KR20120011169A - Composition for prevention or treatment of cancer or HIV - Google Patents

Abstract

PURPOSE: A composition for preventing or treating cancer of HIV is provided to suppress AKT activation and PI3K and/or to activate GSK3beta. CONSTITUTION: A pharmaceutical composition for preventing HIV infection, AIDS, HIV dementia, or HIV neural cell damage contains lancemacide A, lancemacide X, echinocystic acid 3-O-beta-D-glucuronopyranoside, echinocystic acid, compound K, arctigenin, arctiin, naringenin, ginsenoside Rh1, ginsenoside Rh2, kalopanaxsaponin A, tangeretin, nobiletin, apigenin, kaempherol, Codonopsis lanceolata (S. et. Z.) TRAUTV extract, ginseng extract, ginseng fermentation, Arctium lappa extract or Arctium lappa fermeantion as an active ingredient.

Description

Composition for the treatment or prevention of cancer or human immunodeficiency virus (HIB) {Composition for prevention or treatment of cancer or HIV}

The present invention relates to a composition for the treatment (improvement) or prevention of cancer or human immunodeficiency virus, a PI3K inhibitor, an AKT activation inhibitor and a GS3K vector activator, and in particular, cancer or human immunodeficiency virus (HIV) infection, acquired immunity. A pharmaceutical composition, a food composition, a PI3K inhibitor, an AKT activation inhibitor, and a GS3Kbeta activator which are effective in the treatment (improvement) or prevention of one or more selected from deficiency (AIDS), HIV dementia, or HIV neuronal damage.

PI3K, an essential signaling pathway for cancer growth, is essential for a variety of cancers, including breast cancer. PI3K is an enzyme that converts phosphatidylinositol 3,5-bisphosphate (PIP2) into phosphatidylinositol 3,4,5-triphosphate (PIP3), an active signaling intermediate.

PIP3 activates pyruvate dehydrogenase kinase 1 (PDK1) followed by AKT. PI3K consists of p110 and p85, and PTEN is a negative regulator of PI3K that dephosphorylates PIP3 and inhibits the PI3K signaling pathway.

AKT activated by PI3K is a master switch that is involved in the development of all cancers and leukemias. Once AKT signal is switched on, it does not prevent it and stimulates cancer growth. If we can inhibit the AKT enzyme, all cancers and leukemia stop.

Thus, there is a need for the development of AKT inhibitors for the treatment or prevention of cancer.

Human immunodeficiency virus (HIV-1 virus), on the other hand, causes AIDS and has been spreading since it was first isolated in the 1980s.

HIV-1 infects CD4 ⁺ lymphocytes, induces cell death and decreases immune mechanisms, but is not a good hiding place for HIV-1. Representative hides of HIV-1 include macrophages and microglia cells, and the TAT protein of HIV-1 virus activates AKT so that infected cells (macrophage, microglia cells, etc.) can continue to proliferate without dying. Activation of AKT inhibits the activation of GSK3β in cells such as macrophage / microglia, thereby promoting the action of converting to glycolysis, which kills HIV-1 infected cells. It prevents the production of toxic substances and increases neuronal damage, resulting in HIV dementia and HIV neuronal damage as well as acting as HIV-1 latent cells.

Therefore, in order to prevent HIV-1 dementia and HIV-1 neuronal damage, and to completely remove HIV-1 from HIV-1 / AIDS patients, therapies effectively remove HIV reservoir cells that are present in the body in a latent manner. Development is urgent.

The inventors have completed the present invention as a result of research for the development or treatment of cancer, HIV and the like, AKT activation inhibitors, GSK3beta activators, and PI3K inhibitors.

One problem to be solved by the present invention is to provide a composition for the treatment (improvement) or prevention selected from human immunodeficiency virus (HIV) infection, HIV dementia, or HIV neuronal damage.

Another object of the present invention is to provide a composition for the treatment (improvement) or prevention of cancer.

Another object of the present invention is to provide an AKT activation inhibitor.

Another problem to be solved by the present invention is to provide a PI3K inhibitor.

Another object of the present invention is to provide a GSK3beta activator.

The present invention relates to lancemacide A (3-O-β-D-glucuronopyranosyl-3β, 16α-dihydroxyolean-12-en-28-oic acid 28-O-β-D-xylopyranosyl (1 → 3) -β-D-xylopyranosyl- (1 → 4) -α-L-rhamnopyranosyl- (1 → 2) -α-Larabinopyranosyl ester], lancemacide X [echinocystic acid 28- O -β-D- xylopyranosyl (1 → 3) -β-D-xylopyranosyl- (1 → 4) -α-L-rhamnopyranosyl- (1 → 2) -α-L-arabinopyranosyl ester], Ikanocyst acid 3- O- β-D Echinocystic acid 3- O- β-D-glucuronopyranoside (EAG), echinocystic acid (EA), compound K [20-S-β-D-glucopuranosyl -protopanaxadiol], arctigenin [(3R, 4R) -4-[(3,4-dimethoxyphenyl) methyl] -3-[(4-hydroxy-3-methoxyphenyl) methyl] oxolan-2-one], Actinin [(3R, 4R) -4-[(3,4-dimethoxyphenyl) methyl] -3-[[3-methoxy-4-[(2S, 3R, 4S, 5S, 6R) -3, 4,5-trihydroxy-6- (hydroxymethyl) oxan-2-yl] oxyphenyl] methyl] oxolan-2-one], naringenin [5,7-dihydroxy-2- (4- hydroxyphenyl) -2,3-dihydrochromen-4-one], ginsenoside Rh1 ((2R, 3R, 4S, 5S, 6R) -2-[[(3S, 5R, 6S, 8R, 9R, 10R, 12R, 13R, 14R, 17S) -3,12-dihydroxy-17-[(2S) -2-hydroxy-6-methylhept-5-en-2-yl] -4,4,8,10,14 -pentamethyl-2,3,5,6,7,9,11,12,13,15,16,17-dodecahydro-1H-cyclopenta [a] phenanthren-6-yl] oxy] -6- (hydroxymethyl) oxane -3,4,5-triol], ginsenoside Rh2 [2-[[(3S, 8R, 9R, 10R, 12R, 14R, 17S) -12-hydroxy-17-[(2S)- 2-hydroxy-6-methylhept-5-en-2-yl] -4,4,8,10,14-pentamethyl-2,3,5,6,7,9,11,12,13,15,16 , 17-dodecahydro-1H-cyclopenta [a] phenanthren-3-yl] oxy] -6- (hydroxymethyl) oxane-3,4,5-triol], kalopanaxsaponin A [3-O-alpha -L-rhamnopyranosyl (1 → 2) -alpha-L-arabinopyranosyl-Hederagenin], tangeretin [5,6,7,8-tetramethoxy-2- (4-methoxyphenyl) chromen-4-one], nobyl Nobiletin [2- (3,4-dimethoxyphenyl) -5,6,7,8-tetramethoxychromen-4-one], apigenin [5,7-dihydroxy-2- (4-hydroxyphenyl) chromen -4-one], kaempherol [3,5,7-trihydrox] y-2- (4-hydroxyphenyl) chromen-4-one], deodeok extract, Haedongpi extract, ginseng extract, ginseng fermentation, allium extract or allium fermentation Provided is a pharmaceutical composition for treating or preventing one or more selected from human immunodeficiency virus (HIV) infection, acquired immunodeficiency syndrome (AIDS), HIV dementia, or HIV neuronal damage.

