Paris polyphylla extract and application thereof in preparation of marine pathogenic bacterium biofilm inhibitor

Abstract

The paris polyphylla extract is prepared according to the following steps: adding paris polyphylla powder into an ethanol aqueous solution with the volume concentration of 80-95%, performing heating reflux extraction for 1-5 hours, performing suction filtration on an extracting solution (preferably vacuum suction filtration), performing vacuum concentration on filtrate at 65 DEG C until no liquid flows out, and performing vacuum drying to obtain the paris polyphylla extract. Freeze-drying the thick liquid to obtain the paris polyphylla extract. According to the paris polyphylla ethanol extract or the n-butyl alcohol extract of the ethanol extract, the obtaining method is easy to operate, safe, low in cost and convenient to popularize and use, the extract has the advantages of being natural, efficient, low in price, low in toxicity, easy to obtain and the like, formation of a marine vibrio biofilm can be effectively inhibited, and particularly formation of a vibrio alginolyticus biofilm can be inhibited.

Description

(1) Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to an extract of Paris polyphylla, a Chinese herbal medicine, and its application in the preparation of an inhibitor for the formation of marine pathogenic bacteria biofilm. (2) Background Art

[0002] Vibrio alginolyticus is a Gram-negative marine vibrio, belonging to the genus Vibrio of the family Vibrionaceae, suitable for growing in an alkaline environment, and widely existing in seawater and various seafood foods. Vibrio alginolyticus can cause diseases in aquaculture animals, causing huge economic losses to the aquaculture industry. At the same time, consuming seafood foods containing pathogenic bacteria will also pose a hazard to human health. In recent years, studies have found that Vibrio alginolyticus is similar to Vibrio parahaemolyticus and is also a common pathogenic bacterium causing diarrhea in foods in coastal areas. When aquatic products are not adequately processed or eaten raw, the foodborne infection of Vibrio alginolyticus is likely to exist in the form of biofilm, thereby causing harm to humans. At present, antibiotics are the main means of preventing and controlling Vibrio alginolyticus diseases, providing strong technical support for solving human diseases and aquaculture problems caused by bacterial infections. However, with the abuse of antibiotics, a large number of bacteria have developed drug resistance. The emergence of bacterial drug resistance has made antibiotics no longer the main means of treating bacterial infections, thus greatly limiting the treatment of pathogenic bacteria by humans.

[0003] Bacterial biofilm (BF) is a special functional structure formed by bacteria gradually attaching to the surface of inert or active entities during growth to adapt to the natural environment, and is a structural bacterial community composed of bacterial cells and the water-containing polymeric matrix secreted by the bacteria themselves that encloses the bacteria. The formation of biofilm can enhance the resistance of bacteria to adverse environments and is one of the important reasons why most pathogenic bacteria are difficult to remove and cause persistent infections. Therefore, inhibiting the formation of biofilm can effectively control the growth and spread of marine pathogenic bacteria, which has important guiding significance for the prevention and control of bacterial biofilm.

[0004] In 2016, China put forward in the National Action Plan for Containing Bacterial Drug Resistance (2016 - 2020) to implement effective drug resistance prevention and control measures. As a natural drug existing in nature, traditional Chinese medicine has attracted wide attention in the development of drugs for treating bacterial infections due to its advantages such as low toxicity, complex components, and green and pollution-free. Thus, it can be seen that researching and developing natural drug active ingredients that inhibit the biofilm of marine vibrio is an important development direction for realizing green environmental protection and high-efficiency ecology in aquaculture, and is also an important way to solve the problem of bacterial drug resistance and prevent and control bacterial diseases, providing an important reference direction for the development of inhibitors for pathogenic marine vibrio biofilm.

