CN113368107B - Pharmaceutical composition containing berberine and matrine and application thereof in treating or preventing nonalcoholic fatty liver disease - Google Patents

Abstract

The invention relates to a pharmaceutical composition containing berberine and matrine and application thereof in treating or preventing nonalcoholic fatty liver disease NAFLD (nonalcoholic steatohepatitis NASH). Specifically, the pharmaceutical composition of the present invention comprises berberine compounds and matrine compounds as active ingredients. The two active ingredients of the invention have synergistic effect, and can obviously reduce key indexes of NAFLD (containing NASH): triglyceride content, oxidative stress and chronic inflammatory reaction in liver tissue, and also can reduce blood lipid level and liver index.

Description

Pharmaceutical composition containing berberine and matrine and application thereof in treating or preventing nonalcoholic fatty liver disease

Technical Field

The invention relates to a pharmaceutical composition containing berberine and matrine and application thereof in treating or preventing nonalcoholic fatty liver disease, belonging to the field of biological pharmacy.

Background

1. Nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (nonalcoholic fatty liver disease, NAFLD) is a manifestation of metabolic syndrome in the liver, is a metabolic stress liver injury closely related to insulin resistance and genetic susceptibility, and is closely related to high incidence of insulin resistance, metabolic syndrome, type 2 diabetes, arteriosclerotic cardiovascular disease, colorectal tumor, and the like. In the near futureIn bed, primary diagnosis of NAFLD refers to the occurrence of 5% or more of liver fat accumulation in the absence of other recognized factors causing fatty liver (e.g., alcohol, virus, drug, autoimmune). NAFLD is mainly characterized by abnormal accumulation of triglycerides in the liver, defined as steatosis or nonalcoholic fatty liver without inflammation and hepatocyte damage; in the liver of chronic NAFLD patients, lobular inflammation and hepatocyte damage, such as balloon-like changes, fibrosis, may occur, which is known as nonalcoholic steatohepatitis (nonalcoholic steatohepatitis, NASH). NASH is prone to develop into cirrhosis and hepatocellular carcinoma ^[1] 。

2. Incidence of disease

NAFLD is the most common chronic liver disease worldwide, accounting for about 25% of the overall population in the united states, 25% of which progress to NASH. With further accumulation of liver fat, inflammation and formation of liver scars, 25% of NASH patients develop cirrhosis or hepatocellular carcinoma, accounting for 1% -2% of the overall population. Of course, these data are primarily from the united states and the proportion of hepatocellular carcinoma developed remains unknown. However, it is considered that the probability of developing hepatocellular carcinoma per year in patients with cirrhosis caused by NAFLD is 1% -2%. Epidemiological investigation in the united states has shown that NASH is the first leading cause of liver transplantation by 2020 ^[2] 。

In China, NAFLD has become the first major chronic liver disease in China along with the change of dietary structure and life style. Epidemiological investigation results from Shanghai, beijing and other areas show that the prevalence of NAFLD in B-mode ultrasonic diagnosis of common adults increases from 15% to more than 31% over 10 years, whereas the prevalence of type 2 diabetics is higher, up to 70% ^[3] 。

NAFLD is a multi-system disease

Research in the last decade has shown that the clinical burden of NAFLD is not limited to high morbidity and mortality due to liver-related diseases, and that increasing evidence suggests that NAFLD is a multi-system disease affecting multiple extrahepatic organs and related signaling pathways ^[4] And causes the occurrence of chronic complications of extrahepatic organs.

NAFLD is mainly involved in chronic liver disease, cardiovascular disease and type 2 diabetes. A recent Meta analysis showed that NAFLD increased mortality from cardiovascular disease to 57% and increased the risk of developing type 2 diabetes by a factor of approximately 2 ^[5] . In addition, more and more people are focusing on chronic kidney disease associated with NAFLD, and studies have found that NAFLD can increase the prevalence of chronic kidney disease ^[6] . In addition, NAFLD is associated with other chronic diseases such as sleep apnea, colorectal cancer, osteoporosis, psoriasis and various endocrine disorders such as polycystic ovary syndrome ^[7] 。

For many years, studies have found that obesity and type 2 diabetes increase the risk of developing liver cancer ^[8] However, the underlying mechanism of this link is still unclear, that is, it is currently uncertain whether diabetes or obesity can specifically induce the occurrence of liver cancer. NASH is common in obese, insulin resistant and type 2 diabetics and can increase the incidence of liver cancer in patients ^[9] The mechanisms involved are not well understood and may be related to those of liver inflammation, metabolic stress and insulin resistance caused by NASH and type 2 diabetes.

