Caffeic acid bovine serum albumin conjugate as well as preparation method and application thereof

Abstract

The invention discloses a caffeic acid bovine serum albumin conjugate as well as a preparation method and application thereof, the caffeic acid bovine serum albumin conjugate is prepared by coupling amino groups of bovine serum albumin molecules and carboxylic acid groups of caffeic acid molecules through acid-amine condensation reaction, and each bovine serum albumin molecule is coupled with eighteen caffeic acid molecules. The albumin conjugate disclosed by the invention is simple in preparation method, high in safety and good in anti-influenza activity.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a caffeic acid bovine serum albumin conjugate, its preparation method, and its application. Background Technology

[0002] Influenza poses a serious threat to human life and health. During the seasonal influenza season each year, approximately 3 to 5 million people worldwide suffer from severe influenza, accompanied by 300,000 to 650,000 respiratory-related deaths. Currently, the main methods for influenza prevention and treatment are vaccination and anti-influenza medication. Scientists have developed various influenza virus inhibitors, such as oseltamivir, zanamivir, favipiravir, and baloxavir, targeting two important glycoproteins on the surface of the influenza virus and neuraminidase (NA), and considering the life cycle of the influenza virus infecting its host. However, with the emergence of influenza virus mutant strains, the efficacy of drugs like oseltamivir has gradually weakened. Therefore, researchers need to continuously monitor changes in drug sensitivity and strengthen the rational design and development of novel influenza virus inhibitors and anti-influenza materials. Thus, designing and developing drugs or strategies with good anti-influenza activity has become a new option for combating influenza.

[0003] Caffeic acid (CA) is a phenolic acid compound containing two phenolic hydroxyl groups and one carboxyl group in its structure. As an active ingredient in many traditional Chinese medicines, caffeic acid has high medicinal value. Its biological activities are manifested in many key areas such as antibacterial, antiviral, anti-inflammatory, antioxidant, anti-atherosclerotic, immunomodulatory, and anticancer effects. Especially in the field of antiviral activity, caffeic acid shows unique advantages.

[0004] Bovine serum albumin (BSA) possesses good water solubility and biocompatibility. Its widely distributed amino or carboxyl groups often serve as modification sites, allowing for the preparation of bioactive multivalent protein conjugates with small molecule active compounds through simple acid-amine condensation reactions. Existing technologies have synthesized a series of small molecule compounds based on BSA, such as oseltamivir, zanamivir, and sialic acid-modified BSA, and conducted molecular and cellular activity tests against common influenza virus strains and variants. The results show that these modified proteins all possess good anti-influenza activity. However, these modified proteins still have some drawbacks. The synthesis routes for oseltamivir and zanamivir multivalent modified BSA are lengthy and costly, involving frequent protection and deprotection of functional groups, resulting in low yields. Sialic acid multivalent modified BSA requires the use of chemical enzymatic methods to synthesize the monomer, limiting the availability of the final product and affecting its application.

[0005] Based on the principle of multivalent effect, this invention selects bovine serum albumin as the backbone without changing the original activity of caffeic acid, and modifies the surface of caffeic acid in multiple ways through a simple acid-amine condensation reaction to prepare the multivalent protein conjugate CA-BSA, which is then characterized. Subsequently, the anti-influenza activity of the conjugate is examined at the molecular and cellular levels. Summary of the Invention

[0006] The purpose of this invention is to address the technical deficiencies in the prior art by providing a caffeic acid bovine serum albumin conjugate, its preparation method, and its application.

[0007] The technical solution adopted to achieve the purpose of this invention is: A caffeic acid bovine serum albumin conjugate is prepared by coupling the amino group of bovine serum albumin molecule and the carboxylic acid group of caffeic acid molecule through an acid-amine condensation reaction, wherein each bovine serum albumin molecule is coupled with eighteen caffeic acid molecules.

[0008] Another aspect of the present invention includes a method for preparing the caffeic acid bovine serum albumin conjugate, comprising the following steps: The purified caffeic acid was reacted with bovine serum albumin at a molar ratio of 500:1-1000:1 in the presence of a condensing agent and an organic base to carry out an acid-amine condensation reaction. The mixture was further purified by dialysis with deionized water, and then subjected to membrane treatment and freeze drying to obtain the caffeic acid-bovine serum albumin conjugate.

