CN114380776B - Sesquiterpene electrophilic natural product, and separation preparation method and application thereof - Google Patents

Abstract

The invention discloses a sesquiterpene electrophilic natural product, its separation preparation method and application. The semiterpenoid electrophilic natural product has a chemical structural formula as shown in formula (I); the present invention utilizes an electrophilic probe in conjunction with the usual means of separating and extracting natural compounds, and successfully separates it from the crude extract of Ginkgo biloba An electrophilic natural product monomer with a quasi-molecular ion peak [M+H] ⁺ of 263.1275. Through structural characterization, the electrophilic natural product separated and prepared in the present invention is a new type of sesquiterpene electrophilic natural product containing an unsaturated lactone ring and a five-membered furan ring adjacent to carbonyl, and whose core structure is completely different from ginkgolide K. The electrophilic natural product acts on the activity of the Keap1-Nrf2 signaling pathway, has strong anti-inflammatory biological activity, and is an ideal lead compound of a new covalent inhibitor of cysteine residues, which can be used in the treatment of inflammation, tumors, It has great application prospects in neurodegeneration, cardiovascular disease or metabolic disease.

Description

A sesquiterpene electrophilic natural product and its separation and preparation method and application

technical field

The present invention relates to the technical field of biomedicine, in particular, to a sesquiterpene electrophilic natural product and its separation and preparation method and application, more specifically, to the separation of a novel sesquiterpene electrophilic natural product derived from ginkgo biloba Preparation and its application in the preparation of anti-inflammatory drugs.

Background technique

The cysteine residue associated with protein activity, with a highly nucleophilic sulfhydryl group, is an ideal covalent binding site. Therefore, many covalent inhibitors work by modifying cysteine residues of target proteins. Electrophilic natural products (ENP) are an important source of cysteine covalent inhibitors, which play a series of roles such as anti-inflammatory and anti-tumor by modifying protein cysteine residues, and have a good Medicinal properties. However, the development of electrophilic natural products is still at the stage of serendipitous discovery.

Ginkgo extract refers to the active substances extracted from Ginkgo biloba, which contain ginkgo total flavonoids, ginkgolides and other substances. It has the functions of expanding blood vessels, protecting vascular endothelial tissue, regulating blood lipids, protecting low-density lipoprotein, inhibiting PAF (platelet activating factor), inhibiting thrombosis, and scavenging free radicals. It is reported that only ginkgolide K (Beek, T.A.v., Chemical analysis of Ginkgo bilobaleaves and extracts.Journal of Chromatography A 2002, 967, 21 -55; Singh, B.; Kaur, P.; Gopichand; Singh, R.D.; Ahuja, P.S., Biology and chemistry of Ginkgobiloba. Fitoterapia 2008, 79(6), 401-418.); The active natural electrophilic product of , as a lead compound for cysteine covalent inhibitors.

Contents of the invention

The object of the present invention is to overcome the above-mentioned defects and deficiencies in the prior art, and provide a sesquiterpene electrophilic natural product.

Another object of the present invention is to provide a method for the separation and preparation of the sesquiterpenoid electrophilic natural product.

Another object of the present invention is to provide the application of the sesquiterpene electrophilic natural product.

Above-mentioned purpose of the present invention is given to realize by following technical scheme:

A sesquiterpene electrophilic natural product, its chemical structural formula is as shown in formula (I):

The electrophilic natural product described in the formula (I) of the present invention is a novel electrophilic natural product derived from Ginkgo biloba. Novel sesquiterpene electrophilic natural products of ester K. The novel electrophilic natural product acts on the activity of the Keap1-Nrf2 signaling pathway, has strong anti-inflammatory biological activity (EC ₅₀ ≈15 μM), and is an ideal lead compound for a new covalent inhibitor of cysteine residues .

