KR102169141B1 - Compositions for Prevention or Treatment of nonalcoholic fatty liver disease Comprising Manassantin A or Manassantin B - Google Patents

Abstract

The present invention relates to a composition for preventing or treating non-alcoholic fatty liver disease, comprising manassantin A or manassantin B, and manassantin A or manassantin B of the present invention. The composition for preventing or treating non-alcoholic fatty liver disease, including B), does not have various side effects such as cardiovascular, central, hepatic and renal disorders caused by conventional synthetic pharmaceutical compositions, and reduces inflammation caused by non-alcoholic fatty liver disease, non-alcoholic Since it has the effect of inhibiting the progression and metastasis of fatty liver, inhibiting lipid accumulation in the liver, anti-apoptotic effect and promoting autophagy activity of cells, a pharmaceutical composition for preventing and/or treating non-alcoholic fatty liver or non-alcoholic fatty liver and/or It can be usefully used as a health functional food composition for improvement.

Description

Compositions for Prevention or Treatment of Nonalcoholic Fatty Liver Disease Comprising Manassantin A or Manassantin B containing manasantin A or manassantin B}

The present invention relates to a composition for preventing or treating non-alcoholic fatty liver disease comprising manassantin A or manassantin B, and more particularly, to a composition for preventing or treating non-alcoholic fatty liver disease, non-alcoholic fatty liver Non-alcoholic fatty liver disease containing manasanthin A and/or manasanthin B as active ingredients, which have the effect of inhibiting progression and metastasis, inhibiting lipid accumulation in the liver, anti-apoptotic effect, and promoting autophagy activity of fat-accumulated hepatocytes It relates to a composition for prevention or treatment.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic disease and is known to be closely related to type 2 diabetes, obesity and metabolic syndrome. Although there are some differences in frequency from region to region, it has been reported that as few as 6.3%, as many as 33%, and about 20% of patients worldwide have developed this disease, and some of them have nonnalcoholic steatohepatitis (NASH). It has been shown to progress to cirrhosis or liver cancer through stages.

Non-alcoholic fatty liver disease is associated with several factors such as insulin resistance, autophagy, apoptosis, inflammation, metabolic syndrome {obesity, lipotoxicity, mixed hyperlipidemia, type 2 diabetes}, and non-alcoholic fatty liver disease (simple fatty liver). The pathogenesis of non-alcoholic steatohepatitis) has not been fully elucidated since it has been developed by several routes, but the mechanisms of invention for the treatment of these diseases have recently been reported (Min, HK et al , Cell Metab . , 15:665, 2012; Puri, P. et al ., Gastroenterology , 134:568, 2008).

Lipotoxicity caused by large amounts of free fatty acids is known to induce insulin resistance, oxidative stress, hepatocellular damage and inflammation in the liver, leading to the onset of non-alcoholic fatty liver disease (Fuchs, M. , and Sanyal, AJ, Journal of hepatology , 56:291, 2012), in particular saturated fatty acids, such as palmitate, are directly toxic to cells and tumor necrosis factor-α (TNF-α) and interlukin-6 (interlukin-6). -6: It has been reported to induce inflammation and insulin resistance by promoting cytokine production, such as IL-6) (Ajuwon, KM and Spurlock, ME, The Journal of nutrition , 135:1841, 2005).

In addition, free fatty acids function in endoplasmic reticulum (ER) and mitochondria through phosphorylation of JNK (c-Jun N-terminal kinase 3), NF-kB (Nuclear factor-kappa B), and extracellular signal-regulated kinases (ERK) in the liver. Disorders, lipid accumulation, and increased inflammation have been reported to cause NAFLD (Wei, Y. et al. al ., American journal of physiology . Endocrinology and metabolism , 291:E275, 2006), nonalcoholic fatty liver disease and insulin resistance through the gp130 metabolic pathway trigger JAK (Janus activated kinase)-signaling, JAK-STAT3 transcription activation, Src-Ras and PI3 kinase-Akt activation. It is known to induce (Steinberg, GR et al ., Diabetes , 58:829. 2009: Yamauchi-Takihara, K. and Kishimoto, T., Int J Exp Pathol ., 81:1, 2000).

As described above, many studies have been conducted on therapeutic drugs and mechanisms of occurrence for non-alcoholic fatty liver disease, but there are no therapeutic drugs yet, and only diet therapy or exercise therapy is recommended. For this reason, more active drug treatment is needed.

To date, metadoxine, betaine glucuronate, methionine, choline, and lipotropic drugs are used as auxiliary drugs for fatty liver drugs, but these drugs The effect of is not medically proven, and fibrate drugs are generally known to have side effects such as liver dysfunction. Therefore, there is a demand for the development of a new therapeutic agent for the prevention and treatment of non-alcoholic fatty liver that has no side effects and is inexpensive and has excellent therapeutic effect. It is being studied.

On the other hand, manassantin A (manassantin A) or manassantin B (manassantin B) is three hundred seconds ( Saururus chinensis (Lour.) Baill) is a type of lignan derived from manasanthin A and manasanthin B, which are anti-inflammatory (Chang, JS et al ., Journal of pharmacological sciences , 115:84, 2011), anti-human immunodeficiency virus effect (Lee, J. et al) al ., Antiviral research , 85:425, 2010), improvement of hyperpigmentation disease (Seo, CS et al ., Phytotherapy research : PTR , 23:1531, 2009), and various biological activities have been reported.

In addition, manasanthin B inhibited the production of IL-1β in LPS-treated RAW 264.7 cells, inhibited the phosphorylation of ERK1/2 and p38 MAPK, but it was reported that there was no change in the phosphorylation of JNK (Park, HC et al ., Korean journal of anesthesiology , 62:161, 2012), Dependent Cyclooxygenase-2-Prostaglandin D2 (cyclooxygenase) by blocking Fyn-mediated Nuclear factor-kappa B (NF-kB) and MAPK (Mitogen Activated Protein Kinase) pathways in bone marrow-derived mast cells It has been reported to inhibit the production of -2-dependent prostaglandin D2) (Lu, Y. et al ., Biological & pharmaceutical bulletin , 36:1370, 2013).

However, in the prior art, there is no report on the prophylactic or therapeutic effects of manassantin A or manassantin B in non-alcoholic fatty liver disease.

Therefore, in the present invention, as a result of careful efforts to select natural substances for the prevention or treatment of non-alcoholic fatty liver, which has no side effects and has excellent therapeutic effect, manassantin A or manassantin B derived from Sambaekcho A has the effect of reducing inflammation caused by non-alcoholic fatty liver disease, inhibiting the progression and metastasis of non-alcoholic fatty liver, inhibiting lipid accumulation in the liver, anti-apoptotic effect, and promoting autophagy activity of fat-accumulated hepatocytes. It was confirmed that it can be used as a composition for prevention or treatment of, and the present invention was completed.

The present invention has been devised to solve the above problems, a pharmaceutical composition or health functional food for the prevention or treatment of non-alcoholic fatty liver comprising manassantin A or manassantin B as an active ingredient It is to provide a composition.

In order to solve the problems of the present invention described above, the present invention, manassantin A represented by the following formula (1); Manassantin B represented by the following formula (2); And salts thereof; It includes a pharmaceutical composition for the prevention or treatment of non-alcoholic fatty liver comprising one or more compounds selected from, or a health functional food composition for preventing or improving non-alcoholic fatty liver disease.

[Formula 1]

[Formula 2]

According to a preferred embodiment of the present invention, the manassantin A (manassantin A) or manassantin B (manassantin B) is three hundred seconds ( Saururus chinensis ( Lour .) Baill ).

According to another preferred embodiment of the present invention, the composition is any one or more selected from the group consisting of interleukin-6 (IL-6) and interleukin-8 (IL-8). Inhibition of inflammatory cytokine production; Inhibition of c-Jun N-terminal kinases (JNK) activity; And inhibition of nuclear factor-kappaB (NF-κB) activity; Inflammation caused by non-alcoholic fatty liver disease can be suppressed through one or more mechanisms selected from the group consisting of.

According to another preferred embodiment of the present invention, the composition is poly (ADP-ribose) polymerase (Poly ADP ribose polymerase: PARP) cleave inhibition; And caspase-3 cleave inhibition; Apoptosis by non-alcoholic fatty liver can be suppressed through one or more mechanisms selected from the group consisting of.

According to another preferred embodiment of the present invention, the composition inhibits gp130 (Glycoprotein 130) production; And inhibition of STAT3 (signal transducer and activator of transcription 3) activity; It is possible to inhibit the progression and metastasis of non-alcoholic fatty liver disease through one or more mechanisms selected from the group consisting of.

According to another preferred embodiment of the present invention, the composition promotes AMP-activated protein kinase (AMPK) activity; And inhibition of acetyl-CoA carboxylase (ACC) activity; It is possible to reduce the triglyceride content or lipid accumulation in the liver through one or more mechanisms selected from the group consisting of.

According to another preferred embodiment of the present invention, the composition inhibits the production of microtubule-associated protein light chain 3 (LC3)-II or p62 protein involved in autophagy blockages; Promoting AMP-activated protein kinase (AMPK) activity; And inhibition of acetyl-CoA carboxylase (ACC) activity; It is possible to induce autophagy of non-alcoholic fatty liver through one or more mechanisms selected from the group consisting of.

The composition for preventing or treating non-alcoholic fatty liver disease comprising manassantin A or manassantin B of the present invention is the cardiovascular action, central action, liver disorder and kidney disorder caused by conventional synthetic pharmaceutical compositions. There are no side effects, and it has the effect of reducing inflammation caused by non-alcoholic fatty liver disease, inhibiting progression and metastasis of non-alcoholic fatty liver, inhibiting lipid accumulation in the liver, anti-apoptotic effect, and promoting autophagy activity of fat-accumulated hepatocytes. It may be usefully utilized as a pharmaceutical composition for preventing and/or treating non-alcoholic fatty liver or a health functional food composition for improving and/or improving non-alcoholic fatty liver.