[0009] In the above technical solution, the condensing agent is N,N'-dicyclohexylcarbodiimide (DCC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) or 2-(1H-benzotriazo-L-1-yl)-1,1,3,3-tetramethylurea tetrafluoroborate (TBTU), preferably TBTU.

[0010] In the above technical solution, the organic base is triethylamine, N-methylmorpholine or N,N-diisopropylethylamine, preferably N,N-diisopropylethylamine.

[0011] In the above technical solution, the molar ratio of the condensing agent, organic base and the purified caffeic acid is 1:1:1.

[0012] In the above technical solution, the caffeic acid is extracted from hawthorn. The extraction steps are as follows: under ultrasonication, the caffeic acid in the crushed hawthorn is extracted with a solvent, followed by centrifugation to remove solid residue. The supernatant obtained is then treated with a membrane and concentrated to obtain crude caffeic acid.

[0013] In the above technical solution, the solvent is ethanol, propanol or methanol, preferably methanol.

[0014] In the above technical solution, the purification step of the caffeic acid is as follows: the crude caffeic acid is separated by medium-pressure preparative chromatography to obtain purified caffeic acid.

[0015] Another aspect of the present invention includes the use of the caffeic acid bovine serum albumin conjugate in the preparation of influenza virus inhibitors or anti-influenza materials.

[0016] Another aspect of the invention includes an antiviral drug comprising the caffeic acid bovine serum albumin conjugate and pharmaceutically acceptable excipients.

[0017] Compared with the prior art, the beneficial effects of the present invention are: 1. The method for preparing bovine serum albumin conjugates of the present invention is simple to operate, and the target protein conjugate can be obtained in one step. Moreover, the raw materials and reagents are inexpensive and readily available, making it suitable for large-scale production and development.

[0018] 2. The bovine serum albumin conjugate of the present invention has good water solubility and biocompatibility, and high safety.

[0019] 3. The bovine serum albumin conjugate of the present invention exhibits good anti-influenza activity against common influenza virus strains and variants at both the molecular and cellular levels, and the activity is higher than that of caffeic acid alone.

[0020] 4. The bovine serum albumin conjugate of this invention exhibits superior anti-influenza activity compared to caffeic acid alone. This is achieved through a multivalent effect (glycosyl ligands on the cell surface exist in clusters, and protein receptors bind to sugar molecules through multiple sites. Nature utilizes the multivalent binding of multiple glycosyl ligands to multiple protein receptors to achieve stronger binding interactions), resulting in a significant enhancement effect. This provides a scientific basis and novel strategy for the development of new anti-influenza inhibitors and materials. Attached Figure Description

[0021] Figure 1 A schematic diagram of the structure of caffeic acid bovine serum albumin conjugate; Figure 2 This is the 1H NMR spectrum of the caffeic acid isolated and purified in this invention; Figure 3 This is the carbon NMR spectrum of the caffeic acid isolated and purified in this invention; Figure 4 This is a high-resolution mass spectrum of the caffeic acid separated and purified in this invention; Figure 5 This is a MALDI-TOF-MS image of bovine serum albumin, the raw material used in this invention; Figure 6This is a MALDI-TOF-MS image of the caffeic acid bovine serum albumin conjugate in this invention; Figure 7 This is a schematic diagram of the surface plasmon resonance experimental results of the caffeic acid bovine serum albumin conjugate in this invention; Detailed Implementation

[0022] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0023] The influenza virus strains used in the following examples are A / Hecheng, Hunan province / SWL1331 / 2014 (H1N1), A / Huairou, Beijing / 11069 / 2014 (H3N2), and A / Chiken / Beijing / AT609 / 2014 (H9N2); all three influenza virus strains were obtained from the Chinese Center for Disease Control and Prevention.

