Preparation method of dihydromyricetin-lanthanum complex for treating swine pasteurellosis

Abstract

The invention discloses a preparation method of a dihydromyricetin-lanthanum complex for treating swine pasteurellosis. 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 lanthanum salt solution to the dihydromyricetin is 1.1: 3.0; the optimal feeding ratio of the dihydromyricetin to the ethanol-ammonium acetate buffer solution is 1.0 mmol: 60 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 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 an antibiotic substitute therapeutic drug for treating 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-lanthanum complex for treating swine pasteurellosis. Background Art

[0002] Pasteurellosis is a common disease in pig farming, which can cause 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) has a variety of pharmacological effects such as antibacterial, anti-inflammatory and immune enhancement. DMY molecules can be used as good chelating ligands for metal ions, and the biological activity of the complexes formed is often better than DMY and has a stronger antibacterial effect. Dihydromyricelin complexes have broad prospects for development and application. In the process of livestock and poultry production, trace elements have an important impact on the health of livestock and poultry. Lanthanum has some antibacterial activity. Lanthanum has shown broad application prospects in the field of clinical medicine, especially in combination with ligands and other bioactive substances. After lanthanide metals form complexes with ligands, they have good lipid solubility and cell penetrability, and can kill many bacteria. It is predicted that the complexation of dihydromyricetin with lanthanum will have a good application effect in veterinary clinics. The research on dihydromyricetin-lanthanum complex (DMY-La) will become a hot topic, but the preparation process of DMY-La needs to be further improved. The synthesis yield reported by Wang Xiaofeng (Fine Chemical Intermediates, 2017) is 63.59%, and there is no report on its resistance to porcine Pasteurella multocida.

[0004] According to the systematic study of DMY-La, the present invention prepares a DMY-La 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 method for preparing a DMY-La complex for treating porcine pasteurellosis. The prepared DMY-La complex product has high yield and high purity and is suitable for large-scale production. 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, lanthanum 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 yellow-brown precipitate is filtered, washed, and dried to obtain a yellow-brown solid DMY-La complex.

[0007] Preferably, the lanthanum salt in S1 is selected from one or both of lanthanum acetate hydrate, lanthanum sulfate hydrate or lanthanum chloride hydrate, preferably lanthanum chloride heptahydrate.

[0008] Preferably, the molar ratio of lanthanum salt to DMY in step S1 is 1.0-1.2:3.0, preferably 1.1:3.0.

[0009] Preferably, in step S1, the feed ratio of DMY to ethanol-ammonium acetate buffer solution is 3.0 mmol:150-220 mL, preferably 1.0 mmol:60 mL.

[0010] Preferably, the reflux reaction time in step S2 is 20 to 60 min, preferably 30 min.

[0011] In certain embodiments, lanthanum acetate hydrate, lanthanum sulfate hydrate or lanthanum chloride hydrate is used to carry out complex reaction with DMY to prepare the corresponding DMY-La complex with a yield of 90.35-94.22% and a purity of 99.05-99.35%.

[0012] 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-La complex.

[0013] The above technical scheme of the present invention provides a DMY-La 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-La 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 This is a process flow chart for preparing the DMY-La complex of the present invention; Figure 2 is the structural diagram of the DMY-La complex of the present invention; Figure 3 It is the ultraviolet spectrum of DMY and DMY-La complex of the present invention; Figure 4It is the DMY infrared spectrum of the present invention; Figure 5 This is the infrared spectrum of the DMY-La complex of the present invention; Figure 6 This is a high-resolution mass spectrum of the DMY-La 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] 0.96 g (3.0 mmol) DMY, 0.37 g lanthanum chloride heptahydrate (1.0 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 40 min. The obtained yellow-brown precipitate was filtered, washed, and dried to obtain a yellow-brown solid DMY-La complex with a yield of 90.35% and a purity of 99.05%. Example 2

[0018] 0.96 g (3.0 mmol) DMY, 0.37 g lanthanum acetate pentahydrate (1.1 mmol), and 160 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-La complex with a yield of 92.21% and a purity of 99.13%. Example 3

[0019] 0.96 g (3.0 mmol) DMY, 0.45 g lanthanum chloride heptahydrate (1.2 mmol), and 200 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-La complex with a yield of 93.66% and a purity of 99.20%. Example 4

[0020] 0.96 g (3.0 mmol) DMY, 0.41 g lanthanum chloride heptahydrate (1.1 mmol), and 180 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-La complex with a yield of 94.22% and a purity of 99.35%. Example 5

[0021] 0.96 g (3.0 mmol) DMY, 0.78 g lanthanum sulfate octahydrate (1.1 mmol), and 220 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-La complex with a yield of 92.57% and a purity of 99.18%.

