Note: Descriptions are shown in the official language in which they were submitted.
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BLOCKING THE MIGRATION OR METASTASIS OF CANCER CELLS BY
AFFECTING ADHESION PROTEINS AND THE USES OF NEW COMPOUNDS
THEREOF
This application claims priority of PCT/US2007/077273, filed August 30, 2007,
U.S.
Serial No. 60/890,380, filed on February 16, 2007, U.S. No. 60/947,705, filed
on July 3,
2007, and U.S. Serial No.11/683,198, filed on March 7, 2007, which claims
benefit of
U.S. Serial Nos. 60/795,417, filed on April 27, 2006, 60/841,727, filed on
September 1,
2006, 60/890,380, filed on February 16, 2007, and International Application
No.
PCT/US2006/016158, filed April 27, 2006, which claims the benefit of the
priority of the
following applications: (1) U.S. Serial Nos. 11/289142, filed November 28,
2005, and
11/267,523, filed November 4, 2005; (2) International Application No.
PCT/US05/31900,
filed September 7, 2005 (which claims the priority of U.S. Serial Nos.
60/617,379, filed
October 8, 2004, 60/613,811, filed September 27, 2004, and 60/607,858, filed
September 7, 2004); (3) U.S. Serial No. 11/131,551, filed May 17, 2005; and
(4) U.S.
Serial No. 11/117,760, filed April 27, 2005. The contents of these preceding
applications
are hereby incorporated in their entireties by reference into this
application.
FIELD OF THE INVENTION
This invention provides methods and compositions for affecting the gene
expression in
cells as a result that cure diseases, wherein the methods comprise reducing
the
syndrome of diseases. In an embodiment the method comprise inhibition of gene
expression. In an embodiment the method comprises stimulating the gene
expression.
This invention provides methods, processes, compounds and compositions for
modulating the gene expression or secretion of adhesion proteins or their
receptors to
cure disease, wherein the modulating comprises positive and negative
regulating;
wherein comprises inhibiting cancer growth, wherein the adhesion proteins or
receptors
comprise fibronectin, integrins family, Myosin , vitronectin, collagen,
laminin,
Glycosylation cell surface proteins, polyglycans, cadherin, heparin, tenascin,
CD 54,
CAM, elastin and FAK.
BACKGROUND OF THE INVENTION
Metastasis is the late stage of cancer in which cancer cells leave the
original tumor site
and migrate to other parts of the body. The cancer cells break away from the
primary
tumor and attach to the surrounding extracellular matrix and migrate to other
parts of
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the body via bloodstream or the lymphatic system. The adhesion protein play an
essential role in cancer metastasis.
When metastasis occurs, there are numbers of way with which it can be treated,
including radiosurgery, chemotherapy, radiation therapy, biological therapy
hormone
therapy, surgery and laser-immunotherapy. However, these are often not able to
prevent the genesis of metastatic cancer.
SUMMARY OF THE INVENTION
This invention provides a method of modulating the adhesion of cancer cell and
block
their migration, metastasis or inhibit the growth of cancers or anti-
angiogenesis, wherein
the adhesion protein and their receptors comprise fibronectin, integrins
family, Myosin ,
vitronectin, collagen, laminin, Glycosylation cell surface proteins,
polyglycans, cadherin,
heparin, tenascin, CD 54, CAM, elastin and FAK.
This invention provides a method of reducing the adhesion protein in cell and
block the
migration, metastasis of cancer cells or inhibit the growth of cancers or anti-
angiogenesis, wherein the adhesion proteins or its receptors comprise
fibronectin,
integrins family, Myosin, vitronectin, collagen, laminin, Glycosylation cell
surface
proteins, polyglycans, cadherin, heparin, tenascin, CD 54, CAM, elastin and
FAK. In an
embodiment, this invention provides a method of reducing the secretion of
fibronectin.
In an embodiment, this invention provides a method for inhibiting the
expression of
adhesion proteins, wherein the adhesion proteins comprise fibronectin,
integrins family,
Myosin , vitronectin, coliagen, laminin, Glycosylation cell surface proteins,
polyglycans,
cadherin, heparin, tenascin, CD 54, CAM, elastin and FAK. This invention
provides a
method of inhibiting the growth, migration, metastasis of cancer by altering
the
characteristics of membrane of cancer cells, wherein the characteristics
comprise
adhesion protein; wherein the cancers comprise breast, leukocyte, liver,
ovarian,
bladder, prostate, skin, bone, brain, leukemia, lung, colon, CNS, melanoma,
renal and
cervix cancer.
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DETAILED DESCRIPTION OF THE FIGURES
Figure 1: Time studies of inhibition of Fibronectin secretion from cancer
cells (ES2)
after incubation of Xanifolia-Y. Fibronectin released in culture medium was
determined
byWestern blot A: (results of experiment F1) Y is Xanifolia compound Y; B:
(results of
experiment F3); C: (results of experiment F4)
Figure 2: Inhibition of Fibronectin Secretion by Xanifolia-Y (Western Blot).
A: result of experiment F5; B: result of experiment F7; C: result of
experiment F8; D:
result of experiment F11; E: result of experiment F12; F: result of experiment
F13; G:
result of experiment F14B; H: result of experiment 14C
Figure 3: Inhibition of Fibronectin Secretion by Xanifolia-Y (Western Blot).
A: result of experiment F23; B: result of experiment F24; C: result of
experiment F26;
D: result of experiment F27; E: result of experiment F29; F: result of
experiment F28.
Figure 4: Inhibition of Fibronectin Secretion by Xanifolia-Y (Western Blot).
A: result of experiment F30; B: result of experiment F31; C: result of
experiment F32;D:
result of experiment F33A; E: result of experiment F20.
Figure 5: Increase synthesis of Angiopoietin-2 in ES2 cells by Xanifolia-Y
treatment.
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DETAILED DESCRIPTION OF THE INVENTION
This invention provides methods and compositions for modulating the gene
expression
to cure diseases or reduce the syndrome of diseases, wherein the modulating
comprises positive and negative regulating. In an embodiment, the method
comprises
inhibiting the gene expression. In an embodiment the method comprises
stimulating the
gene expression.
This invention provides methods and compositions for inhibiting the migration,
metastasis or growth of cancers or anti-angiogenesis, wherein the methods
comprise
affecting the gene expression, wherein comprise affecting the adhesion
proteins or their
receptors, reducing adhesion protien, or inhibiting the expression or
secretion of
adhesion proteins, wherein the adhesion proteins comprise fibronectin,
integrins family,
Myosin , vitronectin, collagen, laminin, Glycosylation cell surface proteins,
polyglycans,
cadherin, heparin, tenascin, CD 54, CAM, elastin and FAK.
This invention provides methods and compositions for inhibiting the migration,
metastasis or growth of cancers or anti-angiogenesis, wherein the methods
comprise
affecting the gene expression, wherein comprises stimulating the gene
expression.
This invention provides a method for altering the characteristic of cancer
cell membrane
resulting in blocking the migration, metastasis of cancer cells or inhibit the
growth of
cancers or anti-angiogenesis, wherein the method comprises reducing adhesion
protiens or their receptors, wherein the adhesion proteins comprise
fibronectin,
integrins family, Myosin , vitronectin, collagen, laminin, Glycosylation cell
surface
proteins, polyglycans, cadherin, heparin, tenascin, CD 54, CAM, elastin and
FAK.
This invention provides methods, processes, compounds and compositions of
reducing
adhesion protein of cells, wherein the adhesion proteins comprise fibronectin,
integrins
family, Myosin, vitronectin, collagen, laminin, Glycosylation cell surface
proteins,
polyglycans, cadherin, heparin, tenascin, CD 54, CAM, elastin and FAK. In an
embodiment, methods comprise inhibiting the gene expression. In an embodiment,
this
invention provides a method of reducing the secretion of fibronectin. In an
embodiment
the method can block the migration, metastasis of cancer cells or inhibit the
growth of
cancers or anti-angiogenesis, wherein the cancers comprise breast cancer,
leukocyte
cancer, liver cancer, ovarian cancer, bladder cancer, prostate cancer, skin
cancer, bone
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cancer, brain cancer, leukemia cancer, lung cancer, colon cancer, CNS cancer,
melanoma cancer, renal cancer or cervix cancer,
This invention provides a method of altering the characteristic of cancer cell
membrane,
wherein the method comprises altering the secretion of adhesion proteins,
wherein the
adhesion proteins comprise fibronectin, integrins family, Myosin, vitronectin,
collagen,
laminin, Glycosylation cell surface proteins, polyglycans, cadherin, heparin,
tenascin,
CD 54, CAM, elastin and FAK. In an embodiment, the methods, processes,
compounds
and compositions comprises blocking, suppressing or inhibiting the expression
or
secretion of adhesion protein, wherein the adhesion proteins . In an
embodiment, the
methods, processes, compounds and compositions is interacting with adhesion
protein,
wherein the adhesion proteins. In an embodiment the methods, processes,
compounds
or compositions can block the migration, metastasis of cancer cells or inhibit
the growth
of cancers or anti-angiogenesis, wherein the cancers comprise breast,
leukocyte, liver,
ovarian, bladder, prostate, skin, bone, brain, leukemia, lung, colon, CNS,
melanoma,
renal and cervix cancer.
The adhesion proteins help cancer cell adhesion, invasion or metastasis,
wherein the
cancers comprise ovarian cancer. Reducing the adhesion proteins will reduces
the
metastasis of cancers.The fibronectin is one of the key factors in the biology
of epithelial
ovarian cancers. The reducing of fibronectin will inhibit the metastasis of
cancer cells.
This invention provides a method and composition for inhibiting the secretion
of
adhesion protein comprising fibronectin in order to cure the diseases, wherein
the
diseases comprise inhibiting cancer growth, wherein the cancers comprise
breast,
leukocyte, liver, ovarian, bladder, prostate, skin, bone, brain, leukemia,
lung, colon,
CNS, melanoma, renal and cervix cancer.
This invention provides a composition for inhibiting the growth, migration,
metastasis of
cancer and by altering the characteristics of membrane of cancer cell, wherein
the
characteristics comprise adhesion of proteins; wherein comprising the
secretion of
proteins or the adhesion of cells; wherein the characteristic comprise
adhesion ability;
wherein the adhesion proteins comprise fibronectin, integrins family, Myosin ,
vitronectin, collagen, laminin, Glycosylation cell surface proteins,
polyglycans, cadherin,
heparin, tenascin, CD 54, CAM, elastin and FAK; wherein the cancers comprise
breast
cancer, leukocyte cancer, liver cancer, ovarian cancer, bladder cancer,
prostate cancer,
skin cancer, bone cancer, brain cancer, leukemia cancer, lung cancer, colon
cancer,
CNS cancer, melanoma cancer, renal cancer or cervix cancer; wherein the method
is
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administering contacting XanifoliaYO, Y1, Y2, Y, Y7, Y8, Y9, Y10, ACH-Y or a
salt, ester,
metabolite thereof. In an embodiment the composition is the compound selected
from
formulas in this application.
This invention provides a method for altering the adhesion characteristic of
inembrane
of cancer cell, wherein the method compring reducing the adhesion ability,
wherein the
method comprises reducing the secretion of fibronectin. wherein the cancers
comprise
breast cancer, leukocyte cancer, liver cancer, ovarian cancer, bladder cancer,
prostate
cancer, skin cancer, bone cancer, brain cancer, leukemia cancer, lung cancer,
colon
cancer, CNS cancer, melanoma cancer, renal cancer or cervix cancer, wherein
the
method is administering contacting an effective amount of Xanifolia Y0, Y1,
Y2, Y, Y7,
Y8, Y9, Y10, ACH-Y or a salt, ester, metabolite thereof. In an embodiment, the
method
is administering contacting an effectie amount of the compound selected from
formulas
in this application. In an embodiment, the method is inhibiting the growth,
migration,
metastasis of cancer. In an embodiment the compound may be selected from
formula
(1A), (1B), (1C) and (1D). In an embodiment, the compound comprises a
triterpene
backbone, two angeloyl groups and sugar moiety. In an embodiment the
compound(s)
are selected from Xanifolia (x), Escin or Aescin. In an embodiment the
compound(s) are
selected from Compound A to X and Al to X1. In an embodiment the compound(s)
are
selected from Compound Z1 to Z7, in the application.
This invention provides a composition for inhibiting the growth, migration,
metastasis of
cancer by altering the adhesion characteristic of membrane of cancer cell,
wherein the
cancers comprise breast cancer, leukocyte cancer, liver cancer, ovarian
cancer, bladder
cancer, prostate cancer, skin cancer, bone cancer, brain cancer, leukemia
cancer, lung
cancer, colon cancer, CNS cancer, melanoma cancer, renal cancer or cervix
cancer.
This invention provides a method and composition for reducing of adhesion
protein to
cure the diseases, wherein the diseases comprise inhibiting cancer growth,
reducing leg
swelling, symptoms of chronic venous insufficiency, peripheral edema,
antilipemic,
chronic venous disease, varicose vein disease, varicose syndrome, venous
stasis,
expectorant, peripheral vascular disorders, cerebro-organic convulsion,
cerebral
circulation disorder, cerebral edema, psychoses, dysmenorrheal, hemorrhoids,
episiotomies, peripheral edema formation or postoperative swelling; for
reducing
symptoms of leg pain; for treating pruritis, lower leg volume, for reducing
symptoms of
pain; thrombosis, thromophlebitis; for preventing gastric ulcers
antispasmotic,
comprising administering to a subject, in need thereof, an effective amount of
the
composition of this invention.
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In an embodiment, the method comprises interacting with adhesion protien,
wherein the
adhesion proteins comprise fibronectin, integrins family, Myosin, vitronectin,
collagen,
laminin, Glycosylation cell surface proteins, polyglycans, cadherin, heparin,
tenascin,
CD 54, CAM, elastin and FAK. In an embodiment, this invention provides a
method of
reducing the secretion of fibronectin.
In an embodiment, the method comprises reducing the adhesion ability of
adhesion
protein; wherein the adhesion proteins comprise fibronectin, integrins family,
Myosin,
vitronectin, collagen, laminin, Glycosylation cell surface proteins,
polyglycans, cadherin,
heparin, tenascin, CD 54, CAM, elastin and FAK.
In an embodiment, the method comprises modulating the secretion of adhesion
protien,
wherein the adhesion proteins comprise fibronectin, integrins family, Myosin,
vitronectin,
collagen, laminin, Glycosylation cell surface proteins, polyglycans, cadherin,
heparin,
tenascin, CD 54, CAM, elastin and FAK. In an embodiment, the method comprises
blocking the secretion of adhesion protien, wherein the adhesion protein
comprising
fibronectin. In an embodiment the method is administering contacting an
effective
amount of the compound selected from formulas in this application.
In an embodiment the method is administering contacting an effective amount of
the
compound in this application comprising Xanifolia Y0, Yl, Y2, Y, Y7, Y8, Y9,
Y10, ACH-
Y, Xanifolia (x), Escin or Aescin or a salt, ester, metabolite thereof. In an
embodiment
the compound(s) are selected from Compound A to X and Al to Xl. In an
embodiment
the compound(s) are selected from Compound Z1 to Z7, in the application. In an
embodiment the compound may be selected from formula (1A), (113), (1 C) and (1
D).
This invention provides a method and composition for altering the
characteristic of
adhesion protein to cure diseases, wherein the characteristic comprising
adhesion
ability, wherein the method comprises reducing the secretion of fibronectin,
wherein the
diseases comprise inhibiting cancer growth, reducing leg swelling, symptoms of
chronic
venous insufficiency, peripheral edema, antilipemic, chronic venous disease,
varicose
vein disease, varicose syndrome, venous stasis, expectorant, peripheral
vascular
disorders, cerebro-organic convulsion, cerebral circulation disorder, cerebral
edema,
psychoses, dysmenorrheal, hemorrhoids, episiotomies, peripheral edema
formation or
postoperative swelling; for reducing symptoms of leg pain; for treating
pruritis, lower leg
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volume, for reducing symptoms of pain; thrombosis, thromophlebitis; for
preventing
gastric ulcers antispasmotic, comprising administering to a subject, in need
thereof, an
effective amount of the composition of this invention; wherein the adhesion
proteins
comprise fibronectin, integrins family, Myosin , vitronectin, collagen,
laminin,
Glycosylation cell surface proteins, polyglycans, cadherin, heparin, tenascin,
CD 54,
CAM, elastin and FAK In an embodiment the method is administering contacting
an
effective amount in a subject with the compound selected from formulas in this
application.
A isolated, purified or synthesized compound or its salt, ester, metabolite or
derivative
thereof, having the formula of
R15 R1a
R,
"Rz
Rll R1z R
a
R8
R13 =
R5p Rs
Rs` RIo also named (1A),
wherein
R1 is selected from hydrogen, hydroxyl, 0-angeloyl, 0-tigloyl, O-senecioyl, 0-
alkyl, 0-
dibenzoyl, 0-benzoyl, 0-alkanoyl, 0-alkenoyl, O-benzoyl alkyl substituted 0-
alkanoyl,
0-aryl, 0-acyl, 0-heterocylic, 0-heteroraryl, and derivatives thereof;
R2 is selected from hydrogen, hydroxyl, 0-angeloyl, 0-tigloyl, 0-senecioyl, 0-
alkyl, 0-
dibenzoyl, 0-benzoyl, 0-alkanoyl, 0-alkenoyl, O-benzoyl alkyl substituted 0-
alkanoyl,
0-aryl, 0-acyl, 0-heterocylic, 0-heteroraryl, and derivatives thereof;
R4 represents CH2R6 or COR6, wherein R6 is selected from a group consisting of
hydroxyl, 0-angeloyl, 0-tigloyl, O-senecioyl, 0-alkyl, O-dibenzoyl, O-benzoyl,
0-
alkanoyl, 0-alkenoyl, O-benzoyl alkyl substituted O-alkanoyl, 0-aryl, 0-acyl,
0-
heterocylic, 0-heteroraryl, and derivatives thereof;
R3 is H or OH;
R8 is H or OH, particularly OH;
R5 is a hydrogen or sugar moiety(ies), wherein the sugar moiety(ies) is/are
selected
from a group consisting of glucose, galactose, rhamnose, arabinose, xylose,
fucose,
allose, altrose, gulose, idose, lyxose, mannose, psicose, ribose, sorbose,
tagatose,
talose, fructose, alduronic acid, glucuronic acid, galacturonic acid, and
derivatives or
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combination thereof; wherein R9, R10, R11, R12, R13, R14, R15 are
independently
attached a group selecting from CH3, CHZOH, CHO, COOH, COO-alkyl, COO-aryl,
COO-heterocyclic, COO-heteroaryl, CHZOaryI, CH2O- heterocyclic, CHZO-
heteroaryl,
alkyls group, hydroxyl, acetyl group, particularly CH3;
wherein at least two of R1, R2 and R6 are compriseing a group selected from 0-
angeloyl, 0-tigloyl, O-senecioyl, O-dibenzoyl, 0-benzoyl, 0-alkanoyl, 0-
alkenoyl, 0-
benzoyl alkyl substituted 0-alkanoyl, 0-aryl, 0-acyl, 0-heterocylic, 0-
heteroraryl, and
derivatives thereof; or at least one of R1, R2, and R4 is a sugar moiety
substituted with
at least two groups selected from a group consisting of angeloyl, acetyl,
tigloyl,
senecioyl, benzoyl, dibenzoyl, alkanoyl,.alkenoyl, benzoyl alkyl substituted
alkanoyl, aryl,
acyl, heterocylic, heteroraryl, and a derivative thereof; or
wherein R4 is CH2R6; wherein R1 and R2 independently consists an 0-angeloyl
group,
or at least two of R1, R2 and R6 are 0-angeloyl or at least one of R1, R2 or
R6 is a
sugar moiety with two 0-angeloyls;
wherein R5 is/are the sugar moiety(ies) selected from the following sugars and
alduronis acids: glucose, galactose, rhamnose, arabinose, xylose, fucose,
allose,
altrose, gulose, idose, lyxose, mannose, psicose, ribose, sorbose, tagatose,
talose,
fructose, glucuronic acid, galacturonic acid; or their derivatives thereof, or
the
combination thereof; wherein the sugar preferably comprises glucuronic acid,
arabinose
and galactose.
