Application of TLR4 protein in screening of active ingredients of taxus chinensis var mairei fruits

Abstract

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to application of TLR4 protein in screening of active ingredients of taxus chinensis var mairei fruits. The preparation method comprises the following steps: dissolving TLR4 protein in PBS (Phosphate Buffer Solution), diluting into a TLR4 protein solution with the concentration of 50 mu g / ml by adding a sodium acetate buffer solution, and coupling the TLR4 protein solution with a sensor chip to obtain the TLR4 protein fishing chip. Respectively taking a taxus chinensis var mairei fruit test solution and a reference substance solution, putting the solutions into a surface plasmon resonance test pore plate of the TLR4 protein fishing chip, and carrying out SPR detection to screen active ingredients of the taxus chinensis var mairei fruits. The method is high in accuracy, can quickly screen the TLR4 protein-based active components of the taxus chinensis var mairei fruits, and has practical application and popularization values.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical analysis, and specifically relates to the application of TLR4 protein in screening active ingredients of Taxus chinensis var. mairei fruits. Background Art

[0002] Taxus is a general term for plants of the genus Taxus in the family Taxaceae. In 1999, the genus Taxus was listed as a first-class protected wild plant in China. Its variety, Taxus chinensis var. mairei, also known as the beautiful yew, is the most widely distributed species of this genus in China and is known as the "plant gold" and "gold tree". Its seeds (with arils), bark, stems, and branches and leaves have all been recorded for medicinal use. Its seeds are used as medicine, with the effects of eliminating indigestion and killing insects, and diuresis, and can be used for symptoms such as abdominal pain caused by food accumulation; the arils have activities such as antioxidant and blood sugar lowering. At present, Taxus chinensis var. mairei has not been included in the Chinese Pharmacopoeia, and its relevant quality standards are all local standards, and the original sources, characters, content indicators, and other inspection items vary from province to province, and the medicinal parts are all branches and leaves or bark. Therefore, there is currently no quality standard related to the fruit. In addition, the diverse original sources of Taxus chinensis var. mairei also make the fruit sources mixed, and the issues of the effectiveness and safety of its components have not been clarified, which to a certain extent restricts the further development and utilization of Taxus chinensis var. mairei fruits.

[0003] Chinese invention patent CN200510050861.1 discloses a method for extracting paclitaxel and polysaccharides from Taxus fruits and flowers, and its ethanol extraction method improves the yields of polysaccharides and paclitaxel from Taxus fruits. Patent CN109200057B discloses the application of Taxus fruit polysaccharides in whitening and anti-aging, and uses relevant experiments such as melanin synthesis for verification, but the above patents do not involve the relevant analysis of screening active ingredients of Taxus fruits.

[0004] TLR4 protein, a member of the Toll-like receptor (TLR) family, plays a fundamental role in pathogen recognition and innate immune activation. In recent years, studies have found that it is closely related to aging regulation, and Taxus fruits have good anti-aging effects in traditional Chinese medicine theory and folk applications. Therefore, using TLR4 protein to screen active ingredients of Taxus fruits, constructing a TLR4 protein chip, and applying it to the screening of active ingredients in Taxus fruits are of great significance for the modern development and utilization of Taxus fruits and the analysis of active ingredients. Summary of the Invention

[0005] The purpose of the present invention is to provide the application of TLR4 protein in screening active ingredients of Taxus chinensis var. mairei fruits.

[0006] To achieve the purpose of the present invention, the present invention adopts the following technical solutions: Application of TLR4 protein in screening active ingredients of Taxus chinensis var. mairei fruit, which couples TLR4 protein to SPR sensor chip and uses SPR to screen active ingredients of Taxus chinensis var. mairei fruit; the active ingredients are procyanidin B1, ginkgetin, 7-demethylginkgetin, 7-epitaxol.

[0007] A method for screening active ingredients of Taxus chinensis var. mairei fruit based on TLR4 protein, which uses SPR to screen active ingredients of Taxus chinensis var. mairei fruit, specifically including the following steps: 1) Construction of protein chip: Take TLR4 protein, dissolve it with PBS, and then dilute it with sodium acetate buffer solution with pH 4.0 to a TLR4 protein solution of 50 μg / ml. Then take the TLR4 protein solution and couple it with the SPR sensor chip to obtain the TLR4 protein fishing chip. 2) Preparation of test solution: Take 0.1 g of Taxus chinensis var. mairei fruit, ultrasonically extract it with 80 vol% methanol solution, centrifuge the extract, take the upper clear liquid and blow it dry with liquid nitrogen, dissolve it with DMSO, filter it, and mix the filtrate with PBS to obtain the test solution. 3) Preparation of reference solution: Take procyanidin B1, dissolve it with DMSO, and mix the dissolution solution with PBS and PBS containing DMSO to obtain the reference solution. 4) Respectively take the test solution obtained in step 2) and the reference solution obtained in step 3), and place them in the surface plasmon resonance test well plate of the TLR4 protein fishing chip obtained in step 1). SPR detection conditions: Running time: 60 s; Flow rate: 30 μl / min, Dissociation time: 60 s, Temperature: 25 °C, Running buffer: PBS containing 5 vol% DMSO.

