Ratiometric fluorescent probe for rapid detection of sulfur dioxide residue in sulfur fumigation traditional Chinese medicine, preparation method and application

Abstract

The application discloses a ratio type fluorescent probe for rapidly detecting residual amount of sulfur dioxide in sulfur fumigation traditional Chinese medicine, a preparation method and application. By introducing a propionic acid group into a benzothiazole structure of the probe, water solubility of the probe can be effectively improved, environmental errors can be reduced, and detection accuracy can be improved. An olefin bond in the structure of the fluorescent probe can effectively respond to sulfur dioxide derivatives with nucleophilicity, exhibit obvious ratio type fluorescent signal performance, can reduce interference of a detection environment and operation factors, and improve detection accuracy. The probe can realize rapid response, has excellent detection sensitivity (a minimum detection limit is 1.03 nM), and environmental specificity. The fluorescent probe can be applied to quantitative detection of residual amount of sulfur dioxide in traditional Chinese medicine, has the characteristics of simple operation and rapid detection, and the detected residual amount of sulfur dioxide is consistent with a detection result of an acid-base titration method in the 2020 edition of Chinese Pharmacopoeia, and has good practical value.

Description

Technical Field

[0001] This invention relates to the field of drug analysis and traditional Chinese medicine quality evaluation methods, specifically to a ratiometric fluorescent probe for rapid detection of sulfur dioxide residue in sulfur-fumigated traditional Chinese medicine by detecting sulfur dioxide derivatives, its preparation method, and its application. Background Technology

[0002] Sulfur fumigation is a common method for the initial processing of Chinese medicinal herbs at their place of origin. The sulfur dioxide (SO2) generated by heating sulfur can be used to fumigate the herbs to achieve sterilization, mold prevention, insect prevention, and bleaching and color protection. However, there is currently a problem of the overuse or excessive use of sulfur fumigation in Chinese medicine processing, resulting in excessive sulfur dioxide residues and harming human health. Studies have shown that residual sulfur dioxide after fumigation can cause varying degrees of damage to the respiratory tract, liver, gastrointestinal tract, and immune system. Therefore, the content and safety of sulfur dioxide in Chinese medicinal herbs are of great concern. The 2020 edition of the Chinese Pharmacopoeia stipulates that the sulfur dioxide residue of 10 Chinese medicinal herbs, including yam, asparagus, and trichosanthes root, must not exceed 400 mg / kg, while the limit for other herbs is 150 mg / kg. The United States Pharmacopeia specifies limits for SO2 residues in pharmaceutical excipients, ranging from 5 to 80 mg / kg, while the European Pharmacopoeia specifies limits of 20 to 400 mg / kg. The pharmacopoeia lists acid-base titration, gas chromatography, and ion chromatography as methods for detecting sulfur dioxide residues. However, these methods have problems such as cumbersome sample processing, complex detection steps, long processing time, and high detection costs, making them unsuitable for convenient and rapid detection of sulfur dioxide residues in traditional Chinese medicine. Among many analytical methods, the fluorescent probe method based on optical signals stands out due to its advantages such as high sensitivity, good selectivity, good reproducibility, small sample volume, and simple operation, and has been applied in fields such as drug detection, medical diagnosis, and biochemical analysis. Developing a rapid detection method based on fluorescence technology suitable for the qualitative and quantitative detection of sulfur dioxide residues in traditional Chinese medicine is of great significance for controlling the quality of traditional Chinese medicine. Due to the complex composition of traditional Chinese medicine, a fluorescent probe that can quickly and conveniently quantitatively detect sulfur dioxide residues in traditional Chinese medicine needs to have the following characteristics: (1) rapid response; (2) excellent detection sensitivity and obvious response signal, which can accurately respond to trace sulfur dioxide in traditional Chinese medicine; (3) strong response specificity, which can avoid interference from other complex components in traditional Chinese medicine. Since sulfur dioxide in aqueous solution and traditional Chinese medicine is in the form of its derivative bisulfite (HSO3) - ) and / or sulfite (SO3) 2-Sulfur dioxide exists in various forms, therefore, the detection of sulfur dioxide in actual samples currently mainly relies on sulfur dioxide derivatives. While there are reports of fluorescent probes using sulfur dioxide derivatives to detect sulfur dioxide in samples, most examples still have some drawbacks, such as slow response speed, low sensitivity, and low water solubility. In particular, some fluorescent probes exhibit fluorescence quenching, making them unsuitable for the qualitative and quantitative detection of sulfur dioxide residues in traditional Chinese medicine. Furthermore, the synthesis processes of some fluorescent probes for sulfur dioxide detection are quite complex, posing challenges to probe development and application. Therefore, developing a fluorescent probe suitable for detecting complex samples such as traditional Chinese medicine materials / processed herbs is a problem that needs to be solved by those skilled in the art. Summary of the Invention

