Application of TRPC6 / TRPM4 molecular sensory artificial intelligence biosensor in anti-virus pungent-warm key quality attribute identification of negundo chastetree prevention mixture

Abstract

The invention provides application of a TRPC6 / TRPM4 molecular sensory artificial intelligence biosensor in anti-virus pungent-warm key quality attribute identification of a negundo chastetree prevention mixture, and belongs to the technical field of traditional Chinese medicine analysis. According to the application, the medicinal components of the negundo chastetree prevention mixture entering blood and target organs are determined through molecular biology, a TRPC6 / TRPM4 double-target receptor is determined through a biosensor and a UPLC-MS (Ultra Performance Liquid Chromatography-Mass Spectrometry) technology, then the pungent and warm components of the negundo chastetree prevention mixture are determined, and the property and flavor key quality attributes of the negundo chastetree prevention mixture are determined by comparing the medicinal components with the pungent and warm components. In addition, nobiletin in key quality attributes of properties and flavors of the negundo chastetree fruit and prevention mixture has good anti-influenza virus activity, and can be used for preparing drugs for treating anti-influenza A H1N1 viruses.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of traditional Chinese medicine analysis, and particularly relates to an application of a TRPC6 / TRPM4 molecular sensory artificial intelligence biosensor in identification of antiviral pungent and warm key quality attributes of Jingfang mixture. BACKGROUND

[0002] Critical quality attributes (CQAs) of a drug refer to physical, chemical, biological or microbiological properties or characteristics within appropriate limits, ranges or distributions to ensure the quality of a desired product. Nature and taste refer to the nature and smell of a drug, i.e. four natures and five tastes, which are the core part of the nature theory of traditional Chinese medicine. The nature and taste of traditional Chinese medicine are closely related to the efficacy. The existing quality control system of traditional Chinese medicine has the limitation of focusing on the quantitative analysis of single index component and lacking the systematic characterization of nature and taste characteristic component group. In addition, the traditional nature and taste evaluation relies on experience and fails to establish a quantifiable quality control index that is positively correlated with the efficacy, resulting in a serious disconnection between the nature theory and the modern quality control system. Associating the nature and taste of traditional Chinese medicine with the key quality attributes, identifying the nature and taste key quality attributes, establishing a quality control index based on the nature and taste, and returning to the “nature-taste-efficacy” evaluation system are conducive to the intelligent manufacturing upgrade of traditional Chinese medicine with the nature and taste determining the standard and the efficacy controlling the quality.

[0003] With the development of modern research, the discovery of nature and taste receptors provides a good breakthrough point for the identification of nature and taste key quality attributes of traditional Chinese medicine. Most pungent and warm traditional Chinese medicines can exert effects through TRPC6 / TRPM4 family ion channels, and the biological effects and pharmacological effects involving TRPC6 / TRPM4 family ion channels are highly related to the pungent and warm nature and taste of traditional Chinese medicine. The known TRP channels can be divided into seven subfamilies, including TRPA, TRPC, TRPV, TRPM, TRPP, TRPML and TRPN. Except TRPN, the other subfamilies are expressed in the human body and are involved in various complex physiological and pathological processes. For example, the transient receptor potential vanilloid 1 (TRPV1) can be activated by capsaicin in chili (pungent, hot), vanillin in ginger (pungent, slightly warm), imperatorin in Ba Zhi (pungent, warm) and temperature greater than 43℃, but TRPV1 is unstable after being activated and desensitization occurs, and after desensitization, it is not sensitive to various stimuli, thus having great potential in the treatment of pain. However, most of the existing identification methods of nature and taste key quality attributes are based on known nature and taste receptors, which cannot associate nature and taste components with efficacy indicators, and thus cannot identify nature and taste key quality attributes related to efficacy. SUMMARY

[0004] The application aims to provide an application of a TRPC6 / TRPM4 molecular sensory artificial intelligence biosensor in identification of antiviral pungent and warm key quality attributes of Jingfang mixture, so as to solve the problem that the existing identification method of nature and taste key quality attributes cannot identify the nature and taste key quality attributes related to drug efficacy.

[0005] To achieve the above-mentioned purpose, the application adopts the following technical scheme: The application of the TRPC6 / TRPM4 molecular sensory artificial intelligence biosensor in identification of antiviral pungent and warm key quality attributes of Jingfang mixture, wherein the method for identifying the antiviral pungent and warm key quality attributes of Jingfang mixture by the TRPC6 / TRPM4 molecular sensory artificial intelligence biosensor comprises the following steps: S01: screening TRPC6 / TRPM4 receptors with high expression and statistical difference in the lesion tissues of normal mice and diseased mice; wherein the diseased mice are infected with influenza A H1N1 virus.

[0006] Thirty-six SPF mice, 4-6 weeks old, weighing 19±1 g, were randomly divided into a normal control group, a model control group, a normal administration group and a model administration group, 6 mice in each group. After the mice were anesthetized with isoflurane, the mice in the model control group and the model administration group were respectively dripped with influenza A H1N1 virus H1N1 / PR8 strain, 20 μL per mouse, to replicate the mouse influenza virus infection model. The mice in the normal control group and the normal administration group were respectively dripped with the same amount of normal saline.

[0007] After 24 hours of infection, the normal administration group and the model administration group were respectively administered with 0.47 mL of Jingfang mixture, and the normal control group and the model control group were respectively administered with the same amount of ultrapure water every day, for 3 consecutive days. During this period, the macroscopic signs and body weight changes of the mice in each group were observed and recorded at regular time intervals every day. After 24 hours of the last administration, the mice were sacrificed by cervical dislocation, and the lesion tissues of normal mice, diseased mice and Jingfang mixture intervention mice in each group were collected after dissection. The relative expression levels of corresponding TRPC6 / TRPM4 receptor genes and proteins in the lesion tissues were detected based on real-time fluorescence quantitative PCR (RT-qPCR) and Western Blot, and statistical analysis was performed.

