U.S. patent application number 11/204392 was filed with the patent office on 2006-04-27 for compounds from antrodia camphorata and use thereof.
Invention is credited to Masao Hattori, Chia-Chin Sheu, Coolin Yang.
Application Number | 20060089402 11/204392 |
Document ID | / |
Family ID | 34930575 |
Filed Date | 2006-04-27 |
United States Patent
Application |
20060089402 |
Kind Code |
A1 |
Hattori; Masao ; et
al. |
April 27, 2006 |
Compounds from Antrodia camphorata and use thereof
Abstract
The present invention relates to novel compounds from Antrodia
camphorata and the use thereof.
Inventors: |
Hattori; Masao; (Sugitani
Toyama, JP) ; Sheu; Chia-Chin; (Kuei Shan Hsiang,
TW) ; Yang; Coolin; (Jaili Township, TW) |
Correspondence
Address: |
HARNESS, DICKEY & PIERCE, P.L.C.
P.O. BOX 8910
RESTON
VA
20195
US
|
Family ID: |
34930575 |
Appl. No.: |
11/204392 |
Filed: |
August 16, 2005 |
Current U.S.
Class: |
514/424 ;
514/473; 548/546 |
Current CPC
Class: |
A61P 29/00 20180101;
C07D 207/46 20130101; C07D 307/34 20130101; C07D 207/444
20130101 |
Class at
Publication: |
514/424 ;
514/473; 548/546 |
International
Class: |
A61K 31/4015 20060101
A61K031/4015; A61K 31/365 20060101 A61K031/365; C07D 207/40
20060101 C07D207/40 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 17, 2004 |
EP |
04254939.4 |
Claims
1. A compound having the formula ##STR9## its tautomeric forms, its
stereoisomers, its polymorphs, its pharmaceutically acceptable
salts, or its pharmaceutically acceptable solvates, wherein X is N
or O; R.sub.1 is H, hydroxyl, C.sub.1-10 alkyloxy, C.sub.2-10
alkenyloxy, or C.sub.2-10 alkynyloxy; R.sub.2 is H, hydroxyl,
C.sub.1-10 alkyl, C.sub.2-10 alkenyl or C.sub.2-10 alkynyl; R.sub.3
is C.sub.1-10 alkyl, C.sub.2-10 alkenyl or C.sub.2-10 alkynyl;
denotes a single or double bond; provided that if X is 0, R.sub.3
is absent; if denotes a single bond, the compound has the formula:
##STR10##
2. The compound of claim 1, wherein R.sub.1 is C.sub.2-6 alkenyloxy
or C.sub.2-6 alkynyloxy.
3. The compound of claim 2, wherein C.sub.2-6 alkenyloxy is
substituted with C.sub.1-6 alkyl.
4. The compound of claim 1, wherein R.sub.2 is isobutyl.
5. A mixture from Antrodia camphorata, which comprises the
compounds of claim 1.
6. The mixture of claim 5, which is prepared from water or organic
solvent extract of mycelium of Antrodia camphorate.
7. The mixture of claim 6, wherein the organic solvent is
ethanol.
8. The mixture of claim 5, which inhibits factor increasing
reactive oxygen species (ROS) generation.
9. The mixture of claim 5, which inhibits TGF-.beta. mediated
inflammation and fibrosis.
10. The mixture of claim 9, wherein the fibrosis is related to
diabetic nephropathy, liver cirrhosis, idiopathic pulmonary
fibrosis, rheumatoid arthritis, fibrosarcomas, arteriosclerosis,
and scleroderma.
11. The mixture of claim 5, which inhibits nitric oxide
activities.
12. The mixture of claim 11, which is for use in treating or
preventing of epithelial cell carcinogenesis and free radical
damages.
13. The mixture of claim 11, which is for use in treating or
preventing septic shock, ischemia, overexpression of cytokines,
ulcer, ulcerative colitis, diabetes, arthritis, asthma, Alzheimer's
disease, Parkinson's disease, multiple sclerosis, cirrhosis,
allograft rejection, encephalomyelitis, meningitis, pancreatitis,
peritonitis, vasculitis, lymphocytic choriomeningitis,
glomerulonephritis, uveitis, ileitis, liver inflammation, renal
inflammation, hemorrhagic shock, anaphylactic shock, burn, Crohn's
disease or infection.
14. A composition comprising a compound having the formula
##STR11## its tautomeric forms, its stereoisomers, its polymorphs,
its pharmaceutically acceptable salts, or its pharmaceutically
acceptable solvates, wherein X is N or O; R.sub.1 is H, hydroxyl,
C.sub.1-10 alkyloxy, C.sub.2-10 alkenyloxy, or C.sub.2-10
alkynyloxy; R.sub.2 is H, hydroxyl, C.sub.1-10 alkyl, C.sub.2-10
alkenyl or C.sub.2-10 alkynyl; R.sub.3 is absent, H, hydroxyl or
C.sub.1-10 alkyl, C.sub.2-10 alkenyl or C.sub.2-10 alkynyl; denotes
a single or double bond; provided that if X is 0, R.sub.3 is
absent; if denotes a single bond, the compound has the formula:
##STR12##
15. The composition of claim 14, which inhibits factor increasing
reactive oxygen species (ROS) generation.
16. The composition of claim 15, which inhibits TGF-.beta. mediated
inflammation and fibrosis.
17. The composition of claim 16, wherein the fibrosis is related to
diabetic nephropathy, liver cirrhosis, idiopathic pulmonary
fibrosis, rheumatoid arthritis, fibrosarcomas, arteriosclerosis,
and scleroderma.
18. The composition of claim 14, which inhibits nitric oxide
activities.
19. The composition of claim 18, which is for use in treating or
preventing of epithelial cell carcinogenesis and free radical
damages.
20. The composition of claim 18, which is for use in treating or
preventing septic shock, ischemia, overexpression of cytokines,
ulcer, ulcerative colitis, diabetes, arthritis, asthma, Alzheimer's
disease, Parkinson's disease, multiple sclerosis, cirrhosis,
allograft rejection, encephalomyelitis, meningitis, pancreatitis,
peritonitis, vasculitis, lymphocytic choriomeningitis,
glomerulonephritis, uveitis, ileitis, liver inflammation, renal
inflammation, hemorrhagic shock, anaphylactic shock, burn, Crohn's
disease or infection.
Description
PRIORITY STATEMENT
[0001] A claim of priority under 35 U.S.C. .sctn. 119 is made to
European Patent Application 04254939.4 filed on Aug. 17, 2004, the
entire contents of which are hereby incorporated by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to novel compounds from
Antrodia camphorata and the use thereof. The present invention
relates to the composition comprising the compounds of the
invention.
BACKGROUND OF THE INVENTION
[0003] The fruiting body of Antrodia camphorata (Polyporaceae,
Aphyllophorales) is well known in Taiwan as a traditional Chinese
medicine. It grows only on the inner heartwood wall of the endemic
evergreen Cinnamomun kanehirai (Hay) (Lauraceae) in Taiwan. It is
rare and has not been cultivated. The fruiting bodies have been
used for treating of food and drug intoxication, diarrhea,
abdominal pain, hypertension, itchy skin, and liver cancer. Very
few biological activity studies have been reported hitherto.