1 is data showing the results of HPLC (A) and NMR analysis of manasanthin A or manasanthin B isolated from 300 seconds.
FIG. 2 is data confirming the cell viability according to the concentration of manasanthin A or manasanthin B (MNS-A: manasanthin A, MNS-B: manasanthin B).
3 is an HC assay performed to analyze the cytotoxicity and genetic mutation of manasanthin B, and is data showing the relative cell density (A) and fluorescence induction value (B).
Figure 4 is a saturated fatty acid palmitate (palmitate) and/or interleukin-6 (IL-6) treated liver cells treated with manasanthin A or manasanthin B, JNK (c-Jun N-terminal kinases) and The degree of phosphorylation of NF-κB (nuclear factor-kappaB) was confirmed by Western blot (A), and the data obtained by digitizing it (B), and interleukin-8 (IL-8) mRNA expression level (C) and interleukin-6 (IL- 6) Data showing the level of mRNA expression (D).
FIG. 5 shows c-Jun N-terminal kinases (JNK) and NF-κB when hepatocytes treated with tumor necrosis factor-alpha (TNF-α) were treated with manasanthin A or manasanthin B. This is the data obtained by confirming the degree of phosphorylation of (nuclear factor-kappaB) by Western blot (A) and digitizing it (B).
6 is a poly(ADP-ribose) polymerase in order to confirm the degree of apoptosis by treatment of manasanthin A or manasanthin B in palmitate and/or interleukin-6 (IL-6)-treated liver cells. (Poly ADP ribose polymerase: PARP) and caspase-3 (caspase-3) The degree of activity by cleavage was confirmed by Western blot (A), and this data was quantified (B).
FIG. 7 shows the degree of gp130 (Glycoprotein 130) production and the STAT3 (signal transducer and activator of transcription) when palmitate or interleukin-6 (IL-6)-treated liver cells were treated with manasanthin A or manasanthin B. 3) This is the data obtained by confirming the degree of phosphorylation by Western blot (A) and digitizing it (B and C).
8 is a Western blot (A) showing the degree of gp130 production and the degree of STAT3 and Tyk phosphorylation when hepatic cells treated with palmitate and interleukin-6 (IL-6) were treated with manasanthin A or manasanthin B. This is the data that was confirmed and quantified (B).
FIG. 9 is data obtained by confirming the degree of AMPK phosphorylation by Western blot (A) and quantifying it (B) when manasanthin A or manasanthin B is treated in palmitate-treated liver cells.
Figure 10 is when palmitate (palmitate) and/or interleukin-6 (IL-6)-treated liver cells were treated with manasanthin A or manasanthin B, confirming the degree of AMPK and ACC phosphorylation by Western blot (A). This is the data obtained by digitizing (B).
Figure 11 is when palmitate (palmitate) treated liver cells were treated with manasanthin A or manasanthin B, triglyceride content in liver cells (A), using oil-red-O, and This is the data confirming the degree of lipid accumulation (B) and the degree of lipid accumulation (C) using an electron microscope.
12 is data confirming the activity of SITR 1 and the degree of protein expression by manasanthin A or manasanthin B.
13 is a western blot (A) to confirm the level of p62 and LC3-II production in order to confirm the autophagy induction effect by manasanthin A or manasanthin B in palmitate-treated liver cells. This is the data obtained by digitizing (B).
14 is to confirm the autophagy induction effect by manasanthin A or manasanthin B in palmitate or ammonium chloride/leupeptin-treated liver cells, p62 protein and This is the data obtained by confirming the degree of LC3-II generation by Western blot (A) and digitizing it (B: LC3-II, C: p62).
Figure 15 is a palmitate (palmitate) or ammonium chloride (ammonium chloride) / leupeptin (leupeptin) in order to confirm the autophagy induction effect by manasanthin A or manasanthin B in liver cells treated, AMPK and ACC This is the data obtained by confirming the degree of phosphorylation by Western blot (A) and digitizing it (B).
FIG. 16 is a Western blot of the expression levels of protein kinase C (PKCθ) and dephosphorylation enzyme-2 (PP2A) by manasanthin A or manasanthin B in palmitate-treated hepatocytes. This is the data confirmed by (A) and digitized (B and C).
FIG. 17 shows the degree of phosphorylation of AMPK and the degree of phosphorylation of AMPK by manasanthin A or manasanthin B in a palmitate-treated hepatocyte as a western blot (A). This is the data that was confirmed and quantified (B).
18 is data confirming the intracellular AMP/ATP ratio by manasanthin A or manasanthin B in palmitate-treated hepatocytes.
19 is a schematic diagram summarizing the effect of manasanthin A or manasanthin B on non-alcoholic fatty liver.

Hereinafter, the present invention will be described in more detail.

As described above, most non-alcoholic fatty liver diseases are accompanied by complications such as obesity, diabetes, insulin resistance, and hyperlipidemia, and diet or exercise therapy is recommended to treat them, but there are disadvantages that are difficult to implement. There is no need to develop a new therapeutic agent for the prevention and treatment of non-alcoholic fatty liver, which is inexpensive and has excellent therapeutic effect.

The present invention seeks to solve the above-described problem by providing a composition for preventing or treating non-alcoholic fatty liver disease comprising manassantin A or manassantin B. Through this, there are no side effects such as cardiovascular action, central action, liver disorder and kidney disorder, which are side effects that have been seen in conventional pharmaceutical compositions, while reducing inflammation caused by non-alcoholic fatty liver disease, inhibiting progression and metastasis of non-alcoholic fatty liver, and inhibiting lipid accumulation in the liver. , It is effective to provide a pharmaceutical composition for the prevention and treatment of non-alcoholic fatty liver that has anti-apoptotic effect and autophagy activity of fat-accumulated hepatocytes, and a health functional food for the prevention and improvement of non-alcoholic fatty liver. .

Thus, the present invention is manassantin A (manassantin A) represented by the following formula (1); Manassantin B represented by the following formula (2); And salts thereof; It includes a pharmaceutical composition for the prevention or treatment of non-alcoholic fatty liver comprising one or more compounds selected from, or a health functional food composition for preventing or improving non-alcoholic fatty liver disease.

[Formula 1]

[Formula 2]

The term'nonalcoholic fatty liver disease (NAFLD)' of the present invention is classified into nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH). Non-alcoholic fatty liver disease (NAFLD), for example, is a liver disorder characterized by hepatic fat deposits of large aptitude, similar to alcoholic liver disease, with liver tissue findings similar to alcoholic liver disease, although there is no clear drinking history. It is defined as the concept of a disease involving hyperprogressive NASH.

The manassantin A (MNS-A) or manassantin B (MNS-B) is 300 seconds ( Saururus chinensis (Lour.) Baill) can be characterized, and can be extracted by adding a single or mixed solvent such as water, alcohol, ethyl acetate, chloroform, butanol or hexane to 300 seconds, but is usually prepared and/or It is not particularly limited as long as it can be purchased.

It may be extracted by applying a general solvent extraction method using the above solvent, and may also be a fraction purified using column chromatography. The method for preparing the extract according to the present invention can be applied to any plant extraction method known to those of ordinary skill in the art, and preferably, in one embodiment of the present invention, three hundred seconds are extracted using methanol, and then sequentially Fractionation was finally performed to obtain an F1 fraction and an F4 fraction, and as shown in FIG. 1, as a result of analyzing the HPLC and NMR spectrum data, Compound 1 obtained from the F1 fraction was obtained from manassantin A and F4 fractions. One compound 2 was found to be manassantin B.

In the present invention, manassantin A; Manassantin B; And salts thereof; One or more compounds selected from among may be included in the composition of 5 to 150 nM, preferably 10 to 80 nM, more preferably 10 to 40 nM. The solvent of the composition is not particularly limited as long as it does not interfere with the prevention, improvement, or therapeutic effect of non-alcoholic fatty liver of manasanthin A or manasanthin B, but more preferably water, ethanol, propyl alcohol, DMSO, and the like. If a concentration lower than 5 nM of manasanthin A or manasanthin B is included, the preventive or therapeutic efficacy of non-alcoholic fatty liver may decrease, and manasanthin A or manasanthin B at a concentration of 150 nM or more can be used, but the concentration is increased. As a result, the efficacy does not increase significantly, but, rather, cytotoxicity due to manasanthin A or manasanthin B may occur.

In one embodiment of the present invention, it was confirmed whether the cytotoxicity of manasanthin A or manasanthin B was confirmed, and the cells were cultured for 48 hours by treating each of the human liver cells (Human primary hepatocytes) with mana santin A or mana santin B by concentration. As a result of measuring the degree of apoptosis, as shown in FIG. 2, it was confirmed that the cytotoxicity was not observed in the concentration of 0 to 80 nM of manasanthin A or the mana santin B, but affects cell death in the 160 nM concentration.

In addition, as a result of performing a GreenScreen HC assay using S9 metabolism inactivation to confirm the cytotoxic and genotoxic effects of manasanthin B, as shown in FIG. 3, cytotoxicity was observed at 0 to 80 nM concentration. It was not seen, and it was confirmed that the relative cell density at a concentration of 160 nM or higher decreased to 80% or less set as the threshold value compared to the control group. However, in HC analysis, it was confirmed that the fluorescence induction value showed a negative value (threshold value is less than 1.5 times) in manasanthin B 0 to 640 nM, which is slightly cytotoxic at 160 nM concentration of manasanthin B. It means that genotoxicity is not even at 640 nM.

