A method for detecting the key quality attribute of sweetness in traditional Chinese medicine preparations

Abstract

This invention provides a method for detecting key quality attributes of sweetness in traditional Chinese medicine (TCM) preparations, belonging to the field of TCM quality control technology. The method includes the following steps: 1) separating the TCM preparations using preparative liquid chromatography and collecting samples in segments; 2) reacting the collected samples with a sweetness receptor-functionalized biosensor and detecting changes in current; the sweetness receptor-functionalized biosensor is modified with sweetness receptors TAS1R2 and TAS1R3; 3) after the reaction, eluting sequentially with non-specific eluent, TAS1R2-specific eluent, and TAS1R3-specific eluent, collecting specific eluent fractions; 4) using ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS) to detect the collected specific eluent fractions and obtain the key quality attributes of sweetness in the TCM preparations. The method described in this invention is accurate and effective; it provides core indicators for TCM quality control and a methodological reference for screening active sweetness key quality attributes in the TCM manufacturing process.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine quality control technology, and in particular relates to a method for detecting the key quality attribute of sweetness in traditional Chinese medicine preparations. Background Technology

[0002] Traditional Chinese medicine (TCM) preparations are generally made from raw herbal powders containing several medicinal ingredients. One of the core issues in their manufacturing process is quality. Identification of key quality attributes is a fundamental prerequisite for quality control of TCM preparations. Identifying the key quality attributes of TCM herbs that conform to the characteristics of TCM and the needs of TCM manufacturing is the core of quality control in the manufacturing of large-scale TCM products. Developing identification methods is a core scientific problem that urgently needs to be solved behind the technological bottlenecks in TCM manufacturing.

[0003] Sweet-tasting Chinese medicinal herbs play an indispensable role in compound prescriptions and in exerting their efficacy. The key quality attribute of sweetness is one of the core components of the key quality attributes of taste and properties, and is an important indicator for the quality control of compound Chinese medicine preparations. T1Rs receptors belong to the glycoside receptor class, and their main function is to recognize sweetness. T1Rs receptors play an important role in the perception of taste components and the digestion and absorption of nutrients. These substances influence the occurrence of diseases, indicating that sweetness receptors are closely related to both sensory perception and function. Therefore, targeting T1Rs receptors is a crucial breakthrough in exploring the key quality attributes of sweetness in major Chinese medicinal herbs.

[0004] There is currently no accurate and effective method for screening the key quality attribute of sweetness in the complex system of traditional Chinese medicine. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide an accurate and effective method for detecting the key quality attribute of sweetness in traditional Chinese medicine preparations.

[0006] This invention provides a method for detecting the key quality attribute of sweetness in traditional Chinese medicine preparations, comprising the following steps:

[0007] 1) After pretreatment of traditional Chinese medicine preparations, the samples are separated by preparative liquid chromatography and collected in segments;

[0008] 2) The sample solution collected in each segment is reacted with the sweet taste receptor functionalized biosensor, and the change in current is detected; the sweet taste receptor functionalized biosensor is modified with sweet taste receptors TAS1R2 and TAS1R3;

[0009] 3) After the reaction is complete, elute sequentially with non-specific elution buffer, TAS1R2 specific elution buffer and TAS1R3 specific elution buffer, and collect the TAS1R2 specific elution fraction and the TAS1R3 specific elution fraction.

[0010] 4) The key quality attribute of sweetness in traditional Chinese medicine was obtained by detecting the TAS1R2-specific eluent and TAS1R3-specific eluent collected in step 3).

[0011] Preferably, the traditional Chinese medicine includes Tongren Niuhuang Qingxin Wan.

[0012] Preferably, the reaction time between the sample solution and the sweetness receptor-functionalized biosensor is 5–10 min; the injection volume of the sample solution is 50–100 μL.

[0013] Preferably, the sweet taste receptor functionalized biosensor includes a biosensor chip, an electrochemical workstation, and a signal receiving and processing system; the biosensor chip is modified with sweet taste receptors TAS1R2 and TAS1R3.

