KR20130106599A - A labdane-type diterpenoids compounds derived from hedychium coronarium and a use thereof - Google Patents

Abstract

PURPOSE: A Labdane-type Diterpenoids Compound derived from Hedychium coronarium is provided to prevent the formation and activation of LPS-stimulated inflammatory cytokine, thereby treating and preventing sepsis and autoimmune diseases. CONSTITUTION: A Labdane-type Diterpenoids Compound derived from Hedychium coronarium for treating and preventing sepsis and autoimmune diseases contains the extract of Hedychium coronarium which suppresses the generation of LPS-stimulated Pro-inflammatory Cytokines. The LPS-stimulated inflammatory Cytokine is TNF-α, IL-6, or IL-12. The extract of Hedychium coronarium includes compounds as an active ingredient. The sepsis is systemic inflammatory reaction or endotoxic shock reaction.

Description

A rapdan-type diterpenoids compounds derived from hedychium coronarium and a use about

The present invention relates to the use of Hedychium coronarium extract and rabdan-type diterpenoids derived from these compounds, compositions or methods for preventing and treating autoimmune diseases and sepsis containing the same as an active ingredient It is about. More specifically, the present invention relates to Hedium coronarium extracts that inhibit the production of inflammatory cytokines stimulated by lipopolysaccharides (LPS), and the use of these lobular diterpenoid 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, redness, swelling, fever and ultimate loss of function of the infected area. These symptoms are the result of a series of complex interactions between cells of the immune system. The response of the cell results in an interactive network of different groups of inflammatory mediators: proteins (eg cytokines, enzymes (eg proteases, peroxidases), major basic proteins, adhesion molecules (ICAM, VCAM), lipid mediators (e.g. eicosanoids, prostaglandins, leukotriene, platelet activating factor (PAF)), reactive oxygen species (e.g. hydroperoxide, superoxide anion O2-, nitric oxide (NO) However, most of these mediators of inflammation are also regulators of normal cellular activity, thus the host is compromised (ie, infected) uncontrolled due to lack of an inflammatory response, and thus partially due to chronic inflammation. As a result, an inflammatory disease mediated by overproduction of several of the above-mentioned mediators is caused.

In particular, autoimmune diseases, one of the inflammatory diseases, are characterized by the immune system attacking its own organs causing spontaneous reactions. These responses are due to the recognition of auto-antigens by T lymphocytes, leading to humoral (autoantigen production) and cellular (increased lymphocyte and macrophage cytotoxic activity) immune responses. 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 fatal, to date there is no effective treatment method for treating the diseases. Therefore, any drug, medicine or medium that can alleviate or alleviate the disease during the course of the disease will be an important solution for the health of the patient.

Intensive efforts have been made to find appropriate drugs and methods by searching for treatments for 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 these pharmacological therapies poses a problem for various cytotoxic actions, which can expose the patient to risk of infection and cancer by inhibiting the immune system in a nonspecific manner. Since calcineurin and glucocorticoids present another problem due to their nephrotoxicity and diabetes-inducing properties, their use is limited for some clinical symptoms (eg renal failure, diabetes, etc.).

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.

Accordingly, the inventors have confirmed that the extract extracted from Hedychium coronarium and the specific lobdan diterpenoid compounds isolated from them inhibit the production of TNF-α, IL-6 or IL-12, that is, LPS (Lipopolysaccharide) The present invention was completed by elucidating the influence of lobular diterpenoid compounds that inhibit 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 Diterpenoid compound of Formula 1 or Formula 3 and its use.

Still another object of the present invention is to provide a method for inhibiting LPS-stimulated inflammatory cytokine production by using a Labdane-Type Diterpenoid compound of Formula 1 or Formula 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.

In another aspect, the present invention is a novel lobular diterpenoids of the formula (1) or (3) having LPS-stimulated inflammatory cytokine production inhibitory ability, isolated from the extract of the Hedychium coronarium ) Compounds and their use:

[Formula 1]

(3)

That is, as a specific example, containing the extract of the Hedychium coronarium and the lobdan diterpenoid compounds of Formula 1 or 3 isolated from them as an active ingredient, compositions for treating autoimmune diseases and sepsis and using the same Provided are methods for treating autoimmune diseases and sepsis.

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 the Hedychium coronarium of the present invention and the lobdan diterpenoid compound of Formula 1 or 3 isolated therefrom have an effect of inhibiting LPS-stimulated inflammatory cytokine production.

