Method for fluorescently labeling flavone as effective component of traditional Chinese medicine and application of flavone
By covalently combining flavonoids with the fluorescent dye Cy5 and using EDC and NHS activation reactions, the problem of insufficient efficiency and stability of fluorescent labeling methods in the prior art was solved, and the fluorescent labeling effect with high efficiency, stability and high sensitivity was achieved, ensuring the feasibility of pharmacological activity and biological applications.
Patent Information
- Application Number
- CN202510057925.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-14
- Publication Date
- 2025-04-25
AI Technical Summary
The existing fluorescent labeling methods have limitations on the labeling efficiency, stability and sensitivity of the active ingredient of Chinese medicine, and may have an impact on pharmacological activities, making it difficult to meet the experimental needs of high sensitivity and high resolution.
By covalently combining flavonoids with the fluorescent dye Cy5, the reaction is activated by EDC and NHS to form a stable fluorescent labeled compound, and the label is purified by dialysis, centrifugation and filtration, thereby improving its purity and labeling rate.
The efficient, stable and high sensitivity of fluorescently labeled flavonoids is achieved, ensuring that the biological activity of the marker is basically unaffected, and the distribution and metabolic process of flavonoids in vivo and outside can be effectively tracked and quantified.
Smart Images
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of traditional Chinese medicine, and particularly relates to a method for fluorescently labeling flavonoids, an effective component of traditional Chinese medicine, and uses thereof. Background Art
[0002] Traditional Chinese medicine is a natural treasure trove of chemistry, containing macromolecular components such as proteins and polysaccharides, as well as a variety of effective ingredients such as small molecule compounds of various structures. In the process of research and development of traditional Chinese medicine, understanding the in vivo distribution and absorption of the active ingredients of traditional Chinese medicine, pharmacokinetic parameters such as metabolic rate, is helpful in selecting the best route of administration and dosage, but since drug molecules usually have no fluorescent groups or lack ultraviolet absorption, it is difficult to achieve in vivo visualization of drug detection. In recent years, researchers have tried to specifically label the active ingredients of traditional Chinese medicine and then administer them for in vivo detection. Among the methods used for drug labeling and tracing, spectrophotometry, chromatography, fluorescence labeling and isotope labeling are widely used. Among them, the fluorescence labeling method can form fluorescent metabolites by combining with drug molecules through covalent derivatization or physical adsorption, so that they can be quantitatively converted into molecules with fluorescent properties, and the in vivo process information of the drug can be obtained through visualization methods such as fluorescence microscopy, laser confocal microscopy and flow cytometry. Compared with other tracing methods, fluorescence labeling can monitor life-related activities in real time at the cellular and molecular levels. It can label target molecules by covalent derivatization or physical adsorption with drug molecules. It is widely used in in vivo tracing experiments of macromolecules such as proteins, polysaccharides, and peptides, and in life science fields such as detection of intracellular and extracellular substances.
[0003] Among the various active ingredients of traditional Chinese medicine, flavonoids have become a hot topic of research due to their significant biological activity and wide range of pharmacological effects. They have a long history of application in traditional Chinese medicine and have been proven to have multiple functions such as anti-inflammatory, antioxidant, anti-tumor, and immunomodulatory, and play a key role in the treatment of various diseases. Icariin (ICA), as a flavonoid, has significant therapeutic potential, especially in bone and joint diseases, neurodegenerative diseases, cardiovascular diseases, etc. Due to its excellent pharmacological properties, icariin occupies an important position in both traditional Chinese medicine and modern medicine. Therefore, developing a fluorescent labeling method with icariin as the target molecule for key labeling and tracking is very important for tracking and quantifying the distribution and metabolic process of flavonoids, especially icariin, in vivo and in vitro.