[0004] Antrodia camphorata, also known as "niu-chang-chih" or
"niu-chang-ku" in Taiwan, was recently reported as a new fungus
species characterized by the cylindrical shape of its basidiospores
appearing in fruiting bodies, weakly amyloid skeletal hyphae,
bitter taste and light cinnamon resupinate to pileate basidiocarps,
as well as chlamydospores and anthroconidia in pure culture. The
growth of this new fungus species is extremely slow and restricted
to an endemic tree species, Cinnamomum kanehirai Hay (Lauraceae),
as the only host. The detailed characterization and taxonomic
position of Antrodia camphorata were described in Wu, S.-H., et
al., Antrodia cinnamomea ("niu-chang-chih"), New combination of a
medicinal fungus in Taiwan, Bot. Bull. Acad. Sin. 38: 273-275
(1997).
[0005] In Taiwanese folk medicine, the fruiting bodies of Antrodia
camphorata are believed to have certain medical effects. According
to the traditional way, the fruiting bodies are ground into dry
powder or stewed with other herbal drugs for oral uptake to treat
conditions caused by poisoning, diarrhea, abdominal pain,
hypertension, skin itches and liver cancer. However, few
pharmacological or clinical studies in these aspects have appeared
in literature to date. Because of the stringent host specificity
and rarity in nature, as well as the failure of artificial
cultivation, "niu-chang-chih" is very expensive in Taiwan. In
recent years, the fruiting bodies of this fungus with high quality
have been sold at an extremely high price of around U.S.$ 15,000
per kg.
[0006] Oxidative stress including the generation of reactive oxygen
species (ROS) can be implicated as a cause of hepatic fibrosis (M.
Chojkier et al., Stimulation of collagen gene expression by
ascorbic acid in cultured human fibroblasts, A role for lipid
peroxidation, J. Biol. Chem. 264 (1989), pp. 16957-16962. and I.
Shimizu, Antifibrogenic therapies in chronic HCV infection. Curr
Drug Targets Infect Disord 1 (2001), pp. 227-240). It has been
reported that hepatocytes, which are undergoing oxidative stress,
release ROS that stimulate hepatic stellate cell (HSC)
proliferation and transformation into a smooth muscle actin
(.alpha.-SMA)-positive myofibroblast-like cells (GS. Baroni et al.,
Fibrogenic effect of oxidative stress on rat hepatic stellate
cells, Hepatology 27 (1998), pp. 720-726). These HSCs are referred
to as activated cells and are responsible for the abnormal
extracellular matrix (ECM) proteins during hepatic fibrosis to
cirrhosis. Transforming growth factor-.beta. (TGF-.beta. is a major
fibrogenic cytokine, regulating the production, degradation, and
accumulation of ECM proteins in hepatic fibrogenesis (A. Casini et
al., Regulation of extracellular matrix synthesis by transforming
growth factor .beta.1 in human fat-storing cells, Gastroenterology
105 (1993), pp. 245-253). This cytokine induces its own expression
in activated HSCs, thereby creating a self-perpetuating cycle of
events, referred to as an autocrine loop. TGF-.beta. gene
expression correlates with the extent of hepatic fibrosis (A.
Castilla et al., Transforming growth factors .beta.1 and .alpha. in
chronic liver disease. Effects of interferon .alpha. therapy. N.
Eng. J. Med. 324 (1991), pp. 933-940.), and an increased production
of ROS such as H.sub.2O.sub.2 in fibrotic livers is associated with
the up-regulation of TGF-.beta. (E. R. Garcia-Trevijano et al.,
Transforming growth factor .beta.1 induces the expression of
.alpha.1 (I) procollagen mRNA by a hydrogen peroxide-C/EBP
.beta.-dependent mechanism in rat hepatic stellate cells,
Hepatology 29 (1999), pp. 960-970).
[0007] Fibrotic diseases are characterized by excessive scarring
due to excessive production, deposition, and contraction of
extracellular matrix. This process usually occurs over many months
and years, and can lead to organ dysfunction or death. Examples of
fibrotic diseases include diabetic nephropathy, liver cirrhosis,
idiopathic pulmonary fibrosis, rheumatoid arthritis, fibrosarcomas,
arteriosclerosis, and scleroderma (systemic sclerosis; SSc).
Fibrotic disease represents one of the largest groups of disorders
or which there is no effective therapy and thus represents a major
unmet medical need. Often the only redress for patients with
fibrosis is organ transplantation; since the supply of organs is
insufficient to meet the demand, patients often die while waiting
to receive suitable organs.
[0008] Oxidative stress is associated with liver fibrosis and
activation of hepatic stellate cells either directly or through
paracrin stimulation by injured hepatocytes. Factors increasing
reactive oxygen species (ROS) generation may also be involved in
stimulation of excessive matrix production in vivo. Increase in
hydrogen peroxide production leads to activation of a potent
profibrogenic mediator TGF-.beta., supporting the idea that
oxidative stress has important roles in fibrogenesis (Mehmet R. M.,
et al., The effect of taurine treatment on oxidative stress in
experimental liver fibrosis, Hepatology Research, 28, 207-215
(2004).
[0009] TGF-.beta. induces fibroblasts to synthesize and contract
ECM, this cytokine has long been believed to be a central mediator
of the fibrotic response TGF-.beta.1 triggered enhancement of
.alpha.-SMA and collagen type I expression.
[0010] The discovery in 1987 that nitric oxide (NO) accounted for
the bioactivity of endothelium-derived relaxing factor rapidly led
to an explosion of information on the physiological and
pathological roles of this molecule. Although most well known for
its physiological roles in vasorelaxation, neurotransmission,
inhibition of platelet aggregation, and immune defense, NO also
acts as an intracellular messenger for various cells in almost
every system in the body (Peeyush and Chandan, Lancet Oncol 3:149,
2001).
[0011] Nitric oxide, this highly reactive free radical agent is
synthesized from L-arginine by nitric oxide synthase. It acts in
both the intercellular and extracellular environment and is
believed to be a regulatory molecule in a variety of soft tissues
including articular cartilage, ligament, tendon, skeletal muscle
and bone. It is induced during tendon healing in vitro.
[0012] It appears that there is a dose-dependent effect upon its
contribution to fibroblast production of collagen. There is also a
site-specific effect with the anterior cruciate ligament-derived
fibroblasts capable of producing more nitric oxide than from cells
derived from the medial collateral ligament. Manipulation of nitric
oxide production has been thought to help accelerate repetitive
overuse tendon injury and tendinosis. The role of nitric oxide in
the incorporation of an ACL graft remains under investigation
through studies using transfection of cDNA for nitric oxide
synthase (Deehan et al., J Bone Joint Surg 87(7):889, 2005).
[0013] Nitric oxide (NO) is integral to many biological processes
including the control of blood pressure, protection against
microbial infection and neurotransmission. Additionally, it appears
to be a potent cytotoxin to tumor cells. Among its mechanisms of
action on malignant cells, nitric oxide appears to inhibit DNA
synthesis and mitochondrial respiration in vitro. It induces
programmed cell death or apoptosis in these cells. Unfortunately,
NO itself is difficult to administer as it is a highly reactive
gas. It also causes hypotension if administered systemically. These
limitations have prevented its use to date as an antineoplastic
agent.
[0014] Generation of nitric oxide (NO) by inducible nitric oxide
synthase (iNOS) is a cardinal feature of inflamed tissues including
those of the gastrointestinal tract. iNOS overexpression with high
levels of NO generation provides a plausible link between
inflammation and cancer initiation, progression, and promotion. NO
is involved in a number of biological actions including
cytotoxicity of phagocytic cells and cell-to-cell communication in
the central nervous system. NO is also involved in the immune
response (inducible NOS or iNOS), smooth muscle relaxation
(endothelial NOS or eNOS), and neuronal signaling (neuronal NOS or
nNOS).
