A swertia base bacteriostatic composition, and a preparation method and application thereof

Abstract

The application discloses a sabia-based bacteriostatic composition and a preparation method and application thereof, and belongs to the technical field of bacteriostatic agents. The sabia is used as a main bacteriostatic component, the cross-linking of peptidoglycan of a bacterial cell wall is inhibited, the synthesis of the cell wall is destroyed, the integrity of the bacterial cell wall is destroyed, and the growth and reproduction of Staphylococcus aureus is inhibited. Secondly, the sabia is compounded with leonurine, on the one hand, the damage to the cell wall of Staphylococcus aureus is enhanced, and on the other hand, in the glycolysis process of Staphylococcus aureus, the sabia and leonurine have a significant inhibitory effect on hexokinase, so that the growth and reproduction of Staphylococcus aureus is inhibited by interfering with the metabolic process of bacteria. Then, the damage to the biofilm of Staphylococcus aureus is improved by the assistance of nicotinic acid, and the inhibitory effect on Staphylococcus aureus is further enhanced.

Description

Technical Field

[0001] This invention relates to the field of antibacterial agents, specifically to a swert-based antibacterial composition, its preparation method, and its application. Background Technology

[0002] Staphylococcus aureus, belonging to the genus Staphylococcus, is one of the most frequently isolated Gram-positive bacteria in clinical practice. It is widely distributed in nature and can infect both humans and animals, causing zoonotic diseases. In veterinary clinics, Staphylococcus aureus is one of the most common pathogens causing mastitis in lactating animals. Infectious diseases caused by Staphylococcus aureus seriously endanger human health and the development of animal husbandry. Currently, the treatment of Staphylococcus aureus infections in clinical practice mainly relies on the use of antibiotics. However, the widespread use of antibiotics leads to the frequent emergence of drug-resistant strains, and even super-resistant bacteria, increasing the difficulty of treatment. Biofilms are organized bacterial communities encapsulated by extracellular macromolecules, exhibiting strong resistance to antibiotics and host immune defense mechanisms. Bacteria living in biofilms, compared to planktonic bacteria, show high resistance to antibiotics, leading to chronic infections that are difficult to eradicate and causing serious losses to healthcare, the food industry, and other sectors. Staphylococcus aureus is also one of the bacteria most prone to biofilm formation. Biofilm formation leads to a significant increase in the resistance of Staphylococcus aureus to antibiotics. Since biofilm-producing bacteria are less sensitive to or even resistant to traditional antimicrobial drugs, the exploration of new antibiofilm drugs is increasingly being promoted.

[0003] Traditional Chinese medicine (TCM) also has unique advantages in reducing bacterial resistance. *Swertia spp.*, belonging to the Gentianaceae family and mainly distributed in southwestern my country, contains swertia cyclohexene ethers, triterpenoids, ketones, and flavonoids. It is effective in clearing the liver and gallbladder, reducing fever, and treating jaundice-type hepatitis and viral hepatitis. It also exhibits good antibacterial activity against *Staphylococcus aureus*, *Escherichia coli*, and *Salmonella typhi*. However, the inhibitory activity of *Swertia spp.* alone against bacteria is poor, and the minimum inhibitory concentration (MIC) against multidrug-resistant *Staphylococcus aureus* is relatively high. Summary of the Invention

[0004] This invention provides a swert-based antibacterial composition, its preparation method, and its application. It effectively solves the technical problems of weak inhibitory activity of swert alone against bacteria and high minimum inhibitory concentration against multidrug-resistant Staphylococcus aureus. At the same time, it provides a swert-based antibacterial composition that enhances antibacterial activity against Staphylococcus aureus by disrupting bacterial cell walls and interfering with bacterial metabolic processes, inhibiting the formation of Staphylococcus aureus biofilm, and simultaneously removing mature Staphylococcus aureus biofilm.

[0005] The first objective of this invention is to provide a swertiamarin-based antibacterial composition, which is made from the following raw materials in the indicated mass fractions: 70.5%–78.5% swertiamarin aqueous extract, 15.2%–20.3% leonurine, and 1.0%–10.0% nicotinic acid, totaling 100%.

[0006] In a preferred embodiment, the water extract of *Swertia spp.* has a mass fraction of 74.5%–76.0%, the leonurine has a mass fraction of 15.5%–17.0%, and the nicotinic acid has a mass fraction of 7.0%–9.0%, totaling 100%.

[0007] A second objective of this invention is to provide a method for preparing the above-mentioned swertiamarin-based antibacterial composition, comprising the following steps:

[0008] Aqueous extracts of Swertia were obtained from Swertia using a decoction method.

[0009] Leonurine was extracted from Leonurus japonicus using an alcohol extraction method.

[0010] Nicotinic acid was added to the Swertia japonica water extract under stirring, and the mixture was allowed to stand at 25℃~40℃. Then, leonurine was added and mixed well to obtain a Swertia japonica-based antibacterial composition.

[0011] In a preferred embodiment, the swertiamarin-based antibacterial composition contains 70.5%–78.5% swertiamarin water extract, 15.2%–20.3% leonurine, and 1.0%–10.0% nicotinic acid.

