Cyclooxygenase-modified functionalized magnetic bead as well as preparation method and application thereof

Abstract

The invention provides a functionalized magnetic bead modified by cyclooxygenase as well as a preparation method and application of the functionalized magnetic bead, and belongs to the technical field of mass spectrometric detection. According to the invention, the functionalized magnetic bead modified by cyclooxygenase is successfully constructed, and the functionalized magnetic bead has good physical properties, activity, specificity and stability, and can be repeatedly used. The functionalized magnetic bead can be used as a material for screening active ingredients, and is applied to screening of anti-inflammatory active ingredients in meconopsis quintuplina alkaloid. The method for screening the active components by using the cyclooxygenase-modified functional magnetic beads disclosed by the invention provides a certain thought and reference for screening the active components from Chinese herbal medicines, and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of mass spectrometry detection, and in particular relates to cyclooxygenase-modified functionalized magnetic beads and a preparation method and application thereof. Background Art

[0002] Meconopsis quinuplinervia Regel belongs to the genus Meconopsis of the Papaveraceae family. It is a precious Tibetan medicine, mainly distributed in high-altitude cold areas such as the Qinghai-Tibet Plateau. It has a long history of medicinal use. Its dried whole herb is often used to treat diseases such as trauma, rheumatism, pneumonia and high fever, and has significant anti-inflammatory and analgesic effects. Modern research has found that Meconopsis quinuplinervia Regel contains a variety of active ingredients, mainly including alkaloids, flavonoids and phenylpropanoid glycosides. Among them, alkaloids are one of the main active ingredients of Meconopsis quinuplinervia Regel that exert anti-inflammatory and analgesic effects. They have diverse chemical structures and biological activities, such as simple pyrrolizidine alkaloids, indole alkaloids and quinoline alkaloids. These alkaloids can inhibit inflammatory responses and relieve pain through various mechanisms.

[0003] Currently, the following methods are used to identify the anti-inflammatory active ingredients in Meconopsis quinqueneri. Chromatography, including thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC), is a commonly used method. TLC can be used for the preliminary separation and identification of chemical components in Meconopsis quinqueneri. It is simple and rapid to operate, but the separation efficiency is relatively low. HPLC provides higher resolution and sensitivity, allowing for the separation and quantitative analysis of multiple components in complex samples. Furthermore, it can be coupled with mass spectrometry (MS) to identify the structures of compounds. Furthermore, mass spectrometry (MS) is also an important method for identifying the anti-inflammatory active ingredients in Meconopsis quinqueneri. By measuring the molecular ion peaks and fragment ion peaks of a compound, the molecular formula and structural characteristics of the compound can be inferred, and the compound's identity can be determined. Furthermore, nuclear magnetic resonance spectroscopy (NMR) can also be used to elucidate the structure of the active ingredients in Meconopsis quinqueneri, providing more comprehensive molecular structural information and facilitating accurate identification of the compound's structure.

[0004] There are some problems and limitations in the existing technology in identifying the anti-inflammatory active ingredients of Meconopsis quinqueneri. Although traditional chromatography and mass spectrometry methods can provide relatively rich chemical information, they often face challenges such as low separation efficiency, long analysis time, and complex sample pretreatment when faced with complex natural product systems, and it is difficult to achieve rapid and efficient screening and identification of multiple active ingredients at the same time. Although bioassay methods can directly reflect the biological activity of compounds, they usually have low throughput, the screening process is time-consuming and labor-intensive, and are easily interfered by experimental conditions and impurity components, resulting in false positive or false negative results. In addition, existing computer-aided drug design methods such as molecular docking still need to be improved in terms of prediction accuracy, and require a large amount of compound structure information and computing resource support, making it difficult to directly apply to the actual screening of active ingredients of natural products. These problems limit the in-depth research and development of the anti-inflammatory active ingredients of Meconopsis quinqueneri.

[0005] Functionalized magnetic beads have a wide range of applications in the field of detection technology. The basic principle is to chemically modify the surface of the magnetic beads so that they can specifically bind to specific target molecules, and then use a magnetic field to achieve rapid separation and enrichment of the target molecules, thereby improving the sensitivity and specificity of the detection. In terms of biological detection, functionalized magnetic beads can be used for cell separation, protein purification, nucleic acid extraction, etc. For example, by fixing specific antibodies on the surface of magnetic beads, specific cells or proteins can be enriched and separated, facilitating subsequent analysis and research. In the field of drug screening, functionalized magnetic beads can be used to screen compounds with specific biological activities. By fixing biological targets on the surface of magnetic beads, incubating them with a compound library, and then using a magnetic field to separate active compounds bound to the target, the efficiency of drug screening can be improved.

[0006] The literature (DOI: 103724 / SP.J.1123.2024.07003) screened cyclooxygenase (COX-2) inhibitors from Panax notoginseng leaves based on ligand fishing technology. By synthesizing Fe3O4 coated with polydopamine (PDA), Ni was chelated on the surface of PDA. 2+ Using metal ion affinity to immobilize COX-2, a green, environmentally friendly, and highly specific fishing tool was developed, providing a valuable reference for the efficient discovery of anti-inflammatory drugs or lead compounds from complex traditional Chinese medicine systems. However, there are currently no reports on the use of functionalized magnetic beads to screen and identify COX-2-inhibiting components in the total alkaloids of Meconopsis quinqueneri. Summary of the Invention

[0007] In order to solve the deficiencies in the prior art, the present invention aims to provide a cyclooxygenase-modified functionalized magnetic beads and a preparation method and application thereof.

[0008] The invention provides cyclooxygenase-modified functional magnetic beads. The preparation raw materials thereof include amino magnetic beads and cyclooxygenase. The mass ratio of the amino magnetic beads to the cyclooxygenase is 5 mg:0.4 μg to 2.0 μg.

[0009] Furthermore, the mass ratio of the amino magnetic beads to cyclooxygenase is 5 mg:1.2 μg.

