U.S. patent application number 14/864203 was filed with the patent office on 2016-01-14 for pharmaceutical compositions for inhibiting angiogenesis.
This patent application is currently assigned to INDUSTRIAL TECHNOLOGY RESEARCH INSTITUTE. The applicant listed for this patent is INDUSTRIAL TECHNOLOGY RESEARCH INSTITUTE. Invention is credited to Yi-Cheng CHENG, Ching-Huai KO, Zong-Keng KUO, Mei-Wei LIN, Shyh-Horng LIN, I-Huang LU, I-Horng PAN, Tien-Soung TONG, Hsiang-Wen TSENG, Chun-Chung WANG, Hsin-Chieh WU, Hsin-Jan YAO.
Application Number | 20160008319 14/864203 |
Document ID | / |
Family ID | 50931179 |
Filed Date | 2016-01-14 |
United States Patent
Application |
20160008319 |
Kind Code |
A1 |
PAN; I-Horng ; et
al. |
January 14, 2016 |
PHARMACEUTICAL COMPOSITIONS FOR INHIBITING ANGIOGENESIS
Abstract
The disclosure provides a pharmaceutical composition for
inhibiting angiogenesis, including an effective amount of a lignan
as an effective ingredient. The pharmaceutical composition may
further include a pharmaceutically acceptable carrier or salt. The
disclosure also provides a method for inhibiting angiogenesis,
including administering an effective amount of a lignan as an
effective ingredient for inhibiting angiogenesis to a subject in
need thereof.
Inventors: |
PAN; I-Horng; (Hsinchu City,
TW) ; YAO; Hsin-Jan; (Yunlin County, TW) ;
LIN; Mei-Wei; (Zhubei City, TW) ; LU; I-Huang;
(Zhudong Township, TW) ; WU; Hsin-Chieh; (Hsinchu
City, TW) ; TSENG; Hsiang-Wen; (New Taipei City,
TW) ; KO; Ching-Huai; (Changhua City, TW) ;
WANG; Chun-Chung; (Kaohsiung City, TW) ; KUO;
Zong-Keng; (New Taipei City, TW) ; LIN;
Shyh-Horng; (Kaohsiung City, TW) ; CHENG;
Yi-Cheng; (Hsinchu City, TW) ; TONG; Tien-Soung;
(Taichung City, TW) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
INDUSTRIAL TECHNOLOGY RESEARCH INSTITUTE |
Hsinchu |
|
TW |
|
|
Assignee: |
INDUSTRIAL TECHNOLOGY RESEARCH
INSTITUTE
Hsinchu
TW
|
Family ID: |
50931179 |
Appl. No.: |
14/864203 |
Filed: |
September 24, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
14133213 |
Dec 18, 2013 |
|
|
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14864203 |
|
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Current U.S.
Class: |
514/32 ; 514/464;
536/18.1; 549/320 |
Current CPC
Class: |
A61P 9/00 20180101; A61K
36/14 20130101; A61K 31/7048 20130101; A61K 31/7048 20130101; A61K
31/365 20130101; A61P 9/14 20180101; A61P 43/00 20180101; A61P
27/02 20180101; A61P 9/10 20180101; A61P 35/00 20180101; A61K
2300/00 20130101 |
International
Class: |
A61K 31/365 20060101
A61K031/365; A61K 31/7048 20060101 A61K031/7048 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 19, 2012 |
TW |
101148230 |
Claims
1. A pharmaceutical composition for inhibiting angiogenesis,
comprising: an effective amount of a lignan as an effective
ingredient, wherein a formula of the lignin is shown as Formula
(I): ##STR00005## wherein R.sub.1 is --H or --OCH.sub.3, R.sub.2 is
--H or --OH, R.sub.3 is --H, --OH or .beta.-O-glucoside, and
R.sub.4 is --H or --OCH.sub.3.
2. The pharmaceutical composition for inhibiting angiogenesis as
claimed in claim 1, wherein the lignin comprises yatein,
5'-desmethoxyyatein (bursehernin), 7', 7'-dihydroxy bursehernin, 5
'-methoxyyatein, podorhizol or podorhizol
4'-o-.beta.-D-glucopyranoside.
3. The pharmaceutical composition for inhibiting angiogenesis as
claimed in claim 1, further comprising a pharmaceutically
acceptable carrier or salt.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a Divisional of U.S. patent application
Ser. No. 14/133,213 filed on Dec. 18, 2013, which claims priority
under 35 U.S.C. .sctn.119(a) to Patent Application No. 101148230
filed in Taiwan on Dec. 19, 2012, all of which are hereby expressly
incorporated by reference into the present application.
TECHNICAL FIELD
[0002] The technical field relates to pharmaceutical compositions
and method for inhibiting angiogenesis.
BACKGROUND
[0003] Angiogenesis means a process for forming a new blood vessel
near a pre-existing blood vessel. Under a normal physiological
mechanism, during the process of a response which can result from a
stimulation of signal transduction for angiogenesis, such as wound
healing or the menstrual cycle of women, a controlled angiogenesis
will occur and be maintained for about 1-2 weeks. However,
pathological angiogenesis is not controlled by a normal
physiological mechanism. Regulation of angiogenesis plays a very
important balancing role in the human body. Under a strong
angiogenesis effect, it may result in diabetic retinopathy,
rheumatoid arthritis, or may accelerate aggravation or metastasis
of a tumor. In addition, when angiogenesis is over-suppressed, it
may result in occurrence of diseases related to hemorrhage,
apoplexy, cardiovascular diseases, etc. and even affect wound
healing of a patient due to a defective coagulation function.
[0004] At present, about 19 kinds of angiogenesis inhibitors are
used clinically, and indications for these drugs comprise various
solid tumors, age-related macular degeneration, choroidal
neovascularization, diabetic macular edema, diabetic retinopathy,
ocular neoplasm, retinal venous occlusion, telangiectasis, etc.
Since angiogenesis is related to various diseases, development of a
new angiogenesis inhibitor is a very important research direction
and development field at present and in the future.
SUMMARY
[0005] The disclosure provides a pharmaceutical composition for
inhibiting angiogenesis, comprising: an effective amount of an
extract of Juniperus chinensis as an effective ingredient.
[0006] The disclosure also provides a method for inhibiting
angiogenesis, comprising: administering an effective amount of an
extract of Juniperus chinensis as an effective ingredient for
inhibiting angiogenesis to a subject in need thereof.
[0007] Furthermore, the disclosure provides a pharmaceutical
composition for inhibiting angiogenesis, comprising: an effective
amount of a lignan as an effective ingredient, wherein a formula of
the lignin is shown as Formula (I):
##STR00001##
wherein R.sub.1 is --H or --OCH.sub.3, R.sub.2 is --H or --OH,
R.sub.3 is --H, --OH or .beta.-O-glucoside, and R.sub.4 is --H or
--OCH.sub.3.
[0008] The disclosure also provides a method for inhibiting
angiogenesis, comprising: administering an effective amount of a
lignan as an effective ingredient for inhibiting angiogenesis to a
subject in need thereof, wherein a formula of the lignin is shown
as Formula (I):
##STR00002##
wherein R.sub.1 is --H or --OCH.sub.3, R.sub.2 is --H or --OH,
R.sub.3 is --H, --OH or .beta.-O-glucoside, and R.sub.4 is --H or
--OCH.sub.3.
