KR20120125754A - Pharmaceutical composition comprising melittin for treatment of liver disease - Google Patents

Abstract

PURPOSE: A pharmaceutical composition containing melitin ingredients is provided to treat liver diseases. CONSTITUTION: A pharmaceutical composition for treating liver diseases contains 0.001-0.05 wt% of melittin as an active ingredient. Mellitin is isolated from honey bee-derived bee venom. The composition additionally contains pharmaceutically acceptable carrier. Mellitin reduces inflammation-inducing cytokine expression in a liver tissue. The inflammation-inducing cytokine is TNF-alpha, IL-1beta, IL-6, or VCAM. [Reference numerals] (AA) Tumor necrosis factor(TNF-α); (BB) NC control group; (CC) TAA experimental group of liver cirrhosis; (DD) TAA + Mel experimental group of liver cirrhosis + melittin administration

Description

Pharmaceutical composition for the treatment of liver disease containing a melittin component {PHARMACEUTICAL COMPOSITION COMPRISING MELITTIN FOR TREATMENT OF LIVER DISEASE}

The present invention relates to a pharmaceutical composition for treating liver disease comprising a melittin component, and in particular, a melittin isolated from bee venom extracted from bees has a therapeutic effect for treating liver disease, which has the effect of treating liver diseases such as cirrhosis and acute liver damage. It relates to a composition.

Chronic liver disease is an important cause of adult disease and death in Korea. It is important to study the etiology, prevention and treatment of this disease.

In general, the most representative liver diseases include hepatitis, fatty liver, cirrhosis, and liver cancer. Such liver diseases include virus infection, cholestasis, accumulation of iron / Fe, and galactosemia. (Galactosemia) and are known to be due to metabolic diseases such as Tyrosinaemia.

Hepatic stellate cells are activated by many free radicals released from cooper cells corresponding to hepatic macrophages to induce and proliferate hepatic stellate cells with the release of cytokines. As protein synthesis is accelerated, collagen accumulation progresses and eventually the concentration of collagen that causes hardening in liver tissue is increased, leading to liver fibrosis, but the exact molecular mechanism is still unknown. not.

Bee venom has been used as a natural remedy since ancient times, especially for various inflammatory and painful diseases.

Bee venom is a complex composed of about 40 different components of bee venom, consisting of enzymes, peptides, non peptides, and physiologically active amines. have.

The ingredients and efficacy of such bee venom are known as follows.

① Treatment of diseases of the immune system: Bee venom is effective in the treatment of most immune diseases, which enables the bee venom to stimulate the mammal's immune system to fight disease successfully. The two main immune actions of bee venom are to stimulate the organism's biological system and, in the following order, to increase the body's defenses.

② anti-inflammatory action: The main components of bee venom are very effective in inhibiting inflammation. Protease inhibitors and adolamine treatment also inhibited 15 to 40% of edema.

③ neurotoxic effect: stinging bees cause pain and inflammation, and the substances causing them are effectively used to develop avoidance reactions, or painkillers, in stray animals. In particular, it is analgesic by the action of apamin in bee venom, which is strong in various neuralgia, arthritis and rheumatoid arthritis, gout, myalgia, and high therapeutic effect.

④ hemolytic effect: One of the most excellent effect of bee sting is hemolysis. Absorbs and excretes blood, such as bruises and internal bleeding, and supplies new blood, oxygen, and nutrients to the treatment and hemolysis.

⑤ vasodilation effect: the action of histamine substances capillary, small arteries, predetermined veins, especially visceral blood vessels are effective in expanding. Therefore, it treats diseases such as coldness, frostbite, myalgia.

⑥ blood pressure lowering action: by the action of histamine substances to reduce the blood pressure is a powerful action. In particular, there is a blood pressure lowering action at a concentration of 250 million 1, has an excellent effect in the treatment of essential hypertension and the like.

⑦ autonomic nervous system action: If the human body continues to stress, sympathetic nerves and parasympathetic nerves of the autonomic nerves come to this cause of many diseases. The catecholamines and acetylcholine, which are necessary to normalize the autonomic nerve, are contained in bee venom, and since it is a brain cell transporter, it is effective in treating psychotic menopausal stress disorder.

