A plant-based medicine for inhibiting brucella and a preparation method thereof

Abstract

The application discloses a kind of plant-based brucella inhibiting medicine and preparation method thereof, the method comprises the following steps: step one: prepare plant raw materials chebula 2.5 parts, white grass 1 part, madder 1 part, black cloud incense 1 part according to the following proportion, the above-mentioned plant raw materials are crushed and mixed uniformly;Step two: solvent is added to the mixed powder, and boiled for 1.5~2.5h;Step three: the mixed solution of step two is filtered;Step four: heat to make solvent volatilize clean, obtain extract;Step five: water is added to extract, and freeze;Step six: extract the medicine obtained after freeze-drying machine freeze-drying 20~30h is prepared based on plant raw materials C a H b N c O d It has significant effect of inhibiting brucella, and has better safety compared with traditional antibiotics, and opens a new path for the treatment of brucella.

Description

Technical Field

[0001] This invention belongs to the field of Brucella inhibition technology, and specifically relates to a plant-based drug for inhibiting Brucella and its preparation method. Background Technology

[0002] Brucella is a Gram-negative, non-motile bacterium, non-capsulated (except for the smooth-skinned variety which has microcapsules), catalase- and oxidase-positive, an absolute aerobe, capable of reducing nitrates, and an intracellular parasite that can survive in many types of livestock. Brucella causes brucellosis, a zoonotic, chronic infectious disease with significant harm. In my country, the main sources of infection are cattle, sheep, and pigs, with *Brucella melitensis* being the most transmissible, pathogenic, and harmful to humans. Brucellosis primarily damages the reproductive system and joints of both humans and animals, posing a significant threat to livestock development and human health.

[0003] Under mild conditions, Brucella can survive for weeks to months in fur, water, and dry soil. However, it is not very tolerant of high temperature, high humidity, and light: it can be killed in 7 to 9 minutes under dry heat at 100°C, and only about 6 minutes under humid heat at 80°C. It can not survive for more than 4 hours under direct sunlight, but high-temperature sterilization has limitations for animals and humans.

[0004] Currently, the treatment of brucellosis infection typically involves drugs such as doxycycline, rifampin, streptomycin, azithromycin, and levofloxacin. Doxycycline inhibits brucellosis protein synthesis, thus exerting an antibacterial effect; however, liver function monitoring is necessary during its use. Rifampin has broad-spectrum antibacterial activity and is effective against various Gram-positive and Gram-negative bacteria; however, allergic reactions should be monitored when taking rifampin, especially for first-time users. Streptomycin kills bacteria by interfering with bacterial protein synthesis, but should be used with caution in patients with impaired renal function. Azithromycin is a macrolide antibiotic with strong antibacterial activity against Gram-positive bacteria, but should be used with caution in breastfeeding women, pregnant women, and those with impaired liver function. Levofloxacin is a quinolone antibiotic that effectively kills brucellosis, but caution is needed when using it in breastfeeding women. Therefore, antibiotic treatment requires close monitoring and special consideration, and drug resistance often develops in the later stages of treatment.

[0005] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0006] The purpose of this invention is to provide a plant-based drug for inhibiting Brucella and a method for preparing the same, thereby overcoming the deficiencies in the prior art.

[0007] To achieve the above objectives, the present invention provides a plant-based drug for inhibiting Brucella, wherein the main component of the drug is C. a H b N c O d In the main components of the drug, a=14~35, b=6~49, c=0~4, and d=1~14.

[0008] Furthermore, preferably, the oral bioavailability of the drug is ≥30%, and the drug-likeness is ≥0.18.

[0009] This invention also provides a method for preparing a plant-based drug for inhibiting Brucella, comprising the following steps: Step 1: Prepare the following plant materials in the proportions: 2.5 parts Terminalia chebula, 1 part Potentilla chinensis, 1 part Rubia cordifolia, and 1 part Black clover. Crush the above plant materials and mix them evenly. Step 2: Add solvent to the mixed powder and boil for 1.5~2.5 hours; Step 3: Filter the mixed solution from Step 2; Step 4: Heating to evaporate the solvent completely, yielding the extract; Step 5: Add water to the extract and freeze; Step 6: The extracted drug is obtained by freeze-drying for 20-30 hours using a freeze dryer.

[0010] Furthermore, preferably, the solvent in step two is methanol, ethanol, ethyl acetate, or water.

[0011] Furthermore, as a preferred embodiment, the weight ratio of the mixed powder to the solvent in step two is 1:5~10.

[0012] Furthermore, preferably, the extract obtained in step four is C. a H b N c O d The values ​​are a=14~35, b=6~49, c=0~4, and d=1~14.

