Abstract:
The present invention provides a composition of  Antrodia cinnamomea , a mixture of  Antrodia cinnamomea , a fungal mixture and preparation thereof. In particular, the present invention can massively produce fungi in gel mediums.

Description:
FIELD OF THE INVENTION 
       [0001]    The present invention provides a composition of  Antrodia cinnamomea , a mixture of  Antrodia cinnamomea , a fungal mixture and preparation thereof. In particular, the present invention can massively produce fungi in gel mediums. 
       BACKGROUND OF THE INVENTION 
       [0002]      Antrodia cinnamomea  is a perennial fungus under the classification of Aphyllophorales, Polyporaceae, and Antrodia. It is an unique Taiwanese fungus only grown on Taiwanese  Cinnamomum kanehirae  Hay.  Antrodia cinnamomea  is a special and precious medicinal fungus as well as the most expensive wild fungus nowadays in Taiwan. The first publication of  Antrodia cinnamomea  is in 1990, when Zhang and Su presented  Antrodia cinnamomea  fruiting body with contaminated  Ganoderma  spores and grouped it as a new species under  Ganoderma commmphoratum  Zang &amp; Su. In 1995, the second new species reported by Chang and Chou based on morphology and cultivation features of  Antrodia cinnamomea . Particularly, they discovered that  Antrodia cinnamomea  is wood brown rot fungus, and classified it as a new species  Antrodia cinnamomea  Chang T T &amp; W N Chou under  Antrodia . In 1997, the third new species publication renamed this species as  Antrodia cinnamomea  by Wu. In 2004, based on ICBN Article 9. 12 (Greuter et al., 2000), Chang and Chou disregarded the misleading  Ganoderma comphoratum  and  Antrodia camphorate , and recovered to  Antrodia cinnamomea.    
         [0003]    Research has shown that the extract from  Antrodia cinnamomea  fruiting body contains three triterpene compounds which are antcin A, antcin B and antcin C. Three new triterpene compounds has been identified in further research as antrocin, 4,7-dimethoxy-5-methyl-1,3-benzodioxole, and 2,2′,5,5′-tetramethoxy-3,4,3′,4′-bimethyl-enedioxy-6,6′-dimethylbip henyl. In 1996, another four new triterpene compounds have been found such as antcin E, antcin F, methylantcinate G, and methylanticinate H. In 1996, two ergostane based new compounds were identified from fruiting body extract, such as zhankuic acid D and zhankuic acid E; and three lanostane based new compounds were identified from fruiting body extract as 15α-acetyl-dehydro-sulphurenic acid, dehydroeburicoic acid, and dehydro-sulphurenic acid. 
         [0004]    Based on previous research, it is known that  Antrodia cinnamomea  contains complex compounds. Besides triterpene, it contains lots of physiological active substances such as polysaccharides, SOD, adenosine, small molecular protein, vitamin, trace elements, nucleic acid, steroid, blood pressure stabilizing substance, and etc. Previous research revealed that  Antrodia cinnamomea  contains unique triterpene compounds, although their activity of anti-cancer cell growth and neuron cell growth activation is not fully discovered yet. 
         [0005]    Because wild  Antrodia cinnamomea  grows in dark, damp, low temperature condition of middle altitude mountainous regions accompanied with its long growth period (over one year), the time to produce fruiting body is very long.  Cinnamomum kanehirae  Hay. and wild  Antrodia cinnamomea  are reserved plants, and current artificial cultivation method is not able to provide mass cultivation and biological activity simultaneously. Besides, serious stealing issue of wild  Antrodia cinnamomea  brings this species the crisis of extinction. Currently, there are three cultivation methods: liquid fermentation, solid cultivation and wood cultivation. Liquid fermentation is used in early stage mainly in industry and academia which is good for mass production with short cultivation time, high yield, and high carbohydrate content in general. However, this method can not produce the effective triterpene compounds. Therefore, the research is restricted to the function of polysaccharide and its potential is limited. Solid cultivation is filling various grains into plastic bag or container as medium for higher yield. It produces some kinds of triterpene with more complete features than that from liquid fermentation. However, the production rate is not stable, and it is hard to separate fungi from its medium. Therefore, the grain medium is usually harvested simultaneously causing less than 5% recovery of  Antrodia cinnamomea . As a result, total activity is decreasing, and the cultivation time is as long as 3-4 months. Wood cultivation is using  Cinnamomum kanehirae  Hay. to culture  Antrodia cinnamomea , which is used to solve the stealing problem of  Cinnamomum kanehirae  and  Antrodia cinnamomea  without real benefit. The problems with wood cultivation includes 1-2 years of growth period, limited source of wood, and extreme quality variation between each lot leading to unstable product quality. Due to current shortcoming and limitation of  Antrodia cinnamomea  cultivation, the present invention is applying gel medium to cultivate  Antrodia cinnamomea  in corporation with screening platform to obtain  Antrodia cinnamomea  with cancer cell growth inhibition, liver protection and other activities through medium modification. The advantage of the present invention is not only eliminating the medium effect on its function and higher fungal purity, shortening cultivation time (1˜1.5 month), and reducing the contamination chances during long cultivation time, but also improving space circulation for improving unit production rate, and lowering cost. 
