CN113855719A - Application of malva seed extract in preparation of medicine for inhibiting bacterial quorum sensing system - Google Patents

Application of malva seed extract in preparation of medicine for inhibiting bacterial quorum sensing system Download PDF

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CN113855719A
CN113855719A CN202111354196.0A CN202111354196A CN113855719A CN 113855719 A CN113855719 A CN 113855719A CN 202111354196 A CN202111354196 A CN 202111354196A CN 113855719 A CN113855719 A CN 113855719A
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horseradish
seed extract
quorum sensing
extract
sensing system
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郑俊霞
夏志云
陈浩铭
陈婷婷
陈楚汕
黄钰景
方静妍
顾子敏
谢俊涛
刘坤
范红霞
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Guangdong University of Technology
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/31—Brassicaceae or Cruciferae (Mustard family), e.g. broccoli, cabbage or kohlrabi
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/10—Preparation or pretreatment of starting material
    • A61K2236/15—Preparation or pretreatment of starting material involving mechanical treatment, e.g. chopping up, cutting or grinding
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying

Abstract

The invention discloses an application of a horseradish seed extract in preparing a medicament for inhibiting a bacterial quorum sensing system; the horseradish seed extract can effectively inhibit a las signal channel and an pqs signal channel in a pseudomonas aeruginosa quorum sensing system, thereby inhibiting the secretion of elastase and pyocin, and simultaneously not inhibiting the growth of pseudomonas aeruginosa, so that the pseudomonas aeruginosa is not easy to generate drug resistance. In addition, the horseradish seeds are natural substances, the toxicity and the side effect are very low, and the horseradish seeds can be used independently and also can be used as an auxiliary therapeutic agent of antibiotics to achieve the aim of preventing and treating bacterial infection; belongs to the technical field of biological medicine.

Description

Application of malva seed extract in preparation of medicine for inhibiting bacterial quorum sensing system
Technical Field
The invention relates to the field of medicines, in particular to application of horseradish seed extraction in preparation of a bacterial quorum sensing inhibitor.
Background
Antibiotic abuse has made bacterial Resistance (AMR) a major public health threat worldwide, and the search for new drugs or more effective treatment regimens is urgent in the face of the growing problem of antibiotic Resistance worldwide. In 2017, the WHO published the most drug-resistant "superbacteria" in the world that threatened human health the most, with gram-negative bacteria, acinetobacter baumannii, pseudomonas aeruginosa and escherichia coli, listed as "severely lethal" grades. Traditional antibiotics can kill bacteria, so that drug-resistant strains are enriched continuously, and the drug resistance rate of the bacteria is improved continuously. If we want to accelerate the research and development of new targets and new mechanisms of novel drug-resistant bacteria-resistant drugs, otherwise, the development of new targets and new mechanisms of novel drug-resistant bacteria-resistant drugs will fall into the situation of no drug cure.
Quorum sensing system (QS) is a microbial cell-cell communication system, during the growth and reproduction process, bacteria can secrete and sense the concentration of certain signal molecules, when the concentration reaches a certain threshold value, the expression of each gene of quorum sensing system is started, and then the cells are regulated and controlled to make response in a quorum form so as to adapt to the environment, and the viability of the bacterial quorum is improved. In recent years, researches show that the pathogenicity of bacteria is closely related to a quorum sensing system, and the secretion of virulence factors such as pyocyanin, elastase, rhamnolipid, exotoxin, hemolysin and the like of the bacteria is regulated and controlled by the quorum sensing system. Among them, the formation of a biofilm, which provides a protective barrier for bacteria, is an important cause of drug resistance in bacteria. Therefore, the development of a novel quorum sensing inhibitor which can target a quorum sensing system of bacteria, reduce the pathogenic capability of the bacteria and simultaneously does not kill the bacteria can well solve the problem of bacterial drug resistance.
The pseudomonas aeruginosa is a gram-negative bacterium, is the most common clinical conditional pathogenic bacterium which is easy to form biomembrane resistance, and often causes diseases such as wound infection, urinary tract infection, cystic fibrosis and the like. Has extremely high lethality in patients with low immune function, such as cancer and AIDS patients. There are mainly 3 quorum sensing systems for pseudomonas aeruginosa: the las, rhl and PQS systems are mediated by three signal molecules with different chemical properties, namely 3-oxo-C12-HSL, C4-HSL and PQS, and the signal molecules can regulate and control the expression of downstream genes by combining with corresponding receptors, thereby regulating and controlling the expression of pathogenic factors such as elastase, pyocyanea, rhamnolipid and the like and the formation of a biological membrane. By inhibiting the quorum sensing of bacteria, the virulence regulated by the quorum sensing system of the bacteria can be inhibited, the harm of the bacteria to a host is reduced, and the aim of preventing or assisting antibiotics to treat bacterial infection is achieved. Therefore, the bacterial quorum sensing inhibitor has wide application prospect in the aspect of preventing and treating bacterial infection.
Horseradish, the name of Chinese, behenic, the name of Latin, Eutremayunnanense, is a cruciferous plant, a special edible health plant, and is mainly distributed in China and Japan. The history of medicine and food in China is recorded early, and the Chinese patent medicines are also called wasabi, potherb mustard, hollyhock, mallow and the like, and have the functions of immunoregulation and various pharmacological actions such as virus resistance, cancer resistance, oxidation resistance and the like. At present, the research on the malva seeds at home and abroad is less, and the effects of the malva seeds on the aspects of antibiosis and quorum sensing inhibition are not reported in documents.