The present invention also provides a method for separating and preparing the sesquiterpene electrophilic natural product, comprising the steps of:

S1. Form a natural product-probe complex between the electrophilic probe and the crude extract of Ginkgo biloba, and detect it by chromatography-tandem mass spectrometry; select the Ginkgo-probe target pro-probe with the quasi-molecular ion peak [M+H] ⁺ of 612.3103 Electron natural product, minus the molecular weight of electrophilic probe, the quasi-molecular ion peak [M+H] of the target electrophilic natural product obtained ^is 263.1275; the chromatography adopts Thermo Scientific UltiMate 3000 HPLC, liquid phase condition: Hypersil Gold Dim C18 column, 100×2.1mm, 1.9μm, mobile phase: phase A: aqueous solution containing 0.1% formic acid, phase B: acetonitrile; flow rate: 0.3mL/min, gradient elution, gradient elution conditions are shown in Table 1; The mass spectrometry adopts Thermo Fisher QExactive, and the mass spectrometry detection conditions are as shown in Table 2;

S2. Under the same chromatographic and mass spectrometric conditions as step S1, the crude extract of Ginkgo biloba is subjected to high-resolution first- ^level scanning, and the quasi-molecular ion peak [M+H] of the target electrophilic natural product is extracted with a chromatographic peak of 263.1275 to obtain the target electrophilic The retention time of the electric natural product on the chromatographic column is 13.10 minutes;

S3. Under the same chromatographic and mass spectrometric conditions as step S1, the crude extract of Ginkgo biloba is subjected to DAD ultraviolet full-scan liquid-mass detection, and the ultraviolet absorption wavelength range of the target electrophilic natural product is 240-270nm;

S4. Use the preparation solution to separate and prepare the target electrophilic natural product in the crude extract of Ginkgo biloba, and determine that the peak eluting time of the target electrophile natural product under the conditions of the preparation liquid phase is 24.98 to 25.12 minutes; the preparation liquid phase The condition is Agilent ZORBAX SB-C18, 9.4×250mm, 5μm;

S5. The Ginkgo crude extract is separated and purified for the first time by medium and low pressure preparative liquid chromatography; the medium and low pressure preparative liquid chromatography adopts 0.1% formic acid water and methanol elution system, C18 self-packing column, packing 80g, flow rate 12mL /min, the gradient ratio of 0.1% formic acid water is set to 25% to 80%, and the column separation time is 150 minutes. Use the peak time of the target electrophile natural product summarized in the preparation of the liquid phase in step S4 to collect all the samples containing the target electrophile Fractions of natural products were collected by rotary evaporation under reduced pressure; the C18 self-packing column was YMC, packing: ODS-A, micropore diameter 12nm, particle diameter 50 μm;

S6. Perform secondary separation and purification of all fractions containing the target electrophilic natural product in step S6 by thin-layer chromatography; the developer of the thin-layer chromatography is cyclohexane:ethyl acetate with a volume ratio of 2:1, The development time was 45 minutes; all the obtained bands were collected and eluted separately, and each band was analyzed by liquid chromatography-mass spectrometry to obtain the target electrophile with a quasi-molecular ion peak [M+H] ⁺ of 263.1275 Pure products of natural products.

Preferably, the mass spectrometry instrument model of the ultraviolet full-scan liquid mass detection in step S3 is Bruker timsTOF, and the liquid phase instrument model is Thermo U3000.

Preferably, the preparation plate size of the thin layer chromatography described in step S6 is 1mm.

Preferably, the elution in step S6 is elution with acetone.

The novel electrophilic natural product of the present invention acts on the activity of the Keap1-Nrf2 signaling pathway, has strong anti-inflammatory biological activity (EC ₅₀ ≈15 μM), and is an ideal new covalent inhibitor of cysteine residues. lead compound. Therefore, the present invention also provides the use of the sesquiterpene electrophilic natural product in the preparation of anti-inflammatory products.

The present invention also provides the application of the sesquiterpene electrophilic natural product in the preparation of cardiovascular disease medicine.

The present invention also provides an anti-inflammatory product, which contains the sesquiterpene electrophilic natural product represented by the above formula (I).

In the early stage of the present invention, a new type of electrophilic molecular probe (CN112321489A) based on reporter ions and active sulfhydryl reactive groups was established, which can efficiently combine with trace electrophilic natural products in complex matrices such as natural crude extracts or cell extracts to form natural products. - Probe complex. Combined with the detection method of chromatographic tandem mass spectrometry, the Tianchan-probe complex is fragmented in a specific way during the secondary scanning process, and a characteristic reporter ion peak (m/z = 126.1277) and calculate the exact molecular weight (<5ppm) of the product reacted with the corresponding natural product compound and the probe, and the exact molecular weight (<5ppm) of the corresponding natural product; and predict its exact molecular formula according to the above-mentioned exact molecular weight; Quickly and accurately discover trace electrophilic natural products in natural matrices, and use the tracer function of the electrophilic probe to guide the separation of trace electrophilic natural products from complex natural matrices. In the early stage of the present invention, many documents have been reported and documents have not been reported from various natural plant extracts (Cocklebur extract, Ginkgo biloba extract, Melon pedicle extract, Ganoderma lucidum extract, Chrysanthemum chrysanthemum extract and Garcinia cambogia extract) The potential active electrophilic natural products have been verified, and the powerful function of electrophilic probes to efficiently discover potentially active electrophilic natural products has been verified. The separation and preparation method of the above-mentioned sesquiterpenoid electrophilic natural products of the present invention provides an example of a method for using electrophilic probes to guide the separation of electrophilic natural products.

