WO2010090407A2 - A composition comprising an extract of puerariae radix showing immune enhancing activity and the use thereby - Google Patents

A composition comprising an extract of puerariae radix showing immune enhancing activity and the use thereby Download PDF

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WO2010090407A2
WO2010090407A2 PCT/KR2010/000434 KR2010000434W WO2010090407A2 WO 2010090407 A2 WO2010090407 A2 WO 2010090407A2 KR 2010000434 W KR2010000434 W KR 2010000434W WO 2010090407 A2 WO2010090407 A2 WO 2010090407A2
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extract
composition
immune
puerariae radix
immunodeficiency disease
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PCT/KR2010/000434
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English (en)
French (fr)
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WO2010090407A3 (en
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Hyun Park
Sung Yeon Kim
Youn-Chul Kim
Joo Young Lee
Bum Seok Kim
Ok Jin Kim
Young Gab Yun
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Wonkwang University Center For Industry-Academy Cooperation
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    • 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
    • A61K36/488—Pueraria (kudzu)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders

Definitions

  • the present invention relates to a composition
  • a composition comprising an extract of Puerariae radix showing potent immune-enhancing activity for treating, preventing and alleviating immunodeficiency disease and the use thereby.
  • the immune system can be classified into surface barrier, non-specific immune system and specific immune system.
  • the surface barrier (1 st layer barrier) protect organisms from infection, including mechanical, chemical and biological barriers regardless of the sort of in fection and non-specific immune system (innate immune system, secondary barrier) is immediate and non-specific response consisting of humoral barrier such as complement system, chemical barrier, and cellular barrier such as phagocytosis including the action of leucocyte, macrophage and neutrophils etc, which get rid of the entered pathogens through the surface barrier.
  • the specific immune system includes the action of lymphocytes such as B cells and T cells.
  • the system is antigen-specific therefore it allows to mount faster and stronger at tacks each time this pathogen is encountered by dint of immunological memory which can distinguish self and non-self molecules (RichardA. goldsby et al., KUBY Immunology, 2000)
  • Leukocytes are in charge of secondary or 3 rd barriers from the entered pathogens and the control of action of macrophage and lymphocyte plays principle roles in determining the therapeutic efficacy of drugs.
  • Macrophages are versatile cells that reside within tissues and produce a wide array of chemicals including enzymes, complement proteins, regulatory factors such as interleukin 1, and various cytokines and NO which play important role in immune system.
  • LPS Lipopolysaccharide
  • cytokine TNF- ⁇
  • T-cell a leukocyte cells, is differentiated from thymus and has T-cell antigen receptor (TCR).
  • T-CeIIs are divided into two T-cell, i.e., helper T-cells (CD4+ T H cell) are a sub-group of T cells that kill cells infected with viruses and other pathogens, which are activated by recognizing antigens coupled to MHC II class molecules and classified into two sub groups, i.e., T H 1 cell which involves in cellular immune response, activates cell toxicity and inflammatory response and excretes IL-I, IFN- ⁇ etc; and T H 2 cell which excretes IL-4, IL-5, IL-6, IL-9, IL-10, IL-13 etc and produces cytokines stimulating antibody production, especially, IgE production.
  • helper T-cells CD4+ T H cell
  • T H 1 cell which involves in cellular immune response, activates cell toxicity and inflammatory response and excretes IL-I, IFN- ⁇ etc
  • T H 2 cell which excretes IL-4, IL-5, IL-6, IL-9, IL-10
  • T H 1 cell cytokine and T H 2 cell cytokine has been reported to have mutually regulating functions and anti-IL-4 antibody and anti-IFN- ⁇ antibody may change the progression of animals 'infectious disease.
  • IFN- ⁇ injection has been found to improve the syndrome of patients suffering from rheumatic arthritis (Richard A. Goldsby et al., KUBY Immunology, 2000).
  • NF-KB and IRF3 are representative transcription factors which are activated by TLR-4 (Youn et al., Biochem. Pharmacol., Jan. 15:75(2). pp.494-502, 2008).
  • TLR-4 activation gives rise to the clustering of adaptor molecules in the cell, which activates downward signaling molecules and then stimulates the production of immune-mediators.
