Hemicyanine probe compound as well as preparation and application thereof

Abstract

The invention provides a hemicyanin probe compound, and establishes a method for detecting sulfite ions and sulfur ions in a solution by probe molecules under ultraviolet visible and fluorescent conditions. Common anions are respectively added into a C2H5OH-Hepes buffer solution, only sulfite and sulfide ions are changed, the sulfite ions can fade the solution, the fluorescence intensity of the solution is weakened and the anti-interference performance is good, and the sulfide ions can deepen the color of the solution, enhance the fluorescence intensity of the solution and have good anti-interference performance. Meanwhile, the residual quantity of sulfur dioxide in the stoving traditional Chinese medicinal materials is taken as a detection target, the stoving traditional Chinese medicinal materials are treated under an alkaline condition, and ultra-sensitive and rapid detection of the stoving traditional Chinese medicinal materials is realized through a standard curve method. The semi-cyanin probe compound has the advantages of high analysis speed, stable fluorescence performance, real-time detection and the like when being used for determining the content of sulfur dioxide residues in traditional Chinese medicinal materials, and has popularization and application values in sulfur dioxide residue detection in the fields of traditional Chinese medicines, foods and the like.

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 rapid detection of sulfite ions and sulfur-fumigated Chinese medicinal materials. Background Art

[0002] Sulfur fumigation is a common method for processing Chinese herbal medicines. It has been used for a long time in the processing and storage and maintenance of Chinese herbal medicines, foods and agricultural products. Its main purpose is to dry and color Chinese herbal medicines, and it can also achieve the effects of anti-corrosion, mildew prevention, bleaching, and insect prevention. The main residue of sulfite after sulfur fumigation of Chinese herbal medicines causes clinical diseases such as asthma, urticaria, itching, angioedema, and even anaphylactic shock and death. It may also have carcinogenic effects and cause tissue, organ, and nervous system poisoning. At present, sulfur fumigation is mainly used for some Chinese herbal medicines that are difficult to dry or easy to spoil, such as Pueraria root, Chinese yam, Chinese wolfberry and Gastrodia elata. A large amount of SO2 will be produced during the sulfur fumigation process, which exists in Chinese herbal medicines in two forms: free and bound. The free state includes SO2 molecules, sulfites and bisulfites. The bound state is mainly the bound SO2 formed by the reaction of free SO2 with some components in Chinese medicinal materials. Since irreversibly bound sulfites do not dissociate under physiological conditions and will not harm human health, the detection objects are free sulfites and reversibly bound sulfites. The 2020 edition of the Chinese Pharmacopoeia has strict regulations on the detection limit of SO2 residues, among which yam, gastrodia, asparagus, Achyranthes bidentata, Radix Trichosanthis, Atractylodes macrocephala, Bletilla striata, Codonopsis pilosula, Paeonia lactiflora, and Pueraria lobata shall not exceed 400 mg / kg, yam slices shall not exceed 10 mg / kg, and other Chinese medicinal materials shall not exceed 150 mg / kg. However, in order to increase the appearance of Chinese medicinal materials and pursue economic benefits, farmers over-fumigate with sulfur, resulting in excessive SO2 content. In order to speed up the circulation of Chinese medicinal materials in the market, ensure the safety of medication, and reduce potential health risks, it is of great significance to study sensitive, rapid, and real-time detection methods for SO2 residues in sulfur-fumigated Chinese medicinal materials. 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; The second object of the present invention is to provide a probe compound of hemicyanine for detecting SO3 2- and S 2- Application in.

[0004] The third purpose of the present invention is to provide a probe compound of hemicyanine for rapid detection of SO3 in sulfur-fumigated Chinese medicinal materials. 2- application.

[0005] 1. Probe compounds of hemicyanine and their synthesis The hemicyanine probe compound HCY-FN of the present invention has the molecular formula: 25 H 24 N2O6S, the structural formula is as follows: .

[0006] The method for preparing the probe compound of the present invention comprises the following steps: (1) Add p-nitroaniline and hydrochloric acid to water, stir in an ice bath for 20-40 min, then add sodium nitrite solution, continue stirring in an ice bath for 1-2 h, add furanaldehyde acetone solution to the reaction system, 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; Among them, the mass ratio of p-nitroaniline to sodium nitrite is 1:1~2:1; the mass ratio of p-nitroaniline to furanaldehyde is 1:1~2:1; the mass ratio of furanaldehyde to cupric chloride is 1:1~1:2; and the volume ratio of hydrochloric acid to water is 1:2~1:4.