[0008] In step 1), the coupling is amino coupling, and the parameters of the amino coupling are: Sensor chip model: CM7 or CM5, Contact time: 420 s, Flow rate: 5 μl / min, Temperature: 25 °C, Coupling reagent: EDC and NHS, Blocking reagent: Ethanolamine, Running buffer: 1×HBS-EP. Preferably, the sensor chip model is CM5, and the volume ratio of the TLR4 protein solution, EDC and NHS, and ethanolamine is 100:200:120; the volume ratio of EDC and NHS is 1:1.

[0009] In step 2), the mass-volume ratio of the Taxus chinensis var. mairei fruit to the 80 vol% methanol solution is 0.1 g:5 - 25; the ultrasonic extraction time is 20 - 60 min; the volume ratio of the upper clear liquid to DMSO is 1:15 - 25.

[0010] In step 3), the concentration of procyanidin B1 in the dissolution solution is 10 mM; the volume ratio of the dissolution solution, PBS, and PBS containing DMSO is 5:95:900.

[0011] In step 3), the concentration of DMSO in the PBS containing DMSO is 5 vol%.

[0012] The remarkable advantages of the present invention are as follows: The present invention uses the constructed TLR4 protein fishing chip to fish for the chemical components of Taxus chinensis var. mairei fruits. Four active compounds are obtained through the verification of the activity of biological quality markers. Through the optimization and verification of the methodology, a method for screening the active components of Taxus chinensis var. mairei fruits based on TLR4 is established. This method is simple, convenient to operate, highly accurate, and has practical application and promotion value. Description of the Drawings

[0013] Figure 1 : Pre-enrichment and coupling diagram of TLR4 protein on the chip. a is the pre-enrichment of TLR4 protein; b is the coupling diagram of TLR4 protein.

[0014] Figure 2 : Chip specificity investigation diagram. a is the preliminary screening diagram; b is the affinity kinetics diagram of the positive.

[0015] Figure 3 : Fishing system methodology investigation diagram. a is the linear fitting diagram of peak area and number of cycles; b is the linear fitting diagram of peak area and injection concentration.

[0016] Figure 4 : Fishing result diagram of Taxus chinensis var. mairei fruits. a is the schematic diagram of the binding and recovery of the extract of Taxus chinensis var. mairei fruits and TGF-β protein; b is the schematic diagram of the negative control running buffer flowing through the system.

[0017] Figure 5 : Total ion current diagram of the fishing solution of Taxus chinensis var. mairei fruits. a is the blank total ion current diagram; b is the total ion current diagram of the recovered fishing solution. 1 is procyanidin B1, 2 is ginkgetin, 3 is 7-demethylginkgetin, and 4 is 7-epitaxol.

[0018] Figure 6a : Affinity test diagram of the fishing component procyanidin B1. Figure 6b : Affinity test diagram of the fishing component 7-demethylginkgetin. Figure 6c : Affinity test diagram of the fishing component ginkgetin. Figure 6d : Affinity test diagram of the fishing component 7-epitaxol. Detailed Embodiments

[0019] To make the content of the present invention easier to understand, the technical solutions of the present invention will be further described below in conjunction with specific embodiments. However, the present invention is not limited thereto.

[0020] The experimental instruments and reagents involved in the present invention are as follows: Biacore T200 (GE Healthcare, USA), Milli-Q ultrapure water system (Millipore, USA), TLR4 protein (Abcam, UK, ab159717), 10×HBS-EP+ buffer (GE Healthcare, USA, BR-1006-69), 1×HBS-EP+ buffer is obtained by diluting 10×HBS-EP with ultrapure water by 10 times, 10×PBS buffer (GE Healthcare, USA, BR-1006-72), 1×PBS buffer is obtained by diluting 10×PBS buffer with ultrapure water by 10 times, DMSO (VETEC, Germany, V900090), CM5 sensor chip (GE Healthcare, USA, BR-1005-30), sterile water is MilliQ ultrapure water after autoclaving, amine coupling kit (GE Healthcare, USA, BR-1000-50), sodium acetate buffer pH4.0, sodium acetate buffer pH4.5, sodium acetate buffer pH5.0, sodium acetate buffer pH5.5 (GE Healthcare, USA), NaOH 50 (GE Healthcare, USA, BR-1003-58), positive drug procyanidin B1 (Chengdu Mansite Biotechnology Co., Ltd., A0698), negative drug paeoniflorin (Chengdu Mansite Biotechnology Co., Ltd., A0133), Taxus chinensis var. mairei fruit (College of Traditional Chinese Medicine, Fujian University of Traditional Chinese Medicine), methanol, acetonitrile (mass spectrometry grade, Merck, Germany), formic acid (chromatography grade, batch number F190210, Aladdin Reagent Shanghai Co., Ltd.), and the rest of the reagents are of analytical grade.

[0021] Example 1: Quality detection of Taxus chinensis var. mairei fruit of the present invention 1) Construction of protein chip: Take TLR4 protein and dissolve and dilute it with 1×PBS buffer to obtain a protein mother liquor of 500 μg / ml; take 10 μl of the TLR4 protein mother liquor and mix it with 90 μl of 10 mM sodium acetate buffer at pH 4.0 to obtain a TLR4 protein solution of 50 μg / ml.

