A fulvic acid acylated amino acid alcohol ester having antibacterial activity

Abstract

This invention discloses a fulvic acid-acylated amino acid alcohol ester with antibacterial activity and its preparation method. The fulvic acid-acylated amino acid alcohol ester with antibacterial activity of this invention is prepared by reacting compounds such as arginine, histidine, lysine, methanol, ethanol, n-propanol, and fulvic acid. The fulvic acid-acylated amino acid alcohol ester with antibacterial activity prepared by this invention has good water solubility and high antibacterial performance. Its good water solubility can greatly improve the bactericidal performance of the fulvic acid-acylated amino acid alcohol ester. The synthesis process is green, environmentally friendly, safe, non-toxic, and simple.

Description

Technical Field

[0001] This invention belongs to the fields of chemical synthesis and antibacterial drugs, specifically relating to a fulvic acid acylated amino acid alcohol ester with antibacterial activity and its preparation method. Background Technology

[0002] Fulvic acid, CAS number 479-66-3, contains numerous phenolic hydroxyl and carbonyl groups, allowing it to interact with oxides, metal ions, and organic matter, including toxic and harmful substances, thus influencing their environmental chemical behavior, including chemical degradation, photolysis, bioabsorption, migration, and volatilization. Nearly 700 years of traditional Chinese medicine practice and modern medical pharmacology have demonstrated that fulvic acid has multiple physiological effects on the human body. Specifically, it affects blood circulation; red blood cells carry more oxygen in the presence of fulvic acid, leading to feelings of well-being and increased energy after consumption. It also promotes wound healing and cell regeneration. Furthermore, it facilitates the conversion of important minerals; as a natural mineral chelating agent, fulvic acid helps increase the absorption of calcium and other trace elements. Current research on fulvic acid primarily focuses on its application in the adsorption of heavy metals in water.

[0003] Arginine, lysine, and histidine are three essential amino acids that play irreplaceable roles in living organisms. Arginine participates in the urea cycle, helping to eliminate ammonia and maintain nitrogen balance; lysine is a key component in the synthesis of many proteins and is crucial for growth, development, and immune function; histidine can be broken down in the human body to produce histamine, which participates in physiological processes such as immune responses and regulation of gastric acid secretion. They are widely found in animal and plant proteins, and their intake and metabolic balance are of profound significance for maintaining human health. In-depth research into the physiological functions and metabolic mechanisms of these three amino acids is of paramount importance for the development of nutrition, medicine, and related fields.

[0004] This invention prepares fulvic acid-acylated amino acid alcohol esters by fulvic acid-acylated arginine alcohol esters, lysine alcohol esters, and histidine alcohol esters. These esters exhibit good water solubility and, due to the presence of numerous phenolic hydroxyl groups and the amino groups of histidine and imidazole amino acids, can self-protonate in aqueous solutions, resulting in slightly acidic solutions. The fulvic acid-acylated amino acid alcohol esters demonstrate significant antibacterial effects, and the preparation process is simple. Summary of the Invention

[0005] The purpose of this invention is to provide a fulvic acid acylated amino acid alcohol ester with antibacterial activity. The preparation method of this fulvic acid acylated amino acid alcohol ester conforms to the green chemistry concept advocated by the state and has the advantages of low cost, high preparation efficiency, high antibacterial activity and simple operation.

[0006] The fulvic acid acylated amino acid alcohol ester with antibacterial activity described in this invention has the following chemical structural formula: Figure 1As shown, R1 is an amino acid side chain group, and R2 is a C1-C12 alkane group.

[0007] To achieve the above objectives, this patent provides the following solution:

[0008] A fulvic acid acylated amino acid alcohol ester with antibacterial activity, characterized in that R1 is an arginine side chain group, a histidine side chain group, or a lysine side chain group; and R2 is a methyl, ethyl, or n-propyl group.

[0009] A fulvic acid acylated amino acid alcohol ester with antibacterial activity, characterized in that its preparation method includes the following steps:

[0010] (1) Add amino acids to an alcohol solvent, add 2-5 times the mass of amino acids in thionyl chloride, heat to 50-90℃ and stir for 1.0-2.5 h until the amino acids are completely dissolved, evaporate the solvent to dryness, and obtain amino acid alcohol ester hydrochloride;

[0011] (2) Add fulvic acid to an alcohol solvent and vibrate in an ultrasonic-assisted manner until the fulvic acid is completely dissolved. Place it in an ice bath (-5℃) and add 2-5 times the mass of fulvic acid sulfoxide. Mix and stir for 0.2-1h. After raising the temperature to room temperature, filter out the insoluble matter from the solution.