[0024] Example 1 Preparation of caffeic acid bovine serum albumin conjugate CA-BSA (structure as shown) Figure 1 (As shown), including the following steps: Step 1, Extraction of Caffeic Acid: Methanol was used as the extraction solvent. Crushed fresh hawthorn was added at a material-to-liquid ratio of 1:50 (g / mL), and ultrasonic extraction was performed for 3 h at 100 W and 50 ℃. Subsequently, the extract was centrifuged at 2000 rpm for 10 min. The supernatant was then subjected to membrane treatment, followed by rotary evaporation to remove methanol, yielding a preliminarily concentrated crude caffeic acid product.

[0025] Step 2, purification of caffeic acid: Medium-pressure preparative chromatography was used for the purification of caffeic acid. A methanol-water gradient system (containing 0.2% TFA in the aqueous phase) was used as the mobile phase, with a flow rate of 1 mL / min. The mobile phase gradient was set to 10-50% methanol, and the elution time was 30 min. The eluent from 15.55 min to 16.12 min was collected, concentrated, and dried to obtain highly pure caffeic acid, which can be used for the preparation of protein conjugates. The 1H NMR spectrum of the purified caffeic acid is shown below. Figure 2 The carbon NMR spectrum is shown below. Figure 3 High-resolution mass spectrometry can be seen Figure 4 .

[0026] Step 3, Preparation of Caffeic Acid-Bovine Serum Albumin Conjugate: Purified caffeic acid (10 mg, 11 mmol) was weighed and placed in a suitable container, then dissolved in 5 mL of PBS buffer. TBTU (7 mg, 11 mmol) and DIPEA (18 mg, 11 mmol) were added under ice bath conditions, and the reaction was continued at this temperature for 4 h. Bovine serum albumin was added to the reaction system at an equivalent ratio of 500:1. The reaction was stirred overnight. The reaction solution was added to a dialysis bag and dialyzed with deionized water for 2 days. After dialysis, the resulting solution was membrane-treated and then aliquoted into 2 mL EP tubes (1 mL per tube). After freeze-drying, the dry powdered caffeic acid-bovine serum albumin conjugate CA-BSA was obtained. The molecular weight of the conjugate was then quantitatively determined. The MALDI-TOF-MS chromatogram of the conjugate is shown below. Figure 6 The MALDI-TOF-MS image of bovine serum albumin is shown below. Figure 5 Calculations show that 18 caffeic acid molecules were successfully coupled to each bovine serum albumin molecule.

[0027] Experiment Example 2 Surface plasmon resonance experiment of caffeic acid bovine serum albumin conjugate CA-BSA.

[0028] This invention selects influenza virus HA, NA and mutant strain NA (H274Y) as target proteins to conduct surface plasmon resonance experiments, and analyzes the binding affinity between the test compound and the target protein from a kinetic perspective.

[0029] The specific procedures for the surface plasmon resonance experiment are as follows: (1) Prepare buffer solution, sample solution, chip activation reagent, etc. Select CM5 chip and perform pretreatment according to its instructions.

[0030] (2) Install the chip correctly in the flow cell, start the fluid system, and let the buffer solution flow through the chip surface at a constant rate until the baseline is stable. Record the baseline value as a reference.

[0031] (3) The chip was activated using a mixed solution of N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI); the influenza virus protein solution was flowed through the chip at a certain speed and coupled to a suitable amount, and then the uncoupled sites were blocked with the blocking agent ethanolamine to prevent interference with the binding results.

[0032] (4) Set the mobile phase flow rate to 30 μL / min, the binding time to 120 s, the dissociation time to 180 s, and the regeneration time to 30 s. Arrange the analyte solutions in order of increasing concentration and load them for detection. The solutions will flow sequentially through the chip surface to interact with the influenza virus proteins. Record the response unit (RU) value using software.

[0033] (5) Using the Biacore T200 data analysis software built into the instrument, fit the curve and obtain the binding dissociation constant (K). D =kd / ka), and thus determine the strength of the affinity between the analyte and the influenza virus. Generally, the K of the analyte... D Values ​​in the range of 10 -3 Up to 10 -6 M represents a weak bond; 10 -6 Up to 10 -9 M represents a medium-strength bond; while greater than 10 -9 M represents a strong binding.

