A hemicyanin probe compound and its preparation and application

Abstract

The present invention provides a hemicyanin probe compound, and establishes a detection method for sulfite ions and sulfide ions in a solution under ultraviolet-visible and fluorescence conditions using a probe molecule. Common anions are added to a C2H5OH-Hepes buffer solution, and only sulfite and sulfide ions change. Sulfite ions can cause the solution to fade, the fluorescence intensity of the solution to weaken, and the anti-interference performance is good; sulfide ions can cause the solution color to deepen, the fluorescence intensity of the solution to increase, and the anti-interference performance is good. At the same time, taking the amount of sulfur dioxide residue in sulfur-fumigated Chinese medicinal materials as the detection target, the sulfur-fumigated Chinese medicinal materials are treated under alkaline conditions, and ultra-sensitive rapid detection of sulfur-fumigated Chinese medicinal materials is achieved through a standard curve method. The hemicyanin probe compound has the advantages of fast analysis speed, stable fluorescence performance, and real-time detection for the determination of the content of sulfur dioxide residues in Chinese medicinal materials, and has the value of promotion and use in the detection of sulfur dioxide residues in the fields of traditional Chinese medicine and food.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescence analysis, relates to a hemicyanin probe compound and a preparation method thereof, and also relates to the application of the probe compound in the rapid detection of sulfite ions and sulfur-fumigated Chinese medicinal materials. Background Art

[0002] Sulfur fumigation is a common method for processing Chinese medicinal materials (TCMs). It has long been used in the processing, storage, and maintenance of TCMs, foods, and agricultural products. Its primary purpose is to dry and enhance the color of TCMs, while also providing preservation, mildew prevention, bleaching, and insect repellency. Sulfite, the primary residue left after sulfur fumigation of TCMs, can cause clinical symptoms such as asthma, urticaria, itching, angioedema, and even anaphylactic shock and death. It may also be carcinogenic and cause tissue, organ, and nervous system toxicity. Currently, sulfur fumigation is primarily used for certain TCMs that are difficult to dry or prone to spoilage, such as kudzu root, yam, wolfberry, and gastrodia elata. The sulfur fumigation process generates significant amounts of SO₂, which exists in TCMs in both free and bound forms. The free form includes SO₂ molecules, sulfite, and bisulfite. The bound state is primarily SO2, formed by the reaction of free SO2 with certain components of traditional Chinese medicines. Since irreversibly bound sulfites do not dissociate under physiological conditions and pose no health risks, free and reversibly bound sulfites are the targets of detection. The 2020 edition of the Chinese Pharmacopoeia has strict detection limits for residual SO2: limits of 400 mg / kg are permitted for Chinese yam, Gastrodia elata, Asparagus cochinchinensis, Achyranthes bidentata, Radix Trichosanthis, Atractylodes macrocephala, Bletilla striata, Codonopsis pilosula, Paeonia lactiflora, and Pueraria lobata; limits of 10 mg / kg are permitted for Chinese yam slices; and limits for other Chinese medicinal materials are 150 mg / kg. However, to enhance the appearance of Chinese medicinal materials and pursue economic benefits, farmers excessively sulfur-fumigate them, resulting in excessive SO2 levels. To accelerate the market circulation of Chinese medicinal materials, ensure their safe use, and mitigate potential health risks, developing sensitive, rapid, and real-time methods for detecting residual SO2 in sulfur-fumigated Chinese medicinal materials is of great significance. Summary of the Invention

[0003] One of the purposes of the present invention is to provide a hemicyanin probe compound and a preparation method thereof;

[0004] The second object of the present invention is to provide a method for detecting SO3 2- and S 2- Application in.

[0005] The third purpose of the present invention is to provide a probe compound of hemiflorin for rapid detection of SO3 in sulfur-fumigated Chinese medicinal materials. 2- application.

[0006] 1. Hemicyanin Probe Compounds and Their Synthesis

[0007] The hemicyanin probe compound HCY-FN of the present invention has the molecular formula: 25 H 24 N2O6S, the structural formula is as follows: .

[0008] The method for preparing the probe compound of the present invention comprises the following steps:

[0009] (1) Add p-nitroaniline and hydrochloric acid to water and stir in an ice bath for 20-40 min. Then add sodium nitrite solution and continue stirring in an ice bath for 1-2 h. Add furfural acetone solution to the reaction system and then add copper chloride solution. React at 0-5 °C for 10-15 h. After the reaction is completed, cool and filter, vacuum dry, and recrystallize from ethyl acetate to obtain product A.

