Combined metabolic marker for judging degranulation inhibition effect of tea-derived polyphenol substance based on rat basophilic leukemia cells and detection kit thereof

Abstract

The invention relates to a combined metabolic marker for judging the degranulation inhibition effect of tea-derived polyphenol substances based on rat basophilic leukemia cells and a detection kit thereof. The method comprises the following steps: detecting relative concentrations of lipid metabolites fatty acid (FA22: 5), phosphatidylcholine (PC 38: 2) and phosphatidylcholine (PCO-30: 0) in a rat basophilic leukemia cell sample, calculating a combined marker variable P and a determined intercept point value based on a binary logistic regression equation, and judging the degranulation inhibition effect of the tea-derived polyphenol substance. The kit can realize high-sensitivity and high-efficiency detection of the three small molecule metabolites involved in the invention, and has the characteristics of low detection cost and good repeatability. The combined use of the three small molecule metabolites has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical fields of analytical chemistry, biochemistry and molecular biology, and particularly relates to a combined metabolic marker for judging the degranulation inhibition effect of tea-derived polyphenols based on rat basophilic leukemia cells and a detection kit thereof. Background Art

[0002] Food allergy is a specific immune response that develops rapidly and poses serious risks to the body, with its incidence increasing year by year. It has become a global food safety and public health issue. Food allergy involves multiple types of immune responses, with type I allergy (i.e., IgE-mediated) being the predominant form. When food allergens enter the body, they are recognized by antigen-presenting cells, activating T cells and inducing B cells to produce IgE antibodies. The IgE receptors on the surface of mast cells bind to these allergens, placing the body in a sensitized state. Upon further exposure to the allergen, mast cell degranulation is triggered, initiating the allergic pathology. RBL-2H3 cells are a subline of rat-derived basophilic leukemia granulocytes. Because they express the high-affinity FcεRI receptor on their cell surface and possess many characteristics of mast cells, they can rapidly degranulate and release inflammatory mediators upon activation. Therefore, RBL-2H3 cells are often used as a substitute for mast cells and are a classic model cell for studying the mechanisms of allergies.

[0003] Epigallocatechin gallate (EGCG), a flavanol compound, is a white crystalline powder and the primary component of green tea polyphenols. It is the most abundant catechin component, accounting for 9% to 13% of the gross weight of green tea. It has been shown to exhibit significant anti-inflammatory properties in vitro. S-epigallocatechin gallate-theanine conjugate (S-EGCG-cThea), a novel compound recently discovered in tea, has become a hot topic in the field of tea chemistry. In vitro experiments have shown that S-EGCG-cThea exhibits antioxidant activity and inhibits acetylcholinesterase activity and the formation of advanced glycation end products. Therefore, tea-derived polyphenols (S-EGCG-cThea and EGCG) were selected to intervene in cells during the stimulation phase to investigate their effects on degranulation in RBL-2H3 cells and their lipid metabolism regulation mechanisms. This study aimed to further clarify the anti-allergic activity of these tea-derived polyphenols and provide theoretical guidance for drug target discovery and the development of diagnostic methods for allergic diseases. Phenotypic tests based on the IL-4 release, TNF-α release, degranulation efficiency and inhibition rate of the RBL-2H3 cell model have confirmed that S-EGCG-cThea and EGCG have an inhibitory effect on RBL-2H3 cell degranulation and significantly reduce the release of TNF-α and IL-4, while another typical tea polyphenol, Theanine, has no inhibitory effect on degranulation and there is no downward trend in the release of TNF-α and IL-4, indicating that S-EGCG-cThea and EGCG have potential anti-allergic effects ( Figure 1 ).

[0004] Lipidomics is a key branch of metabolomics research. Leveraging technologies such as chromatography-mass spectrometry, lipidomics allows for high-throughput qualitative and quantitative analysis of lipid metabolites in organisms, offering unique advantages in assessing the physiological state of an organism. As essential components of cell membranes, lipids participate in many crucial processes regulating life, such as energy storage, transport, signal transduction, cell development, differentiation, and apoptosis. In recent years, lipid biomarkers have demonstrated numerous successes in the clinical diagnosis and research of important diseases such as cancer, diabetes, and polycystic ovary syndrome.

