Patent Document

BACKGROUND OF THE INVENTION  
       [0001]     Zang Zhi is a kind of fungus. Its academic name is  Antrodia camphorata,  also known as “Zang ku”, “red Zang Zhi”, “niou Zang Zhi” or “niou Zang ku.” In Taiwan, Zang Zhi only grows on the inner wall of the empty rotting trunk of  Cinnamomum kanehirai  in the shape of a platy or bell and has a strong cinnamon flavor. People say that Zang Zhi can tranquilize, prevent or treat a cold, promote vital energy circulation, remove blood stasis, promote blood circulation, relieve dyspepsia, detoxify and promote the subsidence of swelling, calm the nerves to reduce stress, alleviate pain, and is the best anticorrosive and detoxicant. It is considered the most expensive wild fungus in the market on Taiwan. The mechanism of anti-tumor cells of Zang Zhi is different from that of  Ganoderma  sp., which indirectly promotes immunity against tumors. Zang Zhi can directly inhibit or kill cancer cells, and has the capacity to strengthen the heart, adjust immunity, and antagonize the parasympathetic nervous system, and has serous activity. In particular, Zang Zhi can cure a stomachache, bowelache, nausea, diabetes, gout, arthritis, fever, allergy, exorbitant urine proteins, uremia, cirrhosis, hepatomia, flu, etc. Many research institutions have invested human resources and materials in the study of the active constituents of Zang Zhi. Among them, the National Science Council of the Executive Yuan has a special research project to make use of chemical methods, NMR spectrum analysis, and comparative spectrum with known materials to establish the constitutive characteristics of active constituents. Research results of the active constituents show that the water and methanol extracts of Zang Zhi are effective on the inhibition of the growth of  Streptococcus aureus  and  Trichophytone mentagrophytes.  Zhankuic acid A, a methanol extract of Zang Zhi has conspicuous inhibition to the lymphoma cells of a p-388 mouse and agglutination of blood platelets. Zhankuic acid B, however, shows little anticholinergic and anti-serotonin effect.  
         [0002]     It is indicated in Gau Sheau Jy&#39;s masters thesis on the triterpenoid of Zang Zhi that Zang Zhi is a new species of  Ganoderma  discovered in 1990. The extract of the solid fruiting body is obtained by use of acetone, and then is separated and recrystallized with chromatography LC and PLC. The purity of the extract is identified by TLC scanning and HPLC. The configuration test is done with mass spectrum, infrared spectrum, ultraviolet spectrum, H-NMR, and C-NMR. 3,11-dioxo-8,23-dien-26-oic acid can reduce GPT in the blood of a mouse with the acute hepatitis induced by CCl 4 .  
         [0003]     Most of Zang Zhi grows in the broad-leaf forest areas in Taiwan&#39;s East Coast Mountain Range. However, according to the bulletin of the Niou Zang Conservation Notice issued by the Taiwan Forestry Bureau, this mountain range has been listed as a natural resources conservation zone. Since Zang Zhi is a very popular product which most people are willing to purchase in Asia, Zang Zhi growing outside the said zone have been almost completely harvested. In fact, wild Zang Zhi are not available in the local markets.  
         [0004]     With an estimated sale of 500 kg Zang Zhi per month in Taiwan, the requirement to provide an incubation method for solid culture of Zang Zhi has become more important in recent years.  
       SUMMARY OF THE INVENTION  
       [0005]     The main objective of the subject invention is to provide an incubation method for solid culture of Zang Zhi. The cultured Zang Zhi will have the same pharmaceutical efficacy as the wild one does.  
         [0006]     The other objective of this invention is to provide a solid culture of Zang Zhi by use of the inoculums of Zang Zhi spawn preserved in the Food Industry Research and Development Institute (Taiwan), coded CCRC 35398, to incubate the solid culture in accordance with the incubation method.  
         [0007]     Another objective of the invention is to provide the processed products of the solid cultured Zang Zhi.  
         [0008]     Another objective of the invention is to provide a solid cultured Zang Zhi useful as active constituents. 
     