       SUMMARY OF THE INVENTION 
       [0006]    The present invention provides a composition of  Antrodia cinnamomea , a mixture of  Antrodia cinnamomea , a fungal mixture and preparation thereof. In particular, the present invention can massively produce fungi in gel mediums. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0007]      FIG. 1  shows a flow chart of  Antrodia cinnamomea  cultivation method. 
           [0008]      FIG. 2  illustrates a bar graph of inhibition effect of  Antrodia cinnamomea  extracts on human cancer cells. 
           [0009]      FIG. 3  illustrates a bar graph of scavenging effect of  Antrodia cinnamomea  extracts on DPPH (1,1-diphenyl-2-picrylhydrazyl) radicle. 
           [0010]      FIG. 4  illustrates the effect of  Antrodia cinnamomea  extracts and CCl 4  on cell released GOT. 
           [0011]      FIG. 5  illustrates  Antrodia cinnamomea  curing effect on CCl 4  induced liver damage. 
           [0012]      FIG. 6  shows rat liver tissue section of  Antrodia cinnamomea  treated CCl 4  induced liver damage. 
           [0013]      FIG. 7  shows the HPLC chromatogram of  Antrodia cinnamomea  from three different cultivation methods. A: Zhankuic acid A; B: Zhankuic acid B; C: Antcin K. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0014]    Currently, there are three cultivation methods: liquid fermentation, solid cultivation and wood cultivation. Liquid fermentation is used in early stage mainly in industry and academia which is good for mass production with short cultivation time, high yield, and high carbohydrate content in general. However, this method can not produce the effective triterpene compounds. Therefore, the research is restricted to the function of polysaccharide and its potential is limited. Solid cultivation is filling various grains into plastic bag or container as medium for higher yield. It produces some kinds of triterpene with more complete features than that from liquid fermentation. However, the production rate is not stable, and it is hard to separate fungi from its medium. Therefore, the grain medium is usually harvested simultaneously causing less than 5% recovery of  Antrodia cinnamomea . As a result, total activity is decreasing, and the cultivation time is as long as 3˜4 months. Wood cultivation is using  Cinnamomum kanehirae  Hay. to culture  Antrodia cinnamomea , which is used to solve the stealing problem of  Cinnamomum kanehirae  and  Antrodia cinnamomea  without real benefit. The problems with wood cultivation includes 1˜2 years of growth period, limited source of wood, and extreme quality variation between each lot leading to unstable product quality. 
         [0015]    In order to improve this situation for massive production of  Antrodia cinnamomea  with high pharmacological activity, the present invention is invented. 
         [0016]    Therefore, the present invention is related to a gel medium cultivated  Antrodia cinnamomea  mycelium extract which exhibits the HPLC features as shown in  FIG. 7(   c ). 
         [0017]    The present invention is also related to a mixture which comprises the extract of the present invention. 
         [0018]    In preferred embodiment of the mixture, the extract comprises compound as shown in formula I below: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    wherein, R 1  is α-OH or ═O, R 2  is β-OH or H, R 3  is β-OH or ═O, R 4  is α-OH or H, and R 5  is H. 
         [0019]    The mixture of the present invention inhibits cancer cell growth (such as human liver cancer cell) or exhibits liver protection function (such as inhibiting cell released GOT and GPT, reducing the probability of liver fibrosis or liver cell pathogenesis). 
         [0020]    In preferred embodiment of the mixture comprises (a) antcin K, wherein R 1  is α-OH, R 2  is β-OH, R 3  is β-OH, R 4  is H, and R 5  is H; (b) zhankuic acid A, wherein R 1  is O, R 2  is H, R 3  is ═O, R 4  is H, and R 5  is H; (c) zhankuic acid B, wherein R 1  is α-OH, R 2  is H, R 3  is ═O, R 4  is H, and R 5  is H; or (d) zhankuic acid C, wherein R 1  is α-OH, R 2  is H, R 3  is ═O, R 4  is α-OH, and R 5  is H. 
         [0021]    The present invention is also related to a fungal cultivation method, which comprises the following steps: 
         [0000]    a) small scale fungal cultivation on medium for fungal propagation;
 
