阅读说明：本技术 一种7-醛基-9-异丁酰氧基-8-羟基百里酚其在制备抗菌剂或抗菌药物中的应用 (Application of 7-aldehyde-9-isobutyryloxy-8-hydroxythymol in preparation of antibacterial agent or antibacterial drug ) 是由 谭建文 徐巧林 刘少博 钱涛 于 2020-12-23 设计创作，主要内容包括：本发明公开了一种7-醛基-9-异丁酰氧基-8-羟基百里酚其在制备抗菌剂或抗菌药物中的应用。本发明从紫茎泽兰植物中提取分离对革兰氏阳性菌和革兰氏阴性菌均具有有效抑制作用的7-醛基-9-异丁酰氧基-8-羟基百里酚。紫茎泽兰植物材料来源丰富,提取制备方便,且采用入侵植物紫茎泽兰为原料提取还可对该入侵植物推进实现变废为宝地加以利用,可望在取得经济效益的同时还能间接对紫茎泽兰的防控治理具积极促进作用,且该天然来源的化合物稳定、易存放。化合物7-醛基-9-异丁酰氧基-8-羟基百里酚抑菌活性显著,极可能发展为有效安全的新抗菌剂或开发为新的治疗细菌感染类疾病的药物,具有潜在良好的前景。(The invention discloses an application of 7-aldehyde-9-isobutyryloxy-8-hydroxythymol in preparing an antibacterial agent or an antibacterial drug. The invention extracts and separates 7-aldehyde-9-isobutyryloxy-8-hydroxythymol which has effective inhibition effect on gram-positive bacteria and gram-negative bacteria from eupatorium adenophorum. The eupatorium adenophorum plant material has rich sources and convenient extraction and preparation, and the invasive plant eupatorium adenophorum is taken as the raw material for extraction, so that the invasive plant can be promoted to realize the utilization of waste materials, the economic benefit can be obtained, meanwhile, the prevention and control treatment of the eupatorium adenophorum can be indirectly promoted, and the compound from natural sources is stable and easy to store. The compound 7-aldehyde-9-isobutyryloxy-8-hydroxythymol has remarkable bacteriostatic activity, is very likely to be developed into a novel effective and safe antibacterial agent or a novel medicament for treating bacterial infection diseases, and has a potentially good prospect.)

1. 7-formyl-9-isobutyryloxy-8-hydroxythymol represented by the following formula (I):

2. use of 7-aldehyde-9-isobutyryloxy-8-hydroxythymol according to claim 1 for the preparation of an antibacterial agent or an antibacterial drug.

3. The use according to claim 2, wherein the antibacterial agent or drug is an antibacterial agent or drug against gram-pathogenic bacteria.

4. The use according to claim 3, wherein the gram-pathogenic bacterium is Staphylococcus aureus, Bacillus cereus, Bacillus thuringiensis, Escherichia coli or Salmonella.

5. A medicament for the antibacterial or therapeutic treatment of bacterial infectious diseases, which comprises an effective amount of 7-aldehyde-9-isobutyryloxy-8-hydroxythymol or its phenolate salt according to claim 1 and a pharmaceutically acceptable pharmaceutical excipient or carrier.

6. The medicament of claim 5, wherein the medicament is an antibacterial agent or an antibacterial agent against gram-pathogenic bacteria.

7. The medicament of claim 6, wherein the gram-pathogenic bacterium is Staphylococcus aureus, Bacillus cereus, Bacillus thuringiensis, Escherichia coli, or Salmonella. .

8. A method for preparing 7-aldehyde-9-isobutyryloxy-8-hydroxythymol according to claim 1, wherein said 7-aldehyde-9-isobutyryloxy-8-hydroxythymol is isolated from tissue of eupatorium adenophorum.

9. The process according to claim 8, wherein the aerial parts of Eupatorium Adenophorum are pulverized, extracted by soaking in ethanol or aqueous ethanol, the extractive solution is concentrated to remove ethanol, suspended in water, extracted with petroleum ether, the petroleum-extracted part is concentrated to obtain extract, the extract is separated by normal phase silica gel column chromatography, and CHCl is used₃MeOH was eluted from 100: 0-90: 10, v/v gradient and CHCl was collected at 98:2v/v₃-fraction P6 eluted with MeOH followed by normal phase silica gel column chromatography with a petroleum ether-acetone gradient from 100:0 to 80:20, v/v, wherein the molar ratio of petroleum ether-acetone 9: after the subfraction P6-7 is obtained by elution and collection at 1v/v, the mixture is further treated by petroleum ether-acetone 85: eluting at 15v/v to obtain subfraction P6-8, performing Sephadex LH-20 gel column chromatography on the subfraction P6-8, and separating and purifying with methanol as eluent to obtain 7-aldehyde-9-isobutyryloxy-8-hydroxythymol.

