentis coumarin class chemical composition data library, preliminary to sieve in conjunction with software XIC Manager function
Mass deviation is selected less than 5 × 10-6Coumarinoids, then by the ingredient filtered out further in reference substance and document two
Grade fragment compares, and identifies and deduce altogether a variety of coumarin kind compounds from decomposite leaf Radix Angelicae Pubescentis alcohol extract.
In conclusion by adopting the above-described technical solution, the beneficial effects of the present invention are: the present invention uses UHPLC-Q-
TOF/MS, by the optimization to chromatographic condition and Mass Spectrometry Conditions, establishing one kind in the positive-ion mode can efficiently separate simultaneously
The method of a variety of Coumarins ingredients, it is fast from decomposite leaf Radix Angelicae Pubescentis alcohol extract by database rapid screening and second mass analysis
Speed identifies 32 kinds of Coumarins ingredients, and this method has many advantages, such as quick, sensitive and high-resolution, can be used as decomposite leaf Radix Angelicae Pubescentis matter
One of the method for measuring control, while also important references are provided for the research of decomposite leaf Radix Angelicae Pubescentis effective substance and clinical pharmacy research.
Detailed description of the invention
Reference substance total ion current figure under Fig. 1 positive ion mode.
Total ion current figure under Fig. 2 decomposite leaf Radix Angelicae Pubescentis alcohol extracting thing positive ion mode.
Fig. 3 psoralen may lytic pathway and MS/MS.
Fig. 4 bergapten may lytic pathway and MS/MS.
Fig. 5 oxypeucedanin may lytic pathway and MS/MS.
Specific embodiment
In order to more clearly express the present invention, below by way of specific embodiment, the invention will be further described.
One, instrument and material.
1.1 instruments: 5600 type high-resolution mass spectrometer of TripleTOF (American AB SCIEX company)；LC-30AD
Type Ultra Performance Liquid Chromatography instrument (Japanese Shimadzu Corporation)；(Town in Shanghai pavilion scientific instrument are public for TGL-16B type low-temperature and high-speed centrifuge
Department)；BT-125D type electronic balance (German Sartorius company)；SZ-93 type dual pure water distiller (Shanghai Asia Rong Sheng automatically
Change instrument plant)；The ultrapure water purification system of Milli-Q Synthesis (U.S.'s Millipore Products)；KQ-3200DE type
Cnc ultrasonic cleaning instrument (Kunshan Ultrasonic Instruments Co., Ltd.)；DZTW regulating temperature electrothermal cover (Beijing's bright Medical Instruments forever
Co., Ltd)；Water is ultrapure water, and methanol, acetonitrile are that mass spectrum is pure (ACS company, the U.S.)；Formic acid is the pure (U.S. Thermo of mass spectrum
Fisher company)；Other reagents are to analyze pure (Xilong Chemical Co., Ltd).
1.2 materials: psoralen (lot number: FY1031B1332), scopolactone (lot number: FY1155B4449), fingered citron
Mandarin orange lactone (lot number: FY1232B1304), Imperatorin (lot number: FY1495B1859), Osthole (lot number: 110822-
201308), oxypeucedanin (FY2856B1050), angeloyloxy-columbianetin (lot number: FY1223B2424) reference substance
Purchased from Chinese food drug identification research institute and Nantong Fei Yu Biotechnology Co., Ltd.Decomposite leaf Radix Angelicae Pubescentis medicinal material is purchased in Xi Zangshan
The southern area Gongga County town Gang Dui, is identified by pharmaceutical college, Jiangxi University of Traditional Chinese Medicine professor Gong Qianfeng.
Two, the analysis of Coumarins chemical component and identification method in decomposite leaf Radix Angelicae Pubescentis alcohol extract.