The present invention utilizes the tracing function of the electrophilic probe, combined with the usual separation means of natural products, successfully separates and obtains a new type of electrophilic natural product with a quasi-molecular ion peak [M+H] ⁺ of 263.1275 from the crude extract of natural ginkgo biloba. product monomer. The research of the present invention shows that the electrophilic natural product is a new type of sesquiterpene electrophilic natural product that contains a five-membered unsaturated lactone ring and an adjacent carbonyl five-membered furan ring, and whose core structure is completely different from ginkgolide K. It has the structure shown in formula (I). The present invention also verifies that the novel electrophilic natural product derived from ginkgo acts on the activity of the Keap1-Nrf2 signaling pathway, has strong anti-inflammatory biological activity (EC ₅₀ ≈15 μM), and is an ideal novel cysteine residue Lead compounds for covalent inhibitors.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention provides a novel furan sesquiterpene electrophilic natural product derived from Ginkgo biloba, its chemical structural formula is as shown in formula (I), and the electrophilic natural product acts on the activity of Keap1-Nrf2-ARE signaling pathway, and has High activity of activating the Keap1-Nrf2 pathway, strong anti-inflammatory activity (EC ₅₀ ≈15μM), and good water solubility and stability, the new compound is an ideal target for cysteine residues A lead compound for generating new anti-inflammatory drugs based on covalent inhibition as a pharmacological mechanism.

Description of drawings

Figure 1 is the primary scanning chromatogram of ginkgo crude extract. (Top) Total ion chromatogram of Ginkgo biloba extract; (Bottom) Extracted [M+H]+ chromatographic peak of 263.1275.

Figure 2 is the mass spectrum full scan of ginkgo crude extract and [M+H] + is 263.1275 chromatographic peak; (top) mass spectrum full scan of ginkgo crude extract; (bottom) extraction [M+H] + is 263.1275 chromatographic peaks.

Figure 3 Full scan of high-resolution mass spectrometry of ginkgo crude extract.

Fig. 4 is the high-resolution mass spectrum of the chromatographic peak extracted with [M+H] ⁺ being 263.1275.

Figure 5 is a full scan of the DAD mass spectrum of ginkgo crude extract. (1) Full scan of mass spectrum (arrow: target electrophilic natural product peak); (2) Absorption of Ginkgo crude extract at 190-400nm UV wavelength; (3) Absorption of Ginkgo crude extract at 254nm UV wavelength ( Arrow: target electrophilic natural product peak); (4) Absorption of Ginkgo crude extract at 270nm ultraviolet wavelength.

Fig. 6 is the ultraviolet absorption of the target electrophilic natural product at 254, 270 and 260 nm. (Arrows: target electrophilic natural product peaks).

Fig. 7 is a ¹ H NMR spectrum of a novel electrophilic natural product.

Fig. 8 is a ¹³ C NMR spectrum of a novel electrophilic natural product.

Figure 9 is the Dept135 spectrum of a novel electrophilic natural product.

Figure 10 is the Dept 90 spectrum of the novel electrophilic natural product.

Figure 11 is the HMBC spectrum of the novel electrophilic natural product.

Figure 12 is the HSQC spectrum of a novel electrophilic natural product.

Figure 13 is the H-H Noesy spectrum of the novel electrophilic natural product.

Figure 14 is the H-H Cozy spectrum of the novel electrophilic natural product.

Figure 15 is the cLogP value of the novel electrophilic natural product.

Figures 16-17 are the mass spectrograms of the same sample prepared by reacting the novel electrophilic natural product with the electrophilic thiol probe at different time periods.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

The electrophilic probe is prepared according to the patent CN112321489A.

