Application of TRPC6 / TRPM4 molecular sensory artificial intelligence biosensor in identification of antiviral and pungent key quality attributes of jingfang mixture

Abstract

The application provides an application of a TRPC6 / TRPM4 molecular sensory artificial intelligence biosensor in identification of antiviral pungent and warm key quality attributes of Jingfang mixture, and belongs to the technical field of traditional Chinese medicine analysis. In the application, the efficacy components of Jingfang mixture entering blood and target organs are determined through molecular biology, the TRPC6 / TRPM4 double-target receptor is determined through a biosensor and UPLC-MS technology, and then the pungent and warm components of Jingfang mixture are determined, and the pungent and warm key quality attributes of Jingfang mixture are determined by comparing the efficacy components and the pungent and warm components. In addition, nobiletin in the pungent and warm key quality attributes of Jingfang mixture has good anti-influenza virus activity, and can be used for preparing a medicine for treating anti-influenza A H1N1 virus.

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 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 nature and taste, and returning to the “nature-taste-efficacy” evaluation system are conducive to the intelligent manufacturing upgrade of traditional Chinese medicine with “nature-taste-determined standard and efficacy-controlled 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 and hot), vanillin in ginger (pungent and slightly warm), imperatorin in Ba Zhi (pungent and warm), and temperature greater than 43℃. However, TRPV1 is unstable after being activated and will appear desensitization, and after desensitization, it is not sensitive to various stimuli, so it has 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 solutions.

[0006] 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:

[0007] 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 the influenza A H1N1 virus.

[0008] 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 20 μL of the influenza A H1N1 virus H1N1 / PR8 strain per mouse, thereby replicating 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.

[0009] 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 the normal mice, the diseased mice and the Jingfang mixture intervention mice in each group were collected after dissection. The relative expression levels of the 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.

[0010] S02: constructing a TRPC6 / TRPM4-SPE functionalized biosensor by taking the TRPC6 / TRPM4 receptor as a biomolecular recognition element, and screening the pungent and warm components in Jingfang mixture according to the TRPC6 / TRPM4-SPE functionalized biosensor and UPLC-MS / MS technology.

[0011] 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.

[0012] 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.

[0013] 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.

[0014] 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.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] S05: verifying the efficacy of the screened nature and taste key quality attributes through in vitro cell experiments.

[0020] 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 drug for treating anti-H1N1 influenza A virus.

[0021] The present application has the following beneficial effects:

[0022] (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 pungent and warm components in the Jingfang mixture are screened by combining the UPLC-MS / MS technology. Then, the Jingfang mixture drug-containing serum and target organ are collected, and the efficacy components of the Jingfang mixture into the blood and target organ are analyzed based on the UPLC-MS / MS technology; further, the pungent and warm components of the Jingfang mixture are compared and analyzed with the efficacy components, 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.

[0023] (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, the quality control standard of the Jingfang mixture is further improved, and the intelligent manufacturing upgrade of traditional Chinese medicine is realized.

[0024] (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 components of the Jingfang mixture, which can be significantly adjusted by the Jingfang mixture with significantly reduced gene and protein expression levels in the lesion tissues of diseased mice, and the Jingfang mixture is a typical pungent and warm formula. The present application provides a new direction for the research on the material basis of pungent and warm substances.

[0025] (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 drug for treating H1N1 influenza A virus. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1The macroscopic pharmacodynamic index detection chart of the jingfang mixture intervention H1N1 influenza model; among them, A is the weight change rate of each group of mice; B is the lung index of each group of mice; C is the lung virus load of each group of mice; D is the lung tissue morphological change of each group of mice; E is the lung tissue pathological change HE staining chart of each group of mice;

[0027] Figure 2 After the jingfang mixture intervened in the H1N1 influenza model, the relative expression level chart of key TRPC6 / TRPM4 receptor genes and proteins in lung tissue was detected by real-time fluorescence quantitative PCR and Western blot; among them, A is the relative expression amount of TRPC6 / TRPM4 receptor mRNA in lung tissue, and a1 is TRPC6 receptor, a2 is TRPM4 receptor, a3 is TRPM6 receptor, and a4 is TRPV6 receptor; B is the expression level of key TRPC6 / TRPM4 receptor protein in lung tissue, and b1, b2 are TRPC6 receptor, and b1, b3 are TRPM4 receptor;

[0028] Figure 3 It is the process chart of TRPC6 / TRPM4-SPE biosensor chip construction;