Embodiment 1
In present embodiment, the analysis of Coumarins ingredient and identification method in decomposite leaf Radix Angelicae Pubescentis alcohol extract, comprising the following steps:
S1: the effective component in decomposite leaf Radix Angelicae Pubescentis medicinal material is extracted using the method for ethyl alcohol heating ultrasonic extraction, decomposite leaf Radix Angelicae Pubescentis is made and supplies
Test sample solution；Coumarins standard items are weighed, standard solution is configured to；Specific steps are as follows:
The preparation of S1.1 test sample stock solution: decomposite leaf Radix Angelicae Pubescentis pulverizing medicinal materials are crossed into 120 meshes, precision weighs 5g, according to solid-to-liquid ratio
The alcohol solution dipping 20min for being 70% with volumetric concentration for 1:10, is then heated to 45 DEG C, using ultrasonic extraction 1h, subsequent mistake
Leaching filtrate；Filter residue is repeated into aforesaid operations, merges 2 filtrates, is concentrated into no alcohol taste, obtains medicinal extract；By medicinal extract volumetric concentration
It is settled to 25mL for 50% methanol solution, obtains test sample stock solution, 4 DEG C of storages are spare；
The preparation of S1.2 standard items stock solution: precision weighs psoralen, scopolactone, bergapten, Imperatorin, snake
Seven kinds of machine tool element, oxypeucedanin, angeloyloxy-columbianetin each 1mg of standard items, being separately added into 5mL volumetric concentration is
50% methanol dissolution, it is 200 μ g/mL standard items stock solutions that mass concentration, which is made, and 4 DEG C of storages are spare；
The preparation of S1.3 test solution and standard solution: taking above-mentioned test sample stock solution and each 1mL of standard items stock solution, point
It is not transferred in 2mL centrifuge tube, is put into low-temperature and high-speed centrifuge with 13000rmin-1It is centrifuged 8min, is taken respectively after centrifugation
0.5mL supernatant in the sample injection bottle of 1.5mL to get decomposite leaf Radix Angelicae Pubescentis test solution and standard solution；
S2: test solution and standard solution are carried out using ultra performance liquid chromatography-quadrupole time-of-flight mass spec-trometry method
Detection；
Chromatographic condition: chromatographic column specification be 2.1 × 100mm, 1.7 μm；40 DEG C of column temperature, sample volume 4 μ L, flow velocity 0.25mL/min；
Using 0.1% aqueous formic acid as mobile phase A, gradient elution, the program of elution are carried out by Mobile phase B of acetonitrile are as follows: 0~10min,
5%~15%B；10~20min, 15%~25%B；20~30min, 25%~45%B；30~32min, 45%~47%B；32~
35min, 47%B；35~40min, 47%~50%B；40~45min, 50%~55%B；45~50min, 55%~95%B；50~
52min, 95%B；52~52.1min, 95%~5%B；52.1~55min, 5%B；
Mass Spectrometry Conditions: ESI electric spray ion source, positive ion mode detection are used；Mass scan range m/z100~1000, it is spraying
Voltage 5.5kV, removes 100 eV of cluster voltage, collision energy 45eV by 500 DEG C of ion source temperature, and collision energy is superimposed 15eV, gas curtain gas
Pressure 40psi, atomization gas and assist gas pressure power are 50psi, data acquisition time 55min, using TOF-MS-IDA-MS/MS
Mode acquires data, and IDA setting response is more than that 6 tops of 100cps carry out second order ms scanning, and daughter ion scans model
M/z:50~1000 are enclosed, the preferential carry out second level scanning of the condition is met, open dynamic background deduction；
After mass spectral analysis, the total ion current figure, first mass spectrometric information and second order ms information of compound are obtained；
S3: analyzing mass spectrographic data using PeakView1.2 software, passes through database rapid screening and second order ms point
Analysis, from a variety of Coumarins ingredients of decomposite leaf Radix Angelicae Pubescentis alcohol extract Rapid identification.Specifically: being confronted using PeakView1.2 software
The data of spectrum are analyzed；According to domestic and international specialized database Pubmed, ChemSpider, Metlin, Chinese Academy of Sciences's specialty chemical
Database and domestic and international decomposite leaf Radix Angelicae Pubescentis and cumarin research papers collect coumarin kind compound title, molecular formula and two
Grade fragment information, tentatively establishes decomposite leaf Radix Angelicae Pubescentis coumarin class chemical composition data library, in conjunction with software XIC Manager function, just
Step filters out mass deviation less than 5 × 10-6Coumarinoids, then by the ingredient filtered out further with reference substance and document
Middle secondary fragment compares, and identifies and deduce altogether a variety of coumarin kind compounds from decomposite leaf Radix Angelicae Pubescentis alcohol extract.