  • MyD88 and TRIF the main adaptor molecules of TLR-4, specifically activate MyD88-dependent and MyD88-independent downward signaling molecules respectively and the downward signaling molecules have specific kinase and transcription factor to each pathway resulting in inducing the expression of specific targeting genes (Akira S et al., Nat. Rev. Immunol., 4(7], pp.499-511, 2004).
  • Puerariae radix a root of Pueraria lobata (Willd) Ohwi belonging to Leguminosae family, has been known to contain puerarin, daidzein, daidzin, soyasaponin, starch and etc.
  • the present inventors have studied to determine the immune-modulating activity of Puerariae radix, through various experiments, i.e., increasing effect on the release of immune-modulating factors such as NO and TNF- ⁇ ; the stimulating effect on spleen cell proliferation; increasing effect on the production of cytokines such as IFN- ⁇ and TNF- ⁇ and TLR-4 activation in immune cell as well as immune-modulating effect on rotavirus-induced enteritis, rotavirus-specific immune response, rotavirus- infected polyclonal antibody stimulation and immune receptor expression using by C57BL/6 newborn mice together with preventive test on bacterial infection symptom effect using by ICR mice, and finally, confirmed the inventive extract shows potent immune-modulating activity, therefore, it can be useful as a medicament, health functional food and feed composition for treating, preventing and alleviating human and animal disease.
  • the present invention provides a composition comprising an extract of Puerariae radix showing potent immune- enhancing activity for treating, preventing and alleviating immunodeficiency disease and the use thereby.
  • composition comprises a pharmaceutical composition, health care food, veterinary formulation, feed composition and feed additive according to the subject suffering from immunodeficiency disease.
  • immunodeficiency disease comprises the immune-deficiency syndrome caused by the chemo-therapy, radio-therapy to treat various cancer diseases; the immune-deficiency syndrome caused by bone-marrow transplantation; the AIDS caused by impaired immune system; and cancer disease caused by immune deficiency etc.
  • a pharmaceutical composition comprising an extract of Puerariae radix showing potent immune-enhancing activity, as an active ingredient in an amount effective to treat, prevent and alleviate immunodeficiency disease, together with a pharmaceutically acceptable carrier.
  • the crude drug which can be used in the present invention, include the same genus plants which would be apparent to those skilled in the art and have be used for identical or similar purpose and can be substituted for the prevention and treatment of the diseases.
  • composition of the present invention is used in the form of pulverized form thereof, extracted form therefrom or dried extract form thereof.
  • the above extract from the above-described mentioned crude drug can be obtained by extracting the crude drug with distilled water, lower alcohols such as methanol, ethanol and the like, or the mixtures thereof, preferably, water, ethanol and the mixture thereof, more preferably, water.
  • the pharmaceutical composition for treating the purposed diseases could contain about 0.01 to 95 w/w%, preferably 0.5 to 80 w/w% of the above composition of present invention based on the total weight of the composition.
  • An inventive composition may be prepared in accordance with the following preferred embodiment.
  • the present invention also provide a method for preparing the inventive extract comprising the steps of; mixing dried Puerariae radix with 1 to 20-fold, preferably, 10 to 15-fold volume of distilled water, alcohols such as methanol, ethanol and the like, or the mixtures thereof, preferably, distilled water, ethanol or the mixture thereof, more preferably, water at 1 st step; extracting the solution with the extraction method by the extraction with hot water, cold water, reflux extraction, or ultra- sonication extraction, preferably, hot water extraction at the temperature ranging from 7O 0 C-IOO 0 C, preferably, 85°C ⁇ 95°C, for the period ranging from 1 to 24 hours, preferably, 2 to 5 hours at 2 nd step; repeating the above-described extraction process 1 to ten times, preferably, 2 to 5 times to collect the filtrate with filtration, concentrating at the temperature, and drying to obtain dried inventive extract of present invention.
  • alcohols such as methanol, ethanol and the like, or the mixtures thereof
  • It is still another object of the present invention to provide a pharmaceutical composition comprising the crude drug extract obtained by the above described process as an active ingredient for preventing and treating immunodeficiency disease.
  • the inventive composition of the present invention have increasing effect on the release of immune-modulating factors such as NO and TNF- ⁇ ; the stimulating effect on spleen cell proliferation; increasing effect on the production of cytokines such as IFN- ⁇ and TNF- ⁇ and TLR-4 activation in immune cell as well as immune-modulating effect on rotavirus-induced enteritis, rotavirus-specific immune response, rotavirus- infected polyclonal antibody stimulation and immune receptor expression using by C57BL/6 newborn mice together with the preventive test on bacterial infection symptom effect using by ICR mice, therefore, it can be useful as a medicament, health functional food and feed composition for treating, preventing and alleviating human and animal immunodeficiency disease.
