Swertia-based antibacterial composition as well as preparation method and application thereof

Abstract

The invention discloses a swertia-based bacteriostatic composition and a preparation method and application thereof, and belongs to the technical field of bacteriostatic agents. Swertia is used as a main bacteriostatic component, and cross-linking of peptidoglycan of bacterial cell walls is inhibited, so that synthesis of the cell walls is destroyed, the integrity of the bacterial cell walls is destroyed, and the antibacterial effect is improved. Therefore, the growth and reproduction of the staphylococcus aureus are inhibited. Secondly, the false Chinese swertia herb and the leonurine are compounded, so that on one hand, the destructive effect on cell walls of the staphylococcus aureus is enhanced, and on the other hand, in the glycolysis process of the staphylococcus aureus, the false Chinese swertia herb and the leonurine synergistically have a remarkable inhibition effect on hexokinase; therefore, the effect of inhibiting the growth and reproduction of the staphylococcus aureus is achieved by interfering the metabolic process of bacteria. And the destructive effect on a staphylococcus aureus biological membrane is improved through the assistance of nicotinic acid, so that the inhibition effect on staphylococcus aureus is enhanced.

Description

Technical Field

[0001] The present invention relates to the technical field of bacteriostatic agents, and particularly relates to a swertia-based bacteriostatic composition, a preparation method thereof, and an application thereof. Background Art

[0002] Staphylococcus aureus belongs to the genus Staphylococcus and is one of the Gram-positive bacteria with the highest isolation rate clinically. Staphylococcus aureus is widely distributed in nature and can infect both humans and animals, causing zoonotic diseases. In veterinary clinics, Staphylococcus aureus is one of the main pathogenic bacteria causing mastitis in lactating animals. Infectious diseases caused by Staphylococcus aureus seriously endanger human health and the development of the livestock industry. Currently, for the treatment of Staphylococcus aureus infections, the clinical application of antibacterial drugs is mainly used. However, the widespread use of antibacterial drugs has led to the frequent emergence of drug-resistant strains and even the emergence of super drug-resistant bacteria, increasing the treatment difficulty. A biofilm is an organized bacterial community wrapped by extracellular macromolecules of bacteria, which has strong resistance to antibiotics and host immune defense mechanisms. Bacteria living in biofilms show a high degree of drug resistance to antibiotics compared with their planktonic bacteria, resulting in chronic infections that are difficult to eradicate, causing serious losses to the healthcare, food industry, and other fields. Staphylococcus aureus is also one of the bacteria that are most likely to form biofilms. The formation of biofilms leads to a significant increase in the resistance of Staphylococcus aureus to antibiotics. Since biofilm-producing bacteria are less sensitive or even resistant to traditional antibacterial drugs, the exploration of new anti-biofilm drugs has been increasingly promoted.

[0003] Traditional Chinese medicine also has unique advantages in reducing bacterial drug resistance. Swertia belongs to the genus Swertia of the Gentianaceae family and is mainly distributed in the southwestern region of China. It contains active ingredients such as swertiamarinene ether terpenoids, triterpenoids, xanthones, and flavonoids. It has significant effects in clearing the liver and gallbladder, removing various fevers, and treating jaundice hepatitis and viral hepatitis. It has good bacteriostatic activity against Staphylococcus aureus, Escherichia coli, and Salmonella typhi. However, the inhibitory activity of using swertia alone against bacteria is not good, and the minimum inhibitory concentration against multi-drug resistant Staphylococcus aureus is relatively high. Summary of the Invention

[0004] The present invention provides a swertia-based bacteriostatic composition, a preparation method thereof, and an application thereof, effectively solving the technical problem that the inhibitory activity of single swertia against bacteria is not strong and the minimum inhibitory concentration against multi-drug resistant Staphylococcus aureus is relatively high. At the same time, a swertia-based bacteriostatic composition is provided that improves the antibacterial activity against Staphylococcus aureus by destroying the bacterial cell wall and interfering with the bacterial metabolic process, inhibits the formation of Staphylococcus aureus biofilms, and simultaneously removes mature Staphylococcus aureus biofilms.

[0005] The first object of the present invention is to provide a swertia-based antibacterial composition, which is made of raw materials with the following mass fractions: 70.5% - 78.5% of water extract of Swertia, 15.2% - 20.3% of leonurine, 1.0% - 10.0% of nicotinic acid, with a total of 100%.

