KR101449796B1 - A Labdane-Type Diterpenes Compounds derived from Hedychium coronarium and a Use thereof - Google Patents

Abstract

The present invention relates to the use of a hedchium cornarium coronarium extract and a Labdane-Type Diterpenes compound derived therefrom. The present invention relates to a composition or a method for preventing and treating autoimmune diseases and sepsis. More specifically, the present invention relates to a Hodigitum coronarium extract inhibiting the production of inflammatory cytokines stimulated by lipopolysaccharide (LPS) and the use of these derived dipeptane diterpen compounds.

Description

[0002] The present invention relates to a compound of the formula (I), a compound of the formula (I), and a compound of formula (I)

The present invention relates to a use of a Heddichium coronarium extract and a Labdane-Type Diterpenes compound derived therefrom, and it relates to a composition or a method for preventing and treating autoimmune diseases and sepsis, . More specifically, the present invention relates to a Hodigitum coronarium extract inhibiting the production of inflammatory cytokines stimulated by lipopolysaccharide (LPS) and the use of these derived dipeptane diterpen compounds.

Inflammatory disorders are one of the most important health problems in the world. Inflammation is generally a localized protective response of the body tissue to host infiltration by foreign substances or by harmful stimuli. Causes of inflammation include infectious causes such as bacteria, viruses and parasites; Physical causes such as burns or irradiation; Chemicals such as toxins, drugs or industrial formulations; An immune response such as an allergic and autoimmune response, or a condition associated with oxidative stress.

Inflammation is characterized by pain, numbness, swelling, heat and ultimate loss of function in the affected area. These symptoms are the result of a series of complex interactions that take place between cells of the immune system. The reaction of cells results in a network of interaction groups of inflammatory mediators as a result: proteins (eg, cytokines, enzymes (eg proteases, peroxidases), major basic proteins, (E.g., VCAM), lipid mediators (e.g., eicosanoids, prostaglandins, leukotrienes, platelet activating factors (PAF)), reactive oxygen species such as hydroperoxides, superoxide anions O2-, ), Etc. However, most of these mediators of inflammation are also regulators of normal cellular activity. Thus, a deficiency of the inflammatory response causes damage (i.e., infection) without control of the host, Inflammatory diseases mediated by the overproduction of several of the above-mentioned mediators.

In particular, autoimmune diseases, one of the inflammatory diseases, are characterized by the immune system attacking its own organs and causing a spontaneous response. These responses are due to the recognition of auto-antigen by T lymphocytes, which leads to immune responses in the body fluid (autoantigen production) and in the cellular phase (increased lymphocyte and macrophage cytotoxic activity). Examples of autoimmune diseases include rheumatic diseases, psoriasis, systemic dermatomyositis, multiple sclerosis, lupus erythematosus, or aggravation of immune responses by antigens, such as allergies to asthma, drugs or food. These diseases are all limited and chronic diseases, and in some cases, they are fatal, and thus there is no effective treatment method for treating the diseases. Therefore, drugs, medicines or medicines capable of alleviating or alleviating the disease during the course of the disease will be said to be an important solution for the health of the patient.

We have focused our efforts on finding suitable medicines and methods by exploring methods of treating autoimmune diseases. Today, the treatment of autoimmune diseases is mainly based on the use of immunosuppressive drugs such as glucocorticoids, calcineurin inhibitors and antiproliferatives-antimetabolites. However, such pharmacological therapies act on a variety of targets and, as a whole, can reduce immune function. Otherwise, long-term use of this pharmacotherapy can lead to a number of cytotoxic effects, which can lead to exposure of the patient to infection and cancer by inhibiting the immune system in a nonspecific manner. The use of calcineurin and glucocorticoids is limited in the case of some clinical symptoms (eg, renal insufficiency, diabetes, etc.), as they present another problem due to their nephrotoxicity and diabetes-inducing properties.

For this reason, patients with an autoimmune disease or an inflammatory disease of sepsis are of particular interest in the type of treatment that is considered to be "natural" with no major side effects and weak anti-inflammatory effects that can be used as disease prevention and adjunctive therapy. While there are examples of "natural" formulations that exhibit anti-inflammatory properties, these "natural" compounds often have inappropriate biochemical and therefore inhibitory activity. For the above reasons, there is a great need for an anti-inflammatory agent containing a novel natural compound as an active ingredient.

In addition, while Hodigmi coronarium extract and some compounds isolated therefrom are known in the prior art, the molecular mechanisms involved in the production of LPS (Lipopolysaccharide) -stimulated inflammatory cytokines have not been elucidated.

Therefore, the present inventors confirmed that the extracts extracted from Hedychium coronarium and the specific rapamycin diaphene compounds isolated therefrom inhibit the production of TNF-α, IL-6 or IL-12, that is, LPS Lipopolysaccharide). The present inventors completed the present invention by examining the effect of dipeptan diterpene compounds inhibiting the production and activation of the cytokines in stimulated bone marrow derived dendritic cells (DCs).

The present invention aims to provide therapeutic and prophylactic uses for an autoimmune disease and sepsis which contain an extract of Hedychium coronarium .

Another object of the present invention is to provide a Labdane-Type Diterpene compound of the general formulas (1) to (3) and its use.

It is another object of the present invention to provide a method for inhibiting LPS-stimulated inflammatory cytokine production using Labdane-Type Diterpene compounds of Formulas (1) to (3).

In order to accomplish the above object, the present invention provides the use of an extract of Hedychium coronarium having LPS (Lipopolysaccharide) -induced ability to stimulate inflammatory cytokine production.

In particular, the extract of Hedychium coronarium is characterized by comprising the following compounds as active ingredients.