  • immune-modulating factors such as NO and TNF- ⁇
  • TNF- ⁇ and TLR-4 activation in immune cell as well as immune-modulating effect on rotavirus-induced enteritis, rotavirus-specific immune response, rotavirus- infected polyclonal antibody stimulation and immune receptor expression using
  • composition for treating purposed diseases could contain about
  • inventive composition may additionally comprise conventional carrier, adjuvants or diluents in accordance with a using method. It is preferable that said carrier is used as appropriate substance according to the usage and application method, but it is not limited. Appropriate diluents are listed in the written text of Remington's Pharmaceutical Science (Mack Publishing co, Easton PA).
  • the crude drug composition according to the present invention can be provided as a pharmaceutical composition containing pharmaceutically acceptable carriers, adjuvants or diluents, e.g., lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starches, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil.
  • pharmaceutically acceptable carriers e.g., lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starches, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, poly
  • the formulations may additionally include fillers, anti-agglutinating agents, lubricating agents, wetting agents, flavoring agents, emulsifiers, preservatives and the like.
  • the compositions of the invention may be formulated so as to provide quick, sustained or delayed release of the active ingredient after their administration to a patient by employing any of the procedures well known in the art.
  • compositions of the present invention can be dissolved in oils, propylene glycol or other solvents which are commonly used to produce an injection.
  • suitable examples of the carriers include physiological saline, polyethylene glycol, ethanol, vegetable oils, isopropyl myristate, etc., but are not limited to them.
  • the compounds of the present invention can be formulated in the form of ointments and creams.
  • compositions containing crude drug composition may be prepared in any form, such as oral dosage form (powder, tablet, capsule, soft capsule, aqueous medicine, syrup, elixirs pill, powder, sachet, granule), or topical preparation (cream, ointment, lotion, gel, balm, patch, paste, spray solution, aerosol and the like), suppository, or sterile injectable preparation (solution, suspension, emulsion).
  • the crude drug composition of the present invention in pharmaceutical dosage forms may be used in the form of their pharmaceutically acceptable salts, and also may be used alone or in appropriate association, as well as in combination with other pharmaceutically active compounds.
  • the desirable dose of the inventive composition varies depending on the condition and the weight of the subject, severity, drug form, route and period of administration, and may be chosen by those skilled in the art. However, in order to obtain desirable effects, it is generally recommended to administer at the amount ranging 0.01-10 g/kg, preferably, 1 to 5 g/kg by weight/day of the inventive composition of the present invention.
  • the dose may be administered in a single or multiple doses per day.
  • the crude drug composition should be present between 0.01 to 80% by weight, preferably 0.5 to 50% by weight based on the total weight of the composition.
  • composition of present invention can be administered to a subject animal such as mammals (rat, mouse, domestic animals or human) via various routes. All modes of administration are contemplated, for example, administration can be made orally, rectally or by intravenous, intramuscular, subcutaneous, intracutaneous, intrathecal, epidural or intracerebroventricular injection.
  • a health functional food comprising an extract of Puerariae radix showing potent immune-enhancing activity for the prevention or improvement of immunodeficiency disease as an active ingredient in an amount effective to prevent and improve the disease, together with a sitologically acceptable additive.
  • the crude drug composition of inventive health functional food is used in the form of pulverized form thereof, extracted form therefrom or dried extract form thereof.
  • the health functional food composition for preventing and improving purposed diseases could contain about 0.01 to 95 w/w%, preferably 0.5 to 80 w/w% of the above crude drug composition of present invention based on the total weight of the composition.
  • the crude drug composition therein can be added to food, additive or beverage for prevention and improvement of purposed diseases.
  • the amount of above described crude drug composition in food or beverage may generally range from about 0.1 to 15 w/w %, preferably 1 to 10 w/w % of total weight of food for the health food composition and 1 to 30 g, preferably 3 to 10 g on the ratio of 100 m# of the health beverage composition.
  • the health beverage composition of present invention contains above described crude drug composition as an essential component in the indicated ratio
  • the other component can be various deodorant or natural carbohydrate etc such as conventional beverage.