In the present invention, the composition inhibits the production of any one or more inflammatory cytokines selected from the group consisting of interleukin-6 (IL-6) and interleukin-8 (IL-8); Inhibition of c-Jun N-terminal kinases (JNK) activity; And inhibition of nuclear factor-kappa B (NF-κB) activity; It may be characterized in that it suppresses inflammation caused by non-alcoholic fatty liver disease through one or more mechanisms selected from the group consisting of.

Non-alcoholic fatty liver can metastasize to non-alcoholic steatohepatitis, and non-alcoholic steatohepatitis can occur as part of the body's response to hepatocyte-related inflammation, hepatocyte damage, hepatocellular necrosis, and damage. Interleukin-8 (IL-8), interleukin-6 (IL-6), interleukin 1-b (IL1-b), and tumor necrosis factor-α (TNF-α) are inflammatory cytokines in liver cells. Known as pro-inflammatory cytokines, these cytokines are known to induce TNF-α, as well as activating c-Jun N-terminal kinases (JNK) and nuclear factor-kappa B (NF-κB) in hepatocytes. Is known.

Saturated fatty acids are known to induce liver cell damage and inflammation, leading to the onset of non-alcoholic fatty liver disease. In particular, palmitate is a direct cytotoxicity of cytokines such as TNF-α, IL-8, and IL-6. It has been reported that it promotes production to induce inflammation and induces insulin resistance, and when IL-6 is also increased, it is known to promote the expression of various inflammatory cytokines.

In the present invention, in order to check whether there is an effect of preventing and treating inflammation of non-alcoholic fatty liver caused by manasanthin A or manasanthin B, liver cells treated with palmitate and/or interleukin-6 (IL-6), which are saturated fatty acids, are Manasanthin A or manasanthin B was treated respectively.

As shown in FIGS. 4A and 4B, in the liver cells in which inflammation was induced by palmitate or IL-6, the degree of phosphorylation of JNK and NF-κB rapidly increased, whereas in the group treated with manasanthin A or manasanthin B It was confirmed that the degree of phosphorylation of JNK and NF-κB was significantly reduced.

The decrease in the phosphorylation of JNK and NF-κB means that the activity of JNK and NF-κB decreases, thereby reducing the inflammatory response, and manasanthin A or manasanthin B of the present invention significantly reduces the induction of inflammation in liver cells. It was confirmed what to do.

In addition, Figures 4C and 4D confirm the relative expression levels of IL-8 and IL-6 mRNA, and the group treated with manasanthin A or manasanthin B to liver cells inflamed by palmitate or IL-6 treatment. It was confirmed that the expression levels of IL-8 and IL-6 mRNA were significantly decreased, and it was confirmed that manasanthin A or manasanthin B of the present invention significantly reduced the expression of inflammatory cytokines in liver cells.

FIG. 5 shows c-Jun N-terminal kinases (JNK) and NF-κB when manasanthin A or manasanthin B is treated in hepatocytes treated with tumor necrosis factor-alpha (TNF-α). (Nuclear factor-kappaB) is the data confirming the degree of phosphorylation by Western blot.JNK and NF in the group treated with manasanthin A or manasanthin B in liver cells inflamed by TNF-α as shown in the result of FIG. It was confirmed that the degree of phosphorylation of -κB was significantly reduced.

In the present invention, the composition is poly (ADP-ribose) polymerase (Poly ADP ribose polymerase: PARP) cleave inhibition; And caspase-3 cleave inhibition; Cell death by non-alcoholic fatty liver was suppressed through one or more mechanisms selected from the group consisting of.

The apoptosis pathway in non-alcoholic fatty liver disease involves death ligand-induced activation of receptors and intracellular proteins including JNK, PARP, and capase-3. The apoptosis effect of liver cells and kupffer cells promotes liver fibrosis and inflammation through activation of stellate cells and release of cytokines, and apoptosis is important in the development and progression of non-alcoholic fatty liver disease. Plays a role. In addition, caspase-3 is an important inducer involved in apoptosis, and the cleaved nuclear enzyme poly(ADP-ribose) polymerase (PARP) protein is involved in the survival of cells and acts as a biomarker for apoptosis. .

In one embodiment of the present invention, in order to confirm the anti-apoptotic effect of manasanthin A or manasanthin B, palmitate and/or interleukin-6 (IL-6)-treated liver cells are treated with manasanthin A or mana. After each treatment with Xanthine B, the degree of activity by cleavage of Poly ADP ribose polymerase (PARP) and caspase-3 was confirmed by Western blot.

As a result, it was confirmed that the generation of PARP (cleaved-PARP) and caspase-3 (cleaved-caspase-3) cleaved by manasanthin A or manasanthin B significantly decreased, as shown in FIG. It means that A or manasanthin B can inhibit the cleavage of PARP or caspase-3 to inhibit apoptosis caused by non-alcoholic fatty liver disease.

In the present invention, the composition inhibits gp130 (Glycoprotein 130) production; And inhibition of STAT3 (signal transducer and activator of transcription 3) activity; It is possible to inhibit the progression and metastasis of non-alcoholic fatty liver disease through one or more mechanisms selected from the group consisting of.

The Gp130 cell surface receptor is glucoprotein 130, known as gp130, IL6ST, IL6-beta, and CD130, and is a key component of metabolic and other stressful cellular responses. The Gp130-mediated signaling pathway is hematopoietic, immune regulation, and inflammation. And plays an important role in cancer. IL-6 and STAT3 are known to play important roles not only in the progression of ovarian cancer, but also in the mechanism of nonalcoholic fatty liver disease.Gp130/STAT3 is JAK (Janus activated kinase)-signaling and transcription by free fatty acids and IL-6. It is activated through

In one embodiment of the present invention, when palmitate or interleukin-6 (IL-6)-treated liver cells are treated with manasanthin A or manasanthin B, the level of gp130 (Glycoprotein 130) production and STAT3 (signal transducer) and activator of transcription 3) The degree of phosphorylation was confirmed by Western blot, and this was quantified as gp130/β-actin and p-STAT3/STAT3. As a result, as shown in FIG. 7, it was confirmed that STAT3 phosphorylation was significantly reduced when manasanthin A or manasanthin B was treated in liver cells induced by inflammation by IL-6, and the expression level of gp130 protein was manasanthin A. Alternatively, it was confirmed that the expression decreased depending on the concentration of manasanthin B treatment.

In addition, Figure 8 is when palmitate and/or IL-6-treated liver cells were treated with manasanthin A or manasanthin B, the degree of gp130 production, the degree of STAT3 and Tyk phosphorylation was confirmed by Western blot, and data obtained by quantifying this , Palmitate and IL-6, respectively, alone or concurrently to treat inflammation-inducing hepatocytes with manasanthin A or manasanthin B, the expression level of gp130, STAT3 and tyrosine kinase (tyrosine kinase: tyk) phosphorylation are significantly reduced. I confirmed that.

This means that manasanthin A or manasanthin B can inhibit the progression and metastasis of non-alcoholic fatty liver disease by inhibiting gp130 production and STAT3 activity.

In the present invention, the composition promotes AMP-activated protein kinase (AMPK) activity; And inhibition of acetyl-CoA carboxylase (ACC) activity; It may be characterized by reducing the triglyceride content or lipid accumulation in the liver through one or more mechanisms selected from the group consisting of.

Lipid accumulation in the liver is caused by an imbalance of lipid availability (lipid absorption or production of new fat) and reduction of excretion (fatty acid oxidation or triglyceride-rich lipoprotein secretion), resulting in fatty liver, including liver lipotoxicity and damage. Results.

AMPK (AMP-activated protein kinase: 5'AMP-activated protein kinase or 5'adenosine monophosphate-activated protein kinase) plays an important role in cellular molecular regulation and energy homeostasis, as well as in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). As a very important enzyme, it is known to regulate liver autophagy, cholesterol synthesis, fatty acid synthesis, fat production, triglyceride synthesis, fat breakdown of fat cells, fatty acid oxidation and ketone production in the liver. AMPK regulates skeletal muscle fatty acid oxidation, muscle glucose absorption, and insulin secretion by pancreatic beta cells, and is expressed in almost all tissues, and is a protein that regulates other human diseases as well as prevention and treatment of NAFLD.

In addition, ACC (Acetyl-CoA carboxylase) is an enzyme involved in fat synthesis. When ACC is phosphorylated by AMPK, ACC is inactivated, inhibiting fatty acid biosynthesis, and promoting fatty acid oxidation. In other words, as the activity of AMPK increases, the activity of ACC decreases, thereby suppressing fat production.

In one embodiment of the present invention, in order to confirm the effect of inhibiting lipid accumulation or adipogenesis in the liver by manasanthin A or manasanthin B, palmitate-treated liver cells are treated with manasanthin A or manasanthin B. Next, the degree of AMPK phosphorylation was confirmed by Western blot and this was quantified.

As a result, as shown in FIG. 9, the phosphorylation of AMPK decreased in the group treated with palmitate only, whereas the treatment of manasanthin A or manasanthin B in the group treated with palmitate or not treated AMPK phosphorylation ( Activation) was confirmed to increase.

10 is a data obtained by confirming and quantifying the degree of AMPK and ACC phosphorylation by Western blot when palmitate and/or IL-6-treated liver cells were treated with manasanthin A or manasanthin B. Palmitate alone, palmitate In the group treated with Tate and IL-6 at the same time, AMPK phosphorylation was decreased compared to the control group, and as a result, the phosphorylation of ACC was also decreased.