[0029] Figure 4 It is the performance test result chart of TRPC6 / TRPM4-SPE biosensor chip; among them, A is the performance test result of TRPC6-SPE biosensor chip, a1 is stability; a2 is repeatability; a3 is precision; B is the performance test result of TRPM4-SPE biosensor chip, b1 is stability; b2 is repeatability; b3 is precision;

[0030] Figure 5 It is the interaction chart of jingfang mixture and TRPC6 / TRPM4 receptor; among them, A. The interaction of jingfang mixture and TRPC6 receptor. And a1 is I DS - V DS Signal change, a2 is linear range, a3 is K D ; B. The interaction of jingfang mixture and TRPM4 receptor, and b1 is I DS - V DS Signal change, b2 is linear range, b3 is K D ;

[0031] Figure 6 It is the TIC chart of the specificity eluent of the interaction of jingfang mixture and TRPC6 / TRPM4;

[0032] Figure 7 The results of the verification study of the interaction of the pungent and warm components of Jingfang mixture with TRPC6 / TRPM4 receptors are shown in the figure; wherein, A is the interaction of 5-O-methylvisamminol with TRPC6 receptor, 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 receptor, 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 ;

[0033] Figure 8 The total ion flow graphs of blank serum and drug-containing serum of normal mice and influenza mice in positive and negative ion modes are shown in the figure;

[0034] Figure 9 The total ion flow graphs of blank target organs and drug-containing target organs of normal mice and influenza mice in positive and negative ion modes are shown in the figure;

[0035] Figure 10 The intersection graph of the pungent and warm components of Jingfang mixture and the blood and target organ efficacy components of TRPC6 / TRPM4 is shown in the figure;

[0036] Figure 11 The experimental flowchart of CCK8 method for determining the cytotoxicity of CAQs of different natures and flavors is shown in the figure;

[0037] Figure 12 The cytotoxicity detection graph of Jingfang mixture CAQs of different natures and flavors on MDCK cells is shown in the figure;

[0038] Figure 13 The experimental flowchart of CCK8 method for determining the antiviral effect of CAQs of different natures and flavors is shown in the figure;

[0039] Figure 14 The inhibition detection graph of Jingfang mixture CAQs of different natures and flavors on H1N1 virus is shown in the figure. DETAILED DESCRIPTION

[0040] The technical solutions of the present application will be further explained and described below through specific examples.

[0041] Example 1: Screening of TRPC6 / TRPM4 receptors with high expression and statistical difference in the lesion tissues of normal mice and diseased mice

[0042] 1. Animal grouping and intervention

[0043] Thirty-six SPF-grade mice, weighing 19±1g and aged 4-6 weeks, were randomly divided into three groups: a normal control group (NC), an influenza model group (Virus), an oseltamivir phosphate positive control group (OP, 19.5 mg / kg / d equivalent to the clinically equivalent dose), and low, medium, and high dose groups of Jingfang compound (JF, 7.80, 15.6, and 23.4 g / kg / d equivalent to 1, 2, and 3 times the clinically equivalent dose), with six mice in each group. After acclimatization for 3 days, the SPF-grade mice were anesthetized with isoflurane on day 4. For the anesthetized mice, the NC group received 20 μL of physiological saline intranasally, while the other groups received an equal amount of influenza A H1N1 / PR8 strain (1 TCID50) intranasally to establish a mouse model of influenza A H1N1 virus infection. Twenty-four hours after modeling, the mice were given medication. The NC and Virus groups were given 0.3 mL of ultrapure water by gavage twice a day, morning and evening. The OP and JF groups were given an equal volume of oseltamivir phosphate and Jingfang compound by gavage, respectively. The medication was administered for 7 consecutive days. During this period, the macroscopic signs and weight changes of the mice in each group were observed and recorded at regular intervals.

[0044] 2. Sample collection and preprocessing

[0045] Twenty-four hours after the last administration, blood was collected from the eyes of mice by enucleation and stored in EP tubes for later use. Mice were euthanized by cervical dislocation and dissected to obtain tissue samples. The wet weight of the lungs, spleen, and thymus was measured, and the organ index was calculated based on the mouse's body weight. The organ index was calculated as follows: organ wet weight / mouse body weight × 100%.

