WO2009069887A1 - A pharmaceutical composition comprising glabridin or glabridin derivatives for suppression of dendritic cell maturation - Google Patents

A pharmaceutical composition comprising glabridin or glabridin derivatives for suppression of dendritic cell maturation Download PDF

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Publication number
WO2009069887A1
WO2009069887A1 PCT/KR2008/005774 KR2008005774W WO2009069887A1 WO 2009069887 A1 WO2009069887 A1 WO 2009069887A1 KR 2008005774 W KR2008005774 W KR 2008005774W WO 2009069887 A1 WO2009069887 A1 WO 2009069887A1
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Prior art keywords
glabridin
derivatives
composition
cell maturation
dendritic cell
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PCT/KR2008/005774
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French (fr)
Inventor
Hwan Mook Kim
Jong Soon Kang
Song-Kyu Park
Kiho Lee
Jee Youn Kim
Sang Bae Han
Ki Hoon Lee
Chang Woo Lee
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Korea Research Institute Of Bioscience And Biotechnology
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Priority to CN200880117804A priority Critical patent/CN101873856A/en
Publication of WO2009069887A1 publication Critical patent/WO2009069887A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A61K36/484—Glycyrrhiza (licorice)

Definitions

  • the present invention relates to a pharmaceutical composition for suppressing dendritic cell maturation, a composition for immune regulation, a composition for prevention and treatment of autoimmune disease comprising glabridin or glabridin derivatives as an active ingredient and a method for treatment of autoimmune disease using the same .
  • Dendritic cells are powerful antigen presenting cells which induce and regulate adaptive immune response.
  • Dendritic cells are found in various tissues and lymphatic organs and exist as immature cells in the absence of an antigen or inflammation. Such immature dendritic cells are very good at capturing an antigen but poor at activating T-lymphocytes by presenting an antigen. These immature dendritic cells can be matured by inflammation reaction product resulted from bacterial infection such as lipopolysaccharide or CpG and inflammatory cytokine such as TNF-alpha and IL-I. During dendritic cell maturation, the expressions of surface molecules such as MHC (major histocompatibility complex) or CD80, CD86 and CD40 are increased, along which the expressions of adhesion molecule and chemokine receptor are also increased.
  • MHC major histocompatibility complex
  • CD80, CD86 and CD40 are increased, along which the expressions of adhesion molecule and chemokine receptor are also increased.
  • cytokine receptor paves the way for dendritic cells to transfer from peripheral tissues into blood vessels and to lymphatic organs. Dendritic cells transferred into lymphatic organs present an antigen to T- lymphocytes to induce activation of T-lymphocytes .
  • dendritic cells In addition to induce immune response, dendritic cells induce immune tolerance via diverse mechanisms. Because of such counter function of dendritic cells, it is expected that dendritic cells can be involved in the development of autoimmune disease. In fact, when dendritic cells separated from an animal having autoimmune disease or artificially matured dendritic cells were administered to a normal animal, T-lymphocyte activation was strongly induced and as a result autoimmune disease was developed (Dittel BN et al., J. Immunol., 163, 32-39, 1999).
  • Glycyrrhiza uralensis is a perennial plant belonging to Rosales, Fabaceae, which is the most famous medicinal herb used world-widely .
  • Glycyrrhiza uralensis is originated from Asia, South Europe, and Mediterranean and widely distributed in Russia, Spain, Iran and India.
  • the roots of Glycyrrhiza uralensis is reddish brown and growing deep down under the ground. The stem is angular but strait-growing about 1 cm. 1.4-2.5 cm purple flowers blossom in July - August.
  • the roots are sweet, so that they have been used as a sweetening agent.
  • Glycyrrhiza uralensis has been used as an antidote, an abirritant and an expectorant in Asia and Europe. It has also been used as a therapeutic agent for allergic inflammation.
  • Major components of Glycyrrhiza uralensis are glycyrrhizin, glycyrrhetinic acid and glabridin. Glycyrrhizin and glycyrrhetinic acid taste sweet and have anti-inflammation effect.
  • Glabridin is polyphenolic flavonoid that is a major component of hydrophobic fraction of Glycyrrhiza uralensis extract.
  • Glabridin is also known to inhibit melanogenesis , so that it is used as a material of cosmetics for whitening. It has also been reported that glabridin activates macrophages and suppresses secretion of inflammatory cytokines, suggesting that it has anti- inflammation activity (Kang JS et. al., J. Pharm. Exp. Ther., 312, 1187-1194, 2005). However, the involvement of glabridin in activation of dendritic cells has not been reported, yet.
  • the present inventors completed this invention by confirming that the effect of glabridin on the activation of dendritic cells known to be involved in various immune responses .
  • the present invention provides a pharmaceutical composition comprising glabridin or glabridin derivatives as an active ingredient for suppression of dendritic cell maturation and a preparation method of the same.
  • the present invention also provides a composition for immune regulation comprising glabridin or glabridin derivatives as an active ingredient.
  • the present invention also provides a method for immune regulation using glabridin or glabridin derivatives.
  • the present invention also provides a composition for treatment of autoimmune disease comprising glabridin or glabridin derivatives as an active ingredient.
  • the present invention further provides a composition for health food and health beverages for prevention of autoimmune disease comprising glabridin or glabridin derivatives as an active ingredient.
  • the present invention provides a method for treatment of autoimmune disease.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising glabridin or glabridin derivatives as an active ingredient for suppression of dendritic cell maturation.
  • Galbridin has the effects of increasing expressions of surface factors of dendritic cells induced by lipopolysaccharide, phagocytosis of dendritic cells, secretion of IL-12 and activation of T-lymphocytes . Therefore, the pharmaceutical composition of the present invention can be effectively used for treatment of various autoimmune diseases .
  • cytokine One of the most important functions of mature dendritic cells is to secret cytokine.
  • the present inventors investigated the effect of glabridin on the secretion of IL-12, one of important cytokines involved in T-lymphocyte activation.
  • IL-12 secretion was increased in the dendritic cells treated with lipopolysaccharide, compared with the control.
  • IL-12 secretion was reduced glabridin dose-dependently in the experimental group co-treated with lipopolysaccharide and glabridin ( see Figure 3 ) .
  • glabridin suppressed dendritic cell maturation.
  • the most important immune-related function of mature dendritic cells is to activate T-lymphocytes . So, the present inventors investigated the effect of glabridin on the activation of T-lymphocytes by dendritic cells.
  • T-lymphocyte activation was increased in the dendritic cells treated with lipopolysaccharide, compared with the control group.
  • T- lymphocyte activation was significantly suppressed in the experimental group co-treated with lipopolysaccharide and glabridin (see Figure 4).
  • the present inventors also measured the secretions of IFN-Y and IL-4, the cytokines secreted from T-cells.
  • the pharmaceutical composition containing glabridin as an active ingredient is expected to be an effective agent for suppressing dendritic cell maturation.
  • the present invention also provides a pharmaceutical composition containing glabridin or glabridin derivatives as an active ingredient for suppression of dendritic cell maturation or an inhibitory agent of dendritic cell maturation.
  • the glabridin herein is preferably represented by the following formula 1:
  • R and Rl are independently H or alkyl
  • the glabridin derivative is one or more compounds selected from the group consisting of glabrene, Hispaglabridin A, Hispaglabridin B, 4-0-methylglabridin, 2 ' -O-methylglabridin, 2 ' , 4 ' -O-methylglabridin, and 3 1 - hydroxy-4 ' O-methylglabridin, but not always limited thereto and any glabridin derivative known to those in the art can be used.
  • the effect of glabridin alone is described but it is well understood by those in the art that glabridin derivatives have equal effect to glabridin.
  • Glabridin or glabridin derivatives are preferably extracted from Glycyrrhiza uralensis, but not always limited thereto and any commercial compound (glabridin or its derivatives) can be used.
  • the preferable content of glabridin or glabridin derivatives in the pharmaceutical composition for suppression of dendritic cell maturation or the inhibitory agent of dendritic cell maturation of the present invention is 0.1-50 weight% by the total weight of the composition, but not always limited thereto.
  • Glabridin or glabridin derivative can be used as it is or as a form of pharmaceutically acceptable salt.
  • any acceptable non-toxic salt can be used without limitation, for example, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrofluoric acid, hydrobromic acid, formic acid, acetic acid, tartaric acid, lactic acid, citric acid, fumaric acid, maleic acid, succinic acid, metalsulfonic acid, benzenesulfonic acid, toluenesulfonic acid, naphthalenesulfonic acid, etc.
  • the pharmaceutical composition for suppression of dendritic cell maturation of the present invention can be administered alone or together with surgical operation, hormone therapy, chemo-therapy and biological regulators.
  • the pharmaceutical composition for suppression of dendritic cell maturation or the inhibitory agent of dendritic cell maturation of the present invention can include, in addition to glabridin or glabridin derivatives, one or more effective ingredients having the same or similar function to glabridin or glabridin derivatives.
  • glabridin or glabridin derivatives one or more effective ingredients having the same or similar function to glabridin or glabridin derivatives.
  • effective ingredients having the same or similar function to glabridin or glabridin derivatives.
  • saline sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome and a mixture
  • a general additive such as an antioxidant, buffer and an antimicrobial agent can be additionally added.
  • the pharmaceutical composition for suppression of dendritic cell maturation or the inhibitory agent of dendritic cell maturation of the present invention can be prepared for oral or parenteral administration by mixing with generally used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactant.
