WO2021150077A1 - Pharmaceutical composition or health functional food for prevention or treatment of non-alcoholic fatty liver disease - Google Patents

Abstract

The present invention provides a pharmaceutical composition or health functional food for prevention or treatment of a non-alcoholic fatty liver disease, comprising at least one selected from the compounds represented by chemical formulas 1 to 4 as an active ingredient.

Description

Pharmaceutical composition or health functional food for the prevention or treatment of nonalcoholic fatty liver disease

The present invention relates to a pharmaceutical composition or health functional food for preventing or treating non-alcoholic fatty liver disease.

Fatty liver is a disease in which fat is deposited in the liver, and is usually defined as a case in which the weight due to fat deposition consists of 5% or more of the weight in the liver. Non-alcoholic fatty acid liver disease (NAFLD) refers to a case where the cause of fatty liver is not caused by alcohol. It includes all the processes leading to advanced fibrosis and cirrhosis.

Nonalcoholic fatty liver disease has been on the rise for several years, and is known to account for most of chronic liver diseases except for cases with clear causes such as viral or alcoholic. 70~90% of chronic liver diseases are caused by nonalcoholic fatty liver disease. There are reports that it was caused by

Even simple steatosis can progress to steatohepatitis, fibrosis, and cirrhosis, leading to death. In fact, 20-30% of nonalcoholic fatty liver disease histologically showed steatohepatitis (NASH) with fibrosis and inflammation, and if it progresses to steatohepatitis, the risk of developing cirrhosis, liver failure, and hepatocellular carcinoma increases. Although the mechanism of the development of nonalcoholic fatty liver has not yet been fully elucidated, fat accumulation in the liver is considered a feature of insulin resistance along with abdominal obesity, hypertension, type 2 diabetes, and hyperlipidemia, and is known to be closely related to metabolic syndrome. there is.

In the treatment of such nonalcoholic fatty liver disease, it is important to first treat factors related to fatty liver, such as obesity, diabetes, hyperlipidemia, blood pressure, and related drugs. Drugs based on various mechanisms such as weight loss drugs, insulin resistance improving drugs, hepatoprotective drugs, and antioxidants have been developed and clinically tested as drug therapy, but there is no drug approved as a treatment for nonalcoholic fatty liver disease yet. Since most patients with nonalcoholic fatty liver are overweight or obese, active weight loss, appropriate diet, and steady aerobic exercise are the most effective for now.

On the other hand, recently, in order to treat such non-alcoholic fatty liver, various studies for extracting active ingredients from herbal medicines or natural materials have been conducted. Korean Patent Laid-Open No. 2017-0135349 discloses a composition for preventing and treating non-alcoholic fatty liver comprising a ginseng seed extract. Republic of Korea Patent Publication No. 2018-0082362 discloses a composition for the treatment or prevention of non-alcoholic fatty liver disease comprising a steamed deodeok extract as an active ingredient. Republic of Korea Patent Publication No. 2019-0113272 discloses a composition for the treatment or prevention of non-alcoholic fatty liver disease, comprising an extract or fraction of glycolysis as an active ingredient. Korean Patent Laid-Open No. 2019-0017393 discloses a composition for treating or improving non-alcoholic fatty liver comprising a seahorse extract. Korean Patent Registration No. 1759516 discloses a pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease containing astringent persimmon and dermis complex extract.

Deer antlers (Cervi Parvum Cornu) are cut and dried deer antlers that are not ossified, such as Cervus nippon , Cervus elaphus , or Cervus canadensis , belonging to the deer and the genus Cervus.

Deer antler has traditionally been widely used as a representative herbal medicine, and according to Donguibogam et al., it has a tonic action, a blood-keeping action, a gangrene action, an analgesic action, a hematopoietic action, a growth-promoting action, a heart failure treatment action, an enhancement action, a recovery from fatigue, and It is known to have a wide variety of effects, such as enhancing vitality and strengthening the diuretic function of the kidneys. Deer antlers are known to contain various free amino acids, polysaccharides, glycosaminoglycans (GAGs), hyaluronic acid, keratin, sialic acid, cholesterol, fatty acids, phospholipids, and inorganic components.

As a method of taking antler, hot water extraction of deer antler with various kinds of herbal medicines and taking the filtrate, or pulverizing with herbal medicines and taking them as pills are mainly used, but solvent extraction Various studies have been conducted to extract or isolate physiologically active ingredients from deer antlers using a fractionation method.

Korean Patent Laid-Open No. 1999-0044781 discloses that antler (maehwarok) is extracted with chloroform, and the chloroform extract is fractionated using silica gel column chromatography to isolate 5 types of monoacetyldiacylglycerol compounds, among which: Disclosed is the proliferation-promoting activity of 1-Palmitoyl-2-linoleoyl-3-acetyl glycerol (PLAG, hereinafter 'PLA glycerol') represented by the chemical formula. No. 2006-0047447 is an immunomodulatory agent and anticancer agent, Patent Publication No. 2015-0021464 is a composition for inhibiting blood cancer or cancer metastasis, Patent Publication No. 2015-0021465 is a composition for preventing or treating rheumatoid arthritis, Patent Publication No. 2015- No. 0021472 discloses a composition for preventing or treating chronic obstructive pulmonary disease, Patent Publication No. 2017-0005484 discloses a composition for treating leukopenia and thrombocytopenia, and Patent Registration No. 1817552 discloses a composition for preventing or treating hepatitis.