  • natural carbohydrate are monosaccharide such as glucose, fructose etc; disaccharide such as maltose, sucrose etc; conventional sugar such as dextrin, cyclodextrin; and sugar alcohol such as xylitol, and erythritol etc.
  • natural deodorant such as taumatin, stevia extract such as levaudioside A, glycyrrhizin et al., and synthetic deodorant such as saccharin, aspartam et al.
  • the amount of above described natural carbohydrate is generally ranges from about 1 to 20 g, preferably 5 to 12 g in the ratio of 100 m# of present beverage composition.
  • the other components than aforementioned composition are various nutrients, a vitamin, a mineral or an electrolyte, synthetic flavoring agent, a coloring agent and improving agent in case of cheese chocolate et al., pectic acid and the salt thereof, alginic acid and the salt thereof, organic acid, protective colloidal adhesive, pH controlling agent, stabilizer, a preservative, glycerin, alcohol, carbonizing agent used in carbonate beverage et al.
  • the other component than aforementioned ones may be fruit juice for preparing natural fruit juice, fruit juice beverage and vegetable beverage, wherein the component can be used independently or in combination.
  • the ratio of the components is not so important but is generally range from about 0 to 20 w/w% per 100 w/w% present composition.
  • Examples of addable food comprising aforementioned crude drug composition therein are various food, beverage, gum, vitamin complex, health improving food and the like.
  • the present invention also provides veterinary formulations comprising an extract of
  • Puerariae radix showing potent immune-enhancing activity as an active ingredient for preventing and alleviating immunodeficiency disease.
  • the feed composition or veterinary formulation of the present invention further may comprise additional additive well-known in art, for example, ⁇ -glucan, vitamin E, yeast culture or L-carnitine and the like.
  • composition of the present invention has no toxicity and adverse effect therefore can be used with safe.
  • Veterinary formulations containing present composition may be prepared in any form, such as oral dosage form (powder, tablet, capsule, soft capsule, aqueous medicine, syrup, elixirs pill, powder, sachet, granule), or topical preparation (cream, ointment, lotion, gel, balm, patch, paste, spray solution, aerosol and the like), or injectable preparation (solution, suspension, emulsion).
  • oral dosage form powder, capsule, soft capsule, aqueous medicine, syrup, elixirs pill, powder, sachet, granule
  • topical preparation cream, ointment, lotion, gel, balm, patch, paste, spray solution, aerosol and the like
  • injectable preparation solution, suspension, emulsion
  • composition according to the present invention can be provided as a veterinary composition containing pharmaceutically acceptable carriers, adjuvants or diluents, e.g., lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starches, acacia rubber, alginate, gelatin, pyrrolidone, water, methylhydroxy benzoate propylhydroxy benzoate, talc, magnesium stearate and mineral oil.
  • pharmaceutically acceptable carriers e.g., lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starches, acacia rubber, alginate, gelatin, pyrrolidone, water, methylhydroxy benzoate propylhydroxy benzoate, talc, magnesium stearate and mineral oil.
  • the desirable dose of the inventive composition varies depending on the condition and the weight of the subject, severity, drug form, route and the period of administration, and may be chosen by those skilled in art. However, in order to obtain desirable effects, it is generally recommended to administer at the amount ranging from 0.01 mg/kg to 10 g/kg per day, more preferably 1 mg/kg to 1 g/kg of the inventive extract of the present invention. The dose may be administered in single of divided into several times per day.
  • the previous invention can be injected to mammals such as rodents, livestock etc, through variable administrations.
  • the administration can be by any of procedures well known in art, for example, oral, rectal, intravenous, intramuscular injections and the like.
  • a feed composition comprising a dried powder of Puerariae radix or the extract thereof showing potent immune-enhancing activity as an active ingredient for preventing and alleviating immunodeficiency disease.
  • the present invention provides a feed additive comprising an extract of Puerariae radix showing potent immune-enhancing activityas an active main ingredient.
  • inventive feed composition can be formulated into the form of 20-90% high- concentrated liquid, powder or granule in the present invention.
  • At least one of the other ingredients selected from organic acid such as citric acid, fumaric acid, adipic acid, lactic acid, malic acid and the like; phosphate salt such as sodium phosphate, potassium phosphate, acid pyrophosphate, polyphosphate and the like; and natural anti-oxidants such as polyphenol, catechin, alpha-tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, phytic acid and the like may further be added to the active ingredient of the present invention.