[0015] Treatment of overuse tendinopathy using transdermal nitric
oxide-generating agents is disclosed in U.S. Patent Application
Pub. No. 2005171199. Use of nitric oxide scavengers to treat side
effects caused by therapeutic administration of sources of nitric
oxide is disclosed in U.S. Pat. No. 6,596,733. Use of products that
release nitric oxide in vivo to treat or prevent infectious
diseases in humans or animals is described in Germany patent
application Pub. No. DE10303196 A1. Nitric oxide (NO) synthase
inhibitor to treat or prevent Type II diabetes is described in
Australia patent application Pub. No. AU4865800 A. Use of nitric
oxide-releasing agents to treat impotency is disclosed in U.S. Pat.
No. 6,290,981. Modification of nitric oxide activity to treat
fas-induced pathologies is mentioned in PCT publication no.
WO9903462 A1. Combined use of angiotensin inhibitors and nitric
oxide stimulators to treat fibrosis is described in U.S. Pat. No.
6,139,847 A. Blocking induction of tetrahydrobiopterin to block
induction of nitric oxide synthesis is disclosed in U.S. Pat. No.
6,274,581.
SUMMARY OF THE INVENTION
[0016] This invention provides a compound having the formula
##STR1## its tautomeric forms, its stereoisomers, its polymorphs,
its pharmaceutically acceptable salts, or its pharmaceutically
acceptable solvates, wherein X is N or O; R.sub.1 is H, hydroxyl,
C.sub.1-10 alkyloxy, C.sub.2-10 alkenyloxy, or C.sub.2-10
alkynyloxy; R.sub.2 is H, hydroxyl, C.sub.1-10 alkyl, C.sub.2-10
alkenyl or C.sub.2-10 alkynyl; R.sub.3 is C.sub.1-10 alkyl,
C.sub.2-10 alkenyl or C.sub.2-10 alkynyl; denotes a single or
double bond; provided that if denotes a single bond, the compound
has the formula: ##STR2##
[0017] This invention also provides a mixture from Antrodia
camphorate, which is prepared from water or organic solvent extract
of mycelium of Antrodia camphorate.
[0018] This invention further provides a composition comprising a
compound having the formula ##STR3## its tautomeric forms, its
stereoisomers, its polymorphs, its pharmaceutically acceptable
salts, or its pharmaceutically acceptable solvates, wherein X is N
or O; R.sub.1 is H, hydroxyl, C.sub.1-10 alkyloxy, C.sub.2-10
alkenyloxy, or C.sub.2-10 alkynyloxy; R.sub.2 is H, hydroxyl,
C.sub.1-10 alkyl, C.sub.2-10 alkenyl or C.sub.2-10 alkynyl; R.sub.3
is absent, H, hydroxyl or C.sub.1-10 alkyl, C.sub.2-10 alkenyl or
C.sub.2-10 alkynyl; denotes a single or double bond; provided that
if X is 0, R.sub.3 is absent; if denotes a single bond, the
compound has the formula: ##STR4##
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 shows the compounds 1-5 used in the present
invention.
[0020] FIG. 2 shows the ROS assay results.
[0021] FIG. 3 shows the flow cytometry results for ROS assay.
[0022] FIG. 4 shows the ROS quantitative results.
[0023] FIG. 5 shows test results of gel electrophoretic mobility
shift assays.
[0024] FIG. 6 shows test results of SDS-PAGE and Western blotting
assays.
[0025] FIG. 7 shows test results of COX-2 promoter assay.
[0026] FIG. 8 shows test result of Col1A2 promoter assay.
[0027] FIG. 9 shows the influence of compound 2 on mouse liver
collagen. A: negative (TGF-.beta. free), B: positive (pPk9a 10
.mu.g/3 ml i.v.), C: compound-2 1.15 .mu.g/kg, D: compound-2 2.31
.mu.g/kg, and E: compound-2 4.62 .mu.g/kg.
[0028] FIG. 10 shows the Masson's trichrome stain for collagen. A:
negative (TGF-.beta. free), B: positive (TGF-.beta.2 ng/ml), and C:
compound-2 1 .mu.g/kg. Collagen fiber accumulation in the
sinusoidal wall is blue-stained (arrows). The original
magnification is .times.200.
[0029] FIG. 11 shows the effect of the extract on nitric oxide
inhibition.
DETAILED DESCRIPTION OF THE INVENTION
[0030] The present invention provides a compound having the formula
##STR5## its tautomeric forms, its stereoisomers, its polymorphs,
its pharmaceutically acceptable salts, or its pharmaceutically
acceptable solvates, wherein X is N or O; R.sub.1 is H, hydroxyl,
C.sub.1-10 alkyloxy, C.sub.2-10 alkenyloxy, or C.sub.2-10
alkynyloxy; R.sub.2 is H, hydroxyl, C.sub.1-10 alkyl, C.sub.2-10
alkenyl or C.sub.2-10 alkynyl; R.sub.3 is C.sub.1-10 alkyl,
C.sub.2-10 alkenyl or C.sub.2-10 alkynyl; denotes a single or
double bond; provided that if denotes a single bond, the compound
has the formula: ##STR6##
[0031] In the compound of the invention, the preferred R.sub.1 is
C.sub.2-6 alkenyloxy, or C.sub.2-6 alkynyloxy; the more preferred
R.sub.1 is C.sub.2-6 alkenyloxy substituted with C.sub.1-6 alkyl
and the most preferred R.sub.1 is butenyloxy substituted with
methyl. In the compound of the invention, the preferred R.sub.2 is
C.sub.1-6 alkyl, the most preferred R.sub.2 is isobutyl.
[0032] Certain compounds may exist in one or more particular
geometric, optical, enantiomeric, diasteriomeric, epimeric,
stereoisomeric, tautomeric, conformational, or anomeric forms,
including but not limited to, cis- and trans-forms; E- and Z-forms;
c-, t-, and reforms; endo- and exo-forms; R-, S-, and meso-forms;
D- and L-forms; d- and 1-forms; (+) and (-) forms; keto-, enol-,
and enolate-forms; syn- and anti-forms; synclinal- and
anticlinal-forms; .alpha.- and .beta.-forms; axial and equatorial
forms; boat-, chair-, twist-, envelope- and halfchair-forms; and
combinations thereof, hereinafter collectively referred to as
"isomers" (or "isomeric forms").
[0033] If the compound is in crystalline form, it may exist in a
number of different polymorphic forms.
[0034] The above exclusion does not pertain to tautomeric forms,
for example, keto-, enol-, and enolate-forms, as in, for example,
the following tautomeric pairs: keto/enol, imine/enamine,
amide/imino alcohol, amidine/amidine, nitroso/oxime,
thioketone/enethiol, N-nitroso/hyroxyazo, and nitro/aci-nitro.
[0035] Unless otherwise specified, the compounds of the present
invention include all such isomeric forms, including (wholly or
partially) racemic and other mixtures thereof. Methods for the
preparation (e.g., asymmetric synthesis) and separation (e.g.,
fractional crystallization and chromatographic means) of such
isomeric forms are either known in the art or are readily obtained
by adapting the methods taught herein, or known methods, in a known
manner.
[0036] Unless otherwise specified, the compounds of the present
invention include also includes ionic, salt, solvate, and protected
forms of thereof, for example, as discussed below.
[0037] It may be convenient or desirable to prepare, purify, and/or
handle a corresponding salt of the active compound, for example, a
pharmaceutically-acceptable salt. Examples of pharmaceutically
acceptable salts are discussed in Berge et al., 1977,
"Pharmaceutically Acceptable Salts," J. Pharm. Sci., Vol. 66, pp.