[0004] Some studies have tried to use fluorescent dyes or methods to label ICA. Although the use of these methods or dyes has achieved certain results in some applications, there are certain limitations in terms of labeling efficiency, stability, sensitivity, and the effect on the pharmacological activity of ICA. For example, although commonly used fluorescent dyes such as FITC (fluorescein isothiocyanate) and Rhodamine (rhodamine) are widely used in biomarkers, their fluorescence intensity is relatively low and they are easy to fade in certain environments (such as strong acidic and alkaline conditions), resulting in poor stability in long-term observation and dynamic monitoring. In particular, in experiments requiring high sensitivity and high resolution, the performance of FITC and Rhodamine cannot meet the requirements. For example, although the Alexa Fluor series of dyes have good stability and high fluorescence intensity in some cases, they are relatively expensive, and some dyes may have low binding efficiency when reacting with ICA or the dyes may affect the biological activity of ICA. For example, although labeling methods such as ELISA are often used for quantitative analysis, they are less dependent on fluorescent dyes, and the labeling process is complicated, the reaction time is long, and samples are easily lost during multiple washing processes, which limits efficient labeling and visualization. The gold labeling method uses gold particles to label molecules. Although it has strong sensitivity in some applications, it is difficult to accurately locate gold particles inside cells or tissues due to their large particle size, and the gold label may affect the biological activity of the molecules, especially in intracellular tracking applications, which has certain limitations. Summary of the invention
[0005] The object of the present invention is to provide a method for fluorescently labeling flavonoids, an effective ingredient of traditional Chinese medicine, and its use, so as to solve the above-mentioned problems.
[0006] According to one aspect of the present invention, a method for fluorescently labeling flavonoids, an effective ingredient of traditional Chinese medicine, is provided, comprising the following steps:
[0007] (1) dissolving fluorescent dye Cy5 in phosphate buffer to form a Cy5 fluorescent dye solution;
[0008] (2) taking a fluorescent dye solution, adding 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to obtain a mixed solution, and stirring at room temperature to activate the fluorescent marker;
[0009] (3) dissolving flavonoids in methanol, adding the mixture to form a reaction system, continuing to stir the reaction, performing a labeling reaction, and obtaining a fluorescent labeled product;
[0010] (4) dialyzing the fluorescent labeled product to remove unreacted EDC, NHS and small molecule solvent ethanol;
[0011] (5) After dialysis, centrifuge with pure water to remove unreacted fluorescent dye, and remove residual unreacted dye by filtration;
[0012] (6) After the fluorescently labeled product solution is dialyzed in step (4), centrifuged and filtered in step (5), it is mixed with a polyvinyl alcohol-cyclodextrin (PVA-CD) solution, shaken, and then the supernatant is filtered to obtain a purified fluorescently labeled flavonoid solution.
[0013] In some embodiments, the mass fraction of polyvinyl alcohol in the polyvinyl alcohol-cyclodextrin solution is 5%-10%, which is prepared by the following method: dissolving 5g-10g of polyvinyl alcohol-cyclodextrin (PVA-CD) gel in 100mL of water.
[0014] In some embodiments, the method for fluorescently labeling flavonoids, an effective ingredient of traditional Chinese medicine, comprises the following steps:
[0015] (1) Take 1 mL of phosphate buffer and dissolve Cy5 fluorescent dye to form a Cy5 solution;
[0016] (2) Take 400 μL of Cy5 solution, add 2.45 mg of EDC and 1.22 mg of NHS to obtain a mixed solution, and stir at room temperature for 15-25 minutes to activate the Cy5 fluorescent marker;
[0017] (3) Dissolve 13 mg of flavonoid compound icariin (ICA) in 10 mL of methanol, add the mixture to form a reaction system, and continue to stir the reaction at 0-10° C. for 22-26 hours to perform a labeling reaction;
[0018] (4) After the reaction, the product was transferred to a dialysis bag with a molecular weight cutoff of 500 Da and dialyzed for 48 hours to remove unreacted EDC, NHS and small molecule solvent ethanol;
[0019] (5) After dialysis, centrifuge with pure water (10,000 g, 25-35 min) to remove unreacted Cy5, and remove residual unreacted dye by filtration;
[0020] (6) The obtained fluorescently labeled product solution is mixed with a polyvinyl alcohol-cyclodextrin (PVA-CD) solution, shaken overnight, and then the supernatant is taken and filtered through a filter membrane to obtain a purified fluorescently labeled flavonoid solution.
[0021] The method for fluorescently labeling flavonoids, an effective ingredient of traditional Chinese medicine, provided by the present invention forms a stable fluorescent labeling compound by covalently bonding the flavonoid compounds of the traditional Chinese medicine with fluorescent dyes, mainly by covalently bonding the carboxyl group in Cy5 with the hydroxyl group in the flavonoid compounds.
