CN113072442B

CN113072442B - Benzophenone compound in mangrove endophytic fungi and preparation method and application thereof

Info

Publication number: CN113072442B
Application number: CN202110232241.9A
Authority: CN
Inventors: 白猛; 刘永宏; 高程海; 刘锴; 黄炳耀; 唐振洲; 林霄
Original assignee: Guangxi University of Chinese Medicine
Current assignee: Guangxi University of Chinese Medicine
Priority date: 2021-03-02
Filing date: 2021-03-02
Publication date: 2023-03-21
Anticipated expiration: 2041-03-02
Also published as: CN113072442A

Abstract

The invention relates to a benzophenone compound in mangrove endophytic fungi as well as a preparation method and application thereof, in particular to 2 benzophenone compounds or pharmaceutically acceptable salts thereof, which is characterized in that the benzophenone compounds have the structures shown in compounds 1 and 2:

Description

Benzophenone compound in mangrove endophytic fungi and preparation method and application thereof

Technical Field

The invention belongs to the field of secondary metabolites of mangrove endophytic fungi, and particularly relates to a benzophenone compound in mangrove endophytic fungi as well as a preparation method and application thereof.

Background

Marine-derived endophytic fungi are one of the important sources of compounds with novel structures and unique activities. In recent years, some new active compounds have been isolated from the fungus Penicillium citrinum, for example: cytotoxic compounds penibenzophenones a and B, anti-phytopathogen compounds citrinin and emodin, alpha-glucopsidase activity compounds penitimarin J and penitimarin K, and antifungal compounds penicitrinone a and penicitrinol J. The invention provides a benzophenone compound in mangrove endophytic fungus Penicillium citrinum HL-5126 and a preparation method and application thereof.

Disclosure of Invention

The invention provides a benzophenone compound derived from mangrove endophytic fungi or pharmaceutically acceptable salt thereof, which is characterized in that the benzophenone compound has the structure shown by compounds 1 and 2:

another embodiment of the present invention provides a method for simultaneously preparing the above-mentioned

compounds

1 and 2, characterized by comprising the steps of:

(1) Preparing a seed culture medium, inoculating Penicillium citrinum HL-5126 strain into the seed culture medium, and culturing for 3-4 days at 28 ℃ to obtain a seed culture solution;

(2) Inoculating the seed culture solution obtained in the step (1) into a fermentation culture medium, and performing shake culture at 26-28 ℃ for 28 days to obtain a fermented product;

(3) Separating the fermentation liquor and the thalli in the fermentation product obtained in the step (2), extracting the fermentation liquor for 3-5 times by using ethyl acetate with the same volume, combining the extraction liquor, and then concentrating under reduced pressure to obtain an extract;

(4) The extract obtained in the step (3) is subjected to reduced pressure silica gel column chromatography, and is subjected to gradient elution with petroleum ether-ethyl acetate according to the following ratio of 100 ₃ CN:H ₂ O =35, to finally obtain compound 1; merging 20And (2) carrying out Sephadex LH-20 gel column chromatography after eluting the obtained fraction and concentrating, wherein an eluent is petroleum ether, ethyl acetate =1 and 3, and the Sephadex LH-20 gel column chromatography is carried out for preparation by High Performance Liquid Chromatography (HPLC), a chromatographic column is Agilent C18, 9.4X 250mm and 7 mu m, the flow rate is 2mL/min, and a mobile phase is MeOH, H and ₂ o =45, to finally obtain compound 2.

Wherein the ratio of the eluent or the mobile phase is volume ratio; the seed culture medium contains 1.5-3.0% of glucose, 0.1-0.5% of yeast extract, 0.1-0.5% of peptone, 0.11-0.6% of crude sea salt and a proper amount of water; the fermentation culture medium contains 1.6-3.5% of glucose, 0.1-0.5% of yeast extract, 0.1-0.5% of peptone, 0.11-0.6% of crude sea salt and a proper amount of water; the percentages are weight percentages; the seed culture medium and the fermentation culture medium are both sterilized at 120 ℃ for 25-30 minutes.