[0034] Relevant experimental results are as follows Figure 7 As shown, monomeric caffeic acid has a weak binding affinity for hemagglutinin (HA) and neuraminidase (NA) (e.g., Figure 7 (among A and B), its K D The values ​​were 899.3 μM and 480 μM, respectively, indicating that caffeic acid has the potential to bind to two key proteins on the surface of the influenza virus. Compared to the NA protein, the binding affinity of the mutant protein H274Y to caffeic acid was reduced (K... D 924.3 μM, such as Figure 7 (C) confirmed that caffeic acid can bind to neuraminidase on the surface of influenza virus, and the mutation site H274Y has a certain influence on the binding ability.

[0035] In comparison, the protein conjugate CA-BSA exhibits significantly enhanced binding affinity to hemagglutinin and neuraminidase (e.g., Figure 7 (D and E), its K) D The values ​​were 963 nM and 550 nM, respectively. Meanwhile, CA-BSA showed resistance to the NA mutant protein H274Y of the drug-resistant strain (e.g., Figure 7 The binding affinity of caffeic acid to influenza virus surface proteins was also significantly improved. SPR experimental results showed that the multivalent coupling modification of caffeic acid significantly enhanced the binding affinity to influenza virus surface proteins, thereby increasing the anti-influenza activity of the conjugate. Based on this, the present invention further designed and conducted hemagglutination inhibition and neuraminidase inhibition experiments.

[0036] Experimental Example 3 Hemagglutination inhibition assay of caffeic acid bovine serum albumin conjugate CA-BSA.

[0037] The hemagglutination inhibition (HAI) assay of this invention is used to detect the binding ability of viral surface HA to erythrocytes. The specific operating steps of the hemagglutination inhibition assay are as follows: (1) Dilute the three virus strains according to their titers using PBS buffer solution.

[0038] (2) CA-BSA and its control group CA were serially diluted 2 times to a concentration of 10.

[0039] (3) Take a 96-well plate and label it "B", "V", "20"..."29". Add PBS buffer solution to the control well "B", add the labeled influenza virus solution to the virus well "V", and add the test compound in the experimental groups "20"..."29" in a 2-fold serial dilution. Then add equal amounts of the prepared influenza virus solution to the three groups of wells, mix well by blowing and aspirating, and incubate at room temperature for 30 min. Set up 3 parallel controls for each experiment.

[0040] (4) After the test compound and influenza virus have been incubated, chicken blood red blood cells are added to each well, blown and aspirated evenly, and incubated at room temperature for 30 min.

[0041] (5) After incubation is complete, stand the plate and observe and record the phenomenon of chicken blood red blood cell agglutination in the well, that is, the hemagglutination inhibition constant Ki of the compound against influenza virus.

[0042] The results of the blood coagulation inhibition test are shown in Table 1: a Influenza A / Hecheng / Hunan / SWL1331 / 2014 (H1N1) b Influenza A / Huairou / Beijing / 11069 / 2014 (H3N2) c Influenza A / Chicken / Beijing / AT609 / 2014 (H9N2) As shown in Table 1, the low-molecular-weight caffeic acid (CA) exhibits weak inhibitory activity against HA, with an inhibition constant of several hundred micromoles. In contrast, CA-BSA consistently maintained a Ki value of around 1 μM against HA in all three viral strains, representing a significant improvement of two orders of magnitude compared to low-molecular-weight caffeic acid. This result indicates that CA-BSA can significantly enhance the inhibitory effect on influenza virus hemagglutinin (HA). Subsequently, this invention further conducted neuraminidase inhibition experiments to investigate the inhibitory ability of the conjugate against influenza virus neuraminidase.

[0043] Experiment Example 4 Neuraminidase inhibition assay of caffeic acid bovine serum albumin conjugate CA-BSA This invention employs a neuraminidase inhibition assay to verify whether CA-BSA can effectively inhibit the function of influenza virus NA. 4-Methylumbelliferyl-N-acetyl-α-D-neuraminidase sodium (4-MUNANA) can bind to NA on the surface of influenza virus. The umbelliferyl ketone released after NA hydrolysis emits blue fluorescence. Influenza virus is incubated with serially diluted inhibitors, then bound to 4-MUNANA. Changes in fluorescence values ​​are detected using an ELISA reader to calculate the half-maximal inhibitory concentration (IC50) of the influenza virus inhibitor. 50 value.