[0022] Moreover, in the reaction of carboxyl and hydroxyl groups, the role of adding EDC and NHS is mainly to enhance the efficiency and selectivity of the reaction; among them, EDC can react with carboxyl groups to generate an activated amide intermediate (acyl oxide), which is easier to react with hydroxyl groups than unactivated carboxyl groups, thereby promoting the reaction. Therefore, EDC can effectively activate carboxyl groups, making them easier to react and forming carboxylate ester bonds; the addition of NHS can improve the efficiency and specificity of the reaction. When EDC reacts with carboxyl groups to generate acyl oxides, NHS forms a stable NHS ester with the intermediate, which can avoid hydrolysis of the intermediate (for example, the presence of water will make the reaction product worse). NHS is more stable and easier to react with nucleophilic hydroxyl groups, thereby improving the yield and selectivity of the reaction. In summary, the combined use of EDC and NHS can not only improve the efficiency of the reaction of carboxyl and hydroxyl groups, but also reduce the occurrence of side reactions, making the reaction product purer.
[0023] In addition, after the fluorescent labeling reaction of step (3), the product may be mixed with unreacted Cy5 dye, unlabeled icariin, and reaction byproducts, so these impurities should be removed by purification steps to improve the purity and labeling rate of the fluorescent marker, reduce background fluorescence interference, and improve labeling efficiency. Among them, step (6) uses PVA-CD to further purify the product. Since cyclodextrin has a hydrophobic inner cavity and a hydrophilic outer shell, the water solubility of Cy5-labeled icariin can be increased by inclusion. This characteristic is particularly important for icariin, a highly hydrophobic labeled product, which can reduce nonspecific aggregation between molecules, maintain the monomer state of fluorescent molecules, and improve the stability and uniformity of fluorescent signals. Moreover, the byproducts or unreacted substances contained in the product after the fluorescent labeling reaction may interfere with subsequent applications (such as cell labeling or in vivo imaging), especially unbound Cy5 dye may bring high background fluorescence. Through further purification by PVA-CD, these components can be effectively removed, thereby reducing the possibility of nonspecific binding and improving the signal-to-noise ratio of the labeled sample.
[0024] In some embodiments, the molar ratio of Cy5 to flavonoids is 1:1 to 1:10, and the specific selection depends on the desired labeling effect and the degree of retention of biological activity. For example, 1:1 to 1:3 is suitable for situations where high-density labeling is required to ensure that there is at least one Cy5 molecule on each flavonoid molecule; 1:3 to 1:5 is suitable for situations where the labeling density is moderate, which can both ensure the labeling effect and avoid excessive dyes interfering with the biological activity of flavonoids; 1:5 to 1:10 is suitable for situations where a lower labeling density is required, which can ensure that the biological activity of the flavonoids is basically unaffected.
[0025] In some embodiments, the molar ratio of EDC to flavonoids is 1: 1 to 5: 1, and the molar ratio of NHS to EDC is 1: 1 to 2: 1. EDC is used to activate the coupling reaction between the carboxyl group and the amino group, and NHS is used to enhance the effect of the EDC reaction and make the coupling reaction more efficient.
[0026] In some embodiments, the Cy5 fluorescent dye has the following structural formula:
[0027]
[0028] In some embodiments, the structural formula of icariin (ICA) is as follows:
[0029]
[0030] In some embodiments, phosphate buffer is prepared by weighing 8.0 g NaCl, 0.2 g KCl, 1.44 g Na 2 HPO 4 , 0.24g KH 2 PO 4 Dissolve in 800 mL of distilled water, adjust the pH of the solution to about 6.0 with HCl, and finally add distilled water to make up to 1 L.
[0031] In some embodiments, the pH value of the reaction system is maintained between 4.5 and 7.5. This is because the pH value is crucial to the reaction, and the effects of EDC and NHS are better under neutral or weakly acidic conditions. Under weakly acidic conditions, such as pH 4.5-7.5 or 5-6, the carboxyl group is more easily activated by EDC, and the ester intermediate generated by EDC and NHS is more stable; if the pH is less than 4, EDC may be hydrolyzed (i.e., water molecules attack the acyl intermediate), reducing the reaction efficiency; if the pH is greater than 7.5, the amino substances in the reaction may have adverse reactions, such as nonspecific hydrolysis or dissolution of amines.