[0003] To address the shortcomings of existing technologies, the purpose of this invention is to provide a ratiometric fluorescent probe (SZH) based on sulfur dioxide derivative response for rapid quantitative detection of sulfur dioxide residues in sulfur-fumigated Chinese medicinal herbs / processes. This solves the problems of cumbersome sample processing, complex detection steps, and long processing times in current methods for detecting sulfur dioxide residues in Chinese medicinal herbs, achieving rapid detection of sulfur dioxide residues in these herbs. Sulfur dioxide derivatives (HSO3) - SO3 2- Due to its nucleophilicity, it can undergo addition reactions with alkene bonds. Based on this, the fluorescent probe SZH is composed of a coumarin derivative and a benzothiazole chromophore, and it can recognize sulfur dioxide derivatives (HSO3). - SO3 2-The formation of alkene bonds and the introduction of carboxyl groups into the benzothiazole structure effectively improves the water solubility of the fluorescent probe, which is beneficial for the detection of sulfur dioxide residues in traditional Chinese medicine samples present as sulfur dioxide derivatives. At an excitation wavelength of 450 nm, the probe SZH exhibits a significant fluorescence signal at 581 nm in the long-wavelength band. After the alkene bond in the structure undergoes an addition reaction with the nucleophilic sulfur dioxide derivative, the conjugated structure of the probe changes, significantly reducing the fluorescence intensity at 581 nm while producing a significant fluorescence signal at 485 nm. The probe exhibits a typical ratiometric fluorescence signal before and after the response, avoiding interference from environmental and experimental factors, thus providing more accurate measurements. The probe SZH reacts very rapidly and sensitively, reaching response equilibrium in 90 s, with a detection limit in the nanomolar range (1.03 nM). Its rapid response and high sensitivity make it effective for the rapid quantitative detection of trace sulfur dioxide in traditional Chinese medicine samples. Furthermore, this probe has good detection selectivity, enabling specific detection. The application of probe SZP to the quantitative detection of sulfur dioxide in fine powders of traditional Chinese medicine has achieved the expected results. It can quickly and conveniently detect the residual amount of sulfur dioxide in traditional Chinese medicine samples, and the results are consistent with those of the acid-base titration method for detecting sulfur dioxide residues specified in the 2020 edition of the Chinese Pharmacopoeia. Compared with the method included in the determination of sulfur dioxide residues in General Chapter 2331 of the 2020 edition of the Chinese Pharmacopoeia, the detection method of the present invention is simple to operate, rapid in detection, and has good practical value.

[0004] To achieve the above objectives, one aspect of the present invention is to provide a ratiometric fluorescent probe for detecting sulfur dioxide residues in sulfur-fumigated Chinese medicinal herbs / processes by responding to sulfur dioxide derivatives. The structural formula of the fluorescent probe is as follows:

[0005]

[0006] On the other hand, the present invention provides a method for synthesizing the above-mentioned ratiometric fluorescent probe, comprising the following steps:

[0007] (1) 2-methylbenzothiazole and 3-iodopropionic acid were suspended in solvent one, stirred and heated to react, and the reaction solution was treated to obtain compound 1;

[0008] (2) Dissolve p-fluorobenzaldehyde and piperazine in solvent 2, heat and react, and treat the reaction solution to obtain compound 2;

[0009] (3) Dissolve 7-(diethylamino)coumarin-3-carboxylic acid and EDCI in solvent three, stir and react, add compound 2 to react, and treat the reaction solution to obtain compound 3;

[0010] (4) Compound 3 and Compound 1 are suspended in solvent 4, a catalyst is added and the reaction is heated. The reaction solution is treated to obtain the final product, which is the ratiometric fluorescent probe.