4. Pathogenesis of disease

The pathogenesis of NAFLD is complex and multifactorial. The "second hit hypothesis" was originally proposed, i.e., liver lipid accumulation due to bad lifestyle, high fat diet, obesity and insulin resistance is the first hit, further sensitising the liver to injury, activating inflammatory cascades and forming fibrosis, the second hit ^[10] 。

However, this view is too simplistic to generalize the complexity of NAFLD, as there are multiple concurrence factors in genetically susceptible individuals that are closely related to the occurrence and progression of the disease. Thus, there is also proposed a "multiple hit hypothesis" that dietary habits, environmental and genetic factors may lead to insulin resistance, obesity and changes in intestinal flora. Insulin resistance is one of key factors for steatosis and NASH development, and can increase liver new-born fat generation and promote lipolysis of adipose tissue, thereby leading fatty acid in liverIncrease in ^[11] . Insulin resistance also causes adipose tissue dysfunction, thereby increasing the production and secretion of adipose and inflammatory cytokines ^[12] . Fat accumulates in the liver in the form of triglycerides, while increasing fat toxicity due to high levels of free fatty acids, free cholesterol and other lipid metabolites, leading to oxidative stress, leading to mitochondrial dysfunction, production of reactive oxygen species and activation of endoplasmic reticulum stress ^[13] Thereby causing inflammation of the liver. In addition, changes in intestinal flora lead to further production of fatty acids in the intestine, increasing the permeability of the small intestine, leading to bacterial derivatives such as lipopolysaccharides entering the portal circulation, which activate inflammatory small body and endoplasmic reticulum stress and release pro-inflammatory cytokines such as tumor necrosis factor alpha (tumor necrosis factor alpha, TNF-alpha) and interleukin 6 (Interleukin-6, IL-6) ^[14] . Genetic factors or epigenetic modifications affect hepatocyte fat content, enzymatic processes, and the liver inflammation environment, thereby affecting the development of NAFLD.

In addition, activation of certain metabolic or stress pathways, including one-carbon metabolism, activation of B cell k light chain enhancer by nuclear factors (nuclear factor kappa-light-chain-enhancer of activated B cells, NF-kB), homologous phosphatase tensins (phosphatase and tension homolog, PTEN), and microRNAs, are also associated with the development and progression of hepatocellular carcinoma ^[15] . Toll-like receptor 4 (TLR-4), an upstream activator of NF-kB signaling ^[16] Chronic activation can occur by binding to gut flora derived ligands. In addition, intestinal flora regulates bile acid metabolism associated with the development of hepatocellular carcinoma, and intestinal flora can metabolize primary bile acid produced by the liver into secondary bile acid such as deoxycholic acid. In the mouse model of hepatocellular carcinoma, when deoxycholic acid production is blocked by an inhibitor, the progress of hepatocellular carcinoma is inhibited ^[17] This means that the interaction between intestinal flora and bile acid production plays an important role in the pathogenesis of hepatocellular carcinoma. Indeed, alterations in the intestinal flora promote the development of type 2 diabetes and NAFLD and increase the risk of hepatocellular carcinoma, which makes the intestinal flora a factorAn attractive potential new target for treating NAFLD. However, if the treatment of intestinal flora can reduce NAFLD complications such as cirrhosis and hepatocellular carcinoma, further investigation is needed.

5. Current treatment methods

Guide for preventing and treating 2018 non-alcoholic fatty liver disease ^[18] The current treatment for NAFLD is indicated to include 1. Change of bad lifestyle: such as healthy diet, exercise enhancement, alcohol consumption restriction, etc. 2. Drug treatment for metabolic syndrome: such as obesity, hypertension, type 2 diabetes, etc., these drugs have no positive therapeutic effect on NAFLD, particularly liver fibrosis. 3. Weight-loss surgery: asian countries use sleeve gastrectomy most often, but there is currently no adequate evidence to recommend bariatric surgery to treat NAFLD.4. Drug treatment for liver injury: given that it is difficult to reverse NASH, especially liver fibrosis, in weight loss surgery, in view of changing lifestyle and application of drugs for metabolic syndrome, it is necessary to apply liver protecting drugs to protect hepatocytes, antioxidant, anti-inflammatory, and even anti-liver fibrosis. Clinical trial results from the united states show that 800IU/d vitamin E orally administered for 2 years can restore normal serum amino acid transferase to non-diabetic NASH adult humans and significantly improve liver steatosis and inflammatory lesions. However, the pharmacopoeia of China does not have indications for the treatment of chronic hepatitis with large doses of vitamin E, and the safety of long-term large-dose use of vitamin E is worrying. Clinical trial results from the united states show that obeticholic acid can significantly reduce the degree of liver fibrosis in NASH patients, but that this drug has a detrimental effect on lipid metabolism, can cause skin itching, and its role in NASH treatment is not confirmed by clinical trials in japan. At present, the medicines for treating liver injury, such as silybin, matrine, bicyclo alcohol, polyene phosphatidylcholine, glycyrrhizic acid diamine, reduced glutathione, S-adenosylmethionine, ursodeoxycholic acid and the like, which are widely applied in China, have good safety, and part of medicines have relatively definite curative effects in patients with drug-induced liver injury, cholestatic liver diseases and the like, but the curative effects of the medicines on NASH and liver fibrosis still need to be verified by further clinical experiments. 5. Liver transplantation operation: decompensation period due to NASHThere are also increasing cases of end-stage liver diseases such as cirrhosis and hepatocellular carcinoma that require liver transplantation. However, NAFLD recurrence rate after liver transplantation is as high as 50% and there is a high risk of cardiovascular complications.