[0014] Preferably, the biosensor chip includes a surface plasmon resonance biosensor chip, a high electron mobility transistor biosensor chip, or a screen-printed electrode biosensor chip.

[0015] Preferably, the non-specific eluent in step 3) is a PBS solution; the specific eluent for TAS1R2 is a ginsenoside Rg1 solution; and the specific eluent for TAS1R3 is an arginine solution.

[0016] Preferably, the concentration of the ginsenoside Rg1 solution is 0.08–0.12 mM, the number of elutions is 8–12, and the elution volume for each elution is 40–60 μL.

[0017] Preferably, the concentration of the arginine solution is 0.08–0.12 mM, the number of elutions is 8–12, and the elution volume for each elution is 40–60 μL.

[0018] Preferably, in step 4), the detection results of ultra-high performance liquid chromatography-mass spectrometry are compared with all known chemical components of the traditional Chinese medicine to determine the key quality attribute substances of sweetness in the traditional Chinese medicine.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] This invention utilizes TAS1R2 / TAS1R3 functionalized biosensors to enrich substances that bind to sweet taste receptors in traditional Chinese medicine (TCM) preparations. Then, it combines ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS) to identify key sweet taste quality attributes in TCM preparations. This provides core indicators that meet the characteristics of TCM and the needs of TCM manufacturing for quality control, and offers methodological reference and technical support for screening active sweet taste key quality attributes in the TCM manufacturing process.

[0021] Taking Tongren Niuhuang Qingxin Pill as an example, this invention uses a TAS1R2 / TAS1R3 functionalized biosensor to detect the following components: glucose, fructose, sucrose, maltose, glycyrrhizin, apigenin, 5-O-methylvisamidol, ginsenoside Rg1, glycyrrhizic acid, paeonol, paeoniflorin, paeoniflorin, typhain, arginine, 6-hydroxydaidzein, and atractylodes lactone II. Attached Figure Description

[0022] Figure 1 A schematic diagram of the TAS1R2 / TAS1R3 functionalized biosensor.

[0023] Figure 2 The diagram shows the affinity characterization between the TAS1R2 / TAS1R3 functionalized biosensor and the fractionated solution of Tongren Niuhuang Qingxin Pill; where (a1) segment A sample interacts with the TAS1R2 site I. DS -V DS Curve diagram, (a2) interaction between sample segment B and TAS1R2 site I DS -V DS The curve shows the interaction between the sample in segment (a3) ​​and the TAS1R2 site. DS -V DS The graphs show: (b1) linear relationship between sample A and TAS1R2 receptor; (b2) linear relationship between sample B and TAS1R2 receptor; (b3) linear relationship between sample C and TAS1R2 receptor; and (c1) interaction between sample A and TAS1R3 site. DS -V DS Curve diagram, (c2) segment B sample interaction with TAS1R3 site I DS -V DS The curve shows the interaction between the sample in segment (c3) C and the TAS1R3 site. DS -V DS The graphs show the linear relationship between the interaction between sample A and TAS1R3 receptor (d1), sample B and TAS1R3 receptor (d2), and sample C and TAS1R3 receptor (d3).

[0024] Figure 3 The total ion chromatograms for mass spectrometry analysis are shown in the following diagrams: (a1) R2-TNA solution positive ion mode, (a2) R2-TNB solution positive ion mode, (a3) ​​R2-TNC solution positive ion mode, (a4) blank solvent positive ion mode, (b1) R2-TNA solution negative ion mode, (b2) R2-TNB solution negative ion mode, (b3) R2-TNC solution negative ion mode, and (b4) blank solvent negative ion mode.