1-19.
[0038] The present invention also provides a mixture from mycelium
of Antrodia camphorata, which comprises the compound of the
invention. The mixture of the invention is prepared from water or
organic solvent extract of mycelium of Antrodia camphorata. The
organic solvent includes but is not limited to alcohol (such as
CH.sub.3OH, C.sub.2H.sub.5OH, C.sub.3H.sub.7OH), ester (such as
acetyl acetate), alkane (such as hexane) and halogenated alkane
(such as CH.sub.3C.sub.1, C.sub.2H.sub.2Cl.sub.2). The preferred
organic solvent is ethanol or alcoholic solvent without causing any
side effect of human.
[0039] The mixture of the present invention inhibits factor
increasing reactive oxygen species (ROS) generation. The mixture of
the present invention also inhibits TGF-.beta. mediated
inflammation and fibrosis. The fibrosis is not limited but to
include diabetic nephropathy, liver cirrhosis, idiopathic pulmonary
fibrosis, rheumatoid arthritis, fibrosarcomas, arteriosclerosis,
and scleroderma.
[0040] The mixture of the present invention also inhibits nitric
oxide activities. The mixture can be used in treating or preventing
of epithelial cell carcinogenesis and free radical damages.
[0041] Nitric oxide overproduction is associated with a wide range
of disease states and/or indications, such as, for example, septic
shock, ischemia, administration of cytokines, overexpression of
cytokines, ulcers, inflammatory bowel disease (e.g., ulcerative
colitis or Crohn's disease), diabetes, arthritis, asthma,
Alzheimer's disease, Parkinson's disease, multiple sclerosis,
cirrhosis, allograft rejection, encephalomyelitis, meningitis,
pancreatitis, peritonitis, vasculitis, lymphocytic
choriomeningitis, glomerulonephritis, uveitis, ileitis, liver
inflammation, renal inflammation, hemorrhagic shock, anaphylactic
shock, burn, infection (including bacterial, viral, fungal and
parasitic infections), and the like.
[0042] Those of skill in the art recognize that the mixture
described herein can be delivered in a variety of ways, such as,
for example, orally, intravenously, subcutaneously, parenterally,
rectally, by inhalation, and the like.
[0043] Since individual subjects may present a wide variation in
severity of symptoms and each drug has its unique therapeutic
characteristics, the precise mode of administration and dosage
employed for each subject is left to the discretion of the
practitioner.
[0044] Accordingly, the mixture of the present invention could be
applied to treat or prevent septic shock, ischemia, overexpression
of cytokines, ulcer, ulcerative colitis, diabetes, arthritis,
asthma, Alzheimer's disease, Parkinson's disease, multiple
sclerosis, cirrhosis, allograft rejection, encephalomyelitis,
meningitis, pancreatitis, peritonitis, vasculitis, lymphocytic
choriomeningitis, glomerulonephritis, uveitis, ileitis, liver
inflammation, renal inflammation, hemorrhagic shock, anaphylactic
shock, burn, Crohn's disease or infection.
[0045] The present invention also provides a composition, having
the formula ##STR7## its tautomeric forms, its stereoisomers, its
polymorphs, its pharmaceutically acceptable salts, or its
pharmaceutically acceptable solvates, wherein X is N or O; R.sub.1
is H, hydroxyl, C.sub.1-10 alkyloxy, C.sub.2-10 alkenyloxy, or
C.sub.2-10 alkynyloxy; R.sub.2 is H, hydroxyl, C.sub.1-10 alkyl,
C.sub.2-10 alkenyl or C.sub.2-10 alkynyl; R.sub.3 is absent, H,
hydroxyl or C.sub.1-10 alkyl, C.sub.2-10 alkenyl or C.sub.2-10
alkynyl; denotes a single or double bond; provided that if X is 0,
R.sub.3 is absent; if denotes a single bond, the compound has the
formula: ##STR8##
[0046] In the preferred embodiment, the compound is
3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]furan-2,5-dione,
3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]-1H-pyrrol-2,5-dione,
3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]-1H-pyrrol-1-ol-2,5-dione,
3R*,
4S*-1-hydroxy-3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]pyrrolid-
ine-2,5-dione, or 3R*,
4R*-1-hydroxy-3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]pyrrolidine-2-
,5-dione.
[0047] The composition inhibits factor increasing reactive oxygen
species (ROS) generation. Further, the composition inhibits
TGF-.beta. mediated inflammation and fibrosis. The fibrosis is
related to diabetic nephropathy, liver cirrhosis, idiopathic
pulmonary fibrosis, rheumatoid arthritis, fibrosarcomas,
arteriosclerosis, and scleroderma. The present composition can
inhibit nitric oxide activities and can be for use in treating or
preventing of epithelial cell carcinogenesis and free radical
damages. In particular, the present composition could be applied to
treat or prevent septic shock, ischemia, overexpression of
cytokines, ulcer, ulcerative colitis, diabetes, arthritis, asthma,
Alzheimer's disease, Parkinson's disease, multiple sclerosis,
cirrhosis, allograft rejection, encephalomyelitis, meningitis,
pancreatitis, peritonitis, vasculitis, lymphocytic
choriomeningitis, glomerulonephritis, uveitis, ileitis, liver
inflammation, renal inflammation, hemorrhagic shock, anaphylactic
shock, burn, Crohn's disease or infection.
[0048] The compositions of the present invention can be used in the
form of a solid, a solution, an emulsion, a dispersion, a micelle,
a liposome, and the like, wherein the resulting composition
contains one or more of the compounds of the present invention, as
an active ingredient, in admixture with an organic or inorganic
carrier or excipient suitable for enteral or parenteral
applications. The active ingredient may be compounded, for example,
with the usual non-toxic, pharmaceutically acceptable carriers for
tablets, pellets, capsules, suppositories, solutions, emulsions,
suspensions, and any other form suitable for use. The carriers
which can be used include glucose, lactose, gum acacia, gelatin,
mannitol, starch paste, magnesium trisilicate, talc, corn starch,
keratin, colloidal silica, potato starch, urea, medium chain length
triglycerides, dextrans, and other carriers suitable for use in
manufacturing preparations, in solid, semisolid, or liquid form. In
addition auxiliary, stabilizing, thickening and coloring agents and
perfumes may be used.
[0049] The compositions of the invention may be in a form suitable
for oral use, for example, as tablets, troches, lozenges, aqueous
or oily suspensions, dispersible powders or granules, emulsions,
hard or soft capsules, or syrups or elixirs. Compositions intended
for oral use may be prepared according to any method known to the
art for the manufacture of pharmaceutical compositions and such
compositions may contain one or more agents selected from the group
consisting of a sweetening agent such as sucrose, lactose, or
saccharin, flavoring agents such as peppermint, oil of wintergreen
or cherry, coloring agents and preserving agents in order to
provide pharmaceutically elegant and palatable preparations.
Tablets containing the active ingredient in admixture with
non-toxic pharmaceutically acceptable excipients may also be
manufactured by known methods. The excipients used may be, for
example, (1) inert diluents such as calcium carbonate, lactose,
calcium phosphate or sodium phosphate; (2) granulating and
disintegrating agents such as corn starch, potato starch or alginic
acid; (3) binding agents such as gum tragacanth, corn starch,
gelatin or acacia, and (4) lubricating agents such as magnesium
stearate, stearic acid or talc. The tablets may be uncoated or they
may be coated by known techniques to delay disintegration and
absorption in the gastrointestinal tract and thereby provide a
sustained action over a longer period. For example, a time delay
material such as glyceryl monostearate or glyceryl distearate may
be employed. They may also be coated by the techniques described in
the U.S. Pat. Nos. 4,256,108; 4,160,452 and 4,265,874, to form
osmotic therapeutic tablets for controlled release.
