Extraction method of antibacterial clematis tangutica extract as well as obtained product and application of antibacterial clematis tangutica extract

Abstract

The invention belongs to the field of extraction of active ingredients, and particularly relates to an extraction method of an antibacterial clematis tangutica extract, an obtained product and application. The method comprises the following steps: preparing a Clematis tangutica extract through soaking and ultrasonic extraction, extracting the Clematis tangutica extract to prepare petroleum ether, ethyl acetate and a water phase component, and performing experimental methods such as a double dilution method and a co-culture method. And determining the bacteriostatic ability of each component of the Clematis tangutica to escherichia coli, methicillin-resistant staphylococcus aureus, enterococcus faecalis, staphylococcus aureus and pseudomonas aeruginosa. The extraction method provided by the invention is simple and efficient, has little damage to active substances in the Clematis tangutica, can realize the extraction of effective components to the maximum extent, is beneficial to the playing of the effect of the Clematis tangutica, is simple and easy to operate, and is suitable for industrial popularization and application.

Description

Technical Field

[0001] The present invention belongs to the field of extraction of active ingredients, and particularly relates to an extraction method of an antibacterial Clematis tangutica extract, the obtained product, and applications thereof. Background Art

[0002] According to government data, by 2050, 10 million people may die each year from antibiotic resistance, which is one of the main causes of population death. Natural products have unique chemical structures, folding modes, good cell permeability, and target specificity. In the current situation of increasingly serious bacterial resistance, natural products play an important role in the research and development of new antibacterial drugs.

[0003] Compared with traditional synthetic small molecules, natural products usually have larger molecular weights, more sp3-hybridized carbon and oxygen atoms, fewer nitrogen and halogen atoms, more hydrogen bond acceptors and donors, lower lipophilic-hydrophilic partition coefficients (cLogP values, indicating higher hydrophilicity), and higher molecular rigidity. Clematis tangutica ( Clematis tangutica ), is an important wild medicinal plant, and its whole herb can be used as medicine. In the prior art, there is no record of Clematis tangutica in terms of antibacterial activity. Summary of the Invention

[0004] In view of the research and development gap in the prior art, the present invention provides an extraction method of an antibacterial Clematis tangutica extract.

[0005] The present invention also provides an antibacterial Clematis tangutica extract obtained by using the above method.

[0006] Another object of the present invention is to provide the application of the above Clematis tangutica extract in the preparation of antibacterial drugs.

[0007] The technical solution adopted by the present invention to achieve the above object is as follows: The present invention provides an extraction method of an antibacterial Clematis tangutica extract, comprising the following steps: (1) Add pullulan polysaccharide solution to the Clematis tangutica powder, soak overnight, filter, wash, then steam, add the steamed Clematis tangutica to an alcohol solution for soaking extraction, then perform ultrasonic extraction, combine the soaking extraction solution and the ultrasonic extraction solution, and concentrate to obtain a concentrated extraction solution; (2) Extract the concentrated extraction solution successively with petroleum ether and ethyl acetate, concentrate and freeze-dry each extraction solution to obtain a petroleum ether phase, an ethyl acetate phase, and an aqueous phase.

[0008] Preferably, in step (1), the mass concentration of the pullulan polysaccharide solution is 1.5%; the steaming time is 20 - 35 min; preferably, in step (1), the number of soaking times is three, and the soaking times for the three times are 22 days, 26 days, and 10 days respectively; the ultrasonic extraction is carried out at 70 °C and 90 W for 60 min, and the ultrasonic extraction is carried out twice.

[0009] Preferably, in step (2), the volume ratio of petroleum ether to the concentrated extract is 1:1; the volume ratio of ethyl acetate to the concentrated extract is 1:1; both petroleum ether and ethyl acetate extract the concentrated extract three times.

[0010] The present invention also provides a Clematis tangutica extract obtained by using the above extraction method as claimed in the claims.

[0011] Another object of the present invention is to provide the application of the above Clematis tangutica extract in the preparation of antibacterial drugs.

[0012] Preferably, the bacteria are Escherichia coli, methicillin-resistant Staphylococcus aureus, Enterococcus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa.

