阅读说明：本技术 源自真菌代谢产物的抗菌化合物、制备方法及应用 (Antibacterial compound derived from fungal metabolite, preparation method and application ) 是由 胡琳珍 肖雪洋 江雯 童周 周慧 胡天卉 罗梦颖 于 2021-07-20 设计创作，主要内容包括：本发明属于生物医药技术领域,尤其涉及源自真菌代谢产物的抗菌化合物、制备方法及应用。本发明对来源于中国传统中药贯叶连翘的内生真菌西曲霉进行培养发酵,获取其次生代谢产物。从菌株的发酵代谢产物中分离得到了新化合物1和已知化合物2-4。选取革兰氏阴性菌(大肠杆菌、沙门氏菌)和革兰氏阳性菌(表皮葡萄球菌、金黄色葡萄球菌)为研究对象,采用微量肉汤稀释法评价化合物的抗菌活性。发现化合物1-4均有一定的抑菌活性,可以预期发展成为抗菌药物。(The invention belongs to the technical field of biological medicines, and particularly relates to an antibacterial compound derived from a fungus metabolite, a preparation method and application. The invention cultures and ferments the aspergillus West which is an endophytic fungus from the traditional Chinese medicine hypericum perforatum to obtain the secondary metabolite of the aspergillus kawachii. The new compound 1 and the known compounds 2-4 are isolated from fermentation metabolites of the strain. Gram-negative bacteria (escherichia coli and salmonella) and gram-positive bacteria (staphylococcus epidermidis and staphylococcus aureus) are selected as research objects, and the antibacterial activity of the compound is evaluated by adopting a trace broth dilution method. The compounds 1-4 are found to have certain bacteriostatic activity, and can be expected to be developed into antibacterial drugs.)

1. A compound is characterized in that the structural formula of the compound is shown as any one of a compound 1-a compound 4 in the following formula (I),

2. use of a compound as claimed in claim 1 for the manufacture of an antibacterial medicament.

3. An antibacterial agent comprising at least one of the 4 compounds as claimed in claim 1.

4. An antibacterial agent according to claim 3, wherein: also comprises pharmaceutically acceptable auxiliary materials.

5. An antibacterial agent according to claim 3, wherein: the dosage form of the medicine comprises any one of injection, powder injection, oral agent, spray, capsule and liniment.

6. The use according to claim 2, or the antibacterial medicament according to any one of claims 3 to 5, wherein: the antimicrobial includes inhibiting at least one of escherichia coli, salmonella, staphylococcus epidermidis and staphylococcus aureus.

7. A process for the preparation of a compound as claimed in claim 1, comprising the steps of:

inoculating aspergillus westchi TJ23 into a culture medium mixed by rice and water for constant-temperature fermentation; then, leaching the fermentation product by using alcohol liquid, and concentrating the leaching liquor to obtain extract; sequentially adding ethyl acetate: water and petroleum ether: extracting with methanol; recovering solvent from the methanol extraction part to obtain extract; subjecting the methanol extract to normal phase silica gel chromatography, and gradient eluting with dichloromethane-methanol system to obtain 4 fractions Fr.1-Fr.4;

performing gradient elution on the fraction Fr.2 by using a normal phase column in a petroleum ether-ethyl acetate system, and monitoring by using a thin layer chromatography silica gel plate to obtain components Fr.2.1 and Fr.2.2 in sequence; fr.2.2 is separated by reversed phase high performance liquid chromatography and chiral chromatographic column to obtain a compound 4;

fr.3 separating with gel column to obtain two components Fr.3.1 and Fr.3.2; fr.3.1 preparing a compound 3 by using a reverse phase high performance liquid phase;

fr.4 passing through reverse phase C-18 column, eluting with methanol-water to obtain 70% fraction, and eluting with petroleum ether-acetone system by using normal phase column chromatography; then preparing the compound 1 and the compound 2 by high performance liquid chromatography and a P4VP column.

8. A process for the preparation of a compound according to claim 7, characterized in that:

in preparing compound 4, the hplc conditions were mobile phase: methanol and water (90: 10, 2 ml/min); the chiral chromatographic column conditions are IG column, mobile phase: methanol and water (95: 5, 1 ml/min);

when the compound 3 is prepared, the gel column is a pure methanol Sephadex LH-20 gel column, and the conditions of the reversed phase high performance liquid phase are as follows: methanol and water (75: 25,2 ml/min);

in preparing compounds 1 and 2, the hplc conditions were mobile phase: methanol and water (60: 40,2 ml/min); column conditions of P4VP were mobile phase: methanol and water 70:30, 1 ml/min.