[0006] As a preferred embodiment, the mass fraction of the water extract of Swertia is 74.5% - 76.0%, the mass fraction of leonurine is 15.5% - 17.0%, and the mass fraction of nicotinic acid is 7.0% - 9.0%, with a total of 100%.

[0007] The second object of the present invention is to provide a preparation method of the above-mentioned swertia-based antibacterial composition, which includes the following steps:

[0008] The water extract of Swertia is obtained by extracting Swertia with water decoction method.

[0009] Leonurine is obtained by extracting Leonurus with alcohol extraction method.

[0010] Under the stirring state, nicotinic acid is added to the water extract of Swertia, left to stand at 25°C - 40°C, and then leonurine is added and mixed evenly to obtain the swertia-based antibacterial composition.

[0011] As a preferred embodiment, in the swertia-based antibacterial composition, the mass fraction of the water extract of Swertia is 70.5% - 78.5%, the mass fraction of leonurine is 15.2% - 20.3%, and the mass fraction of nicotinic acid is 1.0% - 10.0%.

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

[0013] As a preferred embodiment, the preparation method of leonurine includes the following steps: according to the dosage ratio of 1g:10 - 20mL, anhydrous ethanol is added to the crushed Leonurus, refluxed and extracted at 30°C - 40°C, filtered to obtain a filtrate, the solvent of the filtrate is evaporated, and dried under vacuum conditions of 7.0kPa - 8.0kPa at 80°C - 100°C to obtain leonurine.

[0014] As a preferred embodiment, the preparation method of the water extract of Swertia includes the following steps: according to the dosage ratio of 1g:8mL - 12mL, water is added to the crushed Swertia, soaked at room temperature for 1h - 2h, heated and decocted, filtered to obtain a filtrate and filter residue, the filter residue is decocted with water, and the decoction is repeated 3 times, the filtrates are combined and centrifuged, concentrated, sterilized and diluted to obtain the product.

[0015] As a preferred embodiment, the standing time is 2h - 5h.

[0016] The third object of the present invention is to provide an application of the above-mentioned swertia-based antibacterial composition in inhibiting Staphylococcus aureus.

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

[0018] The swertia-based antibacterial composition provided by the present invention is prepared by water-extracting swertia and using leonurine and nicotinic acid as active ingredients. First, using swertia as the main antibacterial ingredient, by inhibiting the cross-linking of peptidoglycan in the bacterial cell wall, the synthesis of the cell wall is thus disrupted, and the integrity of the bacterial cell wall is damaged, thereby inhibiting the growth and reproduction of Staphylococcus aureus. Secondly, swertia and leonurine are compounded. On the one hand, it enhances the destructive effect on the cell wall of Staphylococcus aureus. On the other hand, during the glycolysis process of Staphylococcus aureus, swertia and leonurus synergistically have a significant inhibitory effect on hexokinase, thereby inhibiting the growth and reproduction of Staphylococcus aureus by interfering with the bacterial metabolic process. The auxiliary effect of nicotinic acid further enhances the destructive effect on the biofilm of Staphylococcus aureus, and further enhances the inhibitory effect on Staphylococcus aureus. Description of the Drawings

[0019] Figure 1 It is a comparison chart of the inhibitory effects of the swertia-based antibacterial compositions of Example 3, Comparative Example 1 to Comparative Example 2 of the present invention and the blank control group of Comparative Example 3 on Staphylococcus aureus.

[0020] Figure 2 It is a comparison chart of the inhibition rates of the swertia-based antibacterial compositions of Example 3, Comparative Example 1 to Comparative Example 2 of the present invention on Strain 1 and Strain 2 respectively.

[0021] Figure 3 It is a comparison chart of the clearance rates of the swertia-based antibacterial compositions of Example 3, Comparative Example 1 to Comparative Example 2 of the present invention on Strain 1 and Strain 2 respectively.

[0022] Figure 4 It is a SEM diagram of the inhibitory effect of the swertia-based antibacterial compositions of Example 3, Comparative Example 1 to Comparative Example 2 of the present invention and the blank control group of Comparative Example 3 on Staphylococcus aureus. Among them, Figure A is Comparative Example 3, Figure B is Comparative Example 2, Figure C is Comparative Example 1, and Figure D is Example 3.

[0023] Figure 5 It is a SEM diagram of the clearance effect of the swertia-based antibacterial compositions of Example 3, Comparative Example 1 to Comparative Example 2 of the present invention and the blank control group of Comparative Example 3 on Staphylococcus aureus. Among them, Figure a is Comparative Example 3, Figure b is Comparative Example 2, Figure c is Comparative Example 1, and Figure d is Example 3.

