KR102078818B1 - Pharmaceutical composition for preventing or treating of hepatitis comprising a compound from Sargassum thunbergii as an active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating hepatitis containing a Sargassum thunbergii-derived compound as an active component, wherein indole-4-carboxaldehyde (STC-3) isolated from a Sargassum thunbergii extract increases the expression of enzymes that break down methylglyoxal in a concentration range which does not show toxicity to human hepatocyte cell lines, thereby suppressing inflammatory responses induced by methylglyoxal. Therefore, the pharmaceutical composition can be usefully used as a composition for preventing, treating or alleviating hepatitis.

Description

Pharmaceutical composition for preventing or treating of hepatitis comprising a compound from Sargassum thunbergii as an active ingredient}

The present invention relates to a pharmaceutical composition for the prevention or treatment of hepatitis containing a compound derived from insects as an active ingredient.

The liver is one of the most important metabolic organs in the body's organs, and plays important functions such as secretion of bile, storage of nutrients, and detoxification. Hepatitis (Hepatitis) refers to the inflammation of the liver cell tissue, it is caused by viral infections, drugs, alcohol, poison, etc. Hepatitis is considered to be acute hepatitis if it lasts for less than 6 months, and chronic hepatitis if it lasts for more than 6 months. Acute hepatitis may have non-specific digestive symptoms such as anorexia, nausea, and vomiting, and may be accompanied by severe helplessness or jaundice, mild fever, headache, muscle pain, and joint pain. hepatic encephalopathy). Chronic hepatitis often results in unspecified symptoms such as anxiety, fatigue, and weakness, and there may be no symptoms at all. Efforts to develop a drug for treating liver diseases including hepatitis have been continued, but only a few drugs have been clinically recognized as effective against liver diseases.

On the other hand, Sargassum thunbergii belongs to the Phaeophyta Fucales and Sargassaceae, and is commonly found on the coast of Korea. do. It has been reported that wormworm has anti-oxidant, anti-inflammatory and antibacterial properties, and substances such as (+)-epiloliolide, (-)-loliolide and apo-9'-fucoxanthinone, which are norisoprenoid compounds as active ingredients. This has been reported separately.

The present inventors have developed a hepatitis treatment using a natural product that is safe for the human body or a component derived from a natural product that has been used for a long time, and STC-3 (indole-4-carboxaldehyde) isolated from an extract of an insect is toxic to a human liver cell line. The present invention was completed by confirming that the expression of an enzyme that decomposes methylglyoxal is increased in a concentration range that does not represent and inhibits the inflammatory response induced by methylglyoxal.

Republic of Korea Registered Patent No. 10-1794114

An object of the present invention is to provide a composition for the prevention, treatment or improvement of hepatitis, which contains a compound derived from insects as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention or treatment of hepatitis containing a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient:

[Formula 1]

.

.

In addition, the present invention provides a health functional food for preventing or improving hepatitis containing the compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:

[Formula 1]

.

.

STC-3 (indole-4-carboxaldehyde) isolated from the insecticidal extract of the present invention increases the expression of an enzyme that decomposes methylglyoxal in a concentration range that does not show toxicity to a human hepatocyte cell line, and accordingly to methylglyoxal By inhibiting the inflammatory response induced by, it can be useful as a composition for the prevention, treatment or improvement of hepatitis.

1 is a graph showing the results of measuring the survival rate of HepG2 cells treated with STC-3 (compared to the control group: * p <0.05, ns, not significant).
FIG. 2 shows the inhibitory effect of TNF-α (A), IFN-γ (B), iNOS (C) and COX-2 (D) mRNA expression induced by methylglyoxal (MGO) of STC-3 in HepG2 cells. It is a graph showing.
3 is a graph showing the effect of STC-3 and sulfolapane (sulforaphane, sulfo) on the increase of glyoxalase 1 (Glo-1) mRNA expression in HepG2 cells.
Figure 4 is a Western blot picture (A) confirming the effect of increasing the expression of Glyoxalase 1 and 2 (Glo-1 and Glo-2) proteins of STC-3 in HepG2 cells and a graph of the quantitative results of each band (B and C). to be.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for the prevention or treatment of hepatitis containing a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient:

[Formula 1]

.

.