[0050] In some cases, compositions for oral use may be in the form
of hard gelatin capsules wherein the active ingredient is mixed
with an inert solid diluent, for example, calcium carbonate,
calcium phosphate or kaolin. They may also be in the form of soft
gelatin capsules wherein the active ingredient is mixed with water
or an oil medium, for example, peanut oil, liquid paraffin, or
olive oil.
[0051] The compositions may be in the form of a sterile injectable
suspension. This suspension may be formulated according to known
methods using suitable dispersing or wetting agents and suspending
agents. The sterile injectable preparation may also be a sterile
injectable solution or suspension in a non-toxic
parenterally-acceptable diluent or solvent, for example, as a
solution in 1,3-butanediol. Sterile, fixed oils are conventionally
employed as a solvent or suspending medium. For this purpose any
bland fixed oil may be employed including synthetic mono- or
diglycerides, fatty acids (including oleic acid), naturally
occurring vegetable oils like sesame oil, coconut oil, peanut oil,
cottonseed oil, etc., or synthetic fatty vehicles like ethyl oleate
or the like. Buffers, preservatives, antioxidants, and the like can
be incorporated as required.
[0052] Compounds contemplated for use in the practice of the
present invention may also be administered in the form of
suppositories for rectal administration of the drug. These
compositions may be prepared by mixing the drug with a suitable
non-irritating excipient, such as cocoa butter, synthetic glyceride
esters of polyethylene glycols, which are solid at ordinary
temperatures, but liquify and/or dissolve in the rectal cavity to
release the drug.
[0053] Since individual subjects may present a wide variation in
severity of symptoms and each drug has its unique therapeutic
characteristics, it is up to the practitioner to determine a
subject's response to treatment and vary the dosages
accordingly.
EXAMPLES
[0054] The examples below are non-limiting and are merely
representative of various aspects and features of the present
invention.
Example 1
General Experimental Procedures
[0055] Melting points were measured on a Yanagimoto micro hot-stage
melting point apparatus and uncorrected. Optical rotations were
measured with a Jasco DIP-360 automatic polarimeter. UV spectra
were measured with a Shimadzu UV-2200 recording spectrophotometer.
IR spectra were measured with a Jasco FT/IR-230 infrared
spectrometer. .sup.1H- and .sup.13C-NMR spectrum were measured with
a Varian Unity Plus 500 spectrometer. EIMS and HR-EIMS were
measured with a Jeol JMS-AX 505 HAD mass spectrometer at an
ionization voltage of 70 eV. Column chromatography was carried out
on silica gel BW-820 MH (normal phase) and Chromatorex-ODS DM1020T
(reversed phase) (Fuji Silysia).
Extraction and Isolation
[0056] Antrodia camphorata mycelia powder (ACM) (60 g), from
Simpson Biotech Co. Ltd., Taiwan, October 2001, were three times
extracted with CHCl.sub.3 for 3 h under reflux. The CHCl.sub.3
extract (5.3 g) was chromatographed on silica gel eluted with
n-hexane-acetone (19:1-14:6), and CHCl.sub.3-MeOH (1:1) to give
nine fractions (Fr. 1-9). Fraction 2 was chromatographed on silica
gel to give compound 1 (8.7 mg). Fraction 4 was chromatographed on
normal and reversed phase silica gel to give compound 2 (13.6 mg).
Fraction 5 was chromatographed on silica gel eluted with
n-hexane-acetone (8:2) to give ergosterol peroxide (35.8 mg).
Fraction 6 gave compound 3 (14.6 mg) by combination of normal and
reversed phase silica gel column chromatography. Fraction 7 yielded
a mixture of compounds 4 and 5 (4:1) by column chromatography. The
mixture of compounds 4 and 5 were subsequently separated by
preparative HPLC [column: Tosoh TSK-gel ODS-80T.sub.M
(21.5.times.300 mm), mobile phase: CH.sub.3OH--H.sub.2O containing
0.1% TFA (70:30)].
[0057]
3-Isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]furan-2,5-dione
(compound 1): yellow oil; UV (MeOH) .lamda..sub.max (log .epsilon.)
227 (4.1), 258 (3.9), 275 (3.8), 355 (3.4) nm; IR (CHCl.sub.3)
.nu..sub.max 1763 cm.sup.-1; .sup.1H-NMR Table 1; .sup.13C-NMR
Table 2; EIMS m/z 314 [M].sup.+ (100), 246 (100), 131 (100);
HR-EIMS m/z 314.1523 (Calcd for C.sub.19H.sub.22O.sub.4,
314.1518).
[0058]
3-Isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]-1H-pyrrole-2,5-dion-
e (2): yellow needles (n-hexane-AcOEt); mp 110-111.degree. C.; UV
(MeOH) .lamda..sub.max (log .epsilon.) 230 (4.3), 272 (3.5), 355
(3.7) nm; IR (CHCl.sub.3) .nu..sub.max 1724 cm.sup.-1; .sup.1H-NMR
Table 1; .sup.13C-NMR Table 2; EIMS m/z 313 [M].sup.+ (8), 245
(100), 203 (77), 131 (28); HR-EIMS m/z 313.1681 (Calcd for
C.sub.19H.sub.23NO.sub.3, 313.1678).
X-ray Crystallography of Compound 2:
[0059] Yellow needles were obtained by crystallization from
n-hexane-AcOEt and selected for data collection. Crystal data:
C.sub.19H.sub.23NO.sub.3; M.sub.r=313.40; dimensions
0.15.times.0.02.times.0.02 mm; triclinic, space group P1 (#2),
a=6.3505(5) .ANG., b=12.205(1) .ANG., c=12.560(2) .ANG.,
.alpha.=64.623(7), .beta.=75.358(4).degree.,
.gamma.84.681(5).degree., V=850.9(2) .ANG..sup.3, Z=2,
D.sub.calc=1.223 g/cm.sup.3, .mu.(MoK.alpha.)=0.82 cm.sup.-1,
F.sub.000=336.00. Measurement was made on a Rigaku RAXIS-RAPID
Imaging Plate diffractometer with graphite monochromated
Mo-K.alpha. (.lamda.=0.71069 .ANG.) radiation at 93 K.
[0060] Of the 8950 reflections that were collected, 4745 were
unique (R.sub.int=0.108); equivalent reflections were merged. The
crystal structure was solved by direct methods (SHELXS86) and
refined by full-matrix least-squares. The non-hydrogen atoms were
refined anisotropically. Hydrogen atoms were included but not
refined. The final indices were R=0.074, R.sub.W=0.099, with GOF
(Guest Observer Facility)=1.06. The maximum and minimum peaks on
the final difference Fourier map corresponded to 0.83 and -0.89
e.sup.-/.ANG..sup.3, respectively.
[0061]
3-Isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]-1H-pyrrol-1-ol-2,5--
dione (compound 3): yellow oil; UV (MeOH) .lamda..sub.max (log
.epsilon.): 232.5 (4.3), 296 (3.7), 374 (3.7) nm; IR (CHCl.sub.3)
.nu..sub.max 1717 cm.sup.-1; .sup.1H-NMR Table 1; .sup.13C-NMR
Table 2; EIMS m/z 329 [M].sup.+ (12), 261 (100), 131 (50); HR-EIMS
m/z: 329.1637 (Calcd for C.sub.19H.sub.23NO.sub.4, 329.1627).