[0012] In a preferred embodiment, the concentration of the Swertia japonica water extract is 20 μg / mL to 30 μg / mL.

[0013] As a preferred embodiment, the preparation method of the leonurine includes the following steps: adding anhydrous ethanol to the pulverized motherwort at a dosage ratio of 1g:10-20mL, reflux extraction at 30℃-40℃, filtering to obtain a filtrate, evaporating the filtrate to remove the solvent, and drying under vacuum conditions of 7.0kPa-8.0kPa at 80℃-100℃ to obtain leonurine.

[0014] As a preferred embodiment, the preparation method of the water extract of *Swertia spp.* includes the following steps: adding water to the crushed *Swertia spp.* at a dosage ratio of 1g:8mL to 12mL, soaking at room temperature for 1 to 2 hours, heating and decocting, filtering to obtain filtrate and residue, adding water to the residue and decocting, repeating the decoction 3 times, combining the filtrates and centrifuging, concentrating, sterilizing and diluting to obtain the final product.

[0015] In a preferred embodiment, the settling time is 2h to 5h.

[0016] A third objective of this invention is to provide the application of the above-mentioned swertiamarin-based antibacterial composition in inhibiting Staphylococcus aureus.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] This invention provides a Swertia-based antibacterial composition, prepared using Swertia water extract and leonurine and nicotinic acid as active ingredients. First, Swertia, as the main antibacterial component, inhibits the cross-linking of peptidoglycan in bacterial cell walls, thereby disrupting cell wall synthesis and damaging cell wall integrity, thus inhibiting the growth and reproduction of Staphylococcus aureus. Second, the combination of Swertia and leonurine enhances the destructive effect on the Staphylococcus aureus cell wall. Furthermore, during Staphylococcus aureus glycolysis, Swertia and leonurine synergistically exhibit significant inhibitory effects on hexokinase, thereby interfering with bacterial metabolism and inhibiting the growth and reproduction of Staphylococcus aureus. Finally, the addition of nicotinic acid further enhances the destructive effect on Staphylococcus aureus biofilm, thus strengthening the inhibitory effect. Attached Figure Description

[0019] Figure 1 This is a comparison chart showing the inhibitory effects of the *Swertia spp.*-based antibacterial compositions of Example 3, Comparative Examples 1 and 2, and the blank control group of Comparative Example 3 on *Staphylococcus aureus*.

[0020] Figure 2 The graph shows a comparison of the inhibition rates of the *Swertia spp.*-based antibacterial compositions of Example 3 and Comparative Examples 1 to 2 against strains 1 and 2, respectively.

[0021] Figure 3 The graph shows a comparison of the clearance rates of strain 1 and strain 2 using the swertiamarin-based antibacterial compositions of Example 3 and Comparative Examples 1 to 2 of the present invention.

[0022] Figure 4 The images show the SEM images of the inhibitory effects of the swertiamarin-based antibacterial compositions of Example 3, Comparative Examples 1 and 2, and the blank control group of Comparative Example 3 on Staphylococcus aureus. In the images, A represents Comparative Example 3, B represents Comparative Example 2, C represents Comparative Example 1, and D represents Example 3.

[0023] Figure 5 The images show the SEM images of the swertiamarin-based antibacterial compositions of Example 3, Comparative Examples 1 and 2, and the blank control group of Comparative Example 3 against Staphylococcus aureus. In the images, a is Comparative Example 3, b is Comparative Example 2, c is Comparative Example 1, and d is Example 3.

[0024] Figure 6 The images show a comparison of the inhibitory effects of the *Swertia spp.*-based antibacterial compositions of Example 3, Comparative Examples 1 and 2, and the blank control group of Comparative Example 3 on *Staphylococcus aureus* biofilm using laser confocal microscopy. Figures A through D show the PI staining results: Figure A is Comparative Example 3, Figure B is Comparative Example 2, Figure C is Comparative Example 1, and Figure D is Example 3. Figures A1 through D1 show the FITC-ConA staining results: Figure A1 is Comparative Example 3, Figure B1 is Comparative Example 2, Figure C1 is Comparative Example 1, and Figure D1 is Example 3.

[0025] Figure 7 The images show a comparison of the swertiamarin-based antibacterial compositions of Example 3, Comparative Examples 1 and 2, and the blank control group of Comparative Example 3 against Staphylococcus aureus biofilm using laser confocal microscopy. Figures a-d show the PI staining results: Figure a represents Comparative Example 3, Figure b represents Comparative Example 2, Figure c represents Comparative Example 1, and Figure d represents Example 3. Figures a1-d1 show the FITC-ConA staining results: Figure a1 represents Comparative Example 3, Figure b1 represents Comparative Example 2, Figure c1 represents Comparative Example 1, and Figure d1 represents Example 3. Detailed Implementation

[0026] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention will be further described below with reference to specific embodiments and accompanying drawings. However, the embodiments described are not intended to limit the invention. Unless otherwise specified, the following test methods and detection methods are conventional methods; unless otherwise specified, the reagents and raw materials are commercially available.