[0009] A detailed description is given in the following embodiments
with reference to the accompanying drawings.
BRIEF DESCRIPTION OF DRAWINGS
[0010] The present disclosure can be more fully understood by
reading the subsequent detailed description and examples with
references made to the accompanying drawings, wherein:
[0011] FIG. 1 shows a preparation process for the fractional
extract, CBT-143-S-F6F7 according to one embodiment of the
disclosure
[0012] FIG. 2 shows a preparation process for isolating and
purifying an active ingredient of the fractional extract,
CBT-143-S-F6F7 by column chromatography and activity-directed
analysis according to one embodiment of the disclosure;
[0013] FIG. 3A is a .sup.1H NMR spectrum of compound JC-5 according
to one embodiment of the disclosure;
[0014] FIG. 3B is a .sup.13C NMR spectrum of the compound JC-5
according to one embodiment of the disclosure;
[0015] FIG. 4 shows the structure of yatein according to one
embodiment of the disclosure;
[0016] FIG. 5 shows activity analysis results for JC-5, yatein,
inhibiting formation of net structure according to one embodiment
of the disclosure;
[0017] FIG. 6A shows the high-performance liquid chromatography
fingerprint of the crude extract, CBT-143-S according to one
embodiment of the disclosure;
[0018] FIG. 6B shows the high-performance liquid chromatography
fingerprint of the fractional extract, CBT-143-S-F6F7 according to
one embodiment of the disclosure;
[0019] FIG. 6C shows the high-performance liquid chromatography
fingerprint of the yatein control standard according to one
embodiment of the disclosure;
[0020] FIG. 7 shows the state of the crude extract, CBT-143-S,
inhibiting the formation of HUVEC net structure according to one
embodiment of the disclosure;
[0021] FIG. 8 shows the state of the fractional extract,
CBT-143-S-F6F7, inhibiting the formation of HUVEC net structure
according to one embodiment of the disclosure;
[0022] FIG. 9 shows the relative relationship between the
concentration of the crude extract, CBT-143-S, and the tube
formation for HUVECs according to one embodiment of the
disclosure;
[0023] FIG. 10 shows the relative relationship between the
concentration of the fractional extract, CBT-143-S-F6F7, and the
tube formation for HUVECs according to one embodiment of the
disclosure;
[0024] FIG. 11 shows the effect of the fractional extract,
CBT-143-S-F6F7 on the mobility of HUVECs according to one
embodiment of the disclosure;
[0025] FIG. 12 shows the effects of the crude extract, CBT-143-S,
and the fractional extract, CBT-143-S-F6F7, on the mobility of
SK-Hep-1 cells according to one embodiment of the disclosure;
[0026] FIG. 13 shows the effects of the crude extract, CBT-143-S,
and the fractional extract, CBT-143-S-F6F7, on in vivo growth
factor-induced angiogenesis. Data are represented by mean
.+-.standard deviation; p<0.05:* (compared with the growth
factor treatment group) according to one embodiment of the
disclosure;
[0027] FIG. 14 shows the effects of the crude extract, CBT-143-S,
and the fractional extract, CBT-143-S-F6F7, on in vivo cell
conditional medium-induced angiogenesis. Data are represented by
mean .+-.standard deviation; p<0.05:* (compared with the
conditional medium treatment group) according to one embodiment of
the disclosure;
[0028] FIG. 15 shows the effects of the fractional extract,
CBT-143-S-F6F7, and the crude extract, CBT-143-S, on angiogenesis
in a living body (1.25.times. magnification). Reference number 1301
in FIG. 15 refers to filter paper containing a test drug according
to one embodiment of the disclosure;
[0029] FIG. 16 shows the effect of the fractional extract,
CBT-143-S-F6F7, on angiogenesis in a living body (4.times. and
8.times. magnification). Reference number 1401 in FIG. 16 refers to
filter paper containing a test drug according to one embodiment of
the disclosure;
[0030] FIG. 17 shows the effects of the fractional extract,
CBT-143-S-F6F7, and the crude extract, CBT-143-S, on cell
proliferation of HUVEC cells according to one embodiment of the
disclosure;
[0031] FIGS. 18A, 18B and 18C show the effect of the compound,
JC-5, on rat corneal neovascularization according to one embodiment
of the disclosure; and
[0032] FIGS. 19A and 19B show the effect of the compound, JC-5, on
laser-induced choroidal neovascularization according to one
embodiment of the disclosure.
DETAILED DESCRIPTION
[0033] In the following detailed description, for purposes of
explanation, numerous specific details are set forth in order to
provide a thorough understanding of the disclosed embodiments. It
will be apparent, however, that one or more embodiments may be
practiced without these specific details. In other instances,
well-known structures and devices are schematically shown in order
to simplify the drawing.
[0034] The disclosure provides a pharmaceutical composition for
inhibiting angiogenesis, which uses an extract of Juniperus
chinensis as a main effective ingredient, and which has an effect
of inhibiting angiogenesis.
[0035] The pharmaceutical composition for inhibiting angiogenesis
mentioned above may comprise, but is not limited to, an extract of
Juniperus chinensis, wherein the extract of Juniperus chinensis is
an active ingredient for inhibiting angiogenesis. In one
embodiment, pharmaceutical composition for inhibiting angiogenesis
of the present disclosure may further comprise a pharmaceutically
acceptable carrier or salt.
[0036] In the present disclosure, an example of Juniperus chinensis
which is used to provide the extract of Juniperus chinensis may
comprise Juniperus chinensis var. Shimpaku, Juniperus chinensis L.
var. sargentii Henry, Juniperus chinensis L. var. chinensis
(proto-variety), Juniperus chinensis L. var. taiwanensis R. P.
Adams & C. F. Hsieh, Juniperus chinensis L. var. kaizuka Hort.
ex Endl., Juniperus chinensis L. var. pyramidalis (Carr.) Hort. ex
Rehd. and/or Juniperus chinensis cv. Pfitzeriana Glauca. In one
embodiment, the Juniperus chinensis which is used to provide the
extract of Juniperus chinensis is Juniperus chinensis L. var.
sargentii Henry.
[0037] The extract of Juniperus chinensis may be extracted from a
root, a stem/trunk, a branch, a leaf, and/or a combination thereof,
but it is not limited thereto. In one embodiment, the extract of
Juniperus chinensis may be extracted from thin branches and leaves
of the Juniperus chinensis.
[0038] In the present disclosure, a solvent which is used to
extract the extract of Juniperus chinensis may comprise alcohol
(such as methanol, ethanol or propanol), ester (such as ethyl
acetate), alkane (such as hexane) or haloalkane (such as
chloromethane, chloroethane), but it is not limited thereto. In one
embodiment, the extract solvent is ethanol.
[0039] In one embodiment, ingredients of the extract of Juniperus
chinensis of the present disclosure may comprise at least one
indicator ingredient, yatein, which has an effect of inhibiting
angiogenesis. In another embodiment, ingredients of the extract of
Juniperus chinensis of the present disclosure may at least comprise
yatein which has an effect of inhibiting angiogenesis.
[0040] In one embodiment, the extract of Juniperus chinensis may be
extracted from Juniperus chinensis L. var. sargentii Henry, and the
extract of Juniperus chinensis is obtained from using ethanol to
extract. In this embodiment, the ingredients of the extract of
Juniperus chinensis mentioned above may at least comprise yatein.