[0046] 3. HE staining to observe pathological changes in the lungs

[0047] The left lungs of mice in each group were fixed in 4% paraformaldehyde, routinely dehydrated, permeabilized with xylene, embedded in paraffin, sectioned, dried, and then stained with hematoxylin and eosin (HE). The lung tissue structure and pathological damage were observed under a microscope. Figure 1 .

[0048] From the appendix Figure 1 As can be seen, compared with the normal control group mice (NC), the influenza model group (Virus) mice showed a significant decrease in body weight, and a significant increase in lung index and viral load in the lungs. P The result was <0.01, indicating that the mouse model of H1N1 influenza A virus infection was successfully replicated. The intervention of Jingfang compound can effectively slow down the trend of significant weight loss in influenza mice, significantly reduce the lung index and viral load in the lungs, and significantly reduce the degree of pathological damage to lung tissue.

[0049] 4. Immunoblotting detection of expression levels of key TRPC6 / TRPM4 receptor proteins in lung tissue

[0050] 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 .

[0051] 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 the expression levels of the genes and proteins tended to be normal after the intervention of Jingfang Mixture. 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 components in Jingfang Mixture.

[0052] Example 2: Constructing a TRPC6 / TRPM4-SPE functional biosensor using TRPC6 / TRPM4 receptors as biomolecular recognition elements, and screening pungent and warm components in Jingfang Mixture according to TRPC6 / TRPM4-SPE functional biosensor and UPLC-MS / MS technology

[0053] 50 μL of a 20 mmol / L solution of 3-mercaptopropionic acid (3-MPA) was dropped onto the surface of a screen-printed gold electrode (SPGE) and covered the working electrode. After reacting at 4 °C for 17–24 h, Au-S bonds were formed on the SPGE device surface, creating a carboxyl self-assembled monolayer. After washing away the free 3-MPA on the SPGE device surface with ultrapure water, a mixed solution was prepared by mixing 20 mmol / L 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 50 mmol / L N-carboxysuccinimide (NHS) in an equal volume ratio. 100 μL of this mixed solution was added dropwise to the SPGE device surface for activation at room temperature for 15 min, generating a stable amine activation product, which was then used to activate the carboxyl groups. TRPC6 and TRPM4 proteins (purchased from MCE) were dissolved in 0.22 μM buffer solution and stored at -80 °C. Add 194.8 μL of 10 mmol·L⁻¹ solution containing 0.1% BSA to 20 μg TRPVC6 protein at a concentration of 3.88 mg / mL. -1 PBS buffer was diluted to a concentration of 0.1 μg / μL for TRPC6 protein, and aliquots were prepared for use. TRPM4 protein was similarly diluted to a concentration of 0.1 μg / μL. 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. The reaction was carried out at 4°C for 2–4 hours. The TRPC6 / TRPM4 protein was covalently modified onto the surface of the SPGE device, resulting in a TRPC6 / TRPM4-SPE functionalized biosensor, as shown in the attached figure. Figure 3 As shown. The prepared TRPC6 / TRPM4-SPE functionalized biosensor is connected to an adapter and integrated with an electrochemical workstation, current signal receiving and processing system, enabling online detection.

[0054] To assess the stability of the TRPC6 / TRPM4-SPE biosensor chip, electrochemical workstations were used to collect samples of the Jingfang compound solution on days 1, 2, 3, 4, and 5 after successful construction of the TRPC6 / TRPM4-SPE biosensor chip. I DS - V DS Specifically, after dividing the same concentration of the Jingfang compound sample solution into 6 equal portions, an electrochemical workstation was used to collect data from each Jingfang compound sample solution. I DS - V DS The detection information was statistically compared to evaluate its repeatability. The Jingfang compound sample solution was collected six times using an electrochemical workstation. IDS - V DS The precision of the six detection signals was evaluated by statistically comparing the differences. Figure 4 The preparation of the Jingfang compound sample solution includes: taking 10 ml of Jingfang compound, centrifuging at 12000 rpm for 10 min, and collecting the supernatant to obtain a Jingfang compound sample solution with an initial concentration of 1 g / mL. This solution is then diluted 16 times in a 10-fold gradient to obtain a concentration of 10... -1 -10 -16 A sample solution of Jingfang compound at g / mL (C1-C16) should be stored at 4℃ protected from light for later use.

