Ethanol extract of antrodia camphorata for inducing apoptosis and preparation method thereof

Abstract

A preparation method for an ethanol extract of the fruiting body of Antrodia camphorata (EEAC) is provided. The preparation method includes steps of: (a) providing the fruiting body of A. camphorata (AC); (b) extracting the fruiting bodies with a first ethanol solution; and (c) obtaining EEAC. EEAC further can be sequentially extracted or fractioned by n-hexane, ethyl acetate and ethanol, and an n-hexane fraction (FC), an ethyl acetate fraction (FA) and an ethanol fraction (FB) respectively are generated. The growth inhibition and apoptosis induction of leukemia cell line HL 60 are effectively mediated by FA product, in which zhankuic acid A is the bioactive marker. The amount of triterpenoid in the fruiting body of AC can be determined by NMR and HPLC analysis.

1 . An ethyl acetate extract of a fruiting body of an  Antrodia camphorata,  comprising at least one characteristic of:
 (a) causing a plurality of methyl signals generated from one of a sterol and a triterpene at δ H  0.5 to 2 and terminal olefinic protons at δ H  4.8 to 5.0 on a first  1 H nuclear magnetic resonance ( 1 H NMR) spectrum when the ethyl acetate extract is solved in a deuterium chloroform (CDCl 3 ); and   (b) causing the signals of terminal olefinic protons at δ H  5.0 to 5.4 on a second  1 H NMR spectrum when the ethyl acetate extract is solved in a pyridine-D5 (C 5 D 5 N).   
     
     
         2 . The ethyl acetate extract according to  claim 1  further comprising at least one compound being one selected from a group consisting of a zhankuic acid A, a dehydroeburicoic acid, an actcin C and a zhankuic acid C. 
     
     
         3 . The ethyl acetate extract according to  claim 2 , wherein the zhankuic acid A has characteristic  1 H NMR signals of two tertiary methyls (CH 3 -18 and CH 3 -19) and terminal olefinic protons (H 2 -28) in 24-exo-methylene-26-oic acid side-chain, and the ethyl acetate extract further has characteristic signals of triterpenoid the same with those of the zhankuic acid A. 
     
     
         4 . The ethyl acetate extract according to  claim 1  being fractionized from an ethanol extract of the fruiting body of the  Antrodia camphorata.    
     
     
         5 . A method for measuring an existence and an amount of triterpenoid of a target object, comprising steps of:
 (a) extracting the target object with a first ethanol solution to obtain an ethanol extract;   (b) sequentially extracting the ethanol extract with an n-hexane and an ethyl acetate to obtain an ethyl acetate extract; and   (c) measuring the existence and the amount of the triterpenoid in the ethyl acetate extract.   
     
     
         6 . The method according to  claim 5 , wherein in the step (c), the existence of the triterpenoid is performed by a nuclear magnetic resonance at a resolution of at least 200 MHz, the ethyl acetate extract is solved in a pyridine-D5 (C 5 D 5 N), and the amount of the triterpenoid is performed by a high performance liquid chromatography (HPLC). 
     
     
         7 . The method according to  claim 5 , wherein the target object is a fruiting body of an  Antrodia camphorata.    
     
     
         8 . An extracting method, comprising steps of:
 (a) providing a fruiting body of an  Antrodia camphorata;      (b) extracting the fruiting body with a first ethanol solution; and   (c) obtaining an ethanol extract of the fruiting body.   
     
     
         9 . The method according to  claim 8 , wherein the step (a) further comprises a step (a1) of grinding the fruiting body. 
     
     
         10 . The method according to  claim 8 , wherein the step (c) further comprises a step (c1) of extracting the ethanol extract with at least one organic solvent to obtain an organic solvent extract. 
     
     
         11 . The method according to  claim 10 , wherein the at least one organic solvent has a total number of species more than one, each of the at least one organic solvent has a polarity, and the step (c1) has a extracting sequence that the polarity of a specific one of the at least one organic solvent is higher than that of a preceding one of the specific organic solvent. 
     
     
         12 . The method according to  claim 10 , wherein the at least one organic solvent is one selected from a group consisting of an n-hexane, an ethyl acetate, a second ethanol solution and a combination thereof. 
     
     
         13 . The method according to  claim 10 , wherein the step (c1) further comprises steps of:
 (c11) extracting the ethanol extract with an n-hexane to obtain an n-hexane extract and a first residue;   (c12) extracting the first residue with an ethyl acetate to obtain an ethyl acetate extract and a second residue; and   (c13) extracting the second residue with a second ethanol solution to obtain a second ethanol extract.   
     
     
         14 . The method according to  claim 13 , wherein the step (c12) further comprises a step (c121) of isolating the ethyl acetate extract with a plurality of trichloromethane-methanols to obtain a zhankuic acid A, wherein the plurality of trichloromethane-methanols have respective ratios of a trichloromethane to a methanol. 
     
     
         15 . The method according to  claim 13 , wherein the step (c12) further comprises a step (c121) of isolating the ethyl acetate extract with a plurality of n-hexane-ethyl acetate-methanols to obtain a plurality of fractions of the ethyl acetate extract, wherein the plurality of n-hexane-ethyl acetate-methanols have respective ratios of an n-hexane-ethyl acetate to a methanol. 
     
     
         16 . The method according to  claim 15 , wherein the step (c121) further comprises at least one step of:
 (c121-1) sequentially isolating a first one of the plurality of fractions with an ethyl acetate-dichloromethane-methanol, a trichloromethane-methanol and a methanol-water to obtain a dehydroeburicoic acid;   (c121-2) sequentially isolating a second one of the plurality of fractions with the ethyl acetate-dichloromethane-methanol and an acetonitrile-water having a first ratio of an acetonitrile to a water to obtain a zhankuic acid A;   (c121-3) sequentially isolating a third one of the plurality of fractions with a dichloromethane-methanol and the acetonitrile-water having a second ratio of an acetonitrile to a water to obtain an actcin C; and   (c121-4) sequentially isolating a fourth one of the plurality of fractions with the dichloromethane-methanol and the acetonitrile-water having a third ratio of an acetonitrile to a water to obtain a zhankuic acid C.   
     
     
         17 . The method according to  claim 8 , wherein the step (b) further generates a third residue, and the method further comprises a step (c1) of extracting the third residue with a water to obtain a water extract. 
     
     
         18 . The method according to  claim 8 , wherein the ethanol extract is used to treat a cell for regulating an expression of a specific protein of the cell. 
     
     
         19 . The method according to  claim 18 , wherein the cell is a leukemia cell, and the specific protein is one selected from a group consisting of a histone, a histone deacetylase, a histone acetylase, a p21, a poly (ADP-ribose) polymerase (PARP), a Bax, a Bcl-2, a death receptor (DR5), a tumor necrosis factor receptor-associated death domain (TRADD), a p50 and a p65 and a combination thereof. 
     
     
         20 . The method according to  claim 8 , wherein the ethanol extract and a 7-[4-(dimethylamino)phenyl]-N-hydroxy-4,6-dimethyl-7-oxohepta-2,4-dienamide (trichostatin A) have a synergistic effect of causing a cell to go into an apoptosis.