CN115040502B - Application of marine aspergillus source compound - Google Patents

Abstract

The invention belongs to the technical field of biomedicine, and in particular relates to the application of a compound derived from marine aspergillus in the preparation of a drug for preventing and treating non-alcoholic fatty liver. Cell experiments and animal experiments have verified that the marine Aspergillus-derived compound has a significant effect on the treatment of non-alcoholic fatty liver, providing more options for the treatment of non-alcoholic fatty liver; and the compound utilizes rich sources of marine Aspergillus, Combined with modern microbial fermentation engineering technology for production, there is no need to worry about raw materials. At the same time, it has the advantages of not destroying the ecological balance and easy industrialization. It is a renewable drug resource with development prospects. Rich raw materials are provided.

Description

Application of a compound derived from marine aspergillus

technical field

The invention belongs to the technical field of biomedicine and relates to new uses of known compounds. More specifically, it relates to the application of a compound derived from natural marine Aspergillus in the preparation of drugs for preventing and treating non-alcoholic fatty liver.

Background technique

Non-alcoholic fatty liver disease (NAFLD) is a metabolic liver disease characterized by excessive accumulation of liver lipids (steatosis), which can gradually progress to non-alcoholic steatohepatitis (NASH), liver fibrosis, liver cirrhosis and hepatocellular cancer. Among them, NASH is considered to be the key decisive link in the progress of chronic liver disease, and it is an irreversible lesion of liver cirrhosis and liver cancer. At present, the number of patients with NAFLD/NASH has increased dramatically around the world, and the number of patients with NASH has surpassed hepatitis B as the world's largest chronic liver disease and a major global public health problem.

In the prior art, the treatment methods for NAFLD/NASH are very limited. The treatment for NAFLD and early NASH is mostly based on diet control and exercise intervention. For middle and late NASH, the clinical effect of weight loss, liver transplantation and other surgical treatments is mediocre. In terms of drug treatment, researchers have discovered multiple therapeutic targets and targeted drugs for each important cell molecular activity of NASH. The NASH therapeutic drugs under clinical research are mainly divided into: farnesoid receptor (FXR) agonists, Peroxisome proliferator-activated receptor (PPAR) agonists, acetyl-CoA carboxylase (ACC) inhibitors, apoptosis enzyme inhibitors, etc. For example, a Chinese patent application discloses a compound for regulating FXR, which has a good effect on regulating farnesoid receptors. In addition, obeticholic acid, an FXR agonist drug, indirectly inhibits the expression of cytochrome 7A1 (CYP7A1) gene, thereby inhibiting bile acid synthesis, and is used for the treatment of primary biliary cirrhosis and nonalcoholic fatty liver disease; but Clinical trials have been terminated one after another due to drug efficacy (inability to effectively target all key cellular molecular activities of NASH) and safety (itching, weight gain, heart failure, etc.). In addition, there are compounds under clinical research, but their safety and long-term efficacy still need to be further verified.

Therefore, in order to solve the current shortage of NASH drugs, it is of great significance to develop drugs with high efficiency, low toxicity, high safety, and good anti-NASH effect.

Contents of the invention

The technical problem to be solved by the present invention is to overcome the defects and deficiencies of the shortage of existing high safety and significant effect NAFLD treatment drugs, and to provide a natural marine Aspergillus source compound with high safety and good NAFLD treatment effect in the preparation and prevention of non-alcoholic fatty acids. Applications in liver medicine.

Another object of the present invention is to provide a preparation method of the marine Aspergillus-derived compound.

The above object of the present invention is achieved through the following technical solutions:

An application of a marine Aspergillus-derived compound in the preparation of a drug for preventing and treating non-alcoholic fatty liver, characterized in that the marine Aspergillus-derived compound has a structure of formula (I):

Further, the non-alcoholic fatty liver includes inhibiting liver lipid accumulation, liver damage, liver inflammation and fibrosis.

Furthermore, the marine Aspergillus-derived compound also includes its pharmaceutically acceptable salts, hydrates, and solvates.