[0044] The specific operating steps of the neuraminidase inhibition experiment in this invention are as follows: (1) The three influenza viruses were diluted to their optimal dilution factor using a buffer with a pH of 5.5, while the multivalent protein conjugate CA-BSA and its control CA were serially diluted 10-fold.

[0045] (2) The influenza virus and the test sample were mixed in equal amounts and added to a 384-well plate and incubated with shaking at room temperature for 30 min. A blank control group and a virus control group were set up at the same time, and three parallel control groups were set up for each experiment.

[0046] (3) After incubation, add the prepared substrate 4-MUNANA solution to each well and mix well. Incubate at 37°C with shaking for 30 min.

[0047] (4) The fluorescence values ​​of each well were monitored using an ELISA reader. The inhibition rate of influenza virus at each concentration of the influenza virus inhibitor was calculated using the formula. Then, the "S"-shaped curve was fitted using the Graphpad software to obtain the half-maximal inhibitory concentration (IC50) of the influenza virus inhibitor. 50 value.

[0048] Note: F E The fluorescence value of the experimental group; F B The fluorescence value is for the blank control group; F V The fluorescence value represents the fluorescence value of the virus control group.

[0049] The results of the neuraminidase inhibition experiment are shown in Table 2: a Influenza A / Hecheng / Hunan / SWL1331 / 2014 (H1N1) bInfluenza A / Huairou / Beijing / 11069 / 2014 (H3N2) c Influenza A / Chicken / Beijing / AT609 / 2014 (H9N2) d Mutant of H3N2 Table 2 shows the half-maximal inhibitory concentrations (IC50) of CA-BSA against the NA and mutant protein H274Y of the three influenza viruses. 50 The values ​​all reached the single-digit micromolar level, compared to the IC50 values ​​of small molecule caffeic acid. 50 The value increased by two orders of magnitude. This improvement is likely due to the fact that multiple binding sites of the caffeic acid bovine serum albumin conjugate can simultaneously interact with the NA protein on the surface of the influenza virus, thereby enhancing the overall inhibitory effect. Furthermore, CA-BSA further restricts the movement range of viral particles through steric hindrance, blocking the infection process of influenza virus into host cells. This result confirms that CA-BSA can indeed effectively inhibit the function of influenza virus NA. Subsequently, this invention designed and conducted virus growth inhibition experiments to verify the anti-influenza activity of CA-BSA at the cellular level.

[0050] Experimental Example 5 Viral growth inhibition experiment of caffeic acid bovine serum albumin conjugate CA-BSA.

[0051] This invention conducts a virus growth inhibition experiment to investigate the anti-influenza activity of CA-BSA at the cellular level. In this experiment, influenza virus and the analyte were added to MDCK cells, and after incubation for 48 h, cell viability was detected using the MTT assay. MTT can be absorbed by the cell membrane of living cells and then reduced to formazan by succinate dehydrogenase in the mitochondria, causing the solution color to change from yellow to blue-purple. After dissolving the formazan in DMSO, the absorbance was measured at 590 nm using an ELISA reader, allowing for the indirect calculation of cell viability. The concentration (EC) of the compound at which the analyte caused half-maximal effective inhibition of the virus was calculated. 50 This demonstrates the inhibitory effect of the test substance on the viral growth cycle.

[0052] The operation steps of the virus growth inhibition experiment in this invention are briefly described as follows: (1) Culture the cells in a 96-well plate to about 90% confluence and wash the cells with PBS buffer solution with a pH of 5.5.

[0053] (2) The three influenza viruses were diluted to their TCID50 using DMEM basal medium, while the multivalent protein conjugate CA-BSA and its control CA were serially diluted 10-fold.

[0054] (3) Add equal amounts of influenza virus and test substance diluent to the well plate, and set it as the experimental group. Incubate in an incubator for 48 h. At the same time, set up a blank control group and a virus control group. Each experiment has three parallel controls.