In another embodiment, the extract of Juniperus chinensis is
obtained from extracting thin branches and leaves of Juniperus
chinensis L. var. sargentii Henry with ethanol. In this embodiment,
the ingredients of the extract of Juniperus chinensis mentioned
above may at least comprise yatein.
[0041] The pharmaceutical composition for inhibiting angiogenesis
of the present disclosure is used for treating a disease related to
angiogenesis. Examples of the disease related to angiogenesis may
comprise solid tumors, age related macular degeneration, choroidal
neovascularization, diabetic macular edema, diabetic retinopathy,
ocular neoplasm, retinal venous occlusion, telangiectasis, etc.,
but they are not limited thereto.
[0042] The disclosure also provides a pharmaceutical composition
for inhibiting angiogenesis, which uses a lignan as a main
effective ingredient. The pharmaceutical composition for inhibiting
angiogenesis mentioned above may comprise, but is not limited to an
effective amount of a lignan, wherein a formula of the lignan is
shown as Formula (I):
##STR00003##
wherein R.sub.1 is --H or --OCH.sub.3, R.sub.2 is --H or --OH,
R.sub.3 is --H, --OH or .beta.-O-glucoside, and R.sub.4 is --H or
--OCH.sub.3.
[0043] In one embodiment, the foregoing pharmaceutical composition
for inhibiting angiogenesis may further comprise a pharmaceutically
acceptable carrier or salt.
[0044] In one embodiment, the lignan mentioned above may comprise,
but is not limited to, yatein, 5'-desmethoxyyatein (bursehernin),
7',7'-dihydroxy bursehernin, 5'-methoxyyatein, podorhizol or
podorhizol 4'-o-.beta.-D-glucopyranoside. In one exemplary
embodiment, the lignan mentioned above is yatein.
[0045] In the preceding pharmaceutical composition for inhibiting
angiogenesis of the present disclosure, the pharmaceutically
acceptable carrier mentioned above may comprise, but is not limited
to, a solvent, a dispersion medium, a coating, an antibacterial and
antifungal agent, or an isotonic and absorption delaying agent,
etc. which is compatible to pharmaceutical administration. The
pharmaceutical composition can be formulated into dosage forms for
different administration routes utilizing conventional methods.
[0046] Moreover, the pharmaceutically acceptable salt mentioned
above may comprise, but is not limited to, inorganic cation salt,
such as alkali metal salts such as sodium salt, potassium salt or
amine salt, such as alkaline-earth metal salt such as magnesium
salt or calcium salt, such as the salt containing bivalent or
quadrivalent cation such as zinc salt, aluminum salt or zirconium
salt. In addition, the pharmaceutically acceptable salt may also
comprise organic salt, such as dicyclohexylamine salt,
methyl-D-glucamine, and amino acid salt such as arginine, lysine,
histidine, or glutamine.
[0047] The pharmaceutical composition of the present disclosure may
be administered orally, parenterally by an inhalation spray, or via
an implanted reservoir. The parenteral method may comprise
subcutaneous, intracutaneous, intravenous, intramuscular,
intra-articular, intra-arterial, intrasynovial, intrasternal,
intrathecal, and intraleaional, as well as infusion techniques.
[0048] An oral composition can comprise, but is not limited to,
tablets, capsules, emulsions, and aqueous suspensions, dispersions
and solutions.
[0049] In addition, the present disclosure also provides a method
for inhibiting angiogenesis. The method for inhibiting angiogenesis
mentioned above may comprise, but is not limited to, administering
an effective amount of an extract of Juniperus chinensis as an
effective ingredient for inhibiting angiogenesis to a subject in
need thereof.
[0050] The extract of Juniperus chinensis is extracted from
Juniperus chinensis, and the Juniperus chinensis may comprise
Juniperus chinensis var. Shimpaku, Juniperus chinensis L. var.
sargentii Henry, Juniperus chinensis L. var. chinensis
(proto-variety), Juniperus chinensis L. var. taiwanensis R. P.
Adams & C. F. Hsieh, Juniperus chinensis L. var. kaizuka Hort.
ex Endl., Juniperus chinensis L. var. pyramidalis (Carr.) Hort. ex
Rehd. and/or Juniperus chinensis cv. Pfitzeriana Glauca, but it is
not limited thereto. In one embodiment, the Juniperus chinensis
mentioned above is Juniperus chinensis L. var. sargentii Henry.
[0051] In the present disclosure, an extracting part for the
Juniperus chinensis may comprise a root, a stem/trunk, a branch, a
leaf, and/or a combination thereof, but it is not limited thereto.
In one embodiment, the foregoing extract of Juniperus chinensis is
extracted from thin branches and leaves of Juniperus chinensis.
[0052] In addition, examples of a solvent which is suitable for
extracting the extract of Juniperus chinensis mentioned above may
comprise alcohol (such as methanol, ethanol or propanol), ester
(such as ethyl acetate), alkane (such as hexane) or haloalkane
(such as dichloromethane, dichloroethane), but it is not limited
thereto. In one embodiment, the extract solvent is ethanol.
[0053] In one embodiment, ingredients of the foregoing extract of
Juniperus chinensis may comprise yatein, which has an effect of
inhibiting angiogenesis.
[0054] In one embodiment, the extract of Juniperus chinensis may be
extracted from Juniperus chinensis L. var. sargentii Henry, and the
extract of Juniperus chinensis is obtained from using ethanol to
extract. In this embodiment, ingredients of the extract of
Juniperus chinensis mentioned above may at least comprise yatein.
In another embodiment, the extract of Juniperus chinensis is
obtained from extracting thin branches and leaves of Juniperus
chinensis L. var. sargentii Henry with ethanol. In this embodiment,
ingredients of the extract of Juniperus chinensis mentioned above
may at least comprise yatein.
[0055] Furthermore, the present disclosure provides another method
for inhibiting angiogenesis. The method for inhibiting angiogenesis
mentioned above may comprise, but is not limited to, administering
an effective amount of a lignan as an effective ingredient for
inhibiting angiogenesis to a subject in need thereof, wherein a
formula of the lignan is shown as Formula (I):
##STR00004##
wherein R.sub.1 is --H or --OCH.sub.3, R.sub.2 is --H or --OH,
R.sub.3 is --H, --OH or .beta.-O-glucoside, and R.sub.4 is --H or
--OCH.sub.3.
[0056] In one embodiment, the lignan mentioned above may comprise,
but is not limited to, yatein, 5' -desmethoxyyatein (bursehernin),
7',7'-dihydroxy bursehernin, 5'-methoxyyatein, podorhizol or
podorhizol 4'-o-.beta.-D-glucopyranoside. In one exemplary
embodiment, the lignan mentioned above is yatein.
EXAMPLES
A. Investigation and Determination for an Active Part of Juniperus
Chinensis L. Var. Sargentii Henry
[0057] About 10 g of dry thin branches and leaves of Juniperus
chinensis L. var. sargentii Henry was immersed in 8- to 10-fold
weight of solvent (such as water, ethanol, propanol, ethyl acetate
or hexane), and the immersed plant material was extracted at the
boiling point of the solvent for 1 hour to obtain an extract
solution. After that, the extract solution was filtered with filter
paper, concentrated to dry, mixed with pure water and then
sonicated to form a suspension. Then, the suspension was
lyophilized.
