阅读说明：本技术 一种夏枯草黄酮的新应用 (New application of selfheal flavone ) 是由 郑俊霞 谢俊涛 黄智霖 陈鸿庚 黄家晋 王立抗 杨超 田文月 黄华容 张蓝月 苏 于 2020-08-10 设计创作，主要内容包括：本发明涉及医药技术领域,尤其涉及一种夏枯草黄酮的新应用。本发明公开了夏枯草黄酮的新应用,特别是在制备抑制细菌群体感应系统药物中的应用、在制备抑制细菌绿脓素药物中的应用、在制备抑制细菌弹性蛋白酶分泌药物中的应用和在制备抑制细菌生物膜形成药物中的应用。本发明夏枯草可以通过抑制群体感应系统达到细菌毒力因子的同时不抑制菌株生长,不易产生细菌耐药性的效果,发挥抑制铜绿假单胞菌的作用。另外,夏枯草黄酮是天然物质,其毒性和副作用很低,夏枯草黄酮可以单独使用,也可以作为抗生素的辅助治疗剂达到防治细菌感染的目的。(The invention relates to the technical field of medicines, in particular to a new application of selfheal flavone. The invention discloses a new application of selfheal flavone, in particular to an application in preparing a medicament for inhibiting a bacterial quorum sensing system, an application in preparing a medicament for inhibiting bacterial pyocyanin, an application in preparing a medicament for inhibiting bacterial elastase secretion and an application in preparing a medicament for inhibiting bacterial biofilm formation. The selfheal can achieve the effect of inhibiting bacterial virulence factors and bacterial strain growth simultaneously by inhibiting a quorum sensing system, is not easy to generate bacterial drug resistance, and plays a role in inhibiting pseudomonas aeruginosa. In addition, the prunella vulgaris flavone is a natural substance, has low toxicity and side effects, can be used independently, and can also be used as an auxiliary therapeutic agent of antibiotics to achieve the purpose of preventing and treating bacterial infection.)

1. Application of Prunellae Spica flavone in preparing medicine for inhibiting bacterial quorum sensing system is provided.

2. The use of claim 1, wherein said prunella flavonoid is used to inhibit the las signaling pathway of the bacterial quorum sensing system.

3. The use of claim 2, wherein said prunella vulgaris flavones are used to inhibit the pqs signal pathway of the bacterial quorum sensing system.

4. Application of Prunellae Spica flavone in preparing medicine for inhibiting bacterial pyocyanin is provided.

5. Application of Prunellae Spica flavone in preparing medicine for inhibiting secretion of bacterial elastase is provided.

6. Application of Prunellae Spica flavone in preparing medicine for inhibiting bacterial biofilm formation is provided.

7. The use according to any one of claims 1 to 6, wherein the bacteria comprise Pseudomonas aeruginosa.

8. The use of claim 7, wherein the medicament further comprises a pharmaceutically acceptable excipient.

9. The use of claim 7, wherein the medicament is in a dosage form for oral administration.

10. The use according to claim 7, wherein the effective amount of selfheal-clomazone in the medicament is 10 to 90 wt%.

Technical Field

The invention relates to the technical field of medicines, in particular to a new application of selfheal flavone.

Background

Because the antibiotics have more advantages, the antibiotics are widely applied in clinic and have more obvious clinical curative effect. However, with the abuse of antibiotics, the problem of bacterial drug resistance is highlighted continuously, such as modification or destruction of the antibiotics by enzyme production, membrane permeability change, change or generation of new target sites, active efflux of antibiotics and the like, so that the search for drugs capable of replacing antibiotics is very important.