On the other hand, Korean Patent Laid-Open No. 2000-0059468 discloses that antler (maehwarok) is extracted with ethanol, and the ethanol extract is fractionated using silica gel column chromatography, and four types of phospholipids including the compound of the following formula are novel. The structure of the compound and its antifungal activity are disclosed.

As described above, deer antler has various pharmacological activities, and its components are also very diverse, so continuous research on pharmacologically active ingredients that are not yet known is required.

The present invention isolates a novel compound useful for the prevention and treatment of nonalcoholic fatty liver disease from deer antlers using solvent extraction and fractionation, and proposes a chemical synthesis method thereof to enable mass production of the novel compound, thereby providing non-alcoholic fatty liver An object of the present invention is to provide a pharmaceutical composition and health functional food that can be used for the prevention and treatment of diseases.

The present invention provides a pharmaceutical composition or health functional food for preventing or treating non-alcoholic fatty liver disease, comprising at least one selected from compounds represented by the following formulas 1 to 4 as an active ingredient.

[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

The compounds of Formulas 1 to 4 may be isolated from antlers or chemically synthesized.

The pharmaceutical composition or health functional food of the present invention may include, for example, a mixture of the compound represented by Formula 1 and the compound represented by Formula 2 above. The compound represented by Formula 1 and the compound represented by Formula 2 are isomers of each other.

As another example, the pharmaceutical composition or health functional food of the present invention may include a mixture of a compound represented by Formula 3 and a compound represented by Formula 4 above. The compound represented by Formula 3 and the compound represented by Formula 4 are isomers of each other.

As another example, the pharmaceutical composition or health functional food of the present invention may include a mixture of compounds represented by Formulas 1 to 4.

The nonalcoholic fatty liver disease may be nonalcoholic steatohepatitis, steatosis, or cirrhosis, but is not limited thereto.

The active ingredient may be to inhibit the production of cytokines in the liver.

The novel compounds of Examples 1 and 2 according to the present invention have no cytotoxicity, and the production of representative cytokines IL-6, IL-1β, TNF-α, MIP2 and CXCL-1 is significantly higher than that of the control in animal experiments. It was confirmed that the cytokine production was significantly inhibited even when compared with the comparative example (PLA glycerol). Therefore, the novel compound according to the present invention can be usefully used as a composition for preventing, improving or treating chronic obstructive pulmonary disease.

1 is a diagram showing an extraction method using three organic solvents from antler according to the present invention.

2 is a diagram illustrating a method for fractionating an extract C2 extracted from deer antler according to the present invention.

3 is mass spectrometry data of fraction C2-2-E-a-Ms according to the present invention.

4 is LC data of fraction C2-2-E-a-Ms according to the present invention.

Figure 5 (A) is UV analysis data of fraction C2-2-Ea-Ms according to the present invention, (B) is UV analysis data of PLA glycerol of Comparative Example, (C) is conjugated linoleic acid (conjugated linoleic acid) of UV analysis data, and (D) is UV analysis data of linoleic acid.

6 is a graph showing the measurement results of cell viability (cell viability) according to the MTT assay of the novel compounds of Examples 1 and 2 of the present invention.

7 is a graph showing the IL-6 production inhibitory effect of the novel compounds of Examples 1 and 2 of the present invention.

8 is a graph showing the IL-1β production inhibitory effect of the novel compounds of Examples 1 and 2 of the present invention.

9 is a graph showing the TNF-α inhibitory effect of the novel compounds of Examples 1 and 2 of the present invention.

10 is a graph showing the NO formation inhibitory effect of the novel compounds of Examples 1 and 2 of the present invention.

11 is a graph showing the degree of grade of steatosis, lobular inflammation and fibrosis in MCDD animal experiments administered with the novel compounds of Examples 1 and 2 of the present invention. .

12 is a graph showing the TNF-α inhibitory effect of the novel compounds of Examples 1 and 2 of the present invention in a fatty liver-induced animal experiment.

13 is a graph showing the TGF-β inhibitory effect of the novel compounds of Examples 1 and 2 of the present invention in a fatty liver-induced animal experiment.

14 is a graph showing the MCP-1 inhibitory effect of the novel compounds of Examples 1 and 2 of the present invention in a fatty liver-induced animal experiment.

In order to develop a pharmaceutical composition for the prevention or treatment of antler-derived inflammatory diseases, the present inventors analyzed and carried out the solvent extraction method and fractionation method of the prior literature from various angles to develop a novel compound having excellent physiological activity in inflammatory diseases by the following method was isolated, and it was confirmed that the novel compound significantly inhibited the production of cytokines, a representative factor of nonalcoholic fatty liver disease, and developed a novel synthesis method for the compound, thereby completing the present invention.

In order to extract the physiologically active ingredients from the antler, as shown in FIG. 1 , the antler (maehwarok) was sequentially extracted using three organic solvents: hexane, chloroform, and 70% ethanol, as shown in FIG. As described above, a novel compound having excellent anti-inflammatory activity was isolated by silica gel column chromatography and thin layer liquid chromatography.

Extraction with organic solvents

Pulverize 2 kg of commercially purchased Cervus nippon, put it in a beaker, add 9 L of n-Hexane as the primary extraction solvent, and then extract twice by heating at 80° C. for 2 hours, then filter and hexane After obtaining the extract, the hexane extract was distilled under reduced pressure and dried to obtain 30.3 g of extract C1.

