A method for detecting the chemical components of Cassytha filiformis

Abstract

The present invention provides a method for detecting and separating the chemical constituents of Cassytha filiformis. By using ultra-high performance liquid chromatography combined with a specific mobile phase and elution program, the present invention can separate 44 compounds from Cassytha filiformis. Further, by using the analysis method of ultra-high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometer, 42 compounds can be quickly identified therefrom, including 13 flavonoid compounds, 8 triterpenoid compounds, 4 proanthocyanidin components, 9 iridoids, 6 phenolic acids and 2 other types of structures. The results show that Cassytha filiformis contains a variety of bioactive components, has strong ecological significance and high development and utilization value. The present invention enriches the chemical constituents of Cassytha filiformis, 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 42 compounds, enriching the sources of the compounds.

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 Cassytha filiformis. Background Art

[0002] Cassytha filiformis L. is a parasitic twining herbaceous plant that attaches itself to host plants by means of disc-shaped haustoria. The whole herb is used medicinally and has the effects of dispelling dampness and reducing swelling, promoting diuresis, and treating nephritis, edema and other diseases. Research shows that Cassytha filiformis contains alkaloids, flavonoids, volatile oils and other components. However, there are few comprehensive and systematic analytical studies on its chemical components at present, which greatly limits the further clarification of the pharmacological activities of Cassytha filiformis and also restricts the further research and development of its drug activities. Ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) can quickly and accurately analyze the chemical components in traditional Chinese medicine, providing a new research approach for clarifying the material basis of the medicinal efficacy of Cassytha filiformis and further extracting and separating bioactive substances. 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 Cassytha filiformis, which can effectively separate and identify the chemical components in Cassytha filiformis.

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

[0005] The conditions for ultra-high performance liquid chromatography are as follows:[[]]

[0006] 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 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%; gradient elution, and the elution gradient is as follows:[[]]

[0007] 0 - 2 min, 99% → 92% mobile phase A, 1% → 8% mobile phase B;[[]]

[0008] 2 - 7 min, 92% → 84% mobile phase A, 8% → 16% mobile phase B;[[]]

[0009] 7 - 10 min, 84% → 80% mobile phase A, 16% → 20% mobile phase B;[[]]

[0010] 10 - 11 min, 80% → 64% mobile phase A, 20% → 36% mobile phase B;[[]]

[0011] 11 - 14 min, mobile phase A changes from 64% to 24%, mobile phase B changes from 36% to 76%;

[0012] 14 - 15 min, mobile phase A changes from 24% to 1%, mobile phase B changes from 76% to 99%;

[0013] 15 - 19 min, 1% mobile phase A, 99% mobile phase B;

[0014] 19 - 20 min, mobile phase A changes from 1% to 99%, mobile phase B changes from 99% to 1%;

[0015] 20 - 23 min, 99% mobile phase A, 1% mobile phase B.

[0016] Among them, before determination by ultra - 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.

[0017] Preferably, the column temperature is 40 °C.

[0018] Preferably, the volume flow rate is 0.25 - 0.35 mL / min, more preferably 0.3 mL / min.

[0019] 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%.

[0020] Preferably, mobile phase B is a formic acid acetonitrile solution with a volume concentration of 0.008 - 0.012%, preferably a formic acid acetonitrile solution with a volume concentration of 0.01%.

[0021] Among them, the mass spectrometry conditions of the mass spectrometer are: The mass spectrometry conditions of the mass spectrometer are: Electrospray ionization source (ESI) is used, and MS data is collected under positive and negative ion conditions. E Data.

[0022] Preferably, the mass spectrometry conditions of the mass spectrometer further include: scanning range m / z 50 - 1200, scanning time 0.2 s, detection time 20 min; low - energy collision voltage (CE) 6 V, high - energy collision voltage 20 - 60 V; capillary voltages in positive and negative ion modes are 3.8 kV and 2 kV respectively, cone - hole voltage 45 V, ion source temperature 120 °C, auxiliary spray ionization and desolvation gas is high - purity N 2 , desolvation temperature 350 °C, cone - hole gas flow rate 50 L·h -1 , desolvation gas flow rates in positive and negative ion modes are 650 L·h -1 and 600 L·h -1 .