[0027] To address the technical problem that Swertia alone has weak inhibitory activity against bacteria and a high minimum inhibitory concentration against multidrug-resistant Staphylococcus aureus, this invention provides a Swertia-based antibacterial composition, its preparation method, and its application.

[0028] The technical solution of the present invention will be analyzed and described in detail below.

[0029] This invention provides a swertiamarin-based antibacterial composition, which is made from the following raw materials in the indicated mass fractions: 70.5%–78.5% swertiamarin aqueous extract, 15.2%–20.3% leonurine, and 1.0%–10.0% nicotinic acid, totaling 100%.

[0030] In the above technical solution, a swertiamarin-based antibacterial composition is prepared using swertiamarin water extract, leonurine, and nicotinic acid as active ingredients. Swertia can inhibit the cross-linking of peptidoglycan in bacterial cell walls, thereby disrupting cell wall synthesis and damaging the integrity of the bacterial cell wall, thus inhibiting the growth and reproduction of Staphylococcus aureus. The combination of swertiamarin and leonurine enhances the destructive effect on the Staphylococcus aureus cell wall. Furthermore, during the glycolysis process of Staphylococcus aureus, swertiamarin and leonurine synergistically exhibit a significant inhibitory effect on hexokinase, thereby interfering with bacterial metabolism to inhibit the growth and reproduction of Staphylococcus aureus. The addition of nicotinic acid further enhances the destructive effect on the Staphylococcus aureus biofilm, thus strengthening the inhibitory effect.

[0031] To further enhance the antibacterial activity against Staphylococcus aureus, the water extract of *Swertia spp.* has a mass fraction of 74.5%–76.0%, the leonurine has a mass fraction of 15.5%–17.0%, and the nicotinic acid has a mass fraction of 7.0%–9.0%, totaling 100%.

[0032] The present invention also provides a method for preparing the above-mentioned swertiamarin-based antibacterial composition, comprising the following steps:

[0033] Aqueous extracts of Swertia were obtained from Swertia using a decoction method.

[0034] Leonurine was extracted from Leonurus japonicus using an alcohol extraction method.

[0035] Nicotinic acid was added to the Swertia japonica water extract under stirring, and the mixture was allowed to stand at 25℃~40℃ for 2~5 hours. Then, Leonurus japonicus alkaloid was added and mixed well to obtain a Swertia japonica-based antibacterial composition.

[0036] To enhance the inhibitory effect against Staphylococcus aureus, the Swertia japonica-based antibacterial composition contains, by mass fraction, 25.3%–50.5% of the water extract of Swertia japonica, 5.0%–10.6% of the leonurine, and 1.0%–6.0% of the nicotinic acid.

[0037] It should be noted that the concentration of the aqueous extract of Swertia japonica is 20 μg / mL to 30 μg / mL.

[0038] To extract leonurine from motherwort, the preparation method of leonurine includes the following steps: adding anhydrous ethanol to pulverized motherwort at a dosage ratio of 1g:10-20mL, reflux extraction at 30℃-40℃, filtering to obtain filtrate, evaporating the filtrate to remove the solvent, and drying under vacuum conditions of 7.0kPa-8.0kPa at 80℃-100℃ to obtain leonurine.

[0039] To obtain the aqueous extract of Swertia japonica, the preparation method of the aqueous extract of Swertia japonica includes the following steps: adding water to the crushed Swertia japonica at a dosage ratio of 1g:8mL~12mL, soaking at room temperature for 1h~2h, heating and boiling, filtering to obtain filtrate and residue, boiling the residue with water, repeating the boiling 3 times, combining the filtrates and centrifuging, concentrating, sterilizing and diluting to obtain the final product.

[0040] The technical effects of the present invention will be described below through specific embodiments and comparative examples.

[0041] Example 1

[0042] A swert-based antibacterial composition, wherein the swert water extract has a mass fraction of 70.5%, the leonurine has a mass fraction of 20.3%, and the nicotinic acid has a mass fraction of 9.2%, totaling 100%.

[0043] The preparation method of the above-mentioned swert-based antibacterial composition includes the following steps:

[0044] Aqueous extracts of Swertia japonica were obtained by decoction: water was added to the pulverized Swertia japonica at a ratio of 1g:10mL, soaked at room temperature for 2 hours, heated and decocted, filtered, and the filtrate and residue were obtained. The residue was decocted with water, and the decoction was repeated 3 times. The filtrates were combined and centrifuged, concentrated, sterilized and diluted to obtain a Swertia japonica aqueous extract with a concentration of 26μg / mL.

[0045] Leonurine was extracted from Leonurus japonicus by alcohol extraction: 1 g: 15 mL of anhydrous ethanol was added to the pulverized Leonurus japonicus, and the mixture was refluxed at 35 °C for extraction. The mixture was filtered to obtain a filtrate. The filtrate was evaporated to remove the solvent and dried at 100 °C under a vacuum of 8.0 kPa to obtain leonurine.

[0046] Nicotinic acid was added to the Swertia japonica water extract under stirring, and the mixture was allowed to stand at 30°C for 4 hours. Then, Leonurus japonicus alkaloid was added and mixed well to obtain a Swertia japonica-based antibacterial composition.