In addition, the present scholars have proposed a scheme for treating NAFLD by using the combination of silybin and lovastatin ^[19,20] Research shows that the combination of the two can improve clinical symptoms, liver functions, blood fat, ultrasonic images and other symptoms of NAFLD patients. However, the side effects of the statin component in this regimen are high, including neuromuscular, urinary, cutaneous and digestive systems, and are most commonly seen as myalgia (muscle pain or weakness, without increased creatine kinase), myositis (with muscle symptoms, and increased creatine kinase) and rhabdomyolysis, which has severe muscle pain, muscle necrosis and myoglobin urine, leading to renal failure and death, especially in asia populations.

6. Meaning of research

In view of the recent decades, the prevalence of NAFLD is increasing year by year. As mentioned above, there is currently no clear treatment other than reasonable exercise and lifestyle changes. With the rapid increase in the incidence of NAFLD, new and effective preventive and therapeutic strategies are necessary.

Disclosure of Invention

The invention provides a novel way for preventing or treating nonalcoholic fatty liver disease (NAFLD) through the combined administration of berberine and matrine.

The inventor discovers that the combination of berberine and matrine can obviously reduce key indexes of NAFLD based on experiments of molecular biology, animal models induced by high-fat feed and the like: triglyceride levels in the liver, oxidative stress and chronic inflammation are superior to those of each component administered alone at high doses. In addition, the composition can also reduce blood lipid level and improve liver index. Therefore, the invention observes that the berberine and matrine are combined for administration, has the prevention and treatment effects on NAFLD (including simple fatty liver SFL and non-alcoholic fatty liver disease NASH) of the non-alcoholic fatty liver disease, and provides experimental data support for the development of novel NAFLD therapeutic drugs, thereby forming the invention.

The mechanism of the present invention is presumed as follows, but the present invention is not limited by the theory. Berberine can reduce the level of leptin and thus reduce the expression level of CD14, while CD14 can promote bacterial endotoxin Lipopolysaccharide (LPS) to enter liver, LPS is one of the important causes of nonalcoholic fatty liver disease NAFLD. The inventor discovers for the first time that matrine can reduce the generation of intestinal LPS, thereby further enhancing the treatment effect of berberine. That is, when berberine and matrine are used in combination, the target points of action are different, but they promote each other, thereby exerting a synergistic effect.

An object of the present invention is to provide a pharmaceutical composition comprising berberine-based compounds and matrine-based compounds as active ingredients.

It is another object of the present invention to provide a pharmaceutical composition comprising therapeutically effective amounts of berberine compounds and matrine compounds, and pharmaceutically acceptable excipients.

In one embodiment, in the above pharmaceutical composition, the berberine compound is berberine or a derivative thereof, such as berberine hydrochloride, berberine sulfate, berberine citrate, berberine phthalate or alcohol type berberine, preferably the berberine compound is berberine, berberine hydrochloride or berberine sulfate.

In one embodiment, in the above pharmaceutical composition, the matrine compound is matrine or its derivative, such as oxymatrine, matrine organic acid salt, matrine double salt (18 α -matrine glycyrrhizate, matrine salvianolic acid B double salt, silybin matrine double salt, silybin two-tablet succinic acid matrine double salt), 13 α -methoxy matrine, 13 α -ethoxy matrine, 13 α -methylaminomatrine, 13 α -ethylamino matrine, 13, 14-dihydroxymatrine, sophocarpine, etc.

In one embodiment, the pharmaceutical composition is capable of providing a ratio of berberine dose in terms of berberine to matrine dose in terms of matrine of 1:4-1:0.25, preferably 1:2-1:0.5, more preferably 1:0.5.

In one embodiment, the pharmaceutical composition is capable of providing a dosage of berberine of 5-30 mg/kg/day, preferably 10-20 mg/kg/day, more preferably 10 mg/kg/day, calculated as berberine; and a matrine dose of 2-15 mg/kg/day, preferably 2.5-7.5 mg/kg/day, more preferably 5 mg/kg/day, calculated as matrine. It is noted that the dose is a dose for humans. When administered to other animals, corresponding dose transitions may be performed, e.g., typically the human dose is one tenth of the mouse dose. This can be routinely determined by those skilled in the art.

In one embodiment, the pharmaceutical composition is for use in the treatment or prevention of non-alcoholic fatty liver disease.

It is still another object of the present invention to provide the use of berberine compounds and matrine compounds in the manufacture of a medicament for the treatment or prevention of non-alcoholic fatty liver disease.

In one embodiment, in the above use, the berberine compound is berberine or a derivative thereof, such as berberine hydrochloride, berberine sulfate, berberine citrate, berberine phthalate or alcohol type berberine, preferably the berberine compound is berberine, berberine hydrochloride or berberine sulfate; and/or the matrine compound is matrine or its derivative, such as oxymatrine, matrine organic acid salt, matrine double salt (18 alpha-matrine glycyrrhizate, matrine salvianolic acid B double salt, silybin matrine double salt, silybin two-tablet succinic acid matrine double salt), 13 alpha-methoxy matrine, 13 alpha-ethoxy matrine, 13 alpha-methylamino matrine, 13 alpha-ethylamino matrine, 13, 14-dihydroxymatrine, sophocarpine, etc.