[0022] The CM5 chip has four Flow Cell channels numbered 1, 2, 3, and 4. Start the surface plasmon resonance instrument, set channel 1 as the reference channel, and channel 2 as the coupling unit (after confirming the chip specificity, channels 3 and 4 are used for subsequent fishing experiments). Take 100 μl of the TLR4 protein solution and couple it to the amino group of the CM5 chip through the built-in program of the Biacore T200 system to obtain the TLR4 protein fishing chip. The coupling parameters are as follows: activation time: 420 s, flow rate: 5 μl / min, temperature: 25 °C, and the coupling reagents are 100 μl each of EDC and NHS (from the amino coupling kit, the volume ratio of EDC to NHS is 1:1). 120 μl of ethanolamine (from the amino coupling kit) is used for blocking; running buffer: 1×HBS-EP.

[0023] 2) Preparation of the test sample solution: Take 0.1 g of Taxus chinensis var. mairei fruit powder, place it in a 25 ml Erlenmeyer flask, add 10 ml of 80 vol% methanol, extract it by ultrasound (power 350 W, frequency 50 kHz) for 30 min, let it cool to room temperature, weigh it again, make up the weight loss with 80 vol% methanol, centrifuge at 4 °C and 12,000 r for 10 min, precisely pipette 1 ml of the supernatant, evaporate it to dryness by nitrogen blowing, dissolve it in 200 μl of DMSO, filter it through a 0.22 μm pore filter membrane, precisely pipette 10 μl, add 190 μl of 1×PBS buffer, mix well to obtain the Taxus chinensis var. mairei fruit solution containing 5 vol% DMSO, which is the test sample solution.

[0024] 3) Preparation of the reference sample solution: Precisely weigh the reference substance of procyanidin B1, prepare a stock solution with a concentration of 10 mM in DMSO; take 5 μl of the procyanidin B1 reference substance stock solution, add 95 μl of 1×PBS buffer and 900 μl of 1×PBS buffer containing 5 vol% DMSO, mix well to obtain the reference sample solution; 4) Respectively take the test sample solution obtained in step 2) and the reference sample solution obtained in step 3), and place them in the surface plasmon resonance test well plate carrying the TLR4 protein fishing chip obtained in step 1); Chip detection conditions: running time: 60 s; flow rate: 30 μl / min; dissociation time: 60 s; temperature: 25 °C; running buffer: 1×PBS buffer containing 5 vol% DMSO.

[0025] The beneficial effects of the present invention are further illustrated by the following test examples.

[0026] Test Example 1 I. Ligand pre-enrichment and coupling of biological mass markers (protein targets) 1 Experimental methods and results 1.1 Ligand pre-enrichment experiment 1) Dissolve and dilute TLR4 protein with 1×PBS buffer to obtain a protein mother solution of 500 μg / ml. Take 5 μl of the TLR4 protein mother solution and add it to 45 μL of 10 mM sodium acetate buffer with different pH values, and mix well to obtain a TLR4 protein solution of 50 μg / ml.

[0027] 2) Inject the sample and detect the response value of TLR4 protein under different pH conditions on the Biacore T200 pre-enrichment system to determine the optimal protein coupling conditions. Protein pre-enrichment Flow rate: 10 μl / min; Flow path: Flow path 2, select the Sample and Reagent Rack1 mode for pre-enrichment; Contact time: 120 s.

[0028] 3) The pre-enrichment results are shown in Figure 1 , and the TLR4 protein has the highest response value under the condition of pH 4.0. Therefore, a buffer with pH 4.0 is selected for coupling in the subsequent experiment.

[0029] 2.2 Coupling of TLR4 protein on the SPR sensor 1) Dissolve and dilute TLR4 protein with 1×PBS buffer to obtain a protein mother solution of 500 μg / ml. Precisely pipette 10 μl of the TLR4 protein mother solution and add it to 90 μl of 10 mM sodium acetate buffer (pH 4.0), and mix well to obtain a TLR4 protein solution of 50 μg / ml for coupling.

[0030] 2) Couple the TLR4 protein solution to the CM5 chip through the built-in program of the BiacoreT200 system. The coupling parameters are set as follows: Chip type: CM5, Flow cells per cycle: 4 (for fishing, all 4 channel units are coupled, and 100 μl of protein solution is used for each channel unit), method: amine amino coupling, ligand: TLR4 protein, coupling method: specify contact time and flow rate, contact time: 420 s, flow rate: 5 μl / min, temperature: 25 °C. The coupling reagents are 100 μl each of EDC and NHS (from the amino coupling kit), and 120 μl of ethanolamine (from the amino coupling kit) is used for blocking.

[0031] 3) Calculate the target coupling amount according to the formula Rmax = MWanalyte × RL × Sm / MWligand. In the formula, Rmax is the maximum binding capacity on the chip surface, which is usually substituted with 100 RU in small molecule related experiments; MWanalyte and MWligand are the molecular weights of the small molecule and the protein respectively, Sm is the stoichiometric ratio; RL is the protein coupling level. The coupling results are shown in Figure 1 , and the coupling amounts of TLR4 protein with the 3 channels of the CM5 chip are 17386 RU, 17297 RU, and 18365 RU respectively, meeting the expectations.