[0062] 3R*,4S*-1-Hydroxy-3-isobutyl-4-[4-(3-methyl-2-butenyloxy)
phenyl] pyrrolidine-2,5-dione (4): colorless oil;
[.alpha.].sub.D.sup.23+2.5.degree. (c 0.2, MeOH); UV (MeOH)
.lamda..sub.max (log .epsilon.): 225 (4.3), 275 (3.3), 283 (3.2)
nm; IR (CHCl.sub.3) .nu..sub.max 1715 cm.sup.-1; .sup.1H-NMR Table
1; .sup.13C-NMR Table 2; EIMS m/z 331 [M].sup.+ (2), 263 (67), 207
(66), 191 (30), 179 (40), 133 (64), 69 (100); HR-EIMS m/z 331.1747
(Calcd for C.sub.19H.sub.25NO.sub.4, 331.1783).
[0063] 3R*,4R*-1-Hydroxy-3-isobutyl-4-[4-(3-methyl-2-butenyloxy)
phenyl] pyrrolidine-2,5-dione (5): colorless oil;
[.alpha.].sub.D.sup.23+3.0.degree. (c 0.2, MeOH); UV (MeOH)
.lamda..sub.max (log .epsilon.): 227 (4.3), 275 (3.4), 283 (3.3)
mm; IR (CHCl.sub.3) .nu..sub.max 1715 cm.sup.-1; .sup.1H-NMR Table
1; .sup.13C-NMR Table 2; EIMS m/z 331 [M].sup.+ (1), 263 (45), 207
(50), 191 (75), 179 (30), 133 (100), 69 (92); HR-EIMS m/z 331.1766
(Calcd for C.sub.19H.sub.25NO.sub.4, 331.1783).
Results and Discussion
[0064] The CHCl.sub.3 extract of the mycelium of Antrodia
camphorata was repeatedly hromatographed on normal and reversed
phase silica gel to afford five new maleic and succinic acid
derivatives (compounds 1-5) together with ergosterol peroxide.
TABLE-US-00001 TABLE 1 Table 1. .sup.1H-NMR Spectral Data of
Compounds 1-5(.delta. ppm, J=Hz)(500MHz, CDCl.sub.3) H 1 2 3 4 5 3
-- -- -- 2.87(1H, m) 3.08(1H, m) 4 -- -- -- 3.52(1H, d, 4.07(1H, d,
J=4.0) J=8.0) 1.51(1H, m) 1.02(1H, m) 1' 2.59(2H, d, 2.51(2H, d,
2.50(2H, d, 1.72.about.1.84 1.42.about.1.48 J=7.0) J=7.0) J=7.0)
(1H) (1H) 2' 2.12(1H, 2.06(1H, 2.05(1H, 1.72.about.1.84
1.42.about.1.48 sep, J=7.0) sep, J=7.0) sep, J=7.0) (1H) (1H) 3'
0.94(6H, d, 0.90(6H, d, 0.88(6H, d, 0.70(3H, d, 0.66(3H, d, J=7.0)
J=7.0) J=7.0) J=6.5) J=6.5) 4' 0.89(3H, d, 0.80(3H, d, J=6.5)
J=6.5) 2'', 6'' 7.50(2H, d, 7.50(2H, d, 7.50(2H, d, 7.07(2H, d,
6.96(2H, d, J=9.0) J=9.0) J=9.0) J=8.5) J=9.0) 3'', 5'' 7.02(2H, d,
6.95(2H, d, 6.98(2H, d, 6.87(2H, d, 6.84(2H, d, J=9.0) J=9.0)
J=9.0) J=8.5) J=9.0) 1''' 4.57(2H, d, 4.56(2H, d, 4.55(2H, d,
4.47(2H, d, 4.47(2H, d, J=6.6) J=6.5) J=6.9) J=6.5) J=6.5) 2'''
5.50(1H, 5.50(1H, 5.49(1H, 5.47(1H, brt, 5.47(1H, brt, brt, J=6.6)
brt, J=6.5) brt, J=6.9) J=6.5) J=6.5) 4''' 1.81(3H, s) 1.81(3H, s)
1.81(3H, s) 1.79(3H, s) 1.79(3H, s) 5''' 1.76(3H, s) 1.76(3H, s)
1.76(3H, s) 1.73(3H, s) 1.73(3H, s)
[0065] TABLE-US-00002 TABLE 2 .sup.13C-NMR Spectral Data for
Compound 1-5(.delta. ppm)(125MHz, CDCl.sub.3) C 1 2 3 4 5 2
166.4(s) 171.7(s) 168.8(s) 174.8(s) 175.1(s) 3 139.8(s)
138.8(s).sup.a) 135.9(s).sup.a) 44.6(d) 40.3(d) 4 140.2(s)
139.2(s).sup.a) 136.0(s).sup.a) 49.8(d) 47.5(d) 5 165.4(s) 171.1(s)
168.1(s) 173.2(s) 173.6(s) 1' 33.6(t) 32.8(t) 33.2(t) 40.4(t)
35.3(t) 2' 27.9(d) 28.1(d) 28.4(d) 25.3(d) 25.2(d) 3' 22.7(q)
22.7(q) 23.0(q) 21.3(q) 21.8(q) 4' 23.0(q) 22.4(q) 1'' 119.9(s)
121.2(s) 120.8(s) 127.9(s) 125.1(s) 2'', 6'' 131.1(d) 130.9(d)
131.0(d) 128.8(d) 130.2(d) 3'', 5'' 115.1(d) 114.9(d) 115.0(d)
115.4(d) 115.0(d) 4'' 160.9(s) 160.1(s) 160.2(s) 158.7(s) 158.7(s)
1''' 65.0(t) 64.9(t) 65.1(t) 64.1(t) 64.8(t) 2''' 118.7(d) 119.3(d)
119.2(d) 119.4(d) 119.3(d) 3''' 139.1(s) 138.6(s).sup.a) 138.9(s)
138.3(s) 138.4(s) 4''' 25.2(q) 25.8(q) 26.1(q) 25.8(q) 25.8(q) 5'''
18.2(q) 18.2(q) 18.5(q) 18.1(q) 18.2(q) .sup.a)Assignments may be
interchangeable.
[0066] The structures of the new compounds were determined as
follows: The molecular formula of compound 1 was assigned as
C.sub.19H.sub.22O.sub.4 by HR-EIMS. The IR spectrum revealed
carbonyl absorption of acid anhydride at 1763 cm.sup.-1. The
.sup.1H-NMR spectrum of compound 1 was similar to that of compound
2, and showed the presence of an isobutyl moiety, a
3-methyl-2-butenyloxy moiety, and a para-substituted benzene ring.
From the HMBC spectrum, compound 1 was demonstrated to have the
same partial structure to compound 2 (FIG. 1), in which the
presence of a maleic anhydride group was deduced on the basis of
the molecular formula compound 1 was consequently determined as
3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]furan-2,5-dione.
