Note: Descriptions are shown in the official language in which they were submitted.
CA 02932277 2016-06-06
185262
SOYBEAN SEED EXTRACT, METHOD FOR PRODUCING THE SAME AND
USES THEREOF
FIELD OF THE INVENTION
[0001] The invention relates to a soybean seeds extract, method for producing
the
same and uses of the soybean seed extract in promoting wound healing,
promoting
neuron cell proliferation and/or treating brain diseases and/or
neurodegenerative
diseases, treating breast cancer, reducing side effects of interfering with
DNA and/or
RNA replication drugs, and/or enhancing pharmaceutical effects of interfering
with
DNA and/or RNA replication drugs.
BACKGROUND OF THE INVENTION
[0002] Glycine max (L.) Merr., including soybean and black soybean, is one of
the
most important sources of oil and proteins in the world. For instance,
soybeans can
be processed to obtain edible oil that is used as salad oil, or for
manufacture of
margarine and shortening. Soybean oil can be also used in the manufacture of
paints,
linoleum, oilcloth, printing inks, soaps, insecticides, and disinfectants.
Besides,
lecithin phospholipids obtained from the by-products of the oil industry, can
be used as
wetting and stabilizing agents in food, cosmetics, pharmaceuticals, leathers,
paints,
plastics, soaps, and detergents. Soy meal is a very protein-rich feeding stuff
for
livestock. In addition, soybean protein can be used in manufacture of
synthetic fibers,
adhesives, textile sizing, waterproofing, fire-fighting foams and so on.
[0003] In medical use, soybeans have been reported to have effects on many
diseases.
[0004] Soybean can be used as a nutritious supplement for regulating the
functions
of bowels, heart, kidney, liver, and stomach. Since soybean oil contains a
high
amount of unsaturated fatty acids, it can be used to combat
hypercholesteremia.
Medical lecithin from soybeans functions as a lipotropic agent. In addition,
- 1 -
stigmasterol known as an antistiffness factor, and sitosterol used as a
replacement for
diosgenin in some antihypertensive drugs, are prepared from soybeans.
Isoflavones and
phyto-oesterogens found in soybeans have been suggested to have a preventive
effect
against various cancers comprising breast, prostate, and colon cancers
(Adlercreutz, H.;
Phyto-oestrogens and cancer. The Lancet Oncology, 2002, Vol. 3, p. 364-373).
Other
literature indicates that in order to achieve the effect on preventing the
occurrence of
breast cancer of isoflavones, at least 100 mg daily dose should be consumed
continually
for a month, and it represents that only by being consumed continually at the
high dose,
isoflavones exhibit anti-cancer effect (Lu LJ, Anderson KE, Grady JJ, Nagamani
M.;
Effects of soya consumption for one month on steroid hormones in premenopausal
women: implications for breast cancer risk reduction. Cancer Epidemiol
Biomarkers Prey.
1996 Jan; 5(1): 6370). Consumption of phytosterol-supplemented margarine is
also
found to lower total plasma cholesterol and LDL-cholesterol concentrations in
older
middle-aged hypercholesterolemic individuals (Matvienko, 0. A., Lewis, D. S.,
Swanson,
M., Aendt, B., Rainwater, D. L., Stewart, J., and Alekel, D. L.; A single
daily dose of
soybean phytosterols in ground beef decreases serum total cholesterol and LDL
cholesterol in young, mildly hypercholesterolemic men. Am J Clin Nutr., 2002,
76, p. 57
64).
[0005] Some
extracts from soybean have been also reported to have pharmaceutical
effects. 1,1-
dipheny1-2-picrylhydrazyl (DPPH) radical-scavenging activity of 70%
aqueous acetone extract from the seed coat of a brown soybean variety, Akita-
Zairai, is
disclosed (Takahata, Y., 0.-Kameyama, M., Furuta, S., Takahashi, M., and Suda,
I.;
Highly polymerized procyanidins in brown soybean seed coat with a high
radical-scavenging activity. J. Agric. Food Chem., 2001, 49, p. 5843 5847). An
extract
from germ extracts, soybean, rice bran, tear grass, sesame, wheat, citron,
green tea, green
leaf extract, and malted rice, which are slowly roasted under a temperature at
less than 60
C and fermented with Aspergillus oryzae over 3 days to transform each
ingredient into
- 2 -
CA 2932277 2018-04-26
=
low molecular weight substances, is found to have antioxidative effects
(Minamiyama, Y.,
Yoshikawa, T., Tanigawa, T., Takahashi, S., Naito, Y., Ichikawa, H., and
Kondo, M.;
Antioxidative effects of a processed grain food. J. Nutr. Sci. Vitaminol.,
1994, 40, P. 467
477). Water extract of black soybean is also reported to effect on
acetaminophen-induced liver injury by measuring serum glutamate-oxalate-
transaminase
(sGOT) and serum glutamate-pyruvate-transaminase (sGPT) activities in rats
(Wu, S.-J.,
Wang, J.-S. and Chang, C.-H.; Evaluation of hepatoprotective activity of
legumes.
Phytomedicine, 2001, Vol. 8(3), p. 213 219).
[0006] Some specific extracts from soybean have been found to be applied in
cosmetics
or pharmaceuticals in treating some skin diseases, A soya extract, which
contains
sphingomyelins and phospholipids in defined ratios is disclosed to be used in
cosmetics
for the treatment of dry skin (U.S. Patent Pub. No. US2002/0009509 Al). Such
extract
is produced by extracting ripe whole soya beans or oil-free soya flour using
aliphatic
alcohols alone or in a mixture with water and followed by the treatment with
aliphatic
hydrocarbons and with aliphatic ketones. Therefore, the extract is
liposoluble.
[0007] An acne medicine, comedo production inhibitor or cosmetic composition
containing one or more plant extracts selected from whey, and a Phellodendron
amurense
Ruprecht extract, and further one or more extracts selected from Scutellaria
baicalensis
Geoegi, Symphytum officinale Linne, and Glycine max (L.) Merrill, is found to
be
effective on preventing or treating skin diseases such as acne or inflammatory
chapped
skin caused by the acne (JP Patent No. 2001097842).
100081 Products of fermenting soybean by microorganisms are also applied as
anti-active oxygen action compositions, agents, foods, cosmetics and medicines
(such as
JP Patent No. 4139132).
[0009] Although many uses of soybeans have been reported, different
applications of
soybean extract are yet to be developed.
- 3 -
CA 2932277 2018-04-26
SUMMARY OF THE INVENTION
[0010]
The invention relates to a soybean seed extract, method for producing the same
and uses of the soybean seed extract in promoting wound healing, promoting
neuron cell
proliferation and/or treating brain diseases and/or neurodegenerative
diseases, treating
breast cancer, reducing side effects of interfering with DNA and/or RNA
replication drugs,
and/or enhancing pharmaceutical effects of interfering with DNA and/or RNA
replication
drugs.
[0011] The invention is to provide an extract composition comprising a soybean
seed
extract, which soybean seed extract is prepared by a process comprising steps
of:
(a) providing soybean seeds and an extracting solution, which extracting
solution is water or an alcohol solution containing alcohol at the
concentration lower
than about 90 wt%;
(b) extracting the soybean seeds with the extracting solution at a barometric
pressure lower than about 1 atm and at a temperature lower than about 60 C to
obtain
an crude extract; and
(c) removing solids from the crude extract to obtain a liquid portion.
[0011a] The present invention is also to provide an extract composition
comprising a
soybean seed extract and a soybean seed vapor fraction, wherein the soybean
seed extract
is prepared by a process consisting of steps of:
(a) providing soybean seeds and an extracting solution, wherein the extracting
solution is water or an alcohol solution containing alcohol at a concentration
lower
than 90 wt%;
(b) extracting the soybean seeds with the extracting solution at a barometric
pressure lower than 1 atm and at a temperature lower than 60 C to obtain a
crude
- 4 -
Date Recue/Date Received 2020-04-23
extract;
(c) removing solids from the crude extract to obtain a liquid portion; and
(d) concentrating the liquid portion obtained in the step (c) to obtain a
concentrated solid portion;
wherein the soybean seed vapor fraction is prepared by a process comprising
steps of:
(i) providing soybean seeds in a vapor extracting solution, wherein the vapor
extracting solution is water or an alcohol solution containing alcohol at a
concentration lower than 15 wt%; and
Hi (ii) extracting the soybean seeds with the vapor extracting solution
at a
barometric pressure lower than 1 atm and at a temperature lower than 110 C and
collecting the vapor fraction.
[0011b] The present invention is also to provide a method for preparing an
extract
composition comprising a soybean seed extract and a soybean seed vapor
fraction,
said method comprising:
preparing the soybean seed extract by a process consisting of steps of:
(a) providing soybean seeds and an extracting solution, wherein the extracting
solution is water or an alcohol solution containing alcohol at a concentration
lower
than 90 wt%;
(b) extracting the soybean seeds with the extracting solution at a barometric
pressure lower than 1 atm and at a temperature lower than 60 C to obtain a
crude
extract;
- 5 -
Date Recue/Date Received 2020-04-23
(c) removing solids from the crude extract to obtain a liquid portion; and
(d) concentrating the liquid portion obtained in step (c) to obtain a
concentrated
solid portion; and
preparing the soybean seed vapor fraction by a process comprising steps of:
(i) providing soybean seeds in a vapor extracting solution, wherein the vapor
extracting solution is water or an alcohol solution containing alcohol at a
concentration lower than 15 wt%; and
(ii) extracting the soybean seeds with the vapor extracting solution at a
barometric pressure lower than 1 atm and at a temperature lower than 110 C and
collecting the vapor fraction;
and
combining the soybean seed extract and the soybean seed vapor fraction to
obtain the
extract composition.
[0012] The present invention is also to provide a method for preparing the
extract
composition as mentioned above comprising a process for preparing the soybean
seed
extract comprising steps of:
(a) providing soybean seeds and an extracting solution, which extracting
solution is water or an alcohol solution containing alcohol at the
concentration lower
than about 90 wt%;
(b) extracting the soybean seeds with the extracting solution at a barometric
pressure lower than about 1 atm and at a temperature lower than about 60 C to
obtain
a crude extract; and
(c) removing solids from the crude extract to obtain a liquid portion.
[0013] The present invention is also to provide use of the extract composition
as
- 5a -
Date Recue/Date Received 2020-04-23
mentioned above in the manufacture of a medicament of promoting wound healing.
[0014] The present invention is also to provide use of the extract composition
as
mentioned above in the manufacture of a medicament of promoting neuron cell
proliferation and/or treating brain diseases and/or neurodegenerative
diseases.
[0014a] The present invention is also to provide the use of the extract
composition as
defined herein in the manufacture of a medicament for promoting neuron cell
proliferation,
improving memory, and/or treating brain diseases and/or neurodegenerative
diseases,
wherein the brain diseases and/or neurodegenerative diseases are selected from
the group
consisting of vascular dementia, Alzheimer's disease, and cerebral vascular
disease.
io [0015] The present invention is also to provide use of the extract
composition as defined
herein in the manufacture of a medicament for treating breast cancer.