[0012] (3) Add amino acid alcohol ester hydrochloride to the flask, adjust the temperature to 70℃-90℃, slowly add fulvic acid alcohol solution, reflux and stir for 2-5 hours. After the reaction is completed, concentrate the reaction solution to 3 / 5 of the original volume using a rotary evaporator, add sodium bicarbonate to the concentrated solution until no more gas is generated in the reaction system, evaporate the alcohol solvent to obtain fulvic acid acylated amino acid alcohol ester.

[0013] A fulvic acid acylated amino acid alcohol ester with antibacterial activity, characterized in that the inhibited bacterial species are Staphylococcus aureus, Escherichia coli, and Porphyromonas gingivalis.

[0014] A method for preparing fulvic acid acylated amino acid alcohol esters with antibacterial activity, characterized in that: the solvent alcohol used in steps (1) and (2) is one of methanol, ethanol, n-propanol, and n-butanol, preferably ethanol.

[0015] A method for preparing a fulvic acid acylated amino acid alcohol ester with antibacterial activity, characterized in that: the solid-liquid ratio in steps (1) and (2) is 1.0g:10~40mL, preferably 1.0g:30mL.

[0016] A method for preparing fulvic acid acylated amino acid alcohol ester with antibacterial activity, characterized in that: in step (3), the reaction feed molar ratio of fulvic acid acylated amino acid alcohol ester is fulvic acid: amino acid alcohol ester hydrochloride = 1:0.9~1.2, preferably 1:1.

[0017] The beneficial effects of the present invention are reflected in the following: the fulvic acid acylated amino acid alcohol ester of the present invention has excellent antibacterial activity and good water solubility, and the raw materials used are safe and non-toxic, the synthesis process is green and environmentally friendly, safe and non-toxic, and the preparation process is simple. Attached Figure Description

[0018] Figure 1 Schematic diagram of the chemical structure of the fulvic acid acylated amino acid alcohol ester described in this invention.

[0019] Figure 2 : Infrared spectrum of fulvic acid acylated histidine ethyl ester

[0020] Figure 3 : Infrared spectrum of fulvic acid acylated lysine ethyl ester

[0021] Figure 4 : Infrared spectrum of fulvic acid acylated arginine ethyl ester

[0022] Figure 5 Graphs showing the inhibitory effects of fulvic acid acylated amino acid alcohol esters on Staphylococcus aureus in various embodiments.

[0023] Figure 6 Graphs showing the inhibitory effects of fulvic acid acylated amino acid alcohol esters on Escherichia coli in various embodiments.

[0024] Figure 7 Graphs showing the inhibitory effects of fulvic acid-acylated amino acid alcohol esters on *Porphyromonas gingivalis* in various embodiments. Detailed Implementation

[0025] Fulvic acid was purchased from Shanghai Yuanye Biotechnology Co., Ltd., and Blue Moon Aloe Vera Hand Sanitizer (the main effective antibacterial ingredient is 0.32%-0.38% salicylic acid) was purchased from Blue Moon (China) Co., Ltd.

[0026] The present invention will be further explained and described below with reference to specific embodiments. It should be understood that these embodiments are preferred implementations of the present invention and are only used for specific illustration.

[0027] Example 1

[0028] (1) Take 1.74 g of histidine into a 100 mL double-necked flask, add 30 mL of ethanol and 4.5 mL of thionyl chloride, adjust the temperature to 70 °C, reflux and heat and stir for 1.5 h, evaporate the solvent to dryness, and obtain histidine ethyl ester hydrochloride.

[0029] (2) Place 1.54g of fulvic acid in a 100mL beaker, add 30mL of ethanol to dissolve it. After the fulvic acid is completely dissolved, place it in an ice bath (-5℃) and add 4.5mL of SOCl2. Mix and stir for 0.5h. After the temperature rises to room temperature, filter the solution to remove undissolved impurities.

