Ginkgo biloba armillaria oral liquid detection method based on supramolecular fluorescence sensing and application thereof

Abstract

The invention belongs to the field of biochemical technology detection, and particularly relates to a gingko armillaria mellea oral solution detection method based on supramolecular fluorescence sensing and application of the gingko armillaria mellea oral solution detection method. According to the invention, a subject-object pair composed of a macrocyclic subject and a dye object is used, and the ginkgo armillaria oral solution is detected through supramolecular fluorescence sensing. The method is simple, convenient and rapid to operate, and the precision, repeatability and stability all meet the requirements. The method can be used for monitoring the consistency of multiple batches of products in the production and product detection of the gingko armillaria mellea oral liquid, and can be used for rapidly monitoring the ratio of the gingko leaf extract to the armillaria mellea powder in the gingko armillaria mellea oral liquid.

Description

Technical Field

[0001] The invention belongs to the field of biochemical technology detection, and in particular relates to a detection method of Ginkgo honey ring oral solution based on supramolecular fluorescence sensing and application thereof. Background Art

[0002] The incidence of cardiovascular and cerebrovascular diseases continues to rise worldwide, bringing an increasingly heavy burden to public health. In recent years, traditional Chinese medicine has shown obvious characteristics and advantages in the prevention and treatment of cardiovascular and cerebrovascular diseases, and has played a vital role in protecting people's health. Ginkgo honey ring oral solution, as a Chinese medicine compound preparation, contains ginkgo leaf extract and Armillaria powder, which has the effect of dilating coronary arteries and protecting cardiovascular and cerebrovascular vessels, and is often used in the clinical prevention and treatment of cardiovascular and cerebrovascular diseases. The current standard of Ginkgo honey ring oral solution is included in the National Drug Western Medicine Standard (WS1-XG-004-2001), and its content inspection items only involve flavonoid compounds quercetin, kaempferol and isorhamnetin. However, the chemical composition of the preparation is complex, and the current method cannot comprehensively evaluate the overall quality of Ginkgo honey ring oral solution. For this reason, it is necessary to conduct in-depth research on the quality evaluation method of Ginkgo honey ring oral solution.

[0003] At present, classical chromatography techniques, including high performance liquid chromatography, gas chromatography and thin layer chromatography, are widely used in the quality evaluation and standardization research of traditional Chinese medicines due to their strong specificity, good stability and good reproducibility, and play an important role in the quality evaluation research of traditional Chinese medicines. However, they still have limitations such as complex operation and long detection time in fully revealing the quality characteristics of some complex traditional Chinese medicine products. With the increasing demand for the quality evaluation and control of traditional Chinese medicines, it is urgent to develop a simpler and more convenient method to achieve rapid analysis of the quality of Ginkgo Biloba Oral Solution. Summary of the invention

[0004] In view of the above technical problems, the present invention provides a method for detecting Ginkgo Honey Ring oral solution based on supramolecular fluorescence sensing and its application. The detection and evaluation method provided by the present invention is simple to operate, can realize rapid detection of Ginkgo Honey Ring oral solution, can be used to evaluate the consistency of Ginkgo Honey Ring oral solution batch by batch, and can be used to construct a fluorescence fingerprint, thereby facilitating rapid analysis and evaluation of the quality of Ginkgo Honey Ring oral solution.

[0005] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0006] The first aspect of the present invention provides a method for detecting Ginkgo honey ring oral liquid based on supramolecular fluorescence sensing: Ginkgo honey ring oral liquid is added to a host-guest pair solution composed of a macrocyclic host and a dye guest, and the Ginkgo honey ring oral liquid is detected by a change in a fluorescence signal; the host-guest pair is selected from at least one of CAC4A (carboxy azocalix [4] arene)·RhB (rhodamine B), SAC5A (sulfonated azocalix [5] arene)·RhB and QAC5A (5,11,17,23,29-penta (trimethylammonium) -31,32,33,34,35-penta (4-methylpentyloxy)calix [5] arene)·EY (eosin Y disodium salt).

[0007] The present invention has found through research that after adding Ginkgo Honey Oral Liquid to the CAC4A·RhB, SAC5A·RhB, or QAC5A·EY host-guest pair solution, the chemical components in the Ginkgo Honey Oral Liquid can compete with the dye guest to bind to the macrocyclic host cavity, displace the dye guest from the host-guest complex, and restore the luminescent performance of the dye guest in the solution, thereby sensitizing the fluorescent signal. Methodological verification shows that the precision, repeatability, and stability of the Ginkgo Honey Oral Liquid detected by the above-mentioned host-guest pair all meet the requirements. Therefore, the present invention detects Ginkgo Honey Oral Liquid with the above-mentioned host-guest pair. Compared with analytical methods such as high performance liquid chromatography, gas chromatography, and thin layer chromatography, this method is simple and quick to operate.

[0008] Preferably, the solvent of the host-guest pair solution is a HEPES buffer solution, the HEPES concentration is 10 mM, and the pH is 7.4.

[0009] Preferably, when the host-guest pair is CAC4A·RhB, the excitation wavelength for detecting the fluorescence signal is 554 nm. Further preferably, the concentration of CAC4A in the host-guest pair solution is 1 μM, and the concentration of RhB is 0.8 μM.

[0010] Preferably, when the host-guest pair is SAC5A·RhB, the excitation wavelength for detecting the fluorescence signal is 554 nm. Further preferably, the concentration of SAC5A in the host-guest pair solution is 1 μM, and the concentration of RhB is 0.8 μM.

[0011] Preferably, when the host-guest pair is QAC5A·EY, the excitation wavelength for detecting the fluorescence signal is 517 nm. Further preferably, the concentration of QAC5A in the host-guest pair solution is 0.4 μM, and the concentration of EY is 0.5 μM.

[0012] Preferably, the host-guest pair is QAC5A·EY. The host-guest pair QAC5A·EY has a strong bonding ability to the representative compounds in the Ginkgo Honey Ring oral solution, and has a better performance in sensing and detecting the main active ingredients in the Ginkgo Honey Ring oral solution.

[0013] The second aspect of the present invention provides the application of the above detection method in monitoring the consistency between batches of Ginkgo Honey Ring Oral Liquid.

[0014] Preferably, the host-guest pairs are CAC4A·RhB, SAC5A·RhB and QAC5A·EY. The three host-guest pairs constitute a ternary calixarene·fluorescent dye sensing array, which can accurately reflect the consistency between different batches of Ginkgo Honey Oral Liquid.

[0015] The above detection method is simple and quick to operate, and can be used for consistency monitoring between multiple batches in the production and product testing of Ginkgo Honey Ring Oral Liquid.

[0016] The third aspect of the present invention provides the use of the above detection method in detecting the ratio of ginkgo leaf extract and Armillaria mellea powder in Ginkgo Honey Ring oral liquid, wherein the host-guest pair is QAC5A·EY.

[0017] The fourth aspect of the present invention provides the application of the above detection method in constructing the fluorescent fingerprint of Ginkgo Honey Ring Oral Liquid.

[0018] The fifth aspect of the present invention provides a method for constructing a fluorescent fingerprint of Ginkgo honey ring oral liquid: adding Ginkgo honey ring oral liquid to a host-guest pair solution composed of a macrocyclic host and a dye guest, and detecting a fluorescent signal; the host-guest pair is CAC4A·RhB, SAC5A·RhB and QAC5A·EY; when the host-guest pair is CAC4A·RhB, the excitation wavelength for detecting the fluorescent signal is 554nm, and the concentration of CAC4A in the host-guest pair solution is 1μM, and the concentration of RhB is 0.8μM; when the host-guest pair is SAC5A·RhB, the excitation wavelength for detecting the fluorescence signal is 554nm, the concentration of SAC5A in the host-guest pair solution is 1μM, and the concentration of RhB is 0.8μM; when the host-guest pair is QAC5A·EY, the excitation wavelength for detecting the fluorescence signal is 517nm, the concentration of QAC5A in the host-guest pair solution is 0.4μM, and the concentration of EY is 0.5μM.

