U.S. patent application number 14/779136 was filed with the patent office on 2016-07-28 for method for inducing pluripotent stem cells and pluripotent stem cells prepared by said method.
The applicant listed for this patent is AMOREPACIFIC CORPORATION, SEOUL NATIONAL UNIVERSITY R&DB FOUNDATION. Invention is credited to Ah Reum KIM, Hyo Soo KIM, Su Na KIM, Yoo Wook KWON, Jae Seung PAEK, Won Seok PARK, Young Bae PARK.
Application Number | 20160215269 14/779136 |
Document ID | / |
Family ID | 52828359 |
Filed Date | 2016-07-28 |
United States Patent
Application |
20160215269 |
Kind Code |
A1 |
KIM; Ah Reum ; et
al. |
July 28, 2016 |
METHOD FOR INDUCING PLURIPOTENT STEM CELLS AND PLURIPOTENT STEM
CELLS PREPARED BY SAID METHOD
Abstract
The present disclosure relates to a method for inducing
pluripotent stem cells by inducing reprogramming and/or
dedifferentiation of differentiated adult cells using shikimic
acid, a plant extract or plant stem cells containing shikimic acid
and an extract of dedifferentiated stem cells (callus), pluripotent
stem cells prepared by the method and a composition containing the
pluripotent stem cells. In accordance with the present disclosure,
ethical concerns implicated with the use of eggs to prepare
pluripotent stem cells such as embryonic stem cell can be resolved.
And, because the plant stem cell extract unharmful to human is
used, pluripotent stem cells with proven safety can be prepared and
they may be used to develop immunocompatible cell therapy agents
suited for individuals. In addition, by pluripotent stem cells from
individuals having diseases, the present disclosure will be very
useful in studying the cause of diseases and devolving therapeutic
strategy.
Inventors: |
KIM; Ah Reum; (Yongin-si,
KR) ; KIM; Su Na; (Yongin-si, KR) ; PARK; Won
Seok; (Yongin-si, KR) ; KWON; Yoo Wook;
(Seoul, KR) ; PARK; Young Bae; (Seoul, KR)
; KIM; Hyo Soo; (Seoul, KR) ; PAEK; Jae Seung;
(Ansan-si, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AMOREPACIFIC CORPORATION
SEOUL NATIONAL UNIVERSITY R&DB FOUNDATION |
Seoul
Seoul |
|
KR
KR |
|
|
Family ID: |
52828359 |
Appl. No.: |
14/779136 |
Filed: |
October 16, 2014 |
PCT Filed: |
October 16, 2014 |
PCT NO: |
PCT/KR2014/009702 |
371 Date: |
September 22, 2015 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2800/74 20130101;
A61K 8/0225 20130101; C12N 2506/02 20130101; A61K 8/9761 20170801;
A61P 17/00 20180101; A61K 8/0216 20130101; A61K 9/0095 20130101;
A61K 8/9789 20170801; A61K 35/28 20130101; A61K 8/9794 20170801;
C12N 5/0696 20130101; A61K 31/191 20130101; A61K 8/9771 20170801;
A61K 9/0019 20130101; A61K 9/0056 20130101; A61K 9/20 20130101;
A61K 2800/91 20130101; A61K 8/06 20130101; A61K 8/365 20130101;
A61K 8/11 20130101; A61K 8/0204 20130101; A23L 2/52 20130101; C12N
2501/73 20130101; C12N 2502/02 20130101; C12N 2533/54 20130101;
A61K 9/14 20130101; A61P 39/06 20180101; A61Q 19/08 20130101; C12N
2501/603 20130101; A23L 33/10 20160801; A61K 36/14 20130101; A23L
33/105 20160801; A61K 8/368 20130101; A61K 2800/78 20130101; C12N
2506/1307 20130101; A61K 8/981 20130101; A61K 2800/92 20130101;
A61P 43/00 20180101; C12N 2500/76 20130101; C12N 2501/115 20130101;
C12N 2500/14 20130101; C12N 2502/13 20130101; A61K 8/9767 20170801;
A61K 9/48 20130101; A23V 2002/00 20130101; A61Q 19/00 20130101 |
International
Class: |
C12N 5/074 20060101
C12N005/074; A61K 36/14 20060101 A61K036/14; A61Q 19/08 20060101
A61Q019/08; A61K 31/191 20060101 A61K031/191; A61K 8/365 20060101
A61K008/365 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 17, 2013 |
KR |
10-2013-0123860 |
Claims
1. A method for preparing stem cells, comprising treating
adult-derived cells with shikimic acid, a plant extract comprising
shikimic acid, a plant stem cell extract comprising shikimic acid,
or a composition comprising one or more of them.
2. The method according to claim 1, wherein the plant stem cell
extract is an extract of dedifferentiated pluripotent plant stem
cells.
3. The method according to claim 1, which comprises injecting the
shikimic acid, the extract or the composition into the
adult-derived cells; and culturing the cells into which the
shikimic acid, the extract or the composition has been
injected.
4. The method according to claim 1, wherein the plant extract
comprising shikimic acid further comprises an extract of one or
more selected from a group consisting of sequoia (Sequoiadendron
giganteum), Iris pseudoacorus, Helianthus tuberosus, Picea pungens,
Picea glauca, Eucalyptus sieberiana, Eucalyptus regnans, Thuja
plicata, Phoenix dactylifera, Dahlia variabilis, Malus baccata,
Pyrus communis, Triticum, Pinus densifloraa, Pinus thunbergii,
Illicium anisatum, Magnolia grandiflora, Houttuynia cordata,
Saxifraga stolonifera, Terminalia arjuna, Pistacia lentiscus, Ribes
aureum, Symphytum officinalis, Actaea pachypoda, Alangium
salvifollium, Gingko biloba, Veratrum viride, Dipsacus laciniatus,
Agastache urticifolia, Inula helenium, Hypericum spp., Commelina
bengalensis, Gymnema sylvestris, Terminalia chebula, Illicium
floridanum, Illicium diffengri, Illicium henryi, Illicium verum,
Illicium lancealatum, Illicium pachyphyllum, Illicium anisatum,
Illicium religiosum, Hemidesmus indicus, Cistus incanus, Sida
acuta, Celastrus paniculata, Glycosmis muricata, Tanacetum
parthenium, Triticum aestivum, Hypericum dolabriforme, Dipsacus
pilosus, Triadenum walteri, Hypericum flondosum and Terminalia
pallid.
5. The method according to claim 1, wherein the plant stem cell
extract is a callus extract.
6. The method according to claim 1, wherein the plant stem cells
are stem cells of sequoia and the sequoia is giant sequoia
(Sequoiadendron giganteum).
7. The method according to claim 1, wherein the composition
comprises the shikimic acid at a concentration of 10 .mu.M to 30 mM
or comprises the extract at a concentration of 0.001 .mu.g/mL to 2
mg/mL, based on the total volume of the composition.
8. The method according to claim 3, which further comprises, before
the injection of the shikimic acid, the extract or the composition,
treating the adult-derived cells with a cell membrane
permeabilizing agent.
9. The method according to claim 8, wherein the cell membrane
permeabilizing agent is streptolysin O or digitonin.
10. The method according to claim 3, which further comprises
culturing the adult-derived cells into which the shikimic acid, the
extract or the composition has been injected after transferring to
a feeder cell layer.
11. The method according to claim 10, wherein the feeder cells are
STO cells.
12. stem cells induced from adult-derived cells by shikimic acid, a
plant extract comprising shikimic acid, a plant stem cell extract
comprising shikimic or a composition containing one or more of
them.
13. The induced pluripotent stem cells according to claim 12, which
are prepared by the method according to claim 1.