Thus, in another aspect, the present invention provides the LPS-stimulated inflammatory cytokine TNF-α, IL-6 or IL- using the Heditchium coronarium extract, preferably the lobdan diterpenoid compound of Formula 1 or Formula 3 It provides a method of suppressing the production of 12.

As such, the present invention is an extract of Hedychium coronarium ( Hedychium coronarium ); And the novel Labdane-Type Diterpenoid compounds of Formula 1 or Formula 3 isolated therefrom and their related use of their LPS-stimulated inflammatory cytokine production inhibitory function.

Extracts of the Hedychium coronarium of the present invention and the novel Labdane-Type Diterpenoid compounds of Formula 1 or Formula 3 derived from Hedichium coronarium, LPS-stimulated inflammatory cytokines TNF-α, Since it inhibits the production and activity of IL-6 or IL-12, it can be usefully used for the prevention and treatment of autoimmune diseases and sepsis.

1 shows the results of analyzing the effects of Compounds 1 to 6 on TNF-α production in DCs stimulated by LPS.
2 shows the results of analyzing the effects of compounds 1 to 6 on IL-6 production in DCs stimulated by LPS.
FIG. 3 shows the results of analyzing the effects of compounds 1 to 6 on IL-12 p40 production 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.

A "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.

A "diluent" is defined as a compound that not only stabilizes the biologically active form of a 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, 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. Sources of tissue or cell samples may include solid tissue from fresh, frozen and / or preserved organ or tissue samples or biopsies or aspirates; 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 do not mix with one another by nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like. For the purposes of the present invention, "section" of a tissue sample means a thin slice of tissue or cells cut from one portion or piece of a tissue sample, eg, a tissue sample. If the present invention is understood to include a method in which the same fragment of a tissue sample is analyzed at both morphological and molecular levels or for both protein and nucleic acid, multiple sections of tissue samples are taken and subjected to the analysis according to the invention. It is understood that it can be done.

As used herein, an "effective amount" is an appropriate amount that affects a beneficial or desirable clinical or biochemical result. An effective amount may be administered one or more times. For the purposes of the present invention, an effective amount is an amount suitable to temporarily alleviate, ameliorate, stabilize, reverse, slow 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, unless defined otherwise, are used in the meaning as commonly understood by those skilled in the art in the related field of the present invention. 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 ) extracts and novel compounds isolated therefrom, and the use of the compounds, specifically, a novel lobdan diterpenoid compound derived from Hedium coronarium and their LPS (Lipopolysaccharide) -stimulated inflammatory cytokine production inhibitory function It is about.

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 embodiment, it can manufacture 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. The preparation method is merely an exemplary method thereof, and may be suitably modified by various methods based on the art. For example, the non-exemplified extraction method according to the present invention may be used. Successful modifications can be made by those 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 Hedithium coronarium extract of the present invention comprises the following rabdan-type diterpenoids (Labdane-Type Diterpenoids) compound as an active ingredient. The structural formulas of the compounds corresponding to the respective substituents are named as chemical formulas (compounds) 1 to 6.

Particularly, among the compounds, Compound 1 and Compound 3 are novel Lapdan-Type Diterpenoids compounds discovered by the present inventors. It relates to a noid compound.

The present invention relates to the function of inhibiting the inflammatory cytokine production of the Heidicium coronarium extract comprising the compound of Formula 1 to 6 and the lobdan diterpenoid compound of Formula 1 or Formula 3 isolated therefrom. In particular, since it inhibits the proinflammatory cytokine production and activity of TNF-α, IL-6 or IL-12, and does not show toxicity in cultured cells, it may be useful for the prevention and treatment of autoimmune diseases and sepsis.

That is, Heditchium coronarium extract comprising the compound of Formulas 1 to 6 of the present invention, and the lobdan diterpenoid compound of Formula 1 or Formula 3 separated therefrom have the following functions.

(1) Heditchium coronarium extract comprising the compound of Formulas 1 to 6 of the present invention and the lobdan diterpenoid compound of Formula 1 or 3 are bone marrow-derived dendritic cells stimulated by lipopolysaccharide (LPS). Marrow-Derived Dendritic Cells) inhibit the production of TNF-α, 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 capable of causing an inflammatory response is applied, expression of inflammatory cytokines such as TNF-α is induced, and the generated inflammatory cytokines stimulate expression of genes encoding iNOS and COX-2, Induces an inflammatory response by producing NO and PGE ₂ substances involved in the inflammatory response.