[0005] Paris polyphylla is a general term for plants of the genus Paris in the family Liliaceae, which are perennial herbs. There are 27 species and more than 10 varieties in this genus globally. So far, 241 compounds have been isolated and identified from Paris polyphylla, including chemical components such as steroidal saponins, phytosterols, triterpenoids, flavonoids, and polysaccharides. Among them, there are 136 steroidal saponins, which are the main active components of Paris polyphylla. In recent years, many scholars have conducted in-depth studies on Rhizoma Paridis Total Saponins (RPTS), showing that RPTS has significant anti-tumor effects and also exhibits good pharmacological activities in aspects such as anti-inflammatory, antioxidant, antibacterial, hemostatic, and analgesic, and the probability of generating drug resistance is much lower than that of antibiotic drugs. There are many species of Paris plants with extensive pharmacological effects, but currently, the research on the volatile components of Paris polyphylla and their antibacterial activities is relatively less. Therefore, in this invention, Paris polyphylla is used as the research object, and it is extracted to prepare an inhibitor that can inhibit the formation of biofilms of the marine pathogenic bacterium Vibrio alginolyticus. (III) Summary of the Invention

[0006] The object of this invention is to provide an extract of the Chinese herbal medicine Paris polyphylla and its application in the preparation of an inhibitor for marine pathogenic bacteria biofilms, which has inhibitory activity against the formation of biofilms of marine pathogenic bacteria, especially Vibrio alginolyticus, providing a green and environmentally friendly alternative to antibiotics for inhibiting marine pathogenic bacteria, and at the same time providing a new option for the problem that the abuse of antibiotics in aquaculture leads to an increase in bacterial drug resistance, making the prevention and control of bacterial diseases more and more difficult.

[0007] The technical solution adopted by this invention is as follows:

[0008] This invention provides an extract of Paris polyphylla, which is prepared according to the following steps: Add Paris polyphylla powder to an ethanol aqueous solution with a volume concentration of 80 - 95%, heat under reflux for 1 - 5 h, after the extract is filtered by suction (preferably vacuum filtration), the filtrate is concentrated under vacuum at 65°C until no liquid flows out, and the thick liquid is freeze-dried to obtain the extract of Paris polyphylla.

[0009] Preferably, the ethanol is an ethanol aqueous solution with a volume concentration of 95%.

[0010] Preferably, the volume of the ethanol aqueous solution used is 10 - 30 L / kg based on the mass of the Paris polyphylla powder, preferably 20 L / kg.

[0011] Preferably, the reflux extraction is carried out as follows: adding Paris polyphylla powder into an ethanol aqueous solution, performing the first reflux extraction for 3 h, filtering with three layers of gauze after cooling to obtain the first extract and the first medicinal residues; reusing the same ethanol aqueous solution to perform the second reflux extraction on the first medicinal residues for 3 h, filtering with three layers of gauze after cooling to obtain the second extract and the second medicinal residues; combining the first extract and the second extract; the volume of the ethanol aqueous solution used for the two extractions is 5 - 15 L / kg based on the mass of the Paris polyphylla powder, and the volume ratio of the ethanol aqueous solution used for the second extraction to that used for the first extraction is 0.5 - 1:1, preferably 1:1.

[0012] Preferably, the freeze-drying is carried out at -4°C and 0 Pa until the mass water content is lower than 5%.

[0013] In order to obtain the effective active substances in the ethanol extract, we use organic solvents with different polarities to extract the ethanol extract, and perform the extraction in turn from low-polarity to high-polarity solvents (petroleum ether < ethyl acetate < n-butanol). Low-polarity solvents can effectively remove inactive substances, and high-polarity solvents can extract the effective substances, thereby obtaining a high-purity extract; among them, petroleum ether and ethyl acetate are low-polarity solvents, which can remove inactive low-polarity impurities during the extraction process; n-butanol is used to extract high-polarity active substances. The method for extraction and purification after freeze-drying the thick liquid is: dissolving and extracting the freeze-dried thick liquid with an organic solvent, collecting the organic phase, concentrating it under vacuum at 65°C until no liquid flows out, and then freeze-drying (freeze-drying at -4°C and 0 Pa until the mass water content is lower than 5%) to obtain the Paris polyphylla extract; the organic solvents include petroleum ether, ethyl acetate, and n-butanol, preferably n-butanol.