In an embodiment,wherein R5 is/are sugar moiety(ies) selected from a group
consisting
of glucose, galactose, arabinose, alduronic acid, glucuronic acid,
galacturonic acid, and
a derivative or combination thereof;
In an embodiment the method is administering contacting the compounds, wherein
the
compound is selected from the following:
a) An isolated, purified or synthesized compound is having structure
Xanifolia(Y),
Y3 O
H O
I ~H
OOH H
H
H OH
OM
H
H
H
OH or chemical name: 3-0-[(3-D-
galactopyranosyl (1->2)]-a-L-arabinofuranosy (1->3)-(3-D-glucuronopyranosyl-
21,22-0-
diangeloyl-3R, 15a, 16a, 21 [3, 22a, 28-hexahydroxyolean-12-ene;
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b) An isolated, purified or synthesized compound is having structure Xanifolia
(Y1),
Y,
Ae
H
OOH H
H
H OH CH~
OH
R
'OH
H
H H
OH or chemical name: 3-0-[[3-D-galactopyranosyl
(1->2)]-a-L-arabinofuranosyl (1->3)-(3-D-glucuronopyranosyl-21-O-(3,4-
diangeloyl)-a-L-
rhamnophyranosyl-22-O-acetyl-3R,16(x, 21R, 22a, 28-pentahydroxyolean-1 2-ene;
c) An isolated, purified or synthesized compound is having structure Xanifolia
(Y2),
YZ
~ o
OOH H
H H
H OH
OH
H
H
OH H or chemical name: 3-0-[(3-D-glucopyranosyl-
(1->2)]-(x-L-arabinofuranosy (1->3)-(3-D-glucuronopyranosyl-21,22-O-diangeloyl-
3R,
15a, 16a, 210, 22a, 240, 28-heptahydroxyolean-12-ene;
d) An isolated, purified or synthesized compound is having structure Xanifolia
(Y8),
O-C
~~
O
Y-8 O-~\
OH p
"'OH
COOH O
OH OH
HO O
OH
HO O
OM
" OH or chemical name: 3-0-[P-glucopyranosyl
(1-a2)]-a-arabinofuranosyl (1->3)-fl-glucuronopyranosyl-21, 22-O-diangeloyl-
3Q, 16a,
21p, 22a, 24,Q, 28-hexahydroxyolean-12-ene;
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e) An isolated, purified or synthesized compound is having structure Xanifolia
(Y9),
O
Y9 'OH
OAC
"'OH
O
O COOH O C-O
O
H `-
CH3
O
HO~
OH O C-O T-T
OH HO O ~(\
OOHO
OH or chemical name: 3-0-[,6-galactopyranosyl
(1-)2)]-a-arabinofuranosyl (1->3)-/3-glucuronopyranosyl-21-O-(3,4-diangeloyl)-
a-
rhamnopyranosyl-28-O-acetyl-3A 16a, 21Q, 22a, 28-pentahydroxyolean-12-ene; and
f) An isolated, purified or synthesized compound is having structure Xanifolia
(Y10),
O-C
O
Y-10 O-C
OH p
"'OH
COOH O
~O
HO OH OH
OH
HO O
OH O
OH or chemical name:
3-0-[/fgalactopyranosyl (1~2)]-a-arabinofuranosyl (1~3)-~glucuronopyranosyl-
21,
22-O-diangeloyl-3,8, 16a, 21,, 22a, 28-pentahydroxyolean-12-ene.
g) An isolated, purified or synthesized compound is having structure Xanifolia
(Y0),
~
"~
0
~ h
I'" o
O o CO " aOM O x
HO HO
HO
aM O
MO O
~H OH or chemical name: 3-0-[R-D-
galactopyranosyl(1->2)]-a-L-arabinofuranosyl(1 ->3)-R-D-glucuronopyranosyl-21-
0-
angeloyl, 22-0-(2-methylpropanoyl)-3[3, 15a, 16a, 21 [i, 22a, 28-
hexahydroxyolean-12-
ene,
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h) An isolated, purified or synthesized compound is having structure Xanifolia
(X),
O
N3C
25 ~CH,
CX,OH O
O OH
21
O
OH O ~'2a
HO HO
HO
O
MO O
OH OH or chemical name: 3-0-{[/j-D-galactopyranosyl
(1->2)]-[a-L-arabinofuranosyl (1->3)]-p-D-glucuronopyranoside butyl ester}-21-
O-acetyl-
22-O-angeloyl- 3A16a,21,13,22a,28-pentahydroxyolean-12-ene.
i) An isolated, purified or synthesized compound is having structure (Y7),
HC
~
LH,
O
5 28 O CH,
11
"=OH O
\ 90- H3C
O O COOH 27 pH
O
O 2 23
HO OH HO
HO
oH O
HO 0
4 OH or chemical name: 3-0-[8-D-
galactopyranosyl-(1->2)]-a-L-arabinofuranosyl-(1 --+3)-,B-D--
glucuronopyranosyl-21-0-
angeloyl-28-O-2-methylbutanoyl-3,6, 15 a, 16a, 21,6, 22a, 28-hexahydroxyolean-
12-ene
j) An isolated, purified or synthesized compound is having structure (ACH-Y):
o-C
u
O
'O-C
OH p
'"OH
HO OH
In an embodiment the method is administering contacting the compound, wherein
the
compound is selected from the following:
k) An isolated, purified or synthesized compound is having a structure:
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H3C
.'{p -~ CH3
o
OHC H
o~
CH=OH 0
~'OH
gi7
~
O o H OH
H
OH O 2 23
HO HO
HO
OH
I) An isolated, purified or synthesized compound is having a structure:
3 ~
~'c~
-C CHa
II
O
ll3CH
~ ......nu,0-C CH3
25 ~ CHZOH p
OH
COOH '
O OH
0 2 2]
HO
OH Q
HO 0
OH
OH
In an embodiment the method is administering contacting the compound, wherein
the
compound is isolated, purified or synthesized having a structure selected from
following
formulas:
,,.
R,
R2
R7
R
6
R R3
R4 (1C)
wherein R1, R2 are individually selected of an 0-acetyl or O-angeloyl; wherein
the R3,
R4, R5, R6, R7 is hydrogen or hydroxyl
In an embodiment the method is administering -contacting the compound in this
application comprising Xanifolia Y0, Yl, Y2, Y, Y7, Y8, Y9, Y10, Xanifolia
(x), Escin or
Aescin or a salt, ester, metabolite thereof. In an embodiment the compound may
be
selected from formulas (1A), (1B), (1C) and (1D). In an embodiment, the
compound
comprises a triterpene backbone, two angeloyl groups and sugar moiety. In an
embodiment the compound(s) are selected from Xanifolia (Y0, Yl, Y2, Y, Y7, Y8,
Y9,
and Y10). In an embodiment the compound(s) are selected from Xanifolia (x),
Escin or
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Aescin. In an embodiment the compound(s) are selected from Compound A to X and
Al
to Xl in the application. In an embodiment the compound(s) are selected from
Compound Z1 to Z7 in the application. In an embodiment the method is
administering
contacting the compound comprise of a triterpene wherein the carbon position
21, 21
has a unsaturated group and sugar moieties at carbon 3.
In an embodiment, compounds of this application reducing the adhesion ability
inhibit
bacteria in colonization and regulate tropism of cells.
In an embodiment, reducing the adhesion ability of cell or viruses in order to
inhibit
viruses binding to host cells, wherein the virus comprise HIV
The composition comprises the bioactive compounds from natural plants or
synthesis.
The majority of the plants are from the Sapindaceae family, which has 140-150
genera
with 1400-2000 species. The program is based on our purification methods and
biological assays including the MTT assay See International Application No.
PCT/US05/31900, filed September 7, 2005, U.S. Serial No. 11/289142, filed
November
28, 2005, and U.S. Serial No. 1 1/1 31 551, filed May 17, 2005, the contents
of which are
incorporated herein by reference
This invention provides the method uses of compositions comprising a
triterpenoidal
saponin. In an embodiment, the saponin has triterpenoid, triterpenoidal or
other
sapogenin, one or more sugar moieties and two angeloyl groups, or at least two
side
groups selected from the following groups: angeloyl groups, tigloyl groups or
senecioyl
groups, wherein the side groups are attached to the sapogenin backbone at
carbon 21
and 22. In an embodiment, at least two of angeloyl, acetyl, tigloyl,
senecioyl, alkyl,
benzoyl, dibenzoyl, alkanoyl, alkenoyl, benzoyl alkyl substituted alkanoyl,
aryl, acyl,
heterocylic or heteroraryl attached to the side groups; wherein the sugar
moiety in the
saponin comprises at least one or more of the following sugars and alduronis
acids:
glucose, galactose, rhamnose, arabinose, xylose, fucose, allose, altrose,
gulose, idose,
lyxose, mannose, psicose, ribose, sorbose, tagatose, talose, fructose,
glucuronic acid,
galacturonic acid; or their derivatives thereof, or the combination thereof;
wherein the
sugar preferably comprises glucuronic acid, arabinose and galactose.
This invention further provides a composition comprising the structures
substituted with
at least two side groups selected from angeloyl, tigloyl or senecioyl groups,
wherein the
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side groups are attached to a triterpenoidal, triterpenoid, triterpenoidal or
other
sapongenin backbone. These structures are obtainable from the natural or
synthesis.
This invention provides a method of preparing the bioactive compounds,
comprising the
steps of:
(a) Extracting roots, kernels, leaves, bark, stem, husks, seeds, seed shells
or fruits of
the plant, or combinations thereof with organic solvents such as ethanol or
methanol to
obtain an organic extract; (b) Collecting the organic extracts; (c) Refluxing
the organic
extract to obtain a second extract; (d) Removing the organic solvent from the
second
extract to obtain a third extract; (e) Drying and sterilizing the third
extract to obtain a
crude extract powder; (f) Fractionating the crude extract powder into
fractions or
components. Fractionation may be achieved by HPLC and FPLC chromatography
with silica gel, C18 or other equivalent solid phase materials; (g) Monitoring
the
fractionating, if using HPLC or FPLC, the absorption wavelength at 207nm to
500nm
may be used; (h) Identifying the bioactive components of the crude extract;
(i) Purifying
one or more bioactive components of the crude extract with FPLC to obtain one
or more
fractions of the bioactive component; and (j) isolating the bioactive
components with
chromatographic techniques that employ preparative columns and HPLC.
In an embodiment, this invention provides the method of MTT Assay TEST
PLATFORM
to test the bioactivities of the saponins or other compounds.
Cells. Human cancer cell lines were obtained from American Type Culture
Collection:
HTB-9 (bladder), HeLa-S3 (cervix), DU145 (prostate), H460 (lung), MCF-7
(breast),
K562 (leukocytes), HCT116 (colon), HepG2 (liver), U2OS (bone), T98G (brain),
SK-
MEL-5 (Skin) and OVCAR-3 (ovary). The cells were grown in following culture
media:
HeLa-S3, DU145, MCF-7, Hep-G2 and T98G are in MEN (Earle's salts); HTB-9,
H460,
K562 and OVCAR-3 in RPMI-1640; HCT-116 and U2OS in McCoy-5A. They are
supplemented with 10% fetal calf serum, glutamine and antibiotics, and
incubated in an
incubator with 5% CO2 humidified at 37 C.
MTT Assay. The procedure for MTT assay followed the method described by
Carmichael et al. (1987) with modifications. The cells were seeded into a 96-
well plate
at concentration of 10,000/well for HTB-9, HeLa, H460, HCT1 16, T98G and OVCAR-
3),
15,000/well for DU145, MCF-7, HepG2 and U20S), and 40,000/well for K562 for 24
hours before drug-treatment. The cells were then exposed to the drugs for 48
hours (72
hours for HepG2 and U2OS, and 96 hours for MCF-7). After the drug-treatment,
MTT
(0.5 mg/mL) was added to cultures and incubated for an hour. The formazan
(product
of the reduction of tetrazolium by viable cells) formed and was dissolved with
DMSO
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and the O.D. at 490nm, and was measured by an ELISA reader. The MTT level of
the
cells before drug-treatment was also measured (TO). The % cell-growth (%G) is
calculated as: %G = (TD-TO / TC-TO) x 100(1), where TC or TD represents O.D.
readings of control or drug-treated cells.
When TO > TD, then the cytotoxicity (LC) expressed as % of the control is
calculated as:
%LC = (TD-TO / TO) x 100(2).
Micro Array: Analysis of gene expression of ES2 cells after Y-treatment by
Microarray
In this invention, the microarray experiments were done in studying the gene
expression.
Total number of 54676 genes has been studied.
Cell culture and drug-treatment. ES2 cells were seeded in a T-25 flask with
4.5 million
cells per flask for 24 hours. Cell culture was replaced with fresh medium with
xanifolia-
Y(Y) or DMSO no-drug -control (D) for 24 hours. Cells were then harvested for
RNA
isolation. Three experiments were done.
RNA extraction, labeling, hybridization, and data analysis. RNA was extracted
from
tumor cells using the Qiagen RNeasy Kit. RNA quality and quantity was checked
by the
Agilent BioAnalyzer and the NanoDrop ND-1000 spectrophotometer respectively
before further manipulation. The first and second cDNA strands were
synthesized from
20 ng of total RNA using the Affymetrix T7 oligo(dT) primer protocol and kit
for the two-
cycle amplificaton. To produce amplified biotin-labeled-cRNA, the cDNA was
reverse
transcribed by in vitro transcription using the MegaScript kit from Ambion.
15.0 pg of the
labeled cRNA was fragmented and re-checked for concentration using the
NanoDrop
ND-1000 spectrophotometer. A hybridization cocktail containing Affymetrix
spike-in
controls and fragmented labeled cRNA was loaded onto the Human U133 Plus 2.0
GeneChip oligonucleotide array. The Affymetrix array (Affymetrix, Inc.Santa
Clara,
CA) is comprised of over 1,300,000 unique oligonucleotide features that
represent
greater than 38,500 well-substantiated human genes. The array was hybridized
for 16
hours at 45 C with rotation at 60 rpm then washed and stained with a
strepavidin, R-
phycoerythrin conjugate stain on the Affymetrix Fluidicis Station 450. Signal
amplification was done using biotinylated antistreptavidin. The arrays were
scanned
using the GeneChip 3000 confocal laser scanner with autoloader. The images
were
analyzed and quality control metrics recorded using Affymetrix GCOS software
version
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1.4. Lastly, the expression value for each gene was calculated using dChip PM-
only
model based or Plier algorithm.
Data Analysis Methods
Pairwise comparisons were made as follows: Treated vs. Control (Y vs. D),
Modified
Drug vs Control (YM/ACY-H vs. D) and Treated vs. Modified Drug (Y vs. YM/ACH-
Y)
Cel files analyzed using the Bioconductor package of R Statistical
programming.
Limma analysis generated a reasonable number of changing genes between the
samples.
The raw data were normalized by the GCRMA method (robust multi-array
analysis). It
is implemented in Bioconductor (http://www.bioconductor.org/). The raw signal
intensity
data were normalized, background corrected and summarized based on certain
statistical models, and an expression value, in loq2-scale, is obtained per
chip per probe
set. Then the null hypothesis was tested that there's no significant changes
in gene
expression between the treatment pairs. This was done by LIMMA and is also
implemented in Bioconductor. It uses empirical Bayes method to estimate the
variance
in gene expression. One comparison was made, namely, High Grade vs. Low Grade.
The raw p-values were adjusted by the Benjamnin-Hochberg method for false
discovery
rate (FDR) control. All data sets contained a significant number of genes with
a p-value
less than 0.05, which is that the probability that a gene is NOT differential
expressed
(false positive) is 1:20.
All expression data is filtered by p-value (0.05).
The raw p-values were adjusted by the Benjamnin-Hochberg method for false
discovery
rate (FDR) control to yield an adjusted p-value.
Western blot
Western blot is applied in this invention as a method to detect the specific
proteins in
treated and untreated cells with compounds in this invention, wherein the
cells are
breast, leukocyte, liver, ovarian, bladder, prostate, skin, bone, brain,
leukemia, lung,
colon, CNS, melanoma, renal and cervix cancer
Cells: targeted cells were grown in RPMI 1640 medium. 1.5 million cells were
seeded in
a T25 flask and grown for 24 hours before drug-treatment.
Drug-treatment: Cells cultures were replaced with fresh RPMI medium containing
either
2.5 ul of DMSO (as control) [D]; or 10, 20, 30, 40, 80 ug/mI of tested
compounds.
After 24 hours, aliquot of culture medium was taken out for Fibronectin
determination
(Western blot method).
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Cell viability at 24 hours was determined by MTT assay. Cultures were replaced
with
RPMI medium (5 ml) with MTT and incubated for an hour. The formation of
formazan
was dissolved in 10 ml of DMSO and OD at 570nm was measured (MTT units).
Western Blot: Spent culture medium was mixed with SDS sample buffer, boiled
for 3
minutes before loading to SDS gel. Samples were applied to a 6-10% SDS gel and
electrophoresis was conducted with 100 volts for 2 hours. Protein was
transferred to a
nitrocellulose membrane electrophoretically. The nitrocellulose blot was
incubated with
the first antibody and second antibody (AP conjugated, Promega S3721). The
immuno-
bands were developed with BCIP/NBT color development system.
Determination of Western band intensity: The band-images of Western blot were
captured with a digital camera and the intensity of bands was determined using
"Image
J" software.
This invention provides a composition comprising an effective amount of
triterpenoidal
saponins named as Xanifolia Y1, Y2, Y, Y7, Y8, Y9, Y10, Y0 or their
derivatives for
modulating the adhesion protein, reducing adhesion protein or reducing the
secretion of
fibronectin, for treating chronic venous insufficiency, peripheral edema,
antilipemic,
chronic venous disease, varicose vein disease, varicose syndrome, venous
stasis,
expectorant, peripheral vascular disorders, cerebro-organic convulsion,
cerebral
circulation disorder, cerebral edema, psychoses, dysmenorrheal, hemorrhoids,
episiotomies, peripheral edema formation or postoperative swelling; for
reducing
symptoms of pain; for reducing symptoms of stomach pain; for reducing symptoms
of
leg pain; for treating pruritis, lower leg volume, thrombosis,
thromophlebitis; for treating
rheumatism; for preventing gastric ulcers antispasmotic; blocking the
migration,
metastasis of cancer cells or inhibiting tumor growth. In an embodiment the
method is
administering contacting the compound in this application comprising Xanifolia
Y0, Y1,
Y2, Y, Y7, Y8, Y9, Y10, Xanifolia (x), Escin or Aescin or a salt, ester,
metabolite thereof.
In an embodiment the compound may be selected from formulas (1A), (1B), (1C)
and
(1D). In an embodiment, the compound comprises a triterpene backbone, two
angeloyl
groups and sugar moiety. In an embodiment the compound(s) are selected from
Compound A to X and Al to Xl in the application. In an embodiment the
compound(s)
are selected from Compound Z1 to Z7 in the application.
This invention provides a method for reducing adhesion proteins or their
receptors on
cells, wherein the adhesion proteins comprise fibronectin, integrins family,
Myosin,
vitronectin, collagen, laminin, Glycosylation cell surface proteins,
polyglycans, cadherin,
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heparin, tenascin, CD 54, CAM, elastin and FAK. In an embodiment the method
can
block the migration, metastasis of cancer cells or inhibit the growth of
cancers or anti-
angiogenesis, wherein the cancers comprise breast cancer, leukocyte cancer,
liver
cancer, ovarian cancer, bladder cancer, prostate cancer, skin cancer, bone
cancer,
brain cancer, leukemia cancer, lung cancer, colon cancer, CNS cancer, melanoma
cancer, renal cancer and cervix cancer.
This invention provides a method for interacting with adhesion proteins or
their
receptors, wherein the adhesion proteins comprise fibronectin, integrins
family, Myosin,
vitronectin, collagen, laminin, Glycosylation cell surface proteins,
polyglycans, cadherin,
heparin, tenascin, CD 54, CAM, elastin and FAK. In an embodiment, this
invention
provides a method of reducing the secretion of fibronectin. Furthermore the
method is
blocking the migration, metastasis of cancer cells or treating a mammal
cancers
comprising administering to said mammal a therapeutically effective amount of
a
pharmaceutical composition comprising a composition comprises the molecular
formula
or compound in this invention. The cancers comprise Leukemia, Lung, Colon,
CNS,
Melanoma, Ovary, Renal, Prostate, Breast, bladder c, cervix, liver, bone ,
brain and
Skin cancer.The compounds comprise Xanifolia Y0, Y1, Y2, Y, Y7,Y8, Y9, Y10, or
a salt,
ester, metabolite or derivative thereof. The compounds of this invention can
be isolated
from natural sources or synthesized.
See experiments results in this application and see PCT/US05/31900, filed
Spetember
7, 2006; U.S. Serial No. 10/906,303, filed February 14, 2005; International
Application
No. PCT/USO4/43465, filed December 23, 2004; International Application No.
PCT/USO4/33359, filed October 8, 2004 and U.S. Serial No. 11/131551, filed May
17,
2005, PCT/US2007/077273, filed August 30, 2007, the contents of which are
incorporated herein by reference.
A salt of compound comprise sodium salt, potassium salt or calcium salt.
A salt of compounds for inhibiting venous insufficiency, particularly
hemorrhoids or
inhibiting leg swelling, or peripheral edema, antilipemic, chronic venous
disease,
varicose vein disease, varicose syndrome, venous stasis, Expectorant,
peripheral
vascular disorders, cerebro-organic convulsion, cerebral circulation disorder,
cerebral
edema, psychoses, dysmenorrheal, hemorrhoids, episiotomies, hamonhoids,
peripheral
edema formation or postoperative swelling; for reducing symptoms of pain; for
reducing
symptoms of stomach pain; for reducing symptoms of leg pain; for treating
pruritis,
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WO 2008/133766 PCT/US2008/002086
lower leg volume, thrombosis, thromophlebitis; for preventing gastric ulcers
antispasmotic.
This invention provides a method of modulating the adhesion proteins or their
receptors,
reducing the adhesion ability of the cancer cells, wherein the modulating
comprises the
positive or negative regulating. In an embodiment, the adhesion proteins
comprise
fibronectin, integrins family, Myosin, vitronectin, collagen, laminin,
Glycosylation cell
surface proteins, polyglycans, cadherin, heparin, tenascin, CD 54, CAM,
elastin and
FAK. In an embodiment, the method is reducing the secretion of fibronectin.