9. Application of aspergillus westerni TJ23 in preparation of salmonella inhibition agents.

10. Use of aspergillus seeger TJ23 in the preparation of an agent that inhibits salmonella and at the same time inhibits at least one of escherichia coli, staphylococcus epidermidis and staphylococcus aureus.

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to an antibacterial compound derived from a fungus metabolite, a preparation method and application.

Background

Staphylococcus epidermidis (Staphylococcus epidermidis) belongs to the family Micrococcaceae, gram-positive cocci, and is present on human skin and mucous membranes. Staphylococcus epidermidis has been considered to be nonpathogenic in humans in the past, and is now considered to be a common important pathogenic bacterium, and the phenomenon of drug resistance is found to be increasingly serious, which causes difficulty in treatment. When the resistance of the human body is reduced or the staphylococcus epidermidis leaves the parts of the skin, mucous membranes and the like which live normally and reaches the abnormal parasitic parts, a plurality of infections can be caused, and the human body is harmed. Drug-resistant staphylococcus epidermidis infections have now become problematic in valvular repair or thoracic surgery.

The extraction of chemical components from plants has been an important means of discovering active compounds in natural medicinal chemistry for the past decades. Hypericum perforatum L is a perennial herb of the genus Hypericum, family Guttiferae, and is antiviral and antitumor when illuminated. However, it may produce photosensitization side effects in light, which limits the use of both biological activities. In recent years, in order to expand the clinical value, the research on the bacteriostatic activity of the compound has been carried out, and the compound can play a role without illumination conditions, thereby proving the safety of the bacteriostatic action. With the progress of research, wild plants available for large-scale research are gradually reduced. The endophytic fungi of the medicinal plants have rich resources, and can generate medicinal plant growth regulating substances and secondary metabolites which are the same as or similar to the host, thereby becoming a research hotspot in recent years. The method for producing the natural product can be repeated infinitely and can be obtained continuously, and the exhaustion and the waste of plant resources are avoided.

In summary, the prior art has the following disadvantages:

1. the extraction of chemical components from plants has been an important means for finding active compounds for decades, but with the progress of research, the number of wild plants available for large-scale research is gradually reduced, and resource shortage and even exhaustion are easily caused.

2. The improper use and even abuse of antibiotics has led to the development of drug-resistant staphylococcus epidermidis, and drug-resistant staphylococcus epidermidis infection has become a formidable problem, and there is a great need for effective drugs to combat drug-resistant antibiotics.

Disclosure of Invention

Aiming at the problems in the prior art, the invention adopts the plant endophytic fungi for culture and fermentation, and the medicinal plant endophytic fungi can generate the same or similar secondary metabolites with the plant, thereby avoiding the exhaustion and waste of plant resources. Furthermore, the present invention provides an antibacterial compound, a preparation method and an application thereof, which aim to solve part of the problems in the prior art or at least alleviate part of the problems in the prior art.

The invention is realized by a compound, the structural formula of which is shown as any one of a compound 1-a compound 4 in the following formula (I),

the invention also provides application of the compound in preparing antibacterial drugs.

The invention also provides an antibacterial drug which comprises at least one of the 4 compounds.

Further, pharmaceutically acceptable auxiliary materials are also included.

Furthermore, the dosage form of the medicine comprises any one of injection, powder injection, oral agent, spray, capsule and liniment.

Further, the antimicrobial includes inhibiting at least one of escherichia coli, salmonella, staphylococcus epidermidis, and staphylococcus aureus.

The invention also provides a preparation method of the compound, which comprises the following steps:

inoculating aspergillus westchi TJ23 into a culture medium mixed by rice and water for constant-temperature fermentation; then, leaching the fermentation product by using alcohol liquid, and concentrating the leaching liquor to obtain extract; sequentially adding ethyl acetate: water and petroleum ether: extracting with methanol; recovering solvent from the methanol extraction part to obtain extract; subjecting the methanol extract to normal phase silica gel chromatography, and gradient eluting with dichloromethane-methanol system to obtain 4 fractions Fr.1-Fr.4;

performing gradient elution on the fraction Fr.2 by using a normal phase column in a petroleum ether-ethyl acetate system, and monitoring by using a thin layer chromatography silica gel plate to obtain components Fr.2.1 and Fr.2.2 in sequence; fr.2.2 is separated by reversed phase high performance liquid chromatography and chiral chromatographic column to obtain a compound 4;

fr.3 separating with gel column to obtain two components Fr.3.1 and Fr.3.2; fr.3.1 preparing a compound 3 by using a reverse phase high performance liquid phase;

fr.4 passing through reverse phase C-18 column, eluting with methanol-water to obtain 70% fraction, and eluting with petroleum ether-acetone system by using normal phase column chromatography; then preparing the compound 1 and the compound 2 by high performance liquid chromatography and a P4VP column.

