Preparation method of dihydromyricetin ferrous complex for treating swine pasteurellosis

Abstract

The invention discloses a preparation method of a dihydromyricetin ferrous complex for treating swine pasteurellosis. According to the method, a synthesis 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 ferrous salt solution to the dihydromyricetin is 1.15: 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 35 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] The invention belongs to the field of complex synthesis, and particularly relates to a method for preparing a dihydromyricetin ferrous complex for treating swine pasteurellosis. Background Art

[0002] Pasteurellosis in pigs can occur all year round and is a common epidemic disease in pig farming, causing large-scale deaths and seriously affecting farming benefits. 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 such as antibacterial, anti-inflammatory and immune enhancement. DMY molecules can serve as good chelating ligands for metal ions. The biological activity of DMY complexes with metal ions is often better than that of DMY, with stronger antibacterial effects and broad prospects for development and application. 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. Ferrous ions play an important role in organisms. They not only participate in basic life processes such as cell death and oxygen transport, but also have biological activities such as antibacterial and antioxidant, which are essential for maintaining the normal progress of life activities. The synthesis and biological activity of ferrous complexes is one of the current hot research fields. By studying the synthesis methods and biological activities of ferrous complexes, researchers can provide a theoretical basis for the design and preparation of more effective ferrous complexes and provide new ideas for the development of new biomedical applications. It is predicted that the complexation of dihydromyricetin with ferrous ions will have a good application effect in veterinary clinical practice, but the current preparation process of dihydromyricetin ferrous complex (DMY-Fe (II)) needs to be further improved. Meng Yan (Master's thesis of Huazhong Agricultural University, 2008), Guo Qingquan (Food Industry Science and Technology, 2006), Yan Zankai (Spectral Laboratory, 2011) and Yuanyong Yao (Frontiers in Chemistry, 2020) did not report the reaction yield and product purity in the preparation of DMY-Fe (II); there was no report on its resistance to porcine Pasteurella multocida.

[0004] According to the systematic study of DMY-Fe (II), the present invention prepares a DMY-Fe (II) complex for treating swine pasteurellosis. The preparation method is simple, easy to control, suitable for large-scale production, high in reaction yield and high in product purity. Summary of the invention

[0005] 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-Fe (II) complex for treating swine pasteurellosis. The prepared DMY-Fe (II) complex product has high yield and high purity and is suitable for large-scale production. In vitro tests have shown that its antibacterial activity against Pasteurella multocida is better than that of DMY.

[0006] 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, ferrous 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 brown solid DMY-Fe (II) complex.

[0007] Preferably, the ferrous salt in S2 is selected from one or two of ferrous acetate hydrate, ferrous sulfate hydrate or ferrous chloride hydrate, preferably ferrous chloride hydrate.

[0008] Preferably, the molar ratio of ferrous salt to DMY in step S1 is 1.0-1.2: 1.0, preferably 1.15: 1.0.

[0009] 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.

[0010] Preferably, the reflux reaction time in step S2 is 20 to 60 min, preferably 35 min.

[0011] In certain embodiments, ferrous acetate hydrate, ferrous sulfate hydrate or ferrous chloride hydrate are used to carry out complex reaction with DMY to prepare the corresponding DMY-Fe (II) complex with a yield of 90.63-93.26% and a purity of 99.02-99.37%.

[0012] In the present invention, the product obtained above is characterized by using UV, Infra-red, high-resolution mass spectrometry and the like, and is determined to be a DMY-Fe (II) complex.

[0013] The above technical scheme of the present invention provides a DMY-Fe (II) complex, which has stable activity and is significantly better than DMY in antibacterial activity against porcine Pasteurella multocida. At the same time, a large-scale preparation method for the above DMY-Fe (II) complex is provided; in addition, the method is simple in process, easy to control, and the product has a high yield and purity.