    
     BRIEF DESCRIPTIONS OF THE DRAWINGS  
       [0009]      FIG. 1  illustrates comparative analysis of the HPLC of constituents of a Zang Zhi, including (1) the fruiting body of cultured Zang Zhi according to the invention, (2) the liquid culture of Zang Zhi and (3) the fruiting body of wild Zang Zhi.  
         [0010]      FIG. 2  illustrates the use of ferrous ions to stimulate the homogenization of mouse brain to cause the free radical peroxidative reaction of lipid, which will result in increasing of the TBARS (peroxidative constituents of lipid). Comparing the triple fold with the eight fold of concentrated extract of Zang Zhi fruiting body cultured according to the invention, we note that the increase inhibition of the peroxidative reaction will vary with the increased of the concentration, that is shown by the percentage of inhibition of per oxidative reaction (n=3).  
         [0011]      FIG. 3  shows the effect of different concentrations of extract of Zang Zhi on the active change of GTP for measurement of liver function. # represents the statistical difference between the normal group and the injury group (P&lt;0.01); *represents the statistical difference between the feed group and the injury group (P&lt;0.01). Value is represented by average ± standard error.  
         [0012]      FIG. 4  shows the effect of different concentrations of Zang Zhi extract on the active change of GOT for the biochemical measurement of liver function. # represents the statistical difference between a normal group and an injury group (P&lt;0.01); * represents the statistical difference between the feed group and the injury group (P&lt;0.01). Value is represented by average ± standard error.  
         [0013]      FIG. 5  shows the effect of an extract of Zang Zhi powder on the growth of bowel cancer cell (COLO 320 HSR). 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0014]     This invention provides a method for the solid culture of Zang Zhi, in order to produce the Zang Zhi fruiting body with the constituents and vitality of wild Zang Zhis. The invention&#39;s method for the solid culture of Zang Zhi is to culture the spawn of Zang Zhi through “Bag Log” cultivation, and then to produce the fruiting body of Zang Zhi in the air.  
         [0015]     Before the phase of mycelium culture, it is usual to greatly multiply the spawn for “Bag Log” cultivation, including the following three steps: (1) taking a piece of medium agar containing hyphae preserved in liquid nitrogen, and transfer it into a fresh medium to culture under constant temperature until the exuberant growth of mycelium appears; (2) inoculating the spawn into the “Bag Log” containing cellulolytic substance (for example kernel or spelk); (3) removing the supra old hyphae until the hyphae overgrows in the “Bag Log”; and (4) then inoculating the multiplied spawn into “Bag Log”.  
         [0016]     Before the phase of mycelium culture, one also can greatly multiply spawn with the liquid culture fermentation, and then inoculate them into “Bag Log”. The formulation of liquid culture fermentation is 1-3% of fructose, 0.01-0.1% of magnesium sulfate, 0.1-1% of yeast extract, and 0.05-0.5% of potassium phosphate. The best formulation of liquid culture fermentation followed was 2% of fructose, 0.05% of magnesium sulfate, 0.5% of yeast extract, and 0.1% of potassium phosphate. (The percentages of the components described in this specification are “weight percentage,” except that the percentages described for the gaseous components are “volume percentages.”)  
         [0017]     After culturing the slide tube, one may inoculate the multiplied spawn into a 5 liter liquid culture for fermentation under 24-26° C, 240 rpm oscillation for 14 days, and then 90 rpm oscillation for 14 days, and then inoculate them into a 20 liter liquid fermentation, stirring culture for 14 days.  
         [0018]     Here, the so-called “Bag Log” is the plastic bag made from 10-70% of cellulolytic material of stem, stalk, fruit, or spelk of any mushroom or plant (especially, the stems, stalks, and fruits of grass plants or even cellulolytic spelk are better), 10-30% of starch resource (especially, potato is better), and 5-15% of millet (rice bran is better), 1-10% of saccharides (glucose is better), 0.5-2% of phosphate (potassium phosphate is better), and 0.1-1% of sulfate salt (magnesium sulfate is better). Relative humidity is maintained at 60-90%, better at 80%. The pH value of the culture medium is adjusted to be neutral.  
         [0019]     The phase of mycelium culture indicated in this text means a period of sixty days after the spawn is inoculated into “Bag Log”. Mycelium will grow at a temperature from 5° to 32° C.; in particular, 28° C. is the best temperature for the growth of mycelium. “Bag Log” cultivation is under way at a relative humidity of about 60-80%, and better at about 80%. As to the condition of air, 0.1-1% of carbon dioxide is better for the culture.  
         [0020]     After the phase of mycelium culture, the phase of the culture of the fruiting body is next. That is to say 61-90 days after the date of the inoculation of the spawn, the plastic bag of “Bag Log” is to be removed to let the sawdust medium be completely exposed to the air. During the phase of the culture of the fruiting body, the difference between day and night temperature has to be under strict control. Day temperature is generally maintained between 20° and 30° C., especially, better at 25±1° C.; night temperature is better maintained at 11±1° C.; the difference between day and night temperature is better maintained at 15° C. The relative humidity at the phase of fruiting body culture is controlled between 90 and 95%. In addition, the fruiting body has to be cultured in the moving air, and the carbon dioxide is less than 1%. Generally, solid culture material will be collected after 90-120 days.  
         [0021]     The strain CCRC35398 spawn used in this invention can be obtained from the Food Industry Research and Development Institute, Taiwan. According to the solid culture method of this invention, a solid culture substance was obtained. The solid culture substance is conoid, about 10-30 cm in diameter, 15-30 cm in height, and 0.2-0.6 kg in weight.  
         [0022]     To use the solid culture method described above, one can get the solid Zang Zhi. The solid Zang Zhi can be produced by any of the processing methods known to those skilled in the art, for example, water or alcohol can be used to extract the active constituents, and then known concentration methods such as vacuum concentration or freeze concentration can be used to produce the concentrated product.  
         [0023]     One can use the dehydrated method to do the dehydration, or by a spray-dry or lyophilization method. However, the lyophilization method is the best one to dehydrate the solid substance, and during dehydration the solid substance will not change its characteristic by the effect of oxygen.  
         [0024]     The microscopic particles produced by the spray-dry or other dehydration method can be agglomerated to increase their diameters. These products are poremeric particles with good wettability.  
         [0025]     The comparative KPLC spectrum of the invention&#39;s solid culture Zang Zhi, known liquid cultured Zang Zhi, and wild Zang Zhi is shown in  FIG. 1 . By analysis of HPLC, the constituents of the fruiting body obtained through the invention&#39;s culture method are similar to those of the wild Zang Zhi and the invention&#39;s Zang Zhi has the activities and functions similar to the wild Zang Zhi&#39;s. These activities and functions include tranquilization, prevention from or treatment of a cold, promoting vital energy circulation, removing blood stasis, promoting blood circulation, relieving dyspepsia, detoxifying and promoting the subsidence of swelling, calming the nerves to reduce stress, alleviating pain, inhibiting bacteria and virus and cancer cells, strengthening the heart, adjusting immunity, and antagonizing the parasympathetic nervous system and serous activity. Zang Zhi can cure a stomachache and bowel ache, nausea, diabetes, gout, arthritis, infection, allergy, exorbitant urine proteins, uremia, cirrhosis, hepatomia, flu and so on. Zang Zhi can regenerate skin and act as human skin or callus material, or as adhesive for bedsore or traumatic skin.  
         [0026]     Furthermore, the invention&#39;s solid cultured substance possesses the abilities to antagonize the free radicals and peroxidation of lipids, and functions to protect the liver and inhibit cancer cells. The invention&#39;s solid cultured substance can be taken as health food to prolong life, and be further developed into new medicines.  
         [0027]     The following working examples will further explain this invention, but not limit this invention. Any application or modification of this invention made by persons skilled in these techniques will fall in the scope of this invention.  
       WORKING EXAMPLES  
       [0028]     The strain CCRC35398 spawn obtained from the Food Industry Research and Development Institute were grown by ferment liquid culture, and then inoculated into the “Bag Log”. The fermented liquid culture contains 2% of fructose, 0.05% of magnesium sulfate, 0.5% of yeast extract, and 0.1% of potassium phosphate. “Bag Log” cultivation is to culture mycelium. “Bag Log” is composed of 65% of the stems, stalks, fruits of grass plants or cellulolytic spelk, 20% of potato, 10% of rice bran, 3.5% of glucose, 1% of potassium phosphate, and 0.5% of magnesium sulfate. Adjust the relative humidity to 80% and pH to 7. Relative humidity was maintained at 60-90%, better at 80%, and the pH of the culture medium was adjusted to being neutral.  
         [0029]     The period of mycelium culture is 60 days after the spawn was inoculated into “Bag Log”. Mycelium will grow at a temperature between 5° and 32° C.; 28° C. is the optimal temperature for the growth of mycelium. “Bag Log” humidity is at 80%, and air condition is 0.2-1% of carbon dioxide.  
         [0030]     After the phase of the mycelium culture, the phase of the culture of the fruiting body is on, that is to say 61-90 days after the date of inoculation of the spawn, the plastic bag of “Bag Log” is to be removed, to let the sawdust medium be completely exposed in the air. During the phase of the culture of the fruiting body, the difference between day and night temperature has to be under strict control. Day temperature is generally maintained between 20° and 30° C.; night temperature is better maintained at 11±1° C.; the difference between day and night temperature is better maintained at 15° EC. The relative humidity of the phase of the fruiting body culture is controlled between 90 and 95%. In addition, the fruiting body has to be cultured in the moving air, and the carbon dioxide is less than 1%.  
