CN108836977B

CN108836977B - Composition for preventing, reducing and/or relieving nonalcoholic steatohepatitis and application thereof

Info

Publication number: CN108836977B
Application number: CN201810530940.XA
Authority: CN
Inventors: 崔国祯; 陈远才; 黄天养; 李惠民; 陈言; 陈雪松
Original assignee: Zhuhai Campus Of Zunyi Medical University; Zhuhai Txy Biotech Holding Co ltd
Current assignee: Zhuhai Campus of Zunyi Medical University; ZHUHAI TXY BIOTECH HOLDING Co.,Ltd.
Priority date: 2018-05-29
Filing date: 2018-05-29
Publication date: 2021-02-19
Anticipated expiration: 2038-05-29
Also published as: CN108836977A

Abstract

The invention provides a composition for preventing, reducing and/or relieving nonalcoholic steatohepatitis and application thereof, wherein the composition comprises beta-glucan and pyrimidine derivatives. The composition exhibits a synergistic effect in the treatment of nonalcoholic steatohepatitis.

Description

Composition for preventing, reducing and/or relieving nonalcoholic steatohepatitis and application thereof

Technical Field

The invention relates to a composition for preventing, reducing and/or relieving nonalcoholic steatohepatitis and application thereof.

Background

Non-alcoholic steatohepatitis (NASH) is a syndrome without excessive drinking, and mainly characterized by steatosis of liver cells, ballooning and diffuse lobular inflammation of liver. It belongs to the Nonalcoholic fatty liver disease (NAFLD), a potentially fatal disease that can lead to cirrhosis and liver cancer. The exact cause of NASH has not been elucidated, but studies have shown that the onset of NASH is closely related to insulin resistance, obesity, hyperlipidemia, metabolic syndrome, and the like. With the increasing incidence of these diseases, the prevalence of NASH is also increasing. At present, the treatment method of clinical trials has no obvious effect on most of the treatments. There is no evidence of evidence for drug treatment of NASH. Therefore, the exploration of a novel safe and effective pharmaceutical composition has important significance for the clinical treatment and improvement of the pharmaceutical composition.

Beta-glucan is a polysaccharide widely existing in various fungi and plants, has various effects of reducing blood fat and blood sugar, protecting liver, regulating immunity and the like, and is considered to be a safe active substance. Ethyl 7-benzyl- (4-hydroxy-3-methoxyphenyl) -5-methyl-4, 7-dihydrotetrazole- [1,5-a ] pyrimidine-6-carboxylate is a pyrimidine derivative. Chinese patent (201810431586.5) describes that the compound has prevention and treatment effects on non-alcoholic fatty liver disease induced by methionine-choline deficiency feed, and especially has remarkable prevention and treatment effects on liver cell damage (such as balloon degeneration). The synergistic effect of the pyrimidine derivative and beta-glucan on preventing and treating non-alcoholic fatty liver disease has not been reported so far.

Disclosure of Invention

The invention provides a composition for preventing, reducing and/or relieving nonalcoholic steatohepatitis, which is combined to be used for showing a synergistic effect in the aspect of treating nonalcoholic steatohepatitis diseases.

The technical scheme of the invention is realized as follows:

a composition for preventing, reducing and/or alleviating nonalcoholic steatohepatitis, which is characterized by comprising beta-glucan and ZY003, wherein the mass ratio of the ZY003 to the beta-glucan is 3-5: 2-4, and the ZY003 has the following structure:

preferably, the mass ratio of said ZY003 to said beta-glucan is 4: 3.

The application of a pharmaceutical composition consisting of beta-glucan and a pyrimidine derivative in preparing a composition for preventing, reducing and/or relieving nonalcoholic steatohepatitis is characterized in that the mass ratio of ZY003 to beta-glucan is 3-5: 2-4, and ZY003 has the following structure:

preferably, the mass ratio of said ZY003 to said beta-glucan is 4: 3.