[0005] In view of the key regulatory role of lipid metabolites in organisms, the present invention adopts a lipidomics research method based on ultra-high performance liquid chromatography-mass spectrometry technology to detect lipid metabolites in RBL-2H3 cells under different states, and screens typical lipid markers of RBL-2H3 cells after intervention with tea-derived polyphenols through bioinformatics analysis. The present invention proposes a new use of the combined use of lipid markers fatty acids (FA22:5), phosphatidylcholine (PC 38:2) and phosphatidylcholine (PC O-30:0) for discriminating the degranulation inhibitory effect of tea-derived polyphenols. Since a single metabolite is affected by many factors and lipid substances have multiple "identities" in living organisms, a combined metabolic marker composed of a few metabolites is screened from the RBL-2H3 cell model, and the "discrimination possibility" P value (Probability) is calculated using a discriminant formula, which helps to improve the discrimination sensitivity and specificity. There is currently no report on the combination of these three lipid metabolites for discriminating the degranulation inhibitory effect of polyphenols. Summary of the Invention

[0006] In view of the problems existing in the prior art, the purpose of the present invention is to design and provide a technical solution of a combined metabolic marker and its application and detection kit for judging the degranulation inhibition effect of tea-derived polyphenols based on rat basophilic leukemia cells.

[0007] The present invention is specifically implemented through the following technical solutions:

[0008] The first aspect of the present invention provides a combined metabolic marker for judging the degranulation inhibitory effect of tea-derived polyphenols based on rat basophilic leukemia cells. The tea-derived polyphenols are S-epigallocatechin gallate-theanine conjugate (S-EGCG-cThea) and epigallocatechin gallate (Epigallocatechingallate, EGCG). The marker includes: fatty acid (FA22:5), phosphatidylcholine (PC 38:2) and phosphatidylcholine (PCO-30:0).

[0009] The second aspect of the present invention provides the use of the above-mentioned combined metabolic marker in the preparation of a detection kit for determining the degranulation inhibitory effect of tea-derived polyphenols.

[0010] The third aspect of the present invention provides a detection kit containing the above-mentioned combined metabolic marker.

[0011] Furthermore, the detection kit comprises:

[0012] A standards: fatty acids (FA22:5), phosphatidylcholine (PC 38:2), and phosphatidylcholine (PC O-30:0);

[0013] B. Pretreatment extract: methanol solution containing internal standard 0.92 μg / mL phosphatidylethanolamine (PE 30:0) and 1.0 μg / mL fatty acid (FAC16:0-d3);

[0014] C eluent: Mobile phase A is an acetonitrile / water solution containing 10 mM ammonium acetate (v / v=6:4), and mobile phase B is an isopropanol / acetonitrile solution containing 10 mM ammonium acetate (v / v=9:1).

[0015] A fourth aspect of the present invention provides a method for determining the degranulation inhibitory effect of tea-derived polyphenols using the combined metabolic marker or the detection kit, wherein the combined marker variable P is used for determination:

[0016] P=1 / [1+e -(0.087a+0.158b-0.231c-37.847) ]

[0017] In the above formula, a is the relative content of fatty acid (FA22:5), b is the relative content of phosphatidylcholine (PC 38:2), and c is the relative content of phosphatidylcholine (PC O-30:0);

[0018] If P>0.5, it is judged that tea-derived polyphenols have an indicative effect on degranulation.

[0019] A fifth aspect of the present invention provides a method for calculating a combined marker variable using the above-mentioned detection kit, comprising the following steps:

[0020] (1) Pretreatment of RBL-2H3 samples: RBL-2H3 cell samples were added to the pretreatment extract, followed by methyl tert-butyl ether and ultrapure water to extract lipid metabolites;

[0021] (2) separating and identifying lipid metabolites of the test sample extracted after treatment in step (1) by ultra-high performance liquid chromatography-mass spectrometry;

[0022] (3) Fatty acid (FA22:5), phosphatidylcholine (PC 38:2), and phosphatidylcholine (PCO-30:0) standards provided in the kit were used to assist in qualitative confirmation of the detected ions;

[0023] (4) For the target fatty acids (FA22:5), phosphatidylcholine (PC 38:2), and phosphatidylcholine (PC O-30:0) in the identified test samples, their chromatographic peak intensities were first corrected for the dry weight of cell sample protein, and then compared with the internal standards phosphatidylethanolamine (PE 30:0) and fatty acid (FA C16:0-d3) to obtain the relative concentrations of the above three metabolites;

[0024] (5) Fatty acids (FA22:5), phosphatidylcholine (PC 38:2) and phosphatidylcholine (PC O-30:0) were calculated by binary logistic regression to obtain the joint marker variable P.