_{After hexane extraction, 9 L of chloroform (CHCl 3} ) was added as a secondary extraction solvent to the residue remaining after extraction with hexane, and then extracted twice by heating at 80° C. for 2 hours, followed by filtration to obtain a chloroform extract, and then the chloroform extract was distilled and dried under reduced pressure. 17.9 g of extract C2 was obtained.

After chloroform extraction, 9L of 70% ethanol as a tertiary extraction solvent was added to the residue remaining after extraction with chloroform, and then extracted twice by heating at 80° C. for 5 hours and filtered to obtain an ethanol extract. Then, the ethanol extract was distilled under reduced pressure and dried to extract C3 89.2 g were obtained.

As a result of the anti-inflammatory activity experiment for each of the extracts C1, C2 and C3 obtained above, it was confirmed that the anti-inflammatory activity effect was high in the extract C2, and the bioactive component was fractionated using silica gel column chromatography for the extract C2.

Silica gel column chromatography

_{A mixed solution of CHCl 3} /MeOH (500:1, v/v) was added to powder silica gel to swell, and then it was filled in an open column. 17.9 g of the obtained extract C2 _{was dissolved in CHCl 3} /MeOH (500:1, v/v) and applied to a column filled with silica gel. _{Elution fractions were received by 50 ml each while flowing CHCl 3} /MeOH (500:2, v/v) as an eluent into the column, _{and then developed with TLC (eluent: CHCl 3} /MeOH=300:1) to develop iodine ( After color development with I ₂ ), 7 main fractions were separated by separation according to the Rf value. Each of the separated fractions was distilled under reduced pressure and dried to obtain fractions C2-1 to C2-7. As a result of the anti-inflammatory activity test for the seven fractions, it was confirmed that the anti-inflammatory activity effect was high in the fraction C2-2, and the bioactive components were re-fractionated using silica gel column chromatography for the fraction C2-2.

1.8 g of the above fraction C2-2 was taken. To 20 g of powdered silica gel, 50 ml of a mixed solution of hexane (n-Hx)/ethyl acetate (EA) (50:1) was added and swollen, and the column was filled. 1.8 g of Fraction C2-2 was dissolved in a minimum amount of n-Hx/EA (50:1, v/v) as an eluent and applied to a silica gel column. While flowing the eluent through the silica gel column, 15 ml of each eluted fraction was received, developed by TLC (eluent: n-Hx/EA=50:1, v/v), developed with iodine, and separated according to Rf, and separated according to Rf. The main fraction was isolated. Each of the separated fractions was distilled under reduced pressure and dried to obtain fractions C2-2-A to C2-2-F. As a result of the anti-inflammatory activity experiment for the six fractions, it was confirmed that the anti-inflammatory activity effect was high in the fraction C2-2-E. fractionated.

212 mg of the above fraction C2-2-E was taken. To 3.5 g of powdered silica gel, 10 ml of a mixed solution of n-Hx/EA/AcOH (20:1:0.5, v/v/v) was added and swollen, and the column was filled. 212 mg of fraction C2-2-E was dissolved in n-Hx/EA/AcOH (20:1:0.5, v/v/v) as an eluent and applied to a silica gel column. While flowing the eluent through the silica gel column, 10 ml of each eluted fraction was received and developed by TLC (eluent: n-Hx/EA/AcOH=20:1:0.5, v/v/v), developed with iodine, and then Rf was separated to separate two main fractions. Each of the separated fractions was distilled under reduced pressure and dried to obtain fractions C2-2-E-a and C2-2-E-b, respectively. As a result of the anti-inflammatory activity test for the two fractions, it was confirmed that the anti-inflammatory activity effect was high in the fraction C2-2-Ea, and the physiologically active ingredients were again recovered using silica gel column chromatography for the fraction C2-2-Ea. fractionated.

137 mg of the fraction C2-2-E-a was taken. Methanol 5ml was added to this C2-2-Ea, mixed, and separated into a methanol-soluble fraction C2-2-Ea-Ms) and a methanol-insoluble fraction C2-2-Ea-Mi), and each fraction was distilled under reduced pressure and dried. and 63 mg of the fraction C2-2-Ea-Ms was obtained. As a result of an anti-inflammatory activity test for the two fractions, it was confirmed that the fraction C2-2-E-a-Ms had a high anti-inflammatory activity.

Anti-inflammatory inhibition evaluation of the fraction was performed through the method according to Experimental Example 2 to be described later, and the results are shown in Table 1 below.

fraction (100㎍/ml)	IL-6 production (pg/ml)	IL-1β production (pg/ml)	TNF-α production (pg/ml)
C2-2	453	59	283
C2-2-E	402	43	233
C2-2-E-a	321	36	197
C2-2-E-a-Ms	253	34	162

As shown in Table 1, it can be seen that the smaller the fraction, the more inhibited the production of inflammatory cytokines IL-6, IL-1β, TNF-α. In order to confirm the active ingredient of the fraction C2-2-Ea-Ms, which has the highest anti-inflammatory activity among the fractions, MALDI-TOF/TOF mass spectrometer (Bruker Ultraflextreme ^TM , German), liquid chromatography (LC) and UV analyzer were used. Component analysis was performed.

Figure 3 is the mass spectrometry data of the fraction C2-2-Ea-Ms, Figure 4 is the LC data of the fraction C2-2-Ea-Ms, Figure 5 (A) is the fraction C2-2-Ea according to the present invention -Ms UV analysis data, (B) is UV analysis data of PLA glycerol of Comparative Example, (C) is UV analysis data of conjugated linoleic acid, (D) is UV analysis data of linoleic acid.