[0032] The method for fluorescently labeling flavonoids, an effective ingredient of traditional Chinese medicine, provided by the present invention has a mild and simple labeling process, can be carried out in an aqueous phase and an organic solvent, has an excellent labeling effect, and the labeled substance has a high fluorescence intensity and stability, and has no significant effect on the biological activity of flavonoids in traditional Chinese medicine. Through this labeling method, the distribution and metabolic process of flavonoids in vivo and in vitro can be effectively tracked and quantified, providing a new tool for drug delivery, efficacy evaluation, and dynamic monitoring of drugs in vivo, which can be widely used in biological research, pharmacodynamic evaluation, targeted drug delivery, and dynamic monitoring of distribution in vivo and in vitro of flavonoid components in traditional Chinese medicine, providing important technical support for the modernization of traditional Chinese medicine. BRIEF DESCRIPTION OF THE DRAWINGS
[0033] Figure 1ICA H 1 NMR spectra.
[0034] Figure 2 For ICA-Cy5 H 1 NMR spectra.
[0035] Figure 3 This is the FTIR graph of ICA.
[0036] Figure 4 This is the FTIR graph of ICA-CYT.
[0037] Figure 5-6 These are images of different fields of view of fluorescently labeled ICA under a confocal microscope. DETAILED DESCRIPTION
[0038] The present invention is further described in detail below in conjunction with specific embodiments. Unless otherwise specified, the following raw materials are commercially available. It should be noted that the "flavonoids" described in the present invention refer to flavonoid compounds.
[0039] Example 1
[0040] A method for fluorescently labeling flavonoids, an effective ingredient of traditional Chinese medicine, comprises the following specific steps:
[0041] 1. Dissolve Cy5: Take 1 mL of phosphate buffer and dissolve Cy5 fluorescent dye to form a Cy5 solution;
[0042] 2. Preparation of reaction system: Take 400 μL Cy5 solution and add it to the reaction system, add 2.45 mg 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and 1.22 mg N-hydroxysuccinimide (NHS) to the system, stir at room temperature for 20 minutes to activate the Cy5 fluorescent marker;
[0043] 3. Add flavonoid compounds: dissolve 13 mg of flavonoid component Icariin (ICA) in 10 mL of methanol, add to the above reaction system, continue to stir and react at 4 ° C for 24 hours to carry out labeling reaction;
[0044] 4. Dialysis to remove small molecules: After the reaction is completed, the product is transferred to a dialysis bag with a molecular weight cutoff of 500Da and dialyzed for 48 hours to remove unreacted EDC, NHS and small molecule solvent (ethanol);
[0045] 5. Centrifugation and filtration: After dialysis, centrifuge with pure water (10,000 g, about 30 minutes) to remove unreacted Cy5, and remove residual unreacted dye by filtration;
[0046] 6. Further treatment: The obtained fluorescently labeled product solution was mixed with a 5% polyvinyl alcohol-cyclodextrin solution, shaken overnight, and then the supernatant was taken and filtered through a filter membrane to obtain a purified fluorescently labeled flavonoid solution.
[0047] Mass spectrometry and infrared spectroscopy were performed on ICA before and after labeling. The mass spectra were as follows: Figure 1-Figure 2 As shown, the results show that the signal of aromatic hydrogen in the Cy5 molecule is in the region of 6.5-8.5ppm. The hydrogen atoms of the benzene ring and phenothiazine ring in the molecule may have multiple peaks due to different chemical environments, usually doublets, triplets or multiplets. The appearance of these peaks reflects the coupling effect between different hydrogen atoms. The signal near 7.5ppm is caused by the hydrogen atoms connected to the phenothiazine ring, while the signal at 6.8-7.2ppm comes from the hydrogen atoms in the benzene ring, indicating that the Cy5 molecule is successfully connected to ICA.
[0048] Infrared spectrum Figure 3-Figure 4 As shown, the results show that at 1700-1750cm - 1 Stretching vibrations of the carboxyl groups of Cy5 and icariin appear; at 1000-1200 cm - 1 The new CN or CO stretching vibration peak appears at 750-900cm - The change of aromatic ring CH bending vibration appeared in 1, indicating that Cy5 molecule was successfully connected to ICA.
[0049] The final fluorescently labeled flavonoid solution was observed under a confocal microscope to evaluate its labeling effect and fluorescence intensity. Figure 5-Figure 6 The results showed that the labeled ICA molecules showed red fluorescence with a fluorescence intensity value of IntDen (528919), indicating that the labeling was successful, the labeling distribution was uniform, and the signal was stable, indicating that the labeling effect was ideal.
