KR20100037866A - A composition for preventing and treating hepatic damages containing glycoprotein from hizikia fusiformis - Google Patents

Abstract

PURPOSE: A pharmaceutical composition containing Hizikia fusiformis-derived glycoprotein as an active ingredient is provided to suppress liver injury and hepatocyte apoptosis and to maintain blood GPT concentration at a normal level. CONSTITUTION: A pharmaceutical composition for preventing and treating liver injury contains Hizikia fusiformis-derived protein as an active ingredient. A food composition having activities of preventing and treating liver injury contains Hizikia fusiformis-derived glycoprotein as an active ingredient. A method for Hizikia fusiformis-derived glycoprotein comprises: a step of washing and heat-drying collected Hizikia fusiformis; a step of pulverizing and maintaining at -20°C; a step of adding 2L of water to 80g of Hizikia fusiformis powder and heat-extracting for three hours; a step of filtering extract and adding three times as extract volume; a step of filtering again and concentrating; a step of adding 80% of ammonium sulfate and maintaining at 4°C; a step of dissolving in water after centrifuging and removing salt using dialyzer; and a step of decompressing.

Description

A composition for preventing and treating hepatic damages containing glycoprotein from Hizikia fusiformis}

The present invention relates to a sugar-derived glycoprotein having hepatic damage prevention and therapeutic activity, and more particularly to a pharmaceutical composition for preventing and treating liver damage, which contains sugar-derived glycoprotein as an active ingredient.

As various physiochemical effects of seaweeds have been verified recently, as the interest in seaweed-derived physiologically active substances increases, the anticancer effect of the extracts extracted from 톳, kelp, seaweed, seaweed, etc., and the improvement of blood lipid metabolism have been reported.

Hizikia fusiformis, which germinate in late summer and early autumn and grow to be visible to the naked eye in the middle of autumn, grows to around 20cm by the end of December and grows rapidly in March to April the following year.

It contains a lot of inorganic salts such as calcium, iodine and iron, which convert blood into alkaline and strengthen cell tissues, preventing aging and activating all organ functions. In addition, it prevents vascular hardening, and when eaten commercially, calcium metabolism is smooth, so it not only increases resistance to diseases, but also makes teeth healthy, hair is healthy, and in pregnant women, it strengthens the bones of the fetus. In addition, the intestinal fluid activity is actively excreted to excrete wastes in the intestine, which is effective in preventing constipation. Therefore, the research on 톳 was mainly done in Japan, and research on 톳 in Korea has been conducted for research on aquaculture and chemical properties.

Acetoaminophen is an anti-inflammatory and analgesic drug that is widely used around the world, causing serious liver damage when overdose. In fact, in the United States and the United Kingdom, acetoaminophen is the leading cause of liver disease-causing liver disease. Therefore, various studies have been conducted to identify the liver damage mechanism caused by acetoaminophen, and to prevent or repair liver damage.

Catlin et al. (Toxicological Sciences 84, 201-208, 2005) reported that PARP activity, one of apoptosis, was observed when acetoaminophen was administered to mice, and Tamara et al .; Toxicological Sciences 76, 229-236, 2003) reported that acetoaminophen causes hepatic lipid peroxidation and causes liver damage.

On the other hand, Sidhartha et al. (Archives of Biochemistry and Biophysics 369, 42-58, 1999) showed that acetoaminophen induced hepatocyte death when oral administration of grape seed extract proanthocyanidin in mice. Francisco et al. Journal of Ethnopharmacology 98, 103-108, 2005, were treated with acetoaminophen-derived hepataphyllum extract, which had been used as a folk remedy in South America. It is reported that damage can be prevented.

Jeong et al. ("Effect of Hizikia fusiformis Extract on Lipid Metabolism and Liver Enzyme Activity in Hyperlipidemic Rats" Journal of the Korean Society of Food Science and Nutrition 30, 1184-1189, 2001) It has been found to have hyperlipidemia, antihypercholesterol and antioxidant effects.

