U.S. patent application number 12/021584 was filed with the patent office on 2008-05-22 for novel compounds from antrodia camphorata.
This patent application is currently assigned to Golden Biotechnology Corporation. Invention is credited to Mao-Tien Kuo, Sheng-Yun Liu, Wu-Che Wen.
Application Number | 20080119565 12/021584 |
Document ID | / |
Family ID | 39417715 |
Filed Date | 2008-05-22 |
United States Patent
Application |
20080119565 |
Kind Code |
A1 |
Liu; Sheng-Yun ; et
al. |
May 22, 2008 |
NOVEL COMPOUNDS FROM ANTRODIA CAMPHORATA
Abstract
The present invention relates to a novel compound and use
thereof, in particular to Antroquinonol B and Antroquinonol C
isolated from Antrodia camphorata extracts which can effectively
inhibit the growth of cancer cells. The compounds from Antrodia
camphorata are first reported, which can be applied not only in
inhibiting growth of breast cancer, lung cancer, hepatic cancer and
prostate cancer, but also in anti-cancer medicinal compositions for
the abovementioned cancer cells.
Inventors: |
Liu; Sheng-Yun; (Danshuei
Township, TW) ; Kuo; Mao-Tien; (Danshuei Township,
TW) ; Wen; Wu-Che; (Danshuei Township, TW) |
Correspondence
Address: |
RABIN & Berdo, PC
1101 14TH STREET, NW
SUITE 500
WASHINGTON
DC
20005
US
|
Assignee: |
Golden Biotechnology
Corporation
Taipei Hsien
TW
|
Family ID: |
39417715 |
Appl. No.: |
12/021584 |
Filed: |
January 29, 2008 |
Current U.S.
Class: |
514/690 ;
568/377 |
Current CPC
Class: |
C07C 403/10 20130101;
C07C 2601/16 20170501; C07C 403/08 20130101; A61P 35/00
20180101 |
Class at
Publication: |
514/690 ;
568/377 |
International
Class: |
A61K 31/12 20060101
A61K031/12; C07C 49/577 20060101 C07C049/577 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 19, 2004 |
TW |
096139241 |
Claims
1. A compound having the following formula, wherein X is hydroxyl
(--OH) or methoxy (--OCH.sub.3). ##STR4##
2. The compound as claimed in claim 1, wherein the compound is
isolated from Antrodia camphorata.
3. The compound as claimed in claim 1, wherein the compound is
Antroquinonol B when x is hydroxyl (OH).
4. The compound as claimed in claim 3, wherein the compound
Antroquinonol B is isolated from Antrodia camphorata.
5. The compound as claimed in claim 1, wherein the compound is
Antroquinonol C when x is methoxy (OCH.sub.3).
6. The compound as claimed in claim 5, wherein the compound
Antroquinonol C is isolated from Antrodia camphorata.
7. A compound as claimed in claim 3 used in inhibiting growth of
cancer cells, wherein the cancer cells are breast cancer cells,
hepatic cancer cells, or prostate cancer cells.
8. The compound as claimed in claim 7, wherein the breast cancer
cells are selected from the group consisting of: breast cancer cell
line MCF-7; and breast cancer cell line MDA-MB-231.
9. The compound as claimed in claim 7, wherein the hepatic cancer
cells are selected from the group consisting of: hepatic cancer
cell line Hep3B; and hepatic cancer cell line HepG2.
10. The compound as claimed in claim 7, wherein the prostate cancer
cells are selected from the group consisting of: prostate cancer
cell line LNCaP; and prostate cancer cell line DU145.
11. A medicinal composition used in inhibiting growth of tumor
cells, which comprises a compound as claimed in claim 3 and a
pharmaceutically-acceptable carrier, wherein the tumor cells are
selected from the group consisting of: breast cancer, hepatic
cancer, and prostate cancer.
12. A compound as claimed in claim 5 used in inhibiting growth of
cancer cells, wherein the cancer cells are breast cancer cells,
hepatic cancer cells, or prostate cancer cells.
13. The compound as claimed in claim 12, wherein the breast cancer
cells are selected from the group consisting of: breast cancer cell
line MCF-7; and breast cancer cell line MDA-MB-231.