⑧ antibacterial effect: antibacterial and antifungal effect is very good and effective in viral tumors. In particular, it has a high therapeutic effect on periodontitis, tonsillitis, stye, purulent diseases.

As described above, the anti-inflammatory and antimicrobial effects of bee venom, and immunity enhancement are reported, but there are no known bee venom-derived components related to liver disease treatment.

An object of the present invention is to provide a pharmaceutical composition for treating liver disease comprising a melittin component having an effect of treating liver diseases such as cirrhosis and acute liver damage among various components isolated from bee venom extracted from bees.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are not intended to limit the invention to the particular embodiments that are described. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are not restrictive of the invention, There will be.

Pharmaceutical composition for treating liver disease of the present invention for achieving the above object includes melittin (Melittin) as an active ingredient.

The melittin is characterized in that it is isolated from bee-derived bee venom.

The pharmaceutical composition for treating liver disease of the present invention is characterized by containing 0.001 to 0.05% by weight of melittin.

In addition, the pharmaceutical composition for treating liver disease may further contain a pharmaceutically acceptable carrier.

The melittin reduces the expression of inflammation-induced cytokines (cytokine) in liver tissue, in particular, tumor necrosis factor (TNF-α) or interleukin-1β (IL-1β) or interleukin 6 (IL-6) or vascular cell adhesion Decreases the expression of one selected from the molecule (VCAM).

In addition, the melittin reduces the expression of liver fibrosis-inducing cytokine (Cytokine), in particular, the expression of one selected from the transforming growth factor (TGF-β1) or α- smooth muscle actin (α-SMA) or fibronectin (Fibronectin) Decreases.

In addition, the melittin also inhibits the activity of caspase-3, a cell death protein of acute liver injury.

As described above, the present invention provides a composition for treating liver disease, which contains a melittin component that exhibits an excellent effect on treating liver disease.

1 is a view showing the liver tissue state of the experimental animal group administered melittin.
Figure 2a is a graph showing the expression level of tumor necrosis factor (TNF-α) contained in the serum of the experimental animal group administered melittin.
Figure 2b is a graph showing the expression level of interleukin-1β (IL-1β) contained in the serum of the experimental animal group administered melittin.
Figure 3a is a diagram showing the expression level of inflammation-inducing factors in the liver of the experimental animal group administered melittin.
Figure 3b is a graph showing the expression level of inflammation-inducing factors in the liver of the experimental animal group administered melittin.
Figure 3c is a diagram showing the expression level of inflammation-inducing factors in the liver tissue of the experimental animal group administered melittin.
Figure 4a is a diagram showing the expression level of hepatic fibrosis inducing factor of the experimental animal group administered melittin.
Figure 4b is a graph showing the expression level of hepatic fibrosis inducing factor of the experimental animal group administered melittin.
Figure 4c is a diagram showing the expression level of hepatic fibrosis inducing factor in liver tissue of the experimental animal group administered melittin.
Figure 5a is a graph showing the survival rate of the acute liver injury experimental animals group administered with melittin.
Figure 5b is a diagram showing the degree of apoptosis of the acute liver injury experimental animals group administered with melittin.
Figure 5c is a diagram showing the effect of reducing apoptosis in acute liver injury experimental animals group administered with melittin.
Figure 6a is a diagram showing the change in the expression of Caspase-3 in liver tissue of acute liver injury experimental animals group administered melittin.
Figure 6b is a diagram showing the change in expression of Cleavage-Caspase-3 in liver tissue of acute liver injury experimental animals group administered melittin.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.

The pharmaceutical composition for treating liver disease according to the present invention includes melittin (Melittin) as an active ingredient, and the melittin is characterized in that it is isolated from bee-derived bee venom.

In particular, melittin is preferably contained 0.001 ~ 0.05% by weight in the pharmaceutical composition for treating liver disease of the present invention.

In this case, when the melittin content is less than 0.001% by weight, the effect on liver disease is inadequate, and when it exceeds 0.05% by weight, problems such as cytotoxicity may occur.

