阅读说明：本技术 一种荧光类化合物及其制备方法与应用 (Fluorescent compound and preparation method and application thereof ) 是由 秦宝福 田均勉 曹让 金苗 魏茜敏 胡仲秋 周乐 高锦明 徐虹 王娜娜 曹劲松 于 2020-07-24 设计创作，主要内容包括：本发明公开了一种荧光类新化合物及其制备方法。分子式为：C<Sub>15</Sub>H<Sub>13</Sub>O<Sub>4</Sub><Sup>+</Sup>,分子量为：257.0805。淡黄色针状结晶,易溶于甲醇；命名为：内生地衣芽孢杆菌菌素A。该化合物从一株盾叶薯蓣内生地衣芽孢杆菌SYt1的发酵液中提取得到。经色谱柱分离,结合核磁,高分辨质谱等多种技术鉴定,确定该化合物为一种新的天然产物。本发明公开的化合物经荧光性能测定,结果显示此化合物具有荧光性,可应用于制作发光材料。本发明还公开创建了一种分离荧光物质的方法,将其命名为荧光关联示踪法。(The invention discloses a fluorescent novel compound and a preparation method thereof. The molecular formula is: c 15 H 13 O 4 + The molecular weight is: 257.0805. light yellow needle crystal, which is easily dissolved in methanol; is named as: endogenous Bacillus licheniformis A. The compound is extracted from fermentation liquor of dioscorea zingiberensis endophytic bacillus licheniformis SYt 1. The compound is determined to be a new natural product by chromatographic column separation and combined with multiple technical identification such as nuclear magnetism, high-resolution mass spectrum and the like. The fluorescence property of the compound disclosed by the invention is measured, and the result shows that the compound has fluorescence and can be applied to the preparation of luminescent materials. The invention also discloses a method for separating fluorescent substances,this was named fluorescence correlation tracing method.)

1. A fluorescent compound has a structural formula as follows:

。

2. the fluorescent compound of claim 1, having a seven-membered ring structure, with a positive charge.

3. The fluorescent compound of claim 1, which is fluorescent.

4. The fluorescent compound as claimed in claim 1, which is isolated from fermentation broth of dioscorea zingiberensis endophyte, wherein the dioscorea zingiberensis endophyte is bacillus licheniformis syb06.11.1, and is stored in CGMCC for a period of time of 2008/1/10, and has a storage number of 2334.

5. The fluorescent compound according to claim 1, wherein the crystal is a light yellow needle crystal.

6. The method for preparing fluorescent compounds according to claim 1, characterized in that the fermentation broth of the dioscorea zingiberensis endophytic bacillus licheniformis Syb06.11.1 is extracted by ethyl acetate and concentrated to obtain a crude product of the target product; and separating the target crude product by a chromatographic column, preparing by thin-layer chromatography, and purifying to obtain the fluorescent compound.

7. The method of claim 6, comprising,

hydrolyzing fermentation liquid of dioscorea zingiberensis endophytic bacillus licheniformis Syb06.11.1 by acid, adjusting pH to be neutral, extracting by using ethyl acetate, and concentrating to obtain ethyl acetate extract;

normal phase column chromatography separation, wherein the eluent is cyclohexane and ethyl acetate which are 10: 1;

separating with Sephadex LH-20 column for the first time, wherein the eluent is methanol; separating with Sephadex LH-20 column, eluting with chloroform and methanol at a ratio of 1: 1;

RP-C18 reverse silica gel column separation, wherein the eluent is methanol and water is 6: 4;

preparing thin layer chromatography;

and (3) performing Sephadex LH-20 gel column chromatography, wherein the eluent is chloroform and methanol which are 1: 1.

8. Use of the fluorescent compound according to claim 1 for the preparation of a luminescent material.

Technical Field

The invention belongs to the technical field of microbial secondary metabolites, relates to separation and purification of a dioscorea zingiberensis endophytic bacillus licheniformis fermentation liquid product, and particularly relates to a fluorescent compound with a novel chemical structure. The invention provides the processes of extraction, separation and identification of the fluorescent compound.