[0023] Using the detection method of the present invention, 42 compounds can be identified from Cassytha filiformis L., including 13 flavonoids, 8 triterpenoids, 4 proanthocyanidins, 9 iridoids, 6 phenolic acids and 2 other types of structures. The identification results are shown in Table 1, and the compound structures are shown in Figure 3 and Figure 4 .

[0024] The second aspect of the present invention is to provide a method for separating the chemical components of Cassytha filiformis L., using Cassytha filiformis L. as the separation object and separating it by ultra-high performance liquid chromatography.

[0025] The conditions for ultra-high performance liquid chromatography are as follows:

[0026] 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 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%; gradient elution, and the elution gradient is as follows:

[0027] 0 - 2 min, 99% → 92% mobile phase A, 1% → 8% mobile phase B;

[0028] 2 - 7 min, 92% → 84% mobile phase A, 8% → 16% mobile phase B;

[0029] 7 - 10 min, 84% → 80% mobile phase A, 16% → 20% mobile phase B;

[0030] 10 - 11 min, 80% → 64% mobile phase A, 20% → 36% mobile phase B;

[0031] 11 - 14 min, 64% → 24% mobile phase A, 36% → 76% mobile phase B;

[0032] 14 - 15 min, 24% → 1% mobile phase A, 76% → 99% mobile phase B;

[0033] 15 - 19 min, 1% mobile phase A, 99% mobile phase B;

[0034] 19 - 20 min, 1% → 99% mobile phase A, 99% → 1% mobile phase B;

[0035] 20 - 23 min, 99% mobile phase A, 1% mobile phase B.

[0036] Among them, before separating by ultra-high performance liquid chromatography, the separation object is extracted with alcohol, and the alcohol is preferably methanol and / or ethanol.

[0037] Preferably, the column temperature is 40 °C.

[0038] Preferably, the volume flow rate is 0.25 - 0.35 mL / min, more preferably 0.3 mL / min.

[0039] 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%.

[0040] Preferably, 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%.

[0041] By using the separation method of the present invention, 44 compounds can be separated from Cassytha filiformis Linn.

[0042] The third aspect of the present invention is to provide a method for preparing a compound, which is one or several of the compounds in the following table:

[0043]

[0044]

[0045] It is separated from Cassytha filiformis Linn. by ultra - performance liquid chromatography, and the conditions of ultra - performance liquid chromatography are as follows:

[0046] 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 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%; gradient elution, and the elution gradient is as follows:

[0047] 0 - 2 min, 99% → 92% mobile phase A, 1% → 8% mobile phase B;

[0048] 2 - 7 min, 92% → 84% mobile phase A, 8% → 16% mobile phase B;

[0049] 7 - 10 min, 84% → 80% mobile phase A, 16% → 20% mobile phase B;

[0050] 10 - 11 min, 80% → 64% mobile phase A, 20% → 36% mobile phase B;

[0051] 11 - 14 min, 64% → 24% mobile phase A, 36% → 76% mobile phase B;

[0052] 14 - 15 min, mobile phase A: 24% → 1%, mobile phase B: 76% → 99%;

[0053] 15 - 19 min, 1% mobile phase A, 99% mobile phase B;

[0054] 19 - 20 min, mobile phase A: 1% → 99%, mobile phase B: 99% → 1%;

[0055] 20 - 23 min, 99% mobile phase A, 1% mobile phase B.

[0056] Among them, before separation by ultra - performance liquid chromatography, the separation object is extracted with alcohol, and the alcohol is preferably methanol and / or ethanol.

[0057] Preferably, the column temperature is 40 °C.

[0058] Preferably, the volume flow rate is 0.25 - 0.35 mL / min, more preferably 0.3 mL / min.

[0059] 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%.

[0060] Preferably, 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%.