[0011] Furthermore, solvent one is anhydrous acetonitrile, solvent two is toluene, solvent three is anhydrous dichloromethane, and solvent four is anhydrous ethanol.

[0012] Preferred,

[0013] In step (1), the molar ratio of 2-methylbenzothiazole to 3-iodopropionic acid is 1:1.5.

[0014] The reaction temperature in step (1) is 80℃ and the reaction time is 24h;

[0015] The steps for processing the reaction solution in step (1) include concentration, filtration, and washing.

[0016] Preferred,

[0017] In step (2), the molar ratio of p-fluorobenzaldehyde and piperazine is 1:1.

[0018] The reaction temperature in step (2) is 80℃ and the reaction time is 8h;

[0019] The reaction solution processing steps in step (2) include vacuum concentration and silica gel column chromatography separation.

[0020] In step (2), the silica gel column chromatography is eluted with petroleum ether and ethyl acetate, with a volume ratio of 6:1.

[0021] Preferred,

[0022] In step (3), the molar ratio of 7-(diethylamino)coumarin-3-carboxylic acid to compound 2 is 1:1;

[0023] In step (3), 7-(diethylamino)coumarin-3-carboxylic acid and EDCI are first stirred and reacted at 0°C for 10 min, and then compound 2 is added and reacted at room temperature for 2 h.

[0024] The steps for processing the reaction solution in step (3) include vacuum concentration, extraction, washing, drying, and silica gel column chromatography separation.

[0025] In step (3), the silica gel column chromatography is eluted with petroleum ether and ethyl acetate, with a volume ratio of 4:1.

[0026] Preferred,

[0027] In step (4), the molar ratio of compound 3 to compound 1 is 1:1;

[0028] The catalyst in step (4) is diethylamine;

[0029] The reaction temperature in step (4) is 60°C and the reaction time is 6 hours.

[0030] The reaction solution processing steps in step (4) include vacuum concentration and dextran gel chromatography column separation.

[0031] In step (4), the dextran gel chromatography column is eluted with methanol.

[0032] On the other hand, the present invention provides the aforementioned fluorescent probe or the synthesized fluorescent probe as a detection tool for the rapid and accurate detection of sulfur dioxide residue in sulfur-fumigated traditional Chinese medicine.

[0033] Furthermore, the processing method for the aforementioned traditional Chinese medicine sample includes the following:

[0034] The Chinese medicinal materials or processed medicinal slices were pulverized and ground into a fine powder. 5g of the fine powder was placed in 20mL of ultrapure water and sonicated for 30min. The mixture was then filtered to obtain the test solution. 1μL of the test solution was placed in 2999μL of PBS buffer containing 3μM fluorescent probe SZH. After reacting for 90s, fluorescence was measured to obtain the fluorescence intensity ratio (IF). 485nm / I 581nm When the SO2 residue is in the range of 0-768.69 mg / kg, the conversion formula between the fluorescence ratio value and the corresponding SO2 residue (mg / kg) in the traditional Chinese medicine is as follows:

[0035] Sulfur dioxide residue (mg / kg) = [([I 485nm / I 581nm [-0.03918) / 4.98126]×M SO2 ×4

[0036] Where I 485nm / I 581nm M is the ratio of the fluorescence intensity at 485 nm to the fluorescence intensity at 581 nm in the fluorescence spectrum of the test solution. SO2 is the relative molecular mass of SO2.

[0037] The beneficial effects of this invention are:

[0038] 1. The fluorescent probe SZH of this invention introduces a propionic acid group into the benzothiazole structure, which can effectively improve the water solubility of the probe. In the detection of sulfur dioxide residues in traditional Chinese medicine, it can reduce environmental errors and improve detection accuracy.

[0039] 2. The alkene bond in the SZH structure of the fluorescent probe of this invention can effectively respond to nucleophilic SO2 derivatives, exhibiting significant ratiometric fluorescence recognition performance. This can reduce interference from detection environment and operational factors, effectively improving detection accuracy. The probe achieves a rapid response (<90s) and possesses excellent detection sensitivity (limit of detection is 1.03 nM) and environmental specificity.