This
invention provides a method of blocking the migration, metastasis of cancer
cells or
inhibiting cancer cell growth comprising administering an effective amount of
a
pharmaceutical composition comprising a composition comprises the molecular
formula
or compound in this invention. The cancers comprise Leukemia cancer, Lung
cancer,
Colon cancer, CNS cancer, Melanoma cancer, ovarian cancer, renal cancer,
Prostate
cancer, Breast cancer, bladder cancer, cervix cancer, liver cancer, bone
cancer, brain
cancer and Skin cancer. The compounds of this invention can be isolated from
natural
sources or synthesized. In an embodiment the method is administering
contacting the
compounds, wherein the compound is selected from the following:
NaC
~
cis
O NaC
CN,
CNiON O
OH
27
O CO H O=N
OH O 2 "==.a
HO HO
HO
ON 0
H O
OH
20 OH (Z1)
3-0-[R-D-galactopyranosyl(1->2)]-a-L-arabinofuranosyl(1->3)-(3-D-
glucuronopyranosyl-
21-0-angeloyl, 22-0-(2-methylpropanoyl)-3(3, 15a, 16a, 21[3, 22a, 28-
hexahydroxyolean-1 2-ene,
N+ CH,
p /~
O NaCHJ
N,C Oo
- C~
CH,OH O
ON
27 =
O ON
OH O 2 '=.p
HO HO
HO
OH O
O
OH
OH (Z2)
CA 02676791 2009-07-27
WO 2008/133766 PCT/US2008/002086
3-0-[8-D-galactopyranosyl-(1->2)]-a-L-arabinofuranosyl-(1->3)-,8-D-
-glucuronopyranosyl-21-O-angeloyl-22-O-(angeloyl-2-methylbutanoyl) -3,8, 15 a,
16a,
21/3, 22a, 28-hexahydroxyolean-12-ene
N,C
29 cN,
O NA
N,
s ze cN,oN o
oN
i7 =
O ooN OM
OH
O O z `'z1
HO HO
HO
ON O
NO O
OH
OH (Z3)
3-0-[(3-D-galactopyranosyl(1 ->2)]-a-L-arabinofuranosyl(1-+3)-(3-D-
glucuronopyranosyl-
21-0-(2-methylpropanoyl), 22-0-(2-methylpropanoyl)-3(3, 15a, 16a, 21R, 22a, 28-
hexahydroxyolean-1 2-ene,
",C
za
O - CN3
O
o
25 26 CH,ON
,,OH
z7 =
O o cooH oN
O z ''zz
HO OH HO
HO
oN O
Ho O
OH
OH (Z4)
3-0-[R-D-galactopyranosyl(1 ->2)]-a-L-arabinofuranosyl(1->3)-R-D-
glucuronopyranosyl-
21-0-angeloyl, 22-O-benzoyl-3(3, 15a, 16a, 21(3, 22cc, 28-hexahydroxyolean-12-
ene,
, H,C
29
O CH!
O
HC
~ ..... O
CHZOH CH2
O
OH
=
O O OOM 27
OH
OH O z ''zs
HO HO
HO
OH O
M O
OH
oH (Z5)
3-0-[R-D-galactopyranosyl(1->2)]-a-L-arabinofuranosyl(1 -+3)-R-D-
glucuronopyranosyl-
20 21-0-angeloyl, 22-O-angeloyl-3(3, 15a, 16a, 21R, 22a, 28-hexahydroxyolean-
12-ene,
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WO 2008/133766 PCT/US2008/002086
HO O
fp /-O I
0-V~
OHa
C/4+OH O
ON
27 :
O Ooom OH
OH p 2 }.n
HO
HO HO
oH O
HO p
OH
pH (Z6)
3-0-[R-D-galactopyranosyl(1->2)]-a-L-arabinofuranosyl(1 ->3)-R-D-
glucuronopyranosyl-
21-0-(2-methylpropanoyl)-O-benzoyl, 22-0-(2-methylpropanoyl)-3[3, 15a, 16a,
21[3,
22a, 28-hexahydroxyolean-12-ene,
H3C H3C
~
O H,C}-\ ..........~ CH3
GH,OH O
OH
O O COOH 27 ~
OH p = '=..
HO HO
HO
oH p
HO p
OH
OH (Z7)
3-0-[(3-D-galactopyranosyl(1->2)]-a-L-arabinofuranosyl(1->3)-R-D-
glucuronopyranosyl-
21-0-(2-methylpropanoyl)-O-angeloyl, 22-0-(2- methylbutanoyl) -3[i, 15a, 16a,
21R,
22a, 28-hexahydroxyolean-12-ene,
~
2B /
O
O
...... m O
26 CHZOH
OH
27 -
O O ~H OH
OH O 2 z~
HO HO
HO
OH 0
HO 0
OH
OH Z8
3-0-[R-D-galactopyranosyl(1->2)]-a-L-arabinofuranosyl(1 ->3)-R-D-
glucuronopyranosyl-
21-O-benzoyl, 22-O-benzoyl-3(3, 15a, 16a, 21 [i, 22a, 28-hexahydroxyolean-12-
ene
This invention provides uses of a compound selected from a compound with
formula
(113), for modulating, regulating or interacting the adhesion protien, wherein
the
adhesion proteins comprise fibronectin, integrins family, Myosin ,
vitronectin, collagen,
laminin, Glycosylation cell surface proteins, polyglycans, cadherin, heparin,
tenascin,
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CD 54, CAM, elastin and FAK. In an embodiment, this invention provides a
method of
reducing the secretion of fibronectin :
3 28
OR,
OR2
25 26
R4
Ra
27 =
R50 R3
23 24 also named as (1 B),
or a salt, ester, metabolite or derivative thereof, wherein R1 comprises a
group selected
from hydrogen, angeloyl, acetyl, tigloyl, senecioyl, alkyl, dibenzoyl,
benzoyl, alkanoyl,
alkenoyl, benzoyl alkyl substituted alkanoyl, acyl, aryl, heterocylic,
heteroraryl and
derivatives thereof; R2 comprises a group selected from hydrogen, angeloyl,
acetyl,
tigloyl, senecioyl, alkyl, benzoyl, dibenzoyl, alkanoyl, alkenoyl, benzoyl
alkyl substituted
alkanoyl, aryl, acyl, heterocylic, heteroraryl and derivative thereof; R4
represents
CH2OR6 or COOR6, wherein R6 is selected from hydrogen, angeloyl, acetyl,
tigloyl,
senecioyl, alkyl, benzoyl, dibenzoyl, alkanoyl, alkenoyl, benzoyl alkyl
substituted
alkanoyl, aryl, acyl, heterocylic, heteroraryl and derivative thereof; R3 is H
or OH;
wherein at least one of R1, R2,and R6 comprises a group selected from
angeloyl, acetyl,
tigloyl, senecioyl, benzoyl, dibenzoyl, alkanoyl, alkenoyl, benzoyl alkyl
substituted
alkanoyl, aryl, acyl, heterocylic, heteroraryl and derivative thereof; R5
comprises a
sugar moiety, wherein the sugar moiety comprises at least one sugar of, but is
not
limited to, D-glucose, D-galactose, L-rhamnose, L-arabinose, D-xylose,
alduronic acid:
D-glucuronic acid, D-galacturonic acid or a derivative thereof, or the
combination thereof.
In an embodiment, R1 comprises a sugar moiety wherein substituted with two
groups
selecting from angeloyl, acetyl, tigloyl, senecioyl, benzoyl, dibenzoyl,
alkanoyl, alkenoyl,
benzoyl alkyl substituted alkanoyl, aryl, acyl, heterocylic heteroraryl and a
derivative
thereof.
In an embodiment, R1 comprises a sugar moiety wherein substituted with at
least one
group selecting from angeloyl, acetyl, tigloyl, senecioyl, benzoyl, dibenzoyl,
alkanoyl,
alkenoyl, benzoyl alkyl substituted alkanoyl, aryl, acyl, heterocylic,
heteroraryl and a
derivative thereof.
In an embodiment, R2 comprises a sugar moiety wherein at least one group is
selected
from angeloyl, acetyl, tigloyl, senecioyl, alkyl, benzoyl, dibenzoyl,
alkanoyl, alkenoyl,
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WO 2008/133766 PCT/US2008/002086
benzoyl alkyl substituted alkanoyl, aryl, acyl, heterocylic, heteroraryl and a
derivative
thereof.
In an embodiment, R2 comprises a sugar moiety or a side chain wherein at least
two
groups are selected from angeloyl, acetyl, tigloyl, senecioyl, alkyl, benzoyl,
dibenzoyl,
alkanoyl, alkenoyl, benzoyl alkyl substituted alkanoyl, aryl, acyl,
heterocylic, heteroraryl
and a derivative thereof.
In an embodiment, R4 comprises CH2OR6 or COOR6 wherein R6 is a sugar moiety
which comprises at least one group selected from angeloyl, acetyl, tigloyl,
senecioyl,
benzoyl, dibenzoyl, alkanoyl, alkenoyl, benzoyl alkyl substituted alkanoyl,
aryl, acyl,
heterocylic, heteroraryl and a derivative thereof.
In an embodiment, R4 comprises CH2OR6 orCOOR6, wherein R6 is a sugar moiety
which comprises at least two groups selected from angeloyl, acetyl, tigloyl,
senecioyl,
benzoyl, dibenzoyl, alkanoyl, alkenoyl, benzoyl alkyl substitutedalkanoyl,
aryl, acyl,
heterocylic, heteroraryl and a derivative thereof.
In an embodiment, R4 comprises CH2OR6 or COOR6, wherein R6 is a sugar moiety
which comprises at least two groups selected from angeloyl, acetyl, tigloyl
and
senecioyl.
In an embodiment, R4 comprises CH2OR6 or COOR6 of formula (1 B), at least two
of R1,
R2 and R6 comprise the group selected from angeloyl, acetyl, tigloyl,
senecioyl, benzoyl,
dibenzoyl, alkanoyl, alkenoyl, benzoyl alkyl substituted alkanoyl, aryl, acyl,
heterocylic,
heteroraryl and a derivative thereof.
In an embodiment, R4 comprises CH2OR6 or COOR6 of formula (1 B), at least two
of R1,
R2 and R6 comprise angeloyl, benzoyl, alkenoyl, or a derivative thereof.
In an embodiment, R4 is a side chain comprising CHZOCOCH3, CH2COO-alkyl,
CH2OH,
COOH, angeloyl, acetyl, tigloyl, senecioyl, alkyl, benzoyl, dibenzoyl,
alkanoyl, alkenoyl,
benzoyl alkyl substituted alkanoyl, aryl, acyl, heterocylic or heteroraryl or
a derivative
thereof.
In a further embodiment, R5 comprises a sugar moiety, wherein the sugar moiety
comprises one or more sugar of, but is not limited to glucose, galactose,
rhamnose,
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arabinose, xylose, fucose, allose, altrose, gulose, idose, lyxose, mannose,
psicose,
ribose, sorbose, tagatose, talose, fructose, or alduronic acid: glucuronic
acid,
galacturonic acid, or derivatives thereof, or the combination thereof.
In an embodiment, R5 comprises a sugar moiety or a group capable of performing
the
function of the sugar moiety.
In an embodiment, the R5 represents H.
In an embodiment, R4 represents H, OH or CH3.
In an embodiment, position C23, C24, C25, C26, C29 and C30 of the compound
independently comprise CH3, CH2OH, CHO, COOH, COOa-lkyl, COO-aryl, COO-
heterocyclic, COO-heteroaryl, CH2OaryI, CH2O- heterocyclic, CH2O- heteroaryl,
alkyls
group, acetyl group or derivatives thereof, particular CH3.
In an embodiment, R1 and R2 independently comprise an angeloyl group.
In an embodiment, R1 is a sugar moiety or a side chain which comprise two
angeloyl
groups.
In an embodiment, R1 and R2 independently comprise a benzoyl group.
In an embodiment, R1 is a sugar moiety which is substituted with two benzoly
groups.
In an embodiment, R3 represents H or OH.
In an embodiment, R8 may be OH
Substitution, deletion and/or addition of any group in the above-described
compounds
by other group(s) will be apparent to one of ordinary skill in the art based
on the
teachings of this application. In a further embodiment, the substitution,
deletion and/or
addition of the group(s) in the compound of the invention does not
substantially affect
the biological function of the compound. A composition comprising an effective
amount
of the compound selected from the above formula or a salt, ester, metabolite
or
derivative thereof as a medicament for regulating or interacting with adhesion
protien,
wherein the adhesion proteins comprise fibronectin, integrins family, Myosin,
vitronectin,
collagen, laminin, Glycosylation cell surface proteins, polyglycans, cadherin,
heparin,
tenascin, CD 54, CAM, elastin and FAK. In an embodiment, this invention
provides a
method of reducing the secretion of fibronectin; wherein the medicament is for
inhibiting
tumor or cancer cell growth and for treating cancer, wherein the cancers
comprise
breast cancer, leukocyte cancer, liver cancer, ovarian cancer, bladder cancer,
prostate
cancer, skin cancer, bone cancer, brain cancer, leukemia cancer, lung cancer,
colon
cancer, CNS cancer, melanoma cancer, renal cancer or cervix cancer.
This invention provides uses of a compound selected from a compound of formula
(1D),
for regulating or interacting with adhesion protien, wherein the adhesion
proteins
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comprise of fibronectin, integrins family, Myosin, vitronectin, coliagen,
laminin,
Glycosylation cell surface proteins, polyglycans, cadherin, heparin, tenascin,
CD 54,
CAM, elastin and FAK. In an embodiment, this invention provides a method of
reducing
the secretion of fibronectin.
3 2p
OR,
MnOR=
28 4
OH
O O R, t7 R
OH O 2 =27 ~
HO HO HO
OR5 and also named as (1 D),
or a salt, ester, metabolite or derivative thereof, wherein R1 comprises a
group selected
from hydrogen, angeloyl, acetyl, tigloyl, senecioyl, alkyl, benzoyl, alkanoyl,
alkenoyl,
benzoyl alkyl substituted alkanoyl, aryl, acyl, heterocylic, heteroraryl and a
derivative
10 thereof; R2 comprises a group selected from hydrogen, angeloyl, acetyl,
tigloyl,
senecioyl, alkyl, benzoyl, alkanoyl, alkenoyl, benzoyl alkyl substituted
alkanoyl, aryl,
acyl, heterocylic, heteroraryl and a derivative thereof; R4 comprises CHZOR6
or
COOR6, wherein R6 comprises a group selected from hydrogen, angeloyl, acetyl,
tigloyl,
senecioyl, alkyl, benzoyl, alkanoyl, alkenoyl, benzoyl alkyl substituted
alkanoyl, aryl,
15 acyl, heterocylic, heteroraryl and a derivative thereof; R3 is H or OH; R5
comprises
sugar moiety, D-glucose or D-galactose; R7 represents COOH; wherein at least
one of
R1 and R2 is an acyl.
In an embodiment, R7 is selected from CH3, CH2OH, COOH and COOalkyl.
20 In an embodiment, R7 is selected from CH3, CHZOH, CHO, COOH, COOalkyl,
COOaryl,
COO-heterocyclic, COO-heteroaryl, CHZOaryl, CHZO- heterocyclic, CH2O-
heteroaryl,
alkyls group, acetyl group and a derivative thereof.
In an embodiment, R1 represents a compound comprising a sugar moiety wherein
the
sugar moiety is substituted with at least two compounds selected from
angeloyl, acetyl,
25 tigloyl, senecioyl, alkyl, benzoyl, alkanoyl, alkenoyl, benzoyl alkyl
substituted alkanoyl,
aryl, acyl, heterocylic , heteroraryl and a derivative thereof;
In an embodiment, R1 represents a compound comprising a sugar moiety
substituted
with at least one selected from angeloyl, acetyl, tigloyl, senecioyl, benzoyl,
alkanoyl,
alkenoyl, benzoyl alkyl substituted alkanoyl, aryl, acyl, heterocylic,
heteroraryl and a
derivative thereof;
In an embodiment, R2 represents a compound comprising a sugar moiety, wherein
the
sugar moiety substituted with at least one selected from angeloyl, acetyl,
tigloyl,
26
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WO 2008/133766 PCT/US2008/002086
senecioyl, benzoyl, alkanoyl, alkenoyl, benzoyl alkyl substituted alkanoyl,
aryl, acyl,
heterocylic, heteroraryl and a derivative thereof;
In an embodiment, R2 represents a compound comprising a sugar moiety or a
compound which substituted with at least two selected from, angeloyl, acetyl,
tigloyl,
senecioyl, benzoyl, alkanoyl, alkenoyl, benzoyl alkyl substituted alkanoyl,
aryl, acyl,
heterocylic, heteroraryl and a derivative thereof;
In an embodiment, R4 comprises a group selected from CH2OR6 and COOR6 wherein
R6 is a sugar moiety which substituted with at least one selected from
angeloyl, acetyl,
tigloyl, senecioyl, benzoyl, alkanoyl, alkenoyl, benzoyl alkyl substituted
alkanoyl, aryl,
acyl, heterocylic, heteroraryl and a derivative thereof;
In an embodiment, R4 comprises a group selected from CH2OR6 and COOR6 wherein
R6 is a sugar moiety which substituted with at least two selected from
angeloyl, acetyl,
tigloyl, senecioyl, benzoyl, alkanoyl, alkenoyl, benzoyl alkyl substituted
alkanoyl, aryl,
acyl, heterocylic, heteroraryl and a derivative thereof;
In an embodiment, R4 comprises a group selected from CH2OR6 and COOR6 wherein
R6 is a sugar moiety which substituted with at least two selected from
angeloyl, acetyl,
tigloyl and senecioyl.
In an embodiment, R4 comprises a group selected from CH2OR6 and COOR6 wherein
R6 is a sugar moiety which substituted with at least two selected from
angeloyl, acetyl,
tigloyl, senecioyl, benzoyl, alkanoyl, alkenoyl, dibenzoyl, benzoyl alkyl
substituted
alkanoyl, aryl, acyl, heterocylic, heteroraryl and a derivative thereof;
In an embodiment, R4 comprises a group seleced from CHZOR6 and COOR6 wherein
at least two of R1, R2 and R6 comprise a group selected from angeloyl, acetyl,
tigloyl,
senecioyl, benzoyl, alkanoyl, alkenoyl, benzoyl alkyl substituted alkanoyl,
aryl, acyl,
heterocylic , heteroraryl and derivative thereof;
In an embodiment, R4 comprises a group selected from CHZOCOCH3, CH2COOalkyl,
CH2OH, COOH, angeloyl, acetyl, tigloyl, senecioyl, alkyl, benzoyl, alkanoyl,
alkenoyl,
benzoyl alkyl substituted alkanoyl, aryl, acyl, heterocylic, heteroraryl and
derivative
thereof.
In a further embodiment, R5 comprises a sugar moiety, glucose, galactose,
rhamnose,
arabinose, xylose, fucose, allose, altrose, gulose, idose, lyxose, mannose,
psicose,
ribose, sorbose, tagatose, talose, fructose, alduronic acid, glucuronic acid
or
galacturonic acid, or derivative thereof, or the combination thereof. In an
embodiment,
R5 comprises a compound capable of performing the function of the sugar
moiety. In a
27
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WO 2008/133766 PCT/US2008/002086
further embodiment, the R5 comprises a H. In a further embodiment, R4
represents H
or OH or CH3.
In an embodiment, position 24 of the compound is CH3 or CH2OH, In a further
embodiment, positions 23, 24, 25, 26, 29, 30 of the compound independently
comprise
CH3, CHZOH, CHO, COOH, COOalkyl, COOaryl, COO-heterocyclic, COO-heteroaryl,
CHZOaryI, CH2O- heterocyclic, CH2O- heteroaryl, alkyls group, acetyl group or
a
derivative thereof.
In an embodiment, R5 comprises a sugar moiety comprising L-glucose, D-
galactose, L-
rhamnose, or/and L-arabinose.
In an embodiment, R1 and R2 independently comprise an angeloyl group; In a
embodiment, R1 is a sugar moiety or rhamnose which comprise two angeloyl
groups.
In an embodiment, R3 represents H or OH; In a further embodiment, the
compounds
canbe isolated from natural sources or synthesized.
A sugar moiety is a segment of a molecule comprising one or more sugar groups.
Substitution, deletion and/or addition of any group in the above-described
compounds
will be apparent to one of ordinary skill in the art based on the teaching of
this
application. In a further embodiment, the substitution, deletion and/or
addition of the
group(s) in the compound of the invention does not substantially affect the
biological
function of the compound.
A method of inhibiting venous insufficiency, particularly hemorrhoids or
inhibiting leg
swelling, or peripheral edema, antilipemic, chronic venous disease, varicose
vein
disease, varicose syndrome, venous stasis, Expectorant, peripheral vascular
disorders,
cerebro-organic convulsion, cerebral circulation disorder, cerebral edema,
psychoses,
dysmenorrheal, hemorrhoids, episiotomies, hamonhoids, peripheral edema
formation or
postoperative swelling; for reducing symptoms of pain; for reducing symptoms
of
stomach pain; for reducing symptoms of leg pain; for treating pruritis, lower
leg volume,
thrombosis, thromophlebitis; for preventing gastric ulcers antispasmotic
comprising
administering to a subject, in need thereof, an effective amount of the
composition of
any one of the above compounds or a compound comprises a triterpene which
comprises any two of angeloyl, tigloyl, senecioyl, perferable two angeloyl
groups, and a
sugar moiety, glucose, galactose, rhamnose, arabinose, xylose, fucose, allose,
altrose,
gulose, idose, lyxose, mannose, psicose, ribose, sorbose, tagatose, talose,
fructose,
alduronic acid, glucuronic acid or galacturonic acid, or a derivative thereof,
or the
28
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WO 2008/133766 PCT/US2008/002086
combination thereof, preferable selected from glucuronic acid, galacturonic
acid,
glucose, galactose and arabinose. The method is regulating or interacting with
adhesion
protien, wherein the adhesion proteins comprise fibronectin, integrins family,
Myosin,
vitronectin, collagen, laminin, Glycosylation cell surface proteins,
polyglycans, cadherin,
heparin, tenascin, CD 54, CAM, elastin and FAK. In an embodiment, the method
is
reducing the secretion of fibronectin.