Ginkgo biloba crude extract was purchased from Baoji Chenguang Biotechnology (Baoji Chenguang 20200501)

Example 1 Application of electrophilic probes to covalently bind Ginkgo biloba crude extracts to form natural natural-probe complexes

1. In a 5mL reaction vessel, add Ginkgo biloba extract, probe compound (1.2eq) and L-ascorbic acid (1.5eq), and use 1mL ultra-dry anhydrous methanol as a solvent (the reaction solvent needs to be deoxygenated before filling with argon After the reaction mixture was fully dissolved by ultrasonication, argon gas was introduced to remove oxygen, and the cap was immediately tightened and sealed with a parafilm. After stirring at room temperature for 22 hours, the stirring was stopped, and the reaction solution was spin-dried immediately. Samples were prepared with mass spectrometry-grade methanol to prepare a 1ppm sample solution, and the Ginkgo natural product-probe complex was analyzed according to the following liquid phase and mass spectrometry methods.

Instrument: ultra-high pressure liquid phase-high resolution mass spectrometer; ultra high performance liquid phase model: Thermo ScientificUltiMate 3000HPLC; high resolution mass spectrometer model: Thermo Fisher QExactive.

Liquid phase conditions: Hypersil Gold Dim C18 column, 100×2.1 mm, 1.9 μm. Mobile phase: Phase A: aqueous solution (containing 0.1% formic acid), phase B: acetonitrile; flow rate: 0.3mL/min, gradient elution.

See Table 1 for the gradient elution conditions of the Ginkgo natural product-probe complex.

Ginkgo natural-probe complex mass spectrometry detection conditions are shown in Table 2

Table 1 Ginkgo natural product-probe complex gradient elution conditions

Retention time: retention time; Flow: flow rate; A: acetonitrile; B: 0.1% formic acid water

Table 2 Ginkgo biloba-probe complex mass spectrometry detection conditions

Results 54 potential active electrophilic natural products were found from the crude extract of Ginkgo biloba, including the known electrophilic product, ginkgolide K. The present invention randomly selects the target natural product-probe complex whose quasi-molecular ion peak [M+H] ⁺ is 612.3103, and deducts the molecular weight of the probe molecule itself, so it can be known that the [M+H] of the target electrophilic natural product ] ⁺ is 263.1275.

2. Under the same chromatographic and mass spectrometric conditions, the crude extract of Ginkgo biloba was scanned at the first level, and the quasi-molecular ion peak [M+H ^] was extracted The chromatographic peak of the target electrophilic natural product of 263.1275, as shown in Figure 1 It can be seen that the retention time of the target electrophilic natural product on the chromatographic column is 13.10 minutes.

3. Immediately afterwards, a DAD ultraviolet full-scan liquid mass detection was performed on the crude extract of ginkgo. The mass spectrometry instrument model was Bruker timsTOF; the liquid phase instrument model was Thermo U3000, and the chromatographic column and chromatographic conditions were the same as above.

The results are shown in Figures 2 to 4, Figure 2 is the full scan of the mass spectrum of the crude extract of Ginkgo biloba and the chromatographic peak with [M+H] ⁺ being 263.1275, and Figure 3 is the full scan of the high-resolution mass spectrum of the crude extract of Ginkgo biloba , Figure 4 is the high-resolution mass spectrum of the chromatographic peak extracted with [M+H] ⁺ being 263.1275, it can be known that peak No. 12 in Figure 3 is the peak of the target electrophilic natural product. The present invention locates the target electrophilic natural product at first and is the No. 12 peak in the full scan figure of Ginkgo biloba crude extract (Fig. 3); As shown in Figures 5-6, it was found that the target electrophilic natural product with [M+H] ⁺ of 263.1275 has ultraviolet absorption, the absorption range is 240-270nm, and the strongest ultraviolet absorption is at 254nm.

4. Considering that the resolution of the target electrophilic natural product on a chromatographic column (Agilent ZORBAX Eclipse Plus C18 column, 2.1mm×50mm, 1.8μm) is ideal, there are basically no interference peaks around. Therefore, it is first considered to expand the separation conditions to the preparative liquid phase (Agilent Technologies PrepStar, Agilent ZORBAX SB-C18, 9.4×250mm, 5μm), and directly separate and prepare the target electrophilic natural product in the crude extract of Ginkgo biloba. Since the components of the ginkgo crude extract are very complex, the separation degree on the preparation column is not enough, so the ginkgo crude extract cannot be directly separated and prepared by the preparative liquid phase. However, in the experiment of separating the target product by using the preparative liquid phase, it was determined that the peak eluting time of the target electrophilic natural product was 24.98-25.12 minutes under the conditions of the preparative liquid phase.