[0014] Other features and advantages of the invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The process flow chart of the preparation of the DMY-Fe (II) complex of the present invention is as follows; Figure 2 The structure diagram of the DMY-Fe (II) complex of the present invention; Figure 3 The UV spectra of DMY and DMY-Fe (II) 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-Fe (II) complex of the present invention; Figure 6 This is a high-resolution mass spectrum of the DMY-Fe (II) complex of the present invention. DETAILED DESCRIPTION

[0016] 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

[0017] 0.64 g (2.0 mmol) DMY, 0.40 g ferrous chloride tetrahydrate (2.0 mmol) and 100 mL ethanol-ammonium acetate buffer solution were added to a three-necked flask in sequence, stirred, and heated under reflux for 50 min. The obtained yellow-brown precipitate was filtered, washed, and dried to obtain a yellow-brown solid DMY-Fe (II) complex with a yield of 90.63% and a purity of 99.02%. Example 2

[0018] 0.64 g (2.0 mmol) DMY, 0.44 g ferrous chloride tetrahydrate (2.2 mmol) and 110 mL ethanol-ammonium acetate buffer solution were added to a three-necked flask in sequence, stirred, and heated under reflux for 40 min. The obtained yellow-brown precipitate was filtered, washed, and dried to obtain a yellow-brown solid DMY-Fe (II) complex with a yield of 91.18% and a purity of 99.15%. Example 3

[0019] 0.64 g (2.0 mmol) DMY, 0.48 g ferrous chloride tetrahydrate (2.4 mmol) and 120 mL ethanol-ammonium acetate buffer solution were added to a three-necked flask in sequence, stirred, and heated under reflux for 30 min. The obtained yellow-brown precipitate was filtered, washed, and dried to obtain a yellow-brown solid DMY-Fe (II) complex with a yield of 91.86% and a purity of 99.22%. Example 4

[0020] 0.64 g (2.0 mmol) DMY, 0.46 g ferrous chloride tetrahydrate (2.3 mmol) and 130 mL ethanol-ammonium acetate buffer solution were added to a three-necked flask in sequence, stirred, and heated under reflux for 35 min. The obtained yellow-brown precipitate was filtered, washed, and dried to obtain a brown solid DMY-Fe (II) complex with a yield of 93.26% and a purity of 99.37%. Example 5

[0021] 0.64 g (2.0 mmol) DMY, 0.44 g ferrous chloride tetrahydrate (2.2 mmol) and 150 mL ethanol-ammonium acetate buffer solution were added to a three-necked flask in sequence, stirred, and heated under reflux for 20 min. The obtained yellow-brown precipitate was filtered, washed, and dried to obtain a yellow-brown solid DMY-Fe (II) complex with a yield of 92.37% and a purity of 99.17%.

[0022] In certain embodiments, ferrous acetate, ferrous sulfate or ferrous nitrate is used instead of ferrous chloride to carry out complex reaction with DMY. Example 6

[0023] 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-Fe (II) complex against porcine Pasteurella multocida were measured to be 1024μg / mL and 256μg / mL, respectively.

[0024] The experiment showed that the antibacterial activity of DMY-Fe (II) 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 7

[0025] Structural characterization 7-1. Determination of the content of metallic element iron (II) in DMY-Fe (II) complex The theoretical value of the content of metallic element iron (II) in the DMY-Fe (II) complex is 14.89%, and the experimental value determined by atomic absorption spectrophotometry is 14.04%. The structure of the complex was preliminarily determined to be one molecule of DMY complexed with one molecule of ferrous ion.

[0026] 7-2. UV spectroscopy analysis of DMY-Fe (II) complex Dissolve DMY and the experimentally prepared DMY-Fe (II) complex in a small amount of DMSO, and then dilute with a large amount of water to the required concentration for the test. Take equal amounts of the above solutions and scan in the range of 200-450nm with a scanning interval of 1nm. The maximum absorption wavelength of the DMY solution is 327.0nm ( Figure 3 ), while the maximum absorption wavelength of DMY-Fe (II) complex solution is 322.0nm ( Figure 3 ), the maximum absorption peak red-shifted by 5nm, and the intensity of the absorption peak decreased significantly, indicating that the structure of DMY changed and DMY formed a complex with Fe (II).

[0027] 7-3. Infrared spectroscopy analysis of DMY-Fe (II) complex Take a small amount of DMY and DMY-Fe (II) 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 400-4000cm -1 , infrared spectroscopy analysis was performed by Fourier transform infrared spectrometer. Infrared spectra of DMY and DMY-Fe (II) complexes ( Figure 4 , Figure 5 )'s main infrared absorption frequencies and their attributions are as follows:

[0028] From the above table, we can see that the hydroxyl groups of DMY and DMY-Fe (II) complexes are at 3392.17 cm -1 、3434.60 cm -1 The characteristic peak at 1295.93cm -1 、1340.28 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-Fe (II) complexes. -1There 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 1051.01cm -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-Fe (II) complex has a peak at 642.18cm -1 A new absorption peak appeared at , indicating that DMY interacted with ferrous ions to form a complex.

