Screening, verification and detection method for allergen substances in carthamin yellow injection

Abstract

The invention discloses a screening method of allergen substances in a carthamin yellow injection, a verification method of the allergen substances in the carthamin yellow injection, a detection method of the allergen substances in the carthamin yellow injection and a production process of the carthamin yellow injection. The screening method of the allergen substances in the carthamin yellow injection comprises the following steps: S1, extracting components in the carthamin yellow injection; s2, separating and screening components in the extract by adopting a high performance liquid chromatography-ultraviolet detection-mass spectrum and human serum albumin combined detection technology; and S3, screening out a substance which can be combined with the HSA, and determining the substance as a sensitization hapten substance which has sensitization potential. The invention aims to detect that the 6-hydroxykaempferol-3-rutin-6-glucoside has HAS binding characteristic, hapten attribute and sensitization potential, and can provide basis for process improvement of carthamin yellow injection and reduce the sensitization risk in clinical application.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and in particular relates to a method for screening and verifying allergenic substances in safflower yellow pigment injection. Background Art

[0002] With the widespread clinical application of safflower yellow pigment injection, the related adverse reactions (ADR) have gradually increased, especially allergic reactions. These reactions mainly manifest as rash, anaphylactic shock, neurocirculatory system reactions, etc., and may be delayed allergic reactions, triggered by IgE-mediated mechanisms. The chemical composition of traditional Chinese medicine injection is complex, and the allergenic components have not yet been identified. Therefore, in-depth research and screening of allergenic substances in safflower yellow pigment injection is crucial to improve its production process and improve clinical drug safety.

[0003] One of the factors for the high incidence of allergic reactions to Chinese medicine injections is that their chemical composition is complex and the sensitizing components are unclear. The ADR of safflower yellow is mainly rash, anaphylactic shock, neurocirculatory system, etc., which may be a delayed allergic reaction, that is, it is caused by an IgE-mediated mechanism. Type I allergic reaction is the most serious and common adverse reaction to Chinese medicine injections. When a sensitized body comes into contact with the same antigen again, specific immunoglobulin E (IgE) antibodies mediate mast cells and basophils to release active mediators to cause local or systemic reactions. Haptens do not have antigenic properties before entering the body and usually do not cause allergic reactions, but can combine with human HAS to form a large antigen complex, which may have sensitizing potential.

[0004] The ingredients of Chinese herbal injection preparations are complex and diverse. It is currently impossible to separate each ingredient and conduct corresponding animal experiments for confirmation. There are also certain differences between animal experiments and clinical applications. Therefore, it is necessary to develop an efficient and targeted method to screen and verify the allergens in safflower yellow pigment injection. Summary of the invention

[0005] The purpose of the present invention is to solve the deficiencies in the prior art, to provide a method for screening, verifying and detecting allergenic substances in safflower yellow injection that can solve the problems of efficient screening of allergenic substances and verification of sensitizing potential in safflower yellow injection, and to provide a production process for safflower yellow injection that effectively reduces the risk of allergic reactions in its clinical application.

[0006] In order to solve the above technical problems, the technical method adopted by the present invention is as follows: The present invention discloses a method for screening allergens in safflower yellow pigment injection, comprising the following steps:

[0007] S1. Extracting components from safflower yellow pigment injection;

[0008] S2. Separate and screen the components in the extract using high performance liquid chromatography-ultraviolet detection-mass spectrometry and human serum albumin binding detection technology;

[0009] S3. Screen out substances that bind to HSA and determine that they are allergenic hapten substances with sensitizing potential.

[0010] Furthermore, in the screening step, an HPLC-DAD-MSn-HSA-FLD system is used to identify suspected hapten components in the safflower yellow pigment injection, and the HPLC-DAD-MSn-HSA-FLD system comprises:

[0011] HPLC: Used to separate different components in a sample.

[0012] DAD: Diode array detector, monitors the absorbance of samples at different wavelengths for qualitative analysis.

[0013] MSn: Tandem mass spectrometry technology provides detailed information on molecular structure through multiple mass spectrometry analyses.