14. The compound as claimed in claim 12, wherein the hepatic cancer
cells are selected from the group consisting of: hepatic cancer
cell line Hep3B; and hepatic cancer cell line HepG2.
15. The compound as claimed in claim 12, wherein the prostate
cancer cells are selected from the group consisting of: prostate
cancer cell line LNCaP; and prostate cancer cell line DU145.
16. A medicinal composition used in inhibiting growth of tumor
cells, which comprises a compound as claimed in claim 5 and a
pharmaceutically-acceptable carrier, wherein the tumor cells are
selected from the group consisting of breast cancer, hepatic
cancer, and prostate cancer.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to novel compounds and use
thereof, in particular to Antroquinonol B and Antroquinonol C
compounds isolated from Antrodia camphorata extracts and their use
in growth inhibition of tumor cells. The compounds from Antrodia
camphorata are first reported, which can be applied not only in
inhibiting growth of breast cancer, lung cancer, hepatic cancer and
prostate cancer, but also in anti-cancer medicinal compositions for
the abovementioned cancer cells.
[0003] 2. The Prior Arts
[0004] Antrodia camphorata is also called Chang-Zhi, Niu Chang-Gu,
red camphor mushroom and the like, which is a perennial mushroom
belonging to the order Aphyllophorales, the family Polyporaceae. It
is an endemic species in Taiwan growing on the inner rotten heart
wood wall of Cinnamomum kanehirae Hay. Cinnamoum kanehirai Hay is
rarely distributed and being overcut unlawfully, which makes
Antrodia camphorata growing inside the tree in the wild became even
rare. The price of Antrodia camphorata is very expensive due to the
extremely slow growth rate of natural Antrodia camphorata that only
grows between June to October.
[0005] The fruiting bodies of Antrodia camphorata are perennial,
sessile, hard and woody, which exhales strong smell of sassafras
(camphor aroma). The appearances are various with plate-like,
bell-like, hoof-like, or tower-like shapes. They are reddish in
color and flat when young, attached to the surface of wood. Then
the brims of the front end become little curled tilted and extend
to the surroundings. The color turns to be faded red-brown or cream
yellow brown, with ostioles all over. This region is of very high
medical value.
[0006] In traditional Taiwanese medicine, Antrodia camphorata is
commonly used as an antidotal, liver protective, anti-cancer drug.
Antrodia camphorata, like general edible and medicinal mushrooms,
is rich in numerous nutrients including triterpenoids,
polysaccharides (such as .beta.-glucosan), adenosine, vitamins
(such as vitamin B, nicotinic acid), proteins (immunoglobulins),
superoxide dismutase (SOD), trace elements (such as calcium,
phosphorus and germanium and so on), nucleic acid, steroids, and
stabilizers for blood pressure (such as antodia acid) and the like.
These physiologically active ingredients are believed to exhibit
effects such as: anti-tumor activities, increasing
immuno-modulating activities, anti-allergy, anti-bacteria,
anti-high blood pressure, decreasing blood sugar, decreasing
cholesterol, and the like.
[0007] Triterpenoids are the most studied component among the
numerous compositions of Antrodia camphorata. Triterpenoids are the
summary terms for natural compounds, which contain 30 carbon atoms
with the pent acyclic or hex acyclic structures. The bitter taste
of Antrodia camphorata is from the component of triterpenoids.
Three novel ergostane-type triterpenoids (antcin A-antcin B-antcin
C) were isolated by Cherng et al. from the fruiting bodies of
Antrodia camphorata (Cherng, I. H., and Chiang, H. C. 1995. Three
new triterpenoids from Antrodia cinnamomea. J. Nat. Prod.
58:365-371). Three new compounds zhankuic acid A, zhankuic acid B
and zhankuic acid were extracted from the fruiting bodies of
Antrodia camphorata with ethanol by Chen et al. (Chen, C. H., and
Yang, S. W. 1995. New steroid acids from Antrodia cinnamomea, --a
fungus parasitic on Cinnamomum micranthum. J. Nat. Prod.