The immunodeficiency virus may be HIV-1.

The human immunodeficiency virus infection may be a latent human immunodeficiency virus latent infection.

The human immunodeficiency virus infection may be one or more cellular infections selected from macrophage or microglia cells.

The acquired immunodeficiency syndrome (AIDS) may be due to HIV infection.

The HIV dementia, or HIV neuronal damage is meant to include neuronal cell damage and / or dementia caused by human immunodeficiency virus latent infection in the body.

In addition, the present invention relates to lancemacide A (3-O-β-D-glucuronopyranosyl-3β, 16α-dihydroxyolean-12-en-28-oic acid 28-O-β-D-xylopyranosyl (1 →). 3) -β-D-xylopyranosyl- (1 → 4) -α-L-rhamnopyranosyl- (1 → 2) -α-Larabinopyranosyl ester], lancemacide X [echinocystic acid 28- O -β- D-xylopyranosyl (1 → 3) -β-D-xylopyranosyl- (1 → 4) -α-L-rhamnopyranosyl- (1 → 2) -α-L-arabinopyranosyl ester], Ikanocyst acid 3- O -β -D-glucuronopyranoside (echinocystic acid 3- O- β-D-glucuronopyranoside (EGA), ecanocystic acid, compound K [20-S-β-D-glucopuranosyl -protopanaxadiol], arctigenin [(3R, 4R) -4-[(3,4-dimethoxyphenyl) methyl] -3-[(4-hydroxy-3-methoxyphenyl) methyl] oxolan-2-one], Actinin [(3R, 4R) -4-[(3,4-dimethoxyphenyl) methyl] -3-[[3-methoxy-4-[(2S, 3R, 4S, 5S, 6R) -3, 4,5-trihydroxy-6- (hydroxymethyl) oxan-2-yl] oxyphenyl] methyl] oxolan-2-one], naringenin [5,7-dihydroxy-2 -(4-hydroxyphenyl) -2,3-dihydrochromen-4-one], ginsenoside Rh1 [(2R, 3R, 4S, 5S, 6R) -2-[[(3S, 5R, 6S, 8R, 9R, 10R, 12R, 13R, 14R, 17S) -3,12-dihydroxy-17-[(2S) -2-hydroxy-6-methylhept-5-en-2-yl] -4,4,8 , 10,14-pentamethyl-2,3,5,6,7,9,11,12,13,15,16,17-dodecahydro-1H-cyclopenta [a] phenanthren-6-yl] oxy] -6- (hydroxymethyl) oxane-3,4,5-triol], ginsenoside Rh2 [2-[[(3S, 8R, 9R, 10R, 12R, 14R, 17S) -12-hydroxy-17- [ (2S) -2-hydroxy-6-methylhept-5-en-2-yl] -4,4,8,10,14-pentamethyl-2,3,5,6,7,9,11,12,13 , 15,16,17-dodecahydro-1H-cyclopenta [a] phenanthren-3-yl] oxy] -6- (hydroxymethyl) oxane-3,4,5-triol], kalopanaxsaponin A [3 -O-alpha-L-rhamnopyranosyl (1 → 2) -alpha-L-arabinopyranosyl-Hederagenin], tangeretin [5,6,7,8-tetramethoxy-2- (4-methoxyphenyl) chromen-4- one], nobiletin [2- (3,4-dimethoxyphenyl) -5,6,7,8-tetramethoxychromen-4-one], apigenin [5,7-dihydroxy-2- (4 -hydroxyphenyl) chromen-4-one], kaempherol [3,5,7-trihy droxy-2- (4-hydroxyphenyl) chromen-4-one], Deodeok Extract, Haedongpi Extract, Ginseng Extract, Ginseng Fermentation, Allium Extract, or Allium Fermented Extract as an active ingredient Provided is a food composition for improving or preventing one or more selected from human immunodeficiency virus (HIV) infection, acquired immunodeficiency syndrome (AIDS), HIV dementia, or HIV neuronal damage.

In the food composition of the present invention, the same applies as long as there is no contradiction mentioned in the pharmaceutical composition of the present invention.

In addition, the present invention is prophylaxis side A (lancemacide A), Ica Ino cyst acid 3- O -β-D- glucuronic in nopi Llano side (echinocystic acid 3- O -β-D -glucuronopyranoside; EAG), Ica Ino It provides a pharmaceutical composition for the treatment or prevention of cancer containing at least one selected from citric acid, actinin, arconiin, kalopanaxsaponin A, deodeok extract, haejupi extract, or allium extract as an active ingredient. do.

The cancer is preferably at least one selected from lung cancer, breast cancer, stomach cancer or colon cancer.

In addition, the present invention is prophylaxis side A (lancemacide A), Ica Ino cyst acid 3- O -β-D- glucuronic in nopi Llano side (echinocystic acid 3- O -β-D -glucuronopyranoside; EAG), Ica Ino Provides a food composition for improving or preventing cancer containing as an active ingredient at least one selected from citric acid (arctiin), actin (arctiin), kalopanaxsaponin A, deodeok extract, thaw skin extract, or alligator extract do.

In the food composition of the present invention, the same applies as long as there is no contradiction mentioned in the pharmaceutical composition of the present invention.