[0067] Compound 2 gave yellow needles, mp 110-111.degree. C., and
the molecular formula C.sub.19H.sub.23NO.sub.3 was assigned by
HR-EIMS. The IR spectrum showed an imide carbonyl absorption at
1724 cm.sup.-1. The .sup.13C-NMR spectrum showed signals of four
methyl carbons, two methylene carbons, and one methine carbon in
the aliphatic region, as well as one benzene ring, one olefinic
group and two carbonyl carbons. The .sup.1H-NMR spectrum showed the
presence of an isobutyl moiety at .delta. 0.90, 2.06, and 2.51, a
3-methyl-2-butenyloxy moiety at .delta.1.76, 1.81, 4.56, and 5.50,
and a para-substituted benzene moiety at .delta. 6.95 and 7.50,
which was further supported by .sup.1H-.sup.1H COSY (cooler
synchrotron) and HMQC (heteronuclear multiple quantum coherence)
experiments. Long-range correlations were observed by HMBC as shown
in FIG. 1. On the basis of the molecular formula and the
.sup.13C-NMR spectrum, this compound was deduced to contain further
CHNO atoms, including one more carbonyl carbon. Thus, this
ambiguous part was speculated to be a maleimide group. This
structure was then established to be
3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]-1H-pyrrole-2,5-dione
by X-ray analysis.
[0068] The molecular formula of compound 3 was assigned as
C.sub.19H.sub.23NO.sub.4 by HR-EIMS. The IR spectrum showed
carbonyl absorption at 1717 cm.sup.-1, assignable to a hydroxy
imide. The 1H- and .sup.13C-NMR spectra were also similar to those
of compounds 1 and 2, and showed the presence of an isobutyl
moiety, a 3-methyl-2-butenyloxy moiety, and a para-substituted
benzene ring. In the HMBC experiment, compound 3 was shown to have
the same partial structure as compound 2 (FIG. 1). Compound 3
contains one more oxygen atom than compound 2, therefore, this
compound was determined to be
(3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]-1H-pyrrol-1-ol-2,5-dione.
[0069] Compounds 4 and 5 had the same R.sub.f values and the same
molecular formula by HR-EIMS (C.sub.19H.sub.25NO.sub.4, found
331.1747 and 331.1766, respectively), however, they could be
separated by preparative HPLC. The IR spectrum of both compounds
showed a hydroxy imide carbonyl absorption at 1715 cm.sup.-1. In
the .sup.1H- and .sup.13C-NMR spectra, both compounds showed the
presence of an isobutyl moiety, a 3-metyl-2-butenyloxy moiety, and
a para-substituted benzene ring, but the isobutyl methylene protons
displayed a multiplet and not a doublet as for compounds 1-3. The
.sup.1H-.sup.1H COSY spectrum indicated that this methylene group
is attached to a --CH--CH-- unit. The .sup.13C-NMR spectra of
compounds 4 and 5 exhibited two additional sp.sup.3 carbon signals,
replacing two sp.sup.2 carbon signals observed for compounds 1-3.
Therefore, compounds 4 and 5 were not N-hydroxy maleimides, but
rather N-hydroxy succinimides, with stereocenters at positions C-3
and C-4 in the succinimide ring. Compounds 4 and 5 were determined
to be trans and cis isomers, respectively, from the coupling
constant between H-3 and H-4 (4.0 and 8.0 Hz for compounds 4 and 5,
respectively). No NOE was observed between H-3 and H-4 in the NOESY
(Nuclear Overhauser Effect Spectroscopy) spectrum of compound 4,
while appreciable NOE was observed in that of compound 5. The
optical rotations of compounds 4 and 5 showed +2.5.degree. and
+3.0.degree., respectively, while their CD spectra showed no Cotton
effects at any wave length, suggesting that both compounds 4 and 5
are racemic mixtures. Resolution of these racemic mixtures by HPLC
using a chiral column with several solvent systems was
unsuccessful. At present, we cannot definitely conclude whether
these compounds are optically active compounds or racemic mixtures.
Thus, their relative structures were determined as 3R*,4S*- and
3R*,4R*-1-hydroxy-3-isobutyl-4-[4-(3-methyl-2-butenyloxy)phenyl]pyrrolidi-
ne-2,5-d ione, respectively.
Example 2
Compound 2 as ROS Scavenger and Down Regulated TGF-.beta. Mediated
Inflammation and Fibrosis
Transfection and Dual-Luciferase Assay:
[0070] HSC-T6 cells (1.5.times.10.sup.5) were plated in each well
of six-well plates. Transient transfection was carried out by the
calcium phosphate precipitation method. The plasmid pRL-TK was
cotransfected to normalize the transfection efficiency. After 12 h
of transfection, the medium was changed, and the cells were
incubated at 37.degree. C. for 24 or 48 h. The cells were washed in
phosphate-buffered saline (137 mM sodium chloride, 2.7 mM potassium
chloride, 10 mM dibasic sodium phosphate, and 2 mM monobasic
potassium phosphate) and the lysates were prepared by scraping the
cells from plates in the presence of 1.times. passive lysis buffer
(Promega). Luciferase assays were performed by using
Dual-Luciferase Assay System (Promega) and a Sirius luminometer
(Berthold Detection System, Pforzheim, Germany).
Preparation of Nuclear Extracts:
[0071] HSC-T6 cells were plated onto 6- or 10-cm cultured dishes
and incubated for 2 days. The cells were washed with 2 ml of
phosphate-buffered saline and collected in 1 ml of
phosphate-buffered saline. The cells were centrifuged at
2,000.times.g for 2 min, and the supernatant was discarded. The
cell pellet was incubated in 400 .mu.l of buffer A (10 mM HEPES (pH
7.9), 1.5 mM magnesium chloride, 10 mM potassium chloride, 0.5 mM
phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol, 2 g/ml
leupeptin, 10 g/ml aprotinin, 50 mM sodium fluoride, and 1 mM
sodium orthovanadate) on ice for 10 min and then gently shaken for
10 s. The pellet of the crude nuclei was collected by
centrifugation at 12,000.times.g for 10 s. The pellet was
resuspended in 100 1 of buffer C (20 mM HEPES (pH 7.9), 25%
glycerol, 420 mM sodium chloride, 1.5 mM magnesium chloride, 0.2 mM
EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol,
2 g/ml leupeptin, 10 g/ml aprotinin, 50 mM sodium fluoride, and 1
mM sodium orthovanadate) by vortex for 15 s, and then incubated on
ice for 20 min. After centrifugation at 12,000.times.g for 2 min,
the supernatant containing the nuclear proteins was collected,
quantified with BCA Protein Assay Reagent (Pierce), and stored at
-70.degree. C. in aliquots.
Measurement of Intracellular ROS:
[0072] For visualization and analysis of intracellular ROS, the
oxidation-sensitive probe DCFH-DA was used, as previously
described. To analyze the net intracellular generation of ROS by
flow cytometry, cells were detached by trypsinizationafter
incubation in the absence or presence of the different factors. The
cellular fluorescence intensity was measured after 30 min
incubation with 5 mM DCFH-DA, by using the same flow cytometer
described above. Propidium iodide (0.005%) was used to detect dead
cells. For each analysis, 10,000 events were recorded. For confocal
microscopy analysis, after incubation of cells in the absence or
presence of the different factors, they were washed twice with PBS
and the cellular fluorescence intensity was visualized after 30 min
of incubation with 5 mM DCFH-DA by using the same confocal
microscopy described above. The analysis results were illustrated
in FIGS. 2-4.