[0040] 3. Sulfur dioxide content is an important indicator for the quality evaluation of Chinese medicinal materials / processed slices. The fluorescent probe SZH of this invention can detect the residual amount of sulfur dioxide in sulfur-fumigated Chinese medicinal materials / processed slices. It has the advantages of convenient operation, rapid detection, sensitivity and accuracy, and has strong practical value. Attached Figure Description

[0041] Figure 1 This is a synthetic route diagram for the fluorescent probe SZH.

[0042] Figure 2 For fluorescent probe SZH 1 H NMR spectrum.

[0043] Figure 3 This is the high-resolution mass spectrum of the fluorescent probe SZH.

[0044] Figure 4 The fluorescent probes SZH (3 μM) and HSO3 - Fluorescence spectra before and after 90 s of reaction at (3 μM).

[0045] Figure 5 The fluorescent probe SZH (3 μM) for HSO3 - Fluorescence response diagram of (3μM) over time (0-120s), with fluorescence emission peaks at 485nm and 581nm.

[0046] Figure 6 SZH is a fluorescent probe for HSO3 - Fluorescence response as a function of concentration. Figure A shows the fluorescence response of probe SZH (3 μM) with different concentrations of HSO3. - Fluorescence emission spectrum of (0-3μM) after 90s reaction, B is the fluorescence intensity ratio (I 485nm / I 581nm ) and HSO3 - Linear relationship graph between (0-3μM) concentrations, the experiment was repeated 3 times.

[0047] Figure 7 This study investigated the specific selectivity of the fluorescent probe SZH. Figure A shows the reaction of probe SZH (3 μM) with HSO3. - (3 μM) or other analytes with a final concentration of 10 μM (K + Zn2+ Na + Ca 2+ HCO3 - ,ClO - NO2 - NO3 - I - HPO4 2- Cl - Fluorescence spectra of H2O2, Hcy, Cys, GSH, Lys, and Pro after co-incubation for 90 s. B represents the fluorescence spectrum of probe SZH (3 μM) after incubation with HSO3. - The ratio of fluorescence intensity after co-incubation with (3 μM) or other analytes (10 μM) for 90 s (I) 485nm / I 581nm (Figure 1 shows the experimental results, which were repeated three times. 1 represents the blank; 2-18 represent K...) + Zn 2+ Na + Ca 2+ HCO3 - ,ClO - NO2 - NO3 - I - HPO4 2- Cl - , H2O2, Hcy, Cys, GSH, Lys, Pro: 10μM; 19 is HSO3 - 3μM. Hcy is homocysteine, Cys is cysteine, GSH is reduced glutathione, Lys is lysine, and Pro is proline. Detailed Implementation

[0048] The specific embodiments of the present invention will be further described in detail below with reference to examples.

[0049] Example 1: Synthesis of fluorescent probe SZH

[0050] Synthetic routes such as Figure 1 As shown, structural identification is as follows Figure 2-3 As shown.

[0051] 1. Synthesis of Compound 1

[0052] 2-Methylbenzothiazole (5.0 mmol) and 3-iodopropionic acid (7.5 mmol) were suspended in anhydrous acetonitrile (100 mL) and heated under reflux at 80 °C for 24 h with stirring. After the reaction was completed, the mixture was cooled to room temperature, concentrated under reduced pressure to remove acetonitrile, and then ethyl acetate was added and stirred at room temperature for 5 min. After filtration, the precipitate was obtained and washed with acetonitrile to give compound 1 (1.42 g, yield 81.6%).

[0053] 2. Synthesis of Compound 2

[0054] p-Fluorobenzaldehyde (5 mmol) and piperazine (5 mmol) were dissolved in 50 mL of toluene and heated and stirred at 80 °C for 8 h. The reaction solution was concentrated under reduced pressure and separated by silica gel column chromatography (petroleum ether / ethyl acetate = 6:1, v / v) to give compound 2 (0.64 g, yield 67.5%).

[0055] 3. Synthesis of Compound 3

[0056] 7-(diethylamino)coumarin-3-carboxylic acid (2.5 mmol) and condensing agent EDCI (5.0 mmol) were dissolved in anhydrous dichloromethane (40 mL). After stirring at 0 °C for 10 min, an anhydrous dichloromethane solution (10 mL) containing compound 2 (2.5 mmol) was added dropwise to the reaction system, and the reaction was stirred at room temperature for 2 h. After the reaction was completed, the solvent (anhydrous dichloromethane) was removed by vacuum concentration. The mixture was extracted with dichloromethane and washed with distilled water to remove the condensing agent. The mixture was then dried over anhydrous Na2SO4, concentrated to remove the solvent, and separated by silica gel column chromatography (petroleum ether / ethyl acetate = 4:1, v / v) to obtain compound 3 (0.87 g, yield 80.3%).