Further, in preparing compound 4, the high performance liquid chromatography conditions were mobile phase: methanol and water (90: 10, 2 ml/min); the chiral chromatographic column conditions are IG column, mobile phase: methanol and water (95: 5, 1 ml/min);

when the compound 3 is prepared, the gel column is a pure methanol Sephadex LH-20 gel column, and the conditions of the reversed phase high performance liquid phase are as follows: methanol and water (75: 25,2 ml/min);

in preparing compounds 1 and 2, the hplc conditions were mobile phase: methanol and water (60: 40,2 ml/min); column conditions of P4VP were mobile phase: methanol and water 70:30, 1 ml/min.

The invention also provides application of the aspergillus westerni TJ23 in preparing a salmonella inhibition reagent.

The invention also provides application of the aspergillus westerni TJ23 in preparing a reagent for inhibiting salmonella and simultaneously inhibiting at least one of escherichia coli, staphylococcus epidermidis and staphylococcus aureus.

Aspergillus (Aspergillus) is the most common filamentous fungus of the Ascomycota genus of the Ascomycota phylum, growing in an oxygen-rich environment. Aspergillus is capable of producing a large number of active secondary metabolites, such as isocoumarin, terpenoids, alkaloids, cytochalasin, butenolide, xanthone, sterols, diphenyl ether, anthraquinone derivatives, etc., and plays an important role in the pharmaceutical and commercial industries. Aspergillus West (Aspergillus western dijkiae) is a fungus with extremely strong sporulation capability, and contains abundant secondary metabolites including alkaloids, terpenoids, polyketones and prenones. Modern pharmacological research shows that isocoumarin compounds generally have various physiological and biological activities such as antibiosis, antiphlogosis, anticancer, protease inhibition and the like, and terpenoid compounds generally have various pharmacological activities such as antibiosis, antiphlogosis, antitumor, antioxidation and the like.

The invention separates and identifies 1 new compound and 3 known compounds by culturing the Hypericum perforatum endophytic fungus Aspergillus westhicus. The structural formula of the compound is shown as a formula (I).

Gram-negative bacteria (escherichia coli and salmonella) and gram-positive bacteria (staphylococcus epidermidis and staphylococcus aureus) are selected as research objects, and the antibacterial activity of the compound is evaluated by adopting a trace broth dilution method. Analysis of bacteriostatic test data shows that the compounds 1-4 have certain inhibitory effect on four bacteria, can inhibit the growth thereof in a dose-dependent manner, and can be expected to be developed into antibacterial drugs.

In summary, the advantages and positive effects of the invention are:

1. through culturing the Hypericum perforatum endophytic fungi, a secondary metabolite which is the same as or similar to the Hypericum perforatum is expected to be obtained, and the current situation of plant resource exhaustion is relieved;

2. pharmacological activity experiment results show that the compounds 1-4 separated from Hypericum perforatum endophytic fungi metabolites have different degrees of antibacterial effects on gram-negative bacteria (escherichia coli and salmonella) and gram-positive bacteria (staphylococcus epidermidis and staphylococcus aureus). The compound 1 is a novel compound, can inhibit the growth of staphylococcus epidermidis, has antibacterial activity, and can be expected to be developed into a medicament for resisting staphylococcus epidermidis infection;

3. the invention can perform infinite amplification fermentation only by subculturing strains according to an experimental scheme to obtain a target compound with antibacterial activity.

Drawings

FIG. 1 is a graph comparing experimental values with calculated values for Electronic Circular Dichroism (ECD) of Compound 1;

FIG. 2 is a graph of the inhibitory effect of compounds 1-4 on bacterial growth.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples, and the equipment and reagents used in the examples and test examples are commercially available without specific reference. The specific embodiments described herein are merely illustrative of the invention and are not intended to be limiting.

Various modifications to the precise description of the invention will be readily apparent to those skilled in the art from the information contained herein without departing from the spirit and scope of the appended claims. It is to be understood that the scope of the invention is not limited to the procedures, properties, or components defined, as these embodiments, as well as others described, are intended to be merely illustrative of particular aspects of the invention. Indeed, various modifications of the embodiments of the invention which are obvious to those skilled in the art or related fields are intended to be covered by the scope of the appended claims.

For a better understanding of the invention, and not as a limitation on the scope thereof, all numbers expressing quantities, percentages, and other numerical values used in this application are to be understood as being modified in all instances by the term "about". Accordingly, unless expressly indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. In the present invention, "about" means within 10%, preferably within 5% of a given value or range.