[0024] Figure 6 To observe the inhibitory effect of the Swertia-based antibacterial composition of Example 3, Comparative Examples 1-2 of the present invention and the blank control group of Comparative Example 3 on Staphylococcus aureus biofilm by laser confocal microscopy. Among them, Figures A-D are the results of PI staining, 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-D1 are the results of FITC-ConA staining, 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 To observe the removal effect of the Swertia-based antibacterial composition of Example 3, Comparative Examples 1-2 of the present invention and the blank control group of Comparative Example 3 on Staphylococcus aureus biofilm by laser confocal microscopy. Among them, Figures a-d are the results of PI staining, 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-d1 are the results of FITC-ConA staining, Figure a1 is Comparative Example 3, Figure b1 is Comparative Example 2, Figure c1 is Comparative Example 1, and Figure d1 is Example 3. Detailed Description of the Invention

[0026] In order to enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific examples and drawings, but the examples given are not intended to limit the present invention. The following test methods and detection methods are all conventional methods unless otherwise specified; the reagents and raw materials are all commercially available unless otherwise specified.

[0027] Aiming at the technical problems that the inhibitory activity of single Swertia against bacteria is not strong and the minimum inhibitory concentration against multi-drug resistant Staphylococcus aureus is relatively high, the present invention provides a Swertia-based antibacterial composition, its preparation method and application.

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

[0029] The present invention provides a Swertia-based antibacterial composition, which is made of the following raw materials by mass fraction: 70.5% - 78.5% Swertia water extract, 15.2% - 20.3% leonurine, 1.0% - 10.0% nicotinic acid, totaling 100%.

[0030] In the above technical solution, a Swertia-based antibacterial composition is prepared by water-extracting Swertia, with leonurine and nicotinic acid as active ingredients. Swertia can inhibit the cross-linking of peptidoglycan in the bacterial cell wall, thereby disrupting the synthesis of the cell wall, damaging the integrity of the bacterial cell wall, and then inhibiting the growth and reproduction of Staphylococcus aureus. The combination of Swertia and leonurine enhances the destructive effect on the cell wall of Staphylococcus aureus on the one hand. On the other hand, during the glycolysis process of Staphylococcus aureus, Swertia and leonurus synergistically have a significant inhibitory effect on hexokinase, thus achieving the effect of inhibiting the growth and reproduction of Staphylococcus aureus by interfering with the bacterial metabolic process. The auxiliary effect of nicotinic acid further enhances the destructive effect on the biofilm of Staphylococcus aureus, and further enhances the inhibitory effect on Staphylococcus aureus.

[0031] To further improve the antibacterial activity against Staphylococcus aureus, the mass fraction of the water extract of Swertia is 74.5% - 76.0%, the mass fraction of leonurine is 15.5% - 17.0%, and the mass fraction of nicotinic acid is 7.0% - 9.0%, with a total of 100%.

[0032] The present invention also provides a preparation method of the above Swertia-based antibacterial composition, which includes the following steps:

[0033] The water extract of Swertia is obtained by extracting Swertia with water decoction method.

[0034] Leonurine is obtained by extracting Leonurus with alcohol extraction method.

[0035] Under stirring, nicotinic acid is added to the water extract of Swertia, and it is left standing at 25°C - 40°C for 2 - 5 h, then leonurine is added and mixed evenly to obtain the Swertia-based antibacterial composition.

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

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

[0038] To obtain leonurine from Leonurus, the preparation method of leonurine includes the following steps: According to the dosage ratio of 1 g:10 - 20 mL, absolute ethanol is added to the crushed Leonurus, and it is refluxed and extracted at 30°C - 40°C, filtered to obtain the filtrate. The solvent of the filtrate is evaporated, and it is dried under vacuum conditions of 7.0 kPa - 8.0 kPa at 80°C - 100°C to obtain leonurine.

[0039] To obtain the water extract of Swertia bimaculata, the preparation method of the water extraction of Swertia bimaculata includes the following steps: according to the dosage ratio of 1 g: 8 mL - 12 mL, add water to the pulverized Swertia bimaculata, soak at room temperature for 1 h - 2 h, heat and decoct, filter to obtain a filtrate and residues, decoct the residues with water, repeat the decoction 3 times, combine the filtrates and centrifuge, and then concentrate, sterilize and dilute to obtain the product.

