Preparation method of dihydromyricetin-cobalt complex for treating swine pasteurellosis
The high yield and purity DMY-Co complexes were prepared by heating and reflux reaction in a three-necked bottle, and the problem of insufficient yield and purity of DMY-Co preparation process in the prior art was solved, which significantly improved the multi-killing ability to pigs The antibacterial activity of Pasteuris has the potential to treat Pasteurisosis in porcine.
Patent Information
- Application Number
- CN202411425602.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-13
- Publication Date
- 2024-12-31
AI Technical Summary
The existing dihydrobamate-cobalt complex (DMY-Co) preparation process has low yield and purity, and no antibacterial effects against Pasteurium porcine have been reported, making it difficult to meet the needs of treating Pasteurium porcine.
By adding dihydrobamate (DMY) and cobalt salt and ethanol-ammonium acetate buffer solution to the three-necked bottle, the heating and reflux reaction was carried out, filtered, washed and dried, and a high yield and high purity DMY-Co complex was prepared. Suitable for large-scale production.
The high yield and high purity preparation of DMY-Co complexes were achieved, and the antibacterial activity against porcine Pasteuris was significantly improved, with potential as a new antibiotic alternative.
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of complex synthesis, and particularly relates to a method for preparing a dihydromyricetin-cobalt 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, including antibacterial, anti-inflammatory and immune-enhancing effects. DMY molecules can serve as good chelating ligands for metal ions, and the biological activity of complexes formed with metal ions is often better than that of DMY, which has 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. The synthesis and biological activity research of cobalt complexes is one of the current hot research fields, and cobalt complexes show broad application prospects in biological activity research. By studying the synthesis method and biological activity of cobalt complexes, researchers can provide a theoretical basis for the design and preparation of more effective cobalt complexes, and provide new ideas for the development of new biomedical applications. It is predicted that the complexation of dihydromyricetin with cobalt will have a good application effect in veterinary clinical practice. At present, the preparation process of dihydromyricetin-cobalt complex (DMY-Co) still needs further research. Li Haixia (Spectrum Laboratory, 2013), Yan Zankai (Spectrum Laboratory, 2011) and YuanyongYao (Frontiers in Chemistry, 2020) did not report the yield and purity in the preparation of DMY-Co; there was no report on its resistance to porcine Pasteurella multocida.
[0004] According to the systematic study of DMY-Co, the present invention prepares a DMY-Co 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] Through further research on the preparation process of DMY-Co complex, the present invention proposes a preparation method of DMY-Co complex for treating swine pasteurellosis, and the prepared DMY-Co 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.
[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: preparing a reaction mixture: sequentially adding DMY, cobalt salt, and ethanol-ammonium acetate buffer solution into a three-necked flask to obtain a reaction mixture; S2: The reaction mixture obtained in S1 is heated and refluxed, and the obtained black precipitate is filtered, washed, and dried to obtain a khaki solid DMY-Co complex.
[0007] Preferably, the cobalt salt in S1 is selected from one or both of cobalt acetate hydrate, cobalt sulfate hydrate or cobalt chloride hydrate, preferably cobalt chloride hexahydrate.
[0008] Preferably, the molar ratio of the cobalt salt to DMY in step S1 is 1.0-1.2:1.0, preferably 1.1:1.0.
[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:60 mL.
[0010] Preferably, the reflux reaction time in step S2 is 20 to 60 min, preferably 40 min.
[0011] In certain embodiments, cobalt acetate hydrate, cobalt sulfate hydrate or cobalt chloride hydrate is used to carry out complex reaction with DMY, respectively, to prepare the corresponding DMY-Co complex with a yield of 90.35-94.22% and a purity of 99.05-99.35%.
[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-Co complex.