Further, the dosage form of the drug for preventing and treating non-alcoholic fatty liver is oral, injection or inhalation.

In addition, the present invention also provides a preparation method of a marine Aspergillus-derived compound, wherein the marine Aspergillus-derived compound is obtained by fermentation and isolation of a marine Aspergillus;

The marine Aspergillus-derived compound has a structure of formula (I):

Further, the marine Aspergillus is a symbiotic fungus isolated from marine hard corals.

Furthermore, the marine Aspergillus includes Aspergillus sp.ITBB-c1, Aspergillus taichungensis ZHN-7-07, and Aspergillus candidus.

Further, the preparation method specifically includes the following steps:

S1, expanding the cultivation of the marine Aspergillus, taking the seed liquid and inoculating it into a medium containing rice for fermentation to obtain a fermentation liquid;

S2. Extract the fermented liquid obtained in step S1 with ethyl acetate. After the extract is concentrated, use the petroleum ether-ethyl acetate system as the eluent, and successively use the petroleum ether-ethyl acetate volume ratio as 100:0, 25 :1, 10:1, 5:1, 2:1, 1:1, 0:1 were separated by normal phase silica gel column chromatography under reduced pressure to obtain 7 fractions of Fr.A～Fr.G;

S3. Collect the fraction Fr.E obtained in step S2 with a petroleum ether-ethyl acetate volume ratio of 2:1, and perform ODS reverse-phase column chromatography with 40-100 vol% methanol aqueous solution as the eluent;

S4. Collect the 75vol% methanol aqueous solution eluted part in step S3 to obtain the fraction Fr.E-6, use methanol as the eluent, complete the Sephadex LH-20 gel column chromatography, concentrate, and obtain the source of the marine Aspergillus compound.

Furthermore, in step S1, the fermentation condition is 25-30°C for 40-50 days.

Further, in step S1, the medium containing rice is prepared from cooked rice and water according to a mass-volume ratio of 1:(1-1.5) g/ml.

The present invention has the following beneficial effects:

The invention provides an application of a compound derived from marine Aspergillus in the preparation of a drug for preventing and treating non-alcoholic fatty liver. Cell experiments and animal experiments have verified that the compound derived from marine Aspergillus has a significant effect on treating non-alcoholic fatty liver. The treatment of non-alcoholic fatty liver provides more options; and the compound is produced by using rich sources of marine Aspergillus, combined with modern microbial fermentation engineering technology, no worries about raw materials, and at the same time, it will not destroy the ecological balance, and it is easy to realize industrialization, etc. The advantage is that it is a promising renewable drug resource, which provides abundant raw materials for the research and development of marine microbial products and marine microbial drugs.

Description of drawings

Fig. 1 is the hydrogen spectrum spectrum ( ¹ H-NMR, 500 MHz) of the compound HN-001 derived from marine Aspergillus obtained in Example 1 of the present invention.

Fig. 2 is the mass spectrogram of the compound HN-001 derived from marine Aspergillus obtained in Example 1 of the present invention.

Fig. 3 is the level of triglyceride (TG) in hepatocytes in Example 2 of the present invention (Fig. 3A) and photos of cells stained with Nile Red at a representative concentration (4-25 μM) (Fig. 3B).

Fig. 4 is the therapeutic effect diagram (Fig. 4A) and the morphological change diagram (Fig. 4B) of each group of hepatocytes and the apoptotic enzyme activity level of each group (Fig. 4C).

Fig. 5 is a data statistical graph of the effect of the compound in Example 4 of the present invention on the content of reactive oxygen species, a marker of lipid oxidative stress in hepatic cells induced by palmitic acid.

Fig. 6 is a line chart of the body weight change of the mouse model in Example 5 of the present invention (Fig. 6A), the statistical chart of the mouse body weight data after 7 weeks of administration (Fig. 6B) and the liver/mouse model of the mouse model after 7 weeks of administration. Body weight comparison chart (Fig. 6C).