As a result of analyzing the mass of the active ingredient of the fraction C2-2-Ea-Ms of the present invention, the mass was the same as the known PLA glycerol (Compound KJ-3 of Patent Publication No. 1999-0044781) isolated from antler, but UV analysis As a result, it showed a completely different UV analysis pattern from PLA glycerol. By comparing the UV pattern of (C) conjugated linoleic acid and (D) UV pattern of linoleic acid in FIG. 5, the compound of the fraction C2-2-Ea-Ms of the present invention is conjugated linoleic acid. linoleoyl) was confirmed to have a novel structure.

The anti-inflammatory bioactive active ingredient according to the present invention is a novel compound represented by the following formulas 1 to 4 in which an acetyl group, palmitoyl, and conjugated linoleoyl are bonded to a glycerol structure. It was only confirmed in the invention.

[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

Chemical synthesis of PCA glycerol represented by Formula 1 and Formula 2

The compounds of Chemical Formulas 1 and 2 (PCA glycerol) according to the present invention can be prepared in high yield according to Scheme 1 below.

[Scheme 1]

PCA glycerol according to the present invention,

(a) reacting palmitic acid with the compound represented by Formula 5 under basic conditions to synthesize 1-palmitoyl glycerol;

(b) reacting the 1-palmitoyl glycerol and acetyl halide under basic conditions to synthesize 1-palmitoyl-3-acetyl glycerol; and

(c) reacting the 1-palmitoyl-3-acetyl glycerol with conjugated linoleic acid under basic conditions to synthesize 1-palmitoyl-2-conjugated linoleoyl-3-acetyl glycerol (PCA glycerol) includes steps.

In step (a), the compound of Formula 5 is a glycerol derivative protected with a 1,3-diol compound. The reaction may be carried out under basic conditions such as triethylamine, and may be carried out through an anhydride reaction by adding pivaloyl halide to maximize the reaction activity of palmitic acid. After the reaction, when deprotected using hydrochloric acid, 1-palmitoyl glycerol is synthesized. The equivalent weight (eq.) of palmitic acid and the compound of Formula 5 is preferably 0.9:1.1 to 1.1:0.9, but is not limited thereto.

The equivalent of 1-palmitoyl glycerol and acetyl halide in step (b) is preferably 1:1 to 1:1.6, but is not limited thereto.

In order to maximize the reaction activity of the conjugated linoleic acid in step (c), it may be carried out through an anhydride reaction by adding a pivaloyl halide. The equivalent (eq.) of 1-palmitoyl-3-acetyl glycerol and conjugated linoleic acid is preferably 0.9:1.1 to 1.1:0.9, but is not limited thereto.

Meanwhile, the compounds of Chemical Formulas 3 and 4 (CPA glycerol) according to the present invention can be prepared in high yield according to the following Reaction Scheme 2.

[Scheme 2]

CPA glycerol according to the present invention,

(a) reacting conjugated linoleic acid with the compound represented by Formula 5 under basic conditions to synthesize 1-conjugated linoleoyl-glycerol;

(b) reacting the 1-conjugated linoleoyl-glycerol with an acetyl halide under basic conditions to synthesize 1-conjugated linoleoyl-3-acetyl glycerol; and

(c) reacting the 1-conjugated linoleoyl-3-acetyl glycerol with palmitic acid under basic conditions to synthesize 1-conjugated linoleoyl-2-palmitoyl-3-acetyl glycerol (CPA glycerol); include Pivaloyl halide may be added for the anhydride reaction in steps (a) and (c).

Hereinafter, a method for synthesizing the novel compound of the present invention will be described in detail through Examples.

Example 1:1 - Palmitoyl -2-conjugated- linoleoyl Preparation of -3-acetyl glycerol (PCA glycerol)

(1) Preparation of 1-Palmitoyl glycerol

After adding 20.0 g of palmitic acid and 17.0 g of triethylamine to 200 ml of methylene chloride (MC), and cooling to 0°C, 10.0 g of pivaloyl chloride was slowly added dropwise, stirred for 2 hours, and then 10.8 g of Solketal and 0.1 g of 4-dimethylaminopyridine were added. After addition, the mixture was stirred at room temperature for 12 hours, purified water 200ml was added, and after layer separation, the organic layer was concentrated in vacuo. 200ml of methanol and 20ml of hydrochloric acid were added to the concentrated mother liquor, and after stirring at room temperature for 10 hours, 300ml of n-hexane and 300ml of purified water were added, stirred for 3 hours, filtered and vacuum dried to obtain 21.9g of the target compound (yield: 85.0%).

(2) Preparation of 1-Palmitoyl-3-acetyl glycerol

20.0 g of 1-Palmitoyl glycerol obtained above, 33.5 g of pyridine, and 0.2 g of 4-dimethylaminopyridine were added to 200 ml of MC and stirred for 1 hour, then 7.1 g of Acetyl chloride was added dropwise and stirred for 5 hours. 200ml of purified water was added, neutralized with dilute hydrochloric acid, and the layers were separated, and the organic layer _{was dehydrated with MgSO 4} , concentrated in vacuo, added 100ml of n-hexane, crystallized at 10° C. or less, filtered and vacuum dried to 18.5 g of the target compound (yield: 82.0%) was obtained.

