Use of biosensing and UPLC-ms technology in identification of critical quality attributes of property and flavor of traditional chinese medicine

Abstract

Use of biosensing and UPLC-MS technology in identification of critical quality attributes of the property and flavor of a traditional Chinese medicine, specifically comprising: (1) using a three-step method to construct a molecular sensory artificial intelligence biosensor by means of taking a taste receptor protein as a research carrier; (2) on the basis of the biosensor, using an electrochemical workstation to measure the interaction strength between a sample under test and the taste receptor protein; (3) using a two-step elution method to elute critical quality attributes bonded to the taste receptor; and (4) enriching the eluent in step 3, using UPLC-MS technology to analyze and identify chemical components in the eluent, and comparing same with all components of said sample to screen critical quality attributes of the property and flavor thereof. By means of taking the taste receptor as an angle of view, the present invention provides the use of biosensing and UPLC-MS technology in identification of critical quality attributes of the property and flavor of a traditional Chinese medicine, and identifies critical quality attributes of the property and flavor of an astragali radix pulse-reinforcing decoction and a stomach benefiting and digestion promoting tablet, thereby providing reference for identification of critical quality attributes of other traditional Chinese medicines.

Description

Application of a biosensor combined with UPLC-MS technology in the identification of critical quality attributes of traditional Chinese medicine properties and flavors

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of Chinese Application No. 2023111287127, filed September 4, 2023. Said application No. 2023111287127 is hereby incorporated by reference in its entirety. Technical Field

[0003] The present invention belongs to the field of traditional Chinese medicine, and specifically relates to an application of biosensor combined with UPLC-MS technology in the identification of key quality attributes of traditional Chinese medicine properties and flavors. Background Art

[0004] The International Council for Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) Guideline Q8 clearly defines critical quality attributes (CQAs) of pharmaceuticals: CQAs are physical, chemical, biological, or microbiological properties or characteristics that should be within appropriate limits, ranges, or distributions to ensure the required product quality. Therefore, CQAs are key elements in current international drug quality assessments. The quality of Traditional Chinese Medicine (TCM) is fundamental to ensuring its stable efficacy, safety, and effectiveness in clinical use. Its standardization, standardization, and modernization are crucial prerequisites for promoting the internationalization of TCM. Identification of CQAs is a crucial component of quality control for TCM preparations. However, in the current modernization of TCM research, there are still issues such as unclear correlations between CQAs and clinical safety and effectiveness. Research on the CQAs of nature and flavor is lacking, and quality control research is relatively weak.

[0005] The theory of medicinal properties in Traditional Chinese Medicine (TCM) is not only a core component of TCM theory but also an important basis for guiding clinical prescriptions. The theory of the five flavors is one of its core components. It not only reflects the efficacy of TCM itself but also plays a crucial role in its clinical application. The five flavors originally referred to the actual taste of a drug. Later, the flavors were linked to its effects, and the effects of drugs were explained and summarized through taste. The main effects of pungent flavors are dispersing and promoting movement, while the main effects of sweet flavors are tonifying, harmonizing, and soothing. The tonifying effect is primarily manifested in tonifying qi, blood, and deficiency. Taste is a sensory modality that guides organisms to identify and consume nutrients while also avoiding toxins. Most pungent TCM herbs act through TRP ion channels, and the pungent taste receptor TRPV1 is widely distributed. Mammals primarily sense taste through taste buds in the oral cavity. The receptors associated with sweet taste are T1R2 and T1R3, which are widely distributed in tissues such as the oral cavity, brain, and internal organs (such as the heart, liver, pancreas, and kidneys).

[0006] In recent years, cardiovascular disease has led the nation's mortality rate. Statistics show that cardiovascular disease ranks first among both urban and rural residents, exceeding 40%. The prevalence of cardiovascular disease is relatively high among middle-aged and elderly people in my country. Coronary heart disease, one of the most common cardiovascular diseases, has a high prevalence and mortality rate, and cannot be ignored. Huangqi Shengmai Yin (Huangqi Shengmai Yin) is used to treat patients with Qi and Yin deficiency, palpitations, and shortness of breath. It is a combination of five herbs: Astragalus, Codonopsis, Ophiopogon, Schisandra, and Schisandrae Chinensis, with a predominantly sweet flavor. Therefore, identifying the key quality attributes of Huangqi Shengmai Yin for preventing and treating coronary heart disease, focusing on sweet taste receptors, is of great scientific value.

[0007] According to statistics, the prevalence of functional dyspepsia in my country, based on the Rome III criteria, ranges from 10.0% to 14.9%, a relatively high rate. Jianweixiaoshi tablets, derived from modified Jianpi pills, are composed of tangerine peel, Pseudostellaria heterophylla, roasted malt, hawthorn, and Chinese yam. The predominantly sweet flavor of the tablets, supplemented by tangerine peel to promote qi circulation, jointly exert their spleen-strengthening and digestion-promoting effects. Therefore, identifying the key quality attributes of Jianweixiaoshi tablets for treating dyspepsia-related conditions, focusing on sweet and pungent taste receptors, is of great scientific value.