[0008] S02: constructing a TRPC6 / TRPM4-SPE functionalized biosensor by taking the TRPC6 / TRPM4 receptor as a biomolecular recognition element, and screening pungent and warm components in Jingfang mixture according to the TRPC6 / TRPM4-SPE functionalized biosensor and UPLC-MS / MS technology.

[0009] The 50 μL of 20 mmol / L 3-mercaptopropionic acid solution (3-MPA) is added dropwise on the surface of the screen-printed gold electrode (SPGE) to cover the working electrode. After the reaction at 4℃ for 17-24 h, the Au-S bond is generated on the surface of the SPGE device to form a carboxyl self-assembled monolayer. After the free 3-MPA on the surface of the SPGE device is washed away by ultrapure water, the 20 mmol / L of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 50 mmol / L of N-carboxy succinimide (NHS) are mixed in a volume ratio of 1:1 to form a mixed solution. The 100 μL of the mixed solution is added dropwise on the surface of the SPGE device to activate the reaction at room temperature for 15 min, so that the stable amine activation product is generated to activate the carboxyl group. The same TRPC6 / TRPM4 receptor standard substance as the TRPC6 / TRPM4 receptor is dissolved in a buffer solution to prepare a TRPC6 / TRPM4 protein solution. The surface of the SPGE device is cleaned by using the 10 mmol / L PBS buffer solution, and the 30 μL of the TRPC6 / TRPM4 protein solution with a concentration of 0.1 μg / μL is added dropwise. The TRPC6 / TRPM4 protein is modified to the surface of the SPGE device by a covalent bond after the reaction at 4℃ for 2-4 h, so that the TRPC6 / TRPM4-SPE functionalized biosensor is obtained. The prepared TRPC6 / TRPM4-SPE functionalized biosensor is connected with the adapter, and the chemical workstation, current signal receiving and processing system are integrated, so that the online detection can be performed.

[0010] After the jingfang mixture is configured into the 10-fold gradient concentration of the sample solution to be detected, the sample solution to be detected is added dropwise on the TRPC6 / TRPM4-SPE functionalized biosensor in the order of low to high concentration, and the reaction is performed at 4℃ for 5 min. The interaction intensity between the sample solution to be detected and the target protein is detected by using the electrochemical workstation. After the reaction is completed, the components not combined with the TRPC6 / TRPM4 receptor on the TRPC6 / TRPM4-SPE functionalized biosensor are eluted by using the PBS buffer solution, and then the specific eluent is used to elute the components combined with the TRPC6 / TRPM4 receptor for 6 times. The eluent is obtained. After the eluent is enriched by using the vacuum concentrator, the UPLC-MS / MS technology is used to identify the pungent and warm components in the jingfang mixture combined with the target protein.

[0011] The UPLC-MS / MS technology conditions are as follows: chromatographic conditions: Hypersil GOLD C 18Chromatographic column, column temperature 30 ℃, sample size 5 μL; mobile phase A is 0.05% formic acid aqueous solution, mobile phase B is 0.05% formic acid acetonitrile solution; flow rate is 0.3 mL / min, elution gradient is: 0 min, 97% A-3% B; 5 min, 80% A-20% B; 25 min, 70% A-30% B; 33 min, 55% A-45% B; 38 min, 0% A-100% B; 40 min, 0% A-100% B; 40.1 min, 97% A-3% B; 45 min, 97% A-3% B.

[0012] Mass spectrometry conditions: electrospray ion source, positive and negative ion mode switching detection, spray voltage is 3 kV; capillary temperature is 350 °C; sheath gas and auxiliary gas are nitrogen, flow rates are 10 mL / min and 3 mL / min respectively; scan mode: Full MS / ddMS2; Full MS resolution is 70000, dd-MS2 resolution is 17500; scan range: m / z 80-1200.

[0013] S03: Collect the drug-containing serum and target organs in mice containing the Jingfang mixture, and analyze the pharmacodynamic components of the Jingfang mixture entering the blood and target organs using the UPLC-MS / MS technique.

[0014] Twenty-four 4-6 week old, 19±1 g SPF mice were randomly divided into normal control group (n=3), model control group (n=3), normal drug administration group (n=9), and model drug administration group (n=9). According to literature and previous work, the corresponding mouse disease model was replicated. After 24 hours of infection, the normal drug administration group and the model drug administration group were given appropriate amounts of Jingfang mixture by gavage every 12 hours, and the normal control group and the model control group were given the same amount of ultrapure water by gavage, for 3 consecutive days. The mice were sacrificed by cervical dislocation at 0.5, 1, and 2 hours after the last administration, and the drug-containing serum and target organs were collected. The drug-containing serum and target organs were pretreated using the protein precipitation method (PPT), enriched using a nitrogen blowing instrument, and then dissolved in methanol and filtered through a 0.22 μm microporous filter to obtain the test solution. The pharmacodynamic components of the Jingfang mixture entering the blood and target organs were analyzed using the UPLC-MS / MS technique.

[0015] S04: Comparative analysis of the pungent and warm components and the pharmacodynamic components to screen the key quality attributes of pungent and warm components associated with pharmacodynamics.

[0016] The intersection of the pungent and warm components in the Jingfang mixture and the pharmacodynamic components entering the blood and target organs was screened using the microbioinformatics online platform, and a Venn diagram was drawn, which screened the key quality attributes of pungent and warm components associated with pharmacodynamics.

[0017] S05: verifying the efficacy of the screened nature and taste key quality attributes through in vitro cell experiments.

[0018] In addition, the nobiletin in the nature and taste key quality attributes of the Jingfang mixture has good anti-influenza virus activity, EC 50 = 6.32 μM, SI = 26.23, and can be used to prepare a medicine for treating anti-H1N1 influenza A virus.