In one embodiment, in the above use, the pharmaceutical composition is capable of providing a ratio of berberine dose in terms of berberine to matrine dose in terms of matrine of 1:4-1:0.25, preferably 1:2-1:0.5, more preferably 1:0.5;

it is preferred that the composition of the present invention,

the pharmaceutical composition can provide berberine dosage of 5-30 mg/kg/day, preferably 10-20 mg/kg/day, more preferably 10 mg/kg/day of berberine in terms of human dosage; and matrine doses of 2-15 mg/kg/day, preferably 2.5-7.5 mg/kg/day, more preferably 5 mg/kg/day, of matrine calculated as a human dose.

It is a further object of the present invention to provide a method of treating or preventing non-alcoholic fatty liver disease, the method comprising administering to a subject a therapeutically effective amount of a berberine compound and a matrine compound, e.g. berberine and matrine.

Advantageous effects

The invention provides a novel way for preventing or treating nonalcoholic fatty liver disease (NAFLD) through the combined administration of berberine and matrine. The combination of berberine and matrine can obviously reduce key indexes of NAFLD: triglyceride content in liver, oxidative stress and chronic inflammation, and can also reduce blood lipid level and improve liver index. The effect is better than the effect of each component when being applied independently, and the synergistic effect between the two components is proved.

Drawings

Fig. 1: liver index in experimental animals after different treatments in the prophylaxis group.

Fig. 2: liver triglyceride content in experimental animals after different treatments in the prophylaxis group.

Fig. 3: plasma cholesterol levels in experimental animals after different treatments in the prophylaxis group.

Fig. 4: plasma low density lipoprotein levels in experimental animals after different treatments in the prophylaxis group.

Fig. 5: liver tumor necrosis factor alpha gene expression levels in experimental animals after various treatments in the prophylaxis group.

Fig. 6: liver interleukin 6 gene expression levels in experimental animals after different treatments in the prophylaxis group.

Fig. 7: liver superoxide dismutase activity in experimental animals after different treatments in the prophylaxis group.

Fig. 8: liver index in experimental animals after different treatments in the treatment group.

Fig. 9: liver triglyceride content in experimental animals after different treatments in the treatment group.

Fig. 10: plasma cholesterol levels in experimental animals after different treatments in the treatment group.

Fig. 11: plasma low density lipoprotein levels in experimental animals after different treatments in the treatment group.

Detailed Description

Nonalcoholic fatty liver disease (NAFLD) refers to a clinical pathological syndrome characterized mainly by excessive deposition of intracellular fat in liver cells, except for alcohol and other clear liver-damaging factors, and is classified according to the degree of pathological changes and whether pathological liver tissue is accompanied with inflammatory reaction and fibrosis, and can be classified as: fatty Liver (SFL), nonalcoholic steatohepatitis (NASH), NASH-related liver fibrosis and liver cirrhosis. The main feature of NAFLD is the abnormal accumulation of triglycerides in the liver.

The invention discovers that the key indexes of NAFLD can be obviously reduced by the combined administration of berberine compounds represented by berberine and matrine compounds represented by matrine: triglyceride content in liver tissue, oxidative stress and chronic inflammation.

Specifically, the invention establishes NAFLD model by feeding high-fat diet feed (H10141, fukang Biotechnology Co., ltd.) to the golden-yellow mice in Syrian. The oral administration mode is adopted, the prevention and the administration are simultaneously carried out, the continuous administration is carried out for 12 weeks, and the continuous administration is carried out for 10 weeks after the treatment and the formation are completed. During the experiment, the body weight and food intake of the animals were recorded weekly; after the administration, the animals were sacrificed by intraperitoneal injection of 1ml of 20% chloral hydrate for anesthesia after the eyeball was collected and the liver was collected for preservation. The blood biochemical index is measured by an automatic biochemical analyzer, the triglyceride and SOD level in the liver is measured by a kit, and the expression level of inflammatory factors such as tumor necrosis factor alpha, interleukin 6 and the like in liver tissues is measured on the mRNA level.

Experimental results show that the berberine and matrine combined administration group has the effects of reducing liver index and triglyceride in liver, and the effects are superior to those of the single administration group. The combined medicine has the effect of reducing the gene level of the liver tumor necrosis factor alpha and the interleukin 6 better than that of a single group, and can increase superoxide dismutase and play an antioxidant role by removing superoxide anion free radicals.

Therefore, the berberine compound and the matrine compound have synergistic effect and can effectively treat nonalcoholic fatty liver disease (NAFLD).

The present invention will be described in detail below.

1. Berberine compounds

In the present invention, the berberine compound comprises berberine or its derivative.

Berberine is berberine, belongs to isoquinoline alkaloid compound, and has molecular formula of C ₂₀ H ₁₈ NO ₄ Molecular weight 336.367; the structural formula is as follows:

the berberine derivative refers to a compound capable of being metabolized into berberine and acting in vivo, and includes various acid salts of berberine and various derivatives obtained by modifying berberine. Acid salts such as berberine hydrochloride, berberine sulfate, berberine citrate, berberine phthalate, and the like. The berberine modified product comprises alcohol type berberine, 13-n-octyl berberine, mangiferin berberine salt, 9-O-aryl substituted berberine, 13-hexyl berberine salt, etc.