[0008] Traditional Chinese medicine (TCM) is a complex system with multiple components and targets. High-performance liquid chromatography (HPLC) is widely used to detect the chemical components of TCM. However, it has disadvantages such as high detection limits, long analysis times, and the inability to correlate components with efficacy indicators, making it difficult to identify the key quality attributes of TCM. Biosensor technology, with its advantages of high sensitivity and high accuracy, can be used to detect the key quality attributes of TCM. This paper, based on the sweet and pungent taste receptors, intends to integrate T1R2 / T1R3 / TRPV1-HEMT biosensors with UPLC-MS technology to establish a method for identifying the key quality attributes of TCM compounds, overcoming the shortcomings of existing technologies.

[0009] Summary of the Invention

[0010] To overcome the deficiencies of the prior art, the present invention provides an application of biosensor combined with UPLC-MS technology in the identification of key quality attributes of traditional Chinese medicine properties and flavors.

[0011] According to some specific embodiments of the present invention, a biosensor combined with UPLC-MS technology is used to identify the key quality attributes of traditional Chinese medicine properties and flavors, and the specific steps are as follows:

[0012] Step 1: Using a three-step approach, we constructed a molecular sensory AI biosensor using taste receptor proteins as a research vehicle.

[0013] Step 2: Based on this biosensor, an electrochemical workstation is used to measure the interaction strength between the test sample and the taste receptor protein;

[0014] Step 3: A two-step elution method is used to elute the key quality attributes bound to the taste receptors;

[0015] Step 4: Enrich the eluate from step 3, analyze and identify the chemical components in the eluate based on UPLC-MS technology, compare it with the full components of the sample to be tested, and screen its key quality attributes of nature and taste.

[0016] According to some specific embodiments of the present invention, a biosensor combined with UPLC-MS technology is used to identify the key quality attributes of the properties and flavors of traditional Chinese medicines. This involves two traditional Chinese medicine compound preparations, Huangqi Shengmai Yin and Jianwei Xiaoshi Pian, which involve sweetness, which is the most common flavor, and pungentness, which is relatively common. Since the binding of sweet and pungent substances to sweet and pungent taste receptors is a prerequisite for their function, the key proteins selected by the present invention are the sweet taste receptor proteins T1R2 and T1R3 and the pungent taste receptor protein TRPV1. The functionally modified biosensor provided is a T1R2 / T1R3 / TRPV1-HEMT biosensor. The specific steps for its construction are as follows:

[0017] (1) A clean quartz glass tube was glued onto the HEMT biosensor chip as a sample pool. 60 μL of 3-mercaptopropionic acid solution was added to the sample pool and soaked at room temperature for 17-24 h to generate Au-S bonds on the surface of the HEMT biosensor chip, forming a self-assembled monolayer.

[0018] (2) After the self-assembled monolayer is formed, the excess thiol reagent in the sample pool is washed away and 20 mmol·L -1 Carbodiimide hydrochloride (EDC) and 50mmol·L -1 N-hydroxysuccinimide (NHS) mixed solution was added to the sample cell and reacted at room temperature for 15 minutes to activate the carboxyl group;

[0019] (3) The HEMT biosensor chip was washed with phosphate buffered saline (PBS), and T1R2, T1R3, and TRPV1 proteins were added respectively, and reacted at 4°C to obtain HEMT biosensors functionally modified with T1R2, T1R3, and TRPV1, respectively;

[0020] (4) The electrical signals of the T1R2-HEMT biosensor, T1R3-HEMT biosensor, and TRPV1-HEMT biosensor were collected using a CHI660e electrochemical workstation to obtain current-voltage curves (IV).

[0021] According to some specific embodiments of the present invention, the electrochemical workstation in step 2 includes but is not limited to 2400, CHI660e devices that provide current and voltage, with a given constant voltage of 2-5V and a current accuracy of μA level.

[0022] The present invention provides the interaction strength between Huangqi Shengmai Yin sample and T1R2 and T1R3 based on T1R2-HEMT biosensor and T1R3-HEMT biosensor, as shown in Figure 1. T1R2 protein interacts with Huangqi Shengmai Yin, and the dissociation constant K D The value is 1.129×10 -8 g·mL -1 , T1R3 protein interacts with Huangqi Shengmai Yin, and the dissociation constant K D The value is 6.463×10 -5 g·mL -1 .

[0023] The present invention provides the interaction strength of Jianweixiaoshi tablet samples with T1R2, T1R3, and TRPV1 based on T1R2-HEMT biosensor, T1R3-HEMT biosensor, and TRPV1-HEMT biosensor, as shown in Figure 2. T1R2 protein interacts with Jianweixiaoshi tablets, and the dissociation constant K D The value is 2.033×10 -5 g·mL -1 ; T1R3 protein interacts with Jianweixiaoshi tablets, and the dissociation constant K D The value is 1.408×10 -6 g·mL -1 TRPV1 protein interacts with Jianweixiaoshi tablets, and the dissociation constant KD value is 2.780×10 -7 g·mL -1 .