[0019] The present application has the following beneficial effects: (1) In the present application, first, the TRPC6 / TRPM4 receptor with high expression and statistical difference is screened in the lesion tissues of normal mice and diseased mice, and the TRPC6 / TRPM4 receptor is used as a biomolecular recognition element to construct a TRPC6 / TRPM4-SPE functional biosensor, and the UPLC-MS / MS technology is combined to screen the pungent and warm ingredients in the Jingfang mixture. Then, the Jingfang mixture drug-containing serum and target organ are collected, and the UPLC-MS / MS technology is used to analyze the efficacy ingredients of the Jingfang mixture into the blood and target organ; further, the pungent and warm ingredients of the Jingfang mixture are compared and analyzed with the efficacy ingredients, and the nature and taste key quality attributes related to efficacy are screened. Finally, the efficacy of the nature and taste key quality attributes is verified based on in vitro cell experiments.

[0020] (2) The pungent and warm key quality attributes of the Jingfang mixture against H1N1 influenza A virus include peucedanin, 5-O-methylvisamminol glycoside, naringin, aurantiamarin, glycyrrhizic acid, nobiletin, and imperatorin. Seven key quality attributes related to nature and taste and efficacy are determined, the "nature and taste-efficacy" evaluation system is returned, and the quality control standard of the Jingfang mixture is further improved, which is conducive to realizing the intelligent manufacturing upgrade of traditional Chinese medicine "determined by nature and taste, and controlled by quality".

[0021] (3) The Jingfang mixture is a complex system with multiple components and multiple targets. The present application breaks through the limitations of traditional TRPV1 pungent receptor research, and the TRPC6 / TRPM4 double target combination is used to identify the anti-viral pungent and warm key quality attribute ingredients of the Jingfang mixture, which provides a new direction for the research on the material basis of pungent and warm substances.

[0022] (4) In the present application, it is first found that nobiletin has good anti-H1N1 influenza A virus effect, and can be used to prepare a medicine for treating H1N1 influenza A virus. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1Figure for detection of macroscopic pharmacodynamic indicators of the jingfang mixture intervention in the H1N1 influenza model; Wherein, A is the body weight change rate of each group of mice; B is the lung index of each group of mice; C is the lung viral load of each group of mice; D is the lung tissue morphological changes of each group of mice; E is the lung tissue pathological changes HE staining of each group of mice; Figure 2 Figure for real-time fluorescence quantitative PCR and Western blot detection of lung tissue key TRPC6 / TRPM4 receptor gene and protein relative expression levels after jingfang mixture intervention in the H1N1 influenza model; Wherein, A is the relative expression amount of TRPC6 / TRPM4 receptor mRNA in lung tissue, and a1 is the TRPC6 receptor, a2 is the TRPM4 receptor, a3 is the TRPM6 receptor, and a4 is the TRPV6 receptor; B is the key TRPC6 / TRPM4 receptor protein expression level in lung tissue, and b1, b2 are TRPC6 receptor, b1, b3 are TRPM4 receptor; Figure 3 Figure for the construction process of TRPC6 / TRPM4-SPE biosensor chip; Figure 4 Figure for the performance evaluation results of TRPC6 / TRPM4-SPE biosensor chip; Wherein, A is the performance evaluation results of TRPC6-SPE biosensor chip, a1 is stability; a2 is repeatability; a3 is precision; B is the performance evaluation results of TRPM4-SPE biosensor chip, b1 is stability; b2 is repeatability; b3 is precision; Figure 5 Figure for the interaction of jingfang mixture and TRPC6 / TRPM4 receptor; Wherein, A. Interaction of jingfang mixture and TRPC6 receptor. And a1 is I DS - V DS Signal change, a2 is the linear range, a3 is the K D ; B. Interaction of jingfang mixture and TRPM4 receptor, and b1 is I DS - V DS Signal change, b2 is the linear range, b3 is the K D ; Figure 6 TIC diagram of specific eluent for the interaction of jingfang mixture and TRPC6 / TRPM4; Figure 7Figure for verification research results of interaction of the Jingfang mixture's pungent and warm components with TRPC6 / TRPM4 receptors; wherein, A is the interaction of 5-O-methylvisamminol with TRPC6 receptors, and a1 is the signal change, a2 is the linear range, and a3 is the signal change in the linear range I DS - V DS signal change, b2 is the linear range, and b3 is the signal change in the linear range K D ; B is the interaction of 5-O-methylvisamminol with TRPM4 receptors, and b1 is the signal change, b2 is the linear range, and b3 is the signal change in the linear range I DS - V DS signal change, b2 is the linear range, and b3 is the signal change in the linear range K D ; Figure 8 Figure for total ion flow of blank serum and drug-containing serum of normal mice and influenza mice in positive and negative ion modes; Figure 9 Figure for total ion flow of blank target organs and drug-containing target organs of normal mice and influenza mice in positive and negative ion modes; Figure 10 Figure for intersection of TRPC6 / TRPM4 Jingfang mixture's pungent and warm components and blood-entering and target organ-entering pharmacodynamic components; Figure 11 Figure for experimental process of CCK8 method for determining cytotoxicity of CAQs of different natures and flavors; Figure 12 Figure for MDCK cell toxicity detection of Jingfang mixture's CAQs of different natures and flavors; Figure 13 Figure for experimental process of CCK8 method for determining antiviral effect of CAQs of different natures and flavors; Figure 14 Figure for inhibition effect detection of Jingfang mixture's CAQs of different natures and flavors on H1N1 virus. DETAILED DESCRIPTION

[0024] The technical solutions of the present application will be further explained and described below through specific examples.