The berberine compound of the present invention is preferably berberine, berberine hydrochloride or berberine sulfate, more preferably berberine hydrochloride, from the viewpoint of availability.

2. Matrine compound

In the present invention, matrine-based compounds include matrine or its derivatives.

Matrine has molecular formula of C ₁₅ H ₂₄ N ₂ O has a molecular weight of 248.36 and a structural formula:

matrine derivatives refer to compounds which are metabolized to matrine and act in vivo and include various derivatives obtained by modifying matrine, such as oxymatrine, matrine organic acid salt, matrine double salt (18 alpha-matrine glycyrrhizate, matrine salvianolic acid B double salt, silybin matrine double salt, silybin two-piece succinic acid matrine double salt), 13 alpha-methoxy matrine, 13 alpha-ethoxy matrine, 13 alpha-methylamino matrine, 13 alpha-ethylamino matrine, 13, 14-dihydroxymatrine, sophocarpine and the like.

The matrine compound of the present invention is preferably matrine from the viewpoint of availability.

3. Pharmaceutical composition

The pharmaceutical composition for preventing or treating the nonalcoholic fatty liver disease comprises the berberine compounds and the matrine compounds, preferably berberine and matrine.

The present invention comprises a pharmaceutically acceptable excipient.

The dosage form of the pharmaceutical composition of the present invention may be a liquid dosage form, a solid dosage form or a semi-solid dosage form. The liquid dosage forms can be solutions (including true solutions and colloidal solutions), emulsions (including o/w type, w/o type and multiple emulsions), suspensions, injections (including water injections, powder injections and infusion solutions), and the like; the solid dosage forms can be tablets (including common tablets, enteric coated tablets, buccal tablets, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets), capsules (including hard capsules, soft capsules and enteric coated capsules), granules, powder, micropills, dripping pills, suppositories, films, patches, aerosol (powder) and sprays; the semisolid dosage form may be an ointment, gel, paste, or the like.

The dosage forms can be prepared into common preparations, slow-release preparations, controlled-release preparations, targeted preparations and various particle administration systems, such as liposome preparations.

The pharmaceutical compositions of the present invention may be prepared according to methods well known in the art. Any dosage form suitable for human or animal use can be made by combining berberine compounds and matrine compounds (preferably berberine and matrine) with one or more pharmaceutically acceptable solid or liquid excipients. The berberine compounds and matrine compounds or the pharmaceutical composition of the present invention containing them may be administered in unit dosage form by intestinal or parenteral route such as oral, intravenous, intramuscular, subcutaneous, nasal, oral mucosa, eye, lung and respiratory tract, skin, vagina, rectum etc.

For example, in order to make the berberine-based compound, matrine-based compound (preferably berberine and matrine) into a tablet, various excipients known in the art, including diluents, binders, wetting agents, disintegrants, lubricants, cosolvents, may be widely used. The diluent can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; the wetting agent can be water, ethanol, isopropanol, etc.; the binder may be starch slurry, dextrin, syrup, mel, glucose solution, microcrystalline cellulose, acacia slurry, gelatin slurry, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methylcellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, etc.; the disintegrating agent can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinylpyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitol fatty acid ester, sodium dodecyl sulfonate, etc.; the lubricant and cosolvent can be talcum powder, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc.

The tablets may be further formulated into coated tablets, such as sugar coated tablets, film coated tablets, enteric coated tablets, or bilayer and multilayer tablets.

For preparing into capsule, berberine compounds and matrine compounds (preferably berberine and matrine) can be mixed with diluent and cosolvent, and the mixture can be directly placed into hard capsule or soft capsule. Or mixing the effective components with diluent, binder, and disintegrating agent, granulating or micropill, and making into hard capsule or soft capsule. The various diluents, binders, wetting agents, disintegrants and co-solvents used in the preparation of the tablets of the invention may also be used in the preparation of capsules of the compounds of the invention.

For the preparation of the pharmaceutical composition into injection, water, ethanol, isopropanol, propylene glycol or their mixture can be used as solvent, and appropriate amount of solubilizer, cosolvent, pH regulator, and osmotic pressure regulator commonly used in the art can be added. The solubilizer or cosolvent can be poloxamer, lecithin, hydroxypropyl-beta-cyclodextrin, etc.; the pH regulator can be phosphate, acetate, hydrochloric acid, sodium hydroxide, etc.; the osmotic pressure regulator can be sodium chloride, mannitol, glucose, phosphate, acetate, etc. For example, mannitol, glucose, etc. can be added as propping agent for preparing lyophilized powder for injection.

In addition, colorants, preservatives, fragrances, flavoring agents, or other additives may also be added to the pharmaceutical formulation, if desired.

The amount of the pharmaceutical composition of the present invention to be administered may vary widely depending on the nature and severity of the disease to be prevented or treated, the individual condition of the patient or animal, the route of administration and the dosage form, etc. Generally, for administration to a human subject, the dosage of berberine compound is 5-30 mg/kg/day, preferably 10-20 mg/kg/day, more preferably 10 mg/kg/day; the dosage of matrine compound is 2-15 mg/kg/day, preferably 2.5-7.5 mg/kg/day, more preferably 5 mg/kg/day. In a preferred embodiment, the ratio (weight ratio) of the dosage of berberine compound to the dosage of matrine compound upon co-administration is 1:4-1:0.25, preferably 1:2-1:0.5, more preferably 1:0.5. It is noted that, in this context, the dosage of the berberine compound is calculated as berberine and the dosage of the matrine compound is calculated as matrine.