[0013] The above technical scheme of the present invention provides a DMY-Co 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-Co 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-Co complex of the present invention is as follows; Figure 2 The structure diagram of the DMY-Co complex of the present invention; Figure 3 It is the ultraviolet spectrum of DMY and DMY-Co complex of the present invention; Figure 4 It is the DMY infrared spectrum of the present invention; Figure 5 It is the infrared spectrum of the DMY-Co complex of the present invention; Figure 6 This is a high-resolution mass spectrum of the DMY-Co 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, 0.50 g cobalt acetate tetrahydrate (2.0 mmol), and 100 mL ethanol-ammonium acetate buffer solution were added to a three-necked flask in sequence, and the mixture was heated under reflux for 50 min. The obtained yellow-brown precipitate was filtered, washed, and dried to obtain a yellow-brown solid DMY-Co complex with a yield of 90.35% and a purity of 99.05%. Example 2
[0018] 0.64 g (2.0 mmol) DMY, 0.55 g cobalt acetate tetrahydrate (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 30 min. The obtained yellow-brown precipitate was filtered, washed, and dried to obtain a yellow-brown solid DMY-Co complex with a yield of 92.21% and a purity of 99.13%. Example 3
[0019] 0.64 g (2.0 mmol) DMY, 0.52 g cobalt chloride hexahydrate (2.2 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 40 min. The obtained yellow-brown precipitate was filtered, washed, and dried to obtain a yellow-brown solid DMY-Co complex with a yield of 94.22% and a purity of 99.35%. Example 4
[0020] 0.64 g (2.0 mmol) DMY, 0.57 g cobalt chloride hexahydrate (2.4 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 20 min. The obtained yellow-brown precipitate was filtered, washed, and dried to obtain a yellow-brown solid DMY-Co complex with a yield of 93.66% and a purity of 99.20%. Example 5
[0021] 0.64 g (2.0 mmol) DMY, 0.56 g cobalt sulfate heptahydrate (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 40 min. The obtained yellow-brown precipitate was filtered, washed, and dried to obtain a yellow-brown solid DMY-Co complex with a yield of 92.68% and a purity of 99.10%. Example 6
[0022] 0.64 g (2.0 mmol) DMY, 0.62 g cobalt sulfate heptahydrate (2.2 mmol), and 150 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-Co complex with a yield of 92.57% and a purity of 99.18%.
[0023] In certain embodiments, cobalt acetate hydrate and cobalt sulfate hydrate are used instead of cobalt chloride hydrate to carry out complex reaction with DMY. Example 7
[0024] 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 & Coboratory 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-Co complex against porcine Pasteurella multocida were measured to be 1024 μg / mL and 512 μg / mL, respectively.
[0025] The experiment showed that the antibacterial activity of DMY-Co complex was greatly improved compared with DMY, and it is expected to become a new, highly effective and low-toxic antibiotic alternative for the treatment of swine pasteurellosis, which is of great significance. Example 8
[0026] Structural characterization 8-1. Determination of the content of metal element cobalt in DMY-Co complex The theoretical value of the cobalt content in the DMY-Co complex is 14.23%, and the experimental value determined by atomic absorption spectrophotometry is 13.86%. The structure of the complex was preliminarily determined to be one DMY molecule complexed with one cobalt ion molecule.
[0027] 8-2. UV spectroscopy analysis of DMY-Co complex Dissolve DMY and the experimentally prepared DMY-Co 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-Co complex solution is 326.0 nm ( Figure 3 ), the maximum absorption peak red-shifted by 1 nm, and the intensity of the absorption peak decreased significantly, indicating that the structure of DMY changed and DMY formed a complex with Co.
[0028] 8-3. Infrared spectroscopy analysis of DMY-Co complex Take a small amount of DMY and DMY-Co complex solid powder and place it in an agate mortar, add KBr powder, grind it thoroughly, press it into a tablet, and set the parameters of the infrared spectrometer with a scanning range of 400-4000 cm -1 , infrared spectroscopy analysis was performed by Fourier transform infrared spectrometer. Infrared spectra of DMY and DMY-Co complex ( Figure 4 , Figure 5 ) and the results are as follows: The main infrared absorption frequencies and attributions of DMY and DMY-Co complexes are as follows:
[0029] From the above table, we can see that the hydroxyl groups of DMY and DMY-Co complex are at 3392.17 cm -1 、3434.60 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-Co complexes. -1 There is a strong absorption peak at 1639.20 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 1639.20 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 -1There is an absorption peak of benzene ring skeleton at 1164.79 cm -1 and 1016.30cm -1 There is an absorption peak at 640.25cm, 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-Co complex has a peak at 640.25cm -1 A new absorption peak appeared at , indicating that DMY reacted with cobalt ions to form a complex.
