Mercury ion anthocyanin fluorescent probe, its preparation method and its application in the detection of traditional Chinese medicinal plants.
By synthesizing a near-infrared fluorescent probe BBN-Hg with an anthocyanin derivative as the fluorophore, the problems of complex process and low biocompatibility in the detection of mercury ions in the existing technology have been solved, realizing highly sensitive and visual detection of mercury ions in the roots of Chinese medicinal plants, and reducing experimental costs and time.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- ANHUI UNIVERSITY OF TRADITIONAL CHINESE MEDICINE
- Filing Date
- 2023-12-06
- Publication Date
- 2026-05-26
Smart Images
Figure CN117777152B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of fluorescence detection technology, specifically relating to a mercury ion anthocyanin fluorescent probe, its preparation method, and its application in the detection of traditional Chinese medicinal plants. Background Technology
[0002] Mercury compounds have high bioaccumulation and persistence, and degrade slowly in organisms. Free Hg 2+ It can exhibit strong binding affinity to proteins containing thiol, sulfur, and selenium groups. However, in aquatic environments, Hg... 2+ It accumulates in aquatic organisms and enters the human body through the nutrient chain, damaging the central nervous system, digestive system, respiratory system, and endocrine system, thus leading to a series of diseases such as Alzheimer's disease, Minamata disease, and brain damage. Furthermore, research has shown that Hg... 2+ It tends to accumulate in plant roots and soil. High concentrations of Hg in the soil 2+ Mercury can cause significant damage to plant growth. To ensure the safety of traditional Chinese medicine (TCM) use, the 2020 edition of the Chinese Pharmacopoeia requires that the mercury content in TCM should not exceed 0.2 mg / kg. Therefore, developing an effective method for monitoring the mercury content in TCM is of great significance.
[0003] In recent years, various analytical methods for detecting mercury ions have been developed. However, these methods suffer from drawbacks such as complex operation procedures, long detection times, and the need for expensive instruments. To overcome these limitations, fluorescent probes have gained widespread attention and application in the medical and biological fields due to their advantages of high sensitivity, good selectivity, low cost, and simple operation. However, among the published literature on mercury ion detection, such as the Dutch journal *Analytica Chimica Acta.* (1192(2022)339353, S. Erdemir, M. Oguz, S. Malkondu, A NIR fluorescent sensor based on thiazoline-isophorone with low cytotoxicity in living cells for Hg), there are still many limitations. 2+ detection through ICT associated hydrogenbonding effect.), American "Analytical Chemistry" (Anal.Chem., 2018,90,4909-4915, J.Hai,FJChen,JXSu,F.Xu,BDWang,PorousWood Members-Based Amplified Colorimetric Sensor for Hg 2+Detection through Hg 2 + The fluorescent probes described in *Triggered Methylene Blue Reduction Reactions* still have some shortcomings, such as cumbersome synthesis processes, weak water solubility, short emission wavelengths, long response times, low biocompatibility, and a lack of research on the detection of mercury ions in plants. These limitations severely restrict their practical applications. Therefore, developing a highly selective fluorescent probe with simple synthesis steps, high detection sensitivity, and excellent photophysical properties is of significant research importance. Summary of the Invention
[0004] In view of the shortcomings of the prior art, the present invention provides a mercury ion anthocyanin fluorescent probe, its preparation method, and its application in the detection of traditional Chinese medicinal plants. This probe is simple to synthesize, easy to operate, highly sensitive and selective for mercury ions, and can visually detect mercury ions in the roots of traditional Chinese medicinal plants.
[0005] In a first aspect, the present invention provides a mercury ion anthocyanin fluorescent probe with the molecular formula: C 30 H 26 ClNO7S has the structure shown in formula (Ⅰ):
[0006]
[0007] In a second aspect, the present invention provides a method for preparing the aforementioned mercury ion anthocyanin fluorescent probe: In the presence of an alkali and an organic solvent, the compound shown in formula (Ia) is contacted with the compound shown in formula (Ib) to react, yielding the compound shown in formula (I), as shown in the following reaction formula:
[0008]
[0009] Wherein, X is a halogen, preferably chlorine.