[0007] (2) Add 2,3,3-trimethylindole and 1,3-propane sultone into toluene and dissolve them. Heat and stir under reflux at 90-110°C for 10-15 h. After the reaction, pour out the toluene solution, clean the residue with ethyl acetate by ultrasonic, and discard the ethyl acetate solution to obtain a purple solid product B. The mass ratio of 2,3,3-trimethylindole to 1,3-propane sultone is 1:1-1:2.

[0008] (3) Compound A and compound B were dissolved in anhydrous ethanol, piperidine was added, and the mixture was heated and stirred at 75-85°C for 5-7 h under reflux. After the reaction was completed, the mixture was cooled and filtered, washed with anhydrous ethanol, and then 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.

[0009] The synthetic route is as follows: 2. Probe compound detection of SO3 2- Applications 1. UV-Vis spectrum of probe compound HCY-FN 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 , and found that with S 2- The UV spectrum showed maximum absorption wavelengths at 350nm and 500nm. 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, S 2- The addition of SO3 2- The addition of makes the color of the probe compound solution fade from yellow, and the maximum absorption wavelength of the probe HCY-FN is max = 350nm, when adding SO3 2- The solution color faded and turned colorless, indicating that the probe molecule can detect SO3 with naked eyes under UV-visible spectrum. 2- and S 2- .

[0010] The results of anti-interference experiments show that the presence of other anions has an important effect on the recognition of SO3 by the probe compound HCY-FN. 2- and S 2- No interference (such as Fig.14 and Fig.16 as shown).

[0011] The results of UV-visible titration experiments showed that the probe compound HCY-FN had an 2 and SO3 2- The probe HCY-FN has the characteristic of high sensitivity. According to the formula 3σ / s, the probe HCY-FN is calculated to be S in the ultraviolet spectrum. 2- The minimum detection limit was 3.05 × 10 - 6 M, for SO3 2- The minimum detection limit was 4.97 × 10 -6 M. (e.g. Figure 8 and Fig.12 shown) 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 proves that the probe molecule HCY-FN can be used to treat S 2- and SO3 2- It can achieve the purpose of rapid detection. Fig.19 b and 20b); 2. Fluorescence properties of the probe compound HCY-FN 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 the probe HCY-FN C2H5OH- Hepes buffer (C2H5OH: Hepes (pH = 4) 1: 9, V / V) and the fluorescence intensity was measured. 2- After that, the fluorescence intensity weakened at the emission wavelength of 600nm, and blue fluorescence was emitted; when S 2- After that, the fluorescence intensity at the emission wavelength of 600nm increases, emitting red fluorescence, and other ions remain unchanged. Figure 4 As shown, the results showed that the probe had a strong affinity for SO3 2- and S 2- Responsive and specific.

[0012] The results of the anti-interference experiment showed that the presence of other anions did not interfere with the probe compound (e.g. Fig.13 and Fig.15 as shown).

[0013] 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 to 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 Fig.10 (shown) Under fluorescence conditions, the probe molecule HCY-FN can recognize S at 20 s and 40 s in the fluorescence spectrum. 2- and SO3 2- , and reached reaction equilibrium within 4 min and 1 min respectively. 2- and S 2- It can realize efficient and rapid detection. Fig.19 a and 20a) 3. Probe compound HCY-FN recognizes SO3 2- Mechanism analysis 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 combination ratio, such as Fig.17 and Fig.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 NMR titration showed that the probe molecules reacted with sulfite ions and sulfur ions by affinity addition reaction (such as Fig.21 High-resolution mass spectrometry also further showed that the probe molecule HCY-FN and SO3 2- The binding ratio is 1:1 (such as Fig. 22 as shown).

[0014] 4. Detection of SO3 by probe compound HCY-FN 2- and S 2- Practical Applications 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. Fig.23 and Fig.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 faded and the color deepened. Fig.24 and Fig.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.