The rotation speed of the shaking culture in the step (2) is preferably 150-180r/min.

The present invention provides a pharmaceutical composition characterized by comprising the above-mentioned

compounds

1 and 2 of the present invention or pharmaceutically acceptable salts thereof as an active ingredient.

The present invention provides a neuronal protection pharmaceutical composition characterized by comprising the above-mentioned

compounds

1 and 2 of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention provides a pharmaceutical composition for preventing and/or treating stroke, which comprises the above-mentioned

compounds

1 and 2 or a pharmaceutically acceptable salt thereof of the present invention as an active ingredient.

The pharmaceutical composition provided by the invention can also comprise other active ingredients; pharmaceutically acceptable adjuvants (preferably pharmaceutically acceptable carriers, diluents or excipients) may also be included. The dosage form of the pharmaceutical composition can be solid preparation, semi-solid preparation or liquid preparation.

Another embodiment of the present invention provides the use of the above-mentioned

compounds

1, 2 or pharmaceutically acceptable salts thereof for controlling agricultural pests. The pest is preferably cotton bollworm.

compounds

1, 2 or pharmaceutically acceptable salts thereof for the preparation of a medicament for the treatment and/or prevention of stroke.

compounds

1, 2 or a pharmaceutically acceptable salt thereof for the preparation of a medicament for neuronal protection.

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 relates to a 'mangrove endophytic fungus Penicillium citrinum HL-5126' which is collected from the root of the horny fruit tree of a mangrove plant in the natural protection area of the mangrove forest of Hongkong nationality of Katsuma by inventor co-workers, separated and purified, and identified as Penicillium (Penicillium citrinum) through morphology and molecular biology; the sequence of the ITS region of this fungus has been submitted to NCBI (ITS Gene Bank: KJ466981.1, LOCUS: KJ 466981). The "Penicillium citrinum HL-5126" of the invention is disclosed in previous research papers "Chin.J.org.chem.2019,39,1479-1482" of the inventors and the collaborators and "CN 106432169A" of the patent, and is always preserved in the Guangxi Marine drug focus laboratory where the inventors and the collaborators are located, and also preserved in the Tropical medicinal resource chemical education department focus laboratory where the inventors and the collaborators are located (great name: tropical medicinal plant chemical education department focus laboratory), and the public can obtain the "Penicillium citrinum HL-5126" of the invention according to the methods described in the research papers of the inventors and the collaborators, or purchase the public Penicillium citrinum laboratory where the inventors and the collaborators are located, and promise to provide the Penicillium citrinum HL-5126 of the invention to the public Penicillium citrinum laboratory where the inventor and the Tropical medicinal resource chemical education laboratory where the inventors are located 20 years from the filing date of the invention.

Drawings

FIG. 1 is a drawing of Compound 1 ¹ H NMR(400MHz,DMSO-d ₆ ) A drawing;

FIG. 2 is a drawing of Compound 1 ¹³ C NMR(100MHz,DMSO-d ₆ ) A drawing;

FIG. 3 is a DEPT diagram of Compound 1;

FIG. 4 is a HMQC plot for Compound 1;

FIG. 5 is a HMBC diagram for compound 1;

FIG. 6 is a drawing of Compound 1 ¹ H- ¹ H COSY picture;

FIG. 7 is a NOESY plot for Compound 1;

FIG. 8 is a drawing of Compound 2 ¹ H NMR(400MHz,DMSO-d ₆ ) A drawing;

FIG. 9 is a drawing of Compound 2 ¹³ C NMR(100MHz,DMSO-d ₆ ) A drawing;

FIG. 10 is a DEPT diagram of Compound 2;

FIG. 11 is a HMQC plot of Compound 2;

FIG. 12 is a HMBC diagram for Compound 2;

FIG. 13 is a high resolution mass spectrum of Compound 1;

fig. 14 is a high resolution mass spectrum of compound 2.