This invention provides a method for inhibiting the growth, migration,
metastasis of
cancer by altering the characteristic of membrane of cancer cell, wherein the
characteristic comprise reducing adhesion protein; wherein the adhesion
proteins
comprise fibronectin, integrins family, Myosin, vitronectin, collagen,
laminin,
Glycosylation cell surface proteins, polyglycans, cadherin, heparin, tenascin,
CD 54,
CAM, elastin and FAK; wherein comprising inhibiting the secretion of
fibronectin,
wherein comprising administering to a subject, in need thereof, an appropriate
amount
of triterpenoidal saponins comprising two or more angeloyl groups, or a
compound
comprises a triterpene which comprises any two of angeloyl, tigloyl,
senecioyl,
perferable two angeloyl groups, and a sugar moiety, glucose, galactose,
rhamnose,
arabinose, xylose, fucose, allose, altrose, gulose, idose, lyxose, mannose,
psicose,
ribose, sorbose, tagatose, talose, fructose, alduronic acid, glucuronic acid
or
galacturonic acid, or a derivative thereof, or the combination thereof,
preferably selected
from glucuronic acid, galacturonic acid, glucose, galactose and arabinose.
This
invention provides a composition comprising an effective amount of the
compound of
any one of compound selected from the above formula or a salt, ester,
metabolite or
derivative thereof as a medicament for reducing adhesion protein; wherein the
adhesion
proteins comprise fibronectin, integrins family, Myosin , vitronectin,
collagen, laminin,
Glycosylation cell surface proteins, polyglycans, cadherin, heparin, tenascin,
CD 54,
CAM, elastin and FAK, for inhibiting the growth, migration, metastasis of
cancer,
wherein the cancers comprise breast cancer, leukocyte cancer, liver cancer,
ovarian
cancer, bladder cancer, prostate cancer, skin cancer, bone cancer, brain
cancer,
leukemia cancer, lung cancer, colon cancer, CNS cancer, melanoma cancer, renal
cancer or cervix cancer.
This invention also provides a composition comprising the above described
compounds
or their derivatives for reducing adhesion protein, wherein the adhesion
proteins
comprise fibronectin, integrins family, Myosin , vitronectin, collagen,
laminin,
Glycosylation cell surface proteins, polyglycans, cadherin, heparin, tenascin,
CD 54,
29
CA 02676791 2009-07-27
WO 2008/133766 PCT/US2008/002086
CAM, elastin and FAK; wherein comprising inhibiting the secretion of
fibronectin,
wherein for treating venous insufficiency, particularly hemorrhoids or
inhibiting leg
swelling, or peripheral edema, antilipemic, chronic venous disease, varicose
vein
disease, varicose syndrome, venous stasis, Expectorant, peripheral vascular
disorders,
cerebro-organic convulsion, cerebral circulation disorder, cerebral edema,
psychoses,
dysmenorrheal, episiotomies, hemonhoids, peripheral edema formation or
postoperative
swelling; for reducing symptoms of pain; for reducing symptoms of stomach
pain; for
reducing symptoms of leg pain; for treating pruritis, lower leg volume,
thrombosis,
thromophlebitis; for preventing gastric ulcers antispasmotic comprising
administering to
a subject, in need thereof, an effective amount of the composition.
In an embodiment of the above, the uses of compositions comprising any one of
triterpenoid saponins with the following formula:
3-0-{[P-D-galactopyranosyl (1 ->2)]-[a-L-arabinofuranosyl (1->3)]-fi-D-
glucuronopyranoside butyl ester}-21-O-acetyl-22-O-angeloyl-3,8,16a,21A22a,28-
pentahydroxyolean-12-ene.
3-0-[(3-D-galactopyranosyl(1 ->2)]-a-L-arabinofuranosyl(1 ->3)-(3-D-
glucuronopyranosyl-
21,22-O-diangeloyl-3[3, 15a, 16a, 21R, 22a, 28-hexahydroxyolean-12-ene,
3-0-[(3-D-galactopyranosyl(1 ->2)]-a-L-arabinofuranosyl(1 ->3)-[3-D-
glucuronopyranosyl -
21-0-(3,4-diangeloyl)-a-L-rhamnophyranosyl-22-O-acetyl-3R,16a, 21(3, 22a, 28-
pentahydroxyolean-1 2-ene,
3-0-[R-D-glucopyranosyl-(1->2)]-a-L-arabinofuranosyI(1->3)-R-D-
glucuronopyranosyl-
21,22-O-diangeloyl-3(3, 15a, 16a, 21R, 22a, 24[i, 28-heptahydroxyolean-12-ene,
3-0-[/3-glucopyranosyl (1 ->2)]-a-arabinofuranosyl (1->3)-fl-
glucuronopyranosyl-21, 22-
O-diangeloyl-3Q, 16a, 21A 22a, 24,8, 28-hexahydroxyolean-12-ene,
3-0-[P-galactopyranosyl (1->2)]-a-arabinofuranosyl (1->3)-/.3-
glucuronopyranosyl-21-O-
(3,4-diangeloyl)-a-rhamnopyranosyl-28-O-acetyl-3,8, 16a, 21A 22a, 28-
pentahydroxyolean-1 2-ene,
3-0-[P-galactopyranosyl (1 ->2)]-a-arabinofuranosyl (1->3)-fl-
glucuronopyranosyl-21,
22-0-diangeloyl-3/3, 16a, 21,Q, 22a, 28-pentahydroxyolean-12-ene,
3-0-[R-D-galactopyranosyl(1 ->2)]-a-L-arabinofuranosyl(1 ->3)-R-D-
glucuronopyranosy I-
21-0-angeloyl, 22-0-(2-methylpropanoyl)-3R, 15a, 16a, 21 R, 22a, 28-
hexahydroxyolean-1 2-ene,
3-0-[P-D-galactopyranosyl-(1 --+2)]-a-L-arabinofuranosyl-(1-+3)-13-D-
-glucuronopyranosyl-21-O-angeloyl-28-0-2-methylbutanoyl-3/3, 15 a, 16a, 21.8,
22a, 28-
hexahydroxyolean-1 2-ene
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This invention provides a composition comprising the compounds as described
above
effective in regulating or reducing adhesion protein, wherein the adhesion
proteins
comprise fibronectin, integrins family, Myosin , vitronectin, collagen,
laminin,
Glycosylation cell surface proteins, polyglycans, cadherin, heparin, tenascin,
CD 54,
CAM, elastin and FAK; inhibiting venous insufficiency, particularly
hemorrhoids or
inhibiting of leg swelling, and inhibiting cancer growth. The cancer includes
but is not
limited to bladder cancer, bone, cancer, skin cancer and ovarian cancer.
This invention also provides a composition for regulating or reducing adhesion
protein,
wherein the adhesion protein comprising fibronectin, integrins family, CD44,
Myosin VI,
vitronectin collagen, laminin, Glycosylation cell surface proteins,
polyglycans and FAK;
inhibiting venous insufficiency, particularly hemorrhoids or inhibition of leg
swelling, or
inhibiting cancer growth comprising any of compounds selected from the
following
compounds:
A) 3-0-[R-D-galactopyranosyl (1->2)]-a-L-arabinofuranosyl (1-)~3)-R-D-
glucuronopyranosyl-21, 22-O-diangeloyl-3R, 15a, 16a, 21R, 22a, 28-
hexahydroxyolean-
12-ene,
B) 3-0-[R-D-galactopyranosyl (1->2)]-a-L-arabinofuranosyl (1->3)-(3-D-
glucuronopyranosyl-21-O-(3, 4-diangeloyl)-a-L-rhamnophyranosyl-22-O-acetyl-
3R,16(X,
21 p, 22a, 28-pentahydroxyolean-12-ene
C) 3-0-[R-D-glucopyranosyl-(1->2)]-a-L-arabinofuranosyl (1->3)-(-D-
glucuronopyranosyl-21, 22-O-diangeloyl-3A 15a, 16a, 21A 22a, 24Q, 28-
heptahydroxyolean-12-ene
D) 3-0-[g-galactopyranosyl (1->2)]-a-arabinofuranosyl (1-->3)-fl-
glucuronopyranosyl-21,
22-O-diangeloyl-3/3, 16a, 21,Q, 22a, 28-pentahydroxyolean-12-ene
E) 3-0-[fl-galactopyranosyl (1->2)]-a-arabinofuranosyl (1-+3)-p-
glucuronopyranosyl-21-
O-(3,4-diangeloyl)-a-rhamnopyranosyl-28-O-acetyl-3A 16a, 21A 22a, 28-
pentahydroxyolean-1 2-ene
F) 3-0-[#-galactopyranosyl (1->2)]-a-arabinofuranosyl (1-+3)-fl-
glucuronopyranosyl-21,
22-O-diangeloyl-3Q, 16a, 21A 22a, 28-pentahydroxyolean-12-ene
G) 3-0-[(3-D-galactopyranosyl (1->2)]-a-L-arabinofuranosyl (1->3)-(3-D-
glucuronopyranosyl -21, 22-O-dibenzoyl-3R, 15a, 16a, 21(3, 22(x, 28-
hexahydroxyolean-
12-ene,
H) 3-0-[(3-D-galactopyranosyl(1->2)]-a-L-arabinofuranosyl(1 ->3)-(3-D-
glucuronopyranosyl-21-O-(3,4- dibenzoyl)-a-L-rhamnophyranosyl-22-O-acetyl-
3R,16a,
21 R, 22a, 28-pentahydroxyolean-12-ene
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I) 3-0-[R-D-glucopyranosyl-(1 -+2)]-a-L-arabinofuranosyi (1->3)-(-D-
glucuronopyranosyl
-21, 22-0- dibenzoyl -3A 15a, 16a, 21A 22a, 24A 28-heptahydroxyolean-12-ene
J) 3-0-[#-galactopyranosyl (1-+2)]-a-arabinofuranosyl (1->3)-fl-
glucuronopyranosyl-21,
22-0- dibenzoyl -3Q, 16a, 21,8, 22a, 28-pentahydroxyolean-12-ene
K) 3-0-[#-galactopyranosyl (1->2)]-a-arabinofuranosyl (1->3)-fl-
glucuronopyranosyl-21-
O-(3,4-dibenzoyl)-a-rhamnopyranosyl-28-O-acetyl-3/3, 16a, 21Q, 22a, 28-
pentahydroxyolean-1 2-ene
L) 3-0-[,B-galactopyranosyl (1->2)]-a-arabinofuranosyl (1->3)-fl-
glucuronopyranosyl-21,
22-0- dibenzoyl -3A 16a, 21,8, 22a, 28-pentahydroxyolean-12-ene
M) 3-0-[P-D-galactopyranosyl (1-+2)]-[3-D-xyopyranosyl (1->3)-[i-D-
glucuronopyranosyl
-21, 22-0-dibenzoyl-3(3, 15a, 16a, 21(3, 22a, 28-hexahydroxyolean-12-ene,
N) 3-0-[R-D-galactopyranosyl(1 ->2)]- R-D-xyopyranosyl (1 -+3)-R-D-
glucuronopyranosyl-21-O-(3,4- dibenzoyl)-a-L-rhamnophyranosyl-22-0-acetyl-
3R,16a,
21 R, 22(x, 28-pentahydroxyolean-1 2-ene
0) 3-0-[(3-D-glucopyranosyl-(1->2)]-(3-D-xyopyranosyi (1->3)-(-D-
glucuronopyranosyl -
21, 22-0- dibenzoyl -3/.3, 15a, 16a, 21A 22a, 24,6, 28-heptahydroxyolean-12-
ene
P) 3-0-[#-D-galactopyranosyl (1-*2)]-[3-D-xyopyranosyl (1->3)-fl-D-
glucuronopyranosyl
-21, 22-0- dibenzoyl -3,6, 16a, 21/3, 22a, 28-pentahydroxyolean-12-ene
Q) 3-0-[/3-galactopyranosyl (1-+2)]- R- xyopyranosyl (1->3)-
,Qglucuronopyranosyl-21-0-
(3,4- dibenzoyl)-a-rhamnopyranosyl-28-0-acetyl-3,8, 16a, 21A 22a, 28-
pentahydroxyolean-12-ene
R) 3-0-[P-galactopyranosyl (1->2)]-R- xyopyranosyl (1->3)-p-glucuronopyranosyl-
21,
22-0- dibenzoyl -3A 16a, 21,6, 22a, 28-pentahydroxyolean-12-ene
S) 3-0-[R-D-galactopyranosyl (1->2)] - R- D-xyopyranosyl (1->3)-(3-D-
glucuronopyranosyl-21, 22-0-diangeloyl-3p, 15a, 16a, 21R, 22a, 28-
hexahydroxyolean-
12-ene,
T) 3-0-[R-D-galactopyranosyl (1->2)] - [i- D-xyopyranosyl (1-+3)-R-D-
glucuronopyranosyl-21-0-(3,4-diangeloyl)-a-L-rhamnophyranosyl-22-0-acetyl-
3(3,16a,
21 R, 22(x, 28-pentahydroxyolean-12-ene
U) 3-0-[R-D-glucopyranosyl-(1-+2)] - [3- D-xyopyranosyl (1-+3)-(-D-
glucuronopyranosyl-
21, 22-0-diangeloyl-3Q, 15a, 16a, 21A 22a, 24Q, 28-heptahydroxyolean-12-ene
V) 3-0-[O-galactopyranosyl (1->2)] - [i- D-xyopyranosyl (1->3)-p-
glucuronopyranosyl-21,
22-0-diangeloyl-3/.3, 16a, 21,6, 22a, 28-pentahydroxyolean-12-ene
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WO 2008/133766 PCT/US2008/002086
W) 3-0-[P--galactopyranosyl (1->2)] - R- D-xyopyranosyl (1->3)-fl-
glucuronopyranosyl-
21-0- (3, 4-diangeloyl)-a-rhamnopyranosyl-28-0-acetyl-3A 16a, 21Q, 22a, 28-
pentahydroxyolean-12-ene
X) 3-0-[#-D-galactopyranosyl (1->2)] - (3- D-xyopyranosyl (1->3)-fi-D-
glucuronopyranosyl-21, 22-0-diangeloyl-3Q, 16a, 21Q, 22a, 28-pentahydroxyolean-
12-
ene
This invention provides a composition for regulating or reducing adhesion
protein,
wherein the adhesion proteins comprise fibronectin, integrins family, Myosin ,
vitronectin, collagen, laminin, Glycosylation cell surface proteins,
polyglycans, cadherin,
heparin, tenascin, CD 54, CAM, elastin and FAK; wherein blocking the
migration,
metastasis of cancer cells inhibiting venous insufficiency, particularly
hemorrhoids or
inhibiting leg swelling, inhibiting cancer growth comprising any of the
compounds
selected from the following:
A1) 3-0-[[3-D-galactopyranosyl (1->2)]-(x-L-arabinofuranosyl(1->3)-(3-D-
glucuronopyranosyl-2l-O-angeloyl,22-O-benzoyl-3R, 15a, 16a, 21(3, 22a, 28-
hexahydroxyolean-1 2-ene,
131) 3-0-[R-D-galactopyranosyl (1 ->2)]-a-L-arabinofuranosyl(1 ->3)-R-D-
glucuronopyranosyl-21-O-(3-angeloyl, 4-benzoyl)-a-L-rhamnophyranosyl-22-0-
acetyl-
3R,16a, 21R, 22a, 28-pentahydroxyolean-12-ene
C1) 3-0-[R-D-glucopyranosyl-(1->2)]-a-L-arabinofuranosyl (1->3)-(-D-
glucuronopyranosyl-21-0-angeloyl,22-O-benzoyl-3A 15a, 16a, 21A 22a, 24A 28-
heptahydroxyolean-1 2-ene
D1) 3-0-[fl-galactopyranosyl (1 ->2)]-a-arabinofuranosyl (1->3)-fl-
glucuronopyranosyl-
21-0-angeloyl, 22-benzoyl-3Q, 16a, 21,6, 22a, 28-pentahydroxyolean-12-ene
El) 3-0-[fl-galactopyranosyl (1-+2)]-a-arabinofuranosyl (1->3)-fl-
glucuronopyranosyl-
21-0-F) (3-angeloyl, 4-benzoyl)-a-rhamnopyranosyl-28-0-acetyl-3/j, 16a, 21/j,
22a, 28-
pentahydroxyolean-12-ene
171) 3-0-[#-galactopyranosyl (1->2)]-a-arabinofuranosyl (1->3)-fl-
glucuronopyranosyl-
21-0-angeloyl, 22-O-benzoyl-3Q, 16a, 21,6, 22a, 28-pentahydroxyolean-12-ene
G1) 3-0-[R-D-galactopyranosyl (1->2)]-a-L-arabinofuranosyl (1->3)-p-D-
glucuronopyranosyl-21 -0-benzoyl, 22-0-angeloyl-3(3, 15a, 16a, 21(3, 22a, 28-
hexahyd roxyolea n-12-ene,
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H1) 3-0-[[3-D-galactopyranosyl(1->2)]-a-L-arabinofuranosyl(1 -+3)-R-D-
glucuronopyranosyl-21-O-(3- benzoyl, 4-angeloyl)-a-L-rhamnophyranosyl-22-O-
acetyl-
3R,16a, 210, 22a, 28-pentahydroxyolean-12-ene
11) 3-0-[R-D-gIucopyranosyl-(1-*2)]-a-L-arabinofuranosyl (1->3)-(-D-
glucuronopyranosyl-21 -0-benzoyl, 22-0-angeloyl-3,6, 15a, 16a, 21A 22a, 24A 28-
heptahydroxyolean-1 2-ene
J1) 3-0-[fl-galactopyranosyl (1->2)]-a-arabinofuranosyl (1-+3)-fl-
glucuronopyranosyl-21
-0-benzoyl, 22-0-angeloyl-3A 16a, 21A 22a, 28-pentahydroxyolean-12-ene
K1) 3-0-[P-galactopyranosyl (1->2)]-a-arabinofuranosyl (1->3)-p-
glucuronopyranosyl-
21-0-(3- benzoyl, 4-angeloyl)-a-rhamnopyranosyl-28-0-acetyl-3A 16a, 21Q, 22a,
28-
pentahydroxyolean-1 2-ene
L1) 3-0-[P-galactopyranosyl (1 ->2)]-a-arabinofuranosyl (1 -+3)-fl-
glucuronopyranosyl-21
-0-benzoyl, 22-0-angeloyl-3A 16a, 21A 22a, 28-pentahydroxyolean-12-ene
M1) 3-0-[{3-D-galactopyranosyl (1->2)]-(3-D-xyopyranosyl (1->3)-R-D-
glucuronopyranosyl-21 -0-angeloyl, 22-0-benzoyl-3[3, 15a, 16a, 21 [3, 22a, 28-
hexahyd roxyolea n-12-ene,
N1) 3-0-[R-D-galactopyranosyl(1->2)]- [3-D-xyopyranosyl (1--->3)-R-D-
glucuronopyranosyl-21-O-(3-angeloyl, 4- dibenzoyl)-a-L-rhamnophyranosyl-22-0-
acetyl-3[3,16a, 21 R, 22(x, 28-pentahydroxyolean-12-ene
01) 3-0-[(3-D-glucopyranosyl-(1->2)]-(3-D-xyopyranosyI (1->3)-(-D-
glucuronopyranosyl-
21 -0-21 -0-angeloyl, 22-0-benzoyl -3,6, 15a, 16a, 21/3, 22a, 24Q, 28-
heptahydroxyolean-1 2-ene,
P1) 3-0-[fl-D-galactopyranosyl (1 -+2)]- [3- D-xyopyranosyl (1->3)-fl- D-
glucuronopyranosyl-2121 -0-angeloyl, 22-0-benzoyl -3Q, 16a, 21A 22a, 28-
pentahydroxyolean-12-ene
Q1) 3-0-[,B-galactopyranosyl (1->2)]- R- xyopyranosyl (1->3)-fl-
glucuronopyranosyl-21-
O-(3-angeloyl, 4- dibenzoyl)-a-rhamnopyranosyl-28-0-acetyl-3Q, 16a, 21/j, 22a,
28-
pentahydroxyolean-12-ene,
R1) 3-0-[P-galactopyranosyl (1->2)]-R- xyopyranosyl (1->3)-/3-
glucuronopyranosyl--
angeloyl, 22-0-benzoyl --3,13, 16a, 21,6, 22a, 28-pentahydroxyolean-12-ene,
S1) 3-0-[(3-D-galactopyranosyl (1-+2)] - p- D-xyopyranosyl (1->3)-R-D-
glucuronopyranosyl-21 -0-benzoyl, 22-0-angeloyl--30, 15a, 16a, 21 R, 22a, 28-
hexahydroxyolean-12-ene,
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T1) 3-0-[(3-D-galactopyranosyl (1->2)] - R- D-xyopyranosyl (1-+3)-R-D-
glucuronopyranosyl-21-O-(3-benzoyl, 4-angeloyl)-(x-L-rhamnophyranosyl-22-O-
acetyl-
3(3, 16a, 210, 22a, 28-pentahydroxyolean-12-ene,
U1) 3-0-[[3-D-glucopyranosyl-(1->2)] - [3- D-xyopyranosyl (1-+3)-(-D-
glucuronopyranosyl-21 -O-benzoyl, 22-O-angeloyl--3p, 15a, 16a, 21A 22a, 24Q,
28-
heptahydroxyolean-1 2-ene
V1) 3-0-[#-galactopyranosyl (1->2)] - [3- D-xyopyranosyl (1->3)-p-
glucuronopyranosyl-
21 -0-benzoyl, 22-O-angeloyl-3A 16a, 21Q, 22a, 28-pentahydroxyolean-12-ene
W1) 3-0-[#-galactopyranosyl (1-->2)] - [3- D-xyopyranosyl (1->3)-fl-
glucuronopyranosyl-
21-0- (3-benzoyl, 4-angeloyl)-a-rhamnopyranosyl-28-0-acetyl-3Q, 16a, 21,(3,
22a, 28-
pentahydroxyolean-1 2-ene
X1) 3-0-[fl-D-galactopyranosyl (1-+2)] - R- D-xyopyranosyl (1->3)-fl-D-
glucuronopyranosyl-21 -O-benzoyl, 22-0-angeloyl-3,6, 16a, 21Q, 22a, 28-
pentahydroxyolean-1 2-ene.