[0055] From the appendix Figure 4 As can be seen, the TRPC6 / TRPM4-SPE biosensor chip exhibited relatively stable current and no significant performance changes within 5 days of placement, demonstrating good stability and allowing for sample detection during this period. Furthermore, the repeatability and precision test results showed RSD values ​​of less than 5%, indicating good repeatability and precision. This demonstrates that the TRPC6 / TRPM4-SPE biosensor chip possesses excellent performance and can be used for subsequent experiments.

[0056] After confirming the good performance of the TRPC6 / TRPM4-SPE biosensor chip, a 1 g / mL solution of Jingfang compound was added dropwise to the TRPC6 / TRPM4-SPE functionalized biosensor, and the reaction was carried out at 4℃ for 5-10 min. After the reaction, the components that did not bind to the TRPC6 / TRPM4 receptor on the TRPC6 / TRPM4-SPE functionalized biosensor were first eluted with PBS buffer. Then, the components that bound to the TRPC6 / TRPM4 receptor were eluted six times with hypericin, which was found to have a strong interaction with the TRPC6 / TRPM4 receptor in literature research, to obtain the eluent. The phosphonate components in the eluent were washed away by solid-phase extraction with methanol as solvent. The solvent was then evaporated using a vacuum concentrator at 30℃ and 1300 rpm / min to obtain the concentrate. The concentrated solution was redissolved in 80% methanol and then filtered through a 0.22 μm microporous membrane. The filtrate was analyzed using UPLC-MS / MS to determine the pungent and warming components in the Jingfang compound, yielding the attached... Figure 5 .

[0057] The UPLC-MS / MS technical conditions were 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.

[0058] 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; scanning mode: Full MS / ddMS2; Full MS resolution is 70000, dd-MS2 resolution is 17500; scanning range: m / z 80-1200.

[0059] By the attached Figure 6 It can be seen that 28 chemical components interacting with TRPC6 are co-eluted from the specific eluent of Jingfang mixture interacting with TRPC6 and TRPM4, mainly including: nodakenin, 5-O-methylvisamminol glycoside, naringin, auraptene, nodakenin, hydrate oxypeucedanin, angelica alcohol G, glycyrrhizic acid, nobiletin, tangeritin, imperatorin, isocitric acid, umbelliferone, aurantiamarin, hesperidin, neohesperidin, 5-O-methylvisamminol, 6'-O-(trans-feruloyl)-nodakenin, epoxyaurapten, isoaurapten, Uralsaponin B, hydroxy-3-butenylphthalide, 3,5,6,7,8,3',4'-heptamethoxyflavone, peucedanol A, tangeritin, isoimperatorin, praeparil A; 21 chemical components interacting with TRPM4, mainly including nodakenin, 5-O-methylvisamminol glycoside, naringin, auraptene, glycyrrhizin, glycyrrhizic acid, nobiletin, imperatorin, isocitric acid, aurantiamarin, hesperidin, neohesperidin, baicalin, haisoaloside, luteolin, trans-dehydroosthol, isoaurapten, saikosaponin A, saikosaponin D, isoimperatorin, praeparil A.

[0060] Based on TRPC6 / TRPM4-SPE biosensor chip integrated UPLC-MS / MS technology, the pungent components of Jingfang mixture were identified, but the interaction of the pungent components obtained by fishing with TRPC6 / TRPM4 receptors still needs to be further verified.

[0061] Specifically: Accurately weigh 4.6 mg of 5-O-methylvisamidol glycoside and 6.0 mg of naringin, add 50 μL of DMSO to dissolve completely, and then dilute with ultrapure water to 10 mL to obtain a concentration of 10. -3 5-O-methylvisamidol glycoside sample solution (W1) and 10 mol / L 5-O-methylvisamidol glycoside sample solution (W1) -3 A sample solution of naringin (Y1) was prepared at a concentration of mol / L and then diluted 11 times in a 10-fold gradient to obtain a concentration of 10. -4 -10 -14 5-O-methylvisamidol glycoside sample solutions (W2-W14) and 10 mol / L -4 -10 -14 A sample solution of naringin (Y2-Y14) at mol / L was used. The dissociation constants of the interactions between 5-O-methylvisamidol and naringin with TRPC6 and TRPM4 were calculated using the same procedure as that used in the UPLC-MS / MS analysis of the pungent and warming components in Jingfang compound. K D The value proves that the fishing results are indeed reliable and credible, and the attached value is obtained. Figure 7 .

