WO2011122805A2 - Composition comprenant de l'ajoène destinée à prévenir ou à traiter une maladie due à la surexpression du lxr-alpha - Google Patents

Composition comprenant de l'ajoène destinée à prévenir ou à traiter une maladie due à la surexpression du lxr-alpha Download PDF

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WO2011122805A2
WO2011122805A2 PCT/KR2011/002094 KR2011002094W WO2011122805A2 WO 2011122805 A2 WO2011122805 A2 WO 2011122805A2 KR 2011002094 W KR2011002094 W KR 2011002094W WO 2011122805 A2 WO2011122805 A2 WO 2011122805A2
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ajoene
lxr
alpha
srebp
extract
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WO2011122805A3 (fr
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Sang Geon Kim
Jae Ha Ryu
Ra Ok Jeon
Chang Yeob Han
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SNU R&DB Foundation
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SNU R&DB Foundation
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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)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/896—Liliaceae (Lily family), e.g. daylily, plantain lily, Hyacinth or narcissus
    • A61K36/8962—Allium, e.g. garden onion, leek, garlic or chives
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives

Definitions

  • the present invention is related to a composition comprising ajoene for preventing and treating a disease caused by over-expression of LXR-alpha and the use thereof.
  • LXR liver X receptor
  • a nuclear hormone receptor plays an important role in controlling a genetic transcription of several genes involved in cholesterol metabolism and homeostasis such as apoE, ABCA1, ABCG1, ABCG5, ABCG8, cholesterol 7alpha-hydroxylase and scavenger receptor class B type I (Biochem. Biophy. Res. Comm., 2000, 274: pp794-802) and LXR controls lipid metabolism by directly acting on SREBP-1c gene (Mol. Cell. Biol., 2001, 21:pp2991-3000).
  • LXR exists as two isoforms, LXR-alpha and LXRbeta, of which LXR-alpha mainly is distributed in liver and LXRbeta in the most of organs.
  • the LXR-alpha is activated by the action of oxysterols, highly abundant saccharides and synthetic ligands such as T0901017, GW3965 et al and in charge of controlling the expression of genes involved in adipogenesis and cholesterol homeostasis.
  • LXR-alpha potently increases the expression and activity of SREBP-1c, a key transcription factor controlling gene expression of adipogenesifunctional by acting as a lipid sensor in hepatic adipogenesis, which stimulates hepatic fatty acid synthesis and increases the level of blood triglyceride in hepatic tissue.
  • SREBP-1c-dependent pathway The activation of LXR-alpha causing to non-alcoholic hepatic lipidosis, can be classified into two pathways, i.e., SREBP-1c-dependent pathway and SREBP-1c-independent pathway.
  • SREBP-1c-dependent fatty liver appears by up-regulating the adipogenesis gene expression through the transcription of activation of LXR-alpha-mediated SREBP-1c.
  • SREBP-1c-independent fatty liver appears by increasing the expression of CD36 protein, a transporter of free fatty acid, which stimulates fatty acid mobilization to liver tissue.
  • LXR-alpha plays important roles in controlling the rennin secretion in kidney.
  • LXR-alpha and LXR-beta are abundantly expressed in juxtaglomerular cell producing rennin and T0901017 or GW3965, a LXR-alpha agonists, increases mRNA expression of rennin in kidney and then increases blood rennin activity (J. Clin. Invest., 2005, 115:pp1913-1922). Excessive increase of blood rennin gives rises to hyperreninemia resulting in hypertension and aldosteronism.
  • LXR inhibitor has been reported to be useful in treating ALD since LEXR controls the gene expression of ABCD2 involved in ALD (adrenoleukodystrophy) disease, a rare disease damaging neuronal cell through VLCFA’s entering into brain (J. Biol. Chem., 2005, 280: pp41243-41251).
  • SREBP Sterol Response Element Binding Protein
  • SRE Sterol Response Element
  • SRE Sterol Response Element
  • SREBP-1c is a transcription factor controlling the transcription of genes mainly involved in adipogenesis while SREBP-2 in cholesterol synthesis.