[0030] (3) Place 1.16 g of histidine ethyl ester hydrochloride in a 100 mL double-necked flask, adjust the temperature to 75 °C, and slowly add fulvic acid ethanol solution dropwise from the mouth of the other flask. The addition is completed in ten minutes. Then, continue stirring the reaction for 3 hours. After the reaction is completed, concentrate the reaction solution to 3 / 5 of the original volume using a rotary evaporator. Add sodium bicarbonate to the concentrated solution until no more gas is produced in the reaction system. Use a rotary evaporator to evaporate the solvent to dryness and obtain a light yellow powder containing 2.35 g of fulvic acid acylated histidine ethyl ester.

[0031] Example 2

[0032] (1) Take 1.74 g of lysine in a 100 mL double-necked flask, add 15 mL of ethanol and 4.5 mL of thionyl chloride, adjust the temperature to 70 °C, reflux and heat and stir for 1.5 h, evaporate the solvent to dryness, and obtain lysine ethyl ester hydrochloride.

[0033] (2) Place 1.54g of fulvic acid in a 100mL beaker, add 35mL of ethanol to dissolve it. After the fulvic acid is completely dissolved, place it in an ice bath (-5℃) and add 4.5mL of SOCl2. Mix and stir for 0.5h. After the temperature rises to room temperature, filter the solution to remove undissolved impurities.

[0034] (3) Place 1.23g of lysine ethyl ester hydrochloride in a 100mL double-necked flask, adjust the temperature to 70℃, and slowly drip fulvic acid ethanol solution from the mouth of the other flask. After the dripping is completed in ten minutes, continue stirring the reaction for 2 hours. After the reaction is completed, concentrate the reaction solution to 3 / 5 of the original volume using a rotary evaporator. Add sodium bicarbonate to the concentrated solution until no more gas is produced in the reaction system. Use a rotary evaporator to evaporate the solvent to dryness and obtain a light yellow powder, 2.27g of fulvic acid acylated lysine ethyl ester.

[0035] Example 3

[0036] (1) Take 1.74 g of arginine in a 100 mL double-necked flask, add 25 mL of ethanol and 4.5 mL of thionyl chloride, adjust the temperature to 70 °C, reflux and heat and stir for 1.5 h, evaporate the solvent to dryness, and obtain arginine ethyl ester hydrochloride.

[0037] (2) Place 1.54g of fulvic acid in a 100mL beaker, add 25mL of ethanol to dissolve it. After the fulvic acid is completely dissolved, place it in an ice bath (-5℃) and add 4.5mL of SOCl2. Mix and stir for 0.5h. After the temperature rises to room temperature, filter the solution to remove undissolved impurities.

[0038] (3) Place 1.20g of arginine ethyl ester hydrochloride in a 100mL double-necked flask, adjust the temperature to 90℃, and slowly drip fulvic acid ethanol solution from the mouth of the other flask. The dripping is completed in ten minutes. Then, continue stirring the reaction for 4 hours. After the reaction is completed, concentrate the reaction solution to 3 / 5 of the original volume using a rotary evaporator. Add sodium bicarbonate to the concentrated solution until no more gas is produced in the reaction system. Use a rotary evaporator to evaporate the solvent to dryness and obtain a light yellow powder, 2.20g of fulvic acid acylated arginine ethyl ester.

[0039] Example 4

[0040] (1) Take 1.74 g of histidine into a 100 mL double-necked flask, add 40 mL of ethanol and 4.5 mL of thionyl chloride, adjust the temperature to 70 °C, reflux and heat and stir for 1.5 h, evaporate the solvent to dryness, and obtain histidine ethyl ester hydrochloride.

[0041] (2) Place 1.54g of fulvic acid in a 100mL beaker, add 35mL of ethanol to dissolve it. After the fulvic acid is completely dissolved, place it in an ice bath (-5℃) and add 4.5mL of SOCl2. Mix and stir for 0.5h. After the temperature rises to room temperature, filter the solution to remove undissolved impurities.