Fig. 7 is the relationship between liver tissue triglyceride (Fig. 7A) and aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) (Fig. 7B) in the blood of the mouse model in Example 5 of the present invention. Statistical diagram of content level; and histopathological staining diagram of mouse model liver, including hematoxylin/eosin staining, oil red O staining, Sirius red staining and Masson staining (Fig. 7C).

Fig. 8 is the result of the cytotoxicity experiment of the compound HN-001 in Example 6 of the present invention on hepatocytes.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

The marine Aspergillus sp.ITBB-c1 used in the present invention is isolated from marine hard corals.

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

Example 1 Preparation of Marine Aspergillus-derived Compound HN-001

The preparation of the marine Aspergillus derived compound HN-001 comprises the following steps:

S1. The marine Aspergillus sp.ITBB-c1 was activated on a PDA solid medium plate, the culture condition was 28°C, and cultured in the dark for 5 days; the colonies in the plate were directly inoculated into 1000mL of ME seed medium containing 200mL In a triangular flask, vibrate and cultivate on a constant temperature shaker for 4 days (160r/min, 28°C) to obtain a seed solution; take 15mL of the seed solution and inoculate it into a tissue culture bottle containing 30g of rice and 30mL of water for fermentation, and ferment a total of 5kg Rice, cultured statically at 28°C for 45 days to obtain fermentation broth;

S2. Extract the fermentation liquid obtained in step S1 with ethyl acetate 4 times, combine the extracts and concentrate under reduced pressure at 45°C to obtain 120 g of ethyl acetate extract; use petroleum ether-ethyl acetate system as the eluent, sequentially Normal-phase silica gel column layer under reduced pressure at volume ratios of petroleum ether-ethyl acetate of 100:0, 25:1, 10:1, 5:1, 2:1, 1:1, 0:1 (gradient elution) Analysis and separation, combined similar fractions under TLC detection to obtain 7 fractions of Fr.A～Fr.G;

S3. Collect the fraction Fr.E obtained in step S2 with a petroleum ether-ethyl acetate volume ratio of 2:1, and perform ODS reverse-phase column chromatography with 40-100 vol% methanol aqueous solution as the eluent;

S4. Collect the 75vol% methanol aqueous solution eluted part in step S3 to obtain the fraction Fr.E-6, use methanol as the eluent, complete the Sephadex LH-20 gel column chromatography, concentrate, and obtain the source of the marine Aspergillus Compound HN-001 (2.0 mg).

Figures 1-2 are the structure confirmation spectrum of the compound HN-001 derived from marine Aspergillus: It can be seen that the quasi-molecular ion peak of the positive source high-resolution mass spectrum (HR-ESI-MS) of the compound HN-001: m/z 381.1336 [M+H ]+, 783.2416[2M+Na]+, the deduced molecular formula is C ₂₂ H ₂₀ O ₆ .

Example 2 The effect of the compound HN-001 derived from marine Aspergillus on the lipid content of hepatocytes induced by palmitic acid (Nile red staining)

In this example, the compound HN-001 derived from marine Aspergillus was used as the test object, and the Nile red staining method was used to verify the effect of HN-001 on the triglyceride content in liver cells.

1. Experimental method:

HepG2 cells in the logarithmic growth phase were uniformly seeded into 48-well plates at a rate of 5.0×10 ⁴ cells/well, and cultured overnight. The medium was replaced with DMEM complete culture solution containing palmitic acid (0.5mM) (DMEM culture solution containing 10% FBS and 1% double antibody), and different concentrations (4, 10, 25 μM) compound HN-001, 37° C., 5% CO ₂ static culture for 24 hours. The experiment set up blank control group, palmitic acid model group and compound HN-001 treatment group. After 24 hours of action, Nile red staining and photographing and triglyceride content analysis were performed on each group.

The specific operation of Nile Red staining is as follows: the cells to be stained were rinsed once with pre-cooled PBS, added with a final concentration of 1 μM Nile Red staining solution and a final concentration of 10 μM Hochest 33342 nucleic acid dye, and stained at room temperature for 15 minutes. After staining, rinse with deionized water for 2 to 3 times at room temperature, and take pictures with an inverted microscope (40 times).