[0010] The present invention also provides a method for preparing the above-mentioned cyclooxygenase-modified functionalized magnetic beads, the method comprising the following steps:

[0011] (1) washing the amino magnetic beads, adding a crosslinker solution for activation reaction, and then washing;

[0012] (2) adding the cyclooxygenase solution to the solution prepared in step (1) for reaction, followed by washing;

[0013] (3) Add a blocking agent to the mixture in step (2) and wash to obtain cyclooxygenase-modified functionalized magnetic beads.

[0014] Furthermore, in step (1), the cross-linking agent is glutaraldehyde; the cleaning reagent is PBS buffer; the number of cleanings is 1 to 5 times; the temperature of the activation reaction is 0 to 8°C, and the time is 1 to 5 hours;

[0015] In step (2), the solvent in the cyclooxygenase solution is PBS buffer; the reaction temperature is 0-8°C and the reaction time is 10-20 hours; the cleaning reagent is Tris-HCl buffer; the number of cleaning times is 1-5 times;

[0016] In step (3), the blocking agent is CE510 blocking agent; the reaction temperature is 0-8°C and the reaction time is 1-3 hours; the cleaning reagent is Tris-HCl buffer; and the number of cleaning times is 1-5 times.

[0017] Furthermore, in step (1), the crosslinking agent solution is a PBS buffer solution containing 1-10% glutaraldehyde, preferably a PBS buffer solution containing 5% glutaraldehyde; the number of washings is 3 times; the activation reaction temperature is 4°C and the time is 3 hours;

[0018] In step (2), the reaction temperature is 4°C and the reaction time is 14 hours; the number of washings is 3 times;

[0019] In step (3), the reaction temperature is 4° C. and the reaction time is 2 h; the number of cleanings is 3 times.

[0020] The present invention also provides the use of the cyclooxygenase-modified functionalized magnetic beads in preparing a reagent for screening anti-inflammatory active ingredients of Chinese herbal medicines.

[0021] Furthermore, the anti-inflammatory active ingredient is an ingredient that inhibits cyclooxygenase.

[0022] Furthermore, the Chinese herbal medicine is Meconopsis quinquenervata.

[0023] Furthermore, the anti-inflammatory active ingredients are alkaloids and flavonoids;

[0024] The alkaloid components are meconopsis quinquefolia isomer 1, purslaneamide E, methoxy yellow crotonine, papaverine, meconopsis quinquefolia isomer 2, protopine, and protopine isomers; the flavonoid components are luteolin, apigenin, wheat flavonoid, and golden celery flavonoid.

[0025] Furthermore, the anti-inflammatory active ingredients are flavinantine, methoxy flavin crotonine, purslanamide E and N-methyl-2,3,6-trimethoxymorphinandien-7-one N-Oxide.

[0026] The present invention also provides a method for screening anti-inflammatory active ingredients in Chinese herbal medicines, wherein the anti-inflammatory active ingredients are ingredients that inhibit cyclooxygenase. The method comprises the following steps: mixing and incubating the above-mentioned cyclooxygenase-modified functionalized magnetic beads and Chinese herbal medicine samples, magnetically separating them, removing the supernatant, and using UPLC-Q-Exactive Orbitrap MS / MS analysis to identify the anti-inflammatory active ingredients.

[0027] Furthermore, the mass ratio of the functionalized magnetic beads to the Chinese herbal medicine sample is 10-30 mg:10-15 μg, preferably 20 mg:12 μg.

[0028] Furthermore, the incubation temperature is 0-5°C, preferably 4°C; and the incubation time is 20-40 min, preferably 30 min.

[0029] The present invention also provides use of N-methyl-2,3,6-trimethoxymorphinandien-7-one N-Oxide and flavinantine in preparing a medicine with anti-inflammatory effect.

[0030] The present invention has achieved the following beneficial effects:

[0031] (1) The present invention successfully constructed functionalized magnetic bead particles (Fe3O4@SiO2-COX-2), which have good physical properties, activity, specificity, and stability and can be reused multiple times.

[0032] (2) Cyclooxygenase (COX-2) is expensive. Directly using the enzyme and the mixture to incubate and screen the active ingredients will result in high enzyme loss costs. The present invention uses Fe3O4@SiO2-COX-2 as a material for screening active ingredients and applies it for the first time in Meconopsis quinqueneri. This technology has the advantages of being able to easily obtain active ingredients from complex components of traditional Chinese medicine, being able to screen and identify active ingredients from complex systems after being combined with UPLC-Q-Exactive Orbitrap MS / MS, being able to concentrate the target ingredients to a great extent, and removing most other interfering components for subsequent research. The Fe3O4@SiO2-COX-2 method for screening active ingredients constructed by the present invention provides certain ideas and references for screening active ingredients from traditional Chinese medicines.

[0033] (3) The present invention uses Fe3O4@SiO2-COX-2 to incubate total alkaloids of Meconopsis quinqueneri, and screens out 7 potential anti-inflammatory alkaloid components, namely Meconopsis quinqueneri alkaloid isomer 1, purslaneamide E, methoxyflavinine, black water papaverine, Meconopsis quinqueneri alkaloid isomer 2, protopine, protopine isomer, and 4 potential anti-inflammatory non-alkaloid components, luteolin, apigenin, wheat flavin (also known as alfalfa), and golden chrysanthemum flavin. The present invention verifies through COX-2 inhibition experiments that purslaneamide E, methoxyflavinine, flavinantine, and N-methyl-2,3,6-trimethoxymorphinandien-7-one N-Oxide screened out by Fe3O4@SiO2-COX-2 are indeed effective anti-inflammatory active ingredients and have broad application prospects in the preparation of drugs with anti-inflammatory effects.

[0034] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0035] The following is a further detailed description of the present invention through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-mentioned content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Total ion currents of (a) potential anti-inflammatory components and (b) Meconopsis quinqueneri samples in positive ion mode.

[0037] Figure 2Extracted ion chromatograms of different substances before and after enzyme addition: (a) celecoxib; (b) huperzine A; (c) flavinantine; (d) methoxyflavin crotonine; (e) purslanamide E; (f) N-methyl-2,3,6-trimethoxymorphinandien-7-one N-Oxide.