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 rabdan diterpenoid compound according to the present invention has a function of inhibiting the production of such inflammatory cytokines.

Although previous studies have shown that Hedicidium coronarium has anti-inflammatory properties (Hwang, Chen, Nines, Shin & Stoner, 2006), rabdan diterpenoid compounds having inhibitory ability to produce inflammatory cytokines, in particular, Formula 1 and The lobdan diterpenoid compound of 3 had not been identified. Therefore, the present invention is characterized by a rabdan diterpenoid compound of the formula (1) or (3), which is a novel compound derived from Hedium coronium.

(2) Heditchium coronarium extract comprising the compound of Formulas 1 to 6 of the present invention and the lobdan diterpenoid compound of Formula 1 or 3 may be used in dendritic cells (DCs) stimulated by lipopolysaccharide (LPS). Inhibit the activity of inflammatory mediators.

Interleukin-12 is a kind of interleukin produced in antigen-providing cells such as dendritic cells and macrophages by antigen stimulation. It acts to stimulate T cells to differentiate into type 1 helper T cells, and NK (natural killer) cells. In addition, it plays an important role in the activity of T lymphocytes. It is a heterodimer form of IL-12A (p35) and IL-12B (p40), the expression of IL-12A (p35) is constitutive, so the biological function of interleukin-12 is mainly IL-12B (p40) Inhibition of interleukin-12 may be helpful in the treatment of autoimmune diseases, especially immune immune systems, which are over-immune responses to cells in the human body. It is becoming.

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 "inflammatory disease" in the present invention is a disease caused by the excessive production of inflammatory cytokines TNF-α, IL-6 or IL-12 induced by lipopolysaccharide (LPS), autoimmune diseases or sepsis Etc. can be mentioned.

More specifically, the autoimmune diseases include dermatitis, allergy, atopic dermatitis, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, ankylosing spondylitis, rheumatic fever , Lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendonitis, hay salt, peritonitis, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome, multiple sclerosis, and acute and chronic inflammatory diseases Sepsis includes fever symptoms with body temperature rising above 38 degrees, hypothermia falling below 36 degrees, respiratory rate increased to more than 24 beats per minute (empty breathing), heart rate above 90 beats per minute (blood pressure), blood tests Systemic inflammatory response syndrome (SIRS) and endotoxin caused by microbial infection, with symptoms of an increase or significant decrease in the number of upper leukocytes It includes shock (endotoxic shock) and the like. More preferably, with autoimmune diseases, i) Rheumatoid Arthritis, in which the immune system attacks tissues of various joints, ii) Autoimmunity of the central nervous system induced by T cells, can lead a relatively normal life. However, in severe cases, multiple sclerosis (MS), which can lead to blindness, paralysis, and premature death, is caused by the destruction of immune cells in the pancreas' insulin-producing cells and the MHC gene plays an important role. Immune-Mediated or Type I Diabetes Mellitus (i) Inflammatory Bowel Diseases, a disease caused by the immune system attacking the intestine, i. Scleroderma, 전신) systemic autoimmunity, accompanied by symptoms of deep fatigue, rash, joint pain, etc. That can cause damage to can be given a systemic lupus (Systemic Lupus Erythematosus, SLE). In addition, sepsis may include systemic inflammatory response syndrome (SIRS) and sepsis (endotoxin) shock, in which microorganisms cause severe inflammatory responses throughout the body. In particular, the endotoxin shock (septicemia shock) is mainly caused by the excessively produced interleukin-6 and TNF-α.

Accordingly, in a specific aspect of the present invention, there is provided a method for preventing and treating autoimmune diseases or sepsis using Hedium coronarium extract or the compound of Formula 1 or 3; 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 include appropriate carriers, excipients and diluents commonly 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 .

Carriers, excipients and diluents that may be included in the compositions of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin , Calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, 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 form preparations for oral administration include tablets, pills, powders, granules, capsules and the like, and such solid form preparations include at least one excipient such as starch, calcium carbonate, sucrose or lactose in the compound. Mixed with gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents, water and liquid paraffin. .