  • SREBP-1 and SREBP-2 are increased at kidney with aging and then the lipogenesis and the accumulation of triglyceride and cholesterol in kidney are increased, which plays important roles in glomerulosclerosis, proteinuria and nephropathy (Kidney Int., 2005, 68(6); pp2608-2620).
  • SREBP-1c targeting the catalyzing enzymes of fatty acid synthetic pathway such as FAS (fatty acid synthase), ACC (acetyl CoA carboxylase), SCD (Stearoyl CoA desaturase) etc in chage of adipogenesis in liver, is reported as a key factor of alcoholic or nonalcoholic liver steatosis (Int. J. Mol. Med., 2008, 21(4): pp507-511).
  • Ajoene is stable unsaturated disulfide compound produced from the sulfoxide rearrangement of allicin. Allin is transformed into allylsulfenic acid by the action of allinase enzyme stored in the vesicle of garlic, which results in forming two allicin.
  • the ajoene compound having allyl vinyl disulfide moiety has been reported to have various physiological activities, for example, anti-tumor, anti-bacterial and anti-platelets aggregation activities (Prostaglandins Leukot Essent Fatty Acids, Vol.49 (2), pp587-595, 1993), immune-modulating activity, hangover treating activity etc (Korea Patent Publication No. 10-2207-0017452).
  • LXR-alpha or SREBP-1 such as alcoholic or nonalcoholic liver steatosis, diabetes, fatty liver, hypertriglyceridemia, hyperreninemia, rennin-induced hypertension, aldosteronism, adrenoleukodystrophy, glomerulosclerosis, proteinuria, nephropathy etc and to study the pharmacological effect of garlic extract or ajoene isolated therefrom through various in vitro test and animal model tests, for example, inhibitory effect on the gene expression of LXR-alpha, the activation of LXR-alpha protein, the gene expression of SREBP-1, the activation of SREBP-1 using by HepG2 hepatic cell line as well as on the over-expressed LXR-alpha induced by high fat diet, the over-expressed SREBP-1 induced by high fat diet, the triglyceride content inhe
  • the present inventors have found that the ajoene-abundant garlic extract or ajoene isolated therefrom is effective in treating and preventing the disease caused by over-expression or hyper-activation of LXR-alpha or SREBP-1.
  • the present invention also provides a use of the ajoene-abundant garlic extract or ajoene isolated therefrom for the manufacture of medicament employed for treating or preventing the disease caused by over-expression or hyper-activation of LXR-alpha or SREBP-1 in human or mammal.
  • the present invention also provides a method for treating the disease caused by over-expression or hyper-activation of LXR-alpha or SREBP-1 in human or mammal comprising administering to said mammal 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 ajoene-abundant garlic extract or ajoene isolated therefrom as an active ingredient for treating or preventing the disease caused by over-expression or hyper-activation of LXR-alpha or SREBP-1.
  • the present invention also provides a health functional food comprising the above-described extract or the compound isolated therefrom for the prevention or improvement of the disease caused by over-expression or hyper-activation of LXR-alpha or SREBP-1 as an active ingredient in an amount effective to preventing and improving the disease caused by over-expression or hyper-activation of LXR-alpha or SREBP-1, together with a sitologically acceptable additive.
  • a pharmaceutical composition comprising the ajoene-abundant garlic extract or ajoene isolated therefrom as an active ingredient for treating or preventing the disease caused by over-expression or hyper-activation of LXR-alpha or SREBP-1, together with a pharmaceutically acceptable carrier.
