Method for detecting chemical components of Ainsliaea delavayi

Abstract

The invention provides a detection method and a separation method for chemical components of Ainsliaea delavayi. According to the method, 38 compounds can be separated from the root of the Aleuristolochia delavayi by combining ultra-high performance liquid chromatography with a specific mobile phase and an elution program, 16 compounds can be rapidly identified from the root of the Aleuristolochia delavayi by further adopting an analysis method that the ultra-high performance liquid chromatography is connected with a quadrupole time-of-flight mass spectrometer in series, and a result shows that the method has the advantages that the method is simple and convenient to operate; the hedyotis diffusa roots are rich in ursane type triterpenes, oleanane type triterpenes and saponins thereof, and in addition, the hedyotis diffusa roots also comprise flavonoid, flavonoid glycoside and other components, so that the hedyotis diffusa roots have very strong ecological significance and relatively high development and utilization values. According to the invention, the chemical components of the hedyotis diffusa are enriched, a foundation is laid for research on pharmacological activity and development and utilization of the hedyotis diffusa, meanwhile, a novel preparation method of 16 compounds is provided, and the sources of the compounds are enriched.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical composition analysis, and particularly relates to a method for detecting the chemical components of Otophora unilocularis. Background Art

[0002] Otophora unilocularis (Leenh.) H.S. Lo is a shrub belonging to the family Sapindaceae and the genus Otophora, and can grow up to 3 meters tall. Since it was discovered in 1935, there has been no further report. In 1997, Otophora unilocularis was listed as a vulnerable species in the IUCN Red List of Endangered Species. It was not until 2013 that Otophora unilocularis was rediscovered again, and preliminary results have been achieved in artificial breeding. As an extinct-level plant, there is currently little research on Otophora unilocularis, and it is difficult to comprehensively understand its biological characteristics. Therefore, conducting research on the related chemical components of Otophora unilocularis is of great significance for understanding the various traits of Otophora unilocularis, analyzing the reasons for its extinction, overcoming the endangered status of Otophora unilocularis, and formulating relevant protection measures for Otophora unilocularis. Summary of the Invention

[0003] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for detecting the chemical components of Otophora unilocularis, which can effectively separate and identify the chemical components in the roots of Otophora unilocularis.

[0004] The first aspect of the present invention is to provide a method for detecting the chemical components of Otophora unilocularis, taking the roots of Otophora unilocularis as the detection object and using an ultra-high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometer for determination.

[0005] The chromatographic column is a 100 mm × 2.1 mm, 1.8 μm Waters ACQUITY UPLC HSS T3 chromatographic column;

[0006] Column temperature: 38 - 42 °C;

[0007] Volume flow rate: 0.2 - 0.4 mL / min;

[0008] Mobile phase: Mobile phase A is an aqueous formic acid solution with a volume concentration of 0.05 - 0.15%; Mobile phase B is an acetonitrile solution of formic acid with a volume concentration of 0.005 - 0.015%;

[0009] The elution gradient is as follows:

[0010]

[0011]

[0012] Preferably, before using the ultra-high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry for determination, the detection object is extracted with alcohol, and the alcohol is preferably methanol and / or ethanol.

[0013] Preferably, the column temperature is 40°C.

[0014] Preferably, the volume flow rate is 0.25 - 0.35 mL / min, more preferably 0.3 mL / min.

[0015] Preferably, mobile phase A is an aqueous solution of formic acid with a volume concentration of 0.08 - 0.12%, more preferably an aqueous solution of formic acid with a volume concentration of 0.1%.

[0016] Preferably, mobile phase B is an acetonitrile solution of formic acid with a volume concentration of 0.008 - 0.012%, more preferably an acetonitrile solution of formic acid with a volume concentration of 0.01%.

[0017] Among them, the mass spectrometry conditions of the mass spectrometer are as follows: an electrospray ionization source (ESI) is used, and under the negative ion (-) condition, MS data is collected in Continuum mode. E data.

[0018] Preferably, the mass spectrometry conditions of the mass spectrometer further include: the scanning range is m / z 50 - 1200 Da, the low-energy collision voltage (CE) is 6 V, and the high-energy collision voltage is 20 - 60 V; the capillary voltage in the negative ion mode is 2.0 kv, the cone voltage is 60 V, and the cone gas flow rate is 50 L / hr.