         [0031]     In 90-120 days, when the conoid fruiting body grows up to 15 cm in diameter and 25 cm in height, the solid-cultured substance will be collected, and then the substance is about 0.4 kg in weight. The HPLC spectrum of major active elements—triterpenoid—is shown in  FIG. 1 ( 1 ), and the measurements are shown in the following Table 1.  
                                                                                                   TABLE 1                           Table 1, Data of HPLC analysis of the invention&#39;s Zang Zhi fruiting body                retention   area       relative           conc       ingredient       No.   time   (uv * sec)   area %   height   height %   conc   unit   basline   name                    1   2.060   27958870   9.3018   1000.279   6.5362   0.0000       PV           2   2.547   6320387   2.1028   999.303   6.5299   0.0000       VV       3   2.663   6333234   2.1070   999.158   6.5289   0.0000       VV       4   2.823   13521140   4.4986   998.886   6.5271   0.0000       VV       5   3.117   3933511   1.3087   328.263   2.1450   0.0000       VV       6   3.413   4614312   1.5352   303.141   1.9808   0.0000       VV       7   3.673   2519874   0.6364   162.126   1.1901   0.0000       VV       8   3.973   2084553   0.6935   144.477   0.9441   0.0000       VV       9   4.367   4265639   1.4192   149.458   0.9766   0.0000       VV       10   5.150   7401131   2.5289   460.320   3.0079   0.0000       VV       11   5.773   1671478   0.5561   71.212   0.4657   0.0000       VB       12   6.043   441685   0.1469   54.184   0.3541   0.0000       VV       13   6.360   1264481   0.4207   62.749   0.4100   0.0000       VV       14   6.597   1359149   0.4522   58.680   0.3834   0.0000       VV       15   7.263   1946283   0.6475   56.407   0.3686   0.0000       VV       16   7.660   517391   0.1721   43.081   0.2815   0.0000       VV       17   8.167   5862269   1.9504   317.837   2.0769   0.0000       VV       18   8.983   932610   0.3103   29.915   0.1955   0.0000       VV       19   9.990   1253931   0.4172   34.691   0.2267   0.0000       VV       20   10.343   538013   0.1790   22.699   0.1496   0.0000       VV       21   10.623   164318   0.0547   12.337   0.0806   0.0000       VV       22   11.327   1233415   0.4104   29.716   0.1942   0.0000       VV       23   12.193   628302   0.2090   19.256   0.1258   0.0000       VV       24   12.990   1259055   0.4189   37.378   0.2442   0.0000       VP       25   14.810   2962581   0.9923   80.403   0.5254   0.0000       PP       26   17.313   2521967   0.8390   68.342   0.4466   0.0000       PV       27   18.113   627346   0.2087   19.934   0.1303   0.0000       VV       28   18.990   117339   0.0390   4.690   0.0306   0.0000       VV       29   19.867   663514   0.2207   20.198   0.1320   0.0000       VV       30   20.867   2932057   0.9755   52.760   0.3448   0.0000       VV       31   22.323   252149   0.0839   9.636   0.0630   0.0000       VV       32   23.080   3131662   1.0419   79.476   0.5193   0.0000       VV       33   23.960   1130632   0.3762   31.023   0.2027   0.0000       VV       34   25.857   4135840   1.3760   78.025   0.5098   0.0000       VV       35   26.910   1070268   0.3561   22.420   0.1465   0.0000       VP       36   28.940   2127664   0.7079   44.217   0.2889   0.0000       PV       37   30.503   15997700   5.3224   304.306   1.9685   0.0000       VP       38   33.793   5256445   1.7488   92.365   0.6036   0.0000       PV       39   35.573   462920   0.1540   6.405   0.0549   0.0000       VV       40   37.360   4416830   1.4695   79.089   0.5168   0.0000       VP       41   39.193   261669   0.0871   5.522   0.0361   0.0000       PV       42   41.267   4411946   1.4678   73.326   0.4791   0.0000       VV       43   42.667   2821209   0.9386   50.936   0.3328   0.0000       VV       44   44.323   36608   0.0122   1.071   0.0070   0.0000       VP       45   46.630   10087950   3.3562   134.950   0.6818   0.0000       PP       46   50.090   333401   0.1109   4.502   0.0294   0.0000       PV       47   52.383   4896649   1.6291   60.376   0.3845   0.0000       PP       48   55.260   124357   0.0414   2.047   0.0134   0.0000       PV       49   56.593   4986938   1.6591   340.488   2.2249   0.0000       VV       50   56.953   7314754   2.4336   625.513   4.0874   0.0000       VV       51   57.290   4586732   1.5260   463.196   3.0267   0.0000       VV       52   57.800   3699586   1.2308   189.756   1.2399   0.0000       VV       53   58.087   1056124   0.3316   116.120   0.7716   0.0000       VV       54   58.477   2468879   0.8214   134.218   0.8770   0.0000       VV       55   56.690   1982234   0.6595   157.526   1.0293   0.0000       VV       56   58.927   1445816   0.4810   124.637   0.8157   0.0000       VV       57   59.210   3138696   1.0442   171.327   1.1195   0.0000       VV       58   59.520   1098725   0.3655   69.794   0.5867   0.0000       VV       59   59.840   1481909   0.4930   102.506   0.6698   0.0000       VV       60   60.093   3406697   1.1334   202.433   1.3228   0.0000       VV       61   60.483   1645084   0.5473   104.449   0.6825   0.0000       VV       62   60.980   10587770   3.5225   757.281   4.9484   0.0000       VV       63   61.403   1626563   0.5412   117.086   0.7651   0.0000       VV       64   61.700   856049   0.2848   79.162   0.5173   0.0000       VV       65   61.907   1498212   0.4984   88.354   0.5773   0.0000       VV       66   62.297   7358936   1.4483   538.464   3.5185   0.0000       VV       67   62.713   1007688   0.3353   74.731   0.5014   0.0000       VV       68   82.980   953127   0.3171   76.873   0.5023   0.0000       VV       69   62.187   1992004   0.6627   69.898   0.4567   0.0000       VV       70   63.603   776093   0.2582   55.283   0.3612   0.0000       VV       71   64.203   6164886   2.0510   515.260   3.3668   0.0000       VV       72   64.537   761873   0.2535   72.303   0.4725   0.0000       VV       73   64.773   2147592   0.7145   124.955   0.6165   0.0000       VV       74   65.287   2333203   0.7762   110.636   0.7229   0.0000       VV       75   65.987   1814540   0.6037   53.283   0.3482   0.0000       VV       76   66.627   1221936   0.4065   56.684   0.3704   0.0000       VV       77   67.103   1666644   0.5545   61.135   0.5302   0.0000       VV       78   67.617   1226534   0.4081   46.888   0.3064   0.0000       VV       79   66.340   3797130   1.2633   106.221   0.7072   0.0000       VV       80   66.910   3980780   1.3244   176.942   1.1562   0.0000       VV       81   69.637   2559917   0.8517   76.359   0.4990   0.0000       VV       82   70.970   680358   0.2264   16.357   0.1069   0.0000       VV       83   71.683   264668   0.0881   12.546   0.0820   0.0000       VV       84   72.387   686349   0.2283   14.902   0.0974   0.0000       VV       85   73.070   513554   0.1709   13.848   0.0905   0.0000       VV       86   73.730   419796   0.1397   9.953   0.0650   0.0000       VV       87   74.950   357733   0.1190   12.698   0.0830   0.0000       VV       88   75.373   465804   0.1550   15.474   0.1011   0.0000       VV       89   76.253   356155   0.1185   10.162   0.0664   0.0000       VV       90   77.137   133340   0.0444   4.645   0.0304   0.0000       VV       91   77.850   272953   0.0905   11.805   0.0771   0.0000       VV       92   78.420   752946   0.2505   30.824   0.2014   0.0000       VV       93   78.690   869866   0.2694   31.809   0.2079   0.0000       VV       94   79.653   66206   0.0220   2.235   0.0146   0.0000       VP       95   80.730   79648   0.0265   2.862   0.0187   0.0000       PP       96   81.753   27502   0.0091   1.070   0.0070   0.0000       PP       97   83.147   186611   0.0621   4.143   0.0271   0.0000       PV       98   83.923   293714   0.0977   9.081   0.0593   0.0000       VV       99   84.520   361025   0.1201   9.061   0.0592   0.0000       VV       100   85.820   820423   0.2730   29.144   0.1804   0.0000       VV       101   86.260   1531752   0.5096   35.708   0.2333   0.0000       VV       102   87.803   1945022   0.6471   51.978   0.3396   0.0000       VV       103   89.500   271109   0.0902   7.353   0.0480   0.0000       VP       104   90.883   26832   0.0089   0.533   0.0035   0.0000       PP       105   92.080   55385   0.0184   1.398   0.0091   0.0000       PP       106   96.443   3758662   1.2505   52.867   0.3455   0.0000       PP       107   100.237   94066   0.0323   1.137   0.0074   0.0000       PP       108   106.650   1435702   0.4777   41.107   0.2686   0.0000       PV       109   107.300   1158655   0.3855   55.895   0.3652   0.0000       VV       110   107.720   1487062   0.4947   72.435   0.4668   0.0000       VV       111   108.190   709049   0.2359   37.254   0.2434   0.0000       VV       112   108.493   818254   0.2722   38.627   0.2524   0.0000       VV       113   109.013   631416   0.2101   35.016   0.2288   0.0000       VV       114   109.507   1010362   0.3361   33.653   0.2199   0.0000       VV       115   109.900   446664   0.1486   27.863   0.1821   0.0000       VV       116   110.160   598899   0.1993   33.360   0.2180   0.0000       VV       117   110.493   840683   0.2797   57.202   0.3738   0.0000       VV       118   111.023   2135668   0.7105   103.717   0.6777   0.0000       VV       119   111.450   547756   0.1822   42.196   0.2757   0.0000       VV       120   111.833   1597280   0.5314   56.890   0.3717   0.0000       VV       121   112.473   967994   0.3220   55.981   0.3658   0.0000       VV       122   112.607   679050   0.2259   59.614   0.3895   0.0000       VV       123   112.820   536941   0.1786   49.913   0.3261   0.0000       VV       124   113.067   987108   0.3284   35.926   0.2348   0.0000       VV       125   113.807   1383823   0.4604   34.665   0.2265   0.0000       VV       126   114.563   1002529   0.3333   36.357   0.2376   0.0000       VV       127   114.970   520199   0.1731   24.136   0.1577   0.0000       VV       128   115.540   1143580   0.3805   28.119   0.1837   0.0000       VV       129   116.577   338258   0.1125   11.209   0.0732   0.0000       VV       130   117.017   176187   0.0586   8.880   0.0580   0.0000       VV       131   117.400   169393   0.0564   8.586   0.0561   0.0000       VV       132   118.133   518740   0.1726   11.014   0.0720   0.0000       VV       133   118.763   134439   0.0447   7.091   0.0463   0.0000       VV       134   119.237   368278   0.1225   10.127   0.0662   0.0000       VP       135   121.140   64584   0.0215   2.560   0.0167   0.0000       PV       136   121.620   185059   0.0616   6.282   0.0410   0.0000       VV       137   122.620   30314   0.0101   1.935   0.0126   0.0000       VV       138   123.090   254238   0.0846   6.468   0.0553   0.0000       VV       139   124.110   141072   0.0469   6.206   0.0406   0.0000       VP       140   125.877   378418   0.1259   7.976   0.0521   0.0000       PP       141   127.977   27554   0.0092   1.016   0.0066   0.0000       PP       142   129.813   187915   0.0625   9.937   0.0649   0.0000       PP           total   300574826       15303.60       0.0000                  
 
