KR101091775B1 - Composition containing Dieckol for treating and preventing neurodegenerative disease - Google Patents

Abstract

The present invention relates to a composition for the prevention and treatment of degenerative neurological diseases containing a Dieckol compound as an active ingredient, and more specifically, to microglia activated by lipopolysaccharide (LPS). The present invention relates to the use of a Dieckol compound derived from brown algae, which inhibits the production of reactive oxygen species (ROS) and the activation of NF-κB. The diecol compound may be usefully used for the treatment and prevention of neurodegenerative diseases.

Eclonia Caba, Dieckol, NF-κB, Degenerative Neuropathy

Description

Composition containing Dieckol for treating and preventing neurodegenerative disease

The present invention relates to a novel use of the Dieckol compound.

Microglia, the glial cells, account for 10-12% of the central nervous system glial cells and were first reported in 1932 by the morphological studies and tissue staining methods of Rio-Hortega. Microglia, which act on the degeneration of neurons and foreign substances in tissues, play an important role in transporting, destroying, removing, and cleaning pathological metabolites. It is considered a macrophage.

In addition, it is known to express characteristic cell surface antigens that are distinguished from neurons and other glial cells (astrocytes, oligodendrocytes) constituting the central nervous system, and perform macrophage-like functions such as phagocytosis. These microglia are known to have differentiated into the central nervous system by monocytes, which are thought to be precursors of these cells, during development. The primary defense against microbial infections and trauma in the central nervous system is that At this time, they move to the site of infection or local proliferation to detect infected microorganisms and digest the remnants of damaged neurons.

Microglial cells perform their intrinsic function of self-defense in the central nervous system, but are produced for defensive purposes such as tumor necrosis factor (TNF), interleukin (IL) -1β, reactive oxygen (ROS) or nitrogen compounds. Excessive secretion of inflammatory mediators or prolonged activation of the cells themselves can lead to serious side effects of nerve tissue damage.

Recently, research reports suggest that hyperglia activation of microglia is involved in neurodegenerative diseases such as Alzheimer's and Parkinson's as well as traumatic and ischemic neuronal damage [Microglia as mediators of inflammatory and degenerative diseases. Gonzalez-Scarano, F and Baltuch, G. Annu. Rev. Neurosci. 22: 219-40 (1999)], therapeutics are being developed that inhibit these activated microglia or prevent the action of proinflammatory substances secreted by the activated microglia.

That is, the neuroinflammatory response represented by the activation of microglia plays an important pathological role in various nerve tissue damages, and ultimately, it is possible to seek ways to inhibit nerve tissue damage by inhibiting the inflammatory activation of the microglia. Therefore, it will be possible to develop clinically applicable preventive and therapeutic agents for neurodegenerative diseases.

Meanwhile, the Eclonia cava is a seaweed belonging to the seaweed family and lives in the southern coastal area including South Korea's East Sea and Jeju Island. Eclonia kava is high in phlorotannin, a polyphenol of many plants. In particular, recently, it has been found that the cause of various adult diseases including aging is free-radical oxidation, and eclonia kava, which contains a large amount of strong antioxidant active substances such as phlorotannin, is attracting attention as a new functional material. . In addition to phlorotannin, Eclonia Kava also contains antioxidants such as alpha-tocopherol.

Recently, studies on the bioactive components of eclonia kava and their use have been conducted. Dietol, one of the major phlorotannins isolated from the Ecklonia species, has been reported to exhibit a variety of biological and pathological activities, for example free radical scavenging activity (Joe et al., 2006), liver Hepatoprotecive activity (Kim, An, Yoon, Nam & Choi, 2005), memory enhancing ability (Myung et al. 2005), bactericidal activity (Nagayama, Iwamura, Shibata, Hirayama & Makamura, 2002), HIV reverse transcription and Protease inhibitory activity (Ahn et al., 2004), tyrosinase inhibitory activity (Kang et al., 2004) and the like are known.

As such, it is known in the prior art that dietol has significant anti-inflammatory and antioxidant properties (Hwang et al., Shin & Stoner, 2006; Myung et al., 2005; Kang et al., 2004). The influence of diecol and its molecular mechanisms on degeneration have not been elucidated.

Accordingly, the present inventors found that dietol, a phlorotannin component extracted from eclonia cama, inhibits NO, PGE ₂ , ROS, iNOS and COX-2 expression, and production of cytokines (TNF-α, IL-1β). The present invention was completed by identifying the effects and molecular mechanisms of dietol inhibiting the production and activation of inflammatory mediators in microglia.

It is an object of the present invention to provide a preventive and therapeutic use of diecol compounds for neurodegenerative diseases.

It is another object of the present invention to provide a pharmaceutical composition for preventing and treating neurodegenerative diseases of the dietol compound.

It is still another object of the present invention to provide a method for inhibiting the production of reactive oxygen species and the activity of NF-κB using a diecol compound.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention and treatment of neurodegenerative diseases containing a diethol compound of formula (1) as an active ingredient.

<Formula 1>

In the above formula, R is hydrogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkenyl, phenyl, phenylalkyl, hydroxyphenyl, or dihydroxyphenyl, preferably hydrogen.

The diecol compound is derived from brown algae, preferably from Eisenia or Ecklonia.

In addition, the dieteck compound (dieckol) compounds are reactive oxygen species production and inhibition of NF-κB activity; Inhibition of translocation of the p65 protein, a subunit of NF-κB; Inhibition of proinflammatory cytokine activity of TNF-α or IL-1β; Inhibition of production of nitrogen monoxide (NO) and PGE ₂ ; inhibition of expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) genes; And inhibits the activity of p38 kinase.

In addition, the present invention provides a method of inhibiting the production of reactive oxygen species (ROS) and the activity of NF-κB using the diecol compound of the formula (1).

The brown algae-derived diecol compound compounds the production and activity of inflammatory mediators such as NO, PGE _2, TNF-α or proinflammatory cytokines of IL-1β produced by activated microglia at the transcription level. Since it inhibits, it can be usefully used for the prevention and treatment of degenerative neurological diseases such as Alzheimer's, Parkinson's disease, stroke and multiple sclerosis.

Hereinafter, the present invention will be described in detail.

The present invention relates to a novel use of the Dieckol compound. Dieckol is a type of phlorotannins, a polyphenolic compound, which acts as an antioxidant and anti-inflammatory.