[0047] Example 2

[0048] A swert-based antibacterial composition, wherein the swert water extract has a mass fraction of 78.5%, the leonurine has a mass fraction of 15.2%, and the nicotinic acid has a mass fraction of 6.3%, totaling 100%.

[0049] The preparation method of the above-mentioned swert-based antibacterial composition includes the following steps:

[0050] Aqueous extracts of Swertia japonica were obtained by decoction: water was added to the pulverized Swertia japonica at a ratio of 1g:10mL, soaked at room temperature for 2 hours, heated and decocted, filtered, and the filtrate and residue were obtained. The residue was decocted with water, and the decoction was repeated 3 times. The filtrates were combined and centrifuged, concentrated, sterilized and diluted to obtain a Swertia japonica aqueous extract with a concentration of 26μg / mL.

[0051] Leonurine was extracted from Leonurus japonicus by alcohol extraction: 1 g: 15 mL of anhydrous ethanol was added to the pulverized Leonurus japonicus, and the mixture was refluxed at 35 °C for extraction. The mixture was filtered to obtain a filtrate. The filtrate was evaporated to remove the solvent and dried at 100 °C under a vacuum of 8.0 kPa to obtain leonurine.

[0052] Nicotinic acid was added to the Swertia japonica water extract under stirring, and the mixture was allowed to stand at 30°C for 4 hours. Then, Leonurus japonicus alkaloid was added and mixed well to obtain a Swertia japonica-based antibacterial composition.

[0053] Example 3

[0054] A swert-based antibacterial composition, wherein the swert water extract has a mass fraction of 75.5%, the leonurine has a mass fraction of 16.5%, and the nicotinic acid has a mass fraction of 8.0%, totaling 100%.

[0055] The preparation method of the above-mentioned swert-based antibacterial composition includes the following steps:

[0056] Aqueous extracts of Swertia japonica were obtained by decoction: water was added to the pulverized Swertia japonica at a ratio of 1g:10mL, soaked at room temperature for 2 hours, heated and decocted, filtered, and the filtrate and residue were obtained. The residue was decocted with water, and the decoction was repeated 3 times. The filtrates were combined and centrifuged, concentrated, sterilized and diluted to obtain a Swertia japonica aqueous extract with a concentration of 26μg / mL.

[0057] Leonurine was extracted from Leonurus japonicus by alcohol extraction: 1 g: 15 mL of anhydrous ethanol was added to the pulverized Leonurus japonicus, and the mixture was refluxed at 35 °C for extraction. The mixture was filtered to obtain a filtrate. The filtrate was evaporated to remove the solvent and dried at 100 °C under a vacuum of 8.0 kPa to obtain leonurine.

[0058] Nicotinic acid was added to the Swertia japonica water extract under stirring, and the mixture was allowed to stand at 30°C for 4 hours. Then, Leonurus japonicus alkaloid was added and mixed well to obtain a Swertia japonica-based antibacterial composition.

[0059] Example 4

[0060] A swert-based antibacterial composition, wherein the swert water extract has a mass fraction of 78.5%, the leonurine has a mass fraction of 20.3%, and the nicotinic acid has a mass fraction of 1.2%, totaling 100%.

[0061] The preparation method of the above-mentioned swert-based antibacterial composition includes the following steps:

[0062] Aqueous extracts of Swertia were obtained from Swertia using a decoction method: water was added to the pulverized Swertia at a ratio of 1g:8mL, the mixture was soaked at room temperature for 1 hour, heated and decocted, filtered, and the filtrate and residue were obtained. The residue was decocted with water, and the decoction was repeated 3 times. The filtrates were combined and centrifuged, concentrated, sterilized and diluted to obtain a Swertia aqueous extract with a concentration of 20μg / mL.

[0063] Leonurine was extracted from Leonurus japonicus using an alcohol extraction method: 1 g: 10 mL of anhydrous ethanol was added to the pulverized Leonurus japonicus, and the mixture was refluxed at 30 °C for extraction. After filtration, the filtrate was evaporated to remove the solvent and dried at 80 °C under a vacuum of 7.5 kPa to obtain leonurine.

[0064] Nicotinic acid was added to the Swertia japonica water extract under stirring, and the mixture was allowed to stand at 40°C for 2 hours. Then, Leonurus japonicus alkaloid was added and mixed well to obtain a Swertia japonica-based antibacterial composition.

[0065] Example 5

[0066] A swert-based antibacterial composition, wherein the swert water extract has a mass fraction of 74.5%, the leonurine has a mass fraction of 16.5%, and the nicotinic acid has a mass fraction of 9.0%, totaling 100%.

[0067] The preparation method of the above-mentioned swert-based antibacterial composition includes the following steps:

[0068] Aqueous extracts of Swertia japonica were obtained by decoction: water was added to the pulverized Swertia japonica at a ratio of 1g:12mL, and the mixture was soaked at room temperature for 1.5h. The mixture was then heated and decocted, filtered, and the filtrate and residue were obtained. The residue was decocted with water, and the decoction was repeated 3 times. The filtrates were combined and centrifuged, concentrated, sterilized, and diluted to obtain a Swertia japonica aqueous extract with a concentration of 30μg / mL.

