CN113173969A

CN113173969A - Marine fungus-derived heteroterpene compound and application thereof in preparation of anti-hepatic fibrosis drugs

Info

Publication number: CN113173969A
Application number: CN202110224821.3A
Authority: CN
Inventors: 崔辉; 苏桃; 唐渝茜; 杨春芳; 赵钟祥
Original assignee: Guangzhou University of Traditional Chinese Medicine
Current assignee: Guangzhou University of Traditional Chinese Medicine
Priority date: 2021-03-01
Filing date: 2021-03-01
Publication date: 2021-07-27
Anticipated expiration: 2041-03-01
Also published as: CN113173969B

Abstract

The invention discloses a heteroterpene compound derived from marine fungi and application thereof in preparing anti-hepatic fibrosis drugs. The invention separates the heteroterpene derivative from the fermentation product of Aspergillus terreus GZU-31-1. The new compound can obviously reduce the expression of TGF-beta 1 induced alpha-SMA, shows stronger activity of inhibiting hepatic stellate cell activation, has clinical application potential of anti-hepatic fibrosis treatment, can be used for preparing anti-hepatic fibrosis medicines, and has good application prospect.

Description

Marine fungus-derived heteroterpene compound and application thereof in preparation of anti-hepatic fibrosis drugs

Technical Field

The invention belongs to the technical field of medical compounds, and particularly relates to a marine fungus-derived heterpene compound and application thereof in preparation of anti-hepatic fibrosis drugs.

Background

Hepatic Fibrosis (HF) is a common pathological process of various chronic liver diseases, and various cytokines secreted by hepatocytes which are damaged for a long time or repeatedly stimulate hepatic stellate cells to be converted into myofibroblasts, and the myofibroblasts repair the damaged hepatocytes by synthesizing extracellular matrixes, but when the synthesis and degradation of the extracellular matrixes are unbalanced, a large amount of extracellular matrixes are deposited to cause hepatic fibrosis, and if timely and effective intervention is not performed, liver cirrhosis, liver cancer and other liver diseases are developed. Since hepatic fibrosis is reversible, effective treatment of hepatic fibrosis is an important treatment for preventing and treating chronic liver disease and liver cirrhosis.

The research shows that transforming growth factor beta 1 (TGF-beta 1) is the most main fibrosis promoting factor, so that the inhibition of TGF-beta 1 signal channel expression has important significance in the treatment of hepatic fibrosis. TGF-beta 1 promotes myofibroblast differentiation, secretes a large amount of extracellular matrix such as alpha-smooth muscle actin (alpha-SMA), and finally causes hepatic fibrosis. Therefore, the degree of hepatic fibrosis can be indirectly reflected by the alpha-SMA. Therefore, the invention takes the alpha-SMA mRNA expression induced by inhibiting TGF-beta 1 as a screening model to screen a lead compound with anti-hepatic fibrosis activity, and provides a target compound for developing a new anti-hepatic fibrosis drug.

At present, no good medicine for treating hepatic fibrosis exists in the first-line clinic, and the common medicines mainly comprise: antiviral and anti-hepatic fibrosis, and a series of adverse reactions and tolerance can be generated after long-term use of the medicine in large quantities. Therefore, in order to solve the tolerance and adverse reaction of the drugs, the search for new anti-hepatic fibrosis drugs becomes a hot spot of international and domestic research.

The marine fungi have abundant chemical diversity of secondary metabolites and high yield, so that the marine fungi become hot spots for researching marine natural medicines. At present, the search for new prodrugs from marine fungi has become a hot spot of research at home and abroad.

Disclosure of Invention

The invention aims to solve the technical problem of providing a heteroterpene compound from marine fungi, and the new compound can inhibit TGF-beta 1 from inducing the expression of alpha-SMA mRNA, has obvious anti-hepatic fibrosis effect and has great potential for developing new drugs.

The invention also aims to provide the application of the heteroterpene compound derived from the marine fungi in preparing the anti-hepatic fibrosis drugs.