Therefore, the present inventors have conducted extensive studies to prevent and treat liver damage caused by acetaminophen, and provide a mechanism of action. As a result, the present invention has been performed by separating glycoproteins isolated from brown algae and confirming their effects. Came to.

It is therefore an object of the present invention to provide novel uses for the prevention and treatment of hepatic damage of VII-derived compounds.

The object of the present invention as described above was achieved by separating glycoproteins from bovine and confirming their inhibitory effect on liver damage by aminoacetophene.

The present invention provides a pharmaceutical composition for the prevention and treatment of liver damage, which contains the 톳 derived glycoprotein as an active ingredient.

In the present invention, the term "active ingredient" refers to a substance or a group of substances (a medicinal agent whose pharmacologically active ingredient is not known, etc.) which is expected to express the efficacy or effect of the medicine directly or indirectly by inherent pharmacological action. It means containing a main component).

In the present invention, the glycoprotein was extracted and separated from the powder, and the effect of inhibiting liver damage by acetoaminophen was examined. In electrophoresis and staining results, the extract was composed of sugar and protein, which is called glycoprotein.These cells were inhibited by the administration of acetoaminophen to hepatocytes and inhibited hepatocellular damage due to acetoaminophen toxicity by the glycoprotein combination treatment. It confirmed that it became.

ERK phosphorylation was found to be involved in the MAPK group proteins that act on the oxidative damage caused by acetoaminophene in order to elucidate the mechanism of action of the glycoprotein derived from the present invention. In other words, ERK phosphorylation was induced by acetoaminophen, and its phosphorylation was further increased by glycoprotein treatment to induce cell survival. Examination of liver damage in rats treated with acetoaminophene and glycoprotein showed that acetoaminophen increased GPT in GOT / GPT in blood and glycoprotein reduced it back to normal levels. In addition, caspase-3 / -9 showed a caspase-3 / -9-dependent caspase activity, and the acetoaminophen poisoning effect was dependent on caspase-3 / -9. Inhibition of acetoaminophen poisoning by reducing -9 activity. Based on these results, the glycoprotein derived from 톳 suppresses acetoaminophen-induced liver damage and hepatic cell death, thereby maintaining blood GPT levels at normal levels, as well as proteins related to cell survival in gastric epidermal cells (PARP, ERK). ·) And caspase activity has been shown to inhibit liver damage by drugs.

The glycoprotein derived from 톳 according to the present invention inhibits liver damage and apoptosis of rats by acetoaminophen to maintain normal blood GPT levels, as well as proteins related to cell survival in hepatocytes (PARP, ERK ·) and Ca It affects the spatase activity and has the effect of inhibiting liver damage by drugs.

Hereinafter, preferred embodiments of the present invention will be described in more detail with reference to Examples. However, the scope of the present invention is not limited to these examples.

Example 1 Preparation of Glycoproteins from Sodium

Ingredients in seaweeds such as falcon, seaweed, and seaweed have increased interest in their role as functional substances in addition to mere nutrients, and have anti-cancer, anti-obesity, anti-cholesterol, immune enhancing and anti-inflammatory effects. Studies on the same physiological activity are being actively conducted. Therefore, in the present embodiment, the algae glycoprotein is extracted and separated to perform the following effect verification experiment.

After washing and hot-air drying 톳 ( Hizikia fusiformis) collected from the offshore of Korea, it is powdered and stored at -20 ℃. 80 80g of powder was put in 2L of water and extracted with hot water at 80 ℃ for 3 hours. Into the filtrate after filtration, three volumes of ethanol were added and filtered again. The filtrate was concentrated, dissolved in water, and ammonium sulfate was added at 80% concentration and left overnight at 4 ° C. Water is added to the residue obtained by centrifugation and dissolved, and then salt is removed using a dialysis membrane. The dialysis solution was concentrated under reduced pressure to use a glycoprotein sample.

