阅读说明：本技术 一种6，7，8-三羟基香豆素的制备方法 (Preparation method of 6, 7, 8-trihydroxy coumarin ) 是由 李笃信 赵磊 张健 朱雪雪 于 2019-10-21 设计创作，主要内容包括：本发明公开了一种6,7,8-三羟基香豆素的制备方法,该方法包括：(1)以秦皮苷或/和秦皮素为起始原料,将所述原料加入第一溶液中使其溶解,获得原料液；(2)动物粪菌制作载体：在无菌条件下收集动物的新鲜粪便,加入生理盐水匀浆,离心后取上清液；(3)厌氧培养：将所述上清液加入到营养肉汤中,厌氧恒温培养后,加入所述原料液,再加入厌氧培养液,厌氧恒温培养,获得第一培养液；(4)产物的提取：萃取所述第一培养液,合并萃取液后减压回收；(5)产物的纯化：利用制备液相色谱进行纯化。本发明转化率可达98.0％以上,步骤简单、速度快,对环境友好,适合工业化生产。(The invention discloses a preparation method of 6, 7, 8-trihydroxy coumarin, which comprises the following steps: (1) taking aesculin or/and aesculin as starting raw materials, and adding the raw materials into the first solution to dissolve the raw materials to obtain a raw material solution; (2) preparing a carrier by using animal dung bacteria: collecting fresh feces of animals under aseptic condition, adding normal saline to homogenate, centrifuging and taking supernatant; (3) anaerobic culture: adding the supernatant into nutrient broth, carrying out anaerobic constant-temperature culture, adding the raw material solution, adding an anaerobic culture solution, and carrying out anaerobic constant-temperature culture to obtain a first culture solution; (4) and (3) extraction of a product: extracting the first culture solution, combining the extract solutions, and recovering under reduced pressure; (5) and (3) purifying a product: purification was performed by preparative liquid chromatography. The invention has the advantages of high conversion rate of over 98.0 percent, simple steps, high speed, environmental protection and suitability for industrial production.)

1. A preparation method of 6, 7, 8-trihydroxy coumarin is characterized by comprising the following steps:

(1) taking aesculin or/and aesculin as starting raw materials, and adding the raw materials into the first solution to dissolve the raw materials to obtain a raw material solution;

(2) preparing a carrier by using animal dung bacteria: collecting fresh feces of animals under aseptic condition, adding normal saline to homogenate, centrifuging and taking supernatant;

(3) anaerobic culture: adding the supernatant into nutrient broth, carrying out anaerobic constant-temperature culture, adding the raw material solution, adding an anaerobic culture solution, and carrying out anaerobic constant-temperature culture to obtain a first culture solution;

(4) and (3) extraction of a product: extracting the first culture solution, combining the extract solutions, and recovering under reduced pressure;

(5) and (3) purifying a product: purification was performed by preparative liquid chromatography.

2. The preparation method of 6, 7, 8-trihydroxy coumarin according to claim 1, wherein the preparation method comprises the following steps: the first solution in the step (1) is physiological saline, phosphate buffer, carbonate buffer, Tris buffer or water.

3. The preparation method of 6, 7, 8-trihydroxy coumarin according to claim 1, wherein the preparation method comprises the following steps: the feces in the step (2) are feces of rats, mice, rabbits, guinea pigs or humans, and are preferably rat feces.

4. The preparation method of 6, 7, 8-trihydroxy coumarin according to claim 1, wherein the preparation method comprises the following steps: the nutrient broth in the step (3) is composed of peptone and beef extract powder, and can be any one or the combination of two components.

5. According toThe process for preparing 6, 7, 8-trihydroxy coumarin as claimed in claim 1, wherein: the anaerobic condition in the step (3) is CO₂、N₂Ar or He atmosphere, and the culture time is 2-10 d.

6. The preparation method of 6, 7, 8-trihydroxy coumarin according to claim 1, wherein the preparation method comprises the following steps: the anaerobic culture solution in the step (3) comprises dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, ammonium sulfate, calcium chloride, magnesium sulfate, resazurin, L-cysteine and L-ascorbic acid; the amount of said anaerobic culture fluid added is 0.2-5 times the volume of said nutrient broth.

7. The preparation method of 6, 7, 8-trihydroxy coumarin according to claim 1, wherein the preparation method comprises the following steps: the solvent for extraction in the step (4) is ethyl acetate, n-butanol, chloroform, dichloromethane or n-hexane, the amount of the extracting agent is 0.5-2 times, and the times are 1-3.

8. The preparation method of 6, 7, 8-trihydroxy coumarin according to claim 1, wherein the preparation method comprises the following steps: the separation in the step (5) is carried out by using any one of silica gel, a C8 column and a C18 column.

Technical Field

The invention belongs to the technical field of microbial transformation, and particularly relates to a preparation method of 6, 7, 8-trihydroxy coumarin.

Background

Fraxins, fraxinin and coumarins are effective components of cortex Fraxini, and fraxins are hydrolysate of fraxins. Bruno M.Bizzarri et al react with aesculin as a substrate and 2-iodoxybenzoic acid as an oxidant to produce 6, 7, 8-trihydroxy coumarin, which has stronger oxidation resistance and antibacterial activity than aesculin. However, the synthesis method has the defects of more byproducts, difficult control of reaction end point and difficult separation of products. Therefore, there is a need to research a novel preparation method of 6, 7, 8-trihydroxy coumarin.