In particular, generation of reactive oxygen species (ROS) and inhibition of NF-κB activity; Inhibition of proinflammatory cytokine activity of TNF-α or IL-1β; Inhibition of production of nitrogen monoxide (NO) and PGE ₂ ; inhibition of expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) genes; And inhibits the activity of p38 kinase and does not exhibit toxicity in experimental animals, and thus may be useful for the prevention and treatment of degenerative neurological diseases such as Alzheimer's, Parkinson's disease, stroke and multiple sclerosis.

The present invention relates to a prophylactic and therapeutic use of a neurodegenerative disease of a diethol compound represented by the following formula (1) in one aspect.

<Formula 1>

Wherein R is hydrogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkenyl, phenyl, phenylalkyl, hydroxyphenyl, or dihydroxyphenyl. Most preferably said R is hydrogen. Exemplary compound 1 includes dibenzo-1,4-dioxin.

"Alkyl" means a linear saturated monovalent hydrocarbon radical or branched saturated monovalent hydrocarbon radical having the carbon number specified in the prefix. For example, C ₁ -C ₆ alkyl means methyl, ethyl, n-propyl, dipropyl, tert-butyl, pentyl and the like.

"Alkenyl" means a linear monovalent hydrocarbon radical or branched monovalent hydrocarbon radical having the number of carbon atoms specified in the prefix and having one or more double bonds but no more than three double bonds. For example, C ₂ -C ₆ alkenyl is meant to include ethenyl, propenyl, 1,3-butadienyl and the like.

Definitions herein In each case (eg, alkyl, alkenyl, alkoxy, aralkyloxy), if the prefix does not specify the number of backbone carbon atoms in the alkyl moiety, the radical or moiety thereof has up to 6 backbone carbon atoms will be.

"Phenyl" is a functional group consisting of carbon and hydrogen, and has a chemical formula of -C ₆ H ₅ , wherein six carbon atoms mean a radical arranged in a cyclic ring structure. The phenyl group is a kind of aryl group. The case where one hydroxy group is bonded to the phenyl group is called hydroxyphenyl, and the case where two hydroxy groups are bonded is called hydroxyphenyl.

The diecol compound of the present invention may be prepared by separating the compound from brown algae, in particular the Eisenia or Ecklonia family.

Specifically for the brown algae, Eisenia bicyclis, Eisenia arborea, Eisenia desmarestioides, Esenia galapagensis, Eisenia masonii, Eclonia kurome, Eclonia cava, Eclonia stolonifera, Eclonia maxima, Eclonia radiata Ecklonia radiata, Eclonia bicyclis, Eclonia biruncinate, Eclonia buccinalis, Eclonia caepaestipes, Eclonia exasperta (Ecklonia exasperta), Eclonia fastigiata, Eclonia brevipes, Eclonia arborea, Eclonia latifolia, Eclonia muratii, Eclonia radicosa, Eclonia richardiana or Eclonia wrightii can be used, most Preferably Ecklonia cava can be used.

The diecol compound may be prepared and used by a method known in the art, a modified method thereof, or a method according to the present invention, and may be purchased and used commercially.

The diecol compound according to the present invention,

(a) extracting the eclonia carba powder with an organic solvent; And

(B) by fractionating the organic solvent extract can be prepared by a method comprising the step of separating and purifying the compound by chromatography.

As the organic solvent in step (a), ethanol, methanol and the like can be used, preferably methanol.

In one embodiment of the present invention E. cava can be used without limitation, such as cultivated or commercially available, it is used to clean and dry. The dried powder of E. cava is extracted with an appropriate amount of an organic solvent, preferably methanol, and the obtained methanol extract is fractionated with ethyl acetate. Then, the methanol compound according to the present invention is separated and purified by chromatography using a stepwise gradient of the solvent.

Accordingly, the present invention relates to a method of preparing the compound of Formula 1 in another aspect. The preparation method is merely an exemplary method thereof, and may be suitably modified by various methods based on the art. For example, the separation and purification of non-exemplified compounds according to the present invention Modifications apparent to those skilled in the art can be successfully carried out, for example, by appropriately protecting the interferer, replacing it with another suitable reagent known in the art, or by customarily changing the reaction conditions.

Those skilled in the art to which the present invention pertains, specific reaction conditions for the preparation of the compound 1 according to the present invention can be confirmed through the production examples and examples to be described later, a detailed description thereof Omit.

The diecol compound of Formula 1 of the present invention obtainable by the above method has the following functions with respect to inflammation-mediated neurodegeneration.

(1) The diecol compound of Formula 1 of the present invention inhibits reactive oxygen species generation and NF-κB activity in microglia stimulated by lipopolysaccharide (LPS).

Microglia act as a primary defense against microbial infections or trauma in the central nervous system, but they are produced by the defense necrosis factor (TNF) -α and interleukin (IL)- Pro-inflammatory cytokines such as 1β; Reactive oxygen species (ROS); Inflammatory substances such as nitric oxide (NO) and prostaglandin E2 (PGE2) can cause serious side effects such as damage to nerve tissue if they are excessively secreted or prolonged with the cells themselves activated. Among them, NO synthase, particularly inducible nitric oxide synthase (iNOS), is overexpressed by stimulation of LPS or the above proinflammatory cytokines and the like to increase the production of NO. It is involved in pathological symptoms related to inflammation in glial cells. In addition, PGE2 is an inflammatory product produced by the activity of COX-2, which is induced by stimulation of mitogen, cytosine and LPS at the site of inflammation in vivo.

Lipopolysaccharide (LPS), an endotoxin, is an inducer of inflammatory mediators in such microglia and induces the activation of NF-κB to promote the production of the inflammatory response.

NF-κB is known as a pleiotropic regulator of various genes related to immune and anti-inflammatory responses, and NF-κB activity plays a decisive factor in the expression of various cytokines and enzymes (Roshak et al. ., 1996). NF-κB binds to the NF-κB site upstream of iNOS and COX-2 promoters and plays an important role in the upregulation of LPS-induced iNOS and COX-2 genes (Lee, Sung, Kim, 2003). As an inactive precursor combined with an inhibitory protein, an IκB-like protein, a heterologous NF-κB complex is sequestered in the cytoplasm and in the nucleus p65 associated with a decrease in cytosolic IκB protein. As the protein increases, LPS induces NF-κB activity, and nuclear transfer of NF-κB occurs to activate genes with NF-κB bound to specific positions (Akira & Takeda, 2004).

That is, when an external stimulus capable of causing an inflammatory response is applied, NF-κB is activated in the cell, thereby inducing the expression of pro-inflammatory cytokines such as TNF-α, IL-1β, and the resulting pro Inflammatory cytokines stimulate the expression of genes encoding iNOS and COX-2, producing NO and PGE ₂ substances involved in the inflammatory response, causing an inflammatory response. In addition to these mechanisms, NF-κB can directly activate the expression of the iNOS gene, can express genes related to other inflammation, and pro-inflammatory cytokines such as TNF-α, IL-1β and IL-6. They also activate other biological responses involved in the inflammatory response.