The present invention also relates to novel Labdane-Type Diterpenes of the formula (I) - (III), which have the ability to inhibit LPS-stimulated inflammatory cytokine production, isolated from the extract of Hedychium coronarium, Compounds and uses thereof:

[Chemical Formula 1]

(2)

(3)

That is, as a specific example, there is provided a composition for treating autoimmune diseases and sepsis, which contains, as an active ingredient, an extract of Hedychium coronarium or a compound of formula (I) Immune diseases and sepsis. Preferably, the compound of the formula (1) and the compound of the formula (2) are contained as an active ingredient.

At this time, the extract of Hedychium coronarium is preferably an organic solvent extract of C1-C4, and most preferably a methanol extract. In addition, the LPS-stimulated inflammatory cytokine includes TNF- [alpha], IL-6 or IL-12, and in particular the overproduction of these cytokines causes autoimmune diseases and sepsis.

The autoimmune disease may be selected from the group consisting of dermatitis, allergy, atopy, asthma, conjunctivitis, periodontitis, rhinitis, otitis, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, ankylosing spondylitis, rheumatic fever, fibromyalgia), psoriatic arthritis, osteoarthritis, rheumatoid arthritis, shoulder inflammation, nephritis, neuritis, perianal inflammation, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome, multiple sclerosis and acute and chronic inflammatory diseases, The sepsis is characterized by fever with body temperature rising to 38 degrees or less, hypothermia falling below 36 degrees, increased respiration rate more than 24 times per minute (minute breath), heart rate more than 90 times per minute (tachycardia) Systemic inflammatory response syndrome (SIRS) and endotoxic shock due to infection of microorganisms with increased or marked reduction of symptoms xic shock, etc.

That is, the extract of Hedychium coronarium according to the present invention and the dipeptan diphter compounds of formulas 1 to 3 isolated therefrom have an effect of inhibiting the production of LPS-stimulated inflammatory cytokine.

Therefore, the present invention provides, from yet another aspect, the use of LPS-stimulated inflammatory cytokines, preferably LPS-stimulated inflammatory cytokines, TNF-alpha, IL-6 or IL-12.

As described above, the present invention relates to an extract of Hedychium coronarium and a novel Labdane-Type Diterpene compound of the general formula (1) to (3) isolated therefrom and their LPS-stimulated inflammatory cytokine production Lt; RTI ID = 0.0 > inhibitory < / RTI >

The extract of Hedychium coronarium of the present invention and the novel Labdane-Type Diterpene compound of Formulas 1 to 3 derived from Hodigitium coronarum are useful for the treatment of LPS-stimulated inflammatory cytokines TNF-α, IL -6 or IL-12, and thus can be effectively used for the prevention and treatment of autoimmune diseases and sepsis.

Figure 1 shows the results of an analysis of the effect of compounds 1 to 10 on TNF- [alpha] production in LPS-stimulated DCs.
Figure 2 shows the results of an analysis of the effect of compounds 1 to 10 on the production of IL-6 in DCs stimulated by LPS.
Figure 3 shows the results of analysis of the effect of compounds 1 to 10 on the production of IL-12 p40 in DCs stimulated by LPS.

The terms used in the present invention are defined as follows.

"Extract" is a crude extract, polar solvent-soluble extract or non-polar solvent-soluble extract of Hodigitium coronarum.

The crude extract is a solvent selected from water containing purified water, a lower alcohol having 1 to 4 carbon atoms such as methanol, ethanol, and butanol, or a mixed solvent thereof, preferably a water and ethanol mixed solvent, more preferably 50 to 100% Methanol or ethanol soluble extract.

The "polar solvent-soluble extract" includes extracts which are soluble in a solvent selected from water, methanol, ethanol or a mixed solvent thereof, preferably methanol.

"Non-polar solvent-soluble extract" includes extracts which are soluble in hexane, chloroform, dichloromethane or ethyl acetate, preferably hexane, chloroform or ethyl acetate, more preferably chloroform or ethyl acetate.

"Pharmaceutical composition" refers to a mixture of other chemical ingredients such as a Hodigitum coronarium extract of the present invention and a diluent or carrier.

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

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

"Subject" or "patient" means any single entity that requires treatment, including human, cow, dog, guinea pig, rabbit, chicken, In addition, any subject who participates in a clinical study test that does not show any disease clinical findings, or who participates in epidemiological studies or used as a control group is included. In one embodiment of the present invention, the present invention was applied to humans.

"Tissue or cell sample" refers to a collection of similar cells obtained from a subject or tissue of a patient. The source of the tissue or cell sample may be a solid tissue from fresh, frozen and / or preserved organ or tissue sample or biopsy or aspirate; Blood or any blood components; It may be a cell at any point in the pregnancy or development of the subject. Tissue samples can also be primary or cultured cells or cell lines.

Tissue samples may include compounds that are not mixed with the tissue by nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like. For purposes of the present invention, a "section" of a tissue sample refers to a portion or piece of tissue sample, eg, a thin slice of tissue or cell that has been cut from a tissue sample. It will be appreciated that multiple sections of a tissue sample may be taken for analysis in accordance with the present invention if the present invention is understood to include methods in which the same section of tissue sample is analyzed both at the morphological and molecular levels, It is understood that it can do.

As used herein, an "effective amount" is an appropriate amount that affects a beneficial or desired clinical or biochemical outcome. An effective amount may be administered one or more times. For purposes of the present invention, an effective amount is an amount sufficient to temporarily alleviate, ameliorate, stabilize, reverse, slow down or slow the progression of a disease state. If the recipient animal is capable of enduring the administration of the composition, or the administration of the composition to the animal is suitable, the composition will be "pharmaceutically or physiologically acceptable ". If the dose administered is physiologically significant, it can be said that the formulation is administered in a "therapeutically effective amount ". The formulation is physiologically relevant if the presence of the formulation results in a physiologically detectable change in the recipient.

