Note: Descriptions are shown in the official language in which they were submitted.
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.
1
CA 02368888 2002-11-28
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
CA 02368888 2002-11-28
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.
3
CA 02368888 2002-O1-22
[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.
4
CA 02368888 2002-O1-22
[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
CA 02368888 2002-O1-22
[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
6
CA 02368888 2002-O1-22
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.
7
CA 02368888 2002-O1-22
(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
CA 02368888 2002-O1-22
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 ,
R1
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
9
CA 02368888 2002-O1-22
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.
CA 02368888 2002-O1-22
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 _
6
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
11
CA 02368888 2002-O1-22
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
105.377 620409 0.259337:0800.2529 0.0000 r7
115.623 713314 0.309537.6600.2670 0.0000
I20,020 617412 0,25833..7970.2321 0.0000 v7
136.367 380678 O.laSi21.9390.1762 0.0000 ':V
is6.e60 622333 0,270124:053O.1,T050.0000 Vv
IS7.060 557303 0.243118:364O.I302 0.0000 v7
I67.710 585765 0.254537:106O.I922 0.0000 vv
I78.353 2762=I 0.1.00I0.95T0.0777 0.0000 V'J
138.867 379887 0.155019:9010.1111 0.0000
191.327 316284 O,I37421.1570.1500 0.0000 :T
~
.09.533 72943 0.03161.91a 0.0348 0.0000 ~J
21:C.393130381 0.052ad.381 0.0467 0.0000 ??
3311.26057846 0.0231...53 0.016. 0.0000 ?V
23Il.Ta732476 O.OIaI~.IS9 0.0156 O.o00o PI
241..253209326 0.090911.4370.0811 0.0000 V'J
231,.937131315 0.03706.603 0.0468 0.0000 v'.7
2513.733270732 0.11767.438 0.0527 O.oo00 v~
2715.300156380 0.06736:54 0.0464 0.0000 ??
,
2S27.313346774 0.130710.3690.0733 0.0000 ?V
.918.27010117770.439623.9040.1837 O.OO00 :V
3019.803298337 0.13967,175 0.0509 0.0000 v?
3121.367237347 O.I0326.713 0.0476 0.0000 vv
3222.657251555 0.11367.129 0.0505 0.0000 TIY
33?3.960419821 0.18248.163 0.0579 0.0000 v?
3425.78010112370.410530.361O.I444 0.0000 ??
3529.32033821091.9172103.9600.7370 0,0000 ??
3532.01769997103.0410136:204I.107a 0.0000 7v
3733.343399131 O.I73a8.733 0.0619 0.0000 v?
3835.05397..3 0.04223.438 0.0173 0.0000 ?v
3937.00730626121.330345.1300.3272 0.0000 v?
4039.387832484 0.301715.925O.1I29 0.0000 7~f
1i10.897-x113031.017630.3340.2132 O.o00o .'?
4213,50394412 0.04102.102 0.0149 0.0000 ??
i3x6.093950329 O.iI2912.9480.0918 0.0000 ??
4447,83029686 0.01290.697 0.0049 0.0000 ?P
i531..7333016 0.00960.305 0.0036 0.0000 ?P
so53.253391511 O.ITOl6.512 0.0461 0.0000 FP
1755.177418079 0.18196.082 0.0431 0.0000 ??
.