The compound represented by Chemical Formula 1 may be derived from an insecticidal extract, and specifically, may be indole-4-carboxaldehyde (STC-3).

The composition may have an inhibitory activity for the expression of inflammatory factors in hepatocytes, and the inflammatory factors include TNF-α (tumor necrosis factor-α), IFN-γ (interferon-γ), iNOS (inducible nitric oxide synthase) and COX- It may be any one or more selected from the group consisting of 2 (cyclooxygenase-2).

The hepatitis may be acute hepatitis or chronic hepatitis.

In a specific embodiment of the present invention, the present inventors increased the expression of glyoxalases 1 and 2, which decompose methylglyoxal in a concentration range in which STC-3 isolated from the insecticidal extract does not exhibit toxicity to the human hepatocyte cell line, (See FIGS. 1, 3 and 4), and thus, it was confirmed that TNF-α, IFN-γ, iNOS and COX-2 mRNA expression, which are inflammatory factors increased by methylglyoxal, were reduced (see FIG. 2). Methyl glyoxal is a by-product of various metabolic processes, including glycolysis. It exhibits cytotoxicity. It has a glyoxalase system to protect it in vivo. Methylglyoxal reacts with glutathione to form hemithioacetal, which is subsequently metabolized by glyoxalase 1 and 2 (Y. Inoue et al., Advances in Microbial Physiology, 37: 177-227, 1995) . It has been reported that methylglyoxal increases in diabetes, renal failure, and arteriosclerosis, and has recently been reported to increase in the early stages of hepatitis, and the association with inflammation has been reported (Wen-Chuang Wang et al., Biomedical Chromatography, 32 (2) : e4039, 2018). Therefore, the STC-3 may be useful for the prevention or treatment of hepatitis.

The pharmaceutical composition according to the present invention may contain 10 to 95% by weight of STC-3 as an active ingredient relative to the total weight of the composition. In addition, the pharmaceutical composition of the present invention may further include one or more active ingredients exhibiting the same or similar function in addition to the active ingredients.

The pharmaceutical composition of the present invention may include carriers, diluents, excipients, or mixtures thereof, which are commonly used in biological agents. Any pharmaceutically acceptable carrier can be used as long as it is suitable for delivering the composition in vivo. Specifically, the carrier is Merck Index, 13th ed., Merck & Co. Inc., a saline solution, sterile water, Ringer's solution, dextrose solution, maltodextrin solution, glycerol, ethanol, or a mixture thereof. In addition, if necessary, conventional additives such as antioxidants, buffers, bacteriostatic agents, etc. can be added.

When formulating the composition, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc., which are usually used may be added.

The composition of the present invention may be formulated as an oral preparation or parenteral preparation. Oral formulations may include solid and liquid formulations. The solid preparation may be a tablet, a pill, a powder, a granule, a capsule or a troche, and the solid preparation may be prepared by adding at least one excipient to the composition. The excipient may be starch, calcium carbonate, sucrose, lactose, gelatin or a mixture thereof. In addition, the solid formulation may include a lubricant, and examples thereof include magnesium stearate and talc. Meanwhile, the liquid formulation may be a suspending agent, an intravenous solution, an emulsion, or a syrup. At this time, the liquid formulation may include excipients such as wetting agents, sweeteners, fragrances, preservatives.

The parenteral preparation may include injections, suppositories, respiratory inhalation powders, spray aerosols, powders and creams. The injection may include a sterilized aqueous solution, a non-aqueous solvent, a suspension solvent, an emulsion, and the like. At this time, as a non-aqueous solvent or a suspension solvent, vegetable oils such as propylene glycol, polyethylene glycol, and olive oil, or injectable esters such as ethyl oleate may be used.

The composition of the present invention may be administered orally or parenterally depending on the desired method. Parenteral administration can include intraperitoneal, rectal, subcutaneous, intravenous, intramuscular or intrathoracic injection.

The composition can be administered in a pharmaceutically effective amount. This may vary depending on the type of disease, severity, drug activity, patient sensitivity to the drug, time of administration, route of administration, duration of treatment, and drugs used simultaneously. However, for a desired effect, the amount of the active ingredient contained in the pharmaceutical composition according to the present invention may be 0.0001 to 100 mg / kg, specifically 0.001 to 10 mg / kg. The administration may be 1 to several times a day.