14. The induced pluripotent stem cells according to claim 12,
wherein the adult-derived cells are derived from an individual to
which the induced pluripotent stem cells will be administered and
the stem cells are specific for the individual.
15. (canceled)
16. A method for cell therapy, activating stem cells, proliferating
skin cells, regenerating skin or anti-aging, comprising a step of
administering shikimic acid, a plant extract comprising shikimic
acid, a plant stem cell extract comprising shikimic or a
composition containing one or more of them to an individual in need
of cell therapy, activation of stem cells, proliferation of skin
cells, skin regeneration or anti-aging.
17. The method according to claim 16, wherein the composition
comprises the shikimic acid at a concentration of 10 .mu.M to 30 mM
or comprises the plant extract or the plant stem cell extract at a
concentration of 0.001 .mu.g/mL to 2 mg/mL, based on the total
volume of the composition.
18. The method according to claim 16, wherein the composition is a
pharmaceutical composition or a cosmetic composition.
19. The method according to claim 16, wherein the plant extract
comprising shikimic acid is the plant extract described in claim 4.
Description
TECHNICAL FIELD
[0001] The present disclosure relates to a method for inducing
pluripotent stem cells by reprogramming and/or dedifferentiating a
differentiated adult cells, pluripotent stem cells prepared by the
method and a composition containing the pluripotent stem cells.
[0002] The present disclosure also relates to a pharmaceutical
composition or a cosmetic composition containing pluripotent stem
cells.
[0003] The present disclosure also relates to a composition for
activating stem cells, proliferating skin cells, regenerating skin
or anti-aging.
BACKGROUND ART
[0004] Stem cells are undifferentiated cells that can differentiate
into various types of cells constituting biological tissues and can
be obtained from the tissues of embryos, fetuses and adults. Among
the different cell types of the stem cell, pluripotent stem cells
refer to the stem cells that can differentiate into any of the
three germ layers, i.e., the endoderm, mesoderm and ectoderm.
[0005] The stem cells can be classified based on their anatomical
sites, cellular functions, antigens presented on the cell surface,
transcription factors, proteins produced by the cells, and specific
cell types that can be derived from the stem cells.
[0006] As a rather clear criterion of classification, the stem
cells can be classified based on their origin. Embryonic stem cells
(ES cells) are isolated from embryos and adult stem cells are
isolated from adult tissues.
[0007] Alternatively, the stem cells can be classified into
pluripotent, multipotent and unipotent stem cells based on their
capacity to differentiate into specialized cell types. In general,
embryonic stem cells (ES cells) can be classified as pluripotent
stem cells and adult stem cells can be classified as multipotent
and unipotent stem cells.
[0008] The embryonic stem cells (ES cells) derived from the inner
cell mass of a blastocyst, an early-stage embryo, are pluripotent
stem cells that can differentiate into all the tissues constituting
the adult body. That is to say, the embryonic stem cells are
undifferentiated cells that can proliferate without limit and can
differentiate into all cell types. Unlike the adult stem cells,
they can be inherited to the next generation because they can form
germ cells.
[0009] However, the pluripotent embryonic stem cells raise serious
religious and ethical concerns implicated with the destruction of
embryos during preparation thereof. In addition, since they are
derived from limited embryos, immune rejection due to lack of
immunocompatability between individuals cannot be avoided. To
overcome these problems, there have been various attempts to
artificially prepare pluripotent stem cells such as induced
embryonic stem cells or embryonic stem cells using the cells
derived from adults.
[0010] Typical examples include somatic cell nuclear transfer
(SCNT), fusion with ES cells and reprogramming by defined factors.
The somatic cell nuclear transfer is very inefficient and there is
an ethical question in that it requires eggs in large quantities.
The fusion with ES cells has a serious problem in terms of cell
stability because the induced cells additionally have two pairs of
genes. The reprogramming by defined factors, which has been
reported most recently, involves the serious problem of
carcinogenesis because it uses oncogene-containing viruses.
[0011] Therefore, a method for preparing induced pluripotent stem
cells with proven stability and safety without raising ethical
problems is needed for the development of a cell therapy agent.
[0012] To satisfy this need, a method for inducing induced
pluripotent stem cells through dedifferentiation by introducing
four genes into somatic cells was studied (Takahashi K, Yamanaka S
(2006). Induction of pluripotent stem cells from mouse embryonic
and adult fibroblast cultures by defined factors. Cell 126:
663-676). This method is free from ethical concerns because adult
cells are used and the immune rejection problem is solved because
autologous cells are used.
[0013] The inventors of the present disclosure have acquired
dedifferentiated stem cells from an extract of animal-derived
induced pluripotent stem cells (iPS) but there are some
limitations.
[0014] First, a large quantity (20 mg or more) of iPSC extract is
necessary for this method. For this reason, induction of
dedifferentiation using an extract of human-derived
dedifferentiated stem cells, which is costly and requires much
labor, has not been successful. For example, to prepare
human-derived dedifferentiated stem cells for obtaining 20 mg of
extract, an expert has to work hard for at least 3 months, which is
very costly.
[0015] Second, when somatic cells to be induced are treated with an
extract of animal stem cells or dedifferentiated stem cells
thereof, if the cells survive in the extract without being
completely destroyed, it is not easy to distinguish the
dedifferentiation-induced cells from the surviving dedifferentiated
stem cells and analysis of genomic DNA is necessary, which is
costly and time-consuming.
[0016] Third, since preparation of human-derived dedifferentiated
stem cells using proteins has been hardly successful, an extract of
human-derived dedifferentiated stem cells prepared using viruses
has to be used. Because the resulting cells may contain oncogenic
substances derived from the viruses, there may be difficulty in
clinical application.
DISCLOSURE
Technical Problem
[0017] In an aspect, the present disclosure is directed to
providing a method for preparing pluripotent stem cells with proven
stability and safety without raising ethical problems in order to
solve the problems in the related art. The present disclosure is
also directed to providing a method for inducing human-derived
dedifferentiated stem cells, which has been hardly successful.
[0018] The inventors of the present disclosure have developed a
method for inducing pluripotent stem cells using cells derived from
an adult, so that the pluripotent stem cells have the same genetic
background as the adult. According to the present disclosure, the
same result can be obtained from adult-derived cells having various
genetic backgrounds. Accordingly, the method of the present
disclosure is suitable for preparation of pluripotent stem
cells.
Technical Solution
[0019] In an aspect, the present disclosure provides a method for
inducing stem cells, including treating adult-derived cells with
shikimic acid, a plant extract containing shikimic acid, a plant
stem cell extract containing shikimic acid, or a composition
containing them.
[0020] Specifically, in an aspect, the present disclosure provides
a method for preparing induced pluripotent stem cells, including
extracting an extract containing active ingredients from plant stem
cells or any type of induced pluripotent plant stem cells induced
by various methods; injecting the extract into adult-derived cells;
and preparing pluripotent cells such as embryonic stem cells by
culturing the cells into which the extract has been injected.
[0021] In another aspect, the present disclosure provides a method
for preparing stem cells, which further includes injecting shikimic
acid, a plant extract containing shikimic acid, a plant stem cell
extract containing shikimic acid, or a composition containing them
into adult-derived cells; and culturing the cells into which the
shikimic acid, the extract or the composition has been
injected.
[0022] The extract may be a callus extract.
[0023] In an exemplary embodiment of the present disclosure, the
method may further include, before the injection of the extract,
treating the adult-derived cells with a cell membrane
permeabilizing agent. The cell membrane permeabilizing agent may
include streptolysin O and digitonin, although not being limited
thereto as long as it allows easy injection of the shikimic acid or
the extract according to the present disclosure through the cell
membrane.
