Preparation method of dihydromyricetin manganese for treating swine pasteurellosis

Abstract

The invention discloses a preparation method of dihydromyricetin manganese for treating swine pasteurellosis. According to the method, a synthetic reaction of a complex is carried out in an ethanol-ammonium acetate buffer solution with the pH value of 3.7; the optimal feeding molar ratio of the manganese salt solution to the dihydromyricetin is 1.1: 1.0; the optimal reflux reaction time is 30 minutes. The compound is stable in activity and good in antibacterial effect on pig pasteurella multocida. The invention also provides a preparation method of the compound, and the method is simple, easy to control, suitable for large-scale production, high in product yield and high in purity, and is expected to become a medicine for replacing antibiotics to treat swine pasteurellosis.

Description

Technical Field

[0001] This invention belongs to the field of complex synthesis, specifically relating to a method for preparing dihydromyricetin manganese for treating porcine pasteurellosis. Background Technology

[0002] Pasteurella multocida infection in pigs can occur year-round and is a highly prevalent disease in pig farming, causing large-scale mortality and severely impacting farming efficiency. The prevalence and infection of Pasteurella multocida remain one of the most serious threats to my country's pig industry. Broad-spectrum antibiotics are the first-line treatment for Pasteurella multocida infection in pigs, but as bacteria develop resistance to antibiotics, the development of more promising new antibiotic alternatives is urgently needed.

[0003] Dihydromyricetin (DMY), a hallmark active ingredient in vine tea, possesses various pharmacological effects, including antibacterial, anti-inflammatory, and immune-enhancing properties. DMY molecules act as excellent chelating ligands for metal ions, and the resulting complexes often exhibit superior bioactivity compared to DMY alone, demonstrating stronger antibacterial effects and promising broad development and application prospects. The coordination chemistry theory of traditional Chinese medicine suggests that forming coordination compounds can reduce the toxicity of active ingredients and increase their activity. Manganese plays a crucial physiological role in animals, significantly impacting livestock health and playing an indispensable role in animal health and disease prevention and treatment. The synthesis and bioactivity research of manganese complexes is currently a hot research area, and manganese complexes show broad application prospects in bioactivity research. By studying the synthesis methods and bioactivity of manganese complexes, researchers can provide a theoretical basis for designing and preparing more effective manganese complexes and offer new ideas for developing new biomedical applications. It is predicted that the complexation of dihydromyricetin with manganese will have good application effects in veterinary clinical practice. However, the preparation process of the dihydromyricetin-manganese complex (DMY-Mn) requires further research. Li Haixia (Journal of Hainan Medical College, 2011) and Yuan Juan (Master's Thesis, Guangdong University of Technology, 2013) reported a synthesis yield of 77.72% and 78%, respectively. Zhang Hui (Master's Thesis, Guangdong University of Technology, 2019) and Guo Qingquan (CN 102334609 B, 2013) et al. did not report the reaction yield and product purity in their reports on the preparation of DMY-Mn. Currently, there are no reports on its anti-swine pasteurellosis activity. Based on the systematic study of DMY-Mn, this invention prepares a DMY-Mn complex for the treatment of swine pasteurellosis. The preparation method is simple, easy to control, suitable for large-scale production, and has high reaction yield and high product purity. Summary of the Invention

[0004] To overcome the immaturity of existing product preparation processes, this invention proposes a method for preparing dihydromyricetin-manganese complex (DMY-Mn) for treating porcine pasteurellosis. The prepared DMY-Mn complex product has high yield and high purity, making it suitable for large-scale production. In vitro experiments show that its antibacterial activity against Pasteurella multocida is better than that of DMY.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solution, the preparation method of which includes the following steps: S1: Preparation of DMY solution: Accurately weigh 0.64 g (2.0 mmol) of DMY, add ethanol, stir and heat to obtain a light green solution; S2: Preparation of manganese salt solution: 2.2 mmol of manganese salt was added to 100 mL of ethanol-ammonium acetate buffer solution to obtain manganese salt solution.

[0006] S3: The manganese salt solution obtained in S2 was slowly added dropwise to the DMY solution obtained in S1 using a constant pressure dropping funnel. The mixture was heated and refluxed. The resulting yellowish-brown precipitate was filtered, washed, and dried to obtain a brown solid DMY-Mn complex.

[0007] Preferably, the ethanol in S1 is 60-100 mL, more preferably 80 mL.

[0008] Preferably, the pH of the ethanol-ammonium acetate buffer solution is 3.7.

[0009] Preferably, the manganese salt in S2 is taken from one or two of manganese acetate hydrate, manganese sulfate hydrate, or manganese chloride hydrate, with manganese sulfate hydrate being preferred.