  • organic acid such as citric acid, fumaric acid, adipic acid, lactic acid, malic acid and the like
  • phosphate salt such as sodium phosphate, potassium phosphate, acid pyrophosphate, polyphosphate and the like
  • natural anti-oxidants such as polyphenol, catechin, alpha-tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, phy
  • inventive feed composition, feed additive and veterinary composition of the present invention could be prepared by the procedure consisting of the steps of: mixing inventive extract of present invention with the combination of various aid component such as amino acid, inorganic salt, vitamin, antibiotic, antibacterial agent, anti-oxidant, anti-fungal agent, live microbial preparation and the like; and grain such as macerated or pulverized wheat, oat, barley, corn or rice; vegetable protein feed such as bean or sunflower seed; animal protein feed such as blood powder, meat powder, bone powder or fish powder; sugar powder or milk product such as various powdered milk or powdered whey and the like together; heating to obtain remaining fluid component, for example, lipid component such as fluidized animal fat, vegetable lipid and the like; mixing the lipid component as a main component with other component such as nutrient supplement, digestive improving agent, growth stimulator, disease preventing agent and the like to prepare purposed inventive composition.
  • various aid component such as amino acid, inorganic salt, vitamin, antibiotic, antibacterial agent, anti-oxidant, anti-fung
  • the above-described animal feed composition can be administrated into animal as a sole or the combinations with other feed additive.
  • inventive extract of the present invention can be administrated into animals through top dressing method, mixing method directly with animal feed or other administration route such as injection, subcutaneous injection or other route with an interval of once a day or several times a day well known in art.
  • the extract may be combined with pharmaceutically acceptable non-toxic edible carrier to make imprompt releasing or sustained releasing preparation.
  • pharmaceutically acceptable non-toxic edible carrier for example, corn starch, lactose, sucrose, bean flake, bean oil, olive oil, sesame oil and propylene glycol may be used as edible carrier of the present invention.
  • solid carrier the dosage form may contain the other aid component such as preservative, stabilizer, humidifier, emulsifier and solubilizer.
  • the animal feed additive of the present invention can be added to animal feed for use as an appetizer, for example, conventionally available optional protein-comprising organic grain such as corn powder, bean powder or the mix therewith.
  • the above-described feed additive can be prepared by any mixing means known to one skilled in the art such as mechanical blending, extrusion, palletizing, and spray drying.
  • the animal feed or feed additive of the present invention can be applied to various animals including mammals, poultry and fish, preferably, commercially important mammals, for example, pig, cow, sheep, goat, experimental rodents such as rat, mouse, hamster, or gerbil mouse, fur animal such as mink or fox, animal in zoological gardens such as monkey, livestock such as cat or dog, poultry such as chicken, turkey, duck, goose, quail and the like and raising fish such as trout and the like.
  • mammals including mammals, poultry and fish, preferably, commercially important mammals, for example, pig, cow, sheep, goat, experimental rodents such as rat, mouse, hamster, or gerbil mouse, fur animal such as mink or fox, animal in zoological gardens such as monkey, livestock such as cat or dog, poultry such as chicken, turkey, duck, goose, quail and the like and raising fish such as trout and the like.
  • the animal feed additive of the present invention can be mixed with animal feed in the amount ranging about 1 to 100 g per lkg of animal feed.
  • inventive feed composition of the present invention could be prepared by the procedure consisting of the steps of: mixing inventive extract of present invention with other feed component to obtain cohesive granule type to be used directly or other type to purpose further processing and packaging steps, for example, adding water to said feed to perform further conventionally necessary procedure such as pellet, expansion or compression etc.
  • the animal feed or feed additive of the present invention can be applied to various animals including mammals, poultry and fish, preferably, commercially important mammals, for example, horse, pig, cow, sheep, goat, raising fish such as red snapper, bastard halibut, flatfish, yellowtail, trout, swellfish, catfish and the like, shellfish such as abalone, scallop, oyster, Crustacea such as lack tiger shrimp, blue crab, poultry such as chicken, turkey, duck, goose, quail and the like and pet animals.
  • mammals preferably, commercially important mammals, for example, horse, pig, cow, sheep, goat, raising fish such as red snapper, bastard halibut, flatfish, yellowtail, trout, swellfish, catfish and the like, shellfish such as abalone, scallop, oyster, Crustacea such as lack tiger shrimp, blue crab, poultry such as chicken, turkey, duck, goose, quail and the like and pet animals.