Triterpenoid saponins with the characteristic structures mentioned above in
this
invention can be used to inhibit venous insufficiency, particularly
hemorrhoids or inhibit
leg swelling, Triterpenoid saponins with the characteristic structures
mentioned above in
this invention can be used to block the migration, metastasis of cancer cells,
reduce or
inhibit cancer growth. The cancers are included but not limited to Leukemia
cancer,
Lung cancer, Colon cancer, CNS cancer, Melanoma cancer, Ovarian cancer, Renal
cancer, Prostate cancer, Breast cancer, bladder cancer, cervix cancer, liver
cancer,
bone cancer, brain cancer and Skin cancer. Triterpenoid saponins with the
characteristic structures mentioned above in this invention can be used to
affect cell
membrane structure and adhesion process.. In an embodiment, it provides a
method of
regulating or reducing adhesion proteins to blocks the migration, metastasis
of cancer
cells, growth of cancers. In an embodiment, the method comprises reducing the
adhesion ability of the cancer cells. In an embodiment, the adhesion proteins
comprise
IgSF CAM, Selectins, Integrin or Cadherins. In an embodiment, the adhesion
proteins
comprise fibronectin, integrins family, Myosin , vitronectin, collagen,
laminin,
Glycosylation cell surface proteins, polyglycans, cadherin, heparin, tenascin,
CD 54,
CAM, elastin and FAK;ln an embodiment, the compound is a triterpenoidal
saponin or
sapogenin, wherein the triterpenoidal saponin comprises at least any one or
two of an
angeloyl group, tigloyl group, or senecioyl group, or their combinations
thereof at carbon
21 and/or 22, or 28, directly attached to the sapogenin or attached to a sugar
moiety
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can be used to to treat varicose vein disease, inhibit venous insufficiency,
particularly
hemorrhoids or inhibit leg swelling, reduce or inhibit cancer growth. In an
embodiment,
the compound is a five ring triterpene saponin comprising at least two
angeloyl groups,
tigloyl group, or senecioyl group, or their combinations thereof and a sugar
moiety. The
angeloyl groups are attached to a side chain at the end of the five rings and
a sugar
moiety is attached to a side chain of the ring at the other end of the five
rings. In an
embodiment, the compound comprises at least two angeloyl groups, a tigloyl
group, or a
senecioyl group, or combinations thereof and a sugar moiety. The angeloyl
groups and
the sugar moiety are attached to the side chains of the backbone of the
compound
respectively. In an embodiment, the angeloyl can be replaced by a functional
group
which functions as an angeloyl group. In an embodiment, a sugar moiety or
chain is at
C3 or other positions, comprising one or more sugar selected from, but is not
limited to
glucose, galactose, rhamnose, arabinose, xylose, fucose, allose, altrose,
gulose, idose,
lyxose, mannose, psicose, ribose, sorbose, tagatose, talose, fructose,
alduronic acid,
glucuronic acid, galacturonic acid, or derivatives thereof, or the combination
thereof
preferably D- glucose, D-galactose, L-rhamnose, L-arabinose, alduronic acids
of D-
glucuronic acid or D-galacturonic acid, or their combinations thereof, or
their derivatives
thereof. In a further embodiment, CH3 or CH2OH or COOH or acetyl group may
attach
at C 23-30 independently. The activities of a saponin compound for regulating
or
inhibiting tumor cell growth are based on or attributed to its structure that
has the
functional group(s) such as angeloyl group, tigloyl group, senecioyl group or
acetyl
group, or their combinations thereof.
This invention provides a composition comprising the compounds with the
structure of:
QOR~ oR' o
(a) ,,,oR2 or (b) OR2
wherein R1 and R2 comprise angeloyl groups, tigloyl groups, senecioyl groups
or acetyl
group or their combinations, preferable wherein the R1 and R2 comprise
angeloyl
groups. In an embodiment, R1 and R2 comprise compounds selected from angeloyl,
acetyl, tigloyl, senecioyl, benzoyl, dibenzoyl, alkanoyl, alkenoyl, benzoyl
alkyl
substituted alkanoyl, aryl, acyl, heterocylic, heteroraryl, or acid with 2 to
5 carbon or
derivative thereof.
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OR,
(c) (C)
wherein R1 and R2 comprise angeloyl groups, tigloyl groups, senecioyl groups
or acetyl
group or their combinations, preferable wherein the Rl and R2 comprise
angeloyl
groups.
In an embodiment, R1 and R2 comprise compounds selected from angeloyl, acetyl,
tigloyl, senecioyl, benzoyl, dibenzoyl, alkanoyl, alkenoyl, benzoyl alkyl
substituted
alkanoyl, aryl, acyl, heterocylic, heteroraryl, or acid with 2 to 5 carbon or
derivative
thereof.
In an embodiment, the compound further comprises a sugar moiety.
In a further embodiment, the sugar moiety comprises glucose, galactose or
arabinose or
combination thereof.
In an embodiment, the sugar moiety comprises at least one sugar, or glucose,
or
galactose, or rhamnose, or arabinose, or xylose, or alduronic acid, or
glucuronic acid, or
galacturonic acid, or their derivative thereof, or the combination thereof.
In an embodiment, the R1 or R 2 may be attached in other position of the
structure.
OR,
OR3
CS(5 .... OR2
(d)
wherein R1, R2 or R3 comprise angeloyl groups, tigloyl groups, senecioyl
groups or
acetyl group or their combinations, preferable wherein at least two of the R1,
R2 and R3
comprise angeloyl groups. In embodiment, at least two of R1, R2 and R3
comprise
compounds selected from angeloyl, acetyl, tigloyl, senecioyl, benzoyl,
dibenzoyl,
alkanoyl, alkenoyl, benzoyl alkyl substituted alkanoyl, aryl, acyl,
heterocylic, heteroraryl,
or acid with 2 to 5 carbon or derivative thereof.
In an embodiment, at least one of R1, R2 and R3 comprise a sugar moiety
comprising
two compounds selected from angeloyl, acetyl, tigloyl, senecioyl, benzoyl,
dibenzoyl,
alkanoyl, alkenoyl, benzoyl alkyl substituted alkanoyl, aryl, acyl,
heterocylic, heteroraryl,
or acid with 2 to 5 carbon or derivative thereof.
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In an embodiment, the compound comprises a sugar moiety. In an embodiment, the
sugar moiety is attached at one end of structure (d), opposite to R1, R2 and
R3. In a
further embodiment, the sugar moiety comprises glucose, galactose or arabinose
or
combination thereof.
In a further embodiment, the sugar moiety comprises at least one sugar, or
glucose, or
galactose, or rhamnose, or arabinose, or xylose, or alduronic acid, or
glucuronic acid, or
galacturonic acid, or their derivative thereof, or the combination thereof.
In a further embodiment, the sugar moiety comprises one or more sugar selected
from,
but is not limited to glucose, galactose, rhamnose, arabinose, xylose, fucose,
allose,
altrose, gulose, idose, lyxose, mannose, psicose, ribose, sorbose, tagatose,
talose,
fructose, alduronic acid, glucuronic acid, galacturonic acid, or derivatives
thereof, or the
combination thereof. In an embodiment, the R1, R 2 and R3 may be attached in
other
position of the structure.
In an embodiment, the compound is triterpenoid saponin comprise comprises at
least
two angeloyl groups, tigloyl groups, senecioyl groups or acetyl group or their
combinations, preferable wherein at least two angeloyl groups.
In an embodiment, at least two compounds selected from angeloyl, acetyl,
tigloyl,
senecioyl, benzoyl, dibenzoyl, alkanoyl, alkenoyl, benzoyl alkyl substituted
alkanoyl, aryl,
acyl, heterocylic, heteroraryl, or acid with 2 to 5 carbon or derivative
thereof.
In an embodiment, at least one of the side bonds comprise a sugar moiety
comprising
two compounds selected from angeloyl, acetyl, tigloyl, senecioyl, benzoyl,
dibenzoyl,
alkanoyl, alkenoyl, benzoyl alkyl substituted alkanoyl, aryl, acyl,
heterocylic, heteroraryl,
or acid with 2 to 5 carbon or derivative thereof.
In an embodiment, the compound comprises a sugar moiety. In a further
embodiment,
the sugar moiety comprises glucose, galactose or arabinose or combination
thereof.
In a further embodiment, the sugar moiety comprises at least one sugar, or
glucose, or
galactose, or rhamnose, or arabinose, or xylose, or alduronic acid, or
glucuronic acid, or
galacturonic acid, or their derivative thereof, or the combination thereof.
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In a further embodiment, the sugar moiety comprises one or more sugar selected
from,
but is not limited to glucose, galactose, rhamnose, arabinose, xylose, fucose,
allose,
altrose, gulose, idose, lyxose, mannose, psicose, ribose, sorbose, tagatose,
talose,
fructose, alduronic acid, glucuronic acid, galacturonic acid, or derivatives
thereof, or the
combination thereof.
In an embodiment, a triterpene comprise the following structure has activities
of
reducing adhesion proteins to blocks the migration, metastasis of cancer
cells, growth of
cancers.
OR,
..,111ORZ
OR3
R50
wherein at least two of R1, R2 and R3 comprise compounds selected from
angeloyl,
acetyl, tigloyl, senecioyl, benzoyl, dibenzoyl, alkanoyl, alkenoyl, benzoyl
alkyl
substituted alkanoyl, aryl, heterocylic, heteroraryl, or acid with 2 to 5
carbon or
derivative thereof. In an embodiment, at least one of R1, R2 and R3 comprise a
sugar
moiety comprising two compounds selected from angeloyl, acetyl, tigloyl,
senecioyl,
alkyl, benzoyl, dibenzoyl, alkanoyl, alkenoyl, benzoyl alkyl substituted
alkanoyl, aryl,
acyl, heterocylic, heteroraryl, or acid with 2 to 5 carbon or derivative
thereof. In
embodiment, R1, R2 or R3 comprise angeloyl groups, tigloyl groups, senecioyl
groups
or acetyl group or their combinations, preferable wherein at least two of the
R1, R2 and
R3 comprise angeloyl groups.
In an embodiment, R5 comprises sugar moiety. In an embodiment, the sugar
moiety
comprises at least one sugar, or glucose, or galactose, or rhamnose, or
arabinose, or
xylose, or alduronic acid, or glucuronic acid, or galacturonic acid, or their
derivative
thereof, or the combination thereof. In an embodiment, the sugar moiety
comprises one
or more sugar selected from, but is not limited to glucose, galactose,
rhamnose,
arabinose, xylose, fucose, allose, altrose, gulose, idose, lyxose, mannose,
psicose,
ribose, sorbose, tagatose, talose, fructose, alduronic acid, glucuronic acid,
galacturonic
acid, or derivatives thereof, or the combination thereof. In an embodiment,
the sugar
moiety comprise glucose, galactose or arabinose, or combination thereof, or
derivatives
thereof. In an embodiment, the sugar moietiy comprise alduronic acids,
galactose and
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arabinose, wherein the alduronic comprise glucuronic acid or galacturonic
acid. In an
embodiment, the sugar moiety comprise alduronic acids, glucose and arabinose,
wherein the alduronic comprise glucuronic acid or galacturonic acid.
In an embodiment, the R1, R 2 and R3 may be attached in other position of the
structure.
In an embodiment, the compound is triterpenoid saponin comprise comprises at
least
two angeloyl groups, tigloyl groups, senecioyl groups or acetyl group or their
combinations, preferable wherein at least two angeloyl groups.
In an embodiment, at least two compounds selected from angeloyl, acetyl,
tigloyl,
senecioyl, benzoyl, dibenzoyl, alkanoyl, alkenoyl, benzoyl alkyl substituted
alkanoyl, aryl,
acyl, heterocylic, heteroraryl, or acid with 2 to 5 carbon or derivative
thereof.
In an embodiment, at least one of the side bonds comprise a sugar moiety
comprising
two compounds selected from angeloyl, acetyl, tigloyl, senecioyl, benzoyl,
dibenzoyl,
alkanoyl, alkenoyl, benzoyl alkyl substituted alkanoyl, aryl, acyl,
heterocylic, heteroraryl,
or acid with 2 to 5 carbon or derivative thereof.
In an embodiment, the compound comprises a sugar moiety. In a further
embodiment,
the sugar moiety comprises glucose, galactose or arabinose or combination
thereof.
In a further embodiment, the sugar moiety comprises at least one sugar, or
glucose, or
galactose, or rhamnose, or arabinose, or xylose, or alduronic acid, or
glucuronic acid, or
galacturonic acid, or their derivative thereof, or the combination thereof.
In a further embodiment, the sugar moiety comprises one or more sugar selected
from,
but is not limited to glucose, galactose, rhamnose, arabinose, xylose, fucose,
allose,
altrose, gulose, idose, lyxose, mannose, psicose, ribose, sorbose, tagatose,
talose,
fructose, alduronic acid, glucuronic acid, galacturonic acid, or derivatives
thereof, or the
combination thereof.
A composition comprising an effective amount of compound selected from the
above
formula or a salt, ester, metabolite or derivative thereof as a medicament for
regulating
or reducing adhesion protein, blocking the migration, metastasis of cancer
cells,
inhibiting tumor or cancer cell growth and for treating cancer, wherein the
cancers
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comprise breast, leukocyte, liver, ovarian, bladder, prostate, skin, bone,
brain, leukemia,
lung, colori, CNS, melanoma, renal and cervix cancer.
In a further embodiment, a compound or sapongenin comprises the structure (d)
or (e)
has anti-cancer or inhibiting virus activities.
A composition for regulating or reducing adhesion protein, blocking the
migration,
metastasis of cancer cells, treating cancers or inhibiting virus, comprising a
compound,
wherein the compound is a triterpene, which comprises at least two side chains
which
comprise angeloyl groups, wherein the side chains are at adjacent carbon in
trans
position. In an embodiment, the side chains are at alternate carbon in cis
position. In an
embodiment, the side chains are at alternate carbon in trans position. In an
embodiment,
the side chains are attached an acyl. In an embodiment, the side chains are
attached an
unsaturated group.
In an embodiment, the side chains are in non-adjacent carbon cis or trans
position. In
an embodiment, the side chains comprise a functional group capable of
performing the
function of angeloyl group.
The above compounds can be used for regulating or reducing adhesion protein,
blocking the migration, metastasis of cancer cells, inhibiting tumor cell
growth, reducing
leg swelling, symptoms of chronic venous insufficiency, peripheral edema,
antilipemic,
chronic venous disease, varicose vein disease, varicose syndrome, venous
stasis,
expectorant, peripheral vascular disorders, by administering to a subject in
need
thereof, an effective amount of the above described compounds.
This invention provides a method for inhibiting tumor cell growth, regulating
cell growth,
reducing inflammation, in a subject, comprising administering to a subject, in
need
thereof, an effective amount of the compound which comprises any of the above
structures to said subject. The cancers are included but not limited to
Leukemia cancer,
Lung cancer, Colon cancer, CNS cancer, Melanoma cancer, Ovarian cancer, Renal
cancer, Prostate cancer, Breast cancer, bladder cancer, cervix cancer, liver
cancer,
bone cancer, brain cancer and Skin cancer.
This invention also provides a method for reducing swelling, reducing symptoms
of
chronic venous insufficiency, peripheral edema, antilipemic, chronic venous
disease,
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varicose vein disease, varicose syndrome, venous stasis, Expectorant,
peripheral
vascular disorders, cerebro-organic convulsion, cerebral circulation disorder,
cerebral
edema, psychoses, dysmenorrheal, hemorrhoids, episiotomies, peripheral edema
formation or postoperative swelling; for reducing symptoms of leg pain; for
treating
pruritis, lower leg volume, for reducing symptoms of pain; thrombosis,
thromophlebitis;
for preventing gastric ulcers antispasmotic, comprising administering to a
subject, in
need thereof, an effective amount of the composition of this invention.
This invention provides a composition comprising the compounds provided in the
invention for treating cancers; for inhibiting virus; for preventing cerebral
aging; for
improving memory; improving cerebral functions, for curing enuresis, frequent
micturition, urinary incontinence,dementia, Alzheimer's disease, autism, brain
trauma,
Parkinson's disease or other diseases caused by cerebral dysfunctions; for
treating
arthritis, rheumatism, poor circulation, arteriosclerosis, Raynaud's syndrome,
angina
pectoris, cardiac disorder, coronary heart disease, headache, dizziness,
kidney
disorder; cerebrovascular diseasea; inhibiting NF-Kappa B activation; for
treating brain
edema, sever acute respiratory syndrome, respiratory viral diseases, chronic
venous
insufficiency, hypertension, chronic venous disease, anti-oedematous, anti
inflammatory,
hemonhoids, peripheral edema formation, varicose vein disease, flu, post
traumatic
edema and postoperative swelling;for inhibiting blood clot, for inhibiting
ethanol
absorption; for lowering blood sugar; for regulating the adrenocorticotropin
and
corticosterone level. This invention provides a composition for AntiMS,
antianeurysm,
antiasthmatic, antibradykinic, anticapillarihemorrhagic, anticephalagic,
anticervicobrachialgic, antieciamptic, antiedemic, antiencaphalitic,
antiepiglottitic,
antiexudative, antiflu, antifracture, antigingivitic, antihematomic,
antiherpetic,
antihistaminic, antihydrathritic, antimeningitic, antioxidant,
antiperiodontic, antiphlebitic,
antipleuritic, antiraucedo, antirhinitic, antitonsilitic, antiulcer,
antivaricose, antivertiginous,
cancerostatic, corticosterogenic, diuretic, fungicide, hemolytic,
hyaluronidase inhibitor,
lymphagogue, natriuretic, pesticide, pituitary stimulant, thymolytic,
vasoprotective, and
venotonic treatment.
Alkenyl means unsaturated linear or branched structures and combinations
thereof,
having 1-7 carbon atoms, one or more double bonds therein. Non-limiting
examples of
alkenyl groups include vinyl, propenyl, isopropenyl, butenyl, s- and t-
butenyl, pentenyl,
hexenyl, butadienyl, pentadienyl, and hexadienyl.
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An aryl is a functional group of organic molecule derived from an aromatic
compound
such as benzene, a 6-14 membered carbocyclic aromatic ring system cpmprising 1-
3
benzene rings. If two or more aromatic rings are present, then the rings are
fused
together, so that adjacent rings share a common bond. Examples include phenyl
and
naphthyl. The aryl group may be substituted with one or more sunstitutes
independnetly
selected from halogen, alkyl or alkoxy.
Acyl is a functional group obtained from an organic acid by the removal of the
carboxyl.
Acyl groups can be written as having the general formula -COR, where there is
a double
bond between the carbon and oxygen. The names of acyl groups typically end in -
yl,
such as formyl, acetyl, propionyl, butyryl and benzoyl.
Benzoyl is one of acyls, C6H5COR, obtained from benzoic acid by the removal of
the
carboxyl.
Heterocyclic compound - a compound containing a heterocyclic ring which refers
to a
non-aromatic ring having 1-4 heteroatoms said ring being isolated or fused to
a second
ring selected from 3- to 7-membered alicyclic ring containing 0-4 heteroatoms,
aryl and
heteroaryl , wherein said heterocyclic comprises pyrrolidinyl , pipyrazinyl
morpholinyl, trahydrofuranyl, imidazolinyl, thiomorpholinyl, and the like.
Heterocyclyl groups derived from heteroarenes by removal of a hydrogen atom
from
any ring atom.
Alkanoyl is the general name for an organic functional group RCO-, where R
represents
hydrogen or an alkyl group. Preferably alkanoyl is selected from acetyl,
propionoyl,
butyryl, isobutyryl, pentanoyl and hexanoyl.
Alkenoyl is alkenylcarbonyl in which alkenyl is defined above. Examples are
pentenoyl(tigloyl) and hexenoyl(angeloyl).
Alkyl is a radical containing only carbon and hydrogen atoms arranged in a
chain,
branched, cyclic or bicyclic structure or their combinations, having 1-18
carbon atoms.
Examples include but are not limited to methyl, ethyl, propyl isopropyl,
butyl, s- and t-
butyl, pentyl, hexyl, heptyl, octyl, nonyl, undecyl, dodecyl, tridecyl,
tetradecyl,
pentadecyl, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
Benzoyl alkyl substituted alkanoyl is refer to straight or branched C1-C6
alkanoyl
substituted with at least one benzoyl and at least one alkyl, wherein the
benzoyl is
attached to a straight or branched C1-C6 alkyl. Preferably a benzoyl alkyl
substituted
alkanoyl is benzoyl methyl isobutanoyl.
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A sugar moiety is a segment of molecule comprising one or more sugars or
derivatives
thereof or aiduronic acid thereof.
Isobutyryl is Synonym of 2-Methylpropanoyl
Y and Y3 represent the same compound.
YM and (ACH-Y) represent the same compound.
This invention provides a method of altering the characteristic of cancer cell
membrane
to block the migration, metastasis of cancer cells or inhibit the growth of
cancers or anti-
angiogenesis.
