阅读说明：本技术 一种海洋真菌来源的penialidins类化合物、制备方法及其应用 (Penialidins compound derived from marine fungi, preparation method and application thereof ) 是由 陈敏 梁朝阳 沈南星 王长云 于 2018-09-30 设计创作，主要内容包括：本发明涉及一种海洋真菌来源的penialidins类化合物、制备方法及其应用,所述penialidins类化合物结构如式I所示：<Image he="360" wi="583" file="DDA0001818945700000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>其中R<Sub>1</Sub>、R<Sub>2</Sub>、R<Sub>3</Sub>各自独立地选自H、任选取代烷基、酰基,“---”表示单键或不存在,且当“---”表示单键时,OR<Sub>3</Sub>不存在。本发明对海洋真菌Penicillium limosum HK1-23,进行大规模发酵,任选进行衍生化可大量获得penialidins类化合物或其药学上可接受的盐,其中penialidin A、penialidin C的发酵产量在260mg/L以上,可为penialidins类化合物药理活性的研究提供丰富的化合物。(The invention relates to a pentalidins compound from marine fungi, a preparation method and application thereof, wherein the structure of the pentalidins compound is shown as a formula I: wherein R is 1 、R 2 、R 3 Each independently selected from H, optionally substituted alkyl, acyl, "- - -" represents a single bond OR is absent, and OR when "- -" represents a single bond 3 Is absent. The invention is used for marine fungus PenicilThe lium limosum HK1-23 is subjected to large-scale fermentation, and a large amount of penilidins or pharmaceutically acceptable salts thereof can be obtained by optionally performing derivatization, wherein the fermentation yield of penilidins A and C is over 260mg/L, and abundant compounds can be provided for research on pharmacological activity of the penilidins.)

1. a process for the preparation of penilidin a, mixture of penilidin C or a pharmaceutically acceptable salt thereof from the marine fungus Penicillium limosum HK1-23, characterized in that it comprises the steps of:

(1) firstly, carrying out strain culture on marine fungus Penicillium limosum HK1-23 in a strain culture medium;

(2) then carrying out fermentation culture on the marine fungus Penicillium limosum HK1-23 in the step (1) in a fermentation culture medium;

(3) separating the fermentation liquor from the thalli in the fermentation product obtained after the fermentation culture in the step (2), extracting the fermentation liquor for 2-4 times by using an organic solvent A, combining the extraction liquor, and concentrating under reduced pressure to obtain a fermentation liquor extract; leaching the thallus for 2-4 times by using an organic solvent B, combining leaching liquor, and concentrating under reduced pressure to obtain a thallus extract; combining the fermentation liquid extract and the thallus extract, and obtaining a crude product through chromatographic separation;

(4) diluting the crude product obtained in the step (3) with an organic solvent A, extracting with a sodium bicarbonate solution for 2-4 times, combining the sodium bicarbonate solutions (inorganic layers), adding concentrated hydrochloric acid to adjust the pH value to 1-3, separating out a yellow precipitate, and filtering, washing and drying to obtain a mixture of penililin A and penililin C;

wherein the strain culture medium contains glucose, yeast extract, peptone, agar, crude sea salt and water, and the fermentation culture medium contains glucose, yeast extract, peptone, crude sea salt and water. The organic solvent A is preferably one or more of ethyl acetate, dichloromethane, chloroform or diethyl ether; the organic solvent B is preferably one or more of methanol, ethanol, THF or acetone; the chromatographic separation is preferably performed by reduced pressure column chromatography, and the filler of the reduced pressure column chromatography is preferably normal phase silica gel or macroporous resin.

2. The method according to claim 1, wherein the culture medium of the strain of step (1) preferably contains glucose 0.2% -5.0%, yeast extract 0.02% -2%, peptone 0.02% -2%, agar 0.2% -3.0%, crude sea salt 0.05% -5%, and a proper amount of water; the percentages are weight percentages; the culture temperature is preferably 15-25 ℃; the culture time is preferably 3-7 days; the fermentation culture medium in the step (2) preferably contains 0.2-5.0% of glucose, 0.02-2% of yeast extract, 0.02-2% of peptone, 0.05-5% of crude sea salt and a proper amount of water; the percentages are weight percentages; the culture temperature is preferably 15-25 ℃; the culture time is preferably 4 to 5 weeks.

3. The preparation method of claim 1, wherein the mobile phase used in the reduced pressure column chromatography is a conventional eluent in the art, preferably one or more of ethyl acetate, petroleum ether, dichloromethane, methanol, ethanol, water or chloroform.

4. A method for producing purified penilidin a and penilidin C products, comprising the steps of:

subjecting the mixture of penilidin A and penilidin C obtained in step (4) of the preparation method according to any one of claims 1 to 3 to normal phase silica gel column chromatography to obtain penilidin C and penilidin A.