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

[0020] The present invention is based on the indicator displacement assay strategy (IDA), taking the chemical components in the Ginkgo honey ring oral solution as the competitive object, adding them to the macrocyclic dye sensing unit CAC4A·RhB, SAC5A·RhB or QAC5A·EY, and using the fluorescence change of the dye in the system to reflect the quantitative change of the sensing chemical components in the Ginkgo honey ring oral solution. The present invention evaluates the bonding constants of the representative components in the Ginkgo honey ring oral solution and the above-mentioned macrocyclic dye sensing unit. The results show that the complex formed by the macrocyclic main body and the representative components in the Ginkgo honey ring oral solution is stable, thereby verifying the feasibility and rationality of the above-mentioned macrocyclic dye sensing unit in the quality evaluation of the Ginkgo honey ring oral solution. The ternary supramolecular sensing array constructed by CAC4A·RhB, SAC5A·RhB and QAC5A·EY can be used for rapid quality evaluation of the Ginkgo honey ring oral solution, for batch consistency detection of different batches of Ginkgo honey ring oral solution, and can also be used to construct the fluorescence fingerprint of the Ginkgo honey ring oral solution. QAC5A·EY can also be used to clearly distinguish Ginkgo biloba sample solutions prepared with different ratios of Ginkgo biloba leaf extract and Armillaria mellea powder, and can be used for process control of Ginkgo biloba oral solution preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The schematic diagram of supramolecular fluorescence sensing of Ginkgo honey ring oral solution based on indicator replacement strategy;

[0022] Figure 2 The effect of Ginkgo honey ring oral solution (YMOS) on the fluorescence spectrum of CAC4A·RhB (1μM / 0.8μM) in Example 1 (λ ex =554nm,λ em =576nm);

[0023] Figure 3 The effect of Ginkgo honey ring oral solution (YMOS) on the fluorescence spectrum of SAC5A·RhB (1μM / 0.8μM) in Example 1 (λ ex =554nm,λ em =576nm);

[0024] Figure 4 The effect of Ginkgo honey ring oral solution (YMOS) on the host-guest fluorescence spectrum of QAC5A·EY (0.4μM / 0.5μM) in Example 1 (λ ex =517nm,λ em =537nm);

[0025] Figure 5 To test the methodological validation results in the example;

[0026] Figure 6 The effect of rutin (QGR) on the fluorescence intensity of QAC5A·EY (0.4μM / 0.5μM) host-guest pair and the results of bond constant determination (λ ex =517nm,λ em =537nm); a is the fluorescence emission spectrum of the host-guest pair of QAC5A·EY (0.4μM / 0.5μM) titrated by MeOH (0~19μL); b is the fluorescence emission spectrum of EY (0.5μM) titrated by rutin solution (0~53.21μM); c is the competitive titration curve of the host-guest pair of rutin solution (0~44.33μM) and QAC5A·EY (0.4μM / 0.5μM); d is the nonlinear fitting curve of the bonding between rutin and QAC5A (n=3);

[0027] Figure 7 The effect of sodium benzoate (SoB) on the fluorescence intensity of the host-guest pair of SAC5A·RhB (1μM / 0.8μM) and the determination results of the bonding constant (λ ex =554nm,λ em =576nm); a is the emission spectrum of sodium benzoate solution (0-1803.28μM) titrated with RhB (0.8μM); b is the fluorescence emission spectrum of sodium benzoate solution (0-1666.67μM) titrated with SAC5A·RhB (1μM / 0.8μM) host and guest; c is the nonlinear fitting curve of sodium benzoate solution and SAC5A (n=3);

[0028] Figure 8 The effect of sodium benzoate on the fluorescence intensity of QAC5A·EY (0.4μM / 0.5μM) host-guest pair and the bond constant determination results (λ ex =517nm,λ em =537nm); a is the fluorescence emission spectrum of sodium benzoate solution (0-340.25μM) titrated with EY (0.5μM); b is the fluorescence emission spectrum of sodium benzoate solution (0-81.53μM) titrated with QAC5A·EY (0.4μM / 0.5μM) host-guest pair; c is the nonlinear fitting curve of the bonding constant of sodium benzoate and QAC5A (n=3);

[0029] Fig. 9 The effect of GinA on the fluorescence intensity of CAC4A·RhB (1 μM / 0.8 μM) host-guest pair and the results of the bond constant determination (λ ex =554nm,λ em=576nm); a is the fluorescence emission spectrum of the host-guest pair of CAC4A·RhB (1μM / 0.8μM) titrated by MeOH (0~29μL); b is the fluorescence emission spectrum of RhB (0.8μM) titrated by Ginkgolide A (0~65.76μM); c is the fluorescence emission spectrum of CAC4A·RhB (1μM / 0.8μM) titrated by Ginkgolide A (0~64.58μM); d is the nonlinear fitting curve of the bonding between Ginkgolide A and CAC4A (n=3);

[0030] Fig.10 The effect of ginkgolide A on the fluorescence intensity of SAC5A·RhB (1μM / 0.8μM) host-guest pair and the bond constant determination results (λ ex =554nm,λ em =576nm); a is the fluorescence emission spectrum of the host-guest pair of SAC5A·RhB (1μM / 0.8μM) titrated by MeOH (0-19μL); b is the fluorescence emission spectrum of the host-guest pair of SAC5A·RhB (1μM / 0.8μM) titrated by Ginkgolide A solution (0-35.57μM); c is the nonlinear fitting curve of the bonding between Ginkgolide A and SAC5A (n=3);

[0031] Fig.11 The effect of Ginkgolide A on the fluorescence intensity of QAC5A·EY (0.4μM / 0.5μM) host-guest pair and the bond constant determination results (λ ex =517nm,λ em =537nm); a is the fluorescence emission spectrum of Ginkgolide A solution (0-78.16μM) titrated with EY (0.5μM); b is the fluorescence emission spectrum of Ginkgolide A solution (0-69.03μM) titrated with QAC5A·EY (0.4μM / 0.5μM) host-guest pair; c is the nonlinear fitting curve of the bonding between Ginkgolide A and QAC5A (n=3);

[0032] Fig.12 The effect of uridine (Uri) on the fluorescence intensity of the host-guest pair of SAC5A·RhB (1μM / 0.8μM) and the determination results of the bonding constant (λ ex =554nm,λ em =576nm); a is the fluorescence emission spectrum of uridine solution (0-4161.29μM) titrated with RhB (0.8μM); b is the fluorescence emission spectrum of uridine solution (0-3975.90μM) titrated with SAC5A·RhB (1μM / 0.8μM) host-guest pair; c is the nonlinear fitting curve of uridine solution and SAC5A bonding (n=3);

[0033] Fig.13 The effect of uridine on the fluorescence intensity of QAC5A·EY (0.4μM / 0.5μM) host-guest pair and the bond constant determination results (λ ex =517nm,λ em =537nm); a is the fluorescence emission spectrum of uridine solution (0-653.20μM) titrated with EY (0.5μM); b is the fluorescence emission spectrum of uridine solution (0-176.47μM) titrated with QAC5A·EY (0.4μM / 0.5μM) host-guest pair; c is the nonlinear fitting curve of uridine and QAC5A bonding (n=3);

[0034] Fig.14 The effect of protocatechuic acid (Pra) on the fluorescence intensity of QAC5A·EY (0.4μM / 0.5μM) host-guest pair and the results of bond constant determination (λ ex =517nm,λ em =537nm); a is the fluorescence emission spectrum of protocatechuic acid solution (0-653.20μM) titrated with EY (0.5μM); b is the fluorescence emission spectrum of protocatechuic acid solution (0-357.29μM) titrated with QAC5A·EY (0.4μM / 0.5μM) host-guest pair; c is the nonlinear fitting curve of the bonding between protocatechuic acid and QAC5A (n=3);

[0035] Fig.15 It is a line graph of the measurement results of 6 batches of qualified Ginkgo Honey Ring oral solution in Example 3; a is a line graph of the measurement results of 6 batches of qualified Ginkgo Honey Ring oral solution by CAC4A·RhB sensor unit (n=3), b is a line graph of the measurement results of 6 batches of qualified Ginkgo Honey Ring oral solution by SAC5A·RhB sensor unit (n=3); c is a line graph of the measurement results of 6 batches of qualified Ginkgo Honey Ring oral solution by QAC5A·EY sensor unit (n=3);

[0036] Fig.16 The P values ​​(I / I0, the ratio of the host-guest pair fluorescence intensity values ​​before (I0) and after (I) the addition of Ginkgo Honey Ring oral solution) of 6 batches of qualified Ginkgo Honey Ring oral solution measured by different sensing units in Example 3;

[0037] Fig.17 It is a radar chart of 6 batches of qualified Ginkgo Honey Ring oral solution in Example 4;

[0038] Fig.18 The batch-to-batch consistency of 6 batches of qualified Ginkgo Honey Ring Oral Solution in Example 4;

[0039] Fig.19The results of the QAC5A·EY sensor unit in Example 5 on the homemade Ginkgo honey ring sample solutions with different proportions (***P<0.001). DETAILED DESCRIPTION

[0040] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work fall within the scope of protection of the present invention.