The above purpose of the invention is realized by the following technical scheme:

a heteroterpene compound derived from marine fungi, which has a structural formula shown as the following formula 1:

the preparation method of the above-mentioned marine fungus-derived hetero-terpene compound is obtained by fermentation production of marine fungus Aspergillus terreus GZU-31-1, specifically is separated from fermentation product of marine fungus Aspergillus terreus GZU-31-1, and the preferable preparation method comprises the following steps:

s1, culturing fungus seed liquid:

inoculating Aspergillus terreus GZU-31-1 into slant culture medium, culturing, inoculating potato glucose water culture medium, and culturing to obtain seed solution;

s2, fungus fermentation culture:

inoculating the seed liquid into a solid rice fermentation culture medium, and culturing to obtain a fermentation product;

s3, crude extraction: extracting the fermentation product obtained in the step S2 with methanol, concentrating the extracting solution, and extracting the obtained concentrated extract with ethyl acetate to obtain an ethyl acetate crude extract;

s4, separation and purification: separating the ethyl acetate crude extract obtained in the step S3 by normal phase silica gel chromatography, and then separating and purifying by silica gel, gel and C-18 reverse phase column chromatography separation technology to obtain the diterpenoid compound.

Preferably, the formula of the slant culture medium in step S1 is: calculated by mass ratio, 0.3 percent of glucose, 0.1 percent of yeast extract, 0.1 to 0.5 percent of peptone, 1.5 to 2.5 percent of agar, 1.5 to 4 percent of sodium chloride and water are added to make up to 100 percent.

More preferably, the formula of the slant culture medium in step S1 is: by mass ratio, glucose 0.3%, yeast extract 0.1%, peptone 0.5%, agar 2.5%, sodium chloride 3%, and water 98%.

Preferably, the potato dextrose aqueous medium formulation in step S1 is: according to the mass ratio, 20 percent of potato, 0.3 percent of glucose, 1.5 to 4 percent of sodium chloride and water are added to make up to 100 percent.

More preferably, the potato dextrose aqueous medium formulation in step S1 is: according to the mass ratio, 20% of potato, 0.3% of glucose, 3% of sodium chloride and water are added to make up to 100%.

Preferably, the culture conditions after the inoculation to the slant culture medium in the step S1 are 28-35 ℃ for 4-10 days, and the culture conditions after the inoculation to the potato dextrose water culture medium are 28-35 ℃ for 3-7 days.

More preferably, the culture conditions after the inoculation of the slant culture medium in the step S1 are 30 ℃ for 6 days, and the culture conditions after the inoculation of the potato dextrose aqueous medium are 30 ℃ for 3 days.

Preferably, the formula of the solid rice fermentation medium in the step S2 is: the weight ratio of the rice to the seawater is 1: 1-2.

More preferably, the formula of the solid rice fermentation medium in the step S2 is: the mass ratio of the rice to the seawater is 1: 1.

Preferably, the culture condition in the step S2 is to be kept still at 25-35 ℃ for 1-2 months.

More preferably, the condition for the cultivation in step S2 is to be left standing at 35 ℃ for 2 months.

Preferably, the number of methanol extractions in step S3 is 2 to 5.

Preferably, the normal phase silica gel chromatography in step S4 is specifically operated as: gradient eluting with petroleum ether/ethyl acetate of 90%, 80%, 70%, 60%, 50%, 40% and 30% volume fraction, and collecting 90-50% petroleum ether/ethyl acetate eluate.

In step S4, the petroleum ether/ethyl acetate refers to a mixture of petroleum ether/ethyl acetate in a volume ratio, and both the petroleum ether and the ethyl acetate are analytically pure reagents.

The application of the heteroterpene compound from the marine fungi in preparing the anti-hepatic fibrosis medicine.

The application of the heteroterpene compound derived from the marine fungi in preparing TGF-beta 1 inhibitors.

In such use, the heteroterpene compounds inhibit the activity of TGF-beta 1.

The activity of the heteroterpene compound for inhibiting TGF-beta 1 to induce cells to express alpha-SMA is provided.

The cells are hepatic stellate cells.

Preferably, the working concentration of the heteroterpene compound is 2.5-10 mu mol/L.

More preferably, the working concentration of the said heteroterpene compound is 6. mu. mol/L.