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.

Example 1

(1) Culture of Penicillium citrinum HL-5126 strain

Preparing a seed culture medium: 80g of glucose, 8g of peptone, 8g of yeast extract, 12g of crude sea salt and 4.0L of water are averagely distributed in 8 conical flasks with 1000mL and are sterilized at 120 ℃ for 25-30 minutes.

Inoculating Penicillium citrinum strain into prepared seed culture medium, and culturing at 28 deg.C for 3 days to obtain seed culture solution;

(2) Fermentation of Penicillium citrinum

Preparing a fermentation medium: 1.1kg of glucose, 100g of peptone, 100g of yeast extract, 150g of sea salt and 50L of water are averagely distributed in 100 conical flasks of 1000mL and sterilized at 120 ℃ for 25-30 minutes.

And (2) taking a proper amount of the seed culture solution obtained in the step (1), inoculating into a conical flask filled with a fermentation culture medium (the inoculation amount is preferably 5-10% by volume), and performing shake cultivation at 26-28 ℃ for 28 days (the rotation speed is 180 r/min) to obtain a fermentation product.

(3) Isolation of

Compounds

1 and 2

Separating the fermentation liquor and the thallus in the fermentation product obtained in the step (2), extracting the fermentation liquor for 3 times by using ethyl acetate with the same volume, combining the extraction liquor, and concentrating under reduced pressure to obtain an extract (37.5 g); the extract is subjected to reduced pressure silica gel column chromatography, eluting with petroleum ether-ethyl acetate in a gradient of 100, 90, 10, 80, 30, 60 ₃ CN:H ₂ O =35, to finally obtain compound 1 (3.5 mg). Combining fractions obtained by gradient elution of 20 ₂ O =45, to finally obtain compound 2 (3.6 mg).

Of compounds 1 and 2 ¹ H and ¹³ C-NMR(400/100MHz,DMSO-d ₆ ) The data are shown in Table 1 below, NMR and MS spectra are shown in FIGS. 1-14.

TABLE 1 preparation of Compounds 1 and 2 ¹ H and ¹³ C-NMR(400/100MHz,DMSO-d ₆ ) Data (ppm)

Example 2

(1) Culture of Penicillium citrinum

Seed medium (5.0L) was prepared: 1.5% of glucose (weight percentage, the same below), 0.5% of yeast extract, 0.1% of peptone, 0.11% of crude sea salt and the balance of water; the mixture is evenly distributed into 8 conical flasks with 1000mL, and is sterilized at 120 ℃ for 25-30 minutes.

Inoculating Penicillium citrinum strain into prepared seed culture medium, and culturing at 28 deg.C for 4 days to obtain seed culture solution;

(2) Fermentation of Penicillium citrinum

Preparing a fermentation medium (100L): 1.6% of glucose (weight percentage, the same below), 0.5% of yeast extract, 0.1% of peptone, 0.11% of crude sea salt and the balance of water; the mixture is evenly distributed into 150 conical flasks with 1000mL and quenched at 120 ℃ for 25-30 minutes.

And (2) taking a proper amount of the seed culture solution obtained in the step (1), inoculating into a conical flask filled with a fermentation culture medium (the inoculum size is 5%), and performing shake culture at 26-28 ℃ for 28 days at a rotation speed of 150r/min to obtain a fermented product.

(3) Isolation of

Compounds

1 and 2

Following a similar isolation procedure as in example 1,

compounds

1 and 2 were obtained, and the structure confirmation data was in agreement with example 1.

Example 3

(1) Culture of Penicillium citrinum

Seed medium (1.0L) was prepared: 3.0 percent of glucose (weight percentage, the same below), 0.1 percent of yeast extract, 0.5 percent of peptone, 0.6 percent of crude sea salt and the balance of water; the mixture is evenly distributed into 3 500mL conical bottles and is sterilized for 25 to 30 minutes at 120 ℃.