[0069] Leonurine was extracted from Leonurus japonicus by alcohol extraction: Anhydrous ethanol was added to the pulverized Leonurus japonicus at a ratio of 1g:20mL, and the mixture was refluxed at 40℃ for extraction. The mixture was filtered to obtain a filtrate. The filtrate was evaporated to remove the solvent and dried at 90℃ under a vacuum of 7.0kPa to obtain leonurine.

[0070] Nicotinic acid was added to the Swertia japonica water extract under stirring, and the mixture was allowed to stand at 35°C for 5 hours. Then, Leonurus japonicus alkaloid was added and mixed well to obtain a Swertia japonica-based antibacterial composition.

[0071] Example 6

[0072] A swert-based antibacterial composition, wherein the swert water extract has a mass fraction of 75.0%, the leonurine has a mass fraction of 20.0%, and the nicotinic acid has a mass fraction of 5.0%, totaling 100%.

[0073] The preparation method of the above-mentioned swert-based antibacterial composition includes the following steps:

[0074] Aqueous extracts of Swertia japonica were obtained by decoction: water was added to the pulverized Swertia japonica at a ratio of 1g:10mL, soaked at room temperature for 2 hours, heated and decocted, filtered, and the filtrate and residue were obtained. The residue was decocted with water, and the decoction was repeated 3 times. The filtrates were combined and centrifuged, concentrated, sterilized and diluted to obtain a Swertia japonica aqueous extract with a concentration of 26μg / mL.

[0075] Leonurine was extracted from Leonurus japonicus by alcohol extraction: Anhydrous ethanol was added to the pulverized Leonurus japonicus at a ratio of 1g:12mL, and the mixture was refluxed at 32℃ for extraction. The mixture was filtered to obtain a filtrate. The filtrate was evaporated to remove the solvent and dried at 100℃ under a vacuum of 8.0kPa to obtain leonurine.

[0076] Nicotinic acid was added to the Swertia japonica water extract under stirring, and the mixture was allowed to stand at 36°C for 3 hours. Then, Leonurus japonicus alkaloid was added and mixed well to obtain a Swertia japonica-based antibacterial composition.

[0077] Example 7

[0078] A swert-based antibacterial composition, wherein the swert water extract has a mass fraction of 72.3%, the leonurine has a mass fraction of 18.7%, and the nicotinic acid has a mass fraction of 9.0%, totaling 100%.

[0079] The preparation method of the above-mentioned swert-based antibacterial composition includes the following steps:

[0080] Aqueous extracts of Swertia were obtained from Swertia using a decoction method: water was added to the pulverized Swertia at a ratio of 1g:9mL, the mixture was soaked at room temperature for 1 hour, heated and decocted, filtered, and the filtrate and residue were obtained. The residue was decocted with water, and the decoction was repeated 3 times. The filtrates were combined and centrifuged, concentrated, sterilized and diluted to obtain a Swertia aqueous extract with a concentration of 25μg / mL.

[0081] Leonurine was extracted from Leonurus japonicus by alcohol extraction: Anhydrous ethanol was added to the pulverized Leonurus japonicus at a ratio of 1g:18mL, and the mixture was refluxed at 38℃ for extraction. The mixture was filtered to obtain a filtrate. The filtrate was evaporated to remove the solvent and dried at 85℃ under a vacuum of 7.0kPa to obtain leonurine.

[0082] Nicotinic acid was added to the Swertia japonica water extract under stirring, and the mixture was allowed to stand at 40°C for 3.5 hours. Then, leonurine was added and mixed well to obtain a Swertia japonica-based antibacterial composition.

[0083] Example 8

[0084] A swert-based antibacterial composition, wherein the swert water extract has a mass fraction of 76.0%, the leonurine has a mass fraction of 17.0%, and the nicotinic acid has a mass fraction of 7.0%, totaling 100%.

[0085] The preparation method of the above-mentioned swert-based antibacterial composition includes the following steps:

[0086] Aqueous extracts of Swertia were obtained from Swertia using a decoction method: water was added to the pulverized Swertia at a ratio of 1g:8mL, the mixture was soaked at room temperature for 1 hour, heated and decocted, filtered, and the filtrate and residue were obtained. The residue was decocted with water, and the decoction was repeated 3 times. The filtrates were combined and centrifuged, concentrated, sterilized and diluted to obtain a Swertia aqueous extract with a concentration of 20μg / mL.

[0087] Leonurine was extracted from Leonurus japonicus using an alcohol extraction method: 1 g: 10 mL of anhydrous ethanol was added to the pulverized Leonurus japonicus, and the mixture was refluxed at 30 °C for extraction. After filtration, the filtrate was evaporated to remove the solvent and dried at 95 °C under a vacuum of 7.5 kPa to obtain leonurine.