Experimental Example 1 SDS-PAGE and Agarose Gel Electrophoresis and Staining

To each fraction obtained in Example 1 was added 2 × Laemmli sample buffer (13.3% SDS, 0.4 M Tris, 0.013% bromophenol blue, 40% glycerol, pH 6.5) to a concentration of 1 ×. And electrophoresed between 12.5% -20% SDS-polyacrylamide gel using a Mighty Small II Apparatus (Hofer Science Instrument, USA).

After electrophoresis, the protein isolated on the gel was confirmed after staining by Coomassie brilliant blue R-250 method and Silver Staining method, and the presence of sugar after the SDS-PAGE electrophoresis, glycoprotein staining kit (Glycoprotein staining kit; Pierce), or agarose gel electrophoresis was confirmed after staining with toluidine blue (see Fig. 1).

As shown in FIG. 1, it can be seen that sugar and protein are present together in the material extracted from the SDS-PAGE and agarose gal electrophoresis results. Therefore, in the present invention 톳 derived material is called glycoprotein (glycoprotein).

Experimental Example 2 Hepatocellular Proliferation Inhibitory Effect by Acetoaminophen Treatment

In this example, two human liver cells, Chang liver cells and HepG2 cells, were used to examine the efficacy of the K-derived glycoprotein. Among them, Chang liver cells are originally derived from normal hepatocytes, and HepG2 cells grow in the same metabolic mechanism as normal hepatocytes, so they are generally used for experiments on liver toxicity and recovery using cells.

Chang liver cells and HepG2 cells were distributed from ATCC (American Type Culture Collection; CCL13, HB8065). 100units / ml of antibiotics (penicillin / streptomycin) was added to MEM medium and filtered through a 0.22μm filter, followed by adding fetal bovine serum (FBS) to a final concentration of 10% before incubation. Cells were incubated in a 37 ° C., 5% CO ₂ incubator. Incubate the cells in culture twice a week, wash with PBS (phosphate buffered saline) when confluent, and centrifuge the cells attached with 0.25% trypsin solution. The cells were mixed well and evenly dispensed so that each cell was evenly distributed.

To examine cell proliferation after acetoaminophen treatment, Chang liver cells and HepG2 cells at 5 × 10 ⁴ cells / well concentration were dispensed in 96-well plates and cultured for 24 hours. After 24 hours, the cells were further incubated with serum-free medium for 24 hours, and then diluted with acetoaminophene in serum-free medium for 24 hours and added to cells. Add 10 μl of MTS / PMS solution (CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay Kit, Promeca Corporation, USA) to acetoaminophen treated cells, and then use the Autometic ELIZA reader within 30 minutes. Absorbance was measured at 490 nm, and cell viability was calculated according to Formula 1. All experiments were repeated three times.

<Calculation Formula 1>

As shown in Figure 2, hepatocellular proliferation (A; Chang liver cells, B; HepG2 cells) was inhibited with increasing acetoaminophen concentration and culture time.

Apoptosis is a phenomenon in which a suicide mechanism inherent in a cell is activated by internal and external stimuli and dies as planned. Unlike cell necrosis, the contents of a dying cell are not released to the outside of the cell. Does not damage. Morphologically, phagocytosis is accompanied by cell specific gravity reduction, cell membrane destruction, chromosome condensation, apoptotic body formation, and biochemically, DNA fragmentation, in which chromosomal DNA is split from large to small fragments. Happens.

Therefore, in order to confirm that the cell proliferation inhibitory effect by acetoaminophen is a phenomenon caused by apoptosis, the cell morphology was observed through Hoechast 33342 staining which is specifically stained with DNA in the cell nucleus. Specifically, the cells were washed with PBS and fixed at room temperature for 10 minutes with 3.7% paraformaldehyde, followed by 30 minutes treatment of the fluorescent substance Hoechast 33342. After 30 minutes, washed with PBS, dehydrated with 100% ethanol and observed by fluorescence microscope.

As shown in FIG. 3 (A; Chang liver cells, B; HepG2 cells), apoptotic bodies were found in the acetoaminophen treatment group, and the number of cells was also significantly reduced compared to the control group.