Disclosure of Invention
In view of the above, the present invention provides a new application of horseradish seed, namely an application of horseradish seed extract in preparing a drug for inhibiting a bacterial quorum sensing system.
The invention takes pseudomonas aeruginosa as a research object to carry out an inhibition experiment of a biological membrane and virulence factors, and the result shows that the horseradish seed ethanol extract, the horseradish seed chloroform extract, the horseradish seed petroleum ether extract, the horseradish seed ethyl acetate extract, the horseradish seed n-butanol extract, the horseradish seed volatile oil and the horseradish seed CO are extracted from the horseradish seed2The supercritical extracts can inhibit the formation of a biofilm of pseudomonas aeruginosa PAO1 and the release of virulence factors, and the research on the inhibitory activity of a bacterial quorum sensing system is carried out by using PAO1-lasB-gfp and PAO1-pqsA-gfp green fluorescent protein reporter strains, so that the wasabi seed extract can inhibit the las and pqs quorum sensing systems of the pseudomonas aeruginosa, and the wasabi seed extract disclosed by the invention can further reduce the formation and the pathogenicity of the bacterial biofilm by inhibiting the quorum sensing system of bacteria, and has the effect of being difficult to generate bacterial drug resistance.
Therefore, the invention provides the application of the horseradish seed extract in preparing the inhibition drugs of the bacterial quorum sensing system.
The technical scheme is as follows:
the horseradish seed extract is horseradish seed ethanol extract, horseradish seed chloroform extract, horseradish seed petroleum ether extract, horseradish seed ethyl acetate extract, horseradish seed n-butanol extract, horseradish seed water extract, horseradish seed volatile oil and horseradish seed CO2At least one of the clinical extracts.
Preferably, the horseradish seed ethanol extract is 45% ethanol extract and/or horseradish seed 75% ethanol extract and/or horseradish seed 95% ethanol extract
Preferably, the preparation method of the malva seed extract is one of the following methods:
the method comprises the following steps: taking dried horseradish seeds, crushing, sieving with a 40-100-mesh sieve, adding an extraction solvent, soaking for 0-12 hours, performing ultrasonic-assisted extraction, removing insoluble substances, and drying to obtain a horseradish seed extract;
the method 2 comprises the following steps: taking dried horseradish seeds, crushing, sieving with a 40-100-mesh sieve, adding an extraction solvent, soaking for 0-12 hours, performing reflux extraction, removing insoluble substances, and drying to obtain a horseradish seed extract;
the method 3 comprises the following steps: taking dried horseradish seeds, crushing, sieving with a 40-100-mesh sieve, adding an extraction solvent, soaking at 4-25 ℃ for 0-72 hours, removing insoluble substances, and drying to obtain a horseradish seed extract;
the method 4 comprises the following steps: taking dry horseradish seeds, crushing, sieving with a 40-100-mesh sieve, adding deionized water, soaking for 0-12 hours, distilling at 100 ℃ for 2-5 hours, collecting an extracting solution, and removing water by using anhydrous sodium sulfate to obtain the horseradish seed volatile oil.
The method 5 comprises the following steps: taking dry horseradish seeds, crushing, sieving with a 40-100-mesh sieve, adding an extraction solvent, placing in a microwave digestion instrument, performing microwave-assisted extraction, removing insoluble substances, and drying to obtain the horseradish seed extract.
The method 6 comprises the following steps: taking dried horseradish seeds, crushing, sieving with a 40-100-mesh sieve, performing supercritical carbon dioxide extraction by using absolute ethyl alcohol as an entrainer, collecting an extract, and freeze-drying to obtain radish seed CO2Supercritical extraction.
Preferably, the extraction solvent is at least one of 30-100% ethanol solution, water, chloroform, petroleum ether, ethyl acetate and n-butanol.
Preferably, the insoluble matter removal is implemented by filtering and collecting filtrate or centrifuging and collecting supernatant.
Preferably, the drying is reduced pressure drying, vacuum drying or freeze drying.
Preferably, the adding amount of the extraction solvent is 10-30 times of the weight of the horseradish seeds, the extraction temperature is 10-150 ℃, the extraction times are 2-6, and the extraction time is 1-72 hours each time.
Furthermore, the results of biomembrane and virulence factor inhibition experiments show that the horseradish seed ethanol extract, the horseradish seed petroleum ether extract, the horseradish seed ethyl acetate extract and the horseradish seed volatile oil have obvious inhibition effects on biomembranes and virulence factors of pseudomonas aeruginosa and drug-resistant pseudomonas aeruginosa. Therefore, the horseradish seed extract is preferably 95% ethanol extract of horseradish seed, petroleum ether extract of horseradish seed, ethyl acetate extract of horseradish seed, and volatile oil of horseradish seedHair oil, wasabi seed CO2Beyond the limit of the extract.
The invention also adopts PAO1-lasB-gfp and PAO1-pqsA-gfp green fluorescent protein reporter strains to carry out corresponding detection, the two reporter strains are respectively inserted the promoters of target regulatory genes corresponding to a Las and PQS quorum sensing system into the upstream of the green fluorescent protein, and finally the activity of the Las and PQS regulatory systems in the bacteria is reflected by detecting the expression level (fluorescence measurement) of the green fluorescent protein in the bacteria.