[0025] Example 1: Screening of TRPC6 / TRPM4 receptors with high expression and statistical difference in lesion tissues of normal mice and diseased mice 1. Animal grouping and intervention Thirty-six SPF mice weighing 19±1g and aged 4-6 weeks were randomly divided into a normal control group (NC), an influenza model group (Virus), an oseltamivir phosphate positive group (OP, 19.5 mg / kg / day, equivalent to the clinical equivalent dose), and low-, medium-, and high-dose Jingfang mixture groups (JF, 7.80, 15.6, and 23.4 g / kg / day, equivalent to 1, 2, and 3 times the clinical equivalent dose), with 6 mice in each group. After acclimation for 3 days, the SPF mice were anesthetized with isoflurane on the 4th day. Anesthetized mice were intranasally instilled with 20 μL of normal saline solution in the NC group and an equivalent dose of influenza A (H1N1 / PR8) virus (1 TCID50) in the other groups to establish an influenza A (H1N1) virus infection model. Drug intervention was performed 24 hours after modeling. The NC group and the Virus group were gavaged with 0.3 mL of ultrapure water every morning and evening, and the OP group and the JF group were gavaged with equal volumes of oseltamivir phosphate and Jingfang mixture, respectively. The drugs were administered continuously for 7 days. During this period, the macroscopic signs and body weight changes of the mice in each group were observed and recorded regularly every day.

[0026] 2. Sample collection and pretreatment Twenty-four hours after the last dose, the mice's eyeballs were removed and blood was collected and stored in EP tubes for later use. Mice were sacrificed by cervical dislocation and dissected. The wet weights of the lungs, spleen, and thymus were weighed. Organ indices were calculated based on the mouse body weight: organ index = organ wet weight / mouse body weight × 100%.

[0027] 3. HE staining to observe lung pathological changes The left lungs of mice in each group were fixed in 4% paraformaldehyde, routinely dehydrated, permeabilized with xylene, embedded in paraffin, sliced, dried, and then HE stained. The lung tissue structure and pathological damage were observed under a microscope. Figure 1 .

[0028] By the attached Figure 1 It can be seen that compared with the normal control group mice NC, the weight of the influenza model group (Virus) mice decreased significantly, and the lung index and lung virus load increased significantly ( P <0.01), indicating that the mouse influenza A (H1N1) virus infection model was successfully replicated. The Jingfang mixture intervention can effectively slow down the trend of significant weight loss in influenza mice, significantly reduce the lung index and lung viral load, and significantly alleviate the degree of pathological damage in lung tissue.

[0029] 4. Immunoblotting to detect the expression levels of key TRPC6 / TRPM4 receptor proteins in lung tissue Take 20 mg of lung tissue from each group of mice respectively, add 200 μL of lysis solution, homogenate and process to obtain homogenate solution, wherein the lysis solution comprises RIPA lysis solution, protease inhibitor and phosphatase inhibitor in equal volume ratio. Lyse each group of homogenate solution in ice bath for 30 min to extract total protein, centrifuge at 12000 rpm for 15 min, and take the supernatant as the protein stock solution. Determine the protein concentration of the protein stock solution by BCA protein quantification kit, dilute to 30 μg / 10 μL, and add quantified 5x loading buffer and β-mercaptoethanol, and heat at 100 ℃ metal bath for 5 min to completely denature the protein, wherein the volume ratio of protein solution, loading buffer and β-mercaptoethanol is 15:4:1. Separate the protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transfer the protein band to a polyvinylidene fluoride (PVDF) membrane activated with methanol. Block the blank binding sites on the PVDF membrane with protein-free quick blocking solution; incubate the PVDF membrane with the corresponding β-actin, TRPC6 and TRPM4 primary antibodies at 4 ℃ overnight. Recover the primary antibody and wash with TBST, then incubate the PVDF membrane with horseradish peroxidase (HRP)-coupled secondary antibody at room temperature for 1 h; discard the secondary antibody and wash with TBST, then add ECL chemiluminescence reagent and image with a full-automatic chemiluminescence image analysis system; analyze the relative expression of the target protein by ImageJ image analysis software to obtain the relative expression of the target protein Figure 2 .

[0030] From the attached Figure 2 It can be seen that after the mice were infected with influenza A H1N1 virus, the expression levels of TRPC6 and TRPM4 genes and proteins were significantly reduced, and after the intervention of Jingfang Mixture, the expression levels of the genes and proteins tended to be normal in mice. Based on this, in the examples of the present application, TRPC64 and TRPM4 are used as biomolecular recognition elements to construct a TRPC6 / TRPM4-SPE biosensor, combined with UPLC-MS / MS technology, to identify the pungent and warm ingredients in Jingfang Mixture.

[0031] Example 2: Constructing a TRPC6 / TRPM4-SPE functional biosensor using TRPC6 / TRPM4 receptors as biomolecular recognition elements, and screening pungent and warm ingredients in Jingfang Mixture according to TRPC6 / TRPM4-SPE functional biosensor and UPLC-MS / MS technology 50 μL of a 20 mmol / L 3-mercaptopropionic acid solution (3-MPA) was dripped onto the surface of a screen-printed gold electrode (SPGE) to cover the working electrode. After reacting at 4°C for 17–24 h, Au-S bonds formed on the SPGE device surface, forming a carboxyl self-assembled monolayer. Free 3-MPA on the SPGE device surface was washed with ultrapure water. Then, 20 mmol / L 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 50 mmol / L N-carboxylsuccinimide (NHS) were mixed in equal volumes to form a mixed solution. 100 μL of this mixed solution was dripped onto the SPGE device surface for activation at room temperature for 15 min, generating a stable amine-activated product that was subsequently used to activate the carboxyl groups. TRPC6 and TRPM4 proteins purchased from MCE were dissolved in 0.22 μM buffer and stored at -80°C. Add 194.8 μL of 10 mmol·L containing 0.1% BSA to 20 μg TRPVC6 protein at a concentration of 3.88 mg / mL. -1 PBS buffer solution was diluted to a concentration of 0.1 μg / μL TRPC6 protein solution and divided for use. TRPM4 protein was diluted to a 0.1 μg / μL TRPM4 protein solution as above. The surface of the SPGE device was washed with 10 mmol / L PBS buffer, and 30 μL of a 0.1 μg / μL TRPC6 / TRPM4 protein solution was added dropwise. The reaction was carried out at 4°C for 2-4 hours. The TRPC6 / TRPM4 protein was covalently modified to the surface of the SPGE device to obtain a TRPC6 / TRPM4-SPE functionalized biosensor. Figure 3 The prepared TRPC6 / TRPM4-SPE functionalized biosensor is connected to an adapter and integrated with an electrochemical workstation and a current signal receiving and processing system for online detection.