5. The present invention changes the separation strategy. First, the ginkgo crude extract is eluted with 0.1% formic acid water and methanol, and the medium and low pressure is used to prepare the liquid phase, and the C18 self-packing column (brand: YMC, filler: ODS-A, micropore diameter 12nm, particle size of 50 μm) for the first separation and purification. Filler 80g, flow rate 12mL/min, gradient ratio of 0.1% formic acid water is set to 25%-80%, column separation time is 150 minutes. For all fractions collected in the present invention, all fractions containing the target electrophilic natural product are collected by using the peak eluting time of the target electrophilic natural product summarized in the preparation liquid phase, and collected by rotary evaporation under reduced pressure. Next, the present invention utilizes the ultraviolet characteristics of the target electrophilic natural product to carry out secondary separation and purification of all fractions containing the target components with a silica gel normal phase preparation plate (preparation plate specification is 1 mm, cyclohexane: ethyl acetate 2:1 developer 30mL). After all the fractions containing the above-mentioned target components are completely separated by normal phase silica gel (the development time is about 45 minutes), all the bands on the preparation plate are collected and eluted with acetone in the present invention, and then each band is All the bands were analyzed by liquid chromatography-mass spectrometry to summarize the position of the target band on the preparation plate, and finally a pure product of the target electrophilic natural product with a quasi-molecular ion peak [M+H]+ of 263.1275 was obtained.

6. Characterize and confirm the structure of the target electrophilic natural product by mass spectrometry, one-dimensional spectrum, two-dimensional spectrum and other detection means. ^{The 1} HNMR spectrum is shown in Figure 7, ¹ H NMR (600MHz, CDCl ₃ ) δ8.06(s, 1H), 7.47(d, J=1.5Hz, 1H), 7.12–7.04(m, 1H), 6.78 (d,J=1.2Hz,1H),5.00(ddd,J=7.5,4.1,2.1Hz,1H),3.51(d,J=3.3Hz,1H),2.86–2.72(m,2H),1.94( d, J＝1.6Hz, 2H), 1.89–1.77(m, 2H), 1.64(ddd, J＝9.1, 6.8, 4.5Hz, 2H), 1.60(s, 3H), 1.58(dd, J＝9.6, 5.0Hz, 1H), 1.54(dd, J＝8.7, 4.4Hz, 1H), 1.52–1.50(m, 1H), 1.28(s, 3H), 1.05(d, J＝6.6Hz, 2H), 0.90( t,J=7.0Hz,1H),0.09(s,1H).;

^{The 13} C NMR spectrum is shown in Figure 8, ¹³ C NMR (151MHz, CDCl ₃ ) δ194.85(s), 174.28(s), 149.18(s), 147.08(s), 144.26(s), 129.79(s ), 127.64(s), 108.62(s), 79.30(s), 40.87(s), 37.86(s), 31.21(s), 29.74(d, J=8.4Hz), 22.70(s), 19.24(s ), 14.13(s), 10.66(s).;

The spectrum of Dept135 is shown in Figure 9, ¹³ C NMR (151MHz, CDCl ₃ ) δ149.17(s), 147.08(s), 144.26(s), 108.62(s), 79.30(s), 77.23(s), 19.24(s), 14.13(s), 10.66(s). 4 quaternary carbons, 8 primary/tertiary carbons, 3 secondary carbons;

The spectrum of Dept 90 is shown in Figure 10, ¹³ C NMR (151MHz, CDCl ₃ ) δ149.18(s), 147.09(d, J=4.2Hz), 144.25(s), 108.61(s), 79.29(s) ,29.76(s).6 tertiary carbons;

HMBC spectrogram as shown in Figure 11, HSQC spectrogram as shown in Figure 12, H-H Noesy spectrogram as shown in Figure 13, H-HCosy spectrogram as shown in Figure 14; The structural formula of the structure of target electrophilic natural product is as shown in formula (I):

是一种母核结构完全不同于已知亲电产物-银杏内酯K的新型呋喃倍半萜亲电天然产物。

It is a new type of furan sesquiterpene electrophilic natural product whose core structure is completely different from the known electrophile product - ginkgolide K.