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

[0041] Example 1

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

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

[0044] Extract the water extract of Swertia bimaculata from Swertia bimaculata by water decoction method: according to the dosage ratio of 1 g: 10 mL, add water to the pulverized Swertia bimaculata, soak at room temperature for 2 h, heat and decoct, filter to obtain a filtrate and residues, decoct the residues with water, repeat the decoction 3 times, combine the filtrates and centrifuge, and then concentrate, sterilize and dilute to obtain a water extract of Swertia bimaculata with a concentration of 26 μg / mL.

[0045] Extract leonurine from Leonurus japonicus by alcohol extraction method: according to the dosage ratio of 1 g: 15 mL, add anhydrous ethanol to the pulverized Leonurus japonicus, reflux and extract at 35°C, filter to obtain a filtrate, evaporate the solvent from the filtrate, and dry at 100°C under a vacuum condition of 8.0 kPa to obtain leonurine.

[0046] Under stirring, add nicotinic acid to the water extract of Swertia bimaculata, let it stand at 30°C for 4 h, and then add leonurine and mix well to obtain the Swertia bimaculata-based antibacterial composition.

[0047] [[ID=2,5]]Example 2

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

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

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

[0051] Leonurine was obtained by alcohol extraction method from Leonurus japonicus: According to the dosage ratio of 1 g:15 mL, anhydrous ethanol was added to the crushed Leonurus japonicus, refluxed and extracted at 35 °C, filtered to obtain the filtrate. The solvent of the filtrate was evaporated, and dried at 100 °C under the vacuum condition of 8.0 kPa to obtain leonurine.

[0052] Under the stirring state, nicotinic acid was added to the aqueous extract of Swertia bimaculata, allowed to stand at 30 °C for 4 h, and then leonurine was added and mixed evenly to obtain the antibacterial composition based on Swertia bimaculata.

[0053] Example 3

[0054] An antibacterial composition based on Swertia bimaculata, wherein the mass fraction of the aqueous extract of Swertia bimaculata is 75.5%, the mass fraction of leonurine is 16.5%, and the mass fraction of nicotinic acid is 8.0%, with a total of 100%.

[0055] The preparation method of the above antibacterial composition based on Swertia bimaculata includes the following steps:

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

[0057] Leonurine was obtained by alcohol extraction method from Leonurus japonicus: According to the dosage ratio of 1 g:15 mL, anhydrous ethanol was added to the crushed Leonurus japonicus, refluxed and extracted at 35 °C, filtered to obtain the filtrate. The solvent of the filtrate was evaporated, and dried at 100 °C under the vacuum condition of 8.0 kPa to obtain leonurine.

[0058] Under the stirring state, nicotinic acid was added to the aqueous extract of Swertia bimaculata, allowed to stand at 30 °C for 4 h, and then leonurine was added and mixed evenly to obtain the antibacterial composition based on Swertia bimaculata.

[0059] Example 4

[0060] An antibacterial composition based on Swertia bimaculata, wherein the mass fraction of the aqueous extract of Swertia bimaculata is 78.5%, the mass fraction of leonurine is 20.3%, and the mass fraction of nicotinic acid is 1.2%, with a total of 100%.

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

[0062] Extract the water extract of swertia from swertia by the decoction method: according to the dosage ratio of 1 g: 8 mL, add water to the crushed swertia, soak at room temperature for 1 h, heat and decoct, filter to obtain the filtrate and filter residue, add water to the filter residue for decocting, repeat the decocting 3 times, combine the filtrates and centrifuge, concentrate, sterilize and dilute to obtain the swertia water extract with a concentration of 20 μg / mL.

[0063] Extract leonurine from motherwort by the alcohol extraction method: according to the dosage ratio of 1 g: 10 mL, add anhydrous ethanol to the crushed motherwort, reflux and extract at 30 °C, filter to obtain the filtrate, evaporate the solvent from the filtrate, dry at 80 °C under the vacuum condition of 7.5 kPa to obtain leonurine.

[0064] Under the stirring state, add nicotinic acid to the swertia water extract, stand at 40 °C for 2 h, then add leonurine and mix evenly to obtain the swertia-based antibacterial composition.

[0065] Example 5

[0066] A swertia-based antibacterial composition, wherein the mass fraction of the water extract of swertia is 74.5%, the mass fraction of leonurine is 16.5%, and the mass fraction of nicotinic acid is 9.0%, with a total of 100%.