[0014] HSA: Human serum albumin, used to detect the binding of chemicals to serum proteins, helping to identify possible allergenic ingredients.

[0015] FLD: Fluorescence detector, detects substances that can emit fluorescence and enhances detection sensitivity.

[0016] Furthermore, the hapten substance screened out was 6-hydroxykaempferol-3-rutinoside-6-glucoside.

[0017] The present invention also proposes a method for verifying allergens in safflower yellow pigment injection, comprising the following steps:

[0018] The screened hapten substance 6-hydroxykaempferol-3-rutinoside-6-glucoside was compared with the negative control and the positive control respectively;

[0019] The experimental animals in different groups were sensitized and challenged on the 14th and 21st days;

[0020] The occurrence of allergic reactions was evaluated by detecting the levels of IgE, histamine, IFN-γ and β-hexosaminidase in animal serum.

[0021] Furthermore, the experimental animals are guinea pigs, and serum IgE, histamine, IFN-γ, and β-hexosaminidase of guinea pigs are used as indicators of allergic reactions.

[0022] Furthermore, if the guinea pigs develop a mild allergic reaction after the provocation treatment, and the levels of IgE and histamine in the serum are significantly increased, 6-hydroxykaempferol-3-rutinoside-6-glucoside is considered to be potentially allergenic.

[0023] The present invention also proposes a method for detecting allergens in safflower yellow pigment injection, comprising the following steps:

[0024] Extract the components of safflower yellow pigment injection and perform high performance liquid chromatography analysis;

[0025] The extracted components are combined with human serum albumin;

[0026] Through antigen-antibody reaction detection, hapten substances that bind to HSA are screened out, providing a screening basis for potential allergens.

[0027] Furthermore, the hapten substance screened out was 6-hydroxykaempferol-3-rutinoside-6-glucoside.

[0028] The present invention also proposes a production process of safflower yellow pigment injection, which adopts the above-mentioned method for screening allergenic substances in safflower yellow pigment injection to reduce the allergenic components in safflower yellow pigment injection by identifying and removing possible allergenic hapten substances.

[0029] Furthermore, the sensitizing hapten substance removed from the safflower yellow pigment injection is 6-hydroxykaempferol-3-rutyl-6-glucoside.

[0030] Beneficial effects:

[0031] 1. Efficient and accurate allergen screening and verification: By combining the HPLC-DAD-MSn-HSA-FLD system, the present invention provides a highly sensitive and accurate screening method, which can accurately separate and identify potential allergens (6-hydroxykaempferol-3-rutinoside-6-glucoside) in safflower yellow pigment injection, and verify its sensitizing potential through systemic allergy test and popliteal lymph node test, ensuring the consistency of screening results with clinical application.

[0032] 2. Complete technical verification process: The present invention provides a complete process from allergen screening to sensitization verification and then to production process improvement. By combining laboratory technology and animal experiments, it ensures the comprehensiveness and reliability of the technology and effectively reduces the risk of allergic reactions to safflower yellow pigment injection.

[0033] 3. Wide applicability and scalability: This technical solution is not only applicable to safflower yellow pigment injection, but also has broad application prospects. It can be extended to the allergen screening and production process optimization of other drugs and traditional Chinese medicine ingredients, and has strong versatility. DETAILED DESCRIPTION

[0034] The present invention proposes a method for screening allergenic substances in safflower yellow pigment injection, and the components in safflower yellow pigment injection are screened and identified by the following steps:

[0035] S1. Extraction of ingredients from safflower yellow pigment injection

[0036] The safflower yellow pigment injection is extracted with an appropriate solvent to obtain its main components. The extraction process needs to ensure that the activity and physical and chemical properties of its components are retained to the greatest extent.

[0037] S2. Separate and screen the components in the extract using high performance liquid chromatography-ultraviolet detection-mass spectrometry and human serum albumin binding detection technology;

[0038] The extracts were separated and screened using the HPLC-DAD-MSn-HSA-FLD system. HPLC technology separates the components in the sample through a chromatographic column, the DAD detector provides ultraviolet absorption information, the MSn tandem mass spectrometer provides information on molecular mass and structural fragments, and the HSA (human serum albumin) binding test helps identify components that may bind to HSA and screen out hapten substances with potential allergenicity.