The composition of the present invention may be administered alone or in combination with other therapeutic agents. In combination administration, administration may be sequential or simultaneous.

In addition, the present invention provides a health functional food for preventing or improving hepatitis containing the compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:

[Formula 1]

.

.

The compound represented by Chemical Formula 1 may be derived from an insecticidal extract, and specifically, may be indole-4-carboxaldehyde (STC-3).

The composition may have inflammatory factor expression inhibitory activity in hepatocytes, and the inflammatory factor may be any one or more selected from the group consisting of TNF-α, IFN-γ, iNOS and COX-2.

The hepatitis may be acute hepatitis or chronic hepatitis.

In a specific embodiment of the present invention, the present inventors increased the expression of glyoxalases 1 and 2, which decompose methylglyoxal in a concentration range in which STC-3 isolated from the insecticidal extract does not exhibit toxicity to the human hepatocyte cell line, (See FIGS. 1, 3 and 4), and thus, it was confirmed that TNF-α, IFN-γ, iNOS and COX-2 mRNA expression, which are inflammatory factors increased by methylglyoxal, were reduced (see FIG. 2). Therefore, the STC-3 may be useful for the prevention or improvement of hepatitis.

The composition of the present invention may be added to food as it is, or used with other foods or food ingredients. At this time, the content of the active ingredient to be added may be determined according to the purpose, and generally may be 0.01 to 90 parts by weight of the total food weight.

The form and type of health functional food are not particularly limited. Specifically, the health functional food may be in the form of tablets, capsules, powders, granules, liquids and pills. The health functional food may include various flavors, sweeteners or natural carbohydrates as additional ingredients. The sweetener may be a natural or synthetic sweetener, and examples of the natural sweetener include tau martin and stevia extract. Meanwhile, examples of synthetic sweeteners include saccharin and aspartame. Further, the natural carbohydrate may be monosaccharide, disaccharide, polysaccharide, oligosaccharide and sugar alcohol.

The health functional food of the present invention, in addition to the above-described additional ingredients, nutrients, vitamins, electrolytes, flavoring agents, colorants, pectans and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives , Glycerin, alcohol, and the like. These ingredients can be used independently or in combination. The proportion of the additive may be selected from 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, the present invention will be described in detail by examples.

However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

Example 1. Preparation of active fraction from worm extract

After collecting and washing the larvae ( Sargassum thunbergii ) on the Jeju coast in winter, they were frozen at -70 ° C for 2 days, and lyophilized using a freeze dryer (LP-20, ILSHINBIOBASE Co. Ltd.). After freeze-dried insects were immersed in 80% methanol and left overnight, the filtrate was collected by filtration with whatman filter paper, and the residue remaining on the filter paper was again immersed in 80% methanol, and the same as above. The process was repeated two more times. The filtrate was concentrated at 40 ° C., chloroform was added and stirred, and then the chloroform fraction was separated and recovered. The chloroform fraction was eluted and fractionated step by step using a mixture of chloroform and methanol (30: 1 → 1: 1) by silica column chromatography. The antioxidant activity of each fraction was confirmed by an in vitro experiment to derive the fraction with the best activity.

Example 2. Isolation and purification of STC-3 from active fractions

The active fraction obtained in Example 1 was fractionated on a column saturated with 100% methanol (Sephadex LH-20 column), a detector (Waters 996 photodiode array detector) and a C18 column (J 'sphere ODS-H80, 250 × A single component was separated using a high-performance liquid chromatography (HPLC) system equipped with 4.6 mm, 4 μm, YMC Co., Japan (Alliance 2690, Waters Co., USA) (absorption wavelength of UV detector, 296 nm; and flow rate, 1 mL / min). The methanol / water mixture solution as a mobile phase was performed under gradient conditions. The structure of the separated compound was analyzed by nuclear magnetic resonance (NMR), and ¹ H-NMR and ¹³ C-NMR data were analyzed (Xu, GH et al. , J. Microbiol. Biotechnol. 20:78) . -81, 2010) it was confirmed that STC-3 (indole-4-carboxaldehyde). The following experiments were performed using the purity of STC-3 separated as a single component of 90% or more.