[0010] Among them, the mass ratio of p-nitroaniline to sodium nitrite is 1:1~2:1; the mass ratio of p-nitroaniline to furfural is 1:1~2:1; the mass ratio of furfural to copper chloride is 1:1~1:2; and the volume ratio of hydrochloric acid to water is 1:2~1:4.

[0011] (2) 2,3,3-Trimethylindole and 1,3-propane sultone were added to toluene and dissolved, and heated at 90-110°C with stirring and reflux for 10-15 h. After the reaction, the toluene solution was poured out, and the residue was ultrasonically cleaned with ethyl acetate, and the ethyl acetate solution was discarded to obtain a purple solid product B; wherein the mass ratio of 2,3,3-trimethylindole to 1,3-propane sultone was 1:1-1:2.

[0012] (3) Compound A and Compound B were dissolved in anhydrous ethanol, piperidine was added, and the mixture was heated at 75-85°C with stirring and reflux for 5-7 h. After the reaction was completed, the mixture was cooled and filtered, rinsed with anhydrous ethanol, and vacuum dried. The mixture was purified by silica gel chromatography to obtain a hemicyanine probe compound. The mass ratio of Compound A to Compound B was 1:1-2:1.

[0013] The synthetic route is as follows:

[0014]

[0015] 2. Detection of SO3 by Probe Compounds 2- Application

[0016] 1. UV-Vis Spectrum of Probe Compound HCY-FN

[0017] In the C2H5OH-Hepes buffer (C2H5OH: Hepes = 1:9, V / V, Hepes buffer pH = 4) of HCY-FN, 50 equiv. of different anions (F - 、SO3 2- Br - , I - 、Ac - 、HPO4 2- 、HSO4 - 、ClO4 - 、NO3 - 、SCN - 、N3 - 、S 2- 、CN - 、PPI、OH - ) to measure its UV-visible absorption spectrum. Figure 3 , it was found that with the S 2- The UV spectrum showed maximum absorption wavelengths at 350nm and 500nm respectively. 2- The UV spectrum of the added ions showed a maximum absorption wavelength at 350nm, and the absorption peak disappeared at 500nm. Under visible light, the solution of the probe compound of other ions was yellow. 2- The addition of SO3 2- The addition of makes the color of the probe compound solution lighter yellow, and the maximum absorption wavelength of the probe HCY-FN is λ max = 350nm, when adding SO3 2- The solution color faded and turned colorless, indicating that the probe molecule can detect SO3 with the naked eye under UV-visible spectroscopy. 2- and S 2- .

[0018] The results of anti-interference experiments showed that the presence of other anions had an impact on the recognition of SO3 by the probe compound HCY-FN. 2- and S 2- No interference (such as Figure 14 and Figure 16 shown).

[0019] The results of UV-visible titration experiments showed that the probe compound HCY-FN had a strong effect on S 2 and SO3 2- The sensitivity of HCY-FN probe to S is high. 2- The minimum detection limit was 3.05 × 10 - 6 M, for SO3 2-The minimum detection limit was 4.97 × 10 -6 M. (as Figure 8 and Figure 12 shown)

[0020] Under UV-visible conditions, the probe molecule HCY-FN can recognize S in 20 s and 40 s, respectively. 2- and SO3 2- , and the reaction equilibrium was reached within 1 min. This experiment proved that the probe molecule HCY-FN could be used to treat S 2- and SO3 2- It can achieve the purpose of rapid detection. Figure 19 (b) and (b) in 20);

[0021] 2. Fluorescence properties of the probe compound HCY-FN

[0022] Various anions (F - 、SO3 2- Br - , I - 、Ac - 、HPO4 2- 、HSO4 - 、ClO4 - 、NO3 - 、SCN - 、N3 - 、S 2- 、CN - ,PPI,OH - ) were added to 5 mL of C2H5OH-Hepes buffer (C2H5OH: Hepes (pH = 4) 1: 9, V / V) of probe HCY-FN, and their fluorescence intensity was measured. 2- After that, the fluorescence intensity at the emission wavelength of 600nm weakened and blue fluorescence was emitted; when S 2- After that, the fluorescence intensity at the emission wavelength of 600nm is enhanced, and red fluorescence is emitted, while other ions do not change. Figure 4 As shown, the results showed that the probe had a strong affinity for SO3 2- and S 2- Responsive and specific.

[0023] The results of the anti-interference experiment showed that the presence of other anions did not interfere with the probe compound (e.g. Figure 13 and Figure 15 shown).