Microbial transformation studies began in the 20 th century and aimed at the co-cultivation of bacteria, fungi or other microorganisms with exogenous substrates to yield valuable products, among which the enzymes produced by the microorganisms play a major role. The traditional Chinese medicine contains various complex active ingredients, each kind of ingredient plays an important role, but the research of the traditional Chinese medicine can not meet the increasing demand of people for medication, so the traditional Chinese medicine is combined with microbial transformation, and the active ingredients in the traditional Chinese medicine are favorably and better extracted and transformed.

Disclosure of Invention

The invention aims to provide a preparation method of 6, 7, 8-trihydroxy coumarin, which solves the problems.

The technical scheme of the invention is as follows: a preparation method of 6, 7, 8-trihydroxy coumarin comprises the following steps: (1) taking aesculin or/and aesculin as starting raw materials, and adding the raw materials into the first solution to dissolve the raw materials to obtain a raw material solution;

(2) preparing a carrier by using animal dung bacteria: collecting fresh feces of animals under aseptic condition, adding normal saline to homogenate, centrifuging and taking supernatant;

(3) anaerobic culture: adding the supernatant into nutrient broth, carrying out anaerobic constant-temperature culture, adding the raw material solution, adding an anaerobic culture solution, and carrying out anaerobic constant-temperature culture to obtain a first culture solution;

(4) and (3) extraction of a product: extracting the first culture solution, combining the extract solutions, and recovering under reduced pressure;

(5) and (3) purifying a product: purification was performed by preparative liquid chromatography.

Further, the first solution in step (1) is physiological saline, phosphate buffer, carbonate buffer, Tris buffer or water.

Further, the feces in the step (2) is feces of a rat, a mouse, a rabbit, a guinea pig or a human, and is preferably rat feces.

Further, the components of the nutrient broth in the step (3) are peptone and beef extract powder, and the components of the nutrient broth can be any one or the combination of two components.

Further, the anaerobic condition in the step (3) is CO2, N2, Ar or He atmosphere, and the culture time is 2-10 d.

Further, the anaerobic culture solution in the step (3) comprises dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, ammonium sulfate, calcium chloride, magnesium sulfate, resazurin, L-cysteine and L-ascorbic acid; the amount of said anaerobic culture fluid added is 0.2-5 times the volume of said nutrient broth.

Further, the solvent for extraction in the step (4) is ethyl acetate, n-butanol, chloroform, dichloromethane or n-hexane, and the amount of the extracting agent is 0.5-2 times, and the times are 1-3.

Further, the separation in the step (5) is carried out by using any one of silica gel, a C8 column and a C18 column.

The invention provides a preparation method of 6, 7, 8-trihydroxy coumarin, which has the advantages that: the method solves the problems of complex production process and low efficiency of the existing 6, 7, 8 trihydroxy coumarin, and has the characteristics of simple operation steps, high conversion efficiency, high conversion rate of more than 98.0 percent, high speed, environmental friendliness and suitability for industrial production.

Drawings

The invention is further described with reference to the following figures and examples:

FIG. 1 is a schematic diagram of the microbial transformation of fraxin and fraxin in an embodiment of the present invention, wherein (A) is fraxin, (B) is fraxin, and (C) is 6, 7, 8-trihydroxycoumarin;

FIG. 2 is a schematic diagram showing the effect of time on the conversion of 6, 7, 8-trihydroxy coumarin, a product obtained in example 2, wherein 1 is aesculin and 2 is 6, 7, 8-trihydroxy coumarin;

FIG. 3 is a schematic diagram showing the effect of time on the conversion rate of 6, 7, 8-trihydroxy coumarin, which is a product obtained in example 3, wherein 1 is aesculin, 2 is aesculin, and 3 is 6, 7, 8-trihydroxy coumarin.

Detailed Description

The invention provides a preparation method of 6, 7, 8-trihydroxy coumarin, which comprises the following steps:

(1) the aesculin and/or the fraxinin are used as starting materials: adding physiological saline or other solution to dissolve;

(2) animal dung bacteria are used as carriers: collecting fresh feces of animals under aseptic condition, wherein the feces can be feces of rat, mouse, human or other animals, preferably rat feces, adding normal saline to homogenate, centrifuging, and collecting supernatant, wherein the centrifugation speed is 2000-;

(3) anaerobic culture: adding the supernatant to a nutrient broth, CO₂、N₂Or culturing at 36.0-38.0 deg.C for 12-36 hr under anaerobic condition, adding raw materials, adding 0.5-1.0 times volume of anaerobic culture solution, and performing anaerobic constant temperature culture;

(4) and (3) extraction of a product: extracting the culture solution with ethyl acetate, n-butanol or other water-immiscible organic solvent, preferably ethyl acetate, for 1-3 times, mixing extractive solutions, and recovering under reduced pressure;

(5) and (3) purifying a product: the separation is carried out with silica gel, ODS or other fillers, preferably silica gel or ODS.

The microbial transformation process is shown in figure 1.

In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below. The invention is not limited to the embodiments listed but also comprises any other known variations within the scope of the invention as claimed.

Reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic may be included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.