[0025] Figure 4 Total ion current chromatogram for mass spectrometry analysis, where (a1) positive ion mode of R3-TNA solution, (a2) positive ion mode of R3-TNB solution, (a3) positive ion mode of R3-TNC solution, (b1) negative ion mode of R3-TNA solution, (b2) negative ion mode of R3-TNB solution, (b3) negative ion mode of R3-TNC solution. Detailed implementation mode

[0026] The present invention provides a method for detecting the key quality attributes of sweet taste in traditional Chinese patent medicines, which includes the following steps: 1) After pretreating the traditional Chinese patent medicine, separate it by preparative liquid chromatography and collect the samples in segments; 2) Prepare the samples collected in each segment into sample solutions with several concentration gradients, and react the sample solutions with the sweet taste receptor-functionalized biosensor in the order of increasing concentration to detect the change in current; the sweet taste receptor-functionalized biosensor is modified with sweet taste receptors TAS1R2 and TAS1R3; 3) After the reaction, elute successively with a non-specific eluent, a TAS1R2-specific eluent, and a TAS1R3-specific eluent, and collect the TAS1R2-specific elution fraction and the TAS1R3-specific elution fraction; 4) Use ultra-high performance liquid chromatography-mass spectrometry to detect the TAS1R2-specific elution fraction and the TAS1R3-specific elution fraction collected in step 3) to obtain the key quality attributes of sweet taste in traditional Chinese patent medicines.

[0027] In the present invention, after pretreating the traditional Chinese patent medicine, separate it by preparative liquid chromatography and collect the samples in segments. The present invention does not have special limitations on the type of the traditional Chinese patent medicine, and any traditional Chinese patent medicine can be used. In the specific implementation process of the present invention, traditional Chinese patent medicine Tongren Niuhuang Qingxin Pills are taken as an example. In the present invention, the pretreatment preferably includes extracting the volatile oil components, water extract components, and alcohol extract components of the traditional Chinese patent medicine. In the specific implementation process of the present invention, the pretreatment preferably includes the following steps: S1) Mix the traditional Chinese patent medicine with water and heat it to collect the volatile oil, centrifuge the water extract to collect the first supernatant and the medicinal residues; S2) Mix the medicinal residues with a methanol aqueous solution and heat under reflux, and centrifuge to collect the second supernatant; S3) Combine the volatile oil, the first supernatant, and the second supernatant, and evaporate to dryness under vacuum to obtain a solid sample. In the present invention, after the pretreatment, the solid sample is dissolved and then separated by preparative liquid chromatography to obtain segmented samples. In the present invention, the preparative liquid chromatography separation preferably uses a reverse silica gel column SW-5222-012-SP, and the collected liquids with similar ultraviolet absorption waves are combined as one segment.

[0028] In this invention, the sample solution collected in each segment is reacted with a sweet taste receptor-functionalized biosensor. In this invention, the sweet taste receptor-functionalized biosensor is modified with sweet taste receptors TAS1R2 and TAS1R3; the sweet taste receptor-functionalized biosensor includes a biosensor chip, an electrochemical workstation, and a signal receiving and processing system; the biosensor chip is modified with sweet taste receptors TAS1R2 and TAS1R3. In this invention, the biosensor chip includes a surface plasmon resonance biosensor chip, a high electron mobility transistor biosensor chip, or a screen-printed electrode biosensor chip.

[0029] In this invention, preferably, the samples collected in each segment are prepared into sample solutions with several concentration gradients. These sample solutions are then reacted with a sweetness receptor-functionalized biosensor in order of increasing concentration. The current signal is detected, and the dissociation equilibrium constant K is calculated. D Value, in K D The numerical values ​​represent the affinity strength between the sample solution and the sweet taste receptors TAS1R2 and TAS1R3.