Inoculating Penicillium citrinum strain into prepared seed culture medium, and culturing at 26-28 deg.C for 4 days to obtain seed culture solution;

(2) Fermentation of Penicillium citrinum

Preparing a fermentation medium (20L): 3.5 percent of glucose (weight percentage, the same below), 0.1 percent of yeast extract, 0.5 percent of peptone, 0.6 percent of crude sea salt and the balance of ultrapure water; the mixture is evenly distributed into 60 conical flasks of 1000mL and is sterilized at 120 ℃ for 25-30 minutes.

And (2) taking a proper amount of the seed culture solution obtained in the step (1), inoculating into a conical flask filled with a fermentation culture medium (the inoculum size is 10 percent), and performing shake culture at 26-28 ℃ for 30 days.

(3) Isolation of

Compounds

1 and 2

Following a similar isolation procedure as in example 1,

compounds

EXAMPLE 4 determination of the anti-insect Activity of

Compounds

1 and 2 of the present invention

(1) The test method comprises the following steps: the activity of the bollworm larvae was tested by taking

compounds

1 and 2.

(2) Cotton bollworm activity assay, artificial feed mixed with samples of varying concentrations (200, 100,50,25, 12.5. Mu.L/well) in equal amounts was added to 6-well plates in triplicate. Setting a positive group, a blank group and a negative group, wherein the positive control group is respectively added with corresponding artificial feed and positive drugs (200, 100,50,25,12.5 mu L/hole), the blank control is equal artificial feed, and the negative control is DMSO (200, 100,50,25,12.5 mu L/hole) with different concentrations. And (3) placing the 6-hole plate at room temperature for continuous culture for one week, observing the growth condition of the cotton bollworm larvae, and observing and recording the growth and death conditions of the cotton bollworms every 2 days. Azadirachtin (azadirachtin) was used as a positive control.

(3) The test results are as follows:

table 2:

in the above table: IC (integrated circuit) ₅₀ : half inhibitory concentration (half inhibitory concentration).

Azadirachtin was used as a positive control.

The test result shows that the two compounds have certain anti-insect activity, and the

compounds

1 and 2 have inhibitory activity and IC (Integrated Circuit) on H ₅₀ The values were 200 and 100. Mu.g/mL, respectively.

Example 5 thisDetermination of anti-stroke activity of

compounds

1 and 2 of the invention (1) test materials: mouse hippocampal neuronal cells (HT 22, north kyo institute of biotechnology of the institute of innovative and allied biotechnology); 90% DMEM-H (Hyclone) +10% FBS (GIBCO); cellTiter

AQueous One Solution Cell promotion Assay (Promega); LDH detection kit (Biyuntian Biotechnology Co., ltd.).

(1) Experimental methods

The method is divided into 5 groups: blank set, model set (H) ₂ O ₂ Lesion group), positive control group (Edaravone, 100. Mu.M), drug group (12.5, 25, 50, 100. Mu.g/ml). Detection of Compound Pair H Using MTS method ₂ O ₂ Effects on cell viability inducing oxidative damage of nerve cells (HT 22). Cells were seeded in 96-well plates at 5X10 ³ Cells per well, 200. Mu.l per well volume. The culture was incubated overnight before use in the experiment. Discarding the medium, gently washing with PBS once, randomly dividing into 8 groups, adding 1% FBS-containing medium to the blank group and the model group, adding 1% FBS-containing medium to the drug group, respectively, adding 12.5, 25, 50, 100. Mu.g/ml of 1% FBS-containing medium to each well, 100. Mu.l each, after 24H of incubation, adding 100. Mu.l of 1% FBS-containing medium to the blank group, and adding H-containing medium to each of the remaining groups ₂ O ₂ (final concentration: 100. Mu.M) 1% FBS medium, cellTiter was added to each well after 24 hours of culture