[0014] The present invention also provides an application of the Paris polyphylla extract in the preparation of a marine pathogenic bacteria biofilm inhibitor, and the marine pathogenic bacteria include Vibrio alginolyticus ATCC33787.

[0015] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0016] The ethanol extract of Paris vietnamensis (Takht.) H. Li or the n-butanol extract of the ethanol extract of Paris vietnamensis (Takht.) H. Li has the advantages of simple, safe and low-cost acquisition method, being convenient for popularization and use, and the extract has the advantages of being natural, highly efficient, low-cost, low toxicity and easy to obtain. The ethanol extract of Paris vietnamensis (Takht.) H. Li or the n-butanol extract of the ethanol extract of Paris vietnamensis (Takht.) H. Li can effectively inhibit the formation of biofilms of Vibrio alginolyticus, especially inhibit the formation of biofilms of Vibrio alginolyticus. The Paris vietnamensis (Takht.) H. Li extract has no antibacterial phenomenon, and can still significantly inhibit the formation of biofilms at low concentrations, has a higher inhibitory effect compared with existing inhibitors, and does not produce drug resistance; the Paris vietnamensis (Takht.) H. Li extract of the present invention is a natural plant extract, and is safer than chemically synthesized inhibitors and will not cause harm to the environment and human body. (IV) BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Effect of ethanol extract of Paris vietnamensis (Takht.) H. Li on biofilm of Vibrio alginolyticus ATCC 33787. A, Plate diagram of 96-well plate after crystal violet staining; blank control group, only 2216E medium was added; positive control group, Vibrio alginolyticus ATCC 33787 dilution + ethanol of the same volume as the added extract + 2216E; experimental group, Vibrio alginolyticus ATCC 33787 dilution + added ethanol extract of Paris vietnamensis (Takht.) H. Li + 2216E; B, Histogram of biofilm amount, the experimental group is the same as A, and the control group is the positive control group.

[0018] Figure 2 Effect of ethanol extract of Paris vietnamensis (Takht.) H. Li extracted by different organic solvents on biofilm of Vibrio alginolyticus ATCC 33787. A, Plate diagram of 96-well plate after crystal violet staining; blank control group, only 2216E medium was added; positive control group, Vibrio alginolyticus ATCC 33787 dilution + ethanol of the same volume as the added extract + 2216E; experimental group, Vibrio alginolyticus ATCC 33787 dilution + different extraction phases of the added ethanol extract of Paris vietnamensis (Takht.) H. Li + 2216E. B, Histogram of biofilm amount, the control group is the positive control.

[0019] Figure 3 Effect of different concentrations of n-butanol extract of Paris vietnamensis (Takht.) H. Li on biofilm of Vibrio alginolyticus ATCC 33787. A, Plate diagram of 96-well plate after crystal violet staining; B, Histogram of biofilm amount. Among them, 1-4 are experimental groups, and the concentrations of 1, 2, 3, and 4 are: 11.933 mg / ml, 5.97 mg / ml, 2.98 mg / ml, 1.49 mg / ml respectively, 5 is the positive control group, and 6 is the blank control group. (V) SPECIFIC EMBODIMENTS

[0020] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto: The Paris polyphylla used in the embodiments of the present invention was collected and prepared in accordance with the Chinese Pharmacopoeia 2015 Edition; among them, the Paris polyphylla was purchased from Yongzhou Yongdian Traditional Chinese Medicine Decoction Pieces Co., Ltd.

[0021] The 2216E liquid medium was purchased from Haibo Biotechnology Co., Ltd. (Qingdao).

[0022] The 2216E plate medium was prepared by adding 15 g / L agar to the 2216E liquid medium.

[0023] Example 1. Preparation of ethanol extract of Paris polyphylla

[0024] 1. Crushing of Paris polyphylla: The Paris polyphylla was crushed using a crusher and passed through a 50-mesh sieve to obtain Paris polyphylla powder.

