CA2368888A1 - Incubation method for obtaining solid culture of zang zhi, solid culture obtained therefrom, processed products and use thereof - Google Patents
Incubation method for obtaining solid culture of zang zhi, solid culture obtained therefrom, processed products and use thereof Download PDFInfo
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- CA2368888A1 CA2368888A1 CA002368888A CA2368888A CA2368888A1 CA 2368888 A1 CA2368888 A1 CA 2368888A1 CA 002368888 A CA002368888 A CA 002368888A CA 2368888 A CA2368888 A CA 2368888A CA 2368888 A1 CA2368888 A1 CA 2368888A1
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- culture
- zang zhi
- fruiting body
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L31/00—Edible extracts or preparations of fungi; Preparation or treatment thereof
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
Abstract
The present invention provides an incubation method for obtaining solid culture of Zang Zhi (Antrodia camphorata) comprising the steps of incubating a stock strain of Zang Zhi in a PP bag for growth of mycelia, followed by an incubation in the air for fruiting the Zang Zhi body. The invention also provides the solid culture of Zang Zhi obtained by the incubation method, and the products processed therefrom and the use thereof.
Description
s CA 02368888 2002-O1-22 PATENT APPLICATION
INCUBATION METHOD FOR OBTAINING SOLID CULTURE OF ZANG ZHI, SOLID
CULTURE OBTAINED THEREFROM, PROCESSED PRODUCTS AND USE THEREOF
INVENTORS: MING-HUANG LAN
LI-YU WU
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" ar "niou Zang ku." In Taiwan, Zang Zhi only grows on the inner wall of the empty rotting trunk of Cinnamomum krrnehirai 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. Zhanlcuic 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.
It is indicated in Gau Sheau Jy's masters thesis on the triterpenoid of Zang Zhi that Zang Zhi is a new species of Ganoderna discovered in 1990. The extract of the solid fruiting body is obtained by use of acetone, and then is separated and re-crystallized 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 CC 14.
Most of Zang Zhi grows in the broad-leaf forest areas in Taiwan'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.
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
The main object of an aspect 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.
The other object of an aspect 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.
INCUBATION METHOD FOR OBTAINING SOLID CULTURE OF ZANG ZHI, SOLID
CULTURE OBTAINED THEREFROM, PROCESSED PRODUCTS AND USE THEREOF
INVENTORS: MING-HUANG LAN
LI-YU WU
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" ar "niou Zang ku." In Taiwan, Zang Zhi only grows on the inner wall of the empty rotting trunk of Cinnamomum krrnehirai 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. Zhanlcuic 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.
It is indicated in Gau Sheau Jy's masters thesis on the triterpenoid of Zang Zhi that Zang Zhi is a new species of Ganoderna discovered in 1990. The extract of the solid fruiting body is obtained by use of acetone, and then is separated and re-crystallized 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 CC 14.
Most of Zang Zhi grows in the broad-leaf forest areas in Taiwan'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.
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
The main object of an aspect 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.
The other object of an aspect 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.
2 Another object of an aspect of the invention is to provide the processed products of the solid cultured Zang Zhi.
Another object of an aspect of the invention is to provide a solid cultured Zang Zhi useful as active constituents.
According to one aspect of the invention, there is provided a method of culturing solid Zang Zhi comprising the use of "Bag Log" inoculated with spawn to culture mycelium and then removing the "Bag Log" to let the sawdust medium be completely exposed in the air for a period of time until the formation of the conoid fruiting body is made, wherein the "Bag Log" contains 10-70% of cellulolytic substance, 10-30% of starch resource, 5-15% of millet, 1-10 % of saccharides, 0.5-2%
phosphate, and 0.1-1% sulfate salt, the relative humidity is maintained at 60-80% and the pH of the cultural medium is adjusted to be neutral.
BRIEF DESCRIPTIONS OF THE DRAWINGS
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.
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).
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<0.01);
*represents the statistical difference between the feed group and the injury group (P
<0.01). Value is represented by average t standard error.
Another object of an aspect of the invention is to provide a solid cultured Zang Zhi useful as active constituents.
According to one aspect of the invention, there is provided a method of culturing solid Zang Zhi comprising the use of "Bag Log" inoculated with spawn to culture mycelium and then removing the "Bag Log" to let the sawdust medium be completely exposed in the air for a period of time until the formation of the conoid fruiting body is made, wherein the "Bag Log" contains 10-70% of cellulolytic substance, 10-30% of starch resource, 5-15% of millet, 1-10 % of saccharides, 0.5-2%
phosphate, and 0.1-1% sulfate salt, the relative humidity is maintained at 60-80% and the pH of the cultural medium is adjusted to be neutral.
BRIEF DESCRIPTIONS OF THE DRAWINGS
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.
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).
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<0.01);
*represents the statistical difference between the feed group and the injury group (P
<0.01). Value is represented by average t standard error.
3 [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< 0.01);
*represents the statistical difference between the feed group and the injury group (P<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 (COLD 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'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 l-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.
represents the statistical difference between a normal group and an injury group (P< 0.01);
*represents the statistical difference between the feed group and the injury group (P<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 (COLD 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'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 l-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.
4 [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 2511°C; night temperature is better maintained at l ltl°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.
S
[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 HPLC spectrum of the invention's solid culture Zang Zhi, known liquid cultured Zang Zhi, and wild Zang Zhi is shown in Figure 1. By analysis of HPLC, the constituents of the fruiting body obtained through the invention's culture method are similar to those of the wild Zang Zhi and the invention's Zang Zhi has the activities and functions similar to the wild Zang Zhi'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'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'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.
j0029] 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 1111°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 Figure 1 ( 1 ), and the measurements are shown in the following Table 1.
Table l, Data of HPLC analysis of the invention's Zang Zhi fruiting body Table 1 No.retentionarea area%relativeh_e:~t%conc conc basline ingredient time (uv*sec) height unit name 1 2.00 2793A709.30Ie1000.279f.33i20.0000 9V
2 2.31763203672.1039999.3036.35990.0000 PI
3 ..66363332742.1070999.1356.32E90.0000 w a S.23 1J3211a0a.19a9!.ff~(.32710.0000 w 1 3.11739333111.307324.2632.11300.0000 w 6 3.413161a3S21.3332303.111l.9sOG0.0000 w 7 3.s73asals7ao.6361lez.lzs1.11010.0000 w t 3.1731041333O.i933Iaa.1770.911 0.0000 w 9 x.3c712681811.119:lal.ase0.17880.0000 w Io3.1507s011312.32!x60.3203.00190.0000 I15.77314711750.536171.27=0.16370.0000 W
1.6.013-1x16130.111931.111-0.33110.0000 Ao 136.36012611A10.1207-12.7x!0.11000.0000 -W
116.59713591490.432238.6800.3A3x0.0000 W
131.2631916.830.617531.1070.36160.0000 161.8605173110.172113.0810.28150.0000 W
I7A.16738822691.9301317.372.01690.0000 W
1d1.9839328100.310329.9130.19550.0000 V?