The diecol compound according to the present invention has a function of inhibiting NF-κB activity as described above in a concentration-dependent manner, and diecol prevents NF-κB activation induced by LPS by preventing degradation of IκB. In addition, transfer of NF-κB to the nucleus requires dependent transcription according to NF-κB activation, and thus, the inhibitory effect of dietol on NF-κB activation is directed to the nucleus of p65 protein, a subunit of NF-κB. This leads to the effect of inhibiting translocation.

Consequently, according to the above mechanism, expression of pro-inflammatory cytokines such as NF-κB inflammatory response-related transcription factors, TNF-α, IL-1β, iNOS and COX-2 is suppressed.

On the other hand, reactive oxygen species (ROS), which cause oxidative damage to cells, are by-products generated by aerobic metabolism of living aerobic respiratory organisms such as mammals when the oxygen partial pressure is high.

Overproduction of ROS stimulates the production of proinflammatory cytokines via the activity of NF-κB. The neuroprotective effect of antioxidants is, in turn, due to increased antioxidant enzymes and decreased ROS (Bastianetto & Quirion 2004; Rosenstock, Carvalho, Jurkiewicz, Frissa-Filho, & Smaili, 2004).

In addition, intracellular ROS can regulate NF-κB signal transduction pathways involved in regulating NF-κB activity. Removal of ROS by antioxidants inhibits NF-κB-dependent production of proinflammatory mediators and thus prevents LPS toxicity (Victor, Rocha, Esplugues, De la Fuente, 2005; Park et al., 2004) )

Since ROS containing anion peroxide, hydroxy radicals, and hydrogen peroxide play a complex role in the development of many neurodegenerative diseases, ROS can function as signaling molecules in microglia. Although previous studies have shown that diecol has significant anti-inflammatory and antioxidant properties (Hwang, Chen, Nines, Shin & Stoner, 2006), the effects and molecular mechanisms of diecol in inflammatory mediated neurodegeneration have not been elucidated. . In addition, it is more desirable to prevent the generation of reactive oxygen species in advance than to eliminate the already produced reactive oxygen species, but research and development on the method of inhibiting the generation of reactive oxygen species is still insufficient. to be. Moreover, it is not easy to selectively inhibit the generation of reactive oxygen species produced by LPS and the activation of NF-κB by reactive oxygen species.

The diecol compound according to the present invention has a function of removing ROS from microglial cells, which is related to the mechanism for the diecol suppression effect on NF-κB activity. That is, inhibition of ROS production by dietol eventually leads to inhibition of NF-κB dependent cytokine, iNOS and COX-2 expression, thereby reducing inflammation in microglia.

(2) Therefore, the diecol compound of Formula 1 of the present invention is monoxide by inhibiting the production of reactive oxygen species and NF-κB activity in microglia stimulated by lipopolysaccharide (LPS). Inhibits the production of nitrogen (NO), prostaglandin E2 (PGE2) and proinflammatory cytokines.

Dietol isolated from brown algae of the present invention can significantly inhibit NO, PGE ₂ in microglia cells in a concentration-dependent manner, and the production of cytokines such as TNF-α and IL-1β, which induces neuronal damage, Because of its concentration-dependent inhibition without cytotoxicity, it exhibits effective therapeutic and prophylactic effects on inflammatory mediated neurodegenerative diseases.

In addition, the diecol inhibits the mRNA and protein expression of iNOS and COX-2, which means that the action of diecol occurs at the level of transcription.

(3) The diecol compound of Formula 1 of the present invention inhibits the activity of inflammatory mediators by inhibiting the activity of p38 kinase in microglia stimulated by lipopolysaccharide (LPS).

Mitogen-activated protein kinase (MAPK) plays a critical role in regulating cell growth and differentiation, is influenced by cellular responses to cytokines and stress, and plays an important role in the activation of NF-κB as well as iNOS. And COX-2 gene expression.

Inhibition of NF-κB activity by dietol according to the present invention may be mediated through the MAPK pathway. That is, the treatment of diecol against ERK-1 / 2, JNK, and p38 kinase phosphorylated by stimulation by LPS significantly inhibits the activity of p38 kinase. In contrast, ERK-1 / 2 and JNK are not affected by dietol.

Therefore, the diecol according to the present invention can also inhibit the activity of NF-κB and inhibit iNOS and COX-2 gene expression through the mechanism of inhibiting the phosphorylation of p38.

Accordingly, the present invention provides ROS, NF-κB signaling pathways in neurodegeneration according to various inflammatory mediators such as NO, PGE ₂ , ROS, iNOS and COX-2 expression, cytokines (TNF-α, IL-1β), and the like. And a novel anti-inflammatory mechanism of dietol based on the inhibition of NF-κB activity determined by the inhibition mechanism of MAPK (p38).

(4) In the neurodegeneration according to the inflammatory mediator, the novel anti-inflammatory mechanism of diecol of formula (I) of the present invention is concentration or dose-dependent.

The dose of dietol compound, which is effective for inflammation-mediated neurodegeneration, is 50 to 300 µg / ml, and if it exceeds 300 µg / ml, cytotoxicity may occur. There is a point.

Within this range, the inhibitory effect of NF-κB activity, which is determined by the inhibition mechanism of ROS, NF-κB signaling pathway and MAPK (p38), increases with the concentration (dose) of the dietol compound.

Unless otherwise stated below, the term "diecol compound" or "compound of formula 1" according to the present invention, the compound itself, pharmaceutically acceptable salts, hydrates, solvates, isomers and prodrugs thereof It is used as a concept that includes all of them.

The term "pharmaceutically acceptable salt" means a formulation of a compound that does not cause severe irritation to the organism to which the compound is administered and does not impair the biological activity and properties of the compound. The terms "hydrate", "solvate" and "isomer" also have the same meanings as above. The pharmaceutical salt is a compound of the present invention, such as hydrochloric acid, bromic acid, sulfuric acid, nitric acid, inorganic acids such as phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid and the like, sulfonic acid, tartaric acid, formic acid, citric acid, acetic acid, trichloro It can be obtained by reaction with organic carboxylic acids such as roacetic acid, trifluoroacetic acid, capric acid, isobutanoic acid, malonic acid, succinic acid, phthalic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, salicylic acid and the like. In addition, the compound of the present invention is reacted with a base, and salts such as alkali metal salts such as ammonium salts, sodium or potassium salts, and alkaline earth metal salts such as calcium or magnesium salts, dicyclohexylamine, and N-methyl-D-glu It may be obtained by forming salts of organic bases such as carmine and tris (hydroxymethyl) methylamine, and amino acid salts such as arginine and lysine.