The term "treating", unless otherwise indicated, refers to reversing, alleviating, inhibiting, or preventing the disease or condition to which the term applies, or one or more symptoms of the disease or disorder . As used herein, the term ' treatment ' refers to treatment and prevention when ' treating ' is defined as above.

Hereinafter, the present invention will be described in detail.

All technical terms used in the present invention are used in the sense that they are generally understood by those of ordinary skill in the relevant field of the present invention unless otherwise defined. Also, preferred methods or samples are described in this specification, but similar or equivalent ones are also included in the scope of the present invention. The contents of all publications referred to herein are incorporated herein by reference.

The present invention relates to the use of a hedchium cornarium coronarium extract and novel compounds isolated therefrom and their use. More particularly, the present invention relates to novel dipeptane diether compounds derived from hordicum coronarum and their LPS (lipopolysaccharide) -stimulated inflammatory cytokine production inhibitory function will be.

The Hedychium coronarium is a perennial plant called 'flower ginger' in the name of the country. It grows in the hot regions of India, Malaysia, China and the Himalayas. In the present invention, the rhizomes of the Hedidium coronarium are used .

The Hodigitum coronarium extract can be prepared by a method known in the art, a modified method thereof or a method according to the present invention.

As one specific example, it can be produced by the following method.

The Hodicum coronarium extract or crude extract of the present invention may be prepared by mixing water, methanol, water, and the like, containing purified water of about 1 to 30 times volume, preferably 5 to 15 times volume (w / v% Preferably 50 to 100% methanol or ethanol, in a solvent selected from the group consisting of water, methanol, ethanol, or a lower alcohol having 1 to 4 carbon atoms such as butanol, or a mixed solvent thereof, preferably water, methanol or ethanol, Extraction with hot water, extraction with ultrasound, reflux with cooling, extraction with hot extraction or the like, preferably extraction with hot water, extraction with hot water, and filtration under reduced pressure, preferably at room temperature for 10 to 60 hours, preferably 30 to 50 hours, The crude extract can be obtained by concentration.

The polar solvent or non-polar solvent-soluble extract of the present invention may be used in an amount of about 1 to 150 times, preferably 5 to 100 times the volume of the crude extract, preferably 50 to 100% methanol or ethanol, v%) is dispersed in water and then fractionated with chloroform (CHCl ₃ ) and ethyl acetate (EtOAc). After removal of solvent in a vacuum, polar solvent and nonpolar solvent soluble extract can be obtained.

In one embodiment of the present invention, polar solvent extracts were prepared.

The present invention includes a method for producing the Hodigitum coronarium extract. For example, the non-exemplified extraction method according to the present invention can be used in a wide range of applications in the art. And can be successfully performed by a person skilled in the art.

As a person skilled in the art to which the present invention pertains, specific reaction conditions and the like for producing the extract of Hodigitum coronarium according to the present invention can be confirmed through the following embodiments, and a detailed description thereof will be omitted .

The Hodicum coronarium extract of the present invention contains the following Labdane-Type Diterpenes compound as an active ingredient. (Compound) 1 to 10 with respect to the structural formula of the compound corresponding to each substituent.

Particularly, among the above compounds, the compounds 1 to 3 are novel Labdane-Type Diterpenes compounds discovered by the present inventors. In another aspect, the present invention provides a compound represented by the following general formula (1) to .

The present invention relates to an extract of Heddicum coronarium comprising the compounds of formulas (1) to (10), and a compound of formula (I), (II) and (III) In particular, it inhibits proinflammatory cytokine production and activity of TNF-a, IL-6 or IL-12 and does not show toxicity in cultured cells, and thus can be usefully used for the prevention and treatment of autoimmune diseases and sepsis

That is, the extract of Heddicum coronarium containing the compounds of formulas (1) to (10) of the present invention and the compounds of formula (1) to (3) separated therefrom have the following functions.

(1) The extract of Heddicum coronarium containing the compounds of the above formulas (1) to (10) of the present invention and the dipeptane compounds of the formulas (1) to (3) can be used as bone marrow-derived dendritic cells stimulated by lipopolysaccharide (LPS) -Derived Dendritic Cells inhibits the production of TNF-a, IL-6 or IL-12.

Lipopolysaccharide (LPS), an endogenous substance, is an inducer of inflammation-inducing substances in resin cells and promotes the production of pro-inflammatory cytokines that cause an inflammatory reaction. That is, when an external stimulus that may cause an inflammatory reaction is applied, the expression of inflammatory cytokines such as TNF-a is induced, and the produced inflammatory cytokines stimulate the expression of genes encoding iNOS and COX-2, It produces NO and PGE ₂ substances involved in the inflammatory reaction and causes an inflammatory reaction.

Therefore, if these inflammatory cytokines of inflammatory cytokines such as TNF- [alpha], IL-6 or IL-12 are excessively secreted or the cells themselves remain active for a long period of time, they cause serious side effects such as nerve tissue damage.

The dipeptan dipeptide compound according to the present invention has a function of inhibiting the production of inflammatory cytokines as described above.

(Hwang, Chen, Nines, Shin & Stoner, 2006), it has been reported that hordicum coronarum has antiinflammatory properties in the prior art, The dipeptide compounds of formula (I) have not been identified. Accordingly, the present invention is characterized mainly by the novel compounds of formulas (1) to (3), which are novel compounds derived from Heddicum coronarum.