In recent years, the bacterial Quorum Sensing (QS) system has become an important target for the study of novel drug-resistant bacterial drugs. The quorum sensing system is a system for overcoming host infection defense mechanism by bacteria, controls and coordinates the behavior of the whole bacterial population by monitoring the concentration of certain signal molecules (also called self-induced molecules), and responds to the surrounding environmental stimulus together, thereby greatly enhancing the viability of the whole bacterial population. Intercellular signaling enables the bacteria to overcome host defense mechanisms. Small amounts of bacterial extracellular virulence factors may cause the host to respond by effectively neutralizing these complexes, however co-expression of the virulence genes of the flora up to a certain density may cause the bacteria to secrete high levels of extracellular virulence factors, thus overcoming the host defense mechanisms.

Therefore, the search for signal pathway inhibitors that inhibit intercellular signaling is one of the important approaches to the resolution of bacterial resistance to antibiotics. The commonly used signal path inhibitors include furanones, pyrrolines, diketopiperazines, substituted acyl homoserine lactones, oligopeptides and hormones, but these have poor inhibitory effect and even have toxic and side effects on human body.

Nearly ten thousand medicinal plants in China have abundant traditional Chinese medicine and natural medicine resources, and provide a foundation for research and development of new medicines, so that many researchers aim at the traditional Chinese medicine resources. Moreover, the traditional Chinese medicine or natural medicine is used for resisting bacteria and diminishing inflammation, adverse reactions and side effects caused by using antibiotics can be reduced, and drug resistance is not easy to generate. The traditional Chinese medicine with antibacterial and anti-inflammatory effects is searched to be used as an alternative product or an auxiliary product of antibiotics, and the world problem of antibiotic resistance can be well solved.

Prunellae Spica (Prunella vulgaris L.) is a plant of Labiatae, and its medicinal part is dry cluster. Mainly produced in Jiangsu, Henan, Hubei, Hunan and other places, and has the effects of clearing heat and purging fire, resolving masses, clearing liver, improving eyesight, relieving pain and reducing swelling. Modern clinical application is mainly used for treating thyromegaly, scrofula, hyperplasia of mammary glands, hypertension, pulmonary tuberculosis, acute icteric infectious hepatitis and other diseases. The flavonoid compound is one of the main active ingredients of the selfheal, and although the flavonoid compound is widely applied to the industries of food, medicine and the like, the research mainly focuses on the aspects of oxidation resistance, free radical resistance and elimination, blood pressure reduction, inflammation diminishing and the like, and the flavonoid compound has no function of inhibiting a bacterial quorum sensing system at present.

Disclosure of Invention

In view of the above, the invention provides a new application of prunella vulgaris flavone, which can achieve the effects of inhibiting bacterial pyocyanin, inhibiting bacterial elastase secretion and forming bacterial biofilms by inhibiting a bacterial quorum sensing system, but does not influence the growth of bacteria and is not easy to generate bacterial drug resistance.

The specific technical scheme is as follows:

the invention provides application of selfheal flavone in preparation of a medicament for inhibiting a bacterial quorum sensing system.

Preferably, the bacteria comprise pseudomonas aeruginosa.

Preferably, the medicament further comprises pharmaceutically acceptable auxiliary materials.

Preferably, the dosage form of the medicine is an oral preparation, and granules, capsules, tablets or dripping pills are preferred.

Preferably, the effective dose of the selfheal clomazone 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%.

Pseudomonas Aeruginosa (PA) can cause various acute and chronic infections and even lead to death of a host, and is one of three general germs of hospital-acquired infections, and currently, the studied and clear quorum sensing regulation and control systems of Pseudomonas aeruginosa mainly include three: including the las, rhl and PQS systems, in which the las and rhl system is a system using homoserine lactone (3-oxo-C12-HSL, C4-HSL) as a signal molecule, and the PQS system is a system using quinolone (PQS) as a signal molecule. In the regulatory network, pqs system plays a key role in the las system and rhl system, on one hand, las and rhl control the generation of pqs, on the other hand, pqs influences the gene expression of las and rhl, and a delicate balance exists between the two, thereby regulating the expression of a series of genes related to virulence factors. lasB is a regulatory gene of the las system, which is involved in the secretion of virulence factors elastase, and the synthesis of the signal molecule PQS requires the involvement of enzymes encoded by operons such as pqsA, pqsB, pqsC, pqsD, pqsE, phnAB, and pqsH.