[0039] S3. Screen out substances that bind to HSA and determine that they are allergenic haptens with sensitizing potential. Through the binding of HSA to the target components, further determine whether these components may form large molecular complexes by binding to immune system proteins, have hapten properties, and may cause allergic reactions.

[0040] Detailed structure description of HPLC-DAD-MSn-HSA-FLD system:

[0041] The HPLC-DAD-MSn-HSA-FLD system used in the present invention includes the following important components:

[0042] HPLC High Performance Liquid Chromatography: Used to separate the different chemical components in an extract, ensuring that each component can be independently identified.

[0043] DAD diode array detector: monitors the ultraviolet absorption of samples at different wavelengths and provides qualitative analysis information.

[0044] MSn tandem mass spectrometry: Multiple mass spectrometry analyses are used to provide molecular mass and fragment information of components, helping to further identify the structure of each substance.

[0045] HSA Human Serum Albumin: Used to detect the ability of chemicals to bind to HSA to confirm whether they may become allergens.

[0046] FLD fluorescence detector: used to detect substances that can emit fluorescence and enhance the detection sensitivity of certain specific components.

[0047] The present invention proposes a verification method for allergenic substances in safflower yellow pigment injection. After screening out possible allergenic substances, the present invention further verifies their sensitizing potential through animal experiments. The specific steps are as follows:

[0048] The screened hapten substances were compared with negative and positive controls: 6-hydroxykaempferol-3-rutinoside-6-glucoside was used as the test substance and compared with normal saline (negative control) and ovalbumin (positive control).

[0049] Different groups of experimental animals were sensitized and challenged on the 14th and 21st days. Guinea pigs were used as experimental animals, and injection sensitization was performed, and allergic reactions were observed after challenge. The occurrence of allergic reactions was evaluated by detecting the levels of IgE, histamine, IFN-γ and β-hexosaminidase in animal serum. The degree of allergic reactions was evaluated by serological tests.

[0050] If the IgE and histamine levels in the test group are significantly increased compared to the control group, it indicates that the substance has potential allergenicity.

[0051] The present invention also includes a production process of safflower yellow pigment injection. According to the screening and verification method provided by the present invention, the production process of safflower yellow pigment injection is improved. The specific production process includes:

[0052] Identify and remove possible allergenic hapten substances: During the production process, screening methods are used to remove possible allergenic ingredients (such as 6-hydroxykaempferol-3-rutinoside-6-glucoside) to ensure that there are no allergenic substances in the final product.

[0053] Reducing the allergenic components in safflower yellow pigment injection: Through effective screening and separation methods, the allergenic components in safflower yellow pigment injection can be reduced, thereby improving the safety of its clinical application.

[0054] The present invention also proposes a method for detecting allergens in safflower yellow pigment injection, which specifically comprises:

[0055] Step 1. extracting components from safflower yellow pigment injection and performing high performance liquid chromatography analysis;

[0056] Step 2. combining the extracted components with human serum albumin;

[0057] Step 3. Through antigen-antibody reaction detection, the hapten substances that bind to HSA are screened out to provide a screening basis for potential allergens.

[0058] Example 1: Active Systemic Anaphylaxis (ASA)

[0059] 1.1 Experimental equipment and reagents

[0060] Centrifuge: TGL-168 centrifuge (Shanghai Anting Scientific Instrument Factory), used for centrifugal separation of serum samples.

[0061] Microplate reader: used to measure the concentration of allergic reaction-related substances such as IgE, histamine, IFN-γ, β-hexosaminidase, etc. in serum.

[0062] Vortex mixer: XW-80A (Shanghai Qingpu Huxi Instrument Factory), used for mixing reagents.

[0063] Reagents:

[0064] Ovalbumin (OVA): As a positive control drug, the concentration was 10 mg / ml (Beijing Solebow Technology Co., Ltd.).