When the melittin is administered to a thioacetamide-induced liver cirrhosis experimental animal model, it can be seen that the expression of inflammation-induced cytokines (Cytokine) in liver tissue is significantly reduced.

In addition, it was confirmed that the effect of reducing the expression of cytokine (Cytokine), a liver fibrosis inducing factor.

These results suggest that melittin acts effectively to reduce inflammatory response in liver tissues, especially tumor necrosis factor (TNF-α) or interleukin-1β (IL-1β) or interleukin 6 (IL-6) or vascular cells. It can be seen that the effect of reducing the expression of one selected from the adhesion molecule (VCAM).

In addition, melittin also plays an effective role in inhibiting the progression of liver fibrosis, and in particular, reduces the expression of one selected from the transforming growth factor (TGF-β1) or α-smooth muscle actin (α-SMA) or fibronectin. It can be seen that the effect of making it.

In addition, melittin is apoptosis protein caspase-3 when administered to lipopolysaccharide (LPS) and D-galactosamine-induced acute liver injury experimental animal model. Activity was inhibited.

As described above, it can be seen that the pharmaceutical composition containing melittin shows a high effect on the treatment of liver disease.

The pharmaceutical composition for treating liver disease may further contain a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier is commonly used in the preparation of lactose, dextrose, sucrose, sorbitol, and mannitol. Starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc , Magnesium stearate, mineral oil and the like, but is not limited thereto.

The pharmaceutical composition for treating liver disease of the present invention may further include a lubricant, a humectant, a sweetener, a flavor, an emulsifier, a suspending agent, a preservative, and the like, in addition to the above components.

The pharmaceutical composition for treating liver disease of the present invention can be administered parenterally, and subcutaneous infusion or topical administration through the skin (transdermal administration) is preferable.

Suitable dosages of the pharmaceutical compositions for the treatment of liver disease of the present invention are determined by factors such as formulation method, mode of administration, patient's age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion and response sensitivity. It can be prescribed in various ways.

On the other hand, the oral dosage of the pharmaceutical composition for treating liver disease of the present invention is preferably 0.1 mg / kg (body weight) per day.

The pharmaceutical composition for treating liver disease of the present invention may be formulated with a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person skilled in the art. It may be made in the form or prepared by incorporation into a multi-dose container.

The formulations here may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of extracts, powders, granules, tablets or capsules, and may further comprise dispersants or stabilizers.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples, but the scope of the present invention is not limited thereto.

Example 1 Isolation of Melitin Component of the Present Invention

Melittin was isolated from bee venom by using bee venom collection device (Chungjin Tech, Korea).

The bee venom was subjected to liquid chromatography using a Superdex-peptide column and a Reverse-Phase column to obtain six reproducible fractions.

The melittin component was then isolated from the bee venom fractions using mass spectrometry (MALDI-TOF, Bruker Daltonics, USA).

<Example 2> Preparation of a pharmaceutical composition for treating liver disease comprising the melittin component of the present invention

300 mg of the composition containing the melittin component of Example 1

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2mg

According to a conventional capsule preparation method, the above components were mixed and filled into gelatin capsules to prepare a pharmaceutical composition for treating liver disease in the capsule form.

<Experimental Example> Confirmation of the therapeutic effect of liver disease of the pharmaceutical composition containing the melatin component of the present invention

1. Experimental material: The melittin isolated in Example 1 was prepared by lyophilization.

2. Subject

(1) Experimental animals

As experimental animals, six-week-old C57BL / 6 mice were purchased from Orient Co., Ltd., and were purified for about one week, followed by observation of general symptoms during the period, and randomly placed healthy animals in each polycarbonate cage for each test group. The experiment was carried out.

During the experimental period, the environmental conditions of breeding were maintained at 22 ± 3 ℃, 50 ± 10% relative humidity, lighting time of 12 hours, and illuminance of 150-300 lux.

Liver cirrhosis model was induced with the most commonly used thioacetamide, and diluted with negative water for 12 weeks.

The acute liver injury experimental animal model induced liver dysfunction by treatment with lipopolysaccharide (LPS; 10 µg / kg) and D-galactosamnine (700 mg / kg).