[0038] Figure 3 The graph shows the percentage of inhibition of COX-2 by the compounds: (a) celecoxib; (b) flavinantine; (c) methoxyflavin crotonine; (d) purslanamide E; (e) N-methyl-2,3,6-trimethoxymorphinandien-7-oneN-Oxide.

[0039] Figure 4 These are the results of investigating the ability of functionalized magnetic beads constructed under different enzyme dosage conditions to bind inhibitors.

[0040] Figure 5 This is the particle size determination result of Fe3O4@SiO2-COX-2.

[0041] Figure 6 These are the scanning electron microscopy results of Fe3O4@SiO2-COX-2 and Fe3O4@SiO2 at different magnifications (Note: a1, a2, a3 are the scanning electron microscopy images of Fe3O4@SiO2 at 5μm, 2μm, and 500nm magnifications, respectively; b1, b2, b3 are the scanning electron microscopy images of Fe3O4@SiO2-COX-2 at 5μm, 2μm, and 500nm magnifications, respectively).

[0042] Figure 7 Infrared spectra of Fe3O4@SiO2-COX-2, Fe3O4@SiO2 and COX-2.

[0043] Figure 8 Magnetic measurement results of Fe3O4@SiO2-COX-2 and Fe3O4@SiO2: (a) Magnetic separation effect diagram of Fe3O4@SiO2-COX-2; (b) hysteresis curve diagram of Fe3O4@SiO2-COX-2 and Fe3O4@SiO2.

[0044] Figure 9 These are the results of enzyme activity investigation of Fe3O4@SiO2-COX-2.

[0045] Figure 10The results of the specificity investigation of Fe3O4@SiO2-COX-2: (a) liquid phase diagram of different magnetic beads bound to celecoxib; (b) liquid phase diagram of different magnetic beads bound to Huperzine-A.

[0046] Figure 11 These are the results of the stability investigation of Fe3O4@SiO2-COX-2. DETAILED DESCRIPTION

[0047] The raw materials and equipment used in the present invention are all known products and are obtained by purchasing commercially available products.

[0048] The reagents and instruments used in the embodiments of the present invention are as follows:

[0049] (1) Experimental reagents

[0050] Table 1 Reagent list

[0051]

[0052]

[0053] Table 2 Instrument List

[0054]

[0055]

[0056] Example 1: Construction of cyclooxygenase-modified functionalized magnetic beads

[0057] First, COX-2 was diluted to 100 μg mL with sterile water. -1 (This solution is the mother liquor used in subsequent operations). Then, 5 mg of amino magnetic beads were placed in a 5 mL centrifuge tube and washed three times with 0.5 mL of PBS buffer. Each time, magnetic separation was performed and the supernatant was discarded. 0.5 mL of PBS buffer containing 5% glutaraldehyde was added to the magnetic beads, shaken for 3 hours at 4°C, magnetically separated, the supernatant was discarded, and washed three times with 0.5 mL of PBS buffer. Then, 500 μL of PBS buffer containing 1.2 μg of COX-2 was added, shaken for 14 hours at 4°C, magnetically separated, the supernatant was discarded, and washed three times with 0.5 mL of Tris-HCl buffer. 500 μL of CE510 blocking agent was added to each particle, shaken for 2 hours at 4°C, and washed three times with 0.5 mL of Tris-HCl buffer to obtain functionalized magnetic beads (i.e., cyclooxygenase-modified functionalized magnetic beads, referred to as Fe3O4@SiO2-COX-2).

[0058] Example 2: Application of functionalized magnetic beads in screening potential anti-inflammatory components in total alkaloids of Meconopsis quinqueneri

[0059] 1. Experimental methods

[0060] Take 20 mg of functionalized magnetic beads and place them in a 5 mL centrifuge tube, add 6 μg mL -1 A 2 mL sample of Meconopsis quinquenervata (total alkaloid content: 19.72%) was mixed with 1 mL of PBS buffer and incubated at 4°C with shaking for 30 minutes. The sample was then allowed to stand and magnetically separated for 5 minutes. The supernatant was removed and washed three times with PBS buffer. The supernatant was then dissociated with 2 mL of methanol for 1 hour. The combined supernatants were spin-dried and dissolved in 200 μL of methanol in a 1 mL centrifuge tube. The sample was centrifuged at 12,000 rpm for 15 minutes at 25°C. The supernatant was then transferred to a liquid chromatography vial containing a 250 μL insert for UPLC-Q-Exactive Orbitrap MS / MS analysis.

[0061] At the same time, an appropriate amount of Meconopsis quinquefolia sample (total alkaloid content of 19.72%) was weighed and dissolved in a trace amount of DMSO and diluted with methanol to a concentration of about 5.0 mg·mL -1 The sample solution was filtered through a 0.22 μm filter membrane and used for UPLC-Q-Exactive Orbitrap MS / MS detection.

[0062] UPLC-Q-Exactive Orbitrap MS / MS analysis conditions: Chromatographic conditions were an ACQUITY UPLC HSS T3 column (2.1 × 100 mm, 1.8 μm), the mobile phase was 0.1% formic acid in water (A)-acetonitrile (B), gradient elution (0-28 min, 5%-22% B; 28-35 min, 22%-90% B), column temperature was 30°C, injection volume was 2 μL, and flow rate was 0.2 mL min. -1 , detection wavelength 200-400 nm. Mass spectrometry conditions were electrospray ionization (ESI), positive ion mode detection, transfer tube temperature 320°C, spray voltage 3.5 kV, auxiliary gas flow rate 3 L·min -1 , auxiliary gas temperature 350℃, cone gas flow rate 12L·min -1 The scanning mode was FullMS / dd-MS2, the Full MS resolution was 70 000, the dd-MS2 resolution was 17 500, and the scanning range was m / z 100-1500.

[0063] 2. Experimental results

[0064] The total ion current of the Meconopsis quinquenervii sample and its potential anti-inflammatory components is shown in the figure. Figure 1 The mass spectrometry data were analyzed and the compounds were identified based on the relative retention time of the chromatographic peaks, quasi-molecular ion peaks, accurate molecular masses, and fragment ions in the secondary mass spectrum. The identification results are shown in Table 3.