This invention provides a method of inhibiting the growth, migration,
metastasis of
cancer by altering the characteristic of membrane of cancer cell, wherein the
characteristic comprise adhesion protein; wherein the cancers comprise breast
cancer,
leukocyte cancer, liver cancer, ovarian cancer, bladder cancer, prostate
cancer, skin
cancer, bone cancer, brain cancer, leukemia cancer, lung cancer, colon cancer,
CNS
cancer, melanoma cancer, renal cancer or cervix cancer, wherein the method is
administering contacting Xanifolia Y0, Yl, Y2, Y, Y7, Y8, Y9, Y10, or a salt,
ester,
metabolite thereof.
This invention provides a composition and method for inhibiting the growth,
migration,
metastasis of cancer by altering the adhesion characteristic of membrane of
cancer cell,
wherein the cancers comprise breast cancer, leukocyte cancer, liver cancer,
ovarian
cancer, bladder cancer, prostate cancer, skin cancer, bone cancer, brain
cancer,
leukemia cancer, lung cancer, colon cancer, CNS cancer, melanoma cancer, renal
cancer or cervix cancer, wherein the method is administering contacting
Xanifolia Y0,
Yl, Y2, Y, Y7, Y8, Y9, Y10, or a salt, ester, metabolite thereof. In an
embodiment the
method is administering contacting the compound selected from formula in this
application.
This application shows Xanifolia-Y is an alternate or supplemental agent to
DNA-
inhibition or microtubule-targeting drugs. It could be beneficial if it is
used singly or in
combination with other drugs of different mechanisms (block M-phase
progression or
DNA synthesis). Our inventions show combined effect of Xanifolia-Y and
paclitaxel on
inhibition of ES2 cells' growth (Detail in Experiment 14 U.S. Serial
Nos.11/683198, filed
on March 7, 2007)
Identify the binding target of Xanifolia-Y of adhesion proteins and signaling
proteins in
ovarian cancer cells.
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In our animal studies, it was shown that Xanifolia-Y extended the life span of
tumor
bearing mice. (See Experiments 7, 8, 9 in U.S. Serial Nos.11/683198, filed on
March 7,
2007,). The animals died sooner if the treatment of Xanifolia-Y was delayed
(comparing
results of treatments started from 1, 4 or 10 days after tumor inoculation).
The results
show that Xanifolia-Y inhibits migration or metastasis of the inoculated
cancer cells.
Ovarian carcinoma cells express high levels of adhesion molecules. Adhesion
proteins
are present in both cancer cells and mesothelial cells. While the lost of
adhesion blocks
of the protein accessibility due to a result of modulating by Xanifolia-Y, In
an
embodiment, the interaction of Xanifolia-Y with membrane alter the adhesion
protein's
binding site(s).
We have shown that Xanifolia-Y are cytotoxic to tumor cells, In an embodiment
it kills
ovarian cancer cells. Our inventions show that Xanifolia-Y inhibits cancer
cell growth
and prolongs life-span of tumor bearing mice. Our studies also indicate that
the sooner
the drug-treatment, the longer the life-span of the tumor bearing animals is
extended.
Xanifolia-Y also has an effect in blocking or inhibiting migration or
metastasis. The
delay of Xanifolia-Y-treatment allows more chances for cancer cells to
metastasize to
the mesothelium lining in the peritoneal cavity which resulted in more tumor
growth and
shorter life span. Adhesive molecules play an important role in cell migration
and
metastasis. It was shown in our studies that Xanifolia-Y inhibits cell
attachment to
culture flasks. Our experiment showed that Xanifolia-Y family inhibits the
secrection of
adhesion protein. Xanifolia-Y interferes with the function of the adhesive
molecules. In
embodiment Xanifolia-Y blocks the function of the adhesive molecules. In an
embodiment, Xanifolia-Y modulates adhesive proteins. It is masking the
adhesive
proteins. In an embodiment, Xanifolia-Y indirectly alters membrane structure
that cause
changes in protein conformation, or locations and result in loss of adhesion
process. In
an embodment, the adhesion proteins comprise fibronectin, integrins family,
Myosin,
vitronectin, collagen, laminin, Glycosylation cell surface proteins,
polyglycans, cadherin,
heparin, tenascin, CD 54, CAM, elastin and FAK, particular fibronectin.
Fibronectin is a kind of glycoprotein that binds to membrane spanning receptor
proteins
comprising the integrins, collagen, fibrin and heparin sulfate. Fibronectin
has been
implicated in tumor development and metastasis. This application provides
methods
and compositions for modulating the gene expression of fibronectin, inhibiting
the
secretion of fibronectin, reducing the receptors of fibronectin, reducing the
adhesion
ability fibronectin, inhibiting the metastasis, or inhibiting cancer growth,
wherein the
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method and composition comprises administering to the said subject as
effective
amount of compounds selected in this appliaction.
Vitronectin is an abundant glycoprotein found in blood plasma and the
extracellular
matix. Vitronectin is involved in hemostasis and tumot malignancy. This
application
provides methods and compositions for modulating the gene expression of
vitonectin,
reducing the receptors of vitronectin, reducing the adhesion ability
Vitronectin, inhibiting
the metastasis, and inhibiting cancer growth, wherein the method and
composition
comprises administering of compounds selected in this appliaction.
Integrins are cell surface receptors that interact with the extracellular
matix. They define
cellular shape, mobility, and regulate the cell cycle. Integrin plays a role
in the
attachment of cells to other cells, and also plays a role in the attachment of
a cell to the
material part of a tissue. Besides the attachment role, integrin also plays a
role in signal
transduction. This application provides method and composition for modulating
the gene
expression of integrins, wherein comprising inhibiting integrins, inhibititng
the adhesion
ability of integrins, inhibiting cancer cell metastasis and inhibiting cancer
growth,
wherein the method and composition comprise administering of compounds
selected in
this appliaction.
Laminins are a family of glycoproteins that are an integral part of the
structural
scaffolding of basement membranes in almost every animal tissue. They are
secreted
and incorporated into cell-associated extrcellular matrices. Inhibiting the
gene
expression of laminins will reduce the adhesion ability of cells in order to
inhibit the cell
migration and metastasis of cancer cells. This application provides method and
composition for modulating the gene expression of laminins, wherein comprising
inhibiting laminins, inhibititng the adhesion ability of laminins inhibiting
cancer cell
metastasis and inhibiting cancer growth, wherein the method and composition
comprise
administering of compounds selected in this appliaction.
CAM (cell adhesion molecules) are proteins located on the cell surface which
inolve
with binding with other cells in an adhesion process. Inhibiting the gene
expression of
CAM will reduce the adhesion ability of cells and further inhibit the cell
migration and
metastasis of cancer cells. This application provides method and composition
for
modulating the gene expression of CAM, wherein comprising inhibiting CAM,
inhibititng
the adhesion ability of CAM, inhibiting cancer cell metastasis and inhibiting
cancer
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growth, wherein the method and composition comprise administering of compounds
selected in this appliaction.
Collagen is the main protein of conective tissue in mammal. Inhibiting the
gene
expression of collagen will reduce the adhesion ability of cells in order to
inhibit the cell
migration and metastasis of cancer cells. This application provides methods
and
compositions for modulating the gene expression of laminins, wherein
comprising
inhibiting laminins, inhibititng the adhesion ability of laminins inhibiting
cancer cell
metastasis and inhibiting cancer growth, wherein the method and composition
comprise
administering of compounds selected in this appliaction.
Tenascin-C (Tn-C) is an extracellular matrix protein on the cell. It is a
positive factor for
cancer growth, invasion and angiogenesis activities. This application provides
methods
and compositions for inhibiting Tenascin-C and inhibiting cancer growth,
wherein the
methods and compositions comprise administering of compounds selected in this
appliaction.
Angiogenesis is a process involving the growth of new blood vessels. It is a
normal
process in growth and development. However, this is also a fundamental step in
the
transition of tumors from a dormant state to a malignant state. The
angiopoietins are
protein growth factors that modulate angiogenesis. The identified
angiopoietins
comprise angiopoietin 1, angiopoietin 2, angiopoietin 3, angiopoietin 4,
angiopoietin 5,
angiopoietin 6, angiopoietin 7, angiopoietin-like 1, angiopoietin-Iike 2,
angiopoietin-Iike 3,
angiopoietin-Iike 4, angiopoietin-Iike 5, angiopoietin-like 6, and
angiopoietin-like 7. In an
embodiment, the angiopoietin 1 is a positive foctor to promote the new blood
vessels. In
embodiment, the angiopoietin 2 is antagonist of angiopoietin 1, which is a
negative
factor for the growth of new blood vessels. This application provides methods
and
compositions for modulating angiopoietin and inhibiting cancer growth; wherein
the
cancers comprise breast, leukocyte, liver, ovarian, bladder, prostate, skin,
bone, brain,
leukemia, lung, colon, CNS, melanoma, renal and cervix cancer, wherein the
methods
and compositions comprise administering to the said subject as effective
amount of
compounds selected in this appliaction. The compounds in this application are
positive
regulating angiopoietin 2. The compounds in this application are negative
regulating the
angiopoietin 1. The results of the micro array experiment showed that compound
Y and
YM (ACH-Y) modulate the gene expression of angiopoietin family in ES2 cells.
They
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promote angiopoietin 2 and inhibit angiopoietin 1 and angiopoietin-Iike 1 and
angiopoietin-Iike 4.
The compounds in this application are anti-angiogenesis, inhibiting cancer
cell
metastasis and inhibiting cancer growth, wherein the compounds comprise
Xanifolia Y0,
Yl, Y2, Y, Y7, Y8, Y9, Y10, ACH-Y or a salt, ester, metabolite thereof and
compounds
selected from formula (1A), (1B), (1C) and (1D). In an embodiment the method
is
administering contacting the compound in this application comprising Xanifolia
Y0, Y1,
Y2, Y, Y7, Y8, Y9, Y10, Xanifolia (x), Escin or Aescin or a salt, ester,
metabolite thereof.
In an embodiment the compound may be selected from formulas (1A), (1B), (1C)
and
(1 D). In an embodiment, the compound comprises a triterpene backbone, two
angeloyl
groups and sugar moiety. In an embodiment the compound(s) are selected from
Compound A to X and Al to Xl in the application. In an embodiment the
compound(s)
are selected from Compound Z1 to Z7 in the application.
Data obtained from our Microarray experiments disclose that Xanifolia Y
modulates
gene expression of the following genes (represented by gene symbol):
Gene Symbol: ABL2, ADAMTS1, AKR1C3, AMIGO2, ANGPT2, ANKRD11, AP2B1,
APEH, APLP2, ARL10C, ARMC4, ARMCX1, ARMCX6, ARNTL2, ARNTL2, ATF3,
ATP6VOE, ATP6V1B2, ATP6V1C1, ATP6V1C1, BCL2A1, BCL6, BRI3, BTD,
C14orf109, C14orf78, C17orf32, C6orf65,C9orf10, C9orf103, CAD, CAV1, CAV2,
CBLL1, CCL20, CD33L3, CEBPB, CEP4, CFH /// CFHL1, CHRDL1, CITED2, CITED2,
CLDN14, CLN8, CLTA, CNAP1, COG6, COL18A1, COL4A2, COL5A1, COL5A2,
COL6A3, COPG, CPM, CPNE3, CPSF1, CSRP2BP, CSTB, CTNS, CXCL2, DDB1,
DDIT3, DDX20, DKFZP564I1171, DKFZP586JO619, DUSP10, DUSP10, DYRK3,
EEF2K, EFEMP1, EMP1, EVC, EVI2A, EXT2, FAM62A, FER1L3, FLJ14466, FLNA,
FN1, FN1, GANAB, GDF15, GEM, GNPDA1, GPAA1, GPC6, GPNMB, GPNMB, GUSB,
H2AFV, H2AFV, HDAC9, HDLBP, HECW2, HMGA2, HMOX1, HSDL2, HSPBAP1,
HSPC196, HYOU1, IDS, IGFBP3, IKBKAP, INSIG1, IPO4, IRS2, JAG1, KDELR3,
KIAA0251, KIAA0586, KIAA1211, KIAA1462, KIAA1706, KIAA1754, KRT18, KRT7,
KRTAP4-7, LAMP2, LEPR, LEPREL1, LHFPL2, LIF, LOC286044, LOC339229,
LOC90693, LRRC8E, MAFG, MAGED2, MCTP1, MGC16291, MGC19764, MGC5618,
MRPS30, MRPS31, MTERFD3, MYH9, NAGA, NAV2, NCSTN, NEK9, NEU1, NFKBIZ,
NMT2, NPC2, NSUN5C, NTNG1, NUP188, OACT2, OS9, P4HA1, P8, PALM2-AKAP2,
PALM2-AKAP2, PARVA, PBX2, PDE4DIP, PDIA4, PDIA6, PEG10, PHF19, PIK4CA,
PLEKHM1, PLOD1, PLOD2, PPP1R15A, PPP1R15A, PRKDC, PRSS23, PRSS23,
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PSEN2, PSMD1, PTPRF, PTPRJ, RAB32, RAB9A, RG9MTD1, RGS4, RHOQ, RND3,
RNF25, RNPEP /// UBE2V1 /// Kua /// Kua-UEV, RNU17D, ROBO4, RRAGC, RRS1,
SEC31L1, SERPINB2, SERPINB7, SESN2, SGEF, SGSH, SKIV2L, SLC25A21,
SLC35A3, SLC3A2, SMARCA1, SNAPC1, SNFILK, SPOCD1, SPTAN1, SQSTM1,
ST3GAL6, STC2, STX3A, TFPI2, TFPI2, TGFBI, TGM2, THRAP1, TLN1, TMEM60,
TNFAIP3, TRIB3, TRIO, TSC2, UAP1L1, UBAP2L, UPP1, URB, USP11, USP5, VDR,
WDR4, YTHDF2, ZCCHC9, ZDHHC20, ZFHX1B, ZNF185, ZNF278, ZNF690, ZNF697
Our experiment disclosed that Xanifolia Y and ACH-Y inhibited genes expression
of the
following genes: FN1, ITGAV, LAMA4, LAMB2, LAMC1, LAMB1, LAMB1, LAMA4,
LAMA5, LAMC1, LAMA2, LAMB1, LAMA3, SCAMP1, TICAM2, SCAMP1, TICAM2,
SCAMP1, SCAMP1, CAMK2B, DL1, ICAM3,CEECAM1,ICAM5,SCAMP1, CAMK1G,
CAMSAP1, MCAM, CAMTA1, CKN1, ALCAM, DCAMKL2, CEACAM3, CAMK2D,
CAMK2B, SCAMP5, CAMK4, NCAM1, CAMK2G, MYH9, MYH10, MYO1D, MYO5A,
MYLK, MYO6, MYO5A, MYO1 C, MYLK, MYO6, MYLC2PL, MYO10, MYO6, TPM3,
MYO1C, BECN1, MYO1E, TPM3, M-RIP, MYO1B, MYO10, MYO5A, M-RIP, MYO10,
MYL6, MYOHD1, BECN1, TPM4, MYLK, MYH10, MYOHD1, LOC221875, LOC402643,
MYO15B, LOC129285, MYH11, MYO1B, MYO1C, MYO9B, CDH13, CTNNAL1, CDH13,
CDH12, CTNNB1, CDH5, CTNND1, CDH2, CTNNA1, CDH2, PCDHB16, CTNNA1,
CELSR2, PCDHB6, PCDHB7, CTNND2, PCDHGC3, PCDHGB4, PCDHGA8,
PCDHGA12, PCDHGC5, PCDHGC4, PCDHGB7, PCDHGB6, PCDHGB5, PCDHGB3,
PCDHGB2, PCDHGB1, PCDHGAII, PCDHGA10, PCDHGA9, PCDHGA7, PCDHGA6,
PCDHGA5, PCDHGA4 , PCDHGA3 , PCDHGA2, PCDHGA1, CTNND1, CDH23,
PCDHB12, PCDHB10, PCDH18, CDH2O, PCDH9, PCDHGA12 , PCDHGAII,
PCDHGA10, PCDHGA6, PCDHGA5, PCDHGA3, PCDH7, CDH18, CDH6, CCBE1,
COL10A1, COL12A1, COL13A1, COL18A1, COL1 A1, COL21 A1, COL4A1, COL4A2,
COL4A5, COL4A6, COL5A1, COL5A2, COL6A1, COL6A2, COL6A3, COL9A1, MMP9,
P4HA1, P4HA2, P4HB, PCOLCE, PCOLCE2, PCOTH, PLOD1, PLOD2, PLOD3, CIB1,
ILK, ITGA2, ITGA3, ITGA4, ITGA6, ITGAV, ITGB1, ITGB1BP1, ITGB2, ITGB5, ITGBL1,
TNC, EMILIN1, ICAM1, HSPG2, HPSE, HS2ST1,SDC2,
This invention provides methods of regulating adhesion proteins which are
important for
blocking migration, metastasis of cancer cells, and inhibiting the growth of
cancers. In
an embodiment, the method comprises reducing the adhesion ability of the
cancer cells..
In an embodiment, the adhesion protein comprise fibronectin, integrins family,
Myosin ,
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vitronectin, collagen, laminin, Glycosylation cell surface proteins,
polyglycans, cadherin,
heparin, tenascin, CD 54, CAM, elastin and FAK, particular for fibronectin.
The following results are obtained from MicroArray experiments:
Y/D is the ratio (in folds) of gene expression in cells treated with compound
Y as
compared with those of the no drug control (D),
YM/D is the ratio of gene expression in cells treated with compound YM (ACH-Y,
Y
without sugar moiety) compared with those of the no drug control (D)
Table 1: Effect of Y and YM on fibronectin expression in ES2 cells
Probe Set ID Y/D YM/D Gene.Symbol Gene.Title
212464 s at -2.7 -1.1 FN1 fibronectin 1
216442 x_at -2.6 -1.1 FN1 fibronectin 1
211719_x at -2.6 -1.2 FN1 fibronectin 1
210495 x at -2.5 -1.1 FN1 fibronectin 1
The results of the microarray experiment showed that compound Y and YM(ACH-Y)
inhibit fibronectin expression; The expression ratio of compound Y/Y3 to the
control are
-2.7, -2.6, -2.6, -2.5 folds detected by gene probes 212464_s_at; 216442 x_at;
211719x at and 210495_x_at, respectively. These results indicate Y/Y3 inhibits
fibronectin expression; wherein the YM/ACH-Y also show minor fibronectin
inhibition
with the inhibiting ratio of -1.1, -1.1, -1.2, -1.1 folds by gene probes
212464_s_at;
216442 x_at; 211719_x at and 210495 x_at, respectively . The results indicate
that
while YM is active but is less potent than Y/Y3.
Table 2. Effects of Y and YM on integrin (vitronectin receptor) expression in
ES2
cells
Probe Set ID Y/D YM/D Gene.Symbol Gene.Title
202351at -1.8 -1.3 1TGAV integrin, alpha V (vitronectin receptor,
alpha polypeptide, antigen CD51)
236251at -1.4 -1.4 ITGAV Integrin, alpha V (vitronectin receptor,
alpha polypeptide, antigen CD51)
The results of the micro array experiment showed that compound Y and YM(ACH-Y)
inhibit integrin (vitronectin receptor) expression; wherein the inhibiting
ratio of compound
Y/Y3 to the control are -1.8, -1.4, folds as detected by different probes;
wherein the
inhibiting ratio of YM (ACH-Y) to the control are -1.3, -1.4 folds.
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Table 3: Effects of Y and YM on laminin expression in ES2 cells
Probe Set ID Y/D YM/D Gene.Symbol Gene.Title
202202_s_at -2.2 -2.0 LAMA4 laminin, alpha 4
216264_s_at -2.0 -2.0 LAMB2 laminin, alpha 5
200770_s_at -1.9 -1.1 LAMC1 laminin, alpha 6
211651_s_at -1.6 -1.7 LAMB1 laminin, alpha 7
201505 at -1.6 -2.0 LAMB1 laminin, beta 1
The results of the micro array experiment showed that compound Y and YM(ACH-Y)
inhibit laminin expression; The expression ratio of compound Y/Y3 to the
control are -
2.2, -2.0, -1.9, -1.6, -1.6 folds as detected by different probes; wherein the
inhibiting
ratio of YM/ACH-Y to the control are -2.0, -2.0, 1.1, -1.7, -2.0 folds.
Table 4: Effects of Y and YM on CAM expression in ES2 cells
Probe Set ID Y/D YM/D Gene.Symbol Gene.Title
201952_at -1.9 -1.4 ALCAM activated leukocyte cell adhesion molecule
201951_at -1.9 -1.7 ALCAM activated leukocyte cell adhesion molecule
212425_at -1.7 -1.5 SCAMP1 Secretory carrier membrane protein 1
240655_at -1.6 -1.3 ALCAM Activated leukocyte cell adhesion molecule
212417_at -1.4 -1.4 SCAMP1 secretory carrier membrane protein 1
239431_at -1.3 -1.3 TICAM2 toll-like receptor adaptor molecule 2
212416_at -1.3 -1.1 SCAMP1 secretory carrier membrane protein 1
228234_at -1.3 -1.3 TICAM2 toll-like receptor adaptor molecule 2
206667_s_at -1.3 -1.5 SCAMP1 secretory carrier membrane protein 1
The micro array experiment showed that compound Y and YM(ACH-Y) inhibit gene
expression related to the adhesion molecule; wherein the inhibiting ratio of
compound
Y/Y3 to the control are -1.3 to -1.9 folds as detected by different probes;
wherein the
inhibiting ration of YM/ACH-Y to the control are -1.1 to -1.7 folds.