Embodiment 2
In present embodiment, the analysis of Coumarins ingredient and identification method in decomposite leaf Radix Angelicae Pubescentis alcohol extract, comprising the following steps:
S1: the effective component in decomposite leaf Radix Angelicae Pubescentis medicinal material is extracted using the method for ethyl alcohol heating ultrasonic extraction, decomposite leaf Radix Angelicae Pubescentis is made and supplies
Test sample solution；Coumarins standard items are weighed, standard solution is configured to；Specific steps are as follows:
The preparation of S1.1 test sample stock solution: decomposite leaf Radix Angelicae Pubescentis pulverizing medicinal materials are crossed into 80 meshes, precision weighs 5g, is according to solid-to-liquid ratio
The ethanol solution that 1:12 volumetric concentration is 70% soaks 60min, is then heated to 50 DEG C, using ultrasonic extraction 2h, then filters to take
Filtrate；Filter residue is repeated into aforesaid operations, merges 2 filtrates, is concentrated into no alcohol taste, obtains medicinal extract；It is 50% by medicinal extract volumetric concentration
Methanol solution is settled to 25mL, obtains test sample stock solution, and 4 DEG C of storages are spare；
The preparation of S1.2 standard items stock solution: precision weighs psoralen, scopolactone, bergapten, Imperatorin, snake
Seven kinds of machine tool element, oxypeucedanin, angeloyloxy-columbianetin each 1mg of standard items, being separately added into 5mL volumetric concentration is
50% methanol dissolution, it is 200 μ g/mL standard items stock solutions that mass concentration, which is made, and 4 DEG C of storages are spare；
The preparation of S1.3 test solution and standard solution: taking above-mentioned test sample stock solution and each 1mL of standard items stock solution, point
It is not transferred in 2mL centrifuge tube, is put into low-temperature and high-speed centrifuge with 13000rmin-1It is centrifuged 15min, is taken respectively after centrifugation
0.5mL supernatant in the sample injection bottle of 1.5mL to get decomposite leaf Radix Angelicae Pubescentis test solution and standard solution；
S2: by test solution and standard solution using ultra performance liquid chromatography-quadrupole time-of-flight mass spec-trometry method into
Row detection；
Chromatographic condition and Mass Spectrometry Conditions are same as Example 1.After mass spectral analysis, obtain compound total ion current figure, one
Grade Information in Mass Spectra and second order ms information；
S3: analyzing mass spectrographic data using PeakView1.2 software, passes through database rapid screening and second order ms point
Analysis, from a variety of Coumarins ingredients of decomposite leaf Radix Angelicae Pubescentis alcohol extract Rapid identification.Specifically: being confronted using PeakView1.2 software
The data of spectrum are analyzed；According to domestic and international specialized database Pubmed, ChemSpider, Metlin, Chinese Academy of Sciences's specialty chemical
Database and domestic and international decomposite leaf Radix Angelicae Pubescentis and cumarin research papers collect coumarin kind compound title, molecular formula and two
Grade fragment information, tentatively establishes decomposite leaf Radix Angelicae Pubescentis coumarin class chemical composition data library, in conjunction with software XIC Manager function, just
Step filters out mass deviation less than 5 × 10-6Coumarinoids, then by the ingredient filtered out further with reference substance and document
Middle secondary fragment compares, and identifies and deduce altogether a variety of coumarin kind compounds from decomposite leaf Radix Angelicae Pubescentis alcohol extract.
Three, compound mass spectral analyses and identification.