  • the composition can further be prepared in suitable forms according to ingredients by following the method represented in Remington' s Pharmaceutical Science (the newest edition), Mack Publishing Company, Easton PA.
  • the pharmaceutical composition for suppression of dendritic cell maturation or the inhibitory agent of dendritic cell maturation of the present invention can be administered orally or parenterally (for example, intravenous, hypodermic, local or peritoneal injection).
  • the effective dosage of the composition can be determined according to weight, age, gender, health condition, diet, administration frequency, administration method, excretion and severity of a disease.
  • the dosage units can contain, for example, 1, 2, 3 or 4 individual doses or 1/2, 1/3 or 1/4 of an individual dose.
  • An individual dose preferably contains the amount of active compound which is administered in one application and which usually corresponds to a whole, 1/2, 1/3 or 1/4 of a daily dose.
  • the pharmaceutical composition for suppression of dendritic cell maturation or the inhibitory agent of dendritic cell maturation of the present invention is evaluated to be a safe substance since its estimated LD50 value is much greater than 5 g/kg in rats, which is confirmed by acute toxicity assay with rats tested via oral administration.
  • the effective dosage is 0.5 - 6 nig/kg per day, preferably 3 nig/kg per day, and administration frequency is preferably 1 - 3 times a day.
  • the dosage is not limited thereto and can be adjusted according to the conditions of a patient and severity of a disease .
  • the present invention also provides a method for suppressing dendritic cell maturation using glabridin or glabridin derivatives.
  • the glabridin or glabridin derivatives herein can be originated from Glycyrrhiza uralensis, but any commercial glabridin or glabridin derivatives can be used.
  • the method for suppressing dendritic cell maturation contains the step of oral administration or parenteral administration of glabridin or glabridin derivatives (for example, intravenous, hypodermic, local or peritoneal injection). At this time, the content of glabridin or glabridin derivatives is 0.1-50 weight part by the total weight of the composition to be administered orally or parenterally , but not always limited thereto.
  • the glabridin or glabridin derivative can be used as it is or as a form of pharmaceutically acceptable salt.
  • any acceptable non-toxic salt can be used without limitation, for example, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrofluoric acid, hydrobromic acid, formic acid, acetic acid, tartaric acid, lactic acid, citric acid, fumaric acid, maleic acid, succinic acid, metalsulfonic acid, benzenesulfonic acid, toluenesulfonic acid, naphthalenesulfonic acid, etc.
  • the pharmaceutical composition for suppression of dendritic cell maturation or the inhibitory agent of dendritic cell maturation of the present invention can be administered alone or together with surgical operation, hormone therapy, chemo-therapy and biological regulators.
  • the present invention also provides a composition for immune regulation comprising glabridin or glabridin derivatives as an active ingredient.
  • the glabridin or glabridin derivative of the present invention can be effectively used as a composition for immune regulation because it effectively suppresses dendritic cell maturation.
  • the glabridin herein is preferably represented by the following formula 1:
  • R and Rl are independently H or alkyl
  • the glabridin derivative is one or more compounds selected from the group consisting of glabrene, Hispaglabridin A, Hispaglabridin B, 4-O-methylglabridin, 2 ' -O-methylglabridin , 2 ' , 4 ' -O-methylglabridin , and 3 ' - hydroxy-4 ' O-methylglabridin, but not always limited thereto and any glabridin derivative known to those in the art can be used.
  • the effect of glabridin alone is described but it is well understood by those in the art that glabridin derivatives have equal effect to glabridin.
  • Glabridin or glabridin derivatives are preferably extracted from Glycyrrhiza uralensis, but not always limited thereto and any commercial compound (glabridin or its derivatives) can be used.
  • the preferable content of glabridin or glabridin derivates is 0.1-50 weight part by the total weight of the composition for immune regulation, but not always limited thereto .
  • the glabridin or glabridin derivative can be used as it is or as a form of pharmaceutically acceptable salt.
  • any acceptable non-toxic salt can be used without limitation, for example, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrofluoric acid, hydrobromic acid, formic acid, acetic acid, tartaric acid, lactic acid, citric acid, fumaric acid, maleic acid, succinic acid, metalsulfonic acid, benzenesulfonic acid, toluenesulfonic acid, naphthalenesulfonic acid, etc.
  • the composition for immune regulation can be administered alone or together with surgical operation, hormone therapy, chemo-therapy and biological regulators.
  • composition for immune regulation of the present invention can include, in addition to glabridin or glabridin derivatives, one or more effective ingredients having the same or similar function to glabridin or glabridin derivatives .
  • effective ingredients having the same or similar function to glabridin or glabridin derivatives .
  • saline sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome and a mixture
  • a general additive such as an antioxidant, buffer and an antimicrobial agent can be additionally added.
  • the pharmaceutical composition for suppression of dendritic cell maturation or the inhibitory agent of dendritic cell maturation of the present invention can be prepared for oral or parenteral administration by mixing with generally used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactant.
  • the composition can further be prepared in suitable forms according to ingredients by following the method represented in Remington's Pharmaceutical Science (the newest edition), Mack Publishing Company, Easton PA.
  • the composition for immune regulation of the present invention can be administered orally or parenterally (for example, intravenous, hypodermic, local or peritoneal injection).
  • the effective dosage of the composition can be determined according to weight, age, gender, health condition, diet, administration frequency, administration method, excretion and severity of a disease.
  • the dosage units can contain, for example, 1, 2, 3 or 4 individual doses or 1/2, 1/3 or 1/4 of an individual dose.
  • An individual dose preferably contains the amount of active compound which is administered in one application and which usually corresponds to a whole, 1/2, 1/3 or 1/4 of a daily dose.
  • the composition for immune regulation of the present invention is evaluated to be a safe substance since its estimated LD50 value is much greater than 5 g/kg in rats, which is confirmed by acute toxicity assay with rats tested via oral administration.
  • the effective dosage is 0.5 - 6 nig/kg per day, preferably 3 nig/kg per day, and administration frequency is preferably 1 - 3 times a day.
  • the dosage is not limited thereto and can be adjusted according to the conditions of a patient and severity of a disease.
  • the present invention also provides a method for immune regulation using glabridin or glabridin derivatives
  • the glabridin or glabridin derivatives herein can be originated from Glycyrrhiza uralensis, but any commercial glabridin or glabridin derivatives can be used.
  • the method for immune regulation contains the step of oral administration or parenteral administration of glabridin or glabridin derivatives (for example, intravenous, hypodermic, local or peritoneal injection). At this time, the content of glabridin or glabridin derivatives is 0.1- 50 weight part by the total weight of the composition to be administered orally or parenterally , but not always limited thereto.
  • the glabridin or glabridin derivative can be used as it is or as a form of pharmaceutically acceptable salt.
  • any acceptable nontoxic salt can be used without limitation, for example, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrofluoric acid, hydrobromic acid, formic acid, acetic acid, tartaric acid, lactic acid, citric acid, fumaric acid, maleic acid, succinic acid, metalsulfonic acid, benzenesulfonic acid, toluenesulfonic acid, naphthalenesulfonic acid, etc.
  • the present invention also provides a composition for treatment of autoimmune disease comprising glabridin or glabridin derivatives as an active ingredient.
  • Dendritic cell maturation has been known to be involved in auto-immune disease. Therefore, suppression of dendritic cell maturation can be an effective treatment method of auto-immune disease.
  • the auto-immune disease herein is exemplified by lupous or Sjogren's syndrome.
  • the glabridin herein is preferably represented by the following formula 1:
  • R and Rl are independently H or alkyl
  • the glabridin derivative is one or more compounds selected from the group consisting of glabrene, Hispaglabridin A, Hispaglabridin B, 4-0-methylglabridin, 2 ' -O-methylglabridin, 2 ' , 4 ' -O-methylglabridin, and 3'- hydroxy-4 ' O-methylglabridin, but not always limited thereto and any glabridin derivative known to those in the art can be used.
  • the effect of glabridin alone is described but it is well understood by those in the art that glabridin derivatives have equal effect to glabridin.
  • Glabridin or glabridin derivatives are preferably extracted from Glycyrrhiza uralensis, but not always limited thereto and any commercial compound (glabridin or its derivatives) can be used.
  • the preferable content of glabridin or glabridin derivates is 0.1-50 weight part by the total weight of the composition for treatment of autoimmune disease, but not always limited thereto.
  • the glabridin or glabridin derivative can be used as it is or as a form of pharmaceutically acceptable salt.
  • any acceptable non-toxic salt can be used without limitation, for example, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrofluoric acid, hydrobromic acid, formic acid, acetic acid, tartaric acid, lactic acid, citric acid, fumaric acid, maleic acid, succinic acid, metalsulfonic acid, benzenesulfonic acid, toluenesulfonic acid, naphthalenesulfonic acid, etc.
  • the composition for treatment of autoimmune disease can be administered alone or together with surgical operation, hormone therapy, chemo-therapy and biological regulators .
  • composition for treatment of autoimmune disease of the present invention can include, in addition to glabridin or glabridin derivatives, one or more effective ingredients having the same or similar function to glabridin or glabridin derivatives.
  • effective ingredients having the same or similar function to glabridin or glabridin derivatives.
  • saline sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome and a mixture
  • a general additive such as an antioxidant, buffer and an antimicrobial agent can be additionally added.
  • the pharmaceutical composition for suppression of dendritic cell maturation or the inhibitory agent of dendritic cell maturation of the present invention can be prepared for oral or parenteral administration by mixing with generally used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactant.