58:1655-1661). In addition, Cherng et al. also found three other
new triterpenoids from the fruiting bodies of Antrodia camphorata,
which are sesquiterpene lactone and 2 biphenyl derived compounds,
4,7-dimethoxy-5-methyl-1,3-benzodioxole and
2,2',5,5'-teramethoxy-3,4,3',4'-bi-methylenedioxy-6,6'-dimethylbiphenyl
(Chiang, H. C., Wu, D. P., Cherng, I. W., and Ueng, C. H. 1995. A
sesquiterpene lactone, phenyl and biphenyl compounds from Antrodia
cinnamomea. Phytochemistry. 39:613-616). In 1996, four novel
ergostane-type triterpenoids (antcins E and F and methyl antcinates
G and H) were isolated by Cherng et al. with the same analytic
methods (Cherng, I. H., Wu, D. P., and Chiang, H. C. 1996.
Triteroenoids from Antrodia cinnamomea. Phytochemistry.
41:263-267). And two ergostane related steroids, zhankuic acids D
and E together with three lanosta related triterpenes, 15
alpha-acetyl-dehydrosulphurenic acid, dehydroeburicoic acid,
dehydrosulphurenic acid were isolated by Yang et al. (Yang, S. W.,
Shen, Y. C., and Chen, C. H. 1996. Steroids and triterpenoids of
Antrodia cinnamomea--a fungus parasitic on Cinnamomum micranthum.
Phytochemistry. 41: 1389-1392).
[0008] Although Antrodia camphorata extracts were reported to have
the anti-tumor effects from previous experiments (such as the
reference mentioned above: Chen & Yang, 1995), further
experiments are needed to identify the effective composition for
tumor inhibition. The application in cancer therapy will be of
great beneficial effects if the real effective compositions are
found.
SUMMARY OF THE INVENTION
[0009] In order to identify the compounds containing anti-tumor
effects from the extracts of Antrodia camphorata, the compound of
formula (1) was isolated and purified in the present invention,
where X can be hydroxyl (--OH) or methoxy (--OCH.sub.3).
##STR1##
[0010] When X is hydroxyl, the structure is as shown in formula
(2). The chemical name is Antroquinonol B, with molecular formula
of C.sub.24H.sub.38O.sub.5 and molecular weight of 406.
##STR2##
[0011] When X is methoxy, the structure is as shown in formula (3).
The chemical name is Antroquinonol C, with molecular formula of
C.sub.25H.sub.40O.sub.5 and molecular weight of 420. ##STR3##
[0012] Compounds Antroquinonol B and Antroquinonol C are purified
from aqueous extraction or organic solvent extraction of Antrodia
camphorata. The organic solvents used include, but not limited to,
alcohols such as methanol, ethanol or propanol, esters such as
ethyl acetate, alkanes such as hexane, or halogenated alkanes such
as chloromethane, chloroethane. Among them, alcohol is preferred,
and ethanol is particularly preferred.
[0013] The abovementioned compounds in the present invention can be
applied in inhibiting tumor cell growth, which can further be used
as a medicinal composition to treat cancer and to enhance the
therapeutic effects. The compounds of the present invention can be
applied in inhibiting a range of cancer cells, including breast
cancer, lung cancer, hepatic cancer and prostate cancer, leading to
a marked slowering of the growth of cancer cells, and further
inhibiting proliferation of cancer cells and decreasing the risk of
malignancy. Therefore they can be used for the treatment of breast
cancer, lung cancer, hepatic cancer, prostate cancer and more.
[0014] On the other hand, the compounds of formula (2) and/or
formula (3) in the present invention can be incorporated into
medicinal compositions for treating breast cancer, lung cancer,
hepatic cancer, and prostate cancer to inhibit the growth of tumor
cells. The medicinal compositions include not only the effective
amounts of Antroquinonol B and Antroquinonol C, but also the
pharmaceutically accepted carries. The carriers include, but are
not limited to, excipients such as water, fillers such as sucrose
or starch, binders such as cellulose derivatives, diluents,
disintegrants, absorption enhancers or sweeteners. The compositions
of the present invention can be manufactured through mixing the
compounds of Antroquinonol B and Antroquinonol C with at least one
of the carriers by means of conventional methods known in the
pharmaceutically technical field, which can be formulated, but are
not limited to, as a powder, tablet, capsule, pellets, granules or
other liquid formulation.