(1) coumarin kind compound cracks law-analysing
Psoralen, scopolactone, bergapten, Imperatorin, Osthole, oxypeucedanin, Columbianetin
Seven kinds of standard items total ion current figures of angelate are shown in Fig. 1, and decomposite leaf extractive of pubescent angelica root coumarin total ion current figure is shown in Fig. 2.The present invention is with a variety of
Cumarin standard items are reference, and the cracking rule of coumarin kind compound is parsed by taking psoralen as an example, see Fig. 3, psoralea corylifolia
Quasi-molecular ion 187 [M+H] is obtained under plain positive ion mode+, on the one hand, 159 [M+H- are formed by sloughing the CO on pyranoid ring
CO]+Then fragment ion continuously sloughs CO, successively form 131 [M+H-2CO]+With 103 [M+H-3CO]+；On the other hand, may be used
By sloughing the CO on pyranoid ring2, form 143 [M+H-CO2]+, this ion continue to take off the CO on furan nucleus generate 115 [M+H-
CO2-CO]+.Similarly it could be speculated that bergapten, which passes through, first sloughs methyl on side chain, continuously slough CO afterwards, form 202,174,
146 equal fragment ions, can also directly slough CO, form the fragment ions such as 189,161, referring to fig. 4；Oxypeucedanin first sloughs side
Chain group (C5H8O), 203 [M+H-C of fragment ion is formed5H8O]+, this ion continues to slough the CO on pyranoid ring2Or on furan nucleus
CO form the fragment ions such as 159,131,175,147, referring to Fig. 5.According to the above-mentioned cracking of coumarin kind compound rule, knot
Mass-to-charge ratio, retention time, fragments characteristic ion and pertinent literature report are closed, identifies 32 kinds altogether from decomposite leaf extractive of pubescent angelica root coumarin
Coumarins ingredient (details are shown in Table 1).
(2) the specific identification method of each compound is as follows:
Reference substance coumarinoids authenticating compound 3,12,16,21,25,27,30 respectively with scopolactone, psoralen,
Bergapten, oxypeucedanin, Imperatorin, Osthole, seven kinds of cumarin reference substances of angeloyloxy-columbianetin
Retention time is consistent with fragment ion, therefore is accredited as this seven kinds of cumarins respectively, sees Fig. 2.
Isomer coumarinoids authenticating compound 13 and psoralen molecular weight having the same and fragment from
Son, retention time is close, and polarity is small compared with psoralen, therefore is accredited as Isopsoralen.In compound 14,20 and bergamot
Ester molecule amount and fragment ion are identical, therefore are accredited as isomer, and according to the literature, bergapten has xanthotoxin
With two kinds of isomers of isobergapten, and xanthotoxin polarity is maximum, and isobergapten polarity is minimum, therefore according to guarantor
The time is stayed, compound 14,20 is accredited as xanthotoxin and isobergapten respectively.Compound 8,19 and oxypeucedanin have
There are identical molecular weight and similar fragment ion, according to the literature, gosferol and different oxypeucedanin are before aoxidizing
Two kinds of isomers of Hu Su, and gosferol polarity is big compared with different oxypeucedanin polarity, therefore compound 8,19 is distinguished
It is accredited as gosferol and different oxypeucedanin.Compound 23,29 and Imperatorin molecular weight having the same and fragment
Ion, therefore it is accredited as isomer, according to the literature, Imperatoria ostruthium is known as Isomperatorin and two kinds of Alloisoimperatorin
Isomer, and Alloisoimperatorin polarity is maximum, Isomperatorin polarity is minimum, therefore according to retention time, compound 23,
29 are accredited as Alloisoimperatorin and Isomperatorin respectively.