[0025] 2. Extraction of Paris polyphylla: Weigh 200 g of Paris polyphylla powder, use 2 L of 95% ethanol aqueous solution by volume for the first reflux extraction for 3 h. After cooling, filter it with three layers of gauze to obtain the first extract and the first residue. The first residue was then subjected to a second reflux extraction for 3 h with 2 L of 95% ethanol aqueous solution by volume. After cooling, filter it with three layers of gauze to obtain the second extract and the second residue. The first extract and the second extract were combined, and vacuum filtered with filter paper (7 cm slow qualitative filter paper) and stored in a conical flask. The collected filtrate was the ethanol extract of Paris polyphylla.

[0026] 3. Concentration of the ethanol extract of Paris polyphylla: The ethanol extract of Paris polyphylla in step 2 was concentrated using a rotary evaporator at 65 °C under vacuum until no liquid flowed out. The thick liquid was freeze-dried at -4 °C and 0 Pa until the mass water content was less than 5% to obtain 3.5 g of the ethanol extract powder of Paris polyphylla.

[0027] Example 2. Biofilm activity test of ethanol extract of Paris polyphylla

[0028] 1. Determination of the minimum inhibitory concentration (MIC) by the 96-well plate bacterial culture method:

[0029] (1) Dilution of the bacterial solution: The Vibrio alginolyticus ATCC33787 was streaked from -80 °C onto the 2216E plate medium and cultured at 28 °C for 24 h for activation; Three single colonies were picked and inoculated into a conical flask containing 20 mL of 2216E liquid medium and cultured overnight at 28 °C; The OD 600 nm value was measured, and the cell concentration was diluted to OD 600 nm = 0.05 with fresh 2216E liquid medium.

[0030] (2) Paris polyphylla alcohol extract dilution: Weigh 0.9951 g of the Paris polyphylla ethanol extract powder extracted by the method of Example 1 (the mass after water content determination is 0.9444 g), dissolve it with 95% ethanol to 944.4 mg / mL, and then perform gradient dilution with 2216E liquid medium to obtain dilution solutions with different concentrations, and the concentrations are 47.22, 23.61, 11.81, 5.90, 2.95, 1.47, 0.74, 0.37 mg / mL respectively;

[0031] (3) Minimum inhibitory concentration: In the first column of the 96-well plate, add 200 μL of 2216E liquid medium to each well as a blank control; in the second column, add 100 μL of 2216E liquid medium containing the same volume of ethanol as the Paris polyphylla ethanol extract dilution (100 μL of the Paris polyphylla ethanol extract dilution is dissolved with 0.6 μL of 95% ethanol, so the volume of 95% ethanol included in 100 μL of the positive control is 0.6 μL) and 100 μL of the diluted bacterial solution in step (1) as a positive control; in the third and fourth columns, take 100 μL of the diluted bacterial solution in step (1) and 100 μL of the Paris polyphylla ethanol extract dilution in step (2) respectively as the experimental groups.

[0032] Leave the 96-well plate in a 28 °C incubator and incubate it statically overnight; OD 600 nm Read the value and record the results. The MIC experiment results show that the Paris polyphylla ethanol extract has an antibacterial effect on Vibrio alginolyticus ATCC33787, and its MIC result is 23.61 mg / ml.

[0033] 2. Detection of the biofilm activity of Paris polyphylla ethanol extract by crystal violet method:

[0034] To avoid the influence of the antibacterial concentration on the biofilm, we select the Paris polyphylla ethanol extract dilution of 5.90 mg / mL (1 / 4 MIC) as the subsequent biofilm activity test. The specific operation is as follows:

[0035] (1) Dilute the bacterial solution: Streak Vibrio alginolyticus ATCC33787 from -80 °C onto a 2216E plate medium and perform activation culture at 28 °C for 24 h; pick three single colonies and inoculate them into a conical flask containing 20 mL of 2216E liquid medium, and culture them overnight at 28 °C; measure the OD 600 nm value, and dilute the bacterial concentration to OD 600 nm = 0.05 with fresh 2216E liquid medium.

[0036] (2) Diluted solution of Paris polyphylla ethanol extract: The Paris polyphylla ethanol extract powder obtained by the method of Example 1 was dissolved in ethanol to 944.4 mg / mL, and then diluted with 2216E liquid medium to obtain a diluted solution of Paris polyphylla ethanol extract at 5.90 mg / mL.