[0015] The present invention is further explained in the following
embodiment illustration and examples. Those examples below should
not, however, be considered to limit the scope of the invention, it
is contemplated that modifications will readily occur to those
skilled in the art, which modifications will be within the spirit
of the invention and the scope of the appended claims.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0016] The mycelia, fruiting bodies or mixture of both from
Antrodia camphorata are first extracted with water or organic
solvents to obtain the aqueous extract or organic solvent extract
of Antrodia camphorata using the methods well known in the arts.
The organic solvents include, but not limited to, alcohols such as
methanol; ethanol or propanol; esters such as Ethyl acetate;
alkanes such as hexane; or halogenated alkanes such as
chloromethane, and chloroethane. Among them, alcohol is preferred,
and ethanol is particularly preferred.
[0017] The aqueous or organic solvent extracts of Antrodia
camphorate were subjected to high-performance liquid chromatography
(HPLC) for isolation and purification. Each fraction was recovered
and applied to anti-tumor assay. The potent fractions with
anti-tumor ability were analyzed for the composition and further
assayed against different cancer cells. The above approach then led
to the identification of compounds of Antroquinonol B and
Antroquinonol C in inhibiting the growth of breast cancer, lung
cancer, hepatic cancer, prostate cancer and more. These compounds
from Antrodia camphorata are regarded as novel compounds since they
have never been reported in any literatures.
[0018] The compounds of Antroquinonol B and Antroquinonol C were
demonstrated with anti-cancer effects using
3-(4,5-dimethylthiazol-2-yl)-2, S-diphenyl tetrazolium bromide
(MTT) assay according to the anti-tumor drugs screening model of
National Cancer Institute (NCI) on cell survival rates using cell
lines of breast cancer, lung cancer, hepatic cancer, prostate
cancer. The above assays had proved that Antroquinonol B and
Antroquinonol C decreased survival rates of breast cancer cell
lines (MCF-7 and MDA-MB-231), lung cancer cell lines (A-549),
hepatocellular carcinoma cell lines (Hep 3B and Hep G2) and
prostate cancer cell lines (LNCaP and DU-145), at the same time
showed relatively low half-maximal inhibitory concentration
(IC.sub.50) values. Therefore Antroquinonol B and Antroquinonol C
can be used in inhibiting cancer cell growth of breast cancer, lung
cancer, hepatic cancer, and prostate cancer, which can further be
applied in cancer treatment of breast cancer, lung cancer, hepatic
cancer, prostate cancer and the like. The details of the examples
are described as follows:
EXAMPLE 1
Isolation of Antroquinonol B and Antroquinonol C
[0019] One hundred grams of mycelia, fruiting bodies or mixture of
both from Antrodia camphorata were placed into a flask. A proper
amount of water and alcohol (70-100% alcohol solution) was added
into the flask and were stirred at 20-25.degree. C. for at least 1
hour. The solution was filtered through a filter and a 0.45 .mu.m
membrane and the filtrate was collected as the extract.
[0020] The filtrate of Antrodia camphorata was subjected to High
Performance Liquid chromatography (HPLC) analysis. The separation
was performed on a RP18 column, the mobile phase consisted of
methanol (A) and 0.1-0.5% acetic acid (B), with the gradient
conditions of 0-10 min in 95%.about.20% B, 10-20 min in
20%.about.10% B, 20-35 min in 10%.about.90% B, 35-40 min in
10%.about.95% B, at the flow rate of 1 ml/min. The column effluent
was monitored with a UV-visible detector.
[0021] The fractions collected at 21.2-21.4 min were collected and
concentrated to yield Antroquinonol B, a product of pale yellow
powder. The analysis of this product showed the molecular formula
of C.sub.24H.sub.38O.sub.5, molecular weight of 406. Investigation
of NMR spectra showed that .sup.1H-NMR (CDCl.sub.3) .delta.
(ppm)=1.21, 1.36, 1.67, 1.71, 1.75, 1.94, 2.03, 2.07, 2.22, 2.25,
3.68, 4.05, 5.71, and 5.56; .sup.13C-NMR (CDCl.sub.3) .delta.
(ppm)=12.31, 16.1, 16.12, 17.67, 25.67, 26.44, 26.74, 27.00, 39.71,
39.81, 4.027, 43.34, 59.22, 60.59, 71.8, 120.97, 123.84, 124.30,
131.32, 134.61, 135.92, 138.05, 160.45, and 197.11.