1 relative molecular mass of side-chain radical isomery Coumarins Components identification compound is 162.0317, compared in Anisodus luridus
Ester lacks 30 Da, main 145 [M+H-H of fragment ion2O]+、135 [M+H-CO]+、117 [M+H-CO-H2O]+Cracking mode
It is similar to scopolactone, it loses a methyl or methoxy less and generates, therefore speculate that compound 1 is on scopolactone basis
Upper to reduce by a methoxyl group composition, umbelliferone meets above-mentioned supposition.7 relative molecular mass of compound is 202.0266Da,
Lack 14Da compared with bergapten, fragment ion and cracking mode are similar to bergapten, all lose a methyl, and pole less
Property is big compared with bergapten, therefore speculates that its structure lacks a methyl compared with bergapten, and Xanthotoxol meets above-mentioned supposition.Chemical combination
18 relative molecular mass of object is 246.0528,30Das more compared with bergapten, 232 [M+H-CH of fragment ion3]+、189 [M+
H-2CH3-CO]+、171 [M+H-CH3-CH3O-CO]+It is similar to bergapten cracking mode, thus it is speculated that it may be by losing more
Methyl or methoxy on side chain and generate, therefore speculate that compound 18 is to increase methoxyl group on the basis of bergapten to constitute,
Isoimpinellin meets above-mentioned supposition.26 relative molecular mass of compound be 300.0998 Da, 30Das more compared with Imperatorin,
233 [M+H-C of main fragment ion5H8]+、218[M+H-C5H8-CH3]+ 、202[M+H-C5H8-CH3O]+, thus it is speculated that it may distinguish
By losing the identical side-chain radical (C of Imperatorin5H8) and methoxyl group and generate, therefore speculate compound 26 be in Imperatorin
Increase methoxyl group in structure basis to constitute, phellopterin meets above-mentioned supposition.
When other 5,10,15,17,31 relative molecular masses of Coumarins Components identification compound, fragment ion and reservation
Between sequence it is consistent with document, therefore be accredited as corresponding coumarin kind compound.Main 203 [the M+H- of fragment ion of compound 32
C10H16]+、185 [M+H-C10H16-H2O]+、175 [M+H- C10H16-CO]+、159 [M+H-C10H16-CO2]+、147[M+H-
C10H16-2CO]+First pass through fracture side chain (C10H16) fragment ion m/z 203.0332 is formed, rear dehydration forms fragment ion m/z
185.0229, it also easily sloughs the molecule CO on pyranoid ring and generates m/z 175.0386, the molecule CO on furan nucleus2It generates
159.0439, therefore authenticating compound 32 is bergamot element.
193 [M+H-C are presented in 2 quasi-molecular ion peak m/z 355.1033 of compound in second order ms figure6H10O5]+、178
[M+H-C6H10O5-CH3]+、165[M+H-C6H10O5-CO]+、150[M+H-C6H10O5-CH3-CO]+Equal fragment ions.Wherein, broken
Piece ion m/z 193.0493 is the generation of one molecule glucose of quasi-molecular ion neutral loss, other fragment ions m/z
178.0261, m/z 165.0545, m/z 150.0312 are identical as scopolactone fragment ion, in conjunction with pertinent literature, identification
Compound 2 is scopolin.
Compound 4 is identical with 6 relative molecular mass of compound, each other isomer, quasi-molecular ion peak m/z
247.0962, main fragment ion m/z 229,187,159,131 is identical as angeloyloxy-columbianetin, average molecular matter
Measure few 82 Da, thus it is speculated that may be the C of side chain connection5H8O2Fatty acid ester linkage breaking, neutral loss C5H6O is obtained, dihydro Ou Shanqin
Pure and mild Marmesin meets above-mentioned supposition.In conjunction with pertinent literature, Columbianetin polarity is bigger, compound 4 and change
It closes object 6 and is identified respectively as Columbianetin and Marmesin.
9 quasi-molecular ion peak of compound is m/z 305.1022,18Das more compared with oxypeucedanin, 203 [M+ of fragment ion
H-C5H10O2]+、175[M+H-C5H10O2-CO]+ 、159[M+H-C5H10O2-CO2]+ 、147[M+H-C5H10O2-2CO] +With oxygen
It is similar to change peucedanin cracking mode, thus it is speculated that compound 9 is to increase a hydroxyl on the basis of Hu Su before oxidation to constitute, aqua oxidation
Peucedanin meets above-mentioned supposition.