Experiments prove that the novel heteroterpenoid derivative from the marine fungi can obviously inhibit the expression of TGF-beta 1 induced alpha-SMA mRNA, has the clinical application potential of anti-hepatic fibrosis treatment, and can be used for preparing anti-hepatic fibrosis medicines. Therefore, the application of the novel heteroterpenoid derivatives derived from the marine fungi in preparing anti-hepatic fibrosis medicines and in preparing medicines capable of inhibiting TGF-beta 1 from inducing alpha-SMA mRNA expression are both within the protection scope of the invention.

The invention has the following beneficial effects:

the novel heteroterpene derivatives are separated from the marine fungus Aspergillus terreus GZU-31-1, have the activity of obviously inhibiting TGF-beta 1 induced alpha-SMA mRNA expression, have the clinical application potential of anti-hepatic fibrosis treatment, can be used for preparing anti-hepatic fibrosis medicaments, and have good application prospects.

Drawings

FIG. 1 is a schematic structural diagram of Compound 1.

FIG. 2 is a graph showing the results of Compound 1 inhibiting the activation of hepatic stellate cells; wherein P < 0.01, compared to the blank group;^#P＜0.05，^##p is less than 0.01, compared with TGF-beta 1 group.

FIG. 3 is a graph showing the results of cytotoxicity evaluation of Compound 1.

Detailed Description

The invention is further described with reference to specific embodiments, but the embodiments are not intended to limit the invention in any way.

Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

EXAMPLE 1 obtaining of the Marine fungus Aspergillus terreus GZU-31-1

The inventor group is obtained by separating and purifying the body of Kadsura longipedunculata in Xuweng coast of Zhanjiang province, and separating to obtain a strain of marine fungus Aspergillus terreus GZU-31-1. The fungus was identified by DNA amplification and sequencing of the ITS region of the fungus, step specific references (Nature protocols,2010,5, 480-: DNA was extracted using DNeasy Plant Mini Kit according to the procedures of the instructions, PCR amplification was performed using ITS as a primer, and the amplified product was sequenced. The sequenced sequence was subjected to similarity search using BLAST database, and compared to 100% with the Aspergillus terreus fungal sequence, the fungus was identified as the marine fungus Aspergillus terreus GZU-31-1. The strain is preserved in Guangdong province microorganism culture collection center (GDMCC) in 2019, 12 months and 19 days, and the preservation number is GDMCC No: 60789, published in the patent application with publication number CN 1111339188A.

Example 2 isolation of the Compounds

A novel hetero-terpenoid derivative is separated from fermentation liquor of Aspergillus terreus GZU-31-1, and the specific method comprises the following steps:

(1) seed culture of the fungus Aspergillus terreus GZU-31-1: inoculating the strain into slant, culturing at 30 deg.C for 6 days, inoculating into potato glucose aqueous medium, and culturing at 30 deg.C for 3 days to obtain seed solution. The slant culture medium comprises the following components in percentage by weight: 0.3 percent of glucose, 0.1 percent of yeast extract, 0.5 percent of peptone, 2.5 percent of agar, 3 percent of sodium chloride and 98 percent of water. The potato dextrose water culture medium comprises the following components in percentage by weight: according to the mass ratio, 20% of potato, 0.3% of glucose, 3% of sodium chloride and water are added to make up to 100%.

(2) Fermentation culture of the fungus Aspergillus terreus GZU-31-1: inoculating the strain in the seed liquid into a fermentation culture medium, and standing at room temperature of 35 ℃ for 2 months; wherein the fermentation medium is a solid rice fermentation medium and comprises the following components in percentage by weight: 1, sea water: 1.

(3) extracting the fermented extract with methanol for 3 times, concentrating the extractive solution, and extracting the obtained concentrated extract with ethyl acetate to obtain ethyl acetate crude extract.

(4) Separating the ethyl acetate crude extract by normal phase silica gel chromatography; gradient eluting with petroleum ether/ethyl acetate with petroleum ether volume fractions of 90%, 80%, 70%, 60%, 50%, 40% and 30%, collecting 90% -50% petroleum ether/ethyl acetate eluate, and sequentially separating and purifying with silica gel, gel and C-18 reverse phase column chromatography to obtain compound 1.

Structural analysis of the Compound of example 3

The following experimental data were obtained by performing structural test analysis on the new compound 1:

novel compound 1: c₂₆H₃₀O₉，HRESI-MS：485.1805[M-H]^-(cald for C₂₆H₂₉O₉ 485.1806)。

The NMR data are shown in Table 1.