The term "hydrate" includes a compound of the present invention comprising a stoichiometric or non-stoichiometric amount of water bound by a non-covalent intermolecular force. Or salts thereof.

The term "solvate" refers to a compound of the present invention or a salt thereof comprising a stoichiometric or nonstoichiometric amount of solvent bound by noncovalent intermolecular forces. Preferred solvents therein are volatile, nontoxic, and / or solvents suitable for administration to humans.

The term "isomer" means a compound of the present invention or a salt thereof that has the same chemical formula or molecular formula, but which is optically or stereoscopically different. For example, the compound according to Formula 1 of the present invention may have an asymmetric center (asymmetric carbon atom) depending on the type of substituents, in which case the compound of Formula 1 is combined with enantiomers and diastereomers. May exist as the same optical isomer.

The term "prodrug" refers to a substance that is transformed into a parent drug in vivo. Prodrugs are often used because, in some cases, they are easier to administer than the parent drug. For example, they may be bioavailable by oral administration, while the parent drug may not. Prodrugs may also have improved solubility in pharmaceutical compositions than the parent drug. For example, prodrugs are esters that facilitate the passage of cell membranes, which are hydrolyzed to carboxylic acids, which are active by metabolism, once the water solubility is detrimental to mobility, but once the water solubility is beneficial. Drug "). Another example of a prodrug may be a short peptide (polyamino acid) that is bound to an acid group that is converted by metabolism to reveal the active site.

The present invention relates to a method of treating and preventing degenerative neurological diseases using the compound 1 in one embodiment. The neurodegenerative disease is characterized by being induced by lipopolysaccharide (LPS), for example, Alzheimer's disease, Parkinson's disease, stroke or multiple sclerosis.

Accordingly, in another aspect of the present invention, the present invention relates to a method of administering an effective amount of a compound of Formula 1 to a patient to reduce or inhibit reactive oxygen species production and NF-κB activity. That is, the method of the present invention provides a method for treating and preventing diseases caused by reactive oxygen species generation and NF-κB activity.

As used herein, the term “treating”, unless stated otherwise, reverses, alleviates, or progresses the disease or condition to which the term applies, or one or more symptoms of the disease or condition. It means to inhibit or prevent. As used herein, the term "treatment" refers to the act of treating when "treating" is defined as above.

Another aspect of the present invention relates to a pharmaceutical composition for the prevention and treatment of degenerative neurological diseases containing a dietary alcohol compound represented by the formula (1) as an active ingredient. The composition may further include a diluent, an excipient, and the like as needed.

The term "pharmaceutical composition" means a mixture of a compound of the present invention with other chemical components such as diluents or carriers.

The pharmaceutical composition facilitates administration of the compound into the organism. There are a variety of techniques for administering compounds, including but not limited to oral, injection, aerosol, parenteral, and topical administration. Pharmaceutical compositions may also be obtained by reacting acid compounds such as hydrochloric acid, bromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.

The term "therapeutically effective amount" means that the amount of the compound administered to alleviate to some extent one or more of the symptoms of the disorder being treated. Thus, a pharmacologically effective amount means (1) to reverse the rate of progression of a disease or (2) to prohibit further progression of the disease, and (3) to some extent one or more symptoms associated with the disease. It means the quantity which has an effect of reducing (preferably removing).

The term "carrier" is defined as a compound that facilitates the addition of a compound into a cell or tissue. For example, dimethyl sulfoxide (DMSO) is a commonly used carrier that facilitates the incorporation of many organic compounds into cells or tissues of an organism.

The term "diluent" is defined as a compound that not only stabilizes the biologically active form of the compound of interest, but also is diluted in water to dissolve the compound. Salts dissolved in buffer solutions are used as diluents in the art. A commonly used buffer solution is phosphate buffered saline, because it mimics the salt state of human solutions. Because buffer salts can control the pH of a solution at low concentrations, buffer diluents rarely modify the biological activity of a compound.

The term "physiologically acceptable" is defined as a carrier or diluent that does not impair the biological activity and the properties of the compound.

The compounds used herein may be administered to human patients as such or as pharmaceutical compositions in combination with other active ingredients or with a suitable carrier or excipient, such as in a combination therapy.

(a) route of administration

Suitable routes of administration include parenteral delivery, including, for example, intramuscular, subcutaneous, intravenous, bone marrow injections, as well as intraoral, intranasal, transmucosal, or enteral septum, direct intraventricular, intraperitoneal, or intraocular injections. Include.

In addition, the compounds may also be administered in a local rather than systemic manner, for example by direct injection into solid tumors, often in immersion or sustained release formulations.

(b) Compositions / Formulations

Pharmaceutical compositions of the invention can be prepared in a known manner, for example, by means of conventional mixing, dissolving, granulating, sugar-making, powdering, emulsifying, encapsulating, trapping and or lyophilizing processes. .

Thus, a pharmaceutical composition for use according to the invention comprises one or more pharmacologically acceptable which comprises excipients or auxiliaries which facilitate the treatment of the active compounds into formulations which can be used pharmaceutically. It may also be prepared by conventional methods using a carrier. Proper formulation is dependent upon the route of administration chosen. Any of the known techniques, carriers and excipients may suitably be used.

For injection, the components of the invention may be formulated in liquid solutions, preferably in pharmacologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer. For mucosal permeation administration, noninvasive agents suitable for the barrier to pass through are used in the formulation. Such non-invasive agents are generally known in the art.

For oral administration, the compounds can be formulated readily by combining the active compounds with pharmacologically acceptable carriers known in the art. Such carriers allow the compounds of the invention to be formulated into tablets, pills, sugars, capsules, liquids, gels, syrups, slurries, suspensions and the like. Pharmaceutical preparations for oral use may be achieved by mixing one or more compounds of the invention with one or more excipients, optionally grinding such mixtures and, if necessary, treating the mixture of granules after permeation of appropriate adjuvants. A tablet or sugar core can be obtained. Suitable excipients include filler corn starch, wheat starch, rice starch, potato starch, gelatin, gum tragakens, methyl cellulose, hydroxypropylmethyl-cellulose, sodium such as lactose, sucrose, mannitol, or sorbitol Cellulose based materials such as carboxymethyl cellulose, and / or polyvinylpyrrolidone (PVP), and the like. If necessary, a disintergrating agent may be added, such as crosslinked polyvinyl pyrrolidone, butadiene, or salts thereof such as alginic acid or sodium alginate.