[0057] 4. Synthesis of fluorescent probe SZH

[0058] Compound 3 (1.5 mmol) and compound 1 (1.5 mmol) were suspended in anhydrous ethanol, and 100 μL of diethylamine was added dropwise. The mixture was heated and stirred at 60 °C for 6 h. After the reaction was complete, the mixture was concentrated under reduced pressure to obtain a crude product. The crude product was then dissolved in methanol and separated by dextran gel chromatography to obtain the final product SZH (0.61 g, yield 64.2%). Structural identification data for probe SZH: 1 H NMR (500MHz, MeOD)δ H 8.17-8.06(m,3H),7.99(s,1H),7.88-7.79(m,3H),7.78-7.68(m,2H),7.48(d,J=7.8Hz,1H),7.07(d,J=7.9Hz,2H),6.79(d,J=8.9Hz,1H ),6.58(s,1H),5.06(d,J=5.5Hz,2H),3.88(s,2H),3.67-3.49(m,10H),3.05(t,J=6.7Hz,2H),1.23(t,J=7.0Hz,6H).HR-ESI-MS:m / z[M] + Calculated value C 36 H 37 N4O5S+ 637.2479, found value 637.2547.

[0059] The above synthesis method and characterization results demonstrate that the fluorescent probe SZH was successfully synthesized. The synthesis method of this fluorescent probe is simple, has a high yield, and is easy to promote.

[0060] Example 2: Detection effect of fluorescent probe SZH

[0061] This invention aims to provide a ratiometric fluorescent probe for the quantitative detection of trace sulfur dioxide residues in traditional Chinese medicine. Since sulfur dioxide typically exists in the form of its nucleophilic derivative HSO3 in medicinal materials / processes and aqueous solutions... - SO3 2- HSO3 exists in the form of - SO3 2- It can recognize alkene bonds, thereby causing changes in the optical properties of the probe and achieving the purpose of detection. This example uses the derivative HSO3. - As a detection target, the detection performance of the probe SZH for identifying sulfur dioxide was examined from multiple aspects to determine its application performance.

[0062] Weigh an appropriate amount of the fluorescent probe SZH and dissolve it in dimethyl sulfoxide (DMSO) to prepare a 3 mM stock solution. The buffer for optical testing is PBS buffer (10 mM, pH 7.4) containing 0.1% (v / v) DMSO, with a fluorescence excitation wavelength set at 450 nm and a test temperature of 37 °C.

[0063] (1) Fluorescent probe and HSO3 - Fluorescence spectrum of the reaction

[0064] First, the fluorescence response performance of the fluorescent probe SZH was examined, using its derivative HSO3. - As the detection target, appropriate amounts of probes SZH and HSO3 are used. - Add to PBS buffer (pH = 7.4, 10 mM) to bring the final probe concentration to 3 μM, HSO3 - The final concentration was 3 μM (a control group with only the SZN probe was also included). After incubation at room temperature for 90 seconds, fluorescence spectroscopy was performed. The results are as follows: Figure 4 As shown, the fluorescence emission peak of the probe SZH itself is located in the long wavelength region of 581 nm, which is similar to that of HSO3. - After the reaction, the intensity of the emission peak decreased significantly, while a new fluorescence emission peak appeared at 485 nm. The experimental results show that the probe SZH can be activated by SO2 derivatives with nucleophilic properties and can be detected by changes in fluorescence signal.

[0065] (2) Reaction time of fluorescent probe

[0066] When performing response time spectroscopy, probes SZH and HSO3 are added to the PBS buffer. - The final concentration of the probe was 3 μM, HSO3 - The final concentration was 3 μM. Fluorescence spectroscopy was then immediately performed (time-of-detection mode selected on the fluorescence spectrophotometer). Results are as follows: Figure 5 As shown, the fluorescence intensity at 581 nm decreased rapidly over time, while the fluorescence intensity at 485 nm increased rapidly. The reaction of the test system reached equilibrium at approximately 90 s, indicating that the response speed of the probe SZH is very fast and can meet the requirements for online rapid detection in actual samples.