[0010] According to a specific embodiment of the present invention, the molar ratio of the compound shown in formula (Ia) to the compound shown in formula (Ib) is 1:0.5 to 2, preferably 1:1.
[0011] According to a specific embodiment of the present invention, the alkali is any one of triethylamine, potassium carbonate, and cesium carbonate, preferably triethylamine; the organic solvent is any one of dichloromethane, anhydrous acetonitrile, and N,N-dimethylformamide, preferably dichloromethane.
[0012] According to a specific embodiment of the present invention, the reaction conditions are 1.5 to 2.5 hours at room temperature, preferably 2 hours at room temperature.
[0013] A third aspect of the present invention provides the application of the aforementioned fluorescent probe in the detection of mercury ions in traditional Chinese medicinal plants; preferably, the traditional Chinese medicinal plant is *Salvia miltiorrhiza*. This probe can visually monitor mercury ions in the roots of *Salvia miltiorrhiza*, and it is low in cost, highly sensitive, and selective, showing promising application prospects.
[0014] In a fourth aspect, the present invention provides a reagent for detecting mercury ions, which contains the aforementioned mercury ion anthocyanin fluorescent probe.
[0015] The beneficial effects of this invention are as follows:
[0016] 1. This invention synthesizes a near-infrared fluorescent probe BBN-Hg using an anthocyanin derivative as the fluorophore and phenyl thiochloroformate as the recognition site. The probe exhibits good spectral performance in a DMSO / PBS (pH = 7.4, v / v = 3:7) system, demonstrating high selectivity and sensitivity for mercury ions, with a low detection limit (53 nM). Its fluorescence intensity shows a good linear relationship with mercury ion concentration in the range of 0-34 μM. This probe boasts high yield, simple operation, and few synthesis steps.
[0017] 2. This invention is the first to apply a fluorescent probe to the detection of heavy metal ions in the roots of traditional Chinese medicine, and to observe the distribution of heavy metal ions in plants using plant imaging. The imaging of exogenous mercury ions by the probe BBN-Hg in the roots of the traditional Chinese medicine *Salvia miltiorrhiza* was studied. The results showed that the fluorescence signal emitted by the untreated *Salvia miltiorrhiza* roots was very weak, while the fluorescence signal in the red channel gradually increased with increasing mercury ion concentration. This indicates that the probe can be successfully used to detect heavy metal mercury ions in the roots of *Salvia miltiorrhiza*. This provides a new direction for the application of fluorescent probe technology.
[0018] 3. This invention is the first to employ a fluorescent probe method for plant imaging of exogenous mercury ions in the roots of the traditional Chinese medicine Salvia miltiorrhiza. This method is simple to operate and allows for direct observation of the mercury ion distribution while simultaneously detecting the mercury ion content in Salvia miltiorrhiza, significantly reducing experimental costs and time. Attached Figure Description
[0019] Figure 1 This is the synthetic route for the fluorescent probe BBN-Hg;
[0020] Figure 2 This is the 1H NMR spectrum of the fluorescent probe BBN-Hg;
[0021] Figure 3 This is the carbon NMR spectrum of the fluorescent probe BBN-Hg;
[0022] Figure 4 This is the mass spectrum of the fluorescent probe BBN-Hg;
[0023] Figure 5The graph shows the changes in the UV absorption spectrum of the fluorescent probe (10 μM) with increasing mercury ion concentration after the addition of different concentrations of mercury ions (0-34 μM); the inset shows photographs of the fluorescent probe (10 μM) under fluorescent light before and after the reaction with the corresponding mercury ions.