To investigate DNA fragmentation, hepatocytes were treated with acetoaminophen for 24 hours, and then mixed with a cell lysate (nuclear lysis buffer, 100 mM NaCl 2, 40 mM Tris-HCl, pH 7.4, 20 mM EDTA, 0.5% SDS). After crushing, RNase A and proteinase K were treated to remove RNA and protein, and isopropanol was treated to precipitate chromosomes. The precipitated DNA was washed with 70% ethanol and dried. After adding TE buffer solution (10mM Tris-HCl, 1mM EDTA, pH8.0) to dissolve the DNA pellet, the absorbance was measured with a spectrophotometer (Amersham Pharmatech) of 260nm and 280nm wavelength to measure DNA purity and concentration. Was measured. 10 μg of DNA was electrophoresed on 2% agarose gel (50V, 2 hours), and then stained with ethidium bromide and observed with ultraviolet light.

As shown in Figure 4 (A; Chang liver cells, B; HepG2 cells), DNA fragmentation was induced by acetoaminophen, it can be seen that the acetoaminophen induces cell death causing liver damage.

Experimental Example 3 Inhibition Effect of Hepatic Glycoprotein from Hepatic Injury by Acetoaminophen I

Most studies of acetoaminophen cytotoxic mechanisms have shown that GSH plays a critical role in inhibiting the poisoning effect of acetoaminophen reaction products. It is well known that many liver models cause intracellular oxidative stress in severe liver injury (Jaeschke and Mitchell, 1989). After 4 hours of acetoaminophen administration, oxidative stress was the result rather than the cause of liver injury because the concentration of free radical formation GSSG (Jaeschke, 1990; Knight et al. , 2001) was increased (Rogers et al. , 2000). Recently published papers report that endogenous and exogenous reactive oxygen species (ROS) regulate mitogen-activated protein kinases (MAPKs) pathways involved in cell proliferation, differentiation and death (Sano et al ., 2001). Jang et al. , 2002). Mitogen-activated protein kinases include extracellular signal-regulated kinase (ERK), p38 kinase and c-Jun N-terminal kinase (JNK). Among them, ERK is activated by growth factors acting on cell division and proliferation and is involved in cell proliferation and differentiation.

On the other hand, JNK and p38 kinase are mainly activated by extracellular stimulation such as UV irradiation, inflammation, cytokines (Kyriakis and Avruch, 2001; Chung et al ., 2003; Kang et al. , 2003). When these proteins are activated, various intracellular reactions occur depending on the cell.

In this example, in order to confirm the apoptotic pathway caused by acetoaminophen as described above, the cytotoxic activity was observed with Chang liver cells and HepG2 cells, and the blood GOT / GPT levels after administration of acetoaminophen to SD rats. The degree of liver damage was examined by measuring caspase activity in liver and liver tissue.

As shown in FIG. 5, cell proliferation was inhibited by acetoaminophen, which can be seen in the Hoechast 33342 stained cell photograph. When the glycoprotein extract was added thereto, cell proliferation inhibited by acetoaminophen was recovered.

First, we examined the level of protein expression involved in the MAPK pathway using HepG2 cells, which were shown to be the more effective of the two cells used earlier to determine which mechanism caused the acetoaminophen toxicity.

As shown in FIG. ERK phosphorylation was increased by acetoaminophen, and it was shown that ERK phosphorylation was further increased by adding the protein extract. In other words, ERK phosphorylation was induced by oxidative damage caused by acetoaminophen, and it can be seen that ERK phosphorylation is more activated for cell survival by the glycoprotein derived from 톳.

Experimental Example 4 Inhibitory Effect of Glucose-Derived Glycoprotein on Liver Damage by Acetoaminophen Ⅱ

Animal experiments were performed to verify the acetoaminophene toxicity inhibitory activity of the γ-derived glycoprotein at the in vivo level.