Table 5: Effects of Y and YM on collagen expression in ES2 cells
Probe Set ID Y/D YM/D Gene.Symbol Gene.Title
217428sat -3.0 -1.2 COL10A1 collagen, type X, alpha 1(Schmid
metaphyseal chondrodysplasia)
231766_s_at -2.8 -2.4 COL12A1 collagen, type XII, alpha 1
201438_at -2.4 -1.5 COL6A3 collagen, type VI, alpha 3
1556138_a_at -2.2 -2.8 COL5A1 Collagen, type V, alpha 1
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211809_x_at -2.0 -1.5 COL13A1 collagen, type XIII, alpha 1
procollagen-proline, 2-oxoglutarate 4-
207543_s_at -2.0 -1.5 P4HA1 dioxygenase (proline 4-hydroxylase), alpha
polypeptide I
213992_at -2.0 -1.9 COL4A6 collagen, type IV, alpha 6
211343_s_at -1.9 -1.7 COL13A1 collagen, type XIII, alpha 1
211966_at -1.8 -1.7 COL4A2 collagen, type IV, alpha 2
procollagen-proline, 2-oxoglutarate 4-
200656sat -1.8 -1.2 P4HB dioxygenase (proline 4-hydroxylase), beta
polypeptide (protein disulfide isomerase-
associated 1)
209081_s_at -1.7 -1.5 COL18A1 collagen, type XVIII, alpha 1
202619sat -1.7 -1.2 PLOD2 procollagen-lysine, 2-oxoglutarate 5-
dioxygenase 2
203325_s_at -1.7 -2.8 COL5A1 collagen, type V, alpha 1
200827at -1.7 -1.2 PLOD1 procollagen-lysine 1, 2-oxoglutarate 5-
dioxygenase 1
221730_at -1.6 -1.6 COL5A2 collagen, type V, alpha 2
202311_s_at -1.6 -3.6 COL1A1 collagen, type I, alpha 1
213110_s_at -1.6 -2.2 COL4A5 collagen, type IV, alpha 5 (Alport syndrome)
212091_s_at -1.6 -1.9 COL6A1 collagen, type VI, alpha 1
213290_at -1.6 -1.5 COL6A2 collagen, type VI, alpha 2
211981_at -1.6 -2.2 COL4A1 collagen, type IV, alpha 1
procollagen-proline, 2-oxoglutarate 4-
200654at -1.6 -1.3 P4HB dioxygenase (proline 4-hydroxylase), beta
polypeptide (protein disulfide isomerase-
associated 1)
212489_at -1.5 -4.1 COL5A1 collagen, type V, alpha 1
202620sat -1.4 -1.3 PLOD2 procollagen-lysine, 2-oxoglutarate 5-
dioxygenase 2
procollagen-proline, 2-oxoglutarate 4-
202733_at -1.4 -1.9 P4HA2 dioxygenase (proline 4-hydroxylase), alpha
polypeptide II
208535_x_at -1.4 -1.2 COL13A1 collagen, type XIII, alpha 1
202185_at -1.3 -1.1 PLOD3 procollagen-lysine, 2-oxoglutarate 5-
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I dioxygenase 3
202465at -1.3 -1.6 PCOLCE procollagen C-endopeptidase enhancer
221729at -1.3 -1.8 COL5A2 collagen, type V, alpha 2
242324xat -1.3 -1.8 CCBE1 collagen and calcium binding EGF domains
-- 1
Procollagen-proline, 2-oxoglutarate 4-
1568611_at -1.3 -2.4 P4HA2 dioxygenase (proline 4-hydroxylase), alpha
polypeptide II
The results of the micro array experiment showed that compound Y and YM(ACH-Y)
inhibit collagen expression. The expression ratio of compound Y/Y3 to the
control range
from -1.3 to -3.0 folds; wherein the expression ratio of YM/ACH-Y to the
control range
from -1.1 to -3.6 folds
Table 6: Effects of Y and YM on integrin expression in ES2 cells
Probe Set ID Y/D YM/D Gene.Symbol Gene.Title
205422sat -1.9 -2.0 ITGBL1 integrin, beta-like 1(with EGF-like repeat
domains)
202351at -1.8 -1.3 ITGAV integrin, alpha V (vitronectin receptor,
alpha polypeptide, antigen CD51)
1557080sat -1.7 -2.5 ITGBL1 Integrin, beta-like 1(with EGF-like repeat
domains)
214927at -1.7 -1.8 ITGBL1 Integrin, beta-like 1 (with EGF-like repeat
- domains)
205885sat -1.7 -2.O ITGA4 integrin, alpha 4 (antigen CD49D, alpha 4
subunit of VLA-4 receptor)
213416at -1.6 -1.7 ITGA4 integrin, alpha 4 (antigen CD49D, alpha 4
subunit of VLA-4 receptor)
215177_s_at -1.6 1.1 ITGA6 integrin, alpha 6
205884at -1.6 -1.7 ITGA4 integrin, alpha 4 (antigen CD49D, alpha 4
subunit of VLA-4 receptor)
integrin, beta 2 (antigen CD18 (p95),
1555349 a at -1.6 -1.4 ITGB2 lymphocyte function-associated antigen 1;
macrophage antigen 1 (mac-1) beta
subunit)
227259_at -1.6 -1.1 CD47 CD47 antigen (Rh-related antigen,
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integrin-associated signal transducer)
201474sat -1.6 -1.5 1TGA3 integrin, alpha 3 (antigen CD49C, alpha 3
subunit of VLA-3 receptor)
214021_x_at -1.5 -2.2 ITGB5 Integrin, beta 5
201656_at -1.5 -1.1 ITGA6 integrin, alpha 6
The results of the micro array experiment showed that compound Y and YM(ACH-Y)
inhibit gene expression related to the integrin family in ES2 cells. The
expression ratio
of compound Y/Y3 to the control are ranging from -1.5 to -1.9 folds; wherein
the
expression ratio of YM/ACH-Y to the control are ranging from -1.1 to -2.5
folds.
Table 7: Effects of Y and YM on myosin expression in ES2 cells
Probe Set
ID Y/D YM/D Gene.Symbol Gene.Title
211926_s_at -2.2 -1.2 MYH9 myosin, heavy polypeptide 9, non-muscle
212372_at -1.7 -1.4 MYH10 myosin, heavy polypeptide 10, non-muscle
212338_at -1.7 -2.1 MYO1 D myosin ID
204527_at -1.6 -1.2 MYO5A myosin VA (heavy polypeptide 12, myoxin)
202555sat -1.6 -1.2 MYLK myosin, light polypeptide kinase /// myosin,
light polypeptide kinase
203215_s_at -1.6 -1.6 MY06 myosin VI
225080_at -1.5 -1.4 MYO1 C Myosin IC
224823_at -1.5 -1.4 MYLK myosin, light polypeptide kinase
The results of the micro array experiment showed that compound Y and YM(ACH-Y)
inhibit gene expression related to the myosin family in ES2 cells. The
expression ratio of
compound Y/Y3 to the control are ranging from -1.5 to -2.2 folds; wherein the
expression ratio of YM/ACH-Y to the control are ranging from -1.2 to -2.1
folds.
Table 8: Effects of Y and YM on cadherins expression in ES2 cells
Probe Set ID Y/D YM/D Gene.Symbol Gene.Title
244091 at -2.0 -1.7 CDH13 Cadherin 13, H-cadherin (heart)
202468sat -1.9 -1.6 CTNNAL1 catenin (cadherin-associated protein),
alpha-like 1
204726 at -1.8 -1.7 CDH13 cadherin 13, H-cadherin (heart)
207149 at -1.7 -2.2 CDH12 cadherin 12, type 2 (N-cadherin 2)
201533 at -1.5 1.2 CTNNB1 catenin (cadherin-associated protein),
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I beta 1, 88kDa
204677at -1.5 -1.1 CDH5 cadherin 5, type 2, VE-cadherin (vascular
epithelium)
208407 s at -1.5 -1.7 CTNND1 catenin (cadherin-associated protein),
- - delta 1
203440at -1.4 1.0 CDH2 cadherin 2, type 1, N-cadherin (neuronal)
210844xat -1.4 -1.2 CTNNAI catenin (cadherin-associated protein),
- - alpha 1, 102kDa
The results of the micro array experiment showed that compound Y and YM(ACH-Y)
inhibit gene expression of cadherins family in ES2 cells.
Table 9: Effects of Y and YM on tenascin-C expression in ES2 cells
Probe Set ID Y/D YM/D Gene.Symbol Gene.Title
201645_at -3.2 1.0 TNC Tenascin C (hexabrachion)
The results of the micro array experiment showed that compound Y inhibit gene
expression of cadherins family in ES2 cells.
Table10: Effects of Y and YM on heparin sulfate expression in ES2 cells
Probe Set ID Y/D YM/D Gene.Symbol Gene.Title
201655sat -2.4 -1.3 HSPG2 heparan sulfate proteoglycan 2
(perlecan)
219403_s_at -1.4 -1.3 HPSE Heparanase
203284 s at -1.3 -1.7 HS2ST1 heparan sulfate 2-0-
- sulfotransferase 1
The results of the micro array experiment showed that compound Y inhibit gene
expression of heparin sulfate family in ES2 cells.
Table 11: Effects of Y and YM on CD54 expression in ES2 cells
Probe Set ID Y/D YM/D Gene.Symbol Gene.Title
intercellular adhesion molecule 1
202638 s at 1.6 2.3 I CAM 1
(CD54), human rhinovirus receptor
The results of the micro array experiment showed that compound Y stimulate
gene
expression of CD54 in ES2 cells.
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Table 12: Effects of Y and YM on angiopoietin expression in ES2 cells
Probe Set ID Y/D YM/D Gene.Symbol Gene.Title
205572_at 3.5 1.4 ANGPT2 angiopoietin 2
211148_s_at 2.5 1.4 ANGPT2 angiopoietin 2
205609_at -1.1 -1.2 ANGPT1 angiopoietin 1
221009_s_at -1.2 -1.4 ANGPTL4 angiopoietin-like 4
227533_at -1.5 -2.3 ANGPTL1 Angiopoietin-like 1
The results of the micro array experiment showed that compound Y and YM(ACH-Y)
modulate the gene expression of angiopoietin family in ES2 cells. There is a
up
regulation of (positive regulating on) angiopoietin 2 and a down regulation of
(negative
regulating on) angiopoietin 1 and angiopoietin-Iike 1 and angiopoietin-like 4.
Fibronectin secretion studies summary:
Reduction of Fibronectin secretion from ES2 cells after xanifolia-Y treatment.
(Results of Fl and F3) In these experiments, we established and described the
basic
phenomenon that Y-treatment of ES2 cancer cells cause inhibition of
fibronectin
secretion. With a Western blot assay, we showed that ES2 cells without drug
treatment
(DMSO control) secret Fibronectin to medium and the amount of Fibronectin
accumulated with time. However, no or only minimally secretion of Fibronectin
was
observed in cell culture treated with Xanifolia-Y. Inhibition of Fibronectin
was observed
as early as 8 hours after drug-treatment.
Inhibition of Fibronectin secretion is physiological and the determination of
its quantity is
based on the following criteria:
1. Fibronectin is secreted from viable cells. Only cell with over 85% viable
cells
after drug-treatment are employed in these experiments. The viable cells were
determined by MTT assay.
2. For comparison, the immuno-band intensity from each samples are normalized
with cell mass. The cell mass was determined by the MTT assay and is
assigned as a MTT unit for each cell sample.
(Results of F4) Under a sub-lethal drug concentration (10 ug/mI Y), Over 95%
of cells
after 18 hours of Y-treatment was viable as determined by MTT assay. Western
Blots
show a reduction of Fibronectin secretion by cells into culture medium after Y-
treatment.
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Scan of Fibronectin Western bands (average 6 pairs of blots) shows that there
is a 40%
reduction of Fibronectin secretion after 18 hours of Y-treatment.
(Results of F5) Similarly, 85% of cells after 24 hours of Y-treatment were
viable as
determined by the MTT assay. Western blot shows a reduction of Fibronectin
bands of
Y-treated samples. Based on 6 pairs of blots and after normalize them to MTT
units, a
31% reduction of Fibronectin band intensity of Y-treated samples was observed.
Accordingly, these results indicate that Fibronectin secretion by cells reduce
31% after
24 hours of Y-treatment.
(Results of F7) Effects of Paclitaxel on Fibronectin secretion by ES2 cells.
To
demonstrate that not all anticancer drugs can inhibit Fibronectin secretion
from cells, we
employed Paclitaxel, a well known anticancer drug that is effective for
ovarian cancer.
Our results showed that there is no inhibition of Fibronectin secretion with
Paclitaxel
treatment in ES2 cells (10 to 50 ng/ml, the IC50 of Paclitaxel is 1.5 ng/ml).
This study
also showed that Fibronectin secreted by ES2 cells reduced 30-40% after Y-
treatment
which agrees with previous results.
(Results of F8) In addition to ES2 cells, another human ovarian cancer cells
(Hey8A)
were employed in this study. It was found that Y-treated Hey8A cells secrete
31%
Fibronectin as compared with the DMSO control, accordingly it has a inhibition
of 69%.
Beside ovarian cancer, other human cancer cells were tested in the following
experiments. These experiments show that the secretion of Fibronectin from
cancer
cells derived from lung, bladder, liver, brain and skin is inhibited by
Xanifolia-Y
treatment.
(F11) For lung carcinoma cells (H460), at concentration of 20ug/ml, there are
inhibitions
of Fibronectin secretion ranged from 20-60%.
(F12A) For bladder carcinoma cells (HTB-9), Xanifolia-Y (10 ug/mI) inhibits
50% of
Fibronectin secretion.
(F15) In liver HepG2 cells. 10 ug/mI of xanifolia-Y inhibits 42% secretion of
Fibronectin.
(F16) Incubation of brain glioblastoma T98G cells with10ug/ml of xanifolia-Y
inhibits
27% Fibronectin secretion and with 20ug/ml Y inhibits74% Fibronectin
secretion.
(F17) For skin SK-MeI-5 cells, the inhibition is 40-57% with 20 ug/mI of
Xanifolia-Y.
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Studies of xanifolia-Y analogs and other saponin on Fibronectin secretion from
ES2 cells.
(F 23) To study the inhibition effect with other saponins, we tested the
compound 054,
a triterpenoid saponin isolated from the same plant. With 054, there is no
inhibition
activity of Fibronectin secretion in ES2 cells, even at higher dose of 40
ug/mI (instead of
the usual effective concentration of 10 ug/mI). This result indicates there is
specificity in
triterpenoid saponin that is responsible for the inhibition effect.
(F21) To research for functional groups that are effective for Fibronectin
inhibition
activity, we tested several derivatives of xanifolia-Y3. In these experiments,
ES2 cells
treated with ACH-Y (Y3 without sugars) and AKOH-Y (Y3 without the C21, C22
angeloyl
group).
With 20 ug/mI Ach-Y, there is a reduction of Fibronectin secretion from ES2
cells
(ranging from 53% - 75% of the control). Inhibition of Fibronectin secretion
was less (or
not observed) when only 10 ug/mI Ach-Y was used. However, no effect was
observed
with AKOH even at 80 ug/mI.
(F 13) We also tested for inhibition activity with beta-Escin, a triterpenoid
saponin with
only one angeloyl group attached at C21.
The results show that 10 and 20 ug/mI of beta-Escin inhibit 7% and 48%,
respectively,
of Fibronectin secretion from ES2 cells. But 10 ug/mI of xanifolia-Y inhibits
49%
Fibronectin secretion. Results indicate that beta-Escin also inhibits
Fibronectin
secretion but has half potency as xanifolia-Y.
(F14) (F24) We have determined the inhibition effect of different analogs of
xanifolia-Y
on ES2 cells. The results are shown in the following table.
ES2 cells (3-ES- X-10 Y0-10 Y1-10 Y3-10 Y7-10 ACH-Y- AKOH-80
10 20
% 19 39 34 41 47 34 48 No effect
inhibition
All samples (except AKOH) tested have effects of inhibition of Fibronectin
secretion
from ES2 cells. With 80 ug/mI of AKOH-Y which is 4 times higher concentration
used in
others saponins (10 ug/mi), it still has no effect on inhibition of
Fibronectin secretion on
ES2 cells.
In conclusion, saponins in general have effects in inhibition of Fibronectin
secretion from
ES2 cells. The fact that AKOH-Y (the Y3 without diangeloyl group) does not
show any
activity, indicating that acylation of C21, 22 positions is important for the
inhibition
activity.
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In addition to ES2 cells, other cancer cells derived from different organs
were also
investigated. Results are shown in following tables.
(F25, 26, 31 B) Liver
HepG2 (3-ES- X-10 Y0- Y1- Y3- Y7- ACH-
10 10 10 10 Y-30
% 44 42 40 33 48 10 21
inhibition
5 (F27,29) Lung
H460 (3-ES- X-20 Y0- Y1- Y3- Y7- ACH-
10 10 10 10 Y-20
% No 37 22 13 19 18 28
inhibition effect
(F28, 30) Bladder
HTB-9 (3-ES- X-10 Y0- Y1- Y3- Y7- ACH-
10 10 10 10 10 Y-30
% 47 38 32 50 51 60 No
inhibition effect
10 F 31, 32 Brain
T98G R-ES= X-20 Y0- Y'F- Y3- Y7- ACH-
20 10 10 10 10 Y-20
% 66 52 22 40 26 24 30
inhibition
(F 33) Skin
SK-MEL- (3-ES- X-20 Y0- Y1- Y3- Y7- ACH-
5 20 10 10 10 10 Y-30
% 17 15 27 10 11 No 21
inhibition effect
(F20) Determination of cellular contents and secretion of Fibronectin after
xanifolia-Y-
15 treatment
Results: This experiment shows that (1) there is a 46% reduction (54% of
control) of
Fibronectin secretion after xanifolia-Y-treatment and (2) the Fibronectin
cellular content
decrease 70% (30% of control) after the Y-treatment; (3) there is no change of
the
cellular beta-actin content in ES2 cells after the Y-treatment.
Up regulation of Angiopoietin 2 (Ang2) in ES2 cells with Xanifolia-Y
treatment.
Meothods: ES2 (human ovarian carcinoma cells) were grew in RPMI 1640 medium.
4.5
million cells were seeded in a T75 flask and grown for 24 hours before drug-
treatment.
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Drug-treatment: Cells cultures were treated with 5, 10 and 15 ug/mI (final
concentration)
of Xanifolia-Y3 [Y3-5, Y3-10, Y3-15]. or DMSO control [D-10]. After 24 hours,
cells were
suspended in 1 ml of SDS sample buffer (cell-extract). Samples (80 uI/lane)
were
applied to 10% SDS gel and electrophoresis was conducted with 100 volts for 2
hours.
Protein was transferred to a nitrocellulose membrane electrophoretically. The
nitrocellulose blot was blocked with 5% non-fat dry milk in PBS. The blot was
then
incubated with the first antibodies (goat anti-Ang2, SIGMA A0851) and second
antibody
(donkey anti-goat AP conjugated, Promega V115A). The immuno-bands were
developed with BCIP/NBT color development system (Promega S3771).
Results: As shown in Figure 5, a Angiopoietin-2 immuno-band (M.W,. 66K) was
observed in cell extract from cells treated with 15 ug/mI Xanifolia-Y . No
detectable or
minimal immuno-band of Angiopoietin-2 was observed in control and low
concentration
of xanifolia-Y under these conditions. This results indicate that treatment of
Xanifolia-Y
in ES2 cells increase the cellular content of Angiopoietin-2. These results
corroborate
the results of Microarray studies.
This invention provides compositions and methods for modulating the gene
expression
in cancer cells, wherein the modulating comprises of positive and negative
regulation,
wherein genes being modulatated are adhesion proteins; wherein modulation
includes
expression, production and secretion of adhesion proteins, wherein the
adhesion
proteins comprise fibronectin, integrins family, Myosin , vitronectin,
collagen, laminin,
cadherin, heparin, tenascin, CD 54, CAM. This invention provides compositions
and
methods for modulating angiopoietins, wherein comprises positive regulating
the
angiopoietin 2, wherein comprises negative regulating angiopoietin 1. The
composition
and method of this invention comprises a triterpene wherein acylation group at
carbon
position 21 and/or 22 of the triterpene is necessary for the function and are
selected
from angeloyl, acetyl, alkanoyl, alkenoyl and acyl group. The sugar moiety
(ies) at
position 5 of the triterpene is important for enchancing activity of these
compounds.
EXPERIMENTAL DETAILS
Experiment details of herb extraction, analysis of extract components by HPLC,
determination of the cell-growth activity effected by Xanifolia Y with cells
derived from
different human organs using MTT Assay, purification of the bioactive
components from
plant extract, fractionation of plant extracts with FPLC, isolation of
component Ys with
preparative HPLC, determination of the chemical structure, cell experiments
and aminal
studings are disclosed in PCT/US05/31900, U.S. Serial No. 11/289142, U.S.
Serial
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10/906303, U.S. Serial No. 11/131551 and U.S. Serial Nos.11/683198, filed on
March 7,
2007, PCT/US2007/077273, filed August 30, 2007, U.S. Serial No. 60/890380,
filed on
February 16, 2007, U.S. Nos. 60/947,705, filed on July 3, 2007, the contents
of which
are incorporated herein by reference.
MicroArray
Experiment I
Analysis of gene expression of ES2 cells after Y-treatment by Microarray
In this invention, the microarray experiments were done in studying the gene
expression.
Total number of 54676 genes has been studied.
Cell culture and drug-treatment.: ES2 cells were seeded in a T-25 flask with
4.5 million
cells per flask for 24 hours. Cell culture was replaced with fresh medium with
xanifolia-
Y(Y) or DMSO no drug control (D) for 24 hours. Cells were then harvested for
RNA
isolation. Three experiments were done.