[0030] In this invention, it is preferable to prepare the initial concentration of each collected sample segment to 0.1 mg / mL (C1), and then perform a 10-fold serial dilution 10 times, with the concentration successively increasing to 10. -2 ~10 -11 mg / mL (C2~C11). In this invention, the reaction time between the sample solution and the sweetness receptor-functionalized biosensor is preferably 5~10 min, and the injection volume of the sample solution is preferably 50~100 μL. In this invention, the methods for detecting current changes include, but are not limited to, linear sweep voltammetry and differential pulse voltammetry, recording I at each concentration. DS -V DS Curve; In this invention, the common logarithm of the sample solution concentration is used as the abscissa, and the relative change in current is used as the ordinate to perform linear fitting, obtaining a linear fitting equation; The dissociation equilibrium constant K of the sample collected in each segment is calculated based on the linear fitting equation and the formula for calculating the dissociation equilibrium constant. D value.

[0031] dissociation constant (K) D The formula for calculating the value is:

[0032]

[0033] K = K A =1 / K D =[A b -A g ] / [A b [A] g ]; (1-2)

[0034] [A g ] / ΔI[A g ] / ΔI Max +K D / ΔI Max (1-3)

[0035] Where [A] b [A] represents the concentration of TAS1R2 or TAS1R3 protein. g [ represents the concentration of the sample, K and K] A As the associative constant, K D Let ΔI be the dissociation constant and ΔI be the change in current. Max This represents the maximum change in current.

[0036] After the reaction is completed, the present invention elutes sequentially with a non-specific eluent, a TAS1R2-specific eluent, and a TAS1R3-specific eluent, collecting the TAS1R2-specific eluent and the TAS1R3-specific eluent. In this invention, the non-specific eluent is preferably a PBS solution with a pH of 7.0–7.2; the TAS1R2-specific eluent is preferably a ginsenoside Rg1 solution; the concentration of the ginsenoside Rg1 solution is preferably 0.08–0.12 mM, more preferably 0.09–0.11 mM, and most preferably 0.1 mM; the number of elutions with the ginsenoside Rg1 solution is preferably 8–12 times, more preferably 9–11 times, and most preferably 10 times; the volume of each elution is preferably 40–60 μL, more preferably 45–55 μL, and most preferably 50 μL. In this invention, the TAS1R3-specific eluent is preferably an arginine solution; the concentration of the arginine solution is 0.08–0.12 mM, more preferably 0.09–0.11 mM, and most preferably 0.1 mM; the elution times of the arginine solution are preferably 8–12 times, more preferably 9–11 times, and most preferably 10 times; the elution volume for each elution is preferably 40–60 μL, more preferably 45–55 μL, and most preferably 50 μL. After collecting the TAS1R2-specific and TAS1R3-specific eluents, the collected components are preferably filtered using a 0.22 μm microporous membrane.

[0037] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0038] Surface plasmon resonance was purchased from Bionavis, Finland.

[0039] The high electron mobility transistors were purchased from the Institute of Semiconductors, Chinese Academy of Sciences.

[0040] The screen printing electrodes were purchased from Glass Carbon Technology (Weihai) Co., Ltd.

[0041] The TAS1R2 / TAS1R3 sweet taste receptors were purchased from Shenzhen Jingdan Biomedical Technology Co., Ltd.

[0042] The CHI660E electrochemical workstation and signal receiving and processing system were both purchased from Shanghai Chenhua Instrument Co., Ltd.

[0043] Example 1

[0044] Construction Method of TAS1R2 / TAS1R3 Functionalized Biosensors

[0045] Using AlGaAs / GaAs high electron mobility transistor chips or screen-printed electrodes as substrates, 3-mercaptopropionic acid solution is added to the gate or working electrode region of the chip, and the mixture is immersed at room temperature for 17–24 h to form gold-sulfur bonds. The chip surface is then cleaned with deionized water to remove residues, and an equal volume of a mixed solution of 20 mM EDC and 50 mM NHS is added. The reaction is carried out at room temperature for 15–20 min to form stable amine activation products for activating carboxyl groups. Finally, the working area of ​​the chip is cleaned with PBS solution, and the TAS1R2 / TAS1R3 sweet taste receptor is added. The mixture is then refrigerated at 4°C for 2–5 h. This mixture is then integrated with a CHI660E electrochemical workstation and signal receiving and processing system to obtain the TAS1R2 / TAS1R3 functionalized biosensor (see illustration). Figure 1 .