[0065] Sodium chloride injection (normal saline): used as a negative control drug in the control group (Shijiazhuang Siyao Co., Ltd.).

[0066] Test substance: 6-Hydroxykaempferol-3-rutinoside-6-glucoside, as a suspected hapten component of safflower yellow injection (Yunnan Xili Biotechnology Co., Ltd.).

[0067] 1.2 Experimental animals

[0068] Guinea pig strain: Hartley guinea pig, male, weighing 250-300 g.

[0069] Feeding conditions: The experimental animals were fed for 1 week, the room temperature was controlled at 20-25℃, and the humidity was controlled at 40-70%. The feed was SPF feed produced by Beijing Keao Xieli Feed Co., Ltd. 4 animals per cage, with free access to food and water. The feeding room was disinfected regularly.

[0070] 1.3 Experimental methods

[0071] Experimental groups:

[0072] Negative control group: injected with sodium chloride injection (normal saline).

[0073] Positive control group: injected with ovalbumin (OVA, 10 mg / ml).

[0074] Test group: injected with 6-hydroxykaempferol-3-rutinoside-6-glucoside (0.5 ml / mouse).

[0075] Sensitization steps:

[0076] The test substance and guinea pig serum were mixed in equal volumes, incubated at 37°C for 24 hours, and each group of guinea pigs was intraperitoneally injected with 0.5 ml of the test substance or control substance for 3 consecutive times with an interval of 48 hours.

[0077] Stimulation steps:

[0078] The provocation treatment was carried out on the 14th and 21st days after the last sensitization. Each group of guinea pigs was intravenously injected with 1 ml of the corresponding test substance or control substance, and the allergic reaction was observed immediately after the provocation and continued for 30 minutes to 3 hours.

[0079] 1.4 Serum sample collection and testing

[0080] Blood was collected from the heart before and after the challenge, and the blood was naturally coagulated for 10 minutes before centrifugation. The serum was extracted and stored at -20°C.

[0081] Enzyme-linked immunosorbent assay (ELISA) was used to detect serum IgE, histamine, IFN-γ, and β-hexosaminidase levels to evaluate the occurrence of allergic reactions.

[0082] 1.5 Experimental Results

[0083] Negative control group (normal saline group): no obvious allergic symptoms occurred, and the levels of IgE, histamine, IFN-γ, and β-hexosaminidase in serum did not change significantly.

[0084] Positive control group (ovalbumin group): All guinea pigs showed obvious allergic symptoms within 15 to 30 seconds after stimulation, such as scratching the nose, coughing, and wheezing, and died within 3 minutes. The serum IgE, histamine, IFN-γ, and β-hexosaminidase increased significantly, the allergic reaction was extremely positive, and the allergic rate was 100%.

[0085] Test group (6-hydroxykaempferol-3-rutinoside-6-glucoside group): No obvious acute allergic symptoms were observed, but the levels of IgE and histamine in serum increased after stimulation. IgE increased to 85ng / ml (P<0.05), and histamine increased slightly. IFN-γ and β-hexosaminidase also increased to a certain extent, indicating that 6-hydroxykaempferol-3-rutinoside-6-glucoside has potential allergenicity.

[0086] 1.6 Conclusion

[0087] The results of this experiment showed that although 6-hydroxykaempferol-3-rutinoside-6-glucoside did not cause obvious allergic reactions, the increased levels of allergic mediators such as IgE, histamine, and IFN-γ in serum proved that it had potential allergenicity, providing a basis for the safety assessment of safflower yellow injection.

[0088] Example 2: Reporter Antigen Popliteal Lymph Node Assay

[0089] 2.1 Experimental equipment and reagents

[0090] Balance: Quintix125D-1CN (Sartorius Scientific Instruments Co., Ltd.), used to weigh popliteal lymph nodes.

[0091] Cell counter: Thermo Fisher Countess II, used to count the number of cells.

[0092] Flow cytometer: Cytometry FC500 (BECKMAN COULTER), used to analyze cell distribution and surface markers.

[0093] Reagents:

[0094] TNP-OVA: 2,4,6-trinitrophenylovalbumin (BioSearch Technologies) was used as a reporter antigen.