  • the ajoene-abundant garlic extract disclosed herein comprise the extract of garlic including Allium sativum LINN, Allium scorodorpasum var. viviparum Regel or and the like, preferably, Allium sativum LINN, for example, which can be prepared by the procedure comprising the steps: washing and crushing garlic to pastes to be left alone for the period ranging from 1 hour to 48 hours, preferably, 2 hours to 28 hours at the temperature ranging from 0°C to 100°C, preferably, from room temperature to 50°C as the 1st fermentation step and subsequently, left alone for the period ranging from 1 hour to 48 hours, preferably, 2 hours to 28 hours at the temperature ranging from 0°C to 100°C, preferably, from 25°C to 50°C as the 2nd fermentation step in order to produce ajoene from alliin in garlic at the 1st step; subjecting to extraction with 5 to 20-fold, preferably, 10 to 15-fold volume of distilled water, alcohols such
  • a health functional food comprising a ajoene-abundant garlic extract or ajoene isolated therefrom for the prevention or improvement of the disease caused by over-expression or hyper-activation of LXR-alpha or SREBP-1 an active ingredient in an amount effective to prevent and improve the disease caused by over-expression or hyper-activation of LXR-alpha or SREBP-1, together with a sitologically acceptable additive.
  • the disease caused by over-expression or hyper-activation of LXR-alpha or SREBP-1 comprises alcoholic or nonalcoholic liver steatosis, diabetes, fatty liver, hypertriglyceridemia, hyperreninemia, rennin-induced hypertension, aldosteronism, adrenoleukodystrophy, glomerulosclerosis, proteinuria, nephropathy etc, preferably, alcoholic or nonalcoholic liver steatosis, diabetes, fatty liver.
  • the garlic 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 purposed diseases.
  • the pharmaceutical composition for treating purposed 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.
  • the above-described extract of garlic or ajoene isolated therefrom can be prepared by following procedure;
  • the garlic including Allium sativum LINN, Allium scorodorpasum var. viviparum Regel or and the like, preferably, Allium sativum LINN is washed and crushed to be pastes to be left alone for the period ranging from 1 hour to 48 hours, preferably, 2 hours to 28 hours at the temperature ranging from 0°C to 100°C, preferably, from room temperature to 50°C as the 1st fermentation step and subsequently, left alone for the period ranging from 1 hour to 48 hours, preferably, 2 hours to 28 hours at the temperature ranging from 0°C to 100°C, preferably, from 25°C to 50°C as the 2nd fermentation step in order to produce ajoene from alliin in garlic at the 1st step; subjected to extraction 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, water and ethanol mixture solvent at the temperature ranging
  • the ajoene-abundant extract is subjected to further purification process such as reverse phase HPLC, Silicagel column chromatography or re-crystallization method to obtain ajoene of the present invention or synthesized from the method well-known in the art, for example the preparation method disclosed in the literature (Bioorg. Med. Chem. Let., 2008, 18: pp5277-5279).
  • further purification process such as reverse phase HPLC, Silicagel column chromatography or re-crystallization method to obtain ajoene of the present invention or synthesized from the method well-known in the art, for example the preparation method disclosed in the literature (Bioorg. Med. Chem. Let., 2008, 18: pp5277-5279).
  • It is the other object of the present invention to provide a method for preparing ajoene-abundant extract from the extract of garlic comprising the steps consisting of; washing and crushing garlic to pastes to be left alone for the period ranging from 1 hour to 48 hours, preferably, 2 hours to 28 hours at the temperature ranging from 0°C to 100°C, preferably, from room temperature to 50°C as the 1st fermentation step and subsequently, left alone for the period ranging from 1 hour to 48 hours, preferably, 2 hours to 28 hours at the temperature ranging from 0°C to 100°C, preferably, from 25°C to 50°C as the 2nd fermentation step in order to produce ajoene from alliin in garlic at the 1st step; subjecting to extraction 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, water and ethanol mixture solvent at the temperature ranging from 0°C to 100°C,
  • 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 fiLXRated 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, 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.
  • 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 fiLXRate 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 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 the disease caused by over-expression or hyper-activation of LXR-alpha or SREBP-1.
  • the inventive composition of the present invention significantly inhibited the gene expression of LXR-alpha, the activation of LXR-alpha protein, the gene expression of SREBP-1, the activation of SREBP-1 using by HepG2 hepatic cell line as well as on the over-expressed LXR-alpha induced by high fat diet, the over-expressed SREBP-1 induced by high fat diet, the triglyceride content in hepatic cell, morphological change in hepatic cell, the weigh-gain induced by high fat diet, and the increased blood glucose level induced by high fat diet etc using by male C57BL/6 mice animal model when the inventive extract or compound of the present invention was orally and intravenously administrated thereto.