I99.99012339310.1723,1.6910.22670.0000 w 2010.3133380130.379012.1990.11960.0000 W
2110.12318a3110.05112.3370.0A060.0000 W
2.11.3271233115O.l0a29.716O.I9120.0000 W
:313.1936283020..09019.231O.I2380.0000 W
2x12.99012390330.111937.37A0.2x120.0000 V7 23I1.~O29823810.192310.1030.625 0.0000 PP
2617.31323219670.839068.3110.41660.0000 ?V
27I8.I136273160.208719.9340.13030.0000 W
21It.99011133D0.0390.890 0.03060.0000 W
2911.8676633110.2301=0.1980.13200.0000 W
30:0.86729320370.973?32.7800.31180.0000 VV
3122.3332321110.083!9.698 0.08300.0000 W
3223.08071318621.011!79.4760.31930.0000 W
3323.96011308320.376=31.0230.20210.0000 W
3x:3.83711338101.376078.0250.50980.0000 W
3321.9101070680.336122.1200.11630.0000 P?
3t28.9x021278810.701941.5110.28890.0000 W
3730.303139917003.3221301.3061.18830.0000 T.' 3833.7935236<x31.T<8A12.3150.60360.0000 , 3133.313as29~01.13108.103 0.039 0.0000 ?v t037.3601116830L.xi9579,089O:S1BA0.0000 ??
x131.193-2814490.08113.1~ 0.03610.0000 PP
12x1.261<x1196I.BT8T3.3Zd0.17910.0000 ' Vv t313.66728212990.93A630.9380.33.'80.0000 W
ax11.3233160 0.01:31.071 0.00700.0000 v9 4548.130100A79303.331213.9300.18180.0000 99 at50.0903331010.11094.30= 0:03910.0000 W
IT32.3831898619I.t2lI40.3710.7!3 0.0000 w t133.28012x351O.Otlx2.041 0.01310.0000 PP
4!58.39319889311.8191310.1882.22190.0000 W
5058.9531317512.1336623.3131.071 0.0000 W
5157.290i5867~51.551083.1983.02670.0000 .
w
[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 2511°C; night temperature is better maintained at l ltl°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.
S
[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 HPLC spectrum of the invention's solid culture Zang Zhi, known liquid cultured Zang Zhi, and wild Zang Zhi is shown in Figure 1. By analysis of HPLC, the constituents of the fruiting body obtained through the invention's culture method are similar to those of the wild Zang Zhi and the invention's Zang Zhi has the activities and functions similar to the wild Zang Zhi'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'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'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.
j0029] 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 1111°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 Figure 1 ( 1 ), and the measurements are shown in the following Table 1.
Table l, Data of HPLC analysis of the invention's Zang Zhi fruiting body Table 1 No.retentionarea area%relativeh_e:~t%conc conc basline ingredient time (uv*sec) height unit name 1 2.00 2793A709.30Ie1000.279f.33i20.0000 9V
2 2.31763203672.1039999.3036.35990.0000 PI
3 ..66363332742.1070999.1356.32E90.0000 w a S.23 1J3211a0a.19a9!.ff~(.32710.0000 w 1 3.11739333111.307324.2632.11300.0000 w 6 3.413161a3S21.3332303.111l.9sOG0.0000 w 7 3.s73asals7ao.6361lez.lzs1.11010.0000 w t 3.1731041333O.i933Iaa.1770.911 0.0000 w 9 x.3c712681811.119:lal.ase0.17880.0000 w Io3.1507s011312.32!x60.3203.00190.0000 I15.77314711750.536171.27=0.16370.0000 W
1.6.013-1x16130.111931.111-0.33110.0000 Ao 136.36012611A10.1207-12.7x!0.11000.0000 -W
116.59713591490.432238.6800.3A3x0.0000 W
131.2631916.830.617531.1070.36160.0000 161.8605173110.172113.0810.28150.0000 W
I7A.16738822691.9301317.372.01690.0000 W
1d1.9839328100.310329.9130.19550.0000 V?
I99.99012339310.1723,1.6910.22670.0000 w 2010.3133380130.379012.1990.11960.0000 W
2110.12318a3110.05112.3370.0A060.0000 W
2.11.3271233115O.l0a29.716O.I9120.0000 W
:313.1936283020..09019.231O.I2380.0000 W
2x12.99012390330.111937.37A0.2x120.0000 V7 23I1.~O29823810.192310.1030.625 0.0000 PP
2617.31323219670.839068.3110.41660.0000 ?V
27I8.I136273160.208719.9340.13030.0000 W
21It.99011133D0.0390.890 0.03060.0000 W
2911.8676633110.2301=0.1980.13200.0000 W
30:0.86729320370.973?32.7800.31180.0000 VV
3122.3332321110.083!9.698 0.08300.0000 W
3223.08071318621.011!79.4760.31930.0000 W
3323.96011308320.376=31.0230.20210.0000 W
3x:3.83711338101.376078.0250.50980.0000 W
3321.9101070680.336122.1200.11630.0000 P?
3t28.9x021278810.701941.5110.28890.0000 W
3730.303139917003.3221301.3061.18830.0000 T.' 3833.7935236<x31.T<8A12.3150.60360.0000 , 3133.313as29~01.13108.103 0.039 0.0000 ?v t037.3601116830L.xi9579,089O:S1BA0.0000 ??
x131.193-2814490.08113.1~ 0.03610.0000 PP
12x1.261<x1196I.BT8T3.3Zd0.17910.0000 ' Vv t313.66728212990.93A630.9380.33.'80.0000 W
ax11.3233160 0.01:31.071 0.00700.0000 v9 4548.130100A79303.331213.9300.18180.0000 99 at50.0903331010.11094.30= 0:03910.0000 W
IT32.3831898619I.t2lI40.3710.7!3 0.0000 w t133.28012x351O.Otlx2.041 0.01310.0000 PP
4!58.39319889311.8191310.1882.22190.0000 W
5058.9531317512.1336623.3131.071 0.0000 W
5157.290i5867~51.551083.1983.02670.0000 .