(1) GOT and GPT in serum

Hepatic indicators, serum GOT and GPT, are enzymes that reflect the degeneration and necrosis of liver cells. GOT / GPT in serum is glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase prepared according to the method of Reitman-Frankel. measured at 505 nm using a spectrophotometer (Ultrospec 2001 pro. Amersham Pharmacia Biotech, UK) using a serum transaminase measurement kit (Shinyang Chemical, South Korea) for measuring trasaminase (GPT) activity, and the results are shown in FIG. 7. It was.

Serum GOT / GPT levels have been reported to increase rapidly during liver damage using carbon tetrachloride (Cho et al ., 2004; Cho et al ., 2003; Kim et al ., 2003). In addition, there have been reports of increased GOT / GPT in serum upon liver injury due to acetoaminophen administration (Lee et al ., 1998; Gujral et al. , 2002). In the experimental results, GPT was significantly increased (p <0.05) when acetoaminophen alone was administered compared to the control group, and the decrease in GPT level was lowered below the control group by the G-derived glycoprotein (FIG. 7). Therefore, it is thought that the hepatocyte-derived glycoprotein inhibited the liver damage by restoring the GPT concentration to the control level by protecting the liver damage caused by acetoaminophen administration.

(2) Caspase activity change in liver tissue

Apoptosis is an important cell death that is important for maintaining homeostasis. Although many studies have been reported on the types of apoptosis caused by acetoaminophene, there is no clear conclusion yet between the natural death of cells and their death.

Therefore, caspase activity was measured to confirm apoptotic cell death by acetoaminophen. To this end, the liver tissue is homogenized with lysis buffer (25 mM HEPES, pH 7.5, 5 mM EDTA, 2 mM DTT, 0.1% CHAPS) containing protease inhibitor, and then 4 ° C., 14,000 rpm. Centrifugation. Take the supernatant and measure protein concentration with the BCA Protein Assay Kit, place 80 μg of protein in a 96-well plate and add DEVD-pNA (Caspase-3 Substrate) and Granzyme B (Caspase-8 Substrate). After the reaction was carried out for 4 hours at 37 ℃. After 4 hours, the absorbance was measured at 405 nm.

As shown in FIG. 8, the caspase-3 activity was increased by acetoaminophen and decreased by the co-administration of the protein, but the caspase-8 activity was significantly increased by the acetoaminophen or co-protein administration. No difference was seen. Meanwhile, Western blot was performed to investigate caspase-9 activation. As with caspase-3 activity, caspase-9 was activated by acetoaminophen and the cleaved caspase-9 band was reduced due to the co-administration of o-derived glycoproteins. Taken together, it can be estimated that apoptosis by acetoaminophen occurs through mitochondria. In other words, acetoaminophen poisoning occurred independently of caspase-8, but caspase-3 / -9, and the glycoprotein derived from oocytes decreased caspase-3 / -9 activity, which was activated by acetoaminophen. It is to inhibit the acetoaminophen poisoning action.

1 is a diagram showing the results of SDS-PAGE and agarose gel electrophoresis.

Figure 2 is a graph showing the effect of inhibiting hepatocyte proliferation according to the increase in acetoaminophen concentration and incubation time.

Figure 3 is a fluorescence micrograph showing the effect of inhibiting cell proliferation by acetoaminophen through Hoechast 33342 staining.

4 is a diagram showing the results of DNA fragmentation induction by acetoaminophene.

5 is a fluorescence micrograph showing the effect of inhibiting cell proliferation by acetoaminophen through Hoechast 33342 staining.

Figure 6 is a diagram showing the protein expression level involved in the MARK pathway using HepG2 cells.

7 is a diagram showing changes in GOT and GPT in serum.

8 is a diagram showing caspase activity.

Claims (3)

톳 Pharmaceutical composition for liver damage prevention and treatment containing glycoproteins as an active ingredient. 식품 Food composition having a liver damage prevention and treatment activity containing the derived glycoprotein as an active ingredient. The method of claim 1, wherein the protein derived from 톳 maintains blood GPT levels at a normal level and inhibits liver damage caused by drugs by affecting cell survival-related proteins (PARP, ERK.) And caspase activity. Characterized by the composition.

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