[0067] (3) Investigation of the detection sensitivity and detection limit of the fluorescent probe

[0068] To explore the application of the fluorescent probe SZH in the qualitative and quantitative detection of trace amounts of sulfur dioxide in traditional Chinese medicine, the probe's response sensitivity and detection limit were investigated. Specifically, the derivative HSO3 was used... - As the detection target, probe SZH and different concentrations of HSO3 were used. - Add to PBS buffer to bring the final probe concentration to 3 μM, HSO3 - Final concentrations were 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.4, 1.8, 2.2, 2.6, and 3 μM. After incubation for 90 seconds, fluorescence spectroscopy was performed. The results are as follows: Figure 6 As shown, with HSO3 - With increasing concentration, the fluorescence intensity of probe SZH gradually decreased at 581 nm, while the fluorescence intensity gradually increased at 485 nm, and the ratio of fluorescence signal intensity (IL) increased. 485nm / I 581nm ) and HSO3 - The concentration (0-3 μM) showed a good linear relationship (R0). 2 =0.9989, [I 485nm / I 581nm ]=4.98126×[HSO3 - +0.03918, [HSO3] - ] indicates HSO3 - (Concentration). Based on the detection limit equation (LOD = 3σ / k), the detection limit of probe SZH was calculated to be 1.03 nM. This indicates that the probe SZH of this invention has excellent detection sensitivity, with a detection limit in the nanomolar range, which can meet the practical requirements for detecting trace sulfur dioxide residues in traditional Chinese medicine.

[0069] Example 3: Detection Selectivity Study of Probe SZH

[0070] The specificity of sulfur dioxide residue detection in traditional Chinese medicine (TCM) is an important condition for evaluating the quality of TCM. Therefore, the selectivity of the fluorescent probe SZH was investigated. In PBS buffer (pH = 7.4, 10 mM), the probe SZP (final concentration 3 μM) was reacted with HSO3. - (final concentration 3 μM) or various analytes with a final concentration of 10 μM (K + Zn 2+ Na + Ca 2+ HCO3 - ,ClO - NO2 - NO3 - I - HPO4 2- Cl - After reacting with H2O2, Hcy, Cys, GSH, Lys, and Pro for 90 seconds, fluorescence spectroscopy was performed. The results are as follows: Figure 7 As shown, other analytes cannot react with the probe SZH, and the fluorescence intensity of the test system (I) 485nm / I 581nm Compared to the probe itself, there was no significant change. This indicates that the probe SZH exhibits specific reactivity only towards sulfur dioxide derivatives, demonstrating high selectivity.

[0071] Example 4: Quantitative Detection of Sulfur Dioxide Residue in Traditional Chinese Medicine Materials

[0072] The fluorescent probe SZH exhibits a rapid response (90s), excellent sensitivity, and strong response specificity. Furthermore, its ratiometric fluorescence characteristic eliminates signal interference caused by factors such as the test solvent, effectively improving detection accuracy. Based on this, this embodiment establishes an application method for the quantitative detection of sulfur dioxide residues in traditional Chinese medicine using the fluorescent probe SZH. The accuracy and consistency of the fluorescent probe spectroscopic detection method are evaluated using the detection results of Method I—acid-base titration—in the 2020 edition of the Chinese Pharmacopoeia as the standard.

[0073] The operation steps of this embodiment are as follows: The prepared Chinese medicinal herbs (white peony root, white atractylodes rhizome, angelica root, astragalus root, achyranthes root, and asparagus root) are pulverized and ground into fine powder. 5g of each powder is weighed and placed in a 50mL Erlenmeyer flask. 20mL of ultrapure water is added, and the mixture is sonicated for 30 minutes. After oscillation and filtration, the test solution is obtained. Then, 1μL of the test solution is placed in 2999μL of PBS buffer (pH = 7.4, 10mM) containing the fluorescent probe SZH to obtain the test solution. The probe concentration is 3μM. After incubation for 90 seconds, fluorescence spectroscopy is performed to obtain the fluorescence ratio (IL). 485nm / I 581nm ), through the above linear formula ([I 485nm / I581nm ]=4.98126×[HSO3 - The concentration of equivalent SO2 in the test solution was calculated using the formula [+0.03918], and the residual amount of sulfur dioxide in the traditional Chinese medicine was then converted using the formula. When the residual SO2 content is in the range of 0-768.69 mg / kg, the conversion formula between the fluorescence ratio and the corresponding residual SO2 content (mg / kg) is as follows:

[0074] Sulfur dioxide residue (mg / kg) = [([I 485nm / I 581nm [-0.03918) / 4.98126]×M SO2 ×4

[0075] Where I 485nm / I 581nm M is the ratio of the fluorescence intensity at 485 nm to the fluorescence intensity at 581 nm in the fluorescence spectrum of the test solution. SO2 is the relative molecular mass of SO2.