(3) Preparation of 1-Palmitoyl-2-C-linoleoyl-3-acetyl glycerol (PCA glycerol)

14.0 g of conjugated linoleic acid (cis-9,trans-11/trans-10,cis-12) and 10.8 g of triethylamine were added to 180 ml of n-Hexane, cooled to 0° C., and 7.0 g of Pivaloyl chloride was slowly added dropwise. After completion of the dropwise addition, after stirring for 1 hour at the same temperature, 18.0 g of 1-Palmitoyl-3-Acetyl glycerol and 1.0 g of 4-Dimethylaminopyridine were added and stirred at room temperature for 10 hours. 180ml of purified water was added, the layers were separated, the organic layer _{was dehydrated with MgSO 4} , concentrated in vacuo, and purified by silica gel column (eluent: n-Hx:EA=20:1, v/v) to 26.1g of the target compound (yield: 85%) was obtained.

Example 2: 1 -Conjugated- lineoyl -2- palmitoyl Preparation of -3-acetyl glycerol (CPA glycerol)

(1) Preparation of 1-C-linoleoyl glycerol

22.0 g of conjugated linoleic acid (cis-9,trans-11/trans-10,cis-12) and 10.8 g of triethylamine were added to 200 ml of n-Hexane, cooled to 0° C., and 7.0 g of Pivaloyl chloride was slowly added dropwise at the same temperature. After stirring for 1 hour, 10.8 g of Solketal and 0.1 g of 4-Dimethylaminopyridine were added, followed by stirring at room temperature for 12 hours, 200 ml of purified water was added, and the organic layer was concentrated in vacuo after layer separation. 200ml of methanol and 20ml of hydrochloric acid were added to the concentrated mother liquor, stirred at room temperature, stirred for 10 hours, then added 300ml of n-hexane and 300ml of purified water, stirred for 3 hours, filtered and vacuum dried to obtain 22.8g of the target compound (yield: 82%). did.

(2) Preparation of 1-C-linoleoyl-3-acetyl glycerol

To 200 ml of MC, 20.0 g of 1-C-linoleoyl glycerol, 33.5 g of pyridine, and 0.2 g of 4-dimethylaminopyridine were added and stirred for 1 hour, then 6.6 g of acetyl chloride was added dropwise and stirred for 5 hours. 200ml of purified water was added, neutralized with dilute hydrochloric acid, and the layers were separated. The organic layer _{was dehydrated with MgSO 4} , concentrated in vacuo, added 100ml of n-hexane, crystallized at 10° C. or lower, filtered, and vacuum dried to 17.7 g of the target compound (yield: 79.0%) was obtained.

(3) Preparation of 1-C-linoleoyl-2-palmitoyl-3-acetyl glycerol (CPA glycerol)

12.2 g of palmitic acid and 10.8 g of triethylamine were added to 180 ml of n-Hexane, cooled to 0° C., and 7.0 g of pivaloyl chloride was slowly added dropwise. After completion of the dropwise addition, after stirring for 1 hour at the same temperature, 18.0 g of 1-C-linoleoyl-3-acetyl glycerol and 1.0 g of 4-dimethylaminopyridine were added and stirred at room temperature for 10 hours. 180ml of purified water was added, the layers were separated, and the organic layer _{was dehydrated with MgSO 4} , concentrated in vacuo, and purified by silica gel column (eluent: n-Hx:EA=20:1, v/v) to 25.9 g of the target compound (yield: 86%) was obtained.

Comparative Example: Preparation of 1-Palmitoyl-2-linoleoyl-3-acetyl glycerol (PLA glycerol)

PLA glycerol was synthesized in the same manner as in Example 1 using Linoleic acid (cis-9,cis-12) instead of conjugated Linoleic acid (cis-9,trans-11/trans-10,cis-12).

Linoleic acid (cis-9,cis-12) 14.0 g and triethylamine 10.8 g were added to 180 ml of n-Hexane, cooled to 0° C., and 7.0 g of pivaloyl chloride was slowly added dropwise. After completion of the dropwise addition, after stirring at the same temperature for 1 hour, 18.0 g of 1-Palmitoyl-3-acetyl glycerol and 1.0 g of 4-dimethylaminopyridine were added and stirred at room temperature for 10 hours. 180ml of purified water was added, the layers were separated, and the organic layer _{was dehydrated with MgSO 4} , concentrated in vacuo, and purified by silica gel column (eluent: n-Hx:EA=20:1, v/v) to 24.9g of the target compound (yield: 81%) was obtained.

The anti-inflammatory activity experiments of the compounds synthesized in Examples 1 and 2 and Comparative Examples were evaluated in the following animal experiments.

A. Experimental animals

As experimental animals, 8-week-old C57BL/6 mice were bred for 7 days at a temperature of 22±2° C., a relative humidity of 65±5%, and a light/dark cycle of 12 hours for 7 days, and then used for the experiment after adapting to the laboratory environment. Solid feed (Samyang feed) and water were sufficiently supplied.