The berberine compound and matrine compound may be administered simultaneously, separately or sequentially. For example, the berberine compound and the matrine compound may be administered simultaneously, or the berberine compound may be administered first, followed by the matrine compound within 3 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 1 hour, or 2 hours after administration, or vice versa.

Furthermore, the above-described dosages may be administered in one dosage unit or in several dosage units, depending on the clinical experience of the physician and the dosage regimen involved in the application of other therapeutic means.

Furthermore, the pharmaceutical composition of the present invention may contain other anti-NAFLD drugs, for example, vitamin E, obeticholic acid, bicyclic alcohol, polyene phosphatidylcholine, glycyrrhizic acid diamine, reduced glutathione, S-adenosylmethionine, ursodeoxycholic acid, etc., as long as they do not impair the combined action of berberine compound and matrine compound.

When the compound of the present invention is present in the pharmaceutical composition of the present invention together with other anti-NAFLD drugs, the dosage thereof should be adjusted according to the actual circumstances.

4. Method of prevention or treatment

In one embodiment, the invention relates to a method of preventing or treating non-alcoholic fatty liver disease (NAFLD) comprising administering to a subject a therapeutically effective amount of a berberine compound and a matrine compound.

As a prophylactic method, the subject is a subject susceptible to non-alcoholic fatty liver disease. Such a subject has, for example, the following features: high working pressure, excessive sleep, reduced exercise, high fat and high protein diet, frying in preferred oil, pungency, sweet food, cold drink, snack before sleep, etc., and has increased body mass index and waist-hip ratio, obesity, hyperlipidemia, hypertension, diabetes, coronary heart disease, genetic and family history.

As a method of treatment, the subject is a subject suffering from non-alcoholic fatty liver disease (NAFLD), for example, a patient suffering from Simple Fatty Liver (SFL), non-alcoholic steatohepatitis (NASH), or NASH-associated cirrhosis. In addition, the subject may be particularly a NAFLD patient intolerant to statins. The present invention provides an effective alternative treatment strategy for these intolerant patients.

Examples

The invention is illustrated below by means of exemplary embodiments.

Example 1

1. Experimental methods and materials

1.1 experimental animals: LVG syrian golden mice, male, 4 week old, weighing 70-90g, were acclimatized in a feeding environment for 2 weeks. Animals were purchased from beijing vernalia laboratory animal technologies limited.

1.2 experimental feed: basal diet (Normal chow diet, blank); high fat diet (model group: 1.0% cholesterol, 0.2% sodium cholate, 10.0% lard, 5.0% egg yolk powder and 83.8% basal diet). Feed was purchased from beijing o synergetic feed limited.

1.3 modes of administration: 1) Preventive group: the medicine is mixed in the high-fat feed and is continuously administered for 12 weeks. 2) Treatment group: the high-fat feed is administered after 8 weeks, and is administered continuously for 10 weeks by adopting a gastric lavage administration mode. Berberine and matrine were purchased from Nanjing sun's biological technology Co.

1.4 experimental grouping and dosing:

1.5 detection index and method

1.5.1 animal treatment: after the end of the administration, 1ml of 20% chloral hydrate was given to each animal for intraperitoneal injection anesthesia to sacrifice, the whole liver was taken, rinsed in physiological saline, the blood was removed, the filter paper was wiped dry, and weighed. The liver was stored at-80℃for subsequent index detection.

1.5.2 liver lipid content and oxidative stress detection: detection of triglycerides in liver: thawing frozen liver tissue at-80deg.C, weighing about 20mg, precisely weighing, adding 9 times of absolute ethanol, homogenizing at low temperature, centrifuging at 5000rpm/min for 10min, and collecting supernatant. Detection of superoxide dismutase in liver: thawing frozen liver tissue at-80deg.C, weighing about 20mg, precisely weighing, adding 9 times of physiological saline, homogenizing at low temperature, centrifuging at 3000rpm/min for 10min, and collecting supernatant. Detection of malondialdehyde in liver: draw 0.3mL of reagent one into a 1.5mL centrifuge tube, add 0.1mL of sample, mix well. Heat-insulating in 95 deg.c water bath for 30min (covering tightly to prevent water loss), cooling in ice bath at 10000rpm/min, centrifuging at 25 deg.c for 10min. 200 μl of supernatant was aspirated into 96-well plates and assayed according to kit instructions. The kit is purchased from Nanjing to build the bioengineering institute.

1.5.3 serological index: animals were bled by orbital extraction and fasted overnight prior to blood collection. The content of cholesterol CHO (mmol/L) and low density lipoprotein LDL-C (mmol/L) in serum was measured by a full-automatic biochemical analyzer. The detection kit was purchased from north control biotechnology limited.