Sugar cores are supplied by appropriate coating. For this purpose, concentrated sugars, which optionally include arabide gum, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures Solutions can be used. Dyestuffs or pigments may be included in the tablets or sugars to characterize the identification of the active compound or to characterize other combinations thereof.

Pharmaceutical preparations that can be used orally may include soft sealing capsules made of gelatin and plasticizers such as glycols or sorbitol, as well as tightly held capsules made of gelatin. The push-fix capsule may contain the active ingredients, as a mixture with a filler such as lactose, a binder such as starch, and / or a lubricant such as talc or magnesium stearate. In soft capsules, the active compounds may be dissolved or dispersed in suitable solvents such as fatty acids, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be included. All preparations for oral administration should be in amounts suitable for such administration.

For buccal administration, the compositions may take the form of tablets or lozenges formulated according to conventional methods.

For administration by inhalation, the compounds of use according to the invention typically use an appropriate propellant such as, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. Thus, it may be delivered in the form of aerosol injection provision from a pressurized pack or nebulisher. For use in inhalants or pulverulents, capsules and cartridges such as, for example, gelatin may be formulated comprising a powdered mixture of the compound and a suitable powder such as lactose or starch.

The compounds may be formulated for parenteral administration by injection, eg, by large pill injection or continuous infusion. Injectable formulations may be presented in unit dose form, for example, as ampoules or multi-dos containers, with preservatives added. The compositions may take the form of suspensions, solutions, emulsions on oily or liquid vehicles, and may include components for formulation such as suspensions, stabilizers and / or dispersants.

Liquid formulations for parenteral administration include liquid solutions of the active compounds in water-soluble form. In addition, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty acids such as sesame oil, synthetic fatty acid esters such as ethyl oleate or triglycerides, liposomes and the like. Liquid injection suspensions may include substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, dextran, and the like. In some cases, suspensions may contain components or stabilizers that increase the solubility of the compound to allow for the preparation of highly concentrated solutions.

The active ingredient may also be in powder form for constitution with a suitable vehicle, such as sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal dosage compositions, such as suppositories or retention enemas, including, for example, conventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described above, the compounds may be formulated as deposits. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular infusion. Thus, the compounds may, for example, be formulated with a suitable polymer or hydrophobic material (such as an emulsion in an acceptable oil), or with an ion exchange resin, or as a low soluble derivative, for example a low soluble salt. have.

The formulation carrier for the hydrophobic compound of the present invention is a cosolvent system composed of benzyl alcohol, nonpolar surfactant, water-miscible organic polymer, and liquid phase. The cosolvent system may be a V palladium cosolvent system. This cosolvent system dissolves hydrophobic compounds well and provides itself with low toxicity upon systemic administration. Naturally, the proportion of cosolvent system may vary considerably without compromising its solubility and toxicological properties. Moreover, the identification of cosolvent components can be varied.

In addition, other delivery systems for hydrophobic drug compounds may be employed. Liposomes and emulsions are known examples of delivery vehicles for hydrophobic agents. Usually organic solvents such as dimethylsulfoxide may be employed, even at the expense of higher toxicity. In addition, the compound may be delivered using a sustained release system, such as a semipermeable matrix of a solid hydrophobic polymer containing a therapeutic ingredient. Various sustained release materials have been developed and are known to those skilled in the art.

Many compounds of the present invention may also be provided as salts with pharmaceutically acceptable counterions. Pharmaceutically acceptable salts may be formed with many acids including, but not limited to, hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, malic acid, succinic acid, and the like. Salts tend to dissolve better in aqueous or proton solutions than their corresponding acid free or base forms.

(c) effective amount

Pharmaceutical compositions suitable for use in the present invention include compositions in which the active ingredients are contained in an amount effective to achieve their intended purpose. More specifically, a therapeutically effective amount means an amount of a compound effective to prolong the survival of the subject to be treated or to prevent, alleviate or alleviate the symptoms of a disease. Determination of a therapeutically effective amount is within the capabilities of those skilled in the art, in particular in terms of the detailed disclosure provided herein. A therapeutically effective amount for any compound used in the methods of the invention can be determined initially from cell culture assays. For example, the dose can be calculated in an animal model to obtain a range of circulating concentrations comprising an IC ₅₀ determined in cell culture. Such information can be used to more accurately determine useful doses in humans.

The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The exact estimate, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. Typically, the dose range of the composition administered to the patient may be about 0.5 to 1000 mg / kg of the patient's body weight. Dosages may be given in a series of two or more, one at a time or in a day or more, depending on the extent required by the patient.

Dosages and intervals may be individually adjusted to provide plasma levels of the active site sufficient to maintain a kinase modulating effect or minimal effective concentration (MEC). The MEC depends on the individual compound, but can also be predicted from ex vivo data, such as, for example, the concentration required to achieve 50-90% inhibition of kinases using the assays described herein. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, HPLC or biological quantification can be used to determine plasma concentration.

Dosage intervals may be determined using MEC values. Compounds should be administered using a dosing regimen that maintains serum levels above the MEC, such that 10-90%, preferably 30-90%, particularly preferably 50-90%, at a time.

Of course, the amount of composition to be administered will depend on the subject to be treated, on the weight of the subject, on the severity of pain, on the manner of administration and on the judgment of the physician.

The invention is illustrated in more detail by the following examples, preparations and experimental examples. However, the scope of the present invention is not limited to the following examples and the like.

The values obtained in the following examples were expressed as mean ± standard error (SEM), and statistical significance was determined by analyzing variable conditions according to the Scheffe test ( p <0.05).

Manufacturing example  One: Eclonia From Kaaba Diecol ( Dieckol ) detach

Brown algae along the coast of Jeju Island, South Korea from October 2005 to March 2006Ecklonia cavaWas taken. The sample was washed three times with tap water to remove salts, epiphytes and sand from the surface, carefully rinsed with water, and then stored frozen at -20 to 0 ° C. The sample was lyophilized and then homogeneously ground with a grinder before extraction.

The E. cava powder (500 g) was extracted three times with 80% MeOH and then filtered. The filtrate was evaporated at 40 ° C. to obtain a methanol extract. The extract was suspended in distilled water and fractionated with ethyl acetate. The ethyl acetate fraction was mixed with celite, then dried and filled into a glass column. Then, eluted with chloride, diethyl ether and methanol in order. For diethyl ether fractions, Sephadex LH-20 column chromatography was performed using a stepwise gradient of chloroform / methanol (2/1 → 0/1) solvent system.