[0024] According to some specific embodiments of the present invention, the nonspecific eluent in step 3 is PBS, the specific eluent for T1R2 protein is sucrose solution, the specific eluent for T1R3 protein is arginine solution, and the specific eluent for TRPV1 protein is ferulic acid solution, and the elution times for each eluent are 3 times.

[0025] According to some specific embodiments of the present invention, the UPLC-MS analysis and identification of the chemical components in the eluate involved in step 4 may be performed as follows:

[0026] (1) For T1R2-HEMT biosensor, PBS was used as a non-specific eluent to elute the sample bound to the T1R2-HEMT biosensor, with each reaction lasting 5 minutes, washed 3 times, and the eluates were combined; further, sucrose solution was used for elution, with each reaction lasting 5 minutes, washed 3 times, and the eluates were combined. For T1R3-HEMT biosensor, PBS was used as a non-specific eluent to elute the sample bound to the T1R3-HEMT biosensor, with each reaction lasting 5 minutes, washed 3 times, and the eluates were combined; further, arginine solution was used for elution, with each reaction lasting 5 minutes, washed 3 times, and the eluates were combined. For TRPV1-HEMT biosensor, PBS was used as a non-specific eluent to elute the sample bound to the TRPV1-HEMT biosensor, with each reaction lasting 5 minutes, washed 3 times, and the eluates were combined; further, ferulic acid solution was used for elution, with each reaction lasting 5 minutes, washed 3 times, and the eluates were combined;

[0027] (2) using solid phase extraction technology to wash away the phosphates in the two eluates, and then enriching the eluates to serve as sample solutions;

[0028] (3) UPLC-MS technology is used to analyze the substances bound to the sample to be tested and the biosensor sensitive element. The specific parameters are as follows:

[0029] Mass spectrometry conditions: electrospray ionization (ESI) positive and negative ion modes; ion source temperature: 350°C; in positive ion mode: ionization source voltage 4.0 kV, capillary voltage 35 V, tube lens voltage 110 V; in negative ion mode: ionization source voltage –3.0 kV, capillary voltage –35 V, tube lens voltage –110 V; collision voltage: 6-10 V; sheath gas flow rate 40 arb, auxiliary gas flow rate 20 arb; drying gas flow rate 15 L / min; the primary mass spectrum of the sample used full scan (mass scan range m / z 100-1200; detection resolution: 30000); data-dependent scanning (DDS) was used for the secondary mass spectrometry data, and the three most abundant primary ions were selected for collision-induced dissociation (CID) fragmentation scanning.

[0030] The total ion currents of Huangqi Shengmai Drink in positive and negative ion modes are shown in Figure 3, and the determination results of the two eluates are shown in Figures 4-5. The chemical component information was compared through the independently constructed Huangqi Shengmai Drink component library, and the chemical components in the two eluates of Huangqi Shengmai Drink were analyzed and identified. The chemical components in the sucrose eluate were analyzed and obtained, including sucrose, quinic acid, caffeic acid, 5-hydroxymethylfurfural, phenylalanine, betaine, calycosin, calycosin-7-O-β-D-glucoside, p-hydroxybenzoic acid, emodin, baicalein, (6αR-11αR)-10-hydroxy-3,9-dimethoxypterocarpone, formononetin, 7,2'-hydroxy-3',4'-dimethoxyisoflavan-7-O-β-D-glucoside, atractylodes lactone I, gomisin E, The chemical components of the arginine eluate were analyzed, including sucrose, quinic acid, caffeic acid, 5-hydroxymethylfurfural, phenylalanine, betaine, calycosin, shikimic acid, ferulic acid, calycosin-7-O-β-D-glucoside, p-hydroxybenzoic acid, emodin, baicalein, naringin, (6αR-11αR)-10-hydroxy-3,9-dimethoxypterocarbamane, formononetin, 7,2'-hydroxy-3',4'-dimethoxyisoflavan-7-O-β-D-glucoside, atractylodesin I, gomisin E, schisandrin, ruscosin, schisandrin A, atractylodesin III, and schisandrin C. Six of the components were compared with the reference substance.

[0031] The total ion chromatogram of Jianweixiaoshi tablets in negative ion mode is shown in Figure 6, and the determination results of the three eluates are shown in Figure 7. By comparing the chemical component information of the whole Jianweixiaoshi tablets, the chemical components in the three eluates of Jianweixiaoshi tablets were analyzed and identified. The chemical components of the sucrose eluate were analyzed to include hesperidin, hesperetin, and nobiletin. The chemical components of the arginine eluate were analyzed to include hesperidin, hesperetin, nobiletin, vecinin-2, vecinin, and isoschaftoside. The chemical components of the ferulic acid eluate were analyzed to include hesperidin, hesperetin, nobiletin, vecinin-2, vecinin, and isoschaftoside. Four of these components were compared with reference substances.