[0016] The present invention is also to provide use of the extract composition
as
mentioned above in the manufacture of a medicament of reducing side effects of
interfering
with DNA and/or RNA replication drugs, and/or enhancing pharmaceutical effects
of
interfering with DNA and/or RNA replication drugs.
[0016a] The present invention is also to provide the use of the extract
composition as
defined herein in the manufacture of a medicament for enhancing tumor volume
reduction
of cyclophosphamide.
[0017] The present invention is described in detail in the following
sections. Other
characteristics, purposes and advantages of the present invention can be found
in the
detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIGs. 1 to 3 show the ion chromatography spectrograms of the soybean
seed
extract (GMA1) according to the invention.
[0019] FIGs. 4 to 6 show the ion chromatography spectrograms of the soybean
seed
vapor fraction (GMC1) according to the invention.
- 5b -
Date Recue/Date Received 2020-04-23
[0020] FIG. 7 shows the high performance liquid chromatography (HPLC)
spectrogram
of the isoflavones standard.
[0021] FIG. 8 shows the HPLC spectrogram of the soybean seed vapor fraction
(GMC1).
[0022] FIG. 9 shows the HPLC spectrogram of the extract composition containing
0.3
part by weight of the soybean seed extract (GMA1) and 1 part by weight of the
soybean
seed vapor fraction (GMC1).
- 5c -
Date Recue/Date Received 2020-04-23
CA 02932277 2016-06-06
185262
[0023] FIG. 10 shows the effects of GMA1 and GMC1 on diabetic wound healing.
To investigate whether GMC1 and GMA1 have effects on improving wound closure,
a
STZ-induced diabetic rat model was utilized. For comparison, two groups
treated
with cream base and CGS-21680 were also conducted. The results show that both
0.3 % GMA1 and GMC1 are effective on would closure, compared to cream base
alone, and 0.3 % GMA1 treated group slightly exhibits better outcome in wound
closure than GMC1.
[0024] FIG. 11 shows the effects of different dosages of GMA1 in combination
with
GMC1 on diabetic wound healing. Experiments were conducted to study the best
m combination of GMA1 and GMC1 in diabetic wound closure. As shown in FIG.
11,
the GMA I was combined with GMC I at different concentrations: 0.009%, 0.045%
and 0.09%, respectively. For comparison, two groups treated with cream base
and
CGS-21680 were also conducted in all experimental studies. The results show
that
for wound healing, the combination effects of the dose levels, 0.009%, 0.045%
and
0.09%, of GMA1 with GMC I are better than GMC1 alone. The most effective
combination treatment is found in the highest dosage of GMA1 (0.09%).
[0025] FIG. 12 shows the effects of combinations of the soybean seed extract
and
soybean seed vapor fraction on HaCaT cell migration.
[0026] FIG. 13 shows the cell viability rates of IMR-32 cells treated with
GMA1.
The data are shown as the means + SEM of seven groups. The viability rates of
IMR-32 cells treated with 25 mg/ml GMA1 and positive control are highly
significantly and significantly increased (GMA1 p=0.0008, positive control
p=0.0246).
The results show that GMA1 promotes cell growth (*** p<0.001).
[0027] FIG. 14 shows the cell viability rates of IMR-32 cells treated with
GMC1.
The data are shown as the means + SEM of seven groups. The viability rates of
IMR-32 treated with 25 mg/ml GMC1 and positive control are highly
significantly and
significantly increased (GMC1 p=0.0255 positive control p=0.0246). The results
show that GMC1 promotes cell growth (*p<0.05).
- 6 -
CA 02932277 2016-06-06
185262
[0028] FIG. 15 shows the cell viability rates of IMR-32 cells treated with
GMA1 and
GMC1 (GM). The data are shown as the means + SEM of seven groups. The
viability rate of IMR-32 cells treated with 25 mg/ml GM and positive control
are
highly significantly and significantly increased (GM p=0.0059, positive
control
p=0.0246). The results show that GM promotes cell growth (** p<0.01).
[0029] FIG. 16 shows the diagram of the radial arm maze.
[0030] FIG. 17 shows the tracking paths of dementia rats in a radial arm maze.
The
tracking path of rats on Day 4 suggests that the rats in the AlC13 + N2 group
arc able to
reach locations of baits with their memory after training, and the rats in the
AlC13
lo group and AlC13+cream base group are not.
[0031] FIG. 18 shows the diagram of the total number of errors (TME) of
dementia
rats in the radial arm maze test. The TME values of A1C13 group and
A1C13+cream
base group show no significant difference (p>0.05), and the TME values on Day
4
after the treatment of the extract composition comprising the soybean seed
extract and
soybean seed vapor fraction are significantly lower than those of A1C13 group
(A1C13+N2 p=0.0128). It shows that the extract composition comprising the
soybean
seed extract and soybean seed vapor fraction has the effect on treating
dementia in rats.
The data are shown as means + SEM. The Student t-test and repeated ANOVA are
used for statistical analysis. When comparing A1C13 group and AlC13+eream base
group, p<0.05 is marked with *, and p<0.01 is marked with**, meaning that
there is a
significant difference in statistics.
[0032] FIG. 19 shows the diagram of the reference (long-term) memory errors
(RME)
of dementia rats in the radial arm maze test. The RME values of AlC13 group
and
AlC13+cream base group show no significant difference (p>0.05), and the RME
values
on Day 4 after the treatment of the extract composition comprising the soybean
seed
extract and soybean seed vapor fraction are significantly lower than those of
AlC13
group (p=0.0046). It shows that the extract composition comprising the soybean
seed
extract and soybean seed vapor fraction has the effect on restoring the long-
term
-7-
CA 02932277 2016-06-06
185262
memory of rats. The data are shown as means + SEM. The Student t-test and
repeated ANOVA are used for statistical analysis. p<0.05 is marked with *, and
p<0.01 is marked with**, meaning that there is a significant difference in
statistics.
1100331 FIG. 20 shows the diagram of the total number of errors (TME) of
vascular
dementia rats treated with cream base (M), GMC1(M1), and GMC1+0.5%GMA I (M3)
in the radial arm maze test. It shows the TME of two-sided carotid arterial
ligature
(2V0) group is significantly higher than Sham group (day 8, p=0. 0013), which
refers
to successful induction of vascular dementia in rats by two-sided carotid
arterial
ligature. The results show that TME values are significantly improved compared
to with 2V0 group after treated by M1 and M3 (day 8, M1 p=0.0019; M3
p=0.0355).
This suggests that M1 and M3 have efficacy for treating vascular dementia in
rats.
The data are shown as means + SEM. The Student t-test and repeated ANOVA are
used for statistical analysis. p<0.05 is marked with *, and p<0.01 is marked
with**,
meaning that there is a significant difference in statistics.
[0034] FIG. 21 shows the diagram of the reference (long-term) memory errors
(RME)
of vascular dementia rats treated with cream base (M), GMCI(M1), and
GMC1+0.5%GMA1 (M3) in the radial arm maze test. It shows that the RME of
two-sided carotid arterial ligature (2V0) group is significantly higher than
Sham group
(day 8, p=0. 0016), which refers to successful induction of vascular dementia
in rats by
two-sided carotid arterial ligature. The results show that RME values are
significantly improved compared to 2V0 group after treated by M1 and M3 (day8,
M1
p=0.0029; M3 p=0.0171). This suggests that M1 and M3 have efficacy for
treating
RME of vascular dementia in rats. The data are shown as means + SEM. The
Student t-test and repeated ANOVA are used for statistical analysis. p<0.05 is
marked with *, and p<0.01 is marked with**, meaning that there is a
significant
difference in statistics.
[0035] FIG. 22 shows the diagram of the working memory (short-term memory)
errors (WME) of vascular dementia rats treated with cream base (M), GMC1(M1),
and
- 8 -
CA 02932277 2016-06-06
185262
GMC1+0.5%GMA1 (M3) in the radial arm maze test. It shows that the WME of
two-sided carotid arterial ligature (2V0) group is significantly higher than
Sham group
(day 8, p=0.0111), which refers to successful induction of vascular dementia
in rats by
two-sided carotid arterial ligature. The
results show that WME values are
significantly improved compared to 2V0 group after treated by M1 and M3 (day8,
M1
p=0.0111; M3 p=0.0139). This suggests that M1 and M3 have efficacy for
treating
WME vascular dementia in rats. The data are shown as means + SEM. The
Student t-test and repeated ANOVA are used for statistical analysis. p<0.05 is
marked with *, and p<0.01 is marked with**, meaning that there is a
significant
difference in statistics.
[0036] FIG. 23 shows the tracking path of vascular dementia (VD) rats in a
radial
arm maze. The tracking path of VD rats on day 8 suggests that sham group, GMC1
(M1) and GMC1+0.5%GMA1 (M3) groups are able to reach the locations of baits
with their memory after training, while 2V0 and M groups are not.
[0037] FIG. 24 shows the statistical diagram of brain injury grades of
vascular
dementia rats. The extent of brain injury in accordance with CT images of rat
brain
is classified to grade 0-4. The results suggest that severe brain injuries of
2V0
(p=0.0000) group is significantly reduced with GMC1 (M1) and GMC1+0.5%GMA1
(M3) treatments (MI p-0.0030; M3 p=0.0238). The data are shown as means
SEM. The Student t-test and repeated ANOVA are used for statistical analysis.
p<0.05 is marked with *, and p<0.01 is marked with**, meaning that there is a
significant difference in statistics.
[0038] CT grade of brain injury:
0: No obvious abnormality on CT scans
1: Small area of abnormal density region in brain tissue
2: Abnormal density regions in over 25% of unilateral brain tissue, or small
area of
abnormal density regions in bilateral brain tissue
3: Abnormal density regions in over 50% of unilateral brain tissue, midline
shift, or
- 9 -
CA 02932277 2016-06-06
185262
abnormal density regions in over 25% of bilateral brain tissue
4: Abnormal density regions in over 50% of bilateral brain tissue, or apparent
midline
shift/distortion.
[0039] FIG. 25 shows the diagram of the tumor growth rate of the nude mice.
The
tumor was induced in the nude mice and the mice were subjected to grouping and
administrated with the extract composition on the tumor area and the whole
back skin
with the dosage of 0.1 g / day when the tumor grew to a determined volume. The
results show that the extract composition can decrease the tumor growth rate
compared
to the tumor control group.
[0040] FIG. 26 shows the diagram of the tumor growth rate of the nude mice.
The
tumor was induced in the nude mice and the mice were subjected to grouping and
administrated with GMC1 and GMA1 on the tumor area and the whole back skin
with
the dosage of 0.1 g / day when the tumor grew to a determined volume. The
results
show that the extract composition can decrease the tumor growth rate compared
to the
tumor control group.
[0041] FIG. 27 shows the diagram of the tumor growth rate of the nude mice.
The
tumor was induced in the nude mice and the mice were subjected to grouping and
administrated with CTX injection and GMC1 and GMA1 on the tumor area and the
whole back skin with the dosage of 0.1 g / day when the tumor grew to a
determined
volume. The results show that the extract composition can decrease the tumor
growth rate compared to the CTX control group.