[0041] The active ingredients of Ginkgo Biloba Oral Solution are Ginkgo Biloba Leaf Extract and Armillaria Armillaria Powder, and the chemical composition is complex. However, the content inspection items in the current Ginkgo Biloba Oral Solution standard only involve flavonoid compounds, which cannot comprehensively evaluate the overall quality of Ginkgo Biloba Oral Solution. The raw material feed ratio is very important for product quality and the safety and effectiveness of clinical medication. Therefore, it is necessary to establish a method that can reflect the content of Ginkgo Biloba Leaf Extract and the raw material feed ratio in Ginkgo Biloba Oral Solution. Existing analytical methods include high performance liquid chromatography, gas chromatography and thin layer chromatography. Although each has different advantages in the quality evaluation of traditional Chinese medicine, it has limitations such as complex operation and long detection time. It is still necessary to develop a simpler and more convenient method to achieve rapid analysis of the quality of Ginkgo Biloba Oral Solution.

[0042] The embodiment of the present invention provides a method for detecting ginkgo leaf oral solution in Ginkgo Honey Loop oral liquid based on an indicator displacement analysis strategy: Ginkgo Honey Loop oral liquid is added to a host-guest pair solution consisting of a macrocyclic host and a dye guest, and the Ginkgo Honey Loop oral liquid is detected by a change in a fluorescence signal; the host-guest pair is selected from at least one of CAC4A·RhB, SAC5A·RhB and QAC5A·EY.

[0043] This method uses the chemical components in the Ginkgo Honey Ring oral solution as the competitive object, and uses the fluorescence change of the dye in the macrocyclic dye sensing unit to reflect the quantitative change of the sensing chemical components in the Ginkgo Honey Ring oral solution, thereby realizing the detection of Ginkgo leaf extract. The principle diagram of this detection method is shown in the figure Figure 1 shown.

[0044] The embodiments of the present invention are further described below with reference to a plurality of embodiments.

[0045] In the following examples, Ginkgo Biloba Oral Solution was provided by Buchang Qionglai Tianyin Pharmaceutical Co., Ltd., with batch numbers 211013, 211014, 211015, 211016, 211018, and 211019, respectively; Ginkgo Biloba Leaf Extract (batch numbers 210607-210804) and Armillaria Armillaria Extract were provided by Qionglai Tianyin Pharmaceutical Co., Ltd.; Rhodamine B (RhB, SHBL2114), Eosin Y disodium salt (EY, MKCG1738), trans-4-[4-(dimethylamino)styryl]-1-methylpyridinium iodide (trans-4-[4-(dimethydamino)styryl]-1-methylpyridinium iodide (DSMI, 07522EE), lucigenin (LCG, MKCL0744), β-cyclodextrin (β-clodextrins, β-CD, Z31J11Y119887), and cucurbit[6]uril (CB[6], BCBW1923) were all purchased from Sigma-Aldrich, USA; sodium benzoate (SoB, 83FCI), protocatechuic acid (Pra, H21J9Z64031), rutin (QGR, Y24F11Y17051), ginkgolide A (GinA, D26J9G66649), and uridine (Uri, 110753-201817) were all of analytical grade and purchased from Shanghai Yuanye Biotechnology Co., Ltd. with a purity greater than 98%; methylene blue (methylene blue, 83FCI) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. blue (MB, K50873270003) was purchased from Merck KGaA, Germany; oxazine 1 (OX1, AAT-89) was purchased from Wuhan Aimejie Technology Co., Ltd.; Tween 80 was purchased from Shanghai Yuanye Biotechnology Co., Ltd. Sulfonated calix[4]arenes (SC4A), sulfonated calix[5]arenes (SC5A), sulfonated azocalix[5]arene (SAC5A), carboxylazocalix[4]arene (CAC4A) and 5,11,17,23,29-penta(trimethylammonium)-31,32,33,34,35-penta(4-methylpentyloxy)calix[5]arene (QAC5A) were from the School of Chemistry, Nankai University. N-(2-Hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES, H05500K101) was of analytical grade and purchased from Tianjin Xiensi Biochemical Technology Co., Ltd.Sodium hydroxide (NaOH, 2021-01-21) was analytically pure and purchased from Tianjin Huihang Chemical Technology Co., Ltd. Methanol (chromatographically pure) was purchased from Sigma-Aldrich, USA. The above reagents and materials can be purchased through commercial channels. Unless otherwise specified, all reagents and materials are used directly after purchase without further treatment. Ultrapure water was prepared using a multifunctional ultrapure water system.

[0046] Example 1

[0047] This example investigates the interference of Ginkgo Biloba Oral Solution on the fluorescence signals of CAC4A·RhB, SAC5A·RhB, QAC5A·EY host-guest pairs and dye molecules.

[0048] 1. Solution preparation

[0049] Preparation of HEPES (10 mM, pH 7.4) buffer solution: weigh 2.38 g HEPES, add 0.9 L ultrapure water to dissolve, and at 25 °C, use sodium hydroxide solution (0.1 mol·L -1 ) The solution pH was adjusted to 7.4 and filled to 1 L with ultrapure water to obtain a 10 mM buffer solution. The buffer pH was measured by a calibrated Sartorius pp-20 precision pH meter.

[0050] Preparation of macrocyclic main body stock solution: Take 1.87 mg, 1.10 mg and 1.15 mg of SAC5A, CAC4A and QAC5A powder respectively, accurately weigh, dissolve in ultrapure water, prepare stock solution, store at 4°C for use.

[0051] Preparation of dye guest stock solution: Take 1.05 mg and 1.38 mg of RhB and EY powder respectively, accurately weigh them, dissolve them in ultrapure water, prepare stock solution, store it at 4°C for later use.

[0052] Preparation of competitive guest solution: Take an appropriate amount of Ginkgo Biloba Oral Solution and place it in an EP tube, store it at 4°C for later use.

[0053] 2. Fluorescence spectroscopy experimental conditions

[0054] A Varian Cary Eclipse fluorescence spectrometer (Agilent Technologies, USA) was used, equipped with a Cary Single-cuvette Peltier temperature control device. The quartz cell (10 × 10 × 45 mm 3 The optical path length of the sample was 10 mm. The titration experiment was carried out at room temperature (25°C).

[0055] 3. Effect of Ginkgo Honey Ring Oral Solution on the Fluorescence Intensity of CAC4A·RhB Host-Guest Pair, SAC5A·RhB Host-Guest Pair, and QAC5A·EY Host-Guest Pair

[0056] The macrocyclic host stock solution and the dye guest stock solution were diluted with HEPES buffer solution, and the host-guest pair solution of the required final concentration was prepared in the fluorescence pool. The fluorescence competitive titration method was used to determine the change in the fluorescence intensity of the host-guest pair before and after the addition of the Ginkgo Honey Ring oral solution containing the dye guest and the macrocyclic host (the concentration of the dye guest and the macrocyclic host in the host-guest pair solution was the same to ensure that the concentration of the dye host-guest in the fluorescence pool remained unchanged during the titration process). The fluorescence emission spectrum when the solution reached equilibrium was recorded, and the fluorescence intensity of the dye at the maximum emission wavelength was I0. After the Ginkgo Honey Ring oral solution was added, the fluorescence emission spectrum when equilibrium was reached was recorded, and the maximum fluorescence intensity of the system was I at this time.