w
5.ST.BOO3699541.2301I9.75d1.=3990.0000 W
5331.0110387210.3316llA.l~0.Ø77180.0000 w Sx31.177216A190.e21113.3180.87700.0000 VV
3331.69019422310.63!!137.326I.02l70.0000 W
St51.92711518 O.xI012x.1310.11370.0000 W
ST39.21031388961.0112171.3:7I.I1930.0000 99 3 59.33010911230.36331.791 0.38670.0000 W
5939.101x819090.1930102.3010.44180.0000 PV
6040.0933x066911.I331302.1331.3=310.0000 w 61x10.1131813010.3173101.1190.48230.0000 Pv 8=10.90103117703.3233T3T.2I1:91110.0000 Vv 6341.10318213830.812111.0140.763!0.0000 W
6161.10013601!0.28179.1620.5173~ 0.0000W
6i61.90111982120.19118.331 0..31730.0000 W
Bt8=.2lT13311382.118333.61 3.51830.0000 W
676..713100161A0.335178.7710.34110.0000 Vv td12:9801331170.311178.1730.30230.0000 W
6963.11119120010.16376f,~! 0.15470.0000 W
~
7013.1037760930.23233.=830.36110.0000 PV
TI6x.30361111182.0310313.3603.384!0.0000 PV
7211.337711A730.233572.7030.17=S0.0000 pp 7311.7733117592D.7113121.1830.11650.0000 9'V
7111.57=3~3?030.776=110.1360.7?I90.0000 w 7568.9A11113100.603153.2130.s1820.0000 w 7686.11712219360.06336.4A10.37010,0000 W
7761.10316666110.351541.1330.5302O.DO00 W
T167.11712265310.081l6.tAa0.301x0.0000 W
7!11.34037171301.2833I01.22I0.70720.0000 W
6081.91031807101.7211176.91=1.138=O.OOOO W
8181.6372339117O.BSI776.3590.19900.0000 Vv 1270.1701107380.228116.7370-_10690.0000 W
1371.611261661Ø011112.3160.01200.0000 W
sx12.7176es31!0.22171x.101o.osTVo.oDOD W
85 5135510.1709I3.tt80.09050:0000 w 73..070 t6 1197960.13979.9530.0650O.o000 w 73.730 1T]1.9103377330.119012.198O.Ot300.0000 w 1t79.3'13Ii3A0t0.153013.1710,10110.0000 w t978.:533361500.111510.1620.066 0.0000 w f077.1371333100.01114.6450.030 0.0000 w 9177.1302729630.090111.050.07710.0000 w 9271.1207329160.250530,1210.20110.0000 w 9378.6908698160.219131.1090.20790.0000 w 9479.65316206 0.02202.2350.01160.0000 V?
95(0.73079611 0.02852.1620.01170.0000 PP
961.733 27501 0.00911.0700.00700.0000 PP
9783.1x71866110.06211.1130.0=710.0000 PV
9883.9:12937110.01779,0110.05930.0000, w 9911,3203610?30.12019.0610.05920.0000 w 10015.1208201:30.275029,111O.I9010.0000 w 10186.26013317520.309633.7080.23330.0000 w 10287.A03191502?0.s17151.9780.33960.0000 w 10319.3002711090.010=7.331-0.0800.0000 VP
10190.1832683? 0.00890.3330.00330.0000 PP
1059=.08035383 0.01111.3980.00910.0000 PP
106!6.13 37386621.230532.4670.34350.0000 PP
107100.23791086 0.03131.1370.00710.0000 PP
!08106.630113570?0.t77711.107o:2see0.0000 Pv 109107.30011516550.3835SS.t950.36320.0000 w 110107.720148'10820.91771.1330.16180.0000 w 111101.1907090190.235937.2310.21310.0000 w 11:108.193x182310.272238.6270:23210.0000 w 113109.01s6311160.2101x.5.0160.2?880.0000 w 111109.50710103920.331133.6330.21990.0000 w 115109.9001116610.1N427.1430.18210.0000 ~ W
116110.16039819!0.199333.3600.21800.0000 w 117110.1931105130.279737.2020.37380.0000 W
118111.02321331410.7105103.7110.67770.0000 w t19I11.1303177380:182212.19a0:27570.0000 w 120111.83315972800.331136.8900.37170.0000 w 121112.1739679910.3:2033.9110.36540.0000 w 122ll=.~OT1790500.223959.1110,31950.0000 w 11311=.1:05369110.171619.9130.32610.0000 w 121113.0679871080.321133.9260.:3180.0000 w 125113.10713838230.460131.6630.22630.0000 w 126111.563100=5~0.333336.3370.13760.0000 W
127111.97032019!0.I73111.1360.13770.0000 w 128115.5401135800.380528.1190.18370.0000 w 129116.37733825A0.112311.=090.07320.0000 w 130117.017l7slt7o.ostrss.seao.oseo0.0000 w 131iaT.4oaIs939ao.o3see.66 x.06610.0000 w 13:11a.133sle7loo.l7xs11.0140.07200.0000 w 133111,7631311390.01177.0910.0s630.0000 w 1sta 9.2x7ss127s0.122510.127o.oss20.0000 w 136121.1x0sisal 0.0n 2.asa0:01670.0000 vv s 131121.6201850390.06166.2120.01100.0000 w 137122.12030311 0.01011.9390.01260.0000 11o' 138123.010231231O.OtIS8.1680.03530.0000 w 139121.11011107=0.04696.2060.01060.0000 v9 110125.1773711110.12597,9760.05:10.0000 P9 111127.97727351 0.00921.0160.00660.0000 PP
112129,1131179130.06331.1370.06190.0000 P1 tOL31 »s7uss as3os.sa 0.0000 [0032] The differences between the invention's Zang Zhi fruiting body, other cultural methods and liquid cultural method were compared by the analysis of I-iPLC.
The HPLC
spectra-are shown in Figure 1 (2) -(3); and the measurements are shown in Table 2 and Table 3. These spectra show that the invention's Zang Zhi fruiting body and wild Zang Zhi have the same shape of peak, and also show that the invention's cultured Zang 2h1 fruiting body and that of wild Zang Zhi have the same activities and functions.