[0024] Fluorescence titration experiments showed that the probe HCY-FN has a strong affinity for SO3 2-The detection of HCY-FN has the characteristics of high sensitivity. According to the formula 3σ / s, the fluorescence spectrum of the probe HCY-FN is calculated to be S 2- The minimum detection limit was 3.73 × 10 -6 M, for SO3 2- The minimum detection limit was 5.62 × 10 -6 M. (as Figure 6 and Figure 10 shown)

[0025] Under fluorescence conditions, the probe molecule HCY-FN can recognize S in 20 s and 40 s respectively in the fluorescence spectrum. 2- and SO3 2- , and the reaction equilibrium was reached within 4 min and 1 min respectively. 2- and S 2- It can realize efficient and rapid detection. Figure 19 (shown in a and 20a)

[0026] 3. Probe compound HCY-FN recognizes SO3 2- Mechanism analysis

[0027] The probe molecules HCY-FN and SO3 were determined by Job-Plot experiment in Hepes buffer solution system using UV absorption spectrum and fluorescence emission spectrum. 2- and S 2- The binding ratio, such as Figure 17 and Figure 18 As shown, an inflection point appears at about 0.5. The experiment shows that the probe molecule HCY-FN and SO3 2- and S 2- The binding ratio was 1:1. The results of nuclear magnetic titration showed that the probe molecules had affinity addition reaction with sulfite ion and sulfur ion respectively (such as Figure 21 High-resolution mass spectrometry also further showed that the probe molecule HCY-FN and SO3 2- The binding ratio is 1:1 (e.g. Figure 22 shown).

[0028] 4. Detection of SO3 using the probe compound HCY-FN 2- and S 2- Practical Application

[0029] 4.1 Take two filter paper strips and immerse them in a solution with a concentration of 2.0 × 10 -3 Soak the HCY-FN in Hepes buffer solution for 30 minutes, then take it out and dry it naturally. Apply SO3 2- and S 2- Solution. Figure 23 and Figure 25 As shown, under the naked eye, it can be observed that SO3 2- and S 2- The yellow filter paper strip of the solution completely fades and the color deepens. Figure 24 and Figure 26 As shown, under the ultraviolet light with a wavelength of 365 nm, it can be observed that the uncoated SO3 2- and S 2- The yellow filter paper strip of the solution emits purple-red fluorescence, while the SO3 2- and S 2- The filter paper strips of the solution emit deep blue fluorescence and deep red fluorescence respectively. This phenomenon further proves that the probe HCY-FN can be used for SO3 2- and S 2- Rapid detection.

[0030] 4.2 Application of Sulfur Fumigation in Rapid Detection of Chinese Medicinal Materials

[0031] 4.2.1 Drawing of Na2SO3 standard curve

[0032] The HCY-FN probe mother solution and Hepes buffer solution were prepared at a ratio of 1:9, and the Na2SO3 standard solution was added dropwise to the HCY-FN probe to a concentration of 2×10 -5 mol / L solution, detect its fluorescence intensity and draw a standard curve. Fluorescence conditions: excitation wavelength 500 nm, emission 600 nm, slit 10 nm / 10 nm, voltage 700 V. The fitting curve y = - 9.54539x +491.42732, SO3 2- The concentration range is 0~3.7μM and the linear relationship is good. 2 = 0.99728.

[0033] 4.2.2 Investigation of Codonopsis Extraction Methods

[0034] Using the SO2 content in sulfur-fumigated Codonopsis as an indicator, three different extraction methods were selected: ultrasound, maceration, and oscillation. The results showed that the oscillation extraction method produced the highest residual SO2 in sulfur-fumigated Codonopsis, so the oscillation method was chosen to extract SO2 from sulfur-fumigated herbs. See Table 1.

[0035]

[0036] Taking the SO2 content in sulfur-fumigated Codonopsis as the investigation index, five different NaOH concentrations of 0.1%, 0.3%, 0.5%, 0.8% and 1% were selected. The results showed that the residual SO2 in sulfur-fumigated Codonopsis was the highest when the NaOH concentration was 1%. Therefore, the extraction solution with a concentration of 1% NaOH was selected to extract SO2 from sulfur-fumigated medicinal materials, see Table 2.

[0037]

[0038] Taking the SO2 content in sulfur-fumigated Codonopsis as the investigation index, six different solid-liquid ratios of 1:10, 1:20, 1:30, 1:40, 1:50 and 1:60 were selected. The results showed that the residual amount of SO2 in sulfur-fumigated Codonopsis was the highest when the solid-liquid ratio was 1:60. Therefore, the solid-liquid ratio of 1:60 was selected to extract SO2 from sulfur-fumigated medicinal materials, see Table 3.