[0032] II. Investigation of chip specificity and methodology investigation of the fishing system 1 Investigation of chip specificity 1.1 Experimental instruments and reagents Biacore T200 (GE Healthcare, USA), Milli-Q ultrapure water system (Millipore, USA), TLR4 protein (Abcam, UK, ab159717), 10×HBS-EP+ buffer (GE Healthcare, USA, BR-1006-69), 1×HBS-EP+ buffer is obtained by diluting 10×HBS-EP with ultrapure water by 10 times, 10×PBS buffer (GE Healthcare, USA, BR-1006-72), 1×PBS buffer is obtained by diluting 10×PBS buffer with ultrapure water by 10 times, DMSO (VETEC, Germany, V900090), CM5 sensor chip (GE Healthcare, USA, BR-1005-30), sterile water is MilliQ ultrapure water after autoclaving, amino coupling kit (GE Healthcare, USA, BR-1000-50), sodium acetate pH4.5 (GE Healthcare, USA, BR-1003-50), sodium acetate pH5.0 (GE Healthcare, USA, BR-1003-51), sodium acetate pH5.5 (GE Healthcare, USA, BR-1003-52), NaOH 50 (GE Healthcare, USA, BR-1003-58), positive drug procyanidin B1 (Chengdu Mansite Biotechnology Co., Ltd., A0698), negative drug paeoniflorin (Chengdu Mansite Biotechnology Co., Ltd., A0133), Taxus chinensis var. mairei fruit (College of Traditional Chinese Medicine, Fujian University of Traditional Chinese Medicine).

[0033] 1.2 Experimental methods and results 1.2.1 Experimental methods (1) Preparation of running buffer: The running buffer is 1×PBS buffer containing 5 vol% DMSO.

[0034] (2) Preparation of positive and negative drugs: Appropriate amount of positive drug procyanidin B1 was taken and prepared into a stock solution with a concentration of 10 mM using DMSO for standby. Appropriate amount of negative drug paeoniflorin was taken and prepared into a stock solution with a concentration of 10 mM using DMSO for standby.

[0035] (3) 5 μl of the procyanidin B1 stock solution was added to 95 μl of 1×PBS buffer and 900 μl of 1×PBS buffer containing 5 vol% DMSO and mixed well. 5 μl of the paeoniflorin stock solution was added to 95 μl of 1×PBS buffer and 900 μl of 1×PBS buffer containing 5 vol% DMSO and mixed well. Both of them and the Running buffer were used as analytes and placed on the surface plasmon resonance test well plate of the TLR4 protein fishing chip. The parameters of the Biacore T200 system were set as follows: flow rate: 30 μL / min; injection time was set to 60 s; dissociation time was set to 60 s, and the binding response value with the TLR4 protein was preliminarily detected. Subsequently, procyanidin B1 was prepared into 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.563 μM, 0.781 μM, 0.391 μM, 0.195 μM, 0.098 μM, 0.049 μM with 1×PBS buffer and 1×PBS buffer containing 5 vol% DMSO. A series of concentration gradients of procyanidin B1 solutions were placed on the surface plasmon resonance test well plate of the TLR4 protein fishing chip. The parameters of the Biacore T200 system were set as follows: flow rate was set to 30 μL / min, injection time was set to 120 s, and dissociation time was set to 80 s. After the program was completed, the data was processed and analyzed using the supporting analysis software Biacore T200 evaluation software, and the model used was the 1:1 steady-state affinity model.

[0036] 1.3 Experimental results The results of the specificity investigation are shown in Figure 2 . The response value of procyanidin B1 with the TLR4 protein was approximately 180 RU, while the response values of paeoniflorin and the Running buffer were similar, both around 0. This result preliminarily confirmed the specificity of the TLR4 protein fishing chip. To further investigate the specificity of the chip, a series of concentration gradients of procyanidin B1 solutions designed were placed on the surface plasmon resonance test well plate of the TLR4 protein fishing chip, and finally the K D value of procyanidin B1 with the TLR4 protein was 8.433 μM. Therefore, procyanidin B1 was selected as the positive small molecule for the subsequent methodological investigation to further construct the fishing system.

[0037] 2 Methodological investigation of the fishing system 2.1 Experimental methods 2.1.1 Number of cycles: Procyanidin B1 at a concentration of 12.5 μM was used for the investigation of linearity and detection limit on the TLR4 protein fishing chip according to 1, 2, 5, 10, 20, 50, and 100 recovery cycles. The binding time was set to 180 s, the flow rate was set to 5 μL / min, 0.5% trifluoroacetic acid (TFA) was used as the dissociation solvent during the fishing process, and 50 mM ammonium bicarbonate (NH4HCO3) was used as the recovery reagent. After each fishing, the recovery solutions in each EP tube were collected together, then dried by nitrogen blowing, redissolved in 100 μL of 50 vol% methanol, mixed well, centrifuged at 12000 rpm for 2 min, and the supernatant was taken for LC-MS analysis to obtain the signal-to-noise ratio (S / N) of the recovered sample, and the correlation between different cycle numbers and peak areas was calculated to analyze the detection limit of the fishing system.