[0029] The UV and IR results show that the UV and IR absorption of the product DMY-Fe (II) complex have changed significantly compared with the raw material (DMY), indicating the formation of the DMY-Fe (II) complex.

[0030] 7-4. High-resolution mass spectrometry analysis of DMY-Fe(II) complex In the high-resolution mass spectrum ( Figure 6 ) It can be seen that the molecular weight of the complex is 375 and the molecular formula is C 15 H 11 FeO8, which is consistent with the structural formula of the complex.

[0031] The above technical scheme of the present invention first provides a DMY-Fe (II) 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-Fe (II) complex is provided; in addition, the method is simple in process, easy to control, and the product has a high yield and purity.

[0032] Comparative Example 1 Weigh 0.16 g (0.5 mmol) of dihydromyricetin DMY, add 25 mL of anhydrous ethanol, heat and stir at 60°C to dissolve; add 0.06 g (0.5 mmol) of anhydrous sodium acetate, heat and dissolve for 0.5 h; add 0.20 g (1 mmol) of ferrous chloride tetrahydrate, stir and reflux for 5 h; cool and filter to obtain a solid; wash it with 70% ethanol 3 times, wash it with distilled water 3 times, and dry it at 60°C for 24 h. The yield is 76.42% and the purity is 82.53%.

[0033] Comparative Example 2 Add 2.5 mL of DMY solution (0.02 mol·L) into the three-necked flask. -1 ), 1.0 mL of ferrous chloride tetrahydrate solution (0.02 mol·L -1 ), 3.0 mL of NaH2PO4-Na2HPO4 buffer solution with pH = 7.4, 3.5 mL of 50% ethanol aqueous solution, stirred at 26°C for 1 h, filtered, washed and dried. The yield was 77.81% and the purity was 85.53%.

[0034] Comparative Example 3 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, 0.40 g (2 mmol) ferrous chloride tetrahydrate 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 83.63% and the purity was 83.51%.

[0035] Comparative Example 4 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, 0.80 g (4 mmol) of ferrous chloride tetrahydrate 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 83.78% and the purity was 83.69%.

[0036] Comparative Example 5 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, 1.20 g (6 mmol) of ferrous chloride tetrahydrate in 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 83.91% and the purity was 84.12%.

[0037] Comparative Example 6 0.64 g (2 mmol) DMY was dissolved in 10 mL water, heated and stirred, 0.60 g of 80% ferrous chloride aqueous solution (weight percentage) was added, stirred, and 5 ml of 5% sodium hydroxide aqueous solution (weight percentage) was added dropwise. After constant temperature reaction for 5 minutes, a black precipitate appeared, and 5% hydrochloric acid aqueous solution (volume percentage) was added dropwise. The pH value of the system was adjusted to 5.5, cooled at room temperature, filtered, and repeatedly washed with water to obtain a black dihydromyricetin-iron chelate. The yield was 81.63% and the purity was 83.14%.

[0038] Comparative Example 7 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 0.38 g (2.2 mmol) of ferrous 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%.

[0039] Comparative Example 8 0.42 g (2.4 mmol) of ferrous 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 82.36% and the purity was 83.22%.

[0040] Comparative Example 9 0.49 g (2.8 mmol) of ferrous acetate was added to 100 mL of anhydrous ethanol containing 0.640 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 80.25% and the purity was 81.43%.

[0041] 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 ferrous complex of dihydromyricetin for treating porcine pasteurellosis, characterized in that: The following steps are involved: (1) Preparing a reaction mixture: Add dihydromyricetin, ferrous 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 yellow-brown precipitate is filtered, washed, and dried to obtain the dihydromyricetin-ferrous iron brown solid complex.

2. The method for preparing a ferrous complex of dihydromyricetin for treating porcine pasteurellosis according to claim 1, characterized in that: The ferrous salt is obtained from one or two of ferrous acetate, ferrous sulfate, ferrous nitrate or ferrous chloride.

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

0.

4. The method for preparing a ferrous complex of dihydromyricetin 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 ferrous complex of dihydromyricetin 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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