b) inoculate fungi containing medium onto a gel medium which is suitable for at least two fungal culture blocks;
 
c) culture gel medium till fungal maturation; and
 
d) harvest fungi from matured fungal medium.
 
         [0022]    The fungi of the present invention is obtained from wild or artificial cultivated  Antrodia cinnamomea.    
         [0023]    The gel medium of the present invention contains basal material (such as agar), carbon source (such as glucose, fructose, galactose, lactose, starch, cellulose, sucrose or any combination from above), nitrogen source (such as peptone, tripeptone, beef extract, malt extract, yeast extract or any combination from above), extender (such as sunflower oil, olive oil, vitamin B6 or any combination from above), and inorganic salt (such as phosphate, sodium chloride, magnesium sulfate, potassium chloride or any combination from above). Inorganic salt comprises but is not limited to K 2 HPO 3 , K 3 PO 3 , KH 2 PO 3 , NaH 2 PO 3 , Na 2 HPO 3  or any combination from above. 
         [0024]    The weight percentage of each component in the gel medium in the present invention is as below: 
         [0000]    The weight percentage of basal material is 0.5˜3%.
 
The weight percentage of carbon source is 0.5˜30%.
 
The weight percentage of nitrogen source is 0.05˜5%.
 
The weight percentage of extender is 0.005˜0.05%.
 
The weight percentage of inorganic salt is 0.01˜2.5%.
 