[0038] 2.1.2 Concentration: Procyanidin B1 at concentrations of 50 μM, 25 μM, 12.5 μM, 6.25 μM, and 3.125 μM was separately used for fishing recovery on the TLR4 protein fishing chip, and the number of cycles for each concentration was set to 10 times. After each fishing, the recovery solutions in each EP tube were collected together, then dried by nitrogen blowing, redissolved in 100 μL of 50 vol% methanol, mixed well, centrifuged at 12000 rpm for 2 min, and the supernatant was taken for LC-MS analysis for the correlation analysis of the concentration of procyanidin B1 and the amount of recovered procyanidin B1.

[0039] 2.2 Experimental results In this study, procyanidin B1, a positive control, was used to replace the compound prescription for the methodological investigation of the fishing system, which is easier to operate. The results of the number of cycles, signal-to-noise ratio, and peak area are shown in Table 1, and the correlation results between the peak area and the number of cycles are shown in Figure 3 . It can be seen from Table 1 that when the number of cycles reaches at least 5, the signal-to-noise ratio is acceptable. Therefore, based on the above experimental results, in order to obtain better results and reduce the influence of complex extracts on proteins (there may be false positive substances in the extracts that cannot be eluted, which will affect subsequent experiments), 10 cycles were selected for subsequent fishing research.

[0040] The peak areas obtained after the recovery of procyanidin B1 at different concentrations are shown in Figure 3 . It can be seen from the figure that as the concentration increases, the peak area has been showing an upward trend, indicating that within a certain range of small molecule substances, the higher the injection concentration, the higher the recovered content. It can be seen from the figure that there are already good peak area results at 6.25 μM. To ensure that the recovered amount can be detected, subsequent fishing experiments were carried out under the condition of 12.5 μM.

[0041] Table 1 Correlation between peak area and number of cycles Number of cycles Signal-to-noise ratio Peak area 1 2.03 133 2 4.46 505 5 13.42 1236 10 36.35 2295 20 47.45 4405 50 104.43 11049 100 197.64 21046 III. Fishing for Target Q-biomarkers of Taxus chinensis var. mairei Fruit and Component Identification Construct a protein fishing chip for the biomarker TLR4, perform chemical component fishing screening on Taxus chinensis var. mairei fruit, and identify the fished components by UPLC-Q-TOF-MS / MS and comparison with reference substances.

[0042] 1 Experimental Instruments and Reagents Biacore T200 (GE Healthcare, USA), Milli-Q ultrapure water instrument (Millipore, USA), TLR4 protein (Abcam, UK, ab159717), 10×HBS-EP+ buffer (GE Healthcare, USA, BR-1006-69), 1×HBS-EP+ buffer was obtained by diluting 10×HBS-EP with ultrapure water by 10 times, 10×PBS buffer (GE Healthcare, USA, BR-1006-72), 1×PBS buffer was obtained by diluting 10×PBS buffer with ultrapure water by 10 times, DMSO (VETEC, Germany, V900090), CM5 sensor chip (GE Healthcare, USA, BR-1005-30), sterile water was MilliQ ultrapure water after autoclaving, amino coupling kit (GE Healthcare, USA, BR-1000-50), sodium acetate pH4.5 (GE Healthcare, USA, BR-1003-50), sodium acetate pH5.0 (GE Healthcare, USA, BR-1003-51), sodium acetate pH5.5 (GE Healthcare, USA, BR-1003-52), NaOH 50 (GE Healthcare, USA, BR-1003-58), positive drug procyanidin B1 (Chengdu Mansite Biotechnology Co., Ltd., A0698), negative drug paeoniflorin (Chengdu Mansite Biotechnology Co., Ltd., A0133), Taxus chinensis var. mairei fruit (College of Traditional Chinese Medicine, Fujian University of Traditional Chinese Medicine), methanol, acetonitrile (mass spectrometry grade, Merck, Germany), formic acid (chromatography grade, batch number F190210, Aladdin Reagent Shanghai Co., Ltd.), and the rest of the reagents were of analytical grade.

[0043] 2 Experimental Methods and Results 2.1 Solution Preparation 2.1.1 Chip Construction and Buffer Preparation The running buffer used in this experiment was 1×HBS-EP, and 10×HBS-EP was diluted with ultrapure water by 10 times to prepare 500 ml of running buffer.

[0044] 2.1.2 Preparation of Test Samples Preparation of the fishing test sample: Take 0.1 g of Taxus chinensis var. mairei fruit powder, place it in a 25-ml Erlenmeyer flask, add 10 ml of 80 vol% methanol, extract it by ultrasound (power 350 W, frequency 50 kHz) for 30 min, let it cool to room temperature, weigh it again, make up the weight loss with 80 vol% methanol, centrifuge at 4 °C and 12,000 r for 10 min, precisely pipette 1 ml of the upper clear liquid, blow it dry with nitrogen, dissolve it again with 200 μl of DMSO, filter it through a 0.22-μm pore filter membrane, precisely pipette 10 μl, and add 190 μl of 1×PBS buffer solution, mix well to obtain a Taxus chinensis var. mairei fruit solution containing 5 vol% DMSO; Preparation of the positive drug procyanidin B1: Weigh an appropriate amount of procyanidin B1 precisely, prepare a stock solution with a concentration of 10 mM using DMSO; take 5 μl of the procyanidin B1 stock solution, add 95 μl of 1×PBS buffer solution and 900 μl of 1×PBS buffer solution containing 5 vol% DMSO, mix well, and prepare a PBS solution containing 5 vol% DMSO with a solution concentration of 50 μM, that is, obtained. The negative drug paeoniflorin is prepared in the same way.

