WO2010041837A2 - A use of the liquiritigenin abundant extract or liquiritigenin derived therefrom for increasing bile flow, choleretic effect, and for preventing and treating cholestatic liver diseases - Google Patents

A use of the liquiritigenin abundant extract or liquiritigenin derived therefrom for increasing bile flow, choleretic effect, and for preventing and treating cholestatic liver diseases Download PDF

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WO2010041837A2
WO2010041837A2 PCT/KR2009/005451 KR2009005451W WO2010041837A2 WO 2010041837 A2 WO2010041837 A2 WO 2010041837A2 KR 2009005451 W KR2009005451 W KR 2009005451W WO 2010041837 A2 WO2010041837 A2 WO 2010041837A2
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liquiritigenin
extract
licorice
abundant
liver diseases
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PCT/KR2009/005451
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French (fr)
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WO2010041837A3 (en
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Sang Geon Kim
Sang Chan Kim
Young Woo Kim
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Snu R&Db Foundation
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Priority to CN2009801472416A priority Critical patent/CN102223888A/en
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Publication of WO2010041837A3 publication Critical patent/WO2010041837A3/en

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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
    • A61K36/484—Glycyrrhiza (licorice)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics

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  • the present invention is related to a use of a liquritigenin- abundant extract of licorice and liquiritigenin derived therefrom as an effective ingredient for increasing bile flow, choleretic effect, and for preventing and treating cholestatic liver diseases; a method for increasing bile flow, choleretic effect using thereby; and the composition comprising the same.
  • Bile juice contains bile acids, phospholipids, bilirubins, glutathione (GSH) and electrolytes, and plays important role in various physiological function involved in the digestion and absorption of lipid such as increasing the absorption of normal lipid at intestine, maintaining the balance of human cholesterol metabolism as well as in performing as a route for the excretion of various toxic chemicals such as drugs, endogenous debris such as bilirubin, and lipid metabolites (Ballatori N. et al., Am. J. Physiol., 263, pp G617-G624, 1992).
  • galactosamine GaIN
  • lipopolysaccharide LPS
  • the galactosamine depletes UTP and UDP-glucuronic acid in liver tissue to inhibit the protein production in liver; depletes GSH, a hepatoprotective substance; and inhibits glucuronidation involved in the waste excretion to induce hepatotoxicity resulting in accompanying abnormal morphologies such as the cell necrosis of substantial tissue and infiltration of inflammatory cell etc (Jonker AM et al, Hepatology, 11, pp622-627, 1990).
  • LPS rapidly increases the influx of endotoxin comprising LPS into venae portalis hepatis; increase TNF- ⁇ (Tumor Necrosis Factor- ) caused by the activation of inflammatory liver cells to give rise to fulminating hepatitis; represses the expression of hepatic transfer protein to induce the disorder in the excretion of bile juice; and increases the expression of hematological indicators of cholestatic liver diseases for example, ALP (Alkaline phosphatase), ⁇ -GTP ( ⁇ -Glutamyl Transpeptidase) etc (Gran M et al., Acta Hepatogastroenterol. (Stuttg) 24, pp64-81, 1977).
  • ALP Alkaline phosphatase
  • ⁇ -GTP ⁇ -Glutamyl Transpeptidase
  • NTCP Na+-dependent taurocholic cotransporting polypeptide
  • OATP-I Organic anion transporting polypeptide- 1
  • Canalicular membrane transporter proteins such as MRP2 (Multidrug resistance protein 2) and BSEP (bile salt export pump) involved in the output of bile juice from liver to biliary duct etc (Keppler D et al., FASEB J., 11, pp509-516, 1997).
  • Licorice root,a root of Glycyrrhiza uralensis FISCH, Glycyrrhiza glabra L. and the like belongs to Leguminosae has been reported to contain triterpene saponin such as glycyrrhizin and several flavonids such as liquiritigenin, liquiritin, neoliquiritin, neoisoliquiritin etc, which has been used to treat cough, gastric ulcer, hypertension, etc.
  • liquiritigenin showed potent treating and preventing effect on the liver disease caused by heavy metal intoxication and acute and chronic hepatic disease induced by hepatotoxic substances (Kim et al., Toxicology, 197, pp 239-251, 2004; Kim et al., Chem. Biol. Interact, 161, ppl25-138, 2006).
  • licorice extract or liquiritigenin there has been not reported or disclosed about the therapeutic effect of licorice extract or liquiritigenin on cholestatic liver diseases in any of above cited literatures, the disclosures of which are incorporated herein by reference.
  • the present inventors have endeavored to find the effective agent for treating cholestatic liver diseases and enhancing bile flow and to study the pharmacological effect of licorice extract and liquiritigenin isolated therefrom through various in vitro test and animal model tests, for example, inhibitory effect on the hematological indicators such as ALT, ALP, ⁇ -GTP etc and on the histo-pathological indicators such as the necrosis of hepatic cell, the depletion of GSH, a hepato-protective indicators in cholestatic liver diseases induced animal model by galactosamine/LPS.
  • inhibitory effect on the hematological indicators such as ALT, ALP, ⁇ -GTP etc
  • histo-pathological indicators such as the necrosis of hepatic cell, the depletion of GSH, a hepato-protective indicators in cholestatic liver diseases induced animal model by galactosamine/LPS.
  • the present invention also provides a use of liquiritgenin abundant extract or the compound isolated therefrom for the manufacture of medicament employed for treating or preventing cholestatic liver diseases and increasing bile flow in human or mammal.
  • the present invention also provides a method for treating cholestatic liver diseases and increasing bile flow in human or mammal comprising administering to said mammal in an effective amount of above-mentioned extract or the compound isolated therefrom, together with a pharmaceutically acceptable carrier thereof.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the extract of licorice or liquiritigenin isolated therefrom as an active ingredient for treating or preventing cholestatic liver diseases and increasing bile flow.
  • the present invention also provides a health functional food comprising the above- described extract or the compound isolated therefrom for preventing or improving cholestatic liver diseases and for increasing bile flow as an active ingredient in an amount effective to prevent and improve cholestatic liver diseases, together with a sito- logically acceptable additive.
  • a pharmaceutical composition comprising the liquiritgenin abundant extract of licorice or liquiritigenin isolated therefrom as an active ingredient for treating or preventing cholestatic liver diseases and increasing bile flow, together with a pharmaceutically acceptable carrier.
  • the term "the liquiritgenin abundant extract of licorice” disclosed herein comprise the extract of glycyrrhiza uralensis, glycyrrhiza glabra L. or the like, preferably, gly- cyrrhiza uralensis, and the liquiritigenin- abundant extract of licorice, for example, which can be prepared by the procedure comprising the steps: washing, drying licorice; subjecting to fractionation with non-polar solvent to remove non-polar solvent soluble substance at 1 st step; adding strong acid to the fraction and performing acid-hydrolysis reaction to obtain hydrolyzed liquiritigenin abundant fraction at 2 nd step; neutralizing the solution with strong base at 3 rd step; and isolating the water-soluble fraction comprising sugar component with water to obtain purposed liquiritigenin- abundant extract of the present invention.
  • a health functional food comprising the liquiritgenin abundant extract of licorice or liquiritigenin isolated therefrom for preventing or improving cholestatic liver diseases and increasing bile flow as an active ingredient in an amount effective to prevent and improve cholestatic liver diseases, together with a sitologically acceptable additive.