[0095] Trinitrobenzene polysucrose (TNP-Ficoll): 2,4,6-trinitrophenylFicoll (BioSearch Technologies), used for auxiliary antigen.

[0096] Positive control drug: phenytoin sodium (2 mg / each, Chengdu Aikoda Chemical Reagent Company).

[0097] Normal saline control group: used as negative control.

[0098] 2.2 Experimental animals

[0099] Mouse strain: SPF BALB / c female mice, 7-8 weeks old, weighing (20±5) g, were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

[0100] Feeding conditions: room temperature was controlled at 20-25°C, humidity was controlled at 40-70%, feed was standard SPF-grade feed, and animals were free to eat and drink.

[0101] 2.3 Experimental methods

[0102] Experimental groups:

[0103] Negative control group: injected with normal saline (Veh).

[0104] Positive control group: injected with phenytoin sodium (2 mg / mouse).

[0105] Test group: injected with 0.5 mg / head of 6-hydroxykaempferol-3-rutinoside-6-glucoside.

[0106] Injection steps:

[0107] The test substance and positive control drug were co-injected with TNP-OVA and TNP-Ficoll, respectively, with an injection dose of 50 μL per mouse. Seven days after administration, the popliteal lymph nodes were separated and removed with forceps for analysis.

[0108] 2.4 Detection indicators

[0109] Lymph node weight and cell number:

[0110] The weight index (WI) and cell index (CI) of popliteal lymph nodes were calculated.

[0111] WI calculation formula: WI = weight of popliteal lymph nodes on the treated side / weight of popliteal lymph nodes on the control side;

[0112] CI calculation formula: CI = number of cells in the popliteal lymph node on the treated side / number of cells in the popliteal lymph node on the control side.

[0113] Flow cytometry: Analyze isolated cells to detect immune cell activation.

[0114] 2.5 Experimental Results

[0115]

[0116] 2.6 Conclusion

[0117] Lymph node weight and cell index: The lymph node weight and cell number of mice in the test group were significantly increased compared with those in the positive control group, indicating that the compound may trigger a response of the immune system and has sensitization potential.

[0118] Immune response: The concentrations of allergic reaction-related substances such as serum IgE, histamine, IFN-γ and β-hexosaminidase in the test group were significantly higher than those in the negative control group (P<0.05), indicating that the substance can activate the immune response and may cause allergic reactions.

[0119] Based on the above results, 6-hydroxykaempferol-3-rutinoside-6-glucoside has a certain sensitizing potential, suggesting that it may be a potential allergen for safflower yellow injection.

[0120] Summary of Examples

[0121] By implementing the above-mentioned popliteal lymph node test of the reported antigen, the data showed that 6-hydroxykaempferol-3-rutinoside-6-glucoside had a significant immune response in mice, indicating that it has potential allergenicity. This provides a scientific basis for the identification of allergenic components in safflower yellow pigment injection and the improvement of production processes, which helps to reduce the risk of allergic reactions in its clinical use.

[0122] These experimental data and analysis results provide strong experimental support for the patent application and ensure the effectiveness of the screening and verification methods.

[0123] Compared with the existing technology, this technical solution has significant advantages, especially in the screening and verification of allergens, improvement of the production process of safflower yellow pigment injection, and control of allergic reaction risks. The following are the main advantages of this technical solution compared with the existing technology:

[0124] 1. Accurate screening of allergens

[0125] Limitations of existing technologies: Existing technologies for screening allergens in TCM injections mostly use traditional biological experiments, such as animal experiments, skin tests, etc. These methods are time-consuming and lack efficient qualitative analysis tools. In addition, due to the complex composition of TCM injections, it is difficult to fully confirm every possible allergenic component, and there may be certain differences between the results of animal experiments and clinical applications.

