Anti-cancer active substance from antrodia camphorata, method for preparing the same and use thereof

Abstract

An anti-cancer active substance from Antrodia Camphorata , produced from mycelium of Antrodia Camphorata by following steps: ( 1 ) culturing mycelium of Antrodia Camphorata by liquid fermentation; ( 2 ) extracting the thus-obtained mycelium with ethanol and concentrating the extracted solution to obtain an ethanol extract; ( 3 ) dissolving the ethanol extract in water, and extracting with equal volume of ethyl acetate to obtain ethyl acetate extract; ( 4 ) purifying the ethyl acetate extract by silica gel column chromatography using hexane/ethyl acetate gradient as the mobile phase to obtain an eluate fraction g; and ( 5 ) purifying the eluate fraction g by silica gel column chromatography using hexane/ethyl acetate gradient as the mobile phase to obtain a specific eluate fraction which could be purified further to obtain the anti-cancer active substance from Antrodia Camphorata.

1 . A bioactive anti-cancer substance purified from  Antrodia camphorata , wherein the purification comprising the steps of:
 Step 1 providing the mycelium pellets of  Antrodia camphorata;      Step 2: extracting the mycelium pellets of  Antrodia camphorata  with organic solvent and then freeze drying;   Step 3 dissolving the freeze dried mycelium extract from step 2 in water and the suspension is extracted with ethyl acetate;   Step 4 purifying the ethyl acetate extract from step 3 by silica gel chromatography, wherein the fraction f is eluted with 80% hexane/20% ethyl acetate to 70% hexane/30% ethyl acetate and the fraction g is eluted with 70% hexane/30% ethyl acetate to 60% hexane/40% ethyl acetate;   Step 5 purifying the fraction g from step 4 by silica gel chromatography by using hexane/ethyl acetate gradient as the mobile phase, wherein the fraction E is eluted with 80% hexane/20% ethyl acetate to 75% hexane/25% ethyl acetate, and the fraction F and G are eluted with 75% hexane/25% ethyl acetate, and the fraction H is eluted with 75% hexane/25% ethyl acetate to 70% hexane/30% ethyl acetate and the fraction I is eluted with 70% hexane/30% ethyl acetate; and   Step 6 purifying the fractions E, F, G, H and I from step 5 by high-performance liquid chromatography (HPLC) to obtain the pure compound 4-acetylantroquinonol B, wherein the ethyl acetate extract from step 3, fraction f and g from step 4, fraction E, F, G, H and I from step 5 and 4-acetylantroquinonol B are the bioactive anti-cancer substance.   
     
     
         2 . The bioactive anti-cancer substance as claimed in  claim 1 , wherein in the step 2 the freeze dried pellets are extracted by organic solvent and wherein a ratio of mycelium pellets to organic solvent is about 0.02 to 0.1 (g/mL). 
     
     
         3 . The bioactive anti-cancer substance as claimed in  claim 1 , wherein in the step 3 the mycelium extract is dissolved in water first, and then the suspension of the mycelium extract is extracted by ethyl acetate and wherein a ratio of the mycelium suspension to ethyl acetate is about 0.4 to 3.0. 
     
     
         4 . The bioactive anti-cancer substance as claimed in  claim 1 , wherein in the step 4 the silica gel chromatography is applied with silica gels for preparative chromatograph. 
     
     
         5 . The bioactive anti-cancer substance as claimed in  claim 1 , wherein in the step 5 the silica gel chromatography is applied with silica gels for preparative chromatograph. 
     
     
         6 . The bioactive anti-cancer substance as claimed in  claim 1 , wherein a column for high-performance liquid chromatography (HPLC) is a normal phase silica gel column and the fractions are eluted with 50% hexane/50% ethyl acetate, 60% hexane/40% ethyl acetate, 70% hexane/30% ethyl acetate, 80% hexane/20% ethyl acetate or 90% hexane/10% ethyl acetate. 
     
     
         7 . The bioactive anti-cancer substance as claimed in  claim 1 , wherein the anti-cancer substance inhibits proliferation of hepatoma liver cancer cells, colon cancer cells, prostate cancer cells, or breast cancer cells. 
     
     
         8 . A pharmaceutical composition used in inhibiting the proliferation of cancer cells, comprising of the bioactive anti-cancer substance as claimed in  claim 1  and pharmaceutical-acceptable excipients. 
     
     
         9 . A pharmaceutical composition used in inhibiting the proliferation of cancer cells as claimed in  claim 8 , wherein the cancer cells are hepatoma liver cancer cells, colon cancer cells, prostate cancer cells, or breast cancer cells. 
     
     
         10 . A pharmaceutical composition used in inhibiting the proliferation of cancer cells as claimed in  claim 8 , wherein the bioactive anti-cancer substance are the ethyl acetate extract from step 3, fraction f and g from step 4, fraction E, F, G, H and I from step 5 and 4-acetylantroquinonol B. 
     
     
         11 . A bioactive anti-cancer compound which is purified from  Antrodia camphorata  is 4-acetylantroquinonol B. 
     
     
         12 . A bioactive anti-cancer compound as claimed in  claim 11 , wherein the bioactive anti-cancer compound has the following structure: 
       
         
           
           
               
               
           
         
       
     
     
         13 . The bioactive anti-cancer compound as claimed in  claim 11 , wherein the cancer cells are hepatoma liver cancer cells, colon cancer cells, prostate cancer cells, or breast cancer cells.