DETAILED DESCRIPTION OF THE INVENTION
[0042] The invention is to provide an extract composition comprising a soybean
seed
extract, which soybean seed extract is prepared by a process comprising steps
of:
(a) providing soybean seeds and an extracting solution, which extracting
solution is water or an alcohol solution containing alcohol at the
concentration
lower than about 90 wt%;
- 10 -
CA 02932277 2016-06-06
185262
(b) extracting the soybean seeds with the extracting solution at a
barometric pressure lower than about 1 atm and at a temperature lower than
about 60 C to obtain an crude extract; and
(c) removing solids from the crude extract to obtain a liquid portion.
[0043] The present invention can be more readily understood by reference to
the
following detailed description of various embodiments of the invention, the
examples,
and the chemical drawings and tables with their relevant descriptions. It is
to be
understood that unless otherwise specifically indicated by the claims, the
invention is
not limited to specific preparation methods, carriers or formulations, or to
particular
modes of formulating the extract of the invention into products or
compositions
intended for topical, oral or parenteral administration, because as one of
ordinary skill
in the relevant arts is well aware, such things can, of course, vary. It is
also to be
understood that the terminology used herein is for the purpose of describing
particular
embodiments only and is not intended to be limiting.
[0044] As utilized in accordance with the present disclosure, the following
terms,
unless otherwise indicated, shall be understood to have the following meaning:
[0045] Often, ranges are expressed herein as from "about" one particular value
and/or to "about" another particular value. When such a range is expressed, an
embodiment includes the range from the one particular value and/or to the
other
particular value. Similarly, when values are expressed as approximations, by
use of
the word "about," it will be understood that the particular value forms
another
embodiment. It will be further understood that the endpoints of each of the
ranges
are significant both in relation to and independently of the other endpoint.
[0046] "Optional" or "optionally" means that the subsequently described event
or
circumstance may or may not occur, and that the description includes instances
where
said event or circumstance occurs and instances where it does not. For
example, the
phrase "optionally comprising an agent" means that the agent may or may not
exist.
- H-
CA 02932277 2016-06-06
185262
[0047] It must be noted that, as used in the specification and the appended
claims,
the singular forms "a," "an" and "the" include plural referents unless the
context
clearly dictates otherwise. Thus, unless otherwise required by context,
singular terms
shall include the plural and plural terms shall include the singular.
[0048] The term "subject" as used herein denotes any animal, preferably a
mammal,
and more preferably a human. The examples of subjects include humans, non-
human
primates, rodents, guinea pigs, rabbits, sheep, pigs, goats, cows, horses,
dogs and cats.
[0049] The term "effective amount" of an active ingredient as provided herein
means
a sufficient amount of the ingredient to provide the desired regulation of a
desired
function. As will be pointed out below, the exact amount required will vary
from
subject to subject, depending on the disease state, physical conditions, age,
sex,
species and weight of the subject, the specific identity and formulation of
the
composition, etc. Dosage regimens may be adjusted to induce the optimum
therapeutic response. For example, several divided doses may be administered
daily
or the dose may be proportionally reduced as indicated by the exigencies of
the
therapeutic situation. Thus, it is not possible to specify an exact "effective
amount."
However, an appropriate effective amount can be determined by one of ordinary
skill
in the art using only routine experimentation.
[0050] The term "treating" or "treatment" as used herein denotes reversing,
alleviating, inhibiting the progress of, or improving the disorder, disease or
condition
to which such term applies, or one or more symptoms of such disorder, disease
or
condition.
[0051] The term "carrier" or "excipient" as used herein refers to any
substance, not
itself a therapeutic agent, used as a carrier and/or diluent and/or adjuvant,
or vehicle
for delivery of a therapeutic agent to a subject or added to a formulation to
improve its
handling or storage properties or to permit or facilitate formation of a dose
unit of the
composition into a discrete article such as a capsule or tablet suitable for
oral
administration. Suitable carriers or excipients are well known to persons of
ordinary
- 12-
CA 02932277 2016-06-06
185262
skill in the art of manufacturing pharmaceutical formulations or food
products.
Carriers or excipients can include, by way of illustration and not limitation,
buffers,
diluents, disintegrants, binding agents, adhesives, wetting agents, polymers,
lubricants,
glidants, substances added to mask or counteract a disagreeable taste or odor,
flavors,
dyes, fragrances, and substances added to improve appearance of the
composition.
Acceptable carriers or excipients include citrate buffer, phosphate buffer,
acetate
buffer, bicarbonate buffer, stearic acid, magnesium stearate, magnesium oxide,
sodium
and calcium salts of phosphoric and sulfuric acids, magnesium carbonate, talc,
gelatin,
acacia gum, sodium alginate, pectin, dextrin, mannitol, sorbitol, lactose,
sucrose,
starches, gelatin, cellulosic materials (such as cellulose esters of alkanoic
acids and
cellulose alkyl esters), low melting wax cocoa butter, amino acids, urea,
alcohols,
ascorbic acid, phospholipids, proteins (for example, serum albumin),
ethylenediamine
tetraacetic acid (EDTA), dimethyl sulfoxide (DMSO), sodium chloride or other
salts,
liposomes, mannitol, sorbitol, glycerol or powder, polymers (such as
polyvinyl-pyrrolidone, polyvinyl alcohol, and polyethylene glycols), and other
pharmaceutically acceptable materials. The
carrier should not destroy the
pharmacological activity of the therapeutic agent and should be non-toxic when
administered in doses sufficient to deliver a therapeutic amount of the agent.
[0052] The extract composition according to the invention comprises the
soybean
seed extract. According to the present invention, depending on the testa color
of the
seeds, the soybean may be referred to yellow soybean, vegetable soybean, white
soybean, peel beans, green bean, black soybean; preferably yellow soybean or
black
soybean. The soybean according to the invention belongs to Fabaceae family,
Glycine genus; preferably, the soybean is Glycine max (L.) Merrill, Glycine
formosana
Hosokawa or Glycine soja auct non Sieb. & Zucc..
[0053] The soybean seed according to the invention preferably refers to the
seed
obtained by removing a shell from a pod. Generally, a soybean fruit is the pod
with
hair, and the shell of the pod covers the seeds. The shell of the pod is very
hard and
- 13 -
CA 02932277 2016-06-06
185262
waterproof for protecting the seeds inside. The manner of obtaining the
soybean
seeds from the soybean fruit, i.e. removing the shell of the pod, is known by
artisans
skilled in this field. Preferably, the soybean seed according to the invention
comprises seed coat, cotyledon and hypocotyl.
[0054] The soybean seed extract according to the invention is prepared by a
process
comprising steps of:
(a) providing soybean seeds and an extracting solution, which extracting
solution is water or an alcohol solution containing alcohol at the
concentration
lower than about 90 wt%;
(b) extracting the soybean seeds with the extracting solution at a
barometric pressure lower than about 1 atm and at a temperature lower than
about 60 C to obtain an crude extract; and
(c) removing solids from the crude extract to obtain a liquid portion.
[0055] The extracting solution for extracting the soybean seeds according to
the
invention is water or an alcohol solution containing alcohol at the
concentration lower
than about 90 wt%. Preferably, the alcohol is Cl to C7 alcohol. The term "Cl
to
C7 alcohol" as used herein refers to linear or branched, substituted or
unsubstituted,
mono- or poly- functional, and saturated or unsaturated alcohol; preferably
unsubstituted, mono-functional and saturated alcohol. In one preferred
embodiment
of the invention, the Cl to C7 alcohol is selected from the group consisting
of
methanol, ethanol, n-propanol, isopropanol, n-butanol, iso-butanol, sec-
butanol,
tert-butanol, 1 -pentanol, 2-pentanol, 3 -
pentanol, 2-methyl-I -butanol,
2-methyl-2-butanol, 3-methyl-2-butanol, 3-methyl -1 -butanol, 2,2-dimethyl-1-
propanol,
1 -hexanol, 2,4-hexadiene-1-ol, 2-methyl-cyclopentanol, cyclohexanol, 1 -
heptanol,
2-heptanol, or cyclohepty-1 alcohol. More preferably, the Cl to C7 alcohol is
methanol or ethanol; most preferably, the Cl to C7 alcohol is ethanol. The Cl
to C7
alcohol can be used solely or in combinations.
- 14 -
CA 02932277 2016-06-06
185262
[0056] The alcohol as used herein is preferably an alcohol solution with a
concentration lower than about 90% (v/v); preferably from about 5% (v/v) to
about
90% (v/v); more preferably from about 30% (v/v) to about 85% (v/v); still more
preferably from about 50% (v/v) to about 75% (v/v).
[0057] The process according to the invention comprises (b) extracting the
soybean
seeds with the extracting solution at a barometric pressure lower than about 1
atm and
at a temperature lower than about 60 C to obtain an crude extract. The manner
of
extracting a part of a seed with a solution is well-known to artisans skilled
in this field.
For example, the crude extract can be obtained by dividing the soybean seeds
into
pieces in any manner such as grinding, stirring, disturbing, cutting or
shredding, and
soaking the pieces in the extracting solution for extraction. The manners for
dividing
the seeds in the field are able to be applied in the invention. In one
preferred
embodiment of the invention, the soybean seeds are grinded into powder. In one
preferred embodiment of the invention, the soybean seeds are soaked in the
extracting
solution for extraction; more preferably, the soybean seeds are soaked in the
extracting
solution and subjected to ultrasonic vibration extraction.
[0058] According to the process of the invention, prior to step (b), the
soybean seeds
are preferably dried.
[0059] According to the invention, the ratio (w/v) of the soybean seeds and
the
extracting solution is not specifically restricted. In one preferred
embodiment of the
invention, the ratio (w/v) of the soybean seeds and the extracting solution is
about 1:1
to about 1:30; more preferably about 1:5 to about 1:20; and most preferably
about
1:10.
[0060] The temperature of extraction in the step (b) according to the
invention is
lower than about 60 C; preferably from about 25 C to about 55 C; more
preferably
from about 30 C to about 50 C; still more preferably about 45 C.
[0061] In one preferred embodiment of the invention, the extraction step (b)
can be
repeated, and the extract is collected by merging.
- 15 -
CA 02932277 2016-06-06
185262
[0062] The process according to the invention comprises the step (c) removing
solids
from the crude extract to obtain a liquid portion. The manner of removing the
solids
to obtain the liquid fraction is well-known to artisans skilled in this field,
and
examples include but not limited to filtration, centrifugation, or
precipitation.
[0063] Preferably, the process according to the invention further comprises a
step (d)
of concentrating the liquid portion obtained in the step (c) to obtain a
concentrated
solid portion. The manner of concentrating is well-known to artisans skilled
in this
field, such as by a reduced-pressure condenser.
[0064] Preferably, the process for according to the invention further
comprises a step
(e) of drying the concentrated solid portion obtained in the step (d). The
manner of
drying is well-known to artisans skilled in this field, such as air-drying or
by a freeze
drier.
[0065] In one preferred embodiment of the invention, the soybean seed extract
is
subjected to an ion chromatography assay with CarboPac PA1 Analytical (4 x
250mm)
column. The mobile phase is 87% water and 13% 500 mM NaOH; the internal
standard is maltose monohydrate. The isocratic elution is applied with the low
rate of
1.0 ml/min and the cycle of 0.5 second. In every cycle, the assay is conducted
with
the relative potential of 0.1 V at 0.00 second to 0.2 second; 0.1 V at 0.2
second to 0.4
second; -2.0 V at 0.41 second to 0.42 second; 0.6 V at 0.43 second; -0.1 V at
0.44
second to 0.5 second, and the total assay duration is 55 minutes.