(2) The extract of Heddicum coronarium containing the compounds of the above formulas (1) to (10) of the present invention and the dipeptane diphterene compounds of the formulas (1) to (3) can be used for dendritic cells (DCs) stimulated by lipopolysaccharide (LPS) Inhibits the activity of inflammatory mediators.

Interleukin-12 is a kind of interleukin that is produced in antigen-presenting cells such as dendritic cells and macrophages by antigen stimulation. It plays a role of stimulating T cells to differentiate into type 1 helper T cells, and NK (natural killer) cells , And plays an important role in the activity of T lymphocytes. IL-12B (p40) is a heterodimer of IL-12A (p35) and IL-12B (p40) and the expression of IL-12A (p35) is always constitutive, , Suggesting that such inhibition of IL-12 may be useful in the treatment of autoimmune diseases, in particular autoimmune diseases mediated by type 1 helper T cells, which are an excess immune response to cells in the human body. .

Therefore, the present invention can be used for the therapeutic or prophylactic use of an inflammatory disease, which is characterized by acting through the activity of inhibiting the production of TNF-a, IL-6 or IL-12.

In particular, the term "inflammatory disease" in the present invention refers to a disease caused by the overproduction of the inflammatory cytokines TNF-a, IL-6 or IL-12 induced by lipopolysaccharide (LPS) Disease or sepsis.

More particularly, the autoimmune disease is selected from the group consisting of dermatitis, allergy, atopy, asthma, conjunctivitis, periodontitis, rhinitis, otitis, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, Sjogren's syndrome, multiple sclerosis, and acute and chronic inflammatory diseases such as psoriatic arthritis, rheumatoid arthritis, osteoarthritis, rheumatoid arthritis, shoulder periitis, tendinitis, hay fever, tendinitis, myositis, hepatitis, cystitis, nephritis, Sepsis is characterized by fever with body temperature rising above 38 degrees, hypothermia down to 36 degrees, increased respiratory rate more than 24 times per minute (ventilation), heart rate more than 90 times per minute (tachycardia) Systemic inflammatory response syndrome (SIRS) due to infection of microorganisms with marked increase or decrease in white blood cell count, It includes shock (endotoxic shock) and the like. More preferably, it is an autoimmune disease. (I) Rheumatoid arthritis, in which the immune system attacks tissues of various joints, (ii) autoimmunity of the central nervous system induced by T cells, Multiple sclerosis (MS), which can lead to blindness, premature death, and severe blindness. (Iii) Immune cells that are produced by the destruction of the pancreatic insulin-producing cells by the immune cells and whose MHC gene plays an important role Inflammatory Bowel Diseases, a disease in which the immune system attacks the intestines, v) inducing thickening of the skin or blood vessels, Scleroderma, vi) Systemic autoimmunity leads to deep fatigue, rash, arthralgia, etc. In severe cases, the immune system may cause kidney, brain, lung, etc. It can be given a systemic lupus that can cause damage (Systemic Lupus Erythematosus, SLE). In addition, sepsis is a systemic inflammatory response syndrome (SIRS) and sepsis (endotoxin) shock in which microorganisms are infected and a serious inflammatory reaction occurs in the whole body. Particularly, the endotoxic shock (sepsis shock) is mainly caused by excess produced interleukin-6 and TNF-a.

Accordingly, as a specific embodiment of the present invention, there is provided a method for preventing or treating autoimmune disease or sepsis, using the extract of Hodigitum coronarium or the compound of Chemical Formulas 1 to 3 above; And pharmaceutical compositions for the prevention and treatment of autoimmune diseases or sepsis comprising these extracts or compounds as active ingredients.

The pharmaceutical composition for the prevention and treatment of autoimmune diseases or sepsis according to the present invention preferably contains 0.1 to 50% by weight of the extract, based on the total weight of the composition.

The pharmaceutical composition may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions. In addition, the composition of the present invention can be formulated or used in combination with medicines such as steroidal drugs, antihistamines, antiinflammatory agents and antibiotics which have already been used.

The pharmaceutical composition according to the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to a conventional method .

Examples of carriers, excipients and diluents that may be contained in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin , Calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used.

Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose or lactose , Gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included .

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of suppository bases include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like.

The amount of the composition of the present invention may vary depending on the age, sex and body weight of the patient, but may be 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg, once to several times per day. The dosage may also be increased or decreased depending on the route of administration, degree of disease, sex, weight, age, and the like. Accordingly, the dosage is not limited in any way to the scope of the present invention.

The pharmaceutical composition may be administered to mammals such as rats, mice, livestock, humans, and the like in a variety of routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection.

The pharmaceutical dosage forms of the compositions of the present invention may also be used in the form of their pharmaceutically acceptable salts and may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable set.

In another specific embodiment of the present invention, there is provided a method for inhibiting inflammatory cytokine production and activity using Rabindane diepenic compounds represented by formulas (1) to (3), preferably Formulas (1) and (2) The inflammatory cytokines include TNF-a, IL-6 or IL-12.

Unless otherwise indicated, the term "levaptan diepene compound according to the present invention" or "compounds of formulas (1), (2) and (3)" refers to a compound itself, a pharmaceutically acceptable salt, hydrate, solvate , Isomers, and prodrugs.

The term " pharmaceutically acceptable salt "means a formulation of a compound that does not cause serious irritation to the organism to which the compound is administered and does not impair the biological activity and properties of the compound. The pharmaceutical salt may be prepared by dissolving the compound of the present invention in an organic solvent such as mineral acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid or phosphoric acid, sulfonic acid such as methanesulfonic acid, ethanesulfonic acid, or p-toluenesulfonic acid, tartaric acid, formic acid, Can be obtained by reacting an organic carboxylic acid such as benzoic acid, benzoic acid, lactic 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. Also, by reacting the compound of the present invention with a base, an alkali metal salt such as an ammonium salt, a sodium or potassium salt, a salt such as an alkaline earth metal salt such as a calcium salt or a magnesium salt, a dicyclohexylamine, Carmine, tris (hydroxymethyl) methylamine and the like, and amino acid salts such as arginine, lysine and the like.