[0022] The fractions collected at 23.7-24 min were collected and
concentrated to yield Antroquinonol C, a product of pale yellow
powder. The analysis of this product showed the molecular formula
of C.sub.25H.sub.40O.sub.5, molecular weight of 420. Investigation
of NMR spectra showed that .sup.1H-NMR (CDCl.sub.3) .delta.
(ppm)=1.21, 1.36, 1.67, 1.71, 1.75, 1.94, 2.03, 2.07, 2.22, 2.25,
3.24, 3.68, 4.05, 5.12, 5.50, and 5.61; .sup.13C-NMR (CDCl.sub.3)
.delta. (ppm)=12.31, 16.1, 16.12, 17.67, 24.44, 26.44, 26.74,
27.00, 37.81, 39.81, 4.027, 43.34, 49.00, 59.22, 60.59, 120.97,
123.84, 124.30, 135.92, 138.05, 160.45, and 197.12.
[0023] The structures of Antroquinonol B and Antroquinonol C were
compared in the chemical compound database. No compound with
similar structure was discovered. Therefore, these compounds are
regarded as novel compounds since they have never been reported
previously.
EXAMPLE 2
Ex Vivo Survival Assay for Anti-Breast Cancer Effects
[0024] The NCI anti-cancer drug screen model was adopted to test
the anti-cancer effect of the isolated compounds from example 1 in
the present invention. The isolated Antroquinonol B and
Antroquinonol C from example 1 were added into the culture media of
human breast-cancer cells, MCF-7 or MDA-MB-231, to test for tumor
cell survival. This survival assay was carried out with the widely
known MTT (3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium
bromide) assay. MCF-7 cell line is originated from primary breast
cancer cells (early stage), sensitive to estrogen and therefore is
estrogen-dependent, while MDA-MB-231 is insensitive to estrogen and
therefore is estrogen-independent, which is a difficult tumor with
a low survival rate.
[0025] MTT assay is commonly used to determine cell proliferation,
percent of viable cells, and cytotoxicity.
3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT)
is a yellow dye, which can be absorbed by the living cells and be
reduced to purplish blue formazan crystals by succinate tetrazolium
reductase in mitochondria. Formazan formation can therefore be used
to assess and determine the survival rate of cells.
[0026] The human breast-cancer cells, MCF-7 (purchased from the
Bioresources Collection and Research Center (BCRC) with the
accession number of BCRC60436) and MDA-MB-231 (purchased from
National Health Research Institutes (NHRI) with the accession
number of CCRC 60425) were cultivated in media containing fetal
calf serum for 24 hours. The proliferated cells were washed once
with PBS, then treated with 1.times. trypsin-EDTA and centrifuged
at 1200 rpm for 5 minutes. The supernatant was discarded and the
cell pellet was resuspended in 10 ml of fresh culture medium by
gently shaking. The cells were placed in a 96-well plate. Total
ethanol extracts of Antrodia camphorata without purification (the
control group), Antroquinonol B or Antroquinonol C (the
experimental group) were added into each of the 96 wells at the
following concentrations: 30, 10, 3, 1, 0.3, 0.1 and 0.03 .mu.g/ml,
respectively. The cells were incubated at 37.degree. C. in a 5%
CO.sub.2 incubator for 48 hours. MTT was added in a concentration
of 2.5 mg/ml into each well in dark and incubated for 4 hours,
followed by the addition of 100 .mu.l of lysis buffer to stop the
reaction. The plates were read on an ELISA reader at wavelength of
570 nm to determine the survival rates. The half maximal inhibitory
concentration (IC.sub.50) values were also calculated and listed in
Table 1. TABLE-US-00001 TABLE 1 Results of ex vivo survival assay
for inhibition of breast cancer cells IC.sub.50(.mu.g/ml) Samples
MCF-7 MDA-MB-231 Control Crude extracts of 11.13 25.81 group A.
camphorata Experiment Antroquinonol B 1.94 8.72 group Antroquinonol
C 1.17 21.9
[0027] Antroquinonol B and Antroquinonol C are capable of
decreasing the survival rate of estrogen-dependent breast cancer
cell MCF-7 from the result of Table 1. The IC.sub.50 value of
Antroquinonol B toward MCF-7 was dropped to 1.94 .mu.g/ml, which
was 82.56% lower than that of crude extracts from A. camphorata
(11.13 .mu.g/ml). And the IC.sub.50 value of Antroquinonol C toward
MCF-7 was dropped to 1.17 .mu.g/ml, which was 89.49% lower than
that of crude extracts from A. camphorata (11.13 .mu.g/ml).
