Preparation method of dihydromyricetin-zinc complex for mixed feeding of pigs

Abstract

The invention discloses a preparation method of a dihydromyricetin-zinc complex for mixed feeding of pigs. According to the method, a synthetic reaction of the 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 zinc salt solution to the dihydromyricetin is 1.1: 1.0; the optimal feeding ratio of the dihydromyricetin to the ethanol-ammonium acetate buffer solution is 1.0 mmol: 65 mL; 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 complex, 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 an antibiotic substitute therapeutic drug for treating swine pasteurellosis.

Description

Technical Field

[0001] The invention relates to the preparation of a dihydromyricetin metal complex, and in particular to application of dihydromyricetin-zinc in treating swine pasteurellosis. Background Art

[0002] Pasteurellosis is a common disease in pig farming, which can easily lead to large-scale deaths and seriously affect the profitability of pig farming. The prevalence and infection of Pasteurellosis remains one of the most serious threats to my country's pig farming industry. Broad-spectrum antibiotics are the first choice for treating Pasteurellosis infection in pigs, but as bacteria develop resistance to antibiotics, it is urgent to explore more potential new antibiotic alternatives.

[0003] Dihydromyricelin (DMY), a signature ingredient of rattan tea, has a variety of pharmacological effects, including antibacterial, anti-inflammatory and immune enhancement. DMY molecules can serve as good chelating ligands for metal ions, and the biological activity of DMY complexes with metal ions is often better than that of DMY. The coordination chemistry theory of traditional Chinese medicine points out that the formation of coordination compounds can reduce the toxicity of active ingredients in traditional Chinese medicine and increase their activity. Zinc is an essential element for animals and is called the "element of life" because of its wide range of physiological and biochemical functions in animals. Zinc plays an important role in improving animal immunity, maintaining lymphocytes and preventing parasite invasion. When coccidia and bacterial infections occur, the zinc content in tissues will be reduced. The synthesis and biological activity research of zinc complexes is one of the current hot research fields, and zinc complexes show broad application prospects in biological activity research. By studying the synthesis method and biological activity of zinc complexes, researchers can provide a theoretical basis for the design and preparation of more effective zinc complexes and provide new ideas for the development of new biomedical applications. It is predicted that the complexation of dihydromyricetin with zinc will have a good application effect in veterinary clinics, but the preparation process of dihydromyricetin-zinc complex (DMY-Zn) needs further study. Fan Luo (Journal of Nanostructure in Chemistry, 2022), Renkai Wang (Biological Trace Element Research, 2024), Yan Zankai (Spectral Laboratory, 2011), Yuanyong Yao (Frontiers in Chemistry, 2020), Jiao Xiaoli (Asia-Pacific Traditional Medicine, 2009), Meng Yan (Master's thesis of Huazhong Agricultural University, 2008) and Yan Gaojian (Master's thesis of Guangdong Pharmaceutical University, 2017) reported no reaction yield in the preparation of DMY-Zn; Yuan Juan (Master's thesis of Guangdong University of Technology, 2013) reported that the yield of the synthetic complex was 83%, Zeng Xianhua (Master's thesis of Guangdong University of Technology, 2014) reported that the yield of the synthetic complex was 85.19%, and Wu Chun (Chemistry and Adhesion, 2009) reported that the yield of the synthetic complex was 78.13%; there is no report on the resistance of DMY-Zn to swine Pasteurellosis multocida. Based on the systematic study of DMY-Zn complexes, the present invention prepares a DMY-Zn complex for mixed feeding of pigs. The preparation method is simple, easy to control, suitable for large-scale production, high reaction yield and high product purity. Summary of the invention

[0004] In order to overcome the immaturity of the preparation process of related products in the prior art, the present invention proposes a preparation method of a DMY-Zn complex for mixed feeding of pigs. The prepared DMY-Zn complex product has high yield and high purity and is suitable for large-scale production. In vitro tests show that its antibacterial activity against Pasteurella multocida is better than that of DMY.

[0005] In order to achieve the above object, the present invention is implemented by the following technical scheme, and its preparation method comprises the following steps: S1: Prepare the reaction mixture: add DMY, zinc salt, and ethanol-ammonium acetate buffer solution (pH=3.7) in a three-necked flask in sequence to obtain a reaction mixture; S2: The reaction mixture obtained in S1 is heated and refluxed, and the obtained yellow-brown precipitate is filtered, washed, and dried to obtain a khaki solid DMY-Zn complex.