[0024] Figure 6 The graph shows the fluorescence spectrum of a 10 μM fluorescent probe as the concentration of mercury ions increases after the addition of different concentrations (0-34 μM); the inset shows photographs of the 10 μM fluorescent probe under 365 nm UV light before and after the reaction with the corresponding mercury ions.
[0025] Figure 7 This is a graph showing the linear relationship between the fluorescence intensity of the fluorescent probe (10 μM) at 650 nm and the concentration of mercury ions;
[0026] Figure 8 This is a graph showing the change in fluorescence intensity of the fluorescent probe (10 μM) over time after the addition of mercury ions (34 μM);
[0027] Figure 9 The fluorescent probe (10 μM) is reacted with mercury ions (34 μM) and different cations and anions (100 μM): Cl - ,ClO - HCO3 - CO3 2- K + Li + Na + NH4 + Ca 2+ Co 2+ Cr 2+ Cu 2+ Fe 2+ Mg 2+ Mn 2+ Zn 2+ Fe 3+ The fluorescence spectrum changes afterward;
[0028] Figure 10 It is a fluorescent probe (10 μM) in different cations and anions (100 μM): Cl - ,ClO - HCO3 - CO3 2- K + Li + Na + NH4 + Ca 2+ Co 2+ Cr 2+ Cu 2+Fe 2+ Mg 2+ Mn 2+ Zn 2+ Fe 3+ Fluorescence spectrum changes after the addition of mercury ions (34 μM) in the presence of mercury;
[0029] Figure 11 This is a graph showing the changes in fluorescence spectra of a fluorescent probe (10 μM) before and after the addition of mercury ions (34 μM) under different pH conditions;
[0030] Figure 12 The images show the fluorescence imaging results after the root tips of Salvia miltiorrhiza seedlings were pretreated with different concentrations of mercury ions (0, 10, 30, 50 μM) for 0.5 h, and then incubated with a fluorescent probe (10 μM) for 0.5 h. Detailed Implementation
[0031] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0032] Example 1: Synthesis of the fluorescent probe BBN-Hg
[0033] Synthesis route diagram as follows Figure 1 As shown, the fluorescent probe molecules were synthesized according to the synthetic route diagram:
[0034] 1. Preparation of intermediate BBN-OH
[0035] 9-Formaldehyde-8-hydroxyjulonidine and 6-hydroxy-1-tetrahydronaphthone were added to a round-bottom flask containing glacial acetic acid at a molar ratio of 1:1. The mixture was stirred at 250 rpm for 2 min with a magnetic stirrer. 2 mL of 70% perchloric acid was slowly added using a syringe. The mixture was refluxed at 90 °C for 2 h. After the reflux was completed, the mixture was cooled to room temperature. 15 mL of a PE / EA = 1 / 1 mixture was added for washing. The mixture was stirred for 30 min, filtered, and washed with a small amount of the PE / EA = 1 / 1 mixture. The mixture was then washed with anhydrous ethanol, and the product was purified using a silica gel column to obtain the intermediate BBN-OH.
[0036] 2. Preparation of fluorescent probe BBN-Hg
[0037] Intermediate BBN-OH (50 mg, 0.1 mmol) and triethylamine (23 mg, 0.2 mmol) were dissolved in analytical grade dichloromethane. After stirring at room temperature for 10 min, phenyl thiochloroformate (24 mg, 0.1 mmol) was added to the system, and the mixture was allowed to react at room temperature for 2 hours. After the reaction was complete, the solvent was evaporated by rotary evaporation, and the product was purified by silica gel column chromatography (DCM / MeOH = 50:1) to obtain a black solid powder BBN-Hg (35 mg), with a yield of 53%. The 1H NMR spectrum, 1C NMR spectrum, and mass spectrum of BBN-Hg are shown below. Figures 2-4 As shown. 1 H NMR (600MHz, DMSO-d6) δ8.47(s,1H),8.21(d,J=9.2Hz,1H),7.59(s,1H),7.56–7.51(m,4H),7.39(d,J=7.4Hz,1H),7.36(d,J=7 .5Hz,2H),3.62(d,J=5.5Hz,4H),3.10(d,J=7.9Hz,2H),3.05(d,J=6.4Hz,4H),2.91(t,J=6.3Hz,2H),2.02(s,2H),1.96(s,2H). 13 C NMR(151MHz,DMSO-d6)δ193.42,158.18,155.90,153.09,152.77,152.31,145.83,143.03,129.95,129.27,127.12,126.95,126.60,125.38, 122.31,121.66,119.96,115.55,104.53,50.78,50.35,31.51,30.36,29.40,26.97,26.11,24.07,19.71,18.84,18.67.HRMS[electrospray ionization(ESI)]:m / z calculated for C 30 H 26 NO3S + [M+H] + ,480.1628;found,480.1621.