Table 2. HPLC spectrum figure of liquid cultured Zang Zhi Table 2 Rio. retention area area% retative height% canc cone basline in~edient time (uv'sec) height ,pit name .. 1.6_0 262998 1.219138.7432.2043 0.0000 ?v 2 1.957 929431238.1242660.e8537.59120.0000 7"r 3 2.3.0 311747625.09637.5.39841.33010,0000 W
3 2.830 15925347.941399.4065.5559 0.0000 Vv 3.790 731157 3.357123.2091.3.03 0.0000 t'3
5331.0110387210.3316llA.l~0.Ø77180.0000 w Sx31.177216A190.e21113.3180.87700.0000 VV
3331.69019422310.63!!137.326I.02l70.0000 W
St51.92711518 O.xI012x.1310.11370.0000 W
ST39.21031388961.0112171.3:7I.I1930.0000 99 3 59.33010911230.36331.791 0.38670.0000 W
5939.101x819090.1930102.3010.44180.0000 PV
6040.0933x066911.I331302.1331.3=310.0000 w 61x10.1131813010.3173101.1190.48230.0000 Pv 8=10.90103117703.3233T3T.2I1:91110.0000 Vv 6341.10318213830.812111.0140.763!0.0000 W
6161.10013601!0.28179.1620.5173~ 0.0000W
6i61.90111982120.19118.331 0..31730.0000 W
Bt8=.2lT13311382.118333.61 3.51830.0000 W
676..713100161A0.335178.7710.34110.0000 Vv td12:9801331170.311178.1730.30230.0000 W
6963.11119120010.16376f,~! 0.15470.0000 W
~
7013.1037760930.23233.=830.36110.0000 PV
TI6x.30361111182.0310313.3603.384!0.0000 PV
7211.337711A730.233572.7030.17=S0.0000 pp 7311.7733117592D.7113121.1830.11650.0000 9'V
7111.57=3~3?030.776=110.1360.7?I90.0000 w 7568.9A11113100.603153.2130.s1820.0000 w 7686.11712219360.06336.4A10.37010,0000 W
7761.10316666110.351541.1330.5302O.DO00 W
T167.11712265310.081l6.tAa0.301x0.0000 W
7!11.34037171301.2833I01.22I0.70720.0000 W
6081.91031807101.7211176.91=1.138=O.OOOO W
8181.6372339117O.BSI776.3590.19900.0000 Vv 1270.1701107380.228116.7370-_10690.0000 W
1371.611261661Ø011112.3160.01200.0000 W
sx12.7176es31!0.22171x.101o.osTVo.oDOD W
85 5135510.1709I3.tt80.09050:0000 w 73..070 t6 1197960.13979.9530.0650O.o000 w 73.730 1T]1.9103377330.119012.198O.Ot300.0000 w 1t79.3'13Ii3A0t0.153013.1710,10110.0000 w t978.:533361500.111510.1620.066 0.0000 w f077.1371333100.01114.6450.030 0.0000 w 9177.1302729630.090111.050.07710.0000 w 9271.1207329160.250530,1210.20110.0000 w 9378.6908698160.219131.1090.20790.0000 w 9479.65316206 0.02202.2350.01160.0000 V?
95(0.73079611 0.02852.1620.01170.0000 PP
961.733 27501 0.00911.0700.00700.0000 PP
9783.1x71866110.06211.1130.0=710.0000 PV
9883.9:12937110.01779,0110.05930.0000, w 9911,3203610?30.12019.0610.05920.0000 w 10015.1208201:30.275029,111O.I9010.0000 w 10186.26013317520.309633.7080.23330.0000 w 10287.A03191502?0.s17151.9780.33960.0000 w 10319.3002711090.010=7.331-0.0800.0000 VP
10190.1832683? 0.00890.3330.00330.0000 PP
1059=.08035383 0.01111.3980.00910.0000 PP
106!6.13 37386621.230532.4670.34350.0000 PP
107100.23791086 0.03131.1370.00710.0000 PP
!08106.630113570?0.t77711.107o:2see0.0000 Pv 109107.30011516550.3835SS.t950.36320.0000 w 110107.720148'10820.91771.1330.16180.0000 w 111101.1907090190.235937.2310.21310.0000 w 11:108.193x182310.272238.6270:23210.0000 w 113109.01s6311160.2101x.5.0160.2?880.0000 w 111109.50710103920.331133.6330.21990.0000 w 115109.9001116610.1N427.1430.18210.0000 ~ W
116110.16039819!0.199333.3600.21800.0000 w 117110.1931105130.279737.2020.37380.0000 W
118111.02321331410.7105103.7110.67770.0000 w t19I11.1303177380:182212.19a0:27570.0000 w 120111.83315972800.331136.8900.37170.0000 w 121112.1739679910.3:2033.9110.36540.0000 w 122ll=.~OT1790500.223959.1110,31950.0000 w 11311=.1:05369110.171619.9130.32610.0000 w 121113.0679871080.321133.9260.:3180.0000 w 125113.10713838230.460131.6630.22630.0000 w 126111.563100=5~0.333336.3370.13760.0000 W
127111.97032019!0.I73111.1360.13770.0000 w 128115.5401135800.380528.1190.18370.0000 w 129116.37733825A0.112311.=090.07320.0000 w 130117.017l7slt7o.ostrss.seao.oseo0.0000 w 131iaT.4oaIs939ao.o3see.66 x.06610.0000 w 13:11a.133sle7loo.l7xs11.0140.07200.0000 w 133111,7631311390.01177.0910.0s630.0000 w 1sta 9.2x7ss127s0.122510.127o.oss20.0000 w 136121.1x0sisal 0.0n 2.asa0:01670.0000 vv s 131121.6201850390.06166.2120.01100.0000 w 137122.12030311 0.01011.9390.01260.0000 11o' 138123.010231231O.OtIS8.1680.03530.0000 w 139121.11011107=0.04696.2060.01060.0000 v9 110125.1773711110.12597,9760.05:10.0000 P9 111127.97727351 0.00921.0160.00660.0000 PP
112129,1131179130.06331.1370.06190.0000 P1 tOL31 »s7uss as3os.sa 0.0000 [0032] The differences between the invention's Zang Zhi fruiting body, other cultural methods and liquid cultural method were compared by the analysis of I-iPLC.
The HPLC
spectra-are shown in Figure 1 (2) -(3); and the measurements are shown in Table 2 and Table 3. These spectra show that the invention's Zang Zhi fruiting body and wild Zang Zhi have the same shape of peak, and also show that the invention's cultured Zang 2h1 fruiting body and that of wild Zang Zhi have the same activities and functions.
Table 2. HPLC spectrum figure of liquid cultured Zang Zhi Table 2 Rio. retention area area% retative height% canc cone basline in~edient time (uv'sec) height ,pit name .. 1.6_0 262998 1.219138.7432.2043 0.0000 ?v 2 1.957 929431238.1242660.e8537.59120.0000 7"r 3 2.3.0 311747625.09637.5.39841.33010,0000 W
3 2.830 15925347.941399.4065.5559 0.0000 Vv 3.790 731157 3.357123.2091.3.03 0.0000 t'3
6 4.133 .71974 1.259911.7130.6664 0.0000 3V
7 4,433 134068 0.71371.210 0.4441 0.0000 7V
B 5.023 134230 0.71154.380 0.2506 0.0000 PJ
9 5.997 .9822 0.13821.632 0.0940 0.0000 7p IO 7.360 150572 0.69755.210 0.2964 0.0000 ?p 2I 3.210 123413 0.57173.311 0.2227 0.0000 ?P
12 11.930 130907 0.60603.363 Q.ZQ27 O.na00 av 13 12.740 37713 0.17471.232 0.0701 0.0000 V?
Ii 15.103 304163 2.3370.11.0480.6386 0.0000 ?P
I3 16.390 113477 0.66473.3.3 O.I998 0.0000 ??
l0 13.350 56860 0.25341.927 0.1039 0.0000 ?P
17 23.510 806878 3.7379I1.27i0.6415 0.0000 ?P .
13 27.157 S20i2 0.42541.234 0,0646 0.0000. P2 ' 19 30.633 114385 0.66812.243 0.127 0.0000 ?p _ 30 33.747 239308 I.3i143.947 0.2243 0.0000 PP
22 38.727 230188 1.1e043.231 0.1839 0.0000 ?P
22 x1.340 61644 0.28360.771 0.0438 0.0000 ??