[0046] A three-stage enrichment and separation method for Tongren Niuhuang Qingxin Pills (A, B, and C stages).

[0047] Take 30g of Tongren Niuhuang Qingxin Pills, cut them into small pieces, and place them in a 1000mL dry round-bottom flask. Add 10 times the amount of pure water and a few boiling stones, shake to mix, and let stand at room temperature for 2 hours. Heat for 5 hours and collect the volatile oil fraction. Centrifuge the water extract in the round-bottom flask at 12000r for 10min and collect the supernatant 1. Place the centrifuged precipitate back into the round-bottom flask, add 10 times the amount of 50% (v / v) methanol aqueous solution, heat and reflux for 2 hours, centrifuge, and collect the supernatant 2. Combine the volatile oil fraction, supernatant 1, and supernatant 2, dispense them into 50mL centrifuge tubes, and then use a vacuum concentrator at 1650r / min, 30℃, and -50℃ cold trap to evaporate to dryness.

[0048] The components obtained by evaporation were separated using a reverse-phase silica gel column (SW-5222-012-SP) with water and ethanol as eluents, ranging from 5% to 95% ethanol ratio, at a flow rate of 10.0 mL / min. The entire wavelength range was collected, with a collection volume of 10–20 mL. Based on the UV results of the preparative liquid chromatography, the collected solutions with similar UV absorption wavelengths were combined and divided into three segments (numbers represent collection bottle numbers): Segment A (002–006), Segment B (007–009), and Segment C (011–014).

[0049] Interaction analysis of three samples (A, B, and C) of Tongren Niuhuang Qingxin Pill with TAS1R2 / TAS1R3 receptors

[0050] The remaining solids from samples A, B, and C were dissolved in PBS solution, and then completely dissolved by water bath heating or sonication. The initial concentration was prepared into a 0.1 mg / mL (C1) solution, which was then serially diluted 10 times in a 10-fold gradient, with each subsequent concentration being 10... -2 ~10 -11 mg / mL (C2~C 11 Sample solutions were injected sequentially into the TAS1R2 / TAS1R3 functionalized biosensor from low to high concentration, with an injection volume of 100 μL and a reaction time of 6 min. After the TAS1R2 / TAS1R3 receptors were fully bound to the solution, Ig at each concentration was recorded using an electrochemical workstation at room temperature. DS -V DS The curve was plotted with a voltage range of 0–2V, repeated three times. The linear relationship between the logarithm of concentration and the relative value of current change was investigated. The detection and analysis results are as follows: Figure 2 As shown.

[0051] Using the common logarithm of the sample solution concentration as the abscissa and the relative change in current as the ordinate, a linear fit was performed to obtain the linear fit equation. Based on the linear fit equation and the formula for calculating the dissociation equilibrium constant, the dissociation equilibrium constant K for each segment of the collected sample was calculated. D value.

[0052] dissociation constant (K) D The formula for calculating the value is:

[0053]

[0054] K = K A =1 / K D =[A b -A g ] / [A b [A] g ]; (1-2)

[0055] [A g] / ΔI[A g ] / ΔI Max +K D / ΔI Max (1-3)

[0056] Where [A] b [A] represents the concentrations of TAS1R2 and TAS1R3 proteins. g [ represents the concentration of the sample, K and K] A As the associative constant, K D Let ΔI be the dissociation constant and ΔI be the change in current. Max This represents the maximum change in current.