[0006] Preferably, the zinc salt in S1 is selected from one or both of zinc acetate hydrate, zinc sulfate hydrate or zinc chloride hydrate, preferably zinc sulfate heptahydrate.

[0007] Preferably, the molar ratio of zinc salt to DMY in step S1 is 1.0-1.2: 1.0, preferably 1.1: 1.0.

[0008] Preferably, in step S1, the feed ratio of DMY to ethanol-ammonium acetate buffer solution is 1.0 mmol: 50-75 mL, preferably 1.0 mmol: 65 mL.

[0009] Preferably, the reflux reaction time in step S2 is 20 to 60 min, preferably 30 min.

[0010] In certain embodiments, zinc acetate hydrate, zinc sulfate hydrate or zinc chloride hydrate is used to carry out complex reaction with DMY to prepare the corresponding DMY-Zn complex with a yield of 89.17-92.29% and a purity of 98.64-99.28%.

[0011] The present invention characterizes the structure of the product obtained above by UV, Infra-red, high-resolution mass spectrometry and the like, and determines that the product is a DMY-Zn complex.

[0012] The above technical scheme of the present invention provides a DMY-Zn complex, which has stable activity and has significantly better antibacterial activity against porcine Pasteurella multocida than DMY. At the same time, a large-scale preparation method for the above DMY-Zn complex is provided; in addition, the method is simple in process, easy to control, and the product has a high yield and purity.

[0013] Other features and advantages of the invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The process flow chart of the preparation of the DMY-Zn complex of the present invention is as follows; Figure 2 The structure diagram of the DMY-Zn complex of the present invention; Figure 3 The UV spectra of DMY and DMY-Zn complexes of the present invention are shown in FIG. Figure 4 It is the DMY infrared spectrum of the present invention; Figure 5 This is the infrared spectrum of the DMY-Zn complex of the present invention; Figure 6 This is a high-resolution mass spectrum of the DMY-Zn complex of the present invention. DETAILED DESCRIPTION

[0015] The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention. Example 1

[0016] 0.64 g (2.0 mmol) DMY, 0.31 g zinc chloride monohydrate (2.0 mmol), and 100 mL ethanol-ammonium acetate buffer solution were added to a three-necked flask in sequence and heated under reflux for 50 min. The obtained yellow-brown precipitate was filtered, washed, and dried to obtain a yellow-brown solid DMY-Zn complex with a yield of 89.17% and a purity of 99.15%. Example 2

[0017] 0.64 g (2.0 mmol) DMY, 0.63 g zinc sulfate heptahydrate (2.2 mmol), and 110 mL ethanol-ammonium acetate buffer solution were added to a three-necked flask in sequence, and the mixture was heated under reflux for 40 min. The obtained yellow-brown precipitate was filtered, washed, and dried to obtain a yellow-brown solid DMY-Zn complex with a yield of 90.32% and a purity of 99.05%. Example 3

[0018] 0.64 g (2.0 mmol) DMY, 0.69 g zinc sulfate heptahydrate (2.4 mmol), and 120 mL ethanol-ammonium acetate buffer solution were added to a three-necked flask in sequence, and the mixture was heated under reflux for 20 min. The obtained yellow-brown precipitate was filtered, washed, and dried to obtain a yellow-brown solid DMY-Zn complex with a yield of 91.58% and a purity of 99.13%. Example 4

[0019] 0.64 g (2.0 mmol) DMY, 0.63 g zinc sulfate heptahydrate (2.2 mmol), and 130 mL ethanol-ammonium acetate buffer solution were added to a three-necked flask in sequence, and the mixture was heated under reflux for 30 min. The obtained yellow-brown precipitate was filtered, washed, and dried to obtain a yellow-brown solid DMY-Zn complex with a yield of 92.29% and a purity of 99.28%. Example 5

[0020] 0.64 g (2.0 mmol) DMY, 0.44 g zinc acetate dihydrate (2.0 mmol), and 140 mL ethanol-ammonium acetate buffer solution were added to a three-necked flask in sequence, and the mixture was heated under reflux for 60 min. The obtained yellow-brown precipitate was filtered, washed, and dried to obtain a yellow-brown solid DMY-Zn complex with a yield of 91.68% and a purity of 98.64%. Example 6