1.5.4 inflammatory factor: RNA extraction: thawing tissue preserved at-80deg.C on ice, weighing about 50mg of tissue into 1ml of RNA extraction solution, crushing with a low-temperature tissue crusher, and extracting total RNA in the tissue with an RNA extraction kit, wherein the method comprises the following steps: 1) Adding 0.2ml chloroform into each tube, covering the tube cover, shaking up and down with force for 15s, standing at room temperature for 5min, centrifuging at 4deg.C for 15min at 12000g, and dividing the sample into three layers; 2) Carefully transferring the upper liquid into another new 1.5ml tube, adding 70% ethanol at equal ratio, mixing, transferring to a separation column with a collecting tube, and centrifuging at 12000g at room temperature for 15s; 3) Discarding the liquid in the collection tube, re-inserting the separation column into the recovery header, adding 700. Mu.L of wash buffer I to the separation column, and centrifuging at 12000g at room temperature for 15s; 4) Discarding the collection tube, inserting the separation column into another new collection tube, adding 500. Mu.L of wash buffer II to the separation column, and centrifuging at 12000g at room temperature for 15s; 5) The same procedure was repeated with wash buffer II; 6) Discarding the liquid in the collecting pipe, re-inserting the separation column into the collecting pipe, centrifuging 12000g at room temperature for 15s to dry the column core; 7) Discarding the collecting tube, inserting the column into 1.5ml recovery tube, adding 30-100 μl RNase-free water to the center of the separation column, incubating for 1min at room temperature, centrifuging for 2min at 12000g at room temperature, discarding the separation column, and collecting the total RNA in the recovery tube. Amplification and detection of genes: the target gene amplification is carried out by using a PCR one-step method kit, and the expression of the target gene is detected by using an ABI 7500Fast real-time fluorescent quantitative PCR instrument. The kits used were all purchased from Thermo company, usa.

1.6 statistics: * For statistical significance compared to the model group, P <0.05, P < 0.01, P < 0.001.

2. Preventive group test results

2.1 liver index

The results of the liver index are shown in fig. 1 and table 1.

TABLE 1

	Blank group	Model group	Berberine group	Matrine group	Berberine matrine group
						Average (%)	4.01	6.30	5.82	5.98	5.51
Standard deviation of	0.24	0.54	0.44	0.48	0.20

Liver index refers to the ratio of wet liver weight to body weight. Compared with the blank group, the liver index of the animal with the high-fat diet model is obviously increased; compared with the model group, berberine and matrine have the tendency of reducing liver index, and the effect of the combined administration group on reducing liver index is better than that of the single administration group, and the ratio of the berberine and matrine to the model group is obviously different (P is less than 0.001).

2.2 liver lipid content

The liver lipid content was expressed as liver triglyceride, and the results are shown in fig. 2 and table 2.

TABLE 2

	Blank group	Model group	Berberine group	Matrine group	Berberine matrine group
						Average value (mmol/g)	9.92	25.51	26.15	27.02	21.26
Standard deviation of	1.54	4.69	3.29	5.20	3.65

Triglyceride in liver of animal with high fat diet model is obviously increased compared with blank group; the berberine and matrine are administered alone without reduction of liver triglyceride, and the combination of the berberine and matrine can significantly reduce liver triglyceride content (< 0.05).

2.3 Total cholesterol, low Density lipoproteins in plasma

The results of the total cholesterol levels in plasma are shown in fig. 3 and table 3.

TABLE 3 Table 3

	Blank group	Model group	Berberine group	Matrine group	Berberine matrine group
						Average value (mmol/L)	3.50	22.30	16.94	14.21	11.80
Standard deviation of	0.25	6.76	4.57	3.29	1.52

The total cholesterol content of the plasma of the animal with the high-fat diet model is obviously increased compared with that of the animal with the blank group; both berberine and matrine have effects of reducing plasma cholesterol levels, but the effect of reducing plasma cholesterol when berberine is combined with matrine is significantly better than that of each single group (< 0.001).

The plasma low density lipoprotein levels are shown in fig. 4 and table 4.

TABLE 4 Table 4

	Blank group	Model group	Berberine group	Matrine group	Berberine matrine group
						Average value (mmol/L)	0.70	11.97	8.15	6.76	4.56
Standard deviation of	0.13	2.94	2.41	2.04	1.27

The plasma low-density lipoprotein of the animal with the high-fat diet model is obviously increased compared with that of a blank group; although berberine and matrine both have the effect of reducing plasma low density lipoprotein, the combined group of berberine and matrine has the effect of reducing plasma low density lipoprotein obviously better than each single group.

2.4 inflammatory factors

2.4.1 liver tumor necrosis factor alpha Gene expression level

The expression level of the hepatic tumor necrosis factor alpha gene is shown in fig. 5 and table 5.

TABLE 5

	Blank group	Model group	Berberine group	Matrine group	Berberine matrine group
						Average value of	0.79	1.00	0.51	0.17	0.11
Standard deviation of	0.46	1.22	0.35	0.07	0.04

The level of the tumor necrosis factor alpha in the liver of the high-fat diet animal is slightly increased compared with that of a blank group; the berberine and matrine have the effect of reducing the level of the tumor necrosis factor alpha, but the effect of reducing the level of the liver tumor necrosis factor alpha gene is better than that of a single group when the berberine and the matrine are combined.