[0037] (3) Value of biofilm quantity:

[0038] Blank control: In the first and second columns of the 96-well plate, 200 μL of 2216E liquid medium was added to each well.

[0039] Positive control group: In the third and fourth columns of the 96-well plate, 100 μL of 2216E liquid medium containing an equal volume of ethanol to the diluted solution of Paris polyphylla alcohol extract (for example, if 100 μL of the diluted solution of Paris polyphylla ethanol extract is dissolved with 0.6 μL of 95% ethanol, then the volume of 95% ethanol included in 100 μL of the positive control is 0.6 μL) and 100 μL of the diluted bacterial solution in step (1) were added to each well;

[0040] Experimental group: In the fifth and sixth columns of the 96-well plate, 100 μL of the diluted bacterial solution in step (1) and 100 μL of the diluted solution of Paris polyphylla ethanol extract in step (2) were taken respectively, and the final concentration of the extract was 2.95 mg / mL.

[0041] The 96-well plate was statically cultured in an incubator at 28 °C. At 6 h and 12 h respectively, the samples were taken out to measure the OD 620nm value and the values were recorded; the bacterial solution was poured out, and the 96-well plate was washed with distilled water 1 - 2 times; 220 μL of 0.1% crystal violet solution was added to each well and stained for 20 min, then the liquid was poured out, and the excess dye was washed away with distilled water (once). 220 μL of absolute ethanol was added to each well to dissolve the crystal violet, and it was left standing for 5 min; finally, the OD 540 nm value was measured. Each group was repeated 2 times, and each repetition had 8 parallels.

[0042] Biofilm quantity OD 540 nm / OD 620 nm = (OD of the experimental group or the positive control group (OD 540 nm ) - OD of the blank control (OD 540 nm ) / (OD of the experimental group or the positive control group (OD 620 nm ) - OD of the blank control (OD 620 nm ). The larger the ratio, the more biofilm is formed.

[0043] The results are shown in Figure 1As shown, the positive control group (adding an equal volume of ethanol instead of the extract volume) of Vibrio alginolyticus ATCC33787 could form an obvious biofilm, but after adding the ethanol extract of Paris polyphylla, the formation of its biofilm was basically inhibited ( Figure 1 in A and B), indicating that the ethanol extract of Paris polyphylla could significantly inhibit the formation of the biofilm of Vibrio alginolyticus ATCC 33787.

[0044] Example 3. Effects of organic solvents on the activity of the extract of Paris polyphylla

[0045] To separate the active ingredients in the ethanol extract of Paris polyphylla, 1 g of the ethanol extract of Paris polyphylla prepared by the method of Example 1 was dissolved with petroleum ether and then extracted. The organic layer was collected and concentrated in vacuo at 65 °C using a rotary evaporator until no liquid flowed out. The thick liquid was freeze-dried at -4 °C and 0 Pa until the mass water content was less than 5%, and 0.28 g of the petroleum ether extract of Paris polyphylla was obtained.

[0046] By the same method, ethyl acetate and n-butanol were used instead of petroleum ether, and 0.15 g of the ethyl acetate extract of Paris polyphylla and 0.33 g of the n-butanol extract of Paris polyphylla were obtained respectively.

[0047] The activity was tested by the method of Example 2. The blank control group had 4 parallels, the positive control group had 3 replicates with 4 parallels for each replicate; the experimental groups had 4 parallels respectively, so as to determine the extraction phase where the active molecule was located. The results are shown in Figure 2 as follows.

[0048] The experimental results showed that the petroleum ether extract and ethyl acetate extract of Paris polyphylla had a promoting effect on the biofilm of Vibrio alginolyticus ATCC 33787, while the n-butanol extract of Paris polyphylla had an obvious inhibitory effect, indicating that the active substance was present in the n-butanol phase.