Preferably, the bacteria comprise pseudomonas aeruginosa PAO 1.
Preferably, the medicament of the invention comprises an effective dose of the horseradish seed extract.
Preferably, the medicament also comprises pharmaceutically acceptable auxiliary materials.
The horseradish seed extract can be directly or indirectly added into various pharmaceutically acceptable common adjuvants such as filler, disintegrant, lubricant, binder, etc. for preparing different dosage forms by those skilled in the art, and can be made into common oral preparation or injection preparation by conventional pharmaceutical preparation method.
Preferably, the dosage form of the medicament is oral preparation, and preferably granules, capsules, tablets or pills.
Preferably, the effective dose of the malva sylvestris seed extract in the medicine is 10-90 wt%, more preferably 10-30 wt%, and the auxiliary material is preferably 10-90 wt%, more preferably 70-90 wt%
In some embodiments, the medicament is an anti-pseudomonas aeruginosa granule prepared from the following raw materials in parts by weight: 5-10 parts of horseradish seed ethanol extract, 5-20 parts of lactose and 0-10 parts of 10% starch slurry.
In some embodiments, the medicament is an anti-pseudomonas aeruginosa capsule prepared from the following raw materials in parts by weight: 10-30 parts of horseradish seed petroleum ether layer extract, 20-40 parts of lactose, 20-40 parts of microcrystalline cellulose and 0-10 parts of talcum powder.
In some embodiments, the medicament is an anti-pseudomonas aeruginosa tablet, and is prepared from the following raw materials in parts by weight: 10-20 parts of horseradish seed ethyl acetate layer extraction, 15-30 parts of lactose, 0-10 parts of 10% starch slurry, 0-10 parts of crospovidone and 0-5 parts of magnesium stearate.
In some embodiments, the medicament is a horseradish seed volatile oil anti-pseudomonas aeruginosa dropping pill prepared from the following raw materials in parts by weight: 5-20 parts of malva seed volatile oil and 20-60 parts of polyethylene glycol.
Compared with the prior art, the invention provides a new application of malva seeds, in particular to an application in preparing a medicament for inhibiting a bacterial quorum sensing system. The experimental results show that the horseradish seed extract is a good quorum sensing inhibitor, in particular to a horseradish seed ethanol extract, a horseradish seed petroleum ether extract, a horseradish seed ethyl acetate extract and a horseradish seed volatile oil, and can effectively inhibit a las signal channel and an pqs signal channel in pseudomonas aeruginosa so as to inhibit secretion of elastase and pyocin and formation of a biological membrane, further reduce bacterial virulence and pathogenicity and prevent the pseudomonas aeruginosa from generating drug resistance. In addition, the horseradish seeds are natural substances, the toxicity and the side effect are low, and the horseradish seeds can be used independently and also can be used as an auxiliary therapeutic agent of antibiotics to achieve the aim of preventing and treating bacterial infection.
Drawings
FIG. 1 shows the inhibition of Pseudomonas aeruginosa biofilm by different extracts of horseradish seed
FIG. 2 shows the inhibition of Pseudomonas aeruginosa elastase by different extracts of horseradish seeds
FIG. 3 shows the inhibition of Pseudomonas aeruginosa pyocin by different extracts of horseradish seed
FIG. 4 Effect of different Horseradish seed extracts on the las System
FIG. 5 Effect of different Horseradish seed extracts on pqs System
FIG. 6 is a graph showing the inhibition of Pseudomonas aeruginosa biofilm by different drug forms
FIG. 7 mouse protection against Pseudomonas aeruginosa infection by different drug formulations
FIG. 8 LC-MS total ion flow diagram of 95% ethanol extract of malva sylvestris seeds
Detailed Description
The claims of the present application are described in further detail below with reference to specific embodiments and the description of the figures.
The raw materials used in the examples of the invention are all commercially or self-made, wherein the PAO1-lasB-gfp green fluorescent protein reporter strain, the PAO1-pqsA-gfp green fluorescent protein reporter strain and the Pseudomonas aeruginosa PAO1 are all obtained from the university of Nanyang science and technology.
Example 1: preparation of 95% ethanol extract of malva seed
(1) Weighing radix Helianthi seed 1k g, pulverizing with a pulverizer, sieving with 60 mesh sieve,
(2) according to the material-liquid ratio of 1: 20, adding 20L of 95% (v/v) ethanol solution, performing ultrasonic-assisted extraction with ultrasonic power of 350W, extracting at 50 deg.C for 30min, repeating the extraction for 3 times,
(3) filtering to remove insoluble substances, mixing the three extractive solutions, and concentrating under reduced pressure to about 100ml
(4) Placing the concentrated solution in a refrigerator at-80 deg.C for pre-freezing, and freeze-drying to obtain powder, i.e. 183g of 95% ethanol extract of radix Semiaquilegiae seed.
Example 2: preparation of 75% ethanol extract of malva seed
(1) Accurately weighing radix Semiaquilegiae seed 1k g, pulverizing with pulverizer, and sieving with 60 mesh sieve
(2) According to the material-liquid ratio of 1: 20, adding 20L of 75% (v/v) ethanol solution, soaking for 8h, heating and refluxing for extraction for 3 times, wherein the extraction temperature is 80 ℃, and the extraction time is 2 h.