[0065] Table 3 Identification of alkaloid components in Meconopsis quinquenervata samples under positive ion mode

[0066]

[0067]

[0068]

[0069]

[0070] Note: * indicates potential anti-inflammatory ingredients.

[0071] Example 3: Isolation and identification of potential anti-inflammatory components

[0072] Depend on Figure 1 It can be seen that in addition to the potential anti-inflammatory alkaloid components identified in the positive ion mode, there are other non-alkaloid potential anti-inflammatory components. Therefore, the present invention has separated and identified other types of components.

[0073] 1. Isolation of potential anti-inflammatory components

[0074] Take 1 g of the total alkaloid part of Meconopsis quinqueneri (total alkaloid content is 19.72%), dissolve it with an appropriate amount of methanol, filter it with a 0.45 μm filter membrane, and then use a semi-preparative high performance liquid chromatography to perform coarse segmentation. The semi-preparative column used is a Huapu Technology C18 column (30×250 mm, 10 μm), the mobile phase is 0.1% phosphoric acid water (A)-acetonitrile (B), and the gradient elution (0-30 min, 10%-15% B; 30-60 min, 15%-25% B). It is divided into 5 sections, which are respectively recorded as section 1, section 2, section 3, section 4, and section 5. The five fractions were analyzed by high performance liquid chromatography using a SWELL Chromplus C18 column (4.6×250 mm, 5 μm), a mobile phase of 0.1% phosphoric acid (A)-acetonitrile (B), a gradient elution (0-15 min, 15% B; 15-25 min, 15%-90% B; 25-30 min, 90% B), a column temperature of 30°C, and a detection wavelength of 220 nm.

[0075] No obvious peak was detected in segment 2 and no treatment was performed. The solutions of the remaining four segments were concentrated separately and then separated using a semi-preparative high performance liquid chromatography (Note: the semi-preparative column used was a Huapu Technology C18 column (30×250 mm, 10 μm)).

[0076] Compound 1 was isolated from segment 1 using a mobile phase of 0.1% phosphoric acid (A)-acetonitrile (B) and a gradient elution procedure (0-30 min, 10%-15% B; 30-60 min, 15% B). After lyophilization, 35 mg of a bright yellow solid was obtained.

[0077] Compound 2 was isolated from segment 3 using a mobile phase of 0.1% phosphoric acid (A)-methanol (B) and a gradient elution procedure (0-30 min, 25%-30% B; 30-60 min, 30% B). After lyophilization, 42 mg of a light yellow solid was obtained.

[0078] Section 4 was separated using a mobile phase of 0.1% phosphoric acid (A)-acetonitrile (B) using a gradient elution procedure (0-30 min, 12%-14% B; 30-60 min, 14% B) to afford compounds 3 and 4. After lyophilization, 8 mg of a brownish-gray solid and 8.5 mg of a light yellow solid were obtained, respectively.

[0079] Compound 5 was isolated from fraction 5 by using a mobile phase of 0.1% phosphoric acid (A)-acetonitrile (B) and an isocratic elution procedure (0-60 min, 15% B). After lyophilization, 1.5 mg of a white solid was obtained.

[0080] 2. Identification of potential anti-inflammatory ingredients

[0081] Compound 5 was not identified due to its low amount. The present invention finally separated and obtained four monomeric substances with high purity, and 1 H-NMR, 13 The identification results of the compounds using C-NMR and high-resolution mass spectrometry are as follows:

[0082] Compound 1: bright yellow solid, soluble in methanol and chloroform, molecular formula C 19 H 21 NO4, HR-ESI-MS m / z: 328.1545[M+H] + .

[0083] 1H-NMR(600MHz,CD3OD)δ:7.03(1H,s,H-4),6.79(1H,s,H-1),6.78(1H,s,H-5 ),6.53(1H,s,H-8),4.40(1H,d,J=6.0Hz,H-9),3.84(3H,s,2-OCH3),3.80(3H ,s,6-OCH3),3.62(1H,m,H-10β),3.38(1H,m,H-10α),3.16(1H,m,H-16α),3.0 3(1H,m,H-16β), 2.87(3H,s,N-CH3), 2.13(1H,m,H-15α), 2.08(1H,m,H-15β).

[0084] 13 C-NMR (150MHz, CD3OD) δ: 181.75 (C-7), 156.83 (C-14), 152.60 (C-6), 149. 04(C-2),147.55(C-3),130.59(C-12),126.87(C-8),125.48(C-11),121.2 4(C-5),113.55(C-4),111.68(C-1),62.44(C-9),56.41(C-17),55.85(C-1 8),47.28(C-16),42.52(C-13),40.81(C-19),38.41(C-15),33.51(C-10).

[0085] The NMR spectrum data of compound 1 is consistent with that of flavinantine, and the structural formula is as follows:

[0086]

[0087] Compound 2: pale yellow solid, soluble in methanol and chloroform, molecular formula C20H23NO4, HR-ESI-MS m / z: 342.1704 [M+H] + .

[0088] 1H-NMR(600MHz,CD3OD)δ:7.15(1H,s,H-4),6.95(1H,s,H-5),6.84(1H,s,H-1),6. 55(1H,s,H-8),4.43(1H,d,J=6.0Hz,H-9),3.87(3H,s,3-OCH3),3.83(3H,s,2-OCH 3),3.83(3H,s,6-OCH3),3.65(1H,m,H-10α),3.41(1H,m,H-10β),3.18(1H,m,H-16 α),3.03(1H,m,16β),2.88(3H,s,N-CH3),2.20(1H,m,H-15α),2.12(1H,m,H-15β).

[0089] 13 C-NMR(150MHz,CD3OD)δ:181.72(C-7),156.71(C-14),152.75(C-6),150.54(C -3),150.33(C-2),130.46(C-12),127.21(C-11),126.91(H-8),121.20(C-5),1 12.09(C-1),110.98(C-4),62.36(C-9),56.95(3-OMe),56.45(6-OMe),55.98(2 -OMe),47.25(C-16),42.71(C-13),40.83(N-OMe),38.24(C-15),33.51(C-10).