Experimental Example 1. Confirmation of STC-3 toxicity to hepatocytes

Whether STC-3 was toxic to hepatocytes was examined using a CCK-8 kit (D-Plus ^TM CCK kit, Dongin LS) in HepG2 cells, a human hepatocyte cell line.

Specifically, HepG2 cells were dispensed into 96-well plates at 2.5 × 10 ⁴ per well, and after 16 hours, 50, 100, or 200 μM STC-3 was treated, and further cultured for 24 hours. Thereafter, the cells were exchanged for 100 μl of a new cell culture medium per well, and 10 μl of CCK-8 was added and reacted for 2 hours, and absorbance was measured at 450 nm with a microplate reader. The cell viability of the drug-treated group was calculated by setting the absorbance of untreated cells to 100%.

As a result, STC-3 showed no hepatotoxicity at all treatment concentrations (FIG. 1).

Experimental Example 2. Confirmation of the inhibitory effect of STC-3 on methylglyoxal-induced hepatitis

To confirm the inhibitory effect of STC-3 on hepatitis, the degree of inhibition of the methylglyoxal-induced inflammatory response of STC-3 in the human hepatocyte cell line HepG2 cells is quantitative real-time polymerase chain reaction (qRT-PCR). ).

Specifically, HepG2 cells were dispensed at a 6-well plate at 2.0 × 10 ⁵ cells per well, and after 16 hours, 100 μM STC-3 was treated for 1.5 hours, and 0.25 mM methylglyoxal was treated for 24 hours. Thereafter, the cells are recovered, and total RNA is extracted with an RNA extraction reagent (RNAiso Plus, Takara Bio Inc.), and cDNA according to the manufacturer's instructions using a cDNA synthesis kit (PrimeScript ^TM cDNA synthesis kit, Takara Bio Inc.). Was prepared. RT-PCR was performed using the cDNA sample, SYBR premix reagent (SYBR Premix Ex Taq ^TM , ROX Plus, Takara Bio Inc.) and the primer pair of Table 1 below (Thermal Cyclers, Bio-rad Laboratories). The relative expression levels of TNF-α, IFN-γ, iNOS, and COX-2 mRNA were quantified based on the expression levels of cyclophilin mRNA.

Primer name Sequence (5 '→ 3') Sequence number TNF-α forward GAG ATC AAT CGG CCC GAC TA One TNF-α reverse ACA GGG CAA TGA TCC CAA AG 2 IFN-γ forward GTA GCG GAT AAT GGA ACT CTT TTC TT 3 IFN-γ reverse AAT TTG GCT CTG CAT TAT TTT TCT G 4 iNOS forward GCA GAA TGT GAC CAT CAT GG 5 iNOS reverse ACA ACC TTG GTG TTG AAG GC 6 COX-2 forward CCT TCC TCC TGT GCC TGA TG 7 COX-2 reverse ACA ATC TCA TTT GAA TCA GGA AGC T 8 Glo-1 forward ATG CGA CCC AGA GTT ACC AC 9 Glo-1 reverse CCA GGC CTT TCA TTT TAC CA 10 Cyclophilin forward TGC CAT CGC CAA GGA GTA G 11 Cyclophilin reverse TGC ACA GAC GGT CAC TCA AA 12

As a result, the relative mRNA levels of TNF-α, IFN-γ, iNOS and COX-2 were significantly increased by treatment with methylglyoxal in HepG2 cells, but decreased to the control level by STC-3 treatment (FIG. 2). .

Experimental Example 3. Confirmation of the increase effect of glyoxalase expression in hepatocytes of STC-3

Whether the effect of inhibiting the methyl glyoxal-induced inflammatory response of STC-3 identified in Experimental Example 2 is due to increased expression of glyoxalase 1 and 2 (Glo-1 and Glo-2), enzymes that degrade methyl glyoxal. To investigate, the following experiments were performed using the human hepatocyte cell line HepG2 cells.

3-1. Effect of STC-3 treatment on the increase of glyoxalase mRNA expression in hepatocytes

HepG2 cells were dispensed at 2.0 x 10 ⁵ per well into 6-well plates and treated with 50, 100 or 200 μM of STC-3, or 10 μM of sulfolapane for 24 hours after 16 hours. Thereafter, cells were recovered and qRT-PCR was performed in the same manner as described in Experimental Example 2, except that the primer pairs (SEQ ID NOs: 9 to 12) described in Table 1 were used.