Therefore Antroquinonol B and Antroquinonol C from Antrodia
camphorata can be applied to inhibit the growth of
estrogen-dependent breast cancer cells MCF-7.
[0028] On the other hand, Antroquinonol B and Antroquinonol C are
capable of decreasing the survival rate of estrogen-independent
breast cancer cell MDA-MB-231. The IC.sub.50 values of
Antroquinonol B and Antroquinonol C toward MDA-MB-231 were 8.72
.mu.g/ml and 21.9 .mu.g/ml respectively, which were 66.21% and
15.15% lower than that of crude extracts from A. camphorata (25.81
.mu.g/ml). The concentration of Antroquinonol B and Antroquinonol C
needed for the half maximal inhibitory concentration of MDA-MB-231
were all lowered than the crude extracts in the control group, and
a better inhibitory effect was found in Antroquinonol B. Therefore
Antroquinonol B and Antroquinonol C from Antrodia camphorata can be
applied to inhibit the growth of estrogen-independent breast cancer
cells MDA-MB-231.
EXAMPLE 3
Ex Vivo Survival Assay for Anti-Lung Cancer Effects
[0029] The NCI anti-cancer drug screen model was also employed to
test the anti-lung cancer effect of the Antroquinonol B and
Antroquinonol C in the present invention. The Antroquinonols
isolated from example 1 were added into the culture media of human
lung cancer cell A549 with the abovementioned MTT assay to analyze
lung cancer cell survival rates.
[0030] Human lung cancer cell A549 was obtained from the
Bioresources Collection and Research Center (BCRC) of the Food
Industry Research and Development Institute with an accession
number of BCRC 60074. A549 cells were cultivated in media
containing fetal calf serum for 24 hours. The proliferated cells
were washed once with PBS, then treated with 1.times. trypsin-EDTA
and centrifuged at 1200 rpm for 5 minutes. The supernatant was
discarded and the cell pellet was resuspended in 10 ml of fresh
culture medium by gently shaking. The cells were placed in a
96-well plate. Total ethanol extracts of Antrodia camphorata
without purification (the control group), Antroquinonol B or
Antroquinonol C (the experimental group) were added into each of
the 96 wells at the following concentrations: 30, 10, 3, 1, 0.3,
0.1 and 0.03 .mu.g/ml, respectively. The cells were incubated at
37.degree. C. in a 5% CO.sub.2 incubator for 48 hours. MTT was
added in a concentration of 2.5 mg/ml into each well in dark and
incubated for 4 hours, followed by the addition of 100 .mu.l of
lysis buffer to stop the reaction. The plates were read on an ELISA
reader at wavelength of 570 nm to determine the survival rates. The
half-maximal inhibitory concentration (IC.sub.50 values) were also
calculated and listed in Table 2. TABLE-US-00002 TABLE 2 Results of
ex vivo survival assay for inhibition of lung cancer cells
IC.sub.50 (.mu.g/ml) Samples A549 Control Crude extracts of 13.21
group A. camphorata Experimental Antroquinonol B 11.9 group
Antroquinonol C 8.21
[0031] From the result of table 2, Antroquinonol B and
Antroquinonol C can effectively decrease the survival rates of
human lung cancer cell line A549. The IC.sub.50 value of
Antroquinonol B and Antroquinonol C toward A549 were 11.9 .mu.g/ml
and 8.21 .mu.g/ml respectively, which were 9.92% and 37.85% lower
than that of crude extracts from A. camphorata (13.21 .mu.g/ml).
Both Antroquinonol B and Antroquinonol C had reduced IC.sub.50
values than the control group, and a better inhibitory effect was
found in Antroquinonol B. Therefore Antroquinonol B and
Antroquinonol C from A. camphorata can be applied to inhibit the
growth of lung cancer cells A549.