The specific steps of the triglyceride content analysis experiment are as follows: the cells to be tested are rinsed twice with pre-cooled PBS. Remove PBS, add deionized solution containing 0.2% Triton X-100, let stand at room temperature for 1h, collect cell suspension, ultrasonically break for 10min, make cells fully lyse, centrifuge to collect supernatant, follow the method of triglyceride detection kit (Roche ) Instructions for determination of triglyceride content.

Wherein, the triglyceride content analysis takes the palmitic acid control group as a contrast (Ctrl), and the multiple relationship is:

The experimental results are the average of three independent experiments, and the results are statistically analyzed according to "mean ± standard deviation".

2. Experimental results:

See Figure 3 for the experimental results. It can be seen from the figure that the compound HN-001 can decrease the triglyceride content of the liver cells in a gradient-dependent manner at a concentration of 4-25 μM. The results of quantitative triglyceride detection showed that the use of compound HN-001 could reduce the level of triglyceride in the liver ( FIG. 3A ). Nile red staining further proved the lipid-lowering activity of the compound HN-001: a large amount of lipids could be clearly seen in the cells of the control group, while the lipid content in the cells of the compound HN-001 treatment group was significantly reduced (Fig. 3B).

Example 3 Protective effect of compound HN-001 derived from marine Aspergillus on palmitic acid-induced apoptosis of hepatocytes (trypan blue staining)

In this example, the compound HN-001 derived from marine Aspergillus was used as the test object, and the protective effect of the compound HN-001 on palmitic acid-induced liver cell apoptosis was verified by the trypan blue staining method.

1. Experimental method:

HepG2 cells in the logarithmic growth phase were uniformly seeded into 6-well plates at 20× ¹⁰⁴ cells/well, and cultured overnight. The medium was replaced with DMEM complete culture solution containing palmitic acid (0.5mM) (DMEM culture solution containing 10% FBS and 1% double antibody), and different concentrations (4, 10, 25 μM) compound HN-001, 37° C., 5% CO ₂ static culture for 24 hours. The experiment set up blank control group, palmitic acid model group and compound HN-001 treatment group. After 24 hours of treatment, the trypan blue staining method was used to detect cell survival and apoptosis, and the kit was used to detect the activity of caspase 3 in each group of cells.

The specific operation of trypan blue staining is as follows: wash the cells to be stained once with pre-cooled PBS, trypsinize to collect the cells, resuspend the cells in 1 mL of phosphate buffer solution containing 4% trypan blue staining solution, and stain at room temperature for 15 minute. After the staining was completed, take 10 μL of the cell suspension to a cell counting plate, observe and count the cell staining under a microscope, and record the number of trypan blue-stained and unstained cells in each group. The experimental results are the average of three independent experiments, and the results are statistically analyzed according to "mean ± standard deviation".

The specific operation of caspase 3 activity detection is as follows: after the cell treatment, the pre-cooled cell lysate was used to lyse the cells on ice for 15 minutes. Add 10 μl of sample supernatant to the Caspase3 antibody coating solution to coat the microwell plate, and incubate at 37°C for 1 hour. Unbound samples were discarded, and the plate was washed 3 times with incubation buffer at room temperature for 1 minute each time. Add freshly prepared Caspase substrate solution, react at 37°C for 3 hours; then perform fluorescence photometric detection, the excitation wavelength is 400nm, and the emission wavelength is 505nm.

2. Experimental results:

The results are shown in FIG. 4 . It can be seen from the figure that the compound HN-001 can increase the survival rate of liver cells in a gradient-dependent manner at a concentration of 4-25 μM and reduce the apoptosis of liver cells induced by palmitic acid ( FIG. 4A ). Cell morphology staining further proved the hepatoprotective activity of compound HN-001. Compared with the cells in the palmitic acid group, the morphology of the cells in the compound HN-001 treatment group was more regular, and the number of shrunken cells decreased (Fig. 4B). At the same time, palmitic acid induction could significantly increase hepatic Caspase 3 activity, while compound HN-001 treatment could significantly reduce palmitic acid-induced hepatic Caspase 3 activity (Fig. 4C).