In the composition of the present invention, the treatment (improvement) or prevention may be due to one or more selected from phosphoinositide 3-kinase (PI3K) inhibition, AKT activation inhibition, or GSK3beta (Glycogen synthase kinase 3 beta) activation. The PI3K is an enzyme that converts phosphatidylinositol 3,5-bisphosphate (PIP2) into an active signaling intermediate, phosphatidylinositol 3,4,5-triphosphate (PIP3), and PI3K inhibition includes the inhibition of enzymatic activity of PI3K. By inhibiting the action of PI3K, activation of PIP2 (conversion to PIP3) can be inhibited, and as a result, activation of pyruvate dehydrogenase kinase 1 (PDK1) can be inhibited. In addition, PI3K inhibition is meant to include the inhibition of PIP2 activation and / or PDK1 activation thereby. The inhibition of AKT activation may be by inhibiting phosphorylation of AKT. The GSK3beta activation may be to restore or maintain the phosphorylation to the ground state by inhibiting phosphorylation.

In the present invention, the extract may be a solvent extract, the solvent may be water, and / or an alcohol having 1 to 4 carbon atoms. In addition, the fermented product may be one obtained by fermenting or extracting natural products such as ginseng, or fermented extract, the fermentation may be lactic acid bacteria fermentation.

The present invention also provides an AKT activation inhibitor containing at least one selected from lancemacide A, compound K, compound decoction, or ginseng fermentation as an active ingredient.

The AKT activation inhibitor can treat, ameliorate or prevent one or more selected from human immunodeficiency virus (HIV) infection, HIV dementia, HIV neuronal damage, acquired immunodeficiency syndrome (AIDS) or cancer by inhibiting AKT activation.

In addition, the present invention provides a PI3K (Phosphoinositide 3-Kinase) inhibitor containing at least one selected from actigenin or allied fermentation as an active ingredient. The inhibitor can treat, ameliorate or prevent one or more selected from human immunodeficiency virus (HIV) infection, HIV dementia, HIV neuronal damage, acquired immunodeficiency syndrome (AIDS) or cancer by PI3K inhibition.

In addition, the present invention GSK3beta (lancemacide A), compound K (compound K), actigenin (arctigenin), deodeok extract, ginseng fermentation or allium fermentation containing one or more selected GSK3beta ( Glycogen synthase kinase 3 beta) activator. The activator can treat, ameliorate or prevent one or more selected from human immunodeficiency virus (HIV) infection, HIV dementia, HIV neuronal damage, acquired immunodeficiency syndrome (AIDS) or cancer.

In the present invention, the food composition may be a health functional food, the formulation is prepared according to a conventional method, and may be encapsulated after drying with a carrier or in the form of other tablets, granules, powders, beverages, porridge and the like. In addition to the above, it can be produced in any food form.

The active ingredient contained in one or more therapeutic (improvement) or preventive compositions selected from human immunodeficiency virus (HIV) infection, acquired immunodeficiency syndrome (AIDS), HIV dementia, or HIV neuronal damage of the present invention is a human immunodeficiency deficiency. It has the effect of treating, ameliorating and / or preventing viral (HIV) infections, acquired immunodeficiency syndrome (AIDS), HIV dementia, and / or HIV neuronal damage, and is contained in compositions for the treatment (improvement) or prevention of cancer. The active ingredient exhibits the effect of treating, improving and / or preventing cancer. In addition, the active ingredient contained in the AKT activation inhibitor of the present invention, the AKT activation inhibitory effect, the active ingredient contained in the PI3K inhibitor, the PI3K inhibitory effect, the active ingredient contained in the GSK3 beta activator shows a GSK3 beta activation effect.

Accordingly, the present invention is effective in the composition for the treatment (improvement) or prophylaxis selected from human immunodeficiency virus (HIV) infection, acquired immunodeficiency syndrome (AIDS), HIV dementia, or HIV neuronal damage of the present invention. Composition for the treatment, amelioration and / or prophylaxis of human immunodeficiency virus (HIV) infection, AIDS, HIV dementia, and / or HIV neuronal damage, treatment (improvement) or prevention of cancer For the treatment, improvement and / or prophylaxis of the contained active ingredient, for inhibiting AKT activation of the active ingredient contained in the AKT activation inhibitor of the present invention, for inhibiting PI3K of the active ingredient contained in the PI3K inhibitor, in the GSK3beta activator It provides a GSK3beta activation of the active ingredient.

In addition, the present invention is effective in contained in the composition for the treatment (improvement) or prevention selected from human immunodeficiency virus (HIV) infection, acquired immune deficiency syndrome (AIDS), HIV dementia, or HIV neuronal damage of the present invention Treating, ameliorating, and / or treating human immunodeficiency virus (HIV) infection, acquired immunodeficiency syndrome (AIDS), HIV dementia, and / or HIV neuronal damage, comprising administering the component to a mammal, including humans. Or administering an active ingredient contained in a prophylactic, cancer therapeutic (improvement) or prophylactic composition to a mammal (including human), the cancer therapeutic, ameliorating and / or prophylactic method, the AKT activation inhibitor of the present invention. AKT activation inhibition comprising administering an active ingredient to a mammal (including humans); PI3K inhibition comprising administering an active ingredient contained in a PI3K inhibitor to a mammal (including humans) The active ingredient contained in, and GSK3 beta activator mammal provides GSK3 beta activation method comprising the step of administering to a (including a human being).

Unless stated otherwise, the descriptions of the compositions, inhibitors, and / or activators of the invention apply equally to the uses, methods of the invention.

The active ingredient in the compositions, inhibitors, and / or activators of the present invention can be administered orally or parenterally to mammals, including humans, and can be formulated and administered in combination with a pharmaceutically acceptable carrier. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants which are commonly used may be used. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid form preparations include at least one excipient such as starch, calcium carbonate, sucrose, lactose, and It is prepared by adding gelatin and the like. Lubricants such as magnesium and talc are also used. Liquid preparations for oral administration include suspensions, solvents, emulsions and syrups, and may include various excipients such as wetting agents, sweeteners, fragrances and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. have. Formulations for parenteral administration include injectable solutions, suspensions, emulsions, lyophilizers, nasal washes and suppositories. Injectable solutions, suspensions and emulsions can be prepared by mixing water, non-aqueous solvents or suspending solvents with the active ingredient. As non-aqueous solvents and suspending solvents, vegetable oils such as propylene glycol, polyethylene glycol and olive oil, and ethyl oleate Injectable esters such as and the like. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol, gelatin and the like can be used. The pharmaceutical composition of the present invention can be administered by subcutaneous injection, intravenous injection or intramuscular injection during parenteral administration.