[0066] The spectrograms obtained are shown in FIGs. 1 to 3. The peak time is
shown in Table 1.
Table 1:
Peak time (minutes)
Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Internal standard
FIG. 1 5.913 6.660 10.120 18.010 20.510 28.390
FIG. 2 5.660 6.420 10.244 18.600 20.784 28.597
FIG. 3 5.857 6.590 10.100 18.167 20.847 28.304
[0067] Preferably, the extract composition according to the invention further
- 16 -
CA 02932277 2016-06-06
185262
comprises a soybean seed vapor fraction, which soybean seed vapor fraction is
prepared by a process comprising steps of:
(i) providing soybean seeds in a second extracting solution, which second
extracting solution is water or an alcohol solution containing alcohol at the
concentration lower than about 15 wt%; and
(ii) extracting the soybean seeds with the second extracting solution at a
barometric pressure lower than about 1 atm and at a temperature lower than
about 110 C and collecting the vapor fraction.
[0068] The second extracting solution for preparing the soybean seed vapor
fraction
according to the invention is water or an alcohol solution containing alcohol
at the
concentration lower than about 15 wt%; preferably water. The kind of the
alcohol
can be the same to that of the extracting solution for preparing the soybean
seed
extract and is not repeated herein.
[0069] The alcohol of the second extracting solution is the alcohol solution
with a
concentration lower than about 15% (v/v); preferably lower than about 10%
(v/v);
more preferably lower than about 5% (v/v).
[0070] The process for preparing the soybean seed vapor fraction according to
the
invention comprises the step (ii) extracting the soybean seeds with the second
extracting solution at a barometric pressure lower than about 1 atm and at a
temperature lower than about 1100C and collecting the vapor fraction. The
manner
of extracting can be the same to that of preparing the soybean seed extract,
provided
that the soybean seed vapor fraction is vaporized at a barometric pressure
lower than
about 1 atm and at a temperature lower than about 110 C. The vapor fraction
can be
collected in a liquid form by chilling the vapor.
[0071] In a preferred embodiment of the invention, a process of vaporizing the
soybean seeds at a given barometric pressure and temperature, and collecting
said
vapor fraction by chilling the vapor can be performed in a rotary evaporator
where the
vapor is evaporated to the condensing tube supplied with cold water, and then
the
- 17 -
CA 02932277 2016-06-06
185262
vapor is chilled by passing through the condensing tube to collect the vapor
fraction in
a liquid form. The manipulation is simple and the cost is low.
[0072] According to the invention, the ratio (w/v) of the soybean seeds and
the
second extracting solution is not specifically restricted. In one preferred
embodiment
of the invention, the ratio (w/v) of the soybean seeds and the second
extracting
solution is about 1:1 to about 1:30; more preferably about 1:5 to about 1:20;
and most
preferably about 1:10.
[0073] The temperature of extraction in the step (ii) according to the
invention is
lower than about 110 C; preferably from about 60 C to about 110 C.
[0074] In one preferred embodiment of the invention, the extraction step (ii)
can be
repeated, and the soybean seed vapor fraction is collected by merging.
[0075] In one preferred embodiment of the invention, the soybean seed vapor
fraction is subjected to an ion chromatography assay with CarboPac PA1
Analytical (4
x 250mm) column. The mobile phase is 87% water and 13% 500 mM NaOH; the
is internal standard is maltose monohydrate. The isocratic elution is
applied with the
low rate of 1.0 ml/min and the cycle of 0.5 second. In every cycle, the assay
is
conducted with the relative potential of 0.1 V at 0.00 second to 0.2 second;
0.1 V at
0.2 second to 0.4 second; -2.0 V at 0.41 second to 0.42 second; 0.6 V at 0.43
second;
-0.1 V at 0.44 second to 0.5 second, and the total assay duration is 55
minutes.
[0076] The spectrograms obtained are shown in FIGs. 4 to 6. The peak time is
shown in Table 2.
Table 2:
Peak time
FIG. 4 2.690
FIG. 5 2.587
FIG. 6 2.664
[0077] In one embodiment of the invention, the content of the soybean seed
extraction based on the extract composition is from about 0.001% wt to about
10% wt;
preferably from about 0.01% wt to about 5% wt; more preferably from about
0.001%
- 18 -
CA 02932277 2016-06-06
185262
Wt to about 1.5% wt. In another aspect, the content of the soybean seed vapor
fraction based on the extract composition is from about 0.04% wt to about
99.999% wt;
preferably from about 10% wt to about 99.9% wt; more preferably from about 30%
wt
to about 99% wt.
[0078] The extraction composition according to the invention is preferably a
pharmaceutical composition, food composition or a cosmetic composition.
[0079] The pharmaceutical composition according to the invention is preferably
administered topically or systemically by any method known in the art,
including, but
not limited to, intramuscular, intradermal, intravenous, subcutaneous,
intraperitoneal,
to intranasal, oral, mucosal or external routes. The appropriate route,
formulation and
administration schedule can be determined by those skilled in the art. In the
present
invention, the pharmaceutical composition can be formulated in various ways,
according to the corresponding route of administration, such as a liquid
solution, a
suspension, an emulsion, a syrup, a tablet, a pill, a capsule, a sustained
release
formulation, a powder, a granule, an ampoule, an injection, an infusion, a
kit, an
ointment, a lotion, a liniment, a cream or a combination thereof. If
necessary, it may
be sterilized or mixed with any pharmaceutically acceptable carrier or
excipient, many
of which are known to one of ordinary skill in the art.
[0080] The external route as used herein is also known as local
administration,
includes but is not limited to administration by insufflation and inhalation.
Examples
of various types of preparation for local administration include ointments,
lotions,
creams, gels, foams, preparations for delivery by transdermal patches,
powders, sprays,
aerosols, capsules or cartridges for use in an inhaler or insufflator or drops
(e.g. eye or
nose drops), solutions/suspensions for nebulisation, suppositories, pessaries,
retention
enemas and chewable or suckable tablets or pellets or liposome or
microencapsulation
preparations.
[0081] Ointments, creams and gels, may, for example, be formulated with an
aqueous or oily base with the addition of suitable thickening and/or gelling
agent
- 19 -
CA 02932277 2016-06-06
185262
and/or solvents. Such bases may thus, for example, include water and/or an oil
such
as liquid paraffin or a vegetable oil such as arachis oil or castor oil, or a
solvent such
as polyethylene glycol. Thickening agents and gelling agents which may be used
according to the nature of the base include soft paraffin, aluminium stearate,
cetostearyl alcohol, polyethylene glycols, woolfat, beeswax,
carboxypolymethylene
and cellulose derivatives, and/or glyceryl monostearate and/or non-ionic
emulsifying
agents.
[0082] Lotions may be formulated with an aqueous or oily base and will in
general
also contain one or more emulsifying agents, stabilising agents, dispersing
agents,
suspending agents or thickening agents.
[0083] Powders for external application may be formed with the aid of any
suitable
powder base, for example, talc, lactose or starch. Drops may be formulated
with an
aqueous or non-aqueous base also comprising one or more dispersing agents,
solubilising agents, suspending agents or preservatives.
[0084] Spray compositions may for example be formulated as aqueous solutions
or
suspensions or as aerosols delivered from pressurised packs, such as a metered
dose
inhaler, with the use of a suitable liquefied propellant. Aerosol compositions
suitable
for inhalation can be either a suspension or a solution. The aerosol
composition may
optionally contain additional formulation excipients well known in the art
such as
surfactants e.g. oleic acid or lecithin and cosolvents e.g. ethanol.
[0085] Topical preparations may be administered by one or more applications
per
day to the affected area; over the skin areas occlusive dressings may
advantageously
be used. Continuous or prolonged delivery may be achieved by an adhesive
reservoir
system.
.. [0086] The cosmetic composition according to the invention may be an
aqueous
phase formulation consisting essentially of water; it may also comprise a
mixture of
water and of water-miscible solvent (miscibility in water of greater than 50%
by
weight at 25 C), for instance lower monoalcohols containing from 1 to 5 carbon
atoms
- 20 -
CA 02932277 2016-06-06
185262
such as ethanol or isopropanol, glycols containing from 2 to 8 carbon atoms,
such as
propylene glycol, ethylene glycol, 1,3-butylene glycol or dipropylene glycol,
C3-C4
ketones and C2-C4 aldehydes, and glycerin. Such an aqueous formulation
preferably
is in a form of aqueous gel or hydrogel formulation. The hydrogel formulation
comprises a thickening agent to thicken the liquid solution. Examples of the
thickening agents include, but are not limited to, carbomers, cellulose base
materials,
gums, algin, agar, pectins, carrageenan, gelatin, mineral or modified mineral
thickeners, polyethylene glycol and polyalcohols, polyacrylamidc and other
polymeric
thickeners. The thickening agents which give the stability and optimal flow
to characteristics of the composition are preferably used.
[0087] The cosmetic composition according to the present invention may be in a
form of emulsion or cream formulation. It can contain emulsifying surfactants.
These surfactants may be chosen from anionic and nonionic surfactants.
Reference
may be made to the document "Encyclopedia of Chemical Technology, Kirk-
Othmer",
volume 22, pp. 333-432, 3rd edition, 1979, Wiley, for the definition of the
properties
and functions (emulsifying) of surfactants, in particular pp. 347-377 of said
reference,
for the anionic and nonionic surfactants.
[0088] The surfactants preferably used in the cosmetic composition according
to the
invention are chosen from: nonionic surfactants: fatty acids, fatty alcohols,
polyethoxylated or polyglycerolated fatty alcohols such as polyethoxylated
stearyl or
cetylstearyl alcohol, fatty acid esters of sucrose, alkylglucosc esters, in
particular
polyoxyethylenated fatty esters of C 1 -C6 alkyl glucose, and mixtures
thereof; anionic
surfactants: C16-C30 fatty acids neutralized with amines, aqueous ammonia or
alkaline salts, and mixtures thereof. Surfactants which make it possible to
obtain an
oil-in-water or wax-in-water emulsion are preferably used.
[0089] The cosmetic composition according to the invention may further
comprise
an effective amount of a physiologically acceptable antioxidant selected from
the
group consisting of butylated p-cresol, butylated hydroquinone monomethyl
ether, and
-21 -
CA 02932277 2016-06-06
185262
a tocopherol.
[0090] The cosmetic composition according to the invention may further
comprise
natural or modified amino acid, natural or modified sterol compound, natural
or
modified collagen, silk protein or soy protein.
s [0091] The cosmetic composition according to the invention is preferably
formulated
for topical application to keratin materials such as the skin, the hair, the
eyelashes or
the nails. They may be in any presentation form normally used for this type of
application, especially in the form of an aqueous or oily solution, an oil-in-
water or
water-in-oil emulsion, a silicone emulsion, a microemulsion or nanoemulsion,
an
Hi aqueous or oily gel or a liquid, pasty or solid anhydrous product.