[0013] Furthermore, as a preferred embodiment, the freezing temperature in step five is -100℃ to -60℃.

[0014] Compared with the prior art, the present invention has the following beneficial effects: This invention relates to a drug C derived from plant-based raw materials. a H b Nc O d It has a significant inhibitory effect on Brucella and has better safety compared with traditional antibiotics, opening up a new path for the treatment of Brucella. Attached Figure Description

[0015] Figure 1 This is a diagram illustrating the antibacterial effect of Example 1 of the present invention; Figure 2 This is a diagram illustrating the antibacterial effect of Example 2 of the present invention; Figure 3 This is a diagram illustrating the antibacterial effect of Example 3 of the present invention; Figure 4 This is a diagram illustrating the antibacterial effect of Example 4 of the present invention. Detailed Implementation

[0016] The specific embodiments of the present invention will be described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments.

[0017] The following provides a brief overview of one or more aspects to offer a basic understanding of them. This overview is not an exhaustive summary of all conceived aspects, nor is it intended to identify key or decisive elements of all aspects, nor to define the scope of any or all aspects. Its sole purpose is to present some concepts of one or more aspects in a simplified form to prepare for the more detailed descriptions that follow.

[0018] A method for preparing a plant-based drug for inhibiting Brucella includes the following steps: Step 1: Weigh out 250g of Terminalia chebula, 100g of Potentilla chinensis, 100g of Rubia cordifolia, and 100g of Aquilaria sinensis. Crush the above-mentioned plant materials and mix them evenly. Step 2: Pour 5g of the mixed powder into a round-bottom flask, add 50mL of methanol solution to the round-bottom flask, and boil for 2 hours using an electric heater. Step 3: Filter the mixed solution from Step 2 using filter paper; Step 4: Heating to 65℃ allows the methanol solution to evaporate completely, yielding extract C. a H b N c O d Where a = 14~35, b = 6~49, c = 0~4, d = 1~14; Step 5: Add 1mL of water to the extract and freeze it in a -80℃ freezer; Step 6: The extracted drug is obtained by freeze-drying for 24 hours using a freeze dryer.

[0019] A method for preparing a plant-based drug for inhibiting Brucella includes the following steps: Step 1: Weigh out 250g of Terminalia chebula, 100g of Potentilla chinensis, 100g of Rubia cordifolia, and 100g of Aquilaria sinensis. Crush the above-mentioned plant materials and mix them evenly. Step 2: Pour 5g of the mixed powder into a round-bottom flask, add 50mL of anhydrous ethanol solution to the round-bottom flask, and boil for 2 hours using an electric heater. Step 3: Filter the mixed solution from Step 2 using filter paper; Step 4: Heating at 70℃ allows the anhydrous ethanol solution to evaporate completely, yielding extract C. a H b N c O d Where a = 14~35, b = 6~49, c = 0~4, d = 1~14; Step 5: Add 1mL of water to the extract and freeze it in a -80℃ freezer; Step 6: The extracted drug is obtained by freeze-drying for 24 hours using a freeze dryer.

[0020] A method for preparing a plant-based drug for inhibiting Brucella includes the following steps: Step 1: Weigh out 250g of Terminalia chebula, 100g of Potentilla chinensis, 100g of Rubia cordifolia, and 100g of Aquilaria sinensis. Crush the above-mentioned plant materials and mix them evenly. Step 2: Pour 5g of the mixed powder into a round-bottom flask, add 50mL of ethyl acetate solution to the round-bottom flask, and boil for 2 hours using an electric heater. Step 3: Filter the mixed solution from Step 2 using filter paper; Step 4: Heating at 75℃ allows the ethyl acetate solution to evaporate completely, yielding extract C. a H b N c O d Where a = 14~35, b = 6~49, c = 0~4, d = 1~14; Step 5: Add 1mL of water to the extract and freeze it in a -80℃ freezer; Step 6: The extracted drug is obtained by freeze-drying for 24 hours using a freeze dryer.

[0021] A method for preparing a plant-based drug for inhibiting Brucella includes the following steps: Step 1: Weigh out 250g of Terminalia chebula, 100g of Potentilla chinensis, 100g of Rubia cordifolia, and 100g of Aquilaria sinensis. Crush the above-mentioned plant materials and mix them evenly. Step 2: Pour 5g of the mixed powder into a round-bottom flask, add 50mL of deionized water to the round-bottom flask, and boil for 2 hours using an electric heater. Step 3: Filter the mixed solution from Step 2 using filter paper; Step 4: Heat to 100℃ to evaporate all water, obtaining extract C. a H b N c O d Where a = 14~35, b = 6~49, c = 0~4, d = 1~14; Step 5: Add 1mL of water to the extract and freeze it in a -80℃ freezer; Step 6: The extracted drug is obtained by freeze-drying for 24 hours using a freeze dryer.