[0024] In another exemplary embodiment of the present disclosure,
the method may further include culturing the adult-derived cells
into which the extract has been injected after transferring to a
feeder cell layer. The feeder cells may include STO cells, although
not being limited thereto.
[0025] In another aspect, the present disclosure provides a method
for preparing induced pluripotent stem cells, including extracting
an extract containing active ingredients from plant stem cells, a
callus or any type of induced pluripotent plant stem cells induced
by various methods; injecting the extract into adult-derived cells;
culturing the cells into which the extract has been injected using
normal cell culture media; and further culturing the cells using
embryonic stem cell culture media after transferring to a feeder
cell layer.
[0026] In another aspect, the present disclosure provides stem
cells prepared by the above-described method.
[0027] In another aspect, the present disclosure provides a
composition containing the stem cells. In another aspect, the
present disclosure provides a method for inducing inducible
pluripotent stem cells (iPSC) by inducing reprogramming and/or
dedifferentiation of differentiated adult cells using shikimic acid
or a plant extract or a plant stem cell extract containing the
same, pluripotent stem cells prepared by the method, and a cell
therapy agent containing the pluripotent stem cells.
[0028] The plant extract or plant stem cell extract containing
shikimic acid used in the present disclosure may further contain an
extract of one or more selected from a group consisting of sequoia
(Sequoiadendron giganteum), Iris pseudoacorus, Helianthus
tuberosus, Picea pungens, Picea glauca, Eucalyptus sieberiana,
Eucalyptus regnans, Thuja plicata, Phoenix dactylifera, Dahlia
variabilis, Malus baccata, Pyrus communis, Triticum, Pinus
densifloraa, Pinus thunbergii, Illicium anisatum, Magnolia
grandiflora, Houttuynia cordata, Saxifraga stolonifera, Terminalia
arjuna, Pistacia lentiscus, Ribes aureum, Symphytum officinalis,
Actaea pachypoda, Alangium salvifollium, Gingko biloba, Veratrum
viride, Dipsacus laciniatus, Agastache urticifolia, Inula helenium,
Hypericum spp., Commelina bengalensis, Gymnema sylvestris,
Terminalia chebula, Illicium floridanum, Illicium diffengri,
Illicium henryi, Illicium verum, Illicium lancealatum, Illicium
pachyphyllum, Illicium anisatum, Illicium religiosum, Hemidesmus
indicus, Cistus incanus, Sida acuta, Celastrus paniculata,
Glycosmis muricata, Tanacetum parthenium, Triticum aestivum,
Hypericum dolabriforme, Dipsacus pilosus, Triadenum walteri,
Hypericum flondosum and Terminalia pallid (Denis V. Bochkov et.
al., Shikimic acid: review of its analytical, isolation, and
purification techniques from plant and microbial sources, J Chem.
Biol. (2012) 5; 5-17).
[0029] In another aspect, the present disclosure provides a
composition for activating stem cells, regenerating skin or
anti-aging, which contains the stem cells prepared by the
above-described method, or a pharmaceutical or cosmetic composition
containing the same.
Advantageous Effects
[0030] In accordance with the present disclosure, an extract of
plant stem cells or any type of induced pluripotent plant stem
cells induced by various methods, shikimic acid, a plant extract
containing shikimic acid, or a composition containing the same may
be used to prepare stem cells. The method of the present disclosure
is applicable to the cells of all species having various genetic
backgrounds, including human. In addition, the method of the
present disclosure is free from ethical concerns because it uses a
plant-derived stem cell extract and allows for preparation of
induced pluripotent stem cells with proven safety.
BRIEF DESCRIPTION OF DRAWINGS
[0031] FIG. 1 schematically describes an experimental procedure of
inducing pluripotent stem cells according to the present
disclosure.
[0032] FIG. 2 shows induced pluripotent stem cells observed on day
5 after treatment with a plant stem cell extract.
[0033] FIG. 3 shows induced pluripotent stem cells observed on day
8 after treatment with a plant stem cell extract and day 2 after
transfer to a feeder cell layer.
[0034] FIG. 4 A shows induced pluripotent stem cells observed on
day 32 after treatment with a plant stem cell extract and after
subculturing for 4 times after transfer to a feeder cell layer.
FIG. 4 B shows typical embryonic stem cells.
[0035] FIG. 5 shows a result of alkaline phosphatase staining of
induced pluripotent stem cells observed on day 32 after treatment
with a plant stem cell extract and after subculturing for 4 times
after transfer to a feeder cell layer.
[0036] FIG. 6 A shows induced pluripotent stem cells observed on
day 50 after treatment with a plant stem cell extract and after
subculturing for 7 times after transfer to a feeder cell layer as
well as a result of alkaline phosphatase staining thereof. FIG. 6 B
shows human pluripotent stem cells induced by using four factor
viruses of Yamanaka as well as a result of alkaline phosphatase
staining thereof.
[0037] FIG. 7 shows gene expression in pluripotent stem cells
induced according to the present disclosure.
[0038] FIG. 8 shows an HPLC result of a sequoia callus extract
according to the present disclosure. Shikimic acid has a structure
of [Chemical Formula 1].
[0039] FIG. 9 schematically describes an experimental procedure of
inducing pluripotent stem cells using shikimic acid or a plant stem
cell extract containing the same according to the present
disclosure.
[0040] FIG. 10 shows expression of the Oct3/4 gene in HDF after
treatment with shikimic acid or a sequoia callus extract.
[0041] FIG. 11 shows expression of the Oct3/4 gene in HDF after
treatment with shikimic acid at different concentrations.
[0042] FIG. 12 shows increased expression of ALP after treatment
with shikimic acid or a sequoia callus extract.
[0043] FIG. 13 shows increased expression of ALP after treatment
with shikimic acid at different concentrations.
[0044] FIG. 14 shows the colony-forming ability of dermal cells
after treatment with shikimic acid or a sequoia callus extract.
[0045] FIG. 15 shows the colony-forming ability of dermal cells
after treatment with shikimic acid different concentrations.
[0046] FIG. 16 shows the colony-forming ability of dermal cells
after treatment with shikimic acid or a sequoia callus extract.
[0047] FIG. 17 shows the colony-forming ability of dermal cells
after treatment with shikimic acid different concentrations.
[0048] FIG. 18 shows increased proliferating ability of dermal
cells after treatment with shikimic acid or a sequoia callus
extract.
BEST MODE
[0049] Korean Patent Application No. 10-2013-0123860, which was
filed on Oct. 17, 2013 is incorporated herein in its entirety for
all purposes. In addition, this application claims the priority of
Korean Patent Application No. 10-2013-0123860 and all the benefits
accruing therefrom, the contents of which in its entirety are
herein incorporated by reference.
[0050] In an aspect, the present disclosure provides a method for
preparing induced pluripotent stem cells, including:
[0051] a) a step of extracting an extract containing active
ingredients from plant stem cells or induced pluripotent plant stem
cells;
[0052] b) a step of injecting the extract into adult-derived cells;
and
[0053] c) a step of preparing pluripotent cells such as embryonic
stem cells by culturing the cells into which the extract has been
injected.
[0054] In another aspect, the present disclosure provides a method
for preparing stem cells, including treating adult-derived cells
with shikimic acid, a plant extract containing shikimic acid, a
plant stem cell extract containing shikimic acid, or a composition
containing them.
[0055] In another aspect, the present disclosure provides a method
for preparing stem cells, including: injecting shikimic acid, a
plant extract containing shikimic acid, a plant stem cell extract
containing shikimic acid, or a composition containing them into
adult-derived cells; and culturing the cells into which the
shikimic acid, the extract or the composition has been
injected.