10. The method according to claim 9, wherein the above-ground material of Eupatorium Adenophorum is dried above-ground material of Eupatorium Adenophorum.

The technical field is as follows:

the invention belongs to the technical field of antibacterial antisepsis and medicines, and particularly relates to a novel thymol derivative compound, namely 7-aldehyde-9-isobutyryloxy-8-hydroxythymol, a preparation method of the novel compound, and application of the compound in preparation of antibacterial agents or antibacterial medicines.

Background art:

pathogenic bacteria are important sources of infectious diseases of human beings, pathogenic bacteria gradually accumulate drug resistance to the existing antibacterial drugs or antibacterial preparations on the market, and form more and more remarkable threats to the health of people, so that more and more realistic and important demands on developing new antibacterial drugs or antibacterial agents for preventing and treating bacterial infectious diseases are promoted.

The eupatorium adenophorum is an exotic invasive plant with huge resource quantity, disease symptoms which are obviously infected by microorganisms exist in an invasive place, and the fact that the eupatorium adenophorum potentially contains active substances which can effectively resist the infection of the microorganisms such as external bacteria and the like is suggested, the active substances potentially have the potential of being developed into novel effective antibacterial agents or antibacterial drugs, and the abundant resource of the eupatorium adenophorum makes the eupatorium adenophorum also expected to be an ideal material for developing novel natural antibacterial agents or antibacterial drugs. The current research on the plant shows that the phytochemical ingredient is very abundant, but the research on the plant antibacterial active chemical ingredient is still needed.

The invention content is as follows:

the invention aims to provide a novel thymol derivative compound, namely 7-aldehyde-9-isobutyryloxy-8-hydroxythymol, namely 7-formol-9-isobutryloxy-8-hydroxythymol, a preparation method for separating the compound from eupatorium adenophorum and application of the compound in preparing antibacterial agents or antibacterial drugs.

The 7-aldehyde-9-isobutyryloxy-8-hydroxythymol disclosed by the invention has a structure shown in a formula (I):

the new compound 7-aldehyde-9-isobutyryloxy-8-hydroxythymol has obvious inhibition effect on gram-positive bacteria and gram-negative bacteria as proved by in vitro pharmacological experiments. For example, it has an inhibitory effect on strains such as Staphylococcus aureus, Bacillus cereus and Suyun close to that of the positive control kanamycin sulfate, and also exhibits moderate inhibitory activity on Escherichia coli and Salmonella. Therefore, the novel compound can be developed to be used for preparing novel antibacterial agents or antibacterial drugs, in particular antibacterial agents against gram-positive bacteria such as staphylococcus aureus, bacillus cereus, bacillus thuringiensis and the like. The present invention provides a gram bacterium that is significantly inhibited by 7-aldehyde-9-isobutyryloxy-8-hydroxythymol, including but not limited to staphylococcus aureus, bacillus cereus, bacillus thuringiensis, escherichia coli, and salmonella as exemplified in the examples below.

The novel compound 7-aldehyde-9-isobutyryloxy-8-hydroxythymol can be combined with pharmaceutically common auxiliary materials or drug carriers to prepare a drug or a drug composition which has the bacteriostatic activity of 7-aldehyde-9-isobutyryloxy-8-hydroxythymol, inhibits bacterial infection and prevents and treats related diseases caused by pathogenic bacterial infection. The medicine or the pharmaceutical composition can adopt wettable powder, tablets, granules, capsules, oral liquid, dripping pills, injection, aerosol and other formulations; controlled or sustained release formulations or nano-formulations well known in the modern pharmaceutical industry may also be employed.

The novel compound 7-aldehyde-9-isobutyryloxy-8-hydroxythymol is obtained by separating from Eupatorium adenophorum or its kindred. Can be the tissue parts of the eupatorium plants, and the material can be dry or fresh, preferably the dry of overground stem leaves.

In particular toThe preparation method comprises pulverizing aerial parts of Eupatorium Adenophorum, soaking in ethanol or ethanol water solution, extracting, concentrating the extractive solution to remove ethanol, suspending in water, extracting with petroleum ether, concentrating the petroleum-extracted part to obtain extract, separating by normal phase silica gel column chromatography, and separating with CHCl₃MeOH was eluted from 100: 0-90: 10, v/v gradient and CHCl was collected at 98:2v/v₃-fraction P6 eluted with MeOH followed by normal phase silica gel column chromatography with a petroleum ether-acetone gradient from 100:0 to 80:20, v/v, wherein the molar ratio of petroleum ether-acetone 9: 1, eluting and collecting to obtain a subfraction P6-7, and then eluting with petroleum ether-acetone 85: eluting by 15 to obtain subfraction P6-8, performing Sephadex LH-20 gel column chromatography on the subfraction P6-8, and separating and purifying by using methanol as an eluent to obtain 7-aldehyde-9-isobutyryloxy-8-hydroxythymol.