[0015] 4.2 Application of sulfur-fumigation in rapid detection of Chinese medicinal materials 4.2.1 Drawing of Na2SO3 standard curve The probe HCY-FN mother solution and Hepes buffer solution were prepared in a ratio of 1:9, and the Na2SO3 standard solution was added dropwise to the probe HCY-FN 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.

[0016] 4.2.2 Investigation of Codonopsis pilosula extraction methods Taking the SO2 content in sulfur-fumigated Codonopsis as the investigation index, three different extraction methods were selected: ultrasound, immersion, and oscillation. The results showed that the residual amount of SO2 in sulfur-fumigated Codonopsis was the highest when the oscillation extraction method was used, so the oscillation method was selected to extract SO2 from sulfur-fumigated medicinal materials. See Table 1.

[0017] 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 measured with 1% NaOH concentration. Therefore, the extraction solution with 1% NaOH concentration was selected to extract SO2 from the sulfur-fumigated medicinal materials, see Table 2.

[0018] 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.

[0019] 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.

[0020] 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 in sulfur-fumigated medicinal materials. 2， See Table 5.

[0021] From the above results, it can be seen that when the extraction method is the oscillation method, the extraction solution is 1% NaOH, the solid-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.

[0022] 4.2.3 Methodological investigation 4.2.3.1 Precision investigation 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.

[0023] 4.2.3.2 Repeatability study The extract of fumigated Codonopsis pilosula was obtained by 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 proved the reliability of the detection method, as shown in Table 7.

[0024] 4.2.3.3 Stability study The extract of fumigated Codonopsis pilosula was obtained by 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 min, as shown in Figure 8.

[0025] 4.2.3.4 Investigation of sample recovery rate 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 determined by 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-fumigation medicinal material extract. 2- The content is shown in Table 9.

[0026] 4.2.3.5 Blank control The extract of unfumigated Codonopsis pilosula was obtained by 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 6 tests was the same as that without the addition of the extract.

[0027] 4.2.4 Results of sulfur dioxide residues in Chinese medicinal materials 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.

[0028] In summary, 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 detected content was the highest and the precision (RSD ≤ 1.76%), repeatability (RSD ≤ 1.85%), and sample recovery rate (RSD ≤ 1.90%) were the best, 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.

[0029] The present invention provides a hepesin probe compound, and establishes a method for detecting sulfite ions and sulfide ions in a solution under ultraviolet visible and fluorescence conditions. Common anions are added to the solution of the (C2H5OH: Hepes = 1: 9, V / V) probe molecule, and only sulfite and sulfide ions change. Sulfite can make the solution fade, and the fluorescence of the solution is quenched, with good anti-interference performance, and sulfide ions can deepen the fluorescence color. The sulfur dioxide residue in sulfur-fumigated Chinese medicinal materials is taken as the detection target, and 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 the standard curve method. The hepesin probe compound has the advantages of fast analysis speed, stable fluorescence performance, real-time detection, etc. for the determination of the content of sulfur dioxide residue in Chinese medicinal materials, and has the value of popularization and use in the detection of sulfur dioxide residues in the fields of traditional Chinese medicine, food, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the hydrogen spectrum (DMSO-d6) of the probe molecule HCY-FN of the present invention; Figure 2 The carbon spectrum of the probe molecule HCY-FN (DMSO-d 6) ; Figure 3 The UV spectra of different anions of the probe molecule HCY-FN of the present invention are shown in Figure 1: Inset: Photo taken with visible light; Figure 4 The fluorescence spectra of different anions of the probe molecule HCY-FN of the present invention; Inset: Photo taken under ultraviolet light with a wavelength of 365 nm; Figure 5 Add S to the (C2H5OH: Hepes = 1:9, V / V) system of the probe molecule HCY-FN of the present invention. 2- Fluorescence titration of; Figure 6 Add S 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 Figure 7 To add S to the (C2H5OH: Hepes = 1:9, V / V) system of the probe molecule HCY-FN of the present invention 2- UV titration; Figure 8 To add S 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 Fig. 9 SO3 was added to the (C2H5OH: Hepes = 1:9, V / V) system of the probe molecule HCY-FN of the present invention. 2- Fluorescence titration of Fig.10 SO3 was added 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 Fig.11 SO3 was added to the (C2H5OH: Hepes = 1:9, V / V) system of the probe molecule HCY-FN of the present invention. 2- UV titration; Fig.12 SO3 was added 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 Fig.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 diagrams of different anions were added; Fig.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; Fig.15 Add S 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 diagrams of different anions were added; Fig.16 Add S 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; Fig.17 The probe molecules HCY-FN and SO3 2- Working curves between them under fluorescence emission spectrum (a) and UV emission spectrum (b); Fig.18 The probe molecules HCY-FN and S 2-Working curves between them under fluorescence emission spectrum (a) and UV emission spectrum (b); Fig.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; Fig. 20 The probe molecule HCY-FN of the present invention is effective for S 2- The corresponding time curves under fluorescence (a) and UV (b) conditions; Fig.21 The probe molecule HCY-FN of the present invention is effective for SO3 2- and S 2- NMR titration spectrum of Fig. 22 The probe molecule HCY-FN of the present invention is for SO3 2- High-resolution mass spectra of Fig.23 The detection test strip of the probe molecule HCY-FN prepared by the present invention is coated with SO3 2- Photos taken in visible light; Fig.24 The detection test strip of the probe molecule HCY-FN prepared by the present invention is coated with SO3 2- The photos were taken under 365nm ultraviolet light. Fig.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; Fig.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