  • the term "the cholestatic liver diseases”disclosed herein comprises hepatic cholestatic jaundice, cholestatic hepatitis, primary cholestatic hepatocirrhosis, acute or chronic cholestatic liver disease, primary sclerosing cholangeitis, cholelithiasis, transport protein depression disease, biliary fibrosis, or biliary cancer, preferably, cholestatic hepatitis, primary cholestatic hepatocirrhosis, acute or chronic cholestatic liver disease, and cholelithiasis,.
  • the herb which can be used in the present invention, but not intent to limit thereto, 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 cholestatic liver diseases.
  • the pharmaceutical composition for treating liver diseases could contain about 0.01 to 95 w/w%, preferably 0.5 to 50 w/w% of inventive extract or compound of present invention based on the total weight of the composition.
  • the above-describedextract of licorice or liquiritigenin isolated therefrom can be prepared by following procedure;
  • the licorice for example, i.e., glycyrrhiza uralensis
  • the solution is enfleuraged at the temperature ranging from 0 to room temperature, preferably room temperature, for the period ranging from 12 hours to 1 week, preferably 48 to 72 hours or heated with reflux extraction at the temperature ranging from 80 to 12O 0 C, preferably above 105 0 C, for the period ranging from 1 to 24 hours, preferably 2 to 5 hours with 2 to 5 times, or extracted by sonication, reflux or conventional extraction
  • the solution is filtered to obtain the crude extract of licorice of the present invention.
  • the crude extract of licorice prepared from the above step is subjected to repeated column chromatography eluted with mixed solvent system to obtain liquiritin- abundant extract of licorice; the purified extract is subjected to acid hydrolysis using by strong acid such as HCl to remove the sugar-moiety of liquiritin; the reactant is neutralized with alkali solution such as NaOH; and the solution is subjected to Silica gel column chromatography eluting with mixture solvent system (CHCl 3 and acetone) to obtain liquiritigenin-abundant extract of the present invention.
  • mixture solvent system CHCl 3 and acetone
  • the liquiritigenin-abundant extract is subjected to further purification process such as Silicagel column chromatography or re-crystallization method to obtain liquiritigenin of the present invention.
  • It is the other object of the present invention to provide a method for preparing liquiritigenin-abundant extract from the extract of licorice comprising the steps consisting of; washing, drying licorice; mixing with 10 to 15-fold volume of distilled water, alcohols such as methanol, ethanol and the like, or the mixtures thereof; enfleuraging the solution at the temperature ranging from 0 to room temperature, 48 to 72 hours; filtering the residue to obtain the crude extract of licorice; subjecting the extract to repeated column chromatography eluted with mixed solvent system to obtain liquiritin- abundant extract of licorice; subjecting the extract to acid hydrolysis using by strong acid to remove the sugar-moiety of liquiritin; neutralizing the solution with alkali solution; subjecting the solution to Silica gel column chromatography eluting with mixture solvent system (CHCl 3 and acetone) to obtain liquiritigenin-abundant extract of the present invention.
  • the present invention provide a method for preparing inventive liquiritigenin- abundant extract comprising the steps of;washing, drying licorice; extracting with 10 to 15-fold volume of non-polar solvent such as n-hexane, chloroform, dichloromethane, or ethylacetate, preferably, dichloromethane or the mixture thereof at the temperature ranging from 10 to 100 0 C, for the period ranging from 1 hour to 7 days, preferably, 24 to 72 hours with the extraction method selected from enfleuraging extraction, hot-water extraction, sonication extraction, SFE extraction method, etc, preferably, sonication extraction; filtering the residue to remove non-polar solvent soluble substance; mixing the residue with 10 to 15-fold volume of distilled water, alcohols such as methanol, ethanol and the like, or the mixtures thereof, preferably, methanol; enfleuraging the solution at the temperature ranging from 10 to 100 0 C, for the period
  • the inventive compound can be transformed into their pharmaceutically acceptable salt and solvates by the conventional method well known in the art.
  • acid- addition salt thereof formed by a pharmaceutically acceptable free acid thereof is useful and can be prepared by the conventional method.
  • the salts are precipitated by the water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile to prepare acid addition salt thereof and further the mixture of equivalent amount of compound and diluted acid with water or alcohol such as glycol monomethylether, can be heated and subsequently dried by evaporation or filtrated under reduced pressure to obtain dried salt form thereof.
  • organic acid or inorganic acid can be used as a free acid of above-described method.
  • organic acid such as methansulfonic acid, /?-toluensulfonic acid, acetic acid, trifluoroacetic acid, citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonylic acid, vanillic acid, hy- droiodic acid and the like, and inorganic acid such as hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid and the like can be used herein.
  • the pharmaceutically acceptable metal salt form of inventive compound may be prepared by using base.
  • the alkali metal or alkali-earth metal salt thereof can be prepared by the conventional method, for example, after dissolving the compound in the excess amount of alkali metal hydroxide or alkali-earth metal hydroxide solution, the insoluble salts are filtered and remaining filtrate is subjected to evaporation and drying to obtain the metal salt thereof.
  • sodium, potassium or calcium salt are pharmaceutically suitable and the corresponding silver salt can be prepared by reacting alkali metal salt or alkali-earth metal salt with suitable silver salt such as silver nitrate.
  • the pharmaceutically acceptable salt of the compound comprise all the acidic or basic salt which may be present at the compounds, if it does not indicated specifically herein.
  • the pharmaceutically acceptable salt of the present invention comprise the salt of hydroxyl group such as the sodium, calcium and potassium salt thereof; the salt of amino group such as the hydrogen bromide salt, sulfuric acid salt, hydrogen sulfuric acid salt, phosphate salt, hydrogen phosphate salt, dihydrophosphate salt, acetate salt, succinate salt, citrate salt, tartarate salt, lactate salt, mandelate salt, methanesulfonate(mesylate) salt and/?-toluenesulfonate (tosylate) salt etc, which can be prepared by the conventional method well known in the art.
  • It is still another object of the present invention to provide a pharmaceutical composition comprising the pulverized form, extracted form or dried extract form of above crude drug extract obtained by above described process as an active ingredient for preventing and treating cholestatic liver diseases.
  • the inventive composition of the present invention significantly increase the gene expression of the transport protein involved in the excretion of bile acids as well as inhibits the increase of the hematological indicators such as ALT, ALP, ⁇ -GTP etc and of the histo-pathological indicators such as the necrosis of hepatic cell, the depletion of GSH, a hepato-protective indicators in cholestatic liver diseases-induced animal model by galactosamine/LPS when the inventive extract or compound of the present invention was orally and intravenously administrated thereto.
  • the inventive extract had no apparent effect on mortality, clinical signs, body weight changes, and gross findings at necropsy.
  • the pharmaceutical composition for treating cholestatic liver diseases could contain about 0.01 to 99.9 w/w%, preferably 0.1 to 90 w/w% of the above crude drug composition of present invention based on the total weight of the composition.
  • the 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).
  • 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, propy- lhydroxy 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, polyvin
  • 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 inventive 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).
  • oral dosage form poowder, tablet, 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
  • suppository sterile injectable preparation
  • inventive 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-lOg/kg, preferably, 1 to 5g/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 the above extract or the compound for the prevention or improvement of cholestatic liver diseases as an active ingredient in an amount effective to prevent and improve cholestatic liver diseases, 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 cholestatic liver diseases could contain about 0.01 to 95 w/w%, preferably 0.5 to 80 w/w% of the above inventive composition of present invention based on the total weight of the composition.