Example 2 Performance Test of Novel Furan Sesquiterpene Electrophilic Natural Products

1. Anti-inflammatory activity test

The specific method is as follows:

(1) Drug & reagent preparation:

1. Dilute the drug to the required working concentration;

2. Reagent & luciferase working substrate preparation:

(1) ATP buffer: prepare 1M Tris, adjust the pH to 7.5 with HCl, and prepare 1M Tris-Cl (pH7.5).

(2) Luciferin buffer: Add 0.34g KH ₂ PO ₄ powder into 500mL ddH ₂ O, and adjust the pH to 7.8 with KOH. This was formulated as 5 mM KH ₂ PO ₄ (PH 7.8).

(3) Luciferase working substrate;

(4) 0.5% NP-40: NP40 is very viscous, NP-40 is first diluted to 20% with 1X PBS, and then 20% NP-40 is diluted to 0.5% with 1XPBS.

(5) 5mg/mL MTT (to be prepared in the dark): After dissolving in 1X PBS, pass through a 0.22um filter to sterilize. Aliquot into 1mL/tube and store at -20°C for a long time.

(2) Experimental steps for detecting luc:

Plank the day before:

1) Cells were centrifuged at 500rpm for 5min, resuspended in 1mL of culture medium, adjusted to 3*10^5 cells/mL and pipetted evenly;

2) Use a row gun to plate the cells, plate a 96-well flat bottom plate, 200ul/well;

On the day of the experiment:

3) Set up three duplicate wells and set up blank control wells and solvent control wells at the same time, add 2ul drug dilutions to each well in sequence, and tap the four sides of the 96-well plate to mix evenly after adding one plate;

4) Cultivate at 37° C., 5% CO ₂ for 6 hours. After 6 hours, the cell growth status was observed under the microscope;

5) Centrifuge at 1600rpm for 5min, discard the solution; (at this time, it can be frozen at -80°C)

6) Add 50ul of 0.5% NP40 to each well, shake on the medium gear of the micro shaker for 20min;

7) Pipette 10ul per well into a pure white 96-well plate;

8) Measure the fluorescence value (ZH LAB, Promega, GLOMAX 96microplate luminometer);

9) Calculate the inhibition rate: (1-drug group/solvent group)*100%

(3) Detection of MTT experimental steps:

Plank the day before:

1) Cells were centrifuged at 500rpm for 5min, resuspended in 1mL of culture medium, adjusted to 1*10^5 cells/mL and pipetted evenly;

2) Use a row gun to plate the cells, plate a 96-well flat bottom plate, 200ul/well;

On the day of the experiment:

3) Set blank control wells, solvent control wells and lysis wells, add 2ul drug dilutions to each well in sequence and tap the edge to mix;

4) Culture at 37°C, 5% CO ₂ for 48 hours, and observe the cell growth status under the microscope after 48 hours;

5) Add 20ul 10X lysis solution to the analysis well, add 10ul MTT to each well in the dark, and tap the edge to mix;

6) Culture at 37°C, 5% CO2 for 4 hours;

7) Centrifuge at 1600rpm for 5min, gently suck off the supernatant with a pipette, and be careful not to suck up the precipitate;

8) Add 150ul DMSO (manufactured by Shanghai Sangong) to each well, and vibrate for 15 minutes in the middle gear of the micro shaker;

9) Measure the absorbance at 490nm;

10) Calculate the lethal rate: (solvent group-drug group)/(solvent group-LYSIS group)*100%

Result: the result of described novel furan sesquiterpene electrophilic natural product activity test is shown in the following table 3, shows that described novel furan sesquiterpene electrophilic natural product acts on the activity of Keap1-Nrf2-ARE signaling pathway, and has relatively Strong anti-inflammatory activity (EC ₅₀ =15 μM).

Table 3 Activity test results of novel furan sesquiterpene electrophilic natural products

2. Water Solubility Test

Test method: import the chemical structural formula of sesquiterpene electrophilic natural product shown in formula (I) into ligandscout software, calculate its cLogP value, the result is as shown in Figure 15, the cLogP calculated value of this natural product is 2.909, and the cLogP value is more The smaller the water solubility, the better, indicating that the natural product has better water solubility.