[0031] The following is a specific example of the preparation of the probe molecule HCY-FN of the present invention and the ultraviolet visible and fluorescent recognition of SO3 2- The application is further described.

[0032] Example 1. Synthesis of probe compound HCY-FN (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 into the three-necked flask with a syringe, and stir in an ice bath for 1 h; weigh 3.17 g of furan carboxaldehyde 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. (2) Weigh 1.5923 g of 2,3,3-trimethylindole and 1.8321 g of 1,3-propane sultone into a 250 ml two-necked flask and dissolve them in 15 ml of toluene. Heat and stir under reflux at 100 °C for 12 h. After the reaction is completed, pour out the toluene solution, ultrasonically clean the residue with ethyl acetate, and discard the ethyl acetate solution to obtain a purple solid product B. The yield is 22.9%.

[0033] (3) Weigh 1.1856 g of compound A and 0.9556 g of compound B, dissolve them in 20 ml of anhydrous ethanol in a 250 ml double-necked flask, and add 0.374 ml of piperidine dropwise into the double-necked flask with a syringe. Heat, stir and reflux at 80°C for 6 h. After the reaction is completed, cool and filter, rinse with anhydrous ethanol several times and then dry in vacuum. Purify the crude product by silica gel chromatography (CH2Cl2: CH3OH= 11: 4, V / V) to obtain a 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).

[0034] Example 2: Fluorescence recognition of SO3 by probe compound HCY-FN 2- and S 2- 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.

[0035] Example 3 Probe compound HCY-FN identifies SO3 with naked eyes 2- and S 2- 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.

[0036] Example 4: Detection of sulfur-fumigated Chinese medicinal materials by probe compound HCY-FN (1) Preparation of sulfur-fumigated Chinese medicinal materials The astragalus samples that had not been treated with sulfur fumigation were used as the research object, and a fumigation box was made of stainless steel with a mesh partition inside. During the experiment, 20 g of unfumigated medicinal materials were evenly spread on the mesh partition (the medicinal materials were soaked in ultrapure water in advance), 50 g of medicinal sulfur was weighed and placed in an enamel tray and placed at the bottom of the fumigation box, the sulfur was ignited, and the fumigation box door was closed to place it in a closed space, and the sulfur fumigation was repeated 4 times.

[0037] (2) Preparation of sulfur-fumigated Chinese medicinal material extracts The sulfur-fumigated Astragalus membranaceus in (1) was crushed into fine powder, and 0.5 g was accurately weighed and placed in a centrifuge tube. 10 mL of 1% NaOH was accurately added. The mixture was extracted by shaking at 30°C for 50 min, and then centrifuged at high speed (4500 rpm for 10 min). The supernatant was collected to obtain the sulfur-fumigated Chinese medicinal material extract.