[0042] (3) Place 1.03 g of histidine ethyl ester hydrochloride in a 100 mL double-necked flask, adjust the temperature to 80 °C, and slowly add fulvic acid ethanol solution dropwise from the mouth of the other flask. The addition is completed in ten minutes. Then, continue stirring the reaction for 5 hours. After the reaction is completed, concentrate the reaction solution to 3 / 5 of the original volume using a rotary evaporator. Add sodium bicarbonate to the concentrated solution until no more gas is produced in the reaction system. Use a rotary evaporator to evaporate the solvent to dryness and obtain a light yellow powder, yielding 1.90 g of fulvic acid acylated histidine ethyl ester.

[0043] Example 5

[0044] (1) Take 1.74 g of histidine into a 100 mL double-necked flask, add 45 mL of ethanol and 4.5 mL of thionyl chloride, adjust the temperature to 70 °C, reflux and heat and stir for 1.5 h, evaporate the solvent to dryness, and obtain histidine ethyl ester hydrochloride.

[0045] (2) Place 1.54g of fulvic acid in a 100mL beaker, add 40mL of ethanol to dissolve it. After the fulvic acid is completely dissolved, place it in an ice bath (-5℃) and add 4.5mL of SOCl2. Mix and stir for 0.5h. After the temperature rises to room temperature, filter the solution to remove undissolved impurities.

[0046] (3) Place 1.1g of histidine ethyl ester hydrochloride in a 100mL double-necked flask, adjust the temperature to 85℃, and slowly add fulvic acid ethanol solution dropwise from the mouth of the other flask. The addition is completed in ten minutes. Then, continue stirring the reaction for 4 hours. After the reaction is completed, concentrate the reaction solution to 3 / 5 of the original volume using a rotary evaporator. Add sodium bicarbonate to the concentrated solution until no more gas is produced in the reaction system. Use a rotary evaporator to evaporate the solvent to dryness and obtain a light yellow powder, yielding 2.15g of fulvic acid acylated histidine ethyl ester.

[0047] Appendix Figure 2-4 The infrared spectra of the prepared fulvic acid acylated histidine ethyl ester, fulvic acid acylated arginine ethyl ester, and fulvic acid acylated lysine ethyl ester are shown.

[0048] Antibacterial test

[0049] Preparation of the solutions in each example: 4g of fulvic acid acylated amino acid alcohol ester prepared in each example was fully dissolved in 10mL of water for later use; Preparation of comparative solution 1: 4g of Blue Moon Aloe Vera Hand Wash was fully dissolved in 10mL of water for later use; Preparation of comparative solution 2: 4g of fulvic acid was fully dissolved in 10mL of water for later use.

[0050] 1. Antibacterial test for Staphylococcus aureus

[0051] Spread 120 μL of the 1000-fold diluted Staphylococcus aureus bacterial solution evenly onto the prepared agar plate and let it stand until the bacterial solution is absorbed.

[0052] Place 10 filter paper discs in a glass petri dish; shake the sample vigorously, quickly add 10 μL of the sample to the filter paper, and then use sterile forceps to place the filter paper discs onto the agar surface coated with bacterial solution. Gently press the filter paper discs to ensure proper contact with the agar. Place three discs on each plate, parallel to each of the three plates, ensuring equal spacing between the discs. After incubating at 37℃ for 24 hours, observe the inhibition zones. Then, measure the diameter of each inhibition zone with calipers and count the average value.

[0053] 2. Antibacterial test of Escherichia coli

[0054] Spread 120 μL of the Escherichia coli bacterial solution diluted 1000 times evenly onto the prepared agar plate and let it stand until the bacterial solution is absorbed.

[0055] Place 10 filter paper discs in a glass petri dish; shake the sample vigorously, quickly add 10 μL of the sample to the filter paper, and then use sterile forceps to place the filter paper discs onto the agar surface coated with bacterial solution. Gently press the filter paper discs to ensure proper contact with the agar. Place three discs on each plate, parallel to each of the three plates, ensuring equal spacing between the discs. After incubating at 37℃ for 24 hours, observe the inhibition zones. Then, measure the diameter of each inhibition zone with calipers and count the average value.

[0056] 3. Antibacterial test of Porphyromonas gingivalis

[0057] Anaerobic blood agar medium was prepared in a clean workbench equipped with an anaerobic incubator. 50 μL of *Porphyromonas gingivalis* bacterial suspension was added to the medium and spread evenly with a sterile spreader. After air drying, sterile perforators were used to punch holes in the medium, and 15 μL of the drug solution prepared in each example and comparative example was added to each hole. The medium was placed in anaerobic culture bags and incubated at 37°C for 16 hours. The diameter of the inhibition zone was observed and recorded. Each drug group was replicated three times, and the average value was taken.