11 quasi-molecular ion peak of compound is m/z 317.1028, and C-8 bit substituent occurs hydrogen and resets, and loses neutral molecule
C5H8O formed 233 fragment ion of m/z, 233 ion of m/z further lose methyl generate 218 ion of m/z, fragment mode with
Xanthotoxin is consistent with bergapten, 218 ion of m/z continuously lose carbonyl formed the fragments such as m/z 190,162,134 from
Son, in conjunction with document, authenticating compound 11 is Byakangelicol.
22 quasi-molecular ion peak of compound is m/z 231.1013, main 187 [M+H-C of fragment ion2H4O] +、175[M+
H-2CO] +、147[M+H-C5H8O] +、119[M+H-C5H8O-CO] +、91[M+H-C5H8O-2CO] +With document osthenol
Fragment ion is consistent, therefore authenticating compound 22 is osthenol.
24 quasi-molecular ion peak of compound is m/z 203.0337, with Xanthotoxol molecular weight having the same and fragment from
Son, and polarity is smaller than Xanthotoxol, quasi-molecular ion generates 175 [M+H-CO] by continuously sloughing CO +、147[M+H-2CO] +、
119[M+H-3CO] +Equal fragment ions, slough 1 molecule CO2Generate 159 [M+H-CO2] +Fragment ion, in conjunction with pertinent literature,
Authenticating compound 24 is bergaptol.
28 quasi-molecular ion peak of compound is m/z 329.1388, relative molecular mass and angeloyloxy-columbianetin
It is identical, main 247 [M+H-C of fragment ion5H6O] +、229[M+H-C5H6O-H2O] +、213[M+H-C5H6O-H2O-CH4] +、
185[M+H-C5H6O-H2O-CO2] +It is all first neutral loss C5H6O, then lose CO2And H2O is obtained, in conjunction with pertinent literature, identification
Compound 28 is decursin.
The present invention in the positive-ion mode, using UHPLC-Q-TOF/MS to the coumarin kind compound in decomposite leaf Radix Angelicae Pubescentis into
Row separation identification, identifies altogether 32 kinds of compounds, wherein 24 are not reported in previous literature, it is that decomposite leaf Radix Angelicae Pubescentis is further
Chemical component, pharmacological research and quantitative study provide certain reference, are conducive to the development and utilization of decomposite leaf Radix Angelicae Pubescentis.
Above description is the detailed description for the present invention preferably possible embodiments, but embodiment is not limited to this hair
Bright patent claim, it is all the present invention suggested by technical spirit under completed same changes or modifications change, should all belong to
In the covered the scope of the patents of the present invention.
Claims (4)
1. the analysis of Coumarins ingredient and identification method in decomposite leaf Radix Angelicae Pubescentis alcohol extract, which comprises the following steps:
S1: the effective component in decomposite leaf Radix Angelicae Pubescentis medicinal material is extracted using the method for ethyl alcohol heating ultrasonic extraction, decomposite leaf Radix Angelicae Pubescentis is made and supplies
Test sample solution；Coumarins standard items are weighed, standard solution is configured to；
S2: test solution and standard solution are carried out using ultra performance liquid chromatography-quadrupole time-of-flight mass spec-trometry method
Detection；
S3: analyzing mass spectrographic data using PeakView1.2 software, passes through database rapid screening and second order ms point
Analysis, from a variety of Coumarins ingredients of decomposite leaf Radix Angelicae Pubescentis alcohol extract Rapid identification.