(2) experimental animal group

Chronic liver injury experimental animals were divided into liver cirrhosis experimental group (TAA), experimental group in which melittin was administered to liver cirrhosis experimental animals (TAA + Mel) and control group (NC). Experimental animals were sacrificed at 12 weeks and used as test subjects. The dose of melittin administered to the TAA experimental group was 0.1 mg / kg and was administered twice a week intraperitoneally for 12 weeks. The blood of the experimental animals was extracted from the heart and the tissue was extracted from the kidney and fixed in 10% neutral formalin solution.

The acute liver injury group was divided into acute liver injury group (GaIN / LPS), acute liver injury group (GaIN / LPS + Mel) and control group (NC) was administered to the experiment.

The GaIN / LPS + Mel experimental group was injected into the abdominal cavity at a dose of 0.1 mg / kg of melittin 30 minutes before inducing acute liver dysfunction. Confirmed.

3. Experimental Method

(1) Method of Observing Cell Morphological Change

Liver tissue was excised and fixed in 10% formalin.

After sufficient fixation, the liver tissue is subjected to a series of tissue treatment processes and embedded in a paraffin embedding center to produce 4 μm thick sections using a microtome, followed by hemotoxiline and eosin ( Hematoxylin & Eosin staining and trichrome staining were used for optical microscopy.

(2) Protein Isolation and Western Blot Analysis

Liver tissues from sacrificed animals were stored at -70 ° C, and the kidneys had IPH lysis buffer (50 mM pH8.0 Tris, 150 mM NaCl, 5 mM EDTA, 0.5% NP40, 100 mM PMSF). , leupeptin 1 mg / ml, aprotinin 1 mg / ml, and 1 M DTT) were added, and the cells were disrupted by a tissue mill, lysed at 4 ° C. for 30 minutes, centrifuged at 12,000 rpm for 30 minutes, and proteins were separated. It was.

The isolated protein was quantified by using a spectrophotometer by the Bradford assay, using the quantitative protein 2 × SDS-loading buffer (100 × pH pH) with DTT added 6.8 Mix with Tris-Cl, glycerol 20%, bromophenol blue 0.2%, SDS 4%, dithiothreitol 200 mM, heat at 98 ° C for 5 min, and electrophoresis with 10% SDS-PAGE gel To the membrane (Immobilon-P-membrane).

Membrane is the primary antibody anti-tumor necrosis factor (anti-TNF-α), anti- interleukin 6 (anti-IL-6), anti-fibronectin, anti-vascular cell adhesion molecule (anti- VCAM), anti-conversion growth factor (anti-TGF-β1), anti-α-smooth muscle actin (anti-α-SMA), anti-Caspase-3 and react with secondary antibodies, respectively. Reacted.

Expression analysis for the antibodies was analyzed using an enhanced chemiluminescence (ECL) Western blot Analysis system expressed by a Horseradish Peroxidase-Linked secondary antibody.

(3) immunohistochemical staining method

Immunohistochemical staining was performed by first cutting the biopsy tissue of paraffin-embedded liver into 4 μm thickness, attaching it to an adhesive-treated slide, deparaffinizing, and hydrated with ethanol and distilled water.

The solution was treated with 3% hydrogen peroxide solution diluted in methanol for 30 minutes to block the reaction to endogenous peroxidase and washed with distilled water.

After the tissue sections are sufficiently submerged in a preheated 95 ° C Dako Epitope Retrieval Solution (0.01 mol / L citrate buffer, pH 6.0), boil for 10 minutes and proceed to epitope retrieval process. After cooling in, washed with PBS.

Anti-tumor necrosis factor (anti-TNF-α), anti-interleukin 6 (anti-IL-6), anti-fibronectin, anti-vascular cell adhesion molecule (anti-VCAM), anti-conversion growth Primary antibodies to factor (anti-TGF-β1), anti-α-smooth muscle actin (anti-α-SMA), anti-Caspase-3 were reacted at 4 ° C. for 16 hours, Reaction with biotinylated anti-goat IgG or anti-mouse IgG for 15 minutes at 37 ℃.