In the compositions, activators, inhibitors, uses and methods of the present invention, it can be used at a dose of 1 mg to 1000 mg / kg per adult on a daily basis.

The active ingredient may be formulated by adding a pharmaceutically or food-acceptable carrier, excipient or diluent, etc., and the formulation may be found in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA et al. Reference may be made.

The active ingredient in the compositions, activators, and inhibitors of the present invention may be included in an amount of 0.1 to 100% by weight based on the total weight.

The active ingredient contained in one or more therapeutic (improvement) or preventive compositions selected from human immunodeficiency virus (HIV) infection, acquired immunodeficiency syndrome (AIDS), HIV dementia, or HIV neuronal damage of the present invention is a human immunodeficiency deficiency. It has the effect of treating, ameliorating and / or preventing viral (HIV) infections, acquired immunodeficiency syndrome (AIDS), HIV dementia, and / or HIV neuronal damage, and is contained in compositions for the treatment (improvement) or prevention of cancer. The active ingredient exhibits the effect of treating, improving and / or preventing cancer. In addition, the active ingredient contained in the AKT activation inhibitor of the present invention, the AKT activation inhibitory effect, the active ingredient contained in the PI3K inhibitor, the PI3K inhibitory effect, the active ingredient contained in the GSK3 beta activator shows a GSK3 beta activation effect.

Fig. 1 shows lanceemaside A, lanceemaside X, EAG, EA, compound K, actigenin, actinin, naringenin, ginsenoside Rh1, ginsenoside Rh2, carlopanaxasponin A, fungeretetin, nobiliretin, It is a graph which confirmed the anti-HIV effect of apigenin and camphorol.
Figure 2 is the result of observing the anti-HIV effect of lanceemaside A, actigenin, compound K by fluorescence microscope.
Figure 3 is a Western blotting result confirming the effect of lancemaside A on the inhibition of AKT activation and the activation of GSK3beta.
Figure 4 is a Western blotting result confirming the effect of compound k on the inhibition of AKT activation and the activation of GSK3beta.
Figure 5 is a Western blotting result confirming the effect on the phosphorylation of actikinin PDK-1 (pyruvate dehydrogenase kinase isozyme 1), AKT, GSK-3beta.
6 is a PIP3 analysis result confirming the effect on the phosphoinositide 3-kinase activity of actigenin.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the following Examples and Preparation Examples are only for illustrating the present invention, and the content of the present invention is not limited by the following Examples or Preparation Examples.

<Manufacture example 1>

The active ingredient was prepared by the following method.

1-1. Lancemaside A, EAG, Lancemaside X, Ikanocyst acid

5 kg of dried Deodeok (Codonopsis lanceolata Trautv) roots (Gyeongdong Market, Seoul, South Korea) was boiled for 3 hours with MeOH and refluxed with a reflux extractor, and the extract was decompressed with a rotary decompressor (Sunil Isla, Eyla KSB 201, Korea). Concentration gave 439.24 g of extract. After dissolving in water and fractional extraction with BuOH to separate the BuOH layer and concentrated under reduced pressure to give an extract of 211g. 14 fractions (dichloromethane: MeOH = 100: 0, 95: 5, 90:10, 85:15, 80:20, 75:25) using silica gel as the stationary phase and the mobile phase of the gradient elution system of dichloromethane and MeOH , 70:30, 65:35, 60:40, 55:45, 50:50, 45:55, 40:60, 30:70 (1 L each) and the 13th fraction of medium pressure liquid chromatography ; Yamazen 540-SY-S2CSC, Japan) (mobile phase: 10% ACN in water to 70% ACN in water) at 4 ml / min for 4 hours to obtain 80 fractions of which 37-42 The bun fraction was concentrated under reduced pressure to yield 620 mg of lanceemaside A. A semaphore is a side 10mg was dissolved in water, where the β-D-glucuronidase (sigma) and the process is concentrated to 7mg echinocystic acid 28- back extracted with butanol bunhaek After 1 hours at 37 degrees Celsius hot water O -β- D-xylopyranosyl (1 → 3) -β-D-xylopyranosyl- (1 → 4) -α-L-rhamnopyranosyl- (1 → 2) -α-L-arabinopyranosyl ester (lancemaside X) was obtained.

In addition, 50 mg of lancemaside A was added to 10ml 3N NaOH, heated in 70 ° C hot water for 10 minutes, neutralized with hydrochloric acid, fractionated and extracted with butanol, and concentrated to 29mg of echinocystic acid 3- O- . β-D-glucuronopyranoside (EAG) was obtained.

In addition, 50 mg of Lansemaside A was added to 10 ml 5N HCl, refluxed in boiling water at 100 degrees Celsius for 3 hours, and the acid degradation product obtained by heating was neutralized with sodium bicarbonate, fractionated with chloroform, and concentrated to 22 mg. Icainocyst acid (EA) was obtained. The compounds separated above were identified by H1-NMR (Bruker, AVANCE digital 400) and 13C-NMR (Bruker, AVANCE digital 400).

1-2. ginsenoside Rh1, ginsenoside Rh2, compound K

Add about 1L of water to immerse the powder (0.5 kg) of dried Ginseng (Panax Ginseng CA Meyer, Kyungdong Market, Seoul, South Korea) in water and extract it for 2 hours in a 100 degree Celsius water bath. An extract was prepared. It was confirmed by NMR that the extract contained ginsenoside Rh1 and ginsenoside Rh2.

Powder (0.5 kg) of dried Ginseng (Panax Ginseng CA Meyer, Kyungdong Market, Seoul, South Korea) was added to about 1L of water to sterilize and sterilized in tryptic soy broth (1 L) for 24 hours. Collected Lactobacillus acidophilus (KCTC, Korea Institute of Bioscience and Technology, Korea), Leuconostoc mesenterodies (KCTC, Korea Institute of Biotechnology, Korea), and Bifidobacterium longum (KCTC, Korea Institute of Bioscience and Technology, Korea) were incubated for 3 to 5 days. The culture solution was lyophilized with a lyophilizer (ELYLA, Japan) to powder or extracted with 50% alcohol (ethanol) to prepare ethanol extract of ginseng fermentation broth.