[0045] Preparation of the running buffer: The running buffer is 1×PBS buffer solution containing 5 vol% DMSO.

[0046] 2.1.3 Fishing and recovery Respectively take the fishing test sample, the test solution of the positive drug procyanidin B1, the negative drug paeoniflorin, and the running buffer as analytes and place them in the surface plasmon resonance test well plate of the TLR4 protein fishing chip. Biacore T200 system parameters are set as follows: contact time: 60 s, flow rate: 30 μl / min, incubation time: 60 s, wash solution: ultrapure water, recovery solution: 0.5% TFA, deposition solution: 50 mM NH4HCO3 (as the collection solution to dissociate the active ingredients bound to the chip surface). After preliminary verification of the activity of Taxus chinensis var. mairei fruit, conduct fishing, and the parameter settings are: contact time: 180 s; flow rate: 5 μL / min; cycle: 10.

[0047] Control group experiment: Take another CM5 chip not conjugated with TLR4 protein as a negative control, and the fishing parameters are the same. After fishing, collect the recovery solutions in each EP tube together, then blow them dry with nitrogen, add 100 μL of 50 vol% methanol for re-dissolution, mix well and centrifuge at 12,000 rpm for 2 min, and take the supernatant for UPLC-Q-TOF-MS analysis to obtain the active ingredients.

[0048] 2.1.3 Fishing and Recovery Results The extracts of Taxus chinensis var. mairei fruits, positive and negative small molecules were used as analytes and placed in the surface plasmon resonance test wells of the TLR4 protein fishing chip respectively. The response value of Taxus chinensis var. mairei fruits and TLR4 protein was about 420 RU, higher than that of procyanidin B1. Subsequently, the fishing and recovery of the extract were carried out. The fishing results of Taxus chinensis var. mairei fruits are shown in Figure 4 。

[0049] 2.3 UPLC-Q-TOF-MS / MS Analysis 2.3.1 Chromatographic Conditions UPLC-Q-TOF-MS / MS analysis: Chromatographic conditions: Chromatographic column: CORTECSTM UPLC C18 (2.1×100 mm, 1.6 μm); Mobile phase A is 0.1% formic acid aqueous solution, and mobile phase B is acetonitrile; Gradient elution (0 - 0.5 min, 5 - 5% B; 0.5 - 5.0 min, 5.0 - 12.0% B; 5.0 - 14.0 min, 12 - 18% B; 14.0 - 19.0 min, 18.0 - 25.0% B; 19.0 - 24.0 min, 25 - 32% B; 24.0 - 30.0 min, 32 - 40% B; 30.0 - 42.0 min, 40 - 50% B; 42.0 - 47.0 min, 50 - 60% B; 47.0 - 49.5 min, 60 - 95% B; 49.5 - 51.5 min, 95 - 95% B; 51.5 - 51.6 min, 95 - 5% B; 51.6 - 52.0 min, 5 - 5% B); Flow rate: 0.25 mL / min; Column temperature: 45 °C; Injection volume: 2 μL.

[0050] 2.3.2 Mass Spectrometry Conditions The time-of-flight mass spectrometry adopts the positive and negative ion modes of electrospray ionization source: Capillary voltage 2.5 kV, nebulizer pressure 0.2 MPa, collision energy 10 - 55 eV. Desolvation gas flow: Nitrogen, flow rate 800 L / h, desolvation temperature 400 °C, cone gas flow: N2, cone voltage 30.0 V, flow rate 50 L / h, ion source temperature 120 °C, quadrupole ion energy 3.0 eV, collision gas: Ar. The mass spectrometry measurement data are collected in the full-scan positive and negative ion modes, and the data collection range is m / z 50 - 1500 Da. It is corrected in real time with sodium formate calibration solution and leucine enkephalin calibration solution (positive ion m / z 556.2771, negative ion m / z 554.2615).

[0051] 2.5 Mass Spectrometry Results A total of 4 chemical components were identified from the fruits of Taxus chinensis var. mairei by UPLC-Q-TOF-MS / MS total ion current. The specific identification method is as follows: First, according to the accurate mass numbers obtained from the total ion current chromatogram of high-resolution mass spectrometry, that is, the molecular ion peak information (MS1) of each chromatographic peak is obtained. Then, in the Tools Elemental Composition software of Masslynx 4.2 software, its accurate molecular formula is calculated within a mass deviation range of ±10 ppm. And the preliminarily identified chemical components are compared with the purchased reference standards to accurately identify each chromatographic peak. Finally, a total of 4 compounds were identified by comparison with reference standards: procyanidin B1, 7-demethylginkgetin, ginkgetin, and 7-epitaxol. The total ion current chromatogram is shown in Figure 5 。

[0052] IV. Verification of the affinity activity of Q-biomarker for fishing components The effects of different concentrations of the selected components on the activity of Q-biomarker were studied, and the kinetic parameters of affinity were calculated to verify the affinity activity.