The term "hydrate" refers to a compound of the present invention comprising a stoichiometric or non-stoichiometric amount of water combined by non-covalent intermolecular forces Or a salt thereof.

The term "solvate" means a compound of the present invention or a salt thereof, comprising a stoichiometric or non-stoichiometric amount of a solvent bound by noncovalent intermolecular forces. Preferred solvents therefor are volatile, non-toxic, and / or solvents suitable for administration to humans.

The term "isomer" means a compound of the present invention or a salt thereof, which has the same chemical or molecular formula but is optically or sterically different. For example, the compound according to Formula 1 of the present invention may have an asymmetric center (asymmetric carbon atom) depending on the kind of substituents. In this case, the compound of Formula 1 may be an enantiomer or diastereomer 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 in some cases because they are easier to administer than parent drugs. For example, they may achieve viability by oral administration, whereas parent drugs may not. Prodrugs may also have improved solubility in pharmaceutical compositions over the parent drug. For example, a prodrug is an ester that facilitates the passage of a cell membrane, which is hydrolyzed to a carboxylic acid that is active by metabolism in a cell whose water solubility is once beneficial, Drug "). ≪ / RTI > Another example of a prodrug may be a short peptide (polyamino acid) that is attached to an acid group that is converted by metabolism so that the peptide reveals its active site.

As described above, the Hedychium coronarium of the present invention coronarium extracts and their derivatives can be safely used for long-term use or use because they have excellent ability to inhibit the production of inflammatory cytokines and have little toxicity and side effects.

Hereinafter, the present invention will be described in more detail with reference to Examples. It should be apparent to those skilled in the art that these embodiments are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples

Values obtained in the following examples are expressed as mean + - SD.

Experiment tool used

Optical rotations were measured with a Jasco DIP-370 automatic polarimeter and FT-IR spectra were obtained from a Jasco Report-100 infrared spectrometer. NMR spectra were recorded on a Bruker DRX 500 spectrometer ( ¹ H, 500 MHz; ¹³ C, 125 MHz). ESI-MS was obtained with an AGILENT 1200 SERIES LC-MSD Trap spectrometer and HR-ESI-MS was obtained using a JEOL JMS-T100LC spectrometer. Column chromatography was carried out using silica gel (Kieselgel 60, 70-230 mesh and 230-400 mesh, Merck) or YMC RP-18 resin (30-50 μm, Fujisilisa Chemical Ltd., Japan) ) Was performed using pre-coated silica gel 60 F ₂₅₄ (0.25 mm, Merck) and RP-18 F ₂₅₄ S plate (0.25 mm, Merck).

< Example  1>

Hedicium Coronarium ( Hedychium coronarium ) Preparation of extracts and separation of compounds

In January 2010, the rhizome of H. coronarium was collected from Sapa, Vietnam. Ninh Khac Ban (Institute of Marine Biochemistry, VAST, Vietnam). Samples (IMBC-HC10) were stored in a plant sampling room of Institute of Marine Biochemistry (VAST, Vietnam).

The rootstock (1.0 kg) of H. coronarium was extracted with methanol three times under reflux for 15 hours to obtain 90.0 g of dark solid extract which was suspended in water and then partitioned with chloroform (CHCl ₃ ) and ethyl acetate (EtOAc) after removing the solvent in _{vacuo, CHCl 3 (HC1, 50.1 g} ), EtOAc (HC2, 25.3 g), and water extract (HC3, 14.6 g) was obtained.

HCl was chromatographed on a silica gel column and eluted with n-hexane-acetone gradient (100: 1 to 1: 1, v / v) to give 4 sub-fractions, HClA (15.0 g), HClB (10.4 g), HClC g), and HClD (6.0 g).

The HC1A fraction was chromatographed on a silica gel column and eluted with n-hexane-ethyl acetate (EtOAc) gradient (40: 1 to 1: 1, v / v) to give compound 8 (100.0 mg) and compound 9 (120.0 mg) Respectively. Chromatography and _{CHCl 3 -MeOH (40: 1,} v / v) to the HC1C fractions on a silica gel column eluted with compound 10 (150.0 mg) was obtained.

The HC1D fraction was further chromatographed on a silica gel column and eluted with CH ₂ Cl ₂ -MeOH (30: 1, v / v) to give three small fractions (HC1D1-HC1D3).

The HC1D1 The fractions were chromatographed on a YMC RP-18 column and eluted with acetone-water (3: 1, v / v) to give compound 4 (5.6 mg) and compound 5 (5.0 mg). Fraction HClD2 was chromatographed on a YMC RP-18 column and eluted with MeOH-water (6: 1, v / v) to give compound 1 (7.0 mg) and compound 3 (2.5 mg). The fractions HC1D3 were chromatographed on a YMC RP-18 column and eluted with MeOH-acetone-water (2: 2: 1, v / v) to give compound 2 (2.5 mg) and compound 6 (8.5 mg).

As a result, a total of ten compounds of the new compounds 1 to 3 and the known compounds 4 to 10 were isolated and purified.

In particular, the NMR data of the compounds of the above-mentioned new compounds 1 to 3 are shown in the following table.