[0039]

[0040] Taking the SO2 content in sulfur-fumigated Codonopsis as the investigation index, five different extraction temperatures of 30℃, 40℃, 50℃, 60℃ and 70℃ were selected. The results showed that the residual amount of SO2 in sulfur-fumigated Codonopsis was the highest when the extraction temperature was 30℃, so the extraction temperature of 30℃ was selected to extract SO2 from sulfur-fumigated medicinal materials, see Table 4.

[0041]

[0042] Taking the SO2 content in sulfur-fumigated Codonopsis as the investigation index, six different extraction times of 10min, 20min, 30min, 40min, 50min and 60min were selected. The results showed that the residual amount of SO2 in sulfur-fumigated Codonopsis was the highest when the extraction time was 50min. Therefore, the extraction time of 50min was selected to extract SO2 from sulfur-fumigated medicinal materials. 2， See Table 5.

[0043]

[0044] From the above results, it can be seen that when the extraction method is oscillation method, the extraction solution is 1% NaOH, the material-liquid ratio is 1:60, the extraction temperature is 30℃, and the extraction time is 50 min, the residual amount of SO2 in the sulfur-fumigated medicinal materials is the highest.

[0045] 4.2.3 Methodological Review

[0046] 4.2.3.1 Precision investigation

[0047] The extract of fumigated Codonopsis pilosula was obtained using the established extraction method, and the probe compound HCY-FN was used to detect the residual SO2 in fumigated Codonopsis pilosula. The average value of 6 tests was 2683.89 mg / Kg, and the RSD was 1.76%. The results proved the reliability of the precision of this method.

[0048]

[0049] 4.2.3.2 Repeatability study

[0050] The extract of fumigated Codonopsis pilosula was obtained using the established extraction method, and the probe compound HCY-FN was used to detect the residual SO2 in fumigated Codonopsis pilosula. The average value of 6 tests was 2539.31 mg / Kg, and the RSD was 1.85%. The results demonstrated the reliability of the detection method, as shown in Table 7.

[0051]

[0052] 4.2.3.3 Stability assessment

[0053] The extract of fumigated Codonopsis pilosula was obtained using the established extraction method, and the probe compound HCY-FN was used to detect the residual SO2 in fumigated Codonopsis pilosula. In the five tests, the fluorescence intensity tended to be stable when the time exceeded 70 minutes, as shown in Figure 8.

[0054]

[0055] 4.2.3.4 Sample recovery rate investigation

[0056] The extract of Codonopsis pilosula was obtained by the established extraction method, and the SO3 in the extract was detected by the probe compound HCY-FN. 2- content, then add an equal amount of SO3 2- The fluorescence intensity was measured using the probe compound HCY-FN, and the calculated recovery rate was within the range of 98.52%-104.02%, with an RSD value of 1.90%. The results showed that this detection method can accurately determine the SO3 in the sulfur-fumigated medicinal material extract. 2- The content is shown in Table 9.

[0057]

[0058] 4.2.3.5 Blank control

[0059] The extract of unfumigated Codonopsis pilosula was obtained using the established extraction method, and the fluorescence intensity corresponding to the unfumigated Codonopsis pilosula was detected by the probe compound HCY-FN. The results showed that the fluorescence intensity of the six tests was the same as that without the addition of the extract.

[0060] 4.2.4 Results of sulfur dioxide residues in Chinese medicinal materials

[0061] Since the sample processing process is an alkaline environment and an acidic environment, the reversibly bound sulfite in the medicinal material can be extracted under alkaline conditions. Therefore, the residual SO2 in the fumigated sulfur astragalus measured by probe HCY-FN is higher than the residual SO2 in the fumigated sulfur astragalus measured by acid-base titration. The value measured by the probe compound is closer to the actual sulfur content of the medicinal material. The results are shown in Table 10.

[0062]

[0063] In summary, the highest content was detected when the oscillation method was used, the extraction time was 50 min, the extraction temperature was 30℃, 1% NaOH solution was used, and the solid-liquid ratio was 1:60. The precision (RSD ≤ 1.76%), repeatability (RSD ≤ 1.85%), and sample recovery rate (RSD ≤ 1.90%) were also good, indicating that this method can be used for the quantitative detection of SO2 residues in sulfur-fumigated Chinese medicinal materials and realize the rapid detection of sulfur-fumigated Chinese medicinal materials.