(3) Filtering to remove insoluble substances, mixing the extractive solutions for 3 times, testing, and concentrating to about 100 mL;
(4) placing the concentrated solution at-80 deg.C for pre-freezing, and freeze-drying to obtain powder, to obtain 226g of 75% ethanol extract of radix Semiaquilegiae seed.
Example 3: preparation of 45% ethanol extract of malva seed
(1) Accurately weighing the malva seed 1k g, crushing by a crusher, and sieving by a 60-mesh sieve;
(2) according to the material-liquid ratio of 1: 30, adding 30L of 45% (v/v) ethanol solution, leaching at 4-25 deg.C for 72h, filtering to remove insoluble substances, and concentrating under reduced pressure to about 100mL
(3) Placing the concentrated solution at-80 deg.C for pre-freezing, and freeze-drying to obtain powder, to obtain 226g of 45% ethanol extract of radix Semiaquilegiae seed.
Example 4: preparation of horseradish seed chloroform, petroleum ether, ethyl acetate and n-butanol extract
(1) Weighing 5kg of malva seeds, fully crushing by using a crusher, sieving by a 60-mesh sieve,
(2) taking 4 clean conical flasks, adding 1kg of horseradish seed powder into each conical flask,
(3) each conical flask is filled with a liquid material according to the ratio of 1: 10, respectively adding 10L of chloroform, petroleum ether, ethyl acetate and n-butanol, placing in a microwave digestion instrument, extracting at the extraction power of 350W and the temperature of 45 ℃ for 60min according to set parameters, and repeating for 3 times;
(4) filtering to remove insoluble substances, mixing the three extractive solutions, and concentrating under reduced pressure to about 100ml
(5) Placing the concentrated solution in a refrigerator at-80 deg.C for pre-freezing, and freeze-drying to obtain powder, to obtain horseradish seed chloroform extract 188g, petroleum ether extract 233g, ethyl acetate extract 216g, and n-butanol extract 176 g.
Example 5: preparation of malva seed volatile oil
(1) Accurately weighing the malva seed 1k g, crushing by a crusher, and sieving by a 60-mesh sieve;
(2) putting horseradish seed powder into a round-bottom flask, and mixing the materials according to a material-liquid ratio of 1: 10 adding 10L of deionized water and adding zeolite;
(3) the round-bottom flask is connected with a volatile oil extractor and a condensing device, and is heated by a constant-temperature electric heating jacket at 100 ℃, distilled for 5 hours, and then an oil layer in the volatile oil extracting solution is collected;
(4) removing excessive water from the above volatile oil extractive solution with sodium sulfate to obtain horseradish seed volatile oil 18.25 g.
Example 6: preparing supercritical extract of radix Helianthi seed CO 2.
(1) Accurately weighing 1kg of dried malva seeds, crushing, sieving with a 60-mesh sieve, loading into an extraction tank, taking absolute ethyl alcohol as an entrainer,
(2) the extraction pressure is 32Mpa, and the extraction temperature is 48 ℃;
(3) the pressure of the analysis I is 8MPa, the pressure of the analysis II is 12MPa, the analysis temperature is 42 ℃, the flow rate of carbon dioxide is 22L/h, and the extraction time is 2 h.
(4) Collecting extractive solution, concentrating with rotary evaporator to obtain extract, and freeze drying to obtain radix Helianthi seed CO2Extract 201g
Example 7: influence of different extracts of malva sylvestris seeds on pseudomonas aeruginosa biofilm formation
Test samples: the 95% ethanol extract of the horseradish seeds in example 1, the 75% ethanol extract of the horseradish seeds in example 2, the 45% ethanol extract of the horseradish seeds in example 3, the chloroform extract of the horseradish seeds, the petroleum ether extract of the horseradish seeds, the ethyl acetate extract of the horseradish seeds, the n-butanol extract of the horseradish seeds in example 4, the volatile oil of the horseradish seeds in example 5, and the CO extract of the horseradish seeds in example 6 are taken2Beyond the limit of the extract. The stock solution was prepared in DMSO solution to 32mg/mL for use.
The experimental method comprises the following steps: inoculating Pseudomonas aeruginosa PAO1 in LB culture medium, culturing in shaking table at 37 deg.C and 200rmp overnight, diluting the bacterial liquid with ABTGC culture medium to OD600And (5) preparing the product for later use, wherein the product is 0.01. Diluting the drug-containing mother liquor with an ABTGC culture medium, adding 100 mu L of the drug-containing culture medium into a 96-well plate, then inoculating 100 mu L of the bacterial liquid into each well, finally enabling the concentration of the horseradish seed extract in the wells to be 32 mu g/mL, setting a positive control group (azithromycin with the concentration of 32 mu g/mL), culturing at 37 ℃ for 24 hours, and determining the formation amount of a biological membrane by adopting a crystal violet method, wherein the specific operation is as follows:
(1) gently sucking away the bacterial liquid by a gun, washing for 2 times by 120 mu L/hole PBS, and drying;
(2) adding 120 μ L methanol into each well, and fixing at room temperature for 20 min;
(3) sucking methanol away, drying, adding 120 μ L of filtered 0.1% crystal violet, and dyeing for 15 min;
(4) the dye was aspirated off, washed three times with PBS;
(5) after drying, 120. mu.L of 33% glacial acetic acid was added to dissolve crystal violet, and OD was measured570，OD570Of valueThe level indirectly reflects the content of the biological membrane.