RNA extraction, labeling, hybridization, and data analysis. RNA was extracted
from
tumor cells using the Qiagen RNeasy Kit. RNA quality and quantity was checked
by the
Agilent BioAnalyzer and the NanoDrop ND-1000 spectrophotometer respectively
before further manipulation. The first and second cDNA strands were
synthesized from
ng of total RNA using the Affymetrix T7 oligo(dT) primer protocol and kit for
the two-
20 cycle amplificaton. To produce amplified biotin-labeled-cRNA, the cDNA was
reverse
transcribed by in vitro transcription using the MegaScript kit from Ambion.
15.0 pg of the
labeled cRNA was fragmented and re-checked for concentration using the
NanoDrop
ND-1000 spectrophotometer. A hybridization cocktail containing Affymetrix
spike-in
controls and fragmented labeled cRNA was loaded onto the Human U133 Ptus 2.0
GeneChip oligonucleotide array. The Affymetrix array (Affymetrix, Inc.Santa
Clara,
CA) is comprised of over 1,300,000 unique oligonucleotide features that
represent
greater than 38,500 well-substantiated human genes. The array was hybridized
for 16
hours at 45 C with rotation at 60 rpm then washed and stained with a
strepavidin, R-
phycoerythrin conjugate stain on the Affymetrix Fluidicis Station 450. Signal
amplification was done using biotinylated antistreptavidin. The arrays were
scanned
using the GeneChip 3000 confocal laser scanner with autoloader. The images
were
analyzed and quality control metrics recorded using Affymetrix GCOS software
version
1.4. Lastly, the expression value for each gene was calculated using dChip PM-
only
model based or Plier algorithm.
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Data Analysis Methods
Pairwise comparisons were made as follows: Treated vs. Control (Y vs. D),
Modified
Drug vs Control (YM/ACY-H vs. D) and Treated vs. Modified Drug (Y vs. YM/ACH-
Y)
Cel files analyzed using the Bioconductor package of R Statistical
programming.
Limma analysis generated a reasonable number of changing genes between the
samples.
The raw data in the CEL files were normalized by the GCRMA method (robust
multi-
array analysis). It is implemented in Bioconductor
(http://www.bioconductor.orQ/). The
raw signal intensity data were normalized, background corrected and summarized
based on certain statistical models, and an expression value, in Iog2-scale,
is obtained
per chip per probe set. Then the null hypothesis was tested that there's no
significant
changes in gene expression between the treatment pairs. This was done by LIMMA
and is also implemented in Bioconductor. It uses empirical Bayes method to
estimate
the variance in gene expression. One comparison was made, namely, High Grade
vs.
Low Grade. The raw p-values were adjusted by the Benjamnin-Hochberg method for
false discovery rate (FDR) control. All data sets contained a significant
number of
genes with a p-value less than 0.05, which is that the probability that a gene
is NOT
differential expressed (false positive) is 1:20.
All expression data is filtered by p-value (0.05).
The raw p-values were adjusted by the Benjamnin-Hochberg method for false
discovery
rate (FDR) control to yield an adjusted p-value.
Results: Please see Table 1 to 12
Inhibition of Fibronectin Secretion by Xanifolia-Y (Western blot)
Experiment 2 (F1)
Methods:
Cells: ES2 cells were grew in T-25 flask with RPMI 1640 medium over night
before
drug-treatment.Drug-treatment: cells cultures were replaced with fresh RPMI
medium
with Xanifolia-Y (10 ug/mI final concentration) or DMSO (as control) at 0
hour.At 1, 2, 4,
8 and 24 hour, aliquot of culture medium was taken out for Fibronectin
determination.
Fibronectin was determined by Western blot with monoclonal antibody (SIGMA)
specific
to human Fibronectin only.
Results (also see Figure 1):
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1. Cells treated with DMSO (as no drug control) secret Fibronectin to medium
and
the amount of Fibronectin accumulated with time. There is no or only minimally
secretion of Fibronectin observed in cell culture treated with Xanifolia-Y.
2. For controls, Fibronectin immunoband was not observed in RPMI medium with
fetal bovine serum, or employing the normal mouse serum (NS1).
Experiment 3 (F3)
Methods:
Cells: ES2 cells were grew in RPMI 1640 medium over night before drug-
treatment.
Drug-treatment: cells cultures were replaced with fresh RPMI medium containing
Xanifolia-Y (10 ug/mI final concentration) or DMSO (as control) at 0 hour. At
4 hours (A)
or 8 hour (B), culture medium was replaced with fresh culture medium without
drug. At
2, 4, 8 and 24 hour, aliquot of culture medium was taken out for Fibronectin
determination. Fibronectin (FN) was determined by Western blot with monoclonal
antibody (SIGMA) specific only to human Fibronectin.
Results (also see Figure 2):
(1) Compare the control and Y-treated cells before drug removal (at 4 and 8
hours),
there is a reduction of FN secretion from Y-treated cells. There is no obvious
cell
morphology change during these times, suggesting cells are alive.
(2) Compare the control and Y-treated cells after the removal of drug at 24
hours, it was
estimated that secretion of FN from Y-treated cells was reduced to over 50%.
(3) The amount of FN secreted by Y-treated cells at 24 hours is higher than
those at 8
hours (before removal of Y) indicating that cells are still alive after Y-
treatment.
Experiment 4 (F4)
Methods:
Cells: ES2 cells were grew in RPMI 1640 medium over night before drug-
treatment.
Drug-treatment: cells cultures were replaced with fresh RPMI medium containing
Xanifolia-Y (10 ug/mI final concentration) or DMSO (as control) at 0 hour. At
2, and 18
hour, aliquot of culture medium was taken out for Fibronectin determination
(Western
blot method). Cell viability at 18 hours was determined by MTT assay. Cultures
were
replaced with RPMI medium with MTT and incubated for an hour. The formation of
formazan was dissolved in DMSO and OD at 570nm was measured.
Results (also see Figure 2):
= Over 95% of cells after 18 hours of Y-treatment were viable as determined by
MTT assay.
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= Western Blots show a reduction of FN secretion by cells into culture medium
after Y-treatment. Scan of FN Western bands (average 5 sets of blots) shows
that there is a 40% reduction of FN secretion after 18 hours of Y-treatment.
Experiment 5 (F5):
Methods:
Cells and Drug-treatment: same as previous experiments. After 7 or 24 hours,
aliquot
of culture medium was taken out for Fibronectin determination (Western blot
method).
Cell viability at 24 hours was determined by MTT assay.
Results:
= 93% and 85% of cells after 7 and 24 hours, respectively, of Y-treated cells
was
viable as determined by MTT assay.
= Change in Fibronectin secretion during the first 7 hours of Y-treatment is
not
noticeable. However, after 24 hours, as compared with the control, the
Fibronectin band of Y-treated samples is reduced. Based on same amount of live
cells, the intensity of the immuno-bands were compared (per MTT O.D. unit).
The
scan of 3 pairs of blots shows a 31% reduction of Fibronectin band.
Accordingly,
these results indicate that Fibronectin secretion by cells reduce 31% after 24
hours of Y-treatment.
Experiment 6 (F 7): Effects of Paclitaxel on Fibronectin secretion by ES2
cells
Methods:
Cells: ES2 cells were grew in RPMI 1640 medium over night before drug-
treatment.
Drug-treatment: cells cultures were replaced with fresh RPMI medium containing
DMSO
(as control) [D]; Xanifolia-Y (10 ug/ml) [Y]; or Paclitaxel 10 or 50 ng/ml
[T10, ro T50].
After 24 hours, aliquot of culture medium was taken out for Fibronectin
determination
(Western blot method). Cell viability at 24 hours was determined by MTT assay.
Results:
Based on the MTT units (cell basis) of treated cells and compared those with
the DMSO
control, 87%, 94% and 91% growth of Y, T10 and T50 cells, respectively, were
viable
after 24 hours of treatment.
The amount of Fibronectin secreted by cells into medium was determined by
Western
blot assay. The amount of Fibronectin secreted per cells basis was determined
by
dividing the Western-band intensity with the MTT unit.
By comparing with the DMSO control, ES2 cells treated with 10 ng/ml or 50
ng/ml Taxel
secret 105% or 97%, respectively, of Fibronectin into medium during 24 hours
of
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treatment. At the same time, Y-treated ES2 cells secreted 62% of control (a
reduction
of 38%).
Experiment 7 (F 8): Hey8A cells treated with Xanifolia-Y
Methods:
Cells: Hey8A (human ovarian carcinoma cells) were grew in RPMI 1640 medium to
90%
confluent before drug-treatment. Drug-treatment: Cells cultures were replaced
with
fresh RPMI medium containing either DMSO (as control) [D1]; or Xanifolia-Y
(10, 15, or
20 ug/mI) [Y1, Y2 and Y3]. Aliquot of medium was removed as 0 hours sample and
no
FN was detected at this time. After 24 hours, aliquot of culture medium was
taken out
for Fibronectin determination (Western blot method). Cell viability at 24
hours was
determined by MTT assay. Cultures were replaced with RPMI medium (5 ml) with
MTT
and incubated for an hour. The formation of formazan was dissolved in DMSO and
OD
at 570nm was measured.
Western Blot: Spent culture medium (0.6 ml) was mixed with SDS sample buffer
(0.2
ml), boiled for 3 minutes before loading to SDS gel. Samples (60 uI/lane) were
applied
to a 6% SDS gel and electrophoresis was conducted with 100 volts for 2 hours.
Protein
was transferred to a nitrocellulose paper electrophoretically (30 min at 100
volts). The
Western blot was incubated with the first antibody (mouse anti-FN, specific to
human
FN, SIGMA F0916) and second antibody (Anti-mouse IgG AP conjugated, Promega
S3721). The immunobands were developed with BCIP/NBT color development system
(Promega S3771).
Results:
= After 24 hours of drug-treatment, cells with 15ug/ml and 20ug/ml were found
dead (floating) and were not further proceeded. Cells with DMSO and 10 ug/mI Y
were processed.
= The MTT assay showed that the growths of cells with Y-treatment (10ug/mI)
are
83% of control.
= The Western blot show that the band intensity of Y-treated samples (Y1) is
much
reduced compare to the DMSO control (D1)
= The average band intensity after corrected with the MTT unit are: 1179 and
366,
for DMSO control and Y-treated samples, respectively. Accordingly, Y-treated
Hey8A cells secrete 31% Fibronectin (FN) as compared with the DMSO control,
or a 69% inhibition.
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Experiment 8 (F 11): Inhibition of Fibronectin secretion by Xanifolia-Y in
Human
Lung Carcinoma cells (H460)
Methods: please see Experiment 8.
Results: Lung cells (H460) are sensitive to Y in inhibition of FN secretion.
Based on
MTT results, cells are still viable at 20 ug/mI Y, but the inhibition of FN is
over 60%.
Experiment 9'(F 12): Inhibition of Fibronectin secretion by Xanifolia-Y in
bladder
Carcinoma cells (HTB-9)
Methods: please see Experiment 8.
Results:
= The MTT assay showed that the growth of cells treated with 10 ug/mI Y
reduced
to 77% - 91 % compared to the DMSO control.
= The Western blot shows that the FN band intensity of Y-treated samples are
reduced. After corrected with the MTT unit (equivalent to cell mass) there is
about 50% reduction of FN band intensity per cell mass.
= These results indicate that Xanifolia-Y (10 ug/mI) inhibit 50% of FN
secretion.
Experiment 10 (F 13): ES2 cells treated with Y and beta-Escin
Methods: please see Experiment 8
Results:
= The MTT assay showed that the growth of cells with Y, EslO and Es20 are 89%,
90% and 82%, respectively as compared to the DMSO control.
= The Western blot show that the FN band intensity of EslO and Es20 samples
are
93% and 52%, respectively, of DMSO control. The band intensity of Y10 sample
is 51% of control.
= These results show that 10 and 20 ug/mi of beta-escin inhibit 7% and 48%,
respectlvely, of FN secretion. But 10 ug/mI of Y inhibits 49% FN secretion.
= Results indicate that beta-escin also inhibits FN secretion but with half
potency
as Xanifolia-Y.
Experiment 11 (F 14): ES2 cells treated with different Xanifolia-Ys
Methods: please see Experiment 8
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= Two experiments were done (FN14B and FN14C). Five gels per each
experiment were run.
Results:
Except for AKOH-Y (the Y3 without diangeloyl group), all samples have some
degrees
of inhibition of FN secretion from ES2 cells. 80 ug/mI of AKOH-Y which is 4
times
higher concentration used in others saponins (10 ug/ml), still have no effect
on inhibition
of FN secretion on ES2 cells.
ES2 cells R-ES-10 X-10 Y1-10 Y3-10 Y7-10 AKOH-80
% inhibition 19 39 41 47 34 No effect
Conclusion:
It seems that saponins in general have effects in inhibition of Fibronectin
secretion from
ES2 cells. However, this experiment found that acylation of C21, 22 positions
is
important for the inhibition activity.
Experiment 12 (F 23): ES2 cells treated with 054
Methods: Please see Experiment 8.
Results: Based on these results, there is no inhibition of FN secretion in ES2
cells with
054 treatment at 40 ug/ml.
Experiment 13 (F 24): ES2 treated with YO and Y5
Methods: please see Experiment 8.
Results: By comparing the immuno band's intensities of these samples the
results of
this experiment indicate that: (1) Y5 has same activity as Y3 for inhibition
of FN
secretion (both inhibit 68% at 10 ug/mI);
(2) Y0 is weaker as compare to Y3 for inhibition of FN secretion.(inhibit 34%
at 10
ug/mI);
(3) Conclusion, both Y0 and Y5 have inhibition activity for FN secretion from
ES2 cells.
Experiment 14 (F 25, 26): HepG2 cells treated with Ys
Methods: Please see Experiment 8.
Results: By comparing the immuno band's intensities of these samples (see
table), it
was found that at concentration of 10 ug/mI. X, ES, Y0, Yl, Y3, and Y5 have
inhibition
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effect on Fibronectin secretion from HepG2 cells. Minimum or No effect was
observed
with Y7, Ach (10 ug/mI) and AKOH (80 ug/mI).
HepG2 P-ES-10 X-10 Y0-10 Y1-10 Y3-10 Y7-10 ACH-Y-
% 44 42 40 33 48 10 21
inhibition
Experiment 15 (F 27, 29): NCI-H460 cells (Lung) treated with Ys
5 Methods: please see Experiment 8
H460 (3-ES- X- Y0- Yl- Y3- Y7- ACH-
20 20 10 10 10 10 Y-20
No 37 22 13 19 18 28
inhibition effect
Experiment 16 (F 28, 30): HTB-9 cells (bladder) treated with Ys
Methods: please see Experiment 8
(FN28, 30) Bladder
HTB-9 (3-ES- X- Y0- Y1- Y3- Y7- ACH-Y-
10 10 10 10 10 10 30
47 38 32 50 51 60 No
inhibition effect
Experiment 17 (F31, 32): T98G (brain) treated with Y2
Methods: please see Experiment 8
G98G YO-10 1-10 Y3-10 7-10 X-20 ES-20 CH-20
% inhibition 2 0 6 24 52 66 30
Experiment 18(F 33): SK-MEL-5 cells treated with Ys
Methods: please see Experiment 8
SK-MEL-5 (3-ES- X-20 Y0-10 Y1-10 Y3-10 Y7-10 ACH-Y-
30
% 17 15 27 10 11 No 21
inhibition effect
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Experiment 19(F 20): Determination of cellular contents and secretion of FN
after
Y3-treatment
Methods:
Cells: ES2 (human ovarian carcinoma cells) were grew in RPMI 1640 medium. 1.5
million cells were seeded in a T25 flask and grown for 24 hours before drug-
treatment.
Drug-treatment: Cells cultures were replaced with fresh RPMI medium containing
either
2.5 ul of DMSO (as control) [D]; or 10 ug/mI (final concentration) of
Xanifolia-Y3 [Y].
After 24 hours, aliquot of culture medium was taken out for Fibronectin
determination
(Western blot method). The attached cells were suspended in 1 ml of SDS sample
buffer (cell-extract).
Western Blot: Spent culture medium (0.6 ml) was mixed with SDS sample buffer
(0.2
ml), and the cell-extract was boiled for 3 minutes before loading to SDS gel.
Samples
(80 uI/lane) were applied to a 6% - 10% SDS gel and electrophoresis was
conducted
with 100 volts for 2 hours. Protein was transferred to a nitrocellulose
membrane
electrophoretically (30 min at 100 volts). The nitrocellulose blot was blocked
with 5%
non-fat dry milk in PBS (1-2 hours). The blot was then incubated with the
first antibodies
(mouse anti-FN, specific to human FN, SIGMA F0916 and mouse anti-beta actin,
SIGMA A5316) and second antibody (Anti-mouse IgG AP conjugated, Promega
S3721).
The immuno-bands were developed with BCIP/NBT color development system
(Promega S3771). Determination of Western band intensity: The band-images of
Western blot were captured with a digital camera (3-5 pictures were taken per
gel) and
the intensity of bands was determined using "Image J" software.
FN concentrations were normalized with the cellular beta-Actin concentrations.
Fibronectin secreted into medium and inside Y-treated cells were determined
and
compare to controls (DMSO-treated cells).
Results: This experiment shows that (1) there is a 46% reduction (54% of
control) of FN
secretion after Y-treatment and (2) the FN cellular content decrease 70% (30%
of
control) after the Y-treatment; (3) there is no change of cellular beta-aecin
concentration
after the Y-treatment.
Experiment 20: Animal Study
= Athymic Nu/Nu mice (2-3 months old) were transplanted sc with ES2 (human
ovarian cancer) cells.
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= Five days after the transplant (day one), mice were divided into two groups
(H and J)
with two animals in each group.
= Group H: On days 1-5, and 8-10 mice received daily drug administration of
Xanifolia-
Y, by i.p. route at dose of 2.5 mg/kg.
= Group J mice received no drug-treatment.
Result:
Group H: Mice received drug-treatment, tumor size is 10 mm in 10 days
Group J: Mice received no drug-treatment, tumor size is 18 mm in 10 days
The tumor size is 45% smaller in mice with drug than the mice with no drug
in 10 days period.
Experiment 21: Aminal Study
Methods
= Athymic Nu/Nu mice (5-6 weeks old) are divided into three groups (0, P and
Q) with
5-6 animals in each group.
= On day 0, all mice were transplanted intra-peritoneally with ES2 (human
ovarian
cancer) cells.
= Group 0: animals received no drug-treatment.
= Group P: On days 4-8, 11-15, 18-22, 25-29, 32-36,39-43, animals received
daily
drug administration of Xanifolia-Y, by i.p. route at dosage of 2.5 mg/kg
= Group Q: On days 10-15, 18-22, 25-29, 32-36, 39-43, animals received daily
drug
administration of Xanifolia-Y, by i.p. route at dosage of 2.5 mg/kg.
Result:
The median survival time of tumor bearing mice without drug-treatment is 24
days. The
median survival time of tumor bearing mice with drug-treatment starting on day
4 after
tumor inoculation is 58 days (extension of life span of 141 %); and The median
survival
time of tumor bearing mice with drug-treatment started on day 10 after tumor
inoculation is
31 days (extension of life span of 29%).
Experiment 22: Inhibition of cell adhesion by Xanifolia-Y.
Methods and Results: ES2 or Hey8A cells were plated in T25 fiasks with medium
containing 5 ug/mi of Xanifolia-Y. Cultures were incubated for 5 hours.
Attached cells were
removed from flasks by trypsinization and the amounts were counted. Compare to
no drug
controls, 86 4 % of ES2 cells and 67 8 % of Hey8A cells were found
attached to flasks
under this condition. At 5 ug/mi Xanifolia-Y, over 90% of unattached cells are
alive as
determined by the trypan Blue exclusion assay and by their ability to re-
attach to flasks
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when plating in medium without Xanifolia-Y. However, with 10 ug/mI Xanifolia-
Y, less than
40% of cells attached to flasks and many of them are dead cells. This
experiment shows
that Xanifolia-Y inhibits cells adhesion process.
Experiment 23: Increase Synthesis of Angiopoietin-2 in ES2 cells by Xanifolia-
Y
Meothods: ES2 (human ovarian carcinoma cells) were grew in RPMI 1640 medium.
4.5
million cells were seeded in a T75 flask and grown for 24 hours before drug-
treatment.
Drug-treatment: Cells cultures were treated with 5, 10 and 15 ug/mI (final
concentration) of
Xanifolia-Y3 [Y3-5, Y3-10, Y3-15]. or DMSO control [D-10]. After 24 hours,
cells were
suspended in 1 ml of SDS sample buffer (cell-extract). Samples (80 uI/lane)
were applied to
a 10% SDS gel and electrophoresis was conducted with 100 volts for 2 hours.
Protein was
transferred to a nitrocellulose membrane electrophoretically. The
nitrocellulose blot was
blocked with 5% non-fat dry milk in PBS. The blot was then incubated with the
first
antibodies (goat anti-Ang2, SIGMA A0851) and second antibody (donkey anti-goat
AP
conjugated, Promega V115A). The immuno-bands were developed with BCIP/NBT
color
development system (Promega S3771).
Results: As shown in this Western Blot, a Angiopoietin-2 immuno-band was
observed in
extract from cells treated with 15 ug/mI Xanifolia-Y. No or minimal immuno-
band of
Angiopoietin-2 was observed in control and low concentration of xanifolia-Y.
This result
indicates that treatment of Xanifolia-Y in ES2 cells increase the cellular
content (or
synthesis) of Angiopoietin-2. These results corroborate the results of
Microarray studies.
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