< Example  2>

Characterization of isolated compounds

(1) Characterization of Compound 1

Compound 1 was obtained in the form of a colorless viscous oil. The basic ion peak on cation ESI-MS was observed at m / z 333 [M + H] ⁺ and the cluster ion peak m / z 333.2409 [M + H] ⁺ (Calcd for C ₂₁ H ₃₃ O ₃ : 333.2430). As a result, it was revealed that the molecular formula was C ₂₁ H ₃₂ O ₃ by HR-ESI-MS (high-resolution electrospray ionization mass spectroscopy).

The ¹ H-NMR spectrum of Compound 1 (in chloroform-d ₁ ) showed the following signals:

three tertiary methyl groups at? _H 0.68, 0.80, and 0.87 (each 3H, s);

one &_lt; / RTI &gt; methoxy group @ delta _H 3.56 (s); And

three olefinic protons at 隆_H 4.54, 4.85, and 6.76 (see Table 1).

^The 21 carbon signals including 4 methyl groups, 8 methylenes, 4 methines and 5 quaternary carbons were identified by ¹³ C-NMR and DEPT (distortionless enhancement by polarization transfer) spectra. ¹ H- and &lt; ¹³ &gt; C-NMR analysis of Compound 1 revealed that this was a dipterene type diterpene compound.

On the other hand, the NMR data of compound 1 was similar to that of coronarin C, but the additional part of the methoxy group was different at C-15. The α, β-unsaturated γ-lactone having a methoxy group in γ-carbon (C-15) in the C ring of compound 1 was confirmed by comparison with ¹ H and ¹³ C data with 15-methoxypinusolidic acid. Also between olefinic proton H-14 (δ _H 6.76) and carbons C-13 (δ _C 139.4), C-15 (δ _C 102.4), and C-16 (δ _C 171.4); (heteronuclear multiple bond correlations) between proton H-15 (δ _H 5.73) and C-14 (δ _C 141.4), C-16 (δ _C 171.4), and methoxy carbon (δ _C 56.8).

In _{addition, C-7 (δ C 38.3} ), C-8 (δ C 148.0) and _C-9 (δ C 56.5) and the two protons _{H-17 (δ H 4.54 and} 4.85 in combination in the HMBC spectrum of the compound 1 ) Means that double bonds are located at C-8 and C-17. Between H-19 (delta _H 0.80) and H-20 (delta _H 0.68); NOESY (nuclear Overhauser enhancement and exchange spectroscopy) correlation between H-5 (δ _H 1.08) and H-9 (δ _H 1.54) confirmed the β sequences of methyl groups and C rings at C-10 and C-9 .

As a result, Compound 1 was determined to be 15-methoxylabda-8 (17), 13-dien-16,15-olide and named coronarin G.

(2) Characterization of Compound 2

Compound 2 was obtained in the form of a colorless viscous oil.

Basic ion peaks were observed at m / z 317 [M + H] ⁺ on cation ESI-MS and m / z 317.2116 [M + H] ⁺ (Calcd for C ₂₀ H ₂₉ O ₃ : 317.2117), the molecular formula is C ₂₀ H ₂₈ O ₃ .

The ¹ H-NMR spectrum of compound 2 (chloroform-d ₁ ) showed the following signals:

three tertiary methyl groups at? _H 0.91, 1.16, and 1.18 (each 3H, s);

one aldehydic group at 隆_H 9.37 (s); and

two olefinic protons at δ _H 5.92 and 6.82 (see Table 1).

¹³ C-NMR and DEPT spectra confirmed 20 carbon signals including 3 methyl groups, 6 methylene, 6 methine and 5 quaternary carbons. Based on the ¹ H- and ¹³ C-NMR analyzes of compound 3, it was found that this was dipeptide compound of the dipole type and its NMR data was similar to that of curcuminol D, but the appearance of a carbonyl group substituted with a methylene group at C-6 Respectively.

Between _H-5 (δ H 2.19) and _{C-6 (δ C 199.2)} , C-7 (δ C 133.2), C-19 (δ C 21.5), and _{C-20 (δ C 14.4)} ; _H-7 (δ H 5.92) and _{C-5 (δ C 64.0)} , C-6 (δ C 199.2), C-8 (δ C 154.7), C-9 (δ C 53.6), and C-17 ( δ _C 41.5), the HMBC correlations confirmed ketone groups and double bonds located at C-6 and C-7 / C-8, respectively. In addition, seven rings were identified by COZY correlations of H-9 / H-11, H-11 / H-12, H-14 / H-15 and H-15 / H-16.

Δ _H 9.37 (H-17) aldehyde proton signal associated with C-12 (δ _C 155.5), C-13 (δ _C 139.0), and C-14 (δ _C 29.3) in the HMBC spectrum of compound 2; Proton signals at δ _H 2.28 and 2.93 (H-14), 2.38 and 2.57 (H-16) associated with C-15 (δ _C 69.8) indicate that the aldehyde and hydroxyl groups and double bonds are C- 15, and C12 / C13.

The NOESY correlations between H-19 (δ _H 1.18) and H-20 (δ _H 0.91), between H-5 (δ _H 2.19) and H-9 (δ _H 3.00) 10 were confirmed to be the alpha and beta sequences, respectively. Based on this fact, the structure of compound 2 was proved and named coronarin H.

(3) Characterization of Compound 3

Compound 3 was obtained in the form of a colorless viscous oil. The basic ion peak on cationic ESI-MS was observed at m / z 337.2 [M + H] ⁺ and the cluster ion peak m / z 337.2378 [M + H] ⁺ (Calcd for C ₂₀ H ₃₃ O ₄ : 337.2379). As a result, it was revealed that the molecular formula was C ₂₀ H ₃₃ O ₄ by HR-ESI-MS (high-resolution electrospray ionization mass spectrometry) analysis.