Background

Dioscorea zingiberensis c.h.wright) is a unique Chinese yam variety, also called yellow ginger, and is a main plant raw material for producing saponin. Diosgenin, known as "medicinal gold" in the medical community, can synthesize thousands of steroid hormone drugs such as corticoids, anabolic hormones, and sex hormones. Peltate yam is one of the important raw materials for obtaining diosgenin. At present, 138 dioscin-containing dioscin plants have been found worldwide, but only about 10% of dioscin plants have industrial application value. With the ever-increasing gap in yam saponin market demand, dioscorea zingiberensis has been subjected to plunder-type mining, so that this precious wild resource is gradually exhausted.

Endophytes (endophytes) are a class of microorganisms that survive within healthy plant tissue for part or all of their life history without causing the host plant to exhibit significant symptoms of infection. Plant endophytes are a very important microbial resource, widely exist in nature, are rich in species and large in quantity, and mainly comprise three major types of endophytes, endophytes and endophytes. The endophyte not only participates in the secondary metabolism of plants and the transformation and synthesis of components thereof, but also can independently generate abundant secondary metabolites due to the long-term co-evolution with the host, thereby providing a huge resource base for the research and development of new drugs (new compounds).

The inventor of the patent selects a dioscorea zingiberensis endophytic bacterium from the rhizome of dioscorea zingiberensis in 2006. The strain is identified to belong to the bacillus by colony characteristics, cell morphology and physiological and biochemical characteristics. The ITS sequence and the 16S rDNA sequence of the strain are determined by a molecular biological method and are compared by blast to find that the 16S rDNA homology of the strain with Bacillus licheniformis ASC585 is 99 percent, so that the strain is determined to belong to the Bacillus licheniformis and is named as Endophytic Bacillus licheniformis Syb06.11.1. For a screening method of the strain and identification and preservation information of the strain, see patent ZL 200810150274.3.

Disclosure of Invention

At present, researches on dioscorea zingiberensis endophytes mainly focus on related researches on eukaryotic endophytes and actinomycetes of the dioscorea zingiberensis endophytes, reports about bacterial endophytes of the dioscorea zingiberensis endophytes are few, and a novel natural product resource library of the dioscorea zingiberensis endophytes is not widely developed. Based on this, the inventors of the present invention have taken the dioscorea zingiberensis endophyte as a research and development direction and have searched for the isolation and purification of novel compounds from the secondary metabolites of the dioscorea zingiberensis endophyte.

The inventor of the patent successfully separates a fluorescent novel compound from a fermentation product of dioscorea zingiberensis endophytic bacillus licheniformis Syb06.11.1, and names the compound as follows: endogenous Bacillus licheniformis A. Through structural identification, the structural formula is as follows:

the endogenous Bacillus licheniformis A is a seven-membered ring de-aromatic compound with a positive charge and a molecular formula C₁₅H₁₃O₄ ⁺The molecular weight is 257.0805, the light yellow needle crystal is easily soluble in methanol, blue fluorescence is developed under the ultraviolet of 365nm, the color is developed by sulfuric acid ethanol, and the strong yellow green fluorescence is obtained under the ultraviolet of 365 nm.

The endophytic bacillus licheniformis A is separated from fermentation liquor of dioscorea zingiberensis endophyte, is bacillus licheniformis Syb06.11.1, is stored in CGMCC, has the storage time of 2008-1-10 months and has the storage number of 2334. For information on that the endophyte of dioscorea zingiberensis is bacillus licheniformis syb06.11.1, see the description of patent ZL 200810150274.3.

The invention provides a preparation method of the fluorescent compound, which is named as a fluorescence correlation tracing method. Extracting fermentation liquor of dioscorea zingiberensis endophytic bacillus licheniformis Syb06.11.1 by using ethyl acetate, and concentrating to obtain a target crude product; and separating the target crude product by a chromatographic column, preparing by thin-layer chromatography, and purifying to obtain the fluorescent compound.