[0019] Preferably, the mass spectrometry conditions of the mass spectrometer further include: the scanning time is 0.2 s, the detection time is 20 min; the ion source temperature is 90 - 110°C, preferably 100°C; the auxiliary spray ionization and desolvation gas is high-purity N2, the desolvation temperature is 450°C, and the desolvation gas flow rate is 600 L / hr.

[0020] Using the detection method of the present invention, 16 compounds can be identified from the roots of Otophora unilocularis (see Table 2).

[0021] The second aspect of the present invention is to provide a method for separating the chemical components of Otophora unilocularis, taking the roots of Otophora unilocularis as the separation object and using ultra-high performance liquid chromatography for separation; the conditions of the ultra-high performance liquid chromatography are as follows:

[0022] The chromatographic column is a 100 mm × 2.1 mm, 1.8 μm Waters ACQUITY UPLC HSS T3 chromatographic column;

[0023] Column temperature: 38 - 42°C;

[0024] Volume flow rate: 0.2 - 0.4 mL / min;

[0025] Mobile phase: Mobile phase A is an aqueous formic acid solution with a volume concentration of 0.05 - 0.15%; mobile phase B is an acetonitrile solution of formic acid with a volume concentration of 0.005 - 0.015%.

[0026] The elution gradient is as follows:

[0027]

[0028] Preferably, before determination by ultra-high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry, the detection object is extracted with alcohol, and the alcohol is preferably methanol and / or ethanol.

[0029] Preferably, the column temperature is 40°C.

[0030] Preferably, the volume flow rate is 0.25 - 0.35 mL / min, more preferably 0.3 mL / min.

[0031] Preferably, mobile phase A is an aqueous formic acid solution with a volume concentration of 0.08 - 0.12%, more preferably an aqueous formic acid solution with a volume concentration of 0.1%.

[0032] Preferably, mobile phase B is an acetonitrile solution of formic acid with a volume concentration of 0.008 - 0.012%, more preferably an acetonitrile solution of formic acid with a volume concentration of 0.01%.

[0033] Using the separation method of the present invention, 38 compounds can be separated from the roots of Otophora unilocularis.

[0034] The third aspect of the present invention is to provide a method for preparing a compound, which is 3-hydroxybenzoic acid, and / or cianidanol, and / or 9-hydroxyoctadecadienoic acid, and / or 12-hydroxy-8,10-heptadecadienoic acid, and / or (-)-pimaric acid, and / or testosterone butyrate, and / or ursolic acid, and / or spirost-5-ene-3-hexopyranoside, and / or 5,13-Bis(3,4-dihydroxyphenyl)-4,12,14-trioxapentacyclo[11.7.1.0~2,11~.0~3,8~.0~15,20~]heneicos-2,8,10,15,17,19-hexol, and / or luteolin-7-O-β-D-neohesperidoside, and / or (3β)-3-{[(2E)-3-(4-hydroxyphenyl)-2-propenoyl]oxy}olean-12-ene-28-oic acid, and / or (3β)-3-hydroxy-27-{[(2E)-3-(4-hydroxyphenyl)-2-propenoyl]oxy}urs-12-ene-28-oic acid, and / or (3β)-3-hydroxy-27-{[3-(4-hydroxyphenyl)acryloyl]oxy}urs-12-ene-28-oic acid, and / or 2,3-dihydroxy-23-{[(2E)-3-(4-hydroxyphenyl)-2-propenoyl]oxy}urs-12-ene-28-oic acid, and / or epigallocatechin (4β,8)-gallocatechin, and / or 2-O-(6-deoxypyranhexosyl)-1-O-{[(2beta,3beta,6beta,22beta)-3-(hydroxyhexopyranosyl)-2,6,23-trihydroxyolean-12-ene-22-yl]carbonyl}pyranopentose, and is separated from the roots of Otophora unilocularis by ultra-high performance liquid chromatography.