B. Isolation of peritoneal macrophages

HBSS was injected intraperitoneally into mice to extract macrophages, and after centrifugation at 3,000 rpm for 5 minutes, 100 units/mL of penicillin/streptomycin was added to DMEM medium supplemented with 10% fetal bovine serum (FBS). was isolated, and was used for the experiment after 24 hours of incubation in an incubator at 37° C., 5% CO ₂

Experimental Example 1: Cytotoxicity evaluation through MTT assay

The cultured peritoneal macrophages were aliquoted at 3×10 ⁵ cells/well in a 96-well plate by 100 uL and cultured overnight. After removing the medium, the compounds of Example 1 (PCA glycerol) and Example 2 (CPA glycerol) were treated in peritoneal macrophages by concentration (10㎍/ml, 100㎍/ml and 200㎍/ml, respectively), and then , 37° C., 5% CO ₂ Incubated for 24 hours in an incubator. After incubation, the medium was removed, 40uL of MTT (5mg/mL) reagent was dispensed, and _{incubated for 4 hours in a CO 2} incubator, MTT reagent was removed, and DMSO reagent was dispensed 600uL each, and then left at room temperature for 30 minutes. Then, the absorbance (OD) was measured at 540 nm with a microplate reader.

Cell viability (cell viability) of the compounds of Examples 1 and 2 according to the MTT assay was measured and shown as a graph in Table 2 and FIG. 6 below. As shown in Table 2 and FIG. 6, it can be seen that there is no significant difference in cell viability even at the highest 200 μg/ml treatment. Therefore, it was confirmed that the novel compound according to the present invention has no cytotoxicity.

	normal group	Example 1 (μg/ml)			Example 2 (μg/ml)
		10	100	200	10	100	200
cell Survival rate (%)	100±1.2	100±2.1	98.3±1.9	98.1±1.2	100±0.9	98.3±1.7	98.0±2.8

Experimental Example 2: Anti-inflammatory activity test (IL-6, IL-1β and TNF-α production inhibition evaluation)

Anti-inflammatory by measuring the secretion of IL-6, IL-1β and TNF-α from mouse peritoneal macrophages induced by lipopolysaccharides (LPS), an inflammatory response inducer, using an ELISA assay (Millipore, USA). Activity was assessed.

In order to obtain a cell culture solution, mouse peritoneal macrophages ^{were adjusted to 3 × 10 5} cells/mL, inoculated in a 96-well plate, and cultured for 24 hours in Example 1 (PCA glycerol), Example 2 (CPA glycerol) and Comparative Example ( PLA glycerol) was treated with each concentration (10 μg/ml, 100 μg/ml and 200 μg/ml, respectively), and LPS (1 μg/ml) was treated. The normal group was untreated, and the control group was treated with LPS (1 μg/ml) only in peritoneal macrophages. After 12 hours of incubation, the supernatant was obtained by centrifugation. For ELISA, anti-mouse IL-6, IL-1β and TNF-α were dispensed on microplates as capture antibodies. Then, it was washed with phosphate buffered saline (PBST) containing 0.05% Tween 20, blocked with 10% FBS, washed with PBST, and the cell culture supernatant was dispensed into the wells and reacted for 2 hours at room temperature. After the reaction, biotinylated anti-mouse IL-6, IL-1β, and TNF-α detection antibody and streptavidin-HRP conjugate (streptavidin-horseradish peroxydase conjugate) were washed and diluted with PBST It was aliquoted and reacted at room temperature for 1 hour. After that, it was washed again with PBST, and an OPD solution was added, followed by dark reaction at room temperature for 30 minutes. After terminating the reaction with 2 NH ₂ SO ₄ , the absorbance was measured at 450 nm using a microplate reader.

The IL-6 production amount of the compounds of Examples 1 and 2 was measured and shown in Table 3 below, and is shown in the graph of FIG. 7 . As shown in Table 3 and Figure 7, it can be seen that the compounds prepared in Examples 1 and 2 significantly reduced IL-6 production compared to the control, and had a significant IL-6 production inhibitory effect even when compared to Comparative Examples there is.

		LPS (1㎍/ml)
	normal group	control	Example 1 (μg/ml)			Example 2 (μg/ml)			Comparative Example (μg/ml)
			10	100	200	10	100	200	10	100	200
IL-6 amount of production (pg/ml)	150±18	834±32	321±22	225±23	180±25	325±13	234±20	179±17	364±16	251±24	203±25

The IL-1β production amount of the compounds of Examples 1 and 2 was measured and shown in Table 4 below, and is shown in the graph of FIG. 8 . As shown in Tables 4 and 8, the compounds prepared in Examples 1 and 2 significantly reduced the amount of IL-1β production compared to the control, and it can be seen that they also had a significant IL-1β production inhibitory effect when compared with the comparative example. there is.

		LPS (1㎍/ml)
	normal group	control	Example 1 (μg/ml)			Example 2 (μg/ml)			Comparative Example (μg/ml)
			10	100	200	10	100	200	10	100	200
IL-1β amount of production (pg/ml)	25±1.8	72±3.5	40±2.9	28±2.3	26±2.8	40±1.7	32±2.4	28±1.7	43±2.2	31±2.4	31±3.1

The TNF-α production amount of the compounds of Examples 1 and 2 was measured and shown in Table 5 below, and is shown in the graph of FIG. 9 . As shown in Table 5 and Figure 9, the compounds prepared in Examples 1 and 2 significantly reduced the amount of TNF-α production compared to the control, and it can be seen that also has a significant TNF-α production inhibitory effect when compared with the comparative example. there is.