         [0032]     The differences between the invention&#39;s Zang Zhi fruiting body, other cultural methods and liquid cultural method were compared by the analysis of HPLC. The HPLC spectra—are shown in  FIG. 1 ( 2 )-( 3 ); and the measurements are shown in Table 2 and Table 3. These spectra show that the invention&#39;s Zang Zhi fruiting body and wild Zang Zhi have the same shape of peak, and also show that the invention&#39;s cultured Zang Zhi fruiting body and that of wild Zang Zhi have the same activities and functions.  
                                                                                                   TABLE 2                           Table 2. HPLC spectrum figure of liquid cultured Zang Zhi                retention   area       relative           conc       ingredient       No.   time   (uv * sec)   area %   height   height %   conc   unit   basline   name                    1   1.620   262998   1.2184   38.745   2.2045   0.0000       PV           2   1.357   8294342   38.4242   660.685   37.5912   0.0000       VV       3   2.520   5417476   25.0969   726.398   41.3301   0.0000       VV       4   2.820   1592534   7.8413   99.406   5.6559   0.0000       VV       5   3.790   731157   3.3871   23.209   1.3205   0.0000       VB       6   4.123   271974   1.2599   11.713   0.6664   0.0000       BV       7   4.623   154068   0.7137   7.810   0.4444   0.0000       VV       8   5.023   154230   0.7145   4.580   0.2606   0.0000       VV       9   5.897   29822   0.1382   1.652   0.0940   0.0000       VP       10   7.360   150572   0.6975   5.210   0.2964   0.0000       PP       11   9.210   123419   0.5717   3.914   0.2227   0.0000       PP       12   11.930   130807   0.6060   3.563   0.2027   0.0000       PV       13   12.740   37712   0.1747   1.232   0.0701   0.0000       VP       14   15.103   504463   2.3370   11.048   0.6286   0.0000       PP       15   16.890   143477   0.6647   3.512   0.1998   0.0000       PP       16   19.350   56860   0.2634   1.827   0.1039   0.0000       PP       17   23.510   806878   3.7379   11.274   0.6415   0.0000       PP       18   27.157   92042   0.4264   1.224   0.0696   0.0000       PP       19   30.633   144285   0.6684   2.243   0.1276   0.0000       PP       20   33.747   239568   1.3414   3.947   0.2245   0.0000       PP       21   38.727   250488   1.1604   3.231   0.1839   0.0000       PP       22   41.340   61644   0.2956   0.771   0.0438   0.0000       PP       23   43.727   29928   0.1386   0.470   0.0267   0.0000       PP       24   57.047   112074   0.5192   9.096   0.5175   0.0000       PV       25   57.433   175711   0.8140   10.275   0.5846   0.0000       VV       26   57.907   107598   0.4985   4.508   0.2565   0.0000       VV       27   58.137   25810   0.1196   2.279   0.1296   0.0000       VP       28   61.380   23245   0.1077   2.024   0.1152   0.0000       PV       29   62.743   196612   0.9108   21.767   1.2385   0.0000       VV       30   64.223   30411   0.1409   2.094   0.1192   0.0000       PV       31   64.717   441196   2.0439   41.620   2.3681   0.0000       VV       32   64.997   48524   0.2248   4.870   0.2771   0.0000       VV       33   65.420   40354   0.1869   3.529   0.2008   0.0000       VV       34   65.833   67766   0.3139   3.823   0.2175   0.0000       VP       35   66.950   21298   0.0987   1.691   0.0962   0.0000       PP       36   67.773   32193   0.1491   1.708   0.0972   0.0000       PP       37   79.013   29414   0.1363   0.920   0.0523   0.0000       PP       38   97.297   21884   0.1014   0.456   0.0260   0.0000       PP       39   100.053   39474   0.1782   0.738   0.0420   0.0000       PP       40   104.457   126686   0.5869   2.358   0.1342   0.0000       PP       41   107.010   57252   0.2652   2.113   0.1202   0.0000       PP       42   111.397   35854   0.1661   4.236   0.2410   0.0000       VV       43   111.813   23337   0.1313   2.512   0.1429   0.0000       VP       44   113.790   25408   0.1177   1.810   0.1030   0.0000       VP       45   115.883   43858   0.2032   3.808   0.2167   0.0000       PV       46   119.590   25451   0.1179   1.656   0.0942   0.0000       PP           total   21586254       1757.553       0.0000                  
 