[0061] The present invention can separate 44 compounds from Cassytha filiformis by using ultra - performance liquid chromatography combined with specific mobile phases and elution procedures. Further, by using the analysis method of ultra - performance liquid chromatography - tandem quadrupole time - of - flight mass spectrometry, 42 compounds can be quickly identified from them, including 13 flavonoids, 8 triterpenoids, 4 proanthocyanidins, 9 iridoids, 6 phenolic acids and 2 other types of structures. The results show that Cassytha filiformis contains a variety of bioactive components, has strong ecological significance and high development and utilization value. The present invention enriches the chemical constituents of Cassytha filiformis, 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 42 compounds, enriching the sources of compounds. Description of the Drawings

[0062] Figure 1 They are the TIC flow charts of Cassytha filiformis in positive and negative ion modes.

[0063] Figure 2 They are the TIC diagrams of Blank in positive and negative ion modes.

[0064] Figure 3 and Figure 4Structural diagram of the compound identified from Cassytha filiformis Detailed implementation mode

[0065] The present invention will be further described below with reference to the accompanying drawings and in conjunction with specific embodiments for better understanding. For those not specifying specific technologies or conditions in the embodiments, they shall be carried out according to the technologies or conditions described in the literature in this field or according to the product specifications. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0066] 1 Instruments and materials

[0067] Instruments: The liquid chromatography-mass spectrometry mainly includes ACQUITY I-Class system, Xevo G2-XS Q-Tof mass spectrometry system, automatic sampler, binary solvent manager (Waters Corporation, USA); ACQUITY UPLC HSS T3 (100×2.1mm 1.8μm) chromatographic column (Waters Corporation, USA), Secura 513-1CN 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 (Eppendorf Centrifuge Corporation, Germany).

[0068] Materials: 0.2μm, 13mm Pall Syringe filter membrane (Pall Corporation, USA), 1mL disposable sterile syringe with needle (Fenglin Medical Instrument Co., Ltd.), Labmed pipette tips 1000μL, 200μL, 20μL (Labmed Biotech Corporation, USA), KG2211W 1.5mL centrifuge tube (KiRGEN Corporation, USA), 2mL transparent screw-cap sample vial (Waters Corporation, USA), Oasis HLB 3cc (60mg) Extraction Cartridges (Waters Corporation, USA).

[0069] Software: Masslynx V4.1 mass spectrometry software (Waters Corporation, USA), Scientific information system (Waters Corporation, USA).

[0070] Reagents: Chromatographic grade methanol, isopropanol (Merck, Germany), mass spectrometry grade acetonitrile, formic acid (Merck, Germany); ACS grade sodium hydroxide (Sigma, USA); leucine enkephalin (Standards Kit for TofG2-S, Waters, USA); distilled water (Guangzhou Watson's Food & Beverage Co., Ltd.); ultrapure water was prepared by a Milli-Q ultrapure water instrument (Millipore, USA); other reagents were all of analytical grade.

[0071] 2 Methods

[0072] 2.1 Preparation of test samples

[0073] All samples for liquid chromatography - mass spectrometry analysis should be freshly prepared and stored in a 4°C refrigerator, with a storage period not exceeding 24 hours.

[0074] Preparation of test solution: Take the sliced Cassytha filiformis material, crush it, and pass through an 80 - mesh sieve. Accurately weigh 1 g of the plant powder, add 20 ml of chromatographic methanol, and extract it by ultrasonic for 30 minutes. Take 1 mL of the extract, perform freezing centrifugation (12000 rpm, 15 minutes, 10°C), take the supernatant and filter it through a 0.2 - μm microporous filter membrane, and store it in a 2 - mL transparent sample vial, labeled as WGT.