The experimental result shows that different extracts of the horseradish seeds can inhibit the formation of a pseudomonas aeruginosa biomembrane, wherein the inhibition effects of the 95% ethanol extract of the horseradish seeds, the petroleum ether extract of the horseradish seeds, the ethyl acetate extract of the horseradish seeds and the volatile oil of the horseradish seeds are obvious, and the inhibition rate is similar to that of a positive medicament or even higher than that of the positive medicament.
Example 8: the 95% ethanol extract of the horseradish seeds in example 1, the 75% ethanol extract of the horseradish seeds in example 2, the 45% ethanol extract of the horseradish seeds in example 3, the chloroform extract of the horseradish seeds, the petroleum ether extract of the horseradish seeds, the ethyl acetate extract of the horseradish seeds, the n-butanol extract of the horseradish seeds in example 4, the volatile oil of the horseradish seeds in example 5, and the CO extract of the horseradish seeds in example 6 are taken2Beyond the limit of the extract. The stock solution was prepared in DMSO solution to 32mg/mL for use.
The experimental method comprises the following steps: inoculating Pseudomonas aeruginosa PAO1 in LB culture medium, culturing at 37 deg.C overnight, diluting the bacterial liquid with ABTGC culture medium to OD600Taking 50mL of the bacterial solution (0.01).)
In a 100mL conical flask, the corresponding compound is added to the desired concentration, placed in 37 ℃ shaking table, 200r/min, cultured for 24 hours. After the bacteria grow for 24 hours, the bacteria are all mixed uniformly, OD600 is measured, 5mL of bacterial liquid is taken, 10000rpm/min is adopted, and centrifugation is carried out for 10min at 4 ℃. And filtering the mixture once by using a 0.22 mu m filter according to the operating steps of a Kit EnzChekfi protein assay Kit E6638, configuring the concentration of the elastin to be 50 mu g/mL by using 1 × reactionbuffer, adding 100 mu L of each of the bacterial supernatant and the prepared substrate, slightly mixing the mixture, putting the mixture into a microplate reader immediately, and measuring fluorescence (excitation wavelength is 400nm and emission wavelength is 450nm) every 10min for 4 hours continuously, wherein the height of the fluorescence content indicates the content of the elastase, and the OD values obtained by detection are divided by OD600 to remove the influence of bacterial density.
The experimental results are as follows: as can be seen from fig. 2, the horseradish seed extract can inhibit secretion of pseudomonas aeruginosa elastase, wherein the horseradish seed 95% ethanol extract, the horseradish seed volatile oil, the horseradish seed petroleum ether extract, the horseradish seed ethyl acetate extract and the horseradish seed 75 ethanol extract have obvious inhibition effect on elastase, so that the horseradish seed extract can inhibit bacterial elastase powder, and further reduce the pathogenicity of the bacterial elastase powder.
Example 9: influence of different extracts of malva seeds on pseudomonas aeruginosa pyocin secretion
Test samples: the 95% ethanol extract of the horseradish seeds in example 1, the 75% ethanol extract of the horseradish seeds in example 2, the 45% ethanol extract of the horseradish seeds in example 3, the chloroform extract of the horseradish seeds, the petroleum ether extract of the horseradish seeds, the ethyl acetate extract of the horseradish seeds, the n-butanol extract of the horseradish seeds in example 4, the volatile oil of the horseradish seeds in example 5, and the CO extract of the horseradish seeds in example 6 are taken2Beyond the limit of the extract. The stock solution was prepared in DMSO solution to 32mg/mL for use.
The experimental method comprises the following steps: inoculating Pseudomonas aeruginosa PAO1 in LB culture medium, culturing at 37 deg.C overnight, diluting the bacterial liquid with ABTGC culture medium to OD6000.01. 50mL of the bacterial solution is taken in a 100mL conical flask, the corresponding compound is added to the conical flask to reach the required concentration, and the conical flask is placed in a 37 ℃ shaking table and cultured for 24 hours at 200 r/min. After the bacteria grow for 24 hours, the bacteria are all mixed uniformly, OD600 is measured, 5mL of bacterial liquid is taken, the bacteria in the conical flask are transferred to a 50mL centrifuge tube, 10000r/min and centrifugation is carried out for 10min at 4 ℃. Centrifuging, collecting supernatant 40mL, extracting with 3mL chloroform, transferring chloroform layer (lower layer) to new tube, adding 0.3 times of chloroform 0.2M HCl, centrifuging, collecting upper layer, and measuring OD520，OD520The content of the pyocin is reflected.
The experimental results are as follows: as can be seen from fig. 3, different extracts of the horseradish seed can inhibit secretion of pseudomonas aeruginosa pyocin to a certain extent, wherein the inhibition effects of the 95% ethanol extract of the horseradish seed, the petroleum ether extract of the horseradish seed and the volatile oil of the horseradish seed are the most obvious, and the secretion of pyocin is regulated and controlled by a bacterial quorum sensing pqs system, which indicates that the horseradish seed extract can inhibit secretion of pyocin by inhibiting a pqs system.
Example 10: effect of different extracts of Malva sylvestris seeds on the las System of Pseudomonas aeruginosa
Test samples: alcohol extract, 45% ethanol extract of horseradish seed in example 3, chloroform extract of horseradish seed, petroleum ether extract of horseradish seed, ethyl acetate extract of horseradish seed, n-butanol extract of horseradish seed in example 4, volatile oil of horseradish seed in example 5, CO extract of horseradish seed in example 62Beyond the limit of the extract. The stock solution was prepared in DMSO solution to 32mg/mL for use.