  • the composition can further be prepared in suitable forms according to ingredients by following the method represented in Remington' s Pharmaceutical Science (the newest edition), Mack Publishing Company, Easton PA.
  • the composition for treatment of autoimmune disease of the present invention can be administered orally or parenterally (for example, intravenous, hypodermic, local or peritoneal injection).
  • the effective dosage of the composition can be determined according to weight, age, gender, health condition, diet, administration frequency, administration method, excretion and severity of a disease.
  • the dosage units can contain, for example, 1, 2, 3 or 4 individual doses or 1/2, 1/3 or 1/4 of an individual dose.
  • An individual dose preferably contains the amount of active compound which is administered in one application and which usually corresponds to a whole, 1/2, 1/3 or 1/4 of a daily dose.
  • the composition for treatment of autoimmune disease of the present invention is evaluated to be a safe substance since its estimated LD50 value is much greater than 5 g/kg in rats, which is confirmed by acute toxicity assay with rats tested via oral administration.
  • the effective dosage is 0.5 - 6 nig/kg per day, preferably 3 mg/ kg per day, and administration frequency is preferably 1 - 3 times a day.
  • the dosage is not limited thereto and can be adjusted according to the conditions of a patient and severity of a disease.
  • the present invention further provides a composition for health food and health beverages for prevention of autoimmune disease comprising glabridin or glabridin derivatives as an active ingredient.
  • the glabridin or glabridin derivative of the present invention can be used as food additive.
  • the glabridin or glabridin derivative can be added as it is or as mixed with other food components according to the conventional method.
  • the mixing ratio of active ingredients can be regulated according to the purpose of use.
  • the glabridin or glabridin derivative is added preferably by 0.1-15 weight part and more preferably by 0.1-10 weight part.
  • the content can be lower than the above but higher content can be accepted as well since the glabridin or glabridin derivative has been proved to be very safe.
  • the food herein is not limited.
  • the glabridin or glabridin derivative can be added to meats, sausages, breads, chocolates, candies, snacks, cookies, pizza, ramyuns, flour products, gums, dairy products including ice cream, soups, beverages, tea, drinks, alcohol drinks and vitamin complex, etc, and in wide sense, almost every food applicable in the production of health food can be included.
  • the composition for health beverages of the present invention can additionally include various flavors or natural carbohydrates, etc, like other beverages.
  • the natural carbohydrates above can be one of monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and glucose alcohols such as xilytole, sorbitol and erythritol .
  • natural sweetening agents such as thaumatin and stevia extract, and synthetic sweetening agents such as saccharin and aspartame can be included as a sweetening agent.
  • the content of the natural carbohydrate is preferably 0.01-0.04 g and more preferably 0.02-0.03 g in 100 m-C of the composition.
  • the glabridin or glabridin derivative of the present invention can include in variety of nutrients, vitamins, minerals, flavors, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acid, protective colloidal viscosifiers , pH regulators, stabilizers, antiseptics, glycerin, alcohols, carbonators which used to be added to soda, etc.
  • the glabridin or glabridin derivative of the present invention can also include natural fruit juice, fruit beverages and/or fruit flesh addable to vegetable beverages . All the mentioned ingredients can be added singly or together. The mixing ratio of those ingredients does not matter in fact, but in general, each can be added by 001-0.1 weight part per 100 weight part of the glabridin or glabridin derivative of the present invention.
  • the present invention provides a method for treatment of autoimmune disease containing the step of administering the pharmaceutical composition for suppressing dendritic cell maturation to a subject.
  • the subject herein can be human or mammals except human and preferably selected from the group consisting of mice, rats, guinea pigs, pigs, rabbits, monkeys and chimpanzees, but not always limited thereto.
  • the composition can be administered orally or parenterally (for example, intravenous, hypodermic, local or peritoneal injection).
  • the effective dosage of the composition can be determined according to weight, age, gender, health condition, diet, administration frequency, administration method, excretion and severity of a disease.
  • the dosage units can contain, for example, 1, 2, 3 or 4 individual doses or 1/2, 1/3 or 1/4 of an individual dose.
  • An individual dose preferably contains the amount of active compound which is administered in one application and which usually corresponds to a whole, 1/2, 1/3 or 1/4 of a daily dose.
  • the auto-immune disease herein is exemplified by lupous or Sjogren's syndrome.
  • the present invention facilitates the preparation of a pharmaceutical composition for suppressing dendritic cell maturation, a composition for immune regulation and a composition for treating auto-immune disease comprising glabridin or glabridin derivatives as an active ingredient.
  • the pharmaceutical composition of the present invention can be effectively used for prevention and treatment of auto-immune disease including lupous and Sjogren's syndrome •
  • Figure 1 is a set of graphs illustrating the inhibitory effect of glabridin on phenotypic changes of dendritic cells caused by the treatment of lipopolysaccharide .
  • Figure 2 is a set of graphs illustrating the inhibitory effect of glabridin on phagocytic activity of dendritic cells induced by the treatment of lipopolysaccharide .
  • Figure 3 is a graph illustrating the inhibitory effect of glabridin on IL-12 secretion of dendritic cells induced by the treatment of lipopolysaccharide.
  • Figure 4 is a graph illustrating the inhibitory effect of glabridin on T-lymphocyte activation activity of dendritic cells induced by the treatment of lipopolysaccharide .
  • Figure 5 is a graph illustrating the inhibitory effect of glabridin on IFN- ⁇ expression in dendritic cells induced by the treatment of lipopolysaccharide.
  • Figure 6 is a graph illustrating the inhibitory effect of glabridin on IL-4 expression in dendritic cells induced by the treatment of lipopolysaccharide.
  • Example 1 Effect of glabridin on dendritic cell maturation induced by lipopolysaccharide
  • mice (Dae Han Biolink Co., Ltd., Chungcheongbuk-do, Korea) were used as test animals.
  • the mice were adapted in an animal lab with providing feeds and water freely under required conditions (temperature: 21 ⁇ 2 ° Q light/dark: 12 hour light/dark cycle) for 7 days.
  • Bone marrow cells were separated from C57BL/6 mice (Orient Bio Inc., Seongnam-si, Korea), followed by culture at the density of 1x106 cells/ml. GM-CSF was added thereto at the concentration of 2 ng/ml, followed by further culture for 8 days to obtain immature dendritic cells .
  • glabridin Effect of glabridin on the expression of cell surface factors of dendritic cells Glabridin (Wako Pure Chemicals, Osaka, Japan) was added at the concentration of 5-20 g/ml to the immature dendritic cells obtained in Example ⁇ l-2>, and then lipopolysaccharide (Sigma-Aldrich, St. Louis, USA) was added thereto at the concentration of 1 g/ml, followed by culture for 48 hours.
  • the cells were recovered, which were treated with antibodies (BD Biosciences, San Jose, USA) such as FITC (Fluorescein Isothiocyanate) -CDlIc, PE (R-Phycoerythrin)-CD ⁇ O , PE-CD86, and PE-MHC II, followed by analysis using a flow cytometer, The results were presented by MFI (Mean Fluorescence Intensity). As MFI value increases, the expression of a surface factor increases and so does dendritic cell maturation.
  • CDlIc is a surface factor selectively expressed in dendritic cells
  • CD80, CD86 and MHC-II are surface factors whose expressions are increased as dendritic cells are maturated.
  • the expression level of MHC-II was 28.86 in the control and 42.55 in the group treated with lipopolysaccharide but the expression was decreased in the experimental group co-treated with lipopolysaccharide and glabridin dose-dependently to 43.48, 40.48, 35.07 and 29.77.
  • the expression level of CD80 was 11.53 in the control but increased to 22.64 in the group treated with lipopolysaccharide but decreased in the experimental group co-treated with lipopolysaccharide and glabridin dose- dependently to 17.38, 14.60, 11.59 and 6.45.
  • Immature dendritic cells have excellent phagocytic activity but once they are matured, the phagocytic activity is reduced.
  • phagocytic activity would be less reduced by the treatment of lipopolysaccharide. So, the present inventors performed following experiments to prove the above.
  • Glabridin was added at the concentration of 5-20 g/ml to the immature dendritic cells obtained in Example ⁇ l-2>, and then lipopolysaccharide was added thereto at the concentration of 1 g/ml, followed by culture for 48 hours. Upon completion of the culture, the cells were recovered, which were treated with FITC-dextran ( Sigma- Aldrich, St. Louis, USA) at the concentration of 0.7 mg/ml for 2 hours. , followed by analysis using a flow cytometer, The cells were washed to eliminate remaining FITC-dextran. Flow cytometry was performed to measure the amount of antigen introduced into the dendritic cells.
  • Double staining was performed using PE-CDlIc selectively binding to dendritic cells, which was to analyze antigen capturing rate after separation of the dendritic cells selectively.
  • antigen capturing rate of the immature dendritic cells was 26.91% and antigen capturing rate of the dendritic cells treated with lipopolysaccharide was 11.48%.
  • antigen capturing rate of the dendritic cells co-treated with lipopolysaccharide and glabridin was reduced dose-dependently to 12.05, 13.78, 15.09 and 17.94 ( Figure 2).
  • IL-12 secretion plays an important role in T- lymphocyte activation by dendritic cells.
  • the immature dendritic cells were treated with lipopolysaccharide and glabridin to investigate cytokine secretion .