[0064] The present invention provides a hemicyanin probe compound, and establishes a method for detecting sulfite ions and sulfide ions in a solution using the probe molecule under ultraviolet-visible and fluorescence conditions. When common anions are added to a solution of the probe molecule (C2H5OH:Hepes = 1:9, V / V), only the sulfite and sulfide ions undergo changes. The sulfite can discolor the solution and quench the solution fluorescence, resulting in good anti-interference performance, while the sulfide ion can deepen the fluorescence color. The sulfur dioxide residue in sulfur-fumigated Chinese medicinal materials is used as the detection target. By treating the sulfur-fumigated Chinese medicinal materials under alkaline conditions, ultrasensitive and rapid detection of sulfur-fumigated Chinese medicinal materials is achieved using a standard curve method. The hemicyanin probe compound has the advantages of fast analysis speed, stable fluorescence performance, and real-time detection for the determination of sulfur dioxide residue in Chinese medicinal materials, and has the value of being promoted and used in the detection of sulfur dioxide residues in the fields of traditional Chinese medicine and food. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 This is the hydrogen spectrum (DMSO-d6) of the probe molecule HCY-FN of the present invention;

[0066] Figure 2 The carbon spectrum of the probe molecule HCY-FN (DMSO-d 6) ;

[0067] Figure 3 UV spectra of different anions of the probe molecule HCY-FN of the present invention: Inset: Photograph taken with visible light;

[0068] Figure 4 Fluorescence spectra of different anions of the probe molecule HCY-FN of the present invention; Inset: Photograph taken under irradiation with a UV light of 365 nm;

[0069] Figure 5 Add S to the probe molecule HCY-FN (C2H5OH: Hepes = 1:9, V / V) system of the present invention 2- Fluorescence titration of;

[0070] Figure 6Add S to the probe molecule HCY-FN (C2H5OH: Hepes = 1:9, V / V) system of the present invention 2 Fluorescence titration fitting curve of

[0071] Figure 7 To add S into the (C2H5OH: Hepes = 1:9, V / V) system of the probe molecule HCY-FN of the present invention 2- UV titration;

[0072] Figure 8 To add S into the (C2H5OH: Hepes = 1:9, V / V) system of the probe molecule HCY-FN of the present invention 2- UV titration fitting curve of

[0073] Figure 9 Add SO3 to the (C2H5OH: Hepes = 1:9, V / V) system of the probe molecule HCY-FN of the present invention 2- Fluorescence titration of

[0074] Figure 10 Add SO3 to the (C2H5OH: Hepes = 1:9, V / V) system of the probe molecule HCY-FN of the present invention 2- Fluorescence titration fitting curve of

[0075] Figure 11 Add SO3 to the (C2H5OH: Hepes = 1:9, V / V) system of the probe molecule HCY-FN of the present invention 2- UV titration;

[0076] Figure 12 Add SO3 to the (C2H5OH: Hepes = 1:9, V / V) system of the probe molecule HCY-FN of the present invention 2 UV titration fitting curve of

[0077] Figure 13 Add SO3 to the (C2H5OH: Hepes = 1:9, V / V) system of the probe molecule HCY-FN of the present invention 2- , on this basis, the fluorescence anti-interference graphs of different anions were added;

[0078] Figure 14 Add SO3 to the (C2H5OH: Hepes = 1:9, V / V) system of the probe molecule HCY-FN of the present invention 2- , on this basis, the UV anti-interference diagrams of different anions were added;

[0079] Figure 15 Add S to the probe molecule HCY-FN (C2H5OH: Hepes = 1:9, V / V) system of the present invention 2- , on this basis, the fluorescence anti-interference graphs of different anions were added;

[0080] Figure 16 Add S to the probe molecule HCY-FN (C2H5OH: Hepes = 1:9, V / V) system of the present invention 2- , on this basis, the UV anti-interference diagrams of different anions were added;

[0081] Figure 17 The probe molecules HCY-FN and SO3 2- Working curves between them under fluorescence emission spectrum (a) and UV emission spectrum (b);

[0082] Figure 18 The probe molecules HCY-FN and S 2- Working curves between them under fluorescence emission spectrum (a) and UV emission spectrum (b);

[0083] Figure 19 The probe molecule HCY-FN of the present invention is effective for SO3 2- The corresponding time curves under fluorescence (a) and UV (b) conditions;

[0084] Figure 20 The probe molecule HCY-FN of the present invention is 2- The corresponding time curves under fluorescence (a) and UV (b) conditions;

[0085] Figure 21 The probe molecule HCY-FN of the present invention is effective for SO3 2- and S 2- NMR titration spectrum of

[0086] Figure 22 The probe molecule HCY-FN of the present invention is SO3 2- High-resolution mass spectrum of

[0087] Figure 23 The detection test strip of the probe molecule HCY-FN prepared by the present invention is smeared with SO3 2- Photos taken in visible light;

[0088] Figure 24 The detection test strip of the probe molecule HCY-FN prepared by the present invention is smeared with SO3 2- The photos were taken under 365nm ultraviolet light;

[0089] Figure 25 The test strip of the probe molecule HCY-FN prepared by the present invention is smeared with S 2- Photos taken in visible light;

[0090] Figure 26 The test strip of the probe molecule HCY-FN prepared by the present invention is smeared with S 2- The photos were taken under 365nm ultraviolet light. DETAILED DESCRIPTION

[0091] The following specific examples are used to describe the preparation of the probe molecule HCY-FN and the UV-visible and fluorescence recognition of SO3 2- The application is further explained.