[0035] The conditions for ultra-high performance liquid chromatography are as follows:

[0036] The chromatographic column is a 100mm×2.1mm, 1.8μm Waters ACQUITY UPLC HSS T3 chromatographic column;

[0037] Column temperature: 38 - 42 °C;

[0038] Volume flow rate: 0.2 - 0.4 mL / min;

[0039] Mobile phase: Mobile phase A is an aqueous formic acid solution with a volume concentration of 0.05 - 0.15%; Mobile phase B is an acetonitrile solution of formic acid with a volume concentration of 0.005 - 0.015%;

[0040] The elution gradient is as follows:

[0041]

[0042] Preferably, before the determination by ultra - performance liquid chromatography - tandem quadrupole time - of - flight mass spectrometry, the test object is extracted with alcohol, and the alcohol is preferably methanol and / or ethanol.

[0043] Preferably, the column temperature is 40 °C.

[0044] Preferably, the volume flow rate is 0.25 - 0.35 mL / min, more preferably 0.3 mL / min.

[0045] Preferably, mobile phase A is an aqueous solution of formic acid with a volume concentration of 0.08 - 0.12%, more preferably an aqueous solution of formic acid with a volume concentration of 0.1%.

[0046] Preferably, mobile phase B is an acetonitrile solution of formic acid with a volume concentration of 0.008 - 0.012%, more preferably an acetonitrile solution of formic acid with a volume concentration of 0.01%.

[0047] The present invention can separate 38 compounds from the roots of Otophora unilocularis by using ultra - performance liquid chromatography combined with a specific mobile phase and elution program. Further, by using the analysis method of ultra - performance liquid chromatography - tandem quadrupole time - of - flight mass spectrometer, 16 compounds can be quickly identified from the roots of Otophora unilocularis (see Table 2). The results show that the roots of Otophora unilocularis are rich in ursane - type triterpenoids (G7.34, G8.57, G9.18), oleanane - type triterpenoids and their saponins (G4.22, G12.85). In addition, there are also components such as flavonoids (G2.18, G2.38, G2.61), flavonoid glycosides (G2.54), etc., which have strong ecological significance and high development and utilization value. The present invention enriches the chemical constituents of Otophora unilocularis, lays a foundation for the research on its pharmacological activities and development and utilization, and at the same time provides a new preparation method for 16 compounds, enriching the sources of compounds. Description of the Drawings

[0048] Figure 1 It is the TIC (The total ion chromatogram) diagram of the negative ion mode of the extract of the roots of Otophora unilocularis. The upper sample is the roots of Otophora unilocularis, and the lower one is Blank.

[0049] Figure 2 It is the structural diagram of the compounds separated and identified from the roots of Otophora unilocularis.

[0050] Figure 3 It is the structural diagram of the compounds separated and identified from the roots of Otophora unilocularis. Detailed Embodiments

[0051] The present invention will be further described below with reference to the accompanying drawings and in combination with specific embodiments to better understand the present invention. For those not specified in the embodiments in terms of specific techniques or conditions, they shall be in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. For the reagents or instruments not specified in terms of the manufacturer, they are all conventional products that can be obtained through commercial procurement.

[0052] 1 Materials

[0053] ① Test samples: Roots of Otophora unilocularis

[0054] The Otophora unilocularis plant samples are from the Danzhou Tropical Medicinal Plant Germplasm Resource Nursery of the Ministry of Agriculture and Rural Affairs. The roots are harvested for analysis.

[0055] ② Instruments: Xevo G2-XS QTof liquid chromatography-mass spectrometry, mainly including ACQUITY I-Class ultra-high performance liquid chromatography system, Xevo G2-XS QTof quadrupole time-of-flight mass spectrometry system, Sample Manage FTN automatic sampler, Binary Solvent Manager binary solvent manager (Waters Corporation, USA), ACQUITY UPLC HSS T3 (100×2.1 mm 1.8 μm) chromatographic column (Waters Corporation, USA). Secura513-1CN precision balance (Sartorius Corporation, Germany), KQ3200DE type numerically controlled ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.), Milli-Q ultrapure water instrument (Millipore Corporation, USA), 5810R bench-top high-speed refrigerated centrifuge (EppendorfCentrifuge Corporation, Germany).