The ₁ H-NMR spectrum of compound 3 (chloroform-d ₁ ) showed the following signals:

_{three tertiary methyl groups at δ H 0.76} , 0.80, and 1.03 (each 3H, s);

three olefinic protons at δ _H 4.72, 5.25, and 5.51 (see Table 1).

¹³ C-NMR and a distortionless enhancement by polarization transfer (DEPT) spectrum show that three methyl groups, seven methylenes (containing two oxygen atoms), six methines (containing three oxygen atoms) and four quaternary carbons were identified. Similar to compounds 1 and 2, compound 3 was also identified as dipeptane-type diaphene compounds by H- and ¹³ C-NMR analysis. The NMR data of compound 3 was similar to that of pacovatinin B, but there was a difference in the lactone ring.

C-11 (? _C 23.2), C-14 (? _C 72.0), and C-16 (? _C 64.3) in H-12 (? _H 5.51); H-15 (delta _H 3.57 and 3.66) to C-13 (delta _C 138.8) and C-14 (delta _C 72.0); The HMBC correlations from H-16 (δ _H 4.00 and 4.13) to C-12 (δ _C 131.8) and C-13 (δ _C 138.8) show that the double bonds, epoxy groups and hydroxyl are C- C-15 / C-16, and C-14. Also, the geometry of 3-substituted olefins in C-12 / C-13 was identified as Z-form from the NOESY correlation between H-12 (δ _H 5.51) and Ha-16 (δ _H 4.00).

HMBC spectrum of Compound 3 _H-3 (δ H 3.23) and _{C-2 (δ C 28.6)} , C-5 (δ C 53.5), C-18 (δ C 28.8), and _C-19 (δ C 16.2 )between; H-7 (delta _H 3.91) and C-5 (delta _C 53.5), C-6 (delta _C 34.2), and C-17 (delta _C 105.4); And showing a correlation between _H-17 (δ H 4.72 and 5.25) and _{C-7 (δ C 74.4)} , C-8 (δ C 151.1), and _{C-9 (δ C 56.5)} , C-8 / C17, C-3, and C-7, respectively, with one double bond and two hydroxy groups.

Also, E geometry isomerism of the C-11 / C-12 double bond was confirmed with a large coupling constant (J11, 12 = 16.0 Hz). H-3: δ _H 3.46 (t, 3.0) and δ _C values for C-1 (39.3), C-3 (76.3), and C-4 (37.3) and 3-hydroxycativic acid [H- 3: δ _H 3.28 (dd, 4.3, 12.0) and δ _C values for C-1 (36.8), C-3 (78.4), and C-4 (38.8) (隆_C 38.2), C-3 (隆_C 79.4), and C-4 (隆_C 40.1), with a large coupling constant of 3 隆_H 3.23 (dd, J = 5.5, 10.0) ) Confirmed the β-equatorial orientation of the 3-hydroxy group.

This was confirmed further by NOE correlations of the _{H-3 (δ H 3.23)} with H-18 (δ H 1.03) and _H-5 (δ H 1.21) and _H-3 (δ H 3.23) combined. Based on the binding constant of H-7 (J6-7 = 5.5, 11.5) and the NOE correlation between H-5 (δ _H 1.21) and H-7 (δ _H 3.91) and hydroxyl groups at C-7, β -equatorial orientation was confirmed.

H-19 (? _H 0.80) and H-20 (? _H 0.76); The NOE correlation between H-5 (δ _H 1.21) and H-9 (δ _H 1.70) confirmed that the C ring at C-9 and the methyl group at C-10 were in the β configuration. As a result, Compound 3 was identified as 3?, 7 ?, 14-trihydroxy-15,16-epoxylabda-8 (17), 12Z-dien and named conorarin I.

(4) Other isolated compounds

In addition to the novel compounds 1 to 3, known compounds were isolated from the methanol extract of the present invention.

By comparing the physical and spectroscopic data in the literature, the respective compounds were identified as follows.

Compound 4-10 was as follows.

coronarin D (compound 4)

coronarin D methyl ether (Compound 5)

hediforrestin C (Compound 6)

(E) -nernolidol (Compound 7)

β-sitosterol (Compound 8)

daucosterol (Compound 9) and

stigmasterol (Compound 10)

These have been identified on the basis of spectral data and chemical evidence and are already reported in the known literature. Compound 4 is described in Itokawa, H .; Morita, H .; Katou, I .; Takeya, K .; Cavalheiro, AJ; Oliveira, RCB; Ishige, M .; Motidome, M. Planta Med. 1988, 54, 311, compounds 5 and 6 are described in Zhao, Q .; Hong, X .; Wang, YS; Zou, C .; Hao, XJ Chin. Chem. Lett. 2003, 14, 1141], compound 7 is described in the literature [Blanc, MC; Bradesi, P .; Casanova, J. Magn. Res. Chem. 2005, 43, 176, compounds 8-10 are described in Kiem, PV; Cuong, NX; Nhiem, NX; Thu, VK; Ban, NK; Minh, CV; Tai, BH; Hai, TN; Lee, SH; Jang, HD; Kim, YH Bioorg. Med. Chem. Lett. 2011, 21, 633].

< Example  3>

Assessment of biological function of isolated compounds

(1) Cell culture

Bone marrow-derived DCs were obtained from wild-type C57BL / 6 mice (Taconic Farm, NY, U.S.A.). Briefly, bone marrow cells were obtained by flushing the mouse shin bone and femur in DMEM (Dulbecco's modified Eagles medium) medium. The cells were seeded in 10% heat-inactivated FBS (Gibco, NY, USA) supplemented with 3% J558L hybridoma cell culture supernatant containing granulocyte-macrophage colony-stimulating factor (GM- , 50 μM β-mercaptoethanol, 2 mM glutamine.