  • Glabridin was added at the concentration of 5-20 g/ml to the immature dendritic cells obtained in Example ⁇ l-2>, and then lipopolysaccharide was added thereto at the concentration of 1 g/ml, followed by culture for 48 hours.
  • the culture solution was obtained and cytokine secreted from the dendritic cells was measured by enzyme-linked immunoassay.
  • the measurement was performed by using Mouse IL- 12 p70 DuoSet, Mouse IFN- DuoSet and Mouse IL-4 DuoSet (R&D Systems, Minneapolis, USA) according to the manufacturer's instructions .
  • Example 2 Effect of glabridin on T-lymphocyte activation activity of dendritic cells induced by lipopolysaccharide ⁇ 2-l> Effect of glabridin on T-lymphocyte activation activity of dendritic cells
  • the most important immune related function of mature dendritic cells is to activate T-lymphocytes . Immature dendritic cells capture an antigen, followed by maturation. Then, they transfer to immune system containing T-cells to induce activation of T-cells. The activated T-cells begin proliferate actively and secrete cytokines. So, the present inventors treated immature dendritic cells with lipopblysaccharide and glabridin to measure the activation of T-lymphocytes .
  • Glabridin was added at the concentration of 20 g/ml to the immature dendritic cells obtained in Example ⁇ l-2>, and then lipopolysaccharide was added thereto at the concentration of 1 g/ml, followed by culture for 48 hours. Upon completion of the culture, the cells were recovered and cultured with allogeneic T cells for 3 days. For the last 16 hours of culture, the cells were treated with [ 3H] -thymidine to introduce radio-isotope into T-cell DNA. Then, cell proliferation was measured.
  • T- lymphocyte proliferation in the group treated with glabridin alone was similar to that in the control.
  • the present inventors also investigated the levels of IFN- ⁇ and IL-4, cytokines secreted in T cells.
  • cytokine secretion was significantly increased in T-lymphocytes co-cultured with the dendritic cells treated with lipopolysaccharide, while cytokine secretion (IFN- ⁇ , Figure 5 and IL-4, Figure 6) in T- lymphocytes co-cultured with the dendritic cells treated with both lipopolysaccharide and glabridin was suppressed.
  • glabridin can suppress dendritic cell maturation induced by lipopolysaccharide and T-lymphocyte activation as well.
  • Example 3 Acute toxicity of glabridin in rats tested via oral administration
  • test compounds did not cause any specific clinical symptoms, weight change, or death in rats . No change was observed in hematological tests, biochemical tests of blood, and autopsy.
  • glabridin used in this experiment is evaluated to be a safe substance since it does not cause any toxic change in rats up to the level of 5 g/kg and its estimated LD50 value is much greater than 5 g/kg in rats.
  • Powders were prepared by mixing all the above components, which were filled in airtight packs according to the conventional method for preparing powders .
  • Magnesium stearate 2 mg Tablets were prepared by mixing all the above components by the conventional method for preparing tablets .
  • Capsules were prepared by mixing all the above components, which were filled in gelatin capsules according to the conventional method for preparing capsules .
  • Pills were prepared by mixing all the above components according to the conventional method for preparing pills. Each pill contained 4 g of the mixture.
  • Foods containing the glabridin of the present invention were prepared as follows .
  • Health enhancing spices for cooking was prepared with 20 ⁇ 95 weight part of the glabridin of the present invention according to the conventional method.
  • Health enhancing tomato ketchup or sauce was prepared by mixing 0.2 ⁇ 1.0 weight part of the glabridin of the present invention with tomato ketchup or sauce according to the conventional method.
  • 0.5 ⁇ 5.0 weight part of the glabridin of the present invention was added to the flour.
  • Health enhancing foods such as bread, cake, cookies, crackers and noodles were prepared with the flour mixture according to the conventional method.
  • Health enhancing ground beef was prepared by mixing 10 weight part of the glabridin of the present invention with ground beef according to the conventional method.
  • Brown rice, barley, glutinous rice and Yulmu (Job's tears) were gelatinized according to the conventional method, dried and pulverized to obtain 60-mesh powders.
  • Black soybean, black sesame and wild sesame were steamed and dried according to the conventional method and pulverized to obtain 60-mesh powders.
  • Sun-Sik was prepared by mixing the dry powders of the grains, seeds and glabridin according to the below ratio.
  • the glabridin of the present invention was mixed with liquid fructose (0.5%), oligosaccharide (2%), sugar (2%), salt (0.5%), and water (75%). After mixing completely, the mixture was sterilized instantly and filled small containers such as glass bottles, pet bottles, etc, to prepare health beverages.
  • Health enhancing vegetable juice was prepared by adding 5 g of the glabridin of the present invention to 1,000 of tomato or carrot juice according to the conventional method.
  • the present invention is effective in the preparation of a pharmaceutical composition for suppressing dendritic cell maturation, a composition for immune regulation and a composition for treating auto- immune disease comprising glabridin or glabridin derivatives as an active ingredient.
  • the said pharmaceutical compositions can be effectively used for the treatment of auto-immune disease.

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Abstract

The present invention relates to a pharmaceutical composition for suppressing dendritic cell maturation comprising glabridin or glabridin derivatives as an active ingredient. Glabridin not only suppresses dendritic cell maturation but also suppresses T-lymphocyte activation. Therefore, glabridin or its derivatives can be an active ingredient for a pharmaceutical composition for suppressing dendritic cell maturation, for a composition for immune regulation and for a composition for preventing and treating auto-immune disease, and can be effectively used for the treatment of auto-immune disease.

Description

[DESCRIPTION]
[invention Title]
A PHARMACEUTICAL COMPOSITION COMPRISING GLABRIDIN OR GLABRIDIN DERIVATIVES FOR SUPPRESSION OF DENDRITIC CELL MATURATION
[Technical Field]
The present invention relates to a pharmaceutical composition for suppressing dendritic cell maturation, a composition for immune regulation, a composition for prevention and treatment of autoimmune disease comprising glabridin or glabridin derivatives as an active ingredient and a method for treatment of autoimmune disease using the same .
[Background Art]
Dendritic cells are powerful antigen presenting cells which induce and regulate adaptive immune response.
Dendritic cells are found in various tissues and lymphatic organs and exist as immature cells in the absence of an antigen or inflammation. Such immature dendritic cells are very good at capturing an antigen but poor at activating T-lymphocytes by presenting an antigen. These immature dendritic cells can be matured by inflammation reaction product resulted from bacterial infection such as lipopolysaccharide or CpG and inflammatory cytokine such as TNF-alpha and IL-I. During dendritic cell maturation, the expressions of surface molecules such as MHC (major histocompatibility complex) or CD80, CD86 and CD40 are increased, along which the expressions of adhesion molecule and chemokine receptor are also increased. The expression of cytokine receptor paves the way for dendritic cells to transfer from peripheral tissues into blood vessels and to lymphatic organs. Dendritic cells transferred into lymphatic organs present an antigen to T- lymphocytes to induce activation of T-lymphocytes .
In addition to induce immune response, dendritic cells induce immune tolerance via diverse mechanisms. Because of such counter function of dendritic cells, it is expected that dendritic cells can be involved in the development of autoimmune disease. In fact, when dendritic cells separated from an animal having autoimmune disease or artificially matured dendritic cells were administered to a normal animal, T-lymphocyte activation was strongly induced and as a result autoimmune disease was developed (Dittel BN et al., J. Immunol., 163, 32-39, 1999). According to the recent reports, constant maturation and activation of dendritic cells play a certain role in the development of autoimmune disease such as lupus and Sjogren's syndrome (DaIl 'era MC et al., Ann. Rheum. Dis . , 64, 1692-1697, 2005; Vogelsang P et al . , Scand. J. Immunol., 64, 219-226, 2006). Therefore, suppression of dendritic cell maturation can be an effective way to treat autoimmune disease (DaIl 'era MC et al., Ann. Rheum. Dis., 64, 1692-1697, 2005).
Glycyrrhiza uralensis is a perennial plant belonging to Rosales, Fabaceae, which is the most famous medicinal herb used world-widely . Glycyrrhiza uralensis is originated from Asia, South Europe, and Mediterranean and widely distributed in Russia, Spain, Iran and India. The roots of Glycyrrhiza uralensis is reddish brown and growing deep down under the ground. The stem is angular but strait-growing about 1 cm. 1.4-2.5 cm purple flowers blossom in July - August. The roots are sweet, so that they have been used as a sweetening agent. Glycyrrhiza uralensis has been used as an antidote, an abirritant and an expectorant in Asia and Europe. It has also been used as a therapeutic agent for allergic inflammation. Major components of Glycyrrhiza uralensis are glycyrrhizin, glycyrrhetinic acid and glabridin. Glycyrrhizin and glycyrrhetinic acid taste sweet and have anti-inflammation effect. Glabridin is polyphenolic flavonoid that is a major component of hydrophobic fraction of Glycyrrhiza uralensis extract. Glabridin is also known to inhibit melanogenesis , so that it is used as a material of cosmetics for whitening. It has also been reported that glabridin activates macrophages and suppresses secretion of inflammatory cytokines, suggesting that it has anti- inflammation activity (Kang JS et. al., J. Pharm. Exp. Ther., 312, 1187-1194, 2005). However, the involvement of glabridin in activation of dendritic cells has not been reported, yet.
The present inventors completed this invention by confirming that the effect of glabridin on the activation of dendritic cells known to be involved in various immune responses .