[0021] 0.64 g (2.0 mmol) DMY, 0.69 g zinc sulfate heptahydrate (2.4 mmol), and 150 mL ethanol-ammonium acetate buffer solution were added to a three-necked flask in sequence and heated under reflux for 30 min. The obtained yellow-brown precipitate was filtered, washed, and dried to obtain a yellow-brown solid DMY-Zn complex with a yield of 92.24% and a purity of 99.19%. Example 7

[0022] Antimicrobial susceptibility testing The drug sensitivity test was conducted in accordance with the "Broth microdilution method" in the "Standards for Drug Susceptibility Tests of Animal-derived Bacteria" (CLSI-M100-S25) issued by the Clinical & Laboratory Standards Institute (CLSI) of the United States. The porcine Pasteurella multocida used in the test was donated by Professor Wu Bin of Huazhong Agricultural University. The minimum inhibitory concentrations (MIC) of DMY and DMY-Zn complex against porcine Pasteurella multocida were measured to be 1024μg / mL and 256μg / mL, respectively.

[0023] The experiment showed that the antibacterial activity of DMY-Zn complex was greatly improved compared with DMY, and it is expected to become a new high-efficiency and low-toxic antibiotic alternative for the treatment of swine pasteurellosis, which is of great significance. Example 8

[0024] Structural characterization 8-1. Determination of the content of metallic element zinc in DMY-Zn complex The theoretical value of the zinc content in the DMY-Zn complex is 15.51%, and the experimental value determined by atomic absorption spectrophotometry is 15.04%. The structure of the complex was preliminarily determined to be one DMY molecule complexed with one zinc ion molecule.

[0025] 8-2. UV spectrum analysis of DMY-Zn complex Dissolve DMY and the experimentally prepared DMY-Zn complex in a small amount of DMSO, and then dilute with a large amount of water to the required concentration for the test. Take an equal amount of the above solution and scan in the range of 200-450 nm with a scanning interval of 1 nm. The maximum absorption wavelength of the DMY solution is 327.0 nm ( Figure 3 ), while the maximum absorption wavelength of DMY-Zn complex solution is 325.0 nm ( Figure 3 ), the maximum absorption peak red-shifted by 2 nm, and the intensity of the absorption peak decreased significantly, indicating that the structure of DMY changed and DMY formed a complex with Zn.

[0026] 8-3. Infrared spectroscopy analysis of DMY-Zn complex Take a small amount of DMY and DMY-Zn complex solid powder and place it in an agate mortar, add KBr powder, grind it thoroughly, press it into tablets, and set the parameters of the infrared spectrometer with a scanning range of 400-4000 cm -1 , infrared spectrum analysis was performed by Fourier transform infrared spectrometer. Infrared spectra of DMY and DMY-Zn complexes ( Figure 4 , Figure 5 )'s main infrared absorption frequencies and their attributions are as follows:

[0027] From the above table, we can see that the hydroxyl groups of DMY and DMY-Zn complexes are at 3392.17 cm -1 、3428.81 cm -1 The characteristic peak at 1295.93cm -1 、1338.36 cm -1 The presence of characteristic peaks at 1643.05 cm-1 indicates that there are phenolic hydroxyl groups in the structures of DMY and DMY-Zn complexes. -1 There is a strong absorption peak at 1637.27 cm-1, which is generated by the carbonyl group at position 4 in the DMY molecule. However, due to the coupling between the hydroxyl group and the carbonyl group at position 4, the carbonyl absorption peak in DMY is lower than the ordinary carbonyl absorption peak wave number. When the complex is formed, the absorption peak shifts to 1637.27 cm-1. -1, indicating that the carbonyl group participated in the reaction. The shift in the absorption peak position was due to the complexation between the metal ion and the carbonyl group and the hydroxyl group, which resulted in a decrease in the density of bonding electrons, deviated from the geometric center of the bond and moved toward the oxygen atom, weakening the carbon-oxygen double bond. -1 、1413.57 cm -1 There is an absorption peak of benzene ring skeleton at 1164.79 cm -1 and 1020.16cm -1 There is an absorption peak at 642.18cm, indicating that the C ring ether bond did not open during the synthesis of the complex; however, in the low-frequency fingerprint region, the DMY-Zn complex has a peak at 642.18cm -1 A new absorption peak appeared at , indicating that DMY interacted with zinc ions to form a complex.