  • composition therein can be added to food, additive or beverage for prevention and improvement of cholestatic liver 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 10Om ⁇ 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 10Om ⁇ 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 composition comprising an extract of licorice or liquir- itigenin derived therefrom of the present invention significantly increases bile flow rate and biliary excretion of bile acid, GSH and bilirubin. Consistent with these results, the composition decreases the blood chemical markers including ALT as a hepato- protective marker, and ALP and g-GTP, as cholestatasis markers, and histochemical markers including central necrosis and inflammation in galactos amine/ lipopolysaccharide-induced hepato-toxicity animal model when the inventive extract or compound of the present invention was orally and intravenously administrated thereto. Accordingly, the inventive compositions according to the present invention are useful in the prevention and treatment of the cholestatic liver diseases and can be used as safe and efficient hepato-protective agent.
  • Fig. 1-A shows the chemical structure of Ml (4 -O-glucuronide) and M2
  • FIG. 1-B shows the change of LQ serum level according to the time in the test in case that liquiritigenin (LQ) was intravenously administrated to rat (50mg/kg);
  • FIG. 1-C shows the change of serum level of LQ metabolites, i.e., Ml and M2 according to the time in the test in case that liquiritigenin (LQ) was intravenously administrated to rat (50mg/kg);
  • Fig. 2- A represents the comparison effect of liquiritigenin treatment with control group on bile acid excretion when 50mg/kg LQ was intraveneously administrated to animal model;
  • Fig. 2-B represents that the choleretic effect of liquiritigenin induces the increase of bile acid excretion in bile juice when 50mg/kg of LQ was intraveneously administrated to animal model;
  • Fig. 2-C represents that the choleretic effect of liquiritigenin induces the increase of GSH excretion in bile juice when 50mg/kg LQ was intraveneously administrated to animal model
  • Fig. 2-D represents that the choleretic effect of liquiritigenin induces the increase of bilirubin excretion in bile juice when 50mg/kg of LQ was intraveneously administrated to animal model
  • Fig. 3-A represents that the intraveneous administration of 15mg/kg of LQ for 3 days increase the level of transporter protein involved in the bile acid excretion within liver tissue (the result of mRNA quantitative analysis verifying the increase of the transporter proteins, i.e., NTCP (Na+-dependent taurocholic cotransporting polypeptide) and OATP-I (Organic anion transporting polypeptide- 1) present in ba- solateral membrane involved in the influx of bile juice into hepatic cell; Canalicular membrane transporter proteins such as MRP2 (Multidrug resistance protein 2) and BSEP (bile salt export pump) involved in the output of bile juice from liver to biliary duct;
  • NTCP Na+-dependent taurocholic cotransporting polypeptide
  • OATP-I Organic anion transporting polypeptide- 1
  • Canalicular membrane transporter proteins such as MRP2 (Multidrug resistance protein 2) and BSEP (bile salt export pump) involved in the output of bile
  • Fig. 3-B represents the result of Western blot analysis reflecting on the increase of MRP2, a main transporter protein involved in the output of bile acid;
  • Fig. 3-C represents the result of immuno-chemical staining analysis reflecting on the increase of MRP2, a main transporter protein involved in the output of bile acid;
  • Fig. 4-A represents the blood biochemical indicator analysis such as ALT (alanine aminotransferase), ALP (Alkaline phosphatase) and -GTP (glutamyltransferase) which proves the hepatoprotective effect on liver injury induced by galactosamine(GalN)/ lipopolysaccharide(LPS) when 15mg/kg of LQ was intraveneously administrated or 50mg/kg of LQ was orally administrated for 3 days;
  • ALT alanine aminotransferase
  • ALP Alkaline phosphatase
  • -GTP glutamyltransferase
  • Fig. 4-B represents the histochemical H&E staining analysis which proves the hepatoprotective effect on liver injury induced by galatosamine/ lipopoly saccharide when 15mg/kg of LQ was intraveneously administrated or 50mg/kg of LQ was orally administrated for 3 days;
  • Fig. 4-C represents the histochemical TUNEL staining analysis which proves the hepatoprotective effect on liver injury induced by galatosamine/ lipopoly saccharide when 15mg/kg of LQ was intraveneously administrated or 50mg/kg of LQ was orally administrated for 3 days;
  • Fig. 5- A presents the recovery of the decreased level of GSH, a representative index, which proves the hepatoprotective effect on liver injury induced by galatosamine/ lipopolysaccharide when 15mg/kg of LQ was intraveneously administrated or 50mg/kg of LQ was orally administrated for 3 days;
  • Fig. 5-B presents the recovery of the increased level of TNF- , a representative inflammatory index, which proves the hepatoprotective effect on liver injury induced by galatosamine/ lipopolysaccharide when 15mg/kg of LQ was intraveneously administrated or 50mg/kg of LQ was orally administrated for 3 days.
  • Example 4 Preparation of inventive liquiritigenin-abundant licorice extract (GEF-I) [127] 2000 ml of IN HCl was added to lkg of licorice extract prepared in Example 1 and reacted at 100 C for 6 hours to hydrolyze the sugar-moiety of liquiritin. The reactant was neutralized with 2000ml of IN NaOH. The hydrolysate was repeatedly washed with distilled water to remove sugar component and salt component in order to obtain 800gof liquiritigenin- abundant licorice extract (designated as GEF-I hereinafter).
  • GEF-I inventive liquiritigenin-abundant licorice extract
  • Liquiritigenin was intravenously administrated into the rats (15mg/kg) once a day for three days and then 1 hour after the treatment, the liver was delivered from the rats.
  • Liquiritigenin was administrated into the rats intravenously three times a day for three days (15mg/kg) or orally three times a day for three days (50mg/kg).
  • galactosamine 600mg/kg
  • LPS l ⁇ g/kg
  • hematological indicators i.e., ALT, a hepatic injury indicator, and -GTP and ALP
  • cholestasis indicators were significantly inhibited ( See Fig. 4), which verify the inhibitory effect of liquiritigenin on the incidence and progress of cholestasis liver disease.
  • Powder preparation was prepared by mixing above components and filling sealed package.
  • Tablet preparation was prepared by mixing above components and entabletting.
  • Tablet preparation was prepared by mixing above components and filling gelatin capsule by conventional gelatin preparation method.
  • Injection preparation was prepared by dissolving active component, controlling pH to about 7.5 and then filling all the components in 2m# ample and sterilizing by conventional injection preparation method.
  • Liquid preparation was prepared by dissolving active component, and then filling all the components in 100Om ⁇ ample and sterilizing by conventional liquid preparation method.
  • Vitamin mixture optimum amount
  • Health beverage preparation was prepared by dissolving active component, mixing, stirred at 85 0 C for 1 hour, filtered and then filling all the components in lOOOm ⁇ ample and sterilizing by conventional health beverage preparation method.
  • the present invention is related to a composition
  • a composition comprising an extract of licorice or liquiritigenin derived therefrom, which significantly increases bile flow rate and biliary excretion of bile acid, GSH and bilirubin.
  • the inventive composition comprising an extract of licorice or liquiritigenin derived therefrom decrease the blood chemical markers including ALT as a hepatoprotective marker, and ALP and ⁇ -GTP, as cholestatasis markers, and histochemical markers including central necrosis and inflammation in galactosamine/lipopolysaccharide-induced hepato- toxicity animal model when the inventive extract or compound of the present invention was orally and intravenously administrated thereto.