[Disclosure] [Technical Problem] It is an object of the present invention to provide a pharmaceutical composition for suppression of dendritic cell maturation, a composition for immune regulation, a composition for prevention and treatment of autoimmune disease comprising glabridin or glabridin derivatives as an active ingredient and a method for treatment of autoimmune disease using the same.
[Technical Solution]
To achieve the above object, the present invention provides a pharmaceutical composition comprising glabridin or glabridin derivatives as an active ingredient for suppression of dendritic cell maturation and a preparation method of the same.
The present invention also provides a composition for immune regulation comprising glabridin or glabridin derivatives as an active ingredient.
The present invention also provides a method for immune regulation using glabridin or glabridin derivatives.
The present invention also provides a composition for treatment of autoimmune disease comprising glabridin or glabridin derivatives as an active ingredient.
The present invention further provides a composition for health food and health beverages for prevention of autoimmune disease comprising glabridin or glabridin derivatives as an active ingredient.
In addition, the present invention provides a method for treatment of autoimmune disease.
The present invention relates to a pharmaceutical composition comprising glabridin or glabridin derivatives as an active ingredient for suppression of dendritic cell maturation. Galbridin has the effects of increasing expressions of surface factors of dendritic cells induced by lipopolysaccharide, phagocytosis of dendritic cells, secretion of IL-12 and activation of T-lymphocytes . Therefore, the pharmaceutical composition of the present invention can be effectively used for treatment of various autoimmune diseases .
Hereinafter, the present invention is described in detail.
To investigate the effect of glabridin on maturation of dendritic cells, immature dendritic cells obtained from C57B/6 mouse were treated with lipopolysaccharide to induce dendritic cell maturation, followed by treatment of glabridin.
First, expression of MHC- J\ one of cell surface factors of dendritic cell, was investigated. As a result, the MHC- π expression was significantly increased in the immature dendritic cells stimulated by lipopolysaccharide, compared with the control group. In the meantime, the MHC- π expression was decreased glabridin dose-dependently in the experimental group co-treated with lipopolysaccharide and glabridin (see Figure 1). Therefore, it was confirmed that the dendritic cell maturation could be suppressed dose-dependently when glabridin was treated to the dendritic cells matured by lipopolysaccharide .
Immature dendritic cells have excellent phagocytic activity, whereas mature dendritic cells have poor phagocytic activity. So, the present inventors investigated the effect of glabridin on phagocytic activity of dendritic cells. As a result, in the dendritic cells matured by lipopolysaccharide, phagocytic activity was decreased compared with that in the control. But, in the experimental group co-treated with lipopolysaccharide and glabridin, the decrease of phagocytic activity was suppressed glabridin dose- dependently (see Figure 2). The above results indicate that the dendritic cell maturation by lipopolysaccharide can be suppressed by glabridin.
One of the most important functions of mature dendritic cells is to secret cytokine. The present inventors investigated the effect of glabridin on the secretion of IL-12, one of important cytokines involved in T-lymphocyte activation.
As a result, IL-12 secretion was increased in the dendritic cells treated with lipopolysaccharide, compared with the control. In the meantime, IL-12 secretion was reduced glabridin dose-dependently in the experimental group co-treated with lipopolysaccharide and glabridin ( see Figure 3 ) .
From the above results, it was confirmed that glabridin suppressed dendritic cell maturation. The most important immune-related function of mature dendritic cells is to activate T-lymphocytes . So, the present inventors investigated the effect of glabridin on the activation of T-lymphocytes by dendritic cells.
As a result, T-lymphocyte activation was increased in the dendritic cells treated with lipopolysaccharide, compared with the control group. In the meantime, T- lymphocyte activation was significantly suppressed in the experimental group co-treated with lipopolysaccharide and glabridin (see Figure 4). The present inventors also measured the secretions of IFN-Y and IL-4, the cytokines secreted from T-cells.
As a result, secretions of those cytokines were greatly increased in the dendritic cells treated with lipopolysaccharide, compared with the control group. In the meantime, secretions of IFN-Y and IL-4 were significantly suppressed in the experimental group co- treated with lipopolysaccharide and glabridin (see Figures 5 and 6 ) .
The above results indicate that glabridin not only suppresses dendritic cell maturation but also suppresses T-lymphocyte activation. So, the pharmaceutical composition containing glabridin as an active ingredient is expected to be an effective agent for suppressing dendritic cell maturation. The present invention also provides a pharmaceutical composition containing glabridin or glabridin derivatives as an active ingredient for suppression of dendritic cell maturation or an inhibitory agent of dendritic cell maturation.
The glabridin herein is preferably represented by the following formula 1:
<Formula 1>
Figure imgf000011_0001
Wherein, R and Rl are independently H or alkyl
The glabridin derivative is one or more compounds selected from the group consisting of glabrene, Hispaglabridin A, Hispaglabridin B, 4-0-methylglabridin, 2 ' -O-methylglabridin, 2 ' , 4 ' -O-methylglabridin, and 31- hydroxy-4 ' O-methylglabridin, but not always limited thereto and any glabridin derivative known to those in the art can be used. In a preferred embodiment of the present invention, the effect of glabridin alone is described but it is well understood by those in the art that glabridin derivatives have equal effect to glabridin.
Glabridin or glabridin derivatives are preferably extracted from Glycyrrhiza uralensis, but not always limited thereto and any commercial compound (glabridin or its derivatives) can be used.
The preferable content of glabridin or glabridin derivatives in the pharmaceutical composition for suppression of dendritic cell maturation or the inhibitory agent of dendritic cell maturation of the present invention is 0.1-50 weight% by the total weight of the composition, but not always limited thereto.
Glabridin or glabridin derivative can be used as it is or as a form of pharmaceutically acceptable salt. In this invention, any acceptable non-toxic salt can be used without limitation, for example, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrofluoric acid, hydrobromic acid, formic acid, acetic acid, tartaric acid, lactic acid, citric acid, fumaric acid, maleic acid, succinic acid, metalsulfonic acid, benzenesulfonic acid, toluenesulfonic acid, naphthalenesulfonic acid, etc.
The pharmaceutical composition for suppression of dendritic cell maturation of the present invention can be administered alone or together with surgical operation, hormone therapy, chemo-therapy and biological regulators.
The pharmaceutical composition for suppression of dendritic cell maturation or the inhibitory agent of dendritic cell maturation of the present invention can include, in addition to glabridin or glabridin derivatives, one or more effective ingredients having the same or similar function to glabridin or glabridin derivatives. For the pharmaceutically acceptable carriers, saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome and a mixture can be used. If necessary, a general additive such as an antioxidant, buffer and an antimicrobial agent can be additionally added. The pharmaceutical composition for suppression of dendritic cell maturation or the inhibitory agent of dendritic cell maturation of the present invention can be prepared for oral or parenteral administration by mixing with generally used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactant. The composition can further be prepared in suitable forms according to ingredients by following the method represented in Remington' s Pharmaceutical Science (the newest edition), Mack Publishing Company, Easton PA.
The pharmaceutical composition for suppression of dendritic cell maturation or the inhibitory agent of dendritic cell maturation of the present invention can be administered orally or parenterally (for example, intravenous, hypodermic, local or peritoneal injection). The effective dosage of the composition can be determined according to weight, age, gender, health condition, diet, administration frequency, administration method, excretion and severity of a disease. The dosage units can contain, for example, 1, 2, 3 or 4 individual doses or 1/2, 1/3 or 1/4 of an individual dose. An individual dose preferably contains the amount of active compound which is administered in one application and which usually corresponds to a whole, 1/2, 1/3 or 1/4 of a daily dose.
The pharmaceutical composition for suppression of dendritic cell maturation or the inhibitory agent of dendritic cell maturation of the present invention is evaluated to be a safe substance since its estimated LD50 value is much greater than 5 g/kg in rats, which is confirmed by acute toxicity assay with rats tested via oral administration. The effective dosage is 0.5 - 6 nig/kg per day, preferably 3 nig/kg per day, and administration frequency is preferably 1 - 3 times a day. However, the dosage is not limited thereto and can be adjusted according to the conditions of a patient and severity of a disease . The present invention also provides a method for suppressing dendritic cell maturation using glabridin or glabridin derivatives.
The glabridin or glabridin derivatives herein can be originated from Glycyrrhiza uralensis, but any commercial glabridin or glabridin derivatives can be used. The method for suppressing dendritic cell maturation contains the step of oral administration or parenteral administration of glabridin or glabridin derivatives (for example, intravenous, hypodermic, local or peritoneal injection). At this time, the content of glabridin or glabridin derivatives is 0.1-50 weight part by the total weight of the composition to be administered orally or parenterally , but not always limited thereto. The glabridin or glabridin derivative can be used as it is or as a form of pharmaceutically acceptable salt. In this invention, any acceptable non-toxic salt can be used without limitation, for example, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrofluoric acid, hydrobromic acid, formic acid, acetic acid, tartaric acid, lactic acid, citric acid, fumaric acid, maleic acid, succinic acid, metalsulfonic acid, benzenesulfonic acid, toluenesulfonic acid, naphthalenesulfonic acid, etc.
The pharmaceutical composition for suppression of dendritic cell maturation or the inhibitory agent of dendritic cell maturation of the present invention can be administered alone or together with surgical operation, hormone therapy, chemo-therapy and biological regulators.
The present invention also provides a composition for immune regulation comprising glabridin or glabridin derivatives as an active ingredient.