  • the inventive extract or compound of the present invention was orally and intravenously administrated thereto.
  • the pharmaceutical composition for treating purposed 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, 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, polyvinyl
  • 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 0.01-10g/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.
  • 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 the disease caused by over-expression or hyper-activation of LXR-alpha or SREBP-1 as an active ingredient in an amount effective to preventing and improving the disease caused by over-expression or hyper-activation of LXR-alpha or SREBP-1, 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.
  • a functional health food defined herein is “the functional food having enhanced functionality such as physical functionality or physiological functionality by adding the extract of the present invention to conventional food to prevent or improve aimed disease in human or mammal”.
  • a health functional food defined herein is the food containing inventive extract of the present invention showing no specific intended effect but general intended effect in a small amount of quantity as a form of additive or in a whole amount of quantity as a form of capsule, pill, tablet etc.
  • a sitologically acceptable additive is “any substance the intended use which results or may reasonably be expected to result-directly or indirectly-in its becoming a component or otherwise affecting the characteristics of any food” for example, thickening agent, maturing agent, bleaching agent, sequesterants, humectant, anticaking agent, clarifying agents, curing agent, emulsifier, stabilizer, thickner, bases and acid, foaming agents, nutrients, coloring agent, flavoring agent, sweetner, preservative agent, antioxidant, etc, which had been well-known in the art.
  • direct additive a substance that becomes part of the food in trace amounts due to its packaging, storage or other handling.
  • Health foods can be contained in food, health beverage, dietary therapy etc, and may be used as a form of powder, granule, tablet, chewing tablet, capsule, beverage etc for preventing or improving aimed disease.
  • 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 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 purposed diseases.
  • 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 100ml 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 100ml 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 inventive extract or compound significantly inhibited the gene expression of LXR-alpha, the activation of LXR-alpha protein, the gene expression of SREBP-1, the activation of SREBP-1 using by HepG2 hepatic cell line as well as on the over-expressed LXR-alpha induced by high fat diet, the over-expressed SREBP-1 induced by high fat diet, the triglyceride content in hepatic cell, morphological change in hepatic cell, the weigh-gain induced by high fat diet, and the increased blood glucose level induced by high fat diet.
  • the inventive compositions according to the present invention are useful in the prevention and treatment of the liver diseases and can be used as safe and efficient hepato-protective agent.
  • Fig. 1 shows the inhibition effect on LXR-alpha expression (Con: control, **: comparison with control group p ⁇ 0.01, #: comparison with the group treated with T0901317 only, p ⁇ 0.05);
  • Fig. 2 shows the inhibition effect on LXRE (Con: control, **: comparison with control group p ⁇ 0.01, #: comparison with the group treated with T0901317 only, p ⁇ 0.05, ##: comparison with the group treated with T0901317 only, p ⁇ 0.01);
  • Fig. 3 shows the inhibition effect on SREBP-1 expression (Con: control, **: comparison with control group p ⁇ 0.01, #: comparison with the group treated with T0901317 only, p ⁇ 0.05, ##: comparison with the group treated with T0901317 only, p ⁇ 0.01);
  • Fig. 4 represents the inhibition effect on increased LXR expression in hepatic tissue caused by high fat diet (ND: normal diet, HFD: High fat diet, Ajo: ajoene, **: comparison with ND group p ⁇ 0.01, #: comparison with the HFD group only, p ⁇ 0.05, ##: comparison with the HFD group only, p ⁇ 0.01);
  • Fig. 5 represents the inhibition effect on increased triglyceride level in hepatic tissue caused by high fat diet (ND: normal diet, HFD: High fat diet, Ajo: ajoene, **: comparison with ND group p ⁇ 0.01, #: comparison with the HFD group only, p ⁇ 0.05, ##: comparison with the HFD group only, p ⁇ 0.01);
  • Fig. 6 represents the treatment protocol of ajoene and the inhibition effect on weight gain change caused by high fat diet (ND: normal diet, HFD: High fat diet, Ajo: ajoene **: comparison with ND group p ⁇ 0.01, #: comparison with the HFD group only, p ⁇ 0.05, ##: comparison with the HFD group only, p ⁇ 0.01);
  • Fig. 7 presents the inhibition effect on the blood glucose level of the mice caused by high fat diet (ND: normal diet, HFD: High fat diet, Ajo: ajoene, **: comparison with ND group p ⁇ 0.01, #: comparison with the HFD group only, p ⁇ 0.05, ##: comparison with the HFD group only, p ⁇ 0.01);
  • Ajoene compound was chemically synthesized by the method disclosed in the literature (Bioorg. Med. Chem. Lett., 2008, 18:pp5277-5279) as follows:
  • Allylpropargyl sulfide was synthesized from ally thiol as a starting material through propargylation and vinylthioacetate was synthesized through regioselective radical addition in the presence with azobisisobutyronitrile (AIBN). Resulting thiolacetate was subjected to deprotection and sulfenylation using by S-allyl-p-toluene sulfonyl thioate to afford vinyl disulfide.