  • the inventive compositions according to the present invention are useful in the prevention and treatment of the cholestatic liver diseases and can be used as safe and efficient hepato-protective agent.

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Abstract

The present invention is related to a composition comprising an extract of licorice or liquiritigenin derived therefrom, which significantly increases bile flow rate and biliary excretion of bile acid, GSH and bilirubin. Consistent with these results, the inventive composition comprising an extract of licorice or liquiritigenin derived therefrom decrease the blood chemical markers including ALT as a hepatoprotective marker, and ALP and γ-GTP, as cholestatasis markers, and histochemical markers including central necrosis and inflammation in galactosamine/lipopolysaccharide-induced hepato-toxicity animal model when the inventive extract or compound of the present invention was orally and intravenously administrated thereto.

Description

A USE OF THE LIQUIRITIGENIN ABUNDANT EXTRACT OR LIQUIRITIGENIN DERIVED THEREFROM FOR INCREASING BILE FLOW, CHOLERETIC EFFECT, AND FOR PREVENTING AND TREATING CHOLESTATIC LIVER DISEASES
The present invention is related to a use of a liquritigenin-abundant extract of licorice and liquiritigenin derived therefrom as an effective ingredient for increasing bile flow, choleretic effect, and for preventing and treating cholestatic liver diseases; a method for increasing bile flow, choleretic effect using thereby; and the composition comprising the same.
Bile juice contains bile acids, phospholipids, bilirubins, glutathione (GSH) and electrolytes, and plays important role in various physiological function involved in the digestion and absorption of lipid such as increasing the absorption of normal lipid at intestine, maintaining the balance of human cholesterol metabolism as well as in performing as a route for the excretion of various toxic chemicals such as drugs, endogenous debris such as bilirubin, and lipid metabolites (Ballatori N. et al., Am. J. Physiol., 263, pp G617-G624, 1992).
Two substances, i.e, galactosamine (GalN) and lipopolysaccharide (LPS), are widely used as representative inducers to cholestatic liver diseases in the experiments. The galactosamine depletes UTP and UDP-glucuronic acid in liver tissue to inhibit the protein production in liver; depletes GSH, a hepatoprotective substance; and inhibits glucuronidation involved in the waste excretion to induce hepatotoxicity resulting in accompanying abnormal morphologies such as the cell necrosis of substantial tissue and infiltration of inflammatory cell etc (Jonker AM et al, Hepatology, 11, pp622-627, 1990). LPS rapidly increases the influx of endotoxin comprising LPS into venae portalis hepatis; increase TNF-α (Tumor Necrosis Factor- ) caused by the activation of inflammatory liver cells to give rise to fulminating hepatitis; represses the expression of hepatic transfer protein to induce the disorder in the excretion of bile juice; and increases the expression of hematological indicators of cholestatic liver diseases for example, ALP (Alkaline phosphatase), γ-GTP (γ-Glutamyl Transpeptidase) etc (Grun M et al., Acta Hepatogastroenterol. (Stuttg) 24, pp64-81, 1977).
Several transporter proteins such as basolateral membrane transporter proteis, i.e., NTCP (Na+-dependent taurocholic cotransporting polypeptide) and OATP-1 (Organic anion transporting polypeptide-1) present in basolateral membrane involved in the influx of bile juice into hepatic cell; Canalicular membrane transporter proteins such as MRP2 (Multidrug resistance protein 2) and BSEP (bile salt export pump) involved in the output of bile juice from liver to biliary duct etc (Keppler D et al., FASEB J., 11, pp509-516, 1997).
Licorice root, a root of Glycyrrhiza uralensis FISCH, Glycyrrhiza glabra L. and the like belongs to Leguminosae has been reported to contain triterpene saponin such as glycyrrhizin and several flavonids such as liquiritigenin, liquiritin, neoliquiritin, neoisoliquiritin etc, which has been used to treat cough, gastric ulcer, hypertension, etc.
Conventionally, UDCA (Ursodeoxychlic acid) has been reported to be a sole medicine to treat cholestatic liver diseases and to increase bile flow till now in Korea. Therefore, the development of novel drug to treat cholestatic liver diseases and to increase bile flow has been still needed till now.
The present inventors proved that liquiritigenin showed potent treating and preventing effect on the liver disease caused by heavy metal intoxication and acute and chronic hepatic disease induced by hepatotoxic substances (Kim et al., Toxicology, 197, pp 239-251, 2004; Kim et al., Chem. Biol. Interact, 161, pp125-138, 2006). However, there has been not reported or disclosed about the therapeutic effect of licorice extract or liquiritigenin on cholestatic liver diseases in any of above cited literatures, the disclosures of which are incorporated herein by reference.
Therefore, the present inventors have endeavored to find the effective agent for treating cholestatic liver diseases and enhancing bile flow and to study the pharmacological effect of licorice extract and liquiritigenin isolated therefrom through various in vitro test and animal model tests, for example, inhibitory effect on the hematological indicators such as ALT, ALP, γ-GTP etc and on the histo-pathological indicators such as the necrosis of hepatic cell, the depletion of GSH, a hepato-protective indicators in cholestatic liver diseases induced animal model by galactosamine/LPS.
Finally, the present inventors have found that the liquiritgenin abundant licorice extract or liquiritigenin isolated therefrom is effective in treating and preventing cholestatic liver diseases and increasing bile flow.
According to one aspect, the present invention also provides a use of liquiritgenin abundant extract or the compound isolated therefrom for the manufacture of medicament employed for treating or preventing cholestatic liver diseases and increasing bile flow in human or mammal.
The present invention also provides a method for treating cholestatic liver diseases and increasing bile flow in human or mammal comprising administering to said mammal in an effective amount of above-mentioned extract or the compound isolated therefrom, together with a pharmaceutically acceptable carrier thereof.
The present invention provides a pharmaceutical composition comprising the extract of licorice or liquiritigenin isolated therefrom as an active ingredient for treating or preventing cholestatic liver diseases and increasing bile flow.
The present invention also provides a health functional food comprising the above-described extract or the compound isolated therefrom for preventing or improving cholestatic liver diseases and for increasing bile flow as an active ingredient in an amount effective to prevent and improve cholestatic liver diseases, together with a sitologically acceptable additive.
Accordingly, it is an object of the present invention to provide a pharmaceutical composition comprising the liquiritgenin abundant extract of licorice or liquiritigenin isolated therefrom as an active ingredient for treating or preventing cholestatic liver diseases and increasing bile flow, together with a pharmaceutically acceptable carrier.
It is another object of the present invention to provide a use of the liquiritgenin abundant extract of licorice or liquiritigenin isolated therefrom for the manufacture of medicament employed for treating or preventing cholestatic liver diseases and increasing bile flow in human or mammal.
It is the other object of the present invention to provide a method for treating cholestatic liver diseases and increasing bile flow in human or mammal comprising administering to said mammal in an effective amount of the liquiritgenin abundant extract of licorice or liquiritigenin isolated therefrom, together with a pharmaceutically acceptable carrier thereof.