[0032] In summary, the present invention provides a component of Huangqi Shengmai Drink combined with sweet proteins T1R2 and T1R3, characterized by including but not limited to sucrose, quinic acid, caffeic acid, 5-hydroxymethylfurfural, phenylalanine, betaine, calycosin, shikimic acid, ferulic acid, calycosin-7-O-β-D-glucoside, p-hydroxybenzoic acid, emodin, baicalein, naringin, (6αR-11αR)-10-hydroxy-3,9-dimethoxypterocarpone, formononetin, 7,2'-hydroxy-3',4'-dimethoxyisoflavan-7-O-β-D-glucoside, atractylodes lactone I, gomisin E, schisandrin B, ruscosapogenin, crotonylgomisin P, schisandrin ester A, atractylodes lactone III, and schisandrin ester C. The present invention provides a composition of a stomach-strengthening and digestion-promoting tablet combined with sweet proteins T1R2, T1R3 and pungent protein TRPV1, characterized by including but not limited to hesperidin, hesperetin, nobiletin, vecinin-2, fennel glycosides, isoschaftoside and quercetin.

[0033] The advantages of the present invention are: from the perspective of taste receptors, a method for identifying the key quality attributes of the properties and flavors of traditional Chinese medicines combining biosensing and UPLC-MS technology is provided, and its application in identifying the key quality attributes of the properties and flavors of two traditional Chinese medicine compound preparations, Huangqi Shengmai Yin and Jianwei Xiaoshi Pian, overcomes the shortcomings of a single technology, and provides a reference for the identification of key quality attributes of other traditional Chinese medicines. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1: Interaction between T1R2 and T1R3 proteins and Huangqi Shengmai Yin (a1-a2) I DS -V DS Signal changes; (b1-b2) Linear fitting results of the binding of T1R2, T1R3 and Huangqi Shengmai Yin; (c1-c2) Dissociation constant results of T1R2, T1R3 and Huangqi Shengmai Yin within the linear concentration range.

[0035] Figure 2: Interactions between T1R2, T1R3, and TRPV1 proteins and Jianweixiaoshi tablets at different concentrations (a1-a3) DS -V DS Signal changes; (b1-b3) Linear fitting results of the binding of T1R2, T1R3, TRPV1 proteins with Jianwei Xiaoshi tablets; (c1-c3) Dissociation constant results of T1R2, T1R3, TRPV1 proteins with Jianwei Xiaoshi tablets within the linear concentration range.

[0036] Figure 3: Total ion current of Huangqi Shengmai Drink concentrate (A) Total ion current in negative ion mode; (B) Total ion current in positive ion mode.

[0037] Figure 4: Total ion current of the sucrose eluate of Huangqi Shengmai Drink sample (A) Total ion current in positive ion mode; (B) Total ion current in negative ion mode.

[0038] Figure 5: Total ion current of the arginine eluate of Huangqi Shengmai Drink sample (A) Total ion current in positive ion mode; (B) Total ion current in negative ion mode.

[0039] Figure 6: Total ion current diagram of Jianweixiaoshi tablets extract in negative ion mode.

[0040] Figure 7: Total ion currents of the eluates from Jianwei Xiaoshi tablets in negative ion mode (A) Total ion current of the eluate from sucrose in negative ion mode; (B) Total ion current of the eluate from arginine in negative ion mode; (C) Total ion current of the eluate from ferulic acid in negative ion mode. DETAILED DESCRIPTION

[0041] The following is a further explanation of the application of a biosensor combined with UPLC-MS technology in the identification of key quality attributes of traditional Chinese medicine properties and flavors with reference to specific examples.

[0042] Example 1: Study on the interaction between Huangqi Shengmai Yin and T1R2 / T1R3 based on T1R2 / T1R3-HEMT biosensor

[0043] (1) Preparation of Huangqi Shengmai Yin sample solution: Take the commercially available Huangqi Shengmai Yin as the stock solution, dilute it 8 times in a 10-fold concentration gradient, and prepare 9 solutions with different concentration gradients as the test samples;

[0044] (2) Determination of blank solution effect: T1R2-HEMT biosensor and T1R3-HEMT biosensor were prepared respectively, and deionized water was used as blank solution. The current intensity (I DS -V DS ), taking this signal as a blank signal;

[0045] (3) Huangqi Shengmai Yin samples were added to the sample cells of T1R2-HEMT biosensor and T1R3-HEMT biosensor in order from low to high concentrations and reacted at 4°C for 6 min. The electrochemical workstation was used to record the I DS -V DS , the results are shown in Figure 1(a1-a2).

[0046] (4) The logarithm of the concentration of Huangqi Shengmai Yin (Lg[A g ]) as the horizontal axis, with concentration ([A g]) / current change (I-I0, ΔI) as the ordinate, linear fitting was performed to determine the linear range of the biosensor. The results are shown in Figure 1 (b1-b2). The dissociation constant K of the interaction between T1R2 protein and Huangqi Shengmai Yin was calculated according to the formula D The value is 1.129×10 -8 g·mL -1 , T1R3 protein interacts with Huangqi Shengmai Yin, and the dissociation constant K D The value is 6.463×10 -5 g·mL -1 .