[0049] Example 4. Effects of the concentration of the n-butanol extract of Paris polyphylla on the biofilm activity

[0050] The Paris polyphylla Smith n-butyl alcohol extract powder was prepared by the method of Example 3, and was fully dissolved with absolute ethanol to obtain a mother liquor (concentration: 238.65 mg / ml). Then it was serially diluted with 2216E liquid medium to obtain diluted solutions with different concentrations, and the concentrations were: 23.865 mg / ml, 11.933 mg / ml, 5.97 mg / ml, 2.98 mg / ml, 1.49 mg / ml, 0.745 mg / ml, 33.8 mg / ml, 16.9 mg / ml. The minimum inhibitory concentration of the Paris polyphylla Smith n-butyl alcohol extract was tested by the method of Example 2. The solvent with an equal volume of the extract was used as the control group (for example, if 100 μL of the Paris polyphylla Smith n-butyl alcohol extract dilution was dissolved with 0.6 μL of absolute ethanol, then the volume of absolute ethanol in 100 μL of the positive control was 0.6 μL). The minimum inhibitory concentration (MIC) was measured by the 96-well plate bacterial culture method, and no antibacterial phenomenon was found.

[0051] Four concentrations (11.933 mg / ml, 5.97 mg / ml, 2.98 mg / ml, 1.49 mg / ml) were selected to test their biofilm activities by the method of Example 2. For each concentration experimental group, blank control group and positive control group, 2 replicates were set, and each replicate had 8 parallels. The experimental results ( Figure 3 ) showed that at the concentrations of 11.933 mg / ml, 5.97 mg / ml, 2.98 mg / ml, and 1.49 mg / ml, the Paris polyphylla Smith n-butyl alcohol extract had a significant inhibitory effect on ATCC 33787. This indicated that the Paris polyphylla Smith n-butyl alcohol extract had a good inhibitory effect on the biofilm of Vibrio alginolyticus ATCC 33787 and had important application value.

Claims

1. A Paris polyphylla extract, characterized in that, The Paris polyphylla extract is prepared according to the following steps: adding Paris polyphylla powder into an ethanol aqueous solution with a volume concentration of 80-95%, heating under reflux for extraction for 1-5 h, filtering the extract by suction, concentrating the filtrate under vacuum at 65 °C until no liquid flows out, and freeze-drying the thick liquid to obtain the Paris polyphylla extract.

2. The Paris polyphylla extract according to claim 1, wherein, The ethanol is an ethanol aqueous solution with a volume concentration of 95%.

3. The Paris polyphylla extract according to claim 1, characterized in that, The volume dosage of the ethanol aqueous solution is 10-30 L / kg based on the mass of the Paris polyphylla powder.

4. The Paris polyphylla extract according to claim 1, wherein, The reflux extraction is carried out according to the following steps: adding Paris polyphylla powder into the ethanol aqueous solution, performing the first reflux extraction for 3 h, filtering with three layers of gauze after cooling to obtain the first extract and the first residue; reusing the same ethanol aqueous solution for the second reflux extraction of the first residue for 3 h, filtering with three layers of gauze after cooling to obtain the second extract and the second residue; combining the first extract and the second extract; the volume of the ethanol aqueous solution used for the two extractions is 5-15 L / kg based on the mass of the Paris polyphylla powder, and the volume ratio of the ethanol aqueous solution used for the second extraction to that used for the first extraction is 0.5-1:

1.

5. The Paris polyphylla extract according to claim 1, wherein The freeze-drying is carried out at -4 °C and 0 Pa until the mass water content is lower than 5%.

6. The Paris polyphylla extract according to claim 1, wherein, The thick liquid is extracted and purified with an organic solvent after freeze-drying. The extraction method is as follows: dissolving and extracting the Paris polyphylla extract with an organic solvent, collecting the organic phase, concentrating under vacuum at 65 °C until no liquid flows out, and freeze-drying to obtain the Paris polyphylla extract; the organic solvent includes petroleum ether, ethyl acetate, and n-butanol.

7. Use of the Paris polyphylla extract according to claim 1 in the preparation of a marine pathogenic bacteria biofilm inhibitor.

8. The application according to claim 7, wherein The marine pathogenic bacteria include Vibrio alginolyticus ATCC33787.

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