         [0033]    
       
         
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                   
               
               
                 Table 3. HPLC spectrum figure of wild Zang Zhi 
               
             
          
           
               
                   
                 retention 
                 area 
                   
                 relative 
                   
                   
                 conc 
                   
                 ingredient 
               
               
                 No. 
                 time 
                 (uv * sec) 
                 area % 
                 height 
                 height % 
                 conc 
                 unit 
                 basline 
                 name 
               
               
                   
               
             
          
           
               
                 1 
                 1.550 
                 1332009 
                 0.5787 
                 162.045 
                 1.1488 
                 0.0000 
                   
                 PV 
                   
               
               
                 2 
                 1.913 
                 12708000 
                 5.5210 
                 797.430 
                 5.6535 
                 0.0000 
                   
                 VV 
               
               
                 3 
                 2.607 
                 7990504 
                 3.4715 
                 374.149 
                 2.6526 
                 0.0000 
                   
                 VV 
               
               
                 4 
                 2.997 
                 3336181 
                 1.4494 
                 151.464 
                 1.0738 
                 0.0000 
                   
                 VV 
               
               
                 5 
                 3.480 
                 827969 
                 0.3597 
                 90.536 
                 0.6419 
                 0.0000 
                   
                 VV 
               
               
                 6 
                 3.687 
                 2100942 
                 0.9127 
                 89.231 
                 0.6326 
                 0.0000 
                   
                 VV 
               
               
                 7 
                 4.110 
                 1565375 
                 0.7235 
                 80.468 
                 0.5705 
                 0.0000 
                   
                 VV 
               
               
                 8 
                 4.590 
                 986857 
                 0.4287 
                 61.543 
                 0.4363 
                 0.0000 
                   
                 VV 
               
               
                 9 
                 4.857 
                 1300649 
                 0.5651 
                 59.445 
                 0.4214 
                 0.0000 
                   
                 VV 
               
               
                 10 
                 5.377 
                 620409 
                 0.2695 
                 37.086 
                 0.2529 
                 0.0000 
                   
                 VV 
               
               
                 11 
                 5.623 
                 712514 
                 0.3095 
                 37.666 
                 0.2670 
                 0.0000 
                   
                 VV 
               
               
                 12 
                 6.020 
                 517412 
                 0.2682 
                 32.787 
                 0.2324 
                 0.0000 
                   
                 VV 
               
               
                 13 
                 6.347 
                 380678 
                 0.1654 
                 24.939 
                 0.1768 
                 0.0000 
                   
                 VV 
               
               
                 14 
                 6.660 
                 622333 
                 0.2704 
                 24.053 
                 0.1705 
                 0.0000 
                   
                 VV 
               
               
                 15 
                 7.060 
                 557302 
                 0.2421 
                 18.364 
                 0.1302 
                 0.0000 
                   
                 VV 
               
               
                 16 
                 7.710 
                 585765 
                 0.2545 
                 27.106 
                 0.1922 
                 0.0000 
                   
                 VV 
               
               
                 17 
                 8.353 
                 276221 
                 0.1200 
                 10.957 
                 0.0777 
                 0.0000 
                   
                 VV 
               
               
                 18 
                 8.867 
                 379887 
                 0.1650 
                 19.904 
                 0.1411 
                 0.0000 
                   
                 VV 
               
               
                 19 
                 9.327 
                 316284 
                 0.1374 
                 21.157 
                 0.1500 
                 0.0000 
                   
                 VV 
               
               
                 20 
                 9.623 
                 72943 
                 0.0315 
                 4.914 
                 0.0348 
                 0.0000 
                   
                 VV 
               
               
                 21 
                 10.593 
                 120581 
                 0.0524 
                 6.584 
                 0.0457 
                 0.0000 
                   
                 PP 
               
               
                 22 
                 11.260 
                 57846 
                 0.0251 
                 2.283 
                 0.0162 
                 0.0000 
                   
                 PV 
               
               
                 23 
                 11.747 
                 32476 
                 0.0141 
                 2.199 
                 0.0156 
                 0.0000 
                   
                 VV 
               
               
                 24 
                 12.263 
                 209326 
                 0.0909 
                 11.437 
                 0.0811 
                 0.0000 
                   
                 VV 
               
               
                 25 
                 12.837 
                 131315 
                 0.0570 
                 6.603 
                 0.0468 
                 0.0000 
                   
                 VV 
               
               
                 26 
                 13.733 
                 270732 
                 0.1176 
                 7.438 
                 0.0527 
                 0.0000 
                   
                 VP 
               
               
                 27 
                 15.300 
                 156380 
                 0.0679 
                 6.542 
                 0.0464 
                 0.0000 
                   
                 PP 
               
               
                 28 
                 17.513 
                 346774 
                 0.1507 
                 10.369 
                 0.0735 
                 0.0000 
                   
                 PV 
               
               
                 29 
                 18.270 
                 1011777 
                 0.4396 
                 25.904 
                 0.1837 
                 0.0000 
                   
                 VV 
               
               
                 30 
                 19.863 
                 298337 
                 0.1296 
                 7.175 
                 0.0509 
                 0.0000 
                   
                 VP 
               
               
                 31 
                 21.567 
                 237547 
                 0.1032 
                 6.715 
                 0.0476 
                 0.0000 
                   
                 VV 
               
               
                 32 
                 22.657 
                 261555 
                 0.1136 
                 7.129 
                 0.0505 
                 0.0000 
                   
                 VV 
               
               
                 33 
                 23.960 
                 419821 
                 0.1824 
                 8.165 
                 0.0579 
                 0.0000 
                   
                 VP 
               
               
                 34 
                 26.780 
                 1014237 
                 0.4405 
                 20.361 
                 0.1444 
                 0.0000 
                   
                 PP 
               
               
                 35 
                 29.320 
                 4482109 
                 1.9472 
                 103.960 
                 0.7370 
                 0.0000 
                   
                 PP 
               
               
                 36 
                 32.017 
                 6999710 
                 3.0410 
                 156.204 
                 1.1074 
                 0.0000 
                   
                 PV 
               
               
                 37 
                 33.343 
                 399151 
                 0.1734 
                 8.733 
                 0.0619 
                 0.0000 
                   
                 VP 
               
               
                 38 
                 35.053 
                 97125 
                 0.0422 
                 2.428 
                 0.0172 
                 0.0000 
                   
                 PV 
               
               
                 39 
                 37.007 
                 3062612 
                 1.3305 
                 45.150 
                 0.3272 
                 0.0000 
                   
                 VP 
               
               
                 40 
                 39.587 
                 832484 
                 0.3617 
                 15.925 
                 0.1129 
                 0.0000 
                   
                 PV 
               
               
                 41 
                 40.897 
                 2411303 
                 1.0476 
                 30.354 
                 0.2152 
                 0.0000 
                   
                 VP 
               
               
                 42 
                 43.503 
                 94412 
                 0.0410 
                 2.102 
                 0.0149 
                 0.0000 
                   
                 PP 
               
               
                 43 
                 46.093 
                 950329 
                 0.4129 
                 12.948 
                 0.0918 
                 0.0000 
                   
                 PP 
               
               
                 44 
                 47.820 
                 29686 
                 0.0129 
                 0.697 
                 0.0049 
                 0.0000 
                   
                 PP 
               
               
                 45 
                 51.273 
                 22016 
                 0.0096 
                 0.505 
                 0.0036 
                 0.0000 
                   
                 PP 
               
               
                 46 
                 53.253 
                 391511 
                 0.1701 
                 6.542 
                 0.0464 
                 0.0000 
                   
                 PP 
               
               
                 47 
                 55.177 
                 418679 
                 0.1819 
                 6.082 
                 0.0431 
                 0.0000 
                   
                 PP 
               
               
                 48 
                 57.243 
                 3988680 
                 1.7329 
                 240.883 
                 1.7078 
                 0.0000 
                   
                 PV 
               
               
                 49 
                 57.500 
                 4155318 
                 1.8053 
                 345.611 
                 2.4503 
                 0.0000 
                   
                 VV 
               
               
                 50 
                 57.787 
                 3545656 
                 1.5404 
                 257.829 
                 1.8279 
                 0.0000 
                   
                 VV 
               
               
                 51 
                 58.010 
                 1206951 
                 0.5244 
                 175.620 
                 1.2451 
                 0.0000 
                   
                 VV 
               
               
                 52 
                 58.257 
                 2986313 
                 1.2974 
                 222.955 
                 1.5807 
                 0.0000 
                   
                 VV 
               
               
                 53 
                 58.400 
                 5093407 
                 2.2128 
                 431.601 
                 3.0599 
                 0.0000 
                   
                 VV 
               
               
                 54 
                 58.673 
                 2540255 
                 1.1036 
                 286.541 
                 2.0315 
                 0.0000 
                   