After reacting with streptoavidin peroxidase at 37 ° C. for 15 minutes, color development was performed with DAB (3,3′-diaminobenzidine tetrahydrochloride), and counterstaining with homotoxylin was performed under an optical microscope.

(4) statistical analysis

Statistical analysis of all the experimental results below was performed by variance analysis to verify whether there is a standard difference in each experimental group, and the diversity analysis was analyzed by Duncan's test.

When the p value was less than 0.05 (p <0.05) after statistical processing, it was determined that the statistical significance, and the experimental results are as follows.

4. Experimental results

(1) Confirmation of the efficacy of the present invention melittin in the liver tissue of experimental animals

The efficacy of the present invention melittin in the liver tissue of the experimental animal was confirmed.

As a result, as shown in FIG. 1, Thioacetamide of the TAA experimental group increased hepatocyte death and inflammatory response in liver tissues through Hematoxylin & Eosin staining.

However, Thioacetamide of TAA + Mel experimental group was confirmed to alleviate hepatocyte death and inflammatory response in liver tissue.

In addition, the expression of collagen increased by cirrhosis through trichrome staining showed that collagen increased around the portal vein of TAA experimental group, but it was confirmed that collagen expression was hardly expressed in TAA + Mel experimental group.

In conclusion, it was found that melittin alleviates the inflammatory response and hepatocellular death in the liver tissues of experimental animals and inhibits the expression of collagen around the portal vein.

(2) Confirmation of TNF-α and IL-1β Production

The amount of TNF-α and IL-1β, one of the causes of inflammation, was measured using an ELISA kit.

As a result, as shown in FIGS. 2A and 2B, TNF-α and IL-1β were expressed in a large amount in the TAA experimental group, but the TAN + Mel experimental group significantly reduced the appearance of TNF-α and IL-1β (p). <0.05).

(3) Inhibition of Inflammatory Factors by Melitin

Inflammatory reactions activated by hepatic Cooper cells induced by liver cirrhosis due to cirrhosis are the most important part of the progression of liver fibrosis. This inflammatory response causes hepatic fibrosis through cytokine, an inflammation-inducing factor.

In the following, the expression levels of the cytokines TNF-α, IL-6, and VCAM were identified.

As a result, the expression levels of TNF-α, IL-6, and VCAM were significantly increased in the TAA experimental group as shown in FIGS. 3A and 3B. However, the expression levels of TNF-α, IL-6, and VCAM were increased in the TAA experimental group. Markedly reduced.

In addition, TNF-α, IL-6, and VCAM in liver tissue of each experimental group, as shown in FIG. 3C, significantly reduced the expression of TNF-α, IL-6, and VCAM in the TAA + Mel experimental group compared to the TAA experimental group. It can be seen that.

(4) Inhibition of TGF-β1, α-SAM and Fibronectin Activity by Melitin

The production amount of liver fibrosis-induced cytokines TGF-β1, α-SAM, and fibronectin were confirmed.

First, the amount of TGF-β1 present in the combined state of ECM and produced in liver cells of the liver, activated hepatic stellate cells, isovascular endothelial cells, hepatocytes and platelets, and played an important role in liver fibrosis was confirmed.

Next, interferon was used to show anti-fibrotic effects. In animal models, interferon alpha inhibited the proliferation of astrocytes, reduced the mRNA of type I collagen, and decreased the expression of α-SAM, an activity marker of astrocytes. .

This confirmed whether melittin affects the expression level of α-SAM.

Finally, Fibronectin, which is known to have a function of chemoattractant for cell migration and inflammatory cells, inhibits function by binding collagen to cells and is involved in ECM production and binds to collagenase. Expression level was confirmed.

The results of confirming the expression level of the TGF-β1, α-SAM, Fibronectin are as follows.

As shown in FIGS. 4A and 4B, the expression levels of TGF-β1, α-SAM, and Fibronectin were significantly increased in the TAA experimental group. Decreased (p <0.05).