[0056] ③ Consumables: 0.2 μm, 13 mm Pall Syringe filter membrane (Pall Corporation, USA), 1 mL disposable sterile syringe with needle (Fenglin Medical Instrument Co., Ltd.), Labmed pipette tips 1000 μL, 200 μL, 20 μL (Labmed Biotech Corporation, USA), KG2211W 1.5 mL centrifuge tube (KiRGEN Corporation, USA), 2 mL transparent screw-cap sample vial (Waters Corporation, USA).

[0057] ④ Reagents: Methanol, isopropanol (for LC, chromatographic grade, Merck, Germany); acetonitrile, formic acid 98%-100% (for LC / MS, mass spectrometry grade, Merck, Germany); sodium hydroxide (ACS grade, ACS reagent ≥97%, Sigma, USA); leucine enkephalin (Standards Kit for TofG2-S, Waters, USA); distilled water (Watsons Food & Beverage Co., Ltd., Guangzhou); ultrapure water was prepared by Milli-Q ultrapure water instrument, and other reagents were all of analytical grade.

[0058] ⑤ MS resolution and calibration check solvents: Sodium formate (NaF) and leucine enkephalin (LE) solutions were prepared in the laboratory.

[0059] ⑥ Software: Masslynx V4.1 mass spectrometry software (Waters, USA), Scientific Information System (Waters, USA).

[0060] 2 Methods

[0061] 2.1 Preparation of test samples

[0062] All samples for LC-MS analysis should be freshly prepared and stored in a 4°C refrigerator. Do not store for a long time.

[0063] Accurately weigh 0.25 g of sample powder, place it in a stoppered glass test tube, add 5 mL of chromatographic methanol to submerge it, heat it in a 50°C water bath, ultrasonically extract for 2 h, shake well and let it stand for 5 min. Take 200 μL of the supernatant of the extraction solution, add 600 μL of 75% acetonitrile for dilution, mix well and then centrifuge at low temperature for 10 min (12000 rpm, 10°C). After taking out the centrifuge tube, place it gently, take 600 - 700 μL of the supernatant and store it in a 2 mL transparent sample vial, labeled as GEM root, to obtain the test sample solution.

[0064] 2.2 UPLC / Q-TOF-MS detection and analysis method

[0065] ① Chromatographic conditions

[0066] Use a Waters ACQUITY UPLC HSS T3 chromatographic column (100 mm × 2.1 mm, 1.8 μm), with a flow rate of 0.3 mL / min, an injection volume of 1 μL, and a column temperature of 40°C; the mobile phase is 0.1% formic acid aqueous solution (A) - 0.01% formic acid acetonitrile solution (B), gradient elution, and the program settings are shown in Table 1.

[0067] Table 1 Gradient elution program

[0068]

[0069]

[0070] ② Mass spectrometry method

[0071] The electrospray ionization source (ESI) is in the negative ion (-) condition, Continuum mode, collecting MS E data. The calibration solution is 200 pg / μL leucine enkephalin and 0.5 mM sodium formate.

[0072] The scanning range is m / z 50 - 1200, the scanning time is 0.2 s, and the detection time is 20 min.

[0073] The low-energy collision voltage (CE) is 6 V, and the high-energy collision voltage is 20 - 60 V; the capillary voltages in the negative ion mode are 2.0 kV respectively, the cone voltage is 60 V, the ion source temperature is 100 °C, the auxiliary spray ionization and desolvation gas are high-purity N2, the desolvation temperature is 450 °C, the cone gas flow rate is 50 L / hr, and the desolvation gas flow rate is 600 L / hr.

[0074] ③ Data processing

[0075] Apply Masslynx V4.1 software to collect, manage and process UPLC / Q TofMS E data. Apply the UNIFI scientific information system for data browsing, storage and comprehensive analysis, etc. By extracting MS and MS / MS mass spectra and related fragment information, based on its built-in mass spectrometry analysis platform, including online databases such as ChemSpider (PubMed, PubChem, MassBank, etc.) and the traditional Chinese medicine database (TCM Chiese [UNIFI1.7]), combined with the literature of compounds and Scifinder data for component discrimination analysis.