[0055] (4) After incubation, wash the cells again with PBS buffer solution with a pH of 5.5.

[0056] (5) Add MTT solution to the well, wrap it with tin foil to protect it from light, and incubate it in a 37°C incubator for 4 h.

[0057] (6) After incubation, discard the supernatant, add DMSO to the well, and incubate at room temperature in the dark for 10 min with shaking.

[0058] (7) Measure the absorbance at 590 nm using an ELISA reader. Calculate the cell viability and virus growth inhibition rate, fit the curves using Graphpad, and obtain the EC value of the analyte. 50 value.

[0059] Note: OD E The absorbance of the experimental group; OD B The absorbance is for the blank control group; OD V The absorbance is the value of the virus control group.

[0060] The results of the virus growth inhibition experiment are shown in Table 3: a Influenza A / Hecheng / Hunan / SWL1331 / 2014 (H1N1) b Influenza A / Huairou / Beijing / 11069 / 2014 (H3N2) c Influenza A / Chicken / Beijing / AT609 / 2014 (H9N2) As shown in Table 3, the results of the virus growth inhibition experiment show the half-maximal inhibitory concentration (IC50) of the multivalent protein conjugate CA-BSA against three influenza viruses at the cellular level. 50 The levels are in the single-digit micromolar range, while CA has an EC value for the virus. 50 The value is in the hundreds of micromoles. The EC of CA-BSA 50The value was significantly lower than that of CA, indicating that the inhibitory activity of the multivalent conjugate against influenza virus was significantly enhanced compared to that of the small molecule.

[0061] The results of the above experimental examples show that the caffeic acid bovine serum albumin conjugate CA-BSA in this invention can exhibit good anti-influenza activity at both the molecular and cellular levels, indicating that this invention provides a novel and referable approach for the design and synthesis of novel influenza virus inhibitors and anti-influenza materials.

[0062] The above description is only a preferred embodiment of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A caffeic acid bovine serum albumin conjugate, characterized in that, It is prepared by coupling the amino group of bovine serum albumin molecule and the carboxylic acid group of caffeic acid molecule through an acid-amine condensation reaction, with each bovine serum albumin molecule coupled with eighteen caffeic acid molecules.

2. The method for preparing the caffeic acid bovine serum albumin conjugate as described in claim 1, characterized in that, Includes the following steps: The purified caffeic acid was reacted with bovine serum albumin at a molar ratio of 500:1-1000:1 in the presence of a condensing agent and an organic base to carry out an acid-amine condensation reaction. The mixture was further purified by dialysis with deionized water, and then subjected to membrane treatment and freeze drying to obtain the caffeic acid-bovine serum albumin conjugate.

3. The preparation method according to claim 2, characterized in that, The condensing agent is DCC, EDCI, or TBTU, preferably TBTU.

4. The preparation method according to claim 2, characterized in that, The organic base is triethylamine, N-methylmorpholine, or N,N-diisopropylethylamine, preferably N,N-diisopropylethylamine.

5. The preparation method according to claim 2, characterized in that, The molar ratio of the condensing agent, organic base, and purified caffeic acid is 1:1:

1.

6. The preparation method according to claim 2, characterized in that, The caffeic acid is extracted from hawthorn. The extraction steps are as follows: under ultrasonication, caffeic acid is extracted from crushed hawthorn using a solvent. Then, the solid residue is removed by centrifugation. The supernatant obtained is treated with a membrane and then concentrated to obtain crude caffeic acid.

7. The preparation method according to claim 6, characterized in that, The solvent is ethanol, propanol, or methanol, preferably methanol.

8. The preparation method according to claim 6, characterized in that, The purification steps for the caffeic acid are as follows: the crude caffeic acid is separated by medium-pressure preparative chromatography to obtain purified caffeic acid.

9. The use of the caffeic acid bovine serum albumin conjugate as described in claim 1 in the preparation of influenza virus inhibitors or anti-influenza materials.

10. A drug for treating influenza virus, characterized in that, Includes the caffeic acid bovine serum albumin conjugate as described in claim 1, and pharmaceutically acceptable excipients.

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