The term “the liquiritgenin abundant extract of licorice”disclosed herein comprise the extract of glycyrrhiza uralensis, glycyrrhiza glabra L. or the like, preferably, glycyrrhiza uralensis, and the liquiritigenin-abundant extract of licorice, for example, which can be prepared by the procedure comprising the steps: washing, drying licorice; subjecting to fractionation with non-polar solvent to remove non-polar solvent soluble substance at 1st step; adding strong acid to the fraction and performing acid-hydrolysis reaction to obtain hydrolyzed liquiritigenin abundant fraction at 2nd step; neutralizing the solution with strong base at 3rd step; and isolating the water-soluble fraction comprising sugar component with water to obtain purposed liquiritigenin-abundant extract of the present invention.
In accordance with one aspect of the present invention, there provided a health functional food comprising the liquiritgenin abundant extract of licorice or liquiritigenin isolated therefrom for preventing or improving cholestatic liver diseases and increasing bile flow as an active ingredient in an amount effective to prevent and improve cholestatic liver diseases, together with a sitologically acceptable additive.
The term “the cholestatic liver diseases”disclosed herein comprises hepatic cholestatic jaundice, cholestatic hepatitis, primary cholestatic hepatocirrhosis, acute or chronic cholestatic liver disease, primary sclerosing cholangeitis, cholelithiasis, transport protein depression disease, biliary fibrosis, or biliary cancer, preferably, cholestatic hepatitis, primary cholestatic hepatocirrhosis, acute or chronic cholestatic liver disease, and cholelithiasis,.
The herb, which can be used in the present invention, but not intent to limit thereto, 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 cholestatic liver diseases.
The pharmaceutical composition for treating liver diseases could contain about 0.01 to 95 w/w%, preferably 0.5 to 50 w/w% of inventive extract or compound of present invention based on the total weight of the composition.
An inventive extract and compound may be prepared in accordance with the following preferred embodiment.
For the present invention, the above-described extract of licorice or liquiritigenin isolated therefrom can be prepared by following procedure;
For example, the licorice, for example, i.e., glycyrrhiza uralensis, is washed, dried, and mixed with 5 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, methanol; the solution is enfleuraged at the temperature ranging from 0 to room temperature, preferably room temperature, for the period ranging from 12 hours to 1 week, preferably 48 to 72 hours or heated with reflux extraction at the temperature ranging from 80 to 120℃, preferably above 105℃, for the period ranging from 1 to 24 hours, preferably 2 to 5 hours with 2 to 5 times, or extracted by sonication, reflux or conventional extraction; the solution is filtered to obtain the crude extract of licorice of the present invention.
To obtain more preferable liquiritigenin-abundant extract of the present invention, the crude extract of licorice prepared from the above step is subjected to repeated column chromatography eluted with mixed solvent system to obtain liquiritin-abundant extract of licorice; the purified extract is subjected to acid hydrolysis using by strong acid such as HCl to remove the sugar-moiety of liquiritin; the reactant is neutralized with alkali solution such as NaOH; and the solution is subjected to Silica gel column chromatography eluting with mixture solvent system (CHCl3 and acetone) to obtain liquiritigenin-abundant extract of the present invention.
To obtain pure liquiritigenin of the present invention, the liquiritigenin-abundant extract is subjected to further purification process such as Silicagel column chromatography or re-crystallization method to obtain liquiritigenin of the present invention.
It is another object of the present invention to provide a process for preparing the above-described extract of licorice and liquiritigenin isolated therefrom as described above for the preparation of composition effective in treating or preventing cholestatic liver diseases.
It is the other object of the present invention to provide a method for preparing liquiritigenin-abundant extract from the extract of licorice comprising the steps consisting of; washing, drying licorice; mixing with 10 to 15-fold volume of distilled water, alcohols such as methanol, ethanol and the like, or the mixtures thereof; enfleuraging the solution at the temperature ranging from 0 to room temperature, 48 to 72 hours; filtering the residue to obtain the crude extract of licorice; subjecting the extract to repeated column chromatography eluted with mixed solvent system to obtain liquiritin-abundant extract of licorice; subjecting the extract to acid hydrolysis using by strong acid to remove the sugar-moiety of liquiritin; neutralizing the solution with alkali solution; subjecting the solution to Silica gel column chromatography eluting with mixture solvent system (CHCl3 and acetone) to obtain liquiritigenin-abundant extract of the present invention.
In an alternative embodiment of the present invention, the present invention provide a method for preparing inventive liquiritigenin-abundant extract comprising the steps of; washing, drying licorice; extracting with 10 to 15-fold volume of non-polar solvent such as n-hexane, chloroform, dichloromethane, or ethylacetate, preferably, dichloromethane or the mixture thereof at the temperature ranging from 10 to 100℃, for the period ranging from 1 hour to 7 days, preferably, 24 to 72 hours with the extraction method selected from enfleuraging extraction, hot-water extraction, sonication extraction, SFE extraction method, etc, preferably, sonication extraction; filtering the residue to remove non-polar solvent soluble substance; mixing the residue with 10 to 15-fold volume of distilled water, alcohols such as methanol, ethanol and the like, or the mixtures thereof, preferably, methanol; enfleuraging the solution at the temperature ranging from 10 to 100℃, for the period ranging from 1 hour to 7 days, preferably, 48 to 72 hours with the extraction method selected from enfleuraging extraction, hot-water extraction, sonication extraction, SFE extraction method, etc, preferably, sonication extraction to obtain the polar solvent soluble fraction which removed the non-polar solvent soluble substances at 1st step; adding 5 to 40-fold, preferably, 15 to 20-fold volume of strong acid such as about 1- 3 N HCl, sulfuric acid etc, preferably, 1N HCl to the fraction and performing acid-hydrolysis reaction at the temperature ranging from 10 to 200℃, preferably, 80 to 100℃, for the period ranging from 2 hour to 10 hours, preferably, 4 to 8 hours to obtain hydrolyzed liquiritigenin abundant fraction at 2nd step; neutralizing the solution with strong base such as sodium hydroxide, potassium hydroxide etc at 3rd step; and isolating the water-soluble fraction comprising sugar component with water to obtain purposed liquiritigenin-abundant extract of the present invention.
The inventive compound can be transformed into their pharmaceutically acceptable salt and solvates by the conventional method well known in the art. For the salts, acid-addition salt thereof formed by a pharmaceutically acceptable free acid thereof is useful and can be prepared by the conventional method. For example, after dissolving the compound in the excess amount of acid solution, the salts are precipitated by the water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile to prepare acid addition salt thereof and further the mixture of equivalent amount of compound and diluted acid with water or alcohol such as glycol monomethylether, can be heated and subsequently dried by evaporation or filtrated under reduced pressure to obtain dried salt form thereof.
As a free acid of above-described method, organic acid or inorganic acid can be used. For example, organic acid such as methansulfonic acid, p-toluensulfonic acid, acetic acid, trifluoroacetic acid, citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonylic acid, vanillic acid, hydroiodic acid and the like, and inorganic acid such as hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid and the like can be used herein.
Further, the pharmaceutically acceptable metal salt form of inventive compound may be prepared by using base. The alkali metal or alkali-earth metal salt thereof can be prepared by the conventional method, for example, after dissolving the compound in the excess amount of alkali metal hydroxide or alkali-earth metal hydroxide solution, the insoluble salts are filtered and remaining filtrate is subjected to evaporation and drying to obtain the metal salt thereof. As a metal salt of the present invention, sodium, potassium or calcium salt are pharmaceutically suitable and the corresponding silver salt can be prepared by reacting alkali metal salt or alkali-earth metal salt with suitable silver salt such as silver nitrate.