The non-adherent cells and loosely adhering DC aggregates were harvested, washed, and resuspended in RPMI 1640 medium supplemented with 5% FBS. The DC Were cultured in 0.5 mL of a 48-well plate containing 1 x 10 ⁵ cells per well, treated with Salmonella minnesota LPS (10 ng) at a concentration of 2.0, 10, 25, and 50 μM for 1 hour / mL, Alexis, NY, USA). A supernatant was obtained at 16 hours after stimulation.

Mouse TNF-α, IL-6, and IL-12 p40 concentrations in the culture supernatant fractions were measured by ELISA analysis (Pharmingen, CA, USA). Data are shown as mean ± standard deviation from independent experiments performed on triplicate at least three times.

(2) Bone marrow-derived In dendritic cells (DCs)  Inflammatory response evaluation

To evaluate the biological functions of the 10 rabbit-type diterpenoid compounds obtained in Example 1, the inflammatory response in the bone marrow-derived dendritic cells (DCs) obtained above was observed.

First, using the colorimetric 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) assay (Sigma, MO, USA) (Data not shown). However, since Compound 5 is cytotoxic, it is decided that the experiment will not proceed further.

In LPS treatment, DCs have been previously known to secrete inflammatory cytokines such as TNF-a, IL-6, and IL-12, and inflammatory responses include such cytokine secretion, activation, and neutro- phils) and subsequent mononuclear cell replenishment.

Based on these facts, in the present experiment, the DCs cultured in a 48-well plate at a density of 2 x 10 ⁵ cells / mL were treated with the compounds at a concentration of 2, 10, 25, and 50 μM for 1 hour, 10 ng / mL). A supernatant was obtained at 16 hours after stimulation.

SB203580, a p38 kinase inhibitor, was used as a positive control, and SB203580 inhibited the production of TNF-a, IL-6, and IL-12: IC ₅₀ of 7.24 0.10, 3.53 0.11, and 5.02 0.24 μM, respectively .

These results are shown in Table 2 and Figs.

Among these compounds, Compound 6 was identified as an excellent inhibitor of LPS-stimulated IL-6 and IL-12 production. This reduced the levels of the cytokines to IC ₅₀ in the range of 3.38 ± 1.25 and 0.19 ± 0.11 μM, respectively.

In particular, Compounds 1 and 2 significantly inhibited the stimulation of TNF-a, IL-6, and IL-12, while other Compounds 7-10 did not significantly inhibit the production of inflammatory cytokines.

From these results, it can be seen that the Hedychium coronarium of the present invention coronarium extract-derived compounds have excellent ability to inhibit the production of inflammatory cytokines.

There are many medicines that inhibit TNF-α and are generally used as immunosuppressants such as cyclosporin A and dexamethasone. However, there is a problem that the range of the effect is somewhat wide and significant toxicity appears. Therefore, a drug having a strong side effect profile and a stronger inhibitory activity against TNF-α is required.

Thus, the present invention, which is a plant-derived anti-inflammatory natural (natural) inhibitor, would be a useful inhibitor of inflammatory cytokine production in response to the above needs and would be particularly useful for the treatment of IL-6, IL-12, TNF- May be used for the effective treatment of autoimmune diseases and sepsis diseases.

Claims (17)

delete delete delete delete delete delete delete Lipopolysaccharide-LPS (Lipopolysaccharide) - having the ability to inhibit stimulated inflammatory cytokine production.
[Chemical Formula 1]

(2)

. 9. The method of claim 8,
Wherein the dipeptide compound of formula (1) or (2) is derived from Hedychium coronarium . 9. The method of claim 8,
Wherein said LPS-stimulated inflammatory cytokine is TNF- [alpha], IL-6 or IL-12. 9. The method of claim 8,
The dipeptide compound of formula (1) or (2)
(a) extracting a rhizome of hodigitium coronarum with an organic solvent; And
(b) fractionating the organic solvent extract and separating and purifying the compound by chromatography. 12. The method of claim 11,
Wherein in step (a) the organic solvent is a C1-C4 lower alcohol. 12. The method of claim 11,
Characterized in that the chromatography is carried out in step (b) using chloroform and ethyl acetate as solvent. A pharmaceutical composition for the treatment and prevention of autoimmune diseases and sepsis, which comprises, as an active ingredient, any one of the following dipeptide compounds selected from the group consisting of the following formulas (1), (2) and (6)
[Chemical Formula 1]

(2)

[Chemical Formula 6]

. 15. The method of claim 14,
The autoimmune disease may be selected from the group consisting of dermatitis, allergy, atopy, asthma, conjunctivitis, periodontitis, rhinitis, otitis, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, ankylosing spondylitis, rheumatic fever, selected from the group consisting of fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendonitis, hay fever, tendinitis, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome, multiple sclerosis and acute and chronic inflammatory diseases A pharmaceutical composition for the treatment and prevention of autoimmune diseases and sepsis characterized by being at least one disease. 15. The method of claim 14,
Wherein the sepsis is a systemic inflammatory reaction or an endotoxic shock caused by infection of a microorganism. The pharmaceutical composition for treating and preventing autoimmune diseases and sepsis. Inhibit the production of TNF-α, IL-6 or IL-12, which are inflammatory cytokines that are LPS-stimulated in vitro, using one of the following dipeptide compounds selected from the group consisting of the following Formulas 1, 2 and 6: How to;
[Chemical Formula 1]

(2)

[Chemical Formula 6]

.

Images