5. The method as claimed in claim 4, wherein the normal phase silica gel column chromatography adopts a stationary phase of 200-300 mesh silica gel and a mobile phase of a mixed solvent of dichloromethane/methanol in a volume ratio of 20/1-10/1 or a mixed solvent of petroleum ether/ethyl acetate in a volume ratio of 1.5/1-1/1.5. And (3) optionally adding a proper amount of organic acid or organic base into the mobile phase, wherein the organic acid is selected from formic acid or acetic acid, and the organic base is selected from triethylamine.

6. Application of marine fungus Penicillium limosum HK1-23 in preparation of penilidins compounds or pharmaceutically acceptable salts thereof; in particular for the preparation of penilidin A, penilidin C or mixtures thereof or pharmaceutically acceptable salts thereof.

7. A penilidins compound shown as formula I or a pharmaceutically acceptable salt thereof, wherein the structure of formula I is as follows:

wherein R is₁、R₂、R₃Each independently selected from H, optionally substituted alkyl (e.g., methyl, ethyl, benzyl), acyl (e.g., acetyl, benzoyl), "- - - -" represents a single bond OR is absent, and OR when "- -" represents a single bond₃Is absent; when "- - -" is absent, R₃Selected from H, optionally substituted alkyl (e.g. methyl, ethyl, benzyl), acyl (e.g. acetyl, benzoyl). Penialidins A, C, E-J compounds are preferred.

8. Use of a penilidins compound of formula I or a pharmaceutically acceptable salt thereof as claimed in claim 7 for the preparation of lead compounds, drug candidates and/or medicaments.

9. The use of a penilidins compound of formula I or a pharmaceutically acceptable salt thereof as claimed in claim 7 for the prevention and treatment of alternaria alternate.

Technical Field

The invention belongs to the field of secondary metabolites of marine fungi, and particularly relates to a pentalidins compound derived from marine fungi, and a preparation method and application thereof.

Background

Penialidins A-C is a polyketone antibacterial active compound first reported by Michael Spiteller et al (Fitoterapia, 2014(98), 209-214). At present, no report is available about chemical synthesis or large-scale fermentation preparation of Penialidins A-C compounds. The invention provides a method for preparing Penialidins A and C in a large scale by using marine fungus Penicillium limosumHK1-23, and a biological activity test is carried out on Penialidins compounds.

Disclosure of Invention

The strain preservation information of the marine fungus Penicillium limosum HK1-23 of the invention is as follows: the name of the depository: china general microbiological culture Collection center; the address of the depository: western road No.1, north west city of township, beijing, institute of microbiology, china academy of sciences; the preservation date is as follows: year 2018, month 2 and day 5; the preservation number is: CGMCC No. 15361; and (3) classification and naming: penicillium limosum.

The invention provides a method for preparing penilidin A, a mixture of penilidin C or pharmaceutically acceptable salts thereof from marine fungus Penicillium limosum HK1-23, which is characterized by comprising the following steps:

(1) firstly, carrying out strain culture on marine fungus Penicillium limosum HK1-23 in a strain culture medium;

(2) then carrying out fermentation culture on the marine fungus Penicillium limosum HK1-23 in the step (1) in a fermentation culture medium;

(3) separating the fermentation liquor from the thalli in the fermentation product obtained after the fermentation culture in the step (2), extracting the fermentation liquor for 2-4 times by using an organic solvent A, combining the extraction liquor, and concentrating under reduced pressure to obtain a fermentation liquor extract; leaching the thallus for 2-4 times by using an organic solvent B, combining leaching liquor, and concentrating under reduced pressure to obtain a thallus extract; combining the fermentation liquid extract and the thallus extract, and obtaining a crude product through chromatographic separation;

(4) and (3) diluting the crude product obtained in the step (3) with an organic solvent A, extracting with a sodium bicarbonate solution for 2-4 times, combining the sodium bicarbonate solutions (inorganic layers), adding concentrated hydrochloric acid to adjust the pH value to 1-3, separating out a yellow precipitate, and filtering, washing and drying to obtain a mixture of the penilidin A and the penilidin C.

Wherein the strain culture medium contains glucose, yeast extract, peptone, agar, crude sea salt and water, and the fermentation culture medium contains glucose, yeast extract, peptone, crude sea salt and water.

In the preparation method of the invention, the organic solvent A is preferably one or more of ethyl acetate, dichloromethane, chloroform or diethyl ether; the organic solvent B is preferably one or more of methanol, ethanol, THF or acetone; the chromatographic separation is preferably performed by reduced pressure column chromatography, and the filler of the reduced pressure column chromatography is preferably normal phase silica gel or macroporous resin.