[0028] From the UV and IR results, we can see that the UV and IR absorption of the product (DMY-Zn complex) have changed significantly compared with the raw material (DMY), indicating the formation of DMY-Zn complex.

[0029] 8-4. High-resolution mass spectrometry analysis of DMY-Zn complex High resolution mass spectrometry ( Figure 6 ), [MH] + is 418.99, the molecular weight of the complex is 420, and the molecular formula is C 15 H 15 ZnO 10 , which is consistent with the structural formula of the complex.

[0030] The above technical scheme of the present invention first provides a DMY-Zn complex, which has stable activity and has a significantly better in vitro antibacterial effect on porcine Pasteurella multocida than DMY. At the same time, a large-scale preparation method for the above DMY-Zn complex is provided; in addition, the method is simple in process, easy to control, and the product has a high yield and purity.

[0031] Comparative Example 1 Weigh 0.64 g (2 mmol) of DMY, add 25 mL of anhydrous ethanol, heat and stir to dissolve to obtain a DMY solution; take another beaker, add 0.58 g (3.6 mmol) of zinc sulfate and 25 mL of water, stir to dissolve to obtain a zinc sulfate solution; add the zinc sulfate solution dropwise to the DMY solution, maintain the pH of the reaction mixture at about 6.4 with 0.1 mol / L NaOH solution, and continue stirring at 70°C for 4 h; cool and filter, wash with water, wash with ethanol, and dry. The yield is 84.65% and the purity is 82.33%.

[0032] Comparative Example 2 Weigh 0.64 g (2 mmol) of DMY and dissolve it in 100 mL of anhydrous ethanol. Heat and stir to dissolve. Add sodium acetate to adjust the pH to 7.5-8. Continue stirring for 0.5 h, then add 0.36 g (2 mmol) of zinc acetate. Stir and reflux in a 70 °C water bath for 6 h. Cool, filter, wash, and vacuum dry at 40 °C for 10 h. The yield is 83.12% and the purity is 81.26%.

[0033] Comparative Example 3 Weigh 0.64 g (2 mmol) of DMY and dissolve it in 50 mL of anhydrous ethanol. Stir and dissolve. Add an appropriate amount of sodium acetate to adjust the pH to 7.4. Then add 0.36 g (2 mmol) of zinc acetate. Reflux in a water bath for 6 h. Cool, filter, wash, and vacuum dry to obtain a yellow-brown powdery solid with a yield of 83.43% and a purity of 82.21%.

[0034] Comparative Example 4 Weigh 0.64 g (2 mmol) of DMY and dissolve it in 100 mL of anhydrous ethanol. Heat and stir to dissolve. Add sodium acetate to adjust the pH to 6.4. Add 0.32 g (2 mmol) of zinc sulfate. Stir and reflux in a 70 °C water bath for 4 h. Cool, filter, wash, and vacuum dry at 40 °C for 10 h. The yield is 84.22% and the purity is 82.45%.

[0035] Comparative Example 5 Weigh 0.16 g (0.5 mmol) of DMY and dissolve it in 25 mL of anhydrous ethanol. Heat and stir at 60°C to dissolve it. Add 0.06 g (0.5 mmol) of sodium acetate and heat and dissolve it for 0.5 h. Add 0.14 g (1 mmol) of zinc chloride and stir and reflux it for 5 h. Cool and filter to obtain a solid. Cool, filter, wash, and dry at 60°C for 24 h. The yield is 84.20% and the purity is 83.15%.

[0036] Comparative Example 6 0.64 g (2 mmol) DMY was dissolved in 100 mL of anhydrous ethanol, heated and stirred to dissolve, an appropriate amount of anhydrous sodium carbonate was added, stirring was continued for 0.5 h, 0.44 g (2 mmol) zinc acetate dihydrate was added, and refluxed at 70 ° C for 1 h. The precipitate was allowed to stand, filtered, washed, and vacuum dried. The yield was 84.73% and the purity was 84.31%.