[0032] In order to detect the stability of the TRPC6 / TRPM4-SPE biosensor chip, the electrochemical workstation was used to collect the Jingfang mixture sample solution on the 1st, 2nd, 3rd, 4th and 5th day after the successful construction of the TRPC6 / TRPM4-SPE biosensor chip. I DS - V DS Specifically, the same concentration of Jingfang mixture sample solution was divided into 6 parts, and the electrochemical workstation was used to collect the I DS - V DS The electrochemical workstation was used to collect the sample solution of Jingfang mixture six times. IDS - V DS Statistical comparison of the six detection signals was performed to evaluate the precision. Figure 4 The preparation of the Jingfang mixture sample solution includes: taking 10 ml of Jingfang mixture, centrifuging at 12000 rpm for 10 minutes, taking the supernatant, and obtaining a Jingfang mixture sample solution with an initial concentration of 1 g / mL. Diluting the mixture 16 times in a 10-fold concentration gradient to obtain a concentration of 10 -1 -10 -16 The sample solution of Jingfang mixture with a concentration of 0.5 g / mL (C1-C16) was stored in the dark at 4°C until use.

[0033] By the attached Figure 4 As can be seen, the current of the TRPC6 / TRPM4-SPE biosensor chip remained relatively stable within 5 days, with no significant performance changes. The chip exhibited excellent stability, allowing for sample testing during this period. Furthermore, the RSD values ​​for both repeatability and precision tests were less than 5%, demonstrating excellent repeatability and precision. This demonstrates that the TRPC6 / TRPM4-SPE biosensor chip has good performance and can be used in subsequent experiments.

[0034] After verifying the good performance of the TRPC6 / TRPM4-SPE biosensor chip, a 1 g / mL Jingfang mixture sample solution was added dropwise to the TRPC6 / TRPM4-SPE functionalized biosensor and allowed to react at 4°C for 5-10 minutes. After the reaction, components not bound to the TRPC6 / TRPM4 receptors on the TRPC6 / TRPM4-SPE functionalized biosensor were first eluted with PBS buffer. Components bound to the TRPC6 / TRPM4 receptors were then eluted six times using hyperforin, a specific eluent found to have a strong interaction with TRPC6 / TRPM4 receptors in the literature, to obtain an eluate. Phosphonates were removed from the eluate using solid-phase extraction (SPE) with methanol as the solvent. The solvent was then evaporated using a vacuum concentrator at 30°C and 1300 rpm / min to obtain a concentrated solution. The concentrate was re-dissolved in 80% methanol and filtered through a 0.22 μm microporous membrane. The filtrate was analyzed by UPLC-MS / MS technology to obtain the pungent and warm components in the Jingfang mixture. Figure 5 .

[0035] Among them, the UPLC-MS / MS technical conditions are: Chromatographic conditions: Hypersil GOLD C 18The chromatographic column was set at a temperature of 30 °C and the injection volume was 5 μL. The mobile phase A was 0.05% formic acid in water and the mobile phase B was 0.05% formic acid in acetonitrile. The flow rate was 0.3 mL / min and the elution gradient was as follows: 0 min, 97% A-3% B; 5 min, 80% A-20% B; 25 min, 70% A-30% B; 33 min, 55% A-45% B; 38 min, 0% A-100% B; 40 min, 0% A-100% B; 40.1 min, 97% A-3% B; 45 min, 97% A-3% B.

[0036] Mass spectrometry conditions: electrospray ion source, positive and negative ion mode switching detection, spray voltage of 3 kV; capillary temperature of 350°C; sheath gas and auxiliary gas were both nitrogen, with flow rates of 10 mL / min and 3 mL / min, respectively; scan mode: Full MS / ddMS2; Full MS resolution of 70,000, dd-MS2 resolution of 17,500; scan range: m / z 80-1200.

[0037] By the attached Figure 6 It can be seen that a total of 28 chemical components that interact with TRPC6 were analyzed from the specific eluates of the Jingfang mixture that interacted with TRPC6 and TRPM4, including: peucedanin, 5-O-methylvisaminol glycoside*, naringin*, hesperidone, peucedanin, hydrated oxypeucedanin, angelicol G, glycyrrhizic acid*, nobiletin*, hesperidin, imperatorin*, isocitric acid, umbelliferone, naringin, hesperidin*, neohesperidin*, 5-O-methylvisaminol, 6'-O-(trans-feruloyl)-peucedanin, epoxyhesperidin, isohesperidone, uralcone B, hydroxy-3-butenylphthalide, 3,5,6,7,8,3',4'-heptamethoxyflavone, peucedanin A, hesperidin, isoimperatorin*, and peucedanin A*; 21 chemical components that interact with TRPM4, mainly including peucedanin, 5-O-methylvisaminol glycoside*, naringin*, hesperidone, liquiritigenin*, glycyrrhizic acid*, nobiletin*, imperatorin, isocitric acid, naringin, hesperidin*, neohesperidin*, baicalin*, chelidonol glycoside*, luteolin*, trans-dehydroosthol, isohesperidone, saikosaponin A*, saikosaponin D*, isoimperatorin*, and peucedanin A*.

[0038] Based on the TRPC6 / TRPM4-SPE biosensor chip integrated with UPLC-MS / MS technology, the pungent and warm components of Jingfang mixture were identified and discovered. However, the interaction between the pungent and warm components obtained by fishing and the TRPC6 / TRPM4 receptors still needs further verification.