[0092] The cosmetic composition according to the invention may be more or less
fluid and may have the appearance of a white or colored cream, an ointment, a
milk, a
lotion, a serum, a paste, a mousse or a gel. It may optionally be topically
applied
onto the skin in the form of an aerosol, a patch or a powder. It may also be
in solid
is form, for example, in the form of a stick. It may be used as care
products and/or as
makeup products for the skin Alternatively, it may be formulated as shampoos
or
conditioners.
[0093] In known fashion, the cosmetic composition according to the invention
may
also contain additives and adjuvants that arc common in cosmetics, such as
20 hydrophilic or lipophilic gelling agents, preservatives, antioxidants,
solvents,
fragrances, fillers, pigments, odor absorbers and dyestuffs.
[0094] The extract composition can be added to a conventional food composition
(i.e.
the edible food or drink or precursors thereof) in the manufacturing process
of the food
composition. Almost all food compositions can be supplemented with the extract
25 composition of the invention. The food compositions that can be
supplemented with
the extract composition of the invention include, but are not limited to,
candies, baked
goods, ice creams. dairy products, sweet and flavor snacks, snack bars, meal
replacement products, fast foods, soups, pastas, noodles, canned foods, frozen
foods,
- 22 -
CA 02932277 2016-06-06
185262
dried foods, refrigerated foods, oils and fats, baby foods, or soft foods
painted on
breads, or mixtures thereof.
[0095] The present invention is also to provide use of the extract composition
as
mentioned above in the manufacture of a medicament of promoting wound healing.
[0096] The invention also provides a method for promoting wound healing in a
subject in need of such promotion comprising administering to said subject an
effective amount of the extract composition as mentioned above and optionally
a
pharmaceutically acceptable carrier or excipient.
[0097] Preferably, the extract composition according to the invention is
promoting
to wound healing by promoting skin cell migration. In one preferred
embodiment of the
invention, the extract composition is able to promoting keratinocyte
migration, and the
skin cell is preferably a keratinocyte. In another aspect, in an animal model
according to the incention, the extract composition is able to promoting wound
healing
in a diabetic patient, and the wound is preferably a diabetic wound.
is [0098] In one embodiment of the invention, the composition comprising
the soybean
seed extract and soybean seed vapor fraction has significant effect (p<0.5) in
would
closure, compared to cream base, and the soybean seed extract slightly
exhibits better
outcome in wound closure than the soybean seed vapor fraction.
[0099] In one another embodiment of the invention, different dose levels of
the
20 soybean seed extract are used in combined with the soybean seed vapor
fraction for
investigating the effect on diabetic wound healing. The results show that for
wound
healing, the combination effects of the soybean seed extract and the soybean
seed
vapor fraction are better than the soybean seed vapor fraction alone. The most
effective combination treatment is found in the highest dosage of the soybean
seed
25 extract.
[0100] In one embodiment of the invention, the soybean seed extract and
soybean
seed vapor fraction are used alone or in comination for assaying the effects
on
promoting skin keratinocyte migration. The results show that for promoting
skin
- 23 -
CA 02932277 2016-06-06
185262
=
keratinocyte migration, the combination effects of the soybean seed extract
and the
soybean seed vapor fraction are better than the soybean seed extract or the
soybean
seed vapor fraction alone.
[0101] The present invention is also to provide use of the extract composition
as
mentioned above in the manufacture of a medicament of promoting neuron cell
proliferation and/or treating brain diseases and/or neurodegenerative
diseases.
[0102] The invention also provides a method for promoting neuron cell
proliferation
and/or treating brain diseases and/or neurodegenerative diseases in a subject
in need of
such promotion and/or treatment comprising administering to said subject an
effective
amount of the extract composition as mentioned above and optionally a
pharmaceutically acceptable carrier or excipient.
[0103] Preferably, the neuron cell is a central nervous system cell; more
preferably is
a brain neuron cell; still more preferably is a neuroblastoma cell.
[0104] The brain diseases and/or neurodegenerative diseases according to the
invention are preferably selected from the group consisting of vascular
dementia,
Alzheimer's disease, Parkinson's disease, cerebral vascular disease,
inflammatory brain
injury, brain damage surrounding inflammatory response, brain lesions,
cerebral
hematoma, swelling ventricle and ventricular dilatation.
[0105] In one embodiment of the invention, the soybean seed extract, soybean
seed
vapor fraction and extraction composition comprising the soybean seed extract
and
soybean seed vapor fraction are used to treat human neuroblastoma cell. The
results
show that the cell viability rate is significantly increased after treated
with the soybean
seed extract, soybean seed vapor fraction and extraction composition
comprising the
soybean seed extract, soybean seed vapor fraction. Therefore, the soybean seed
extract and soybean seed vapor fraction and extraction composition comprising
the
soybean seed extract and soybean seed vapor fraction have effects on promoting
neuron cell proliferation.
[0106] In one another embodiment of the invention, in a radial arm maze test
of a
- 24 -
CA 02932277 2016-06-06
185262
dementia rat animal model, after the treatment of the extract composition
comprising
the soybean seed extract and soybean seed vapor fraction, total memory errors
and
reference memory errors are significantly lower than those of an untreated
group. It
shows that the extract composition comprising the soybean seed extract and
soybean
s seed vapor fraction has the effect on treating dementia rats.
[0107] In one another embodiment of the invention, in a radial arm maze test
of a
dementia rat animal model, after the treatment of the extract composition
comprising
the soybean seed extract and soybean seed vapor fraction, total memory errors
and
reference memory errors are significantly lower than those of an untreated
group. It
shows that the extract composition comprising the soybean seed extract and
soybean
seed vapor fraction has the effect on treating dementia rats.
[0108] In a vascular dementia model, the results show that the memory
impairment
caused by vascular dementia is significantly improved after the extract
composition
treatments according to the invention. The soybean seed vapor fraction and the
extract composition comprising the soybean seed extract and soybean seed vapor
fraction both improve the working memory errors, reference memory errors and
total
memory errors. Compared to the soybean seed vapor fraction alone, the extract
composition comprising the soybean seed extract and soybean seed vapor
fraction has
better effect on treating memory impairment caused by vascular dementia.
[0109] The present invention is also to provide use of the extract composition
as
mentioned above in the manufacture of a medicament of treating breast cancer.
[0110] The invention also provides a method for treating breast cancer in a
subject in
need of such treatment comprising administering to said subject an effective
amount of
the extract composition as mentioned above and optionally a pharmaceutically
acceptable carrier or excipient.
[0111] Preferably, the breast cancer is the breast cancer with p53 mutant
type.
[0112] In one preferred embodiment of the invention, a p53 mutant type of
human
breast cancer cell line is used to establish an animal model to simulate the
situation in
- 25 -
CA 02932277 2016-06-06
185262
the primary site for observing the tumor growth in mice. In the case of
simulating the
administration in human, different concentrations of the extract composition
according
to the present invention is administrated. It shows that the extract
composition
according to the invention has effect on inhibiting tumor growth rate in the
tumor-bearing mice.
[0113] The present invention is also to provide use of the extract composition
as
mentioned above in the manufacture of a medicament of reducing side effects of
interfering with DNA and/or RNA replication drugs, and/or enhancing
pharmaceutical
effects of interfering with DNA and/or RNA replication drugs.
to [0114] The invention also provides a method for reducing side effects of
interfering
with DNA and/or RNA replication drugs, and/or enhancing pharmaceutical effects
of
interfering with DNA and/or RNA replication drugs in a subject in need of such
reduction and enhancement comprising administering to said subject an
effective
amount of the extract composition as mentioned above and optionally a
pharmaceutically acceptable carrier or excipient.
[0115] The term "interfering with DNA and/or RNA replication drugs" as used
herein refers to drugs which kill a tumor cell by preventing the process of
DNA and/or
RNA replication essential in mitosis, Preferably, the interfering with DNA
and/or
RNA replication drugs are cyclophosphamide, temozolomide, hexamethylmelamine,
platinum complexes, Neomycin, vinblastine, vincristine, paclitaxel, docetaxel,
folic
acid antagonists, purine antagonists, or pyrimidine antagonists.
[0116] In one preferred embodiment of the invention, a human breast cancer
cell line
is used to establish an animal model to simulate the situation in the primary
site.
Compared to the tumor-bearing mice administrated with cyclophosphamide, the
extract composition according to the invention has enhancing effect of the
chemotherapy drug on inhibiting tumor growth rate in the tumor-bearing mice.
The
extract composition according to the invention inhibits tumor growth, has
enhancing
effect of the chemotherapy drug on eliminating tumor size, and relieves pain
by
- 26 -
CA 02932277 2016-06-06
185262
reducing tumor compression, thus the patient can have better life quality.
[0117] The following examples are provided to aid those skilled in the art in
practicing the present invention.
EXAMPLES
Soybean seed extract (GMA1)
[0118] Seeds of soybean (Glycine max (L.)Merr.) were grinded into power, and
70%
by weight of ethanol or distilled water was applied as an extracting solution;
the ratio
(w/v) of the soybean seeds and the extracting solution was about 1:10. The
soybean
seeds were extracted at a barometric pressure about 1 atm and at a temperature
of
about 45'C to obtain an crude extract. The solids were removed from the crude
extract to obtain a liquid portion. The liquid portion was further
concentrated by a
reduced-pressure condenser to obtain a concentrated solid portion. The
concentrated
solid portion was further dried at 70 C.
Soybean seed vapor fraction (GMC1)
[0119] Seeds of soybean (Glycine max (L.)Merr.) were grinded into power, and
2%
by weight of ethanol or distilled water was applied as a second extracting
solution; the
ratio (w/v) of the soybean seeds and the second extracting solution was about
1:10.
The vapor fraction was obtained by vaporizing the soybean seeds in a rotary
evaporator (EYELA N-1000S, 1000S-W) at a pressure of lower than 1 atm and a
temperature of 90 C, and passing through a condensing tube supplied with cold
water.
Analysis of the soybean seed extract (GMA1) and soybean seed vapor fraction
(GMC1)
[0120] The obtained soybean seed extract (GMA1) and soybean seed vapor
fraction
(GMC1) were subjected to an ion chromatography assay with CarboPac PA1
Analytical (4 x 250mm) column. The mobile phase was 87% water and 13% 500
mM NaOH; the internal standard is maltose monohydrate. The isocratic elution
was
applied with the low rate of 1.0 ml/min and the cycle of 0.5 second. In every
cycle,
- 27 -
CA 02932277 2016-06-06
185262
the assay was conducted with the relative potential of 0.1 V at 0.00 second to
0.2
second; 0.1 V at 0.2 second to 0.4 second; -2.0 V at 0.41 second to 0.42
second; 0.6 V
at 0.43 second; -0.1 V at 0.44 second to 0.5 second, and the total assay
duration was 55
minutes.
s 101211 The spectrograms of the soybean seed extract (GMA1) are shown in
FIGs. 1
to 3. The peak time is shown in Table 1.
[0122] The spectrograms of the soybean seed vapor fraction (GMC1) are shown in
FIGs. 4 to 6. The peak time is shown in Table 2.