In addition, 50% alcohol extract of ginseng culture extract was extracted with butanol, and only the butanol layer was separated and concentrated under reduced pressure to obtain an extract. The silica gel was used as the stationary phase and dichloromethane and MeOH were separated into six fractions using the mobile phase of the gradient elution system (dichloromethane: MeOH = 100: 0, 90:10, 80:20, 60:40, 40:60, 20:80). 2 L each), and the third fraction was separated using MPLC for 4 hours at 4 ml / min of mobile phase (10% acetonitrile in water to 90% acetonitrile in water) to obtain 80 fractions. Ginsenoside Rh1 and ginsenoside Rh2 were obtained by concentrating under reduced pressure from fraction 35 to fraction 42 and fraction 55 to fraction 61 respectively.

The fourth fraction was separated using MPLC for 4 hours at 4 ml / min of mobile phase (10% acetonitrile in water to 90% acetonitrile in water), and 80 fractions were obtained. Concentration gave compound K. The separated compounds were identified by H1-NMR (Bruker, AVANCE digital 400) and 13C-NMR (Bruker, AVANCE digital 400).

1-3. Actini, Actigenin

Soak 2 kg of Allium (Arctium Lappa, Kyungdong Market, Seoul, South Korea) in Hexane for 1 day to remove oil and extract it in MeOH for 1 day. Extract is extracted with rotary decompressor (Sunil Isla, Eyla KSB 201, Korea) Concentrated under reduced pressure) to obtain 150 g of extract. After dissolving this in 1L of water, 3L MeOH was added and filtered with 5mm filter paper, and only the supernatant was concentrated with a rotary vacuum concentrator to obtain 80g of extract. Using silica gel as a stationary phase, chloroform and MeOH were mixed into 8 fractions (1 L each) using a solvent mixed with 30: 1. The fifth fraction of the chloroform and MeOH was further separated using silica gel as a stationary phase. This 20: 1 solvent was used to divide the solution into 5 fractions (1 L each), and only the third fraction was concentrated under reduced pressure to obtain 100 mg of arctiin. 50mg of the obtained acti was added to about 1L of water and sterilized and incubated in tryptic soy broth (1 L) for 24 hours, and then collected. Institute of Korea, Korea) and then incubated for 3 to 5 days, the solution was centrifuged at 7000 rpm for 20 minutes to precipitate the cells and concentrated under reduced pressure using silica gel as a stationary phase. The solvent was mixed with 4 fractions (1 L each), and the second fraction was concentrated under reduced pressure to obtain 20 mg of arctigenin. The compounds separated above were identified by H1-NMR (Bruker, AVANCE digital 400) and 13C-NMR (Bruker, AVANCE digital 400).

1-4. Naringenin, nobilithin, tengeretin

500g of dried dermis (Aurantii Nobilis pericarpium, Kyungdong Market, Seoul, South Korea) was soaked in nucleic acid for one day to remove oil components, dried, ground, powdered, 2L of water, extracted at 40 ° C for 5 hours, and concentrated under reduced pressure. 185g An extract of was obtained. The silica gel was used as a stationary phase, and the solvent was mixed with benzene and acetone at 6: 1, and then divided into 8 fractions (1 L each). The second fraction was concentrated to Tangeretin, and the third fraction to Nobiletin. The fifth fraction was concentrated to give Naringenin. The compounds separated above were identified by H1-NMR (Bruker, AVANCE digital 400) and 13C-NMR (Bruker, AVANCE digital 400).

1-5. Preparation of kalopanaxsaponin A

Bark of Dried Oak Tree (Kalopanax pictus NAKAI) {thawpi; Gyeongdong Market, Seoul, South Korea} 3 kg of reflux was boiled with MeOH for 3 hours to extract reflux, and the extract was concentrated under reduced pressure to obtain 110 g of extract. After dissolving in water and fractional extraction with BuOH to separate the BuOH layer and concentrated under reduced pressure to obtain an extract of 18g. The silica gel was used as a stationary phase, and the fractions of seven fractions (dichloromethane: methanol = 10: 1, 7: 1, 5: 1, 4: 1 3: 1, 2: 1) were obtained using the mobile phase of the gradient elution system of dichloromethane and MeOH. , 1: 1, 300 ml each), the fifth fraction (2.8 g) was used for 4 hours at 4 ml / min using MPLC (mobile phase: 10% ACN in water to 90% ACN in water). As a result, 80 fractions were obtained, and fractions 61-69 were concentrated under reduced pressure to obtain 69 mg of kalopanaxsaponin A.

The separated components were identified by H1-NMR (Bruker, AVANCE digital 400), 13C-NMR (Bruker, AVANCE digital 400).

1-6. Kaempherol, apigenin

The ingredients were purchased from Sigma (USA) and prepared.

Example 1 Anti-HIV Effect Confirmation: HIV tat induced human microglia cell death rate assay

Lancemaside A, Lancemaside X, EAG, EA, Compound K, Actigenin, Actinin, Naringenin, Ginsenoside Rh1, Ginsenoside Rh2, Carlophanax saponin A, Tengeretine, Nobililet, Apigenin, And anti-HIV effects were measured by measuring the cell death rate of CHME5 cells (human brain-derived microglia cells into which the tat protein gene of HIV virus was inserted), each of which was a human microglia cell induced by HIV tat.

pTat CHME5 cells (Professor Baek Kim, Department of Microbiology and Immunology, University of Rochester Medical Center, New York, USA) were unwound at 10 ⁵ cells / ml in 24well, and after 24 hours each of the active ingredients prepared or prepared in Preparation Example 1 Dissolve in DMSO (Dimethyl sulfoxide) to 10 mM and dilute with water for treatment. The final concentration of the active ingredient was treated to 10uM, DMSO was brought to a final concentration of 0.1%.

After 30 minutes, LPS (Lipopolysaccharide) and cycloheximide are respectively treated to the final concentrations of 50 μg / ml and 10 μg / ml. After 24 hours, the supernatant is stored separately and the cells on the bottom are trypsin-treated and combined with the supernatant. Centrifuge at 13000 rpm for 3 minutes and discard the supernatant. The cells are suspended in 40ul of Dulbecco's Modified Eagle's Media (DMEM) medium. At this time, pipette the cells to spread evenly. Put 60ul of trypan blue again. After 5 minutes, the ratio of cells stained with trypan blue in the total cells was measured under a microscope. The results are shown in FIG.