Example 4 Inhibitory effect of compound HN-001 derived from marine Aspergillus on palmitic acid-induced oxidative stress in hepatocytes (detection of cellular reactive oxygen species (ROS) content by flow cytometry)

In this example, the compound HN-001 derived from marine Aspergillus was used as the test object, and flow cytometry was used to verify the effect of compound HN-001 on the level of reactive oxygen species in liver cells induced by palmitic acid.

3. Experimental method:

HepG2 cells in the logarithmic growth phase were uniformly seeded into 6-well plates at 20× ¹⁰⁴ cells/well, and cultured overnight. The medium was replaced with DMEM complete culture solution containing palmitic acid (0.5mM) (DMEM culture solution containing 10% FBS and 1% double antibody), and different concentrations (4, 10, 25 μM) compound HN-001, 37° C., 5% CO ₂ static culture for 24 hours. The experiment set up blank control group, palmitic acid model group and compound HN-001 treatment group. After 24 hours of action, the cells were collected to detect the level of cellular reactive oxygen species by flow cytometry.

The specific operation for detecting the level of reactive oxygen species is as follows: Rinse the cells to be stained once with pre-cooled PBS, digest and collect the cells with trypsin, resuspend the cells in 1 mL of medium, add 10 μM DCFH-DA reactive oxygen probe, and stain at 37°C for 15 minutes . After the staining was completed, the cells were collected by centrifugation, and 500 μL of phosphate buffer was added to resuspend the cells, and flow cytometry was used to detect the level of reactive oxygen species in the cells. The experimental results are the average of three independent experiments, and the results are statistically analyzed according to "mean ± standard deviation".

4. Experimental results:

See Figure 5 for the results. It can be seen from the figure that the induction of palmitic acid (PA) can significantly increase the level of reactive oxygen species in hepatocytes, and the compound HN-001 can decrease the level of reactive oxygen species in hepatocytes in a gradient-dependent manner at a concentration of 4-25 μM (Figure 5).

Example 5 The effect of compound HN-001 derived from marine Aspergillus on non-alcoholic fatty liver

In this example, the compound HN-001 derived from Aspergillus sp.c1 was used as a test subject to conduct an in vivo verification experiment on a mouse model of non-alcoholic fatty liver induced by a high-fat and high-cholesterol diet.

1. Experimental method:

(1) Construction of non-alcoholic fatty liver model:

Taking 8-week-old male C57BL/6J mice (body weight 18-20 g) as objects, they were fed with high-fat and high-cholesterol diet (HFC, containing 60% fat and 1.2% cholesterol, purchased from Research Diet, USA) or normal diet (purchased from from the Experimental Animal Center of Guangdong Province) and fed for 10 weeks.

(2) Preparation of compound HN-001 solution:

A solution of compound HN-001 with a concentration of 2 mg/mL was prepared by using physiological saline solution containing 2.5% PEG-400 and 5% castor oil, and mixed well to dissolve.

(3) Intervention treatment with vehicle or compound HN-001:

HFC diet mice were randomly divided into two groups (HFC model control group and HN-001 administration group), 10 in each group. Mice with normal diet continued to be fed with normal diet, mice in HFC model control group and HN-001 group continued to be fed with HFC diet. Among them, the HN-001 group was given the HN-001 solution prepared in step (2) by intraperitoneal injection at a dose of 20 mg/kg, and the control group was given vehicle treatment. The mice were treated once every two days, and the food intake and body weight of the mice were observed and recorded. Seven weeks after administration, all mice were anesthetized for blood collection, and sacrificed by cervical dislocation and dissected. The body weight and liver weight of mice in each group were recorded.

(4) Analysis of mouse blood samples:

The mouse blood sample collected in step (3) was centrifuged at 3000 rpm for 10 min, and the supernatant was taken. Detect serum aspartate aminotransferase, alanine aminotransferase and other indicators.