         [0025]    The gel medium in the present invention is incubated at 16˜32C for 20˜45 days. 
         [0026]    The present invention has been examined by anti-cancer cell growth assay, anti-free radical assay, inhibition of cell released GOT and GPT assay, and in vivo liver protection assay and has demonstrated significant curing effect in above assays. 
         [0027]    Features and advantages of the present invention are revealed in the following preferred embodiments and claims. 
       EXAMPLES 
       [0028]    The following examples illustrate the present inventions as material and method guidance but not limited to the same. 
       Example 1 
     Cultivation Method 
       [0029]      FIG. 1  shows a flow chart of  Antrodia cinnamomea  cultivation method in preferred embodiment of the present invention. As shown in  FIG. 1 , the first step is precultivation of  Antrodia cinnamomea , such as on a slant medium. In this step,  Antrodia cinnamomea  is obtained wildly or artificially cultivated. The purpose of precultivaiton is to reserve  Antrodia cinnamomea  strain as a source for later artificial cultivation. Precultivation condition and method can be used as any known method. 
         [0030]    After that, inoculate  Antrodia cinnamomea  on a gel medium. In other words, take part of the precultivated  Antrodia cinnamomea  and inoculate it on a gel medium for cultivation. The gel medium of the present invention comprises a basal material, a carbon source, a nitrogen source, an extender and an inorganic salt. The weight percentage of basal material such as agar is 0.5˜3% relative to the 100% total weight of gel medium. 
         [0031]    Besides, carbon source of the gel medium comprises glucose or dextrose, fructose, galactose, lactose, starch, cellulose, sucrose and any combination from above. The weight percentage of carbon source is 0.5˜30% relative to the 100% total weight of gel medium. 
         [0032]    In addition, nitrogen source of the gel medium comprises peptone, tripeptone, beef extract, malt extract, yeast extract and any combination from above. The weight percentage of nitrogen source is 0.05˜5% relative to the 100% total weight of gel medium. 
         [0033]    Other than that, extender of the gel medium comprises sunflower oil, olive oil, vitamin B6 and any combination from above. The weight percentage of extender is 0.005˜0.05% relative to the 100% total weight of gel medium. 
         [0034]    Besides, inorganic salt of the gel medium comprises phosphate, sodium chloride, magnesium sulfate, potassium chloride and any combination from above. The phosphate comprises K 2 HPO 3 , K 3 PO 3 , KH 2 PO 3 , NaH 2 PO 3 , Na 2 HPO 3  and any combination from above. The weight percentage of inorganic salt is 0.01˜2.5% relative to the 100% total weight of gel medium. 
         [0035]    Temperature for  Antrodia cinnamomea  cultivation in gel medium is between 16˜32° C. The shaking condition for gel medium is standing still. The cultivation time is between 20˜45 days. 
         [0036]    The cultivated  Antrodia cinnamomea  exhibits the same physiological activity as fruiting body of wild  Antrodia cinnamomea . Therefore, it can be used for inhibiting human cancer cell growth and lowering GOT and GPT rising from liver damage. 
         [0037]    Test results are listed below to demonstrate that cultivated  Antrodia cinnamomea  based on the present invention has the ability to inhibit human cancer cell growth and lower GOT and GPT rising from liver damage. 
       Example 2 
     Anti-Human Cancer Cell Growth Assay 
       [0038]    This assay is based on anti-cancer drug screen assay developed by National Cancer Institution (NCI). The assay is performed with human cancer cell line (Hep 3B) in vitro. Human cancer cell line (Hep 3B) is cultured in fetal serum medium for 24 hours before replacing it with new medium, and then testing samples are added to culture for 72 hours. MTT assay is used to evaluate cell survival rate. Cultured  Antrodia cinnamomea  obtained based on the present invention is used in human liver cancer cell inhibition assay by calculating the cell survival rate in 96-well microtiter plate and evaluating it with MTT assay. 
         [0039]    MTT Assay: 
         [0040]    MTT(3-[4,5-Dimethylthialzol-2-yl]-2,5-diphenyltetrazolium bromide) is tetrazolium, a yellow pigment, which can be adsorbed by living cells and reduced to blue formazan by succinate-tetrazolium reductase in mitochondria. It is usually used for screening chemical effect on cell growth and proliferation. 
         [0041]      FIG. 2  shows that gel medium cultured  Antrodia cinnamomea  exhibits higher activity than liquid or solid medium cultured ones on inhibiting human liver cancer cell growth, and its activity is the most similar to that from wild  Antrodia cinnamomea.    
       Example 3 
     Anti-Free Radical Assay 
       [0042]    This assay is performed by DPPH clearance ability in vitro. Add DPPH solution into different concentration of  Antrodia cinnamomea  ethanol extract, mix it thoroughly and let it react in the dark for 30 minutes before detecting OD 517 nm with a spectrophotometer. The lower the OD value represents stronger clearing ability. 
         [0043]      FIG. 3  shows that gel medium cultured  Antrodia cinnamomea  has higher activity than liquid or solid medium cultured  Antrodia cinnamomea  and wild ones on DPPH clearance ability. 
       Example 4 
     Inhibition of Cell Released GOT Assay 
       [0044]    This assay is performed with human liver cancer cell line (Hep G2) in vitro. Human liver cancer cell line (Hep G2) is cultured in medium containing fetal bovine serum for 24 hours before replacing it with new medium, and then testing sample and CCl 4  are added for 1 hour. Mix supernatant with 4% MTS serum free medium and detect OD 490 nm value by ELISA reader after 40 minutes. This assay is performed with cultured  Antrodia cinnamomea  obtained based on the present invention for inhibition of cell released GOT, and calculated by the OD value in 24-well microtiter plate. Lower OD value represents better inhibition effect. 
         [0045]      FIG. 4  shows gel medium cultured  Antrodia cinnamomea  (CAC101) has better inhibition activity on cell release GOT than alternative cultivated  Antrodia cinnamomea  (CJ50005, CT50013) and wild ones (AC). 
       Example 5 
     In Vivo Liver Protection Activity Assay 
       [0046]    The animal model is performed with CCl 4  induced rat chronic liver damage. 
         [0047]    Animal model is performed with 12-week-old, 250-300 g weight male WISTAR animal. Domesticated laboratory animals are grouped into 6 groups for orally received and CCl 4  induced liver disease before tube feeding and  Antrodia cinnamomea  treatment to test the curing effect on CCl 4  induced liver damage as shown in Table 1. 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Treatment of each group 
               