		LPS (1 μg/ml)
	normal group	control	Example 1 (μg/ml)			Example 2 (μg/ml)			Comparative Example (μg/ml)
			10	100	200	10	100	200	10	100	200
TNF-α production (pg/ml)	113±11	375±16	170±8	153±11	121±10	175±7	151±10	123±7	184±21	155±12	137±7

Experimental Example 3: Anti-inflammatory activity test (NO formation inhibition evaluation)

The cultured peritoneal macrophages were suspended in DMEM containing 10% FBS, and ^{then aliquoted at 5×10 5} cells/well in a 96-well plate and cultured at 37° C., 5% CO _{2 in an} incubator for 24 hours, and fresh DMEM medium. After exchanging with peritoneal macrophages, each of the compounds of Examples 1, 2 and Comparative Example was treated with each concentration (10㎍/ml, 100㎍/ml and 200㎍/ml, respectively) in peritoneal macrophages and LPS (1㎍/ml) After treatment, incubated for 24 hours. After incubation, the supernatant was separated and centrifuged at 3000 rpm for 5 minutes, and the separated supernatant was dispensed into a new microplate. The normal group was untreated, and the control group was treated with LPS (1 μg/ml) only in peritoneal macrophages.

The same amount of Griess reagent (1% sulfanilamide, 0.1% naphthyl-ethylenediamine dihydrochloride, 2% phosphoric acid) was treated and reacted at room temperature for 10 minutes. After reacting the culture medium with the Griess solution for 5 minutes, absorbance (OD) was measured at 540 nm.

10 is a graph showing the NO production inhibitory effect of the novel compounds of Examples 1 and 2 of the present invention.

The nitrogen monoxide (NO) production rates of the compounds of Examples 1 and 2 were measured and shown in Table 6 below, and shown in the graph of FIG. 10 . As shown in Tables 6 and 10, the compounds prepared in Examples 1 and 2 significantly reduced the NO production rate compared to the control, and it can be seen that they have a significant NO production inhibitory effect even when compared to the comparative example.

		LPS (μg/ml)
	normal group	control	Example 1 (μg/ml)			Example 2 (μg/ml)			Comparative Example (μg/ml)
			10	100	200	10	100	200	10	100	200
NO production rate (%)	25±2	100±4	52±3	43±3	37±4	50±2	44±3	34±1	56±3	45±2	41±3

Example 4. Nonalcoholic fatty liver disease (NASH) animal model experiment

It is well known that methionine choline-deficient diet (MCDD) induces nonalcoholic steatohepatitis (NASH) in a mouse MCDD diet model. Methionine and choline play an important role in beta-oxidation and very low-density lipoprotein (VLDL) synthesis in the liver, and deficiency of these methionine and choline causes fatty liver in mice. make it The MCDD diet model is widely used as a model for observing the improvement effect of nonalcoholic fatty liver disease because histopathologically, inflammation in the hepatic lobules is severely induced.

In order to confirm the preventive effect of the compounds of Examples 1 and 2 on the development of nonalcoholic steatohepatitis (NASH), an animal model in which MCDD was ingested for 6 weeks was used.

Common microscopic findings of NASH include steatosis, lobular inflammation, and fibrosis. The Clinical Research Network (CRN), a NASH clinical research institute, devised a detailed grading system for lesions corresponding to NASH disease and proposed a NASH disease activity score (Nonalcoholic fatty liver disease activity score, NAS). is widely used in

In the present invention, based on the classification system of CRN, the grade of steatosis, lobular inflammation, and fibrosis were scored in the liver tissue observation results of the animal model, and the compounds of the present invention were The effect on the prevention or progression of nonalcoholic fatty liver disease was evaluated.

8-week-old C57BL/6 mice were divided into 4 groups, and the experimental group was (i) a control group induced by NASH by supplying only MCDD for 6 weeks (Control), (ii) MCDD + 50 mg/kg/day of the compound of Example 1 a group administered together for 6 weeks, (iii) a group administered orally with MCDD + 50 mg/kg/day of Example 2 for 6 weeks, (iv) MCDD + 50 mg/kg/day of the compound of Comparative Example together The administration group was administered orally for 6 weeks. After the end of the diet experiment, each mouse liver tissue was collected and observed under a microscope to determine the degree of steatosis, intralobular inflammation and fibrosis, and grade of steatosis and lobular inflammation according to the NAS system And the degree of fibrosis was evaluated and shown in Table 7 and FIG. 11 .

	control	Example 1	Example 2	comparative example
Grade of steatosis	1.92±0.27	0.97±0.29	1.03±0.36	1.12±0.21
Lobular inflammation	2.58±0.19	0.43±0.16	0.41±0.24	0.71±0.26
Fibrosis	2.16±0.28	0.57±0.11	0.51±0.18	0.89±0.31

As shown in Table 7 and FIG. 11, the administration of the compound of Example 1 and the compound of Example 2 was a control group in all levels of grade of steatosis, lobular inflammation and fibrosis. It can be confirmed that there is a significant improvement compared to the compound of Comparative Example, it was confirmed that a significant improvement effect compared to.

Experimental Example 5: Evaluation of inhibition of cytokine expression in fatty liver tissue

In order to determine the degree of inflammation in the fatty liver tissue induced by MCDD, mice were sacrificed, the liver was removed, washed with PBS, crushed, and then filtered using a 70 μm mesh cell strainer in PBS. The filtrate was centrifuged at a speed of 2,000 rpm for 5 minutes to collect the supernatant, and the expression inhibitory effect of TGF-α, TNF-β and MCP-1 was evaluated using an ELISA assay (Millipore, USA).