The structural formula of compound 1 is shown below:

TABLE 1 NMR data (CDCl) for Compound 1₃，400MHz/101MHz,ppm)

EXAMPLE 4 evaluation of anti-hepatic fibrosis Activity and toxicity screening of Compound 1

1. Evaluation of anti-hepatic fibrosis Activity

(ii) culture and treatment of cells

Rat hepatic stellate cell HSC-T6 cells (ATCC) were cultured in DMEM medium (containing penicillin 60U/mL, streptomycin 100. mu.g/mL) containing 10% (v/v) serum and placed in 5% CO₂The temperature was set at 37 ℃ in the incubator of (1), and conventional maintenance culture and passage were carried out. Cells in logarithmic growth phase were selected for all experiments.

② intervention of compounds

Digesting adherent cells with pancreatin, collecting in a centrifuge tube, centrifuging, re-suspending with culture medium, and performing 3 × 10⁵Cells were seeded in 6-well plates at cell/mL density until cells attached for 12 h. 10 mu g/mL of TGF-beta 1 mother solution is diluted 1000 times by using blank DMEM, the blank group is changed into blank DMEM, and 10ng/mL of TGF-beta 1 is added into the other groups for pre-stimulation for 1 h. Then, the blank group is changed into blank DMEM, the other groups are added with DMEM containing 10ng/mL TGF-beta 1 and prepared into test drug culture solutions with different concentrations to act for 24 hours, and meanwhile, a positive control group (luteolin with the concentration of 10 mu mol/L) is arranged. After each group of cells are treated by the drug, the culture medium is sucked off, and the cells are washed for 1 time by ice PBS; subsequently, 1mL of Trizol reagent was added to each well of the 6-well plate, and RNA extraction was performed according to the instructions of the total RNA extraction kit of encorui bioengineering ltd, han, hannan. The concentration and purity of the extracted total RNA were determined, and then Q-PCR experiments were performed using a reverse transcription kit and a real-time fluorescent quantitative PCR kit of the same company. After the experiment was finished, the number 2^-ΔΔCtThe method calculates the gene expression of α -SMA in the cell sample, and GAPDH was used as an internal reference gene for data normalization. The experimental primers adopt base sequences with more reports in literature, and are synthesized by Huada genes after the reliability of the experimental primers is verified by BLAST.The Q-PCR primer sequences are shown in the following table.

TABLE 2Q-PCR primer sequences

(iii) results of the experiment

The results are shown in figure 2, TGF- β 1 causes significant upregulation of α -SMA levels in cells. Whereas, after administration of compound 1, α -SMA expression decreased with increasing drug concentration. Luteolin (Luteolin) is a natural flavonoid compound with activities of resisting oxidation and inflammation. Recent studies have shown that luteolin couples to CCl₄Induced liver fibrosis in mice has therapeutic effect, and is taken as a positive drug considering the in vitro efficacy of the compound 1. The experimental result shows that the compound 1 can inhibit the activation of hepatic stellate cells, the effect of the compound is equivalent to that of positive luteolin, but the compound 1 high-dose group has no significant difference with the positive drug group.

2. Cytotoxicity assessment

The principle is as follows: the chemical name of MTT is 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazole bromide salt determined by an MTT method, and the commercial name is as follows: thiazole blue. The detection principle is as follows: the live and dead cells differ in that the presence of succinate dehydrogenase in the mitochondria in live cells reduces exogenous MTT to the water-insoluble blue crystalline formazan. Dimethyl sulfoxide (DMSO) can solubilize the formazan deposited within the cells, whose absorbance, measured at 490 or 540nm wavelength depending on the intervening and positive control groups to which the compound was added, can indirectly reflect the number of cells surviving.