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. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

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 aspect of the present invention, a method of inhibiting inflammatory cytokine production and activity using the rabdan diterpenoid compound of the formula (1) or (3) is mentioned. The inflammatory cytokines include TNF-α, IL-6 or IL-12.

Unless otherwise stated, the term "rabdan diterpenoid compound" or "compound of formula 1 or formula 3" according to the present invention, the compound itself, pharmaceutically acceptable salts, hydrates, solvates, isomers thereof And it is used as a concept that includes both 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. 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" 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 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, 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 than 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 bound to an acid group that is converted by metabolism to reveal the 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. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be 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 using 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 HC1B fractions were chromatographed on a silica gel column and eluted with CHCl ₃ -MeOH (40: 1, v / v) to afford compound 2 (8.0 mg) and compound 6 (6.4 mg). The HC1C 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 (HC1C1 to HC1C3).

The HC1C2 fraction was chromatographed on a YMC RP-18 column and eluted with acetone-water (5: 1, v / v) to afford compound 4 (2.6 mg) and compound 5 (6.0 mg). Fraction HC1C2 was chromatographed on YMC RP-18 column and eluted with MeOH-water (3: 1, v / v) to give compound 1 (7.0 mg) and compound 3 (8.5 mg). .

In particular, the NMR data of novel compounds 1 and 3 are shown in the table below.

< 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 electrospray ionization mass spectrometry (ESI-MS) was observed at m / z 331 [M + H] ⁺ , and the cluster ion peak m / z 331.2268 [M + H] ⁺ (Calcd for Analysis of high-resolution electrospray ionization mass spectroscopy (HR-ESI-MS) with C ₂₁ H ₃₁ O ₃ : 331.2273 revealed that the molecular formula was C ₂₁ H ₃₀ O ₃ .

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

three tertiary methyl groups at δ _H 0.84, 0.87, and 0.89 (each 3H, s);

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

five olefinic protons at δ _H 4.48, 4.76, 6.10, 6.78, and 6.96 (see Table 1)

^Thirteen C-NMR and distortionless enhancement by polarization transfer (DEPT) spectra identified 21 carbon signals comprising four methyl groups, six methylenes, six methines, and five quaternary carbons. The ¹ H- and ¹³ C-NMR data of Compound 1 suggested that this was a terdan type diterpenoid compound. However, it was present as an epimer mixture at C-15.

On the other hand, NMR data of Compound 1 were similar to 15-hydroxylabda-8 (17), 11,13-trien-16,15-olide, but the additional part of the methoxy group at C-15 was different. Α, β-unsaturated γ-lactone having a methoxy group on the γ-carbon (C-15) in the branched chain of Compound 1, 3,19-dihydroxy-15-methoxy-ent-labda-8 (17), ¹ H and ¹³ C data with 11,13-trien-16,15-olide. Also between olefinic proton H-14 (δ _H 6.78) and carbons C-13 (δ _C 131.98 / 132.00), C-15 (δ _C 101.94), and C-16 (δ _C 169.56 / 169.57); Heteronuclear multiple bond correlations between proton H-15 (δ _H 5.76) and C-14 (δ _C 139.20 / 139.23), C-16 (δ _C 169.56 / 169.57), and methoxy carbon (δ _C 56.76 / 56.82) ) Was also confirmed.

In addition, the E geometry isomerism of C-11 / C-12 double bonds was confirmed with a large coupling constant (J _{11 , 12} = 16.0 Hz).

In addition, δ _H coupled with two protons H-17 (C-7 (δ _C 36.74), C-8 (δ _C 149.18), and C-9 (δ _C 62.28 / 62.30) in the HMBC spectrum of Compound 1 4.48 and 4.76) mean that an exocyclic double bond is located at C-8 / 17.

Nuclear Overhauser Enhancement and Exchange Spectroscopy (NOESY) Correlation Between H-19 (δ _H 0.84) and H-20 (δ _H 0.87) and H-5 (δ _H 1.09) and H-9 (δ _H 2.38) Confirmed the β-orientation of the methyl group and the branch chains at C-10 and C-9, respectively.

As a result, Compound 1 was determined to be 15-methoxylabda-8 (17), 11E, 13-trien-16,15-olide and named hedycoronen A.