On the other hand, in the group treated with manasanthin A or manasanthin B, as AMPK phosphorylation increased, phosphorylation of ACC increased, indicating that ACC was inactivated, inhibiting fat synthesis, while promoting the fat oxidation process. it means.

In addition, in another embodiment of the present invention, when palmitate-treated liver cells are treated with manasanthin A or manasanthin B, triglyceride (TG) content and lipid accumulation in liver cells are oil- It was confirmed by the oil-red-O method, and as a result, as shown in FIG. 11, in the group treated with palmitate only, the content and lipid accumulation of triglycerides in liver cells increased sharply compared to the control group, whereas mana It was confirmed that the triglyceride content and lipid accumulation in liver cells were significantly reduced in the group treated with Xanthine A or Mana Xanthine B.

12 is data confirming the activity of SIRT 1 (related to fat metabolism, glucose metabolism, and hepatocyte proliferation) and protein expression level by manasanthin A or manasanthin B, and increases SIRT1 activity by manasanthin A or manasanthin B treatment Was not observed. In addition, it was confirmed that the expression of the SIRT1 protein was decreased by palmitate, but no increase in the expression of the SIRT1 protein by the treatment of manasanthin A or manasanthin B was observed.

That is, it was confirmed that the effect of reducing fat metabolism, hepatocyte proliferation, triglyceride content, and lipid accumulation by manasanthin A and manasanthin B was not an effect by promoting SIRT1 protein expression or promoting activity.

In the present invention, the composition inhibits the production of microtubule-associated protein light chain 3 (LC3)-II or p62 protein involved in autophagy blockages; Promoting AMP-activated protein kinase (AMPK) activity; And inhibition of acetyl-CoA carboxylase (ACC) activity; Characterized in that it induces the autophagy of non-alcoholic fatty liver through one or more mechanisms selected from the group consisting of.

Autophagy (autophagy or autophagy) is a basic intracellular mechanism that removes unnecessary components such as proteins, lipids, and organelles through lysosomes, and plays a very important role in liver lipid metabolism, insulin sensitivity, and liver tissue damage. Have. Fatty liver in humans is known to induce an imbalance of autophagy, thereby reducing liver lipid accumulation and autophagy function involved in the development and progression of nonalcoholic steatohepatitis (NASH).

LC3 (Microtubule-associated protein light chain 3)-II or p62 protein is known to be involved in autophagy blockages, and saturated fatty acids, ammonium chloride and leupeptin are LC3-II and The p62 protein expression is regulated to induce autophagy arrest, thereby increasing the number and accumulation of autophagosomes (APs).

In one embodiment of the present invention, in order to confirm the autophagy induction effect by manasanthin A or manasanthin B, palmitate-treated liver cells are treated with manasanthin A or manasanthin B, and then, The expression levels of LC3-II and p62 proteins were confirmed by Western blot, and these were quantified. As a result, as shown in FIG. 13, it was confirmed that the palmitate-treated liver cells decreased LC3-II and p62 protein expression according to the concentration of manasanthin A or manasanthin B. In particular, p62 is manasanthin A and manasanthin B. It was confirmed that both showed the level of expression of LC3-II and p62 proteins similar to those of the control at a concentration of 20nM.

In another embodiment of the present invention, palmitate or ammonium chloride/leupeptin-treated liver cells are treated with manasanthin A or manasanthin B, followed by LC3-II and p62 protein expression. The degree was confirmed by Western blot and this was quantified. As a result, as shown in FIG. 14, the group treated with palmitate alone and palmitate/ammonium chloride/leupeptin at the same time rapidly increased the level of LC3-II protein expression compared to the control group, whereas manasanthin A or manasanthin B was treated. One group confirmed that the expression level of the LC3-II protein decreased, and the p62 protein was also confirmed that the expression level of the p62 protein decreased in the group treated with manasanthin A or manasanthin B, like the LC3-II protein.

In addition, when AMPK is phosphorylated and activated, it is known to induce autophagy, and in another embodiment of the present invention, manasanthin in liver cells treated with palmitate or ammonium chloride/leupeptin After treatment with A or manasanthin B, the degree of phosphorylation of AMPK and ACC was confirmed by Western blot, and this was quantified. As a result, as shown in FIG. 15, the group treated with palmitate alone and palmitate/ammonium chloride/leupeptin at the same time sharply decreased AMPK phosphorylation (activation) compared to the control group, whereas manasanthin A or manasanthin B It was confirmed that AMPK phosphorylation was significantly increased in the treated group, and AMPK phosphorylation was increased compared to the control group in both manasanthin A and manasanthin B.

In the case of ACC, the group treated with palmitate alone and palmitate/ammonium chloride/leupeptin at the same time decreased or increased ACC phosphorylation (inactivation) compared to the control group, but in the group treated with manasanthin A or manasanthin B. It was confirmed that ACC phosphorylation was increased.

In another embodiment of the present invention, protein kinase C (PKCθ), dephosphorylation enzyme-2 in order to determine whether or not manasanthin A or manasanthin B has a cellular function effect on palmitate-treated hepatocytes. The expression levels of (protein phosphatase-2A; PP2A) and extracellular signal-regulated kinases 1/2 (pERK1/2) were confirmed.

In the palmitate-treated hepatocytes, PP2A protein activity and expression were not increased by manasanthin A or manasanthin B treatment, whereas PKCθ phosphorylation (FIG. 16) decreased.

In addition, as a result of confirming ERK 1/2 phosphorylation, ERK1/2 phosphorylation was significantly reduced (Figs.17A and 17B) by treatment with manasanthin A or manasanthin B in palmitate-treated hepatocytes, which is palmitate. ERK1/2 phosphorylation was increased, but ERK1/2 phosphorylation was significantly reduced by the addition of manasanthin A or manasanthin B, and AMPK activity was increased due to decreased ERK1/2 phosphorylation. U0126 and PD184352 were used as positive controls, and both are known to inhibit ERK 1/2 activity.

That is, although PP2A expression is not regulated by manasanthin A or manasanthin B of the present invention, c-Raf/MEK/ERK pathway activity can be inhibited by inhibiting phosphorylation of PKCθ and ERK1/2, thereby AMPK activity It was confirmed that this increases.

In addition, AMPK is known to increase its activity by recognizing AMP (adenosine monophosphate).In the present invention, in order to confirm whether the effect of increasing AMPK activity by manasanthin A or manasanthin B is related to AMP concentration, intracellular AMP and The ATP ratio was measured. As a result, as shown in FIG. 18, it was confirmed that the AMP/ATP ratio increased to a level similar to that of the control group in the case of the mana santin A or mana santin B treatment group. This means that the AMPK activity increases as the intracellular AMP concentration increases by the treatment of manasanthin A or manasanthin B.

In the present invention, in order to confirm the preventive or therapeutic effect of non-alcoholic fatty liver disease by manasanthin A or manasanthin B, human liver cells are treated with palmitate, and then manasanthin A or manasanthin B is treated. The therapeutic effect of non-alcoholic fatty liver disease as shown in FIGS. 4 to 18 of the present invention was confirmed. In addition, in the present invention, unlike the above method, treatment with manasanthin A or manasanthin B, and then treatment with palmitate, confirmed the preventive effect of non-alcoholic fatty liver disease, and all the results of the preventive effect were the same as the data confirming the treatment effect. It was confirmed that the results were shown.

That is, it was confirmed that the composition for preventing or treating non-alcoholic fatty liver disease comprising manassantin A or manassantin B of the present invention has no cytotoxicity and genotoxicity, as shown in FIG. , Manassantin A or manassantin B

1) inhibition of the production of one or more inflammatory cytokines selected from the group consisting of interleukin-6 (IL-6) and interleukin-8 (IL-8); Inhibition of c-Jun N-terminal kinases (JNK) activity; And inhibition of inflammation caused by non-alcoholic fatty liver disease through inhibition of the activity of nuclear factor-kappaB (NF-κB);

2) inhibition of apoptosis due to non-alcoholic fatty liver disease by inhibiting the production of poly (ADP-ribose) polymerase (Poly ADP ribose polymerase: PARP) or caspase-3;

3) inhibition of gp130 (Glycoprotein 130) production or STAT3 (signal transducer and activator of transcription 3) activity to inhibit progression and metastasis of non-alcoholic fatty liver disease;

4) reduction of triglyceride content or lipid accumulation in liver cells or liver by promoting AMP-activated protein kinase (AMPK) activity or inhibiting acetyl-CoA carboxylase (ACC) activity;

5) Autophagy blockages involved in microtubule-associated protein light chain 3 (LC3)-II or p62 protein production, or by promoting AMP-activated protein kinase (AMPK) activity. Judo; And

6) Inhibition of PKCθ (protein kinase C theta) or ERK1/2 (Extracellular signal-regulated kinases 1/2) activity related to non-alcoholic fatty liver disease;

Since it has one or more mechanisms (effects) selected from the group consisting of, it can be usefully utilized as a pharmaceutical composition for preventing and/or treating non-alcoholic fatty liver or a health functional food composition for improving and/or improving non-alcoholic fatty liver.

The pharmaceutical composition for preventing or treating non-alcoholic fatty liver comprising manasanthin A or manasanthin B according to the present invention as an active ingredient may additionally contain other natural substances or compounds having preventive and/or therapeutic efficacy for non-alcoholic fatty liver. In addition, the pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, humans, and the like through various routes including oral, transdermal, subcutaneous, intravenous, or intramuscular. In addition, the pharmaceutical composition for preventing or treating non-alcoholic fatty liver comprising manasanthin A or manasanthin B according to the present invention as an active ingredient may be formulated into various formulations. In the case of formulation, it can be formulated using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, and capsules, and such solid preparations include at least one excipient (e.g., starch, sucrose, lactose And gelatin) and the like may be mixed and prepared. In addition, lubricants other than simple excipients may also be used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, fragrances and preservatives are included. Can be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

The dosage of the pharmaceutical composition for preventing and treating non-alcoholic fatty liver comprising manasanthin A or manasanthin B according to the present invention may be increased or decreased according to the route of administration, the degree of disease, sex, weight, age, and the like. Therefore, the dosage does not limit the scope of the present invention in any way.