  • the inventive composition of the present invention has increasing effect on the release of immune-modulating factors such as NO and TNF- ⁇ ; the stimulating effect on spleen cell proliferation; increasing effect on the production of cytokines such as IFN- ⁇ and TNF- ⁇ and TLR-4 activation in immune cell as well as immune-modulating effect on rotavirus-induced enteritis, rotavirus-specific immune response, rotavirus- infected polyclonal antibody stimulation and immune receptor expression using by C57BL/6 newborn mice together with the preventive test on bacterial infection symptom effect using by ICR mice, therefore, it can be useful as a medicament, health functional food and feed composition for treating, preventing and alleviating human and animal immunodeficiency disease.
  • immune-modulating factors such as NO and TNF- ⁇
  • the stimulating effect on spleen cell proliferation increasing effect on the production of cytokines such as IFN- ⁇ and TNF- ⁇ and TLR-4 activation in immune cell as well as immune-modulating effect on rotavirus-induced enteritis,
  • Fig. 1 shows the increasing effect on NO reproduction and TNF- ⁇ release in RAW264.7 cell after the treatment with an extract of Puerariae radix;
  • Fig. 2 shows the proliferating effect of the extract of Puerariae radix on splenocyte
  • Fig. 3 shows the increasing effect of the extract of Puerariae radix on the reproduction of IFN- ⁇ and TNF- ⁇ in splenocyte;
  • Fig. 4 shows the increasing effect of the extract of Puerariae radix on the gene expression of COX-2 and IFN ⁇ ; and the activations of NFKB and IRF3;
  • Fig. 5 presents the reducing effect of TLR4 and TRIF or MyD88, downward signaling molecules caused by each dominant-negative mutant, on the increased gene expression of COX-I due to the treatment of inventive extract;
  • Fig. 6 presents the reducing effect of TPCK as a NF- KB inhibitor and IkBa mutant as a super-repressor, SB203580 as a p38 pathway inhibitor, on the increased gene expression of COX-I due to the treatment of inventive extract;
  • Fig. 7 represents the reducing effect of an extract of Puerariae radix on rotavirus- induced enteritis and the level of virus NSP3 gene;
  • Fig. 8 represents the enhancing effect of an extract of Puerariae radix on IFN- ⁇ reproduction induced by RV-specific immune response in splenocyte;
  • Fig. 9 depicts the enhancing effect of an extract of Puerariae radix on IFN- ⁇ reproduction and lymphocyte proliferation in lymph organ;
  • Fig. 10 depicts the increasing effect of an extract of Puerariae radix on the gene expressions of TLR2 and TLR4 as congenital immune receptors in RV-induced animal model;
  • Fig. 11 depicts the reducing effect of an extract of Puerariae radix on the mortality rate in E. co/i-infected animal model.
  • RAW264.7 cell ATCC The Global Bioresource CenterTM was in oculated into
  • 96-well plate with adjusting cell concentration to Ix 10 5 cells/well and various concentrations of the extract prepared in Example 1, i.e., 50, 100 and 500 microgram/ml, were added the retoto incubate for 24 hours at 37 0 C under 5% CO 2 atmosphere.
  • 0.1 microgram/ml of LPS was added thereto to incubate for 16 hours and 100 microliter of supernatant was added with 100 microliter of Griess reagent [Griess reagent, 0.1% (w/v)N-(l-naphthyl) ethylenediamine dihydrochloride+ 1% (w/v) sulfanilamide in 5% (v/v) phosphoric acid] to react with each other for 10 mins at room temperature in the dark.
  • the absorbance was determined at 550 nm using by Ultraspec 4000 spectrophotometer (Pharmacia biotech Co. Ltd., Uppsala, Sweden).
  • the amount of NO in culture medium was determined using by dose dependent standard curve of sodium nitrite.
  • TNF- ⁇ tumor necrosis factor- ⁇
  • ELISA examination was performed using by kit (R&D Systems Quantikine mouse TNF- ⁇ /TNFSFIA kit)(Vilcek J. et al., J. Biol. Chem., 266, p.7313, 1991).
  • test sample potently increased the level of NO 2 - by 4.2 folds compared with control group in RAW264.7 cell, which confirms that the inventive extract increases the release of NO.