As a result, STC-3 increased the relative mRNA level of Glo-1 in a concentration-dependent manner in HepG2 cells, and the STC-3 200 μM-treated group showed an effect similar to that of the positive control sulforaphane-treated group (FIG. 3). .

3-2. Effect of STC-3 treatment to increase the expression of glyoxalase protein in hepatocytes

HepG2 cells were dispensed at 6 x well plates at 2.0 x 10 ⁵ per well, and after 16 hours, 25, 50 or 100 μM of STC-3 was treated for 24 hours. Thereafter, the cells were recovered and subjected to western blot analysis.

Specifically, the recovered cells were lysed buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.25% sodium deoxycholate, 1% triton-X 100) and protease inhibitor (protease inhibitor cocktail, GenDEPOT Inc.)) for 20 minutes on ice. After centrifuging the cell lysate at 4 ℃ and 16,000 rpm for 15 minutes, the supernatant was mixed with 5 × sample buffer, electrophoresed on a SDS-PAGE (Sodium dodecyl sulfate polyacrylamide gel electrophoresis) gel, and the separated protein was PVDF ( polyvinylidene difluoride) membrane. After reacting the membrane with a primary antibody (anti-Glo-1, Santa Cruz Biotechnology; anti-Glo-2, Santa Cruze Biotechnology; and anti-GAPDH, Abcam), a secondary antibody bound with horseradish peroxidase (HRP) After the reaction, the HRP conjugate was detected with an enhanced chemiluminescence (ECL) buffer to analyze the blot image. Blot band quantitation was performed with the Image J program (ImageJ, https://imagej.nih.gov/ij/).

As a result, STC-3 increased protein expression of Glo-1 and Glo-2 in a concentration-dependent manner in HepG2 cells (FIG. 4).

From Experimental Examples 3-1 and 3-2, STC-3 can increase the mRNA and protein expression of Glo-1 and Glo-2 in HepG2 cells, thereby promoting the decomposition of methylglyoxal, which is the cause of hepatitis. As confirmed, this suggests that STC-3 can be usefully used as a substance for preventing or treating hepatitis.

<110> Gachon University of Industry-Academic cooperation Foundation <120> Pharmaceutical composition for preventing or treating of          hepatitis comprising a compound from Sargassum thunbergii as an          active ingredient <130> 2018P-06-020 <160> 12 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TNF-alpha forward <400> 1 gagatcaatc ggcccgacta 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TNF-alpha reverse <400> 2 acagggcaat gatcccaaag 20 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> IFN-gamma forward <400> 3 gtagcggata atggaactct tttctt 26 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> IFN-gamma reverse <400> 4 aatttggctc tgcattattt ttctg 25 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> iNOS forward <400> 5 gcagaatgtg accatcatgg 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> iNOS reverse <400> 6 acaaccttgg tgttgaaggc 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> COX-2 forward <400> 7 ccttcctcct gtgcctgatg 20 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> COX-2 reverse <400> 8 acaatctcat ttgaatcagg aagct 25 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Glo-1 forward <400> 9 atgcgaccca gagttaccac 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Glo-1 reverse <400> 10 ccaggccttt cattttacca 20 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Cyclophilin forward <400> 11 tgccatcgcc aaggagtag 19 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Cyclophilin reverse <400> 12 tgcacagacg gtcactcaaa 20

Claims (8)

A pharmaceutical composition for the prevention or treatment of hepatitis containing the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula 1]

.
The pharmaceutical composition of claim 1, wherein the compound represented by Formula 1 is derived from an insecticidal extract.
The pharmaceutical composition of claim 1, wherein the composition has an inhibitory activity for inflammatory factor expression in hepatocytes.
The method according to claim 3, wherein the inflammatory factor is from a group consisting of tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2). The pharmaceutical composition of any one or more selected.
Health functional food for prevention or improvement of hepatitis containing the compound represented by the following formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula 1]

.
According to claim 5, The compound is a dietary supplement, which is derived from the insecticidal extract.
According to claim 5, The health functional food is to have an inhibitory activity of inflammatory factors in the liver cells, health functional food.
The health functional food according to claim 7, wherein the inflammatory factor is at least one selected from the group consisting of TNF-α, IFN-γ, iNOS and COX-2.

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