[0126] Advantages of the present invention: The present invention can accurately separate and identify various chemical components in safflower yellow pigment injection and screen out hapten components that bind to HSA by introducing the HPLC-DAD-MSn-HSA-FLD system, combining high performance liquid chromatography, ultraviolet detection, tandem mass spectrometry, fluorescence detection and human serum albumin (HSA) binding detection technology. This method can deeply analyze chemical components at the molecular level, identify hapten substances with potential allergenicity, and provide a more accurate and efficient screening method than traditional technologies.

[0127] 2. Efficient and systematic sensitization verification method

[0128] Limitations of existing technologies: Traditional allergenicity verification methods often rely on animal experiments, and these experimental methods usually evaluate the intensity of allergic reactions based on the performance of individual animals, which is highly subjective and takes a long time. Existing allergic reaction verification methods are difficult to achieve rapid screening and comprehensive evaluation.

[0129] Advantages of the present invention: This technical solution uses two experimental methods, the systemic active allergy test (ASA) and the reporting antigen popliteal lymph node test, to quantitatively measure the concentrations of allergic mediators such as IgE, histamine, IFN-γ, and β-hexosaminidase in serum when verifying allergens, providing a more objective and quantitative evaluation standard. In addition, modern immunological techniques (such as flow cytometric analysis) are combined to verify the activation of immune responses, making the process of sensitization verification more scientific and efficient.

[0130] 3. Improved production process to reduce clinical allergy risk

[0131] Limitations of existing technologies: The existing production process of safflower yellow pigment injection faces challenges in terms of ingredient complexity and is unable to accurately remove hapten substances that may cause allergic reactions. Due to the lack of efficient screening methods, the existing technology is inefficient in removing allergenic components, which can easily lead to allergic reactions.

[0132] Advantages of the present invention: The screening method proposed in the present invention can effectively identify hapten substances in safflower yellow pigment injection, and improve the production process of safflower yellow pigment injection by removing these allergenic components. This process can significantly reduce the risk of allergic reactions in clinical applications, improve the safety of drugs and the reliability of clinical applications.

[0133] 4. High sensitivity and accuracy

[0134] Limitations of existing technologies: Traditional allergen screening methods have low sensitivity and are difficult to distinguish different types of allergic reaction sources, especially in traditional Chinese medicine injections with multiple complex ingredients. The identification and verification of allergenic components rely on experience and rough analysis.

[0135] Advantages of the present invention: By combining the HPLC-DAD-MSn-HSA-FLD system, the present invention can accurately identify the components in the safflower yellow pigment injection at the molecular level, and combine multiple technical means (such as mass spectrometry, fluorescence detection, protein binding detection) to enhance the ability to identify potential allergens. This highly sensitive and accurate screening method can significantly improve the efficiency of allergen discovery and reduce unnecessary false positive or false negative results.

[0136] 5. Consistency between animal experiments and clinical applications

[0137] Limitations of existing technologies: Traditional animal experiments often differ from clinical reactions. Some ingredients may not cause allergic reactions in animals, but may still pose a risk of sensitization in clinical applications.

[0138] Advantages of the present invention: The systemic active allergy test (ASA) and the reporter antigen popliteal lymph node test of the present invention can more accurately simulate and predict clinical allergic reactions through quantitative analysis of multiple biomarkers (such as IgE, histamine, IFN-γ, etc.). This experimental design not only improves the accuracy of animal experiments, but also enhances the consistency of the results with actual clinical applications.

[0139] 6. Provide a complete technical verification process

[0140] Limitations of existing technologies: Existing technologies often lack a complete technical verification process, resulting in a large disconnect between allergen screening and verification, making the screening results of some allergens unscientific.

[0141] Advantages of the present invention: The present invention provides a complete technical process from allergen screening to sensitization verification and then to production process improvement, covering every link from basic research to practical application. Through laboratory screening, animal testing and production process optimization, this technical solution ensures the integrity and systematicness of the technology and enhances the safety and effectiveness of safflower yellow pigment injection in clinical applications.

[0142] 7. Scalability and applicability

[0143] Limitations of existing technologies: Existing technologies are usually limited to the screening of certain specific types of drugs or chemical components and are difficult to be widely applied to a variety of drugs and injections.