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

[0068] Extract the water extract of swertia from swertia by the decoction method: according to the dosage ratio of 1 g: 12 mL, add water to the crushed swertia, soak at room temperature for 1.5 h, heat and decoct, filter to obtain the filtrate and filter residue, add water to the filter residue for decocting, repeat the decocting 3 times, combine the filtrates and centrifuge, concentrate, sterilize and dilute to obtain the swertia water extract with a concentration of 30 μg / mL.

[0069] Extract leonurine from motherwort by the alcohol extraction method: according to the dosage ratio of 1 g: 20 mL, add anhydrous ethanol to the crushed motherwort, reflux and extract at 40 °C, filter to obtain the filtrate, evaporate the solvent from the filtrate, dry at 90 °C under the vacuum condition of 7.0 kPa to obtain leonurine.

[0070] Under the stirring state, add nicotinic acid to the swertia water extract, stand at 35 °C for 5 h, then add leonurine and mix evenly to obtain the swertia-based antibacterial composition.

[0071] Example 6

[0072] A swertia-based antibacterial composition, wherein the mass fraction of the water extract of swertia is 75.0%, the mass fraction of leonurine is 20.0%, and the mass fraction of nicotinic acid is 5.0%, with a total of 100%.

[0073] The preparation method of the above swertia-based antibacterial composition comprises the following steps:

[0074] Extract the water extract of swertia from swertia by the water decoction method: according to the dosage ratio of 1 g:10 mL, add water to the crushed swertia, soak at room temperature for 2 h, heat and decoct, filter to obtain the filtrate and filter residue, decoct the filter residue with water, repeat the decoction 3 times, combine the filtrates and centrifuge, concentrate, sterilize and dilute to obtain a swertia water extract with a concentration of 26 μg / mL.

[0075] Extract leonurine from leonurus by the alcohol extraction method: according to the dosage ratio of 1 g:12 mL, add anhydrous ethanol to the crushed leonurus, reflux and extract at 32 °C, filter to obtain the filtrate, evaporate the solvent from the filtrate, and dry at 100 °C under a vacuum condition of 8.0 kPa to obtain leonurine.

[0076] Under the stirring state, add nicotinic acid to the swertia water extract, let it stand at 36 °C for 3 h, then add leonurine and mix evenly to obtain the swertia-based antibacterial composition.

[0077] Example 7

[0078] A swertia-based antibacterial composition, wherein the mass fraction of the water extract of swertia is 72.3%, the mass fraction of leonurine is 18.7%, and the mass fraction of nicotinic acid is 9.0%, with a total of 100%.

[0079] The preparation method of the above swertia-based antibacterial composition comprises the following steps:

[0080] Extract the water extract of swertia from swertia by the water decoction method: according to the dosage ratio of 1 g:9 mL, add water to the crushed swertia, soak at room temperature for 1 h, heat and decoct, filter to obtain the filtrate and filter residue, decoct the filter residue with water, repeat the decoction 3 times, combine the filtrates and centrifuge, concentrate, sterilize and dilute to obtain a swertia water extract with a concentration of 25 μg / mL.

[0081] Extract leonurine from leonurus by the alcohol extraction method: according to the dosage ratio of 1 g:18 mL, add anhydrous ethanol to the crushed leonurus, reflux and extract at 38 °C, filter to obtain the filtrate, evaporate the solvent from the filtrate, and dry at 85 °C under a vacuum condition of 7.0 kPa to obtain leonurine.

[0082] While stirring, nicotinic acid was added to the aqueous extract of Swertia bimaculata, and the mixture was allowed to stand at 40 °C for 3.5 h. Then, leonurine was added and mixed evenly to obtain the Swertia bimaculata-based antibacterial composition.

[0083] Example 8

[0084] A Swertia bimaculata-based antibacterial composition, wherein the mass fraction of the aqueous extract of Swertia bimaculata is 76.0%, the mass fraction of leonurine is 17.0%, and the mass fraction of nicotinic acid is 7.0%, with a total of 100%.

[0085] The preparation method of the above-mentioned Swertia bimaculata-based antibacterial composition comprises the following steps:

[0086] The aqueous extract of Swertia bimaculata was obtained by water decoction method: according to the dosage ratio of 1 g:8 mL, water was added to the crushed Swertia bimaculata, soaked at room temperature for 1 h, heated and decocted, filtered to obtain the filtrate and the filter residue. The filter residue was decocted with water, and the decoction was repeated 3 times. The filtrates were combined and centrifuged, concentrated, sterilized and diluted to obtain the aqueous extract of Swertia bimaculata with a concentration of 20 μg / mL.