The invention has the beneficial effects that:

the invention takes the composition of beta-glucan and ZY003 as materials, and researches the prevention and improvement effects of the composition on nonalcoholic steatohepatitis model mice. After 50 male mice of C57BL/6 were acclimatized in a breeding environment for 1 week, they were randomly divided by weight into a control group, a model group (MCD group), a ZY003 group, a β -glucan group and a ZY003+ β -glucan group. A model of mouse NASH was established using methionine-choline deficient (MCD) feed. The normal group was fed methionine-choline replete (MCS) feed, and the remaining groups were fed MCD feed. Daily conditions of the mice were regularly observed and recorded, and the mice were intraperitoneally injected with a drug solution, 40mg/kg ZY003, 30mg/kg beta-glucan, 40mg/kg ZY003+30mg/kg beta-glucan, respectively, at regular times daily. Mice were sacrificed 8 weeks after feeding and blood and liver specimens were collected. The pathological condition of the liver was observed by HE and oil red staining and the level of serum TNF-alpha was determined. After 8 weeks of combined treatment with ZY003 and beta-glucan of NASH model mice induced by MCD feed, the degree of steatosis and balloon-like lesions of the liver was significantly reduced compared to the model group (MCD group) and the single drug treatment group (ZY003 or beta-glucan) after feed feeding. The experimental results prove that the combined use of the beta-glucan and the ZY003 shows a synergistic effect in the treatment of the nonalcoholic steatohepatitis disease.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a chemical formula of compound ZY003 of the present invention;

FIG. 2 is a microscopic image (400X) of HE staining and oil red staining of liver of each group of mice (200X) provided in the examples of the present invention.

FIG. 3 shows the pathological scores of liver tissue inflammation, steatosis, cell ballooning and synthesis for each group of mice provided by the example of the present invention.

FIG. 4 serum TNF- α expression levels in various groups of mice provided by the examples of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

ZY003 of the present invention has the structure shown in FIG. 1.

The composition for preventing, reducing and/or relieving nonalcoholic steatohepatitis and the using method thereof comprise the following steps:

the compounds provided by the present methods can be formulated into compositions using methods known in the art for pharmaceutical compounds, and these compositions can include one or more sweetening compounds, coloring compounds, preserving compounds, and flavoring compounds to provide pharmaceutically suitable formulations.

For example, tablets, pills, hard gelatin capsules, powders or granules may be used as oral solid compositions. In these compositions, suitable excipients or diluents which are acceptable in non-toxic medicaments, such as sucrose, starch or lactose, are admixed. These compositions may contain substances other than diluents, such as lubricants, for example magnesium stearate, talc or hard fatty acids.

For example, pharmaceutically acceptable solutions, emulsions, syrups, suspensions and elixirs, such as water or liquid paraffin, containing inert diluents, may be used as liquid compositions for oral administration. These compositions may also contain flavouring, lubricating or sweetening agents other than diluents.

The active compounds ZY003 and beta-glucan of the present invention may be administered in the form of suppositories or capsules for rectal administration, mixed with suitable non-irritating excipients such as cocoa butter, polyethylene glycols or semi-synthetic glycerides.

Suitable dosage forms for the ZY003 and β -glucan compositions of the invention include, but are not limited to: a tablet; a caplet; capsules such as soft elastic gelatin capsules; a cachet; a lozenge; a lozenge; a dispersant; suppositories; an ointment; pastes (poultices); ointment; powder; a dressing agent; a cream; a plaster; a solution; pasting a piece; aerosol; gelling; liquid dosage forms suitable for oral or mucosal administration to an individual, including suspensions (e.g., aqueous or non-aqueous liquid suspensions, oil-in-water emulsions, or water-in-oil liquid emulsions), solutions and elixirs; a liquid dosage form suitable for parenteral administration to an individual; and sterile solids (e.g., crystalline or amorphous solids) that can be reconstituted to provide a liquid dosage form suitable for parenteral administration to an individual.