  • AIBN azobisisobutyronitrile
  • the disulfide was oxidized with m-CPBA (meta-chloroperoxybenzoic acid) to afford ajoene compound, which was confirmed by comparing the spectral analysis such as MS and H1-NMR spectroscopy with that disclosed in literature.
  • m-CPBA metal-chloroperoxybenzoic acid
  • mice Male C57BL/6 mice (25 to 30g) were purchased from Charles River Orient Co. (Seoul, Korea) and used in the experiment and were allowed to access to feed 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.
  • mice The morphological changes of mice have been checked once a week and the mice were divided into two groups, one group fed with high fat feed (Dyets Inc., Bethlehem) to induce fatty liver and another group with normal feed.
  • cDNA was obtained from 2 microgram of total RNA extracted from mouse liver, d(T) 16 primer and AMV reverse transcriptase.
  • the relative contents of genes was quantitatively determined according to realtime RT-PCR method using by CyBrgreen dye and real time RT-PCR was performed by using Light-cycler2.0 (Roche,Mannheim,Germany).
  • PCR was performed according the manufacture’s manual and relative amount of each gene was analyzed using Light-cycler software 4.0 program.
  • SDS-PAGE Sodium dodecylsulfate-polyacrylamide gel electrophoresis was performed by using Might Smal II SE 250 apparatus according to the method well-known in the art (Laemmli UK method, 1970).
  • Dissolution fraction of each sample was diluted in sample dilution buffer containing 63mM Tris (pH. 6.8), 10% glycerol, 2% SDS, 0.0013% bromophenol blue, and 5% beta-mercaptoethanol and subjected to electrophoresis using 7.5% and 9% gel in electrode buffer (containing 15g of Tris, 72g of glycine and 5g of SDS in 1 L).
  • the finished gel was electroblotted onto nitrocellulose membrane in buffer [25mM Tris, 192mM glycine and 20% v/v methanol, pH 8.3] with 190mAmps for 1 hour.
  • Anti-SREBP-1 was reacted as a primary antibody and horse radish peroxidase-conjugated goat anti-rabbit IgG was reacted for 1 hour as a secondary antibody.
  • the result was developed using by ECL chemi-luminecence system (Amersham, Gaithesberg, MA) and the homogenecity of protein content between each sample was confirmed using by anti-beta-actin antibody (Sigma St. Louis, MO).
  • test groups All the result was analyzed by using pharmacological calculation.
  • the significance between the test groups was evaluated by ANOVA (one-way analysis of variance; Fisher R.A., statistical Methods for Research Workers, Edinburgh: Oliver & Boyd., 1925) and determined by Newmann-Keuls test method (*p ⁇ 0.05, **p ⁇ 0.01; Norman GR et al., Biostatistics: The Bare Essentials, 2000).
  • LXR-alpha is bound to specific region existing in target gene promoter (LXRE) to regulate the gene expression.