The aerial part material of the eupatorium adenophorum

The antibacterial agent (medicine) which has effective inhibition effect on gram-positive bacteria and gram-negative bacteria is extracted and separated from the eupatorium adenophorum, the plant material source is rich, the extraction and preparation are convenient, the invasive plant eupatorium adenophorum is adopted as the raw material for extraction, the invasive plant can be promoted to realize the utilization of changing waste into valuable, the economic benefit is expected to be obtained, meanwhile, the active promotion effect on the prevention and control treatment of the eupatorium adenophorum can be indirectly realized, and the compound from natural sources is stable and easy to store. The compound 7-aldehyde-9-isobutyryloxy-8-hydroxythymol has remarkable bacteriostatic activity, is very likely to be developed into a novel effective and safe antibacterial agent or a novel medicament for treating bacterial infection diseases, and has a potentially good prospect.

Description of the drawings:

FIG. 1 is an HR-ESI-MS spectrum of compound 7-aldehyde-9-isobutyryloxy-8-hydroxythymol;

FIG. 2 preparation of compound 7-formyl-9-isobutyryloxy-8-hydroxythymol¹A HNMR map;

FIG. 3 preparation of compound 7-formyl-9-isobutyryloxy-8-hydroxythymol¹³A C NMR (DEPT) spectrum;

FIG. 4, HSQC spectra of compound 7-aldehyde-9-isobutyryloxy-8-hydroxythymol;

FIG. 5 preparation of compound 7-aldehyde-9-isobutyryloxy-8-hydroxythymol¹H-¹H COSY map;

FIG. 6, HMBC chromatogram of compound 7-aldehyde-9-isobutyryloxy-8-hydroxythymol;

Detailed Description

The following examples are further illustrative of the present invention and are not intended to be limiting thereof, and all simple modifications made to the invention in light of the spirit thereof are intended to be included within the scope of the present invention as claimed.

Example 1: preparation of 7-aldehyde-9-isobutyryloxy-8-hydroxythymol in Eupatorium adenophorum

1.1 plant origin and identification

An overground part sample of plant material eupatorium adenophora (Ageratina adenophora) for extraction is collected from suburb of Kunming of Yunnan in 2009 at 7 months, and is identified by doctor of inventor's Tantan Jianwen.

1.2 extraction and separation

The method comprises soaking and extracting aerial tissue material (5kg) of Eupatorium Adenophorum with 95% ethanol water at room temperature for 3 times (3 × 10L, 24 hr each time). The ethanol extracts were combined and concentrated under reduced pressure to remove ethanol, then suspended in water with the addition of an appropriate amount of water and extracted with petroleum ether (3X 2L), and concentrated under reduced pressure to give a petroleum-extracted fraction (46.5 g).

Separating the petroleum ether extract by normal phase silica gel column chromatography, and separating with CHCl₃Elution with a gradient of-MeOH (100: 0-90: 10, v/v) afforded 12 fractions (P1-P12). Collection of 98:2(v/v) CHCl₃Fraction P6(10.3g) eluted with MeOH, and normal phase silica gel column chromatography using a petroleum ether-acetone gradient (100: 0-80: 20, v/v) elution system to give 9 subfractions (P6-1-P6-9) of petroleum ether-acetone 9: 1, eluting and collecting to obtain a subfraction P6-7, and then eluting with petroleum ether-acetone 85: 15 to obtain a subfraction P6-8(170.0mg), and separating and purifying the subfraction P6-8 by Sephadex LH-20 gel column chromatography (methanol elution) to obtain a pure compound 1(3.4mg) of the formula (I).

Structural identification of 1.37-aldehyde-9-isobutyryloxy-8-hydroxythymol

The obtained compound 1 is a pale yellow oily substance with a molecular formula of C₁₄H₁₈O₅；[α]_D ²⁰–2.0(c 0.65,CHCl₃)；IR(KBr)ν_max 3424,1695,1612,1579cm^–1；UV(MeOH)λ_max(logε)nm:222(4.11),260(3.88),317(3.36)；ESI-MS m/z 571[2M+K]⁺,555[2M+Na]⁺,567[2M+Cl]^–,289[M+Na]⁺,301[M+Cl]^–,265[M–H]^–；HR-ESI-MS m/z 289.1056[M+Na]⁺(calcd.for C₁₄H₁₈O₅Na,289.1052)；¹H(CDCl₃600MHz) and¹³C NMR(CDCl₃150MHz) data are shown in Table 1:

TABLE 1 NMR data for the compound 7-formyl-9-isobutyryloxy-8-hydroxythymol

¹HNMR(in CDCl₃ at 600MHz),¹³C NMR(in CDCl₃ at 150MHz)

According to the comprehensive analysis (figure 1-6) of the infrared, ultraviolet, mass spectrum, one-dimensional and two-dimensional nuclear magnetism and other spectrum data, the chemical structure of the new compound 1 is deduced and analyzed to be 7-aldehyde-9-isobutyryloxy-8-hydroxythymol, and the specific structural formula is shown as the formula (I).