In the following examples of the present invention, the temperature is not particularly limited, and all of the conditions are normal temperature conditions. The normal temperature refers to the natural room temperature condition in four seasons, no additional cooling or heating treatment is carried out, and the normal temperature is generally controlled to be 10-30 ℃, preferably 15-25 ℃.

The invention discloses an antibacterial compound derived from fungal metabolites, a preparation method and application thereof, and Aspergillus westerjkiae related to the invention is derived from plant hypericum perforatum, has the name of Aspergillus western jkiae, and is disclosed in Chinese invention patent with the application number of CN201710951372.6, and is called Aspergillus westerj 23 for short. The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.

EXAMPLE 1 preparation of antibacterial Compounds

1. Fermentation and extraction

Aspergillus West stored at 4 deg.C was inoculated onto potato dextrose agar medium (PDA medium) sterilized at high temperature, and activated in an incubator at 28 deg.C for 7d to serve as a seed plate. Mixing 10.0kg rice with water at a ratio of 1:1, sterilizing at 121 deg.C under 15psi for 30 min, cooling to room temperature, inoculating Aspergillus, sealing with sterile sealing film, fermenting at constant temperature of 25 deg.C for 30d, and adding ethyl acetate to terminate the fermentation. The fermented product is extracted by 20L of 95 percent ethanol, the extracting solution is decompressed and concentrated to obtain 200g of crude extract, and then the crude extract is sequentially extracted by equal volume of ethyl acetate: water and petroleum ether: extracting with 95% methanol, and recovering solvent from 95% methanol extraction part to obtain 40g extract.

2. Separation of

40g of the methanol fraction extract was purified by normal phase silica gel chromatography on dichloromethane: after gradient elution with methanol at 200:1-1:1, TLC run and after combining the same fractions 4 fractions a-D (fr.1-fr.4) were obtained.

Component fr.2 uses a positive phase column with 100: 1-1:1, performing gradient elution by using a petroleum ether-ethyl acetate system, and monitoring by using a thin layer chromatography silica gel plate to obtain components Fr.2.1 and Fr.2.2. Fr.2.2 compound 4 was isolated by reverse phase high performance liquid chromatography (mobile phase: methanol: water: 90:10, 2ml/min), chiral chromatography (IG column, mobile phase: methanol: water: 95:5, 1 ml/min).

Fr.3 was separated using a Sephadex LH-20 gel column (pure methanol) to give two fractions Fr.3.1 and Fr.3.2. Fr.3.1 compound 3 was prepared using reverse phase high performance liquid phase (mobile phase: methanol: water: 75:25,2 ml/min).

Fr.4 is subjected to reverse phase C-18 column, and eluted with methanol-water to obtain 70% fraction, and then subjected to normal phase column chromatography and eluted with petroleum ether-acetone system. Then, the new compound 1 and the known compound 2 were prepared by high performance liquid chromatography (mobile phase: methanol: water 60:40,2ml/min), P4VP column (mobile phase: methanol: water 70:30, 1 ml/min).

EXAMPLE 2 structural characterization of the Compounds

The structures of the compounds 1 to 4 were obtained by Nuclear Magnetic Resonance (NMR) and ECD calculation methods.

1. Physical and chemical data

2' S-formatic acid a (compound 1): a brown powder; HRESIMS [ M + H ]]⁺m/z 213.0175 (calculated value C)₁₀H₉O₅,213.0763)；；UV(CH₃OH)λ_max(logε)＝225(4.726)、241(4.538)、350(4.122)nm；IR(KBr)ν_max 3237,2957,2874,1649,1588,1477,1410cm^–1；¹H and¹³c NMR data, see Table 1.

8-Methoxyjelllein (Compound 2): brown needle-like crystals; calculated m/z value C₁₁H₁₂O₃：192.0786；¹H and¹³c NMR data, see Table 1.