                 VV 
               
               
                 55 
                 58.943 
                 5661787 
                 2.4597 
                 471.025 
                 3.3394 
                 0.0000 
                   
                 VV 
               
               
                 56 
                 59.233 
                 2400643 
                 1.0430 
                 197.347 
                 1.3991 
                 0.0000 
                   
                 VV 
               
               
                 57 
                 59.543 
                 3531683 
                 1.5343 
                 200.445 
                 1.4211 
                 0.0000 
                   
                 VV 
               
               
                 58 
                 59.850 
                 2228945 
                 0.9684 
                 164.165 
                 1.1639 
                 0.0000 
                   
                 VV 
               
               
                 59 
                 60.060 
                 2828869 
                 1.2290 
                 335.781 
                 2.3806 
                 0.0000 
                   
                 VV 
               
               
                 60 
                 60.410 
                 17547630 
                 7.6235 
                 994.818 
                 7.0529 
                 0.0000 
                   
                 VV 
               
               
                 61 
                 60.743 
                 13833800 
                 6.0101 
                 994.628 
                 7.0516 
                 0.0000 
                   
                 VV 
               
               
                 62 
                 60.920 
                 9930270 
                 4.3142 
                 941.167 
                 6.6726 
                 0.0000 
                   
                 VV 
               
               
                 63 
                 61.343 
                 3601150 
                 1.5645 
                 265.761 
                 1.8842 
                 0.0000 
                   
                 VV 
               
               
                 64 
                 61.723 
                 6048523 
                 2.6278 
                 525.238 
                 3.7238 
                 0.0000 
                   
                 VV 
               
               
                 65 
                 62.127 
                 2249845 
                 0.9774 
                 160.527 
                 1.1381 
                 0.0000 
                   
                 VV 
               
               
                 66 
                 62.357 
                 2849680 
                 1.2380 
                 152.131 
                 1.0786 
                 0.0000 
                   
                 VV 
               
               
                 67 
                 63.207 
                 11335680 
                 4.9248 
                 886.220 
                 6.2830 
                 0.0000 
                   
                 VV 
               
               
                 68 
                 63.460 
                 3758158 
                 1.6227 
                 214.751 
                 1.5225 
                 0.0000 
                   
                 VV 
               
               
                 69 
                 64.087 
                 17403430 
                 7.5609 
                 992.676 
                 7.0377 
                 0.0000 
                   
                 VV 
               
               
                 70 
                 64.807 
                 6379895 
                 2.7717 
                 332.965 
                 2.3606 
                 0.0000 
                   
                 VV 
               
               
                 71 
                 65.487 
                 1105522 
                 0.4803 
                 48.756 
                 0.3457 
                 0.0000 
                   
                 VV 
               
               
                 72 
                 66.060 
                 1061718 
                 0.4613 
                 41.562 
                 0.2947 
                 0.0000 
                   
                 VV 
               
               
                 73 
                 66.490 
                 1903467 
                 0.8270 
                 65.469 
                 0.4642 
                 0.0000 
                   
                 VV 
               
               
                 74 
                 67.563 
                 1016818 
                 0.4418 
                 30.915 
                 0.2192 
                 0.0000 
                   
                 VV 
               
               
                 75 
                 68.387 
                 1511594 
                 0.6567 
                 37.104 
                 0.2631 
                 0.0000 
                   
                 VV 
               
               
                 76 
                 69.390 
                 10743430 
                 4.6675 
                 526.207 
                 3.7306 
                 0.0000 
                   
                 VV 
               
               
                 77 
                 70.583 
                 358833 
                 0.1559 
                 13.087 
                 0.0928 
                 0.0000 
                   
                 VV 
               
               
                 78 
                 71.257 
                 2732845 
                 1.1873 
                 127.212 
                 0.9019 
                 0.0000 
                   
                 VV 
               
               
                 79 
                 72.317 
                 237862 
                 0.1033 
                 7.948 
                 0.0553 
                 0.0000 
                   
                 VV 
               
               
                 80 
                 72.730 
                 245290 
                 0.1066 
                 6.784 
                 0.0481 
                 0.0000 
                   
                 VV 
               
               
                 81 
                 73.783 
                 87523 
                 0.0380 
                 3.200 
                 0.0227 
                 0.0000 
                   
                 VV 
               
               
                 82 
                 74.250 
                 127009 
                 0.0552 
                 4.136 
                 0.0293 
                 0.0000 
                   
                 VV 
               
               
                 83 
                 74.890 
                 86115 
                 0.0374 
                 2.980 
                 0.0211 
                 0.0000 
                   
                 VP 
               
               
                 84 
                 75.973 
                 29738 
                 0.0125 
                 1.165 
                 0.0083 
                 0.0000 
                   
                 PP 
               
               
                 85 
                 77.503 
                 92275 
                 0.0401 
                 1.898 
                 0.0135 
                 0.0000 
                   
                 PP 
               
               
                 86 
                 80.060 
                 508744 
                 0.2210 
                 11.408 
                 0.0809 
                 0.0000 
                   
                 PP 
               
               
                 87 
                 82.200 
                 28158 
                 0.0122 
                 0.871 
                 0.0062 
                 0.0000 
                   
                 PP 
               
               
                 88 
                 84.293 
                 79909 
                 0.0347 
                 1.534 
                 0.0109 
                 0.0000 
                   
                 PP 
               
               
                 89 
                 87.500 
                 1092447 
                 0.4746 
                 24.011 
                 0.1702 
                 0.0000 
                   
                 PV 
               
               
                 90 
                 87.530 
                 300046 
                 0.1304 
                 24.553 
                 0.1741 
                 0.0000 
                   
                 VV 
               
               
                 91 
                 87.893 
                 382295 
                 0.1661 
                 25.603 
                 0.1815 
                 0.0000 
                   
                 VV 
               
               
                 92 
                 88.107 
                 224523 
                 0.0975 
                 21.631 
                 0.1534 
                 0.0000 
                   
                 VV 
               
               
                 93 
                 88.397 
                 717983 
                 0.3119 
                 37.094 
                 0.2630 
                 0.0000 
                   
                 VV 
               
               
                 94 
                 89.010 
                 518852 
                 0.2254 
                 22.242 
                 0.1577 
                 0.0000 
                   
                 VV 
               
               
                 95 
                 89.237 
                 307090 
                 0.1334 
                 21.551 
                 0.1528 
                 0.0000 
                   
                 VV 
               
               
                 96 
                 89.473 
                 302635 
                 0.1315 
                 19.158 
                 0.1358 
                 0.0000 
                   
                 VV 
               
               
                 97 
                 90.007 
                 4050733 
                 1.7598 
                 326.533 
                 2.3150 
                 0.0000 
                   
                 VV 
               
               
                 98 
                 91.020 
                 376589 
                 0.1636 
                 16.743 
                 0.1187 
                 0.0000 
                   
                 VV 
               
               
                 99 
                 91.427 
                 152969 
                 0.0665 
                 11.423 
                 0.0810 
                 0.0000 
                   
                 VV 
               
               
                 100 
                 91.543 
                 205346 
                 0.0892 
                 11.033 
                 0.0782 
                 0.0000 
                   
                 VV 
               
               
                 101 
                 92.013 
                 142333 
                 0.0618 
                 11.613 
                 0.0823 
                 0.0000 
                   
                 VV 
               
               
                 102 
                 92.157 
                 268901 
                 0.1168 
                 11.479 
                 0.0814 
                 0.0000 
                   
                 VV 
               
               
                 103 
                 92.633 
                 124915 
                 0.0543 
                 8.073 
                 0.0572 
                 0.0000 
                   
                 VV 
               
               
                 104 
                 93.083 
                 908307 
                 0.3946 
                 88.376 
                 0.6266 
                 0.0000 
                   