             
          
           
               
                   
                 Induced chronic 
                   
               
               
                 group 
                 liver damage (1)   
                 Oral feeding 
               
               
                   
               
               
                 Group 1 (mock) 
                 Olive oil 
                 Water 
               
               
                 Group 2 (negative control) 
                 6% CCl 4 /olive oil 
                 Water 
               
               
                 Group 3 (positive control) 
                 6% CCl 4 /olive oil 
                 Silymarin (2) /2% CMC 
               
               
                 Group 4 
                 6% CCl 4 /olive oil 
                 Low dose (3)   
               
               
                 Group 5 
                 6% CCl 4 /olive oil 
                 Moderate dose (4)   
               
               
                 Group 6 
                 6% CCl 4 /olive oil 
                 High dose (5)   
               
               
                   
               
               
                   (1) twice a week, 2.5 ml/kg each time 
               
               
                   (2) 200 mg/kg everyday 
               
               
                   (3) 0.15 g/kg everyday 
               
               
                   (4) 0.45 g/kg everyday 
               
               
                   (5) 1.35 g/kg everyday 
               
             
          
         
       
     
         [0048]    Sacrifice all rats at week 8, and examine GOT and GPT of inferior vena cava blood. Empty the blood, take right liver leaf, sample the same portion of 1×1 cm liver tissue and put it into 10% neutral formalin for pathological section. 
         [0049]      FIG. 5  demonstrates the curing effect of  Antrodia cinnamomea  on CCl 4  induced liver damage. It shows that gel medium cultured  Antrodia cinnamomea  can effectively inhibit CCl 4  induced GOT and GPT rising in damaged liver. Liver section is obtained from sacrificed animals. As shown in  FIG. 6 , group 1 (mock) exhibits normal liver tissue; group 2 (negative control) exhibits extensive liver cell dysplasia and fibrillar connective tissue cutting into liver lobule; group 3 (positive control) shows small amount of liver cell dysplasia and less fibrillar connective tissue cutting into liver lobule; group 4 (low dose) is shown the least liver cell dysplasia; group 5 (moderate dose) is shown extensive liver cell dysplasia and small amount of fibrillar connective tissue cutting into liver lobule; and group 6 (high dose) is shown huge amount of liver dysplasia. It demonstrates that gel medium cultured  Antrodia cinnamomea  obtained based on the present invention can reduce the rising of GOT, GPT, probability of liver fibrosis, and liver cell pathogenesis. 
       Example 6 
       Antrodia cinnamomea  Extract Preparation 
       [0050]      Antrodia cinnamomea  extract is obtained by grinding 100 g of  Antrodia cinnamomea  dry powder, adding 2 L of ethanol, stirring it for 1-4 hours for extraction, filtering the extracted solution for concentration and finally harvesting 15%  Antrodia cinnamomea  extract. Its HPLC chromatogram is shown in  FIG. 7 . The HPLC column used here is Cosmosil Paked Column 5C18-AR, 4.6×250 mm, and HPLC mobile phase is listed in the table below. The flow rate of HPLC is 1 ml/min, and detector is DAD with wavelength setting at 210, 243, 254, and 280 nm. 
         [0000]    
       
         
               
               
               
             
               
               
               
             
           
               
                   
               
               
                 Time (min) 
                 0.01% H 3 PO 4  (%) 
                 CH 3 CN (%) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 0 
                 70 
                 30 
               
               
                 65 
                 53 
                 47 
               
               
                 110 
                 53 
                 47 
               
               
                 140 
                 0 
                 100 
               
               
                 170 
                 0 
                 100 
               
               
                 175 
                 70 
                 30 
               
               
                 200 
                 70 
                 30