Gel Electrophoretic Mobility Shift Assays (EMSA):
[0073] The EMSA used the following oligonucleotides: consensus SpI
(f), 5'-GTT GCG GGG CGG GGC CGA GTG-3'; consensus SpI (r), 3'-AAC
GCC CCG CCC CGG CTC ACG-5'. 30 pmol of each of the forward and
reverse oligonucleotides placed in a volume of 23 .mu.l of
1.times.Klenow (DNA polymerase) buffer were heated at 94.degree. C.
for 2 min and annealed at room temperature for 30 min. The annealed
double-stranded oligonucleotides were end-labeled by a fill-in
reaction using DNA polymerase (Klenow) (Promega). One unit of the
DNA polymerase (Klenow) and 40 Ci of (-32P)dCTP (PerkinElmer Life
Sciences) were added into the annealed oligonucleotides and the
mixture was incubated at 30.degree. C. for 15 min. The labeled
oligonucleotides were purified by Sephadex G-50 columns (Amersham
Biosciences). Cold double-stranded oligonucleotides were used as
competitors. The DNA binding reaction was conducted at 4.degree. C.
for 30 min in a mixture containing 3 g of nuclear extract, 10 mM
Tris-Cl (pH 7.5), 50 mM sodium chloride, 0.5 mM dithiothreitol, 0.5
mM EDTA, 1 mM magnesium chloride, 4% glycerol, 0.05 g
poly(dI-dC)-poly(dI-dC) (Amersham Biosciences) and 2.times.10.sup.4
cpm of 32P-labeled double-stranded oligonucleotides. In supershift
assays, antibodies were incubated with the reaction mixture at
4.degree. C. for 30 min before the addition of the SP-1 probes.
Samples were analyzed on a 4% polyacrylamide gel
(acrylamide/bisacrylamide 29:1 in 0.5.times. Tris borate-EDTA
buffer) at 10 V/cm for 2.5 h. The gel was dried and analyzed by
autoradiography. The test results were illustrated in FIG. 5.
Western Blot Analysis:
[0074] Liver samples (0.5 g) were lysed (Reporter lysis buffer,
Promega, Madison, Wis., USA), the protein were boiled in Laemmli's
sample buffer and samples were subjected to 10% SDSPAGE followed by
western immunoblotting. Anti-cyclooxygenase was used as the first
antibody, followed by secondary HRP-conjugated goat anti-rabbit
antibodies (Santa Cruz). Immunoreactive bands were detected using
the enhanced chemiluminescence (ECL) reagent using SuperSignal West
Pico Chemiluminescent Substrate (Pierce, Rockford, Ill., USA). The
light emitted by the chemical reaction was detected by exposure to
Hyperfilm ECL (Amersham Biosciences, Uppsala, Sweden). The analysis
results were illustrated in FIG. 6.
Method and Material
Cell Model:
[0075] HSC-T6 fibrosis was induced TGF-.beta.2 ng/ml in MEM medium
these cells by three experimental procedures to gauge how compound
2 influences ROS, collagen 1 A2 COX-2 promoter assay, and stains
alpha-smooth muscle actin, smad4, smad3.
[0076] The influences of hepacin on mice-derived once-activated
HSC-T6 line were studied. HSC-T6 was directed to with a focus on
the collagen alpha2 (I) (COL1A2) promoter expression. Plasmid
containing 353 nt length of COL1A2 promoter linked to firefly
luciferase gene and its various 5'-deletions were transiently
transfected to HSC-T6. The luciferase activity was determined with
or without 10,5,1 nM of hepacin in the absence or presence of 2
ng/ml of transforming growth factor TGF-.beta.. The effects of
hepacin on generation of intracellular reactive oxygen species
(ROS) in HSC-T6 were also analyzed.
[0077] As shown in FIG. 8, HSC-T6 significantly (P<0.05)
suppressed the COL1A2 promoter expression in the absence or
presence of TGF-.beta..
Animal Model:
[0078] Liver fibrosis was induced in these animals by hydrodynamic
method; within 7 second injection mouse tail vein 3 ml ringer'
solution containing 10 .mu.g plasmid of pPK9a which would be
generated TGF-.beta. induced by 100 uM zinc sulfate in water
unlimited feed to gauge how compound2 influences liver collagen.
The test results were illustrated in FIGS. 9 and 10.
Conclusion
[0079] Given the above tests, it demonstrated that the compounds of
the invention could be regarded as a strong antioxidants to
scavenge ROS and down regulated TGF-.beta. mediated inflammation
and fibrosis
Example 3
Inhibition of Nitric Oxide by Extract from Example 1
Cell Subculture Steps
Manipulation
[0080] RAW264.7 cells were incubated at 37.degree. C. in a 5%
CO.sub.2 and 90% relative humidity incubator in DMEM (Dulbucco's
Modified Eagle Medium) containing 2 mM L-glutamine, 100 U/ml
penicillin G, 100 .mu.g/ml streptomycin sulfate, 10% fetal bovine
serum (FBS) with growing 80.about.90% confluence, then passaged by
different test purposes.
Assay and Analysis of Nitrite
Material and Reagent
[0081] (1) Griess reagent solution (1% sulfanilamide, 0.1%
naphthlethylenediamine dihydrochloride in 2.5% phosphoric
acid).
[0082] (2) Sodium nitrite
Protocol
[0083] (1) Cells were treated with drugs in appropriate dosage and
incubated at 37.degree. C. in a 5% CO.sub.2 incubator. [0084] (2)
After treating cells with various dosages over specific time, they
were taken out from the incubator and transferred 100 .mu.l medium
into a 96 well plate, respectively. [0085] (3) A calibration curve
was made by sodium nitrite, and each well was added with 100 .mu.l
by serial dilution. [0086] (4) Each sample and well was further
added with 10011 Griess reagent solution over 10 minutes. [0087]
(5) Absorbance was read at 570 nm using an ELISA reader directly
and compared with calibration curve to get the concentration of
nitrite in the samples.
[0088] The test results were illustrated in FIG. 11. Given the
above tests, it demonstrated that the extract of Antrodia
camphorata actually inhibited nitric oxide activity. The inhibition
of nitric oxide activity was increasing depended on the higher
concentration of the extract.
[0089] While the invention has been described and exemplified in
sufficient detail for those skilled in this art to make and use it,
various alternatives, modifications, and improvements should be
apparent without departing from the spirit and scope of the
invention.
[0090] One skilled in the art readily appreciates that the present
invention is well adapted to carry out the objects and obtain the
ends and advantages mentioned, as well as those inherent therein.
The cell lines, embryos, animals, and processes and methods for
producing them are representative of preferred embodiments, are
exemplary, and are not intended as limitations on the scope of the
invention. Modifications therein and other uses will occur to those
skilled in the art. These modifications are encompassed within the
spirit of the invention and are defined by the scope of the
claims.
[0091] It will be readily apparent to a person skilled in the art
that varying substitutions and modifications may be made to the
invention disclosed herein without departing from the scope and
spirit of the invention.
[0092] All patents and publications mentioned in the specification
are indicative of the levels of those of ordinary skill in the art
to which the invention pertains. All patents and publications are
herein incorporated by reference to the same extent as if each
individual publication was specifically and individually indicated
to be incorporated by reference.
[0093] The invention illustratively described herein suitably may
be practiced in the absence of any element or elements, limitation
or limitations, which are not specifically disclosed herein. The
terms and expressions which have been employed are used as terms of
description and not of limitation, and there is no intention that
in the use of such terms and expressions of excluding any
equivalents of the features shown and described or portions
thereof, but it is recognized that various modifications are
possible within the scope of the invention claimed. Thus, it should
be understood that although the present invention has been
specifically disclosed by preferred embodiments and optional
features, modification and variation of the concepts herein
disclosed may be resorted to by those skilled in the art, and that
such modifications and variations are considered to be within the
scope of this invention as defined by the appended claims.
[0094] Other embodiments are set forth within the following claims.
Sequence CWU 1
1
2 1 21 DNA Artificial Consensus Sp1 (f) 1 gttgcggggc ggggccgagt g
21 2 21 DNA Artificial consensus Sp1 (r) 2 aacgccccgc cccggctcac g
21
* * * * *