Routes of administration of the ZY003 and beta-glucan compositions of the present invention include, but are not limited to, parenteral administration, e.g., intravenous, intradermal, subcutaneous, intramuscular, subcutaneous, oral, buccal, sublingual, inhalation, intranasal, transdermal, topical, transmucosal, intratumoral, intrasynovial and rectal administration. In particular embodiments, the compositions are formulated according to conventional procedures as pharmaceutical compositions suitable for intravenous, subcutaneous, intramuscular, oral, intranasal, or topical administration to humans. In embodiments, the pharmaceutical composition is formulated for subcutaneous administration to a human according to conventional procedures.

The ZY003 and beta-glucan composition can be used for treating nonalcoholic steatohepatitis and relieving steatosis, liver lobular inflammation and liver tissue ballooning.

Examples

1 materials and methods

1.1 materials

Feed: methionine-choline deficient (MCD) feed (cat # TP3006R) and methionine-choline sufficient (MCS) feed (cat # TP3006S2) were purchased from Nantong Turofen feed technologies, Inc.

Detection reagent: ELISA kit for detecting TNF-alpha was purchased from BD company.

Experimental animals: c57BL/6 male mice are 7-8 weeks old, 23 + -2 g in body weight, SPF grade, purchased from Guangdong province medical laboratory animal center, protected from environmental animal house, 30% -60% in relative humidity, 12h in light/12 h in darkness, and freely fed and drunk by animals.

1.2 methods

Animal grouping and intervention methods: after 50 male mice of C57BL/6 were acclimatized in a breeding environment for 1 week, they were randomly divided by weight into a control group (MCS group), a model group (MCD group), a ZY003 group, a β -glucan group and a ZY003+ β -glucan group. After one week of adaptive feeding, a mouse NASH model was established using MCD feed. The normal group is fed with MCS feed, and the other groups are fed with MCD feed. Daily conditions of the mice were regularly observed and recorded, and the mice were intraperitoneally injected with a drug solvent, 40mg/kgZY003, 30mg/kg beta-glucan, 40mg/kgZY003+30mg/kg beta-glucan, respectively, at regular times daily.

Sample collection and index detection: weighing every week, feeding mice 8 weeks later, fasting for 12h, intraperitoneal injecting anesthetized animals with chloral hydrate, collecting blood, standing overnight in a refrigerator at 4 ℃, centrifuging for 10min at 1000g in a centrifuge at 4 ℃, collecting supernatant, and measuring serum TNF-alpha by using mouse serum ELISA kit instructions.

Taking 50mg of liver tissues, placing the liver tissues in 4% paraformaldehyde, fixing for 24h, dehydrating for 12h, embedding, slicing, carrying out HE staining, and then carrying out liver histoscopy pathology detection and grade scoring. Pathological scoring criteria for NASH were evaluated according to NASH pathological diagnosis criteria using NAFLD activity score guidelines established by the national institutes of health clinical research network pathology committee at 2005.

Steatosis, lobular inflammation, cell ballooning and syndromatosis were scored. (1) For hepatic steatosis (steatosis), less than 5% of hepatic steatosis is 0 point; 5% -33%, 1 minute; 33% -66%, 2 min; over 66%, 3 points. (2) For liver Lobular inflammation (Lobular inflammation, 20 fields counted randomly), no inflammation, score 0; <2, 1 min; 2-4, 2 min; 4, 3 points. (3) Balloon-like changes of hepatocytes (balloon), none, 0 min; rare, 1 point; 2 fen, mostly seen. The scores for hepatic steatosis, lobular inflammation and hepatocellular ballooning were added to obtain a composite score (Totalscore).