[0047] Example 2: Identification of the Key Quality Attributes of Huangqi Shengmai Drink's Properties and Taste Based on UPLC-MS Technology

[0048] (1) Instruments and methods:

[0049] Ultimate 3000 ultra-high performance liquid chromatograph, LTQ-Orbitrap XL mass spectrometer (equipped with an electrospray ionization source and Xcalibur 2.1 ChemStation, ThermoFisher Scientific, USA), CV200 vacuum concentrator (Beijing Ji'am Technology Co., Ltd.); Grace Pure TM SPE C 18 -low solid phase extraction cartridge (500 mg / 3 mL), formic acid, and acetonitrile (mass spectrometry grade, Thermo Fisher Scientific Inc.).

[0050] (2) Protein eluate sample pretreatment

[0051] The protein eluate was used as the research carrier and solid phase extraction technology was adopted. 5 mL of methanol was used to rinse the 1 mL solid phase extraction column for column activation. Sample was loaded by first using 5 mL of methanol at 1 mL min. -1 Elute at a flow rate of 1 mL min to remove PBS from the sample; then add 5 mL of water and elute at a flow rate of 1 mL min -1 The sample was eluted at a flow rate of 100 μg / min, evaporated using a centrifugal concentrator, and re-dissolved in 0.5 mL of the initial mobile phase for later use.

[0052] (3) Identification and analysis of eluate components based on UPLC-MS technology

[0053] Chromatographic conditions: The chromatographic column was a Waters AQUITY HSS T3 column (2.1 mm × 100 mm, 1.8 μm); the mobile phase was 0.1% formic acid in water (A)-acetonitrile (B), with a gradient elution program of 0–2 min, 2% B; 2–8 min, 2%–25% B; 8–21 min, 25–55% B; 21–26 min, 55–75% B; 26–27 min, 75–95% B; 27–28 min, 95% B; 28–29 min, 95–2% B; 29–30 min, 2% B; the flow rate was 0.3 mL min -1 ; Column temperature was 30℃; Injection volume was 2μL.

[0054] Mass spectrometry conditions: electrospray ionization (ESI), ionization source temperature: 350°C; in positive ion mode: ionization source voltage 4.0 kV, capillary voltage 35 V, tube lens voltage 110 V; in negative ion mode: ionization source voltage –3.0 kV, capillary voltage –35 V, tube lens voltage –110 V; collision voltage: 6–10 V; sheath gas flow rate 40 arb, auxiliary gas flow rate 20 arb; drying gas flow rate 15 L / min; primary mass spectrum using full scan (mass scan range m / z 100–1200; detection resolution: 30,000); secondary mass spectrometry data using data-dependent scanning (DDS), and the three most abundant primary ions were selected for collision-induced dissociation (CID) fragmentation scanning.

[0055] Figures 3-5 show the total ion currents of the Huangqi Shengmai Yin concentrate in positive and negative ion modes, as well as those of the two eluents. Chemical components were analyzed using the Xcalibur 2.1 Chemistry Workstation. A total of 50 chemical components were identified, as shown in Table 1. Twenty-five components binding to T1R2 and T1R3 were identified, including calycosin, calycosin-7-O-β-D-glucoside, atractylodes lactone I, ruscosapogenin, and schisandrin A, as shown in Table 2. Some fragmentation patterns are as follows:

[0056] The quasi-molecular ion peak of the compound with a retention time of 8.53 min is m / z 283.06033, and its molecular formula is estimated to be C 16 H 12 O5, with an error of 0.813ppm. In its secondary mass spectrum, fragment ions such as m / z 255.04002 and 268.32452 were generated. m / z 268.32452 was generated by the removal of a molecule of CH3 from m / z 283.06033, and m / z 255.04002 was generated by the removal of a molecule of CO from m / z 283.06033. Combined with literature reports, this compound was identified as calycosin (C 16 H 12O5).

[0057] The quasi-molecular ion peak of the compound with a retention time of 9.91 min is m / z 447.12808, and its molecular formula is estimated to be C 22 H 22 O 10 , with an error of -1.103ppm. In its secondary mass spectrum, fragment ions such as m / z 270.13382 and 285.11362 were generated. m / z 285.11362 was generated by removing a molecule of glucose from m / z 447.12808, and then removing a molecule of CH3 to obtain the fragment ion m / z 270.13382. Combined with literature reports, this compound was identified as calycosin-7-O-β-D-glucoside (C 22 H 22 O 10 ).

[0058] The quasi-molecular ion peak of the compound with a retention time of 20.81 min is m / z 231.13815, and its molecular formula is estimated to be C 15 H 18 O2, with an error of 0.838ppm. In its secondary mass spectrum, fragment ions such as m / z 156.93694, 184.96791, and 213.04501 were generated. m / z 184.96791 was generated by the removal of one molecule of HCOOH from m / z 231.13815, and m / z 156.93694 was generated by the removal of one molecule of HCOOH and one molecule of C2H4 from m / z 231.13815. Combined with literature reports, this compound was identified as atractylodes lactone I (C 15 H 18 O2).