Conidiogenone C (compound 3): a yellow oily liquid; calculated m/z value C₂₀H₃₀O₂：302.2246；¹H and¹³c NMR data, see Table 2.

n-hexos-5, 8, 11-trienic acid (compound 4): a colorless oily liquid; calculated m/z value C₂₆H₄₆O₂：490.3498；¹H and¹³c NMR data, see Table 2. In addition to the data in the table, some failed to assign one to another due to similar chemical shifts:¹H NMR(400MHz)：δ_H 1.60(4H,m,2CH₂)，δ_H 1.29(24H,m,12CH₂)。¹³C NMR(400MHz)：δ_C34.3(CH₂)、δ_C 32.1(CH₂)、δ_C 31.7(CH₂)、δ_C 30.0(CH₂)、δ_C 29.9(CH₂)、δ_C29.8(CH₂)、δ_C 29.7(CH₂)、δ_C 29.6(CH₂)、δ_C 29.5(CH₂)、δ_C 29.4(CH₂)、δ_C 29.3(CH₂)、δ_C 29.2(CH₂)、δ_C 27.4(CH₂)、δ_C 27.4(CH₂)、δ_C 27.4(CH₂)、δ_C 25.8(CH₂)、δ_C 24.9(CH₂)、δ_C 22.9(CH₂)、δ_C 22.8(CH₂)。

table 1.¹H NMR data for compounds 1,and 2(400MHz,J in Hz).

^aMeasured in DMSO-d₆；^bMeasured in CDCl₃

Table 2.¹H NMR data for compounds 1,and 2(400MHz,J in Hz).

^aMeasured in DMSO-d₆；^bMeasured in CDCl₃

2. The absolute configuration of the compound 1 was confirmed by the method of calculating ECD. The experimental measured Cotton effect of ECD (electron circular dichroism) was matched with the calculated value, as shown in figure 1.

The resulting compounds 1-4 were finally determined to have the formula (I):

EXAMPLE 3 study of the use of Compounds having antibacterial Activity

Gram-negative bacteria (escherichia coli and salmonella) and gram-positive bacteria (staphylococcus epidermidis and staphylococcus aureus) are selected as action targets, and the antibacterial activity of the compound is evaluated by adopting a trace broth dilution method.

Inoculating Escherichia coli, Salmonella, Staphylococcus epidermidis and Staphylococcus aureus to 10ml of culture solution, and diluting with NB broth to 0.5 in McGrego turbidity after the bacteria have entered logarithmic growth phase, wherein the bacteria count is 10⁸CFU/mL, diluting at a ratio of 1:1000, treating with compound 1, 2, 3 or 4, culturing in 37 deg.C incubator for 16-24 hr, and observingAnd (6) obtaining the result.

And (3) bacterial culture: coli, salmonella, staphylococcus epidermidis and staphylococcus aureus are grown in NB broth. Culturing the bacteria in a constant temperature shaking table at 37 ℃ and 220rpm for 6-8 h; it was diluted with NB broth to a turbidity of 0.5, at which time the bacterial count was 10⁸CFU/mL, then according to 1:1000 dilution, inoculated to 96 hole plate, divided into blank control group, negative control group, administration group, each group set up 6 multiple holes.

The detailed experimental procedure is as follows:

experimental grouping and treatment:

blank control (blank control): no addition of bacteria liquid and compound;

negative control group (negative control): adding only bacteria liquid without adding compound;

administration group (compounds 1, 2or 3): inoculating the diluted bacteria liquid at the ratio of 1:1000 to a 96-well plate, and adding compounds with different concentrations respectively.

And (3) antibacterial experiment: and placing the blank control group, the negative control group and the administration group into a constant-temperature incubator at 37 ℃ for culturing for 16-24h, detecting the OD value at the wavelength of 600nm by using an enzyme-labeling instrument, and calculating the bacteriostasis rate of 128 mu g/mL. The experiment was repeated 3 times and the mean value was taken for analysis.

The results are shown in FIG. 2: data analysis of bacteriostatic tests shows that the compounds 1-4 have bacteriostatic effects of different degrees on four microorganisms, and the more prominent compounds comprise: the inhibitory rate of compound 1 against Staphylococcus epidermidis was 15.68% at a inhibitory concentration (MIC) of 128. mu.g/mL. The compound 2 has an inhibitory rate of 45.48% at a concentration of 128. mu.g/mL against Escherichia coli, 52.83% at a concentration of 128. mu.g/mL against Salmonella, 34.44% at a concentration of 128. mu.g/mL against Staphylococcus aureus, and 50.60% at a concentration of 128. mu.g/mL against Staphylococcus epidermidis; the inhibitory rate of the compound 3 to Staphylococcus epidermidis when the inhibitory concentration (MIC) is 128 mug/mL is 49.34%; the compound 4 had an inhibitory rate of 43.34% at a concentration of 128. mu.g/mL against Escherichia coli, 41.16% at a concentration of 128. mu.g/mL against Salmonella, 41.83% at a concentration of 128. mu.g/mL against Staphylococcus aureus, and 48.76% at a concentration of 128. mu.g/mL against Staphylococcus epidermidis. The compounds 1-4 have certain bacteriostatic action and are dose-dependent, and can be expected to be developed into antibacterial drugs.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.