[0076] 3 Results and analysis

[0077] 2.1 Identification of chemical components in the extract of Alangium platanifolium roots

[0078] Based on the UPLC-Q-TOF-MS technology, the mass spectrometry peak appearance and ion response in the positive and negative ion modes were investigated to qualitatively analyze the chemical components of the roots of Alangium platanifolium. It was found by comparison that the separation degree in the (-) ESI-MS mass spectrometry ion current diagram is better and the response is higher. On the premise of optimized chromatographic conditions, the test sample was qualitatively analyzed, and the total ion current diagram (TIC) is as Figure 1 shown.

[0079] Based on the overall peak appearance, the data in the negative ion mode was imported into UNIFI, and relevant method parameters were set. According to the accurate relative molecular mass given by the mass spectrometry, the mass spectrometry fragment information was extracted to conduct qualitative analysis on the chemical components of the sample. In combination with relevant literature and databases for inference, the identification results are shown in Table 2. The results show that 38 compounds were analyzed from the roots of Gua Mu'er, and the structures of 16 of them were identified, mainly including oleanane-type triterpenoids (G7.34, G8.57, G9.18), oleanane-type triterpenoids and their saponins (G4.22, G12.85), and in addition, flavonoids (G2.18, G2.38, G2.61), flavone glycosides (G2.54), etc.

[0080]

[0081]

[0082]

[0083]

[0084] The specific embodiments of the present invention have been described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to this utility are also within the scope of the present invention. Therefore, all equivalent transformations and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. A method for detecting the chemical components of Otophora unilocularis, characterized in that, Taking the roots of Otophora unilocularis as the detection object, an ultra-high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometer was used for determination; The conditions of ultra-high performance liquid chromatography were as follows: The chromatographic column was a 100 mm×2.1 mm, 1.8 μm Waters ACQUITY UPLC HSS T3 chromatographic column; Column temperature: 38 - 42 °C; Volume flow rate: 0.2 - 0.4 mL / min; Mobile phase: Mobile phase A was an aqueous solution of formic acid with a volume concentration of 0.05 - 0.15%; Mobile phase B was an acetonitrile solution of formic acid with a volume concentration of 0.005 - 0.015%; The elution gradient was as follows:

2. The chemical composition detection method according to claim 1, characterized in that, Before determination by ultra-high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry, the detection object was extracted with alcohol, and the alcohol was preferably methanol and / or ethanol.

3. The chemical composition detection method according to claim 1, characterized in that, The column temperature was 40 °C; the volume flow rate was 0.25 - 0.35 mL / min, preferably 0.3 mL / min; Mobile phase A was an aqueous solution of formic acid with a volume concentration of 0.08 - 0.12%, preferably an aqueous solution of formic acid with a volume concentration of 0.1%; Mobile phase B was an acetonitrile solution of formic acid with a volume concentration of 0.008 - 0.012%, preferably an acetonitrile solution of formic acid with a volume concentration of 0.01%.

4. The chemical composition detection method according to claim 1, characterized in that, The mass spectrometry conditions of the mass spectrometer were as follows: electrospray ionization source (ESI) was used, and MS data was collected in Continuum mode under negative ion conditions. E Data.

5. The chemical composition detection method according to claim 4, wherein The mass spectrometry conditions of the mass spectrometer also included: scanning range m / z 50 - 1200 Da, low-energy collision voltage (CE) was 6 V, high-energy collision voltage was 20 - 60 V; the capillary voltage in the negative ion mode was 2.0 kv, the cone voltage was 60 V, and the cone gas flow rate was 50 L / hr; Scanning time 0.2 s, detection time 20 min; the ion source temperature was 90 - 110 °C, preferably 100 °C; the auxiliary spray ionization and desolvation gas was high-purity N2, the desolvation temperature was 450 °C, and the desolvation gas flow rate was 600 L / hr.

