CN110960553B

CN110960553B - Application of malonic acid modified fullerene C70 in preparation of medicine for treating non-alcoholic fatty liver disease

Info

Publication number: CN110960553B
Application number: CN201911380288.9A
Authority: CN
Inventors: 李红良; 折志刚; 邹头君
Original assignee: Wuhan Sailaiya Biotechnology Co ltd
Current assignee: Wuhan Sailaiya Biotechnology Co ltd
Priority date: 2019-12-27
Filing date: 2019-12-27
Publication date: 2023-10-03
Anticipated expiration: 2039-12-27
Also published as: CN110960553A

Abstract

The invention discloses an application of malonic acid modified fullerene C70 in preparation of a medicine for treating non-alcoholic fatty liver disease. The medicine for preventing, relieving and/or treating the nonalcoholic fatty liver disease and related diseases by taking the C70-malonic acid derivative as an active ingredient has the following advantages: 1. the drug effect is obvious, the C70-malonic acid derivative can obviously inhibit lipid accumulation in primary hepatocytes of mice, and obvious change can be seen at 10 mu M. In addition, the C70-malonic acid derivative has obvious improving effect on liver lipid accumulation and fibrosis induced by HFHC diet. 2. The malonic acid modified fullerene C70 can effectively reduce the damage of a carbon cage in the modification process of the fullerene, so that the pharmacodynamic activity of the fullerene body is better reserved. 3. The C70-malonic acid derivative has a definite molecular structure, successfully solves the problems of no definite structure and difficult quality control of other water-soluble fullerene derivatives, and is beneficial to patent medicine.

Description

Application of malonic acid modified fullerene C70 in preparation of medicine for treating non-alcoholic fatty liver disease

Technical Field

The invention relates to the field of nano biological medicine, in particular to application of malonic acid modified fullerene C70 in preparation of a medicine for treating non-alcoholic fatty liver disease.

Background

With the improvement of the living standard and the change of the living style of people, the number of patients with non-alcoholic fatty liver disease (nonalcoholic fatty liver disease, NAFLD) in China is increased year by year, and the non-alcoholic fatty liver disease has become an important cause of chronic liver disease. According to statistics, the current global population has more than 10 hundred million non-alcoholic fatty liver disease patients, and the incidence rate of NAFLD in adults in China also reaches about 20 percent [1-3]. NAFLD patients often have no history of excessive alcohol consumption and are characterized by diffuse hepatocyte bullous steatosis and fat accumulation. Furthermore, if nonalcoholic fatty liver deficiency is treated in time, it may also progress to nonalcoholic steatohepatitis (nonalcoholic steatohepatitis, NASH), liver fibrosis, cirrhosis or even liver cancer [4]. However, no ideal preventive and therapeutic drug exists in clinic at present, and the situation is very serious.

Fullerenes are another allotrope of carbon elements other than graphite, diamond, and amorphous carbon, and are a cage-like hollow molecule composed entirely of carbon. C70 is one of the most common fullerenes. The fullerene material has rich biological activities of scavenging free radicals, resisting oxidation, resisting aging, inhibiting apoptosis and the like, and the water-soluble derivative thereof has good biological application prospect [5-7]. However, most of the current water-soluble fullerene derivatives are obtained by introducing water-soluble groups such as hydroxyl groups, amino groups or amino acids on the surface of the fullerene through strong acid, strong alkali or hydrogen peroxide. The preparation method has harsh conditions, the fullerene bulk structure is seriously damaged, and the activity of the fullerene is easily reduced and even toxicity is generated. In addition, the fullerene derivative generally has no definite structure or molecular formula, so that the product batches are different, the quality control is difficult, the medicine is difficult to prepare, and the medicinal value of the fullerene derivative in the medical field is limited.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide the application of the C70-malonic acid derivative with a defined structure in preparing the medicine for treating the nonalcoholic fatty liver disease.

In order to achieve the purpose, the invention provides application of malonic acid modified fullerene C70 in preparation of a medicine for treating non-alcoholic fatty liver disease.

Preferably, the structural formula of the C70-malonic acid derivative is as follows:

wherein R is selected from H and Na, and n has a value of 1-70.

Further, the C70-malonic acid derivative has an important role in regulating lipid metabolism of hepatocytes: treatment of the primary hepatocytes of mice with C70-malonic acid derivatives and the NASH model of C57BL/6 mice revealed that the C70-malonic acid derivatives were able to significantly inhibit lipid accumulation of liver cells.

Furthermore, the C70-malonic acid derivative is used as an active ingredient for preparing medicines for preventing, relieving and/or treating nonalcoholic fatty liver diseases.