[0057] It can be seen from the results that after adding the Ginkgo Honey Oral Solution containing RhB (0.8 μM) to the RhB solution (0.8 μM, final concentration), the fluorescence signal did not change significantly; after adding 5 μL of the Ginkgo Honey Oral Solution containing the CAC4A·RhB host-guest pair (1.0 μM / 0.8 μM) to the CAC4A·RhB host-guest pair solution (1.0 μM / 0.8 μM, final concentration), the fluorescence signal changed significantly (|ΔI|=I-I0, 288). The obvious change in the fluorescence signal is due to the competition between the analyte in the Ginkgo Honey Oral Solution and RhB for binding to the CAC4A cavity, displacing RhB from the CAC4A·RhB host-guest complex, and RhB recovering its luminescence performance in the solution, and the fluorescence signal is sensitized (Switch-on), such as Figure 2 As shown; when the main body of the macrocycle is SAC5A, the fluorescence signal changes more significantly (|ΔI|=I-I0,538), as shown Figure 3 As shown, it indicates that SAC5A with sulfonic acid substitution and enlarged cavity has stronger binding with the analyte of Ginkgo Biloba Oral Solution.

[0058] The EY stock solution was diluted with HEPES buffer solution to prepare a 0.5 μM EY solution, which was placed in a fluorescence cell. The Ginkgo honeysuckle oral solution (5 μL) containing EY (0.5 μM) was added dropwise to the EY solution (0.5 μM, final concentration). The EY fluorescence signal did not change ( Figure 4 ), indicating that the Ginkgo Armillaria oral solution had little effect on the fluorescence signal of the dye.

[0059] The QAC5A stock solution and the EY stock solution were diluted with HEPES buffer solution to prepare a QAC5A·EY (0.4μM / 0.5μM) host-guest pair solution, which was placed in a fluorescence cell. The Ginkgo Biloba Oral Solution containing the QAC5A·EY host-guest pair (0.4μM / 0.5μM) was added to the QAC5A·EY host-guest pair solution (0.4μM / 0.5μM). The analyte of the Ginkgo Biloba Oral Solution competed with EY for binding to the cavity of QAC5A, displacing EY from the QAC5A·EY host-guest complex, and EY restored its luminescent properties in the solution, and the fluorescence signal was sensitized (|ΔI|=I-I0, 128), as shown in FIG. Figure 4 shown.

[0060] It can be seen from the above results that in HEPES (10 mM, pH 7.4, 25°C) solution, CAC4A·RhB, SAC5A·RhB, and QAC5A·EY host-guest pairs can be used as sensing units to detect Ginkgo biloba oral solution.

[0061] Test Example 1

[0062] This test example methodologically investigated the method by which the Ginkgo Biloba Oral Solution in Example 1 interfered with the fluorescence signals of the host-guest pairs of CAC4A·RhB, SAC5A·RhB, and QAC5A·EY.

[0063] 1. Intraday precision test

[0064] According to the method of Example 1, the macrocyclic host stock solution and the dye guest stock solution were diluted with HEPES buffer solution to prepare the required final concentration in the fluorescence cell, and the intra-day precision of the same Ginkgo honey ring oral solution was investigated using CAC4A·RhB (1μM / 0.8μM), SAC5A·RhB (1μM / 0.8μM) and QAC5A·EY (0.4μM / 0.5μM) host-guest pair solutions, respectively. P=I / I0 was used as the evaluation index. The obtained average values ​​and RSD values ​​are shown in Table 1. Figure 5 As shown in Figure (a), the RSD values ​​of the six measurement data of the three sensor units were all less than 1%, indicating that the intra-day precision and instrument precision of this method were good.

[0065] Table 1 Intra-day precision test results (n=6)

[0066]

[0067] 2. Daytime precision test

[0068] According to the method of Example 1, the macrocyclic host stock solution and the dye guest stock solution were diluted with HEPES buffer solution to prepare the required final concentration in the fluorescence cell, and the host-guest pair solutions of CAC4A·RhB (1.0μM / 0.8μM), SAC5A·RhB (1.0μM / 0.8μM) and QAC5A·EY (0.4μM / 0.5μM) were used to investigate the daytime precision of the same Ginkgo honey ring oral solution for 3 consecutive days. P=I / I0 was used as the evaluation index. The obtained average values ​​and RSD values ​​are shown in Table 2. Figure 5 As shown in Figure (b), the RSD values ​​of the six measurement data of the three sensor units were all less than 6%, indicating that the method had good inter-day precision.

[0069] Table 2 Results of inter-day precision test (n=3)

[0070]

[0071] 3. Repeatability test

[0072] According to the method of Example 1, the macrocyclic host stock solution and the dye guest stock solution were diluted with HEPES buffer solution to prepare the required final concentration in the fluorescence cell, and CAC4A·RhB (1.0μM / 0.8μM), SAC5A·RhB (1.0μM / 0.8μM) and QAC5A·EY (0.4μM / 0.5μM) host-guest pair solutions were used to measure 6 Ginkgo honey ring oral solutions prepared in parallel, and P=I / I0 was used as the evaluation index. The obtained average values ​​and RSD values ​​are shown in Table 3, Figure 5 As shown in Figure (c), the RSD values ​​of the six measurement data of the three sensor units were all less than 6%, indicating that the method had good repeatability.

[0073] Table 3 Repeatability test results (n=6)

[0074]

[0075] 4. Stability test

[0076] According to the method of Example 1, the macrocyclic host stock solution and the dye guest stock solution were diluted with HEPES buffer solution to prepare the required final concentration in the fluorescence cell, and the host-guest pair solutions of CAC4A·RhB (1.0μM / 0.8μM), SAC5A·RhB (1.0μM / 0.8μM) and QAC5A·EY (0.4μM / 0.5μM) were used to investigate the sample stability of the same Ginkgo honey ring oral solution at the same time point, with P=I / I0 as the evaluation index. The obtained average values ​​and RSD values ​​are shown in Table 4. Figure 5 As shown in Figure (d), the RSD values ​​of the six measurement data of the three sensor units are all less than 5%, indicating that the sample has good stability.

[0077] Table 4 Stability test results (n=6)

[0078]

[0079] Example 2

[0080] This example investigates the binding between the representative components of the Ginkgo Honey Ring oral solution and the macrocyclic host.

[0081] Based on the study of the chemical basis of Ginkgo Biloba Oral Solution, the ingredients and additives with higher content in the preparation were selected as representative ingredients: rutin was selected from flavonoid compounds, ginkgolide A was selected from terpenoid lactone compounds, protocatechuic acid was selected from organic acid compounds, uridine was selected from nucleoside compounds, and sodium benzoate was selected from preservatives and flavoring agents. The bonding constants (Associate constant, K) between the main molecule and the above chemical components were investigated respectively. a / M -1 ). The bonding constant is the physical constant between the host and guest pairs in supramolecular chemistry. The larger the bonding constant is, the stronger the bonding ability between the macrocyclic host and the fluorescent molecule or compound is, and the more stable the complex formed by the host-guest inclusion complex is.

[0082] 1. Solution preparation

[0083] The preparation of HEPES buffer solution, macrocyclic host stock solution, and dye guest stock solution is the same as in Example 1.

[0084] Preparation of competitive guest solution: accurately weigh the reference substances sodium benzoate, protocatechuic acid, rutin, ginkgolide A, and uridine, dissolve rutin and ginkgolide A in methanol, and dissolve the remaining compounds in water to prepare a stock solution, which is prepared before use.

[0085] 2. Fluorescence spectroscopy experimental conditions

[0086] Same as Example 1.

[0087] 3. Determination of the bonding constant between rutin and QAC5A

[0088] The QAC5A stock solution and the EY stock solution were diluted with HEPES buffer solution to prepare a QAC5A·EY (0.4μM / 0.5μM) host-guest pair solution, which was placed in a fluorescence cell. Methanol (0-19μL) containing EY (0.5μM) was added dropwise, and the effect of methanol on the fluorescence signal of the QAC5A·EY host-guest pair was recorded. The results are shown in FIG. Figure 6 As shown in Figure a. When 19 μL of methanol was added, the fluorescence intensity of the host-guest pair (λ em =537nm) changes little, therefore, the amount of methanol added to the titration solution should be controlled within 19μL during the titration process.