[0088] Nicotinic acid was added to the Swertia japonica water extract under stirring, and the mixture was allowed to stand at 30°C for 4 hours. Then, Leonurus japonicus alkaloid was added and mixed well to obtain a Swertia japonica-based antibacterial composition.

[0089] To further illustrate the technical effects of the present invention, comparative examples are also provided, as follows:

[0090] Comparative Example 1

[0091] The difference compared to Example 3 is that leonurine is not added.

[0092] A swert-based antibacterial composition, wherein the swert water extract has a mass fraction of 91.5%, the nicotinic acid has a mass fraction of 8.5%, and the total is 100%.

[0093] The preparation method of the above-mentioned swert-based antibacterial composition includes the following steps:

[0094] Aqueous extracts of Swertia japonica were obtained by decoction: water was added to the pulverized Swertia japonica at a ratio of 1g:10mL, soaked at room temperature for 2 hours, heated and decocted, filtered, and the filtrate and residue were obtained. The residue was decocted with water, and the decoction was repeated 3 times. The filtrates were combined and centrifuged, concentrated, sterilized and diluted to obtain a Swertia japonica aqueous extract with a concentration of 26μg / mL.

[0095] Nicotinic acid was added to the Swertia japonica water extract under stirring, and the mixture was allowed to stand at 30°C for 4 hours. Then, Leonurus japonicus alkaloid was added and mixed well to obtain a Swertia japonica-based antibacterial composition.

[0096] Comparative Example 2

[0097] The difference compared to Example 1 is that nicotinic acid is not added.

[0098] A swert-based antibacterial composition, wherein the water extract of swert has a mass fraction of 81.5%, and the leonurine has a mass fraction of 18.5%, totaling 100%.

[0099] The preparation method of the above-mentioned swert-based antibacterial composition includes the following steps:

[0100] Aqueous extracts of Swertia japonica were obtained by decoction: water was added to the pulverized Swertia japonica at a ratio of 1g:10mL, soaked at room temperature for 2 hours, heated and decocted, filtered, and the filtrate and residue were obtained. The residue was decocted with water, and the decoction was repeated 3 times. The filtrates were combined and centrifuged, concentrated, sterilized and diluted to obtain a Swertia japonica aqueous extract with a concentration of 26μg / mL.

[0101] Leonurine was extracted from Leonurus japonicus by alcohol extraction: 1 g: 15 mL of anhydrous ethanol was added to the pulverized Leonurus japonicus, and the mixture was refluxed at 35 °C for extraction. The mixture was filtered to obtain a filtrate. The filtrate was evaporated to remove the solvent and dried at 100 °C under a vacuum of 8.0 kPa to obtain leonurine.

[0102] While stirring, leonurine was added to the aqueous extract of Swertia japonica, mixed well, and allowed to stand at 30°C for 4 hours to obtain a Swertia-based antibacterial composition.

[0103] The properties of the swertiamarin-based antibacterial compositions prepared in Examples 1 to 8 and Comparative Examples 1 to 5 of the present invention were characterized, and the characterization process and results are as follows.

[0104] 1. Source of strain

[0105] The strains No. 1 and No. 2 used in this invention are both standard strains. The strain No. 1 is Staphylococcus aureus ATCC 25923 and the strain No. 2 is Staphylococcus aureus ATCC 29213, which were purchased from Suzhou Qianshe Biotechnology Co., Ltd.

[0106] 2. Main Reagents and Instruments

[0107] Swertia japonica was purchased from Xueyu Pharmacy in Lhasa, Tibet Autonomous Region; MH culture medium was purchased from Qingdao High-tech Park Haibo Biotechnology Co., Ltd.; crystal violet, analytical grade, was purchased from Guangdong Guanghua Technology Co., Ltd.; propidium iodide (PI) and FITC-labeled concanavalin A (FITC-ConA) were purchased from Shanghai Fushen Biotechnology Co., Ltd.; 2.5% glutaraldehyde was purchased from Nanjing Senbeijia Biotechnology Co., Ltd.; DNP-9162 microbial incubator was purchased from Shanghai Jinghong Experimental Equipment Co., Ltd.; TDZ5 benchtop low-speed centrifuge was purchased from Hunan Hexi Instrument Equipment Co., Ltd.; FC microplate reader was purchased from Thermo Fisher Scientific; Nano SEM-450 scanning electron microscope was purchased from Fidacon Pharmaceuticals, Inc.; OLS5000 laser confocal microscope was purchased from Olympus Corporation.

[0108] Effect of 3 Swertia-based antibacterial composition on the growth curve of Staphylococcus aureus

[0109] Add 10 mL of LB liquid culture medium to each sterilized test tube. Set up comparative examples 2, 1, and 3 (swampys-based antibacterial compositions) and a blank control group (comparative example 3). Add 0.1 mL of bacterial suspension to each tube and mix well. OD is measured immediately. 600 OD was then measured every 2 hours. 600 Plot the growth curve, such as Figure 1 As shown.

[0110] 4. Inhibitory and scavenging effects of swert-based antibacterial composition on Staphylococcus aureus biofilm.