[0076] The residual sulfur dioxide content of the above-mentioned traditional Chinese medicines was determined using the acid-base titration method, Method 1, for the determination of sulfur dioxide residues, in Part IV (General Chapter 2331) of the 2020 edition of the Chinese Pharmacopoeia. The accuracy of the fluorescent probe SZH fluorescence detection method and the feasibility of the detection method were verified by comparing the test results. 10g of each of the finely powdered traditional Chinese medicines (white peony root, white atractylodes rhizome, angelica root, astragalus root, achyranthes root, and asparagus root) were accurately weighed and placed in a two-necked round-bottom flask. 300mL of water was added, and the reflux condenser valve was opened to supply water. Then, the rubber gas delivery tube connected to the upper end of the condenser was placed at the bottom of a 100mL conical flask containing 50mL of absorption liquid (3% hydrogen peroxide solution), with the end of the rubber gas delivery tube below the surface of the absorption liquid. Before use, 3 drops of methyl red ethanol solution indicator (2.5mg / mL) were added to the absorption liquid, and titration was performed with sodium hydroxide titrant (0.01mol / L) until a yellow color (representing the endpoint) was obtained. Continue to purge with nitrogen gas (approximately 0.2 L / min), then open the stopcock of the separatory funnel on the two-necked flask to allow 10 mL of hydrochloric acid solution (6 mol / L) to flow into the distillation flask. Immediately heat the solution in the two-necked flask to a gentle boil. After the water in the flask has been gently boiling for 1.5 hours, stop heating. Allow the absorbent to cool, then place it on a magnetic stirrer and stir continuously. Continue titrating with sodium hydroxide titrant (0.01 mol / L) until the yellow color persists for 20 seconds. Correct the titration result with a blank experiment. The residual sulfur dioxide in the traditional Chinese medicine sample is calculated using the following formula:

[0077] Sulfur dioxide residue (μg / g) = [(AB) × c × 0.032 × 10] 6 ] / W

[0078] Where A is the volume (mL) of sodium hydroxide titrant consumed by the test solution, B is the volume (mL) of sodium hydroxide titrant consumed by the blank solution, c is the molar concentration of sodium hydroxide titrant (mol / L), 0.032 is the mass (g) of sulfur dioxide equivalent to 1 mL of sodium hydroxide titrant (1 mol / L), and W is the weight (g) of the traditional Chinese medicine sample.

[0079] The detection results of fluorescence probe spectroscopy and acid-base titration are shown in Table 1. The results show that the sulfur dioxide residues of the six traditional Chinese medicines detected by fluorescence probe spectroscopy are consistent with the detection results of acid-base titration. Compared with acid-base titration, the fluorescence detection method of probe SZH of this invention has the advantages of less sample requirement, simple operation and rapid detection. It can meet the rapid quantitative detection of sulfur dioxide residues in traditional Chinese medicines and can be applied to the rapid assessment of sulfur dioxide residues in commercially available traditional Chinese medicinal materials / processed slices, showing good application potential.

[0080] Table 1 Comparison of results of sulfur dioxide residue detection in traditional Chinese medicine samples by fluorescent probe method and acid-base titration method.

[0081]

[0082] Note: Each Chinese medicine sample was tested in parallel three times.

[0083] Although the above embodiments have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the above descriptions are merely embodiments of the present invention and do not limit the scope of patent protection of the present invention. Any equivalent structural or procedural transformations made using the content of the present invention's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.