[0010] Preferably, the dropping rate of the manganese salt solution in step S3 is 2.0–3.0 mL / min. -1 2.5 mL / min is preferred. -1 .

[0011] Preferably, the molar ratio of manganese salt to DMY in step S3 is 1.0 to 1.2:1.0, more preferably 1.1:1.0.

[0012] Preferably, the reflux reaction time in step S3 is 20 to 60 minutes, preferably 30 minutes.

[0013] In some embodiments, DMY was complexed with manganese acetate hydrate, manganese sulfate hydrate, or manganese chloride hydrate, respectively, to prepare the corresponding DMY-Mn complexes with yields of 90.13–92.25% and purities of 98.95–99.36%.

[0014] The present invention characterized the structure of the above-obtained products by UV, Infrared, and high-resolution mass spectrometry, and determined them to be DMY-Mn complexes.

[0015] The present invention provides a DMY-Mn complex, which exhibits stable activity and significantly superior antibacterial activity against Pasteurella multocida in porcines compared to DMY. Furthermore, a method for the large-scale preparation of the aforementioned DMY-Mn complex is provided; moreover, this method is simple, easy to control, and yields products with high purity.

[0016] Other features and advantages of the invention will be described in detail in the following detailed description section. Attached Figure Description

[0017] Figure 1 This is a flow chart of the preparation process of the DMY-Mn complex of the present invention; Figure 2 This is a structural diagram of the DMY-Mn complex of the present invention; Figure 3 The following are the ultraviolet spectra of DMY and DMY-Mn complexes of this invention; Figure 4 This is the DMY infrared spectrum of the present invention; Figure 5 The infrared spectrum of the DMY-Mn complex of this invention; Figure 6 This is a high-resolution mass spectrum of the DMY-Mn complex of the present invention. Detailed Implementation

[0018] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention. Example

[0019] Add 0.64 g (2.0 mmol) DMY and 60 mL of ethanol sequentially to a three-necked flask, stir, heat to dissolve, and then add 2.0 mmol of manganese salt solution (0.34 g manganese sulfate monohydrate dissolved in 100 mL of ethanol-ammonium acetate buffer solution) dropwise using a constant pressure dropping funnel at a rate of 2.0 mL / min. -1 After the addition was complete, the mixture was heated under reflux for 50 min. The resulting yellowish-brown precipitate was filtered, washed, and dried to obtain a brown solid DMY-Mn complex with a yield of 90.13% and a purity of 98.95%. Example

[0020] Add 0.64 g (2.0 mmol) DMY and 70 mL of ethanol sequentially to a three-necked flask, stir, heat to dissolve, and then add 2.2 mmol of manganese salt solution (0.37 g manganese sulfate monohydrate dissolved in 100 mL of ethanol-ammonium acetate buffer solution) dropwise using a constant pressure dropping funnel at a rate of 3 mL / min. -1 After the addition was complete, the mixture was heated under reflux for 40 min. The resulting yellowish-brown precipitate was filtered, washed, and dried to obtain a brown solid DMY-Mn complex with a yield of 90.86% and a purity of 99.17%. Example

[0021] Add 0.64 g (2.0 mmol) DMY and 90 mL of ethanol to a three-necked flask, stir, heat to dissolve, and then add 2.4 mmol of manganese salt solution (0.41 g manganese sulfate monohydrate dissolved in 100 mL of ethanol-ammonium acetate buffer solution) dropwise using a constant pressure dropping funnel at a rate of 2.5 mL / min. -1 After the addition was complete, the mixture was heated under reflux for 20 min. The resulting yellowish-brown precipitate was filtered, washed, and dried to obtain a brown solid DMY-Mn complex with a yield of 91.53% and a purity of 99.27%. Example

[0022] Add 0.64 g (2.0 mmol) DMY and 80 mL of ethanol to a three-necked flask, stir, heat to dissolve, and then add 2.2 mmol of manganese salt solution (0.37 g manganese sulfate monohydrate dissolved in 100 mL of ethanol-ammonium acetate buffer solution) dropwise using a constant pressure dropping funnel at a rate of 2.5 mL / min. -1 After the addition was complete, the mixture was heated under reflux for 30 min. The resulting yellowish-brown precipitate was filtered, washed, and dried to obtain a brown solid DMY-Mn complex with a yield of 92.25% and a purity of 99.36%. Example

[0023] Add 0.64 g (2.0 mmol) DMY and 100 mL ethanol sequentially to a three-necked flask, stir, heat to dissolve, and then add 2.2 mmol manganese salt solution (0.37 g manganese sulfate monohydrate dissolved in 100 mL ethanol-ammonium acetate buffer solution) dropwise using a constant pressure dropping funnel at a rate of 2.0 mL / min. -1 After the addition was complete, the mixture was heated under reflux for 60 min. The resulting yellowish-brown precipitate was filtered, washed, and dried to obtain a brown solid DMY-Mn complex with a yield of 91.68% and a purity of 99.19%.