[0039] Specifically, 4.6 mg of 5-O-methylvisaminol glycoside and 6.0 mg of naringin were accurately weighed, 50 μL of DMSO was added to fully dissolve them, and then diluted to 10 mL with ultrapure water to obtain a concentration of 10 -3 mol / L 5-O-methylvisaminol glycoside sample solution (W1) and a concentration of 10 -3 mol / L naringin sample solution (Y1), and then diluted 11 times in a 10-fold concentration gradient to obtain a concentration of 10 -4 -10 -14 mol / L 5-O-methylvisamidoside sample solution (W2-W14) and 10 -4 -10 -14 mol / L naringin sample solutions (Y2-Y14). The same process as that used in the analysis of the pungent and warm components in Jingfang mixture was used to calculate the dissociation constants of the interaction between 5-O-methylvisaminol and naringin with TRPC6 and TRPM4, respectively ( K D ) value, proving that the fishing results are reliable and reliable. Figure 7 .

[0040] By the attached Figure 7 It can be seen that 10 -16 mol / L to 10 -5 mol / L 5-O-methylvisaminol glycosides have good I DS - V DS Signal changes, with TRPC6 at 10 -14 mol / L to 10 -9 There is a good linear relationship within the mol / L concentration range. K D The value is 4.16×10 -12 ; and TRPM4 in 10 -13 mol / L to 10 -9 There is a good linear relationship within the mol / L concentration range. K D The value is 1.60×10 -12 , the affinity strength is very strong, indicating that the fishing results are reliable.

[0041] Example 3: Collection of drug-containing serum and drug-containing target organs containing Jingfang mixture, and analysis of the pharmacological components in the drug-containing serum and drug-containing target organs using UPLC-MS / MS technology 1. Animal grouping and intervention Twenty-four SPF mice, 4-6 weeks old, weighing 19±1 g, were randomly divided into normal control group (n=3), model control group (n=3), normal administration group (n=9) and model administration group (n=9). After the mice were anesthetized with isoflurane, the mice in the model administration group were dropped with influenza A virus H1N1 / PR8 strain, 1TCID50, 20 μL per mouse, to form a mouse influenza virus infection model; the normal group of mice was dropped with the same amount of normal saline. After 24 h of infection, drug intervention was performed, wherein the normal administration group and the model administration group of mice were each given 0.47 mL of jingfang mixture by gavage every 12 h, 23.4 g / kg equivalent to 3 times the clinical equivalent dose; the normal control group and the model control group of mice were each given the same amount of ultrapure water by gavage, continuously for 3 d. At 0.5, 1 and 2 h after the last administration, 3 mice were taken from each group, the eyeballs were removed, and the blood was taken into centrifuge tubes. After standing at room temperature for 2 h, the blood serum was collected by centrifugation at 4°C, 4000 rpm·min -1 for 15 min, and the serum was collected. The serum of the same group of mice was combined and stored at -80°C for testing. The mice were sacrificed by cervical dislocation, and the lung tissue was dissected and stored in a centrifuge tube at -80°C for testing.

[0042] 2. Solution preparation Jingfang mixture test solution: 2.0 mL of jingfang mixture was accurately measured and placed in a 10.0 mL volumetric flask, and methanol was added to the mark. After ultrasonic treatment for 30 min, it was cooled; the volume was adjusted to the mark with methanol, shaken well, and left overnight. After filtration with a 0.45 μm microporous filter, the first 3 drops were discarded, and the filtrate was taken to obtain the jingfang mixture test solution, which was stored at 4°C in the dark for future use.

[0043] Serum test solution: 600 μL of serum from the normal control group and the model control group, and 800 μL of serum from the normal administration group and the model administration group were taken, respectively. Four times the amount of methanol was added to precipitate the protein, vortexed for 5 min, and centrifuged at 12000 rpm·min -1 for 10 min at 4°C. The supernatant was taken, dried under nitrogen at 37°C. 240 μL of 80% methanol was added to the residue for redissolution, vortexed for 5 min, and centrifuged again for 10 min after instantaneous separation. The supernatant was taken, filtered with a 0.22 μm microporous filter, the first 3 drops were discarded, and the filtrate was taken to obtain the serum test solution of each group.

[0044] Lung tissue test solution: lung tissue samples from each group were taken, rinsed with pre-cooled normal saline, and the water was absorbed with filter paper. The lung tissue samples were weighed and mixed with normal saline at a mass-volume ratio of 1:2, and homogenized. After centrifugation at 12000 rpm·min -1 for 10 min at 4°C, the supernatant was taken. All the homogenate supernatants of the same group were combined, 4 times the amount of methanol was added to precipitate the protein, vortexed for 5 min, and centrifuged at 12000 rpm·min -1Centrifugation 10 min, take supernatant, 37℃ nitrogen blow dry. Residue is added to 240 μL 80% methanol, vortex 5 min, centrifugation 10 min again, take supernatant, 0.22 μm microporous filter membrane filtration, the first 3 drops are discarded, take the filtered solution, get each group of lung tissue test solution.

[0045] Control solution: take control sample menthofuran, chlorogenic acid, neochlorogenic acid, cimifugin, cimifugin glycoside, 5-O-methylvisamminol glycoside, huairenol, qianghuai alcohol, imperatorin, osthole, dihydroimperatorin angelica acid ester, zanthotoxin, psoralen, prunella glycoside, ferulic acid, ligustilide, saikosaponin A, saikosaponin D, bungeanin, prunella A, prunella B, anisatin, naringenin, naringin, neohesperidin, coffee acid, glycyrrhizin, glycyrrhizic acid, glycyrrhetic acid, glycyrrhizol A, daidzin, genistein, isofraxidin, quercetin, luteolin, isorhamnetin control, accurately weighed, respectively, in a 10 mL volumetric flask, dissolved with 80% methanol and diluted to the mark, shake, get each control solution. Take each control solution 200 μL in a 10 mL volumetric flask, and re-diluted to the mark with 80% methanol, shake, get mixed control solution, 4℃ light protection, ready for use.

[0046] 3. Component characterization of Jingfang mixture in drug-containing serum UPLC-MS / MS technology was used to detect each serum test solution, and the total ion chromatogram of each group in positive and negative ion mode was obtained, as shown in Figure Figure 8 Among them, the components that exist in Jingfang mixture and drug serum samples but not in control serum samples are considered to be potential Jingfang mixture efficacy substances.