[0062] From the appendix Figure 7 It can be seen that 10 -16 mol / L to 10 -5 Both mol / L 5-O-methylvisamidolol and the TRPC6 / TRPM4-SPE biosensor showed good performance. I DS - V DS Signal changes, with TRPC6 at 10 -14 mol / L to 10 -9 There is a good linear relationship within the concentration range of mol / L. K D The value is 4.16 × 10 -12 ; with TRPM4 in 10 -13 mol / L to 10 -9 There is a good linear relationship within the concentration range of mol / L. K D The value is 1.60 × 10 -12 Both showed strong affinity, indicating that the fishing results were reliable.

[0063] Example 3: Collect drug-containing serum and drug-containing target organs containing Jingfang compound, and analyze the pharmacodynamic components in the drug-containing serum and drug-containing target organs using UPLC-MS / MS technology.

[0064] 1. Animal grouping and intervention

[0065] 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 and placed at room temperature for 2 h. After centrifugation at 4°C, 4000 rpm·min -1 for 15 min, the serum was collected, and 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 placed in a centrifuge tube for storage at -80°C for testing.

[0066] 2. Solution preparation

[0067] Jingfang mixture test solution: accurately measure 2.0 mL of jingfang mixture and place it in a 10.0 mL volumetric flask, add methanol to the mark, ultrasonic for 30 min, and cool; use methanol to re-determine the volume to the mark, shake well, stand overnight, filter with a 0.45 μm microporous membrane, discard the first 3 drops, take the filtered solution, and obtain the jingfang mixture test solution, store at 4°C away from light for standby use.

[0068] Serum test solution: take 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, add 4 times the amount of methanol to precipitate the protein, vortex for 5 min, centrifuge at 12000 rpm·min -1 for 10 min at 4°C, take the supernatant, and dry it under nitrogen at 37°C. Add 240 μL of 80% methanol to the residue, vortex for 5 min, centrifuge again for 10 min after instant separation, take the supernatant, filter with a 0.22 μm microporous membrane, discard the first 3 drops, take the filtered solution, and obtain the serum test solution of each group.

[0069] Lung tissue test solution: take the lung tissue samples of each group, rinse with pre-cooled normal saline, absorb the water with filter paper, and weigh. Mix the lung tissue samples of each group with normal saline according to the mass-volume ratio of 1:2, and homogenize thoroughly. Centrifuge at 12000 rpm·min -1 for 10 min at 4°C, combine the supernatant of all lung homogenates in the same group, add 4 times the amount of methanol to precipitate the protein, vortex for 5 min, and centrifuge at 12000 rpm·min-1 Centrifugation 10 min, take supernatant, 37℃ nitrogen blow dry. Residue added 240 μL 80% methanol, vortex 5 min, centrifugation 10 min again, take supernatant, 0.22 μm microporous filter membrane filtration, the first 3 drops discarded, take the filtered solution, get each group of lung tissue test solution.

[0070] Control solution: take control sample menthone, chlorogenic acid, neochlorogenic acid, cimifugin, cimifugin glycoside, 5-O-methylvisamminol glycoside, huairenol, imperatorin, osthole, dihydroimperatorin angelica acid, amyrin, psoralen, decursinogen, ferulic acid, ligustilide, saikosaponin A, saikosaponin D, bungeanin, prunelliin A, prunelliin B, anisomalin, naringenin, naringin, neohesperidin, coffee acid, glycyrrhizin, glycyrrhizic acid, glycyrrhetinic acid, glycyrrhizol A, daidzin, genistin, isofraxidin, quercetin, luteolin, isorhamnetin control sample, accurately weighed, respectively, in a 10 mL volumetric flask, dissolved with 80% methanol and constant volume to the mark, shake, get each control solution. Take each control solution 200 μL in 10 mL volumetric flask, and reconstant volume to the mark with 80% methanol, shake, get mixed control solution, 4℃ light protection, ready for use.

[0071] 3, the composition of jingfang mixture in drug-containing serum

[0072] 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 the following figure: Figure 8 Among them, the components existing in jingfang mixture and drug serum samples but not in control serum samples are considered to be potential jingfang mixture efficacy substances.

[0073] From the above Figure 7 It can be seen that the above detection results are compared with the control solution, and combined with the relevant literature reports, 20 prototype components are identified in the normal drug serum of jingfang mixture, and 35 prototype components are identified in the model drug serum, and the composition of jingfang mixture in drug-containing serum is shown in table 1.