[0024] In some embodiments, manganese acetate hydrate and manganese chloride hydrate are used instead of manganese sulfate hydrate to undergo complexation reactions with DMY. Example

[0025] Drug sensitivity test Antimicrobial susceptibility testing was conducted according to the "Broth microdilution method" in the Clinical and Laboratory Standards Institute (CLSI) Guidelines for Antimicrobial Susceptibility Testing of Animal-Derived Bacteria (CLSI-M100-S25). The *Pasteurella multocida* used in the test was donated by Professor Wu Bin of Huazhong Agricultural University. The minimum inhibitory concentrations (MICs) of DMY and the DMY-Mn complex against *Pasteurella multocida* were determined to be 1024 μg / mL and 512 μg / mL, respectively.

[0026] Experiments show that the DMY-Mn complex has significantly improved antibacterial activity compared to DMY, and is expected to become a new, highly effective, and low-toxic antibiotic alternative for the treatment of porcine pasteurellosis, which is of great significance. Example

[0027] Structural characterization 7-1. Determination of manganese content in DMY-Mn complex The theoretical value of the manganese content in the DMY-Mn complex is 12.33%, while the experimental value determined by atomic absorption spectrophotometry is 12.21%. The structure of the complex is preliminarily determined to be one molecule of DMY complexed with one molecule of manganese ion.

[0028] 7-2. Ultraviolet Spectroscopic Analysis of DMY-Mn Complexes DMY and the experimentally prepared DMY-Mn complex were dissolved separately in a small amount of DMSO, and then diluted with a large amount of water to the required concentration for testing. Equal volumes of the above solutions were taken and scanned in the range of 200–450 nm, with a scan interval of 1 nm. The maximum absorption wavelength of the DMY solution was 327.0 nm. Figure 3 The maximum absorption wavelength of the DMY-Mn complex solution is 320.0 nm. Figure 3 The maximum absorption peak redshifted by 7 nm, indicating that the structure of DMY has changed and DMY has formed a complex with Mn.

[0029] 7-3 Infrared Spectroscopic Analysis of DMY-Mn Complexes A small amount of DMY and DMY-Mn complex solid powder was placed in an agate mortar, KBr powder was added, and the mixture was thoroughly ground and pressed into a tablet. The parameters of the infrared spectrometer were set, and the scanning range was 400–4000 cm⁻¹. -1 Infrared spectroscopy analysis was performed using a Fourier transform infrared spectrometer. The main infrared absorption frequencies and their assignments for DMY and the DMY-Mn complex are as follows:

[0030] As shown in the table above, the hydroxyl groups of DMY and its DMY-Mn complex are at 3392.17 cm⁻¹. -1 3448.10 cm -1 The characteristic peak at 1295.93 cm⁻¹ -1 1340.28 cm -1 The presence of characteristic peaks at 1643.05 cm⁻¹ indicates the presence of phenolic hydroxyl groups in both the DMY and DMY-Mn complex structures. -1 A strong absorption peak exists at this position, which is generated by the carbonyl group at position 4 in the DMY molecule. However, due to the coupling effect between the hydroxyl group and the carbonyl group at position 4, the carbonyl absorption peak in DMY has a lower wavenumber than the ordinary carbonyl absorption peak. When a complex is formed, the absorption peak shifts to 1637.27 cm⁻¹. -1 This indicates that the carbonyl group participated in the reaction. The shift in the absorption peak position is due to the complexation of the metal ion with the carbonyl and hydroxyl groups, resulting in a decrease in the bonding electron density. The electrons shift away from the geometric center of the bond, moving towards the oxygen atom, thus weakening the carbon-oxygen double bond. The peaks at 1473.35 cm⁻¹ are shown below. -1 1415.40 cm -1 The presence of absorption peaks at 1164.79 cm⁻¹ indicates that both DMY and the DMY-Mn complex possess a benzene ring structure; the absorption peaks of the DMY and DMY-Mn complexes at 1164.79 cm⁻¹ are also present. -1 and 1040.15 cm -1 The presence of an absorption peak at 642.18 cm⁻¹ indicates that the C-ring ether bond did not undergo ring-opening during the synthesis of the complex; however, in the low-frequency fingerprint region, the DMY-Mn complex, compared to DMY, shows a higher absorption peak at 642.18 cm⁻¹. -1 The appearance of a new absorption peak indicates that DMY interacted with manganese ions to form a complex.

[0031] The results of ultraviolet and infrared spectroscopy analysis show that the ultraviolet and infrared absorption of the product (DMY-Mn complex) have changed significantly relative to the raw material (DMY), indicating the formation of the DMY-Mn complex.