[0058] The lyophilized powder was dissolved in an appropriate
solvent and an in vitro anti-angiogenesis was performed thereto.
The ability of the extract for inhibiting net structure formation
of human umbilical vein endothelial cells (HUVEC) in extracellular
matrix (ECM) or growth factor reduced Matrigel was analyzed, and
the forming level of the tube structure thereof was integrated to
be used as an indicator for quantification. The results are shown
in Table 1.
TABLE-US-00001 TABLE 1 Content of indicator ingredients and
activities for inhibiting net structure formation of human
umbilical vein endothelial cells of the extracts of thin branches
and leaves Juniperus chinensis L. var. sargentii Henry extracted
with different solvents Plant part: Thin branches and leaves Yatein
Net structure formation Sample name (mg/g) (%) Note CBT143-LWH N.D
101.1 Water extract CBT143-LEH 2.02 0.3 Ethanol extract CBT143-LAH
2.42 0 Propanol extract CBT143-LTH 2.63 0.2 Ethyl acetate extract
CBT143-LHH 2.73 0.2 n-Hexane extract N.D: Lower than detection
limitation
[0059] The results showed that water extract is not capable of
inhibiting formation of net structure (tube formation), whereas the
other solvent extracts are all capable of inhibiting tube formation
of endothelial cells.
B. Preparation of Ethanol Extract of Juniperus Chinensis L. var.
Sargentii Henry
1. Extraction Process for Crude Extract (CBT-143-S)
[0060] According to the results shown above, thin branches and
leaves of Juniperus chinensis L. var. sargentii Henry were selected
as a target for extraction, and ethanol was selected as the extract
solvent. The obtained crude extract was named CBT-143-S, and the
preparation process thereof is shown in the following:
[0061] (1) 10 g of dry thin branches and leaves of Juniperus
chinensis L. var. sargentii Henry was immersed in 8- to 10-fold
weight of 95% ethanol solvent;
[0062] (2) The immersed plant material of step (1) was extracted at
a temperature of the boiling point of the solvent for 1 hour, and
then filtered with a metal sieve (pore size: 230 mesh) to obtain an
extraction solution;
[0063] (3) The extraction solution obtained from step (2) was
filtered with filter paper [Type: 5A (diameter: 9 cm; pore size: 7
.mu.tm,; thickness: 0.22 mm); TOYO ROSHI CO., LTD];
[0064] (4) The filtrate obtained from step (3) was concentrated to
dry by a rotary evaporator to obtain an extract concrete;
[0065] (5) The extract concrete obtained from step (4) was mixed
with pure water and sonucated to form a suspension.
[0066] (6) The suspension obtained from step (5) was lyophilized by
liquid nitrogen.
[0067] (7) After lyophilization, the product was collected and the
product was CBT-143-S.
2. Preparation for Fractional Extract (CBT-143-S-F6F7)
[0068] In order to increase the activity of the crude extract,
isolation and purification were performed to the crude extract,
CBT-143-S. Isolation and purification process are shown in the
following, and the preparation process for the fractional extract
is shown in FIG. 1
[0069] (1) 30 g of CBT-143-S sample was fixed through dry packing
(i.e. CBT-143-S was dissolved by an appropriate solvent, and then
2-fold weight (60 g) of silica gel was added thereto. After being
mixed well, CBT-143-S was uniformly fixed on the surface of silica
gel by a rotary evaporator.) (Step S1);
[0070] (2) CBT-143-S sample was isolated through a column which
contains 15-fold weight of silica gel to CBT-143-S sample (silica
gel was 450 g and filled into a glass column with a diameter of 6
cm. After being filled into the glass column, the silica gel was 28
cm in height, and the dry packing matrix was 4.5 cm in height)
(Step S2).
[0071] (3) Acetone:n-hexane (1:2) was used as an initial mobile
phase to elute the column for 3300 mL, and every 20 mL of elution
solution was collected by a tube, fractionally (Step S3)
(F1.about.F7 tube).
[0072] (4) The column as washed with acetone (Step S3') (F8
tube);
[0073] (5) The column as washed with methanol (Step S3'') (F9
tube)
[0074] (6) The fractionally collected elution solutions were
analyze through normal phase thin layer chromatography, and
acetone:n-hexane (2:3) was used as a mobile phase to spread the
fractionally collected elution solutions. After being spread, the
fractionally collected elution solutions were stained with 10%
H.sub.2SO.sub.4 (EtOac) and heated to 105.quadrature. to color, and
R.sub.f values of main color spots therefrom were calculated;
[0075] (7) The elution solutions of that R.sub.f values were
between 0.35 and 0.55 were collected (Step S4);
[0076] (8) The elution solutions mentioned above were combined and
concentrated to dry by a rotary evaporator to obtain a fractional
extract concrete;
[0077] (9) The fractional extract concrete was added to equal
weight of pure water, and then sonicated to suspend, and
lyophilized by liquid nitrogen; and
[0078] (10) After lyophilization, the product, lyophilized powder,
was CBT-143-S-F6F7.
3. Isolation, Purification and Identification of Active
Ingredient
[0079] The active ingredient of the fractional extract,
CBT-143-S-F6F7, was isolated and purified by column chromatography
and activity-directed analysis. The preparation process is shown in
FIG. 2. See FIG. 2. CBT-143-S-F6F7 was used as a raw material, and
isolated through a condition P1, and then examined, concentrated
and dried through a condition P', and a fraction containing JC-5
active ingredient, CBT-143-S-F6F7-A2, can be obtained thereby.
After the fraction was isolated through a condition P2, and then
examined, concentrated and dried through a condition P', a fraction
containing JC-5 active ingredient, CBT-143-S-F6F7-B2, can be
obtained. The fraction CBT-143-S-F6F7-B2 was further isolated
through a condition P3, examined, concentrated and dried through a
condition P', and then re-crystallized through a condition P4, and
a pure substance JC-5 can be obtained thereby. Explanations for
conditions P1 to P4 and condition P' is shown in the following:
[0080] P1: Flash performance liquid chromatography, FPLC
[0081] Column: Silica gel column (0.0150.about.0.040 .mu.m); 2.0 cm
(inner diameter)* 30 cm (length); Mobile phase: ethyl acetate
(EtOAc):n-hexane =1:1; Flow rate: 5 mL/minute; Fraction collector:
10 mL/tube.
[0082] P2: Flash performance liquid chromatography, FPLC
[0083] Column: Silica gel column (0.015.about.0.040 .mu.m); 2.0 cm
(inner diameter)* 30 cm (length); Mobile phase: ethyl acetate
(EtOAc):n-hexane =1:1.5; Flow rate: 5 mL/minute; Fraction
collector: 10 mL/tube.
[0084] P3: Flash performance liquid chromatography, FPLC
[0085] Column: Silica gel column (0.015.about.0.040 .mu.m); 1.5 cm
(inner diameter)* 30 cm (length); Mobile phase: ethyl acetate
(EtOAc):n-hexane=1:1.5; Flow rate: 5 mL/minute; Fraction collector:
10 mL/tube.