  • Luciferase reporter gene assay was performed to observe whether LXRE binding affinity is changed or not.
  • LXRE comprising vector was transfected to HepG2 cell using by lipofectamine (Invitrogen, USA). 12 hours after the transfection, 10 micromole and 30 micromole ajoene (Ajo) were pretreated to HepG2 cell respectively, for 1 hour and 10 micromole T0901317, LXR-alpha activator was pretreated for 12 hours. Cell lysate was obtained using by passive lysis buffer (Promega, USA) and Luciferase activity was determined using by Luminometer.
  • mRNA and protein of SREBP-1 in case with the group treated with T0901317 and the group treated with T091317 and ajoene, was compared. 10 micromole and 30 micromole Ajoene were pretreated to HepGe cell line, for 1 hour and 10 micromole T0901317, a LXR-alpha activator was treated thereto for 12 hours. Protein was isolated and the protein change of SREBP-1 was determined by Western blotting method. mRNA was isolated from the hepatic cell line. cDNA was synthesized through RT-PCR method and realtime PCR was performed using by specific primers (Human SREBP-1, sense: 5’-CGACATCGAAGACATGCTTCAG-3’(SEQ. I.D.3); ant-sense: 5’-GGAAGGCTTCAAGAGAGGAGC-3’(SEQ. I.D.4)).
  • LXR-alpha a cellular lipid sensor
  • the expression of LXR-alpha was analyzed by real time PCR using by specific primer after cDNA synthesis.
  • the mice were bred with high fat feed and normal feed for 8 weeks and the LXR-alpha expression of hepatic cell in the group treated with high fat feed and ajoene (10 or 30 mg/kg, 5 times/week) for 4 weeks was determined.
  • mRNA was isolated from the hepatic cell line. cDNA was synthesized through RT-PCR method and realtime PCR was performed using by specific primers (mouse LXR, sense: 5’-TGCCATCAGCATCTTCTCTG-3’(SEQ. I.D.5); anti-sense: 5’-GGCTCACCAGCTTCATTAGC-3’(SEQ. I.D.6)).
  • Relative LXR-alpha mRNA in the group treated with high fat feed or both of high fat feed and ajoene was shown where that in the group treated with normal diet (ND) is set to 1.
  • mRNA was isolated from the hepatic cell line. cDNA was synthesized through RT-PCR method and realtime PCR was performed using by specific primers (mouse SREBP-1, sense: 5’-AACGTCACTTCCAGCTAGAC-3’(SEQ. I.D.7); anti-sense: 5’-CCACTAAGGTGCCTACAGAGC-3’(SEQ. I.D.8)).
  • Relative SREBP-1 mRNA in the group treated with high fat feed or both of high fat feed and ajoene was shown where that in the group treated with normal diet (ND) is set to 1.
  • the triglyceride level in hepatic tissue is an indicator of fatty liver.
  • the mice were bred for 8 weeks with high fat diet and normal diet and ajoene was administrated the mice for final 4 weeks 5 times a week, at the dose of 10mg/kg and 30mg/kg.
  • the triglyceride level in hepatic tissue caused by with high fat feed was observed.
  • the triglyceride level of hepatic tissue in the group treated with 10mg/kg and 30mg/kg of ajoene was sharply reduced to 36mg/g (liver) and 31mg/g(liver) respectively, whereas that of the group treated with high fat diet and normal diet had been increased to 61mg/g (liver) and 27mg/g (liver), respectively.
  • the hepatic tissues prepared in Experimental Example 5 were fixed with 10% formalin solution, dehydrated and embedded with paraffin.
  • the hepatic tissue was sliced to 4 micrometer of width to be stained with dye (Oil Red O).
  • the morphological change of hepatic tissue was observed by optical spectroscopy.
  • mice were bred for 8 weeks with high fat diet (4 animals) and normal diet (4 animals) and ajoene was administrated the mice for final 4 weeks 5 times a week, at the dose of 10mg/kg and 30mg/kg. The weight gain of each mouse caused by high fat feed was observed.