In the preparation method of the invention, the strain culture medium preferably contains 0.2-5.0% of glucose, 0.02-2% of yeast extract, 0.02-2% of peptone, 0.2-3.0% of agar, 0.05-5% of crude sea salt and a proper amount of water; the percentages are weight percentages; the culture temperature is preferably 15-25 ℃; the culture time is preferably 3 to 7 days. The fermentation culture medium preferably contains 0.2-5.0% of glucose, 0.02-2% of yeast extract, 0.02-2% of peptone, 0.05-5% of crude sea salt and a proper amount of water; the percentages are weight percentages; the culture temperature is preferably 15-25 ℃; the culture time is preferably 4-5 weeks; the mobile phase adopted in the reduced pressure column chromatography is a conventional eluent in the field, and preferably one or a mixture of more of ethyl acetate, petroleum ether, dichloromethane, methanol, ethanol, water or chloroform.

Another embodiment of the present invention provides a process for the preparation of purified penilidin a and penilidin C, characterized in that it comprises the steps of:

and (3) subjecting the mixture of the penilidin A and the penilidin C obtained in the step (4) of the preparation method to normal phase silica gel column chromatography to obtain the penilidin C and the penilidin A.

The stationary phase adopted by the normal phase silica gel column chromatography is 200-300 mesh silica gel, and the mobile phase is a mixed solvent with the volume ratio of dichloromethane/methanol being 20/1-10/1 or a mixed solvent with the volume ratio of petroleum ether/ethyl acetate being 1.5/1-1/1.5. And (3) optionally adding a proper amount of organic acid or organic base into the mobile phase, wherein the organic acid is selected from formic acid or acetic acid, and the organic base is selected from triethylamine.

Another embodiment of the invention provides the use of the marine fungus Penicillium limosum HK1-23 for the preparation of a penilidins compound or a pharmaceutically acceptable salt thereof; in particular for the preparation of penilidin A, penilidin C or mixtures thereof or pharmaceutically acceptable salts thereof.

The penilidins compounds provided by the invention refer to compounds with the same frameworks as penilidin A and penilidin C and different only in individual substituent groups, and the structure is shown as formula I:

wherein R is₁、R₂、R₃Each independently selected from H, optionally substituted alkyl (e.g., methyl, ethyl, benzyl), acyl (e.g., acetyl, benzoyl), "- - - -" represents a single bond OR is absent, and OR when "- -" represents a single bond₃Is absent; when "- - -" is absent, R₃Selected from H, optionally substituted alkyl (e.g. methyl, ethyl, benzyl), acyl (e.g. acetyl, benzoyl). Penialidins A, C, E-J compounds are preferred.

In another embodiment of the present invention, the use of the penilidins compound with the structure of formula I or the pharmaceutically acceptable salt thereof in preparing hypoglycemic lead compounds, candidate drugs and drugs is provided.

In another embodiment, the present invention provides the use of a penilidins compound of formula I as described above or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the prevention and/or treatment of diseases mediated by glucokinase.

In another embodiment of the invention, the application of the penilidins compound with the structure shown in the formula I or the pharmaceutically acceptable salt thereof in preventing and treating the alternaria alternate disease is provided.

In another embodiment, the invention provides the use of a penilidins compound with the structure of formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for resisting alternaria alternate.

The marine fungus Penicillium limosum HK1-23 is isolated from the rhizosphere soil of the Morus alba in the Hongkong forest protection area of Shanghai, Haikai.

The term "pharmaceutically acceptable salts" as used herein refers to non-toxic inorganic or organic acid and/or base addition salts, as described in "Salt selection for basic drugs", int.J.pharm. (1986),33, 201-217.

The invention provides a preparation example of penilidin A, penilidin C or a mixture thereof, and also provides a preparation example of methylation and acetylation of the penilidin A and the penilidin C, and other penilidin compounds or pharmaceutically acceptable salts thereof can be prepared from the penilidin A, the penilidin C or derivatives thereof by a chemical derivatization method or a salt preparation method which are conventional in the field, and belongs to the technical means which are conventional in the field.

The invention has the advantages that the marine fungus Penicillium limosum HK1-23 adopted in the invention can be artificially fermented in a large scale without being limited by resources, the culture medium and the culture conditions are adopted for fermentation, and the peniliadins compound or the pharmaceutically acceptable salt thereof can be obtained in a large scale by optionally performing derivatization, the fermentation yield of the peniliadins A and C is more than 260mg/L, and abundant compounds can be provided for the research of the pharmacological activity of the peniliadins compound.

It is to be understood that within the scope of the present invention, the above-described technical features of the present invention and the technical features specifically described below (e.g., examples) may be combined with each other to constitute a new or preferred technical solution. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 is of penilidin A¹H NMR chart;

FIG. 2 is of penilidin C¹H NMR chart;

FIG. 3 is of penilidin C¹³C NMR chart.

Detailed Description

In order to facilitate a further understanding of the invention, the following examples are provided to illustrate it in more detail. However, these examples are only for better understanding of the present invention and are not intended to limit the scope or the principle of the present invention, and the embodiments of the present invention are not limited to the following.