[0013] The present invention prepares a Clematis tangutica extract by soaking and ultrasonic extraction, extracts the Clematis tangutica extract to prepare petroleum ether, ethyl acetate and aqueous components, and measures the antibacterial ability of each component of Clematis tangutica against Escherichia coli, methicillin-resistant Staphylococcus aureus, Enterococcus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa by experimental methods such as the two-fold dilution method and the co-culture method. The minimum inhibitory concentration (MIC) of the ethyl acetate of Clematis tangutica provided by the present invention against Staphylococcus aureus (ATCC25923) is 1.25 mg / mL, the MIC against Pseudomonas aeruginosa (ATCC27853) is 1.25 mg / mL, and the minimum bactericidal concentration (MBC) is between 2.5 mg / mL. The MIC against methicillin-resistant Staphylococcus aureus (ATCC43300) is 62.5 μg / mL, and the MBC is 250 μg / mL.

[0014] The beneficial effects of the present invention are as follows: (1) The extraction method provided by the present invention is simple and efficient, and has little damage to the active substances in Clematis tangutica, can achieve the extraction of effective components to the greatest extent, helps to exert the function of Clematis tangutica, and the method is simple and easy to operate, and is suitable for industrial promotion and application; (2) Through the research on the antibacterial activity of the Clematis tangutica extract, the present invention has a significant inhibitory effect on a variety of common bacteria, confirms its potential application value in the field of antibacterial, provides a theoretical basis and experimental support for the development of new natural antibacterial drugs, and lays a foundation for the development of new natural antibacterial drugs. Brief Description of the Drawings

[0015] Figure 1 Antibacterial effects of various parts of Clematis tangutica Maxim. on five kinds of bacteria; Figure 2 Influence of various parts of Clematis tangutica Maxim. on the replication ability of five kinds of bacteria; Figure 3 Determination of MIC and MBC of EA against four kinds of bacteria. Detailed Implementation Modes

[0016] The technical solutions of the present invention will be further explained and described below through specific embodiments.

[0017] The Clematis tangutica Maxim. used in the present invention is obtained by artificial cultivation.

[0018] Example 1 1. Test Method 1.1 Material Preparation Wash, dry and crush the above-ground parts of Clematis tangutica Maxim.

[0019] Prepare the bacterial strains required for the experiment, including Escherichia coli (ATCC35218), Methicillin-resistant Staphylococcus aureus (ATCC43300), Enterococcus faecalis (ATCC51299), Staphylococcus aureus (ATCC25923), Pseudomonas aeruginosa (ATCC27853). The bacterial strains are all from the American Type Culture Collection (ATCC).

[0020] 1.2 Medicinal Material Extraction (1) Add 1.5% pullulan polysaccharide solution (just covering) to the powder of Clematis tangutica Maxim., soak overnight (12 h), filter, wash, and then steam for 30 min. Soak and extract the 4.9 kg of steamed Clematis tangutica Maxim. three times with 95% ethanol according to the solid-liquid ratio of 1:10 (soak and extract for 22 days, 26 days, and 10 days respectively), and then ultrasonically extract twice according to the same solid-liquid ratio (70 °C, 90 W, 60 min). Combine the soak extract and the ultrasonic extract, and finally concentrate to obtain about 2.4 L of extract; (2) Extract the extract three times with petroleum ether and ethyl acetate in turn (first extract three times with petroleum ether, petroleum ether is on the upper layer, and the remaining extract in the lower layer is then extracted three times with ethyl acetate), petroleum ether: extract (V / V) = 1:1, ethyl acetate: extract (V / V) = 1:1. Concentrate and freeze-dry each extract to obtain a petroleum ether phase (68 g), an ethyl acetate phase (118 g), and an aqueous phase (339 g).

[0021] Effect Example (I) Antibacterial Activity Test The antibacterial activities of the total extract (Total), petroleum ether fraction (PE), ethyl acetate fraction (EA), and water fraction (Water) against five bacteria (Escherichia coli (ATCC35218), methicillin-resistant Staphylococcus aureus (ATCC43300), Enterococcus faecalis (ATCC51299), Staphylococcus aureus (ATCC25923), Pseudomonas aeruginosa (ATCC27853)) were verified using a co-culture method. After dissolving the four fractions with DMSO, 15 μL was taken and added to a 1.5 mL EP tube, and 135 μL of the five bacterial solutions cultured for 12 h was added to each tube to make the final concentration of the sample 50 mg / mL. After incubating for 12 h, 50 μL of the bacterial solution was taken out from each well and diluted 10-fold or 100-fold. 200 μL of the diluted bacterial solution was added to the solid agar medium, and after culturing for 48 h, the colonies on the medium were counted to evaluate the antibacterial activities of the four fractions.