6. A method for separating the chemical constituents of Otophora unilocularis, characterized in that, Taking the roots of Otophora unilocularis as the separation object, ultra-high performance liquid chromatography was used for separation; the conditions of ultra-high performance liquid chromatography were as follows: The chromatographic column was a 100 mm×2.1 mm, 1.8 μm Waters ACQUITY UPLC HSS T3 chromatographic column; Column temperature: 38 - 42 °C; Volume flow rate: 0.2 - 0.4 mL / min; Mobile phase: Mobile phase A was an aqueous solution of formic acid with a volume concentration of 0.05 - 0.15%; Mobile phase B was an acetonitrile solution of formic acid with a volume concentration of 0.005 - 0.015%; The elution gradient was as follows:

7. The separation method according to claim 6, characterized in that, Before separation by ultra-high performance liquid chromatography, the detection object was extracted with alcohol, and the alcohol was preferably methanol and / or ethanol.

8. The separation method according to claim 6, characterized in that, The column temperature was 40 °C; the volume flow rate was 0.25 - 0.35 mL / min, preferably 0.3 mL / min; Mobile phase A was an aqueous solution of formic acid with a volume concentration of 0.08 - 0.12%, preferably an aqueous solution of formic acid with a volume concentration of 0.1%; Mobile phase B was an acetonitrile solution of formic acid with a volume concentration of 0.008 - 0.012%, preferably an acetonitrile solution of formic acid with a volume concentration of 0.01%.

9. A method for preparing a compound, characterized in that, The compounds are 3-hydroxybenzoic acid, and / or cianidanol, and / or 9-hydroxyoctadecadienoic acid, and / or 12-hydroxy-8,10-heptadecadienoic acid, and / or (-)-pimaric acid, and / or testosterone butyrate, and / or ursolic acid, and / or spirost-5-ene-3-hexopyranoside, and / or 5,13-Bis(3,4-dihydroxyphenyl)-4,12,14-trioxatricyclo[11.7.1.0~2,11~.0~3,8~.0~15,20~]heneicos-2,8,10,15,17,19-hexol, and / or luteolin-7-O-β-D-neohesperidoside, and / or (3β)-3-{[(2E)-3-(4-hydroxyphenyl)-2-propenoyl]oxy}olean-12-ene-28-oic acid, and / or (3β)-3-hydroxy-27-{[(2E)-3-(4-hydroxyphenyl)-2-propenoyl]oxy}urs-12-ene-28-oic acid, and / or (3β)-3-hydroxy-27-{[3-(4-hydroxyphenyl)acryloyl]oxy}urs-12-ene-28-oic acid, and / or 2,3-dihydroxy-23-{[(2E)-3-(4-hydroxyphenyl)-2-propenoyl]oxy}urs-12-ene-28-oic acid, and / or epigallocatechin (4β,8)-gallocatechin, and / or 2-O-(deoxyhexopyranosyl)-1-O-{[(2beta,3beta,6beta,22beta)-3-(hydroxyhexopyranosyl)-2,6,23-trihydroxyolean-12-ene-22-yl]carbonyl}pentopyranose, which are isolated from the roots of Otophora unilocularis by ultra-high performance liquid chromatography; the conditions of the ultra-high performance liquid chromatography are as follows: The chromatographic column is a 100 mm×2.1 mm, 1.8 μm Waters ACQUITY UPLC HSS T3 chromatographic column; Column temperature: 38 - 42 °C; Volume flow rate: 0.2 - 0.4 mL / min; Mobile phase: Mobile phase A is an aqueous solution of formic acid with a volume concentration of 0.05 - 0.15%; Mobile phase B is an acetonitrile solution of formic acid with a volume concentration of 0.005 - 0.015%; The elution gradient is as follows:

10. The preparation method according to claim 9, characterized in that, Before separation by ultra-high performance liquid chromatography, the detection object is extracted with alcohol, and the alcohol is preferably methanol and / or ethanol; The column temperature is 40 °C; the volume flow rate is 0.25 - 0.35 mL / min, preferably 0.3 mL / min; Mobile phase A is an aqueous solution of formic acid with a volume concentration of 0.08 - 0.12%, preferably an aqueous solution of formic acid with a volume concentration of 0.1%; Mobile phase B is an acetonitrile solution of formic acid with a volume concentration of 0.008 - 0.012%, preferably an acetonitrile solution of formic acid with a volume concentration of 0.01%.

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