[0087] Leonurine was obtained by ethanol extraction method from Leonurus japonicus: according to the dosage ratio of 1 g:10 mL, anhydrous ethanol was added to the crushed Leonurus japonicus, refluxed and extracted at 30 °C, filtered to obtain the filtrate. The solvent of the filtrate was evaporated, and dried at 95 °C under the vacuum condition of 7.5 kPa to obtain leonurine.

[0088] While stirring, nicotinic acid was added to the aqueous extract of Swertia bimaculata, and the mixture was allowed to stand at 30 °C for 4 h. Then, leonurine was added and mixed evenly to obtain the Swertia bimaculata-based antibacterial composition.

[0089] To further illustrate the technical effects of the present invention, the present invention also sets a comparative example as follows:

[0090] Comparative Example 1

[0091] Compared with Example 3, the difference is that leonurine is not added.

[0092] A Swertia bimaculata-based antibacterial composition, wherein the mass fraction of the aqueous extract of Swertia bimaculata is 91.5%, and the mass fraction of nicotinic acid is 8.5%, with a total of 100%.

[0093] The preparation method of the above-mentioned Swertia bimaculata-based antibacterial composition comprises the following steps:

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

[0095] Under stirring conditions, nicotinic acid is added to the aqueous extract of Swertia bimaculata, and the mixture is allowed to stand at 30 °C for 4 h. Then, leonurine is added and mixed evenly to obtain the Swertia bimaculata-based antibacterial composition.

[0096] Comparative Example 2

[0097] Compared with Example 1, the difference lies in that nicotinic acid is not added.

[0098] A Swertia bimaculata-based antibacterial composition, wherein the mass fraction of the aqueous extract of Swertia bimaculata is 81.5%, and the mass fraction of leonurine is 18.5%, with a total of 100%.

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

[0100] The aqueous extract of Swertia bimaculata is obtained by water decoction method: According to the dosage ratio of 1 g:10 mL, water is added to the crushed Swertia bimaculata, soaked at room temperature for 2 h, heated and decocted, filtered to obtain the filtrate and filter residue. The filter residue is decocted with water, and the decoction is repeated 3 times. The filtrates are combined and centrifuged, concentrated, sterilized and diluted to obtain an aqueous extract of Swertia bimaculata with a concentration of 26 μg / mL.

[0101] Leonurine is obtained by alcohol extraction method from Leonurus japonicus: According to the dosage ratio of 1 g:15 mL, anhydrous ethanol is added to the crushed Leonurus japonicus, refluxed and extracted at 35 °C, filtered to obtain the filtrate. The solvent of the filtrate is evaporated, and dried at 100 °C under a vacuum condition of 8.0 kPa to obtain leonurine.

[0102] Under stirring conditions, leonurine is added to the aqueous extract of Swertia bimaculata, mixed evenly, and allowed to stand at 30 °C for 4 h to obtain the Swertia bimaculata-based antibacterial composition.

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

[0104] 1 Strain source

[0105] Both Strain 1 and Strain 2 used in the present invention are standard strains. Strain 1 is Staphylococcus aureus ATCC 25923, and Strain 2 is Staphylococcus aureus ATCC 29213, purchased from Suzhou Qianshe Biotechnology Co., Ltd.

[0106] 2 Main reagents and instruments

[0107] Swertia was purchased from Xueyu Pharmacy in Lhasa, Tibet Autonomous Region; MH medium was purchased from Qingdao Haibo Biotechnology Co., Ltd.; crystal violet, analytical pure, was purchased from Guangdong Guanghua Sci-Tech 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 desktop low-speed centrifuge was purchased from Huxi Instrument and Equipment Co., Ltd., Hunan; FC microplate reader was purchased from Thermo Fisher Scientific; Nano SEM-450 scanning electron microscope was purchased from FEI Company, USA; OLS5000 laser confocal microscope was purchased from Olympus Corporation.

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

[0109] Add 10 mL of LB liquid medium to each sterilized test tube. Respectively set the Swertia-based antibacterial composition groups of Comparative Example 2, Comparative Example 1 and Example 3, and the blank control group of Comparative Example 3. Add 0.1 mL of bacterial suspension to each tube and mix well. Immediately measure OD 600 , and then measure OD every 2 h 600 , and draw a growth curve, as Figure 1 shown.