The pharmaceutically acceptable salt of the compound comprise all the acidic or basic salt which may be present at the compounds, if it does not indicated specifically herein. For example, the pharmaceutically acceptable salt of the present invention comprise the salt of hydroxyl group such as the sodium, calcium and potassium salt thereof; the salt of amino group such as the hydrogen bromide salt, sulfuric acid salt, hydrogen sulfuric acid salt, phosphate salt, hydrogen phosphate salt, dihydrophosphate salt, acetate salt, succinate salt, citrate salt, tartarate salt, lactate salt, mandelate salt, methanesulfonate(mesylate) salt and p-toluenesulfonate (tosylate) salt etc, which can be prepared by the conventional method well known in the art.
It is still another object of the present invention to provide a pharmaceutical composition comprising the pulverized form, extracted form or dried extract form of above crude drug extract obtained by above described process as an active ingredient for preventing and treating cholestatic liver diseases.
The inventive composition of the present invention significantly increase the gene expression of the transport protein involved in the excretion of bile acids as well as inhibits the increase of the hematological indicators such as ALT, ALP, γ-GTP etc and of the histo-pathological indicators such as the necrosis of hepatic cell, the depletion of GSH, a hepato-protective indicators in cholestatic liver diseases-induced animal model by galactosamine/LPS when the inventive extract or compound of the present invention was orally and intravenously administrated thereto. When the oral acute toxicity of the extract was tested, the extract had no apparent effect on mortality, clinical signs, body weight changes, and gross findings at necropsy.
The pharmaceutical composition for treating cholestatic liver diseases could contain about 0.01 to 99.9 w/w%, preferably 0.1 to 90 w/w% of the above crude drug composition of present invention based on the total weight of the composition.
The 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).
Hereinafter, the following formulation methods and excipients are merely exemplary and in no way limit the invention.
The 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. 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.
For example, the 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. For topical administration, the compounds of the present invention can be formulated in the form of ointments and creams.
Pharmaceutical formulations containing inventive 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 inventive 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 0.01-10g/kg, preferably, 1 to 5g/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. In terms of composition, 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.
The pharmaceutical 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.
In accordance with one aspect of the present invention, there provided a health functional food comprising the above extract or the compound for the prevention or improvement of cholestatic liver diseases as an active ingredient in an amount effective to prevent and improve cholestatic liver diseases, 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 cholestatic liver diseases could contain about 0.01 to 95 w/w%, preferably 0.5 to 80 w/w% of the above inventive composition of present invention based on the total weight of the composition.
Above described composition therein can be added to food, additive or beverage for prevention and improvement of cholestatic liver diseases. For the purpose of preventing and improving cholestatic liver diseases, wherein, 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㎖ of the health beverage composition.
Providing that the health beverage composition of present invention contains above described crude drug composition as an essential component in the indicated ratio, there is no particular limitation on the other liquid component, wherein the other component can be various deodorant or natural carbohydrate etc such as conventional beverage. Examples of aforementioned 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. As the other deodorant than aforementioned ones, natural deodorant such as taumatin, stevia extract such as levaudioside A, glycyrrhizin et al., and synthetic deodorant such as saccharin, aspartam et al., may be useful favorably. 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㎖ 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.
It will be apparent to those skilled in the art that various modifications and variations can be made in the compositions, use and preparations of the present invention without departing from the spirit or scope of the invention.
As mentioned above, the composition comprising an extract of licorice or liquiritigenin derived therefrom of the present invention significantly increases bile flow rate and biliary excretion of bile acid, GSH and bilirubin. Consistent with these results, the composition decreases the blood chemical markers including ALT as a hepatoprotective marker, and ALP and g-GTP, as cholestatasis markers, and histochemical markers including central necrosis and inflammation in galactosamine/lipopolysaccharide-induced hepato-toxicity animal model when the inventive extract or compound of the present invention was orally and intravenously administrated thereto. Accordingly, the inventive compositions according to the present invention are useful in the prevention and treatment of the cholestatic liver diseases and can be used as safe and efficient hepato-protective agent.
The above and other objects, features and other advantages of the present invention will more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which;
Fig. 1-A shows the chemical structure of M1 (4 -O-glucuronide) and M2 (7-O-gucuronide);
Fig. 1-B shows the change of LQ serum level according to the time in the test in case that liquiritigenin (LQ) was intravenously administrated to rat (50mg/kg);
Fig. 1-C shows the change of serum level of LQ metabolites, i.e., M1 and M2 according to the time in the test in case that liquiritigenin (LQ) was intravenously administrated to rat (50mg/kg);
Fig. 2-A represents the comparison effect of liquiritigenin treatment with control group on bile acid excretion when 50mg/kg LQ was intraveneously administrated to animal model;
Fig. 2-B represents that the choleretic effect of liquiritigenin induces the increase of bile acid excretion in bile juice when 50mg/kg of LQ was intraveneously administrated to animal model;
Fig. 2-C represents that the choleretic effect of liquiritigenin induces the increase of GSH excretion in bile juice when 50mg/kg LQ was intraveneously administrated to animal model;
Fig. 2-D represents that the choleretic effect of liquiritigenin induces the increase of bilirubin excretion in bile juice when 50mg/kg of LQ was intraveneously administrated to animal model;
Fig. 3-A represents that the intraveneous administration of 15mg/kg of LQ for 3 days increase the level of transporter protein involved in the bile acid excretion within liver tissue (the result of mRNA quantitative analysis verifying the increase of the transporter proteins, i.e., NTCP (Na+-dependent taurocholic cotransporting polypeptide) and OATP-1 (Organic anion transporting polypeptide-1) present in basolateral membrane involved in the influx of bile juice into hepatic cell; Canalicular membrane transporter proteins such as MRP2 (Multidrug resistance protein 2) and BSEP (bile salt export pump) involved in the output of bile juice from liver to biliary duct;
Fig. 3-B represents the result of Western blot analysis reflecting on the increase of MRP2, a main transporter protein involved in the output of bile acid;
Fig. 3-C represents the result of immuno-chemical staining analysis reflecting on the increase of MRP2, a main transporter protein involved in the output of bile acid;
Fig. 4-A represents the blood biochemical indicator analysis such as ALT (alanine aminotransferase), ALP (Alkaline phosphatase) and -GTP (glutamyltransferase) which proves the hepatoprotective effect on liver injury induced by galactosamine(GalN)/ lipopolysaccharide(LPS) when 15mg/kg of LQ was intraveneously administrated or 50mg/kg of LQ was orally administrated for 3 days;
Fig. 4-B represents the histochemical H&E staining analysis which proves the hepatoprotective effect on liver injury induced by galatosamine/ lipopolysaccharide when 15mg/kg of LQ was intraveneously administrated or 50mg/kg of LQ was orally administrated for 3 days;
Fig. 4-C represents the histochemical TUNEL staining analysis which proves the hepatoprotective effect on liver injury induced by galatosamine/ lipopolysaccharide when 15mg/kg of LQ was intraveneously administrated or 50mg/kg of LQ was orally administrated for 3 days;
Fig. 5-A presents the recovery of the decreased level of GSH, a representative index, which proves the hepatoprotective effect on liver injury induced by galatosamine/ lipopolysaccharide when 15mg/kg of LQ was intraveneously administrated or 50mg/kg of LQ was orally administrated for 3 days;
Fig. 5-B presents the recovery of the increased level of TNF- , a representative inflammatory index, which proves the hepatoprotective effect on liver injury induced by galatosamine/ lipopolysaccharide when 15mg/kg of LQ was intraveneously administrated or 50mg/kg of LQ was orally administrated for 3 days.