.3 13.727 29928 O,I3850.470 0.0307 0.0000 PP
24 57.047 112074 0.51929.096 0.5175 0.0000 ?v 25 37.433 173711 0.914010.2730.3846 0.0000 VV
26 57.907 107398 0,49853.308 0.2365 O.Q000 W
.7 53.137 23810 O.I195..379 0.1296 0.0000 v7 .
28 61.380 23243 '0.10772.024 0.1153 0.0000 7V
29 62.743 19o6I~ 0.910821.7671.2385 0.0000 W
30 v1.223 30411 O.I4093.094 0.119. 0.0000 ?w 31 61.721 441196 2.0439x1,6203.36A1 0.0000 W
3. 84.997 48324 0,22489.870 0.2771 O.OOGO W
33 65.42a 40354 0.18693.329 0.2008 0.0000 VY
3i 65.833 67766 0.31393.923 0.2173 0.0000 V?
35 66.950 31238 0.09871.691 0.0963 O.Q000 ?P
36 67.773 32193 0.14911.708 0.0972 0.0000 ?P
37 79.013 29411 0.13630.920 0.0523 0.0000 PP
38 97:.97 21384 O.IOIi0.456 0.0260 0.0000 PP
39 100.05339:7a O,i7S20.738 0,020 0.0000 ' ?P
40 104.457135636 0.38692,338 O,I3i2 0.0000 ?P
41 107.01057253 0.26533.113 0.1Ø 0.0000 Pp i3 111.39733834 O.la6I4.236 0.2410 0.0000 V'f 43 111.813.9337 0.13132.513 0.1429 0.0000 r?
as 113.790.5408 0.11771.810 O.I030 0.0000 V7 a5 115.88343858 0..03,3.808 0.2167 0.0000 ?V
i6 119.390=3431 0.1179=,630 0.0942 0.0000 ?P
LOL31 213A6254 1T57,553 0.0000 Table 3. HPLC spectrum figure of wild Zang Zhi Table 3 No. cetention area area% relative heigEtt% conc conc basline ingredient time (uv'sec) hei~ttt unit name 1 1.550 13320090.3787162.0451.1488 0.0000 PV
2 1.913 127080005.5.10797:4305.6335 0.0000 vv 3 2.607 79905043.1715374.1432.6525 O.OO00 w 1 ..997 33361511.4494131.1641.0738 0.0000 vv 3.480 827969 0.339790:5300.6419 0.0000 6 3.687 21009120.912789.3310.6326 0.0000 ~'Y
7 1,110 15653780.7.33BO.a680.5703 0.0000 tt
B 5.023 134230 0.71154.380 0.2506 0.0000 PJ
9 5.997 .9822 0.13821.632 0.0940 0.0000 7p IO 7.360 150572 0.69755.210 0.2964 0.0000 ?p 2I 3.210 123413 0.57173.311 0.2227 0.0000 ?P
12 11.930 130907 0.60603.363 Q.ZQ27 O.na00 av 13 12.740 37713 0.17471.232 0.0701 0.0000 V?
Ii 15.103 304163 2.3370.11.0480.6386 0.0000 ?P
I3 16.390 113477 0.66473.3.3 O.I998 0.0000 ??
l0 13.350 56860 0.25341.927 0.1039 0.0000 ?P
17 23.510 806878 3.7379I1.27i0.6415 0.0000 ?P .
13 27.157 S20i2 0.42541.234 0,0646 0.0000. P2 ' 19 30.633 114385 0.66812.243 0.127 0.0000 ?p _ 30 33.747 239308 I.3i143.947 0.2243 0.0000 PP
22 38.727 230188 1.1e043.231 0.1839 0.0000 ?P
22 x1.340 61644 0.28360.771 0.0438 0.0000 ??
.3 13.727 29928 O,I3850.470 0.0307 0.0000 PP
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26 57.907 107398 0,49853.308 0.2365 O.Q000 W
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28 61.380 23243 '0.10772.024 0.1153 0.0000 7V
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3. 84.997 48324 0,22489.870 0.2771 O.OOGO W
33 65.42a 40354 0.18693.329 0.2008 0.0000 VY
3i 65.833 67766 0.31393.923 0.2173 0.0000 V?
35 66.950 31238 0.09871.691 0.0963 O.Q000 ?P
36 67.773 32193 0.14911.708 0.0972 0.0000 ?P
37 79.013 29411 0.13630.920 0.0523 0.0000 PP
38 97:.97 21384 O.IOIi0.456 0.0260 0.0000 PP
39 100.05339:7a O,i7S20.738 0,020 0.0000 ' ?P
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41 107.01057253 0.26533.113 0.1Ø 0.0000 Pp i3 111.39733834 O.la6I4.236 0.2410 0.0000 V'f 43 111.813.9337 0.13132.513 0.1429 0.0000 r?
as 113.790.5408 0.11771.810 O.I030 0.0000 V7 a5 115.88343858 0..03,3.808 0.2167 0.0000 ?V
i6 119.390=3431 0.1179=,630 0.0942 0.0000 ?P
LOL31 213A6254 1T57,553 0.0000 Table 3. HPLC spectrum figure of wild Zang Zhi Table 3 No. cetention area area% relative heigEtt% conc conc basline ingredient time (uv'sec) hei~ttt unit name 1 1.550 13320090.3787162.0451.1488 0.0000 PV
2 1.913 127080005.5.10797:4305.6335 0.0000 vv 3 2.607 79905043.1715374.1432.6525 O.OO00 w 1 ..997 33361511.4494131.1641.0738 0.0000 vv 3.480 827969 0.339790:5300.6419 0.0000 6 3.687 21009120.912789.3310.6326 0.0000 ~'Y
7 1,110 15653780.7.33BO.a680.5703 0.0000 tt
8 4.590 986857 0.423761.5430.4363 0.0000 vv
9 4.867 13006190.565159.3450.4314 0.0000 W
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LOL81 230177336 14103.03 0.0000 [0033] The following Analysis Examination and Pharmacology Examination demonstrate that according to the elements and functions of the concentrated product of the invention's cultured Zang Zhi fruiting body, its major elements include triterpenoid compounds, water-soluble polysaccharides and chitin.
Water Soluble Polysaccharides [0034] Examination by the Food Analysis CetYter in Japan revealed that solid Zang Zhi fruiting body obtained by the invention's culture method contains a high volume of water soluble polysaccharides; each 1008 fruiting body having 1.2 to 6.5g of water soluble polysaccharides.
Content of Heaw Metals (0035] Atomic Spectrum Analysis made by the Mircoanalysis Laboratory of Atomic Science Department of National Ching Hwa University reveals the invention's solid-cultured Zang Zhi has a content of heavy metals as follows:
Na : 48.7ppm Mn : 10.9ppm Cd : ND
Mg : 767 ppm Fe : 100 ppm Cr : ND
Al : 41.6ppm Zn : 16.4 ppm Ca : 605 ppm As : ND
K : 0.560% Hg : ND
Total Antioxidant Activity [0036] A comparison among one gram of the concentrated powder of the invention'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's solid Zang Zhi substance 1180 ug ascorbic acid/ g [0037) The result shows that the total antioxidant activity of the invention's solid Zang Zhi substance is far higher than that of the fruiting body of wild Zang Zhi and that of Ganderma sp.