The soybean seed extract (GM41) contains very small amount of isoflavones and
the
soybean seed vapor fraction (GMC1) contains no isoflavones
[0123] A high performance liquid chromatography (HPLC) was applied to assay if
the soybean seed extract (GMA1) and soybean seed vapor fraction (GMC1)
contained
isoflavones.
[0124] The condition of HPLC was listed as follows:
Apparatus: Hitachi HPLC CM5000 Series; pump: CM5110; detector: CM5430 (DAD);
automatic feeding system: CM5210; column oven: CM5310; software: OpenLab.
Column: RP C18, 4.6x250 mm Sun; detection wavelength: UV 254nm; flow rate: 0.8
min/ml; column oven temperature: 30 C; gradient: as shown in Table 3.
Table 3:
Solvent (%)
Time (minute)
Elution 1 Elution 2
0 95 5
10 85 15
77 23
72 28
20 80
20 80
46 95 5
25 55 95 5
Elution 1: 1% formic acid+0.01% trifluoroacetic acid (TFA) in water
- 28 -
CA 02932277 2016-06-06
185262
Elution 2: 1% formic acid +0.01% trifluoroacetic acid in acetonitrile
[0125] The HPLC spectrogram of the isoflavones standard is shown in FIG. 7,
and
peaks occurred at retention time of 18.853 minutes and 24.693 minutes.
[0126] The HPLC spectrogram of the soybean seed vapor fraction (GMC1) is shown
S in FIG. S. and no peaks occurred.
[0127] The HPLC spectrogram of an ointment containing 0.3 part by weight of
the
soybean seed extract (GMA1) and 1 part by weight of the soybean seed vapor
fraction
(GMC1) is shown in FIG. 9, and peaks occurred at retention time of 17.307
minutes,
19.313 minutes, 20.267 minutes, 20.853 minutes, 24.307 minutes and 25.247
minutes.
to [0128] By comparing FIGs 7 to 9, it shows that no peaks occur in the HPLC
spectrogram of the soybean seed vapor fraction (GMC1) in FIG. 8, and thus the
soybean seed vapor fraction (GMC 1) contains no isoflavones; the peaks
corresponding
to the isotlavones fingerprint absorption peaks occur at a very small amount
in the
HPLC spectrogram of the extract composition containing 0.3 part by weight of
the
15 soybean seed extract (GMA1) and 1 part by weight of the soybean seed
vapor fraction
(GMC1) in FTG 9 After conversion, the content of daidzin is 4.8 p.g/m1 and the
content of genistin is 8.23 ug/ml, which contents are far away from the
pharmaceutically effective amount of isoflavones. Therefore, the soybean seed
extract (GMA1) only contains a very small amount of isoflavones, and the
20 pharmaceutical effect thereof is not exhibited by the very small amount
of isoflavones.
Extract composition for promoting wound healing
Material and method:
[0129] Approximately 8 weeks old, with body weight rages of 250 g to 300 g SD
male rats were obtained from National Laboratory Animal Center, Taiwan. After
7
25 days of quarantine, the rats were moved until the body weight over 350
g. The rats
were identified by ear notch of the animals. Each cage tag was labeled with
the cage
number, study number, sex and group name. The rats were housed 2 per cage in
polycarbonate cage in the AAALAC accredited animal facility.
- 29 -
CA 02932277 2016-06-06
185262
[0130] The environment conditions were: temperature: 21 2 C; humidity: 50
20%; light cycle: in light for 12 hours and in dark for 12 hours. Laboratory
Rodent
Diet 5001 (PMIR Nutrition International, Inc., MO, USA) were supplied ad
libitum
throughout the study period. Tap water was supplied ad libitum via bottles
attached
to the cages.
Experimental methods:
[0131] Samples (test articles) were prepared according to Table 4.
Table 4:
Experimental 1
group animal/number/sex Test article and dose
1 Diabetic rats/5/male Cream base
2 Diabetic rats/5/male CGS-21680 1011g/wound
3 Diabetic rats/5/male GMC1
4 Diabetic rats/5/male 0.3% GMA1
Experimental 2
group animal/number/sex Test article and dose
1 Diabetic rats/5/male Cream base
2 Diabetic rats/5/male CGS-21680 6.7ps/wound
3 Diabetic rats/5/male GMC1
4 Diabetic rats/5/male UMC1+0.009% GMA1
Diabetic rats/5/male GMC1+0.045% GMA1
6 Diabetic rats/5/male GMC1+0.09% GMA I
[0132] CGS-21680: Specific adenosine A2A subtype receptor agonist, prepared by
adding 1.67 mg of CGS-21680 in 248.4 g of cream base.
[0133] Diabetic rats: The rats with body weight over 300g were administrated
with
one dose of Streptozotocin (STZ, 65mg/kg) by intravenous injection. The
STZ-induced rats with high blood sugar (over 300mg/dL) for 2 months were
selected
to conduct the wound closure test.
[01341 Trauma surgeries for diabetic rats: The diabetic rats with weight lower
than
300 g were eliminated and the rest were randomized into 6 groups. The rats
were
then anesthetized with pentobarbital and hairs on surgical area (dorsal area)
were
removed. Three skins on the dorsal medium areas (4, 6 and 8 cm from the
midpoint
- 30 -
CA 02932277 2016-06-06
185262
of two scapulas) in each rat were excised (full thickness) using round cutting
blades.
[0135] Medication and wound closure measurement: The animals were weighed
and the area of wounds was measured every other day for evaluation of wound
closure.
Medications were applied to each wound twice a day and the wounds were covered
with cheesecloth. The hoods were worn on the necks of the rats to prevent the
wounds from receiving animal scratches. For wound closure measurement, the
wound pictures were taken at day 4, 6, 8, 10, 12 and 14. When taking pictures,
a
standard ruler was placed beside the wounds. Before analyzing the wounds with
Image pro (Media cybernetics), length was standardized with the standard ruler
in the
to picture to avoid the errors caused by different picturing distances.
[0136] Data analysis and statistics: Three wound areas on the rats backs were
analyzed by Image pro. The original wound areas were the areas of day zero.
The
original wound areas minus the wound areas at different time points and then
divided
by the original wound areas to get the wound closure percentages. The mean of
three
wound closure percentages of each rat represents the wound closure of each
rat. The
data was shown as mean standard error (SFM). The p-values of the testing
results
were calculated by t-test in statistics software (SYSTAT, Systat software
Inc).
P<0.05 means there is a significant difference, and it is marked with *.
P<0.01
means there is a very significant difference, and it is marked with **.
P<0.001
means there is an extremely significant difference, and it is marked with ***.
Result:
[0137] After medication, wound areas were measured at different time points
and
wound closure percentages were analyzed with statistic software and shown in
Table 5
and FIG. 10. On day 4, the wound closure percentages for groups cream base,
CGS-21680, GMC1, and 0.3% GMA1 are -83.33 6.60 (%), 21.87 5.61 (%), -11.44
4.34 (%) and -7.09 3.65 (%), respectively. All treatment groups are
significantly
different from the cream base group during the whole experimental period. On
day 8
to day 10, the wound closure percentages are mostly enhanced with GMC I and 3%
-31-
CA 02932277 2016-06-06
185262
GMA1 treated groups, changed from -4.10 3.04 (%) to 40.15 +6.47 ( /0) and
21.21
5.52 (%) to 62.19 3.85 (%), respectively. On day 14, the wound closure
percentages of GMC1 and 3% GMA1 are significantly enhanced, 86.20 + 1.88 (%)
and 91.21 + 2.23(%), respectively, and compared to cream base, 58.92 13.14
(%).
Table 5: Comparison of wound closure percentages of diabetic rats treated with
GMC1 and 0.3% GMA1
Cream base CGS-21680 GMC1 0.3% GMA1
Day
Mean+SEM Mean+SEM Mean SEM Mean+SEM
*** *** ***
4 -83.33+6.60 21.87+5.61 -11.44+4.34 -7.09+3.65
*** ***
6 -65.84+11.19 25.45+4.46 6.74+4.86 4.15+3.19
**
8 -63.32+23.22 45.88+4.28*** 4.10+3.04 21.21+5.52
***
-10.08+16.87 93.80+2.79 40.15+6.47 62.19+3.85 **
12 30.85+20.08 91.15+2.72** 74.36+2.04 82.71+2.82
14 58.92+13.14 98.74+0.60** 86.20+1.88 91.21+2.23
Remark: Each value represents the mean SEM. The comparison values between
two groups are significantly different at **p<0.01 and ***p<0.001 by Student's
t test.
10 [0138] The results showed that both 0.3% GMA1 and GMC1 are effective in
would
closure, compared to cream base alone, and 0.3% GMA1 treated group slightly
exhibits better outcome in wound closure than GMC1.
[0139] The optimal combination of GMA1 and GMC1 in promoting diabetic wound
closure was investigated. Different dose levels of GMA1, 0.009%, 0.045% and
0.09% were used in combined with GMC1, For comparison, cream base and
CGS-21680 groups were also incorporated.
[0140] After medication, wound areas were measured at different time points
and
wound closure percentages were analyzed with statistic software and shown in
Table 6
and FIG. 11. As shown in Table 6 and FIG. 11, by comparing to cream base
groups,
an increase in statistical significance is observed with increasing GMA1 dose
levels in
combination drug groups, 0.009%, 0.045% and 0.09% of GMA1. The results show
- 32 -
CA 02932277 2016-06-06
185262
that for wound healing, the combination effects of the dose levels, 0.009%,
0.045%
and 0.09%, of GMA1 with GMC I are better than GMC1 alone. The most effective
combination treatment is found in the highest dosage of GMA1 (0.09%).
Table 6: Comparison of wound closure percentages of diabetic rats treated with
0.009% ¨ 0.09% of GMA1 in combined with GMCI
Cream base CGS-21680 GMC1 GMC1+0.009%GMA1
GMC1+0.045%GMA1 GMC1+0.09%GMA1
Days
Mean:SEM Meani,SEM Mean:SEM MeanISEM Mean SEM Mean:SEM
4 -4.4517.92 40.0215.03 ** -4.4718.88 3.6617.39
9.15111.71 18.90:4.66 *
6 4.3 4.42 63.09 2.59 === 13.59 5.05* 21.97 4.69
28.33 4.50 *** 32.99 5.40'"
8 40.8516.00 77.21 2.44"' 28.61+7.35 46.5214.08
30.25110.69 50.1115.00
46.511435 80.9312.08 *** 513514.86 73.5714.18 = 71.0915.62 *
86.68:0.93 ***
12 76.051:2.30 96.911:1.08*" 76.84 5.98 85.78 2.38"
84.23 2.55" 90.4911.27 ***
14 91.39:1.23 99.17 0.54*** 82.58:3,01* 93.6112.39
88.57 2.44 95.02:0.65*
Remark: Each value represents the means + SEM. The comparison values between
two groups are significantly different at ** p<0.01 and *** p<0.001 by
student's t test.
Extract composition for promoting skin cell migration
10 [01411 HaCaT cells (human skin keratinocytes) were used for assaying the
effect of
the extract composition on promoting skin cell migration.