When administering to the human body a pharmaceutical composition for preventing or treating non-alcoholic fatty liver containing manasanthin A or manasanthin B of the present invention as an active ingredient, since it is a natural extract, there is less fear of side effects than other synthetic drugs, so Can be used.

In addition, the present invention provides a health functional food composition for the prevention or improvement of non-alcoholic fatty liver comprising the mana santin A or mana santin B as an active ingredient. The health functional food composition for preventing or improving non-alcoholic fatty liver of the present invention has the effect of preventing or improving non-alcoholic fatty liver as described above. The types of health functional foods including the health functional food composition for preventing or improving non-alcoholic fatty liver comprising manasanthin A or manasanthin B of the present invention as an active ingredient are not particularly limited, and for example, meat, sausage, bread, It may be chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes.

The health food is used with other foods or food additives in addition to the manasanthin A or manasanthin B, and may be appropriately used according to a conventional method. For example, a beverage for preventing non-alcoholic fatty liver that includes manasanthin A or manasanthin B as an active ingredient includes calcium, gasoline concentrate, liquid fructose, purified water, etc., in addition to manasanthin A or manasanthin B as an active ingredient. After adding and mixing, filling in a drink bottle, sterilizing, and cooling to room temperature, a beverage can be prepared. In addition, the health supplement for the prevention of non-alcoholic fatty liver comprising manasanthin A or manasanthin B as an active ingredient is a nutritional supplement component (vitamins B1, B2, B5, B6, E and acetic acid esters, Nicotinic acid amide), oligosaccharide, 50% ethanol, and purified water are added and mixed, molded into granules, dried in a vacuum dryer, passed through a 12-14 mesh, uniformly produced, and extruded in an appropriate amount to be purified or It can be made into powder or filled into hard capsules to produce hard capsule products.

The effective dose of manasanthin A or manasanthin B contained in the health food can be used in accordance with the effective dose of the pharmaceutical composition, and the mixed amount of the active ingredient will be appropriately determined according to the purpose of use, such as prophylactic or therapeutic treatment. I can. In the case of long-term intake for the purpose of health and hygiene or for the purpose of health control, it may be less than the above range.

Hereinafter, the present invention will be described in detail with reference to preferred embodiments so that those of ordinary skill in the art can easily implement the present invention. However, the present invention may be implemented in various different forms and is not limited to the embodiments described herein.

Manasanthin A( manassantin A) or Manasanthin B( manassantin B) Separation and purification

In the present invention, manasanthin A and manasanthin B were separated and purified from Sambaekcho.

First, 2 kg of Sambaek sec was dried and pulverized at 50° C. or lower, extracted with 2,000 ml of methanol, and then concentrated and dried to obtain 326 g of powder.

Then, the methanolic extract was suspended in distilled water and then sequentially fractionated with n-hexane. The water fraction was successively fractionated with ethyl acetate (EtOAc), and the EtOAC fraction was filtered seven times with magnesium sulfate to remove moisture.

Thereafter, the EtOAC extract was subjected to a flash silica gel column (6.5 x 45 cm; 1.07734.9025, Merck, Germany), and then F1 to F4 using 4 types of n-hexane-acetone (hexane: ethyl acetate, 2:1, 1:1, 1:2, 1:4) solvent. Fractionated.

Of the above fractions, the F1 fraction and the F4 fraction were applied to Sephatex LH-20 column chromatography (17-0090-02, GE Healthcare Biosciences AB, Sweden) and ODS Sepak cartridge (5122487, Grace Davison Discovery Sciences, India). After that, it was eluted with methanol to obtain compound 1 and compound 2.

Compound 1 and Compound 2 are HPLC (High Performance Liquid Chromatograph System; L-2455, Hitachi, Japan) and 1H/13C-NMR spectrum (nuclear magnetic resonance spectra; nuclear magnetic resonance spectra (JNM ECA-600, Jeol, Japan)) As a result, as shown in FIG. 1, compound 1 was found to be manassantin A represented by the following formula (1), and compound 2 was found to be manassantin B represented by the following formula (2). Became.

[Formula 1]

[Formula 2]

Manasanthin A and Manasanthin B toxicity measurement

In the present invention, in order to confirm the cytotoxicity and genotoxicity of manasanthin A or manasanthin B obtained in Example 1, human liver cells (Human primary hepatocytes, Hu4248, Invitrogen, USA) were treated with manasanthin A or manasanthin B. After treatment by concentration, the cell viability was measured.

First, human liver cells were inoculated into 6 wells to become 3×10 ⁴ cells, and then, using a plating medium (cat.no, A1217601 and CM3000, invitrogen, USA), at 37°C and 5% CO ₂ for 24 hours. During incubation. Then, 1 µl each was added so that the concentration of manasanthin A or manasanthin B obtained in Example 1 was 0, 10, 40, 80, 160 nM, and cultured at 37°C and 5% CO ₂ for 24 hours. , Cell viability was measured using a trypan blue exclusion assay known in the art.

As a result, the cytotoxicity was not observed at 0 to 80 nM concentration of manasanthin A or mana santin B, but at 160 nM concentration, cell viability decreased to 85% or less, which is a critical value compared to the control, which affects cell death. Confirmed.

In addition, a green screen HC assay without S9 metabolic activity (GreenScreen HC assay, Gentronix Limited, BioHub at Alderley Park, Alderley Edge, Cheshire SK10 4TG, UK) was requested to confirm the cytotoxic and genotoxic effects of manasanthin B. .

As a result, as shown in FIG. 3, cytotoxicity was not observed at 0 to 80 nM concentration, and it was confirmed that the relative cell density at 160 nM or higher was reduced to 80% or less set as a threshold value compared to the control group.

However, in HC analysis, it was confirmed that the fluorescence induction value is negative (threshold value is less than 1.5 times (50%)) in manasanthin B of 0 to 640 nM, which means that manasanthin B is cytotoxic within the above range. And no genotoxicity.

Manasanthin A and Manasanthin Confirmation of B's inhibitory activity against inflammation by non-alcoholic fatty liver

3-1: JNK And NF -KB phosphorylation degree check

In the present invention, in order to check whether there is an effect of preventing and treating inflammation of non-alcoholic fatty liver caused by manasanthin A or manasanthin B, liver cells treated with palmitate and/or interleukin-6 (IL-6), which are saturated fatty acids, are Manasanthin A or manasanthin B was treated respectively.

Palmitate and IL-6 were used to induce inflammation in liver cells, and palmitate promotes the production of cytokines such as TNF-α and IL-6 to induce inflammation and induce insulin resistance, and IL-6 is It is known to promote the expression of various inflammatory cytokines.

First, human liver cells (Huh-7, hepato cellular carcinoma) were inoculated into 6-wells to become 8×10 ⁵ cells, and then cultured for 24 hours at 37°C and 5% CO ₂ using DMEM medium. . 1 μl each was added so that the concentration of manasanthin A or manasanthin B obtained in Example 1 was 20 nM, and incubated for 1 hour under conditions of 37° C. and 5% CO ₂ , and then palmitate (0.5 mM, P9767). , Sigma-Aldrich, USA), followed by incubation for 10-12 hours at 37°C and 5% CO ₂ conditions, and then interleukin-6 (IL-6; 20 ng/ml, 9954, Cell signaling technology, USA) After each treatment was incubated for 15 minutes at 37 ℃, 5% CO ₂ conditions.

The group that was not treated with anything was used as a control group, and IL-6 treatment group, palmitate treatment group, palmitate/IL-6/manasanthin A treatment group, palmitate/IL-6/manasanthin B treatment group and palmitate/ The treatment was divided into IL-6 treatment groups.

The cells cultured in the above were prepared by a known method in which a protein degradation inhibitor (P8340, Sigma-Aldrich, USA) and a phosphorylation deinhibitor (P0044, Sigma-Aldrich, USA) were added to a buffer of RIPA (R0278, Sigma-Aldrich, USA). The protein was isolated using, and the isolated protein concentration was measured using a BCA protein assay kit (Thermo Scientific, USA).

Then, SDS-PAGE was performed by loading 10 to 20 μg of protein per well, and the protein was electrotransferred to a PVDF membrane (Millipore, USA) by a method known in the art, and then PVDF The membrane was blocked at room temperature for 1 hour by treatment with a TBST (tris buffered saline-0.1% Tween 20) buffer containing 5% skim milk powder. Then, a primary antibody (anti-pJNK, 9251; anti-tJNK, 9258; anti-pNFκB, 3033; anti-NFκB, 8242; β-actin, 4970; Cell signaling technology, USA) was treated overnight (approximately Time; 8-12 hours), followed by washing with TBST.

Thereafter, a blocking solution containing a horseradish peroxidase conjugated secondary antibody was treated and reacted at room temperature for 1 hour, followed by washing with TBST. The washed membrane was analyzed according to the manual using an enhanced chemiluminescence kit (Thermo Scientific, USA), and FlurChem M (Proteinsimple, USA) was used to quantify the expression levels of pJNK and pNF-κB among Western blot results. After scanning, it was quantified using the imageJ (National Institutes of Health Government Agency, USA) program.