  • test sample potently increased the level of TNF- ⁇ by 5.1 folds compared with control group in RAW 264.7 cell, which confirms that the inventive extract increases the release of TNF- ⁇ ( See Fig.1).
  • spleen cell collected from C57BL/6N mouse was inoculated into 96-well plate with adjusting cell concentration to 1 x 10 6 cells/well and 100 microgram/ml of the extract prepared in Example 1 was added thereto to incubate for 24 hours at 37 0 C and MTS method (Cell Titer 96TM, Aqueous Assay) was performed to determine the cell pro life ration of spleence 11.
  • test sample more potently increased the proliferation of spleen cell by 149% compared with control group which is not treated with test sample, which confirms that the inventive extract induced the proliferation of spleen cell ( See Fig.2).
  • the spleen cell collected from C57BL/6N mouse was inoculated into 96- well plate with adjusting cell concentration to 1 x 10 6 cells/well and 50 and 100 microgram/ml of the extract prepared in Example 1 was added thereto to incubate for 24 hours at 37 0 C and the increasing effect on cytokine release was performed using by IFN- ⁇ ELISA kit (mouse IFN- ⁇ ELISA kit, BD) and TNF- ⁇ ELISA kit (Quantikine Mouse TNF- ⁇ /TNFSFHA kit, R&D Systems).
  • test sample more potently increased the release of IFN- ⁇ and TNF- ⁇ compared with control group which is not treated with test sample, which confirms that the inventive extract enhanced the expression of cytokines ( See Fig.3).
  • Each lucif erase reporter plasmid having COX-2-promotor, IFN ⁇ -promotor, NF- ⁇ B binding site and IRF3 binding site was inoculated into mouse macrophage of 1 x 10 6 cells/well and then 50 and 100 microgram/ml of the extract prepared in Example 1 was added thereto to incubate for 8 hours at 37 0 C.
  • the gene expression of COX-2 and IFN ⁇ and the activation of NF- ⁇ B and IRF3 in the cell extract were determined by using gene analysis kit (luciferase reporter assay system TM, Promega Co., Madison, WI).
  • test sample potently increased gene expression of COX-2 and IFN ⁇ and the activation of NF- ⁇ B and IRF3 in a dose dependent manner, which confirms that the inventive extract showed increasing effect on the transcription factors involved in congenital immune system and the expression of immune proteins resulting in activating congenital immune system ( See Fig.4).
  • Lucif erase reporter plasmid having COX-2-promotor and each dominant-negative mutant for TLR4, TRIF and MyD88 was inoculated into macrophage of 1 x 10 5 cells/ well and then 500 microgram/ml of the extract prepared in Example 1 was added thereto to incubate for 8 hours at 37 0 C.
  • the gene expression of COX-2 and the inhibition effect on the immunocyte activation caused by inventive extract of each dominant-negative mutant in the cell extract were determined by using gene analysis kit (luciferase reporter assay system TM, Promega Co., Madison, WI).
  • Luciferase reporter plasmid having COX-2-promotor was inoculated into macrophage of 1 x 10 5 cells/well and then 100 and 500 microgram/ml of the extract prepared in Example 1 were added thereto to incubate for 8 hours at 37 0 C. NF- ⁇ B and p38 pathway inhibitor were added thereto prior to the treatment of test sample.
  • the immunocyte activation due to the treatment of inventive extract in the cell extract was determined through the determination of gene expression of COX-2 by using gene analysis kit (luciferase reporter assay system TM, Promega Co., Madison, WI).
  • rotavirus wild-type murine rotavirus, EC strain, Harry B. Greenberg Prof, of Medical College in Stanford Univ. (USA); Korea national veterinary Research & Quarantine Service, Jmport Approval No.2007-56
  • IFN- ⁇ ELISA mouse IFN- ⁇ ELISA kit, BD
  • rotavirus wild-type murine rotavirus, EC strain, Harry B. Greenberg Prof, of Medical College in Stanford Univ. (USA); Korea national veterinary Research
  • the lymph organ treated with inventive extract showed the remarkably increased activity of IFN- ⁇ production and lymphocyte proliferation ( See Fig.9).
  • rotavirus wild-type murine rotavirus, EC strain, Harry B. Greenberg Prof, of Medical College in Stanford Univ. (USA); Korea national veterinary Research & Quarantine Service, Import Approval No. 2007-56.