Further purification using an HPLC system yielded Dieckol, a type of phlorotannin (Formula 1), and compared 1H and 13C NMR data for the diecol compound.

1 H NMR (400 MHz, methanol- d 4) δ 6.15 (1H, s), 6.13 (1H, s), 6.09 (1H, d, 2.9 Hz), 6.06 (1H, d, 2.9 Hz), 6.05 (1H, d, 2.9 Hz), 5.98 (1H, d, 2.8 Hz), 5.95 (1H, d, 2.8 Hz), 5.92 (3H, m); 13C NMR (100 MHz, methanol- d 4) δ 161.8, 160.1, 157.8, 155.9, 154.5, 152.4, 147.3, 147.2, 147.1, 146.9, 144.3, 144.1, 143.4, 143.3, 138.6, 138.5, 126.5, 126.2, 125.6, 125.5, 124.9, 124.6, 124.5, 99.9, 99.7, 99.5, 99.4, 97.6, 96.2, 95.8, 95.7, 95.3.

Preparation Example 2 Preparation of BV2 Microglia

Rat BV2 cell lines (obtained from the Department of Microbiology, College of Medicine, Inje University) were placed in Dulbeco's Modified Essential Medium (DMEM) medium supplemented with 10% FBS, 100 U / ml penicillin and 100 μg / ml streptomycin, and at 37 ° C. and 5% CO _2. Cultured in a wet wet incubator. After treatment with trypsin when the cells were confluent, the cells were washed twice with warm DMEM without phenol red. It was then transferred to serum-free medium and cultured.

In all the examples, LPS (1 μg / ml) was added to the cells, followed by treatment with diecol for a defined time.

Example 1: NO and PGE ₂  Effect of Diecol on Generation

Various concentrations of diecol (0, 50, 100, 300 µg / ml) were treated for 2 hours and stimulated with LPS for 24 hours prior to addition of LPS (1 µg / ml) to ⁵ × 10 ⁵ cells / well BV2 microglia. .

The concentration of NO in the culture supernatant was measured using a Griess reagent as nitrite, which is an oxidation product of main NO. 100 μl of each cell culture medium was added to the same volume (100 μl) of Griess reagent [1% sulfanilamide / 0.1% N- (1-naphthyl) -naphthylethylenediaminedihydorchlorid / 2.5% phosphoric acid. ]. The level of nitrite was measured colorimetrically at 540 nm using an ELIZA plate reader and the nitrite concentration was calculated by reference to a nitrite standard curve constructed from known sodium nitrite concentrations.

As a result, as shown in FIG. 1, pre-treatment of various doses of LPS and dietol resulted in a significant decrease in NO formation (FIG. 1A). According to the NO detection assay, after 24 hours of LPS stimulation, NO increased significantly by 5.7 times compared to basal level in BV2 microglia, and this increase was due to treatment of dietol in a dose-dependent manner. It was confirmed that inhibition (inhibition).

On the other hand, to determine whether dietol can inhibit the production of PGE ₂ induced by LPS in BV2 microglia, cells were pretreated with diethol for 2 hours and stimulated with LPS (1 μg / ml). After 24 hours of incubation, cell culture medium was collected and the PGE ₂ product was measured with a Cayman Chemical ELISA kit (Ann Arbor, MI, USA).

As a result, pretreatment of diecol (0, 50, 100, 300 μg / mL) and stimulation of LPS showed a significant dose-dependent decrease in PGE ₂ production (FIG. 1B).

These results show that diecol pretreatment significantly inhibits the expression of proinflammatory mediators stimulated by LPS.

Reference Example 1: Cell viability assay

In order to determine whether the inhibition of NO and PGE2 production in Example 1 was caused by cytotoxicity due to diecol treatment, MTT analysis was performed on BV2 microglia treated with diecol for 24 h.

Colorless 3- (4,5-dimethyl-thiazol-2-yl) -2,5-diphenyl tetrazolium bromide (3- (4,5-dimethyl- thiazol-) by mitochondrial dehydrogenases Cell viability was assessed by measuring the amount metabolized from 2-yl) -2,5-diphenyl tetrazolium bromide) (MTT) [Sigma (St Louis, MO, USA)] and digested with blue formazan.

BV-2 cells at ⁵ × 10 ⁵ cells / well were washed after 24-hour preincubation in 24-well plates. The diecol obtained in Preparation Example 1 was treated with various concentrations of LPS (derived from Escherichia coli serotype 0111) for 24 hours and incubated in 0.5 mg / ml MTT solution. After 3 hours, the supernatant was removed and formazen formation was measured with a microplate reader (Dynatech MR-7000; Dynatech Laboratories, Chantilly, VA, USA) at 540 nm.

As a result, dietol did not affect cell viability at the concentration used, i.e., it was confirmed that the NO and PGE2 production inhibitory activity of dietol was not due to any cytotoxic action in BV2 microglia ( 1 C).

Example  2: iNOS  And COX For the expression of -2 proteins Diecole  effect

To determine whether inhibition of NO and PGE2 production was due to reduced levels of iNOS and COX-2, the effect of dietol on iNOS and COX-2 proteins was examined through Western blot.

BV2 microglia obtained in Preparation Example 2 were washed three times with Cells were washed three times with PBS and dissolved in cytolysis buffer (1% Triton X-100, 1% deoxycholate, 0.1% NaN 3). Equal amounts of protein were separated on 10% SDS-polyacrylamide minigel and transferred to Immobilon polyvinylidene difluoride membranes (Millipore, Billerica, Mass., USA). After incubation with a suitable primary antibody, the membrane was incubated with a secondary antibody bound to horseradish peroxidase (HRP) for 1 hour at room temperature. Three washes with Tris-Buffered Saline Tween-20 (TBST) and immunoreactive bands were visualized using an ECL detection system (Pierce, Rockford, IL, USA).

As a result, the expression of iNOS and COX-2 protein was hardly detected in BV2 microglia which were not stimulated, but its expression was markedly increased after 24 h of LPS treatment. However, dietol significantly relieved the expression of iNOS and COX-2 proteins in LPS-stimulated BV2 microglia (FIG. 2A).

Example 3: Effect of Dietol on iNOS and COX-2 mRNA Levels

The effect of dietol on iNOS and COX-2 mRNA levels was evaluated.