The following Examples and Experimental Examples are intended to further illustrate the present invention without limiting its scope.
Example 1. Preparation of inventive extract of licorice (GE-1)
3kg of Glycyrrhiza uralensis purchased from Kyung-dong market located in Seoul were washed, dried and 15 liter of 100% methanol was added thereto. The solution was left alone for 72 hours at room temperature and the extract was filtered with filter paper. The filtrate was concentrated with vacuum concentrator (N-21NS, ELYA) to obtain120g of brown colored gel-type crude extract of licorice (designated as GE-1 hereinafter).
Example 2. Preparation of hydrolyzed licorice extract (GE-2)
2l of 1N HCl was added to 120g of the extract (GE-1) prepared in Example 1 and performed to acid hydrolysis for 6 hrs at 100 ℃. The solution was neutralized with 2l of 1N NaOH solution to obtain about 90g of hydrolyzed licorice extract (designated as GE-2 hereinafter).
Example 3. Preparation of liquiritigenin (LQ)
The hydrolysate prepared in Example 2 was subjected to Silicagel column chromatography (60cm, 230-400 mesh) with eluting solution starting from CHCl3 to mixture solvent (CHCl3―MeOH= 50:1→15:1) to obtain 43g of purified fraction. The fraction was further subjected to Silicagel column chromatography (50cm, 230-400 mesh) with eluting solution of mixture solvent (CHCl3―acetone= 20:1→1:1) to obtain 17g of pale yellow colored liquiritin-abundant fractions, i.e., fractions 22-45 [Rf= 0.7, developing solvent: CHCl3―MeOH= 4:1].
The hydrolysate prepared in Example 2 was subjected to Silicagel column chromatography (60cm, 230-400 mesh) with eluting solution starting from CHCl3 to mixThe collected fractions comprising abundant liquiritin were further subjected to Silicagel column chromatography (50cm, 230-400 mesh) with eluting solution of mixture solvent (CHCl3―MeOH= 50:1→15:1) and performed to re-crystalyzation with dichloromethane to obtain 12g of white-powdered liquiritigenin extract (designated as LQ hereinafter).
Example 4. Preparation of inventive liquiritigenin-abundant licorice extract (GEF-1)
2000 ml of 1N HCl was added to 1kg of licorice extract prepared in Example 1 and reacted at 100 C for 6 hours to hydrolyze the sugar-moiety of liquiritin. The reactant was neutralized with 2000ml of 1N NaOH. The hydrolysate was repeatedly washed with distilled water to remove sugar component and salt component in order to obtain 800g of liquiritigenin-abundant licorice extract (designated as GEF-1 hereinafter).
Reference Example. Preparation of Experiment
Reagent and experimental animals
Galactosamine (Sigma Chemical Co.) and lipopolysaccharide (Sigma Chemical Co.) were purchased from commercial company to use in experimental. Polyethylene glycol #400 (Yakuri Co.) was diluted with physiological solution.
Male Sprague-Dawley rats (Samtako Co. Korea) weighing 140-160g were used in the experiment and were allowed to access to feed (Harlan, teklan, USA) and drinking water. All animals were maintained in a controlled environment with temperatures at 22±2 C and humidity at 55±5% with 12 hours of light and dark cycles for at least one week prior to use.
Statistics
All the result was analyzed by using pharmacological calculation. The significance between the test groups was evaluated by ANOVA (one-way analysis of variance) and determined by Newmann-Keuls test method (*p<0.5, **p<0.01).
Experimental Example 1. Effect of intravenously-administrated inventive extract and compound on bile juice excretion in rat model
In order to investigate the inhibitory effect of the inventive extract and compound obtained in Examples on bile juice excretion, following experiment was performed in the procedure.
Liquiritigenin dissolved in 40% PEG solution prepared in Reference Example in distilled water (50 mg/kg) were intravenously administrated into the rats once a day and bile juice was collected at every 1 hour interval after the end of treatment to analyze the component of bile juice.
At the result, the liquiritigenin intravenously administrated into the rats (50mg/kg) was transformed into its metabolites, i.e., M1 (4’-O-glucuronide) and M2 (7-O-glucuronide). The blood level of M1 and M2 reached to maximum within 5 ~ 30 mins and maintained till 3~6 hours ( See Fig.1).
After investigating the change of bile juice excretion and liquiritigenin metabolism, the excretion of bile juice was significantly increased within 0 ~ 2 hours and the excreted level of bile juice ingredients, i.e., bile juice, glutathione (GSH) and bilirubin were also increased after liquiritigenin (50mg/kg) was intravenously administrated ( See Fig.2).
Experimental Example 2. Effect of intravenously-administrated inventive extract and compound on the increase of transport protein expression in rat model
In order to investigate the inhibitory effect of the inventive extract and compound obtained in Examples on the increase of transporting protein expression which is closely involved in bile juice increasing effect, following experiment was performed in the procedure.
Liquiritigenin was intravenously administrated into the rats (15mg/kg) once a day for three days and then 1 hour after the treatment, the liver was delivered from the rats.
At the result, the increase of the serum level of two basolateral membrane proteins involved in the bile acid influx into hepatic cells, i.e., OATP-1 and NTCP as well as bile canaliculus membrane proteins involved in the bile acid excretion from liver to bile duct, i.e., MRP2 and BSEP was analyzed with mRNA analysis and the expression of mRNA in the group treated with liquiritigenin was sharply increased ( See Fig.3-A).
The level of MRP2, a main proteins correlated in bile excretion was significantly increased not only in mRNA analysis but also in protein and immuno-chemical staining analysis ( See Fig. 3-B and 3-C).
Experimental Example 3. Inhibitory effect of intravenously-administrated inventive extract and compound on galactosamine/LPS-induced cholestasis liver disease in rat model
In order to investigate the inhibitory effect of the inventive extract and compound obtained in Examples on galactosamine/LPS-induced cholestasis liver disease in rat model, following experiment was performed in the procedure.
Liquiritigenin was administrated into the rats intravenously three times a day for three days (15mg/kg) or orally three times a day for three days (50mg/kg). In order to give rise to cholestasis liver disease, galactosamine (600mg/kg) and LPS (1㎍/kg) were intraperitoneally administrated once and then 24 hours after the treatment, the blood and liver were delivered from the abdominal cavity of rats.
At the result, in both of different treatment test groups, the increase of hematological indicators, i.e., ALT, a hepatic injury indicator, and -GTP and ALP, cholestasis indicators were significantly inhibited ( See Fig. 4), which verify the inhibitory effect of liquiritigenin on the incidence and progress of cholestasis liver disease.
Furthermore, the inhibitory effect of liquiritigenin on the cholestatic hepatotoxicity was determined with H&E staining method and TUNEL staining method. It had been confimed that liquritigenin significantly reduced the central necrosis and apoptosis of hepatic cell induced by galactosamine/LPS, of which result is consistent with that in hematological analysis and directly verifies the inhibitory effect of liquritigenin on the incidence and progress of hepatic injury ( See Fig. 4).
Additionally, the amount of TNF-α, an inflammation indicators as well as an etiological cell apoptosis and that of GSH showing hepato-protective activity were determined. At the result, it has been confirmed that liquiritigenin effectively inhibited the increase of TNF-α and the decrease of GSH in the experiment ( See Fig. 5).
Hereinafter, the formulating methods and kinds of excipients will be described, but the present invention is not limited to them. The representative preparation examples were described as follows.