[0075] 2.2 UPLC - Q - TOF - MS detection conditions

[0076] (1) Chromatographic conditions

[0077] Waters ACQUITY UPLC HSS T3 chromatographic column (100 mm × 2.1 mm, 1.8 μm), flow rate 0.3 mL·min -1 , injection volume 1 μL, column temperature 40°C; the mobile phase is 0.1% formic acid aqueous solution (A) - 0.01% formic acid acetonitrile solution (B), gradient elution, and the elution gradient is as follows:

[0078] 0 - 2 min, 99% → 92% mobile phase A, 1% → 8% mobile phase B;

[0079] 2 - 7 min, 92% → 84% mobile phase A, 8% → 16% mobile phase B;

[0080] 7 - 10 min, 84% → 80% mobile phase A, 16% → 20% mobile phase B;

[0081] 10 - 11 min, 80% → 64% mobile phase A, 20% → 36% mobile phase B;

[0082] 11 - 14 min, 64% → 24% mobile phase A, 36% → 76% mobile phase B;

[0083] 14 - 15 min, mobile phase A changes from 24% to 1%, and mobile phase B changes from 76% to 99%;

[0084] 15 - 19 min, 1% mobile phase A, 99% mobile phase B;

[0085] 19 - 20 min, mobile phase A changes from 1% to 99%, and mobile phase B changes from 99% to 1%;

[0086] 20 - 23 min, 99% mobile phase A, 1% mobile phase B.

[0087] (2) Mass spectrometry conditions

[0088] Electrospray ionization source (ESI) collects MS data under positive and negative ion conditions respectively. E Data.

[0089] Scanning range m / z 50 - 1200, scanning time 0.2 s, detection time 20 min.

[0090] Low - energy collision voltage (CE) is 6 V, high - energy collision voltage is 20 - 60 V; capillary voltages for positive and negative ion modes are 3.8 kV and 2 kV respectively, cone - hole voltage is 45 V, ion source temperature is 120 °C, auxiliary spray ionization and desolvation gas are high - purity N 2 , desolvation temperature is 350 °C, cone - hole gas flow rate is 50 L·h -1 , desolvation gas flow rates for positive and negative ion modes are 650 L·h -1 and 600 L·h -1 .

[0091] 2.3 Data processing

[0092] Adopt the Masslynx V4.1 software intelligent control system to collect UPLC / Q - TOF - MS data; with the help of the UNIFI scientific information system, integrate the reported data in the literature, and combine with TCM Chinese [UNIFI1.7], ChemSpider online database to identify compound components. E Data; with the help of the UNIFI scientific information system, integrate the reported data in the literature, and combine with TCM Chinese [UNIFI1.7], ChemSpider online database to identify compound components.

[0093] 3 Results and analysis

[0094] Based on the UPLC / Q - TOF - MS technology, under the premise of optimized chromatographic conditions, the mass spectrometry peak appearance and ion response in positive and negative ion modes are investigated, and qualitative analysis is carried out on the test samples. By observing the total ion current chromatogram (TIC), it is found that in the negative ion mode, the separation degree of the mass spectrometry ion current chromatogram is better and the response is higher ( Figure 1 ).

[0095] Based on the overall peak emergence situation, the data was imported into UNIFI, relevant method parameters were set, and according to the accurate relative molecular mass given by the mass spectrometry, the mass spectrometry fragment information was extracted for qualitative analysis of the chemical components of the sample. In combination with relevant literature and databases for inference, the identification results are shown in Table 1. More than 400 compounds with response values exceeding 100,000 were analyzed and collected from the LC-MS data of this material. Through literature retrieval, fragmentation pattern analysis, chemical structure inference, error value calculation, matching degree comparison, etc., 44 compounds were obtained from Cassytha filiformis, and the structures of 42 of them were identified ( Figure 3 and Figure 4 , and the numbers below the compound structural formulas in the figure correspond to No. in Table 1). Among them, there are 13 flavonoid compounds, 8 triterpenoid compounds, 4 proanthocyanidin components, 9 iridoids, 6 phenolic acids and 2 other types of structures. Flavonoid components are the main components of Cassytha filiformis, including flavones, flavonoid glycosides, dihydroflavonoid aglycones and their glycosides. Flavonoid components have obvious antioxidant, antibacterial and anti-inflammatory functions. Characteristic proanthocyanidin components were also identified in this material, mainly B-type catechins formed by the connection of catechin and epicatechin through a C-C bond. Proanthocyanidins are natural antioxidants, indicating that Cassytha filiformis has strong antioxidant activity and good development and application prospects. Since this material is derived from the vine of a plant, a large amount of phenolic acids will be produced during the biosynthesis process. The phenolic acids are then combined to form proanthocyanidins, and the anthocyanins are combined to form the tanning components of the vine. Therefore, a series of proanthocyanidin dimers and trimers were identified in this material. Iridoid components have good protective effects on the nervous system and also have the effect of enhancing immunity, and are also one of the main components of Cassytha filiformis. There is also a large class of triterpenoid components in Cassytha filiformis. In this study, 8 triterpenoid components were identified, mainly oleanane-type triterpenoids. Triterpenoids are a major class of plant secondary metabolites with multiple effects of anti-inflammatory, whitening and liver protection.