[0022] For the fractions with better antibacterial activities, their MIC and MBC were determined. The fractions with better activities were prepared into a stock solution of 50 mg / mL, and were successively diluted to 4 different concentration solutions of 50, 25, 12.5, and 6.25 mg / mL using the two-fold dilution method. After dilution 10-fold, the final concentrations of the components were 5, 2.5, 1.25, and 0.625 mg / mL, and after dilution 100-fold, the final concentrations of the components were 500, 250, 125, and 62.5 μg / mL, and DMSO was used as the control group. According to the above method, 100 μL of the diluted bacterial solution was spread on the solid agar medium, and after observing and recording for 20 h, MIC and MBC were calculated based on the dilution factor.

[0023] (II) Antibacterial effects of various parts of Clematis tangutica on five bacteria As Figure 1 and Figure 2 shown, the EA fraction had antibacterial effects on Escherichia coli (35218), methicillin-resistant Staphylococcus aureus (43300), Staphylococcus aureus (25923), and Pseudomonas aeruginosa (27853), while the antibacterial effects of other components were not significant; when not soaked in pullulan polysaccharide solution and not steamed, and other steps were the same as in Example 1, the antibacterial effect of the EA fraction on Escherichia coli decreased by approximately 5.8% and that on methicillin-resistant Staphylococcus aureus (43300) decreased by 3.3%.

[0024] 2.2 Determination of MIC and MBC of EA against four bacteria The EA had the best antibacterial activity. Further studies were conducted on the MIC and MBC of EA against Escherichia coli (35218), methicillin-resistant Staphylococcus aureus (43300), Staphylococcus aureus (25923), and Pseudomonas aeruginosa (27853). The experimental results are as Figure 3As shown, EA has the strongest ability to inhibit methicillin-resistant Staphylococcus aureus (43300), and its MIC value against methicillin-resistant Staphylococcus aureus (43300) is 62.5 μg / mL. The antibacterial activity of EA against Escherichia coli (35218) is slightly worse than that against other bacteria.

[0025] Table 1 MIC and MBC values of EA against three kinds of bacteria

Claims

1. A method for extracting an antibacterial Clematis tangutica extract, characterized in that: The following steps are involved: (1) adding a pullulan solution to the powder of Clematis tanguticus, soaking it overnight, filtering, washing, and then steaming it, adding the steamed Clematis tanguticus to an alcohol solution for soaking extraction, and then ultrasonic extraction, combining the soaking extract and the ultrasonic extract, and concentrating them to obtain a concentrated extract; (2) The concentrated extract was extracted with petroleum ether and ethyl acetate in turn, and each extract was concentrated and freeze-dried to obtain a petroleum ether phase, an ethyl acetate phase and an aqueous phase.

2. The extraction method according to claim 1, characterized in that In step (1), the mass concentration of the pullulan solution is 1.5%; the steaming time is 20-35 min; the solid-liquid ratio of the Clematis tanguticus and the alcohol solution is 1:10; and the alcohol solution is a 95% ethanol aqueous solution.

3. The extraction method according to claim 1 or 2, characterized in that In step (1), the soaking is performed three times, and the soaking time is 22 days, 26 days, and 10 days respectively; the ultrasonic extraction is performed at 70°C and 90 W for 60 minutes, and the ultrasonic extraction is performed twice.

4. The extraction method according to any one of claims 1 to 3, characterized in that In step (2), the volume ratio of petroleum ether to the concentrated extract is 1:1; the volume ratio of ethyl acetate to the concentrated extract is 1:1; and the concentrated extract is extracted three times with both petroleum ether and ethyl acetate.

5. A Clematis tangutica extract obtained by the extraction method according to any one of claims 1 to 4.

6. Use of the Clematis tangutica extract as claimed in claim 5 in the preparation of antibacterial drugs.

7. The use according to claim 6, characterized in that: The bacteria are Escherichia coli, methicillin-resistant Staphylococcus aureus, Enterococcus faecalis, Staphylococcus aureus, and Pseudomonas aeruginosa.

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