Still further, the non-alcoholic fatty liver disease comprises: simple liver fatty liver degeneration, nonalcoholic steatohepatitis, liver fibrosis, liver cirrhosis and liver cancer.

The medicine for preventing, relieving and/or treating the nonalcoholic fatty liver disease and related diseases by taking the C70-malonic acid derivative as an active ingredient has the following advantages:

1. the drug effect is obvious, the C70-malonic acid derivative can obviously inhibit lipid accumulation in primary hepatocytes of mice, and obvious change can be seen at 10 mu M. In addition, the C70-malonic acid derivative has obvious improving effect on liver lipid accumulation and fibrosis induced by HFHC diet.

2. The malonic acid modified fullerene C70 can effectively reduce the damage of a carbon cage in the modification process of the fullerene, so that the pharmacodynamic activity of the fullerene body is better reserved.

3. The C70-malonic acid derivative has a definite molecular structure, successfully solves the problems of no definite structure and difficult quality control of other water-soluble fullerene derivatives, and is beneficial to patent medicine.

Drawings

Figure 1 is a diagram of a constitution of a C70-malonic acid derivative hydrolysis precursor prepared in example I

FIG. 2 is a graph showing the results of oil red staining of C70-malonic acid derivative pretreated mice primary hepatocytes at different concentrations after 12 hours of treatment with PA/OA

FIG. 3 weight measurement results of mice in the control group and mice in the group given C70-malonic acid derivative (p.ltoreq.0.05).

FIG. 4 liver weight detection results (p.ltoreq.0.05) of mice from the control group and mice from the group given C70-malonic acid derivative.

FIG. 5 results of fat weight measurements of mice in the control group and mice given the C70-malonic acid derivative group (p.ltoreq.0.01).

FIG. 6H & E staining pattern of liver of control mice and mice given C70-malonic acid derivative (x 20).

FIG. 7A is a graph of the masson staining of the livers of mice from the control group and mice given the C70-malonic acid derivative group (x 20).

Detailed Description

A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification taken in conjunction with the drawings. The examples provided are merely illustrative of the methods of the present invention and are not intended to limit the remainder of the disclosure in any way whatsoever

[ example 1 ] C70-malonic acid derivative C ₇₀ [C(COONa) ₂ ] ₄ Is prepared from

50mg of C70 are dissolved in 10ml of toluene, and 0.8ml of diethyl bromomalonate and 0.7ml of DBU are added under nitrogen. The reaction was stirred at room temperature for about 2h. Separating and purifying by silica gel column chromatography (ethyl acetate: toluene=1:99), taking the 4 th color band, and concentrating under reduced pressure. The obtained product is hydrolyzed by adding NaH under the protection of nitrogen, and the reaction is stirred for about 6 hours at 60 ℃. After the reaction, methanol was added to quench excess NaH and the mixture was centrifuged to obtain a precipitate. Washing the precipitate with ethanol for three times to obtain tan C ₇₀ [C(COONa) ₂ ] ₄ The mass spectrum of the product is shown in figure 1.

[ example 2 ] C70-malonic acid derivative C ₇₀ [C(COONa) ₂ ] ₄ Pretreatment reduces hepatocyte fat accumulation

Isolation and culture of mouse primary hepatocytes:

the primary hepatocytes of the mice were isolated by type iv collagenase digestion. The mice are subjected to ether inhalation anesthesia, a portal vein is punctured by an indwelling needle, the liver is subjected to in-situ perfusion (SC-1 and SC-2 are alternately perfused) until the liver is completely digested, the liver is taken down, repeatedly blown and filtered, and a hepatocyte suspension is collected.

And (3) coating a culture dish: after dilution of absolute ethanol to 30% ethanol with sterilized ultrapure water, filtration was performed with a 0.22 μm filter, and then 100× rat tail gum was diluted to 1×. 200 μl of diluted rat tail gel was added to each well of the six well plate, and the six well plate was rotated to spread the gel over the entire plate bottom. Uncapping and blowing at a clean bench overnight.

And (3) paving: isolated primary hepatocytes were counted by trypan blue followed by 6-well plates with a cell number of 1×10 per well ⁶ The cells were attached for about 6-8 hours.

The primary hepatocytes of mice were divided into 2 groups, respectively PA/OA group, C70-malonic acid derivative 10. Mu.M PA/OA group, and C70-malonic acid derivative 20. Mu.M PA/OA group. After cell attachment, PBS, 10. Mu. M C70-malonic acid derivative, 20. Mu. M C70-malonic acid derivative were added and treated for 3 hours, followed by treatment with 0.2/0.4mM PA/OA for 12 hours, followed by oil red O staining. The staining results are shown in FIG. 2, where the amount of red fat droplets was significantly reduced in the C70-malonic acid derivative-pretreated group compared to the PA/OA group. The results demonstrate that pretreatment with the C70-malonic acid derivative significantly inhibited hepatocyte lipid accumulation.