[0089] The EY stock solution was diluted with HEPES buffer solution to prepare a 0.5 μM EY solution, which was placed in a fluorescence cell. The rutin solution (0-53.21 μM) containing EY (0.5 μM) was added dropwise to the EY solution. There was no obvious change in the EY fluorescence signal, indicating that rutin (0-53.21 μM) had no interference with the EY fluorescence signal. Figure 6 As shown in Figure b.

[0090] The QAC5A stock solution and the EY stock solution were diluted with HEPES buffer solution to prepare a QAC5A·EY (0.4μM / 0.5μM) host-guest pair solution, which was placed in a fluorescence cell, and a rutin solution (0-44.33μM, total volume 30μL, containing 18μL methanol) containing QAC5A·EY (0.4μM / 0.5μM) was added dropwise. Rutin competed EY out of the QAC5A cavity in the QAC5A·EY host-guest complex, and EY restored its luminescent properties in the solution, and the fluorescence signal was sensitized (Switch-on). The titration was measured three times in parallel. The fluorescence emission spectrum is shown in the figure. Figure 6 As shown in Figure c.

[0091] The EY fluorescence intensity value and rutin concentration value were nonlinearly fitted using the host-guest 1:1 competitive inclusion model fitting formula (oneHost_oneGuest_oneCompetitor) to obtain the bonding constant K between QAC5A and rutin. a =(9.68±0.95)×10 5 M -1 ,like Figure 6 As shown in Figure d.

[0092] 4. Determination of bonding constants between sodium benzoate and SAC5A and QAC5A

[0093] (1) Determination of the bonding constant between sodium benzoate and SAC5A

[0094] The RhB stock solution was diluted with HEPES buffer solution to prepare a 0.8 μM RhB solution, which was placed in a fluorescence cell. A sodium benzoate solution (0-1803.28 μM) containing RhB (0.8 μM) was added dropwise to the RhB solution. The RhB fluorescence signal did not change, indicating that sodium benzoate (0-1803.28 μM) had no interference with the RhB fluorescence signal. Figure 7 As shown in Figure a.

[0095] The SAC5A stock solution and the RhB stock solution were diluted with HEPES buffer solution to prepare a SAC5A·RhB (1.0μM / 0.8μM) host-guest pair solution, which was placed in a fluorescence cell, and a sodium benzoate solution (0-1666.67μM) containing SAC5A·RhB (1.0μM / 0.8μM) was added dropwise. Sodium benzoate competed with RhB for binding to the cavity of SAC5A. As the concentration increased, the dye RhB was gradually displaced from the macrocyclic molecule SAC5A. RhB restored the fluorescence signal in the solution, and the signal was enhanced. The fluorescence emission spectrum was shown in the figure below. Figure 7 As shown in Figure b.

[0096] The RhB fluorescence intensity value and the sodium benzoate concentration value were nonlinearly fitted using the host-guest 1:1 competitive inclusion model fitting formula to obtain the SAC5A·sodium benzoate bonding constant K a =(6.86±0.48)×10 3 M -1 ,like Figure 7 As shown in Figure c.

[0097] (2) Determination of the bonding constant between sodium benzoate and QAC5A

[0098] The EY stock solution was diluted with HEPES buffer solution to prepare a 0.5 μM EY solution, which was placed in a fluorescence cell. The sodium benzoate solution (0-340.25 μM) containing EY (0.5 μM) was added dropwise to the EY solution. The EY fluorescence signal did not change, indicating that sodium benzoate (0-340.25 μM) had no interference with the EY fluorescence signal. Figure 8 As shown in Figure a.

[0099] The QAC5A stock solution and the EY stock solution were diluted with HEPES buffer solution to prepare the QAC5A·EY (0.4μM / 0.5μM) host-guest pair solution, which was placed in a fluorescence cell. A sodium benzoate solution (0-81.53μM) containing QAC5A·EY (0.4μM / 0.5μM) was added dropwise. Sodium benzoate competes with EY for binding to the cavity of QAC5A, displacing EY from the QAC5A·EY host-guest complex. EY restored its luminescent properties in the solution, and the fluorescence signal was enhanced. The measurement was repeated three times. The fluorescence emission spectrum is shown in the figure below. Figure 8 As shown in Figure b.

[0100] The EY fluorescence intensity value and the sodium benzoate concentration value were nonlinearly fitted using the host-guest 1:1 competitive inclusion model fitting formula to obtain the QAC5A·sodium benzoate bonding constant K a =(2.46±0.70)×10 5 M -1 ,like Figure 8 As shown in Figure c.

[0101] 5. Determination of the bonding constants of Ginkgolide A with CAC4A, SAC5A and QAC5A

[0102] (1) Determination of the bonding constant between Ginkgolide A and CAC4A

[0103] CAC4A stock solution and RhB stock solution were diluted with HEPES buffer solution to prepare CAC4A·RhB (1.0μM / 0.8μM) host-guest pair solution, which was placed in a fluorescence cell. Methanol (0-29μL) containing RhB (0.8μM) was added dropwise, and the effect of methanol on the fluorescence signal of the CAC4A·RhB host-guest pair was recorded. The results are shown in FIG. Fig. 9 As shown in Figure a. When 29 μL of methanol was added, the fluorescence intensity of the host-guest pair (λ em =576nm) changes little, therefore, the amount of methanol added to the titration solution should be controlled within 29μL during the titration process.

[0104] The RhB stock solution was diluted with HEPES buffer solution to prepare a 0.8 μM (final concentration) RhB solution, which was placed in a fluorescence cell. The Ginkgolide A solution (0-65.76 μM, total volume 51 μL, containing 20.4 μL methanol) containing RhB (0.8 μM) was added dropwise to the RhB solution (0.8 μM, final concentration). The RhB fluorescence signal did not change, indicating that Ginkgolide A had no effect on the RhB dye fluorescence signal. Fig. 9 As shown in Figure b.

[0105] CAC4A stock solution and RhB stock solution were diluted with HEPES buffer solution to prepare CAC4A·RhB (1.0μM / 0.8μM) host-guest pair solution, which was placed in a fluorescence cell, and ginkgolide A solution (0-64.58μM, total volume 44μL, containing 26.4μL methanol) containing CAC4A·RhB (1.0μM / 0.8μM) was added dropwise. Ginkgolide A competes with RhB for binding to the cavity of CAC4A, displacing RhB from the CAC4A·RhB host-guest complex. RhB restores its luminescent properties in the solution, and the fluorescence signal is sensitized. The measurement was repeated three times. The fluorescence emission spectrum is shown in the figure below. Fig. 9 As shown in Figure c.

[0106] The fluorescence intensity value of RhB and the concentration value of Ginkgolide A were fitted nonlinearly using the host-guest 1:1 competitive inclusion model fitting formula to obtain the CAC4A·Ginkgolide A bonding constant K a =(4.43±0.77)×10 4 M -1 ,like Fig. 9 As shown in Figure d.

[0107] (2) Determination of the bonding constant between Ginkgolide A and SAC5A

[0108] The SAC5A stock solution and the EY stock solution were diluted with HEPES buffer solution to prepare a SAC5A·RhB (1.0μM / 0.8μM) host-guest pair solution, which was placed in a fluorescence cell. Methanol (0-19μL) containing RhB (0.8μM) was added dropwise, and the effect of methanol on the fluorescence signal of the SAC5A·RhB host-guest pair was recorded. The results are shown in FIG. Fig.10 As shown in Figure a. When 19 μL of methanol was added, the fluorescence intensity of the host-guest pair (λ em =576nm) changes little, therefore, the amount of methanol added to the titration solution should be controlled within 19μL during the titration process.

[0109] The SAC5A stock solution and the RhB stock solution were diluted with HEPES buffer solution to prepare a SAC5A·RhB (1.0μM / 0.8μM, final concentration) host-guest pair solution, which was placed in a fluorescence cell, and a solution of ginkgolide A (0-35.57μM, total volume 24μL, containing 14.4μL methanol) containing SAC5A·RhB (1μM / 0.8μM) was added dropwise. Ginkgolide A competed with RhB to bind to the cavity of SAC5A, displacing RhB from the SAC5A·RhB host-guest complex. RhB restored its luminescent properties in the solution, and the fluorescence signal was enhanced. The measurement was repeated three times, and the fluorescence emission spectrum is shown in the figure below. Fig.10 As shown in Figure b.