[0090] The NMR spectrum data of compound 2 is consistent with that of O-Methylflavinantine, and the structural formula is as follows:

[0091]

[0092] Compound 3: brown-gray solid, soluble in methanol and chloroform, molecular formula C 12 H 13 NO3, HR-ESI-MS m / z: 220.0970[M+H] + .

[0093] 1H-NMR(600MHz,CD3OD)δ:6.55(1H,s,H-7),6.55(1H,s,H-10),4.72(1H,t,J=6.0Hz,H-13),4.09(1H,m,H-5β),3 .05(1H,m,H-1β),3.05(1H,m,H-5α),2.74(2H,m,H-6),2.61(1H,m,H-2β),2.39(1H,m,H-2α),1.77(1H,m,H-1α).

[0094] 13 C-NMR(150MHz,CD3OD)δ:175.9(C-3),145.6(C-9),145.5(C-8),129.8(C-11),125.6(C-12 ),116.3(C-7),112.4(C-10),58.3(C-13),38.7(C-5),32.7(C-2),28.8(C-1),28.7(C-6).

[0095] The NMR spectrum data of compound 3 is consistent with that of purslanamide E, and the structural formula is as follows:

[0096]

[0097] Compound 4: pale yellow solid, soluble in methanol and chloroform, molecular formula C20H23NO5, HR-ESI-MS m / z: 358.1653 [M+H] + .

[0098] 1 H-NMR(600MHz,CD3OD)δ:7.17(1H,s,H-4),6.95(1H,s,H-1),6.81(1H,s,H-5 ),6.54(1H,s,H-8),4.74(1H,d,J=6.0Hz,H-9),3.87(3H,s,3-OCH3),3.83(3H ,s,2-OCH3),3.83(3H,s,6-OCH3),3.62(3H,s,N-CH3),3.62(1H,m,H-10β),3. 56(2H,m,H-16), 3.38(1H,m,H-10α), 2.43(1H,m,H-15α), 2.14(1H,m,H-15β).

[0099] 13C-NMR(150MHz,CD3OD)δ:181.56(C-7),156.43(C-14),152.76(C-6),150.67(C -3),150.57(C-2),130.72(C-12),127.98(C-11),125.38(C-8),120.50(C-5),1 12.19(C-1),111.03(C-4),76.18(C-9),60.30(C-16),56.92(N-OMe),56.46(2- OMe),56.21(3-OMe),56.01(6-OMe),41.97(C-13),37.42(C-15),35.06(C-10).

[0100] The NMR spectrum data of compound 4 are consistent with N-methyl-2,3,6-trimethoxymorphinandien-7-oneN-Oxide, and the structural formula is as follows:

[0101]

[0102] These results demonstrate that the present invention ultimately isolated four highly pure monomeric substances, all of which are known alkaloid components. Among them, N-methyl-2,3,6-trimethoxymorphinandien-7-one N-Oxide is isolated from Meconopsis quinqueneri for the first time. All four compounds are isoquinoline alkaloids.

[0103] Example 4: Verification of the activity of potential anti-inflammatory ingredients

[0104] 1. Experimental methods

[0105] (1) Molecular docking studies

[0106] The enzyme's three-dimensional structure was obtained in pdb format from the Uniprot database, and the compound's three-dimensional structure was drawn using Chem 3D. Molecular docking was performed using AutoDock Vina, and the binding energy was calculated.

[0107] (2) Intensity decay mass spectrometry research

[0108] The present invention uses 100 μg·mL -1 COX-2 is used as the target for interaction with alkaloid molecules. The appropriate amount of alkaloid compounds (compounds 1, 2, 3, and 4), celecoxib, and huperzine A samples were accurately weighed, dissolved and diluted with chromatographic methanol to obtain a concentration of 100 μg mL -1Take 200 μL of the sample solution and centrifuge it at 4°C, 10000 rpm for 15 min. Then take the supernatant as the test solution for subsequent liquid quality testing. Take another 2 mL of 100 μg mL -1 The sample solution was dried by nitrogen blower, dissolved in 100 μL of chromatographic methanol and then diluted to 2 mL with Tris-HCl buffer to obtain another aliquot with a concentration of 100 μg mL -1 Take 200 μL of the sample solution and add 40 μL of COX-2 solution. Mix thoroughly, incubate at 37°C for 30 minutes, transfer to a 30 kDa ultrafiltration centrifuge tube, and centrifuge at 4°C, 10,000 rpm, for 15 minutes to remove the enzyme and its complexes. Drain the bottom of the tube with nitrogen and reconstitute with 00 μL of chromatographic methanol. Centrifuge the solution at 4°C, 10,000 rpm, for 5 minutes. The supernatant is used as the test solution for subsequent LC / MS analysis. The peak areas of the alkaloid molecules before and after the enzyme addition reaction are recorded to compare the binding capacity of the alkaloid molecules with the enzyme. Table 4 lists all samples to be tested.

[0109] UPLC-Q-Exactive Orbitrap MS / MS analysis conditions:

[0110] Samples No. 1 to No. 8 were analyzed using the same chromatographic column, mobile phase, and elution conditions: an ACQUITY UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm), a mobile phase of 90% acetonitrile-10% water (containing 0.1% formic acid), a column temperature of 30°C, a detection wavelength of 210–410 nm, an injection volume of 2 μL, and a flow rate of 0.2 mL min. -1 .

[0111] Samples No. 9 and No. 10 used the same chromatographic column, mobile phase, and elution conditions: ACQUITY UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm), mobile phase 85% methanol-15% water, column temperature 30°C, detection wavelength 210-410 nm, injection volume 2 μL, and flow rate 0.2 mL min-1.

[0112] Samples No. 11 and No. 12 were eluted using the same chromatographic column, mobile phase, and elution conditions: an ACQUITY UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm), a mobile phase of 85% acetonitrile-15% water (containing 0.1% formic acid), a column temperature of 30°C, a detection wavelength of 210–410 nm, an injection volume of 2 μL, and a flow rate of 0.2 mL min-1. 1 .

[0113] Mass spectrometry conditions: electrospray ionization (ESI), positive ion mode detection, transfer tube temperature 325°C, spray voltage 3.5 kV, auxiliary gas flow rate 10 arb, auxiliary gas temperature 350°C, cone gas flow rate 40 arb, scan mode Full MS / dd-MS 2 , Full MS resolution 70 000, dd-MS 2 Resolution 17500, scanning range m / z 100~1000.