[0086] P4: Re-crystallizing by methanol
[0087] P': Thin layer chromatography, TLC:
[0088] A thin layer chromatography aluminum sheet (TLC Silica gel
60 F254, Merck) was cut to 5 cm in length and 7 cm in width, and 1
uL of sample was loaded into the cut aluminum sheet at a position 1
cm from the bottom of the cut aluminum sheet by a quantitative
capillary. After the sample was loaded into the cut aluminum sheet
and the cut aluminum sheet is dried, the cut aluminum sheet was
placed in a spreading tank to spread the sample for 4 cm (spreading
solution: ethyl acetate:n-hexane=1:1). After the spreading process
was completed and the cut aluminum sheet was dried, a coloring
agent (10% H.sub.2SO.sub.4(EtOAc) was spread uniformly on the cut
aluminum sheet and heated (105for 5 minutes). After the coloring
process was completed, the region containing JC-5 (R.sub.f value:
0.55.about.0.61, black spot) was observed, and a part of the sample
containing the spot was collected and concentrated and dried.
[0089] The pure substance, JC-5, was identified by nuclear magnetic
resonance spectroscopy, NMR. The results are shown in FIG. 3A and
3B. FIG. 3A is a .sup.1HNMR spectrum of compound JC-5, and this
spectrum shows the number of hydrogen atoms which contained by the
structure of the compound JC-5. According to the positions
occurring hydrogen atoms (.delta. value) and ratio of integral
values, it could be extrapolated that there were a total of 24
hydrogen atoms in the structure. FIG. 3B is a .sup.13C NMR spectrum
of the compound JC-5, and this spectrum shows that there are a
total of 22 carbon atoms in the structure of the compound JC-5.
[0090] The data obtained from FIG. 3A and FIG. 3B were compared
with the description related to yatein of the literature [Ikeda,
R.; Nagao, T.; Okabe, H.; Nakano, Y.; Matsunaga, H.; Katano, M.;
Mori, M. Chem. Pharm. Bull. 1998, 46, 871-874.], and the data are
sorted in Table 2. According to the comparison result mentioned
above, the compound
[0091] JC-5 was identified as yatein. Moreover, the structure of
yatein is shown in FIG. 4.
TABLE-US-00002 TABLE 2 Distribution of the hydrogen atoms and
carbon atoms contained in the structure of yatein Data from the
experiment (600 MHz, CDCl.sub.3) Position C H (J in Hz) 1 131.47 2
121.45 6.45, dd(7.8, 1.8) 3 108.21 6.68 d(7.8) 4 146.32 5 147.83 6
108.68 6.44 d(1.8) 7 38.24 2.56, m 2.56, m 8 40.94 2.47, m 9 71.09
4.15, t(9.6); 3.85, t(8.4) 1' 133.26 2' 106.15 6.34, s 3' 153.17 4'
136.78 5' 153.17 6' 106.15 6.34, s 7' 35.16 2.91, dd(14.4, 5.4);
2.86, dd(14.4, 5.4) 8' 46.35 2.58, m 9' 178.44 OCH.sub.2O 101.00
5.90, d(1.8); 5.91, d(1.8) C-3', OCH.sub.3 56.01 3.81, s C-4',
OCH.sub.3 60.76 3.80, s C-5', OCH.sub.3 56.01 3.81, s
[0092] Furthermore, by using Matrigel (BD, Cat. No. 356231) as a
matrix and observing the state of human umbilical vein endothelial
cells (HUVECs) constructing a net structure on the matrix, state of
JC-5 (yatein) inhibiting the formation of HUVEC net structure was
analyzed. Analysis standards and conditions are described in the
following. The total length of the net structure was calculated
through NIS element image analysis software (Nikon; Agent: Lin
Trading Co., Ltd. Taiwan), wherein by using the total length of a
net structure formed from a group without a drug addition as 100%,
the state of JC-5 inhibiting the formation of HUVEC net structure
was analyzed.
[0093] According to activity analysis results for of inhibiting the
formation of net structure as mentioned above, it was found that
50% inhibiting concentration (IC.sub.50) of yatein for inhibiting
tube formation was 0.335 tM (FIG. 5). Therefore, yatein was
determined to be an active ingredient.
4. High-Performance Liquid Chromatography (HPLC) Fingerprint of the
Crude Extract (CBT-143-S) and the Fractional Extract
(CBT-143-S-F6F7)
[0094] Analysis method:
[0095] Sample preparation: 10 mg of powder of CBT-143-S and 10 mg
of powder of CBT-143-S-F6F7 were placed in respective 10 mL
volumetric flasks. After that, 95% ethanol was added to the 10 mL
volumetric flasks to quantify the volume to 10 mL, and the 10 mL
volumetric flasks were sonicated to completely dissolve the powder
therein.
[0096] Conditions for high-performance liquid chromatography:
[0097] Type of chromatography column: Cosmosil 5C18-MS-II 4.6*
250;
[0098] Flow rate: 0.8 ml/minute;
[0099] Observation wave length: 280 nm;
[0100] Mobile phase: A: 0.1% H.sub.3PO.sub.4; B: CH.sub.3CN; C:
MeOH;
[0101] Gradient: A/B/C=65/25/10 (60 minutes) .fwdarw.A/B/C=25/60/15
(1 minute).fwdarw.A/B/C=65/25/10 (9 minutes).
[0102] Analysis results:
[0103] FIG. 6A and FIG. 6B show the high-performance liquid
chromatography graphs for quality control analysis for the crude
extract (CBT-143-S) and the fractional extract (CBT-143-S-F6F7),
respectively, and can used as a reference for quality control of
the extract of the present disclosure.
[0104] By using the purified yatein (JC-5) pure substance as a
control standard, high-performance liquid chromatography
qualitative/quantitative analysis methods for the crude extract
(CBT-143-S) and the fractional extract (CBT-143-S-F6F7) were
developed, and the high-performance liquid chromatography graphs
for the crude extract (CBT-143-S) and the fractional extract
(CBT-143-S-F6F7) were compared with the high-performance liquid
chromatography fingerprint of the yatein control standard (see FIG.
6C), respectively. According to FIG. 6A and FIG. 6B, the
high-performance liquid chromatography graphs for the crude extract
(CBT-143-S) and the fractional extract (CBT-143-S-F6F7) all present
a main peak for the JC-5 active ingredient. In addition, the
extracts of thin branches and leaves of Juniperus chinensis L. var.
sargentii Henry extracted by different solvents were also analyzed,
and the yatein contents thereof are shown in above Table 1.
5. In Vitro Evaluation for Activities of the Crude Extract
(CBT-143-S) and the Fractional Extract (CBT-143-S-F6F7)
(1) Inhibition of Blood Vessel Tube Formation
[0105] By using Matrigel (BD, Cat. No. 356231) as a matrix and
observing the state of human umbilical vein endothelial cells
(HUVECs) constructing a net structure on the matrix, the states of
the crude extract (CBT-143-S) and the fractional extract
(CBT-143-S-F6F7) inhibiting the formation of HUVEC net structure
were analyzed. The analysis standards and conditions are described
in the following. The total length of the net structure was
calculated through NIS element image analysis software (Nikon;
Agent: Lin Trading Co., Ltd. Taiwan), wherein by using the total
length of a net structure formed from a group without a drug
addition as 100%, the states of the crude extract (CBT-143-S) and
the fractional extract (CBT-143-S-F6F7) inhibiting the formation of
HUVEC net structure was analyzed.