The experimental method comprises the following steps: the PAO1-lasB-gfp strain was inoculated in LB medium and after overnight culture, diluted to OD in ABTGC medium600After diluting the horseradish seed extract of the present invention with ABTGC medium 0.01, 100 μ L was taken in a 96-well plate, and finally 100 μ L of drug-containing ABTGC medium was contained in each well. Next, 100. mu.L of the bacterial suspension was added to each well of a 96-well microtiter plate to give a final concentration of the compound of the invention per well of 32. mu.g/mL. And simultaneously setting a control group. The 96-well microtiter plates were incubated at 37 ℃ in a TecanInfinate 200Pro plate reader for at least 16 hours, with the following parameters measured every 15 minutes: OD600, GFP fluorescence signal (excitation 485nm, emission 535 nm). Inhibition assays for all test samples and controls were performed in triplicate.
The experimental results are as follows: as can be seen from FIG. 4, the horseradish seed extract can inhibit the expression of green fluorescent protein in PAO1-lasB-gfp, and in the report strain PAO1-lasB-gfp, the selected target regulatory gene is a lasB gene, namely an elastase gene mainly regulated by a Las system, and the initiation of the target regulatory gene is mainly regulated by a transcription factor LasR, so that the fluorescent expression amount in the PAO1-lasB-gfp can directly reflect the LasR activity in bacteria. The 95% ethanol extract of the horseradish seed, the petroleum ether extract of the horseradish seed, the ethyl acetate extract of the horseradish seed, the volatile oil of the horseradish seed and the 75% ethanol extract of the horseradish seed have good inhibitory activity, so the horseradish seed extract can further inhibit the secretion of elastase and reduce the pathogenicity of bacteria by inhibiting a las system in a pseudomonas aeruginosa quorum sensing system.
Example 11: effect of different extracts of Malva sylvestris seeds on Pseudomonas aeruginosa pqs system
Test samples: alcohol extractionThe extract is prepared from 45% ethanol extract of Hibiscus sabdariffa seed in example 3, chloroform extract of Hibiscus sabdariffa seed, petroleum ether extract of Hibiscus sabdariffa seed, ethyl acetate extract of Hibiscus sabdariffa seed, n-butanol extract of Hibiscus sabdariffa seed in example 4, volatile oil of Hibiscus sabdariffa seed in example 5, and CO of Hibiscus sabdariffa seed in example 62Beyond the limit of the extract. The stock solution was prepared in DMSO solution to 32mg/mL for use.
The experimental method comprises the following steps: same as example 4
The experimental results are as follows: as can be seen from FIG. 5, the horseradish seed extract can inhibit the expression of green fluorescent protein in PAO1-pqsA-gfp, in the report strain PAO1-pqsA-gfp, the selected target regulation gene is pqsA which is the first gene regulated and controlled by a pqs system, and can code the generation of pyocyanin virulence factors, and the fluorescent expression quantity in the PAO1-pqsA-gfp after the compound treatment can directly reflect the expression activity of the pqsA in bacteria, so that the influence of the compound on the pqs system is judged. Therefore, the horseradish seed extract can further inhibit the secretion of pyocin and reduce the pathogenicity of bacteria by inhibiting a bacterial quorum sensing pqs system.
Example 12: malva sylvestris seed 95% ethanol extract anti-pseudomonas aeruginosa quorum sensing granule
The present example provides a pseudomonas aeruginosa resistant quorum sensing particle, which has the following formula:
Figure BDA0003353731230000121
Figure BDA0003353731230000131
the preparation steps of the pseudomonas aeruginosa resistant granules are as follows:
(1) firstly, uniformly mixing 95% ethanol extract of malva seed with lactose, mannitol and CMS-Na;
(2) then adding the prepared 10% starch slurry into the mixture, and sieving the mixture by a 16-mesh sieve to prepare a soft material;
(3) drying at 60 ℃ in a vacuum drying oven, and finishing granules to obtain the anti-pseudomonas aeruginosa quorum sensing granules.
Example 13: malva sylvestris seed petroleum ether extract anti-pseudomonas aeruginosa quorum sensing capsule
The embodiment of the invention provides a pseudomonas aeruginosa-resistant capsule, which comprises the following formula:
Figure BDA0003353731230000132
the preparation method of the pseudomonas aeruginosa resistant capsule comprises the following steps:
(1) firstly, mixing the malva seed petroleum ether extract with lactose for 5-15 minutes;
(2) adding microcrystalline cellulose and mixing for 5-15 minutes;
(3) adding talcum powder and mixing for 3-6 min;
(4) and filling the mixture into a gelatin capsule shell, namely the pseudomonas aeruginosa resistant capsule.