The glabridin or glabridin derivative of the present invention can be effectively used as a composition for immune regulation because it effectively suppresses dendritic cell maturation.
The glabridin herein is preferably represented by the following formula 1:
<Formula 1>
Figure imgf000016_0001
Wherein, R and Rl are independently H or alkyl,
The glabridin derivative is one or more compounds selected from the group consisting of glabrene, Hispaglabridin A, Hispaglabridin B, 4-O-methylglabridin, 2 ' -O-methylglabridin , 2 ' , 4 ' -O-methylglabridin , and 3 ' - hydroxy-4 ' O-methylglabridin, but not always limited thereto and any glabridin derivative known to those in the art can be used. In a preferred embodiment of the present invention, the effect of glabridin alone is described but it is well understood by those in the art that glabridin derivatives have equal effect to glabridin.
Glabridin or glabridin derivatives are preferably extracted from Glycyrrhiza uralensis, but not always limited thereto and any commercial compound (glabridin or its derivatives) can be used.
The preferable content of glabridin or glabridin derivates is 0.1-50 weight part by the total weight of the composition for immune regulation, but not always limited thereto .
The glabridin or glabridin derivative can be used as it is or as a form of pharmaceutically acceptable salt. In this invention, any acceptable non-toxic salt can be used without limitation, for example, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrofluoric acid, hydrobromic acid, formic acid, acetic acid, tartaric acid, lactic acid, citric acid, fumaric acid, maleic acid, succinic acid, metalsulfonic acid, benzenesulfonic acid, toluenesulfonic acid, naphthalenesulfonic acid, etc. The composition for immune regulation can be administered alone or together with surgical operation, hormone therapy, chemo-therapy and biological regulators.
The composition for immune regulation of the present invention can include, in addition to glabridin or glabridin derivatives, one or more effective ingredients having the same or similar function to glabridin or glabridin derivatives . For the pharmaceutically acceptable carriers, saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome and a mixture can be used. If necessary, a general additive such as an antioxidant, buffer and an antimicrobial agent can be additionally added. The pharmaceutical composition for suppression of dendritic cell maturation or the inhibitory agent of dendritic cell maturation of the present invention can be prepared for oral or parenteral administration by mixing with generally used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactant. The composition can further be prepared in suitable forms according to ingredients by following the method represented in Remington's Pharmaceutical Science (the newest edition), Mack Publishing Company, Easton PA. The composition for immune regulation of the present invention can be administered orally or parenterally (for example, intravenous, hypodermic, local or peritoneal injection). The effective dosage of the composition can be determined according to weight, age, gender, health condition, diet, administration frequency, administration method, excretion and severity of a disease. The dosage units can contain, for example, 1, 2, 3 or 4 individual doses or 1/2, 1/3 or 1/4 of an individual dose. An individual dose preferably contains the amount of active compound which is administered in one application and which usually corresponds to a whole, 1/2, 1/3 or 1/4 of a daily dose.
The composition for immune regulation of the present invention is evaluated to be a safe substance since its estimated LD50 value is much greater than 5 g/kg in rats, which is confirmed by acute toxicity assay with rats tested via oral administration. The effective dosage is 0.5 - 6 nig/kg per day, preferably 3 nig/kg per day, and administration frequency is preferably 1 - 3 times a day. However, the dosage is not limited thereto and can be adjusted according to the conditions of a patient and severity of a disease.
The present invention also provides a method for immune regulation using glabridin or glabridin derivatives, The glabridin or glabridin derivatives herein can be originated from Glycyrrhiza uralensis, but any commercial glabridin or glabridin derivatives can be used. The method for immune regulation contains the step of oral administration or parenteral administration of glabridin or glabridin derivatives (for example, intravenous, hypodermic, local or peritoneal injection). At this time, the content of glabridin or glabridin derivatives is 0.1- 50 weight part by the total weight of the composition to be administered orally or parenterally , but not always limited thereto. The glabridin or glabridin derivative can be used as it is or as a form of pharmaceutically acceptable salt. In this invention, any acceptable nontoxic salt can be used without limitation, for example, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrofluoric acid, hydrobromic acid, formic acid, acetic acid, tartaric acid, lactic acid, citric acid, fumaric acid, maleic acid, succinic acid, metalsulfonic acid, benzenesulfonic acid, toluenesulfonic acid, naphthalenesulfonic acid, etc.
The present invention also provides a composition for treatment of autoimmune disease comprising glabridin or glabridin derivatives as an active ingredient. Dendritic cell maturation has been known to be involved in auto-immune disease. Therefore, suppression of dendritic cell maturation can be an effective treatment method of auto-immune disease.
The auto-immune disease herein is exemplified by lupous or Sjogren's syndrome.
The glabridin herein is preferably represented by the following formula 1:
<Formula 1>
Figure imgf000021_0001
Wherein, R and Rl are independently H or alkyl
The glabridin derivative is one or more compounds selected from the group consisting of glabrene, Hispaglabridin A, Hispaglabridin B, 4-0-methylglabridin, 2 ' -O-methylglabridin, 2 ' , 4 ' -O-methylglabridin, and 3'- hydroxy-4 ' O-methylglabridin, but not always limited thereto and any glabridin derivative known to those in the art can be used. In a preferred embodiment of the present invention, the effect of glabridin alone is described but it is well understood by those in the art that glabridin derivatives have equal effect to glabridin.
Glabridin or glabridin derivatives are preferably extracted from Glycyrrhiza uralensis, but not always limited thereto and any commercial compound (glabridin or its derivatives) can be used.
The preferable content of glabridin or glabridin derivates is 0.1-50 weight part by the total weight of the composition for treatment of autoimmune disease, but not always limited thereto.
The glabridin or glabridin derivative can be used as it is or as a form of pharmaceutically acceptable salt. In this invention, any acceptable non-toxic salt can be used without limitation, for example, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrofluoric acid, hydrobromic acid, formic acid, acetic acid, tartaric acid, lactic acid, citric acid, fumaric acid, maleic acid, succinic acid, metalsulfonic acid, benzenesulfonic acid, toluenesulfonic acid, naphthalenesulfonic acid, etc. The composition for treatment of autoimmune disease can be administered alone or together with surgical operation, hormone therapy, chemo-therapy and biological regulators .
The composition for treatment of autoimmune disease of the present invention can include, in addition to glabridin or glabridin derivatives, one or more effective ingredients having the same or similar function to glabridin or glabridin derivatives. For the pharmaceutically acceptable carriers, saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome and a mixture can be used. If necessary, a general additive such as an antioxidant, buffer and an antimicrobial agent can be additionally added. The pharmaceutical composition for suppression of dendritic cell maturation or the inhibitory agent of dendritic cell maturation of the present invention can be prepared for oral or parenteral administration by mixing with generally used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactant. The composition can further be prepared in suitable forms according to ingredients by following the method represented in Remington' s Pharmaceutical Science (the newest edition), Mack Publishing Company, Easton PA. The composition for treatment of autoimmune disease of the present invention can be administered orally or parenterally (for example, intravenous, hypodermic, local or peritoneal injection). The effective dosage of the composition can be determined according to weight, age, gender, health condition, diet, administration frequency, administration method, excretion and severity of a disease.
The dosage units can contain, for example, 1, 2, 3 or 4 individual doses or 1/2, 1/3 or 1/4 of an individual dose.
An individual dose preferably contains the amount of active compound which is administered in one application and which usually corresponds to a whole, 1/2, 1/3 or 1/4 of a daily dose.
The composition for treatment of autoimmune disease of the present invention is evaluated to be a safe substance since its estimated LD50 value is much greater than 5 g/kg in rats, which is confirmed by acute toxicity assay with rats tested via oral administration. The effective dosage is 0.5 - 6 nig/kg per day, preferably 3 mg/ kg per day, and administration frequency is preferably 1 - 3 times a day. However, the dosage is not limited thereto and can be adjusted according to the conditions of a patient and severity of a disease.
The present invention further provides a composition for health food and health beverages for prevention of autoimmune disease comprising glabridin or glabridin derivatives as an active ingredient.
The glabridin or glabridin derivative of the present invention can be used as food additive. In that case, the glabridin or glabridin derivative can be added as it is or as mixed with other food components according to the conventional method. The mixing ratio of active ingredients can be regulated according to the purpose of use. In general, to produce health food or beverages, the glabridin or glabridin derivative is added preferably by 0.1-15 weight part and more preferably by 0.1-10 weight part. However, if long term administration is required for health and hygiene or regulating health condition, the content can be lower than the above but higher content can be accepted as well since the glabridin or glabridin derivative has been proved to be very safe.
The food herein is not limited. For example, the glabridin or glabridin derivative can be added to meats, sausages, breads, chocolates, candies, snacks, cookies, pizza, ramyuns, flour products, gums, dairy products including ice cream, soups, beverages, tea, drinks, alcohol drinks and vitamin complex, etc, and in wide sense, almost every food applicable in the production of health food can be included. The composition for health beverages of the present invention can additionally include various flavors or natural carbohydrates, etc, like other beverages. The natural carbohydrates above can be one of monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and glucose alcohols such as xilytole, sorbitol and erythritol . Besides, natural sweetening agents such as thaumatin and stevia extract, and synthetic sweetening agents such as saccharin and aspartame can be included as a sweetening agent. The content of the natural carbohydrate is preferably 0.01-0.04 g and more preferably 0.02-0.03 g in 100 m-C of the composition.