[0114] Table 4 Samples analyzed by UPLC-Q-Exactive Orbitrap MS / MS

[0115]

[0116] (3) Enzyme inhibition activity study

[0117] Accurately weigh an appropriate amount of compound sample, dissolve it in a small amount of DMSO, and then dilute it with ultrapure water to obtain a total of 6 sample solutions at 100, 50, 25, 12.5, 6.25, and 3.125 nM. Accurately draw an appropriate amount of celecoxib (positive drug) sample provided by the kit and dilute it with ultrapure water to obtain a total of 6 sample solutions at 100, 50, 25, 12.5, 6.25, and 3.125 nM. Accurately weigh an appropriate amount of huperzine A (negative drug) sample, dissolve it in a small amount of DMSO, and then dilute it with ultrapure water to obtain a total of 3 sample solutions at high, medium, and low doses of 100, 50, and 10 nM.

[0118] The prepared sample solution was operated according to the COX-2 inhibitor screening kit method to obtain the results, and the sample concentration-inhibition percentage graph was drawn using Origin 2024 software.

[0119] 2. Experimental results

[0120] (1) Molecular docking analysis

[0121] The docking results are shown in Table 5, which show that all four compounds have good affinity with COX-2, among which flavinantine has the strongest binding to COX-2.

[0122] Table 5 Molecular docking results

[0123]

[0124] (2) Intensity decay mass spectrometry analysis

[0125] See the results Figure 2After integration and calculation, the binding rates of COX-2 to celecoxib and huperzine A were 100% and 15.33%, respectively, indicating that the functionalized magnetic beads have good specificity; the binding rates to flavinantine, methoxyflavin crotonine, purslanamide E, and N-methyl-2,3,6-trimethoxymorphinandien-7-one N-Oxide were 83.88%, 77.28%, 89.59%, and 72.31%, respectively.

[0126] (3) Enzyme inhibition activity results

[0127] The inhibition percentages of the negative drug Huperzine A at high, medium, and low concentrations were 1.75%, 0, and 0, respectively, which showed almost no inhibition on the enzyme. The fitting curves R of the positive drug celecoxib and compound samples flavinantine, methoxymorphinidine, purslanamide E, and N-methyl-2,3,6-trimethoxymorphinandien-7-one N-Oxide were 2 The values ​​are 0.9953, 0.9950, 0.9996, 0.9986, and 0.9986, respectively, to determine the IC 50 The values ​​were 60.54, 15.82, 27.30, 37.98, and 81.13 nM, respectively. The results showed that compared with the positive drugs, the compounds methoxy-flavinyl crotonine, purslanamide E, and flavinantine had greater inhibitory strength on the enzyme, while N-methyl-2,3,6-trimethoxymorphinandien-7-oneN-Oxide had a weaker inhibitory effect on the enzyme. Figure 3 .

[0128] In summary, the present invention found that purslanamide E, methoxyflavin crotonine, flavinantine and N-methyl-2,3,6-trimethoxymorphinandien-7-one N-Oxide all have good binding energy with COX-2, among which purslanamide E has the highest binding rate with COX-2. Purslanamide E, methoxyflavin crotonine, flavinantine and N-methyl-2,3,6-trimethoxymorphinandien-7-one N-Oxide all showed strong anti-inflammatory ability. The IC values ​​of each compound were obtained using the COX-2 (human) inhibitor screening kit. 50The values ​​were 15.82, 27.30, 37.98 and 81.13 nM respectively. The anti-inflammatory effects of flavinantine and N-methyl-2,3,6-trimethoxymorphinandien-7-one N-Oxide were first discovered in the present invention.

[0129] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0130] Experimental Example 1: Screening of conditions for constructing cyclooxygenase-modified functionalized magnetic beads

[0131] 1. Experimental methods

[0132] The effect of binding inhibitors on functionalized magnetic beads constructed with different amounts of COX-2 (0.4 μg, 1.2 μg, 2.0 μg) was investigated. The specific operation was as follows:

[0133] Referring to the preparation method of Example 1, 0.01 mg·mL -1 600 μL of celecoxib solution and 1 mL of PBS buffer were incubated with shaking at 4°C for 30 minutes, allowed to stand, and subjected to magnetic separation for 5 minutes. The supernatant was removed and washed three times with PBS buffer. After dissociation with 700 μL of methanol for 1 hour, the supernatant was injected into HPLC for analysis, and the celecoxib peak area in different eluates was compared. Functionalized magnetic beads were prepared using the optimal enzyme dosage, washed three times with Tris-HCl buffer, and stored at 4°C for subsequent experiments.

[0134] Celecoxib HPLC analysis conditions: WondaSil C18-WR column (4.6×150 mm, 5 μm), column temperature 30°C, detection wavelength 254 nm, mobile phase 65% methanol-35% water, flow rate 1 mL min -1 The celecoxib peak time is about 14 minutes, and the peak shape is good.

[0135] 2. Experimental results

[0136] See the results Figure 4When the enzyme dosage increased from 0.4μg to 1.2μg, the amount of Fe3O4@SiO2-COX-2 bound to the selective inhibitor increased, while when the enzyme dosage increased from 1.2μg to 2.0μg, the amount of Fe3O4@SiO2-COX-2 bound to the selective inhibitor decreased. At an enzyme dosage of 0.4μg, there were still a large number of available bonding sites and spaces on the magnetic bead surface. Increasing the enzyme dosage also enhanced the ability to bind the selective inhibitor. At an enzyme dosage of 2.0μg, the available bonding sites on the magnetic bead surface approached saturation. Increasing the enzyme dosage resulted in overly tight crosslinking, which in turn weakened the ability to bind the selective inhibitor. Therefore, the optimal enzyme dosage for constructing functionalized magnetic beads is 1.2μg.