[0059] The quasi-molecular ion peak of the compound with a retention time of 23.54 min is m / z 401.19586, and its molecular formula is estimated to be C 23 H 28 O6, with an error of -0.013ppm. In its secondary mass spectrum, fragment ions such as m / z 370.19778 and 386.31177 were generated. m / z 386.31177 was generated by the removal of one molecule of CH3 from m / z 401.19586, and m / z 370.19778 was generated by the removal of one molecule of CH3O from m / z 401.19586. Combined with literature reports, the compound was identified as schisandrin B (C 23 H 28 O6).

[0060] The quasi-molecular ion peak of the compound with a retention time of 24.38 min is m / z 431.31705, and its molecular formula is estimated to be C27 H 42 O4, with an error of 3.394ppm. In its secondary mass spectrum, fragment ions such as m / z 367.25256 and 413.29555 were generated. m / z 413.29555 was generated by the removal of one molecule of H2O from m / z 431.31705, and m / z 367.25256 was generated by the removal of two molecules of H2O and one molecule of CO from m / z 431.31705. Combined with literature reports, this compound was identified as ruscosapogenin (C 27 H 42 O4).

[0061] The quasi-molecular ion peak of the compound with a retention time of 24.52 min is m / z 537.20978, and its molecular formula is estimated to be C 30 H 32 O9, with an error of -3.963ppm. In its secondary mass spectrum, fragment ions such as m / z 371.25568 and 415.27136 were generated. m / z 415.27136 was generated by the removal of one molecule of benzoic acid (C7H6O2) from m / z 537.20978, and m / z 371.25568 was generated by the removal of one molecule of C7H6O2 and one molecule of CH3CHO from m / z 537.20978. Combined with literature reports, the compound was identified as schisandra chinensis ester methyl (C 30 H 32 O9).

[0062] The quasi-molecular ion peak of the compound with a retention time of 25.03 min is m / z 537.20923, and its molecular formula is estimated to be C 28 H 34 O9, with an error of -0.510ppm. In its secondary mass spectrum, fragment ions such as m / z 371.18774, 385.36475, and 415.2352 were generated. m / z 415.2352 was generated by the removal of one molecule of C4H6COOH from m / z 537.20923, m / z 385.36475 was generated by the removal of one molecule of C4H6COOH and one molecule of CH2O from m / z 537.20923, and m / z 371.18774 was generated by the removal of one molecule of C4H6COOH and one molecule of C2H4O from m / z 537.20923. Combined with literature reports, the compound was identified as schisandra chinensis ester propionate (C 28 H 34 O9).

[0063] Table 1 Identification of chemical components of Huangqi Shengmai Decoction Note: a: Astragalus; b: Codonopsis; c: Ophiopogon; d: Schisandra chinensis; e: Schisandra chinensis. The compounds marked with an asterisk "*" are the components compared with the reference substance.

[0064] Table 2 Identification of chemical components of Huangqi Shengmai Decoction eluate

[0065] Example 3: Identification of the Key Quality Attributes of Nature and Taste of Jianweixiaoshi Tablets Based on T1R2 / T1R3 / TRPV1-HEMT Biosensor Combined with UPLC-MS Technology

[0066] (1) Preparation of Jianweixiaoshi Tablets sample solution: Weigh 2.5261 g of yam raw material powder, add 40 mL of distilled water to a constant temperature water bath at 60°C for 30 min, then ultrasonically extract at 60°C for 30 min, 8000 r·min -1 Centrifuge for 5 minutes, take the supernatant, add 1.4742g of extract powder, shake in a 60℃ constant temperature water bath to dissolve thoroughly, and shake at 8000r·min -1 Centrifuge for 5 minutes, take the supernatant, filter through a 0.8 μm microporous membrane, and obtain the mother liquor. Dilute the mixture in a tenfold concentration gradient to obtain a concentration of 10 -1 ~10 -9 g·mL -1 Nine sample solutions of , as the samples to be tested;

[0067] (2) Determination of blank solution effect: HEMT biosensors functionalized with T1R2, T1R3, and TRPV1 proteins were prepared respectively. Deionized water was used as blank solution, and the current intensity (I DS -V DS ), taking this signal as a blank signal;

[0068] (3) The Jianweixiaoshi tablet samples were added to the sample cells of the T1R2-HEMT biosensor, T1R3-HEMT biosensor, and TRPV1-HEMT biosensor in order from low to high concentrations for 6 min. The signals were collected using an electrochemical workstation, as shown in Figure 2 (a1-a3).

[0069] (4) The logarithm of the concentration of Jianweixiaoshi tablets (Lg[A g ]) as the horizontal axis, with concentration ([A g ]) / current change (I-I0, ΔI) as the vertical axis, linear fitting was performed to determine the linear range of the biosensor. The results are shown in Figure 1 (b1-b3). The dissociation constant K of the interaction between T1R2 protein and Jianweixiaoshi tablets was calculated according to the formula D The value is 2.033×10 -5 g·mL -1; T1R3 protein interacts with Jianweixiaoshi tablets, and the dissociation constant K D The value is 1.408×10 -6 g·mL -1 TRPV1 protein interacts with Jianweixiaoshi tablets, and the dissociation constant K D The value is 2.780×10 -7 g·mL -1 .