Hedycoronen A (1): A colorless oil, [α] _D 25: + 19.3 ° (c = 0.5 in CHCl ₃ ); positive ESI-MS m / z: 331 [M + H] ⁺ , HR-ESI-MS Found m / z: 331.2268 [M + H] ⁺ ; Calcd C ₂₁ H ₃₁ O ₃ for 331.2273), ¹ H- and ¹³ C-NMR: (See Table 1)

(2) Characterization of Compound 3

Compound 3 was obtained in the form of a colorless viscous oil.

A basic ion peak was observed at m / z 331 [M + H] ⁺ on the cationic ESI-MS, and m / z 331.2256 [M + H] ⁺ (Calcd for C) by HR-ESI-MS analysis. Cluster ion peak at ₂₁ H ₃₁ O ₃ : 331.2273) revealed that the molecular formula was C ₂₁ H ₃₀ O ₃ .

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

three tertiary methyl groups at δ _H 0.85, 0.87, and 0.90 (each 3H, s);

one methoxy group at δ _H 3.47 (s); and

five olefinic protons at δ _H 4.37, 4.43, 5.89, 6.28 / 6.29, and 6.48 / 6.50 (see Table 1).

¹³ C-NMR and DEPT spectra identified 21 carbon signals comprising 4 methyl groups, 6 methylenes, 6 ethine and 5 quaternary carbons. The ¹ H- and ¹³ C-NMR data of compound 3 suggested that this was a terdan type diterpenoid compound, and compound 3 was isolated as an epimer mixture at C-16.

The NMR data of compound 3 were similar to 16-hydroxylabda-8 (17), 11,13-trien-15,16-olide, but the alternative part of the hydroxy group by the methoxy group at C-16 was different. Compound 13 has an α, β-unsaturated γ-lactone having a methoxy group on the γ-carbon (C-16), 16-methoxy-6-oxo-7,11,13-labdatrien-15,16- It was confirmed from comparative data of ¹ H and ¹³ C with olide.

C-8 (δ _C 148.83 / 148.87), C-9 (δ _C 61.96 / 62.05), C-10 (δ _C 39.50 / 39.58), C-12 (from olefinic proton H-11 (δ _H 6.48 / 6.50) δ _C 122.71 / 122.76) and C-13 (δ _C 159.18); olefinic proton H-14 (δ _H 5.89) to C-13 (δ _C 159.18), C-15 (δ _C 170.68), and C-16 (δ _C 102.57 / 102.66); proton H-16 (δ _H 6.00) to C-14 (δ _C 116.52 / 116.55), C-15 (δ _C 170.68), and methoxy carbon (δ _C 54.30); HMBCs from two olefinic protons H-17 (δ _H 4.37 and 4.43) to C-7 (δ _C 36.61), C-8 (δ _C 148.83 / 148.87), and C-9 (δ _C 61.96 / 62.05) were 3 Epoxy bridges and carbonyl groups at two double bonds, C8 / 17, C11 / 12, C13 / 14, C15 / 16, and C-15.

In addition, the E geometry isomerism of the C-11 / C-12 double bond was confirmed with a large binding constant (J _{11 , 12} = 16.0 Hz).

As a result, compound 3 was determined as 16-methoxylabda-8 (17), 11E, 13-trien-15,16-olide, and named hedycoronen B.

Hedycoronen B (2): A colorless oil, [α] _D 25: + 22.1 ° (c = 0.5 in CHCl ₃ ); positive ESI-MS m / z: 331 [M + H] &lt; + &gt;, HR-ESI-MS Found m / z: 331.2256 [M + H] ⁺ ; Calcd C ₂₁ H ₃₁ O ₃ for 331.2273), ¹ H- and ¹³ C-NMR: see Table 1

(3) other isolated compounds

In addition to the novel compounds 1 and 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.

labda-8 (17), 11,13-trien-16 (15) -olide (Compound 2) [Nakatani N., Kikuzaki H., Yamaji H., Yoshio K., Kitora C., Okada K., Padolina WG , Phytochemistry, 37, 1383-1388 (1994)]

16-hydroxylabda-8 (17), 11,13-trien-15,16-olide (Compound 4) [Mohamad H., Lajis NH, Abas F., Ali AM, Sukari MA, Kikuzaki H., Nakatani N., J. Nat. Prod., 68, 285-288 (2005)]

coronarin A (Compound 5) [Itokawa H., Morita H., Katou I., Takeya K., Cavalheiro AJ, de Oliveira RC, Ishige M., Motidome M., Planta Med., 54, 311-315 (1988) ], And

coronarin E (Compound 6) [Itokawa H., Morita H., Takeya K., Motidome M., Chem. Pharm. Bull., 36, 2682-2684 (1988)].