[0137] Experimental Example 2: Characterization and Performance Determination of Functionalized Magnetic Beads

[0138] 1. Experimental methods

[0139] (1) Physical characterization

[0140] At 25°C, the particle size analyzer was used to determine the average particle size and distribution of the functionalized magnetic beads. The superconducting quantum interference magnetometer was used to determine the magnetic properties of amino magnetic beads and functionalized magnetic beads. After the amino magnetic beads and functionalized magnetic beads were adhered to the conductive glue and sprayed with gold using a sputtering coater, the surface morphology was determined using a scanning electron microscope. The Fourier transform infrared spectrometer used the potassium bromide tablet method to determine the functional groups on COX-2, amino magnetic beads, and functionalized magnetic beads. The mass ratio of the sample to potassium bromide was 1:100, and after mixing, the tablets were pressed into transparent sheets of about 0.5 mm. The specific operation for determining COX-2 was to first take 100 mg of potassium bromide and press it into a transparent sheet, then add 1 drop of COX-2 solution on its surface to evenly disperse it before determining it. The acquisition times were 32 and the resolution was 4.0 cm. -1 A standard curve was drawn using enzyme solutions of different concentrations against the absorbance value. After the magnetic beads and enzyme were fixed, magnetic separation was performed, and the supernatant was collected. The absorbance value at 450 nm was measured using a microplate reader to calculate the enzyme loading capacity of the functionalized magnetic beads.

[0141] Enzyme loading mg·mg -1 =(enzyme concentration before fixation - enzyme concentration after fixation) * solution volume / magnetic bead mass

[0142] (2) Determination of enzyme activity of functionalized magnetic beads

[0143] Take 5 mg of amino magnetic beads and functional magnetic beads and place them in 5 mL centrifuge tubes respectively, add 0.01 mg mL -1 600 μL of celecoxib solution and 1 mL of PBS buffer were incubated with shaking at 4°C for 30 minutes, allowed to stand, and magnetically separated for 5 minutes. The supernatant was removed and washed three times with PBS buffer. After dissociation in 700 μL of methanol for 1 hour, the supernatant was injected into HPLC for analysis. The celecoxib peak area in the different eluates was compared. Each experiment was repeated three times.

[0144] (3) Investigating the specificity of functionalized magnetic beads

[0145] The experiment was divided into a positive group (selective inhibitor celecoxib) and a negative group (huperzine A).

[0146] The positive group was treated with the same weight (5 mg) of magnetic microspheres, functionalized magnetic beads, and inactivated functionalized magnetic beads (prepared by boiling in boiling water for 10 min) and 0.01 mg mL -1 Celecoxib 600 μL and 1 mL PBS buffer were shaken and incubated at 4 ° C for 30 minutes. The negative group was treated with the same weight (5 mg) of magnetic microspheres, functionalized magnetic beads, and inactivated functionalized magnetic beads (boiled in boiling water for 10 minutes) and 0.1 mg mL -1 600 μL of Huperzine A and 1 mL of PBS buffer were incubated at 4°C with shaking for 30 minutes. After incubation, both groups of particles were allowed to stand and magnetically separated for 5 minutes. They were then washed three times with PBS buffer and dissociated with 700 μL of methanol for 1 hour. The supernatant was then injected into an HPLC analyzer to compare the celecoxib peak area in the different eluates. Each experiment was repeated three times.

[0147] HPLC analysis conditions for Huperzine A: WondaSil C18-WR column (4.6×150 mm, 5 μm), column temperature 30°C, detection wavelength 310 nm, mobile phase 15% acetonitrile-85% water (containing 0.1% formic acid), flow rate 1 mL min -1 The peak time of Huperzine A is about 5 minutes, and the peak shape is good.

[0148] (4) Investigate the stability of functionalized magnetic beads

[0149] Take 5 mg of functionalized magnetic beads and place them in a 5 mL centrifuge tube, add 0.01 mg mL -1 600 μL of celecoxib solution and 1 mL of PBS buffer were incubated at 4°C with shaking for 30 minutes, allowed to stand, and magnetically separated for 5 minutes. The supernatant was removed and washed three times with PBS buffer. After dissociation with 700 μL of methanol for 1 hour, the supernatant was collected and stored at 4°C. The functionalized magnetic beads were washed twice with PBS buffer, and the above process was repeated four times. The five collected supernatants were injected into HPLC for analysis, and the celecoxib peak areas in the different eluates were compared. The RSD% was calculated by integrating the peak areas.

[0150] 2. Experimental results

[0151] (1) Particle size determination of Fe3O4@SiO2-COX-2

[0152] The particle size distribution of Fe3O4@SiO2-COX-2 is shown in Figure 5 The particle size range is 2304~5559nm, with an average particle size of 3600nm. The particle size is increased compared with amino magnetic beads (1μm), which is related to the addition of enzymes and blocking agents on the surface of amino magnetic beads.

[0153] (2) Scanning electron microscopy results of magnetic beads

[0154] The morphologies of Fe3O4@SiO2-COX-2 and Fe3O4@SiO2 are as follows Figure 6 As shown, the amino magnetic beads are irregular spheres with uneven surfaces. After the enzyme is immobilized, the particle size increases and square spherical substances are attached to the surface.

[0155] (3) Infrared characterization of magnetic beads

[0156] The infrared characterization results of magnetic beads are shown in Figure 7 The infrared spectrum shows that around 575cm -1 The stretching vibration absorption peak of Fe-O bond appears at about 1087cm -1 The strong absorption peak at about 3415 cm is the stretching vibration absorption peak of Si-O bond. -1 The broad absorption peaks at about 1642 and 1541 cm indicate the HO vibration of the carboxyl group in the protein. -1 The peaks at represent the stretching vibration of the C=O bond and the bending vibration of the NH bond in the peptide bond, respectively, indicating that COX-2 was successfully immobilized on the amino magnetic beads.

[0157] (4) Magnetic characterization of magnetic beads

[0158] The magnetic characterization results of the magnetic beads are shown in Figure 8 First, preliminary experiments showed that the magnetic bead suspension could be clarified in 5 minutes under the magnetic attraction of a strong magnet, and the magnetic separation effect was good. Secondly, according to the hysteresis curve, the saturation magnetization of Fe3O4@SiO2 and Fe3O4@SiO2-COX-2 was 31.90emu·g -1 and 29.73emu·g -1 This indicates that the magnetic beads still have good magnetism after loading COX-2. The lower magnetism than amino magnetic beads may be due to the immobilization of non-magnetic enzyme on the surface.