Toxicit~Test [0038] 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'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 mglkg.
Ames Test [0039] Under the Ames test, the invention'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 examplesFeed dosage x PeriodTest result Salmonella Five mutant tested concentrationWhether under ~ big typhimurium isolates required(ug/plate) mouse liver enzyme Histidine 312.5, 625, 1250, metabolic system of 2500 and (S9) TA97, TA98, 5000 plate mixed or not, alI tested assay for TA100, TA102 triplicate. The assayconcentrations was show and TA1535 under constant temperatureno significant increase 37~ 1 EC for 48-72 of Ames test mutant hours, and the mutant coloniescolonies.
calculated. Test result is negative.
Anti-Lipid Peroxidation Abilit~Test [0040] 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 Fe2+ introduction and induced the kinetics of generation of lipid-derived free radical. Figure 2 shows that while increasing the concentration of triple and 8 fold the invention's Zang Zhi solid substance concentrate, it has a trend to inhibit lipid peroxidation and reduce the generation of free radical.
Protection of Liver Function Experiment [0041] 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% CCl4/olive oil (0.3 mg/100g0(control) 6 BVV) C As above SO mg(triple)/Kg6 D As above 50 mg(8 fold)/Kg6 [0043] The mice of Groups A and B to D were under a hypodermic injection of olive oil (0.1 ml/100g BW), and 40% of CCI~/Olive oil (0.3 ml/100g BV~ 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'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's Zang Zhi have the capacity to inhibit the increase of GTP and GOT induced by CCl4. 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 CCl4 after six weeks, the liver tissue treated with CCl4 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 CC14 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 InflammationFat Cell BiliaryCalcific necrosis of liver degener- Prolifer-ation ation -ation Local CosecutiveTuberous necrosisnecrosis transform-ation 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 (COLD 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 Cancer Cell Lines Rate of Cell Growth Inhibition (%) Average (%) t SD
Hela 83 t 3.1 AGS 84 ~ 2.7 HepG2 75 ~ 2,4 MCF-7 87 t 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 (Newt, New2, New3, New4 and NewS), 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 Figure 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%.
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'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's Zang Zhi to damage the cellular chromosome. Three treatments are given as follows: the first treatment is to add the invention'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 ExperimentalExperimental Feed Period Result Target Examples Chinese Inoculate The tested concentrationsResults of the third each group hamster 60 mm cell of the first and under treatment ovary second reveal cells; ATCC,cultured plategroups are 0, 312.5 that treatment of 625, Chinese repository according 1250, 2500 and 5000 hamster ovary cells No. to the . with CCL-61 tested ug/ml. the invention's Zang Zhi, CHO-KI concentrations; before or after metabolic each The tested concentrationsactivities, does not show concentrationof the third groups significant increase has are 0, of the two plates; 30, I00, 1000, and frequency of these 3000 samples are ug/ml. chromosomal divided into configuration mutation of three groups After culture under Chinese hamster ovary according different conditions,cells.
to the cells different with scattered uniformly treatments. I 8-21 chromosomes The experimental are result is selected for observation.negative.
Analysis of Micronuclei in Animal Body [0049] The clastogenicity of the invention'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's Zang Zhi to circumference blood micronuclei of mouse is negative.
Table 9. Analvsis of Micronuclei in Animal Bodv ExperimentalExperimentalFeed period Experimental Results Target Samples ICR mouse Samples 1s' - 4~' groups 1. There is no difference are: of between 1. Solvent mice respectivelytested dosage group and solvent control were fed on 0, control group.
group 500, 2. High 1000, 2000 mglKg 2. The rate of reticular red blood dosage controldosage; cell analysis proves that high group Mice of positive dosage would not be toxic to 3. Medium control group mouse marrow.
were dosage controlfed on 1.0 mg/Kg 3. There is no tendency of group MMC. dosage reaction to micronuclei 4. Low dosageThe blood was rate and the comparison of the control collected and control group shows no group the 5. Positivereticular red significant increase.
blood Thus control cells were observedanalysis of the reaction group of 6. Each under fluorescentmicronuclei of mouse group has five microscopy. circumference blood to mice. Zang Zhi shows negative reaction.
(0050] According to the pharmacological data described above, the invention'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'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.
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LOL81 230177336 14103.03 0.0000 [0033] The following Analysis Examination and Pharmacology Examination demonstrate that according to the elements and functions of the concentrated product of the invention's cultured Zang Zhi fruiting body, its major elements include triterpenoid compounds, water-soluble polysaccharides and chitin.
Water Soluble Polysaccharides [0034] Examination by the Food Analysis CetYter in Japan revealed that solid Zang Zhi fruiting body obtained by the invention's culture method contains a high volume of water soluble polysaccharides; each 1008 fruiting body having 1.2 to 6.5g of water soluble polysaccharides.
Content of Heaw Metals (0035] Atomic Spectrum Analysis made by the Mircoanalysis Laboratory of Atomic Science Department of National Ching Hwa University reveals the invention's solid-cultured Zang Zhi has a content of heavy metals as follows:
Na : 48.7ppm Mn : 10.9ppm Cd : ND
Mg : 767 ppm Fe : 100 ppm Cr : ND
Al : 41.6ppm Zn : 16.4 ppm Ca : 605 ppm As : ND
K : 0.560% Hg : ND
Total Antioxidant Activity [0036] A comparison among one gram of the concentrated powder of the invention'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's solid Zang Zhi substance 1180 ug ascorbic acid/ g [0037) The result shows that the total antioxidant activity of the invention's solid Zang Zhi substance is far higher than that of the fruiting body of wild Zang Zhi and that of Ganderma sp.
Toxicit~Test [0038] 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'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 mglkg.
Ames Test [0039] Under the Ames test, the invention'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 examplesFeed dosage x PeriodTest result Salmonella Five mutant tested concentrationWhether under ~ big typhimurium isolates required(ug/plate) mouse liver enzyme Histidine 312.5, 625, 1250, metabolic system of 2500 and (S9) TA97, TA98, 5000 plate mixed or not, alI tested assay for TA100, TA102 triplicate. The assayconcentrations was show and TA1535 under constant temperatureno significant increase 37~ 1 EC for 48-72 of Ames test mutant hours, and the mutant coloniescolonies.
calculated. Test result is negative.
Anti-Lipid Peroxidation Abilit~Test [0040] 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 Fe2+ introduction and induced the kinetics of generation of lipid-derived free radical. Figure 2 shows that while increasing the concentration of triple and 8 fold the invention's Zang Zhi solid substance concentrate, it has a trend to inhibit lipid peroxidation and reduce the generation of free radical.