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

[0111] 4.1 Detection by crystal violet staining

[0112] Dilute the bacterial suspension with a turbidity of 0.5 McFarland 1:100 with LB culture medium. Take an appropriate amount and add it to a 96-well culture plate, and then add an equal volume of the Swertia-based antibacterial composition of Comparative Example 2, Comparative Example 1 and Example 3. In addition, use the bacterial suspension as a positive control and LB medium as a negative control. Each group of drugs is repeated 6 times. Stain with 1% crystal violet for 5 min, wash 3 times with PBS buffer, and measure the OD 590 value, and repeat 3 times. The difference between the scavenging effect and inhibitory effect of the Swertia-based antibacterial composition on Staphylococcus aureus biofilm is that after the biofilm culture is completed, wash 3 times with PBS buffer, air dry and then add the Swertia-based antibacterial composition. The inhibitory effect is as Figure 2 shown, and the scavenging effect is as Figure 3 shown.

[0113]

[0114] 4.2 Observation by scanning electron microscope

[0115] Appropriately take an appropriate amount of the diluted bacterial suspension of 4.1 and add it to a 24-well plate with a sterilized cover slip for cultivation. Then, successively add equal volumes of the Swertia-based antibacterial composition of Comparative Example 2, Comparative Example 1, and Example 3. At the same time, set a blank control group for Comparative Example 3. After culturing at 37°C for 48 h, take it out, discard the liquid in each well, rinse the cover slip with PBS 3 times, add an appropriate amount of 2.5% glutaraldehyde, and fix it overnight at 4°C. Discard the fixing solution and wash it 3 times with PBS. Dehydrate it with 30%, 50%, 70%, 80%, 90%, and 100% ethanol solutions for 10 min each. Take out the cover slip, perform critical point drying and gold plating, and then observe it under a scanning electron microscope, as Figure 4 and Figure 5 shown. The difference between the scavenging effect and inhibitory effect of Swertia on the biofilm of Staphylococcus aureus lies in adding the Swertia-based antibacterial composition after the biofilm culture ends.

[0116] 4.3 Observation with a laser confocal microscope

[0117] Use a 24-well plate to culture the biofilm. After diluting the bacterial solution with a turbidity of 0.5 McFarland 1:100, take an appropriate amount and add it to the 24-well culture plate. Then, successively add equal volumes of the Swertia-based antibacterial composition of Comparative Example 2, Comparative Example 1, and Example 3. Only add the bacterial suspension without adding drugs as a blank control. After culturing at 37°C for 48 h, take it out, discard the liquid in the wells, gently wash the cover slip with sterilized PBS buffer 3 times, add 2 mL of 2.5% glutaraldehyde solution to each well, and fix it at 4°C for 1 h. After the culture ends, remove the fixing solution and wash it 3 times with PBS. Stain it with FITC-ConA and PI, and observe it with a laser confocal microscope, as Figure 6 and Figure 7 shown. The difference between the scavenging effect and inhibitory effect of Swertia on the biofilm of Staphylococcus aureus lies in adding the Swertia-based antibacterial composition after the biofilm culture ends.

[0118] 5 Data statistics

[0119] Use SPSS 22.0 statistical software to perform statistical analysis on the data, and the results are expressed as mean ± standard deviation. Perform a t-test for significant analysis, and take P < 0.05 as having statistical significance.

[0120] Select the No. 1 isolate to determine the effects of the Swertia-based antibacterial compositions of Comparative Example 2, Comparative Example 1, and Example 3 on its growth. The results are as Figure 1 shown. It can be seen from Figure 1 that the Swertia-based antibacterial compositions of Comparative Example 2, Comparative Example 1, and Example 3 can all inhibit the growth of Staphylococcus aureus. Especially under the action of the Swertia-based antibacterial composition of Example 3, the growth of Staphylococcus aureus is completely inhibited.

[0121] The inhibitory and clearance effects of the Swertia-based antibacterial composition of Comparative Example 2, Comparative Example 1, and Example 3 on the biofilms of 2 strains of Staphylococcus aureus with strong biofilm-forming ability were determined by crystal violet staining method. The results are shown in Figure 2 and Figure 3 . Under the action of the Swertia-based antibacterial compositions of Comparative Example 2, Comparative Example 1, and Example 3, the formation of biofilms of 2 strains of Staphylococcus aureus with strong biofilm-forming ability was inhibited. After the biofilms were cultured and matured, they could be cleared. For strain 1, the Swertia-based antibacterial compositions of Example 3 and Comparative Example 1 had a clearance effect on the mature biofilms, while the Swertia-based antibacterial composition of Comparative Example 2 did not have a clearance effect. Strain 2 could clear the mature biofilms of Staphylococcus aureus under the action of the Swertia-based antibacterial compositions of Comparative Example 2, Comparative Example 1, and Example 3.