[0142] Samples of the cell migration assay were:
A I -0.3: containing 0.3 [tg/mL of GMA1
A1-0.3-CSTC-2500X: containing 2500-fold diluted GMC1 and 0.3 iug/mI, of GMA1
Cell line: HaCaT
Medium: DMEM containing 10% fetal bovine serum
Positive control: CGS-21680
Cell culture: HaCaT cells were cultured in DMEM containing 10% fetal bovine
serum
at 37 C and 5% CO2, and subcultured twice weekly.
101431 Wound healing assay: Oris Cell Migration Assay kit was applied. The
cells were planted into a 96-well plate (4x 104 cells/well) with a stopper and
cultured in
a carbon dioxide incubator. After cultured overnight, the stopper was pulled
out to
produce wounds and new medium or medium containing test articles or CGS-21680
(positive control) were added. After treatment for 0, 16 and 24 hours, the
cells were
- 33 -
CA 02932277 2016-06-06
185262
observed under an optical microscope at 100 times magnification observation
and
photographs were taken for monitoring wound healing.
[0144] Analysis: Software of image j was applied to quantify the wound healing
area. Area at 0 hour was taken as the original size of the wound. After a
period of
time, the wound area was gradually reduced, and the extent of wound healing
was
quantified by calculating the wound area relative to the original area at 0
hour (healing
rate = difference between the area at 116 or T24 time point and that at TO /
TO area), to
assess the effect of the drugs on HaCaT cell migration activity.
[0145] Statistics: Each group was repeated at least four times. The data was
to shown as mean standard error (SEM). The significance of the testing
results was
calculated by t-test.
Result:
[0146] HaCaT cell migration assay is used for comparing the effects of GMC1
and
GMA1 on promoting wound healing. The combination effects of 2500-fold diluted
GMC1 and 0.3 ug/mL of GMA1 (A1-0.3-CSTC-2500X) are better than A1-0.3 alone
on promoting I IaCaT cell migration (FIG 12).
Extract composition for promoting neuron cell proliferation
Material and method:
[0147] Cell line: Human neuroblastoma cell IMR-32 (purchased from Food
Industry
Research and Development Institute, Taiwan)
[0148] Reagents: HyCloneTM DMEM/High Glucose Media (Thermo Scientific), Fetal
Bovine Serum (FBS, Gibco0), HyClone Phosphate Buffered Saline (PBS, Thermo
Scientific), Antibiotic Antimycotic Solution (Pen/Strep/Fungizone 100x; Thermo
Scientific), Trypan blue, 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyl
tetrazolium
bromide (MTT), and Dimethyl sulfoxide (DMSO, Sigma).
[0149] Equipment: Laminar Flow (VERTICAL HF-4BH).
[0150] Cell Culture: IMR-32 cells were cultured in DMEM/High Glucose Media
(HyCloneTM) supplemented with 10% fetal bovine serum in 37 C, humidified air
with
- 34 -
CA 02932277 2016-06-06
185262
5% CO2.
[0151] IMR-32 cells were seeded to 96-well plate at the density of 5 x 104
with 100
1.A1 culture medium supplemented with 10% FBS for 24 hours. Upon dosing, the
culture medium was changed to 100 u.L medium supplemented with 5%FBS then, for
each group, formulated to 25 mg/mL GMC1, 25 mg/mL GMA1, GMC1+0.3% GMA1
(GM) or PBS (control group) and 100 pt medium supplemented with 10% FBS
(positive control group). Each group was repeated at least seven times in a 96-
well
plate and incubated for 72 hours for the MTT assay.
[0152] MIT assay: Removed culture dishes from incubator and transferred into
to laminar flow hood. Replaced the medium with 100 uL serum free medium
with 0.5
mg/ml MIT reagent for each well and incubated for 30-60 minutes. Add equal
volume of DMSO to each well and shake for 5 minutes. Measured the absorbance
at
570 nm in an ELISA reader.
[0153] Data analysis and statistics: The results were analyzed with GraphPad
Prism 6 and T-test was used for statistical analysis. The data shown was the
percentage rate of cell viability+SEM.
Results:
[0154] The cell viability rate is shown in Tables 7 to 9 and FIGs. 13 to 15.
The cell
viability rate is significantly increased after treated with 25 mg/mL GMA1
(Table 7
and FIG. 13), 25 mg/mL GMC1 (Table 8 and FIG. 14) or GMC1-l-0.3% GMAl(GM,
Table 9 and FIG. 15) compared to the PBS control group.
Table 7:
Control 25 mg/mL Positive Control
GMA1 Group
Mean = SEM Mean SEM Mean + SEM
1 99.2+17.2 106.7+8.3 137.9+9.8
2 110.1+6.9 119.7+6.7
128.7+13.8
3 86.3+5.6 123.2+6.3 ** 94.5 2.8
4 98.3+1.8 130.2+7.8 ** 119,0+7.7 *
5 88.6+2.6 114.2 5.8 ** 90.7+1.6
- 35 -
CA 02932277 2016-06-06
185262
6 94.8+1.9 111.4+6.1 * 105.7+3.1 *
7 92.1+5.7 105.7+10.2 128.2 2.9***
Average 95.6+3.0 115.9 3.4*** 115.0 6.9*
Table 8:
Control 25 mg/mL Positive Control
GMC1 Group
Mean SEM Mean SEM Mean SEM
1 99.2+17.2 94.1+3.3 137.9+9.8
2 110.1+6.9 144.1 8.2* 128.7+13.8
3 86.315.6 120.011.0 * 94.52.8
4 98.3+1.8 149.2 6.4*** 119.0 7.7*
88.6+2.6 106.4+7.2 90.7+1.6
6 94.8+1.9 101.5+3.1 105.7+3.1 *
7 92.1+5.7 107.8+6.3 128.2 2.9***
Average 95.6+3.0 117.6+8.1 * 115.0 6.9*
Table 9:
GM Group Control 25 mg/mL Positive Control
Mean SEM Mean SEM Mean SEM
1 99.2+17.2 117.2+11.6 137.9+9.8
2 110.1+6.9 115.1+12.5 128.7+13.8
3 86.3+5.6 103.0+6.0 94.5+2.8
4 98.3+1.8 118.2+10.1 119.0 7,7*
5 88.6+2.6 106.1+5.5 * 90.7+16
6 94.8+1.9 99.9+2.0 105.7+3.1 *
7 92.1+5.7 105.7+1.6* 128.2+2.9***
Average 95.6+3.0 109.3 2.8** 115.0 6.9*
Remark of Tables 7 to 9: Each value represents the means+SEM. The comparison
between different dosages of two groups was statistically analyzed using T-
test.
5 p<0.05 means there is a significant difference, and it is marked with *.
p<0.01 means
there is a highly significant difference, and it is marked with *= p<0.001
means there
is a very highly significant difference, and it is marked with ***.
Extract composition for treating brain diseases and/or neurodegenerative
diseases
(I) Induced dementia:
- 36 -
=
Material and method:
[0155] Approximately 6 weeks old, with body weight ranges of 250 g to 300 g
male
rats were obtained from BioLASCO, Taiwan. After 7 days of quarantine, rats
were
moved until the body weight over 300 g. The rats were identified by tail
tattoos. Each
cage tag was labeled with the cage number, study number, sex and group name.
The
rats were housed 2 per cage in polycarbonate cage in the animal facility.
[0156] The environment conditions were: temperature: 25 1 C; humidity: 60
5%;
light cycle: in light for 12 hours and in dark for 12 hours. Altromin 1324
FORTI
(Germany) was supplied ad libitum throughout the study period. Tap water was
supplied ad libitum via bottles attached to the cages.
[0157] The groups were shown in Table 10.
Table 10
Groups Dementia induction Test article
Normal None None
A1C13 AlC13 AlC13 control
A1C13+cream base AlC13 Cream base
AlC13+N2 AlC13 0.5% GMA 1+GMC1
[0158] Dementia induction:
[0159] Eight weeks old rats were fed with daily oral administration of 15
mg/mL A1C13
solution at a dose of 100 mg/kg for 11 weeks to induce the mimic symptoms of
dementia.
The rats were divided into groups as shown in Table 10 and the test articles
were
topically applied to head and neck area with massaging for 30 seconds, and to
nasal
mucosa on week 5 to week 11. The training started on week 11 and the memory
ability
was then evaluated by a radial arm maze on week 11. The rats were continuously
fed
with AlC13 during the treatment.
[0160] Radial arm maze: Radial arm maze is one of the most common methods to
measure spatial learning and memory in rats. The radial arm maze was designed
by
Olton in 70s. It is based on forcing hungry rats to check food at the end of
each arm,
training them to remember locations of food in the maze in a period of time.
This can
- 37 -
CA 2932277 2018-04-26
CA 02932277 2016-06-06
185262
measure the working memory (referring to short-term memory) and reference
memory
(referring to long-term memory) of rats at the same time. The radial arm maze
is
shown in FIG. 16. Size: (A) 122 cm; (B) 47 cm; (C) 30 cm; (D) 10 cm; (E) 20
cm.
[0161] The rats were habituated to the environment for 1 week. Weighed each
rat
and let rats fast for 24 hours. Prior to the experiment, kept the rats' body
weight to be
80-85% of the original body weight; 60% of the normal diet was given after
daily
training (twice a day). On day 1 and day 2, the baits were scattered on the
arms and
central platform. Four rats were placed on the central platform at the same
time and
allowed to explore the maze for 10 minutes. On day 3 and 4, baits were placed
on the
end of each arm. Placed each rat separately on central platform and allow the
rat to
explore the maze until food finished. If the rat didn't finish all food within
10
minutes, training stopped. On day 5-14, baits were placed on the end of four
fixed
arms. Placed each rat on central platform and allowed to explore the maze
until baits
on four arms were finished. If the rat didn't finish all food within 10
minutes,
training stopped. The arm entries were recorded and analyzed automatically.
Each
rat was trained twice each day, and there was one hour apart between two
trainings.
[0162] The following three indicators were analyzed:
a. Working memory (short-term memory) errors (WME): number of reentries into
baited arms.
b. Reference memory (long-term memory) errors (RME): number of reentries into
unbaitcd arms.
c. Total memory errors (TME): WME+RME
[0163] These three indicators stand for the abilities of learning and memory
of rats,
which would significantly increase along with the level of brain injuries.
[0164] Data analysis and statistics: The Student t-test and repeated ANOVA
were
used for statistical analysis. Data shown was mean results SEM of each
group.
Data of A1C13 group was compared with normal group to calculate the
statistical
significance. Data of AlC13 group was also compared with A1C13+N2 group to
- 38-
CA 02932277 2016-06-06
185262
calculate the statistical significance. If p<0.05, it is marked with *. If
p<0.01, it is
marked with **.
Results:
[01651 It has been reported that heavy metal-induced dementia is similar to
Alzheimer's disease. Both of them induce the formation of amyloid precursor
proteins, and theresults in the formation of senile plaques and
neurofibrillary tangles.