[0070] (5) Identification and analysis of eluate components based on LC-MS / MS

[0071] Chromatographic conditions: The chromatographic column was a Waters AQUITY HSS T3 column (2.1 mm × 100 mm, 1.8 μm); the mobile phase was 0.1% formic acid aqueous solution (A)-acetonitrile (B), and the gradient elution program was: 0-2 min, 2% B; 2-25 min, 2%-95% B; 25-27 min, 95% B; the flow rate was 0.3 mL min -1 Flow rate: 0.3 mL min -1 ; Column temperature was 30℃; Injection volume was 2μL.

[0072] Mass spectrometry conditions: electrospray ionization (ESI) negative ion mode; ionization source voltage –3.0 kV, capillary voltage –35 V, tube lens voltage –110 V; collision voltage: 6-10 V; sheath gas flow rate 40 arb, auxiliary gas flow rate 20 arb; drying gas flow rate 15 L / min; primary mass spectrometry used Fourier transform high-resolution full scan, the mass scan range m / z was 100-1200, and the detection resolution was 30,000; secondary mass spectrometry data used data-dependent scanning (DDS), and the three ions with the highest primary abundance were selected for collision-induced dissociation (CID) fragmentation scanning.

[0073] The total ion current (TIC) of the Jianweixiaoshi tablet extract in negative ion mode and the total ion current of the three eluents in negative ion mode are shown in Figures 6-7. A total of 16 chemical components were identified, as shown in Table 3. Six components were identified that bind to T1R2 and T1R3, including hesperidin, hesperetin, valerianin, vecinin-2, isoschaftoside, and nobiletin. Seven components were identified that bind to TRPV1, including hesperidin, hesperetin, valerianin, vecinin-2, isoschaftoside, quercetin, and nobiletin, as shown in Table 4. Some fragmentation patterns are as follows:

[0074] The quasi-molecular ion peak of the compound with a retention time of 8.36 min is m / z 593.15588, and its molecular formula is estimated to be C 27 H 30 O 15, with an error of 9.750ppm. In its secondary mass spectrum, fragment ions such as m / z 575.31, m / z 473.19, m / z 455.18, m / z 383.07 and m / z 353.16 were generated. Among them, m / z 353.16 was generated by the removal of C4H9O4-C4H9O4 from m / z 593.15588. Combined with literature reports, this compound was identified as vetsenin-2(C 16 H 14 O6).

[0075] The quasi-molecular ion peak of the compound with a retention time of 9.11 min is m / z 563.14502, and its molecular formula is estimated to be C 26 H 28 O 14 , with an error of 9.746ppm. In its secondary mass spectrum, fragment ions such as m / z 473.18, m / z 443.19, m / z 383.12 and m / z 353.10 were generated. Among them, m / z 473.18 was generated by the removal of one molecule of C3H6C3 from m / z 563.14502, m / z 443.19 was generated by the removal of one molecule of C4H8C4 from m / z 563.14502, m / z 383.12 was generated by the removal of two molecules of C3H6C3 from m / z 563.14502, and m / z 353.10 was generated by the removal of one molecule of C3H6C3 and one molecule of C4H8C4 from m / z 563.14502. Combined with literature reports, the compound was identified as isoschaftoside (C 26 H 28 O 14 ).

[0076] The quasi-molecular ion peak of the compound with a retention time of 10.30 min is m / z 579.17651, and its molecular formula is estimated to be C 27 H 32 O 14 , with an error of 9.804ppm. In its secondary mass spectrum, fragment ions such as m / z 459.21 and m / z 271.04 were generated. Combined with literature reports, the compound was identified as nobiletin (C 27 H 32 O 14 ).

[0077] The quasi-molecular ion peak of the compound with a retention time of 10.64 min is m / z 609.18689, and its molecular formula is estimated to be C 28 H 34 O 15, with an error of 9.018ppm. In its secondary mass spectrum, fragment ions such as m / z 301.09 were generated. m / z 301.09 is produced by removing a molecule of rhamnose and glucose from m / z 609.18689. Combined with literature reports, this compound was identified as hesperidin (C 28 H 34 O 15 ).

[0078] The quasi-molecular ion peak of the compound with a retention time of 10.70 min is m / z 301.03473, and its molecular formula is estimated to be C 15 H 10 O7, with an error of 1.498ppm. In its secondary mass spectrum, fragment ions such as m / z 257.09 were generated. m / z 257.09 is generated by the removal of a molecule of CO-O from m / z 301.03473. Combined with literature reports, the compound was identified as quercetin (C 15 H 10 O7).