[0106] The experimental results showed that by increasing the
testing concentration, the inhibition effects of the crude extract
(CBT-143-S) and the fractional extract (CBT-143-S-F6F7) on tube
formation were also increased, and those showed significant
dose-dependent effects (FIG. 7 and FIG. 8).
[0107] Furthermore, after calculation, it was determined that 50%
inhibiting concentrations (IC.sub.50) of the crude extract
(CBT-143-S) and the fractional extract (CBT-143-S-F6F7) for
inhibiting tube formation were 0.221 .mu.g/mL and 0.0123 .mu.g/mL
(FIG. 9 and FIG. 10). The results showed that through a
purification process, the activity of the fractional extract
(CBT-143-S-F6F7) could be increased 20.3-fold, compared with that
of the crude extract (CBT-143-S).
(2) Evaluation for Inhibition of Mobility of Vascular Endothelial
Cells (HUVEC Migration Assay)
[0108] In a BD transwell system, the state of HUVEC migrating
downward to pass through the membrane in the presence of nutrition
for driving HUVEC was observed. By using a group without serum for
driving as a negative control and using a group with serum for
driving as a positive control, the state of the extract inhibiting
migration of the endothelial cells, wherein the cells stained by
crystal violet allowed the migration thereof to be easily observed.
The results showed that the fractional extract (CBT-143-S-F6F7) was
indeed capable of inhibiting migration of the endothelial
cells.
(3) Evaluation for Inhibition of Mobility of Hepatocellular
Carcinoma Cells (Wound Healing Migration Assay)
[0109] Hepatocellular carcinoma cell line, SK-Hep-1, which has high
mobility, was selected to be tested. First, the cells were
inoculated in a 6-well plate. After attaching to the plate, a part
of the cells were scraped to form a line on the plate. After the
cells scraped from the line were washed out, the state of the cells
migrating to mend the line was observed at 24 hours and 48 hours
(FIG. 12). The results showed that, after treatment with different
concentrations of the crude extract (CBT-143-S) and the fractional
extract (CBT-143-S-F6F7), the cell migration of the cells were all
reduced.
[0110] At 48 hours, the observation was terminated, and an MTT
assay was immediately performed on the cells in the plate to
determine the viability of the cells. Through comparing the cell
mobility with cell viability, it could be determined whether the
crude extract (CBT-143-S) and the fractional extract
(CBT-143-S-F6F7) had the effect of inhibiting migration of
SK-Hep-1.
[0111] According to the results of the analysis, it was determined
that the effect of inhibiting migration of SK-Hep-1 of the crude
extract (CBT-143-S) and the fractional extract (CBT-143-S-F6F7)
increased with time.
6. In Vivo Anti-Angiogenesis Experiments for the Crude Extract
(CBT-143-S) and the Fractional Extract (CBT-143-S-F6F7)
[0112] Through a subcutaneous injection to BALB/c mice to form
Matrigel plugs, an evaluation was made of whether a test substance
was capable of inhibiting angiogenesis induced by vascular
endothelial growth factor or Hep-3B conditional medium (CM).
(1) Matrigel Plugs Assay
[0113] Matrigel is extracted from the Engelbreth-Holm-Swarm (EHS)
mouse sarcoma and contains rich extracellular matrix proteins and
contains various factors need by vascular growth. In a condition of
room temperature and 37.quadrature., Matrigel is capable of
polymerize as a solid gel from, and can provide a better growth
environment for vascular endothelial cells.
[0114] Matrigel and vascular endothelial growth factors (FGFb+VEGF)
or Hep-3B conditional medium (CM) were mixed, and then a test
substance was added thereto and mixed.
[0115] The experimental method is described as follows:
[0116] (a) Animal strain: Female BALB/c mice (BALB/cAnNCrlBltw,
purchased from BioLasco Taiwan Co., Ltd), 6-8 weeks old.
[0117] (b) A group treated with Matrigel mixed with PBS was used as
a negative control group, and a group treated with Matrigel mixed
with FGFb (500 ng/ml)+VEGF (500 ng/ml) or mixed with Hep-3B
conditional medium (CM) was used as a positive control group. A
test substance was mixed with Matrigel containing vascular
endothelial growth factors and then injected to BALB/c mice by
subcutaneous injection to observe whether the test substance is
capable of inhibiting angiogenesis in a Matrigel.
[0118] (c) Hemoglobin content in Matrigel was quantified by a
QuantiChrom.upsilon. Hemoglobin Assay Kit (DIHB-250) to evaluate
the amount of neo-blood vessels in Matrigel.
[0119] After subcutaneous injection to BALB/c mice for 14 days, the
Matrigel plugs in the subcutaneous tissue of the mouse was taken
out and equal amount of dispase was added therein to form a mixture
and reacted at 37.quadrature. for 16 hours to dissolve
intercellular substance gel. After that, the mixture was
centrifuged at 14,000 rpm for 10 minutes, and transferred the
supernatant to another microcentrifuge tube. 50 .mu.L of
supernatant and 200 .mu.L of reaction reagent contained by the
Hemoglobin Assay Kit are mixed. After the supernatant was reacted
with the reagent for 5 minutes, the optical density (O.D.) at 400
nm therefrom was determined. Optical density (O.D.) values were
converted into a unit concentration according to the formula shown
in the following:
((OD.sub.SampleOD.sub.Blank)/(OD.sub.CalibrationOD.sub.Blank))100n
(mg/dL),
wherein 100 was a calibrator concentration, and n is
dilution-fold.
[0120] The experimental results:
[0121] (a) Assay for evaluation of in vivo growth factor-induced
angiogenesis
[0122] In the assay for evaluation of in vivo growth factor induced
angiogenesis, in the negative control group which was only treated
with PBS, the hemoglobin concentration of the Matrigel plug
implanted in the subcutaneous tissue of the mouse for 14 days was
close to a background value, 17.68.+-.11.64 (mg/dL). However, after
addition of FGFb (500 ng/ml)+VEGF (500 ng/mL), significant
angiogenesis could be found in the Matrigel plug, and the
hemoglobin concentration could reach 52.64.+-.26.18 (mg/dL). In the
group orally administered with Nexavar every day (30 mg/kg/day),
the hemoglobin concentration was significantly reduced to
27.09.+-.5.88 (mg/dL). In the group treated with the crude extract
(CBT-143-S) (100 .mu.g/mL), the hemoglobin concentration was
27.92.+-.9.62 (mg/dL). Moreover, in the groups treated with 30
.mu.g/mL and 100 .mu.g/mL of the fractional extract
(CBT-143-S-F6F7), the hemoglobin concentrations were 56.70.+-.5.58
(mg/dL) and 25.08.+-.9.59 (mg/dL), respectively.
[0123] The results showed that at a higher concentration, the crude
extract (CBT-143-S) and the fractional extract (CBT-143-S-F6F7)
have the potential for inhibiting angiogenesis (FIG. 13).