The above-mentioned embodiments are only used for illustrating the technical solutions of the present invention, and not for limiting the same; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Example 14: malva sylvestris seed ethyl acetate anti-pseudomonas aeruginosa quorum sensing tablet
The invention provides a pseudomonas aeruginosa resistant tablet, which comprises the following components in percentage by weight:
15mg of an ethyl acetate layer extract of malva seed;
20mg of lactose;
6mg of 10% starch slurry;
9mg of crospovidone;
magnesium stearate 3 mg;
the preparation steps of the pseudomonas aeruginosa resistant tablet are as follows:
(1) mixing the horseradish seed ethyl acetate layer extract with lactose for 5-15 min;
(2) adding 10% starch slurry to make soft material, sieving with 14 mesh sieve, granulating, drying, sieving with 12 mesh sieve, and grading;
(3) then adding the crospovidone and the magnesium stearate, and mixing for 3-6 minutes;
(4) and tabletting after uniformly mixing to obtain the pseudomonas aeruginosa resistant tablet.
Example 15: horseradish seed volatile oil anti-pseudomonas aeruginosa dropping pill
The invention provides an anti-pseudomonas aeruginosa dropping pill, which comprises the following formula:
10mg of lotus plumule volatile oil;
50mg of polyethylene glycol;
the preparation method of the anti-pseudomonas aeruginosa dropping pill comprises the following steps:
(1) respectively weighing horseradish seed volatile oil and polyethylene glycol, heating the polyethylene glycol to be molten, adding the lotus plumule volatile oil under stirring, and uniformly mixing to obtain a mixed solution;
(2) dripping pills: adding the mixed solution into a dripping device of a dripping pill machine, dripping into condensate through a dripper, adjusting a temperature control system of the dripping pill machine, keeping the temperature of the dripper of the dripping pill machine at 80 ℃, the gradient temperature of the condensate at 35 ℃, and the inner diameter of a dripping pipe opening at 3 mm;
(3) wiping pills: taking out the pills, and removing surface condensate to obtain the anti-pseudomonas aeruginosa dropping pills.
EXAMPLE 164 inhibition of biofilms by different forms of a drug that is quorum sensing against Pseudomonas aeruginosa
Test samples: the method for testing the inhibitory activity of the horseradish seed volatile oil on the pseudomonas aeruginosa biofilm is the same as that in example 4 except that the horseradish seed 95% ethanol extract anti-pseudomonas aeruginosa quorum sensing granule in example 12 is taken, the horseradish seed petroleum ether extract anti-pseudomonas aeruginosa quorum sensing tablet prepared in example 13 is taken, the horseradish seed ethyl acetate extract anti-pseudomonas aeruginosa quorum sensing tablet prepared in example 14 is taken, and the horseradish seed volatile oil anti-pseudomonas aeruginosa quorum dropping pill prepared in example 15 is taken.
The experimental results are as follows: as shown in figure 6, the 4 formulations of medicines for resisting pseudomonas aeruginosa quorum sensing have obvious biofilm inhibition activity and have equivalent effect with positive medicines, which shows that the horseradish seed extract medicine for resisting pseudomonas aeruginosa quorum sensing has obvious curative effect, can further inhibit the generation of bacterial biofilms by inhibiting a quorum sensing system of bacteria, can be singly used, and can also be used as an auxiliary therapeutic agent of antibiotics to achieve the purpose of preventing and treating bacterial infection.
Example 17: 4 preparations have the protective effect on the infected mice against pseudomonas aeruginosa medicaments.
Test samples: the method comprises the steps of taking the horseradish seed 95% ethanol extract anti-pseudomonas aeruginosa quorum sensing granules in the embodiment 12, taking the horseradish seed petroleum ether extract anti-pseudomonas aeruginosa quorum sensing tablets prepared in the embodiment 13, taking the horseradish seed ethyl acetate extract anti-pseudomonas aeruginosa quorum sensing tablets prepared in the embodiment 14, taking the horseradish seed volatile oil anti-pseudomonas aeruginosa quorum dropping pill prepared in the embodiment 15, and preparing mother liquor with the effective concentration of 32mg/mL by using DMSO solution for later use.
The experimental method comprises the following steps: inoculating Pseudomonas aeruginosa to LB culture medium containing the above medicine, culturing overnight, centrifuging to collect thallus, resuspending with PBS thallus and diluting to 1 × 108CFU/mL, with DMSO as a negative control group, dividing 60 male mice of 7 weeks of age into 6 groups at random, each group containing 10 mice, injecting 100 μ L of bacterial resuspension liquid treated with different drug formulations into the abdominal cavity of each group of mice, injecting pure PBS solution as a blank control group, and observing the mortality of the mice within 1 week.
The experimental results are as follows: as shown in fig. 7, the survival rate of the mice in the blank control group is 100%, the survival rate of the mice in the negative control group is only 20%, and different radish seed anti-pseudomonas aeruginosa drugs have a certain protection effect on infected mice, wherein the effect of the radish seed 95% ethanol extract anti-pseudomonas aeruginosa quorum sensing granules and the effect of the radish seed petroleum ether extract anti-pseudomonas aeruginosa quorum sensing capsules are the best, and the survival rates of the mice respectively reach 90% and 80%.
Example 18: LC-MS analysis of Malva Asiatica seed extract
The experimental method comprises the following steps: qualitative analysis of the horseradish seed extract from example 1 was performed by Q-active quadrupole-electrostatic orbitals high resolution mass spectrometer using a hyperspil GOLD C18 column, mobile phase a: 0.1% aqueous formic acid, mobile phase B: acetonitrile solution, chromatographic conditions: 0-5 min, 5% → 15% acetonitrile, 5-15 min, 15% → 30% acetonitrile, 15-25 min, 30% → 100% acetonitrile, 25-30 min, 100% acetonitrile, the mass spectrum scanning mode is a positive and negative ion scanning mode, the scanning range is as follows: 70-1000m/z, experimental results: as shown in fig. 8.