[0159] (5) Study on enzyme loading and enzyme activity

[0160] The enzyme loading capacity of the functionalized magnetic beads was calculated to be 0.0002345 mg·mg -1 The results of enzyme activity test of functionalized magnetic beads are as follows: Figure 9As shown, Fe3O4@SiO2-COX-2 significantly adsorbed a certain amount of celecoxib and had good ligand recognition activity.

[0161] (6) Specificity studies

[0162] Celecoxib is a selective COX-2 inhibitor, and Huperzine A is a negative drug in the present invention. Figure 10 It can be seen that Fe3O4@SiO2-COX-2 does not adsorb Huperzine A, but has a good adsorption capacity for celecoxib. However, the adsorption capacity of celecoxib by Fe3O4@SiO2-COX-2 after high-temperature inactivation is significantly lower than that before inactivation. This adsorption capacity, along with the adsorption capacity of Fe3O4@SiO2, is nonspecific adsorption and can be ignored. This result indicates that Fe3O4@SiO2-COX-2 has good specificity for COX-2 ligand screening.

[0163] (7) Stability study

[0164] The stability test results are as follows Figure 11 As shown in the figure, by comparing the peak areas of celecoxib in the five eluates, it can be seen that after two association-dissociation cycles, the binding ability of Fe3O4@SiO2-COX-2 to celecoxib began to decrease significantly; after four association-dissociation cycles, its binding ability was still retained above 50%, with an RSD value of 18.5373%, indicating good stability, as shown in Table 6.

[0165] Table 6 Stability investigation results of Fe3O4@SiO2-COX-2

[0166]

[0167] The above results show that the COX-2 modified Fe3O4@SiO2-COX-2 was successfully prepared in the present invention. The particle size increased after the enzyme was immobilized, and the average particle size was 3600nm. The magnetic beads loaded with COX-2 had good magnetic properties, and the enzyme loading capacity of the functionalized magnetic beads was 0.0002345mg·mg -1 , Fe3O4@SiO2-COX-2 has good specificity and stability for COX-2 ligand screening.

[0168] In summary, the present invention provides a cyclooxygenase-modified functionalized magnetic beads, and a preparation method and application thereof. The present invention successfully constructed cyclooxygenase-modified functionalized magnetic beads, which have good physical properties and activity, specificity, and stability, and can be reused many times. The functionalized magnetic beads can be used as a material for screening active ingredients and applied to the screening of anti-inflammatory active ingredients in Meconopsis quinqueneri alkaloids. The method for screening active ingredients using cyclooxygenase-modified functionalized magnetic beads constructed by the present invention provides certain ideas and references for screening active ingredients from Chinese herbal medicines, and has broad application prospects.

Claims

1. A cyclooxygenase-modified functionalized magnetic bead, characterized in that: The preparation raw materials include amino magnetic beads and cyclooxygenase; the mass ratio of the amino magnetic beads to the cyclooxygenase is 5mg:0.4μg-2.0μg.

2. The functionalized magnetic beads according to claim 1, characterized in that The mass ratio of the amino magnetic beads to cyclooxygenase is 5 mg:1.2 μg.

3. A method for preparing the cyclooxygenase-modified functionalized magnetic beads according to claim 1 or 2, characterized in that: The method comprises the following steps: (1) washing the amino magnetic beads, adding a crosslinker solution for activation reaction, and then washing; (2) adding the cyclooxygenase solution to the solution prepared in step (1) for reaction, followed by washing; (3) Add a blocking agent to the mixture in step (2) and wash to obtain cyclooxygenase-modified functionalized magnetic beads.

4. The method according to claim 3, characterized in that In step (1), the cross-linking agent is glutaraldehyde; the cleaning reagent is PBS buffer; the number of cleanings is 1 to 5 times; the temperature of the activation reaction is 0 to 8° C., and the time is 1 to 5 hours; In step (2), the solvent in the cyclooxygenase solution is PBS buffer; the reaction temperature is 0-8°C and the reaction time is 10-20 hours; the cleaning reagent is Tris-HCl buffer; the number of cleaning times is 1-5 times; In step (3), the blocking agent is CE510 blocking agent; the reaction temperature is 0-8°C and the reaction time is 1-3 hours; the cleaning reagent is Tris-HCl buffer; and the number of cleaning times is 1-5 times.

5. The method according to claim 4, characterized in that In step (1), the crosslinking agent solution is a PBS buffer solution containing 1-10% glutaraldehyde, preferably a PBS buffer solution containing 5% glutaraldehyde; the number of washings is 3 times; the activation reaction temperature is 4° C. and the time is 3 hours; In step (2), the reaction temperature is 4°C and the reaction time is 14 hours; the number of washings is 3 times; In step (3), the reaction temperature is 4° C. and the reaction time is 2 h; the number of cleanings is 3 times.

6. Use of the cyclooxygenase-modified functionalized magnetic beads according to claim 1 or 2 in the preparation of a reagent for screening anti-inflammatory active ingredients of Chinese herbal medicines.

7. The use according to claim 6, characterized in that The anti-inflammatory active ingredient is an ingredient that inhibits cyclooxygenase.

8. The use according to claim 6, characterized in that The Chinese herbal medicine is Meconopsis quinquenervata.

9. A method for screening anti-inflammatory active ingredients of Chinese herbal medicines, wherein the anti-inflammatory active ingredients are ingredients that inhibit cyclooxygenase, characterized in that: The method comprises the following steps: mixing and incubating the cyclooxygenase-modified functionalized magnetic beads according to claim 1 or 2 and a Chinese herbal medicine sample, performing magnetic separation, removing the supernatant, and analyzing and identifying the anti-inflammatory active components using UPLC-Q-Exactive Orbitrap MS / MS.

10. Use of N-methyl-2,3,6-trimethoxymorphinandien-7-one N-Oxide and flavinantine in the preparation of drugs with anti-inflammatory effects.

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