EXAMPLE 4
Ex Vivo Survival Assay for Anti-Hepatic Cancer Effects
[0032] The NCI anti-cancer drug screen model was employed to test
the anti-cancer effect of the Antroquinonol B and Antroquinono C in
the present invention. The Antroquinonos isolated from example 1
were added into the culture media of human hepatic-cancer cells,
Hep 3B or Hep G2, for tumor cell survival assay. Hepatic-cancer
cells Hep 3B (BCRC 60434) and Hep G2 (BCRC 60025), were obtained
from the Bioresources Collection and Research Center (BCRC) of the
Food Industry Research and Development Institute.
[0033] The human hepatic-cancer cells, Hep 3B and Hep G2, were
cultivated in media containing fetal calf serum for 24 hours. The
proliferated cells were washed once with PBS, then treated with
1.times. trypsin-EDTA and centrifuged at 1200 rpm for 5 minutes.
The supernatant was discarded and the cell pellet was resuspended
in 10 ml of fresh culture medium by gently shaking. The cells were
placed in a 96-well plate. Total ethanol extracts of Antrodia
camphorata without purification (the control group), Antroquinonol
B or Antroquinonol C (the experimental group) were added into each
of the 96 wells at the following concentrations: 30, 10, 3, 1, 0.3,
0.1 and 0.03 .mu.g/ml, respectively. The cells were incubated at
37.degree. C. in a 5% CO.sub.2 incubator for 48 hours. MTT was
added in a concentration of 2.5 mg/ml into each well in dark and
incubated for 4 hours, followed by the addition of 1001 of lysis
buffer to stop the reaction. The plates were read on an ELISA
reader at wavelength of 570 nm to determine the survival rates. The
half-maximal inhibitory concentration (IC.sub.50) values were also
calculated and listed in Table 3. TABLE-US-00003 TABLE 3 Results of
ex vivo survival assay for inhibition of hepatic cancer cells
IC.sub.50 (.mu.g/ml) Samples Hep3B HepG2 Control Crude extracts of
5.10 18.63 group A. camphorata Experimental Antroquinonol B 1.21
3.32 group Antroquinonol C 1.32 5.12
[0034] From the result of table 3, Antroquinonol B and
Antroquinonol C can effectively decrease the survival rates of
human hepatic cancer cell line Hep 3B and Hep G2. The IC.sub.50
value of Antroquinonol B toward Hep 3B were 1.21 .mu.g/ml, which
was 76.27% lower than that of crude extracts from A. camphorata
(5.10 g/ml). The IC.sub.50 value of Antroquinonol C toward Hep 3B
were 1.32 .mu.g/ml, which was 74.12% lower than that of crude
extracts from A. camphorata. Both compounds showed remarkedly
decrease IC.sub.50 values than the control group in Hep3B cells.
The IC.sub.50 values of Antroquinonol B and Antroquinonol C toward
Hep G2 were 3.32 .mu.g/ml and 5.12 .mu.g/ml respectively, which
were 82.18% and 72.52% lower than that of crude extracts from A.
camphorata (18.63 .mu.g/ml). Both compounds showed remarkedly
decrease IC.sub.50 values than the control group in HepG2 cells.
Therefore Antroquinonol B and Antroquinonol C from A. camphorata
can be applied to inhibit the growth of hepatic cancer cells.
[0035] In addition, hepatic cancer cells treated with Antroquinonol
B showed lower IC.sub.50 values than cells treated with
Antroquinonol C, that is, lower concentration of Antroquinonol B
was needed in inhibiting growth the hepatic cancer cells Hep 3B and
Hep G2. Antroquinonol B has better inhibiting effects than
Antroquinonol C toward the growth of hepatic cancer cells.
EXAMPLE 5
Ex Vivo Survival Assay for Anti-Prostate Cancer Effects
[0036] The NCI anti-cancer drug screen model was also employed to
test the anti-cancer effect of the Antroquinonols in the present
invention. Antroquinonol B and Antroquinonol C isolated from
example 2 were added into the culture media of human prostate
cancer cells, LNCaP or DU-145, and prostate cancer cell survival
rates were analyzed with the abovementioned MTT assay. LNCaP is
originated from epithelial cells of prostate gland, which depends
on androgen in the early stage and thus is androgen-dependent
cancer cell. DU-145 is recurrent type prostate cancer cell, which
does not rely on androgen for growth and therefore is
androgen-independent. This recurrent type prostate cancer has no
effective treatment so far. Both LNCaP and DU-145 were ordered from
NHRI with accession numbers of CCRC 60088 and CCRC 60348
respectively.