[0110] The RhB fluorescence intensity value and the ginkgolide A concentration value were nonlinearly fitted using the host-guest 1:1 competitive inclusion model fitting formula to obtain the SAC5A·ginkgolide A bonding constant K a =(6.35±2.45)×10 5 M -1 ,like Fig.10 As shown in Figure c.

[0111] (3) Determination of the bonding constant between Ginkgolide A and QAC5A

[0112] The EY stock solution was diluted with HEPES buffer solution to prepare a 0.5 μM EY solution, which was placed in a fluorescent cell. The Ginkgolide A solution (0-78.16 μM, total volume 61 μL) containing EY (0.5 μM) was added dropwise to the EY solution. The EY fluorescence signal did not change, indicating that Ginkgolide A had no effect on the EY dye fluorescence signal. Fig.11 As shown in Figure a.

[0113] The QAC5A stock solution and the EY stock solution were diluted with HEPES buffer solution to prepare a QAC5A·EY (0.4μM / 0.5μM) host-guest pair solution, which was placed in a fluorescence cell. A solution of ginkgolide A (0-69.03μM, total volume 28μL, containing 14μL methanol) containing QAC5A·EY (0.4μM / 0.5μM) was added dropwise. Ginkgolide A competed with EY for binding to the cavity of QAC5A, displacing EY from the QAC5A·EY host-guest complex. EY restored its luminescent properties in the solution, and the fluorescence signal was enhanced. The measurement was repeated three times. The fluorescence emission spectrum is shown in the figure. Fig.11 As shown in Figure b.

[0114] The EY fluorescence intensity value and the concentration value of Ginkgolide A were fitted by nonlinear fitting using the host-guest 1:1 competitive inclusion model fitting formula to obtain the QAC5A·Ginkgolide A bonding constant K a =(5.50±1.52)×10 4 M -1 ,like Fig.11 As shown in Figure c.

[0115] 6. Determination of the bonding constants of uridine with SAC5A and QAC5A

[0116] (1) Determination of the binding constant between uridine and SAC5A

[0117] The RhB stock solution was diluted with HEPES buffer solution to prepare a 0.8 μM RhB solution, which was placed in a fluorescence cell. The uridine solution (0-4161.29 μM) containing RhB (0.8 μM) was added dropwise to the RhB solution. The RhB fluorescence signal did not change, indicating that uridine had no effect on the RhB dye fluorescence signal. Fig.12 As shown in Figure a.

[0118] The SAC5A stock solution and the RhB stock solution were diluted with HEPES buffer solution to prepare a SAC5A·RhB (1.0μM / 0.8μM) host-guest pair solution, which was placed in a fluorescence cell, and a uridine solution (0-3975.90μM, final concentration) containing SAC5A·RhB (1.0μM / 0.8μM) was added dropwise. Uridine competed with RhB for binding to the cavity of SAC5A, displacing RhB from the SAC5A·RhB host-guest complex. RhB restored its luminescent properties in the solution, and the fluorescence signal was enhanced. The results were measured three times in parallel. The fluorescence emission spectrum is shown in the figure. Fig.12 As shown in Figure b.

[0119] The RhB fluorescence intensity value and uridine concentration value were nonlinearly fitted using the host-guest 1:1 competitive inclusion model fitting formula to obtain the SAC5A·uridine bonding constant K a =(2.07±0.30)×102 M -1 ,like Fig.12 As shown in Figure c.

[0120] (2) Determination of the binding constant between uridine and QAC5A

[0121] The EY stock solution was diluted with HEPES buffer solution to prepare a 0.5 μM EY solution, which was placed in a fluorescence cell. A uridine solution (0-653.20 μM, total volume 61 μL, containing 24.4 μL methanol) containing EY (0.5 μM) was added dropwise to the EY solution. The EY fluorescence signal (λ ex =517nm) did not change, indicating that uridine did not interfere with the fluorescence signal of EY dye. Fig.13 As shown in Figure a.

[0122] The QAC5A stock solution and the EY stock solution were diluted with HEPES buffer solution to prepare a QAC5A·EY (0.4μM / 0.5μM) host-guest pair solution, which was placed in a fluorescence cell. Uridine (0-176.47μM) containing QAC5A·EY (0.4μM / 0.5μM) was added dropwise. Uridine competed with EY for binding to the cavity of QAC5A, displacing EY from the QAC5A·EY host-guest complex. EY restored its luminescent properties in the solution, and the fluorescence signal was enhanced. The solution was measured three times in parallel. The fluorescence emission spectrum is shown in the figure. Fig.13 As shown in Figure b.

[0123] The EY fluorescence intensity value and uridine concentration value were nonlinearly fitted using the host-guest 1:1 competitive inclusion model fitting formula to obtain the QAC5A·uridine bonding constant K a =(1.77±0.30)×10 5 M -1 ,like Fig.13 As shown in Figure c.

[0124] 7. Determination of the bonding constant between protocatechuic acid and QAC5A

[0125] The EY stock solution was diluted with HEPES buffer solution to prepare a 0.5 μM (final concentration) EY solution, which was placed in a fluorescence cell. A protocatechuic acid solution (0-653.20 μM) containing EY (0.5 μM) was added dropwise to the EY solution. The EY fluorescence signal (λ ex =517nm,λ em =525~700nm) had no change, indicating that protocatechuic acid (0~653.20μM) had no interference with the EY fluorescence signal. Fig.14 As shown in Figure a.

[0126] The QAC5A stock solution and the EY stock solution were diluted with HEPES buffer solution to prepare a QAC5A·EY (0.4μM / 0.5μM) host-guest pair solution, which was placed in a fluorescence cell, and a protocatechuic acid solution (0-357.29μM) containing QAC5A·EY (0.4μM / 0.5μM) was added dropwise. Protocatechuic acid competed with EY for binding to the cavity of QAC5A, displacing EY from the QAC5A·EY host-guest complex. EY restored its luminescent properties in the solution, and the fluorescence signal was enhanced. The measurement was repeated three times in parallel. The fluorescence emission spectrum is shown in the figure. Fig.14 As shown in Figure b.

[0127] The EY fluorescence intensity value and uridine concentration value were nonlinearly fitted using the host-guest 1:1 competitive inclusion model fitting formula to obtain the QAC5A·protocatechuic acid bonding constant K a =(9.11±0.73)×10 4 M -1 ,like Fig.14 As shown in Figure c.

[0128] The bonding constants (K) of the five representative analytes, rutin, ginkgolide A, uridine, protocatechuic acid, and sodium benzoate, with the three macrocyclic molecules a / M -1 ) See Table 5. The three macrocyclic hosts showed different degrees of binding ability to rutin, ginkgolide A, uridine, protocatechuic acid, and sodium benzoate, which is related to the non-covalent interaction between the macrocyclic host molecules and the guest molecules. Among them, the QAC5A·EY host-guest has a stronger bonding ability to the five compounds. The performance of the QAC5A·EY (0.4μM / 0.5μM) host-guest pair in sensing the main active ingredients in the Ginkgo Honey Ring oral solution is better.

[0129] Table 5 Bonding constants (K) of analytes to the macrocyclic host a / M -1 )

[0130]

[0131] Comparative Example 1

[0132] This comparative example provides the fluorescence changes after adding Ginkgo Honey Oral Solution to other macrocyclic host molecules and dye guests.

[0133] 1. Solution preparation

[0134] The preparation solution of HEPPS buffer solution is the same as that in Example 1.

[0135] Preparation of macrocyclic main body stock solution: Take appropriate amount of SC4A, SC5A, CB[6] and β-CD powder, weigh accurately, dissolve in ultrapure water, prepare stock solution, store at 4°C for later use.

[0136] Preparation of dye guest stock solution: Take appropriate amount of LCG, MB, DSMI and OX1 powder respectively, weigh accurately, dissolve with ultrapure water, prepare stock solution, store at 4℃ for later use.

[0137] Preparation of competitive guest solution: same as in Example 1.

[0138] 2. Fluorescence spectroscopy conditions

[0139] Same as Example 1.