[0038] (3) Determination of SO2 Residue in Sulfur Fumigated Chinese Medicinal Materials Probe compound method: Take the probe compound HCY-FN in Hepes buffer solution (C HCY-FN = 2 × 10 -54The sample was placed in a 5 mL colorimetric tube, and sulfur-fumigated Astragalus membranaceus extract was added. The sample was placed at 30 °C for 40 min, and 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 concentration of sulfite ions in the Chinese medicinal material solution was obtained according to the regression equation: y = - 9.54539x + 491.42732, and the residual sulfur dioxide in Astragalus membranaceus was calculated to be 5435.15 according to the mass of the Chinese medicine sample.

[0039] Comparative Example Acid-base titration: According to the acid-base titration method of the fourth volume of the 2020 edition of the Chinese Pharmacopoeia (General Rule 2331), the residual SO2 in sulfur-fumigated Astragalus was determined. Take 5g of the Astragalus powder in Example 4, accurately weigh it, put it in a two-necked round-bottom flask, add 300mL of ultrapure water and 10mL of 6mol / L hydrochloric acid solution, connect the condenser, connect the air guide tube at the upper end of the condenser, and place the other end of the air guide tube in a conical flask. Add 50mL of 3% H2O2 solution and 3 drops of 2.5mg / mL methyl red ethanol solution indicator as absorption liquid in the conical flask, introduce nitrogen to the bottom of the conical flask and control the flow rate to 0.2L / min, heat the solution in the two-necked flask to boiling, stop heating after 1.5h, and titrate with 0.01mol / LNaOH after the absorption liquid is cooled to determine the residual SO2 in Astragalus, which is 3532.82 mg / kg.

[0040] The fluorescence method proposed in the present invention was used for determination, and the results are shown in Table 10. Each group of experiments was repeated 6 times. The results show that the fluorescence method proposed in the present invention was successfully used for the determination of sulfur dioxide in sulfur-fumigated traditional Chinese medicines. Compared with the pharmacopoeia method, the method is accurate and reliable, and the method is feasible.

Claims

1. A hemicyanine probe compound, the structural formula of which is: 。 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, stir in an ice bath for 20-40 min, then add sodium nitrite solution, continue stirring in an ice bath for 1-2 h, add furanaldehyde acetone solution to the reaction system, 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) Add 2,3,3-trimethylindole and 1,3-propane sultone into toluene and dissolve them. Heat and stir under reflux at 90-110°C for 10-15 h. After the reaction is completed, pour out the toluene solution, wash the residue with ethyl acetate by ultrasonic, and discard the ethyl acetate solution 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 then 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, characterized in that: In step (1), the mass ratio of p-nitroaniline to sodium nitrite is 1:1-2:1; the mass ratio of p-nitroaniline to furanaldehyde is 1:1-2:1; the mass ratio of furanaldehyde to cupric 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, characterized in that: 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, characterized in that: 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 under detection is characterized by: In the C2H5OH-Hepes buffer of the hemicyanin probe compound, add F - 、SO3 2- Br - ,I - ,Ac - 、HPO4 2- 、HSO4 - 、ClO4 - 、NO3 - 、SCN - 、N3 - , S 2- , CN - , PPI, OH - The addition of sulfite ions changes the color of the probe compound solution from yellow to colorless; the addition of sulfite ions changes the color of the probe compound solution from yellow to dark yellow, realizing 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 under detection is characterized by: In the C2H5OH-Hepes buffer of the hemicyanin probe compound, add F - 、SO3 2- Br - ,I - ,Ac - 、HPO4 2- 、HSO4 - 、ClO4 - 、NO3 - 、SCN - 、N3 - , S 2- , CN - , PPI, 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. An application of the hemicyanine probe compound according to claim 1 in rapid detection of sulfite ions in sulfur-fumigated Chinese medicinal materials, characterized in that: (1) Extracting sulfur dioxide from sulfur-fumigated Chinese medicinal materials, adding 0.8-1% NaOH to the sulfur-fumigated Chinese medicinal materials, with a solid-liquid ratio of 1:40-1:60, extracting at 30-40°C, shaking for 30-50 min, centrifuging, and taking the supernatant to obtain a sulfur-fumigated Chinese medicinal material extract; (2) Add sulfur-fumigated Chinese medicinal material extract to the C2H5OH-Hepes buffer of the hemicyanin probe compound and measure the fluorescence intensity. 2- The standard curve of was used to obtain the concentration of sulfite ions in sulfur-fumigated Chinese medicinal materials, so as to realize the 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 is in mol / L.

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