                 VV 
               
               
                 105 
                 93.590 
                 210904 
                 0.0916 
                 15.423 
                 0.1093 
                 0.0000 
                   
                 VV 
               
               
                 106 
                 94.063 
                 323059 
                 0.1404 
                 22.173 
                 0.1572 
                 0.0000 
                   
                 VV 
               
               
                 107 
                 94.547 
                 63428 
                 0.0275 
                 3.102 
                 0.0220 
                 0.0000 
                   
                 VV 
               
               
                 108 
                 95.123 
                 29222 
                 0.0127 
                 2.059 
                 0.0146 
                 0.0000 
                   
                 VP 
               
               
                 109 
                 95.963 
                 45135 
                 0.0196 
                 2.967 
                 0.0210 
                 0.0000 
                   
                 VP 
               
               
                 110 
                 98.950 
                 58309 
                 0.0253 
                 3.644 
                 0.0258 
                 0.0000 
                   
                 VP 
               
               
                 111 
                 100.273 
                 20525 
                 0.0089 
                 0.713 
                 0.0051 
                 0.0000 
                   
                 PP 
               
               
                 112 
                 105.563 
                 38299 
                 0.0166 
                 1.804 
                 0.0128 
                 0.0000 
                   
                 PP 
               
               
                 113 
                 112.263 
                 55710 
                 0.0242 
                 1.468 
                 0.0104 
                 0.0000 
                   
                 PP 
               
               
                 114 
                 126.417 
                 44605 
                 0.0194 
                 0.861 
                 0.0061 
                 0.0000 
                   
                 PP 
               
               
                   
                 total 
                 230177556 
                   
                 14105.03 
                   
                 0.0000 
               
               
                   
               
             
          
         
       
     
         [0034]     The following Analysis Examination and Pharmacology Examination demonstrate that according to the elements and functions of the concentrated product of the invention&#39;s cultured Zang Zhi fruiting body, its major elements include triterpenoid compounds, water-soluble polysaccharides and chitin.  
         [0000]     Water Soluble Polysaccharides  
         [0035]     Examination by the Food Analysis Center in Japan revealed that solid Zang Zhi fruiting body obtained by the invention&#39;s culture method contains a high volume of water soluble polysaccharides; each 100 g fruiting body having 1.2 to 6.5 g of water soluble polysaccharides.  
         [0000]     Content of Heavy Metals  
         [0036]     Atomic Spectrum Analysis made by the Mircoanalysis Laboratory of Atomic Science Department of National Ching Hwa University reveals the invention&#39;s solid-cultured Zang Zhi has a content of heavy metals as follows:  
                                                           Na: 48.7 ppm   Mn: 10.9 ppm   Cd: ND           Mg: 767 ppm   Fe: 100 ppm   Cr: ND           Al: 41.6 ppm   Zn: 16.4 ppm           Ca: 605 ppm   As: ND           K: 0.560%   Hg: ND                      
 
 Total Antioxidant Activity 
 
         [0037]     A comparison among one gram of the concentrated powder of the invention&#39;s solid Zang Zhi substance, the powder of wild Zang Zhi, and the concentrated powder of  Ganderma  sp. reveals their equivalent of the number of micrograms of Vitamin C as follows:  
                                       Content of one gram powder/           equivalent to the number of       Source   micrograms of Vitamin C                   Powder of wild Zang Zhi    633 ug ascorbic acid/g       Concentrated Powder of  Ganderma  sp.    854 ug ascorbic acid/g       Concentrated Powder of the invention&#39;s   1180 ug ascorbic acid/g       solid Zang Zhi substance                  
 
         [0038]     The result shows that the total antioxidant activity of the invention&#39;s solid Zang Zhi substance is far higher than that of the fruiting body of wild Zang Zhi and that of  Ganderma  sp.  
         [0000]     Toxicity Test  
         [0039]     According to the experiment of the acute oral toxicity test LD50, the tested mice were each fed with 2,000 mg/kg of the solid powder of the invention&#39;s Zang Zhi, while the other mice were fed with the specially distilled water as control. The results show that the tested mice neither act abnormally nor die. Hence, the volume of the acute oral toxicity test LD50 is higher than 2000 mg/kg.  
         [0000]     Ames Test  
         [0040]     Under the Ames test, the invention&#39;s Zang Zhi is examined by the plate mixed assay. Five tested bacterial isolates are  Salmonella tiyphimurium  TA97, TA98, TA100, TA102 and TA1535. Each isolate is under five concentrations analysis of 312.5, 625, 1250, 2500 and 5000 ug/plate, respectively. Coincidentally, a negative control group and a positive control group specific to bacterial isolate are included, and each group is triplicate. Test results show that negative control group falls in the acceptable range, and positive control group reflects an increase of mutant bacterial colonies. Whether or not the bacterial isolates are treated under big mouse liver enzyme metabolic system (S9), all tested concentrates do not show an obvious increase of colonies of Ames tested isolates, as shown in table 4.  
                                 TABLE 4                             Salmonella typhimurium  Ames Test            Test Target   Test examples   Feed dosage × Period   Test result                 Salmonella     Five mutant   tested concentration   Whether under big         typhimurium     isolates required   (ug/plate)   mouse liver enzyme           Histidine of   312.5, 625, 1250, 2500 and   metabolic system (S9)           TA97, TA98,   5000 plate mixed assay for   or not, all tested           TA100, TA102   triplicate. The assay was   concentrations show           and TA1535   under constant temperature   no significant increase               37 ± 1° C. for 48-72 hours,   of Ames test mutant               and the mutant colonies calculated.   colonies.                   Test result is negative.                  
 
 Anti-Lipid Peroxidation Ability Test 
 
         [0041]     Anti-lipid peroxidation ability and anti free radical ability of the solid cultured Zang Zhi solid concentrate was tested, by the lipid peroxidation in mouse brain homogenization solution. Mouse brain homogenization solution exposed to an oxidative stress was stimulated by Fe 2+  introduction and induced the kinetics of generation of lipid-derived free radical.  FIG. 2  shows that while increasing the concentration of triple and 8 fold the invention&#39;s Zang Zhi solid substance concentrate, it has a trend to inhibit lipid peroxidation and reduce the generation of free radical.  
         [0000]     Protection of Liver Function Experiment  
         [0042]     An experimental model using carbon tetrachloride to cause slow damage to the liver of a mouse to discover the effect of different concentrates of Zang Zhi on the slow damage to a mouse liver was established. Twenty-four mice (ICR strain) are divided into 4 groups as shown in Table 5.  
                                 TABLE 5                           Zang Zhi Test Dosage and Animal Group Division            Group   Carbon Tetrachloride Dosage   Dosage   No. of Mice               A   0(control)   0(control)   6       B   40% CCl 4 /olive oil   0(control)   6           (0.3 mg/100 g BW)       C   As above   50 mg(triple)/Kg   6       D   As above   50 mg(8 fold)/Kg   6                  
 