[0038] Example 2: Spectral response of fluorescent probe to mercury ions
[0039] The detection method steps are as follows:
[0040] 1. Probe BBN-Hg against Hg 2+ UV / fluorescence spectral changes
[0041] The fluorescent probe prepared in Example 1 was used to prepare a probe stock solution with a concentration of 1 mmol / L using DMSO; HgCl2 was dissolved in deionized water to prepare a mercury ion stock solution with a concentration of 10 mmol / L; and a spectral solution of DMSO / PBS (pH=7.4, v / v=3:7) was prepared.
[0042] Accurately transfer 3 mL of the spectral solution into a quartz cuvette. After adding 30 μL of the probe stock solution to the cuvette, add different volumes of mercury ion stock solution in sequence. After reacting for 15 min, use a UV-Vis absorption spectrometer and a fluorescence absorption spectrometer to test the spectra of the probe and the probe plus mercury ion solution.
[0043] from Figure 5 and Figure 6 It can be seen that adding different concentrations of Hg... 2+ After (0-34μM), the main absorption peak of the probe BBN-Hg (10μM) shifted from 560nm to 606nm, and the solution color changed from purple to blue. Figure 5 (Illustration). Simultaneously, a new fluorescence emission peak was observed at 650 nm using a fluorescence spectrophotometer, and this peak increased with Hg. 2+ As the concentration increases, the fluorescence gradually intensifies, changing from a pale red to a bright red. Figure 6 (Illustration). From Figure 7 and Figure 8 It can be seen that in the range of 0-34 μM Hg 2+ Within the range, there is a good linear relationship between the fluorescence intensity and the probe fluorescence intensity (R). 2 =0.9276). The detection limit LOD was calculated using the formula LOD = 3δ / k, and the effect of the probe BBN-Hg on Hg was calculated. 2+ The detection limit was 53 nM. Meanwhile, the fluorescence at 650 nm gradually increased with increasing reaction time (0-15 min).
[0044] 2. Selectivity and anti-interference experiments
[0045] Accurately transfer 3 mL of the spectral solution into a quartz cuvette. After adding 30 μL of the probe stock solution to the cuvette, add Hg sequentially. 2+ (34 μM) and other cations and anions (100 μM): Cl - ,ClO - HCO3 - CO3 2- K + Li + Na + NH4 + Ca 2+ Co 2 + Cr 2+ Cu2+ Fe 2+ Mg 2+ Mn 2+ Zn 2+ Fe 3+ Perform spectral testing.
[0046] like Figure 9 As shown, the fluorescence intensity changes very little with the addition of other ions, only with the addition of Hg... 2+ It exhibits strong red fluorescence, indicating that the probe is effective in recognizing Hg. 2+ It has good single selectivity.
[0047] Accurately transfer 3 mL of the spectral solution into a cuvette. After transferring 30 μL of the probe stock solution into a quartz cuvette, add the other cations and anions (100 μM) sequentially: Cl... - ,ClO - HCO3 - CO3 2- K + Li + Na + NH4 + Ca 2+ Co 2+ Cr 2+ Cu 2+ Fe 2 + Mg 2+ Mn 2+ Zn 2+ Fe 3+ Then, continue to add Hg separately. 2+ The concentration was measured at 34 μM.