[0056] In the present disclosure, the extract may be a callus
extract.
[0057] In an aspect of the present disclosure, the plant extract or
the plant stem cell extract may contain shikimic acid, caffeic acid
or ferulic acid.
[0058] In the present disclosure, `shikimic acid` may be
represented by Chemical Formula 1 and is used in a broad concept,
including its precursors, derivatives, etc. The shikimic acid may
have a molecular weight of 174.15 g/mol.
##STR00001##
[0059] In the present disclosure, `caffeic acid` may be represented
by Chemical Formula 2 and is used in a broad concept, including its
precursors, derivatives, etc. It may have a molecular weight of
180.16 g/mol. Caffeic acid is a phenolic compound contained in
fruits including coffee bean and pear and medicinal plants such as
basil, thyme, banana, tarragon, oregano, dandelion, etc.
##STR00002##
[0060] In the present disclosure, `ferulic acid` may be represented
by Chemical Formula 3 and is used in a broad concept, including its
precursors, derivatives, etc. It may have a molecular weight of
191.18 g/mol. Ferulic acid is a precursor to lignin constituting
plant cell walls and is abundant in plant cell walls. It can be
found in plant seeds of wheat, oats, coffee, apple, orange, peanut,
etc.
##STR00003##
[0061] In the present disclosure, the term `stem cell` refers to a
master cell that can proliferate without limit so as to form cells
specialized for various tissues and organs. The stem cells are
developable pluripotent or multipotent cells. A stem cell may
divide into two daughter stem cells or into one daughter stem cell
and one transit cell. Afterwards, they proliferate as mature and
complete cells of the tissues.
[0062] In the present disclosure, the term `embryonic stem cell`
refers to a pluripotent cell which is isolated from the inner cell
mass of a blastocyst, an early-stage embryo, after fertilization
and then cultured.
[0063] In the present disclosure, the term `dedifferentiated stem
cell or induced pluripotent stem cell (iPS) extract` refers to a
substance obtained by finely chopping dedifferentiated stem cells
or induced pluripotent stem cells (iPS), which have been induced
and cultured by various methods, in a test tube using
physical/chemical methods and then separating through
centrifugation, etc.
[0064] In the present disclosure, the term `plant stem cell` refers
to a plant stem cell derived from a cambium. In particular, it
includes physically intact pure cambial meristematic cells (CMC),
found in the cambium at a boundary between the xylem and the
phloem. In the present disclosure, the term `plant stem cell` is
used in the broad concept, including any type of induced
pluripotent plant stem cells induced by various methods.
[0065] In the present disclosure, the term `callus` refers to a
mass of unorganized parenchyma cells, a typical example of which is
a tumor tissue formed from a meristematic tissue around a plant
wound. The plant tissues are largely divided into the meristematic
tissues which show cell division and the permanent tissues which do
not. When the cells of a meristematic issue is cultured in a
nutrient medium, a callus is formed initially. Then, an
adventitious embryo is formed and it is differentiated into a plant
organ. The callus is commonly called `plant stem cells`. The callus
used in the present disclosure is not limited in its kind.
[0066] In the present disclosure, the term `pluripotent stem cell`
refers to a stem cell having the pluripotency to differentiate into
any of the three germ layers of an organism, i.e. endoderm,
mesoderm and ectoderm. Traditionally, embryonic stem cells are the
stem cells in this category.
[0067] In the present disclosure, the term `induced pluripotent
stem cell` refers to a pluripotent stem cell which is genetically
identical to a donor cell used to prepare the induced pluripotent
stem cell. That is to say, the induced pluripotent stem cell
originates from the donor cell.
[0068] In the present disclosure, the terms `induced pluripotent
stem cell,` `dedifferentiated stem cell` and inducible pluripotent
stem cell can be used interchangeably.
[0069] In the present disclosure, the term `adult-derived cell`
refers, as opposed to an embryonic cell, to a cell which is derived
from a surviving adult.
[0070] In the present disclosure, the term `differentiation` refers
to a process by which the morphology or function of a cell is
specialized during it divides and proliferates. The morphology or
function of the cell changes so as to perform specific functions of
the cell, tissue, etc. of an organism. In general, it refers to a
phenomenon by which a relatively simple system is divided into two
or more qualitatively different sub-systems. That is to say,
occurrence of differences from an essentially identical part of an
organism or division to qualitatively different systems as a result
thereof, such as formation of head or body parts from an egg during
ontogenesis or differentiation of muscle cells, neural cells, etc.,
is called differentiation.
[0071] In an aspect of the present disclosure, the plant may be
sequoia.
[0072] In an aspect of the present disclosure, the plant stem cells
may be a callus.
[0073] In an aspect of the present disclosure, the plant stem cells
may be sequoia stem cells.
[0074] In an aspect of the present disclosure, the plant stem cell
extract may be a callus extract.
[0075] In an aspect of the present disclosure, the extract may be a
sequoia callus extract.
[0076] In an aspect of the present disclosure, the sequoia may be
giant sequoia (Sequoiadendron giganteum).
[0077] In an aspect of the present disclosure, the extract or the
composition may contain shikimic acid.
[0078] In an aspect of the present disclosure, the composition may
contain the shikimic acid at a concentration of 10 .mu.M to 30 mM
based on the total volume of the composition. In another aspect of
the present disclosure, the composition may contain the shikimic
acid, the plant extract or the plant stem cell extract at a
concentration of 10 .mu.M or higher, 20 .mu.M or higher, 30 .mu.M
or higher, 50 .mu.M or higher, 100 .mu.M or higher, 1 mM or higher,
5 mM or higher, 10 mM or higher, 20 mM or higher or 30 mM or
higher, and 1 M or lower or 100 M or lower, based on the total
volume of the composition. When the content of the shikimic acid
contained in the composition is 10 .mu.M or lower, it may be
difficult to achieve the effect of the present disclosure. And,
when it is 30 mM or higher, skin irritation may be caused.
Specifically, the shikimic acid may be contained at a concentration
of 0.1 mM or higher, 0.5 mM or higher, 0.6 mM or higher, 0.7 mM or
higher, 0.8 mM or higher or 0.9 mM or higher, and 5 mM or lower, 4
mM or lower, 3 mM or lower, 2 mM or lower, 1.5 mM or lower, 1.4 mM
or lower, 1.3 mM or lower, 1.2 mM or lower or 1.1 mM or lower,
based on the total volume of the composition. More specifically,
the shikimic acid may be contained at a concentration of 0.8-1.2
mM, based on the total volume of the composition. Most
specifically, the shikimic acid may be contained at a concentration
of 1 mM, based on the total volume of the composition.
[0079] In an aspect of the present disclosure, the plant extract or
the plant stem cell extract may contain the shikimic acid at a
concentration of 0.0001-45% (w/v) based on the total volume of the
extract. When the shikimic acid is contained in this range,
superior Oct3/4 gene expressing effect, ALP expressing effect and
fibroblast proliferation promoting effect may be achieved. In this
aspect, in the present disclosure, the plant extract or the plant
stem cell extract may contain the shikimic acid at a concentration
of 0.001% (w/v) or higher, 0.01% (w/v) or higher, 0.1% (w/v) or
higher, 1% (w/v) or higher, 5% (w/v) or higher, 10% (w/v) or
higher, 15% (w/v) or higher, 20% (w/v) or higher, 25% (w/v) or
higher or 30% (w/v) or higher, and 45% (w/v) or lower, 40% (w/v) or
lower, 38% (w/v) or lower, 36% (w/v) or lower, 34% (w/v) or lower,
33% (w/v) or lower or 32% (w/v) or lower, based on the total volume
of the extract. Specifically, in the present disclosure, the plant
extract or the plant stem cell extract may contain the shikimic
acid at a concentration of 32-34% (w/v), more specifically 32.75%
(w/v), based on the total volume of the extract.