[0057] The concentration range for segment A is 0.1–10. -7 At mg / mL, the common logarithm of the concentration shows a good linear relationship with the corresponding relative change in current, and the linear fitting equation is y = -0.0193x - 0.1903(R²). 2 =0.9976); Calculate the K value of the interaction between the Tongren Niuhuang Qingxin Pill A sample solution and TAS1R2. D The value is 2.18 × 10 -7 g / mL;

[0058] The concentration range for segment B is 10. -2 ~10 -10 At a concentration of mg / mL, the common logarithm of the concentration shows a good linear relationship with the corresponding relative change in current, and the linear fitting equation is y = -0.0072x - 0.0603(R²). 2 =0.9105); Calculate the K value of the interaction between the Tongren Niuhuang Qingxin Pill B sample solution and TAS1R2. D The value is 3.37 × 10 -8 g / mL;

[0059] The concentration range in segment C is 10. -2 mg / mL~10 -9 At mg / mL, the common logarithm of the concentration shows a good linear relationship with the corresponding relative change in current, and the linear fitting equation is y = 0.0007x + 0.0035(R²). 2 =0.9712);

[0060] The K value of the interaction between the C-segment sample solution of Tongren Niuhuang Qingxin Pill and TAS1R2 was calculated. D The value is 4.80 × 10 -8 g / mL.

[0061] The above experiments revealed that Tongren Niuhuang Qingxin Pills bind more easily to the TAS1R3 receptor, and that segments B and C are more easily identified by biosensors than segment A. This confirms that the key quality attribute of sweetness, which can activate the TAS1R2 / TAS1R3 receptor, exists in all three segments of Tongren Niuhuang Qingxin Pills.

[0062] Identification of the key quality attribute of sweetness in Tongren Niuhuang Qingxin Pills

[0063] 1) Elution process: After the A, B, and C solutions of Tongren Niuhuang Qingxin Pill were combined with the TAS1R2 / TAS1R3 functionalized biosensor, the unbound components were eluted with PBS solution. Then, the compounds bound to the TAS1R2 receptor in the three samples of A, B, and C were eluted with 0.1 mM ginsenoside Rg1 solution, a TAS1R2-specific elution buffer, 50 μL at a time, repeated 10 times. The eluted solutions were labeled as R2-TNA, R2-TNB, and R2-TNC, respectively. The compounds bound to the TAS1R3 receptor in the three samples of A, B, and C were specifically eluted with 0.1 mM arginine solution, 50 μL at a time, repeated 10 times. The eluted solutions were labeled as R3-TNA, R3-TNB, and R3-TNC, respectively. The solutions were then filtered through a 0.22 μm microporous membrane.

[0064] 2) Chromatographic conditions

[0065] WaterACQUITY An HSS T3 column (2.1 × 100 mm, 1.8 μm) was used at a column temperature of 35 °C, with an injection volume of 2 μL. The mobile phase ratios and flow rates were 0.1% formic acid aqueous solution (phase A) and acetonitrile (phase B), respectively. The elution gradient and flow rate are shown in Table 1.

[0066] Table 1 Elution gradient and flow rate of mobile phase

[0067]

[0068]

[0069] 3) Mass spectrometry conditions

[0070] Measurements were performed using an LTQ-Orbitrap XL mass spectrometer in both positive and negative modes, with an ESI ion source at a temperature of 350°C. In positive ion mode: ionization source voltage 4 kV, capillary voltage 25 V, and tube lens voltage 110 V; in negative ion mode: ionization source voltage -3 kV, capillary voltage -35 V, and tube lens voltage -110 V; collision energy 35 eV; sheath gas (N2) flow rate: 40 arb; auxiliary gas (N2) flow rate: 20 arb; capillary temperature 350°C. The primary mass spectra were obtained using full-scan mode (mass scan range m / z 50–1400; detection resolution 70,000). Secondary mass spectrometry employed parentlist (PL) scanning and collision-induced dissociation (CID) fragmentation.

[0071] 4) Mass Spectrometry Data Analysis: Through literature retrieval and full-formula mass spectrometry analysis, chemical component information of all traditional Chinese medicines in Tongren Niuhuang Qingxin Pill was collected, including compound name, molecular formula, relative molecular mass, molecular structure, and structural type. Xcalibur 2.1 software was used to process and analyze the mass spectrometry data, utilizing primary and secondary spectra information (mass error ±5×10⁻⁶). 6 The method involves collecting secondary fragment information using data acquisition modes such as retention time, fragmentation fragments, and PL, and then combining this information with literature, reference standards, and databases to identify and analyze the compound, inferring the fragmentation mode of the obtained compound; and using the lipid-water partition coefficient (AlogP) value to infer the isomers of the compound.