[0030] From the UV and IR results, we can see that the UV and IR absorption of the product (DMY-Co complex) have changed significantly compared with the raw material (DMY), indicating the formation of the DMY-Co complex.
[0031] 8-4. High-resolution mass spectrometry analysis of DMY-Co complex In the high-resolution mass spectrum ( Figure 6 ) It can be seen that the molecular weight of the complex is 414.00 and the molecular formula is C 15 H 14 CoO 10 , which is consistent with the complex structure.
[0032] The above technical scheme of the present invention first provides a DMY-Co 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-Co complex is provided; in addition, the method is simple in process, easy to control, and the product has a high yield and purity.
[0033] Comparative Example 1 Weigh 0.5 mmol (0.16 g) of dihydromyricetin DMY, add 25 mL of anhydrous ethanol, heat and stir at 60 °C to dissolve; add 0.5 mmol (0.06 g) of anhydrous sodium acetate, heat and dissolve for 0.5 h; add 1 mmol (0.24 g) of cobalt chloride hexahydrate, stir and reflux for 5 h; cool and filter to obtain a solid; wash it with 70% ethanol 3 times, distilled water 3 times, and then dry it at 60 °C for 24 h. The yield is 74.56% and the purity is 83.35%.
[0034] Comparative Example 2 Add DMY solution (0.02 mol·L -1 ) 2.5 mL, cobalt chloride hexahydrate solution (0.02 mol·L -1) 1.0 mL, NaH2PO4-Na2HPO4 buffer solution with pH = 7.4 3.0 mL, 50% ethanol aqueous solution 3.5 mL, stirred at 26 ° C for 1 h, filtered, washed and dried. The yield was 78.42% and the purity was 86.47%.
[0035] Comparative Example 3 In a three-necked flask, add 0.64 g (2.0 mmol) DMY, add an appropriate amount of 95% ethanol, stir, heat to dissolve, add 55 mL alkaline solution [sodium acetate dissolved in water-ethanol solution (50%, v / v), pH = 8.2], stir for 5 minutes, add 0.39 g (2.2 mmol) cobalt acetate (dissolved in an appropriate amount of 95% ethanol), heat and reflux for 2 hours, cool to room temperature, precipitate, filter, wash with water, and dry. The yield is 81.15% and the purity is 83.52%.
[0036] 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-cobalt complex for treating porcine pasteurellosis, characterized in that The following steps are involved: (1) Preparing a reaction mixture: Add dihydromyricetin, cobalt 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-cobalt ochre-yellow solid complex.
2. The method for preparing a dihydromyricetin-cobalt complex for treating porcine pasteurellosis according to claim 1, characterized in that: The cobalt salt is taken from one or both of cobalt acetate hydrate, cobalt sulfate hydrate or cobalt chloride hydrate.
3. The method for preparing a dihydromyricetin-cobalt complex for treating swine pasteurellosis according to claim 1, characterized in that: The feeding molar ratio of cobalt salt to dihydromyricetin is 1.0-1.2:1.
0.
4. The method for preparing a dihydromyricetin-cobalt complex for treating porcine pasteurellosis according to claim 1, characterized in that: The feeding ratio of dihydromyricetin to ethanol-ammonium acetate buffer solution is 1.0 mmol:50-75 mL.
5. The method for preparing a dihydromyricetin-cobalt complex for treating swine pasteurellosis according to claim 1, characterized in that: The reflux reaction time is 20 to 60 min.