[0025] Furthermore, in this method, fatty acid (FA22:5), phosphatidylcholine (PC 38:2) and phosphatidylcholine (PC O-30:0) standards are used to assist in qualitative confirmation of the detected ions, specifically including:

[0026] i) The fatty acid (FA22:5), phosphatidylcholine (PC 38:2), and phosphatidylcholine (PC O-30:0) standards in the kit were analyzed by ultra-performance liquid chromatography-mass spectrometry to determine the chromatographic retention time, measured mass-to-nuclear ratio, and secondary mass spectrometry characteristic ions of the standards. Combined with the Lipid Maps database and the fatty acid (FA22:5), phosphatidylcholine (PC 38:2), and phosphatidylcholine (PC O-30:0) standards in the kit, the secondary mass spectrometry bond breakage patterns of fatty acids and phosphatidylcholine were determined. In positive ion mode, phosphatidylcholine produces a phosphorylcholine characteristic ion with an m / z of 184. In negative ion mode, fatty acids are synergistically lost from glycerol, resulting in a characteristic ion with an m / z of 329.24860 and the corresponding fatty acid radical ion.

[0027] ii) In the test cell samples, the chromatographic peaks of fatty acid (FA22:5), phosphatidylcholine (PC 38:2), and phosphatidylcholine (PC O-30:0) with theoretical mass-to-nuclear ratios of 329.24860, 814.63202, and 692.55890 were extracted respectively under a reasonable mass-to-nuclear ratio tolerance threshold;

[0028] iii) Check whether the MS ions of the above chromatographic peaks conform to the MS bond breakage rules of fatty acids and phosphatidylcholine respectively, and confirm the target fatty acid (FA22:5), phosphatidylcholine (PC 38:2) and phosphatidylcholine (PC O-30:0) based on the chromatographic retention behavior.

[0029] The principles of the present invention are specifically as follows:

[0030] (1) Using ultra-high performance liquid chromatography-quadrupole and orbitrap hybrid Fourier transform ultra-high resolution mass spectrometry, lipid metabolic profiles of RBL-2H3 cells in the negative control group (Veh), allergic group (AG), effective intervention group (tea-derived polyphenols), and ineffective intervention group (Theanine) were analyzed to obtain qualitative and quantitative analysis results of lipid metabolites.

[0031] (2) Based on the results of the phenotypic test of IL-4 release, TNF-α release, degranulation efficiency and inhibition rate in the RBL-2H3 cell model, the allergic group was compared with the two administration methods of the effective intervention group and the ineffective intervention group. The two administration methods were co-incubation (c) and pre-administration (s). The co-incubation group first added the drug solvent as a solvent control for reaction, and then added the drug and DNP-BSA for reaction; the pre-intervention group first added the drug for reaction, and then added the drug solvent and DNP-BSA for reaction. At the same time, based on the results of univariate analysis, differential lipid compounds were screened, and potential markers were discovered by combining the differential fold change and random forest machine learning algorithm. The specific methods include:

[0032] i) First, based on the results of phenotypic tests of IL-4 release, TNF-α release, degranulation efficiency, and inhibition rate in the RBL-2H3 cell model, EGCG inhibited RBL-2H3 cell degranulation and significantly decreased TNF-α and IL-4 release. Therefore, in the comparison of lipid metabolic profiles between the allergic group (AG) and the effective intervention group 1 (EGCG), lipids were screened that met the requirements of a significant difference (p-Value < 0.05) in the univariate nonparametric test and a change ratio greater than 1.5 times (ratio > 3 / 2 or < 2 / 3) between the two groups, and that either administration method had an intervention effect;

[0033] ii) The trend of change in the effective intervention group 1 (EGCG) was consistent with that in the negative control group (Veh). Furthermore, in the phenotypic test of IL-4 release, TNF-α release, degranulation efficiency, and inhibition rate in the RBL-2H3 cell model, it was confirmed that Theanine had no inhibitory effect on degranulation and that there was no downward trend in TNF-α and IL-4 release. Therefore, there were no significant differences (p-Value > 0.05) in lipids between the ineffective intervention group (Theanine) and the allergic group (AG).