[0053] 1 Instruments and reagents Biacore T200 (GE Healthcare, USA), Milli-Q ultrapure water instrument (Millipore, USA), TLR4 protein (Abcam, UK, ab159717), 10×HBS-EP+ buffer (GE Healthcare, USA, BR-1006-69), 1×HBS-EP+ buffer was obtained by diluting 10×HBS-EP with ultrapure water by 10 times, 10×PBS buffer (GE Healthcare, USA, BR-1006-72), 1×PBS buffer was obtained by diluting 10×PBS buffer with ultrapure water by 10 times, DMSO (VETEC, Germany, V900090), CM5 sensor chip (GE Healthcare, USA, BR-1005-30), sterile water was Milli-Q ultrapure water after autoclaving, amine coupling kit (GE Healthcare, USA, BR-1000-50), sodium acetate pH 4.5 (GE Healthcare, USA, BR-1003-50), sodium acetate pH 5.0 (GE Healthcare, USA, BR-1003-51), sodium acetate pH 5.5 (GE Healthcare, USA, BR-1003-52), NaOH 50 (GE Healthcare, USA, BR-1003-58), positive drug procyanidin B1 (Chengdu Mansite Biotechnology Co., Ltd., A0698), negative drug paeoniflorin (Chengdu Mansite Biotechnology Co., Ltd., A0133), fruits of Taxus chinensis var. mairei (College of Traditional Chinese Medicine, Fujian University of Traditional Chinese Medicine), methanol, acetonitrile (mass spectrometry grade, Merck, Germany), formic acid (chromatography grade, batch number F190210, Aladdin Reagent Shanghai Co., Ltd.), and the rest of the reagents are of analytical grade.

[0054] 2. Experimental methods and results 2.1 Solution Preparation 2.1.1 Preparation of Chip Construction Buffer The buffer used in this experiment is 1×HBS-EP. Dilute 10×HBS-EP 10-fold with ultrapure water to obtain 500 ml of 1×HBS-EP, which is the protein pre-enrichment and coupling running buffer.

[0055] 2.1.2 Preparation of Running Buffer for SPR Screening and Affinity Analysis Dilute 10×PBS buffer 10-fold with ultrapure water to obtain 1×PBS buffer. Take 1×PBS buffer and add DMSO to make a PBS solution containing 5 vol% DMSO, which is used as the running buffer for Biacore protein chip screening.

[0056] 2.1.3 Preparation of Test Samples Accurately weigh appropriate amounts of procyanidin B1, 7-epitaxol, 7-demethylginkgetin, and ginkgetin reference substances, and prepare a stock solution with a concentration of 10 mM in DMSO. Take 5 μl of the reference substance stock solution, add 95 μl of 1×PBS and 900 μl of 1×PBS containing 5 vol% DMSO, mix well, and prepare a PBS solution containing 5 vol% DMSO with a solution concentration of 50 μM, and that's it.

[0057] 2.1.4 Preparation of Solvent Calibration Solution First, dilute pure DMSO with 1×PBS to 1×PBS solutions containing 4.5 vol% DMSO and 5.8 vol% DMSO respectively, and then prepare the solvent calibration solution according to Table 2.

[0058] Table 2 Preparation of Solvent Calibration Solution <![CDATA[Buffer / Volume μL > 1 2 3 4 5 6 7 8 4.5% DMSO 0 100 200 300 400 500 600 700 5.8% DMSO 700 600 500 400 300 200 100 0 Total volume 700 μL 700 μL 700 μL 700 μL 700 μL 700 μL 700 μL 700 μL 2.2 Detection of SPR of Fishing Components on TLR4 For the test solutions of the 4 fishing components (procyanidin B1, ginkgetin, 7-demethylginkgetin, 7-epitaxol) detected and identified, the positive drug procyanidin B1, the negative drug paeoniflorin, and the blank solvent, accurately pipette 100 μl each and place them in the surface plasmon resonance test well plate of the TLR4 protein fishing chip. The contact time of the sample during chip detection is 120 s; the flow rate is 30 μl / min, the dissociation time is 80 s, and the temperature is 25°C. Running Buffer: 1×PBS containing 5 vol% DMSO) Replace the corresponding test tube rack and adjust the position of the EP tube according to the operation panel. Finally, 4 components with positive results were screened out: procyanidin B1, ginkgetin, 7-demethylginkgetin, 7-epitaxol. See specifically Figures 6a to 6d .

[0059] SPR Affinity KD Analysis of 2.34 Fishing Components Perform affinity analysis on the four selected components. Prepare test solutions containing 5 vol% DMSO in 1×PBS with concentrations of 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.5625 μM, 0.7813 μM, 0.3906 μM, 0.1953 μM, 0.0977 μM, and 0.0488 μM in sequence.