In addition to the ingredients mentioned above, the glabridin or glabridin derivative of the present invention can include in variety of nutrients, vitamins, minerals, flavors, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acid, protective colloidal viscosifiers , pH regulators, stabilizers, antiseptics, glycerin, alcohols, carbonators which used to be added to soda, etc. The glabridin or glabridin derivative of the present invention can also include natural fruit juice, fruit beverages and/or fruit flesh addable to vegetable beverages . All the mentioned ingredients can be added singly or together. The mixing ratio of those ingredients does not matter in fact, but in general, each can be added by 001-0.1 weight part per 100 weight part of the glabridin or glabridin derivative of the present invention.
In addition, the present invention provides a method for treatment of autoimmune disease containing the step of administering the pharmaceutical composition for suppressing dendritic cell maturation to a subject.
The subject herein can be human or mammals except human and preferably selected from the group consisting of mice, rats, guinea pigs, pigs, rabbits, monkeys and chimpanzees, but not always limited thereto.
The composition can be administered orally or parenterally (for example, intravenous, hypodermic, local or peritoneal injection). The effective dosage of the composition can be determined according to weight, age, gender, health condition, diet, administration frequency, administration method, excretion and severity of a disease. The dosage units can contain, for example, 1, 2, 3 or 4 individual doses or 1/2, 1/3 or 1/4 of an individual dose. An individual dose preferably contains the amount of active compound which is administered in one application and which usually corresponds to a whole, 1/2, 1/3 or 1/4 of a daily dose. The auto-immune disease herein is exemplified by lupous or Sjogren's syndrome.
[Advantageous Effect]
The present invention facilitates the preparation of a pharmaceutical composition for suppressing dendritic cell maturation, a composition for immune regulation and a composition for treating auto-immune disease comprising glabridin or glabridin derivatives as an active ingredient. The pharmaceutical composition of the present invention can be effectively used for prevention and treatment of auto-immune disease including lupous and Sjogren's syndrome •
[Description of Drawings] The application of the preferred embodiments of the present invention is best understood with reference to the accompanying drawings, wherein:
Figure 1 is a set of graphs illustrating the inhibitory effect of glabridin on phenotypic changes of dendritic cells caused by the treatment of lipopolysaccharide .
Figure 2 is a set of graphs illustrating the inhibitory effect of glabridin on phagocytic activity of dendritic cells induced by the treatment of lipopolysaccharide .
Figure 3 is a graph illustrating the inhibitory effect of glabridin on IL-12 secretion of dendritic cells induced by the treatment of lipopolysaccharide. Figure 4 is a graph illustrating the inhibitory effect of glabridin on T-lymphocyte activation activity of dendritic cells induced by the treatment of lipopolysaccharide .
Figure 5 is a graph illustrating the inhibitory effect of glabridin on IFN-γ expression in dendritic cells induced by the treatment of lipopolysaccharide.
Figure 6 is a graph illustrating the inhibitory effect of glabridin on IL-4 expression in dendritic cells induced by the treatment of lipopolysaccharide.
[Mode for Invention]
Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples. However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.
Example 1: Effect of glabridin on dendritic cell maturation induced by lipopolysaccharide
<1-1> Preparation of experiment animal
C57BL/6 female mice having the weight of 18-22 g
(Dae Han Biolink Co., Ltd., Chungcheongbuk-do, Korea) were used as test animals. The mice were adapted in an animal lab with providing feeds and water freely under required conditions (temperature: 21±2°Q light/dark: 12 hour light/dark cycle) for 7 days.
<l-2> Preparation of dendritic cell
Bone marrow cells were separated from C57BL/6 mice (Orient Bio Inc., Seongnam-si, Korea), followed by culture at the density of 1x106 cells/ml. GM-CSF was added thereto at the concentration of 2 ng/ml, followed by further culture for 8 days to obtain immature dendritic cells .
<l-3> Effect of glabridin on the expression of cell surface factors of dendritic cells Glabridin (Wako Pure Chemicals, Osaka, Japan) was added at the concentration of 5-20 g/ml to the immature dendritic cells obtained in Example <l-2>, and then lipopolysaccharide (Sigma-Aldrich, St. Louis, USA) was added thereto at the concentration of 1 g/ml, followed by culture for 48 hours. Upon completion of the culture, the cells were recovered, which were treated with antibodies (BD Biosciences, San Jose, USA) such as FITC (Fluorescein Isothiocyanate) -CDlIc, PE (R-Phycoerythrin)-CDδO , PE-CD86, and PE-MHC II, followed by analysis using a flow cytometer, The results were presented by MFI (Mean Fluorescence Intensity). As MFI value increases, the expression of a surface factor increases and so does dendritic cell maturation. CDlIc is a surface factor selectively expressed in dendritic cells, and CD80, CD86 and MHC-II are surface factors whose expressions are increased as dendritic cells are maturated.
As a result, the expression level of MHC-II was 28.86 in the control and 42.55 in the group treated with lipopolysaccharide but the expression was decreased in the experimental group co-treated with lipopolysaccharide and glabridin dose-dependently to 43.48, 40.48, 35.07 and 29.77. The expression level of CD80 was 11.53 in the control but increased to 22.64 in the group treated with lipopolysaccharide but decreased in the experimental group co-treated with lipopolysaccharide and glabridin dose- dependently to 17.38, 14.60, 11.59 and 6.45. The expression level of CD86 was 7.59 in the control but increased to 43.32 in the group treated with lipopolysaccharide but decreased in the experimental group co-treated with lipopolysaccharide and glabridin dose- dependently to40.15, 39.67, 13.75, and 6.83 (Figure 1). The above results indicate that when glabridin is treated to the dendritic cells maturated by lipopolysaccharide, the dendritic cell maturation is suppressed glabridin dose-dependently. <l-4> Effect of glabridin on phagocytic activity of dendritic cells
Immature dendritic cells have excellent phagocytic activity but once they are matured, the phagocytic activity is reduced. As shown in the result of Example <l-3>, as glabridin suppressed the dendritic cell maturation induced by lipopolysaccharide, phagocytic activity would be less reduced by the treatment of lipopolysaccharide. So, the present inventors performed following experiments to prove the above.
Glabridin was added at the concentration of 5-20 g/ml to the immature dendritic cells obtained in Example <l-2>, and then lipopolysaccharide was added thereto at the concentration of 1 g/ml, followed by culture for 48 hours. Upon completion of the culture, the cells were recovered, which were treated with FITC-dextran ( Sigma- Aldrich, St. Louis, USA) at the concentration of 0.7 mg/ml for 2 hours. , followed by analysis using a flow cytometer, The cells were washed to eliminate remaining FITC-dextran. Flow cytometry was performed to measure the amount of antigen introduced into the dendritic cells. Double staining was performed using PE-CDlIc selectively binding to dendritic cells, which was to analyze antigen capturing rate after separation of the dendritic cells selectively. As a result, after 2 hour culture at 37 °Q antigen capturing rate of the immature dendritic cells (control group) was 26.91% and antigen capturing rate of the dendritic cells treated with lipopolysaccharide was 11.48%. In the meantime, antigen capturing rate of the dendritic cells co-treated with lipopolysaccharide and glabridin was reduced dose-dependently to 12.05, 13.78, 15.09 and 17.94 (Figure 2). The above results indicate that the dendritic cell maturation induced by lipopolysaccharide can be suppressed by glabridin. When the cells were cultured at 4 "Q antigen capturing of the cells was completely stopped and antigen capturing rate of the cells was as low as 4.94-7.59%. This indicates that the antigen capturing is an immune response undergoing actively.
<l-5> Effect of glabridin on the cytokine generation by dendritic cells
One of the most important functions of mature dendritic cells is to induce cytokine secretion. In particular, IL-12 secretion plays an important role in T- lymphocyte activation by dendritic cells. Thus, the immature dendritic cells were treated with lipopolysaccharide and glabridin to investigate cytokine secretion . Glabridin was added at the concentration of 5-20 g/ml to the immature dendritic cells obtained in Example <l-2>, and then lipopolysaccharide was added thereto at the concentration of 1 g/ml, followed by culture for 48 hours. Upon completion of the culture, the culture solution was obtained and cytokine secreted from the dendritic cells was measured by enzyme-linked immunoassay. The measurement was performed by using Mouse IL- 12 p70 DuoSet, Mouse IFN- DuoSet and Mouse IL-4 DuoSet (R&D Systems, Minneapolis, USA) according to the manufacturer's instructions .
As a result, IL-12 was hardly secreted in the control but IL-12 secretion was strongly increased in the group treated with lipopolysaccharide. In the meantime, in the group co-treated with both lipopolysaccharide and glabridin, IL-12 secretion was suppressed glabridin dose- dependently (Figure 3). The above results indicate that dendritic cell maturation is suppressed by glabridin. IL- 12 secretion in the group treated with glabridin alone was similar to that in the control.
Example 2: Effect of glabridin on T-lymphocyte activation activity of dendritic cells induced by lipopolysaccharide <2-l> Effect of glabridin on T-lymphocyte activation activity of dendritic cells The most important immune related function of mature dendritic cells is to activate T-lymphocytes . Immature dendritic cells capture an antigen, followed by maturation. Then, they transfer to immune system containing T-cells to induce activation of T-cells. The activated T-cells begin proliferate actively and secrete cytokines. So, the present inventors treated immature dendritic cells with lipopblysaccharide and glabridin to measure the activation of T-lymphocytes .