  • mice were bred for 8 weeks with high fat diet (4 animals) and normal diet (4 animals) and ajoene was administrated the mice for final 4 weeks 5 times a week, at the dose of 10mg/kg and 30mg/kg. 0.01 ml of blood sample was collected from the tail vein of each mouse caused by high fat feed and the blood glucose level was determined by kit (Accucheck blood glucose monitoring apparatus, Roche Co.).
  • 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 2ml ample and sterilizing by conventional injection preparation method.
  • Liquid preparation was prepared by dissolving active component, and then filling all the components in 1000ml ample and sterilizing by conventional liquid preparation method.
  • Vitamin A acetate 70mg
  • Vitamin E 1.0mg
  • Vitamin B 1 0.13mg
  • Vitamin B 6 0.5mg
  • Vitamin B 12 0.2mg
  • Vitamin A 0.2g
  • Vitamin B 1 0.25g
  • Vitamin B 2 0.3g
  • Health beverage preparation was prepared by dissolving active component, mixing, stirred at 85°C for 1 hour, filtered and then filling all the components in 1000ml ample and sterilizing by conventional health beverage preparation method.
  • the inventive extract or compound significantly inhibited the gene expression of LXR-alpha, the activation of LXR-alpha protein, the gene expression of SREBP-1, the activation of SREBP-1 using by HepG2 hepatic cell line as well as on the over-expressed LXR-alpha induced by high fat diet, the over-expressed SREBP-1 induced by high fat diet, the triglyceride content in hepatic cell, morphological change in hepatic cell, the weigh-gain induced by high fat diet, and the increased blood glucose level induced by high fat diet.
  • the inventive compositions according to the present invention are useful in the prevention and treatment of the liver diseases and can be used as safe and efficient hepato-protective agent.

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Abstract

La présente invention concerne une composition comprenant, comme principe actif, de l'extrait d'ail concentré en ajoène, ou de l'ajoène qui en a été extrait, destinée à traiter ou à prévenir la maladie due à la surexpression ou à l'hyper-activation du LXR-alpha ou du SREBP-1, conjointement avec un transporteur pharmaceutiquement acceptable.
PCT/KR2011/002094 2010-04-01 2011-03-25 Composition comprenant de l'ajoène destinée à prévenir ou à traiter une maladie due à la surexpression du lxr-alpha Ceased WO2011122805A2 (fr)

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KR1020100029984A KR101176618B1 (ko) 2010-04-01 2010-04-01 아조엔을 유효성분으로 포함하는 엘엑스알-알파 과다 발현으로 인한 질병의 예방 및 치료용 조성물
KR10-2010-0029984 2010-04-01

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104887644A (zh) * 2015-05-20 2015-09-09 青岛博恩高科生物技术有限公司 一种阿藿烯胶囊
JP2017538695A (ja) * 2014-11-25 2017-12-28 ニーム バイオテク リミティド アホエンを製造するための方法
CN115166085A (zh) * 2022-07-07 2022-10-11 王超 一种测定软胶囊类保健食品中阿藿烯的方法

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997025031A1 (fr) 1996-01-11 1997-07-17 Davidson John B Procedes d'utilisation de modulateurs des integrines
JP2001302508A (ja) * 2000-04-24 2001-10-31 Nagoya Seiraku Kk 抗糖尿病剤
KR100622100B1 (ko) * 2005-09-12 2006-09-11 (주)에이티 랩 아호엔을 함유하는 화장료 조성물

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2017538695A (ja) * 2014-11-25 2017-12-28 ニーム バイオテク リミティド アホエンを製造するための方法
CN104887644A (zh) * 2015-05-20 2015-09-09 青岛博恩高科生物技术有限公司 一种阿藿烯胶囊
CN115166085A (zh) * 2022-07-07 2022-10-11 王超 一种测定软胶囊类保健食品中阿藿烯的方法
CN115166085B (zh) * 2022-07-07 2023-12-12 王超 一种测定软胶囊类保健食品中阿藿烯的方法

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