[ example 3] C70-malonic acid derivative C ₇₀ [C(COONa) ₂ ] ₄ Inhibiting the progression of nonalcoholic fatty liver disease

1 laboratory animal and raising

Experimental animals: 8-10 weeks old, with a weight of 23.5-27.5g, male C57BL/6 mice were used as subjects.

Feeding environment: SPF-grade laboratory animal center, SPF-grade mouse feed was purchased from Beijing Fukang Biotechnology Co. Feeding conditions: the room temperature is between 22 and 24 ℃, the humidity is between 40 and 70 percent, the lighting time of light and dark alternation is 12 hours, and the food intake is realized by free drinking water.

Model 2 construction and drug administration

C57 mice (20 total) were fed for 10 weeks with High Fat High Cholesterol (HFHC) and NASH model was established. Thereafter, the mice were divided into two groups (10 each, administration group and control group, respectively), the control group was injected with physiological saline by intraperitoneal injection, and the administration group was injected with C70-malonic acid derivative C by intraperitoneal injection ₇₀ [C(COONa) ₂ ] ₄ (10 mg/kg) (dosing frequency: once daily) while continuing HFHC feeding for 4 weeks. Liver weights and white fat weights of two groups of mice were measured, and the liver of the mice was HE stained and masson stained.

3 pathology detection

3.1 preparation of Paraffin sample sections

The main operation procedure comprises: trimming liver, embedding frame treatment, running water flushing, dehydration, transparency, wax dipping, embedding, slicing, spreading, airing or baking for later use.

3.2 hematoxylin-eosin (H & E) staining

The method mainly comprises the following steps: baking paraffin sample slices at 55 ℃ for 30min, xylene for 5min,3 times, 100% alcohol for 1min, 95% alcohol for 1min, 70% alcohol for 1min, double distilled water for 1min, hematoxylin solution for 5min, water washing for 1min, 1% hydrochloric acid alcohol for 1-3s, water washing for 1min, scott solution for 1min, water washing for 1min, eosin solution for 3-5min, washing off floating color by distilled water, 70% alcohol for 1s, 95% alcohol for 1s, 100% alcohol for 30s,3 times, xylene for 2min,3 times, sealing the slices immediately before xylene is dried in a fume hood, and photographing by a microscope.

3.3 Masson (Masson) staining

The method mainly comprises the following steps: taking paraffin specimen slices, baking at 55 ℃ for 30min, carrying out xylene 5min,3 times of 100% alcohol 1min, carrying out 95% alcohol 1min, carrying out 70% alcohol 1min, carrying out double steaming for 1min, carrying out hematoxylin solution 7min, carrying out tap water 5min, carrying out 1% hydrochloric acid alcohol differentiation for 1-3s, carrying out running water flushing for 5min, carrying out double steaming for 1min, carrying out ponceau acid fuchsine solution 7min, carrying out double steaming for 5min, carrying out phosphomolybdic acid water solution differentiation for 5min, carrying out aniline blue dyeing for 5min, carrying out 1% glacial acetic acid 1min multiplied by 2 times, carrying out 70% alcohol 1s, carrying out 95% alcohol 1s, carrying out 100% alcohol 30s,3 times, carrying out xylene 2min, carrying out 3 times while the xylene is not dried, immediately sealing the xylene, carrying out blow drying in a fume hood, and carrying out microscopic photographing.

The results of the changes in the body weight, liver weight and white blood fat weight of the mice in the two groups are shown in FIGS. 3, 4 and 5, and the body weight, liver weight and white blood fat weight of the mice in the administration group are significantly lower than those in the control group. H&As shown in FIGS. 6 and 7, it was observed that the liver cells of the mice in the control group were aliphatically denatured, vacuolated and fused into pieces, the morphology of the liver cells was severely destroyed, the morphology of the liver cells of the mice in the administration group was significantly improved, and the liver fibrosis was also alleviated. The above results illustrate C70-malonic acid derivative C ₇₀ [C(COONa) ₂ ] ₄ Has good therapeutic effect on nonalcoholic fatty liver disease.

Claims

1. An application of malonic acid modified fullerene C70 in preparing a medicine for treating non-alcoholic fatty liver disease, which is characterized in that:

the structural general formula of the C70-malonic acid derivative is as follows:

wherein R is selected from Na, and n has a value of 4;

the non-alcoholic fatty liver disease comprises: simple liver fatty liver degeneration or nonalcoholic steatohepatitis.