[0092] Example 1. Synthesis of probe compound HCY-FN

[0093] (1) Weigh 4.1 g of p-nitroaniline, dissolve 43 ml of hydrochloric acid and 150 ml of water in a 250 ml three-necked flask and stir in an ice bath for 30 min. Weigh 2.5 g of sodium nitrite and dissolve it in 50 ml of water in a 50 ml beaker, add it dropwise to the three-necked flask with a syringe, and stir in an ice bath for 1 h; weigh 3.17 g of furfural and dissolve it in 80 ml of acetone in a 100 ml beaker and add it to the reaction system; weigh 5.2 g of copper chloride and dissolve it in 50 ml of water in a 50 ml beaker and add it to the reaction system, and react at 0°C for 12 h. After the reaction is completed, cool and filter, vacuum dry, and recrystallize from ethyl acetate to obtain product A with a yield of 23.9%, a melting point (mp) of 222-227°C, and MS m / z 217.05.

[0094] (2) 1.5923 g of 2,3,3-trimethylindole and 1.8321 g of 1,3-propane sultone were weighed and added to a 250 ml two-necked flask. The mixture was dissolved in 15 ml of toluene and heated at 100°C with stirring under reflux for 12 h. After the reaction, the toluene solution was poured out, and the residue was ultrasonically cleaned with ethyl acetate. The ethyl acetate solution was discarded to obtain a purple solid product B. The yield was 22.9%.

[0095] (3) Weigh 1.1856 g of compound A and 0.9556 g of compound B and dissolve them in 20 ml of anhydrous ethanol in a 250 ml two-necked flask. Add 0.374 ml of piperidine dropwise to the two-necked flask using a syringe. Heat and stir under reflux at 80°C for 6 h. After the reaction is complete, cool and filter, rinse several times with anhydrous ethanol, and dry in vacuo. Purify the crude product by silica gel chromatography (CH2Cl2: CH3OH = 11:4, V / V) to obtain the pure product. The yield is 53%. 1 H NMR (400 MHz, DMSO- d 6 ) δ8.56(d, J = 8.7 Hz, 1H),8.43 (s, 1H),8.23 – 8.35 (dd, J = 20.1, 8.9 Hz, 2H), 8.00(d, J =8.9 Hz, 1H),7.87 (d, J = 7.0 Hz, 1H),7.72 (d, J = 3.5 Hz, 1H), 7.35(d, J = 3.5 Hz,1H), 7.02 (d, J = 6.2 Hz, 1H), 6.77 (d, J = 7.7 Hz, 1H), 6.64(s, 1H), 4.88(s, 1H), 2.73(s, 1H), 2.22(s, 1H), 1.87 –1.96 (s, 3H), 1.79 (s, 2H), 1.16 – 1.28 (s, 3H), 1.04 – 1.11 (s, 2H).

[0096] Example 2: Fluorescence recognition of SO3 by probe compound HCY-FN 2- and S 2-

[0097] Pipette 2 mL of the probe compound HCY-FN in C2H5OH-Hepes buffer solution (C2H5OH: Hepes = 1:9, V / V) (c HCY-FN = 2×10 -4 M) in a series of colorimetric tubes, add F - 、SO3 2- Br - , I - 、Ac - 、HPO4 2- 、HSO4 - 、ClO4- 、NO3 - 、SCN - 、N3 - 、S 2- 、CN - ,PPI,OH - If the fluorescence intensity of the probe compound HCY-FN solution weakens and emits blue fluorescence, it means that SO3 2- If the fluorescence intensity of the probe compound HCY-FN solution increases and emits red fluorescence, it means that S 2- If the fluorescence of the Hepes buffer solution of the probe compound HCY-FN does not change significantly, it means that other anions have been added.