[0034] iii) retaining substances with RSD < 10% of lipid metabolites in the quality control (QC) samples;

[0035] iv) Obtain the lipid metabolites from the above three data analysis methods and take the union of different administration methods;

[0036] v) Repeat the experiment in the above pool, use different correction methods to correct, and still meet the retention of lipid metabolites;

[0037] Based on the random forest machine learning algorithm, the combined marker combination of fatty acid (FA22:5), phosphatidylcholine (PC 38:2) and phosphatidylcholine (PC O-30:0) was finally selected.

[0038] The beneficial effects of the present invention are:

[0039] The present invention relates to a novel application of the combined use of lipid metabolites fatty acids (FA22:5), phosphatidylcholine (PC 38:2), and phosphatidylcholine (PC O-30:0) in RBL-2H3 cells as markers for determining the degranulation inhibition effect of tea-derived polyphenols in rat basophilic leukemia cells. The present invention also relates to a detection kit for determining degranulation inhibition. The detection kit can achieve highly sensitive and efficient detection of the three small molecule metabolites disclosed herein, and has the characteristics of low detection cost and good reproducibility. The combined use of the three small molecule metabolites has promising application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The figures show the phenotypic test results of IL-4 release, TNF-α release, and degranulation efficiency and inhibition rate based on the RBL-2H3 cell model. (A) IL-4 release, administered by co-incubation (c); (B) IL-4 release, administered by pre-administration (s); (C) TNF-α release, administered by co-incubation (c); (D) TNF-α release, administered by pre-administration (s); (E) Degranulation efficiency and inhibition rate, administered by co-incubation (c); (F) Degranulation efficiency and inhibition rate, administered by pre-administration (s).

[0041] Figure 2 Shown are the structural formulas and secondary fragmentation characteristics of three lipid markers.

[0042] Figure 3 This is the feature importance graph of the random forest algorithm.

[0043] Figure 4 The levels of lipid markers and combined lipid markers in each group of samples are expressed as mean ± standard deviation; S-EGCG-cThea is abbreviated as S, co-incubation is abbreviated as c, and pre-administration is abbreviated as s; * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0044] Figure 5 ROC curve analysis. (A) ROC curve of the combined markers in the discovery set (AG vs. EGCG & Veh); (B) ROC curve of the combined markers in the validation set (AG vs. S-EGCG-cThea & Veh). DETAILED DESCRIPTION

[0045] The present invention is further described below with reference to the following examples.

[0046] Example 1

[0047] The detection kit used in this embodiment is as follows:

[0048] Standards: (1) fatty acids (FA22:5), phosphatidylcholine (PC 38:2) and phosphatidylcholine (PC O-30:0) (purchased from Avanti Polar Lipids). These standards were used for the auxiliary qualitative analysis of lipid metabolites in the RBL-2H3 cell core. (2) cell sample pretreatment extract: a methanol solution containing 0.92 μg / mL internal standard phosphatidylethanolamine (PE 30:0) and 1.0 μg / mL isotope internal standard fatty acid (FAC16:0-d3). The protein dry weight combined with the internal standard method was used to calibrate the fatty acids (FA22:5), phosphatidylcholine (PC 38:2) and phosphatidylcholine (PC O-30:0). (3) eluent: mobile phase A is acetonitrile / water solution containing 10 mM ammonium acetate (v / v=6:4), mobile phase B is isopropanol / acetonitrile solution containing 10 mM ammonium acetate (v / v=9:1).

[0049] 1. Cell Sample Collection

[0050] This sample set included a negative control group (Veh), an allergic group (AG), and intervention group 1 (EGCG). The two dosing methods in intervention group 1 were co-incubation (c) and pre-dosing (s), and both were incubated for 3 h.

[0051] i) Negative control (Veh) and allergic group (AG): RBL-2H3 cells were incubated with 200 ng / mL IgE for 18 hours. The medium was then aspirated and discarded. The negative control and allergic groups were incubated with the corresponding solvents for 3 hours. Following incubation, the medium was aspirated and discarded. The negative control group was incubated with the control solvent, and the allergic group was incubated with 500 ng / mL DNP-BSA.