As a result, lanceemaside A showed 22.5% cell death rate, lanceemaside X was 18.2%, icanocystic acid 3- O- β-D-glucuronopyranoside (EAG) was 10.8%, Icainocyst acid (EA) is 7.6%, Compound K is 18.1%, Actigenin is 18.2%, Actiin is 12.2%, Naringenin is 13.8%, Ginsenoside R. 1 is 15%, Ginsenosides Egg etch 2 was 13.6%, carlophanax saponin A was 14.5%, tungeretein was 12.7%, nobiliretin was 8.1%, apigenin was 12.7%, and camperol was 16%.

From the above results, it can be seen that lanceemaside A and the like have anti-HIV action. That is, the TAT protein of the HIV-1 virus activates AKT to continue to proliferate without infecting infected cells (macrophages, glia, etc.). Lancemaside A, Compound K, Actinigenin, etc. act on these activated cells, and latent by inhibiting AKT activation, which prevents the death of infected cells (macrophage, glia, etc.) while HIV-1 is infected and latent. It can be seen that effectively removing HIV-1 by removing the host cells of HIV-1 by killing the host cells in this way.

As a result, lanceemaside A, compound K, and actigenin may be used for the treatment (improvement) or prevention of human immunodeficiency virus (HIV) infection, AIDS, HIV dementia, or HIV neuronal damage. It can be seen that it is effective.

In addition, it contains deodeok extract containing lanceemaside A, thaw skin extract containing carlopanax saponin A, ginseng fermentation containing compound K, ginsenoside R. 1 and / or ginsenoside R. 2 The ginseng extract, alligator extract containing actin and alligator ferment containing actinigen are also believed to have the above effects.

Example 2 Confirmation of Anti-HIV Effect: Confirmation of HIV tat Induced Human Microglia Cell Death Using Fluorescence Microscopy

Lancemaside A, Compound K, and Actigenin are CHME5 cells (human microglia cells derived from HIV tat), which are brain-derived microglia cells of humans that have inserted the tat protein gene of the HIV virus. The anti-HIV effect was examined by fluorescence microscopy to observe the effect on the killing of HIV latent infected microglia.

After dissolving pTat CHME5 cells in 24 wells at 10 ⁵ cells / ml, 24 hours later, the active ingredients prepared in Preparation Example 1, lanceemaside A, compound K, and actinigen, were dissolved in DMSO to 10 mM and diluted with water, respectively. . The final concentration of the active ingredient was treated to 10uM, DMSO was brought to a final concentration of 0.1%.

After 30 minutes, LPS (Lipopolysaccharide) and cycloheximide were respectively treated to a final concentration of 50 μg / ml and 10 μg / ml, and after 12 hours, washed three times with PBS (phosphate buffered saline) and calcein 1: 8000, and ethidium. After diluting in PBS at a ratio of 1: 4000, 200ul per well was reacted for 40 minutes. After washing three times with PBS again, it was observed by fluorescence microscope (Carl Zeiss). In addition, the cells treated in the same manner except for LPS treatment were observed separately, and the cells not treated with the active ingredient were observed as a control.

The results are shown in FIG. Figure 2 shows the anti-HIV effect of lanceemaside A, Compound K, Actigenin fluorescence microscopy, red cells are cells.

As a result, it can be seen that the dead cells (cells stained in red) increase when treated with Lancemaside A, Compound K, and Actigenin. That is, it can be confirmed that lanceemaside A, compound K, and actinigen effectively induce the death of cells which did not die by TAT treatment.

From the above results, it can be seen that lanceemaside A, compound K, and actigenin exhibit anti-HIV action. As a result, Lancemaside A, Compound K, and Actinin are effective in the treatment (improvement) or prevention of human immunodeficiency virus (HIV) infection, AIDS, HIV dementia, or HIV neuronal damage. It can be seen that.

In addition, deodeok extract containing lanceemaside A, ginseng fermentation containing compound K and allied fermentation containing actigenin are also expected to exhibit the above effects.

Example 3 Anticancer Effect Confirmation

Cells {Lung cancer cell line A549 (Korea Cell Line Bank, Seoul National University), breast cancer cell line MCF-7 (Korea Cell Line Bank, Seoul National University), gastric cancer cell line KATO-III (Korea Cell Line Bank, Seoul National University)} per 1 well After dispensing ⁵ x ¹ × ¹⁰ incubation in a CO ₂ incubator for 24 hours, and treated with LPS (200 ng / ml), and the components treated in the concentration of 0, 10, 20, 50, 100uM in Table 1, LPS The experiment shown in Table 1 was divided into groups treated with concentrations of 0, 10, 20, 50, and 100 uM without treatment. The components were dissolved in DMSO and diluted with water and treated separately. Three wells in each group were tested, and after 24 hours of incubation after drug treatment, the medium was removed, washed, and the remaining amount of cells was measured by crystal violet method. The 50% cell growth concentration (uM) was calculated from the results, and the results are shown in the following table.

[Table 1]

As shown in the table, it can be seen that lanceemaside A, EAG, EA, actin, kalopanaxsaponin A has an anticancer effect. Cancer cells do not die more when there is a stimulus such as external LPS, and the PI3K / AKT survival signal pathway is activated and does not die.However, when treated with PI3K / AKT inhibitors lanceemaside A, EAG, EA, actin, and kalopanaxsaponin A It can be seen that the cancer cell killing effect is increased.

In addition, deodeok extract containing lanceemaside A, thaw skin extract containing carlopanax saponin A, and alligator extract containing actiniin are also expected to exhibit the above effects.