The invention adopts PAO1-lasB-gfp and PAO1-pqsA-gfp green fluorescent protein reporter strains to research the influence of selfheal flavone on las and pqs channels of a pseudomonas aeruginosa quorum sensing system. Experimental results show that the prunella vulgaris flavone has an inhibition effect on expression of lasB-gfp and pqsA-gfp, but does not inhibit growth of pseudomonas aeruginosa, so that the pseudomonas aeruginosa is not easy to generate drug resistance. Therefore, in the application of the selfheal flavone in preparing the medicament for inhibiting the bacterial quorum sensing system, the selfheal flavone is used for inhibiting a lasB signal channel and a pqsA signal channel.

The invention also provides application of the selfheal flavone in preparation of a medicine for inhibiting bacterial pyocyanin.

Preferably, the bacteria comprise pseudomonas aeruginosa.

Preferably, the medicament further comprises pharmaceutically acceptable auxiliary materials.

Preferably, the dosage form of the medicine is an oral preparation, and granules, capsules, tablets or dripping pills are preferred.

Preferably, the effective dose of the selfheal clomazone 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%.

The pseudomonas aeruginosa pqsA can regulate the expression of the pyocin, so the invention also researches the influence of the selfheal flavone on the pyocin secretion of the pseudomonas aeruginosa. Experimental results show that the prunella vulgaris flavone inhibits the secretion of pyocyanin and reduces the virulence and pathogenicity of pseudomonas aeruginosa by inhibiting the expression of pqsA.

The invention also provides application of the prunella vulgaris flavone in preparing a medicine for inhibiting secretion of bacterial elastase.

Preferably, the bacteria comprise pseudomonas aeruginosa, more preferably pseudomonas aeruginosa PAO 1.

Preferably, the medicament further comprises pharmaceutically acceptable auxiliary materials.

Preferably, the dosage form of the medicine is an oral preparation, and granules, capsules, tablets or dripping pills are preferred.

Preferably, the effective dose of the selfheal clomazone 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%.

Pseudomonas aeruginosa LasB can regulate and control the expression of elastase, so the invention also researches the influence of selfheal flavone on the secretion of elastase of Pseudomonas aeruginosa. Experimental results show that the prunella vulgaris flavone inhibits the secretion of elastase by inhibiting the expression of LasB, so that the toxicity and the pathogenicity of pseudomonas aeruginosa are reduced.

The invention also provides application of the selfheal flavone in preparation of a medicament for inhibiting bacterial biofilm formation.

Preferably, the bacteria comprise pseudomonas aeruginosa, more preferably pseudomonas aeruginosa PAO 1.

Preferably, the medicament further comprises pharmaceutically acceptable auxiliary materials.

Preferably, the dosage form of the medicine is an oral preparation, and granules, capsules, tablets or dripping pills are preferred.

Preferably, the effective dose of the selfheal clomazone 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%.

The invention also researches the influence of the selfheal on the formation of the pseudomonas aeruginosa biofilm. The experimental result shows that the prunella vulgaris flavone has a good inhibition effect on the formation of pseudomonas aeruginosa biofilm.

In the technical scheme of the invention, the prunella vulgaris flavone is obtained by sequentially extracting prunella vulgaris with ethanol and purifying through macroporous resin chromatography, and the preparation steps are as follows:

step 1: crushing the selfheal to obtain crushed selfheal;

step 2: reflux-extracting pulverized Prunellae Spica with solvent, and removing insoluble substances to obtain first extract;

and step 3: separating the first extract by macroporous resin column chromatography to obtain purified Prunellae Spica flavone.