[0140] 3. Fluorescence changes of macrocyclic host-guest pairs before and after adding Ginkgo honey ring oral solution

[0141] Take the dye guest stock solution, dilute it with HEPES buffer and place it in the fluorescence pool; take the Ginkgo Honey Ring oral solution, add the dye guest to make the dye guest concentration the same as the dye guest concentration in the fluorescence pool, and ensure that the dye guest concentration in the fluorescence pool remains unchanged during the titration process. The fluorescence change before and after the addition of the Ginkgo Honey Ring oral solution is measured by fluorescence competition titration at the maximum emission wavelength of the dye (MB: 688nm, OX1: 668nm, DSMI: 582nm, LCG: 505nm).

[0142] Cyclodextrin, cucurbituril[6], and calixarene were used as macrocyclic hosts, and appropriate host-guest pairs were selected, β-CD·MB (1μM / 10μM), CB[6]·DSMI (8μM / 1μM). When 5μL of Ginkgo honey ring oral solution was added to the above host-guest pair solutions, the fluorescence signal of the system did not change significantly, so it is not suitable for sensing Ginkgo honey ring oral solution.

[0143] Theoretically, Ginkgo honey oral solution can compete with the dyes OX1, LCG, MB, and DSMI for the cavities of SC4A, SC5A, β-CD, and CB[6], respectively. The dyes are released from the cavities, and the fluorescence signals of the macrocyclic host·dye host-guest pair change significantly (SC4A·OX1 (enhanced), SC5A·LCG (enhanced), β-CD·MB (weakened), CB[6]·DSMI (weakened)). However, the corresponding phenomenon did not occur in the experiment. The reason may be that the chemical components of Ginkgo honey oral solution are weakly bound to the macrocyclic host and fail to displace OX1 and LCG. Therefore, the host-guest pairs of SC4A·OX1 (20μM / 1μM), SC5A·LCG (1μM / 1μM), β-CD·MB (1μM / 10μM), and CB[6]·DSMI (8μM / 1μM) are not suitable for sensing Ginkgo honey oral solution.

[0144] Comparative Example 2

[0145] This comparative example provides the situations of other macrocyclic host molecules and dye guests in the detection of rutin, sodium benzoate, uridine and protocatechuic acid.

[0146] 1. Solution preparation

[0147] The preparation of HEPES buffer, macrocyclic host solution and dye guest solution is the same as in Example 1.

[0148] Preparation of competitive guest solution: same as in Example 2.

[0149] 2. Fluorescence spectroscopy experimental conditions

[0150] Same as Example 1.

[0151] 3. Determination of bonding constants between macrocyclic host molecules and rutin, sodium benzoate, uridine and protocatechuic acid

[0152] In HEPES buffer, the bonding constant between the dye guest and the macrocyclic host was determined by direct fluorescence titration. The dye guest stock solution was diluted with HEPES buffer and placed in a fluorescence pool; the macrocyclic host stock solution was diluted with HEPES buffer and added with the dye guest at the same concentration as in the fluorescence pool to ensure that the dye guest concentration in the fluorescence pool remained unchanged during the titration process, which was used as the macrocyclic host titration solution. With the addition of the macrocyclic host titration solution, the fluorescence signal increased or decreased due to the host-guest complexation. The fluorescence intensity at the maximum emission wavelength of the dye (RhB: 576nm) and the macrocyclic host concentration value were fitted using the host-guest 1:1 inclusion model fitting formula (oneHost_oneGuest) to obtain the host-guest bonding constant. All bonding constants are the average values ​​of the fitting data of three parallel titration experiments.

[0153] The bonding constants of the macrocyclic host with rutin, sodium benzoate, uridine and protocatechuic acid were determined by fluorescence competitive titration. Cyclodextrin, cucurbituril and calixarene were used as the macrocyclic host, and appropriate host-guest pairs were selected, CAC4A·RhB (1μM / 0.8μM) and SAC5A·RhB (1μM / 0.8μM). Rutin, sodium benzoate, uridine and protocatechuic acid titration solutions were added dropwise to the above host-guest pair solutions, but no satisfactory bonding constants were obtained, which was not enough to realize the sensing detection of rutin, sodium benzoate, uridine and protocatechuic acid.

[0154] Example 3

[0155] This embodiment provides a method for evaluating the batch consistency of Ginkgo Biloba Oral Solution based on a "ternary host-guest sensor array".

[0156] 1. Solution preparation

[0157] The preparation of HEPES buffer, macrocyclic host solution and dye guest solution is the same as in Example 1.

[0158] 2. Fluorescence spectroscopy experimental conditions

[0159] Same as Example 1.

[0160] 3. Determination of qualified samples of Ginkgo Honey Ring Oral Solution

[0161] Take 6 batches of qualified Ginkgo honey ring oral solution (batch numbers are 211013, 211014, 211015, 211016, 211018, 211019), dilute the macrocyclic host and dye stock solution with HEPES (10mM, pH=7.4) buffer, prepare appropriate final concentrations in the fluorescence pool: CAC4A·RhB (1μM / 0.8μM), SAC5A·RhB (1μM / 0.8μM) and QAC5A·EY (0.4μM / 0.5μM), the total volume of 2.5mL host-guest pair solution, seal the glass capillary and stir evenly to make the solution reach the host-guest complex equilibrium, and then test. Scan rate 600nm / min, smooth filter size (smooth) 9. Record the fluorescence emission spectrum when the solution reaches equilibrium. The fluorescence intensity of the dye at the maximum emission wavelength is I0. After adding Ginkgo Honey Oral Solution (5μL), record the fluorescence emission spectrum when it reaches equilibrium. At this time, the maximum fluorescence intensity of the system is I, and record the fluorescence spectrum. Repeat the measurement 3 times for each sample, and take the average value to evaluate the quality of the batch of Ginkgo Honey Oral Solution. The line graph of the measurement results of 6 batches of qualified Ginkgo Honey Oral Solution is shown in the figure. Fig.15 shown.

[0162] Taking P=I / I0 as the evaluation index, the P values ​​of 6 batches of qualified Ginkgo Honey Ring Oral Solution measured by different sensor units were 1.94, 1.67 and 3.87 respectively, and the RSD values ​​among the 6 batches were calculated to be 0.78%, 0.32% and 0.97% respectively, which all indicated that the reproducibility among different batches of Ginkgo Honey Ring Oral Solution was good. The P value results of 6 batches of qualified Ginkgo Honey Ring Oral Solution measured by different sensor units are shown in the figure below. Fig.16 shown.

[0163] Example 4

[0164] This embodiment provides a fluorescent fingerprint of Ginkgo Biloba Honey Ring oral solution and a method for constructing the same.

[0165] The fluorescence emission spectra of 6 batches of qualified Ginkgo Honey Ring oral solution were calculated according to the method of Example 3, and the fluorescence response value (P=I / I0) of each sensing unit to the Ginkgo Honey Ring oral solution was calculated, and the average value and relative standard deviation (RSD) data table of the P value of the same batch of samples were obtained. The spider web diagram of the fluorescence response of the three sensing host-guest pairs to 6 batches of qualified Ginkgo Honey Ring oral solution was drawn in the OriginPro 2022 software.

[0166] The “spider web” mode is used to visualize the response of different sensor units to multiple batches of samples. To express this concisely, the I / I0 (P value) measured by each sensor unit is designated as A m-k , divided by the maximum fluorescence intensity ratio of the corresponding sensor unit (A k ( max ))Get E m-k As shown in formula (1-1), k represents different batches and m represents different sensor units. m-k Used to construct the "spider web" model of different dimensions in formula (1-2) (p i ). The angle between two dimensions is marked as α (α = 360° / n, n = 3). See the visualization representation based on the “spider web” pattern. Fig.17 , Formulas 1-1 and 1-2 calculate the spider web area (S).

[0167] E m-k =A m-k / A k(max) (1-1)

[0168]

[0169] The results are shown in Table 6 and Fig.18 shown.

[0170] Table 6 Visual representation of the "spider web" of 6 batches of qualified Ginkgo Honey Ring oral solution measured by different sensor units (n=3)

[0171]

[0172] Example 5

[0173] This example provides the determination of Ginkgo honey ring sample solutions with different Ginkgo extract contents.