[0048] like Figure 10 As shown, the addition of other ions helps the probe recognize Hg. 2+ The fluorescence signal showed very little change, indicating that the probe detected Hg. 2+ It has good anti-interference ability.
[0049] 3. Effects of different pH values on fluorescent probes
[0050] like Figure 11 As shown, the fluorescence intensity of the fluorescent probe is weak at pH values between 2 and 8. The addition of mercury ions significantly increases the fluorescence intensity, covering a range that is within the physiological pH range.
[0051] Example 3: Detection of mercury ions in the roots of Salvia miltiorrhiza using fluorescent probes
[0052] A fluorescent probe prepared in Example 1 was used for detection, targeting the root tips of *Salvia miltiorrhiza* seedlings. The detection and imaging method was as follows: Sealed seeds were washed three times with deionized water. They were then soaked in water for 24 hours. The seeds were then evenly spread on moistened filter paper, placed in petri dishes, and incubated at 25°C for 6-7 days. After germination, the seeds were transferred to 96-well plates containing deionized water for further growth. The seedling roots were then immersed in solutions containing different concentrations of Hg. 2+ Seedling roots were incubated in 0, 10, 30, and 50 μM aqueous solutions for 0.5 h, and then transferred to a probe-containing (10 μM) aqueous solution for 0.5 h to obtain different concentrations of seedling root fluorescence imaging groups. Before fluorescence imaging, the seedling roots were carefully washed three times and then placed on a microscope slide. Fluorescence imaging of the seedling roots in each group was performed using an Olympus FV1000 laser confocal microscope.
[0053] from Figure 12 As can be seen, the red fluorescence clearly shows the distribution of mercury ions in the roots of Salvia miltiorrhiza.
[0054] In summary, the fluorescent probe provided by this invention has good selectivity and high sensitivity for mercury ions, and can intuitively observe the distribution of mercury ions, which is of great significance for the visual detection of mercury ions in plant roots.
[0055] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details of the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.
Claims
1. A mercury ion anthocyanin fluorescent probe, characterized in that, It has the structure shown in equation (Ⅰ): Equation (Ⅰ).
2. A method for preparing the mercury ion anthocyanin fluorescent probe according to claim 1, characterized in that, The preparation method is as follows: in the presence of a base and an organic solvent, the compound shown in formula (Ia) is contacted with the compound shown in formula (Ib) to react and obtain the compound shown in formula (I), as shown in the following reaction formula: Where X is a halogen.
3. The preparation method according to claim 2, characterized in that, X is chlorine.
4. The preparation method according to claim 2, characterized in that, The molar ratio of the compound shown in formula (Ia) to the compound shown in formula (Ib) is 1:0.5~2.
5. The preparation method according to claim 4, characterized in that, The molar ratio of the compound shown in formula (Ia) to the compound shown in formula (Ib) is 1:
1.
6. The preparation method according to claim 2, characterized in that, The alkali is any one of triethylamine, potassium carbonate, and cesium carbonate; the organic solvent is any one of dichloromethane, anhydrous acetonitrile, and N,N-dimethylformamide.
7. The preparation method according to claim 6, characterized in that, The base is triethylamine.
8. The preparation method according to claim 6, characterized in that, The organic solvent is dichloromethane.
9. The preparation method according to claim 2, characterized in that, The reaction was carried out at room temperature for 1.5 to 2.5 hours.
10. The preparation method according to claim 9, characterized in that, The reaction was carried out at room temperature for 2 hours.
11. The application of the fluorescent probe according to claim 1 in the detection of mercury ions in traditional Chinese medicinal plants.
12. The application according to claim 11, characterized in that, The medicinal plant mentioned is Salvia miltiorrhiza.
13. A reagent for detecting mercury ions, characterized in that, It contains the mercury ion anthocyanin fluorescent probe as described in claim 1.