  • mice [191] E. coli (Escherichia coli; K88ab; the college of veterinary medicine in Seoul national university (Korea)) and 6 weeks aged male ICR mouse (Samtako Co., Korea) were used in the experiment. 30 mice were divided into three groups, i.e., negative control group (I) treated with nothing, positive control group (II) treated with E.coli, and test sample group (HI) treated with inventive extract after E.coli infection and each group consists of 10 mice.
  • negative control group I
  • II positive control group
  • HI test sample group
  • the extract prepared in Example 1 was orally administrated in to the groups (2) and (3) everyday in an amount of 500 mg/kg per once, twice a day and 24 hours after the treatment, 0.5 ml of the culture medium with E.coli (10 MLD) was peritoneally administrated into each mouse to induce active inoculation. 4 days after the inoculation, the number of mortality for 4 days was recorded and the prevention rate of inventive extract from the bacterial infection was investigated.
  • the group (III) treated with inventive extract showed more reduced mortality rate of 80%, i.e., 6 mice at 1 st day, 2 mice at 2 nd day and 3 rd day were died respectively and 2 mice were survived at the end of the experiment.
  • the positive control group (II) treated with E. coli showed maximum mortality rate of 100% at 1 st day (Dl), which confirmed that the inventive extract showed potent preventive activity from bacterial infection ( See Fig.11).
  • inventive extract for administration was set to 2000 mg/kg and diluted into 5 mg/kg, 50 mg/kg and 500 mg/kg to be used as test samples.
  • the test sample was orally administrated and the symptom of toxicity was observed for 4 weeks such as the change of weight, the hematological analysis and histological test.
  • Feed composition preparation was prepared by mixing above components.
  • Powder preparation was prepared by mixing above components and filling sealed package.
  • Tablet preparation was prepared by mixing above components and entabletting.
  • Capsule preparation was prepared by mixing above components and filling gelatin capsule by conventional gelatin preparation method.
  • Injection preparation was prepared by dissolving the components in 2m# ample and sterilizing by conventional injection preparation method.
  • Liquid medicine was prepared by dissolving the components to distilled water with a proper dose of lemon scent, mixing, adjusting to 100ml with distilled water in brown bottle and sterilizing by conventional liquid medicine preparation method.
  • the inventive composition of the present invention have increasing effect on the release of immune-modulating factors such as NO and TNF- ⁇ ; the stimulating effect on spleen cell proliferation; increasing effect on the production of cytokines such as IFN- ⁇ and TNF- ⁇ and TLR-4 activation in immune cell as well as immune-modulating effect on rotavirus -induced enteritis, rotavirus-specific immune response, rotavirus -infected polyclonal antibody stimulation and immune receptor ex- pression using by C57BL/6 newborn mice together with the preventive test on bacterial infection symptom effect using by ICR mice, therefore, it can be useful as a medicament, health functional food and feed composition for treating, preventing and alleviating immunodeficiency disease in human and animal.
  • immune-modulating factors such as NO and TNF- ⁇
  • TNF- ⁇ and TLR-4 activation in immune cell as well as immune-modulating effect on rotavirus -induced enteritis, rotavirus-specific immune response, rotavirus -infected polyclon

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PCT/KR2010/000434 2009-02-09 2010-01-22 A composition comprising an extract of puerariae radix showing immune enhancing activity and the use thereby WO2010090407A2 (en)

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CN103704714A (zh) * 2014-01-10 2014-04-09 吉首大学 一种山楂葛粉香醋含片的制作方法

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KR101297574B1 (ko) * 2010-10-08 2013-08-19 고려대학교 산학협력단 갈근 추출물을 함유하는 안드로스테논 분해 촉진용 조성물
KR101409762B1 (ko) 2012-03-05 2014-06-19 주식회사한국야쿠르트 류코노스톡 락티스 ｈｙ６００１을 이용한 기능성 칡즙 발효물 및 이를 유효성분으로 함유하는 제품
KR101429379B1 (ko) * 2013-01-23 2014-08-12 충북대학교 산학협력단 수지상세포 활성화를 위한 갈근 조성물의 용도
KR102287633B1 (ko) * 2015-04-03 2021-08-10 한국 한의학 연구원 갈화 추출물을 유효성분으로 함유하는 선천면역 증진 및 항바이러스용 조성물

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