[0080] In an aspect of the present disclosure, the composition may
contain the plant extract or the plant stem cell extract at such a
concentration that the concentration of the shikimic acid contained
in the extract is that described above based on the total volume of
the composition. For example, the plant extract or the plant stem
cell extract may be contained in the composition at a concentration
of 0.001 .mu.g/mL or higher, 0.01 .mu.g/mL or higher, 0.1 .mu.g/mL
or higher, 1 .mu.g/mL or higher, 10 .mu.g/mL or higher, 50 .mu.g/mL
or higher, 100 .mu.g/mL or higher, 0.3 mg/mL or higher, 0.4 mg/mL
or higher, 0.5 mg/mL or higher, 0.6 mg/mL or higher, 0.7 mg/mL or
higher, 1 mg/mL or higher, 1.5 mg/mL or higher, 2 mg/mL or higher,
5 mg/mL or higher, 10 mg/mL or higher, 20 mg/mL or higher or 50
mg/mL or higher, and 1 g/mL or lower or 10 g/mL or lower, based on
the total volume of the composition.
[0081] In the present disclosure, the concentration of the shikimic
acid contained in the plant extract or the plant stem cell extract
may be measured using the Waters 1525.mu. Binary HPLC Pump and the
Gemini 5u C18 110 A column (5 .mu.m, 4.60 mm.times.250 nm,
Phenomenex), using solvent A (water, containing 0.1% TFA) and
solvent B (acetonitrile, containing 0.1% TFA) as mobile phases.
[0082] In the present disclosure, `sequoia` includes redwood
(Sequoia sempervirens), giant sequoia (Sequoiadendron giganteum) or
metasequoia (Metasequoia glyptostroboides).
[0083] Redwood (Sequoia sempervirens) is a tree of the family
Cupressaceae, order Pinales. It grows only in the northwestern
coastal California and the southwestern coastal Oregon within the
United States and in New Zealand. It lives about 2500-3000 years
and is the tallest tree in the world, reaching up to 112 m. It is
2.5-4.5 m in diameter and 50-100 m in height and the bark can be
very thick, up to 20-30 cm. The leaves, which are similar to those
of yew trees, are 1-3 cm long, with pointed tips and distinct main
veins. They are dark green above and whitish below.
[0084] Giant sequoia (Sequoiadendron giganteum) is the sole living
species in the genus Sequoiadendron. It grows in the western slopes
of the Sierra Nevada Mountains of California, at 1500-2500 m above
the sea level. It reaches 3.5-6 m in diameter and 60-90 m in height
and the diameter near the root is around 10 m. The leaves are
similar to those of cedar. They are about 1 cm long and arranged
spirally. But, the leaves on mature branches are scale-shaped.
[0085] Metasequoia (Metasequoia glyptostroboides) is the sole
living species in the genus Metasequoia, family Cupressaceae. It
grows up to 35 m in height. The grayish brown bark is vertically
fissured. The branches spread sideways and the leaves are opposite,
10-23 mm in length and 1.5-2 mm in width. The pointed leaves turn
reddish brown in fall. The flowers bloom moneciously in February to
March. The male flowers hang in racemes from the tip of the
branches at the axils and have 20 stamens. The female flowers hang
on the tip of the branches and bloom in March. The cones are
globose, 18-25 mm in length. They ripen brown and produce winged
oval seeds. The deciduous conifer tree is native to Sichuan and
Hubei provinces of China and is distributed in Korea, China, etc.
It is mainly planted as park trees.
[0086] A callus refers to a mass of undifferentiated, unorganized
parenchyma cells, a typical example of which is a tumor tissue
formed from a meristematic tissue around a plant wound. The plant
tissues are largely divided into the meristematic tissues which
show cell division and the permanent tissues which do not. When the
cells of a meristematic issue is cultured in a nutrient medium, a
callus is formed initially. Then, an adventitious embryo is formed
and it is differentiated into a plant organ. The callus is commonly
called "plant stem cells".
[0087] In the present disclosure, the `extract` includes any
substance extracted from a natural product, regardless of
extraction method, extraction solvent, extracted components or type
of extract. It is used in a broad concept, including the substance
obtained by processing or treating otherwise the extract.
[0088] In the present disclosure, the sequoia may be used in the
form of an extract, a pulverization product of the plant or a dried
pulverization product of the plant, although not being limited
thereto. Besides, the sequoia used in the present disclosure is not
limited as to how it is obtained. It may be either cultivated or
purchased commercially and may be the aerial part, underground part
or both of sequoia. The aerial part may include the fruit, leaf and
stem of sequoia and the underground part may include the root,
although not being limited thereto.
[0089] In the present disclosure, the `plant extract` includes any
substance extracted from the plant such as sequoia, regardless of
extraction method, extraction solvent, extracted components or type
of extract. It is used in a broad concept, including the substance
extracted by treating with heat, an acid, a base, an enzyme, etc.
as well as the substance obtained by processing or treating
otherwise the extract of sequoia.
[0090] In the present disclosure, the `plant stem cell extract`
includes any substance obtained by culturing the plant stem cells
of sequoia, etc. and extracting its active ingredients, regardless
of extraction method, extraction solvent, extracted components,
type of extract, etc. It is used in a broad concept, including any
substance extracted from the active ingredients of the plant stem
cells of sequoia, etc. by treating with heat, an acid, a base, an
enzyme, etc. as well as the substance obtained by processing or
treating otherwise the extract of sequoia.
[0091] The method of the present disclosure includes a step of
culturing plant stem cells or induced pluripotent plant stem cells
induced by various methods and preparing an extract therefrom,
which may be conducted by a method known in the art. For example,
after culturing plant stem cells or induced pluripotent plant stem
cells induced by various methods, they may be treated with a
protease and then the supernatant may be collected. Alternatively,
plant stem cells may be incubated at 65.degree. C. for 2 hours and
then filtered to extract the proteins derived from each cell or
shikimic acid. In an exemplary embodiment of the present
disclosure, a callus powder may be used.
[0092] In the present disclosure, the sequoia callus extract may be
obtained through i) a step of inducing a callus from sequoia; ii)
step of establishing a stem cell line by culturing the callus in a
solid medium; iii) a step of producing active ingredients in large
scale by suspension culturing the cell line; and iv) a step of
extracting the produced active ingredients.
[0093] In the present disclosure, the extraction of the active
ingredients from the sequoia callus may be performed by culturing
of a cell line derived from a tissue explant of the plant as
described in Korean Patent Publication No. 2007-0113193.
Specifically, a stabilized plant cell line derived from sequoia may
be extracted using a mixture of a C.sub.5 or lower alcohol,
although not being limited thereto.
[0094] Alternatively, the sequoia callus extract or the sequoia
callus powder used in in the present disclosure may be purchased
commercially.
[0095] In the present disclosure, the sequoia callus extract may be
prepared by dissolving a sequoia callus powder in a solvent. The
solvent may include one or more selected from a group consisting of
water, an organic solvent and a mixture of water and an organic
solvent. The water may include distilled water or purified water
and the organic solvent may include an alcohol such as a
C.sub.1-C.sub.5 lower alcohol, one or more selected from a group
consisting of acetone, ether, ethyl acetate, diethyl ether, methyl
ethyl ketone and chloroform, hexane, methylene chloride, ethyl
acetate, n-butanol, a mixture solvent of butylene glycol (BG) and
ethanol (EtOH), dimethyl sulfoxide (DMSO), etc., although not being
limited thereto.