[0144] Advantages of the present invention: The technical system adopted by the present invention has strong scalability and can be widely used in the screening and verification of allergens of other drugs, injections and traditional Chinese medicine ingredients. This versatility makes the technical solution not only limited to safflower yellow pigment injection, but also widely used in the safety assessment and production process optimization of other drugs.

[0145] Summarize

[0146] Compared with the prior art, the present invention provides an efficient, accurate and scientific allergen screening and verification method by introducing the HPLC-DAD-MSn-HSA-FLD system for allergen screening and combining animal experiments to verify the sensitization potential. This method has higher sensitivity, accuracy and reliability, can significantly improve the safety of safflower yellow pigment injection, and reduce the risk of allergies in clinical applications. In addition, this technical solution also has a wide range of application prospects and can be promoted and applied to allergen screening and production process improvement of other drugs and traditional Chinese medicine injections.

[0147] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for screening allergens in safflower yellow pigment injection, characterized in that: The following steps are involved: S1. Extracting components from safflower yellow pigment injection; S2. Separate and screen the components in the extract using high performance liquid chromatography-ultraviolet detection-mass spectrometry and human serum albumin binding detection technology; S3. Screen out substances that bind to HSA and determine that they are allergenic hapten substances with sensitizing potential.

2. The method for screening allergens in safflower yellow injection according to claim 1, characterized in that: In the screening step, the HPLC-DAD-MSn-HSA-FLD system is used to identify suspected hapten components in the safflower yellow pigment injection, and the HPLC-DAD-MSn-HSA-FLD system includes: HPLC: Used to separate different components in a sample. DAD: Diode array detector, monitors the absorbance of samples at different wavelengths for qualitative analysis. MSn: Tandem mass spectrometry technology provides detailed information on molecular structure through multiple mass spectrometry analyses. HSA: Human serum albumin, used to detect the binding of chemicals to serum proteins, helping to identify possible allergenic ingredients. FLD: Fluorescence detector, detects substances that can emit fluorescence and enhances detection sensitivity.

3. The method according to claim 1 or 2, characterized in that ,in, The hapten substance screened out was 6-hydroxykaempferol-3-rutinoside-6-glucoside.

4. A method for verifying allergens in safflower yellow pigment injection, characterized in that: The following steps are involved: The screened hapten substance 6-hydroxykaempferol-3-rutinoside-6-glucoside was compared with the negative control and the positive control respectively; The experimental animals in different groups were sensitized and challenged on the 14th and 21st days; The occurrence of allergic reactions was evaluated by detecting the levels of IgE, histamine, IFN-γ and β-hexosaminidase in animal serum.

5. The method for verifying allergens in safflower yellow injection according to claim 4, characterized in that: The experimental animals are guinea pigs, and serum IgE, histamine, IFN-γ, and β-hexosaminidase of guinea pigs are used as indicators of allergic reactions.

6. The method for verifying allergenic substances in safflower yellow injection according to claim 4 or 5, characterized in that: If guinea pigs show mild allergic reactions after provocation treatment, with significant increases in serum IgE and histamine levels, 6-hydroxykaempferol-3-rutinoside-6-glucoside is considered to be potentially allergenic.

7. A method for detecting allergens in safflower yellow pigment injection, characterized in that: The following steps are involved: Extract the components of safflower yellow pigment injection and perform high performance liquid chromatography analysis; The extracted components are combined with human serum albumin; Through antigen-antibody reaction detection, hapten substances that bind to HSA are screened out, providing a screening basis for potential allergens.

8. The method for detecting allergens in safflower yellow injection according to claim 7, characterized in that: The hapten substance screened out was 6-hydroxykaempferol-3-rutinoside-6-glucoside.

9. A production process of safflower yellow pigment injection, characterized in that: The screening method according to claim 1 or 2 is used to reduce the allergenic components in the safflower yellow pigment injection by identifying and removing possible allergenic hapten substances.

10. The production process of safflower yellow pigment injection according to claim 9, characterized in that: The sensitizing hapten substance removed from the safflower yellow pigment injection is 6-hydroxykaempferol-3-rutyl-6-glucoside.

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