[0050] Of course, in the step of labeling icariin with Cy5, the reaction parameters can also be modified within a certain range based on Example 1, for example, the reaction temperature in step (3) can also be 0°C, 2°C, 6°C, 8°C, 10°C, etc., and the reaction time can also be 22 hours, 23 hours, 25 hours, 26 hours, etc.; the pH value of the reaction system can be 4.5, 5, 5.5, 6, 6.5, 7, 7.5, etc. These situations are all within the scope of protection of the present invention.
[0051] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements can be made without departing from the creative concept of the present invention, which all belong to the protection scope of the present invention.
Claims
1. A method for fluorescently labeling flavonoids, an effective ingredient of traditional Chinese medicine, characterized in that: The steps include: (1) dissolving fluorescent dye Cy5 in phosphate buffer to form a fluorescent dye Cy5 solution; (2) taking a fluorescent dye Cy5 solution, adding 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to obtain a mixed solution, and stirring at room temperature; (3) dissolving flavonoids in methanol, adding the mixture to form a reaction system, and continuing to stir the reaction to obtain a fluorescent labeled product; (4) dialyzing the fluorescent labeled product; (5) After the dialysis of the fluorescent labeled product is completed, centrifugation is performed, and then the supernatant after centrifugation is filtered; (6) After the fluorescently labeled product solution is dialyzed in step (4), centrifuged and filtered in step (5), it is mixed with the polyvinyl alcohol-cyclodextrin solution, shaken, and then the supernatant is filtered to obtain a purified fluorescently labeled flavonoid solution.
2. The method for fluorescently labeling flavonoids as an effective ingredient of traditional Chinese medicine according to claim 1, characterized in that: The steps include: (1) taking phosphate buffer and dissolving Cy5 fluorescent dye to form a Cy5 solution; (2) taking a Cy5 solution, adding 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to obtain a mixed solution, and stirring at room temperature; (3) dissolving the flavonoid compound icariin in methanol, adding the mixture to form a reaction system, and continuing to stir the reaction at 0-10° C. for 22-26 hours to perform a labeling reaction; (4) After the reaction is completed, the product is transferred to a dialysis bag with a molecular weight cutoff of 500 Da for dialysis; (5) After dialysis, centrifuge using pure water; (6) The obtained fluorescently labeled product solution is mixed with the polyvinyl alcohol-cyclodextrin solution, shaken overnight, and then the supernatant is taken and filtered through a filter membrane to obtain a purified fluorescently labeled flavonoid solution.
3. The method for fluorescently labeling flavonoids, an effective ingredient of traditional Chinese medicine, according to claim 1, characterized in that: The molar ratio of Cy5 to flavonoids is 1:1 to 1:
10.
4. The method for fluorescently labeling flavonoids, an effective ingredient of traditional Chinese medicine, according to claim 1 or 2, characterized in that: The molar ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to flavonoids is 1:1 to 5:1, and the molar ratio of N-hydroxysuccinimide to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 1:1 to 2:
1.
5. The method for fluorescently labeling flavonoids, an effective ingredient of traditional Chinese medicine, according to claim 1 or 2, characterized in that: The mass fraction of polyvinyl alcohol in the polyvinyl alcohol-cyclodextrin solution is 5%-10%.
6. The method for fluorescently labeling flavonoids, an effective ingredient of traditional Chinese medicine, according to claim 1 or 2, characterized in that: Phosphate buffer was prepared by the following method: 8.0 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, and 0.24 g KH2PO4 were weighed and dissolved in 800 mL distilled water, the pH of the solution was adjusted to 7.4 with HCl, and finally the volume was fixed to 1 L with distilled water.
7. The method for fluorescently labeling flavonoids, an effective ingredient of traditional Chinese medicine, according to claim 1 or 2, characterized in that: The pH value of the reaction system was maintained between 4.5 and 7.
5.
8. The method for fluorescently labeling flavonoids, an effective ingredient of traditional Chinese medicine, according to claim 1 or 2, characterized in that: The structural formula of Cy5 fluorescent dye is as follows:
9. The method for fluorescently labeling flavonoids, an effective ingredient of traditional Chinese medicine, according to claim 1 or 2, characterized in that: The icariin structural formula is as follows:
10. Use of the method for fluorescently labeling flavonoids, an effective ingredient of traditional Chinese medicine, as claimed in claim 1 or 2 in tracking and quantifying the distribution and metabolic process of flavonoids in vivo and in vitro.