3. Stability test

The two mass spectrum data shown in Figure 16 and Figure 17 all come from the same sample prepared by the reaction of the semiterpenoid electrophilic natural product and the electrophilic thiol probe of the present invention, and 263.1275 is the quasi-molecular ion peak [M+H of the natural product monomer ] ⁺ , the two spectra are the data obtained from the injection of the same sample at different time periods (2021.09.14, ^2021.09.27 ). Degradation occurs over time, which shows that the stability of this natural product is relatively good.

Claims (7)

1. A sesquiterpenoid electrophilic natural product, characterized in that its chemical structural formula is as shown in formula (I):

(I). 2. the separation and preparation method of sesquiterpene electrophilic natural product as claimed in claim 1, is characterized in that, comprises the steps: S1. Form a natural product-probe complex between the electrophilic probe and the crude extract of Ginkgo biloba, and detect it by chromatography-tandem mass spectrometry; select the Ginkgo-probe target pro-probe with the quasi-molecular ion peak [M+H] ⁺ of 612.3103 Electron natural product, minus the molecular weight of electrophilic probe, the quasi-molecular ion peak [M+H] of the target electrophilic natural product obtained ^is 263.1275; the chromatography adopts Thermo Scientific UltiMate 3000 HPLC, liquid phase condition: Hypersil Gold Dim C18 column, 100×2.1 mm, 1.9 µm, mobile phase: phase A: aqueous solution containing 0.1% formic acid, phase B: acetonitrile; flow rate: 0.3mL/min, gradient elution; the mass spectrometer uses Thermo Fisher QExactive; The chemical structural formula of the electrophilic probe is

; S2. Under the same chromatographic and mass spectrometric conditions as step S1, the crude extract of Ginkgo biloba is subjected to high-resolution first- ^level scanning, and the quasi-molecular ion peak [M+H] of the target electrophilic natural product is extracted with a chromatographic peak of 263.1275 to obtain the target electrophilic The retention time of the electric natural product on the chromatographic column is 13.10 minutes; S3. Under the same chromatographic and mass spectrometric conditions as step S1, perform DAD ultraviolet full-scan liquid mass detection on the ginkgo crude extract, and obtain the ultraviolet absorption wavelength range of the target electrophilic natural product in the range of 240-270 nm; S4. Use the preparation solution to separate and prepare the target electrophilic natural product in the crude extract of Ginkgo biloba, and determine that the peak eluting time of the target electrophile natural product under the conditions of the preparation liquid phase is 24.98 to 25.12 minutes; the preparation liquid phase The condition is Agilent ZORBAX SB-C18, 9.4×250 mm, 5 µm; S5. Adopt medium and low pressure preparative liquid chromatography to separate and purify Ginkgo crude extract for the first time; said medium and low pressure preparative liquid chromatography adopts 0.1% formic acid water and methanol elution system, C18 self-packing column, packing 80g, flow rate 12mL /min, the gradient ratio of 0.1% formic acid water is set to 25% to 80%, and the column separation time is 150 minutes. Using the peak time of the target electrophile natural product summarized in the preparation of the liquid phase in step S4, collect all the samples containing the target electrophile Fractions of natural products were collected by rotary evaporation under reduced pressure; the C18 self-packed column was YMC, packing: ODS-A, micropore diameter 12 nm, particle diameter 50 µm; S6. Perform secondary separation and purification of all fractions containing the target electrophilic natural product in step S5 by thin-layer chromatography; the developer of the thin-layer chromatography is cyclohexane:ethyl acetate with a volume ratio of 2:1, The development time was 45 minutes; all the obtained bands were collected and eluted separately, and each band was analyzed by liquid chromatography-mass spectrometry to obtain the target electrophile with a quasi-molecular ion peak [M+H] ⁺ of 263.1275 Pure products of natural products. 3. The preparation method according to claim 2, wherein the mass spectrometry instrument model of the ultraviolet full-scan liquid mass detection in step S3 is Bruker timsTOF, and the liquid phase instrument model is Thermo U3000. 4. The preparation method according to claim 2, characterized in that the specification of the thin-layer chromatography preparation plate in step S6 is 1 mm. 5. The preparation method according to claim 2, characterized in that the elution in step S6 is elution with acetone. 6. The application of the sesquiterpene electrophilic natural product according to claim 1 in the preparation of anti-inflammatory products. 7. An anti-inflammatory product, characterized in that it contains the sesquiterpene electrophilic natural product according to claim 1.

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