[0072] The total ion current was finally analyzed by mass spectrometry. Figure 3 and Figure 4 The components identified by the TAS1R2 / TAS1R3 functionalized biosensor include glucose, fructose, sucrose, maltose, glycyrrhizin, apigenin, 5-O-methylvisamidol, ginsenoside Rg1, glycyrrhizic acid, paeonol, paeoniflorin, paeoniflorin, typhain, arginine, 6-hydroxydaidzein, and atractylodes lactone II. These substances are the key quality attributes of the sweet taste of Tongrentang Niuhuang Qingxin Pill. The specific analysis results are shown in Tables 2 and 3.

[0073] Table 2. Analysis results of key quality attributes of sweetness bound to the TAS1R2 receptor.

[0074]

[0075]

[0076]

[0077] Table 3. Analysis results of key quality attributes of sweetness bound to the TAS1R3 receptor.

[0078]

[0079]

[0080] As can be seen from the above embodiments, the present invention, based on T1R2 / T1R3 functionalized biosensors combined with mass spectrometry technology, has realized the identification of the key quality attribute of sweetness in Tongren Niuhuang Qingxin Pill, providing feasible technical support and reference ideas for the identification of key quality attributes of sweetness in major Chinese medicine products in the field of Chinese medicine manufacturing.

[0081] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for detecting the key quality attribute of sweetness in traditional Chinese medicine preparations, characterized in that, Includes the following steps: 1) After pretreatment of traditional Chinese medicine preparations, the samples are separated by preparative liquid chromatography and collected in segments; 2) The sample solution collected in each segment is reacted with a sweet taste receptor-functionalized biosensor; the sweet taste receptor-functionalized biosensor is modified with sweet taste receptors TAS1R2 and TAS1R3; 3) After the reaction is complete, elute sequentially with non-specific elution buffer, TAS1R2 specific elution buffer and TAS1R3 specific elution buffer, and collect the TAS1R2 specific elution fraction and the TAS1R3 specific elution fraction. 4) The key quality attribute of sweetness in traditional Chinese medicine was obtained by detecting the TAS1R2-specific eluent and TAS1R3-specific eluent collected in step 3). The reaction time between the sample solution and the sweetness receptor-functionalized biosensor is 5–10 min; the injection volume of the sample solution is 50–100 μL. The sweet taste receptor-functionalized biosensor includes a biosensor chip, an electrochemical workstation, and a signal receiving and processing system; the biosensor chip is modified with sweet taste receptors TAS1R2 and TAS1R3; The non-specific elution buffer mentioned in step 3) is PBS solution; the specific elution buffer for TAS1R2 is ginsenoside Rg1 solution. The specific elution buffer for TAS1R3 is an arginine solution; The concentration of the ginsenoside Rg1 solution is 0.08–0.12 mM, the number of elutions is 8–12, and the elution volume for each elution is 40–60 μL. The concentration of the arginine solution is 0.08–0.12 mM, the number of elutions is 8–12, and the elution volume for each elution is 40–60 μL.

2. The method according to claim 1, characterized in that, The traditional Chinese medicine mentioned includes Tongren Niuhuang Qingxin Wan.

3. The method according to claim 1, characterized in that, The biosensor chip includes a surface plasmon resonance biosensor chip, a high electron mobility transistor biosensor chip, or a screen-printed electrode biosensor chip.

4. The method according to claim 1 or 2, characterized in that, In step 4), the results of ultra-high performance liquid chromatography-mass spectrometry are compared with all known chemical components of traditional Chinese medicine to determine the key quality attribute of sweetness in traditional Chinese medicine.

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