[0124] (b) Assay for evaluation of in vivo Hep-3B hepatocellular
carcinoma cell conditional medium-induced angiogenesis
[0125] In the assay for evaluation of in vivo Hep-3B hepatocellular
carcinoma cell conditional medium-induced angiogenesis: in the
negative control group, which was only treated with PBS, the
hemoglobin concentration of the Matrigel plug implanted in the
subcutaneous tissue of the mouse for 14 days was 16.23.+-.11.64
(mg/dL). However, after the addition of a conditional medium,
significant angiogenesis could be found in the Matrigel plug, and
the hemoglobin concentration could reach 52.61.+-.13.14 (mg/dL). In
the group orally administered with Nexavar every day (30
mg/kg/day), the hemoglobin concentration was significantly reduced
to 25.05.+-.17.59 (mg/dL). In the group treated with the crude
extract (CBT-143-S) (100 .mu.g/mL), the hemoglobin concentration
was 22.91.+-.15.95 (mg/dL). Moreover, in the groups respectively
treated with 30 .mu.g/mL and 100 .mu.g/mL of the fractional extract
(CBT-143-S-F6F7), the hemoglobin concentrations were 51.49.+-.8.97
(mg/dL) and 20.61.+-.7.52 (mg/dL), respectively.
[0126] The results showed that at a higher concentration, the crude
extract (CBT-143-S) and the fractional extract (CBT-143-S-F6F7)
have the potential for inhibiting angiogenesis (FIG. 14).
(2) In Vivo Anti-Angiogenesis Experiment (Chorioallantoic Membrane
(CAM) Assay)
[0127] Filter paper absorbed a test drug or PBS, and then was
placed on a chorioallantoic membrane (CAM) of a chicken embryo, and
vascular formation was observed.
[0128] The chicken embryo of a specific-pathogen-free (SPF) White
Leghorn chicken was placed laterally in an incubator
(37.3.quadrature., RH 55-60%). On day 4 (D4) of the incubation
period, 2.5 mL of albumin was sucked out with a 20 G needle and a
fake air chamber was made on the embryo. After that, the needle
hole and the fake air chamber were sealed with 3M breathable tape.
On day 7, a test drug was dissolved with DMSO and then diluted with
PBS. For each group, including a PBS control group, the DMSO final
concentration was 1%. Circular Advantec.RTM. Filter paper
(diameter: 6mm) (Toyo Roshi Kaisha, Ltd.) was used to absorb the
diluted test drug, and the absorbing volume was 25 .mu.L. Each test
drug was prepared for 3 dosages, which were 50, 25, and 10
.mu.g/embryo. Moreover, on day 7 (D7), the filter paper was placed
on a chorioallantoic membrane, and then on day 9 (D9), the
chorioallantoic membrane was photographed with a dissecting
microscope, SZX16 (Olympus) at 1.25.times. objective lens. By using
the filter paper as the center, 4 concentric circles were marked on
the photograph (the diameters thereof were 7, 8, 9 and 10 mm; the
total circumference of the four concentric circles was 106.8 mm,
and the total circumference represented the region close to the
filter paper. The amount of vessel crossed with the concentric
circles (namely, the vascular density index, or VDI) was determined
by the naked eye) to evaluate the state of angiogenesis. The
vascular density index for the same photograph was determined by 3
people and the mean value thereof was adopted. The vascular density
index for each drug treatment group was compared with the control
group to observe whether there was a significant difference or not.
An unusual region for vascular morphology was observed and
photographed using 4.times. and 8.times. objective lenses.
[0129] The experimental results:
[0130] According to Table 2 and FIG. 15, the fractional extract
(CBT-143-S-F6F7) (25 g/embryo) and the crude extract (CBT-143-S)
(50 .mu.g/embryo) all resulted in a significant decrease in the
angiogenesis of the chorioallantoic membrane. In FIG. 15, reference
number 1301 refers to filter paper containing a test drug.
Furthermore, according to FIG. 16, it was found that moderate and
high dosage groups of the fractional extract (CBT-143-S-F6F7)
indeed resulted in conformation change for capillary vessels. In
FIG. 16, reference number 1401 refers to filter paper containing a
test drug.
TABLE-US-00003 TABLE 2 Results of in vivo anti-angiogenesis
experiment (Chorioallantoic membrane (CAM) assay) VDI (in 106.8 mm)
Number Stan- Test drug Death/ N dard p value (in 1% .mu.g/ test
(for Aver- devi- (t-test DMSO) embryo embryo VDI) age ation CV vs
PBS) CBT-143-S- 50 4/5 1 112.3 -- -- -- F6F7 CBT-143-S- 25 0/6 6
119.1 30.1 0.25 0.023 F6F7 CBT-143-S- 10 0/5 5 149.3 40.3 0.27
0.506 F6F7 CBT-143-S 50 1/6 5 124.5 20.1 0.16 0.020 CBT-143-S 25
1/6 5 146.5 22.1 0.15 0.258 CBT-143-S 10 1/5 4 184.6 9.2 0.05 0.131
PBS -- 0/6 5 163.7 22.7 0.14 --
7. Toxicity Tests for the Crude Extract (CBT-143-S) and the
Fractional Extract (CBT-143-S-F6F7) to Normal Cells
[0131] In order to investigate whether the inhibiting effects
mentioned above of the crude extract (CBT-143-S) and the fractional
extract (CBT-143-S-F6F7) are due to the toxicity thereof or not,
cell viability assay for normal cells, PBMCs, and growth inhibition
assays for HUVECs were performed to the crude extract (CBT-143-S)
and the fractional extract (CBT-143-S-F6F7), and a commercial
clinical drug, sorafenib, were used for comparison as a
reference.
(1) Toxicity Tests for Normal Cells, PBMCs (Alamarblue Cell
Viability Assay)
[0132] Alamarblue is a plant dye that is usually blue. The dye
decomposes during cell proliferation and reduces from blue to pink,
and a rise in O.D. at 570 nm can be determined. Conversely, the
more dead cells there are, the lower the O.D. value is determined
to be.
[0133] PBMC cells (1.times.10.sup.5 cells/well) were inoculated in
a 96-well plate, and the crude extract (CBT-143-S) and the
fractional extract (CBT-143-S-F6F7) samples, etc. with different
concentrations (300, 100, 30, 10, 3 and 1 .mu.g/mL) were added to
the plate. After incubating for 72 hours, Alamarblue was added to
the plate and incubated at 37.quadrature. for 24 hours, and then
the O.D. at 570 nm/600 nm thereof was determined by an ELISA
reader. After that, the obtained O.D. values were substituted into
the formal calculation as follows:
[0134] Percent difference in reduction=
( OX ) .lamda. 2 A .lamda. 1 - ( OX ) .lamda. 1 A .lamda. 2 of test
agents dilution ( OX ) .lamda. 2 A 0 .lamda. 1 - ( OX ) .lamda. 1 A
0 .lamda. 2 untreated positive growth control .times. 100
##EQU00001##
[0135] .lamda..sub.1=570
[0136] .lamda..sub.2=600
[0137] (.epsilon..sub.OX).lamda..sub.2=117,216 (OX represents
oxidation)
[0138] (.epsilon..sub.OX).lamda..sub.1=80,586
[0139] A.lamda..sub.1=0.65 Observed absorbance reading for the test
well
[0140] A.lamda..sub.2=0.36 Observed absorbance reading for the test
well
[0141] A.lamda..sub.2=0.78 Observed absorbance reading for the
positive control well
[0142] A.lamda..sub.1=0.19 Observed absorbance reading for the
positive control well
[0143] The results showed that when the concentration of the
fractional extract (CBT-143-S-F6F7) was 30 ug/ml, cell viability of
PBMCs was still higher than 50%, and that showed that IC.sub.50 of
the fractional extract (CBT-143-S-F6F7) was greater than 30 ug/mL
(Table 3).