[0074] Table 1: composition of jingfang mixture in blood

[0075]

[0076]

[0077]

[0078]

[0079] Note: A. Schizonepeta; B. Saposhnikovia divaricata; C. Notopterygium incisum; D. Angelica pubescens; E. Bupleurum chinense; F. Peucedanum praeruptorum; G. Ligusticum chuanxiong; H. Citrus aurantium; I. Poria cocos; J. Platycodon grandiflorus; K. Glycyrrhiza uralensis.

[0080] 4. Characterization of the components of Jingfang compound in drug-containing target organs

[0081] UPLC-MS / MS was used to analyze the test solutions of lung tissue from each group of mice, and the total ion chromatograms for each group under positive and negative ion modes were obtained, as shown in the attached figure. Figure 9 Among them, the components that are present in both the Jingfang compound solution and the administered serum sample but not in the control serum sample are considered to be potential active ingredients of the Jingfang compound.

[0082] From the appendix Figure 9 As can be seen, by comparing the above detection results with those of the control solution and in conjunction with relevant literature reports, a total of 6 prototype components were identified in the lung tissue test solution under normal administration and a total of 29 prototype components were identified in the lung tissue test solution under model administration. The component characterization of Jingfang compound in the drug-containing target organs is shown in Table 2.

[0083] Table 2: Characterization of the target organ components of Jingfang Compound

[0084]

[0085]

[0086]

[0087]

[0088] Note: A. Schizonepeta; B. Saposhnikovia divaricata; C. Notopterygium incisum; D. Angelica pubescens; E. Bupleurum chinense; F. Peucedanum praeruptorum; G. Ligusticum chuanxiong; H. Citrus aurantium; I. Poria cocos; J. Platycodon grandiflorus; K. Glycyrrhiza uralensis.

[0089] Example 4: Comparative analysis of pungent and warming components and medicinal components to screen key quality attributes of Jingfang Compound related to medicinal efficacy.

[0090] As attached Figure 10 As shown, by using the MicroBio Information online platform to extract the intersection of the pungent and warm components targeted by TRPC6 / TRPM4 protein with the pharmacodynamic components that enter the blood and target organs and plotting the Venn diagram, seven key quality attributes of pungent and warm properties associated with pharmacodynamic effects can be screened out: imperatorin, 5-O-methylvisamidol, naringin, hesperidin, glycyrrhizic acid, nobiletin, and imperatorin.

[0091] Example 5: Drug Efficacy Testing

[0092] 1. MDCK cell culture

[0093] 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.

[0094] 2. Virus infection titer determination

[0095] Logarithmic growth phase MDCK cells were inoculated in a 96-well plate at a seeding rate of 5 x 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 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.

[0096] 3. CCK8 method for determining the cytotoxicity of flavor CAQs

[0097] 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 x 10 5The cells were inoculated in 96-well plates at an inoculation rate of 1×10 Figure 11 The highest concentration at which the cell survival rate was ≥90% was defined as the maximum non-toxic concentration of the CAQs on MDCK cells CC 0, and the median toxic concentration was calculated CC 50 Cell survival rate = [(OD of the drug-treated well - OD of the blank well) / (OD of the control well - OD of the blank well)] × 100%.

[0098] As shown in the attached Figure 12 table, the highest concentration at which the cell survival rate was ≥90% was defined as the maximum non-toxic concentration of the CAQs on MDCK cells. The CC0of nodakenin was 1000 μM, the CC0of 5-O-methylvisamminol was 4000 μM, the CC0of naringin was 400 μM, the CC0of auraptene was 383.3 μM, the CC0of glycyrrhizic acid was 125 μM, the CC0of nobiletin was 229.7 μM, the CC0of imperatorin was 165.8 μM, the CC0of osthole was 12.5 μM, and the CC0of tangeritin was 137.4 μM. 50 50 50 50