[0122] The effects of the Swertia-based antibacterial compositions of Comparative Example 2, Comparative Example 1, and Example 3 on the biofilm morphology were observed by scanning electron microscopy. The results are as shown in Figure 4 . It can be seen from Figures A to D of Figure 4 that the blank control group formed a multi-layered network-like biofilm, which intersected with each other, and the bacteria adhered to each other or were wrapped in it, and the bacterial morphology was relatively complete. After treatment with the Swertia-based antibacterial compositions of Comparative Example 2, Comparative Example 1, and Example 3, the bacterial biofilms were significantly loosened, the structural integrity of the biofilms was damaged, a small number of colonies aggregated, and the number of bacteria also decreased. Individual bacteria were scattered and the number was significantly reduced, indicating that the Swertia-based antibacterial composition had an inhibitory effect on the biofilms of Staphylococcus aureus with strong biofilm-forming ability. Similarly, for the clearance effect of the Swertia-based antibacterial composition on the biofilms, as shown in Figures a to d of Figure 5 , the changes in bacteria also had a similar trend, but compared with the inhibitory effect group in the same group, the number of colonies was relatively large, indicating that the Swertia-based antibacterial composition could also show a clearance effect on the mature biofilms.

[0123] The effects of different concentrations of Swertia on the biofilms of Staphylococcus aureus were observed by laser confocal microscopy. The results of its inhibitory and clearance effects are shown in Figure 6 and Figure 7As shown in the figure. In the figure, the red fluorescence is the result of PI staining, representing dead bacteria; the green fluorescence is the staining result of FITC-ConA, indicating the distribution of the polysaccharide matrix. After treatment with the Swertia-based antibacterial compositions of Comparative Example 2, Comparative Example 1, and Example 3, the green fluorescence area decreased continuously in the order of Comparative Example 2 - Comparative Example 1 - Example 3, indicating a decrease in the production of polysaccharides, while the red fluorescence area increased continuously in the order of Comparative Example 2 - Comparative Example 1 - Example 3, indicating an increasing number of dead bacteria. The above results show that the Swertia-based antibacterial compositions of Comparative Example 2, Comparative Example 1, and Example 3 can effectively inhibit the formation of Staphylococcus aureus biofilms, and have a certain effect on clearing mature biofilms. Among them, the Swertia-based antibacterial composition of Example 3, which contains the water extract of Swertia, leonurine, and nicotinic acid, has the best inhibitory and clearing effects on Staphylococcus aureus. The effect of Comparative Example 1, which contains the water extract of Swertia and leonurine, is second, and the effect of Comparative Example 2, which contains the water extract of Swertia and nicotinic acid, is the worst.

[0124] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention also intends to include these changes and modifications.

Claims

1. A Swertia-based antibacterial composition, characterized in that, The Swertia-based antibacterial composition is prepared from the following raw materials by mass fraction: 70.5% - 78.5% of Swertia water extract, 15.2% - 20.3% of leonurine, 1.0% - 10.0% of nicotinic acid, with a total of 100%.

2. The swertia-based antibacterial composition according to claim 1, wherein The mass fraction of the Swertia water extract is 74.5% - 76.0%, the mass fraction of leonurine is 15.5% - 17.0%, and the mass fraction of nicotinic acid is 7.0% - 9.0%, with a total of 100%.

3. A preparation method of the swertia-based antibacterial composition according to claim 1, characterized in that, It includes the following steps: Adopt the water decoction method to extract Swertia water extract from Swertia; Adopt the alcohol extraction method to extract leonurine from Leonurus japonicus; Under the stirring state, add nicotinic acid to the Swertia water extract, let it stand at 25°C - 40°C, then add leonurine, and mix evenly to obtain the Swertia-based antibacterial composition.

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

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

6. The preparation method according to claim 3, wherein, The preparation method of the leonurine includes the following steps: According to the dosage ratio of 1 g:10 - 20 mL, add anhydrous ethanol to the crushed Leonurus japonicus, reflux and extract at 30°C - 40°C, filter to obtain the filtrate, evaporate the solvent from the filtrate, and dry it at 80°C - 100°C under the vacuum condition of 7.0 kPa - 8.0 kPa to obtain leonurine.

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

8. The preparation method according to claim 3, characterized in that, The standing time is 2 h - 5 h.

9. Application of the Swertia-based antibacterial composition according to claim 1 or 2 in inhibiting Staphylococcus aureus.

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