[0022] Antibacterial test: The drugs in Examples 1-3 were dissolved in DMSO as a solvent, and the drug in Example 4 was dissolved in sterile distilled water as a solvent, and each was prepared into a drug solution with a concentration of 100 mg / mL. The concentration of Brucella cells was adjusted to 0.5 McFarland turbidity using a bacterial turbidimeter, and the bacterial suspension was then diluted 1:100 with Brucella broth medium for later use. The drug was diluted to 32 mg / mL with Brucella broth. 200 μL of the drug diluted to 32 mg / mL was transferred to a 96-well plate. 100 μL of the drug was then taken from the 96-well plate, and another 100 μL of Brucella broth was transferred to the 96-well plate to dilute the drug to 16 mg / mL. Repeat step (3) to prepare drugs at concentrations of 16 mg / mL, 8 mg / mL, 4 mg / mL, 2 mg / mL, 1 mg / mL, and 0.5 mg / mL; prepare at least two groups for each concentration for testing; Add 100uL of bacterial suspension from step (2) to the drug in step (4); The drug containing the bacterial suspension was placed in a CO2 incubator and incubated for 48 hours. The antibacterial effect was then tested, and the results are as follows: Figure 1-4 As shown.

[0023] Figure 1 The horizontal axis represents different concentrations of antibiotics and methanol-extracted drugs, with — indicating no addition. The vertical axis represents bacterial growth. * indicates significance (p), **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05. Specifically, **** p represents the values ​​with and without antibiotics (10 μg / mL), * p represents the values ​​with and without drug (2 mg / mL), *** p represents the values ​​with and without drug (4 mg / mL), and ** p represents the values ​​with and without drug (8 mg / mL). *p < 0.05 indicates a statistically significant difference.

[0024] Figure 2 The horizontal axis represents different concentrations of antibiotics and ethanol-extracted drugs, with — indicating no addition. The vertical axis represents the growth of bacteria. In the figure, **** p represents the value with no antibiotics added and with 10 ug / mL antibiotics added, * p represents the value with no drug added and with 2 mg / mL or 8 mg / mL drug added, and *** p represents the value with no drug added and with 4 mg / mL drug added. Figure 3 The horizontal axis represents different concentrations of antibiotics and drugs extracted with ethyl acetate, with — indicating no addition. The vertical axis represents the growth of the bacteria. In the figure, **** p represents the value with no antibiotics added and with 10 ug / mL antibiotics added, ** p represents the value with no drug added and with 4 mg / mL drug added, and *** p represents the value with no drug added and with 8 mg / mL drug added. Figure 4 The horizontal axis represents different concentrations of antibiotics and water-extracted drugs, with — indicating no addition. The vertical axis represents the growth of bacteria. In the figure, *** p represents the value with no antibiotics added and with 10 ug / mL antibiotics added, and ** p represents the value with no drug added and with 8 mg / mL drug added.

[0025] The test results show that the drug obtained by this invention has a good inhibitory effect on Brucella. The drug extracted with methanol or ethanol has the best antibacterial effect at a concentration of 4 mg / mL, while the drug extracted with ethyl acetate or water has the best antibacterial effect at a concentration of 8 mg / mL.

[0026] In addition, the oral bioavailability and drug-likeness of the extract were tested. The oral bioavailability was ≥30% and the drug-likeness was ≥0.18, which means it can be used as a drug to inhibit Brucella. Compared with traditional antibiotics, it has better safety and opens up a new path for the treatment of Brucella.

[0027] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A plant-based drug for inhibiting Brucella, characterized in that: The preparation method of the drug includes the following steps: Step 1: Prepare the following plant materials in the proportions: 2.5 parts Terminalia chebula, 1 part Potentilla chinensis, 1 part Rubia cordifolia, and 1 part Black clover. Crush the above plant materials and mix them evenly. Step 2: Add solvent to the mixed powder and boil for 1.5~2.5 hours; Step 3: Filter the mixed solution from Step 2; Step 4: Heating to evaporate the solvent completely, yielding the extract; Step 5: Add water to the extract and freeze; Step 6: The extracted drug is obtained by freeze-drying in a freeze dryer for 20-30 hours; The solvent in step two is methanol, ethanol, ethyl acetate, or water.

2. The plant-based drug for inhibiting Brucella according to claim 1, characterized in that: In step two, the weight ratio of the mixed powder to the solvent is 1:5~10.

3. The plant-based drug for inhibiting Brucella according to claim 1, characterized in that: The freezing temperature in step five is -100℃ to -60℃.

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