Mu.l of the AQueous One Solution was incubated for 2 hours, the culture was terminated, and the light absorption (OD) of each well was measured at 490 nm. The experiment was repeated 3 times. Each set is provided with 6 multiple holes. Analysis of variance was performed using SPSS software, and the results are expressed as mean. + -. Standard deviation

(3) Results of the experiment

TABLE 3

Compounds

1 and 2 vs. H ₂ O ₂ Effect of cell viability inducing oxidative damage of HT 22: (

n＝6)

Note: in comparison to the blank set, the data is, ^# P<0.05， ^## P<0.01; in comparison to the set of models, ^* P<0.05， ^** P<0.01

H ₂ O ₂ after 24h of induction of HT22 oxidative damage, cell viability is obviously reduced (P)<0.01). Cell viability was improved by incubation for 24h with different concentrations of drug (12.5, 25, 50, 100 μ g/ml), with

compounds

1 and 2 significantly improving cell viability (P) at

doses

25, 50, 100 μ g/ml compared to the model group<0.05 or P<0.01). The results are shown in Table 1. These data indicate that benzophenone compounds 1 and 2 have a better protective effect on the cell viability of oxidative damage of neuronal cells (HT 22).

Claims

1. A benzophenone compound or a pharmaceutically acceptable salt thereof derived from mangrove endophytic fungi, which is characterized in that the benzophenone compound has a structure shown as a compound 1:

2. a process for the simultaneous preparation of compounds 1 and 2, characterized by comprising the steps of:

(1) Preparing a seed culture medium, inoculating Penicillium citrinum HL-5126 strain into the seed culture medium, and culturing at 28 ℃ for 3-4 days to obtain a seed culture solution;

(4) The extract obtained in step (3) is subjected to reduced pressure silica gel column chromatography, petroleum ether-ethyl acetate is used for gradient elution according to the following ratio of 100, 90, 10, 80, 20, 30, 60, 50 ₃ CN:H ₂ O =35, to finally obtain compound 1; combining fractions obtained by gradient elution of 20 ₂ O =45, to finally obtain compound 2;

the seed culture medium contains 1.5-3.0% of glucose, 0.1-0.5% of yeast extract, 0.1-0.5% of peptone, 0.11-0.6% of crude sea salt and a proper amount of water; the fermentation culture medium contains 1.6-3.5% of glucose, 0.1-0.5% of yeast extract, 0.1-0.5% of peptone, 0.11-0.6% of crude sea salt and a proper amount of water; the percentages are weight percentages; the seed culture medium and the fermentation culture medium are sterilized at 120 ℃ for 25-30 minutes;

the structures of the compounds 1 and 2 are as follows:

3. a neuroprotective pharmaceutical composition characterized by having the compound 1, 2 or a pharmaceutically acceptable salt thereof prepared by the process of claim 2 as an active ingredient.

4. A pharmaceutical composition for preventing and/or treating stroke, characterized by comprising the compound 1, 2 or a pharmaceutically acceptable salt thereof prepared by the process of claim 2 as an active ingredient.

5. The use of compounds 1, 2 or pharmaceutically acceptable salts thereof prepared by the process of claim 2 for controlling an agricultural pest selected from the group consisting of cotton bollworm.

6. The use of compound 1, 2 or a pharmaceutically acceptable salt thereof prepared by the process of claim 2 for the preparation of a medicament for the treatment and/or prevention of stroke.

7. Use of compound 1, 2 or a pharmaceutically acceptable salt thereof, prepared by the process of claim 2, in the preparation of a medicament for neuronal protection.

8. A pharmaceutical composition according to any one of claims 3 to 4, characterized in that it further comprises other active ingredients.

9. The pharmaceutical composition of claim 8, further comprising a pharmaceutically acceptable excipient.

10. The pharmaceutical composition of claim 9, wherein the pharmaceutical composition is in a dosage form selected from the group consisting of a solid formulation, a semi-solid formulation, and a liquid formulation.