[0096] Those skilled in the art will easily understand that a
high-concentration protein extract can be prepared using the
existing protein extraction method. The concentration of the
shikimic acid or the protein extract derived from the plant may be
specifically 10 .mu.g/mL to 1 mg/mL, more specifically about 500
.mu.g/mL, based on the total volume of the composition containing
the extract. When the concentration of the protein extract is
outside the above range, the efficiency of inducing the induced
pluripotent stem cells may decrease or the cells treated with the
extract may die.
[0097] The method of the present disclosure includes a step of
injecting a protein extract isolated from plant stem cells or
induced pluripotent plant stem cells induced by various methods
into adult-derived cells.
[0098] The adult-derived cells may include human dermal fibroblasts
or neonatal human dermal fibroblasts.
[0099] For the injection, the cells may be permeabilized by
treating with a cell membrane permeabilizing agent (e.g.,
streptolysin O or digitonin), so that the extract can be introduced
into the cells. Following the permeabilization, the shikimic acid,
the plant extract, the plant stem cell extract, the induced
pluripotent plant stem extract or the composition is injected into
the adult-derived cells through incubation.
[0100] In an aspect of the present disclosure, a method for
preparing pluripotent stem cells may include:
[0101] 1. a step of preparing shikimic acid, a plant extract
containing shikimic acid, a plant stem cell extract or a
composition containing the same according to the present
disclosure;
[0102] 2. a step of injecting the shikimic acid, the extract or the
composition into adult-derived cells;
[0103] 3. a step of culturing the adult-derived cells using a
normal culture medium;
[0104] 4. a step of further culturing the cells after transferring
to a feeder cell layer; and
[0105] 5. a step of recovering the cultured pluripotent stem
cells.
[0106] Specifically, the step of injecting the shikimic acid, the
extract or the composition into the adult-derived cells may
include:
[0107] 1. a step of separating adult-derived cells into individual
cells and transferring to a tube after resuspending them;
[0108] 2. a step of centrifuging the cells and resuspending the
resulting cell pellets in a water bath;
[0109] 3. a step of adding a cell membrane permeabilizing
agent;
[0110] 4. a step of performing reaction while mixing the sample in
the water bath up and down;
[0111] 5. a step of centrifuging the sample;
[0112] 6. a step of resuspending the cell pellets using the
shikimic acid, the extract or the composition;
[0113] 7. a step of performing reaction while mixing the sample in
the water bath up and down using an ATP regeneration system;
[0114] 8. a step of adding an ES culture medium and performing
incubation in an incubator; and
[0115] 9. a step of cleansing and resuspending the cell pellets in
an embryonic stem cell culture medium and then seeding onto a
dish.
[0116] The method of the present disclosure includes a step of
preparing pluripotent cells such as embryonic stem cells by
culturing the cells into which the shikimic acid, the extract or
the composition has been injected.
[0117] The method of the present disclosure may further include,
after culturing the adult-derived cells into which the shikimic
acid, the extract or the composition has been injected using a
normal cell culture medium, a step of further culturing them after
transferring to a feeder cell layer
[0118] More specifically, after injecting the shikimic acid, the
extract or the composition, the adult-derived cells may be cultured
using a normal cell culture medium (DMEM, 5-15% FBS, 10-100 U/mL
penicillin, 20-80 mg/mL streptomycin) until a colony is formed.
After the colony has been formed, the cells may be transferred to a
feeder cell layer and then subcultured in an embryonic stem cell
culture medium with 5-8 day intervals while replacing the medium
every day. The feeder cells used in the present disclosure may
include STO cells, although not being limited thereto.
[0119] The pluripotent stem cells induced by the extract may be
cultured in DMEM (Dulbecco's modified Eagle's medium)/F12
supplemented with 15-25% KSR (knockout serum replacement), 1-4 mM
L-glutamine, 0.05-0.2 mM nonessential amino acids, 0.05-0.2 mM
.beta.-mercaptoethanol, 30-70 U/mL penicillin, 30-70 mg/mL
streptomycin and 1-30 .mu.g/mL bFGF. Those skilled in the art can
easily recognize that the concentration of the compounds added to
the DMEM can vary within the range at which the effect of the
present disclosure can be achieved.
[0120] The present disclosure also provides a method for inducing
induced pluripotent stem cells, including:
[0121] a) a step of preparing an extract by isolating proteins from
plant stem cells or induced pluripotent plant stem cells induced by
various methods;
[0122] b) a step of treating adult-derived cells with a cell
membrane permeabilizing agent and then injecting the extract into
the adult-derived cells; and
[0123] c) a step of culturing the extract-injected cells in a
normal cell culture media and then culturing in an embryonic stem
cell culture medium after transferring to a feeder cell layer.
[0124] The method of the present disclosure is characterized in
that pluripotent stem cells which are hardly distinguishable from
embryonic stem cells can be prepared from adult-derived cells using
shikimic acid, a plant extract, plant stem cells, an extract of
induced pluripotent plant stem cells induced by various methods or
a composition containing the same.
[0125] The inventors of the present disclosure have confirmed that
pluripotent stem cells are induced by the method of the present
disclosure.
[0126] Specifically, the pluripotent stem cells induced by the
method of the present disclosure are almost identical to embryonic
stem cells in shape (see FIG. 4). In addition, investigation of the
expression of the genes specific for embryonic stem cells (Nanog,
Oct4) revealed that the genes are expressed in the pluripotent stem
cells induced by the method of the present disclosure as in the
embryonic stem cells (see FIG. 7 and FIG. 10).
[0127] In another aspect, the present disclosure provides induced
pluripotent stem cells induced by the method according to an aspect
of the present disclosure.
[0128] The inventors of the present disclosure have confirmed
self-renewal, which is characteristic of stem cells, by performing
subculturing 8-12 times using the method of the present disclosure
(see FIG. 6).
[0129] In another aspect, the present disclosure provides a
composition containing the pluripotent stem cells prepared by the
method according to an aspect of the present disclosure.
[0130] In an aspect, the present disclosure provides a composition
containing one or more of shikimic acid, a plant extract containing
shikimic acid and a plant stem cell extract containing shikimic
acid as an active ingredient.
[0131] In an aspect of the present disclosure, the composition may
be a pharmaceutical composition, a food composition or a cosmetic
composition.
[0132] In an aspect of the present disclosure, the composition may
be a composition for activating stem cells, regenerating skin or
anti-aging.
[0133] In an aspect, the present disclosure may relate to a method
for activating stem cells, including a step of administering
shikimic acid, a plant extract containing shikimic acid and a plant
stem cell extract containing shikimic acid or a composition
containing one or more of them to an individual in need of
activation of stem cells. The administration may be performed
according to the administration method or administration dose
described in the present disclosure.
[0134] In an aspect, the present disclosure may relate to a method
for regenerating skin, including a step of administering shikimic
acid, a plant extract containing shikimic acid and a plant stem
cell extract containing shikimic acid or a composition containing
one or more of them to an individual in need of skin
regeneration.
[0135] In an aspect, the present disclosure may relate to a method
for anti-aging, including a step of administering shikimic acid, a
plant extract containing shikimic acid and a plant stem cell
extract containing shikimic acid or a composition containing one or
more of them to an individual in need of anti-aging.
[0136] In an aspect, the present disclosure may relate to a use of
shikimic acid, a plant extract containing shikimic acid and a plant
stem cell extract containing shikimic acid or a composition
containing one or more of them for activation of stem cells, skin
regeneration or anti-aging.