Example 2: antibacterial activity detection of compound 7-aldehyde-9-isobutyryloxy-8-hydroxythymol

3.1 species of bacteria for experiments

Gram-positive bacteria: staphylococcus aureus (staphylococcus aureus), Bacillus cereus (Bacillus cereus), Bacillus thuringiensis (Bacillus thuringiensis);

gram-negative bacteria: escherichia coli (Escherichia coli), Salmonella (Salmonella enterica).

3.2 Experimental drugs

Positive control: kanamycin sulfate (kanamine sulfate)

Negative control: MeOH

Test compounds: the novel compound 7-aldehyde-9-isobutyryloxy-8-hydroxythymol (i.e. compound 1) of the present invention is prepared from the above experimental examples, and two thymol compounds with similar structures, i.e. 7, 9-diisobutyroniyloxy-8, 10-dehydrothymol (i.e. compound 2) and 7, 9-diisobutyroniyloxy-8-methoxythymol (i.e. compound 3) are used as the reference for comparing the activities of the same compounds, and the structures of compounds 1-3 are shown in the following figures:

3.3 Experimental methods:

7-aldehyde-9-isobutyryloxy-8-hydroxythymol (and compound 2 or 3) and kanamycin sulfate were prepared from methanol (MeOH) as 1mg/ml solutions, respectively. The following determination of the Minimum Inhibitory Concentrations (MIC) of the compound 7-aldehyde-9-isobutyryloxy-8-hydroxythymol against five bacterial strains was performed by the Resazurin color reaction method through a 96-well cell culture plate.

First 100. mu.g/mL of resazurin indicator was added to the 11 th well of a 96 well cell culture plate, and then 7.5mL of 100. mu.g/mL resazurin solution and 5mL of bacteria (10)⁶cfu/mL, OD ═ 0.07) were mixed, and then 100 μ L of the mixture of resazurin and the bacterial culture solution was added to each of the culture wells of columns 1 to 10 and 12, respectively. Then 100. mu.L of the test sample at a concentration of 1mg/mL is added to the first well of each column, mixed, 100. mu.L of the solution is aspirated and applied to the second well, and so on, up to the tenth well, and finally 100. mu.L is removed. The concentration change of the test sample was: 500. mu.g/mL, 250. mu.g/mL, 125. mu.g/mL, 62.5. mu.g/mL, 31.2. mu.g/mL, 15.6. mu.g/mL, 7.8. mu.g/mL, 3.9. mu.g/mL and 1.9. mu.g/mL, 0.9. mu.g/mL. The plate was then placed in an incubator at 37 ℃ and the test sample wells were not changed to bluePink for inhibitory effect, blue to pink for no inhibitory effect, the concentration of the sample in the last well that did not change from blue to pink was the Minimum Inhibitory Concentration (MIC) of the test sample until column 12 when the culture fluid changed from blue to pink (about 5-6 hours), and MIC values of compound 7-aldehyde-9-isobutyryloxy-8-hydroxythymol (and compound 2 or 3) against each bacterium were observed (Rahman, m.m.,&gray, A.I., A benzoin derivative and carbazole analogs from Murraya koenigii and the antimicrobial activity. phytochemistry,2005,66: 1601-. Three replicates per experiment.

3.4 Experimental data see Table 2:

TABLE 2.7 MIC values (μ g/ml) for aldehyde-9-isobutyryloxy-8-hydroxythymol and the reference compound for five bacteria

KS＝Kanamycin sulfate

3.5 conclusion of the experiment:

experiments show that the compound 7-aldehyde-9-isobutyryloxy-8-hydroxythymol has obvious inhibiting effect on gram-positive bacteria (including staphylococcus aureus, bacillus thuringiensis and the like) and gram-negative bacteria (including escherichia coli, salmonella, dysentery bacillus and the like), has obvious inhibiting activity on five test strains, particularly has the inhibiting activity close to kanamycin sulfate serving as a positive control on three gram-positive bacteria, is remarkably superior to the control compounds (2 and 3) with similar structures, shows that the remarkable antibacterial activity of the 7-aldehyde-9-isobutyryloxy-8-hydroxythymol has characteristics, and is expected to be used for preparing effective and safe novel antibacterial agents or novel medicines for treating bacterial infection diseases, and the source plant of the eupatorium adenophorum is rich in resources, and the application and development potential is superior.