[0047] As shown in Figure Figure 7 It can be seen that by comparing the above detection results with the control solution and combining with the relevant literature reports, 20 prototype components are identified in the normal Jingfang mixture drug serum, and 35 prototype components are identified in the model drug serum, and the component characterization of Jingfang mixture in drug-containing serum is obtained, as shown in Table 1.

[0048] Table 1: Component characterization of Jingfang mixture into blood Note: A. Schizonepeta; B. Qianhuang; C. Qianghuang; D. Duhuo; E. Radix Bupleuri; F. Radix Peucedani; G. Chuanxiong; H. Fructus Aurantii; I. Poria cocos; J. Platycodon; K. Glycyrrhiza.

[0049] 4. Characterization of the components of Jingfang mixture in drug-containing target organs The lung tissue test solution of each group of mice was detected by UPLC-MS / MS technology, and the total ion current of each group in positive and negative ion modes was obtained, as shown in the attached figure. Figure 9 Among them, the components that exist in the Jingfang mixture solution and the administered serum samples but not in the control serum samples are considered to be potential active substances of the Jingfang mixture.

[0050] By the attached Figure 9 It can be seen that by comparing the above test results with the reference solution and combining with relevant literature reports, a total of 6 prototype components were identified in the test solution of normal lung tissue, and a total of 29 prototype components were identified in the test solution of model lung tissue, and the component characterization of Jingfang mixture in the drug-containing target organ was obtained, as shown in Table 2.

[0051] Table 2: Characterization of target organ components of Jingfang mixture Note: A. Schizonepeta tenuifolia; B. Saposhnikovia divaricata; C. Notopterygium wilfordii; D. Angelica dahurica; E. Bupleurum chinense; F. Peucedanum peucedanum; G. Ligusticum chuanxiong; H. Citrus aurantium; I. Poria; J. Platycodon grandiflorum; K. Glycyrrhiza uralensis.

[0052] Example 4: Comparative analysis of pungent and warm components and medicinal components to screen the key quality attributes of the properties and taste of Jingfang mixture associated with medicinal efficacy.

[0053] As attached Figure 10 As shown in the figure, by using the Weishengxin online platform, the intersection of the pungent and warm components targeting the target TRPC6 / TRPM4 protein and the pharmacological components entering the blood and target organs was taken and a Venn diagram was drawn, and 7 pungent and warm key quality attributes associated with each pharmacodynamic were screened out: peucedanum rutin, 5-O-methylvisamidoside, naringin, hesperidone, glycyrrhizic acid, nobiletin, and imperatorin.

[0054] Example 5: Drug efficacy detection 1. MDCK cell culture MDCK (Madin-Darby Canine Kidney cells) were cultured in DMEM high glucose basal medium containing 10% FBS (Fetal Bovine Serum) and 1% P / S (Penicillin-streptomycin) at 37°C in a 5% CO2 incubator, and the medium was changed every other day. The logarithmic growth phase MDCK cells were used for experiments. The MDCK cell maintenance medium was a virus isolation serum-free medium containing 1.5 µg / mL TPCK trypsin.

[0055] 2. Virus infection titer determination Logarithmic growth phase MDCK cells were inoculated in a 96-well plate at a seeding rate of 5×10 4 cells / mL, 100 μL per well, 6 replicates, and placed in a 37°C, 5% CO2 incubator for about 12 h of pre-culture. The H1N1 / PR8 virus solution was diluted to 10 -1 , 10 -2 , 10 -3 , … 10 -9 series of concentrations using the cell maintenance medium. When the cell confluence reached about 50%, the 96-well plate was removed, the medium was discarded and washed twice with PBS. The control group was added with 100 μL of MDCK cell maintenance medium, and the virus group was added with 100 μL of virus solution at different concentrations. The culture was incubated in a 35°C, 5% CO2 incubator, and the cytopathic effect (CPE) was observed daily. When no CPE was observed in the wells with the lowest dilution of H1N1 / PR8 virus solution under a microscope, the number of wells with CPE at each dilution was recorded. The H1N1 / PR8 virus titer was calculated according to the Reed-Muench method. TCID 50 The virus titer was 10 -5.34 / 100 μL.

[0056] 3. CCK8 method for determining the cytotoxicity of flavor CAQs As shown in the attached Figure 11 , logarithmic growth phase MDCK cells were inoculated in a 96-well plate at a seeding rate of 1×10 5The cells / mL inoculation rate was inoculated into a 96-well plate, 100 μL per well, 6 replicates, and pre-cultured in a 37°C, 5% CO2 constant temperature incubator for 12-24 hours. When the cell confluence reached 80-90%, the 96-well plate was removed, the culture medium was discarded, and the cells were rinsed twice with PBS. 100 μL of MDCK cell maintenance medium was added to each well of the blank group and the control group, and 100 μL of drugs of different concentrations were added to each well of the drug-treated group. The cells were placed in a 37°C, 5% CO2 incubator for further culture for 48 hours. The 96-well plate was removed, the culture medium was discarded, and the cells were rinsed twice with PBS. 110 μL of DMEM high-glucose basal medium containing 10% CCK-8 was added to each well in the form of a replacement medium. The cells were placed in a 37°C, 5% CO2 incubator for further incubation for 1 hour. At this time, the OD value of the control group was 1.0-1.5. The absorbance OD at 450 nm was measured with a microplate reader to calculate the cell survival rate and obtain the attached cell viability. Figure 11 The highest concentration with a cell survival rate of ≥90% was considered the maximum non-toxic concentration of the flavor CAQs on MDCK cells. CC 0, and calculate the median toxic concentration CC 50 Cell viability = [(OD of the drug-treated well - OD of the blank well) / (OD of the control well - OD of the blank well)] × 100%.