[0037] Comparative Example 7 0.64 g (2 mmol) DMY was dissolved in 100 mL anhydrous ethanol, heated and stirred to dissolve, an appropriate amount of anhydrous sodium carbonate was added, stirring was continued for 0.5 h, 0.88 g (4 mmol) zinc acetate ethanol solution was added, and refluxed at 70 ° C for 1 h. The precipitate was allowed to stand, filtered, washed, and vacuum dried. The yield was 84.69% and the purity was 84.56%.

[0038] Comparative Example 8 0.64 g (2 mmol) DMY was dissolved in 100 mL of anhydrous ethanol, heated and stirred to dissolve, an appropriate amount of anhydrous sodium carbonate was added, stirring was continued for 0.5 h, 1.32 g (6 mmol) of zinc acetate in ethanol solution was added, and refluxed at 70 ° C for 1 h. The precipitate was allowed to stand, filtered, washed, and dried in vacuum. The yield was 84.51% and the purity was 84.39%.

[0039] Comparative Example 9 0.64 g (2 mmol) DMY was dissolved in 100 mL of anhydrous ethanol, heated and stirred to dissolve, and an appropriate amount of sodium acetate was added until the pH was about 7.5. Stirring was continued for 0.5 h, and 0.44 g (2 mmol) zinc acetate ethanol solution was added. The reaction was refluxed at 70 ° C for 2 h. The precipitate was allowed to stand, filtered, washed, and vacuum dried to obtain a yellow-brown powder solid. The yield was 83.57% and the purity was 84.45%.

[0040] Comparative Example 10 0.44 g (2.4 mmol) zinc acetate was added to 100 mL anhydrous ethanol containing 0.640 g (2 mmol) 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 82.78% and the purity was 83.42%.

[0041] Comparative Example 11 0.51 g (2.8 mmol) zinc acetate was added to 100 mL anhydrous ethanol containing 0.640 g (2 mmol) 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 80.16% and the purity was 81.31%.

[0042] Comparative Example 12 0.48 g (2.6 mmol) of zinc acetate was added to 100 mL of anhydrous ethanol containing 0.640 g (2 mmol) of DMY, the pH was adjusted to 7, and the mixture was stirred and refluxed at 60°C for 6 h. The precipitate was allowed to stand, filtered, washed, and dried. The yield was 81.63% and the purity was 82.11%.

[0043] Comparative Example 13 Add an appropriate amount of 95% ethanol to 0.64 g (2.0 mmol) of DMY, stir, heat to dissolve, add 55 mL of alkaline solution [sodium acetate dissolved in water-ethanol solution (50%, v / v), pH = 8.2], stir for 5 minutes, add 0.40 g (2.2 mmol) of zinc acetate (dissolved in an appropriate amount of 95% ethanol), heat to reflux for 2 hours, cool to room temperature, precipitate, filter, wash with water, and dry. The yield is 83.24% and the purity is 84.61%.

[0044] The above shows and describes 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 above embodiments and descriptions only describe the principles of the present invention. The present invention may be subject to various changes and improvements without departing from the spirit and scope of the present invention. These changes and improvements fall within the scope of the present invention. The scope of protection claimed by the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for preparing a dihydromyricetin-zinc complex for mixed feeding of pigs, characterized in that The following steps are involved: (1) Preparing a reaction mixture: Add dihydromyricetin, zinc salt, and ethanol-ammonium acetate buffer solution (pH = 3.7) in a three-necked flask in sequence to obtain a reaction mixture; (2) The reaction mixture is heated and refluxed, and the obtained black precipitate is filtered, washed, and dried to obtain the dihydromyricetin-zinc yellow-brown solid complex.

2. The method for preparing a dihydromyricetin-zinc complex for mixed feeding of pigs according to claim 1, characterized in that: The zinc salt is taken from one or both of zinc acetate hydrate, zinc sulfate hydrate or zinc chloride hydrate.

3. The method for preparing a dihydromyricetin-zinc complex for mixed feeding of pigs according to claim 1, characterized in that: The molar ratio of zinc salt to dihydromyricetin is 1.0-1.2: 1.

0.

4. The method for preparing a dihydromyricetin-zinc complex for mixed feeding of pigs according to claim 1, characterized in that: The feed ratio of dihydromyricetin to ethanol-ammonium acetate buffer solution is 1.0 mmol: 50-75 mL.

5. The method for preparing a dihydromyricetin-zinc complex for mixed feeding of pigs according to claim 1, characterized in that: The reflux reaction time is 20 to 60 min.

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