[0058] The experimental results are attached. Figure 5 , 6 As shown in Figure 7

[0059] Figure 5 The inhibition effect of fulvic acid acylated amino acid alcohol esters on Staphylococcus aureus in each embodiment is shown in the figure. The three antibacterial strengths are: Example 5 > Example 1 > Example 4 > Example 3 > Example 2 > Comparative drug solution 1 > Comparative drug solution 2 > Distilled water.

[0060] Figure 6 The inhibition effect of fulvic acid acylated amino acid alcohol esters on Escherichia coli in each example is shown in the figure. The three antibacterial strengths are: Example 5 > Example 1 > Example 4 > Example 3 > Example 2 > Comparative drug solution 1 > Comparative drug solution 2 > Distilled water.

[0061] Figure 7 : Inhibitory effect of fulvic acid acylated amino acid alcohol esters on Porphyromonas gingivalis in each embodiment. The three antibacterial strengths are: Example 5 > Example 1 > Example 4 > Example 3 > Example 2 > Comparative drug solution 1 > Comparative drug solution 2 > Distilled water.

[0062] In summary, the antibacterial effect of fulvic acid acylated amino acid alcohol esters is significantly better than that of Blue Moon Aloe Vera Hand Sanitizer, and far exceeds that of fulvic acid at the same concentration.

[0063] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications or equivalent changes made to the above embodiments based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A fulvic acid acylated amino acid alcohol ester with antibacterial activity, characterized in that, The structure of fulvic acid acylated amino acid alcohol esters is shown in the following formula: R1 is an amino acid side chain group, and R2 is a C1-C12 alkane group.

2. The fulvic acid acylated amino acid alcohol ester with antibacterial activity according to claim 1, characterized in that, R1 is an arginine side chain group, a histidine side chain group, or a lysine side chain group; R2 is a methyl, ethyl, or n-propyl group.

3. The fulvic acid acylated amino acid alcohol ester with antibacterial activity according to claim 1, characterized in that, Its preparation method includes the following steps: (1) Add amino acids to an alcohol solvent, add 2-5 times the mass of amino acids in thionyl chloride, heat to 50-90℃ and stir for 1.0-2.5 h until the amino acids are completely dissolved, evaporate the solvent to dryness, and obtain amino acid alcohol ester hydrochloride; (2) Add fulvic acid to an alcohol solvent and shake it in an ultrasonic-assisted manner until the fulvic acid is completely dissolved. Place it in an ice bath (-5℃) and add 2-5 times the mass of fulvic acid sulfoxide. Mix and stir for 0.2-1h. After raising the temperature to room temperature, filter out the insoluble matter from the solution. (3) Add amino acid alcohol ester hydrochloride to the flask, adjust the temperature to 70℃-90℃, slowly add fulvic acid alcohol solution, reflux and stir for 2-5 hours. After the reaction is completed, concentrate the reaction solution to 3 / 5 of the original volume using a rotary evaporator, add sodium bicarbonate to the concentrated solution until no more gas is generated in the reaction system, evaporate the alcohol solvent to obtain fulvic acid acylated amino acid alcohol ester.

4. The fulvic acid acylated amino acid alcohol ester with antibacterial activity according to claim 1, characterized in that: The fulvic acid acylated amino acid alcohol ester is used to inhibit Staphylococcus aureus, Escherichia coli, and Porphyromonas gingivalis.

5. The method for preparing the fulvic acid acylated amino acid alcohol ester with antibacterial activity according to claim 3, characterized in that: The alcohol solvent used in steps (1) and (2) is one of methanol, ethanol, n-propanol, and n-butanol.

6. The method for preparing the fulvic acid acylated amino acid alcohol ester with antibacterial activity according to claim 3, characterized in that: The solid-liquid ratio in steps (1) and (2) is 1g:10~35mL.

7. The method for preparing the fulvic acid acylated amino acid alcohol ester with antibacterial activity according to claim 3, characterized in that: In step (3), the molar ratio of fulvic acid to amino acid alcohol ester is fulvic acid: amino acid alcohol ester hydrochloride = 1: 0.9~1.2.

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