2. the analysis of Coumarins ingredient and identification method in decomposite leaf Radix Angelicae Pubescentis alcohol extract according to claim 1, special
Sign is, the specific practice in the step S1 are as follows:
The preparation of S1.1 test sample stock solution: decomposite leaf Radix Angelicae Pubescentis pulverizing medicinal materials are crossed into 80-120 mesh, precision weighs 5g, according to solid-liquid
Than the alcohol solution dipping 20min-60min for being 70% with volumetric concentration for 1:10-12, it is then heated to 45-50 DEG C, using super
Sound extracts 1-2h, then filters to take filtrate；Filter residue is repeated into aforesaid operations, merges 2 filtrates, is concentrated into no alcohol taste, obtains medicinal extract；
Medicinal extract is settled to 25mL with volumetric concentration for 50% methanol solution, obtains test sample stock solution, 4 DEG C of storages are spare；
The preparation of S1.2 standard items stock solution: precision weighs psoralen, scopolactone, bergapten, Imperatorin, snake
Seven kinds of machine tool element, oxypeucedanin, angeloyloxy-columbianetin each 1mg of standard items, being separately added into 5mL volumetric concentration is
50% methanol dissolution, it is 200 μ g/mL standard items stock solutions that mass concentration, which is made, and 4 DEG C of storages are spare；
The preparation of S1.3 test solution and standard solution: taking above-mentioned test sample stock solution and each 1mL of standard items stock solution, point
It is not transferred in 2mL centrifuge tube, is put into low-temperature and high-speed centrifuge with 13000rmin-1It is centrifuged 8-15min, is taken respectively after centrifugation
0.5mL supernatant in the sample injection bottle of 1.5mL to get decomposite leaf Radix Angelicae Pubescentis test solution and standard solution.
3. the analysis of Coumarins ingredient and identification method in decomposite leaf Radix Angelicae Pubescentis alcohol extract according to claim 1, special
Sign is, in the step S2, using ultra performance liquid chromatography-quadrupole time-of-flight mass spec-trometry method respectively to decomposite leaf Radix Angelicae Pubescentis
Test solution and standard solution are detected, and determination condition is as follows:
Chromatographic condition: chromatographic column specification be 2.1 × 100mm, 1.7 μm；40 DEG C of column temperature, sample volume 4 μ L, flow velocity 0.25mL/min；
Using 0.1% aqueous formic acid as mobile phase A, gradient elution, the program of elution are carried out by Mobile phase B of acetonitrile are as follows: 0~10min,
5%~15%B；10~20min, 15%~25%B；20~30min, 25%~45%B；30~32min, 45%~47%B；32~
35min, 47%B；35~40min, 47%~50%B；40~45min, 50%~55%B；45~50min, 55%~95%B；50~
52min, 95%B；52~52.1min, 95%~5%B；52.1~55min, 5%B；
Mass Spectrometry Conditions: ESI electric spray ion source, positive ion mode detection are used；Mass scan range m/z100~1000, it is spraying
Voltage 5.5kV, removes 100 eV of cluster voltage, collision energy 45eV by 500 DEG C of ion source temperature, and collision energy is superimposed 15eV, gas curtain gas
Pressure 40psi, atomization gas and assist gas pressure power are 50psi, data acquisition time 55min, using TOF-MS-IDA-MS/MS
Mode acquires data, and IDA setting response is more than that 6 tops of 100cps carry out second order ms scanning, and daughter ion scans model
M/z:50~1000 are enclosed, the preferential carry out second level scanning of the condition is met, open dynamic background deduction；
Obtain total ion current figure, first mass spectrometric information and the second order ms information of compound.
4. the analysis of Coumarins ingredient and identification method in decomposite leaf Radix Angelicae Pubescentis alcohol extract according to claim 1, special
Sign is, in the step S3, by database rapid screening and second mass analysis, quickly reflects from decomposite leaf Radix Angelicae Pubescentis alcohol extract
Fixed a variety of Coumarins ingredients method particularly includes: mass spectrographic data are analyzed using PeakView1.2 software；According to state
Inside and outside specialized database Pubmed, ChemSpider, Metlin, Chinese Academy of Sciences's specialty chemical database and domestic and international decomposite leaf Radix Angelicae Pubescentis and
Cumarin research papers collect coumarin kind compound title, molecular formula and secondary fragment information, it is only tentatively to establish decomposite leaf
Coumarins chemical composition data library living, in conjunction with the function of software XIC Manager, preliminary screening mass deviation less than 5 ×
10-6Coumarinoids, then by the ingredient filtered out further compared with secondary fragment in reference substance and document, from splitting
A variety of coumarin kind compounds are identified and deduce altogether in leaf Radix Angelicae Pubescentis alcohol extract.
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