[0111] 4.1 Detection using crystal violet staining method

[0112] The bacterial suspension with a turbidity of 0.5 McFarland was diluted 1:100 with LB medium, and an appropriate amount was added to a 96-well plate. An equal volume of the swertiamarin-based antibacterial composition from Comparative Example 2, Comparative Example 1, and Example 3 was then added. The bacterial suspension served as a positive control, and LB medium as a negative control. Each drug was repeated six times. The plates were stained with 1% crystal violet for 5 min, washed three times with PBS buffer, and the OD was measured using a microplate reader. 590 The value was repeated 3 times. The difference between the swertiamarin-based antibacterial composition's scavenging and inhibitory effects on Staphylococcus aureus biofilms lies in the fact that after biofilm culture, the biofilm was washed 3 times with PBS buffer, air-dried, and then the swertiamarin-based antibacterial composition was added. The inhibitory effect was as follows: Figure 2 As shown, the cleaning effect is as follows Figure 3 As shown.

[0113]

[0114] 4.2 Scanning electron microscopy observation

[0115] An appropriate amount of the 4:1 diluted bacterial suspension was added to a 24-well plate with sterile coverslips for incubation. Then, equal volumes of the swertiamarin-based antibacterial compositions from Comparative Example 2, Comparative Example 1, and Example 3 were added sequentially. A blank control group for Comparative Example 3 was also included. After incubation at 37°C for 48 hours, the plates were removed, the liquid in each well was discarded, the coverslips were washed three times with PBS, and an appropriate amount of 2.5% glutaraldehyde was added. The plates were fixed overnight at 4°C. The fixative was discarded, and the plates were washed three times with PBS. The plates were then dehydrated for 10 minutes each with 30%, 50%, 70%, 80%, 90%, and 100% ethanol solutions. The coverslips were removed, subjected to critical point drying and gold plating, and observed under a scanning electron microscope. Figure 4 and Figure 5 As shown. The difference between the scavenging and inhibitory effects of Swertia japonica on Staphylococcus aureus biofilm lies in the addition of a Swertia japonica-based antibacterial composition after the biofilm culture is completed.

[0116] 4.3 Observation using laser confocal microscopy

[0117] Biofilms were cultured in 24-well plates. A bacterial suspension with a 0.5 McFarland turbidity was diluted 1:100, and an appropriate amount was added to the 24-well plate. Equal volumes of the swertiamarin-based antibacterial compositions from Comparative Example 2, Comparative Example 1, and Example 3 were then added sequentially. A blank control was prepared by adding only the bacterial suspension without the drug. After incubation at 37°C for 48 hours, the plates were removed, the liquid in the wells was discarded, and the coverslips were gently washed three times with sterile PBS buffer. 2 mL of 2.5% glutaraldehyde solution was added to each well for fixation at 4°C for 1 hour. After incubation, the fixative was removed, and the plates were washed three times with PBS. The plates were stained with FITC-ConA and PI, and observed using a laser confocal microscope. Figure 6 and Figure 7 As shown. The difference between the scavenging and inhibitory effects of Swertia japonica on Staphylococcus aureus biofilm lies in the addition of a Swertia japonica-based antibacterial composition after the biofilm culture is completed.

[0118] 5. Data Statistics

[0119] Data were statistically analyzed using SPSS 22.0 software, and results are expressed as mean ± standard deviation. A t-test was used for significance analysis, with P < 0.05 considered statistically significant.

[0120] The effect of the *Swertia spp.*-based antibacterial compositions from Comparative Example 2, Comparative Example 1, and Example 3 on the growth of isolate No. 1 was determined, and the results are as follows: Figure 1 As shown. By Figure 1 It can be seen that the swampyr-based antibacterial compositions of Comparative Example 2, Comparative Example 1 and Example 3 can all inhibit the growth of Staphylococcus aureus. In particular, under the action of the swampyr-based antibacterial composition of Example 3, the growth of Staphylococcus aureus is completely inhibited.

[0121] The inhibitory and scavenging effects of the swertiamarin-based antibacterial compositions of Comparative Examples 2, 1, and 3 on the biofilms of two strains of Staphylococcus aureus with strong biofilm-producing capabilities were determined using the crystal violet staining method. The results are shown in [Figure Number]. Figure 2 and Figure 3 Under the action of the swertiamarin-based antibacterial compositions of Comparative Examples 2, 1, and 3, the formation of biofilms from two strains of Staphylococcus aureus with strong biofilm-producing capacity was inhibited. After the biofilms matured, they could be removed. For strain 1, the swertiamarin-based antibacterial compositions of Examples 3 and 1 had a removal effect on the mature biofilm, while the swertiamarin-based antibacterial composition of Comparative Example 2 did not. Strain 2 could remove the mature Staphylococcus aureus biofilm under the action of the swertiamarin-based antibacterial compositions of Comparative Examples 2, 1, and 3.