Example 4 Inhibition of AKT Activation and / or Confirmation of GSK3beta Activation Effect

After dissolving pTat CHME5 cells in 24 wells at 10 ⁵ cells / ml, 24 hours later, the active ingredients prepared in Preparation Example 1, lanceemaside A, compound K, and actinigen, were dissolved in DMSO to 10 mM and diluted with water, respectively. . The final concentration of the active ingredient was treated to 5, 10, 20uM, DMSO was brought to a final concentration of 0.1%. After 30 minutes, LPS treatment and lysis of cells with RIPA buffer (+ PI) for 20 minutes. Centrifuge for 3 minutes at 2000 rpm. Take the supernatant and perform electrophoresis on SDS 10% (w / v) polyacrylamide gel for 1 hour 30 minutes (sample, 50ug). Electrophoresis was transferred to nitrocellulose paper at 100 V and 400 mA for 1 hour and 10 minutes. The transferred nitrocellulose was blocked with 5% skim milk for 30 minutes, washed three times with PBS-Tween for 5 minutes each, and the primary antibody (Santa Cruz Biotechnology, USA) was reacted 1: 100 overnight. Wash three times for 10 minutes. The secondary antibody (Santa Cruz Biotechnology, USA) is reacted at 1: 1000 for 1 hour 20 minutes. It is washed three times for 15 minutes and developed by fluorescence.

Cells treated in the same manner were used as controls, except that the active ingredient and LPS were not treated.

The results are shown in Figures 3-4.

3 is a Western blotting result confirming the effect of lancemaside A on the inhibition of AKT activation and activation of GSK3beta, Figure 4 is a Western blotting result confirming the effect of compound k on the inhibition of AKT activation and activation of GSK3beta to be.

As shown in Figures 3 and 4, it can be seen that lanceemaside A or compound k inhibits the phosphorylation of AKT and GSK3beta in a concentration-dependent manner. Since AKT is phosphorylated to be activated and GSK3beta is inactivated by phosphorylation, it can be seen that lancemaside A inhibits the activity of AKT and activates GSK3beta. As a result, Lansemaside A or Compound K has anticancer and anti-HIV effects, which can be used to treat human immunodeficiency virus (HIV) infection, acquired immunodeficiency syndrome (AIDS), HIV dementia, HIV neuronal cell damage, and cancer. It can be seen that it is effective for (improvement) or prevention.

In addition, deodeok extract containing lanceemaside A, ginseng fermentation containing the compound K is also believed to have the above effect.

Example 5 Confirmation of Arctigenin PI3K Inhibition, AKT Activation Inhibition and / or GSK3beta Activation Effect

pTat CHME5 cells were loosened in 24 wells at 10 ⁵ cells / ml, and after 24 hours, the active ingredient prepared in Preparation Example 1 was dissolved in DMSO to 10 mM, diluted with water, and treated. The final concentration of the active ingredient was treated to 5, 10, 20uM, DMSO was brought to a final concentration of 0.1%. After 30 minutes, LPS treatment and lysis of cells with RIPA buffer (+ PI) for 20 minutes. Centrifuge for 3 minutes at 2000 rpm. Take the supernatant and perform electrophoresis on SDS 10% (w / v) polyacrylamide gel for 1 hour 30 minutes (sample, 50ug). Electrophoresis was transferred to nitrocellulose paper at 100 V and 400 mA for 1 hour and 10 minutes. The transferred nitrocellulose was blocked with 5% skim milk for 30 minutes, washed three times with PBS-Tween for 5 minutes each, and the primary antibody (Santa Cruz Biotechnology, USA) was reacted 1: 100 overnight. Wash three times for 10 minutes. The secondary antibody (Santa Cruz Biotechnology, USA) is reacted at 1: 1000 for 1 hour 20 minutes. It is washed three times for 15 minutes and developed by fluorescence.

Cells treated in the same manner as above were used as a control group (CON), except that the active ingredient and LPS were not treated. In addition, cells treated in the same manner as described above were used as negative controls (LPS / CHX) except that the active ingredients were not treated.

The results are shown in Fig.

Figure 5 is a Western blotting result confirming the effect on the phosphorylation of actikinin PDK-1 (pyruvate dehydrogenase kinase isozyme 1), AKT, GSK-3beta.

As shown in Figure 5, it can be seen that actigenin inhibits the phosphorylation of PDK-1, AKT and GSK3 beta in a concentration-dependent manner. Since AKT is phosphorylated and activated, GSK3beta is inactivated by phosphorylation. As a result, it can be seen that actigenin inhibits AKT activity and activates GSK3beta. As a result, actigenin exhibits anti-cancer and anti-HIV effects, resulting in the treatment (improvement) or prevention of human immunodeficiency virus (HIV) infection, AIDS, HIV dementia, HIV neuronal damage, cancer, etc. It can be seen that it is effective for.

In addition, since activation of PDK-1 is by PIP3 and PIP3 is converted from PIP2 by PI3K, it can be seen that actigenin may be associated with PI3K inhibition.

In addition, alligator fermentation containing actigenin is also believed to have the effect.

Example 6 Confirmation of Arctigenin Inhibitory Effect of PI3K

pTat CHME5 cells were loosened in 24 wells at 10 ⁵ cells / ml, and after 24 hours, the active ingredient prepared in Preparation Example 1 was dissolved in DMSO to 10 mM, diluted with water, and treated. The final concentration of the active ingredient was treated to 20uM, DMSO was brought to a final concentration of 0.1%. After 30 minutes LPS treatment on the sample, Phosphatidylinositol (3,4,5) -trisphosphate (PI (3,4,5) P3 using Echelon # K-2400 Kit (Echelon, USA) according to the manufacturer's instructions ) Was extracted and measured. Specifically, the mixture is left for 5 minutes after the addition of cold 0.5M TCA, the cells are scraped off, and centrifuged at 1500 rpm for 5 minutes, the supernatant is discarded. After adding 1 mM EDTA to the TCA, the solution is vortexed and centrifuged at 1500 rpm for 5 minutes. Discard the supernatant (repeat twice), add a buffer containing MeOH and CHCl ₃ in a ratio of 2: 1, and leave it at room temperature for 10 minutes (vortexing three times while standing), centrifuge at 1500 rpm for 5 minutes, and remove the supernatant. Discard (2 times), add MeOH, CHCl ₃ and HCl in a 80: 40: 1 buffer, leave for 15 minutes (vortexing 4 times while standing), centrifuge at 1500 rpm for 5 minutes, and remove CHCl ₃ from the supernatant. To the supernatant is added 0.1N HCl. After centrifugation at 1500rpm for 5 minutes, the organic solvent layer was vacuum-dried in an eppentube and PIP3 was measured. That is, add CHCl3, MeOH and H2O 1: 2: 0.8 buffer to the dry, vortexing for 30 seconds, sonication in a cold water bath for 5 minutes, a small amount of samples on the strip and repeat about 5 times, Add strips to the buffer solution containing 3% BSA in PBS-T, shake slowly at room temperature for 1 hour, and place the detector in the buffer solution containing 3% BSA in PBS-T, shake slowly for 1 hour, and PBS-T After washing, put the antibody in the buffer solution added 3% BSA to PBS-T and shake slowly for 45 minutes, wash, put the antibody in the buffer solution added 3% BSA to PBS-T, shake slowly and wash for 45 minutes Develop.