Therefore, we employed the heavy metal-induced dementia rat as the animal
model to
understand the effect of the extract composition containing the soybean seed
extract
and soybean seed vapor fraction on Alzheimer's - like diseases. The results
are
shown in FIGs. 17 to 19. Referring to FIG. 17, the tracking path of rats on
Day 4
suggests that the rats of the AlC13 + N2 group are able to reach the locations
of baits
with their memory after training, and the rats of the AlC13 group and
AlC13+cream
base group are not. Referring to FIG. 18, the TME values of A1C13 group and
AlC13+cream base group show no significant difference (p>0.05), and the TME
values
on Day 4 after the treatment of the extract composition comprising the soybean
seed
extract and soybean seed vapor fraction are significantly lower than those of
A1C13
group (A1C13+N2 p=0.0128). It shows that the extract composition comprising
the
soybean seed extract and soybean seed vapor fraction has the effect on
treating
dementia rats. Referring to FIG. 19, the RME values of A1CI3 group and
A1C13+cream base group show no significant difference (p>0.05), and the RME
values
on Day 4 after the treatment of the extract composition comprising the soybean
seed
extract and soybean seed vapor fraction are significantly lower than those of
AlC13
group (p=0.0046). It shows that the extract composition comprising the soybean
seed
extract and soybean seed vapor fraction has the effect on restoring the long-
term
memory of rats. The results show that the errors of A1C13 group are
significantly
higher than the control group in the radial arm maze test, which refers to
successful
induction of dementia by AlC13 in rat. The memory impairment of reference
memory
is also significantly improved after treated by the extract composition
comprising the
- 39 -
CA 02932277 2016-06-06
185262
soybean seed extract and soybean seed vapor fraction. This indicates that the
extract
composition comprising the soybean seed extract and soybean seed vapor
fraction has
the efficacy in treating dementia.
(II) Vascular dementia:
Material and method:
[0166] The groups were shown in Table 11.
Table 1 I :
Groups Dementia induction Test article
Sham none sham
2V0 two-sided carotid arterial ligature two-sided carotid arterial
ligature control
group
two-sided carotid arterial ligature Cream base
M1 two-sided carotid arterial ligature GMC1
M3 two-sided carotid arterial ligature GMC1+0.5% GMA I
[0167] Rats and animal facility are as described in (I) induced dementia.
[0168] Rats were anesthetized with 1:1.5 ketamine-Rompun mixtures (0.1
mL/100g,
i.p.) and were fixed on the surgery plate. Both common carotid arteries were
exposed
via a midline cervical incision in the dorsal neck region and were double-
ligated with
silk sutures. The wounds were sutured and the rats were placed under warm
light
until they recovered.
[0169] The cream base (M), GMC1(M1), and GMC1+0.5%GMA1 (M3) were
topically applied (2 g / rat) to head and neck area with massaging for 30
seconds, and
to nasal mucosa. Treatment continued for four weeks, and memory of rats was
measured by radial arm maze in the last two weeks. The radial arm maze test is
as
described in (I) induced dementia.
[0170] Data analysis and statistics: The unpaired Student t-test and two-way
ANOVA were used for statistical analysis. Data shown was mean results SEM of
each group. Data of Sham group was compared with 2V0 group to calculate the
statistical significance. Drug treated groups was compared with 2V0 group to
- 40 -
CA 02932277 2016-06-06
185262
calculate the statistical significance. p<0.05 means there is a significant
difference,
and it is marked with *. p<0.01 means there is a very significant difference,
and it is
marked with **.
Results:
s [0171] The number of errors of 2V0 group is significantly higher than
Sham group
(FIGs. 20 to 23), which refers to successful induction of vascular dementia in
rats by
two-sided carotid arterial ligature, and the memory impairment caused by
vascular
dementia is significantly improved after two weeks of M1 and M3 treatments.
According to the results of Computed Tomography scan of the VD rat brains,
bilateral
io occlusion of both common carotids arteries (2V0) caused swelling,
softening,
histolysis of brain tissues, or even blood clot formation, and thus induced
the dementia.
The brain injuries are significantly decreased after M1 and M3 treatments.
Extract composition for treating breast cancer and reducing side effects of
interfering
with DNA and/or RNA replication drugs, and/or enhancing pharmaceutical effects
of
15 interfering with DNA and/or RNA replication drugs
Material:
[0172] Cell line and reagent: MDA-MB-231 (purchased from Food Industry
Research and Development Institute, Taiwan); Penicillin-Streptomycin-Neomycin
Mixture (100x), fetal bovine scrum (FBS) and Dulbecco's Modified Eagle Medium
20 (DMEM) were purchased from Gibcog; cyclophosphamide (CTX, EndoxanR).
[0173] Animal facility: Approximately 6 to 8 week-old
BALB/cAnN.Cg-Foxn1"/CrlNarl female mice were obtained from National
Laboratory Animal Center, Taiwan. The environment conditions were:
temperature:
25 1 C; humidity: 60 5%; independent air condition; controlled light
cycle.
25 Water and food was supplied ad libitum throughout the study period. The
mice were
housed according to the standard procedure of the laboratory animal committee.
[0174] Tumor-bearing mice induction: MDA-MB-231 cells (human breast cancer
cells) were cultured until 5x106 cells, the cells required in injection of
female nude
- 41 -
CA 02932277 2016-06-06
185262
mice were collected with trypsin, and resuspended in PBS solution for tumor
induction.
The tumor of nude mice was induced by subcutaneous injected with 100 ill of
cell
solution and released back into the cages to be a normal diet. When the tumor
was
about 300 mm3, the mice were subjected to topical administration of different
doses of
the extract composition and grouping into CTX group and non-CTX group. The
weight, appetite, blood sugar and tumor size growth were observed every week
to
assess the appearance of the normal physiological state of nude mice to
understand the
efficacy of the extract composition.
[0175] Grouping the tumor-bearing mice and treatment: When the tumor was
about 300 mm3, the mice were subjected to grouping as shown in Table 12. The
chemotherapy drug (an injection a week) was administrated with the extract
composition. The treating group was topically applied to the tumor area, skin
around
the tumor, and the whole back skin with the dosage of 0.1 g / day. After
treatment
for 5 weeks, the weight, appetite, blood sugar and tumor size growth of the
nude mice
were observed.
Table 12:
Group Tumor induction Chemotherapy drug Test article
Normal None None None
Tumor MDA-MB-231 induction None None
J1 MDA-MB-231 induction None GMC1
J2 MDA-MB-231 induction None 0.3%GMA1
NO CTX+ L5 MDA-MB-231 induction None GMC1+0.5%GMA1
CTX MDA-MB-231 induction CTX/4 injections None
CTX+ LI MDA-MB-231 induction CTX/4 injections Cream base
CTX+ L5 MDA-MB-231 induction CTX/4 injections GMC1+0,5%GMA1
[0176] Clinical assessment of tumor-bearing mice: The physiological state and
tumor size of the tumor-bearing mice were assessed including weight, appetite,
blood
sugar and tumor size. The tumor size was calculated according to: tumor volume
(1=3) =ab2/2, to assess the tumor growth rate.
Results:
- 42 -
CA 02932277 2016-06-06
185262
[0177] In the model of tumor-bearing nude mice induced by human breast cancer
cell MDA-MB-231, after the treatment, the extract compositions J1 and J2 show
effect
on inhibiting tumor growth in nude mice (Table 13 and FIG. 25). In the
physiological state assessment, the extract composition groups show better
appetite
and blood sugar value.
Table 13:
Tumor size (mm3)
Week 0 Week 1 Week 2 Week 3
J1 0.0 63.40 126.23 369.51
J2 0.0 -2.02 26.83 182.19
Tumor 10.00 72.69 380.71 696.90
[0178] In GMC1+0.5%GMA1 (NO CTX+ L5) group, it shows that the extract
composition inhibits the tumor growth in nude mice (FIG. 26).
[0179] Compared to the normal group, the physiological state and tumor growth
of
the tumor + chemotherapy drug group (CTX group), extract composition +
chemotherapy drug group (CTX+Ll and CTX+L5 groups) was assessed. Referring
to Table 14 and FIGs. 26 and 27, the results of CTX+Ll and CTX+L5 groups show
enhancing effect of the chemotherapy drug on eliminating tumor in the tumor-
bearing
mice administrated with the chemotherapy drug without affecting the body
weight, the
is average food intake and the average blood sugar (Tables 15 to 17).
Table 14: tumor size elimination (mm3)
4/28 5/2 5/5 5/9 5/12 5/16 5/19 5/23 5/26
5/30
Normal 0.00 0.00 0.00 0.00 0,00 0.00 0.00 0.00
0.00 0.00
Tumor 0.00 108.05 364.31 428.36 627.45 874.62 1295.63 1961.51 2727.56 2938.30
CTX 0.00 99.61 78.95 -19.52 -45.11 -157.57 -176.41 -264.85 -293.68 -391.42
CTX1-L1 0.00 89.22 113.36 21.27 -46.75 -138.97 -179.55 -224.38 -294.03 -384.43
CTX+L5 0.00 149.85 173.28 48.54 -56.91 -170.01 -220.34 -274.15 -338.79 -
510.04
Table 15: average body weight (g)
4/28 5/2 5/5 5/9 5/12 5/16 5/19 5/23 5/26
5/30
Normal 20.61 21.34 20.91 21.16 20.31 21.13 20.41
21.90 21.63 21.96
Tumor 19.64 19.11 20.03 18.75 18.57 20.22 20.48
21.36 21.74 22.67
- 43 -
CA 02932277 2016-06-06
185262
CTX 19.64 20.04 18.46 19.00 19.51 20.99 20.42
21.16 20.21 22.18
CTX+1,1 20.05 20.59 18.72 19.34 18.79 19.23 19.98
20.08 19.94 21.31
CTX+L5 19.12 19.51 18.45 19.09 19.23 19.61 19.93
19.69 19.03 20.52
Table 16: average food intake (g)
5/5-5/9 5/9-5/12 5/12-5/16 5/16-5/19 5/19-5/23 5/23-5/26 5/26-5/30
Normal 4.95 4.75 5.18 4.69 4.52 4.37 4.50
Tumor 4.94 4.48 4.56 4.75 4.25 4.65 4.20
CTX 4.89 5.61 5.38 5.21 4.70 4.76 5.22
CTX+Ll 4.45 4.58 4.88 4.64 4.08 4.01 4.46
CTX+L5 j 4.60 5.12 4.47 5.09 4.36 4.21 4.62
Table 17: average blood sugar (dL)
5/2 5/9 5/16 5/23 5/30
Normal 110 84 99 97 98
Tumor 60 66 82 89 74
CTX 113 93 109 122 108
CTX+Ll 110 93 92 99 101
CTX+L5 , 110 109 104 106 102
[0180] Clinically, one of the main reasons for abandonment of cancer treatment
is
cancer pain caused by cancer. It is found that cancer pain is caused in part
by the
tumor, mainly due to the inflammation and nerve compression caused by tumor
invasion to the internal organs, peripheral nerves, and bones. The
extract
composition can effectively inhibit tumor growth, and also suppress cancer
pain
caused by cancer in part, and is helpful in the treatment.
[0181] While the present invention has been described in conjunction with the
specific embodiments set forth above, many alternatives thereto and
modifications and
variations thereof will be apparent to those of ordinary skill in the art. All
such
alternatives, modifications and variations are regarded as falling within the
scope of
the present invention.
- 44 -