Protection of Liver Function Experiment [0041] 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% CCl4/olive oil (0.3 mg/100g0(control) 6 BVV) C As above SO mg(triple)/Kg6 D As above 50 mg(8 fold)/Kg6 [0043] The mice of Groups A and B to D were under a hypodermic injection of olive oil (0.1 ml/100g BW), and 40% of CCI~/Olive oil (0.3 ml/100g BV~ 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'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's Zang Zhi have the capacity to inhibit the increase of GTP and GOT induced by CCl4. 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 CCl4 after six weeks, the liver tissue treated with CCl4 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 CC14 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 InflammationFat Cell BiliaryCalcific necrosis of liver degener- Prolifer-ation ation -ation Local CosecutiveTuberous necrosisnecrosis transform-ation 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 (COLD 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 Cancer Cell Lines Rate of Cell Growth Inhibition (%) Average (%) t SD
Hela 83 t 3.1 AGS 84 ~ 2.7 HepG2 75 ~ 2,4 MCF-7 87 t 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 (Newt, New2, New3, New4 and NewS), 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 Figure 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%.
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'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's Zang Zhi to damage the cellular chromosome. Three treatments are given as follows: the first treatment is to add the invention'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 ExperimentalExperimental Feed Period Result Target Examples Chinese Inoculate The tested concentrationsResults of the third each group hamster 60 mm cell of the first and under treatment ovary second reveal cells; ATCC,cultured plategroups are 0, 312.5 that treatment of 625, Chinese repository according 1250, 2500 and 5000 hamster ovary cells No. to the . with CCL-61 tested ug/ml. the invention's Zang Zhi, CHO-KI concentrations; before or after metabolic each The tested concentrationsactivities, does not show concentrationof the third groups significant increase has are 0, of the two plates; 30, I00, 1000, and frequency of these 3000 samples are ug/ml. chromosomal divided into configuration mutation of three groups After culture under Chinese hamster ovary according different conditions,cells.
to the cells different with scattered uniformly treatments. I 8-21 chromosomes The experimental are result is selected for observation.negative.
Analysis of Micronuclei in Animal Body [0049] The clastogenicity of the invention'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's Zang Zhi to circumference blood micronuclei of mouse is negative.
Table 9. Analvsis of Micronuclei in Animal Bodv ExperimentalExperimentalFeed period Experimental Results Target Samples ICR mouse Samples 1s' - 4~' groups 1. There is no difference are: of between 1. Solvent mice respectivelytested dosage group and solvent control were fed on 0, control group.
group 500, 2. High 1000, 2000 mglKg 2. The rate of reticular red blood dosage controldosage; cell analysis proves that high group Mice of positive dosage would not be toxic to 3. Medium control group mouse marrow.
were dosage controlfed on 1.0 mg/Kg 3. There is no tendency of group MMC. dosage reaction to micronuclei 4. Low dosageThe blood was rate and the comparison of the control collected and control group shows no group the 5. Positivereticular red significant increase.
blood Thus control cells were observedanalysis of the reaction group of 6. Each under fluorescentmicronuclei of mouse group has five microscopy. circumference blood to mice. Zang Zhi shows negative reaction.
(0050] According to the pharmacological data described above, the invention'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'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.
Claims (49)
1. A method of culturing solid Zang Zhi comprising the use of "Bag Log"
inoculated with spawn to culture mycelium and then removing the "Bag Log" to let the sawdust medium be completely exposed in the air for a period of time until the formation of the conoid fruiting body is made, wherein the "Bag Log" contains 10-70% of cellulolytic substance, 10-30% of starch resource, 5-15% of millet, 1-10 % of saccharides, 0.5-2% phosphate, and 0.1-1%
sulfate salt, the relative humidity is maintained at 60-80% and the pH of the cultural medium is adjusted to be neutral.
inoculated with spawn to culture mycelium and then removing the "Bag Log" to let the sawdust medium be completely exposed in the air for a period of time until the formation of the conoid fruiting body is made, wherein the "Bag Log" contains 10-70% of cellulolytic substance, 10-30% of starch resource, 5-15% of millet, 1-10 % of saccharides, 0.5-2% phosphate, and 0.1-1%
sulfate salt, the relative humidity is maintained at 60-80% and the pH of the cultural medium is adjusted to be neutral.
2. The method according to Claim 1, wherein the "Bag Log" is composed of 60-70% of the cellulolytic substance, 20% of starch source, 10% of millet, 3.5% of glucose, 1% of potassium phosphate, and 0.5% of magnesium sulfate, the relative humidity is at 80% and pH
is adjusted to be neutral.
is adjusted to be neutral.
3. The method according to Claim 1, wherein the cellulolytic substance is mushroom or stem, stalk, fruit, or spelk of the plant.
4. The method according to Claim 2, wherein the cellulolytic substance is mushroom or stem, stalk, fruit, or spelk of the plant.
5. The method according to Claim 1, wherein the source of the starch is potato.
6. The method according to Claim 2, wherein the source of the starch is potato.
7. The method according to Claim 1, wherein the millet is rice bran.
8. The method according to Claim 2, wherein the millet is rice bran.
9. The method according to Claim 1, wherein the "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.
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.
10. The method according to Claim 2, wherein the "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.
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.
11. The method according to Claim 1, wherein the temperature for the culture of mycelium is between 5°C and 28°C.
12. The method according to Claim 11, wherein the temperature for the culture of mycelium is 28°C.
13. The method according to Claim 1, wherein the humidity of "Bag Log" is 60-80%.
14. The method according to Claim 1, wherein the humidity of "Bag Log" is 80%.
15. The method according to Claim 1, wherein the condition of air for the culture of mycelium is of 0.2-1% carbon dioxide.
16. The method according to Claim 1, wherein the culture of the fruiting body is done in the moving air.
17. The method according to Claim 16, wherein the culture of the fruiting body is being done at the temperature of 20-30°C during the day and at the temperature of 8-14°C at night.
18. The method according to Claim 17, wherein the culture of the fruiting body is done at the temperature of 24-26°C during the day and at the temperature of 10-12°C at night.
19. The method according to Claim 18, wherein the temperature difference between the day and the night is 15°C.
20. The method according to Claim 1, wherein the air humidity for the culture of the fruiting body is at 90-95%.
21. The method according to Claim 20, wherein the air is of less than 1% of carbon dioxide.
22. The method according to Claim 1, wherein the culture of mycelium takes 50-80 days; the culture of the fruiting body takes 20-50 days.
23. A solid fruiting body of Zang Zhi obtained from the method according to Claim 1, which has the same characteristics of physiological activities of triterpenoid as that of wild Zang Zhi.