[0144] Toxicity tests for the crude extract (CBT-143-S) and the
fractional extract (CBT-143-S-F6F7) to normal cells, PBMCs
TABLE-US-00004 IC.sub.50 of a test drug for PBMCs CBT-143-S (ug/mL)
CBT-143-S-F6F7 (ug/mL) Sorafenib (uM) IC.sub.50 >30 >30 10.75
.+-. 0.95
(2) Inhibition Analysis for HUVEC Growth
[0145] Similarly, an Alamarblue assay was used to evaluate cell
viability. HUVEC cells (1.times.10.sup.4 cells/well) were
inoculated in a 96-well plate. The crude extract (CBT-143-S) and
the fractional extract (CBT-143-S-F6F7) samples, etc. with
different concentrations (300, 100, 30, 10, 3 and 1 .mu.g/mL) were
added to the plate, and absorbance at 0 hours and 48 hours was
record to evaluate whether cell growth was affected by the test
drug or not. The results showed that 50% growth inhibition
concentration (GI.sub.50) of the crude extract (CBT-143-S) and the
fractional extract (CBT-143-S-F6F7) were 0.52 ug/mL and 0.006 ug/mL
(FIG. 17). According to the above-mentioned, the inhibition
phenomenon of Juniperus chinensis L. var. sargentii Henry extract
to the growth of HUVEC was very obvious.
8. Rat Corneal Neovascularization Assay
[0146] Pellets made of the hydron polymer (polyHEME) and a test
drug or DMSO were implanted on the corneal surface of rats, and
vascular formation thereof was observed.
[0147] A hydron polymer (polyHEME) was dissolved in absolute
ethanol (12% w/v) in a rotator at 37.degree. C. overnight, and then
stored at room temperature before pellet making. Each pellet for
the corneal pocket assay contained 60 ng of bFGF, or 1 ug JC-5 and
20 ug of sucralfate in 3 uL of casting gel, which was constituted
as a 50:50 (vol/vol) mixture of hydron gel and
factor-sucralfate-PBS. The casting gel was promptly pipetted onto
an autoclaved, sterilized 20.times.20-mm piece of nylon mesh with
an approximate pore size of 2.times.2 mm. The pellets were prepared
the day before corneal surgery in a laminar flow hood under sterile
conditions. Subsequently, the fibers of the mesh were pulled apart,
and uniformly sized pellets of 2.times.2.times.0.4 mm.sup.3 were
selected for implantation. All procedures were performed in sterile
conditions. Such pellets can be stored frozen at -20.degree. C. for
several days without loss of bioactivity. Each group contained six
eyes. Male rats (Sprague Dawley, purchased from National Laboratory
Animal Center, Taiwan.) were anesthetized with ketamine, eyes were
topically anesthetized with 0.5% proparacaine. Using an operative
microscope, we performed a central intrastromal linear keratotomy
(.about.2.5 mm in length) with a surgical knife at the 12 o'clock
position. A lamellar micropocket was dissected to 2 mm near the
limbus. The pellet was advanced to the end of the pocket.
Antibiotic ointment (erythromycin) was applied once to the surgical
eye to prevent infection and to decrease irritation of the
irregular ocular surface. 7 and 14 days after pellet implantation,
the rats were anesthetized with ketamine. The eyes were exposed,
and the maximum vessel length (VL) of the neovascularization zone,
extending from the base of the limbal vascular plexus toward the
pellet, was measured. Photographs were taken. The contiguous
circumferential zone of neovascularization (CN) was measured in
clock hours with a 360.degree. reticule (where 30.degree. of arc
equals 1 clock hour).
[0148] The experimental results:
[0149] Pellets containing the slow-release polymer hydron with PBS
alone, bFGF alone, or bFGF plus JC-5 were implanted in rat corneas.
Pellets containing PBS alone (N=6 eyes in all groups) did not
induce neovascularization (FIGS. 18A, 18B and 18C, PBS). Pellets
containing 60 ng of bFGF induced neovascularization on
postoperative day 7 (FIGS. 18A, 18B and 18C, bFGF). The neovascular
response was inhibited in implanted cornea with 60 ng of bFGF plus
1 ug JC-5 on day 7 (FIGS. 18A, 18B and 18C, bFGF+JC-5). These
results showed JC-5 inhibited corneal neovascularization in a rat
corneal pocket assay.
9. Laser-Induced Choroidal Neovascularization (CNV) Assay
[0150] Male rats (brown Norway rats, purchased from BioLASCO Co.,
Taiwan) were used. CNV lesions were induced in rat eyes by laser
photocoagulation. Briefly, after the rats were anesthetized, their
pupils were dilated with 1% tropicamide. A piece of 18.times.24
mm.sup.2 standard cover glass served as a contact lens for
application of photocoagulation. Argon laser irradiation was
delivered through a slit lamp. Laser parameters were set as
follows: spot size of 50 .mu.m; power of 120 mW; and exposure
duration of 0.1 second. An attempt was made to break Bruch's
membrane, as clinically evidenced by central bubble formation, with
or without intraretinal or choroidal hemorrhage. Four to six
lesions were created between the major retinal vessels in each
fundus. One day prior to laser treatment, animals received
bilateral intravitreal injection of 2 ul JC-5 (1 ug/eye, N=6) or 2
ul vehicle (DMSO, N=6). Fourteen days later, a self-retaining eye
speculum was placed in the eye to facilitate administration.
Intravitreal injections of 2 ul of JC-5 or vehicle were
administered per eye using a 30-gauge needle. Intravitreal
injections were followed by topical administration of 1-2 drops of
Vigamox (0.5% moxifloxacin hydrochloride) antibacterial ophthalmic
solution. Characterization of laser-induced CNV on fluorescein
angiograms was performed using a digital imaging system. Rats were
analyzed under general anesthesia after dilation of the pupil and
subsequent intraperitoneal injection of 0.5 ml of 2.5% fluorescein
sodium (Alcon, CITY, Germany). Each investigation included early
phase (1-3 min after injection) images. For quantitative analysis
of fluorescein leakage, the area of fluorescein leakage, determined
as area of hyperfluorescence in which no normal retinal blood
vessels were observed, was measured for each burn using ImageJ
software (NIH, USA).
[0151] The experimental results:
[0152] The therapeutic efficacy of JC-5 (1 .mu.g/2 ul in DMSO) over
time was evaluated by using fluorescein angiography to analyze
laser-induced CNV after JC-5 treatment. JC-5-treated eyes had less
fluorescent dye uptake and extent of CNV (FIG. 19A). In addition,
CNV lesions areas in JC-5-treated rats were significantly decreased
on day 7 and day 14 compared to DMSO-treated vehicles (FIGS. 19A
and 19B). These results indicated that topical JC-5 application
attenuated the severity of experimental CNV.
[0153] It will be apparent to those skilled in the art that various
modifications and variations can be made to the disclosed
embodiments. It is intended that the specification and examples be
considered as exemplary only, with a true scope of the disclosure
being indicated by the following claims and their equivalents.
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