[0060] Set the affinity analysis parameters: Flow path: 2-1, Chip type: CM5, Contact time: 120 s. Flow rate: 30 μl / min, Dissociation time: 80 s, Temperature: 25 °C. After the program is completed, use the supporting analysis software Biacore T200 evaluation software to process and analyze the data. The model used is the 1:1 steady-state affinity model.

[0061] The experimental results are shown in Table 3. According to the results in the table, it can be seen that the order of magnitude of the K D values of the four components is the same. The K D value of ginkgetin is the smallest, and it may be a component with better activity. The graphs of the affinity test results of each component are shown in Figures 6a to 6d .

[0062] Table 3 Affinity Kinetics K D Values (Mol) Name <![CDATA[K D Value( Mol )]]> 1 Procyanidin B1 <![CDATA[1.390×10 -5 > 2 Ginkgetin <![CDATA[1.386×10 -5 > 3 7-Demethylginkgetin <![CDATA[1.778×10 -5 > 4 7-Epitaxol <![CDATA[2.002×10 -5 > In this study, an SPR fishing chip was constructed with TLR4 protein. By investigating its specificity, linearity, and detection limit, various fishing parameters for fishing Taxus chinensis var. mairei fruits were determined, providing a basis for exploring the active ingredients of Taxus chinensis var. mairei fruits in the future. After constructing the fishing system, the extract of Taxus chinensis var. mairei fruits was fished and recovered to identify potential active ingredients, and then the affinity screening of potential active ingredients was carried out to further determine the accuracy of its active ingredients.

[0063] In summary, the present invention provides the construction of a TLR4 protein chip and its application in the active ingredients of Taxus chinensis var. mairei fruits, which is simple and convenient to operate, has high accuracy, and has practical application and promotion value.

[0064] The above are only the preferred embodiments of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope covered by the present invention.

Claims

1. Application of TLR4 protein in screening active ingredients of Taxus chinensis var. mairei fruits.

2. The application according to claim 1, wherein: The active ingredients are procyanidin B1, ginkgetin, 7-demethylginkgetin, and 7-epitaxol.

3. The application according to claim 1, wherein: Couple TLR4 protein to an SPR sensor chip and use SPR to screen the active ingredients of Taxus chinensis var. mairei fruits.

4. A method for screening active ingredients from Taxus chinensis var. mairei fruits based on TLR4 protein, characterized in that: Using SPR to screen the active ingredients of Taxus chinensis var. mairei fruits specifically includes the following steps: 1) Construction of the protein chip: Dissolve TLR4 protein in PBS, then dilute it with sodium acetate buffer at pH 4.0 to a TLR4 protein solution with a concentration of 50 μg / ml. Then couple the TLR4 protein solution to the SPR sensor chip to obtain the TLR4 protein fishing chip. 2) Preparation of the test sample solution: Take 0.1 g of Taxus chinensis var. mairei fruits, extract them by ultrasonic extraction with 80 vol% methanol solution. Centrifuge the extract, take the supernatant, evaporate it to dryness with nitrogen blowing, redissolve it with DMSO, filter it, and mix the filtrate with PBS to obtain the test sample solution. 3) Preparation of the reference substance solution: Dissolve procyanidin B1 in DMSO, and mix the dissolution solution with PBS and PBS containing DMSO to obtain the reference substance solution. 4) Respectively take the test sample solution obtained in step 2) and the reference substance solution obtained in step 3), and place them in the surface plasmon resonance test well plate of the TLR4 protein fishing chip obtained in step 1). SPR detection conditions: Running time: 60 s; Flow rate: 30 μl / min, Dissociation time: 60 s, Temperature: 25 °C, Running buffer: PBS containing 5 vol% DMSO.

5. The screening method for active ingredients of Taxus chinensis var. mairei fruits according to claim 4, characterized in that: In step 1), the coupling is amino coupling, and the parameters of the amino coupling are: Sensor chip model: CM7 or CM5, Contact time: 420 s, Flow rate: 5 μl / min, Temperature: 25 °C, Coupling reagents: EDC and NHS, Blocking reagent: Ethanolamine, Running buffer: 1×HBS-EP.

6. The screening method for the active ingredients of Taxus chinensis var. mairei fruits according to claim 5, characterized in that: The sensor chip model is CM5, and the volume ratio of the TLR4 protein solution, EDC, NHS, and ethanolamine is 100:200:120; the volume ratio of EDC and NHS is 1:

1.

7. The screening method for the active ingredients of Taxus chinensis var. mairei fruits according to claim 4, wherein: In step 2), the mass-volume ratio of Taxus chinensis var. mairei fruits to 80 vol% methanol solution is 0.1 g:5 - 25; the ultrasonic extraction time is 20 - 60 min; the volume ratio of the supernatant to DMSO is 1:15 - 25.

8. The screening method for the active ingredients of Taxus chinensis var. mairei fruits according to claim 4, characterized in that: In step 3), the concentration of procyanidin B1 in the dissolution solution is 10 mM; the volume ratio of the dissolution solution, PBS, and PBS containing DMSO is 5:95:

900.

9. The screening method for active ingredients of Taxus chinensis var. mairei fruits according to claim 4, characterized in that: In step 3), the concentration of DMSO in the PBS containing DMSO is 5 vol%.

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