[0122] The effects of the swertiamarin-based antibacterial compositions of Comparative Example 2, Comparative Example 1, and Example 3 on biofilm morphology were observed using scanning electron microscopy. The results are as follows: Figure 4 As shown. By Figure 4 As shown in Figures A through D, the blank control group formed a multi-layered network-like biofilm, with bacteria intertwined or encapsulated within it, exhibiting relatively intact bacterial morphology. After treatment with the Swertia-based antibacterial compositions of Comparative Examples 2, 1, and 3, the bacterial biofilms became significantly looser, their structural integrity was disrupted, a small number of colonies aggregated, and the bacterial count decreased, with individual bacteria scattered and significantly reduced in number. This indicates that the Swertia-based antibacterial composition has an inhibitory effect on the biofilm of Staphylococcus aureus, which has a strong biofilm-producing capacity. Similarly, regarding the biofilm-clearing effect of the Swertia-based antibacterial composition, such as... Figure 5 Figures a through d show similar trends in bacterial changes, except that the number of colonies is relatively higher compared to the inhibition group in the same group, indicating that the Swertia-based antibacterial composition can also show a clearing effect on mature biofilms.

[0123] The effects of different concentrations of *Swertia spp.* on *Staphylococcus aureus* biofilm were observed using laser confocal microscopy. The results of its inhibitory and scavenging effects are as follows: Figure 6 and Figure 7As shown in the figure, red fluorescence represents the results of PI staining, indicating dead bacteria; green fluorescence represents the results of FITC-ConA staining, indicating the distribution of the polysaccharide matrix. After treatment with the swertiamarin-based antibacterial compositions of Comparative Example 2, Comparative Example 1, and Example 3, the area of ​​green fluorescence decreased continuously in the order of Comparative Example 2-Comparative Example 1-Example 3, indicating a decrease in polysaccharide production, while the area of ​​red fluorescence increased continuously in the order of Comparative Example 2-Comparative Example 1-Example 3, indicating a continuous increase in the number of dead bacteria. The above results indicate that the swertiamarin-based antibacterial compositions of Comparative Example 2, Comparative Example 1, and Example 3 can effectively inhibit the formation of Staphylococcus aureus biofilm and have a certain removal effect on mature biofilms. Among them, the swertiamarin-based antibacterial composition of Example 3, which contains swertiamarin aqueous extract, leonurine, and nicotinic acid, has the best inhibitory and removal effect on Staphylococcus aureus. The effect of Comparative Example 1, which contains swertiamarin aqueous extract and leonurine, is second best, and the effect of Comparative Example 2, which contains swertiamarin aqueous extract and nicotinic acid, is the worst.

[0124] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A swert-based antibacterial composition, characterized in that, The Swertia-based antibacterial composition is made from the following raw materials in the indicated mass fractions: 70.5%~78.5% Swertia water extract, 15.2%~20.3% Leonurus tinctoria alkaloid, 1.0%~10.0% nicotinic acid, totaling 100%.

2. The swertiamarin-based antibacterial composition according to claim 1, characterized in that, The water extract of *Swertia zedoaria* has a mass fraction of 74.5% to 76.0%, the leonurine has a mass fraction of 15.5% to 17.0%, and the nicotinic acid has a mass fraction of 7.0% to 9.0%, totaling 100%.

3. A method for preparing the swertiamarin-based antibacterial composition according to claim 1, characterized in that, Includes the following steps: Aqueous extracts of Swertia were obtained from Swertia using a decoction method. Leonurine was extracted from Leonurus japonicus using an alcohol extraction method. Nicotinic acid was added to the Swertia japonica water extract under stirring, and the mixture was allowed to stand at 25°C to 40°C. Then, Leonurus japonicus alkaloid was added and mixed well to obtain a Swertia japonica-based antibacterial composition.

4. The preparation method according to claim 3, characterized in that, In the Swertia-based antibacterial composition, the water extract of Swertia has a mass fraction of 70.5% to 78.5%, the leonurine has a mass fraction of 15.2% to 20.3%, and the nicotinic acid has a mass fraction of 1.0% to 10.0%.

5. The preparation method according to claim 4, characterized in that, The concentration of the aqueous extract of *Swertia zebrina* is 20 μg / mL to 30 μg / mL.

6. The preparation method according to claim 3, characterized in that, The preparation method of the leonurine includes the following steps: according to the dosage ratio of 1g:10~20mL, anhydrous ethanol is added to the pulverized motherwort, and the mixture is extracted by reflux at 30℃~40℃, filtered, and the filtrate is obtained. The solvent is removed by evaporation of the filtrate, and the mixture is dried under vacuum conditions of 7.0kPa~8.0kPa at 80℃~100℃ to obtain leonurine.

7. The preparation method according to claim 3, characterized in that, The preparation method of the water extract of Swertia japonica includes the following steps: add water to the crushed Swertia japonica according to the dosage ratio of 1g:8mL~12mL, soak at room temperature for 1h~2h, heat and decoct, filter to obtain filtrate and filter residue, add water to the filter residue and decoct, repeat the decoction 3 times, combine the filtrates and centrifuge, concentrate, sterilize and dilute to obtain the final product.

8. The preparation method according to claim 3, characterized in that, The settling time is 2 to 5 hours.

9. The use of the swertiamarin-based antibacterial composition according to claim 1 or 2 in the preparation of a product inhibiting Staphylococcus aureus.

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