[0052] ii) Pre-drug treatment group: RBL-2H3 cells were incubated with 200 ng / mL IgE for 18 h, the medium was discarded, and 200 μM of the corresponding drug was added and incubated for 3 h. After the incubation, the medium was discarded, and the control solvent and 500 ng / mL DNP-BSA were added.

[0053] iii) Co-incubation group: RBL-2H3 cells were incubated with 200 ng / mL IgE for 18 hours, the medium was aspirated, and the corresponding solvent was added as a solvent control for 3 hours. After the incubation, the medium was aspirated, and 200 μM of the corresponding drug and 500 ng / mL DNP-BSA were added.

[0054] iv) After incubation for 3 hours, the culture medium was aspirated and the cells were quickly rinsed three times with DPBS. The biochemical reaction was terminated with liquid nitrogen, and the culture dish was sealed with sealing film and immediately stored at -80°C until use.

[0055] 2. Sample Pretreatment

[0056] Sample pretreatment was performed on ice. First, 1 mL of a methanol solution containing 0.92 μg / mL phosphatidylethanolamine (PE 30:0) and 1.0 μg / mL fatty acid (FA C16:0-d3) isotope internal standard was added. The cells were scraped with a cell scraper and transferred to a 5 mL centrifuge tube. 2.5 mL of MTBE was added, vortexed for 30 seconds, and shaken at 1000 rpm and 10°C for 30 minutes. Subsequently, 750 μL of ultrapure water was added, vortexed for 1 minute, and centrifuged at 6°C and 12,000 g for 10 minutes. After phase separation, the upper hydrophobic phase was quantitatively collected and vacuum-dried in a refrigerated centrifugal concentrator. The resulting lyophilized sample was stored at -80°C.

[0057] Before the lipid freeze-dried sample was detected by ultra-performance liquid chromatography-mass spectrometry, it was re-dissolved in a methanol / dichloromethane mixture (v / v = 1:2) and then diluted with acetonitrile / isopropanol / water (v / v / v = 65:30:5, containing 5 mM ammonium acetate) for injection.

[0058] 3. Data Collection

[0059] (1) Ultra-high performance liquid chromatography conditions: A Thermo Fisher Vanquish Neo (UPLC, Thermo, USA) system was used, and the chromatographic column was an ACQUITY UPLC BEH C8 column (2.1×100 mm×1.7 μm, Waters, USA). During the separation process, the column temperature was set to 55°C, and the injection chamber temperature was controlled at 6°C. Mobile phase A was acetonitrile: ultrapure water = 6:4 (containing 10 mM ammonium acetate), and mobile phase B was isopropanol: acetonitrile = 9:1 (containing 10 mM ammonium acetate). A gradient elution mode was used, with the following gradient: first, 32% B was maintained for 1.5 min, then linearly increased to 85% B from 1.5 to 15.5 min, then linearly increased to 97% B within 0.1 min and maintained for 18 min, and then decreased to 32% B within 0.1 min until equilibrium was reached for the next injection. The flow rate was maintained at 0.26 mL / min throughout the entire process.

[0060] (2) Mass spectrometry conditions: A quadrupole and orbitrap hybrid Fourier transform ultra-high resolution mass spectrometer (Q-Exactive, Thermo, USA) was used, and the mass spectrometry analysis was performed in negative ion mode using an electrospray ionization source. The mass spectrometry parameters were set as follows: spray voltage 3.5 kV, sheath gas flow rate 50 arb, auxiliary gas flow rate 15 arb, auxiliary gas temperature 400°C, capillary temperature 325°C, acquisition range 200-1800 m / z, acquisition rate 1.7 spectra / s, and secondary analysis of the top 10 parent ions in HCD fragmentation mode with a collision energy of 15-45 eV. During the acquisition process, Xcalibur software (Thermo, USA) was used to record the total ion current and MS spectra.