2 results

Pathological examination

FIG. 2 is a microscopic view (400X) of HE staining and oil red staining of mouse liver, wherein the hepatic lobules of MCS group have clear and complete structure, and the hepatocytes are arranged radially with the central vein as the center, without balloon-like change of hepatocytes and without obvious large lipid droplets. The MCD group has serious pathological changes, abnormal hepatic lobule structure and different sizes of cells in the liver, lipid in the hepatic cells is dissolved by alcohol and xylene and then is removed to form a plurality of vacuoles, red lipid drops are large and dense, and a plurality of inflammatory cells are infiltrated. Compared with the MCD group, the mice in the ZY003 treatment group have lighter liver lesion degree, a small amount of vacuoles appear in liver cells, lipid droplets are reduced, and the hepatic lobule structure tends to be normal; vacuoles in liver tissues of mice in a beta-glucan treatment group are obviously reduced, the lobular structure of the liver is basically normal, and hepatic blood sinuses are obvious; the liver cells in the ZY003+ beta-glucan treatment group are remarkably reduced in vacuoles, the hepatic lobule structure is recovered to be normal, the hepatic chordae structure is basically normal and radial, a large number of lipid droplets are reduced, and hepatic blood sinuses are obvious.

Pathology scoring

Figure 3 is an assessment of the liveness score of pathological sections of mouse liver, in which the MCD group with intrahepatic lobular inflammation score was significantly higher than the MCS group of mice (P < 0.05). Mice in the ZY003, beta-glucan and ZY003+ beta-glucan groups had significantly lower intrahepatic lobular inflammation scores (P <0.05) compared to the MCD group.

The steatosis score MCD group was significantly higher than the MCS group mice (P < 0.05); mice in the ZY003+ beta-glucan group had significantly lower steatosis scores (P <0.05) compared to the MCD group.

The MCD group showed significantly higher cell ballooning scores than the MCS group mice (P <0.05), and the ZY003+ beta-glucan group showed significantly lower cell ballooning scores than the beta-glucan group (P < 0.05).

The overall score of the MCD group was significantly higher than that of the MCS group mice (P < 0.05). Mice in the ZY003, beta-glucan and ZY003+ beta-glucan groups had significantly lower intrahepatic lobular inflammation scores (P <0.05) compared to the MCD group. The mice in the ZY003+ β -glucan group had significantly lower overall scores (P <0.05) compared to the ZY003 or β -glucan groups. The research results show that the ZY003 compound and the beta-glucan have the effect of preventing and treating NASH synergistically.

Effect of ZY003 on TNF-alpha content in mouse serum

The serum TNF-alpha content of mice in MCD group is obviously higher than that of mice in MCS group (P < 0.05). Mice in ZY003, β -glucan and ZY003+ β -glucan groups had significantly reduced serum TNF- α levels (P <0.05) compared to MCD groups. Compared with ZY003 group or beta-glucan group, the mouse with ZY003+ beta-glucan group has obviously reduced serum TNF-alpha content (P < 0.05). The prophylactic and therapeutic effects of ZY003 and β -glucan on NASH may be associated with a reduction in serum levels of TNF- α.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. A composition for preventing, reducing and/or alleviating non-alcoholic steatohepatitis, which is characterized by consisting of beta-glucan and a pyrimidine derivative; the pyrimidine derivative is ZY003, and the mass ratio of the ZY003 to the beta-glucan is 3-5: 2-4; the structural formula of ZY003 is as follows:

2. the composition of claim 1, wherein the mass ratio of ZY003 to beta-glucan is 4: 3.

3. The application of a pharmaceutical composition consisting of beta-glucan and a pyrimidine derivative in preparing a composition for preventing, reducing and/or relieving nonalcoholic steatohepatitis is characterized in that the pyrimidine derivative is ZY003, and the mass ratio of ZY003 to beta-glucan is 3-5: 2-4; the structural formula of ZY003 is as follows:

4. the use of claim 3, wherein the mass ratio of ZY003 to β -glucan is 4: 3.