As a result, as shown in FIGS. 4A and 4B, liver cells in which inflammation was induced by palmitate or/and IL-6 rapidly increased the degree of phosphorylation of JNK and NF-κB, whereas manasanthin A or manasanthin B It was confirmed that the degree of phosphorylation of JNK and NF-κB was significantly reduced in the group treated with.

The decrease in the phosphorylation of JNK and NF-κB means that the activity of JNK and NF-κB decreases, thereby reducing the inflammatory response, and manasanthin A or manasanthin B of the present invention significantly reduces the induction of inflammation in liver cells. It was confirmed what to do.

3-2: IL -8 and IL -6 mRNA Confirmation of expression change

In order to confirm the expression levels of IL-8 and IL-6 mRNA by manasanthin A or manasanthin B, experiments were conducted in the same manner as in Example 3-1, and then cultured cells were obtained to obtain Trizol (Trizol, 15596-026, Invitrogen, USA) was used to obtain total RNA. Then, cDNA was synthesized using a CDNA synthesis kit (cDNA synthesis kit, 11146024, Invitrogen, USA), and then PCR was performed using the primers shown in Table 1 below.

Name sequence(5' -> 3') Sequence number IL-6 Forward ACC TCA GAT TGT TGT TGT SEQ ID NO: 1 Reverse GTC CTA ACG CTC ATA CTT SEQ ID NO: 2 IL-8 Forward CTA GGA CAA GAG CCA GGA AG SEQ ID NO: 3 Reverse GGT GGA AAG GTT TGG AGT ATG SEQ ID NO: 4 GAPDH Forward ACA GTC AGC CGC ATC TTC SEQ ID NO: 5 Reverse CTC CGA CCT TCA CCT TCC SEQ ID NO: 6

Real-time PCR was performed under the following conditions; After 10 minutes reaction at 95°C, 40 cycles were repeated for 15 seconds at 94°C, 30 seconds at 60°C, and 30 seconds at 80°C. Data analysis was performed using the CFX96 Real-Time system (Bio -Rad, USA) software was used.

The relative mRNA expression level was calculated using the Δ CT method, The mRNA level of each gene was normalized using GAPDH .

As a result, as shown in Figs. 4C and 4D, IL-8 and IL-6 mRNA expression in the group treated with manasanthin A or manasanthin B in liver cells inflamed by palmitate or IL-6 treatment It was confirmed that the amount was remarkably decreased, and it was confirmed that manasanthin A or manasanthin B of the present invention significantly reduced the expression of inflammatory cytokines in liver cells.

3-3: TNF -α on treated liver cells Manasanthin A or Manasanthin B according to treatment JNK And NF -KB phosphorylation degree check

To confirm JNK and NF-κB phosphorylation when hepatic cells treated with tumor necrosis factor-α (TNF-α) in addition to palmitate or IL-6 were treated with manasanthin A or manasanthin B. For this, cells were cultured in the same manner as in Example 3-1, and then treated with 50 ng/ml of TNF-α (8902, Cell signaling technology, USA) instead of palmitate or IL-6, and then manasanthin A or Manasanthin B was treated with 20 nM, and all experiments for performing western blot were performed in the same manner as in Example 3-1.

A group that was not treated with anything was used as a control group, and treated by dividing into a TNF-α treatment group, a TNF-α / manasanthin A treatment group, and a TNF-α / manasanthin B treatment group.

As a result, as shown in FIG. 5, the degree of phosphorylation of JNK and NF-κB was remarkable in the group treated with manasanthin A or manasanthin B to liver cells induced by TNF-α, as in the results of FIG. 4 It was confirmed that it decreased significantly.

Manasanthin A and Manasanthin Confirmation of inhibition of apoptosis by non-alcoholic fatty liver of B

In the present invention, in order to confirm the anti-apoptotic effect of manasanthin A or manasanthin B, manasanthin A or manasanthin B is added to palmitate and/or interleukin-6 (IL-6)-treated liver cells, respectively. After treatment, the degree of activity by cleavage of Poly ADP ribose polymerase (PARP) and caspase-3 was confirmed by Western blot.

First, cells were cultured in the same manner as in Example 3-1, and then palmitate or/and IL-6 were treated, and then manasanthin A or manasanthin B was treated with 10 nM, and proteins were separated to perform western blot. I did. When performing Western blot, the primary antibodies are anti-PARP (anti-PARP; 9532, Cell signaling technology, US), anti-cPARP (anti-cPARP; 5625, Cell signaling technology, US), anti-CASP3 (anti-CASP3; 9665 Cell signaling technology, US), anti-cCASP3 (anti-cCASP3; 9665, Cell signaling technology, US), and anti-β-actin (anti-β-actin; 4970, Cell signaling technology, US) were used.

The group that was not treated with anything was used as a control group, and IL-6 treatment group, palmitate treatment group, palmitate/IL-6/manasanthin A treatment group, palmitate/IL-6/manasanthin B treatment group and palmitate/ The treatment was divided into IL-6 treatment groups.

As a result, it was confirmed that the production of cleaved-PARP (PARP) and cleaved-caspase-3 (cleaved-caspase-3) cleaved by manasanthin A or manasanthin B was significantly reduced, as shown in FIG. It means that manasanthin A or manasanthin B can inhibit the cleavage of PARP or caspase-3 to inhibit apoptosis caused by non-alcoholic fatty liver disease.

Manasanthin A and Manasanthin Confirmation of inhibition of progression and metastasis of non-alcoholic fatty liver by B

5-1: Palmitate On liver cells treated alone Manasanthin A or Manasanthin B according to treatment gp130 Create and STAT3 Check the degree of phosphorylation

In the present invention, when manasanthin A or manasanthin B is treated, the degree of gp130 (Glycoprotein 130) production and STAT3 (signal transducer and activator of transcription 3) phosphorylation, which are related to the progression and metastasis of non-alcoholic fatty liver, were confirmed.

First, cells were cultured in the same manner as in Example 3-1, and then palmitate was treated, then manasanthin A or manasanthin B was treated with 10 nM and 20 nM, respectively, and proteins were separated to perform western blot. . When performing Western blot, the primary antibodies are anti-gp130 (anti-gp130; 3732, Cell signaling technology, US), anti-pStat3 (anti-pStat3; 9145, Cell signaling technology, US), and anti-Stat3 (anti-Stat3; 4904, Cell signaling technology, US) and anti-β-actin (anti-β-actin; 4970, Cell signaling technology, US) were used.

In the experiment to confirm the expression level of gp130, the group that was not treated with anything was used as the control group, and the manasanthin A 10 nM treatment group, the manasanthin A 20 nM treatment group, the palmitate treatment group, palmitate/manasanthin A 10 nM treatment group, Experiment by dividing into palmitate/manasanthin A 20 nM treatment group, manasanthin B 10 nM treatment group, manasanthin B 20 nM treatment group, palmitate/manasanthin B 10 nM treatment group, palmitate manasanthin B 20 nM treatment group Was performed.

In the experiment to confirm the degree of STAT3 phosphorylation, the group that was not treated with anything was used as a control group, and the IL-6 treatment group, IL-6/manasanthin A 10 nM treatment group, IL-6/manasanthin A 20 nM treatment group, and IL- The experiment was performed by dividing into 6/manasanthin B 10 nM treatment group and IL-6/manasanthin B 20 nM treatment group.

The relative expression level of gp130 was calculated by comparing it with the expression level of β-actin, and the degree of STAT3 phosphorylation was calculated by comparison with the expression level of non-phosphorylated general STAT3.

As a result, as shown in FIG. 7, it was confirmed that STAT3 phosphorylation was significantly reduced when manasanthin A or manasanthin B was treated in liver cells induced by inflammation by IL-6, and the expression level of gp130 protein was manasanthin A. Alternatively, it was confirmed that the expression decreased depending on the concentration of manasanthin B treatment.

5-2: Palmitate / IL -6 on treated liver cells Manasanthin A or Manasanthin B according to treatment gp130 produce, STAT3 And Tyk Check the degree of phosphorylation

When palmitate and/or IL-6-treated liver cells were treated with manasanthin A or manasanthin B, the degree of gp130 production, STAT3 and Tyk phosphorylation were confirmed by Western blot.

First, cells were cultured in the same manner as in Example 3-1, and then treated with palmitate and/or IL-6, and then treated with manasanthin A or manasanthin B. In the group for confirming gp130 production, manasanthin A or manasanthin B was each treated with 20 nM, and a group for confirming the degree of STAT3 and Tyk phosphorylation was treated with 10 nM, respectively, and proteins were separated to perform western blot. When performing Western blot, the same antibody as in Example 5-1 was used as the primary antibody, and additionally, anti-tyk2 (anti-tyk2; 14193, Cell signaling technology, USA) and anti-ptyk2 (anti-ptyk2; 9321, Cell signaling technology (USA) was used.

The group that was not treated with anything was used as a control group, and IL-6 treatment group, palmitate treatment group, palmitate/IL-6/manasanthin A treatment group, palmitate/IL-6/manasanthin B treatment group and palmitate/ The treatment was divided into IL-6 treatment groups.

The relative expression level of gp130 was calculated by comparing the expression level of β-actin, and the degree of phosphorylation of STAT3 and tyk2 was calculated by comparing the level of expression of normal STAT3 and tyk2 without phosphorylation.

As a result, as shown in FIG. 8, when the hepatocytes induced inflammation by treatment with palmitate and IL-6 alone or simultaneously were treated with manasanthin A or manasanthin B, the expression level of gp130, STAT3 and tyrosine kinase (tyrosine kinase: tyk) phosphorylation was significantly reduced.

This means that manasanthin A or manasanthin B can inhibit the progression and metastasis of non-alcoholic fatty liver disease by inhibiting gp130 production and STAT3 activity.