[0061] 4. Data Preprocessing

[0062] Raw mass spectrometric data obtained with the ULPC-Q Exative were imported into MSDIAL software for peak identification, extraction, and alignment (http: / / prime.psc.riken.jp / compms / index.html), excluding peaks with a signal-to-noise ratio below 10. The resulting peak table was used to identify lipids using Xcalibular, and fatty acid (FA22:5), phosphatidylcholine (PC 38:2), and phosphatidylcholine (PC O-30:0) standards were used to assist in qualitative confirmation of the detected ions. The identified lipids met the following three criteria:

[0063] (1) Accurate mass of primary mass spectrometry (theoretical value ± 5ppm);

[0064] (2) Secondary mass spectrometry MS / MS fragmentation rules;

[0065] (3) Chromatographic retention time pattern.

[0066] Accurately identified lipid compounds were imported into TraceFinder (Thermo, USA) for compound quantification. Subsequently, chromatographic peak intensity was calibrated based on cell protein weight and internal standard.

[0067] 5. Results Analysis

[0068] This sample set was used for biomarker discovery and included a negative control group (Veh), an allergic group (AG), and two administration methods (EGCG-c & EGCG-s) in intervention group 1. The structural formulas and secondary mass spectra of the three lipid biomarkers are shown in Figure 2. Figure 2 As shown. Random forest algorithm feature importance is as follows Figure 3 The contents of lipid markers and combined lipid markers in each group of samples are shown in Figure 4 shown.

[0069] Using the statistical software SPSS, fatty acids (FA22:5), phosphatidylcholine (PC 38:2), and phosphatidylcholine (PC O-30:0) were further calculated by binary logistic regression to obtain the joint marker P. The regression equation is as follows:

[0070] P=1 / [1+e -(0.087a+0.158b-0.231c-37.847) ]

[0071] Wherein, a is the relative content of fatty acid (FA22:5), b is the relative content of phosphatidylcholine (PC 38:2), and c is the relative content of phosphatidylcholine (PC O-30:0).

[0072] EGCG, AG, and Veh samples of all administration methods were included in the discrimination comparison. Based on the discriminant variable P obtained from the combined marker, the area under the ROC curve (AUC value) for discrimination was 1.000. At a cut-off value of 0.5, the sensitivity and specificity were both 100% (Table 1, Figure 5 ), the above results indicate that the combined marker has good discriminative potential and can be used to discriminate the degranulation inhibitory effect of tea-derived polyphenols (EGCG).

[0073] Example 2

[0074] 1. Cell Sample Collection

[0075] Samples were collected from the negative control group (Veh), the allergy group (AG), and intervention group 2 (S-EGCG-cThea) under different dosing methods. The two dosing methods in intervention group 2 were co-incubation (c) and pre-dosing (s). This sample set was used for marker validation. Cell collection was the same as in Example 1.

[0076] 2. Sample Pretreatment

[0077] Same as Example 1.

[0078] 3. Data Collection

[0079] Same as Example 1.

[0080] 4. Data Preprocessing

[0081] Same as Example 1.

[0082] 5. Results Analysis

[0083] Based on the comparison between the AG group and the intervention group 2 (S-EGCG-cThea), Example 2 was used to verify the feasibility of the combined use of the above three lipid markers to further confirm the discriminant effect of the degranulation inhibitory effect of tea-derived polyphenols. The S-EGCG-cThea group, Veh group, and AG group of all administration methods were included in the discriminant comparison. Based on the discriminant variable P obtained by the combined marker, the area under the ROC curve for discrimination was 0.948, the sensitivity was 100%, and the specificity was 91.7% ( Figure 5 , Table 1), the above results further confirmed that the combined use of the above three lipid markers is effective in discriminating the degranulation inhibitory effect of tea-derived polyphenols.

[0084] Table 1. ROC discriminant analysis results of markers

[0085]

Claims

1. A combined metabolic marker for determining the degranulation inhibitory effect of tea-derived polyphenols on rat basophilic leukemia cells, wherein the tea-derived polyphenols are S-epigallocatechin gallate-theanine conjugate and epigallocatechin gallate, characterized in that: The markers include: fatty acids (FA22:5), phosphatidylcholine (PC 38:2) and phosphatidylcholine (PC O-30:0).

2. Use of the combined metabolic marker according to claim 1 in the preparation of a detection kit for determining the degranulation inhibitory effect of tea-derived polyphenols.