In addition, as shown in Figure 4c TGF-β1, α-SAM, Fibronectin in the liver tissue of each experimental group is significantly reduced TGF-β1, α-SAM, Fibronectin of the TAA + Mel test group compared to the TAA experimental group (P <0.05).

In conclusion, as a result of the administration of melittin in the experimental group TAA, as in the TAA + Mel experimental group, it was found that the cytokine-related expression levels were reduced.

(5) Efficacy of melittin in acute liver injury experimental animal group

As shown in FIG. 5A, the survival rate of GaIN / LPS experimental group rapidly decreased from 6 hours, and all died at 96 hours.

In particular, the survival rate was very low at 20% at 8 and 12 hours, but the GaIN / LPS + Mel experimental group with melittin showed 80% survival at 8 hours and was maintained for 96 hours.

Analysis of chromosomal DNA is a representative method for confirming apoptosis. As shown in FIG. 5B, DNA fragmentation was clearly observed in the GaIN / LPS experimental group, but DNA fragmentation was observed in the GaIN / LPS + Mel experimental group. Did.

In addition, as a result of analysis through Tunel staining to specifically stain the cells inducing apoptosis, GaIN / LPS + Mel test group to which the melittin was administered as shown in Figure 5c confirmed that the cells causing the apoptosis was sharply reduced It was.

(6) Effect of melittin on apoptosis related protein of acute liver injury

In the GaIN / LPS experimental group that induced acute liver injury, it was confirmed that the expression changes of proteins related to apoptosis are generally clear.

Expression of Caspase-3, a representative protein for melittin apoptosis, is divided into activated form (Cleavage-caspase-3) and inactive form (Pro-caspase-3). Known.

Based on this, as shown in Figures 6a and 6b, the expression change of Caspase-3 was confirmed in the protein and liver tissue.

In the GaIN / LPS experimental group that induced acute liver injury, the activation of Caspase-3 was induced, but the activation of Caspase-3 was inhibited in the GaIN / LPS + Mel experimental group administered with melittin.

Based on the above experimental results, melittin decreases the expression level of Cytokine related to liver disease and inhibits the activity of Caspase-3, which is an apoptosis protein, to confirm that it is effective in treating liver disease.

The present invention has been described with reference to the preferred embodiments, and those skilled in the art to which the present invention pertains to the detailed description of the present invention and other forms of embodiments within the essential technical scope of the present invention. Could be. The scope of the present invention is defined by the appended claims, and all differences within the scope of the claims are to be construed as being included in the present invention.

Claims (9)

Melittin (Melittin) as an active ingredient,
Pharmaceutical composition for the treatment of liver disease.
The method of claim 1,
The melittin is isolated from bee-derived bee venom,
Pharmaceutical composition for the treatment of liver disease.
The method of claim 1,
The composition contains 0.001 to 0.05% by weight of melittin,
Pharmaceutical composition for the treatment of liver disease.
The method of claim 1,
The composition further contains a pharmaceutically acceptable carrier,
Pharmaceutical composition for the treatment of liver disease.
The method of claim 1,
The melittin is characterized in that to reduce the expression of inflammation-induced cytokines (Cytokine) in liver tissue,
Pharmaceutical composition for the treatment of liver disease.
6. The method of claim 5,
The inflammation-inducing cytokine (Cytokine) is any one selected from tumor necrosis factor (TNF-α) or interleukin-1β (IL-1β) or interleukin 6 (IL-6) or vascular cell adhesion molecule (VCAM) doing,
Pharmaceutical composition for the treatment of liver disease.
The method of claim 1,
The melittin is characterized in that to reduce the expression of liver fibrosis-induced cytokine (Cytokine),
Pharmaceutical composition for the treatment of liver disease.
8. The method of claim 7,
The liver fibrosis-induced cytokine (Cytokine) is characterized in that any one selected from the transforming growth factor (TGF-β1) or α-smooth muscle actin (α-SMA) or fibronectin,
Pharmaceutical composition for the treatment of liver disease.
The method of claim 1,
The melittin inhibits the activity of caspase-3 (Caspase-3), an apoptosis protein of acute liver injury,
Pharmaceutical composition for the treatment of liver disease.

Images