Total RNA was isolated using TRIzol reagent (Invitrogen, CA, USA). After preparing cDNA using M-MLV reverse transcriptase (Promega, Madison, WI, USA) for the cells obtained in Preparation Example 2, the total RNA (1.0 μg) reverse transcription was obtained. Antibodies against iNOS, COX-2, and p65 were purchased from Santa Cruz Biotechnology (Santa Cuz, CA, USA), and RT-generated cDNA encoding iNOS, COX-2 gene was amplified using PCR. The selective primers of the mice used for PCR were as follows. After amplification, the PCR reactions were electrophoresed on agarose gels.

Sequence of PCR primers gene order iNOS
 5'-ATGTCCGAAGCAAACATCAC-3 '(SEQ ID NO: 1)  5'-TAATGTCCAGGAAGTAGGTG-3 '(SEQ ID NO: 2) COX-2
 5'-CAGCAAATCCTTGCTGTTCC-3 '(SEQ ID NO: 3)  5'-TGGGCAAAGAATGCAAACATC-3 '(SEQ ID NO: 4)

As a result, iNOS and COX-2 mRNA expression was shown to be inhibited equal to their protein levels (FIG. 2B). These results indicate that exposure to LPS increases the expression of mRNAs of iNOS and COX-2, but dietol effectively inhibits the induction of proinflammatory mediators stimulated by LPS through transcriptional inhibition.

Example  4: Proinflammatory  For the generation of cytokines Diecole  effect

Next, the influence of dietol on the production of proinflammatory cytokines such as TNF-α and IL-1β was examined.

BV2 microglia were incubated with dietol (0, 50, 100, 300 μg / ml) in the presence or absence of LPS for 24 hours, and the cytokine levels in the culture medium were subjected to an enzyme-linked immunosorbent assay (ELISA). Was measured. IL-1β and TNF-α were measured with the ELISA kit (Minneapolis, MN, USA) of the R & D system, PGE ₂ was measured with the Cayman Chemical kit (Ann Arbor, MI, USA), and 450 using a microplate reader. Absorbance at nm was measured.

As a result, as shown in FIG. 3A, TNF-α and IL-1β levels were increased in culture medium of BV2 microglia stimulated by LPS, and this increase was according to the concentration-dependent manner of dietol. Markedly decreased.

Meanwhile, RT-PCR was performed to determine whether dietol inhibited the expression of the cytokine at the level of transcription.

RT-generated cDNA encoding IL-1β and TNF-α genes were amplified using PCR. The selective primers of the mice used for PCR were as follows. After amplification, the PCR reactions were electrophoresed on agarose gels.

Sequence of Selective Primers Used for PCR gene order IL-1β
5'-ATGGCAACTGTTCCTGAACTCAACT-3 '(SEQ ID NO: 5) 5'-TTTCCTTTCTTAGATATGGACAGGAC-3 '(SEQ ID NO: 6) TNF-α
5'-ATGAGCACAGAAAGCATGATC-3 '(SEQ ID NO: 7) 5'-TACAGGCTTGTCACTCGAATT-3 '(SEQ ID NO: 8)

As a result, as shown in B of Figure 3, the diet was treated with BV2 microglia at various concentrations for 2 hours prior to LPS stimulation, the mRNA of TNF-α and IL-1β was dose-dependently reduced. These results suggest that dietol negatively regulates the accumulation of proinflammatory cytokines at the level of transcription.

Example 5: Effect of Diecol on NF-κB Activity

NF-κB binding activity was analyzed by EMSA (electrophoretic mobility shift assay) to elucidate the molecular mechanism for the inhibition of dietol at iNOS and COX-2 protein levels.

First, nuclear extracts were prepared using NE-PER nuclear extraction reagent (Pierce) according to the manufacturer's protocol. Oligonucleotides comprising an immunoglobulin κ-chain binding site [κB, 5'-CCGGTTAACAGAGGGGGCTTTCCGAG-3 '(SEQ ID NO: 9)] as a probe for gel retardation assay were synthesized. A non-radioactive method was used, in which the 3 ′ end of the probe was labeled with biotin.

The binding reaction was performed with 10 μg of nuclear protein, buffer (10 mM Tris, pH 7.5, 50 mM KCl, 5 mM MgCl ₂ , 1 mM dithiothreitol, 0.05% Nonidet P-40, and 2.5% glycerol), 50ng poly (dI-dC), and 20 fM of biotin-labeled DNA were used. After incubation at room temperature for 20 min at a final volume of 20 μl, the reaction mixture was analyzed by electrophoresis on a 11.5% polyacrylamide gel in 0.5 × Tris-borate buffer. The reaction was transferred to a nylon membrane and biotin-labeled DNA was analyzed using a light shift chemiluminescent EMSA kit (Pierce). DNA-binding specificity was confirmed using 50-fold greater than biotin-unlabeled NF-κB (cold κB) probes relative to the reaction mixture prior to labeled probe addition.

As a result, LPS treatment significantly increased the DNA binding activity of NF-κB, and in contrast, the treatment of diecol significantly inhibited NF-κB activity induced by LPS (FIG. 4A).

The effect of NF-κB p65 intranuclear migration induced by LPS was determined by Western blot analysis. As a result, as shown in FIG. 5, 15 minutes after LPS stimulation, it was confirmed that NF-κB p65 was expressed in the nucleus, but the p65 protein was significantly decreased in the nucleus of cells exposed to LPS and dietol. This means that dietol inhibits the nuclear migration of p65 protein.

In addition, in order to clarify the effect of diecol on NF-κB p65 intracellular migration, NF-κB p65 intranuclear migration in BV2 microglia was observed by immunofluorescence assay.

First, BV2 microglia were cultured directly on glass coverslips for 24 hours in 24-well plates. The cells were stimulated with 1 μg / ml LPS and / or 300 μg / ml dietol and then fixed with 4% paraformaldehyde in PBS. And permeability was imparted with 0.2% Triton X-100 in PBS and blocked with 1.5% normal donkey serum (Sigma). After reacting with polyclonal antibody against NF-κB p65 (1 μg / well) for 1 hour, fluorescein isothiocyanate bound with donkey anti rabbit IgG (Jackson ImmunoResearch Laborities, Inc., West Grove, PA, USA) ) Was incubated for 1 hour. After washing with PBS, the cover slip was mounted on Fluoromount-G (Southern Biotechnology Associates Inc., Birmingham, AL, USA), and the fluorescent material was visualized using a Zeiss LSM 510 laser scanning confocal device and observed at 400x magnification. .