[0037] The human prostate-cancer cells, LNCaP and DU-145, were
cultivated in media containing fetal calf serum for 24 hours. The
proliferated cells were washed once with PBS, then treated with
1.times. trypsin-EDTA and centrifuged at 1200 rpm for 5 minutes.
The supernatant was discarded and the cell pellet was resuspended
in 10 ml of fresh culture medium by gently shaking. The cells were
placed in a 96-well plate. Total ethanol extracts of Antrodia
camphorata without purification (the control group), Antroquinonol
B or Antroquinonol C (the experimental group) were added into each
of the 96 wells at the following concentrations: 30, 10, 3, 1, 0.3,
0.1 and 0.03 .mu.g/ml, respectively. The cells were incubated at
37.degree. C. in a 5% CO.sub.2 incubator for 48 hours. MTT was
added in a concentration of 2.5 mg/ml into each well in dark and
incubated for 4 hours, followed by the addition of 1001 of lysis
buffer to stop the reaction. The plates were read on an ELISA
reader at wavelength of 570 nm to determine the survival rates. The
half-maximal inhibitory concentration (IC.sub.50) values were also
calculated and listed in Table 4. TABLE-US-00004 TABLE 4 Results of
ex vivo survival assay for inhibition of prostate cancer cells
IC.sub.50 (.mu.g/ml) Samples LNCaP DU-145 Control Crude extracts of
11.49 41.39 group A. camphorata Experimental Antroquinonol A 5.67
10.12 group Antroquinonol J 8.72 12.21
[0038] From the result of table 4, the cell survival rates of LNCaP
and DU-145 were effectively reduced through the activities of
Antroquinonol B or Antroquinonol C. The IC.sub.50 values of
Antroquinonol B and Antroquinonol C toward LNCaP were 5.67 .mu.g/ml
and 8.72 .mu.g/ml respectively, which were 50.65% and 24.11% lower
than the IC.sub.50 value of the control group (11.49 .mu.g/ml). The
IC.sub.50 values of Antroquinonol B and Antroquinonol C toward
DU-145 were 10.12 .mu.g/ml and 12.21 .mu.g/ml respectively, which
were 75.55% and 70.5% lower than the IC.sub.50 value of the control
group (41.39 .mu.g/ml). Therefore the half maximal inhibitory
concentrations of these two Antroquinonols from A. camphorata were
remarkedly reduced toward prostate cancer cells LNCaP and DU-145 in
comparison to the crude extracts in the control group. In addition,
LNCaP prostate cancer cells treated with Antroquinonol B showed a
much lower IC.sub.50 values than Antroquinonol C treated cells,
that is, a better inhibitory effect was found in Antroquinonol B
for inhibiting prostate cancer cells.
[0039] On the other hand, both Antroquinonol B and Antroquinonol C
can effectively inhibit the growth of LNCaP and DU-145 prostate
cancer cells with two different characteristics. Therefore
Antroquinonol B and Antroquinonol C can be applied in inhibiting
not only the growth of androgen-dependent prostate cancer cells
LNCaP, but also androgen-independent prostate cancer cells DU-145.
This is beneficial to the therapy of prostate cancer and recurrent
prostate cancer.
[0040] In summary, Antroquinonol B and Antroquinonol C purified
from extracts of A. camphorata can effectively inhibit the growth
of breast cancer, lung cancer, hepatic cancer and prostate cancer.
These compounds can be incorporated into medicinal compositions for
treating the abovementioned cancer cells. The medicinal
compositions include not only the compounds of Antroquinonol B and
Antroquinonol C, but also the pharmaceutically accepted carries.
The carriers include, but are not limited to, excipients such as
water, fillers such as sucrose or starch, binders such as cellulose
derivatives, diluents, disintegrants, absorption enhancers or
sweeteners. The inventive composition can be manufactured through
mixing the compounds of Antroquinonol B or Antroquinonol C with at
least one of the carriers by means of conventional methods known in
the pharmaceutically technical field, which can be formulated, but
are not limited to, as a powder, tablet, capsule, pellets, granules
or other liquid formulation.
* * * * *