24. The method according to Claim 1, wherein before the mycelium culture, there is the culture of a large quantity of bacteria comprising (1) a sample piece of medium agar containing hyphae preserved in the liquid nitrogen and transferred into a fresh medium to culture at the constant temperature; (2) until the exuberant growth of mycelium appears;
inoculate the spawn into the "Bag Log" containing a cellulolytic substance;
(3) wait for the overgrowth of hyphae in the "Bag Log"; remove the supra-old hyphae, and then inoculate an amount of spawn into the "Bag Log."
inoculate the spawn into the "Bag Log" containing a cellulolytic substance;
(3) wait for the overgrowth of hyphae in the "Bag Log"; remove the supra-old hyphae, and then inoculate an amount of spawn into the "Bag Log."
25. The method according to Claim 1, wherein before mycelium culture, there is the culture of a large quantity of bacteria comprising the culture of a large quantity of spawn with liquid fermentation. That is to say, after culturing them in the slide tube, inoculate the mass-cultured spawn into 5 liters of liquid fermentation, at the temperature of 24-26°C, under 240 rpm oscillation for 14 days, and then 90rpm oscillation for 14 days, after that inoculate into 20 liters of liquid fermentation, stir to culture for 14 days. The formula of liquid fermentation is 1-3% of fructose, 0.01- 0.1% of magnesium sulfate, 0.1-1% of yeast extract, 0.05-0.5% of potassium phosphate.
26. The method according to Claim 25, wherein the formula of liquid fermentation is 2% of fructose, 0.05% of magnesium sulfate, 0.5% of yeast extract, and 0.1% of potassium phosphate.
27. A solid fruiting body of Zang Zhi, which use the spawn coded CCRC 35398 as deposited in the Food Industry and Development Institute, and obtained from the method according to Claim 1, wherein the cultured solid Zang Zhi fruiting body has the characteristics of physiological activities of triterpenoid the same as those wild Zang Zhi, which is conoid, 10-20 cm in diameter, 20-30 cm in height, and 0.3-0.5 kilogram in weight.
28. A Zang Zhi fruiting body product, comprising the solid fruiting body of Zang Zhi according to Claim 27 ground into tiny pieces and then homogenized with water or alcohol to concentrate them to become sticky so as to get a concentrated liquid.
29. The product according to Claim 28, which can be dehydrated to make dry powder.
30. The product according to Claim 29, wherein the powder can be re-wetted and then re-dried to make products of granule.
31. The product according to Claim 28, which is used as food composition.
32. The solid fruiting body of Zang Zhi according to Claim 27, which has the function for liver protection.
33. The solid fruiting body of Zang Zhi according to Claim 27, which can cure or inhibit the growth of cancer cells.
34. The solid fruiting body of Zang Zhi according to Claim 33, wherein the cancer cells are cervix cancer (HeLa), stomach cancer (AGS), breast cancer (MCF- 7) liver cancer (HePG2) and bowel cancer (COLO 320 HSR).
35. The solid fruiting body of Zang Zhi according to Claim 27, which is used to inhibit peroxide reaction.
36. The solid fruiting body of Zang Zhi according to Claim 27, which is used for anti-free radical and anti-lipid peroxide reaction.
37. A food composition comprising the solid fruiting body of Zang Zhi according to Claim 27 or the processed products thereof.
38. A food composition according to Claim 37 for use in liver protection.
39. A food composition according to Claim 37 for use in curing or inhibiting the growth of cancer cells.
40. A food composition according to Claim 37 for use in inhibiting peroxide reaction.
41. A food composition according to Claim 37 which exhibits anti-free radical or anti-lipid peroxide activity.
42. A pharmaceutical composition which is made of the solid fruiting body of Zang Zhi according to Claim 27 or the processed products thereof.
43. A pharmaceutical composition according to Claim 42 for use in liver protection.
44. A pharmaceutical composition according to Claim 42 for use in curing or inhibiting the growth of cancer cells.
45. A pharmaceutical composition according to Claim 42 for use in inhibiting a peroxide reaction.
46. A pharmaceutical composition according to Claim 42 which exhibits anti-free radical or anti-lipid peroxide activity.
47. The solid fruiting body of Zang Zhi according to Claim 27 which has the same function as that of wild Zang Zhi, including tranquilization, prevention from or treatment of a cold, promoting vital energy circulation, removing blood stasis, promoting blood circulation, relief of dyspepsia, detoxifying and promoting the subsidence of swelling, calming the nerves to reduce stress, alleviating pain, inhibiting bacteria, 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, and flu.
48. The solid fruiting body of Zang Zhi according to Claim 27, which has skin-regeneration function and can be used as recovery material or body skin.
49. The solid fruiting body of Zang Zhi according to Claim 45, which can be made as a tab to cure bedsores and skin wounds.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA002368888A CA2368888A1 (en) | 2002-01-22 | 2002-01-22 | Incubation method for obtaining solid culture of zang zhi, solid culture obtained therefrom, processed products and use thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA002368888A CA2368888A1 (en) | 2002-01-22 | 2002-01-22 | Incubation method for obtaining solid culture of zang zhi, solid culture obtained therefrom, processed products and use thereof |
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| Publication Number | Publication Date |
|---|---|
| CA2368888A1 true CA2368888A1 (en) | 2003-07-22 |
Family
ID=27626473
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002368888A Abandoned CA2368888A1 (en) | 2002-01-22 | 2002-01-22 | Incubation method for obtaining solid culture of zang zhi, solid culture obtained therefrom, processed products and use thereof |
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| CA (1) | CA2368888A1 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102204689A (en) * | 2011-02-12 | 2011-10-05 | 黄清标 | Food containing glutinous rice steamed with blood and poultry meat and preparation method thereof |
| CN105124588A (en) * | 2015-09-23 | 2015-12-09 | 厦门市宝纤体生物科技有限公司 | Combined enzyme with detoxifying function and preparation method thereof |
| CN108624509A (en) * | 2017-03-16 | 2018-10-09 | 浙江泛亚生物医药股份有限公司 | The circulation utilization method of solid medium in a kind of artificial incubation of cicada fungus |
-
2002
- 2002-01-22 CA CA002368888A patent/CA2368888A1/en not_active Abandoned
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102204689A (en) * | 2011-02-12 | 2011-10-05 | 黄清标 | Food containing glutinous rice steamed with blood and poultry meat and preparation method thereof |
| CN102204689B (en) * | 2011-02-12 | 2013-03-20 | 黄清标 | Food containing glutinous rice steamed with blood and poultry meat and preparation method thereof |
| CN105124588A (en) * | 2015-09-23 | 2015-12-09 | 厦门市宝纤体生物科技有限公司 | Combined enzyme with detoxifying function and preparation method thereof |
| CN108624509A (en) * | 2017-03-16 | 2018-10-09 | 浙江泛亚生物医药股份有限公司 | The circulation utilization method of solid medium in a kind of artificial incubation of cicada fungus |
| CN108624509B (en) * | 2017-03-16 | 2022-01-28 | 浙江泛亚生物医药股份有限公司 | Recycling method of solid culture medium in artificial culture process of cordyceps sobolifera |
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