[0174] 1. Solution preparation

[0175] The preparation of HEPES buffer solution, macrocyclic host stock solution, and dye guest stock solution is the same as in Example 1.

[0176] Ginkgo Honey Ring Oral Solution is composed of Ginkgo Leaf Extract (GBE) and Armillaria Powder (AMP), and the company's ratio is 3:100 (mass ratio). In the actual production process of the company, 100g of Armillaria Powder can be extracted into 30g of Armillaria Extract (AME), that is, GME:AME=3:30 (mass ratio) in the company's ratio.

[0177] Preparation of homemade Ginkgo Honey Ring sample solution: Prepare Ginkgo Honey Ring sample solutions with different ratios (as shown in the table below). The specific steps are: add ultrapure water to dilute Tween 80 to 5g / L, slowly add ginkgo leaf extract in an 80℃ water bath, stir while adding, and keep warm until it is completely dissolved; add appropriate amount of ultrapure water to dilute Armillaria extract, stir for 30min in a 70℃ water bath, filter while hot and collect the filtrate, cool the above two solutions and transfer them to volumetric flasks according to the preparation ratio, add ultrapure water to make up to volume, shake well, and you are done. Prepare three parallel copies of each ratio of Ginkgo Honey Ring sample solution, store at 4℃ for later use.

[0178] Table 7 Composition ratio of homemade Ginkgo honey ring sample solution

[0179]

[0180]

[0181] The QAC5A stock solution and the EY stock solution were diluted with HEPES buffer solution to prepare a QAC5A·EY (0.4 μM / 0.5 μM, final concentration) host-guest pair solution, which was placed in a fluorescence cell to detect the self-made Ginkgo honey ring sample solution. The results are shown in Table 8 and Fig.19 As shown. Fig.19 It can be seen that the fluorescence response of QAC5A·EY to the homemade Ginkgo honey ring sample solutions with different proportions of Ginkgo leaf extract is statistically different, and the P value is linearly correlated with the proportion of Ginkgo leaf extract in the homemade Ginkgo honey ring sample solution (y=0.5065x+3.4129, R 2 =0.9681). It can be seen that QAC5A·EY can be used to detect the content of Ginkgo biloba extract in Ginkgo honey ring oral solution, and can be used to monitor the raw material feed ratio on the Ginkgo honey ring oral solution production line and the finished product.

[0182] Table 8 The results of the QAC5A·EY sensor unit on the self-made Ginkgo honey ring sample solutions with different proportions (n=3)

[0183]

[0184]

[0185] The results showed that QAC5A could well distinguish the homemade oral solutions with different contents of Ginkgo biloba extract and could be used for the content detection of Ginkgo biloba extract in Ginkgo honey ring oral solution. The detection results could reflect the feeding ratio of Ginkgo biloba extract in Ginkgo honey ring oral solution.

[0186] Example 6

[0187] This embodiment provides a method for monitoring the feeding ratio of ginkgo leaf extract in Ginkgo Honey Ring Oral Liquid:

[0188] 1. Solution preparation

[0189] Same as Example 5.

[0190] Preparation of Ginkgo Honey Ring control solution: Add Tween 80 to ultrapure water to dilute to 5g / L, slowly add Ginkgo leaf extract in an 80℃ water bath, stir while adding, and keep warm until it is completely dissolved; add appropriate amount of ultrapure water to dilute Armillaria extract, stir for 30min in a 70℃ water bath, filter while hot and collect the filtrate, cool the above two solutions and transfer them to volumetric flasks in different proportions, add ultrapure water to make up to volume, shake well, and get it. Prepare three parallel copies of each ratio of Ginkgo Honey Ring sample solution, store at 4℃ for standby use.

[0191] 2. Fluorescence spectroscopy experimental conditions

[0192] Same as Example 1.

[0193] 3. Using QAC5A·EY host-guest pair to detect the feed ratio of ginkgo leaf extract in Ginkgo Honey Ring Oral Liquid:

[0194] The QAC5A stock solution and the EY stock solution were diluted with HEPES buffer solution to prepare QAC5A·EY (0.4μM / 0.5μM) host-guest pair solution, which was placed in a fluorescence cell. The above-mentioned Ginkgo honey ring control solution was detected, and the fluorescence response value of each Ginkgo honey ring control solution was calculated (P=I / I0). A standard curve was drawn with the proportion of Ginkgo extract in the Ginkgo honey ring control solution as the horizontal axis and the fluorescence response value as the vertical axis to obtain the regression equation.

[0195] The QAC5A stock solution and the EY stock solution were diluted with HEPES buffer solution to prepare a QAC5A·EY (0.4 μM / 0.5 μM) host-guest pair solution, which was placed in a fluorescence cell and tested with the Ginkgo Biloba Oral Solution. The obtained fluorescence response value (P=I / I0) was substituted into the above regression equation to calculate the proportion of Ginkgo extract in the Ginkgo Biloba Oral Solution.

[0196] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting Ginkgo honey ring oral solution based on supramolecular fluorescence sensing, characterized in that: Adding ginkgo honey ring oral liquid to a host-guest pair solution consisting of a macrocyclic host and a dye guest, detecting the ginkgo honey ring oral liquid by changing a fluorescence signal; the host-guest pair is selected from at least one of CAC4A·RhB, SAC5A·RhB and QAC5A·EY.

2. The method for detecting the Ginkgo honey ring oral solution according to claim 1, characterized in that: The solvent of the host-guest pair solution is HEPES buffer solution, the HEPES concentration is 10 mM, and the pH is 7.

4.

3. The method for detecting the Ginkgo honey ring oral solution according to claim 1 or 2, characterized in that: When the host-guest pair is CAC4A·RhB, the excitation wavelength for detecting the fluorescence signal is 554 nm; and / or When the host-guest pair is SAC5A·RhB, the excitation wavelength for detecting the fluorescence signal is 554 nm; and / or When the host-guest pair is QAC5A·EY, the excitation wavelength for detecting the fluorescence signal is 517 nm.

4. The method for detecting the Ginkgo honey ring oral solution according to claim 3, characterized in that: When the host-guest pair is CAC4A·RhB, the concentration of CAC4A in the host-guest pair solution is 1 μM, and the concentration of RhB is 0.8 μM; and / or When the host-guest pair is SAC5A·RhB, the concentration of SAC5A in the host-guest pair solution is 1 μM, and the concentration of RhB is 0.8 μM; and / or When the host-guest pair is QAC5A·EY, the concentration of QAC5A in the host-guest pair solution is 0.4 μM, and the concentration of EY is 0.5 μM.

5. Use of the Ginkgo Biloba Oral Solution Detection Method according to any one of claims 1 to 4 in monitoring the batch-to-batch consistency of Ginkgo Biloba Oral Solution.

6. Use of the method for detecting the Ginkgo Honey Ring oral solution according to any one of claims 1 to 4 in detecting the ratio of Ginkgo biloba leaf extract and Armillaria mellea powder in the Ginkgo Honey Ring oral solution.

7. The use according to claim 6, characterized in that: The host-guest pair is QAC5A·EY.

8. Use of the detection method for Ginkgo Biloba Oral Solution according to any one of claims 1 to 4 in constructing a fluorescent fingerprint of Ginkgo Biloba Oral Solution.

9. A method for constructing a fluorescent fingerprint of Ginkgo Honey Oral Liquid, characterized in that: Ginkgo honey ring oral liquid is added to a host-guest pair solution composed of a macrocyclic host and a dye guest, and a fluorescence signal is detected; the host-guest pair is CAC4A·RhB, SAC5A·RhB and QAC5A·EY; when the host-guest pair is CAC4A·RhB, the excitation wavelength for detecting the fluorescence signal is 554nm, the concentration of CAC4A in the host-guest pair solution is 1μM, and the concentration of RhB is 0.8μM; when the host-guest pair is SAC5A·RhB, the excitation wavelength for detecting the fluorescence signal is 554nm, the concentration of SAC5A in the host-guest pair solution is 1μM, and the concentration of RhB is 0.8μM; when the host-guest pair is QAC5A·EY, the excitation wavelength for detecting the fluorescence signal is 517nm, the concentration of QAC5A in the host-guest pair solution is 0.4μM, and the concentration of EY is 0.5μM.

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