[0099] 4. CCK8 method for determining the antiviral effect of the CAQs

[0100] As shown in the attached Figure 13 table, the log-phase MDCK cells were inoculated at a concentration of 1×10 5 ​​​​The cells / mL inoculum was seeded into 96-well plates at a rate of 100 μL per well, with 6 replicates, and pre-cultured in a 37°C, 5% CO2 incubator for 16-24 h. When cell confluence reached 80-90%, the 96-well plates were removed, the culture medium was discarded, and the plates were washed twice with PBS. For the control group, 100 μL of MDCK cell maintenance medium was added to each well, and for the virus group, 100 μL of 100% CO2 maintenance medium was added to each well. TCID 50 Virus solution, 50 μL added to each well of the drug administration group. TCID 50 Virus solution and 50 μL of different concentrations of drug were incubated in a 37℃, 5% CO2 incubator for 48 h. The 96-well plates were then removed, the culture medium was discarded, and the plates were washed twice with PBS. 110 μL of DMEM high-glucose basal medium containing 10% CCK-8 was added to each well, and the plates were incubated in a 37℃, 5% CO2 incubator for another 1 h. At this point, the OD value of the control group was 1.0-1.5. The absorbance (OD) at 450 nm was measured using a microplate reader, and the virus inhibition rate and half-maximal inhibitory concentration (IC50) were calculated. EC 50 and treatment index SI , received Figure 13 Wherein, virus inhibition rate = [(virus well OD - drug well OD) / (virus well OD - control well OD)] × 100%, SI = CC 50 / EC 50 .

[0101] From the appendix Figure 14 It is evident that baloxavir, a positive control drug, exhibits the strongest antiviral activity against H1N1 virus, with an EC50 of 2.48 nM and an SI of 40.32. The key quality-associated component, norihesperidin, also demonstrates good antiviral activity, with an EC50 of 2.48 nM and an SI of 40.32. 50 =6.32μM, SI=26.23.

[0102] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. Application of TRPC6 / TRPM4 molecular sensory artificial intelligence biosensor in identification of antiviral pungent and warm key quality attributes of Jingfang mixture; 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: Screening of 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; Using the TRPC6 / TRPM4 receptors as biomolecular recognition elements to construct a TRPC6 / TRPM4-SPE functional biosensor, and screening pungent and warm components in Jingfang mixture according to the TRPC6 / TRPM4-SPE functional biosensor combined with UPLC-MS / MS technology; Collecting drug-containing serum and drug-containing target organs of mice containing Jingfang mixture, and analyzing the pharmacodynamic components of Jingfang mixture entering the blood and target organs by UPLC-MS / MS technology; Comparative analysis of the pungent and warm components and the pharmacodynamic components to screen the nature and taste key quality attributes related to the pharmacodynamic effect of Jingfang mixture; Verifying the pharmacodynamic effect of the screened nature and taste key quality attributes through in vitro cell experiments; The nature and taste key quality attributes of Jingfang mixture include decursin, 5-O-methylvisamminol, naringin, auraptene, glycyrrhizic acid, nobiletin and imperatorin.

2. Use according to claim 1, characterized in that, The nobiletin is used for preparing a medicine for treating influenza A H1N1 virus.

3. Use according to claim 1, characterized in that, The screening of TRPC6 / TRPM4 receptors with high expression and statistical difference in the lesion tissues of normal mice and diseased mice comprises the following steps: Collecting the lesion tissues of normal mice, diseased mice and Jingfang mixture intervention mice based on SPF mice replicating corresponding disease models; Detecting the relative expression levels of corresponding TRPC6 / TRPM4 receptor genes and proteins in the lesion tissues of normal mice, diseased mice and Jingfang mixture intervention mice based on real-time fluorescent quantitative PCR and Western blotting, and performing statistical analysis.

4. Use according to claim 1, characterized in that, Collecting drug-containing serum and drug-containing target organs of mice containing Jingfang mixture, and analyzing the pharmacodynamic components of Jingfang mixture entering the blood and target organs by UPLC-MS / MS technology, comprising the following steps: Collecting drug-containing serum and drug-containing target organs of normal mice and diseased mice after 24 hours of drug intervention based on SPF mice replicating corresponding disease models; Pretreating the drug-containing serum and drug-containing target organs by protein precipitation, enriching the drug-containing serum and drug-containing target organs by nitrogen blowing instrument, redissolving with methanol and filtering with 0.22 μm microporous filter membrane to obtain the test sample solution; Analyzing the pharmacodynamic components of Jingfang mixture entering the blood and target organs by UPLC-MS / MS technology.

5. The use according to claim 1, characterized in that, Comparative analysis of the pungent and warm components and the pharmacodynamic components to screen the nature and taste key quality attributes related to the pharmacodynamic effect of Jingfang mixture, comprising the following steps: Using the micro bioinformatics online platform to take the intersection part of the pungent and warm components and the pharmacodynamic components and draw a Venn diagram to screen the pungent and warm key quality attributes.

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