Manasanthin A and Manasanthin Confirmation of reduction of lipid accumulation in liver cells by B

6-1: Palmitate On liver cells treated alone Manasanthin A or Manasanthin B according to treatment AMPK Check the degree of phosphorylation (activation)

In the present invention, in order to confirm the effect of inhibiting lipid accumulation or fat production in the liver by manasanthin A or manasanthin B, palmitate-treated liver cells are treated with manasanthin A or manasanthin B, and then AMPK phosphorylation. To the extent, Western blot was performed.

First, cells were cultured in the same manner as in Example 3-1, and then palmitate was treated, and then manasanthin A or manasanthin B were each treated with 10 nM, and proteins were separated to perform western blot. When performing Western blot, the primary antibodies are anti-AMPK (anti-AMPK; 2603, Cell signaling technology, US), anti-pAMPK (anti-pAMPK; 2531, Cell signaling technology, US), and anti-β-actin (anti- β-actin; 4970, Cell signaling technology, USA) was used. The degree of AMPK phosphorylation was calculated by comparing with the expression level of non-phosphorylated general AMPK.

The group that was not treated with anything was used as a control group, and the palmitate treatment group, manasanthin A treatment group, manasanthin B treatment group, palmitate treatment group, palmitate/manasanthin A treatment group, and palmite/manasanthin B treatment group. It was divided and processed.

As a result, as shown in FIG. 9, the group treated with palmitate only reduced phosphorylation of AMPK compared to the control group that did not treat anything, whereas the group treated with manasanthin A or manasanthin B was the control and palmitate. It was confirmed that AMPK phosphorylation (activation) was significantly increased compared to the treatment group.

6-2: Palmitate / IL -6 on treated liver cells Manasanthin A or Manasanthin B according to treatment AMPK Phosphorylation (activation) and ACC Check the degree of phosphorylation (inactivation)

When palmitate and/or IL-6-treated liver cells were treated with manasanthin A or manasanthin B, the degree of phosphorylation of AMPK and ACC (acetyl CoA carboxylase; Acetyl-CoA carboxylase) was examined by Western blot. .

First, cells were cultured in the same manner as in Example 3-1, and then palmitate and/or IL-6 were treated, and then manasanthin A or manasanthin B was treated with 20 nM, respectively, and proteins were separated and western blot Was performed. When performing Western blot, the same antibody as in Example 6-1 was used as the primary antibody, and additionally, anti-ACC (anti-ACC; 3676, Cell signaling technology, USA), anti-pACC (anti-pACC; 11818, Cell signaling technology, USA) and anti-FASN (3189, Cell signaling technology, USA) were used.

The group that was not treated with anything was used as a control group, and IL-6 treatment group, palmitate treatment group, palmitate/IL-6/manasanthin A treatment group, palmitate/IL-6/manasanthin B treatment group and palmitate/ The treatment was divided into IL-6 treatment groups.

As a result, as shown in FIG. 10, the group treated with palmitate alone, palmitate and IL-6 at the same time decreased AMPK phosphorylation compared to the control group, resulting in a decrease in ACC phosphorylation, which was an increase in ACC activity. It means that synthesis is accelerated.

On the other hand, it was confirmed that the phosphorylation of ACC increased as the phosphorylation of AMPK increased in the group treated with manasanthin A or manasanthin B. This means that ACC is inactivated, inhibiting fat synthesis and promoting fat oxidation.

Manasanthin A and Manasanthin Confirmation of triglyceride content and lipid accumulation in liver cells by B

7-1: Confirmation of triglyceride and lipid accumulation

When palmitate-treated liver cells were treated with manasanthin A or manasanthin B, triglyceride (TG) content and lipid accumulation in hepatic cells were determined by oil-red-O. It was measured by optical microscopy by the method and direct electron microscopy in liver cells.

First, cells were cultured in the same manner as in Example 3-1, and then palmitate was treated, and then manasanthin A or manasanthin B was treated with 20 nM, respectively, and L-Type TG M kit (Wako chemicals Co. , Japan) was used to quantify triglycerides. The level of triglyceride accumulation for each treatment was normalized by measuring the amount of protein (BCA measurement method).

In addition, the intracellular fat staining method (Oil-red-O) was observed using a fat staining kit {Lipid (Oil Red O) Staining kit, K580-24, BioVision, USA}. All methods were performed according to the manufacturer's manual, and after staining, an optical microscope (Nikon microscope, Nikon, Japan) was used to determine the degree of lipid accumulation in liver cells.

Electron microscopy was observed to investigate fat accumulation in liver cells. After treatment with palmitate, manasanthin A or manasanthin B was treated with 20 nM, respectively, and trypsin-EDTA (T4049, Sigma-Aldrich, USA) was treated, followed by centrifugation to collect cells. After receiving the request, it was commissioned to the State Virginia Medical Center (VCU Medical Center, Richmond, VA, USA) and the Microscopy Laboratory.

As a result, as shown in FIG. 11, in the group treated with palmitate only, the content and lipid accumulation of triglycerides in liver cells increased sharply compared to the control group, whereas the group treated with manasanthin A or manasanthin B was neutral in liver cells. It was confirmed that the fat content and lipid accumulation significantly decreased.

7-2: local metabolism, Glucose Metabolism and hepatocyte proliferation related protein SIRT1 Expression level Confirm

In the present invention, in order to confirm whether the prophylactic/treatment effect of non-alcoholic fatty liver by manasanthin A and manasanthin B is related to the regulation of the expression of SIRT 1, a protein related to fat metabolism, glucose metabolism, and hepatocyte proliferation, manasanthin A or manasanthin The SIRT 1 activity was measured by reacting B directly with the SIRT1 substrate. Resveratrol was used as a positive control, and resveratrol is an antioxidant found in plants and is known to be involved in various metabolic processes such as lipolysis by increasing the expression of SIRT 1.

SIRT 1 activity was measured using a SIRT1 direct fluorescent screening assay kit (SIRT1 Direct Fluorescent Screening Assay Kit, Cat no. 10010401, USA), and as shown in FIG. 12A, it was observed that the SIRT 1 activity rapidly increased by treatment with resveratrol. Although it was confirmed, it was observed that the activity was not increased by manasanthin A or manasanthin B.

Next, the expression level of the SIRT 1 protein was confirmed by Western blot.

First, cells were cultured in the same manner as in Example 3-1, and then palmitate was treated, and then manasanthin A or manasanthin B was treated with 20 and 40 nM, respectively, and proteins were separated to perform western blot. . When performing Western blot, the primary antibodies were anti-SIRT1 (anti-SIRT 1; cat. no, 2496, Cell signaling technology, USA) and anti-β-actin (anti-β-actin; 4970, Cell signaling technology, USA). Was used.

The group that was not treated with anything was used as a control group, manasanthin A 20 nM treatment group, manasanthin B 20 nM treatment group, palmitate treatment group, palmitate/manasanthin A 20 nM treatment group, palmitate/manasanthin B 20 nM Experiments were performed by dividing into treatment groups.

As a result, as shown in FIGS. 12B and 12C, it was confirmed that the expression of the SIRT 1 protein was decreased by palmitate, but no increase in the expression of the SIRT 1 protein by the treatment of manasanthin A or manasanthin B was observed.

That is, it was confirmed that the effects of manasanthin A and manasanthin B on fat metabolism, glucose metabolism, and hepatocyte proliferation are not effects of SIRT 1 protein expression or activity promotion.

Manasanthin A and Manasanthin Autophagy in non-alcoholic fatty liver by B ( autophagy ) Judo

8-1: Palmitate On liver cells treated alone Manasanthin A or Manasanthin B according to treatment LC3 -Ⅱ and p62 Confirmation of protein expression level

In the present invention, in order to confirm the autophagy-inducing effect of manasanthin A or manasanthin B, palmitate-treated liver cells are treated with manasanthin A or manasanthin B, and then LC3-II and The level of p62 protein expression was confirmed by Western blot.

First, cells were cultured in the same manner as in Example 3-1, and then palmitate was treated, and then manasanthin A or manasanthin B was treated with 10, 20, and 40 nM, respectively, and proteins were separated to obtain western blots. Performed. When performing Western blot, the primary antibodies are anti-LC3-II (anti-LC3-I, II; 4108, Cell signaling technology, USA), anti-p62 (anti-p62; 8025, Cell signaling technology, USA) and anti- β-actin (anti-β-actin; 4970, Cell signaling technology, USA) was used.

No treatment was used as a control group, palmitate treatment group, palmitate/manasanthin A 10 nM treatment group, palmitate/manasanthin A 20 nM treatment group, palmitate/manasanthin A 40 nM treatment group, palmitate The experiment was performed by dividing into / manasanthin B 10 nM treatment group, palmitate / manasanthin B 20 nM treatment group, and palmitate / manasanthin B 40 nM treatment group.

As a result, as shown in FIG. 13, it was confirmed that the palmitate-treated liver cells reduced LC3-II and p62 protein expression depending on the concentration of manasanthin A or manasanthin B. In particular, p62 is manasatin A and manasanthin B. It was confirmed that both showed the level of expression of LC3-II and p62 proteins similar to those of the control at a concentration of 20nM.

8-2: Palmitate ( palmitate ) Or ammonium chloride ( ammonium chloride )/Leupeptin ( leupeptin ) To the treated liver cells Manasanthin A or Manasanthin B according to treatment LC3 -II and p62 protein expression level confirmation

In the present invention, palmitate or ammonium chloride (ammonium chloride: NH ₄ Cl) / leupeptin (leupeptin: Leup) treated liver cells were treated with manasanthin A or manasanthin B, followed by LC3-II and p62 proteins. The expression level was confirmed by Western blot.