[0032] 7-4. High-resolution mass spectrometry analysis of DMY-Mn complexes In the high-resolution mass spectrum ( Figure 6 It can be seen that the molecular weight of the complex is 446, and the molecular formula is C. 15 H 19 MnO 12 This is consistent with the structural formula of the complex.

[0033] The present invention first provides a DMY-Mn complex, which exhibits stable activity and significantly superior in vitro antibacterial effect against Pasteurella multocida in porcine compared to DMY. Simultaneously, a method for the large-scale preparation of the aforementioned DMY-Mn complex is provided; furthermore, this method is simple, easy to control, and yields products with high purity.

[0034] Comparative Example 1 Weigh 0.64 g (2 mmol) of DMY, add 100 mL of anhydrous ethanol, heat and stir until completely dissolved, then add anhydrous sodium acetate to adjust the pH to 3, and continue stirring for 0.5 h; add 0.35 g (2 mmol) of manganese acetate, stir and reflux in a water bath at 70 °C for 6 h; cool to room temperature, filter, wash, and vacuum dry at 40 °C for 10 h, with a yield of 81.41% and a purity of 82.60%.

[0035] Comparative Example 2 Weigh 0.64 g (2 mmol) of DMY, add 100 mL of anhydrous ethanol, heat and stir until completely dissolved, then add anhydrous sodium acetate to adjust the pH to 3.7, and continue stirring for 0.5 h; add 0.35 g (2 mmol) of manganese acetate, stir and reflux in a water bath at 70 °C for 6 h; cool to room temperature, filter, wash, and vacuum dry at 40 °C for 10 h, with a yield of 83.53% and a purity of 82.57%.

[0036] Comparative Example 3 Weigh 0.64 g (2 mmol) of DMY, add 100 mL of anhydrous ethanol, heat and stir until completely dissolved, then add anhydrous sodium acetate to adjust the pH to 4.5, and continue stirring for 0.5 h; add 0.35 g (2 mmol) of manganese acetate, stir and reflux in a water bath at 70 °C for 6 h; cool to room temperature, filter, wash, and vacuum dry at 40 °C for 10 h, with a yield of 82.79% and a purity of 83.23%.

[0037] Comparative Example 4 0.42 g (2.4 mmol) of manganese acetate was added to 100 mL of anhydrous ethanol containing 0.64 g (2 mmol) of DMY, the pH was adjusted to 6.5, and the mixture was stirred and refluxed at 50 °C for 4 h. The precipitate was allowed to stand, filtered, washed, and dried. The yield was 77.83%, and the purity was 84.42%.

[0038] Comparative Example 5 0.48 g (2.8 mmol) of manganese acetate was added to 100 mL of anhydrous ethanol containing 0.64 g (2 mmol) of DMY, the pH was adjusted to 7.5, and the mixture was stirred and refluxed at 70 °C for 10 h. The precipitate was allowed to stand, filtered, washed, and dried. The yield was 76.12%, and the purity was 83.66%.

[0039] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention. The scope of protection claimed by the appended claims and their equivalents is defined.

Claims

1. A method for preparing dihydromyricetin manganese for treating porcine pasteurellosis, characterized in that: Includes the following steps: (1) Preparation of dihydromyricetin solution: accurately weigh dihydromyricetin, add ethanol, stir, heat and dissolve to obtain dihydromyricetin solution; (2) Preparation of manganese salt solution: 1.1 mmol of manganese salt was added to 50 mL of ethanol-ammonium acetate buffer solution to obtain manganese salt solution.

2. (3) The manganese salt solution was slowly added dropwise to the dihydromyricetin solution using a constant pressure dropping funnel, and the mixture was stirred, heated, and refluxed. The resulting yellowish-brown precipitate was filtered, washed, and dried to obtain the dihydromyricetin-manganese brown solid complex.

3. The method for preparing dihydromyricetin manganese for treating porcine pasteurellosis according to claim 1, characterized in that: Manganese salts are derived from one or two of manganese acetate hydrate, manganese sulfate hydrate, or manganese chloride hydrate.

4. The method for preparing dihydromyricetin manganese for treating porcine pasteurellosis according to claim 1, characterized in that: The dropping rate of the manganese salt solution is 2.0–3.0 mL / min. -1 .

5. The method for preparing dihydromyricetin manganese for treating porcine pasteurellosis according to claim 1, characterized in that: The molar ratio of manganese salt to dihydromyricetin is 1.0–1.2:1.

0.

6. The method for preparing dihydromyricetin manganese for treating porcine pasteurellosis according to claim 1, characterized in that: The reflux reaction time is 20–60 min.

Images