[0096] Table 1 UPLC / Q-TOF-MS / MS Mass Spectrometry Data and Identification of WJT

[0097]

[0098]

[0099]

[0100] 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 made 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 Rhizoma Cynanchum, characterized in that: The rootless vine was used as the detection object and was determined by ultra-performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry; The conditions for ultra-high performance liquid chromatography were: The chromatographic column was a 100 mm×2.1 mm, 1.8 μm Waters ACQUITY UPLC HSS T3 column; column temperature: 38-42°C; volume flow rate: 0.2-0.4 mL / min; mobile phase: mobile phase A was a 0.05-0.15% volume concentration of formic acid in water; mobile phase B was a 0.005-0.015% volume concentration of formic acid in acetonitrile; gradient elution, the elution gradient was as follows: 0-2 min, 99%→92% mobile phase A, 1%→8% mobile phase B; 2-7 min, 92%→84% mobile phase A, 8%→16% mobile phase B; 7-10 min, 84%→80% mobile phase A, 16%→20% mobile phase B; 10-11 min, 80%→64% mobile phase A, 20%→36% mobile phase B; 11-14 min, 64%→24% mobile phase A, 36%→76% mobile phase B; 14-15 min, 24%→1% mobile phase A, 76%→99% mobile phase B; 15-19 min, 1% mobile phase A, 99% mobile phase B; 19-20 min, 1%→99% mobile phase A, 99%→1% mobile phase B; 20-23 min, 99% mobile phase A, 1% mobile phase B; The mass spectrometry conditions of the mass spectrometer are as follows: using an electrospray ion source to collect MS under positive and negative ion conditions E data.

2. The method for detecting chemical components according to claim 1, characterized in that: The test objects were extracted with alcohol before being measured by ultra-performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry.

3. The method for detecting chemical components according to claim 2, characterized in that: The alcohol is methanol and / or ethanol.

4. The method for detecting chemical components according to claim 1 or 2, characterized in that: In the conditions of ultra-high performance liquid chromatography, the column temperature is 40°C; the volume flow rate is 0.25-0.35 mL / min; the mobile phase A is a formic acid aqueous solution with a volume concentration of 0.08-0.12%; and the mobile phase B is a formic acid acetonitrile solution with a volume concentration of 0.008-0.012%.

5. The method for detecting chemical components according to claim 4, characterized in that: The volume flow rate is 0.3 mL / min; mobile phase A is a 0.1% by volume formic acid aqueous solution; and mobile phase B is a 0.01% by volume formic acid acetonitrile solution.

6. The method for detecting chemical components according to claim 1, characterized in that: Scan range m / z The ionization temperature was 120 °C, the auxiliary spray ionization and desolvation gas was high-purity N2, the desolvation temperature was 350 °C, and the cone gas flow rate was 50 L·h -1 The desolvation gas flow rates in the positive and negative ion modes were 650 L·h -1 and 600 L·h -1 .

7. The method for detecting chemical components according to claim 1, characterized in that: The chemical composition is one or more of the compounds in the following table: 。

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