[0137] In an aspect, the present disclosure may relate to shikimic
acid, a plant extract containing shikimic acid and a plant stem
cell extract containing shikimic acid or a composition containing
one or more of them for use in activation of stem cells, skin
regeneration or anti-aging.
[0138] In an aspect of the present disclosure, the composition may
be a cell therapy agent.
[0139] Specifically, the cell therapy agent may be used for
generation of hepatocytes, adipocytes, bone cells, cartilage cells,
muscle cells, neurons, cardiac muscle cells, vascular endothelial
cells, etc.
[0140] In an aspect, the present disclosure may relate to a method
for cell therapy, including a step of administering shikimic acid,
a plant extract containing shikimic acid and a plant stem cell
extract containing shikimic acid or a composition containing one or
more of them to an individual in need of cell therapy.
[0141] In an aspect, the present disclosure may relate to a use of
shikimic acid, a plant extract containing shikimic acid and a plant
stem cell extract containing shikimic acid or a composition
containing one or more of them for cell therapy.
[0142] In an aspect, the present disclosure may relate to shikimic
acid, a plant extract containing shikimic acid and a plant stem
cell extract containing shikimic acid or a composition containing
one or more of them for use in cell therapy.
[0143] As used in the present disclosure, the term `cell therapy
agent` refers to cells or tissues isolated from human and prepared
as a medication through culturing and special operation for use in
treatment, diagnosis and prevention (USFDA definition). It is used
for the purpose of treatment, diagnosis and prevention by
proliferating and screening living autologous, homologous or
heterologous cells ex vivo or otherwise changing the biological
properties of the cells to restore the function of cells or
tissues. The cell therapy agents are largely classified into
somatic cell therapy agents and stem cell therapy agents based on
the degree of differentiation of the cells. The present disclosure
relates particularly to the stem cell therapy agent.
[0144] In an aspect, the present disclosure provides a food
composition, which contains the shikimic acid or the plant extract,
the plant stem cell extract or the pluripotent stem cells
containing shikimic acid according to an aspect of the present
disclosure. In an aspect of the present disclosure, the composition
may contain other ingredients within a range not negatively
affecting the main effect desired by the present disclosure. For
example, additives such as a fragrance, a pigment, a sterilizer, an
antioxidant, a preservative, a humectant, a thickener, a mineral,
an emulsifier, a synthetic polymer, etc. may be further included
for improvement of physical properties. In addition, other adjuvant
ingredients such as a water-soluble vitamin, an oil-soluble
vitamin, a polypeptide, a polysaccharide, a seaweed extract, etc.
may be further included. These ingredients may be adequately
selected by those skilled in the art depending on the formulation
type or purpose of use and the addition amount may be determined
within a range not negatively affecting the purpose and effect of
the present disclosure. For example, those ingredients may be added
in an amount of 0.01-5 wt %, more specifically 0.01-3 wt %, based
on the total weight of the composition. In an aspect of the present
disclosure, the food composition may include a health food
composition, a functional food composition, a nutritional
supplement composition, a processed food composition, a food
additive composition, etc., although not being limited thereto.
[0145] In an aspect, the present disclosure provides a cosmetic
composition, which contains the shikimic acid or the plant extract,
the plant stem cell extract or the pluripotent stem cells
containing shikimic acid according to an aspect of the present
disclosure. The cosmetic composition contains a cosmetically or
dermatologically acceptable medium or matrix. The cosmetic
composition may be in any form which is topically applicable, e.g.,
a solution, a gel, a solid, an anhydrous paste, an oil-in-water
emulsion, a water-in-oil emulsion, a multiemulsion, a suspension, a
microemulsion, a microcapsule, an ionic (liposome) or nonionic
vesicular dispersion, a foam, an aerosol further containing a
compressed propellant or a patch. These compositions may be
prepared according to the methods commonly employed in the art.
[0146] The cosmetic composition may further contain, in addition to
the above-described substances, other ingredients that may provide
a synergic effect to the main effect within a range not negatively
affecting the main effect. The other ingredients may be selected by
those skilled in the art without difficulty depending on the
formulation type or purpose of use. For example, the cosmetic
composition of the present disclosure may contain, in addition to
the active ingredient, other ingredients commonly mixed in a
cosmetic composition. Examples include a fat, an oil, a humectant,
an emollient, a surfactant, an organic or inorganic pigment, an
organic powder, a UV absorbent, a preservative, a sterilizer, an
antioxidant, a stabilizer, a thickener, glycerin, a pH control
agent, an alcohol, a pigment, a fragrance, a blood circulation
accelerator, a coolant, an antiperspirant, purified water, etc.
However, the other ingredients that may be contained in the
cosmetic composition are not limited thereto and the amount thereof
may be determined within a range not negatively affecting the
purpose and effect of the present disclosure.
[0147] The formulation type of the cosmetic composition is not
particularly limited and may be selected adequately depending on
purposes. For example, it may be prepared into one or more
formulation selected from a group consisting of a softening lotion,
a nourishing lotion, an essence, a nourishing cream, a massage
cream, a pack, a gel, a makeup base, a foundation, a powder, a
lipstick, a patch, a spray, an eye cream, an eye essence, a
cleansing cream, a cleansing foam, a cleansing water, a cleanser, a
hair shampoo, a hair conditioner, a hair treatment product, a hair
essence, a hair lotion, a scalp and hair tonic, a scalp essence, a
hair gel, a hair spray, a hair pack, a body lotion, a body cream, a
body oil and a body essence, although not being limited
thereto.
[0148] In an aspect, the present disclosure provides a
pharmaceutical composition, which contains the shikimic acid or the
plant extract, the plant stem cell extract or the pluripotent stem
cells containing shikimic acid according to an aspect of the
present disclosure. The pharmaceutical composition may further
contain, in addition to the active ingredient, pharmaceutical
adjuvants or other therapeutically useful substances such as a
preservative, a stabilizer, a wetting agent, an emulsifier, a salt
and/or buffer for control of osmotic pressure, a diluent (e.g.,
lactose, dextrose, sucrose, mannitol, sorbitol, cellulose or
glycine), a lubricant (e.g., silica, talc, stearic acid and a
magnesium or calcium salt thereof or polyethylene glycol), a binder
(e.g., magnesium aluminum silicate, starch paste, gelatin,
tragacanth, methyl cellulose, sodium carboxymethyl cellulose or
polyvinylpyrrolidone), etc. In some occasions, it may further
contain other pharmaceutical additives such as a disintegrant,
e.g., starch, agar, alginic acid or a sodium salt thereof, an
absorbent, a colorant, a flavor, a sweetener, etc.
[0149] The pharmaceutical composition may be prepared into
formulations for oral or parenteral administration according to
commonly employed methods.
[0150] Formulations for oral administration may include, for
example, a tablet, a fill, a hard/soft capsule, a liquid, a
suspension, an emulsion, a syrup, a powder, a dust, a fine granule,
a granule, a pellet, etc. These formulations may contain, in
addition to the active ingredient, a surfactant, a diluent (e.g.,
lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and
glycine), a lubricant (e.g., silica, talc, stearic acid and a
magnesium or calcium salt thereof and polyethylene glycol). A
tablet may contain a binder such as magnesium aluminum silicate,
starch paste, gelatin, tragacanth, methyl cellulose, sodium
carboxymethyl cellulose and polyvinylpyrrolidone and may further
contain, in some cases, pharmaceutical additives, e.g., a
disintegrant such as starch, agar, alginic acid or a salt thereof,
an absorbent, a colorant, a flavor, a sweetener, etc. The tablet
may be prepared by commonly employed mixing, granulation or coating
methods.