         [0043]     The mice of Groups A and B to D were under a hypodermic injection of olive oil (0.1 ml/100 g BW), and 40% of CCl 4 /Olive oil (0.3 ml/100 g BW) twice a week. The mice of Groups A and D were fed with physiological saline through a stomach tube, while the mice of Groups C and D were fed with two different dosages of extract of the invention&#39;s cultured Zang Zhi for five weeks. Then, the eyepit blood of all mice was collected to examine the biochemical measurement related to liver function. After that, the tested mice were sacrificed and the largest liver leaf was taken out to proceed with the hitopathogenic observation, such as damage to the liver cells, change of lipid, necrosis, cellulose and degree of these changes, and so on. Biochemical tests include activities of GTP and GOT, which are conducted according to the standards of International Clinic Chemistry. The experimental results show that GTP and GOT in the blood of a mouse liver damaged by carbon tetrachloride obviously increased after five weeks. GTP of about 850 units is 20-22 fold above the normal, and GOT of about 1700 units is 40-42 fold above the normal ( FIG. 3-4 ). In addition, mice fed with triple and eight fold of concentrated extract of the invention&#39;s Zang Zhi have the capacity to inhibit the increase of GTP and GOT induced by CCl 4 . Particularly, when the GTP and GOT approach the peak, the triple concentrated extract can inhibit the increase of GTP by 37% and the increase of GOT by 65%.  
         [0044]     When comparing the liver tissue of a normal mouse with the liver tissue of the mouse fed on CCl 4  after six weeks, the liver tissue treated with CCl 4  conspicuously shows tumefacient and gray rough surface. However, the liver tissue of the mouse fed on different concentrated Zang Zhi extracts shows no tumefacient and gray rough surface. Besides, by staining, one can see the pile of lipid granules, the tumid liver cells around the middle vein, macrophage and other irritated white blood cells infiltrating the liver cell space, and partial necrosis cells showing nodular transformation. However, the liver tissue of the mouse fed on different concentrated Zang Zhi extracts has no pathogenic changes as described above.  
         [0045]     Table 6 shows the semi-quantification analysis of pathogenic mouse liver tissue. N represents the normal group, D represents CCl 4  damaged group, X3 represents the group fed on triple concentrated extract, X8 represents the group fed on eight fold concentrated extract. It is proved that the concentrated Zang Zhi extract can significantly improve the pathogenic changes.  
                                                               TABLE 6                           Semi-Quantification Analysis of Mouse Pathogenic Liver Tissue                Cell necrosis                    Inflammation   Fat   Local   Cosecutive   Tuberous   Biliary               of liVer   degeneration   necrosis   necrosis   transformation   Proliferation   Calcification               N   0   0   0-1   0   0   0   0       D   2-3   2-3   2   2   0-1   1   0-1       X3   1   1-2   0-1   1-2   0-1   0-1   0-1       X8   0-1   0-1   0-1   1   0   0-1   0-1                  
 
       Cancer Cells Inhibition Experiment  
       [0046]     The Food Industry Research and Development Institute is entrusted to conduct an experiment to find out how the Zang Zhi solid cultured concentrate of this invention can inhibit cell growth of cervix cancer (HeLa), stomach cancer (AGS), breast cancer (MCF-7), liver cancer (HepG2), bowel cancer (COLO 320 HSR), etc. The results of cell growth inhibition are shown in Table 7. The Zang Zhi solid cultured concentrate of this invention can significantly inhibit the growth of tumor cells (up to 75% to 87%), especially the cells of breast cancer.  
                             TABLE 7                           Results of Cancer Cell Inhibition by Extract of Solid Cultured       Zang Zhi                    Rate of Cell Growth Inhibition (%)           Cancer Cell Lines   Average (%) ± SD                       Hela   83 ± 3.1           AGS   84 ± 2.7           HepG2   75 ± 2.4           MCF-7   87 ± 1.4                         *The final concentration of the tested sample is 10% of the original concentration.             
 
         [0047]     In addition, an experiment is conducted to find out the results of inhibiting bowel cancer growth by using 5 powder specimens (New1, New2, New3, New4 and New5), 11 liquid specimens (20L, New2, New3, 520, 521, 522, 523, 524, 525, 526 and 527) as well as their 1:10 specimen dilution by microculture tetrazolium assay. The results as shown in  FIG. 5  reveal that in the final concentration 1/10 of stock solution can at least inhibit 30% of cancer cell growth, and the specimen 20L can inhibit the growth of cancer cells up to 90%.  
         [0000]     Analysis of Chromosome Mutation in Vitro  
         [0048]     Further, an analysis of chromosome configuration mutation in vitro was conducted to treat Chinese hamster ovary cells with the invention&#39;s Zang Zhi. The cells at middle mitosis were analyzed to observe and measure the frequency of abnormal chromosomes. Analysis of the configuration mutation of chromosome can estimate the ability of the invention&#39;s Zang Zhi to damage the cellular chromosome. Three treatments are given as follows: the first treatment is to add the invention&#39;s Zang Zhi to treat the cells for three hours, without adding the active mouse liver enzymes system (S9); the second one is to add S9 to treat the cells for 3 hours; and the third one is to treat the cells without adding S9 for 20 hours. All tested Zang Zhi concentrations under these three treatments cause no increase of the frequency of chromosomal configuration mutation. The experimental results are shown in the following Table 8.  
                                 TABLE 8                           Analysis of Chromosomal Configuration Mutation            Experimental   Experimental               Target   Examples   Feed Period   Result               Chinese   Inoculate each   The tested concentrations   Results of the third group       hamster ovary   60 mm cell   of the first and second   under treatment reveal       cells; ATCC,   cultured plate   groups are 0, 312.5 625,   that treatment of Chinese       repository No.   according to the   1250, 2500 and 5000 ug/ml.   hamster ovary cells with       CCL-61   tested   The tested concentrations   the invention&#39;s Zang Zhi,       CHO-KI   concentrations;   of the third groups are 0,   before or after metabolic           each   30, 100, 1000, and 3000 ug/ml.   activities, does not show           concentration has   After culture under   significant increase of the           two plates; these   different conditions, cells   frequency of           samples are   with scattered uniformly   chromosomal           divided into   18-21 chromosomes are   configuration mutation of           three groups   selected for observation.   Chinese hamster ovary           according to the       cells.           different       The experimental result is           treatments.       negative.                  
 
         [0049]     The clastogenicity of the invention&#39;s Zang Zhi in the animal body was examined. Further, the risk estimation of human hereditary toxicity was made according to the formation of micronuclei in reticular mouse red blood cells. Twenty-five mice were divided into 5 groups in this experiment. Analysis of the micronuclei reveals that the reaction of invention&#39;s Zang Zhi to circumference blood micronuclei of mouse is negative.  
                                 TABLE 9                           Analysis of Micronuclei in Animal Body            Experimental   Experimental               Target   Samples   Feed period   Experimental Results               ICR mouse   Samples are:   1 st -4 th  groups of   1. There is no difference between           1. Solvent   mice respectively   tested dosage group and solvent           control group   were fed on 0, 500,   control group.           2. High   1000, 2000 mg/Kg   2. The rate of reticular red blood           dosage control   dosage;   cell analysis proves that high           group   Mice of positive   dosage would not be toxic to           3. Medium   control group were   mouse marrow.           dosage control   fed on 1.0 mg/Kg   3. There is no tendency of           group   MMC.   dosage reaction to micronuclei           4. Low dosage   The blood was   rate and the comparison of the           control group   collected and the   control group shows no           5. Positive   reticular red blood   significant increase. Thus           control group   cells were observed   analysis of the reaction of           6. Each group   under fluorescent   micronuclei of mouse           has five mice.   microscopy.   circumference blood to Zang Zhi                   shows negative reaction.                  
 
         [0050]     According to the pharmacological data described above, the invention&#39;s solid Zang Zhi substance not only possesses active constituents similar to wild Zang Zhi, for example, polysaccharide and tertripoid, but also possess the same function of anti-lipid peroxidation, anti-free radicals and anti-oxidation. Besides, the invention&#39;s solid Zang Zhi substance possesses the function to protect the liver and fight against cancer.  
         [0051]     While this invention has been described in detail with particular reference to its preferred embodiments, the principles and modes of operation of the invention have also been described in this specification. The invention should not be construed as being limited to the particular forms disclosed, which are illustrative rather than restrictive. Modifications, variations, and changes may be made by those skilled in the art without departure from the spirit and scope of the invention as described by the following claims.

Technology Category: 1