Anti-mouse TGF-α, TNF-β and MCP-1 capture antibodies were aliquoted into microplates. Then, it was washed with phosphate buffered saline (PBST) containing 0.05% Tween 20, blocked with 10% FBS, washed with PBST, and the supernatant was dispensed into the wells and reacted for 2 hours at room temperature. After the reaction, biotinylated anti-mouse TGF-α, TNF-β and MCP-1 detection antibody and streptavidin-HRP conjugate (streptavidin-horseradish peroxydase conjugate) were washed and diluted with PBST It was aliquoted and reacted at room temperature for 1 hour. After that, it was washed again with PBST, and an OPD solution was added, followed by dark reaction at room temperature for 30 minutes. After terminating the reaction with 2N H ₂ SO ₄ , the absorbance was measured at 450 nm using a microplate reader.

The TGF-β, TNF-α and MCP-1 production amounts of the compounds of Examples 1 and 2 were measured, respectively, and shown in Tables 8 to 10 below, and shown in the graphs of FIGS. 12 to 14 .

	-	MCDD
	normal group	control	Example 1	Example 2	comparative example
TNF-α amount of production (pg/ml)	9.3±1.7	108.6±25.9	35.3±10.6	37.4±8.5	62.2±13.1
Inhibition rate (%)	-	-	%	%	%

As shown in Table 8 and FIG. 12, the amount of TNF-α production in the COPD-induced control group was 151.6±10.6 pg/ml, which was significantly increased compared to 7.1±1.1 pg/ml in the normal group, but the compound administered group of Example 1 is 42.3±8.5 pg/ml, 72% inhibition compared to the control group, and the compound administered group of Example 2 was 39.8±6.7 pg/ml, 74% inhibition rate, which was significant in TNF-α production compared to the comparative example (61% inhibition rate). It was confirmed that there is a reduction effect.

	-	MCDD
	normal group	control	Example 1	Example 2	comparative example
TNF-β amount of production (pg/ml)	2.9±1.6	125.4±18.6	15.4±3.5	24.3±11.2	25.7±8.6
Inhibition rate (%)	-	-	%	%	%

As shown in Table 9 and FIG. 13, the amount of MIP2 production in the COPD-induced control group was 301.4±16.7 pg/ml, which was significantly increased compared to 62.1±7.1 pg/ml in the normal group, but the compound administered group of Example 1 was 91.2 70% inhibition rate compared to the control group at ±11.5 pg/ml, the compound administered group of Example 2 had a significant reduction effect in MIP2 production compared to the comparative example (63% inhibition rate) with a 68% inhibition rate at 95.3±5.6 pg/ml This was confirmed.

	-	MCDD
	normal group	control	Example 1	Example 2	comparative example
MCP-1 amount of production (pg/ml)	50.4±12.1	354.4±28.2	76.4±12.0	75.5±24.1	101.6±28.3
Inhibition rate (%)	-	-	%	%	%

As shown in Table 10 and FIG. 14, the amount of CXCL-1 produced in the control group induced by COPD was 250.4±18.7 pg/ml, which was significantly increased compared to 64.2±8.1 pg/ml in the normal group, but the group administered with the compound of Example 1 is 77.3±12.0 pg/ml, with a 69% inhibition rate compared to the control group, and the compound administered group of Example 2 was 84.9±8.6 pg/ml with a 66% inhibition rate, which was significant in the amount of CXCL-1 production compared to the comparative example (64% inhibition rate). It was confirmed that there is a reduction effect.

In the present invention, the compounds of Formulas 1 to 4 may be included as an active ingredient in a pharmaceutical composition.

In the present invention, the pharmaceutical composition is in the form of a conventional pharmaceutical composition such as tablets, pills, powders, granules, capsules, suspensions, internal solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-drying agents, etc. and may be in various oral or parenteral formulations. In the case of formulation, additives used in pharmaceutical formulations, such as diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, and the like, commonly used may be included.

The composition of the present invention can be administered in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is dependent on the subject's type and severity, age, sex, and disease. The type, activity of the drug, sensitivity to the drug, administration time, administration route and excretion rate, treatment period, factors including concurrent drugs and other factors well known in the medical field may be determined. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. and may be administered single or multiple.

In the present invention, the compounds of Formulas 1 to 4 may be included as an active ingredient of a health functional food.

In the present invention, health functional food is a food used as a non-pharmaceutical, and there is no particular limitation on the type of food, for example, tablets, pills, powders, granules, capsules, suspensions, internal solutions, emulsions, syrups, sterilization It may be in the form of conventional pharmaceutical compositions such as aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-drying agents, etc.

The present invention relates to a pharmaceutical composition or health functional food for preventing or treating non-alcoholic fatty liver disease.

Claims (6)

1. A pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease, comprising as an active ingredient at least one selected from the compounds represented by the following formulas 1 to 4.

   [Formula 1]

   

   [Formula 2]

   

   [Formula 3]

   

   [Formula 4]

   
2. According to claim 1,

   The active ingredient is a mixture of the compound represented by Formula 1 and the compound represented by Formula 2, a pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease.
3. According to claim 1,

   The active ingredient is a mixture of the compound represented by Formula 3 and the compound represented by Formula 4, a pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease.
4. According to claim 1,

   The non-alcoholic fatty liver disease is non-alcoholic steatohepatitis, steatosis or cirrhosis, a pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease.
5. According to claim 1,

   The active ingredient is a pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease, which inhibits the production of cytokines in the liver.
6. A health functional food for preventing or improving non-alcoholic fatty liver disease, comprising a compound represented by the following formulas 1 to 4 as an active ingredient.

   [Formula 1]

   

   [Formula 2]

   

   [Formula 3]

   

   [Formula 4]

   

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