2.4.2 liver interleukin 6 Gene expression level

The results of the liver interleukin 6 gene expression amount are shown in fig. 6 and table 6.

TABLE 6

	Blank group	Model group	Berberine group	Matrine group	Berberine matrine group
						Average value of	0.85	1.00	0.91	0.62	0.50
Standard deviation of	0.48	0.59	0.46	0.22	0.15

The interleukin 6 level in the liver of the high-fat diet animals is slightly increased compared with that in the blank group. The single use group has the tendency of reducing the level of interleukin 6, and when the two are combined, the expression of interleukin 6 genes in the liver can be obviously reduced (P is less than 0.05), and the action intensity is better than that of the single use group.

2.5 liver oxidative stress

Liver oxidative stress is expressed in terms of liver superoxide dismutase activity, the results of which are shown in fig. 7 and table 7.

TABLE 7

	Blank group	Model group	Berberine group	Matrine group	Berberine matrine group
						Average value (U/g)	52.56	37.65	25.82	37.09	42.87
Standard deviation of	6.73	10.15	10.37	8.08	10.60

Superoxide dismutase can play an antioxidant role by scavenging superoxide anion radicals. After high-fat diet, the activity of superoxide dismutase in the liver of experimental animals is obviously reduced compared with that of a blank group, and berberine and matrine are not used for resisting oxidation when being singly administered, but the activity of superoxide dismutase can be obviously increased when the berberine and the matrine are combined, and the antioxidant effect is obviously better than that of each single group.

3. Experimental results in treatment group

3.1 liver index

The results of the liver index are shown in fig. 8 and table 8.

TABLE 8

	Blank group	Model group	Berberine group	Matrine group	Berberine matrine group
						Average (%)	3.37	6.51	5.87	6.63	5.68
Standard deviation of	0.22	0.44	0.38	0.37	0.46

Compared with the model group, berberine has the effect of reducing liver index, and matrine has no effect of reducing liver index; and the effect of the combined administration of the two drugs on reducing liver index is superior to that of the single administration.

3.2 liver lipid content

The liver lipid content is expressed as liver triglycerides, and the results are shown in fig. 9 and table 9.

TABLE 9

	Blank group	Model group	Berberine group	Matrine group	Berberine matrine group
						Average value (mmol/g)	9.61	21.54	20.10	22.38	18.65
Standard deviation of	6.02	5.16	4.23	6.13	3.77

Triglyceride in liver of animals of high fat diet model was significantly elevated compared to the blank group; the berberine and matrine have no effect of reducing triglyceride in liver when administered alone, and the reduction of triglyceride content in liver can be observed when the berberine and matrine are combined, and the action intensity is superior to that of the single group.

3.3 Total cholesterol, low Density lipoprotein levels in plasma

The results of the total cholesterol levels in plasma are shown in fig. 10 and table 10.

Table 10

	Blank group	Model group	Berberine group	Matrine group	Berberine matrine group
						Average value (mmol/L)	5.51	17.63	11.54	13.56	10.05
Standard deviation of	1.09	5.70	0.87	3.36	1.39

The total cholesterol content of the plasma of the animal with the high-fat diet model is obviously increased compared with that of the animal with the blank group; the berberine and matrine have certain effect of reducing the cholesterol content of blood plasma, but the effect of the berberine and matrine is obviously better than that of a single group when the berberine and the matrine are used together.

The plasma low density lipoprotein levels are shown in fig. 11 and table 11.

TABLE 11

	Blank group	Model group	Berberine group	Matrine group	Berberine matrine group
						Average value (mmol/L)	2.02	6.06	4.34	4.71	3.83
Standard deviation of	0.49	2.07	2.37	1.07	1.26

The plasma low-density lipoprotein of the animal with the high-fat diet model is obviously increased compared with that of a blank group; although berberine and matrine both have the effect of reducing plasma low density lipoprotein, the effect of reducing plasma low density lipoprotein is superior to that of each single group when the berberine and matrine are used together.

From the above results, it can be obtained that the pharmaceutical composition of the present invention can significantly reduce key indexes of NAFLD: the triglyceride content, oxidative stress and chronic inflammation in liver tissue are derived from the drug combination and are superior to those of high-dose drugs singly. The medicine composition can also reduce the blood lipid level and improve the liver index.

Industrial applicability

The invention provides a novel way for preventing or treating nonalcoholic fatty liver disease (NAFLD) through the combined administration of berberine and matrine. The combination of berberine and matrine can obviously reduce key indexes of NAFLD: triglyceride content in liver; oxidative stress; chronic inflammation, and can also reduce blood lipid level and improve liver index. The effect is better than the effect of each component when being applied independently, and the synergistic effect between the two components is proved. The invention can also effectively treat statin intolerant non-alcoholic fatty liver disease patients, and provide effective drug replacement treatment strategies for the intolerant patients. In addition, berberine and matrine can be extracted from plants, and has good safety.

Reference (e.g. patent/paper/standard)

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Claims (1)

1. Use of a pharmaceutical composition of berberine and matrine for the manufacture of a medicament for the treatment or prevention of non-alcoholic fatty liver disease, characterized in that the ratio of berberine dose to matrine dose of the pharmaceutical composition is 1:0.5.