[0098] Example 3 Naked Eye Recognition of SO3 by Probe Compound HCY-FN 2- and S 2-

[0099] Pipette 2 mL of the probe compound HCY-FN in C2H5OH-Hepes buffer solution (C2H5OH: Hepes = 1:9, V / V) (c HCY-FN = 2×10 -4 M) in a series of colorimetric tubes, add F - 、SO3 2- Br - , I - 、Ac - 、HPO4 2- 、HSO4 - 、ClO4 - 、NO3 - 、SCN - 、N3 - 、S 2- 、CN - ,PPI,OH - If the color of the probe compound HCY-FN solution changes from yellow to colorless, it means that SO3 2- If the color of the probe compound HCY-FN solution becomes darker yellow, it means that S 2- If the color of the probe compound HCY-FN solution does not change significantly, it means that other anions have been added.

[0100] Example 4: Detection of sulfur-fumigated Chinese medicinal materials using the probe compound HCY-FN

[0101] (1) Preparation of sulfur-fumigated Chinese medicinal materials

[0102] Unfumigated astragalus samples were used as the research subjects. A stainless steel fumigation chamber with mesh partitions was used. During the experiment, 20 g of unfumigated herbs were evenly spread on the mesh partitions (the herbs had been pre-soaked in ultrapure water). 50 g of medicinal sulfur was weighed and placed in an enamel tray at the bottom of the fumigation chamber. The sulfur was ignited, and the chamber door was closed to create a sealed space. The fumigation process was repeated four times.

[0103] (2) Preparation of sulfur-fumigated Chinese medicinal material extracts

[0104] The sulfur-fumigated Astragalus root in (1) was crushed into fine powder, 0.5 g was accurately weighed and placed in a centrifuge tube, 10 mL of 1% NaOH was accurately added, and the mixture was shaken and extracted at 30°C for 50 min. The mixture was then centrifuged at high speed (4500 rpm, 10 min) and the supernatant was collected to obtain the sulfur-fumigated Chinese medicinal material extract.

[0105] (3) Determination of SO2 residues in sulfur-fumigated Chinese medicinal materials

[0106] Probe compound method:

[0107] Take the probe compound HCY-FN in Hepes buffer solution (C HCY-FN = 2×10 -54 M) was placed in a 5 mL colorimetric tube, and the sulfur-fumigated Astragalus membranaceus extract was added. The tube was incubated at 30°C for 40 min. The fluorescence intensity was measured using a 1 cm*1 cm quartz sample cell with an incident and exit slit width of 10 nm, an excitation wavelength of 500 nm, and a voltage of 700 V. The sulfite ion concentration in the Astragalus membranaceus solution was determined using the regression equation: y = - 9.54539x + 491.42732. The residual sulfur dioxide in the Astragalus membranaceus was calculated to be 5435.15 based on the mass of the Chinese medicine sample.

[0108] Comparative Example

[0109] Acid-base titration:

[0110] According to the acid-base titration method of Part IV (General Chapter 2331) of the 2020 edition of the Chinese Pharmacopoeia, the residual SO2 in sulfur-fumigated Astragalus was determined. 5 g of the Astragalus powder in Example 4 was accurately weighed and placed in a two-necked round-bottom flask. 300 mL of ultrapure water and 10 mL of 6 mol / L hydrochloric acid solution were added. A condenser was connected, and an air guide tube was connected to the upper end of the condenser. The other end of the air guide tube was placed in a conical flask. 50 mL of 3% H2O2 solution and 3 drops of 2.5 mg / mL methyl red ethanol solution indicator were added to the conical flask as absorption liquid. Nitrogen was introduced to the bottom of the conical flask at a flow rate of 0.2 L / min. The solution in the two-necked flask was heated to boiling. Heating was stopped after 1.5 h. After the absorption liquid was allowed to cool, it was titrated with 0.01 mol / L NaOH to determine the residual SO2 in the Astragalus, which was 3532.82 mg / kg.

[0111] The fluorescence method proposed in the present invention was used for determination. The results are shown in Table 10. Each experiment was repeated 6 times. The results show that the fluorescence method proposed in the present invention was successfully used for determination of sulfur dioxide in sulfur-fumigated traditional Chinese medicine. Compared with the pharmacopoeial method, the method is accurate, reliable and feasible.