Claims

1. A ratiometric fluorescent probe for detecting sulfur dioxide residue in sulfur-fumigated Chinese medicinal herbs / processes by responding to sulfur dioxide derivatives, characterized in that, The structural formula of the fluorescent probe is as follows: 。 2. A method for synthesizing a ratiometric fluorescent probe as described in claim 1, characterized in that, Includes the following steps: (1) 2-methylbenzothiazole and 3-iodopropionic acid were suspended in solvent one, stirred and heated to react, and the reaction solution was treated to obtain compound 1; (2) Dissolve p-fluorobenzaldehyde and piperazine in solvent 2, heat and react, and treat the reaction solution to obtain compound 2; (3) Dissolve 7-(diethylamino)coumarin-3-carboxylic acid and EDCI in solvent three, stir and react, add compound 2 to react, and treat the reaction solution to obtain compound 3; (4) Compound 3 and compound 1 are suspended in solvent 4, a catalyst is added and the reaction is heated, and the reaction solution is treated to obtain the final product, which is the ratiometric fluorescent probe. The structural formula of compound 1 is: ; The structural formula of compound 2 is: ; The structural formula of compound 3 is: .

3. The synthesis method according to claim 2, characterized in that, Solvent one is anhydrous acetonitrile, solvent two is toluene, solvent three is anhydrous dichloromethane, and solvent four is anhydrous ethanol.

4. The synthesis method according to claim 2, characterized in that, In step (1), the molar ratio of 2-methylbenzothiazole to 3-iodopropionic acid is 1:1.5; The reaction temperature in step (1) is 80℃ and the reaction time is 24 h; The steps for processing the reaction solution in step (1) include concentration, filtration, and washing.

5. The synthesis method according to claim 2, characterized in that, In step (2), the molar ratio of p-fluorobenzaldehyde and piperazine is 1:1; The reaction temperature in step (2) is 80℃ and the reaction time is 8 h; The reaction solution treatment steps in step (2) include vacuum concentration and silica gel column chromatography separation; In step (2), the silica gel column chromatography is eluted with petroleum ether and ethyl acetate, with a volume ratio of 6:

1.

6. The synthesis method according to claim 2, characterized in that, In step (3), the molar ratio of 7-(diethylamino)coumarin-3-carboxylic acid to compound 2 is 1:1; In step (3), 7-(diethylamino)coumarin-3-carboxylic acid and EDCI are first stirred and reacted at 0°C for 10 min, and then compound 2 is added and reacted at room temperature for 2 h. The steps for processing the reaction solution in step (3) include vacuum concentration, extraction, washing, drying, and silica gel column chromatography separation. In step (3), the silica gel column chromatography is eluted with petroleum ether and ethyl acetate, with a volume ratio of 4:

1.

7. The synthesis method according to claim 2, characterized in that, In step (4), the molar ratio of compound 3 to compound 1 is 1:1; The catalyst in step (4) is diethylamine; In step (4), the reaction temperature is 60℃ and the reaction time is 6 h; The reaction solution processing steps in step (4) include vacuum concentration and dextran gel chromatography column separation. In step (4), the dextran gel chromatography column is eluted with methanol.

8. The application of a fluorescent probe as described in claim 1 or a fluorescent probe synthesized according to any one of claims 2-7, characterized in that, As a detection tool, it is used to quickly and accurately detect the residual amount of sulfur dioxide in traditional Chinese medicine fumigated with sulfur.

9. The application according to claim 8, characterized in that, The processing methods for traditional Chinese medicine samples include the following: The Chinese medicinal materials or processed medicinal slices were pulverized and ground into a fine powder. 5 g of the fine powder was placed in 20 mL of ultrapure water and sonicated. The mixture was then filtered to obtain the test solution. 1 μL of the test solution was placed in 2999 μL of PBS buffer containing a 3 μM fluorescent probe. After reacting for 90 s, fluorescence was measured to obtain the fluorescence intensity ratio (Ig). 485 nm / I 581 nm When the SO2 residue is in the range of 0-768.69 mg / kg, the conversion formula between the fluorescence ratio value and the corresponding SO2 residue (mg / kg) in the traditional Chinese medicine is as follows: Sulfur dioxide residue (mg / kg) = [([I 485 nm / I 581 nm [ - 0.03918) / 4.98126]×M SO2 ×4 Where I 485 nm / I 581 nm M is the ratio of the fluorescence intensity at 485 nm to the fluorescence intensity at 581 nm in the fluorescence spectrum of the test solution. SO2 is the relative molecular mass of SO2.

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