The concrete steps are as follows: HSC-T6 cells were cultured in DMEM medium (penicillin 60U/mL, streptomycin 100. mu.g/mL) containing 10% serum and placed in 5% CO₂The temperature was set at 37 ℃. After the cells had stabilized after one generation of culture, they were plated in 96-well plates at a concentration of 5X 10³Cells/well, adherent for 24 h. The compound mother liquor was dissolved in DMSO and diluted with DMEM medium. Samples of compounds at different concentration gradients (0-15 μ M) were applied to 96-well plates (200 μ L). After the culture is carried out for 24 hours,mu.L of MTT solution (5mg/mL in PBS) was added to each well, incubation was continued for 4 hours, the incubation was terminated, the supernatant carefully aspirated, 150. mu.L of DMSO was added to each well, and the mixture was shaken for 10min to dissolve the crystals sufficiently. The 490nm wavelength absorption was detected by a microplate reader and the results recorded. Cell viability was calculated by the following formula: cell viability (%) - (A)_n-A₀)/(A-A₀]X 100% where A_nIs the absorbance of the sample, A₀Is the absorbance of wells with medium and MTT solution without cells, and A is the absorbance of blank control.

Results are shown in FIG. 3

The results show that: compound 1 does not exhibit cytotoxicity at the onset concentrations (2.5, 5, 10. mu.M) with a significant safety margin.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. A heteroterpene compound derived from marine fungi, which is characterized in that: the structural formula is shown as the following formula 1:

2. the process for producing a heteroterpene compound derived from a marine fungus according to claim 1, wherein: the method comprises the following steps:

s1, culturing fungus seed liquid:

s2, fungus fermentation culture:

inoculating the seed liquid into a solid rice fermentation culture medium, and culturing to obtain a fermentation liquid;

3. The method for producing a marine fungus-derived diterpene compound according to claim 2, which is characterized by comprising:

the formula of the slant culture medium in the step S1 is as follows: calculated by mass ratio, 0.3 percent of glucose, 0.1 percent of yeast extract, 0.1 to 0.5 percent of peptone, 1.5 to 2.5 percent of agar, 1.5 to 4 percent of sodium chloride and water are added to 100 percent;

the formula of the potato dextrose water culture medium in the step S1 is as follows: according to the mass ratio, 20 percent of potato, 0.3 percent of glucose, 1.5 to 4 percent of sodium chloride and water are added to make up to 100 percent;

the culture conditions after the inoculation to the slant culture medium in the step S1 are 28-35 ℃ for 4-10 days, and the culture conditions after the inoculation to the potato dextrose water culture medium are 28-35 ℃ for 3-7 days;

the formula of the solid rice fermentation medium in the step S2 is as follows: according to the mass ratio, the ratio of rice to seawater is 1: 1-2;

the culture condition in the step S2 is to stand for 1-2 months at 25-35 ℃.

4. The method for producing a marine fungus-derived diterpene compound according to claim 3, which is characterized by comprising:

the formula of the slant culture medium in the step S1 is as follows: by mass ratio, glucose 0.3%, yeast extract 0.1%, peptone 0.5%, agar 2.5%, sodium chloride 3%, and water to 100%;

the formula of the potato dextrose water culture medium in the step S1 is as follows: according to the mass ratio, 20% of potatoes, 0.3% of glucose, 3% of sodium chloride and water are complemented to 100%;

the culture conditions after the inoculation to the slant culture medium in the step S1 are 30 ℃ and 6 days, and the culture conditions after the inoculation to the potato dextrose water culture medium are 30 ℃ and 3 days;

the formula of the solid rice fermentation medium in the step S2 is as follows: according to the mass ratio, the ratio of rice to seawater is 1: 1;

the condition for the culture described in step S2 was 35 ℃ for 2 months.

5. The method for producing a marine fungus-derived diterpene compound according to claim 2, which is characterized by comprising:

the normal phase silica gel chromatography in the step S4 is specifically operated as follows: gradient eluting with petroleum ether/ethyl acetate of 90%, 80%, 70%, 60%, 50%, 40% and 30% volume fraction, and collecting 90-50% petroleum ether/ethyl acetate eluate.

6. Use of the marine fungus-derived heteroterpene compound of claim 1 for the preparation of an anti-hepatic fibrosis medicament.

7. Use of the marine fungus-derived heteroterpene compound according to claim 1 for the preparation of TGF- β 1 inhibitors.

8. Use according to claim 7, characterized in that:

in the application, the heteroterpene compound derived from the marine fungi inhibits the activity of TGF-beta 1.

9. Use according to claim 7, characterized in that:

in the application, the heteroterpene compound derived from the marine fungi inhibits the activity of TGF-beta 1 for inducing cells to express alpha-SMA.

10. Use according to claim 7, characterized in that:

the cells are hepatic stellate cells.