i857.21339886801.7329.40.8831.7078 0:0000 ?V
4937.50041353181.8033343.6112.4503 0.0000 v'~
3037.78735150381.3401257.8291.8279 0.0000 w
SISA.oiO12069310.324a175.6201.2x5! 0.0000 r~
5.38.2372986313I.29T42.2.9351.3807 0.0000 ttV
5358.40030934072.2128a3i.e0i3,0399 O.O000 v'.t
3i38.6732340255I.I036286.5112,0315 0.0000 v~t
3338.94356617871.1397<7i.0233.3394 0.0000 vv
5659,23324006431.0430197.3471.3991 0.0000 v'f
3739,3x33331583.1.33x3200.aa31.x211 O.OOOO v't
5859,83022289430.968416x.1651.1639 0.0000 vv
3960,060282AA691.2290333.7812.3606 0.0000 ><t
6060.410173<76307.6233994.9287.0329 0.0000 w'
12
CA 02368888 2002-O1-22
61 50.7:3138338006.01:199a.6?.87.05150.0000
62 60.92099302704.31:2941.1676.67260.0000 W
63 .61.31336011501.56:5.55.7611.88420.0000 w
5i 41.72350495232.6274525.2383.72380.0000 VV
55 6=.13722498:50.977:160.5371.13810.0000 V'J
65 53.35729196801.2380152:1311.07960.0000 VV
57 53.20711335fi804.9348986.2.06.29300.0000 '.T'J
58 43.36037581581.6.32721::7512.52.50.0000 W
59 64,087174034307.5609992.5757.03770.0000
70 61.80763798952.7717332.9653.30060.0000 W
71 65.:8711035220.:803:8.7360.3457O.QOOC w
72 6fi.06010617180.4613:1.5620.29470.0000 TJ
73 56.:901903467O.S27055::590.:6420.0000 W
7a 57.5631016818.O.iilB30.9130.21920.0000 w
75 58.387151159:0.656737.10:0.26310.0000 VV
75 69.390107:34304.5673335:2073.73060.0000 w
T7 70.583358833 0.155913.0870.09280.0000 W
7S 71.25727328451.1873127.2120.90190.0000 W
79 72.3172378fi30.20337.928 0.05530.0000 W
80 72.730245290 O.10a55.781 0.04810.0000 W
s1 73.783ST523 O.D3803.200 0'.02::70.0000 W
B2 7x.350127009 0.05524.136 0.02930.0000 W
33 74,89086113 0.037:2:980 0.02110.0000 V?
8x 75.97325738 O.O1=31.165 0.00830.0000 PP
85 77.50392_'75 O.Oa011.598 0.0135O.OODO ?P
B5 80.0505087:x 0.221011.3080.08090.0000 ??
~
a7 82.200291S8 o.oI22D.a o.oofi2o.ooao ?~
n
9E 81.29379909 0.03471.531 0.01090.0000 ??
89 87.50010924x7O.i7x6:x.0110.170 0.0000 Pv
90 87.530300045 0.130:21.5530.1741O.o00o W
9I 87.893382295 0.156125.6030.19150.0000 W
92 88.107224523 0.097321.fi310.153x0.0000 W
93 88.397717983 0.311937.09x0..5300.0000 W
3a 89.010518852 0.225123:3:20.15770.0000 ~J
95 89.237307090 0.133x21.5510.15280.0000 W
96 89.473302535 0.131319.158O.I3580.0000 W
97 90.007~ 40507331.7598325:5332.31300.0000 W
98 91.020376589 0.153613.7430.11870.0000 W
99 91.427152969 0.066511.x230.08100.0000 VV
1009I.Sx3205346 O.OB9211.0330.07820.0000 W
10193.0131x3333 0.0615II.6130.08230.0000 W
10292.157268901 0.116812.:79O.OB140.0000 w
10392.6331_'4915O.OSa38.073 0.05 0.0000 W
7
10i93.083908307 0.394688:3760.62560.0000 VV
10593,590210901 0.091:15.:230.10930.0000 W
10594.063323059 0.1x0421:1730.15720.0000 W
10794.5x763428 0.02733.102 0.02200.0000 W
10895..2329222 0.01272:059 O.Ola60.0000 V?
10995.953:5135 0.0i9fi2.957 0.02100.0000 J?
11098.95058309 0.02533.6xx 0Ø580.0000 =?
111100..7320525 0.00890.711 0.00510.0000 PP
112103.56338.99 0.01641.804 0.01280.0000 PP
113112,25355710 0.02:21.x68 0.01040.0000 ?P
1I4126.4174603 0.01940.861 O.OOfil0.0000 PP
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.
I3
CA 02368888 2002-O1-22
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.
14
CA 02368888 2002-O1-22
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
CA 02368888 2002-O1-22
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
16
CA 02368888 2002-O1-22
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
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
17
CA 02368888 2002-O1-22
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),
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
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
18
CA 02368888 2002-O1-22
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.
19
CA 02368888 2002-O1-22
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.