Claims

1. A hemicyanine probe compound having the structural formula: 。 2. The method for preparing the hemicyanine probe compound according to claim 1, comprising the following steps: (1) Add p-nitroaniline and hydrochloric acid to water and stir in an ice bath for 20-40 min. Then add sodium nitrite solution and continue stirring in an ice bath for 1-2 h. Add furfural acetone solution to the reaction system and then add copper chloride solution. React at 0-5°C for 10-15 h. After the reaction is completed, cool and filter, vacuum dry, and recrystallize from ethyl acetate to obtain product A. The structural formula of product A is: ; (2) 2,3,3-Trimethylindole and 1,3-propane sultone were added to toluene and dissolved, and heated at 90-110°C with stirring and reflux for 10-15 h. After the reaction was completed, the toluene solution was poured out, and the residue was ultrasonically cleaned with ethyl acetate, and the ethyl acetate solution was discarded to obtain a purple solid product B. The structural formula of product B is: ; (3) Compound A and compound B were dissolved in anhydrous ethanol, piperidine was added, and the mixture was heated at 75-85°C with stirring and reflux for 5-7 h. After the reaction was completed, the mixture was cooled and filtered, rinsed with anhydrous ethanol, and vacuum dried. The mixture was purified by silica gel chromatography to obtain a hemicyanine probe compound.

3. The method for preparing the hemicyanine probe compound according to claim 2, wherein: In step (1), the mass ratio of p-nitroaniline to sodium nitrite is 1:1-2:1; the mass ratio of p-nitroaniline to furfural is 1:1-2:1; the mass ratio of furfural to copper chloride is 1:1-1:2; and the volume ratio of hydrochloric acid to water is 1:2-1:

4.

4. The method for preparing the hemicyanine probe compound according to claim 2, wherein: In step (2), the mass ratio of 2,3,3-trimethylindole to 1,3-propane sultone is 1:1 to 1:

2.

5. The method for preparing the hemicyanine probe compound according to claim 2, wherein: In step (3), the mass ratio of compound A to compound B is 1:1 to 2:

1.

6. A method for preparing a hemicyanine probe compound according to claim 1 in SO3 2- and S 2- Application in detection.

7. The hemicyanine probe compound according to claim 6 is in SO3 2- and S 2- The application in detection is characterized by: In the C2H5OH-Hepes buffer of the hemicyanin probe compound, F - 、SO3 2- Br - , I - 、Ac - 、HPO4 2- 、HSO4 - 、ClO4 - 、NO3 - 、SCN - 、N3 - 、S 2- 、CN - OH - The addition of sulfite ions changes the color of the probe compound solution from yellow to colorless; the addition of sulfur ions changes the color of the probe compound solution from yellow to dark yellow, enabling naked eye detection of SO3 2- and S 2- ; In C2H5OH-Hepes buffer, the volume ratio of C2H5OH to Hepes buffer is 1:

9.

8. The hemicyanine probe compound according to claim 6 is in SO3 2- and S 2- The application in detection is characterized by: In the C2H5OH-Hepes buffer of the hemicyanin probe compound, F - 、SO3 2- Br - , I - 、Ac - 、HPO4 2- 、HSO4 - 、ClO4 - 、NO3 - 、SCN - 、N3 - 、S 2- 、CN - OH - , SO3 2- The addition of S weakens the fluorescence intensity of the probe compound solution and emits blue fluorescence; 2- The addition of the probe compound solution enhances the fluorescence intensity, emitting red fluorescence, thus realizing the fluorescence detection of SO3 2- and S 2- ; In C2H5OH-Hepes buffer, the volume ratio of C2H5OH to Hepes buffer is 1:

9.

9. A method for preparing SO3 based on the hemicyanine probe compound according to claim 1 2- and S 2- Application in test strips.

10. A use of the hemicyanine probe compound according to claim 1 in the rapid detection of sulfite ions in sulfur-fumigated Chinese medicinal materials, characterized in that: (1) Extract sulfur dioxide from sulfur-fumigated Chinese medicinal materials by adding 0.8-1% NaOH to the sulfur-fumigated Chinese medicinal materials at a material-liquid ratio of 1:40-1:

60. The mixture was shaken and extracted at 30-40°C for 30-50 min. The mixture was centrifuged and the supernatant was collected to obtain the sulfur-fumigated Chinese medicinal materials extract. (2) Add sulfur-fumigated Chinese herbal medicine extract to the C2H5OH-Hepes buffer of the hemicyanin probe compound and measure the fluorescence intensity. 2- The standard curve of sulfite ion was used to obtain the concentration of sulfite ion in sulfur-fumigated Chinese medicinal materials, thereby realizing rapid quantitative detection of sulfur-fumigated Chinese medicinal materials; in C2H5OH-Hepes buffer, the volume ratio of C2H5OH to Hepes buffer was 1:9; SO3 2- The ion concentration range is 0-3.7×10 -5 mol / L, SO3 2- The linear equation of the standard curve is: y = -9.54539x + 491.42732, R 2 = 0.99795, where y is the fluorescence intensity and x is SO3 2- The concentration of the substance is expressed in mol / L.

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