[0057] By the attached Figure 12 It can be seen that the maximum non-toxic concentrations CC0 of the CAQs of nature and flavor in MDCK cells were as follows: CC0 of peucedanum rutin was 1000 μM, CC0 of 5-O-methylvisaminol glycoside was 4000 μM, CC0 of naringin was 400 μM, CC0 of hesperidin was 200 μM, CC0 of naringin was 300 μM, CC0 of naringin was 1000 μM, CC0 of naringin was 2 ... 50 is 383.3 μM, CC0 of glycyrrhizic acid is 125 μM, CC 50 is 229.7 μM, CC0 of nobiletin is 100 μM, CC 50 was 165.8 μM, the CC0 of imperatorin was 12.5 μM, and the CC 50 is 137.4 μM.

[0058] 4. CCK8 assay to determine the antiviral effects of nature and flavor CAQs As attached Figure 13 As shown, MDCK cells in logarithmic growth phase were cultured at a rate of 1×10 5 Cells / mL were inoculated into 96-well plates, 100 μL per well, and 6 replicates were placed in a 37°C, 5% CO2 constant temperature incubator for pre-incubation for 16-24 hours. When the cell confluence reached 80-90%, the 96-well plate was removed, the culture medium was discarded, and the plate was rinsed twice with PBS. 100 μL of MDCK cell maintenance medium was added to each well of the control group, and 100 μL of 100 cells / mL was added to each well of the virus group.TCID 50 Virus liquid, 50 μL of 200 TCID 50 Virus liquid and 50 μL of different concentrations of drugs, placed in a 37℃, 5% CO2 incubator for continuous culture for 48h, then take out the 96-well plate, discard the culture medium and rinse twice with PBS, in the form of replacement liquid, add 110 μL of DMEM high glucose basic culture medium containing 10% CCK-8 per well, placed in a 37℃, 5% CO2 incubator for continuous incubation for 1h, at this time, the OD value of the control group is 1.0-1.5, the absorbance OD at 450nm is measured by the enzyme marker, the virus inhibition rate and the half inhibitory concentration are calculated EC 50 and the therapeutic index SI , get the attached Figure 13 Among them, the virus inhibition rate = [(virus well OD-drug well OD) / (virus well OD-control well OD)] x 100%, SI = CC 50 / EC 50 .

[0059] From the attached Figure 14 It can be seen that the positive drug baloxavir has the strongest anti-H1N1 virus activity, EC50=2.48nM, SI=40.32; the key quality attribute component of xiaochenpi is good in anti-virus activity, EC 50 =6.32 μM, SI=26.23.

[0060] The above only describes the preferred embodiments of the present application and is not used to limit the present application. For those skilled in the art, the present application can have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Application of TRPC6 / TRPM4 molecular sensory artificial intelligence biosensor in the identification of key quality attributes of antiviral pungent and warm properties of Jingfang mixture.

2. The use according to claim 1, characterized in that The method for the TRPC6 / TRPM4 molecular sensory artificial intelligence biosensor to identify the key quality attributes of the antiviral pungent and warm Jingfang mixture includes: Screening for highly expressed TRPC6 / TRPM4 receptors with statistically significant differences in lesion tissues of normal mice and infected mice; wherein the infected mice are infected with influenza A (H1N1) virus; A TRPC6 / TRPM4-SPE functionalized biosensor was constructed using the TRPC6 / TRPM4 receptor as a biomolecular recognition element, and the pungent and warm components in the Jingfang mixture were screened using the TRPC6 / TRPM4-SPE functionalized biosensor combined with UPLC-MS / MS technology; collecting drug-containing serum and drug-containing target organs of the Jingfang mixture from mice, and using the UPLC-MS / MS technology to analyze the pharmacological components of the Jingfang mixture that have entered the blood and target organs; Comparative analysis of the pungent and warm components and the medicinal ingredients to screen the key quality attributes of nature and taste associated with the medicinal efficacy of the Jingfang mixture; The efficacy of the screened key quality attributes of nature and taste was verified through in vitro cell experiments.

3. The use according to claim 2, characterized in that The key quality attributes of the nature and taste of the Jingfang mixture include peucedanin, 5-O-methylvisamin glycoside, naringin, hesperidin, glycyrrhizic acid, nobiletin, and imperatorin.

4. The use according to claim 3, characterized in that The nobiletin is used for preparing medicine for treating influenza A (H1N1) virus.

5. The use according to claim 2, characterized in that The TRPC6 / TRPM4 receptors that are highly expressed and statistically significantly different in the lesion tissues of normal mice and infected mice include: The corresponding disease model was established using SPF mice, and lesion tissues of normal mice, infected mice, and mice treated with Jingfang mixture were collected; Based on real-time fluorescence quantitative PCR and protein immunoblotting, the relative expression levels of corresponding TRPC6 / TRPM4 receptor genes and proteins in the lesion tissues of normal mice, infected mice and mice treated with Jingfang mixture were detected and statistical analysis was performed.

6. The use according to claim 2, characterized in that The drug-containing serum and drug-containing target organs of the Jingfang mixture are collected, and the pharmacological components of the Jingfang mixture entering the blood and target organs are analyzed using the UPLC-MS / MS technology, including: The corresponding disease model was established in SPF mice. Drug intervention was carried out 24 hours after infection. Drug-containing serum and drug-containing target organs of normal and infected mice were collected. The drug-containing serum and drug-containing target organs were pretreated by protein precipitation method, enriched by nitrogen blowdown instrument, re-dissolved in methanol and filtered through 0.22μm microporous membrane to obtain the test solution; The UPLC-MS / MS technology is used to analyze the pharmacological components of the Jingfang mixture that enter the blood and target organs.

7. The use according to claim 2, characterized in that Comparative analysis of the pungent and warm ingredients and the medicinal ingredients to screen for the key quality attributes of properties and flavors associated with medicinal effects include: The intersection of the pungent and warm components and the medicinal ingredients was taken using the Weishengxin online platform and a Venn diagram was drawn to screen out the pungent and warm key quality attributes.

Images