(5) Analysis of mouse liver tissue samples:

Weigh a certain amount of mouse liver tissue collected in step (3), ultrasonically crush the tissue on ice, centrifuge at 3000 rpm for 10 min, take the supernatant, and detect the levels of indicators such as triglyceride in the supernatant. At the same time, liver tissue samples were subjected to histopathological analysis to evaluate the protective effect of compound HN-001 treatment on the liver.

2. Experimental results:

The results are shown in Figures 6-7, and it can be seen from Figure 6 that the intraperitoneal injection of the compound HN-001 in mice can effectively inhibit the long-term HFC diet-induced weight gain in mice. After 7 weeks of compound HN-001 administration, compared with the model control group mice, the mice in the HN-001 group lost about 19.8% of their body weight, indicating that the compound HN-001 significantly improved the obesity of the mice. Compared with the mice in the model control group, the liver weight of the mice in the HN-001 group was significantly reduced, and the liver/body weight ratio of the mice in the HN-001 group was effectively reduced.

It can be seen from Figure 7 that compared with the model control group mice, the HN-001 group significantly reduced the level of triglycerides in the liver tissue of the mice. Blood biochemical analysis showed that compared with the model control group mice, the HN-001 group significantly reduced the levels of aspartate aminotransferase and alanine aminotransferase in the blood of the model mice. The results of pathological staining showed that compound HN-001 can significantly protect liver cells, reduce liver lipid levels, and inhibit the generation and accumulation of collagen fibers and muscle fibers in liver tissue.

Example 6 Marine Aspergillus-derived compound HN-001 is toxic to liver cells

In this example, the compound HN-001 derived from Aspergillus sp.c1 was used as a test subject to detect the toxic effect of HN-001 on liver cells.

1. Experimental method:

HepG2 cells in the logarithmic growth phase were uniformly seeded into 96-well plates at 5×10 ³ cells/well, and cultured overnight. Compound HN-001 was diluted in the culture medium to different concentrations of the action solution (0.1, 0.5, 1, 5, 10, 25, 50, 100 μM), and the drug action solution of the corresponding group was added to each well of the 96-well plate, 100 μL/well, 37 C, 5% CO ₂ static culture for 24 hours. The experiment set up control group, DMSO control group and compound HN-001 treatment group. After 24 hours of action, the cell viability was detected by the CCK-8 method.

The specific method for detecting cell viability by CCK-8 method is as follows: after the compound acts for 24 hours, the medium is discarded, and 100 μL of cell culture medium containing 10% CCK-8 working solution is added to each well, and cultured statically at 37°C and 5% CO ₂ After 4 hours, the absorbance value (OD) at 450 nm was measured with a microplate reader.

Wherein, the cell viability analysis takes the vehicle (DMSO) control group as the control (Ctrl), and the multiple relationship is:

The experimental results are the average of three independent experiments, and the results are statistically analyzed according to "mean ± standard deviation".

3. Experimental results:

The results are shown in Figure 8. Compared with the cells in the control group, the compound HN-001 incubated hepatocytes for 24 hours had no inhibitory activity on the cell viability and was highly safe.

In summary, through in vivo and in vitro experiments, it can be effectively demonstrated that the compound HN-001 can inhibit the hepatotoxicity induced by palmitic acid, and effectively protect the liver in vivo, thereby effectively improving or treating non-alcoholic fatty liver, and the compound itself Less toxic.

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

Claims (4)

1. Use of a marine aspergillus source compound in the preparation of a medicament for the treatment of non-alcoholic fatty liver disease, characterized in that the marine aspergillus source compound has the structure of formula (I):

（I）。

2. the use according to claim 1, wherein said treatment of non-alcoholic fatty liver disease comprises inhibition of liver lipid accumulation, liver injury, liver inflammation and fibrosis.

3. The use according to claim 1, wherein the marine aspergillus source compound is also a pharmaceutically acceptable salt thereof.

4. The use according to any one of claims 1 to 3, wherein the medicament for treating non-alcoholic fatty liver disease is in the form of an oral or injectable preparation.

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