Cells treated in the same manner as above were used as a control group (CON), except that the active ingredient and LPS were not treated. ), Except that the wortmanin, a potent inhibitor of PI3K, was treated instead of the active ingredient, cells treated in the same manner as above were used as positive controls (L + W).

The results are shown in FIG.

In the arctigenin treatment group, the PIP3 content was significantly lower than in the treatment group. These results show a strong inhibitory effect compared with wortmanin, a potent inhibitor of PI3K. The decrease in the amount of PIP3 means that the action of PI3K, an enzyme that converts PIP2 to PIP3, is inhibited. Therefore, actigenin inhibits the conversion of PIP2 to PIP3 by inhibiting the action of PI3K and, consequently, inhibits the activation of PDK1, thereby exhibiting anticancer activity and anti-HIV action, resulting in human immunodeficiency virus (HIV) infection, acquired It can be seen that it is effective in the treatment (improvement) or prevention of immunodeficiency syndrome (AIDS), HIV dementia, HIV neuronal damage, cancer and the like.

In addition, alligator fermentation containing actigenin is also believed to have the effect.

Claims (16)

A semaphore is a side (lancemacide A), X is the side-Sema (lancemacide X), Ica Ino cyst acid 3- O -β-D- glucuronic nopi Llano side (echinocystic acid 3- O -β-D -glucuronopyranoside) a, Echinocystic acid, compound K, compoundgen, arctigenin, arctiin, naringenin, ginsenoside Rh1, ginsenoside Rh2 ), Kalopanaxsaponin A, tangeretin, nobiletin, apigenin, apigenin, camphorol, deodeok extract, haejupi extract, ginseng extract, ginseng fermentation, allium extract Or one or more therapeutic or prophylactic pharmaceutical compositions selected from human immunodeficiency virus (HIV) infection, acquired immunodeficiency syndrome (AIDS), HIV dementia, or HIV neuronal cell damage, containing at least one selected from allied fermented products. . The composition of claim 1, wherein the human immunodeficiency virus infection is a latent infection of human immunodeficiency virus latent infection. The composition of claim 1, wherein the human immunodeficiency virus infection is at least one cellular infection selected from macrophages or microglia cells. The composition of claim 1, wherein the treatment or prevention is by at least one selected from PI3K inhibition, AKT activation inhibition or GSK3beta (Glycogen synthase kinase 3 beta) activation. A semaphore is a side (lancemacide A), X is the side-Sema (lancemacide X), Ica Ino cyst acid 3- O -β-D- glucuronic in nopi Llano side (echinocystic acid 3- O -β-D -glucuronopyranoside; EAG ), Echinocystic acid, compound K, compoundgen, arctigenin, arctiin, naringenin, ginsenoside Rh1, ginsenoside Rh2 (ginsenoside Rh1) ginsenoside Rh2), kalopanaxsaponin A, tangeretin, nobiletin, apigenin camphorol, deodeok extract, haejupi extract, ginseng extract, ginseng fermented product, For the improvement or prevention of one or more selected from human immunodeficiency virus (HIV) infection, acquired immunodeficiency syndrome (AIDS), HIV dementia, or HIV neuronal cell damage containing at least one selected from extracts or allied fermentation as an active ingredient. Food composition. The composition of claim 5, wherein the human immunodeficiency virus infection is a latent infection that is latent infected in the body. The composition of claim 5, wherein the human immunodeficiency virus infection is at least one cellular infection selected from macrophages or microglia cells. The method according to any one of claims 5 to 7, wherein the treatment or prevention is due to one or more selected from inhibition of Phosphoinositide 3-Kinase (PI3K), inhibition of AKT activation, or GSK3beta (Glycogen synthase kinase 3 beta) activation. Composition. A semaphore is a side (lancemacide A), Ica Ino cyst acid 3- O -β-D- glucuronic in nopi Llano side (echinocystic acid 3- O -β-D -glucuronopyranoside), Ica Ino cyst acid (echinocystic acid), A pharmaceutical composition for treating or preventing cancer containing at least one selected from arctiin, kalopanaxsaponin A, deodeok extract, haejupi extract, or ovarian extract as an active ingredient. A semaphore is a side (lancemacide A), Ica Ino cyst acid 3- O -β-D- glucuronic in nopi Llano side (echinocystic acid 3- O -β-D -glucuronopyranoside), Ica Ino cyst acid (echinocystic acid), A food composition for improving or preventing cancer, comprising one or more selected from actiniin, kalopanaxsaponin A, deodeok extract, thawed bark extract, or allium extract as an active ingredient. An AKT activation inhibitor containing at least one selected from lancemacide A, compound K, compound extract, or ginseng fermentation as an active ingredient. The inhibitor of claim 11, which is for treatment, amelioration or prevention of one or more selected from human immunodeficiency virus (HIV) infection, HIV dementia, HIV neuronal damage, acquired immunodeficiency syndrome (AIDS) or cancer. PI3K (Phosphoinositide 3-Kinase) inhibitor containing at least one selected from actigenin or allied fermentation as an active ingredient. The inhibitor of claim 13, which is for treatment, amelioration or prevention of one or more selected from human immunodeficiency virus (HIV) infection, HIV dementia, HIV neuronal damage, acquired immunodeficiency syndrome (AIDS) or cancer. GSK3 beta (Glycogen synthase kinase 3 beta) containing as an active ingredient at least one selected from lancemacide A, compound K, arctigenin, deodeok extract, ginseng fermentation, or allium fermentation. Activator. The activator according to claim 15, which is for treatment, amelioration or prevention of one or more selected from human immunodeficiency virus (HIV) infection, HIV dementia, HIV neuronal damage, acquired immunodeficiency syndrome (AIDS) or cancer.

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