Claims (10)

1. Application of Malva sylvestris seed extract in preparing medicine for inhibiting bacterial quorum sensing system is provided.
2. The use of the wasabi seed extract as claimed in claim 1 in the preparation of a medicament for inhibiting a bacterial quorum sensing system, wherein the wasabi seed extract is prepared by the steps of:
1) taking dried horseradish seeds, crushing, sieving with a 40-100-mesh sieve, and mixing according to a feed liquid mass ratio of 1: 15-25 adding an extraction solvent;
2) repeatedly extracting for 30min at 50 deg.C and ultrasonic power of 350W for 3 times, filtering to remove insoluble substances, mixing extractive solutions, and concentrating under reduced pressure;
3) placing the concentrated solution obtained in the step 2) in a refrigerator for pre-freezing, and freeze-drying to obtain powder, namely the horseradish seed extract.
Or:
the horseradish seed extract is prepared by the following steps:
1) taking dried horseradish seeds, crushing, sieving with a 40-100-mesh sieve, and mixing according to a feed liquid mass ratio of 1: 15-25 adding an extraction solvent;
2) soaking for 8h, heating and reflux-extracting for 3 times at 80 deg.C for 2h, and repeating the extraction for 3 times; filtering to remove insoluble substances, mixing the extractive solutions for 3 times, and concentrating under reduced pressure;
3) placing the concentrated solution obtained in the step 2) in a refrigerator for pre-freezing, and freeze-drying to obtain powder, namely the horseradish seed extract.
Or: the horseradish seed extract is prepared by the following steps:
1) taking dried horseradish seeds, crushing, sieving with a 40-100-mesh sieve, and mixing according to a feed liquid mass ratio of 1: 25-35 adding an extraction solvent;
2) leaching for 72 hours at 4-25 ℃, filtering to remove insoluble substances, and concentrating under reduced pressure;
3) placing the concentrated solution obtained in the step 2) in a refrigerator for pre-freezing, and freeze-drying to obtain powder, namely the horseradish seed extract.
3. Use of the wasabi seed extract according to claim 2, wherein the solvent is 45% -95% (v/v) ethanol.
4. The use of the wasabi seed extract as claimed in claim 1 in the preparation of a medicament for inhibiting a bacterial quorum sensing system, wherein the wasabi seed extract is prepared by the steps of:
1) taking dried horseradish seeds, crushing, sieving with a 40-100-mesh sieve, and mixing according to a feed liquid mass ratio of 1: adding extraction solvent 8-12 times, placing in a microwave digestion instrument, performing microwave-assisted extraction with extraction power of 300-,
2) filtering to remove insoluble substances, mixing the three extractive solutions, and concentrating under reduced pressure;
3) placing the concentrated solution obtained in the step 2) in a refrigerator for pre-freezing, and freeze-drying to obtain powder, namely the horseradish seed extract.
5. The use of the wasabi seed extract in the preparation of a medicament for inhibiting a bacterial quorum sensing system according to claim 4, wherein the solvent is one of chloroform, petroleum ether, ethyl acetate, and n-butanol.
6. The use of the wasabi seed extract as claimed in claim 1 in the preparation of a medicament for inhibiting a bacterial quorum sensing system, wherein the wasabi seed extract is prepared by the steps of:
1) taking dried horseradish seeds, crushing, sieving with a 40-100-mesh sieve, and mixing according to a feed liquid mass ratio of 1: 8-12 deionized water, and adding zeolite;
2) soaking for 8h, placing in a round bottom flask, connecting volatile oil extractor and condensing device, heating with constant temperature electric heating jacket at 80-100 deg.C, distilling for 1-5h, and collecting oil layer in the volatile oil extractive solution;
3) removing excessive water from the above volatile oil extractive solution with sodium sulfate to obtain horseradish seed volatile oil. (ii) a
7. The use of the wasabi seed extract as claimed in claim 1 in the preparation of a medicament for inhibiting a bacterial quorum sensing system, wherein the wasabi seed extract is prepared by the steps of:
1) taking dried horseradish seeds, crushing, sieving with a 40-100-mesh sieve, and filling into an extraction tank;
2) carrying out supercritical carbon dioxide extraction by using absolute ethyl alcohol as an entrainer, wherein the extraction pressure is 30-36 Mpa, and the extraction temperature is 45-48 ℃; the desorption pressure is 9-20 Mpa, the desorption temperature is 40-46 ℃, the flow rate of carbon dioxide is 18-30L/h, and the extraction time is 1-3 h.
8. Use of horseradish seed extract as claimed in claim 1 in the manufacture of a medicament for inhibiting the quorum sensing system in bacteria, wherein the quorum sensing system signal pathway is the las or/and pqs pathway.
9. Use of a horseradish seed extract according to claim 1 in the manufacture of a medicament for inhibiting a bacterial quorum sensing system, wherein the bacteria in the bacterial quorum sensing system are pseudomonas aeruginosa.
10. The use of the wasabi seed extract as claimed in claim 1 in the preparation of a medicament for inhibiting a bacterial quorum sensing system, wherein the wasabi seed extract is prepared into an oral preparation by using pharmaceutically acceptable excipients; wherein the effective dose of the malva sylvestris seeds in the oral preparation is 10-90 wt%.
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