Glabridin was added at the concentration of 20 g/ml to the immature dendritic cells obtained in Example <l-2>, and then lipopolysaccharide was added thereto at the concentration of 1 g/ml, followed by culture for 48 hours. Upon completion of the culture, the cells were recovered and cultured with allogeneic T cells for 3 days. For the last 16 hours of culture, the cells were treated with [ 3H] -thymidine to introduce radio-isotope into T-cell DNA. Then, cell proliferation was measured.
As a result, strong proliferation of T-lymphocytes was observed in the dendritic cells treated with lipopolysaccharide. But, in the dendritic cells co- treated with lipopolysaccharide and glabridin, T- lymphocyte proliferation was suppressed (Figure 4A). T- lymphocyte proliferation in the group treated with glabridin alone was similar to that in the control. The present inventors also investigated the levels of IFN-γ and IL-4, cytokines secreted in T cells.
As a result, cytokine secretion was significantly increased in T-lymphocytes co-cultured with the dendritic cells treated with lipopolysaccharide, while cytokine secretion (IFN-γ, Figure 5 and IL-4, Figure 6) in T- lymphocytes co-cultured with the dendritic cells treated with both lipopolysaccharide and glabridin was suppressed.
The above results indicate that glabridin can suppress dendritic cell maturation induced by lipopolysaccharide and T-lymphocyte activation as well.
Example 3 : Acute toxicity of glabridin in rats tested via oral administration
The following experiments were performed to see weather glabridin had acute toxicity in rats. 6-week old SPF SD line rats were used in the tests for acute toxicity. Glabridin was suspended in 0.5% methylcellulose solution and orally administered once to rats at the dosage of 5 g/kg/15
Figure imgf000036_0001
Death, clinical symptoms, and weight change in rats were observed, hematological tests and biochemical tests of blood were performed, and any abnormal signs in the gastrointestinal organs of chest and abdomen were checked with the naked eye during autopsy.
The results showed that the test compounds did not cause any specific clinical symptoms, weight change, or death in rats . No change was observed in hematological tests, biochemical tests of blood, and autopsy.
Therefore, glabridin used in this experiment is evaluated to be a safe substance since it does not cause any toxic change in rats up to the level of 5 g/kg and its estimated LD50 value is much greater than 5 g/kg in rats.
The Manufacturing Examples of the composition for the present invention are described hereinafter.
Manufacturing Example 1 : Preparation of pharmaceutical formulations
<1-1> Preparation of powders
Glabridin 2 g Lactose 1 g
Powders were prepared by mixing all the above components, which were filled in airtight packs according to the conventional method for preparing powders .
<l-2> preparation of tablets
Glabridin 100 nig
Corn starch 100 mg
Lactose 100 mg
Magnesium stearate 2 mg Tablets were prepared by mixing all the above components by the conventional method for preparing tablets .
<l-3> Preparation of capsules Glabridin 100 mg
Corn starch 100 nig
Lactose 100 mg
Magnesium stearate 2 nig
Capsules were prepared by mixing all the above components, which were filled in gelatin capsules according to the conventional method for preparing capsules .
<l-4> Preparation of pills 4-O-methyl glabridin 1 g
Lactose 1.5 g
Glycerin 1 g
Xylitol 0.5 g Pills were prepared by mixing all the above components according to the conventional method for preparing pills. Each pill contained 4 g of the mixture.
<l-5> Preparation of granules
Hispaglabridin B 150 mg Soybean extract 50 mg Glucose 200 rag
Starch 600 rag
All the above components were mixed, to which 100 rag of 30% ethanol was added. The mixture was dried at 60°C and the prepared granules were filled in packs.
Manufacturing Example 2 : Preparation of foods
Foods containing the glabridin of the present invention were prepared as follows .
<2-l> Preparation of spices for cooking
Health enhancing spices for cooking was prepared with 20 ~ 95 weight part of the glabridin of the present invention according to the conventional method.
<2-2> Preparation of tomato ketchup and sauce
Health enhancing tomato ketchup or sauce was prepared by mixing 0.2 ~ 1.0 weight part of the glabridin of the present invention with tomato ketchup or sauce according to the conventional method.
<2-3> Preparation of flour food
0.5 ~ 5.0 weight part of the glabridin of the present invention was added to the flour. Health enhancing foods such as bread, cake, cookies, crackers and noodles were prepared with the flour mixture according to the conventional method.
<2-4> Preparation of soups and gravies 0.1 - 5.0 weight part of the glabridin of the present invention was added to soups and gravies . Health enhancing meat products, soups and gravies were prepared with this mixture by the conventional method.
<2-5> Preparation of ground beef
Health enhancing ground beef was prepared by mixing 10 weight part of the glabridin of the present invention with ground beef according to the conventional method.
<2-6> Preparation of dairy products
5 ~ 10 weight part of the glabridin of the present invention was added to milk. Health enhancing dairy products such as butter and ice cream were prepared with the milk mixture according to the conventional method.
<2-7> Preparation of Sun-Sik
Brown rice, barley, glutinous rice and Yulmu (Job's tears) were gelatinized according to the conventional method, dried and pulverized to obtain 60-mesh powders. Black soybean, black sesame and wild sesame were steamed and dried according to the conventional method and pulverized to obtain 60-mesh powders.
Sun-Sik was prepared by mixing the dry powders of the grains, seeds and glabridin according to the below ratio.
Grains (brown rice: 30 weight part, Yulmu: 15 weight part, barley: 20 weight part),
Seeds (wild sesame: 7 weight part, black soybean: 8 weight part, black sesame: 7 weight part), Glabridin (3 weight part),
Ganoderma lucidum ( 0.5 weight part),
Rehmannia glutinosa (0.5 weight part)
Manufacturing Example 3 : Preparation of beverages <3-l> Preparation of health beverages
The glabridin of the present invention was mixed with liquid fructose (0.5%), oligosaccharide (2%), sugar (2%), salt (0.5%), and water (75%). After mixing completely, the mixture was sterilized instantly and filled small containers such as glass bottles, pet bottles, etc, to prepare health beverages.
<3-2> Preparation of vegetable juice
Health enhancing vegetable juice was prepared by adding 5 g of the glabridin of the present invention to 1,000
Figure imgf000042_0001
of tomato or carrot juice according to the conventional method.
<3-3> Preparation of fruit juice Health enhancing fruit juice was prepared by adding 1 g of the glabridin of the present invention to 1,000 ml of apple or grape juice according to the conventional method.
[industrial Applicability]
The present invention is effective in the preparation of a pharmaceutical composition for suppressing dendritic cell maturation, a composition for immune regulation and a composition for treating auto- immune disease comprising glabridin or glabridin derivatives as an active ingredient. The said pharmaceutical compositions can be effectively used for the treatment of auto-immune disease.
Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.

Claims

[CLAIMS]
[Claim l]
A composition for suppressing dendritic cell maturation comprising glabridin or glabridin derivatives as an active ingredient.
[Claim 2]
The composition according to claim 1, wherein the glabridin is represented by the following formula 1 : <Formula 1>
Figure imgf000044_0001
Wherein, R and Rl are independently H or alkyl
[Claim 3] The composition according to claim 1, wherein the glabridin derivative is one or more compounds selected from the group consisting of glabrene, Hispaglabridin A, Hispaglabridin B, 4-0-methylglabridin, 2 ' -0- methylglabridin, 2 ' , 4 ' -O-methylglabridin, and 3 ' -hydroxy- 4 ' O-methylglabridin .
[Claim 4]
The composition according to claim 1, wherein the glabridin or glabridin derivatives are extracted from Glycyrrhiza uralensis.
[Claim 5]
The composition according to claim 1, wherein the content of the active ingredient is 0.1-50 weight part by the total weight of the composition.
[Claim 6l
The composition according to claim 1, wherein the composition additionally contains a pharmaceutically acceptable carrier, an excipient or a diluent.
[Claim 7]
A method for suppressing immature dendritic cell maturation containing the step of contacting glabridin or glabridin derivatives to dendritic cells.
[Claim 8]
An inhibitory agent of dendritic cell maturation comprising glabridin or glabridin derivatives as an active ingredient .
[Claim 9]
A use of glabridin or glabridin derivatives for immune regulation or for the production of a therapeutic agent for auto-immune disease.
[Claim lθ]
A composition for immune regulation comprising glabridin or glabridin derivatives as an active ingredient.
[Claim ll]
A method for immune regulation containing the step of administering glabridin or glabridin derivatives to a subject.
[Claim 12]
A composition for treating auto-immune disease comprising glabridin or glabridin derivatives as an active ingredient.
[Claim 13]
The composition for treating auto-immune disease according to claim 12, wherein the auto-immune disease is lupous or Sjogren's syndrome. [Claim 14]
A method for treating autoimmune disease containing the step of administering glabridin or glabridin derivatives to a subject.
[Claim 15]
The method for treating auto-immune disease according to claim 14, wherein the subject is human or any other mammals .
[Claim 16]
The method for treating auto-immune disease according to claim 14, wherein the auto-immune disease is lupous or Sjogren's syndrome.
[Claim 17]
A health food for preventing auto-immune disease comprising glabridin or glabridin derivatives as an active ingredient.
[Claim 18]
A composition for health beverages for preventing auto-immune disease comprising glabridin or glabridin derivatives as an active ingredient.
PCT/KR2008/005774 2007-11-26 2008-10-01 A pharmaceutical composition comprising glabridin or glabridin derivatives for suppression of dendritic cell maturation WO2009069887A1 (en)

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