As one of the preferable embodiments of the method for preparing the fluorescent compound, it comprises,

hydrolyzing fermentation liquid of dioscorea zingiberensis endophytic bacillus licheniformis Syb06.11.1 by acid, adjusting pH to be neutral, extracting by using ethyl acetate, and concentrating to obtain ethyl acetate extract;

normal phase column chromatography separation, wherein the eluent is cyclohexane and ethyl acetate which are 10: 1;

separating with Sephadex LH-20 column for the first time, wherein the eluent is methanol; separating with Sephadex LH-20 column, eluting with chloroform and methanol at a ratio of 1: 1;

RP-C18 reverse silica gel column separation, wherein the eluent is methanol and water is 6: 4;

preparing thin layer chromatography;

and (3) performing Sephadex LH-20 gel column chromatography, wherein the eluent is chloroform and methanol which are 1: 1.

The compound has a large pi bond in the molecular structure, forms a closed-loop structure, has fluorescence, and can be used for preparing organic luminescent materials. The fluorescence property of the fluorescent compound is measured, and the result shows that the compound has fluorescence and can be applied to the preparation of luminescent materials. Based on this, the invention claims all the applications of the fluorescent compound in preparing luminescent materials.

The fluorescent compound and the preparation method and the application thereof have at least the following beneficial effects or advantages.

The invention separates a fluorescent new compound from the fermentation product of the peltate yam endophytic bacillus licheniformis Syb06.11.1 for the first time, and the compound is determined to be a new natural product through chromatographic column separation and multiple technical identifications such as nuclear magnetism, high-resolution mass spectrometry and the like. It is a seven-membered ring dearomatized compound, as seen in its structural formula, and carries a positive charge. The fluorescence property determination shows that the compound has fluorescence and can be applied to the preparation of luminescent materials. The invention enriches natural product resource library and provides a foundation for research and development of new drugs.

Drawings

FIG. 1 shows a process for separating fluorescent compounds according to the present example.

FIG. 2 is a graph showing the results of the normal phase silica gel column chromatography separation and the TLC detection of no color development in this example.

FIG. 3 is a diagram showing the results of the chromatographic separation of the normal phase silica gel column and the color development by thin layer chromatography.

FIG. 4 is a graph showing the results of two Sephadex LH-20 gel column chromatographies separated and not developed by thin layer chromatography.

FIG. 5 is a diagram showing the results of thin layer chromatography detection and color development of two Sephadex LH-20 gel column chromatographies described in this example.

FIG. 6 is a graph showing the results of preparative thin layer chromatography described in this example.

FIG. 7 is a graph showing the results of detection of no color development by thin layer chromatography using preparative thin layer chromatography described in this example.

FIG. 8 is a graph showing the results of color development by thin layer chromatography detection using preparative thin layer chromatography described in this example.

FIG. 9 is a diagram showing the results of Sephadex LH-20 gel column chromatography purification and TLC detection of no color development described in this example.

FIG. 10 is a diagram showing the results of Sephadex LH-20 gel column chromatography purification and thin layer chromatography detection of color development described in this example.

FIG. 11 is a crystal diagram of the fluorescent compound according to the present example.

FIG. 12 shows the fluorescent compounds of this example¹H-NMR spectrum.

FIG. 13 shows a schematic representation of a fluorescent compound according to this example¹³C-NMR spectrum.

FIG. 14 shows DEPT spectra of fluorescent compounds of this example.

FIG. 15 shows the HMBC spectrum of the fluorescent compound of this example.

FIG. 16 shows HSQC spectra of fluorescent compounds of this example.

FIG. 17 is a spectrum of H1-H1COSY of the fluorescent compound of this example.

FIG. 18 is a low-resolution mass spectrum of the fluorescent compound of this example.

FIG. 19 is a high-resolution mass spectrum of the fluorescent compound of this example.

FIG. 20 is a graph showing the measurement of the maximum excitation wavelength of the fluorescent compound according to the present example.

FIG. 21 is a graph showing the measurement of the maximum emission wavelength of the fluorescent compound according to the present example.

Detailed Description

In order to facilitate understanding of the objects, technical solutions and effects of the present invention, the present invention will be further described in detail with reference to examples.