Preparation of powder
GEF-1 25mg
Corn Starch 20mg
Lactose 30mg
Mg stearate optimum amount
Powder preparation was prepared by mixing above components and filling sealed package.
Preparation of tablet
GEF-1 100mg
Corn Starch 10mg
Lactose 50mg
Magnesium stearate optimum amount
Tablet preparation was prepared by mixing above components and entabletting.
Preparation of capsule
GEF-1 10mg
Crystalline cellulose 3mg
Lactose 14.8mg
Magnesium stearate 0.2 mg
Tablet preparation was prepared by mixing above components and filling gelatin capsule by conventional gelatin preparation method.
Preparation of injection
GEF-1 10mg
Mannitol 180mg
Na2HPO4-12H2O 26mg
Distilled water for injection 1974mg
Injection preparation was prepared by dissolving active component, controlling pH to about 7.5 and then filling all the components in 2㎖ ample and sterilizing by conventional injection preparation method.
Preparation of liquid
GEF-1 20mg
Sugar 20g
Mannitol 5g
Distilled water optimum amount
Liquid preparation was prepared by dissolving active component, and then filling all the components in 1000㎖ ample and sterilizing by conventional liquid preparation method.
Preparation of health food
GEF-1 1000mg
Vitamin mixture optimum amount
Vitamin A acetate 70mg
Vitamin E 1.0mg
Vitamin B1 0.13mg
Vitamin B2 0.15mg
Vitamin B6 0.5mg
Vitamin B12 0.2mg
Vitamin C 10mg
Biotin 10mg
Amide nicotinic acid 1.7mg
Folic acid 50mg
Calcium pantothenic acid 0.5mg
Mineral mixture optimum amount
Ferrous sulfate 1.75mg
Zinc oxide 0.82mg
Magnesium carbonate 25.3mg
Monopotassium phosphate 15mg
Dicalcium phosphate 55mg
Potassium citrate 90mg
Calcium carbonate 100mg
Magnesium chloride 24.8mg
The above mentioned vitamin and mineral mixture may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention.
Preparation of health beverage
GEF-1 1000mg
Vitamin C 15g
Vitamin E(powder) 100g
Vitamin A 0.2g
Vitamin B1 0.25g
Vitamin B2 0.3g
Amide nicotinic acid 3.5g
Zinc oxide 3.5g
Ferrous lactate 19.75g
Distilled water optimum amount
Health beverage preparation was prepared by dissolving active component, mixing, stirred at 85 ℃ for 1 hour, filtered and then filling all the components in 1000㎖ ample and sterilizing by conventional health beverage preparation method.
The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.
The present invention is related to a composition comprising an extract of licorice or liquiritigenin derived therefrom, which significantly increases bile flow rate and biliary excretion of bile acid, GSH and bilirubin. Consistent with these results, the inventive composition comprising an extract of licorice or liquiritigenin derived therefrom decrease the blood chemical markers including ALT as a hepatoprotective marker, and ALP and γ-GTP, as cholestatasis markers, and histochemical markers including central necrosis and inflammation in galactosamine/lipopolysaccharide-induced hepato-toxicity animal model when the inventive extract or compound of the present invention was orally and intravenously administrated thereto. Accordingly, the inventive compositions according to the present invention are useful in the prevention and treatment of the cholestatic liver diseases and can be used as safe and efficient hepato-protective agent.

Claims (8)

  1. A pharmaceutical composition comprising a liquiritgenin abundant extract of licorice or liquiritigenin isolated therefrom as an active ingredient for treating or preventing cholestatic liver diseases and increasing bile flow, together with a pharmaceutically acceptable carrier.
  2. The pharmaceutical composition according to claim 1, wherein said liquiritgenin abundant extract is prepared by the procedure comprising the steps of: washing, drying licorice; subjecting to fractionation with non-polar solvent to remove non-polar solvent soluble substance at 1st step; adding strong acid to the fraction and performing acid-hydrolysis reaction to obtain hydrolyzed liquiritigenin abundant fraction at 2nd step; neutralizing the solution with strong base at 3rd step; and isolating the water-soluble fraction comprising sugar component with water to obtain purposed liquiritigenin-abundant extract of the present invention.
  3. The pharmaceutical composition according to claim 1, wherein said liquiritgenin abundant extract is liquiritigenin-abundant extract of licorice, which is prepared by the procedure consisting of the steps of: purifying the extract of licorice with repeated column chromatographic method to obtain purified liquiritin fraction; treating to acidic hydrolysis and neutralizing the pH of the solution with alkali; and subjecting column chromatography to obtain the purposed liquiritigenin-abundant extract.
  4. The pharmaceutical composition according to claim 1, wherein said the cholestatic liver diseases is hepatic cholestatic jaundice, cholestatic hepatitis, primary cholestatic hepatocirrhosis, acute or chronic cholestatic liver disease, primary sclerosing cholangeitis, cholelithiasis, transporting protein depression disease, biliary fibrosis, or biliary cancer.
  5. A use of the liquiritgenin abundant extract of licorice or liquiritigenin isolated therefrom for the manufacture of medicament employed for treating or preventing cholestatic liver diseases and increasing bile flow in human or mammal.
  6. A method for treating cholestatic liver diseases and increasing bile flow in human or mammal comprising administering to mammal in an effective amount of the liquiritgenin abundant extract of licorice or liquiritigenin isolated therefrom, together with a pharmaceutically acceptable carrier thereof.
  7. A health functional food comprising the liquiritgenin abundant extract of licorice or liquiritigenin isolated therefrom for preventing or improving cholestatic liver diseases and for increasing bile flow as an active ingredient in an amount effective to prevent and improve cholestatic liver diseases, together with a sitologically acceptable additive.
  8. A method for preparing inventive liquiritigenin-abundant extract comprising the steps of; washing, drying licorice; extracting with 10 to 15-fold volume of non-polar solvent such as n-hexane, chloroform, dichloromethane, or ethylacetate, at the temperature ranging from 10 to 100℃, for the period ranging from 1 hour to 7 days, with the extraction method selected from enfleuraging extraction, hot-water extraction, sonication extraction, SFE extraction method, etc,; filtering the residue to remove non-polar solvent soluble substance; mixing the residue with 10 to 15-fold volume of distilled water, alcohols such as methanol, ethanol and the like, or the mixtures thereof,; enfleuraging the solution at the temperature ranging from 10 to 100℃, for the period ranging from 1 hour to 7 days, with the extraction method selected from enfleuraging extraction, hot-water extraction, sonication extraction, SFE extraction method, etc, to obtain the polar solvent soluble fraction which removed the non-polar solvent soluble substances at 1st step; adding 5 to 40-fold volume of strong acid to the fraction and performing acid-hydrolysis reaction at the temperature ranging from 10 to 200℃, for the period ranging from 2 hour to 10 hours, to obtain hydrolyzed liquiritigenin abundant fraction at 2nd step; neutralizing the solution with strong base such as sodium hydroxide, potassium hydroxide etc at 3rd step; and isolating the water-soluble fraction comprising sugar component with water to obtain purposed liquiritigenin-abundant extract of the present invention.
PCT/KR2009/005451 2008-10-08 2009-09-24 A use of the liquiritigenin abundant extract or liquiritigenin derived therefrom for increasing bile flow, choleretic effect, and for preventing and treating cholestatic liver diseases WO2010041837A2 (en)

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