In the step 1 of the invention, the prunella vulgaris is preferably crushed by a universal crusher.

In step 2 of the invention, the mass ratio of the prunella vulgaris to the solvent is 1: 10, selecting 60% ethanol as a solvent, removing insoluble substances by filtration or centrifugation, performing reflux extraction at 85 ℃ for 1.5h for 2 times;

after removing insoluble matter and before obtaining the first extract, the method further comprises: drying under reduced pressure.

In the step 3 of the invention, the macroporous resin is D101 type, the macroporous resin is firstly soaked in absolute ethyl alcohol for 24 hours for activation, and then the macroporous resin is washed by purified water until no alcohol smell exists after being filled into a column. And (3) sampling, washing with 2BV purified water, eluting with 4BV 80% ethanol solution to obtain an eluent, and finally drying under reduced pressure to obtain the selfheal flavone.

According to the technical scheme, the invention has the following advantages:

the invention provides a new application of selfheal flavone, in particular to an application in preparing a medicament for inhibiting a bacterial quorum sensing system, an application in preparing a medicament for inhibiting bacterial pyocyanin, an application in preparing a medicament for inhibiting bacterial elastase secretion and an application in preparing a medicament for inhibiting bacterial biofilm formation. The experimental result shows that the prunella vulgaris flavone can effectively inhibit a lasB signal channel and a pqsA signal channel in pseudomonas aeruginosa, and can also inhibit secretion of pseudomonas aeruginosa pyocin and elastase and formation of a pseudomonas aeruginosa biomembrane without inhibiting growth of a strain. The selfheal can achieve the effect of inhibiting bacterial virulence factors and bacterial strain growth simultaneously by inhibiting a quorum sensing system, is not easy to generate bacterial drug resistance, and plays a role in inhibiting pseudomonas aeruginosa. In addition, the prunella vulgaris flavone is a natural substance, has low toxicity and side effects, can be used independently, and can also be used as an auxiliary therapeutic agent of antibiotics to achieve the purpose of preventing and treating bacterial infection.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without inventive exercise.

FIG. 1 is a liquid chromatogram-mass spectrum of Prunella vulgaris flavone in example 1 of the present invention;

FIG. 2 is a graph showing the effect of Prunella vulgaris flavone on P.aeruginosa PAO1-lasB-gfp expression in example 2 of the present invention;

FIG. 3 is a graph showing the effect of Prunella vulgaris flavone on the growth of Pseudomonas aeruginosa PAO1-lasB-gfp in example 2 of the present invention;

FIG. 4 is a graph showing the statistical results of the IC50 value (5.5h) of Prunella vulgaris flavones inhibiting the expression of Pseudomonas aeruginosa PAO1-lasB-gfp provided in example 2 of the present invention;

FIG. 5 is a graph showing the effect of Prunella vulgaris flavone on P.aeruginosa PAO1-pqsA-gfp expression in example 3 of the present invention;

FIG. 6 is a graph showing the effect of Prunella vulgaris flavone on the growth of Pseudomonas aeruginosa PAO1-pqsA-gfp in example 3 of the present invention;

FIG. 7 is a graph showing the statistical results of the inhibition of P.aeruginosa PAO1-pqsA-gfp IC50 values (3.75h) by Prunella vulgaris flavone in example 3 of the present invention;

FIG. 8 is a graph showing the results of the effect of Prunella vulgaris flavones on Pseudomonas aeruginosa pyocin secretion in example 4 of the present invention;

FIG. 9 is a graph showing the effect of Prunella vulgaris flavones on the secretion of Pseudomonas aeruginosa elastase in example 5 of the present invention;

FIG. 10 is a graph showing the results of the effect of Prunella vulgaris flavones on biofilm inhibition in example 6 of the present invention.

Detailed Description

In order to make the objects, features and advantages of the present invention more obvious and understandable, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it should be apparent that the embodiments described below are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

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.