In particular, the inventors first identified the isolation and chemical analysis of Compound 4 from Hedychium species.

< 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). Supernatants were obtained 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 expressed as mean ± standard deviation were obtained from independent experiments performed on at least three triplicates.

(2) bone marrow-derived In dendritic cells (DCs)  Inflammatory Response Evaluation

In order to evaluate the biological function of the six lobdan-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, compound 4 was not going to further experiment because of cytotoxicity (cytotoxicity).

In LPS treatment, DCs are already known to secrete inflammatory cytokines such as TNF-α, IL-6, and IL-12, and inflammatory responses are such cytokine secretion, activation and neutrophils. rapid accumulation of phils) and subsequent monocyte 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.

A p38 kinase inhibitor, SB203580, was used as a positive control, and SB203580 inhibited TNF-α, IL-6, and IL-12 production: IC ₅₀ was 7.2 ± 0.1, 3.5 ± 0.1, and 5.0 ± 0.2 μM, respectively. .

These results are shown in Table 2 and Figs.

Of these compounds, compounds 1-3 were identified as good inhibitors of reducing LPS-stimulated IL-6 and IL-12 production. They reduced the levels of the cytokines to IC _50s ranging from 4.1 ± 0.2 to 9.1 ± 0.3 μM. In particular, compounds 1 and 3 exhibited moderate inhibitory activity, showing IC ₅₀ values of 46.0 ± 1.3 and 12.7 ± 0.3 μM, respectively, in TNF-α production capacity. Other compounds showed an inactivity of IC ₅₀ > 100 μM.

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)

A composition for the treatment or prevention of autoimmune diseases and sepsis, containing an extract of Hedychium coronarium having the ability to inhibit the production of Lipopolysaccharide (LPS) -stimulated pro-inflammatory cytokines. The method of claim 1,
The LPS-stimulated inflammatory cytokine TNF-α, IL-6 or IL-12 characterized in that the composition for the treatment or prevention of autoimmune diseases and sepsis. The method of claim 1,
The extract of Hedychium coronarium ( Hedychium coronarium ) composition for the treatment or prevention of autoimmune diseases and sepsis, characterized in that it comprises the following compounds as an active ingredient:

. The method of claim 1,
The autoimmune diseases include dermatitis, allergy, atopic dermatitis, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia ( fibromyalgia), psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendinitis, hay salt, periarthritis, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome, multiple sclerosis, and acute and chronic inflammatory diseases Composition for the treatment or prevention of autoimmune diseases and sepsis, characterized in that at least one disease. The method of claim 1,
The sepsis is a system for the treatment or prevention of autoimmune diseases and sepsis, characterized in that the systemic inflammatory response or endotoxin shock symptoms caused by the infection of the microorganism. The method of claim 1,
The extract of Hedidium coronarium is a composition for the treatment or prevention of autoimmune diseases and sepsis, characterized in that from the Heidirium coronarium rhizome (Rhizome). The method of claim 1,
The extract of Hedidium coronarium is a composition for the treatment or prevention of autoimmune diseases and sepsis, characterized in that the methanol extract. Lopdan diterpenoid compounds of Formula 1 or 3, having the ability to inhibit Lipopolysaccharide (LPS) -stimulated inflammatory cytokine production:
[Chemical Formula 1]

. 9. The method of claim 8,
The lobdan diterpenoid compound is Hedychium Coronium ( Hedychium coronarium ) derived from the compound. 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 lobdan diterpenoid compound is
(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. Claim 8 rabdan diterpenoid compound containing as an active ingredient, autoimmune diseases and sepsis treatment and prevention compositions. 15. The method of claim 14,
The autoimmune diseases include dermatitis, allergy, atopic dermatitis, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia ( fibromyalgia), psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendinitis, hay salt, periarthritis, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome, multiple sclerosis, and acute and chronic inflammatory diseases At least one disease. 15. The method of claim 14,
The sepsis is a composition characterized in that the systemic inflammatory response or endotoxin shock symptoms caused by the infection of the microorganism. A method for inhibiting the production of LPS-stimulated inflammatory cytokines TNF-α, IL-6 or IL-12 using the lobdan diterpenoid compound of claim 8.

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