3. A detection kit containing the combined metabolic marker according to claim 1.

4. The detection kit according to claim 3, wherein include: A standards: fatty acids (FA22:5), phosphatidylcholine (PC 38:2), and phosphatidylcholine (PC O-30:0); B. Pretreatment extract: methanol solution containing internal standard 0.92 μg / mL phosphatidylethanolamine (PE 30:0) and 1.0 μg / mL fatty acid (FAC16:0-d3); C eluent: Mobile phase A is an acetonitrile / water solution containing 10 mM ammonium acetate (v / v=6:4), and mobile phase B is an isopropanol / acetonitrile solution containing 10 mM ammonium acetate (v / v=9:1).

5. A method for determining the degranulation inhibitory effect of tea-derived polyphenols using the combined metabolic marker according to claim 1 or the detection kit according to claim 3, characterized in that: Use the joint marker variable P to make judgments: P=1 / [1+e -(0.087a+0.158b-0.231c-37.847) ] In the above formula, a is the relative content of fatty acid (FA22:5), b is the relative content of phosphatidylcholine (PC 38:2), and c is the relative content of phosphatidylcholine (PC O-30:0); If P>0.5, it is judged that tea-derived polyphenols have an indicative effect on degranulation.

6. A method for calculating a combined marker variable using the detection kit according to claim 3 or 4, characterized in that: The following steps are involved: (1) Pretreatment of RBL-2H3 samples: RBL-2H3 cell samples were added to the pretreatment extract, followed by methyl tert-butyl ether and ultrapure water to extract lipid metabolites; (2) separating and identifying lipid metabolites of the test sample extracted after treatment in step (1) by ultra-high performance liquid chromatography-mass spectrometry; (3) Fatty acid (FA22:5), phosphatidylcholine (PC 38:2), and phosphatidylcholine (PC O-30:0) standards provided in the kit were used to assist in qualitative confirmation of the detected ions; (4) For the target fatty acids (FA22:5), phosphatidylcholine (PC 38:2), and phosphatidylcholine (PC O-30:0) in the identified test samples, their chromatographic peak intensities were first corrected for the dry weight of cell sample protein, and then compared with the internal standards phosphatidylethanolamine (PE 30:0) and fatty acid (FAC16:0-d3) to obtain the relative concentrations of the above three metabolites; (5) Fatty acids (FA22:5), phosphatidylcholine (PC 38:2) and phosphatidylcholine (PC O-30:0) were calculated by binary logistic regression to obtain the joint marker variable P.

7. The method according to claim 6, wherein Fatty acid (FA22:5), phosphatidylcholine (PC 38:2), and phosphatidylcholine (PC O-30:0) standards were used to assist in the qualitative confirmation of the detected ions. Specifically, the following are included: i) The fatty acid (FA22:5), phosphatidylcholine (PC 38:2), and phosphatidylcholine (PC O-30:0) standards in the kit were analyzed by ultra-performance liquid chromatography-mass spectrometry to determine the chromatographic retention time, measured mass-to-nuclear ratio, and secondary mass spectrometry characteristic ions of the standards. Combined with the Lipid Maps database and the fatty acid (FA22:5), phosphatidylcholine (PC 38:2), and phosphatidylcholine (PC O-30:0) standards in the kit, the secondary mass spectrometry bond breakage patterns of fatty acids and phosphatidylcholine were determined. In positive ion mode, phosphatidylcholine produces a phosphorylcholine characteristic ion with an m / z of 184. In negative ion mode, fatty acids are synergistically lost from glycerol, resulting in a characteristic ion with an m / z of 329.24860 and the corresponding fatty acid radical ion. ii) In the test cell samples, the chromatographic peaks of fatty acid (FA22:5), phosphatidylcholine (PC 38:2), and phosphatidylcholine (PC O-30:0) with theoretical mass-to-nuclear ratios of 329.24860, 814.63202, and 692.55890 were extracted respectively under a reasonable mass-to-nuclear ratio tolerance threshold; iii) Check whether the MS ions of the above chromatographic peaks conform to the MS bond breakage rules of fatty acids and phosphatidylcholine respectively, and confirm the target fatty acid (FA22:5), phosphatidylcholine (PC 38:2) and phosphatidylcholine (PC O-30:0) based on the chromatographic retention behavior.

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