[0022] In certain embodiments, lanthanum acetate hydrate and lanthanum sulfate hydrate are used instead of lanthanum chloride hydrate 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 (MICs) of DMY and DMY-La complex against porcine Pasteurella multocida were measured to be 1024 μg / mL and 512 μg / mL, respectively.

[0024] The experiment showed that the antibacterial activity of DMY-La 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 7

[0025] Structural characterization 7-1. Determination of the content of metallic element lanthanum in DMY-La complex The mass fraction of lanthanum was determined by EDTA titration method, and the experimental value was 11.81%. Based on the content of lanthanum ions in the complex, the structure of the complex was preliminarily determined to be three molecules of DMY complexed with one molecule of lanthanum ion.

[0026] 7-2. UV spectroscopy analysis of DMY-La complex Dissolve DMY and the experimentally prepared DMY-La 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-La complex solution is 325.0 nm ( Figure 3), the 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 La.

[0027] 7-3. Infrared spectroscopy analysis of DMY-La complex Take a small amount of DMY and DMY-La 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 ( Figure 4 , Figure 5 ). The main infrared absorption frequencies and attributions of DMY and DMY-La complexes are as follows:

[0028] From the above table, we can see that the hydroxyl groups of DMY and DMY-La complex are at 3392.17 cm -1 、3448.10 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-La complex. -1 There is a strong absorption peak at 4, 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 , 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 、1417.42 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 646.04 cm-1, 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-La complex has a peak at 646.04 cm-1 compared with DMY. -1 A new absorption peak appeared at , indicating that DMY interacted with lanthanum ions to form a complex.

[0029] From the UV and IR results, we can see that the UV and IR absorption of the product (DMY-La complex) have changed significantly compared with the raw material (DMY), indicating the formation of the DMY-La complex.

[0030] 7-4. High-resolution mass spectrometry analysis of DMY-La complex High resolution mass spectrometry ( Figure 6 ), m / e is 1169.09, and the molecular formula is C 45 H 40 LaO 28 , which is consistent with the complex structure.

[0031] The above technical scheme of the present invention first provides a DMY-La 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-La 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 Accurately weigh 0.37 g (1.0 mmol) LaCl 3 •7H 2 O was dissolved in 20 mL of ethanol; 0.96 g (3.0 mmol) of DMY was weighed and dissolved in 60 mL of ethanol and then slowly added dropwise to the previous solution, and its pH was adjusted to 5.4 with sodium acetate. The reaction was continued for 3 h, and the solution was allowed to stand at room temperature for 24 h. The solution was filtered, washed, and dried. The yield was 64.82% and the purity was 85.71%.

[0033] Comparative Example 2 Accurately weigh 0.41 g (1.1 mmol) LaCl 3 •7H 2 O was dissolved in 20 mL of ethanol; 0.96 g (3.0 mmol) of DMY was weighed and dissolved in 120 mL of ethanol and then slowly added dropwise to the previous solution, and its pH was adjusted to 5.4 with sodium acetate. The solution was heated and refluxed for 30 min, and allowed to stand at room temperature for 24 h. The solution was filtered, washed, and dried. The yield was 82.57% and the purity was 88.63%.

[0034] 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-lanthanum complex for treating swine pasteurellosis, Features The following steps are involved: (1) Preparing a reaction mixture: adding dihydromyricetin, lanthanum 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-lanthanum yellow-brown solid complex.

2. A method for preparing a dihydromyricetin-lanthanum complex for treating swine pasteurellosis according to claim 1, Features: The lanthanum salt is selected from one or both of lanthanum acetate hydrate, lanthanum sulfate hydrate and lanthanum chloride hydrate.

3. A method for preparing a dihydromyricetin-lanthanum complex for treating swine pasteurellosis according to claim 1, Features: The molar ratio of lanthanum salt to dihydromyricetin is 1.0-1.2:3.

0.

4. A method for preparing a dihydromyricetin-lanthanum complex for treating swine pasteurellosis according to claim 1, Features: The feeding ratio of dihydromyricetin to ethanol-ammonium acetate buffer solution is 3.0 mmol:150-220 mL.

5. The method for preparing a dihydromyricetin-lanthanum complex for treating swine pasteurellosis according to claim 1, Features: The reflux reaction time is 20 to 60 min.

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