From the confocal image, NF-κB p65 is normally isolated into the cytoplasmic region (B, Medium panel in FIG. 4) and the nuclear accumulation of NF-κB p65 was strongly induced after stimulation of LPS (B, LPS in FIG. 4). panel). The migration of NF-κB p65 induced by LPS completely disappeared when the cells were pretreated with dietholol (FIG. 4B, LPS + dieckol panel). Migration of NF-κB p65 was not induced even when pre-treated with diecol alone without LPS stimulation (FIG. 4B, dieckol panel).

In addition, the cytoplasmic level of I-κBα was confirmed by Western blot analysis in order to determine whether NF-κB DNA binding inhibition by diet is associated with the degradation of I-κBα. Antibodies against IκB were purchased from Cell Signaling Technology (Beverly, 15 MA, USA) and used.

As a result, pretreatment of BV2 microglia with dietecol prevented the degradation of I-κBα induced by LPS (FIG. 5). I-κBα protein repair in BV2 microglia is evidence that dietol inhibits the activation of NF-κB.

These results show that dietol inhibits the migration of NF-κB p65. It also suggests that inhibition of NF-κB activity by dietol may be a mechanism associated with NO, PGE2 and proinflammatory cytokines in BV2 microglia.

Example 6 Effect of Dietol on Mitogen-activated Protein Kinase (MAPK)

We attempted to elucidate a series of signaling cascades associated with mitogen-activated protein kinase (MAPK), which drives the expression of iNOS and COX-2 genes in BV2 microglia in response to stimulation of LPS.

To determine whether inhibition of NF-κB activity by diecol is mediated through the MAP kinase pathway, ERK-1 / 2, JNK and p38 kinases induced by LPS in BV2 microglia using Western blot analysis The effect of dietol on phosphorylation of was investigated.

As a result, as shown in FIG. 6, ERK-1 / 2, JNK and p38 kinases were phosphorylated by stimulation by LPS. In addition, dietol (300 µg / ml) significantly inhibited the activity of p38 kinase, but did not affect the activities of ERK-1 / 2 and JNK. This result makes it possible to predict a series of mechanisms in which NF-κB activity is inhibited by dietary inhibition of p38 phosphorylation and iNOS and COX-2 expression is inhibited.

Example 7: Effect of Dietol on the Generation of Reactive Oxygen Species

We examined whether dietol inhibited the production of reactive oxygen species (ROS).

Known Method ( Cho et al ., Toxicology and applied pharmacology , vol . 168 , p. 64-71, 2000) was modified to measure ROS. The BV2 microglia were treated with diecol (0, 50, 100, 300 µg / ml) and cultured in the presence or absence of LPS for 24 hours. BV2 microglia were washed with PBS, and the cells were incubated with PBS containing 5 μM of 2,7-dichlorofluorescein diacetate (DCFDA) (Molecular Probes, Eugene, OR, USA) for 4 hours at 37 ° C. ROS was labeled. The cells were then immediately observed through FACS analysis (fluorescence-activated cell sorting analysis) (BD Biosciences, Rutherford, NJ, USA).

As a result, as shown in Figure 7, BV2 microglia stimulated by LPS showed an increase in ROS production. In contrast, the pretreatment of cells with dieticol showed significant removal of ROS production by LPS. When the cells were treated with dietol (300 µg / ml) alone and cultured, the concentration of ROS was maintained at the same level as the surroundings, similar to the sample without stimulation.

1 is a result of analyzing the effect of diethol on NO production (A), PGE ₂ production (B) and cell viability (C) in BV2 microglia stimulated by LPS.

Figure 2 shows the results of RT-PCR analysis (B) of protein expression of iNOS and COX-2 induced by LPS in BV2 microglia (A) and mRNA of iNOS and COX-2.

Figure 3 shows the results of analyzing the effect of diethol on the TNF-α and IL-1β production induced by LPS in BV2 microglia.

Figure 4 shows the results of analysis of NF-κB binding activity in LPS-stimulated BV2 microglia cells by electrophoretic mobility shift assay (EMSA) (A) and immunofluorescence assay results for NF-κB p65 nuclear transfer (B) to be.

5 is a Western blot analysis of the effect of NF-κB p65 intranuclear migration induced by LPS.

FIG. 6 shows Western blot analysis of ERK-1 / 2, JNK and p38 kinases induced by LPS in BV2 microglia.

7 is a FACS analysis of ROS (reactive oxygen species) production in BV2 microglia stimulated by LPS.

<110> Industry-Academic Cooperation Foundation, Chosun University <120> Composition containing Dieckol for treating and preventing          neurodegenerative disease <160> 8 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> iNOS primer 1 <400> 1 atgtccgaag caaacatcac 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> iNOS primer2 <400> 2 taatgtccag gaagtaggtg 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> COX2 primer 1 <400> 3 cagcaaatcc ttgctgttcc 20 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> COX2 primer2 <400> 4 tgggcaaaga atgcaaacat c 21 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> IL-1beta primer1 <400> 5 atggcaactg ttcctgaact caact 25 <210> 6 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> IL-1beta primer2 <400> 6 tttcctttct tagatatgga caggac 26 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> TNF-a primer 1 <400> 7 atgagcacag aaagcatgat c 21 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> TNF-a primer2 <400> 8 tacaggcttg tcactcgaat t 21  

Claims (10)

It contains a diecol compound of the formula (1) as an active ingredient, The diecol compound is included in the amount of 50 ~ 100㎍ / ㎖, The diecol compound was prepared by adding methanol to Eclonia Cava powder to prepare a methanol extract, and then suspended in distilled water, and then ethyl acetate was added to obtain an ethyl acetate fraction, followed by chloride, diethyl ether, and methanol. In the fractions eluted sequentially, characterized in that the purified through the diethyl ether fraction, Pharmaceutical compositions for the prevention and treatment of inflammatory mediated neurodegenerative diseases induced in microglia: <Formula 1>

In said formula, R is hydrogen. delete delete delete delete The pharmaceutical composition according to claim 1, wherein the neurodegenerative disease is induced by lipopolysaccharide (LPS). The pharmaceutical composition of claim 6, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, stroke and multiple sclerosis. The composition of claim 1, wherein the composition comprises reactive oxygen species production and NF-κB activity; Translocation of the p65 protein, a subunit of NF-κB; Production of proinflammatory cytokines, production of nitric oxide (NO); expression of an inducible NO synthase (iNOS) gene; Production of prostaglandin E2 (PGE ₂ ); And at least one of p38 kinase activity. The pharmaceutical composition of claim 8, wherein the proinflammatory cytokine is TNF-α or IL-1β. According to claim 8, wherein the composition is a pharmaceutical composition, characterized in that to inhibit the expression of the COX-2 (cyclooxygenase-2) gene.

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