First, after culturing the cells in the same manner as in Example 3-1, palmitate or ammonium chloride (20mM, ammonium chloride, A9434, Sigma-Aldrich, USA) / Leupeptin (100uM, leupeptin, Sigma-Aldrich, USA) After treatment, mana santin A or mana santin B was treated with 20 nM, respectively, and the protein was separated to perform western blot.

When performing Western blot, the same antibody as in Example 8-1 was used as the primary antibody, and the relative expression levels of LC3-II and p62 were calculated by comparing with the expression level of β-actin.

The group that was not treated with anything was used as a control group, and manasanthin A treatment group, manasanthin B treatment group, palmitate treatment group, palmitate/manasanthin A treatment group, palmitate/manasanthin B treatment group, palmitate/NH ₄ The experiment was performed by dividing into a Cl/Leup treatment group, a palmitate/NH ₄ Cl/Leup/manasanthin A treatment group, and a palmitate/NH ₄ Cl/Leup/manasanthin B treatment group.

As a result, as shown in FIG. 14, the group treated with palmitate alone and palmitate/ammonium chloride/leupeptin at the same time rapidly increased the level of LC3-II protein expression compared to the control group, whereas manasanthin A or manasanthin B was treated. One group confirmed that the expression level of the LC3-II protein decreased.

As with the LC3-II protein, the p63 protein also showed that the group treated with palmitate/ammonium chloride/leupeptin at the same time and the group treated with manasanthin A or manasanthin B decreased the expression of p63 protein compared to the control group.

8-3: Palmitate ( palmitate ) Or ammonium chloride ( ammonium chloride )/Leupeptin ( leupeptin ) To the treated liver cells Manasanthin A or Manasanthin B according to treatment AMPK Phosphorylation (activation) and ACC Check the degree of phosphorylation (inactivation)

In the present invention, palmitate or ammonium chloride/leupeptin-treated liver cells were treated with manasanthin A or manasanthin B, and then AMPK and ACC phosphorylation levels were confirmed by Western blot.

First, cells were cultured in the same manner as in Example 8-2, and then proteins were separated to perform Western blot.

When performing Western blot, the same antibody as in Example 6-2 was used as the primary antibody, and the degree of phosphorylation of AMPK and ACC was calculated by comparing the expression levels of general AMPK and ACC, respectively.

As a result, as shown in Figure 15, palmitate alone treatment, palmitate / ammonium chloride / leupeptin treatment group significantly decreased AMPK phosphorylation (activation) compared to the control group, whereas manasantin A or manasanthin B were treated together. The group confirmed that AMPK phosphorylation was significantly increased, and both manasanthin A and manasanthin B showed increased AMPK phosphorylation compared to the control group.

In the case of ACC, the palmitate-treated group and the palmitate/ammonium chloride/leupeptin-treated group decreased or increased ACC phosphorylation (inactivation) compared to the control group, but the ACC phosphorylation was higher in the group treated with manasanthin A or manasanthin B. It was confirmed that it increased.

In non-alcoholic fatty liver Manasanthin A and Manasanthin Inhibition of cell function decline by B ( cell dysfunction ) Check the effect

In the present invention, protein kinase C (PKCθ), dephosphorylation enzyme-2 (phosphatase-2; PP2A), and extracellular signals in order to determine whether manasanthin A or manasanthin B inhibits cell function reduction. The expression level of extracellular signal-regulated kinases 1/2 (pERK) was confirmed.

9-1: PKC θ and PP2A Expression confirmation

In the present invention, palmitate-treated liver cells were treated with manasanthin A or manasanthin B, and then the expression levels of PKCθ and PP2A were confirmed by Western blot.

First, cells were cultured in the same manner as in Example 3-1, and then palmitate was treated, and then manasanthin A or manasanthin B was treated with 20 nM, respectively, and proteins were separated to perform western blot. When performing Western blot, the primary antibodies are anti-PP2A (anti-PP2A; cat.no 2041; Cell signaling technology, USA), anti-PKCθ (anti-PKCθ; cat. no 12206; Cell signaling technology, USA), and anti-PKCθ. pPKCθ (anti-pPKCθ; cat. no 9377; Cell signaling technology, USA) and anti-β-actin (anti-β-actin; 4970, Cell signaling technology, USA) were used.

The group that was not treated with anything was used as a control group, and the experiment was performed by dividing into a palmitate treatment group, a palmitate/manasanthin A 20 nM treatment group, and a palmitate/manasanthine B 20 nM treatment group.

As a result, as shown in Figure 16, the palmitate-treated group and the control group did not change the PP2A protein expression (Fig. 16A), it was confirmed that the PP2A protein activity increased in the palmitate-treated group (Fig. 16B). In addition, it was confirmed that there was no change in the PP2A protein activity and expression of the palmitate/manasanthin A 20 nM treatment group and the palmitate/manasanthin B 20 nM treatment group (FIGS. 16A and 16B ).

On the other hand, it was confirmed that the PKCθ phosphorylation was increased in the palmitate-treated group, and the PKCθ phosphorylation was significantly decreased in the group treated with manasanthin A or manasanthin B.

9-2: ERK 1/2 activity inhibition effect confirmed

In the present invention, palmitate-treated liver cells were treated with manasanthin A or manasanthin B, and then the degree of phosphorylation of ERK 1/2 was confirmed by Western blot. U0126 and PD184352 were used as positive controls, and both are known to inhibit ERK 1/2 activity.

First, cells were cultured in the same manner as in Example 3-1, and then palmitate was treated, and manasanthin A or manasanthin B were each treated with 20 nM, and proteins were separated to perform western blot. When performing Western blot, the primary antibody was anti-ERK 1/2 (anti-ERK 1/2; cat. no 9102; Cell signaling technology, USA), anti-pERK 1/2 (anti-pERK 1/2; cat. no 9101; Cell signaling technology, United States), anti-AMPK (anti-AMPK; 2603, Cell signaling technology, United States), anti-pAMPK (anti-pAMPK; 2531, Cell signaling technology, United States), and anti-β-actin ( anti-β-actin; 4970, Cell signaling technology, USA) was used.

The group that was not treated with anything was used as a control group, palmitate treatment group, palmitate/U0126 25 μM treatment group, palmitate/PD184352 1 μM treatment group, palmitate/manasanthin A 20 nM treatment group, palmitate/manasanthin B 20 nM Experiments were performed by dividing into treatment groups.

As a result, as shown in FIG. 17, it was confirmed that the palmitate-only treatment group increased ERK1/2 phosphorylation and decreased AMPK phosphorylation compared to the control group, whereas the positive control treatment groups (U0126 and PD184352) were palmitate-only treatment groups. Compared to that, it was confirmed that ERK1/2 phosphorylation decreased and AMPK phosphorylation increased.

In addition, it was confirmed that the ERK1/2 phosphorylation decreased and the AMPK phosphorylation increased at a level similar to that of the positive control U0126 treatment group in the group treated with manasanthin A or manasanthin B.

That is, it was confirmed that the c-Raf/MEK/ERK pathway activity can be inhibited by inhibiting the phosphorylation of ERK1/2 by manasantin A or manasanthin B of the present invention, thereby increasing AMPK activity.

9-3: AMP / ATP Check the percentage

AMPK is known to increase its activity by the level of AMP (adenosine monophosphate) production.

In the present invention, in order to determine whether the effect of increasing AMPK activity by manasanthin A or manasanthin B is related to the ratio of AMP and ATP concentration, the ratio of intracellular AMP and ATP was measured.

First, cells were cultured in the same manner as in Example 3-1, and then palmitate was treated, and then manasanthin A or manasanthin B was treated with 20 nM, respectively, and intracellular AMP (AMP-Glo assay kit, promega , USA) and ATP (The ENLITEN ATP assay kit, promega, USA) concentration was measured using each assay kit.

The group that was not treated with anything was used as a control group, and the experiment was performed by dividing into a palmitate treatment group, a palmitate/manasanthin A 20 nM treatment group, and a palmitate/manasanthine B 20 nM treatment group.

As a result, as shown in FIG. 18, the palmitate alone treatment group decreased the AMP/ATP ratio compared to the control group, whereas the AMP/ATP ratio increased to a level similar to that of the control group in the case of the manasanthin A or manasanthin B treatment group. Confirmed.

That is, it was confirmed that the AMPK activity increased as the intracellular AMP concentration increased by the treatment of manasanthin A or manasanthin B.

As described above, specific parts of the present invention have been described in detail, and for those of ordinary skill in the art, these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereby It will be obvious. Therefore, it will be said that the practical scope of the present invention is defined by the appended claims and their equivalents.

<110> liver disease Comprising Manassantin A or Manassantin B <120> Compositions for Prevention or Treatment of nonalcoholic fatty liver disease Comprising Manassantin A or Manassantin B <130> 1062916 <160> 6 <170> KopatentIn 2.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> IL-6 forward primer <400> 1 acctcagatt gttgttgt 18 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> IL-6 reverse primer <400> 2 gtcctaacgc tcatactt 18 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IL-8 forward primer <400> 3 ctaggacaag agccaggaag 20 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> IL-8 reverse primer <400> 4 ggtggaaagg tttggagtat g 21 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GAPDH forward primer <400> 5 acagtcagcc gcatcttc 18 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GAPDH reverse primer <400> 6 ctccgacctt caccttcc 18

Claims (14)

delete In vitro, a method of controlling the expression level of gp130 (Glycoprotein 130) / expression level of β-actin to 0.65 by treating liver cells with 10 nM of manassantin A represented by the following formula (1).
[Formula 1]

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