[0079] The quasi-molecular ion peak of the compound with a retention time of 10.71 min is m / z 301.07394, and its molecular formula is estimated to be C 16 H 14 O6, with an error of 10.879ppm. In its secondary mass spectrum, fragment ions such as m / z 286.04, m / z 257.09, m / z 242.09 and m / z 151.00 were generated. m / z 286.04 is produced by the removal of a molecule of CH3 from m / z 301.07394, m / z 257.09 is produced by the removal of a molecule of CO2 from m / z 301.07394, m / z 242.09 is produced by the removal of a molecule of CH3 from m / z 257.09, and m / z 151.00 is produced by RDA cleavage of m / z 301.07394. Combined with literature reports, this compound was identified as hesperidin (C 16 H 14 O6).

[0080] The quasi-molecular ion peak of the compound with a retention time of 12.19 min is m / z 593.19238, and its molecular formula is estimated to be C 28 H 34 O 14 , with an error of 9.943ppm. In its secondary mass spectrum, fragment ions such as m / z 285.04 were generated. m / z 285.04 is produced by removing one molecule of glucose and rhamnose from m / z 593.19238. Combined with literature reports, this compound was identified as fennelside (C 28 H 34 O14 ).

[0081] Table 3 Identification of chemical components of Jianweixiaoshi tablets Note: a: Pseudostellariae Radix; b: Crataegus pinnatifida; c: Roasted malt; d: Tangerine peel. The compounds marked with an asterisk "*" are the components compared with the reference substances. Table 4 Identification of chemical components of the eluate of Jianwei Xiaoshi tablets

Claims

1. An application of biosensor combined with UPLC-MS technology in the identification of key quality attributes of traditional Chinese medicine properties and flavors, characterized in that: The specific steps are as follows: Step 1: Use a three-step approach to construct a molecular sensory artificial intelligence biosensor using taste receptor proteins as research vectors; Step 2: Based on this biosensor, an electrochemical workstation is used to measure the interaction strength between the sample to be tested and the taste receptor protein; Step 3: Use a two-step elution method to elute the key quality attributes bound to the taste receptors; Step 4: Enrich the eluate in step 3, analyze and identify the chemical components in the eluate based on UPLC-MS technology, compare it with the full components of the sample to be tested, and screen its key quality attributes of nature and taste.

2. The application of a biosensor combined with UPLC-MS technology in the identification of key quality attributes of traditional Chinese medicine properties and flavors according to claim 1, characterized in that: The functionally modified proteins of the biosensor in step 1 are sweet and spicy taste receptor proteins, and the biosensors include but are not limited to surface plasmon resonance biosensors, high electron mobility field effect transistor biosensors, and microfluidic chip biosensors.

3. The ingredients of Huangqi Shengmai Yin combined with sweet proteins T1R2 and T1R3 include but are not limited to sucrose, quinic acid, caffeic acid, 5-hydroxymethylfurfural, phenylalanine, betaine, calycosin, shikimic acid, ferulic acid, calycosin-7-O-β-D-glucoside, p-hydroxybenzoic acid, rhamnolide, baicalein, naringin, (6αR-11αR)-10-hydroxy-3,9-dimethoxypterocarpone, formononetin, 7,2'-hydroxy-3',4'-dimethoxyisoflavan-7-O-β-D-glucoside, atractylodes lactone I, gomisin E, schisandrin B, ruscosapogenin, crotonylgomisin P, schisandrin A, atractylodes lactone III, and schisandrin C.

4. The key quality attributes of Huangqi Shengmai Drink in terms of nature and taste include but are not limited to calycosin, calycosin-7-O-β-D-glucoside, atractylodes lactone I, ruscosapogenin, and schisandra chinensis ester A.

5. Application of the key quality attributes of Huangqi Shengmai Drink in the preparation of preparations for the prevention and treatment of coronary heart disease.

6. A preparation for preventing and treating coronary heart disease according to claim 5, characterized in that: It includes the key quality attributes of Huangqi Shengmai Drink and pharmaceutical excipients as described in claim 4, and the preparation form is injection, tablet, capsule, aerosol, suppository, film, pill, ointment, sustained release, controlled release or nano preparation.

7. The ingredients of Jianweixiaoshi tablets combined with sweet proteins T1R2 and T1R3 include but are not limited to hesperidin, hesperidin, nobiletin, galangin, vicinin-2, and isoschaftoside. The ingredients of Jianweixiaoshi tablets combined with pungent protein TRPV1 include but are not limited to hesperidin, hesperidin, nobiletin, vicinin-2, isoschaftoside, quercetin, and nobiletin.

8. The key quality attributes of the nature and taste of Jianweixiaoshi tablets include, but are not limited to, hesperidin, hesperidin, valerianin, vesacerin-2, isoschaftoside, quercetin, and nobiletin.

9. Application of the key quality attributes of nature and taste of Jianweixiaoshi tablets in the preparation of preparations for the treatment of indigestion-related diseases.

10. A preparation for treating dyspepsia-related diseases according to claim 9, characterized in that: The invention comprises the key quality attributes of the properties and taste of the Jianweixiaoshi tablets and pharmaceutical excipients as described in claim 8, and the preparation is in the form of injection, tablet, capsule, aerosol, suppository, film, pellet, ointment, sustained release agent, controlled release agent or nano preparation.

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