[0151] Formulations for parenteral administration may include, for
example, an injection, a drop, an ointment, a lotion, a gel, a
cream, a spray, a suspension, an emulsion, a suppository, a patch,
etc., although not being limited thereto.
[0152] The pharmaceutical composition according to an aspect of the
present disclosure may be administered parenterally, e.g.,
rectally, topically, transdermally, subcutaneously, etc. The
composition is prepared to be suitable for each formulation. In
particular, a composition for intravenous injection is prepared
with very high purity by excluding any unsuitable additive.
[0153] An administration dosage of the active ingredient will vary
depending on the age, gender and body weight of a subject to be
treated, the particular disease or pathological condition to be
treated, administration route or the discretion of a diagnoser. The
determination of the administration dosage considering these
factors is within the level of those skilled in the art.
Specifically, a general administration dose is from 30 .mu.g/mL to
1 mg/mL, although not being limited thereto.
[0154] Specifically, in an aspect of the present disclosure, a
pharmaceutical composition containing the pluripotent stem cells
prepared by the method according to the present disclosure may be
used as an injection. The pluripotent stem cells prepared by the
method according to the present disclosure may be injected into the
skin similarly to Botox, which is used to remove wrinkles, to
activate skin stem cells and promote proliferation of skin cells,
thereby resulting in skin generation, anti-aging, improvement of
skin elasticity and improvement of wrinkles.
[0155] In another aspect, the present disclosure provides a reagent
or medium composition for use in experiments, which contains the
shikimic acid or the plant extract, the plant stem cell extract or
the pluripotent stem cells containing shikimic acid according to an
aspect of the present disclosure.
[0156] Hereinafter, the present disclosure will be described in
detail through examples. However, the following examples are for
illustrative purposes only and it will be apparent to those of
ordinary skill in the art that the scope of the present disclosure
is not limited by the examples.
Example 1
Injection of Plant Cell Extract into Adult-Derived Cells
[0157] A callus powder was used as a plant-derived stem cell
extract. The callus powder that can be used in the present
disclosure is not particularly limited and a commercially available
one may also be used.
[0158] A sequoia callus extract is obtained by inducing a callus
from the leaf of sequoia, establishing a stem cell line by
culturing the callus in a solid medium and producing active
ingredients in large quantities through suspension culturing and
then extracting the same. The extraction of the active ingredients
from the sequoia callus can be performed by culturing of the cell
line derived from the tissue explant of the plant as described in
Korean Patent Publication No. 2007-0113193. Specifically, a
stabilized plant cell line derived from sequoia may be extracted by
dissolving in a mixture of a C.sub.5 or lower alcohol, although not
being limited thereto.
[0159] In this example, a sequoia callus powder commercially
purchased from BIO-FD&C was used. Specifically, human dermal
fibroblasts were separated into individual cells using trypsin-EDTA
and then washed with cold PBS (phosphate buffered saline). The
resulting cell pellets were resuspended in cold Ca.sup.2+- and
Mg.sup.2+-free HBSS (Hank's balanced salt solution) with 100,000
cells/100 .mu.L and then transferred to a 1.5-mL tube. After
centrifuging at 120 g for 5 minutes at 4.degree. C. using a
horizontal swing-out rotor, the supernatant was discarded and the
remaining cell pellets resuspended again in 97.7 .mu.L of cold HBSS
and incubated in a water bath at 37.degree. C. for 2 minutes. Then,
2.3 .mu.L of streptolysin O (SLO, 100 g/mL stock solution diluted
1:10 in cold HBSS) was added (final SLO concentration: 230 ng/mL).
Alternatively, digitonin (20 .mu.g/mL) and 200 .mu.L of a transport
solution (110 mM potassium acetate, 5 mM sodium acetate, 2 mM
magnesium acetate, 1 mM EGTA, 2 mM DTT, protease inhibitor
cocktail, 20 mM HEPES, pH 7.3) may be added instead of the SLO. The
sample was incubated in a water bath at 37.degree. C. for 50
minutes while mixing up and down with 10-minute intervals. After
the incubation, the sample was placed on ice and centrifugation was
carried out at 120 g for 5 minutes at 4.degree. C. using a
horizontal swing-out rotor after adding 200 .mu.L of cold HBSS.
After this permeabilization process, the resulting cell pellets
were resuspended in 200 .mu.L of a plant stem cell extract with
1000 cells/1 .mu.L. A callus powder was used as the plant stem cell
extract (500 .mu.g/mL). Then, after adding 1 mM nucleotide
triphosphate and ATP regeneration system (10 mM creatine phosphate
and 25 g/mL creatine kinase) to the suspended cell pellets,
incubation was conducted in a water bath at 37.degree. C. for 1
hour while mixing up and down with 10-minute intervals. After the
incubation, 1 mL of an ES medium containing 2 mM CaCl.sub.2 was
added and incubation was conducted in a 37.degree. C. incubator for
2 hours for reconstitution of the plasma membrane. After washing
with PBS, the cell pellets were resuspended in an embryonic stem
cell culture medium and then seeded onto a dish coated with 0.1%
gelatin.
Example 2
Preparation of Pluripotent Cells Such as Embryonic Stem Cells by
Culturing Extract-Injected Cells
[0160] The extract-injected cells were incubated in a normal cell
culture medium wherein DMEM (Dulbecco's modified Eagle's medium)
was supplemented with 10% FBS, 50 U/mL penicillin and 50 mg/mL
streptomycin in an incubator maintained at 37.degree. C. and 5%
CO.sub.2. The adult-derived cells (human-derived dermal
fibroblasts) into which the plant stem cell extract (callus powder)
was injected were cultured on a dish coated with 0.1% gelatin. The
medium was replaced after the first two days. After culturing for
10 days while replacing the medium every day, the cells were
transferred to a feeder cell (STO cell) layer treated with
mitomycin C (MMC) at a ratio of 1:2. Then, the cells were
transferred to a new feeder cell layer with 7-day intervals while
replacing DMEM (Dulbecco's modified Eagle's medium)/F12
supplemented with 20% KSR (knockout serum replacement), 2 mM
L-glutamine, 0.1 mM nonessential amino acids, 0.1 mM
3-mercaptoethanol, 50 U/mL penicillin, 50 mg/mL streptomycin and 10
.mu.g/mL bFGF every day. It took about 21 days on average to
culture induced stem cells necessary for analysis.
[0161] FIG. 1 schematically describes an experimental procedure of
inducing pluripotent stem cells according to the method of the
present disclosure. FIGS. 2-4 show induced pluripotent stem cells
observed on days 5, 10 and 32 after culturing, respectively. And,
FIGS. 5 and 6 show a result of alkaline phosphatase staining on
days 32 and 50 after culturing, respectively. The alkaline
phosphatase staining was carried out using a commonly used staining
kit.
[0162] As seen from FIG. 6, the pluripotent stem cells induced
according to the method of the present disclosure showed a positive
result (violet) for the alkaline phosphatase staining, which is
characteristic of embryonic stem cells.
Example 3
Characterization of Induced Pluripotent Stem Cells (Gene Expression
Analysis)
[0163] The cultured cells were recovered and total RNA was
separated by using the TRIzol reagent (Invitrogen). After
synthesizing cDNA through reverse transcription polymerase chain
reaction (RT-PCR), PCR was conducted using primers specific for the
Nanog and Oct3/4 genes and the GAPDH gene as a control gene. The
expression of these genes was analyzed by electrophoresing the PCR
product on an agarose gel. The result is shown in FIG. 7.
[0164] As seen from FIG. 7, the pluripotent stem cells (hiPS)
induced by the method of the present disclosure showed expression
of the Nanog and Oct3/4 genes, which are characteristic of
embryonic stem cells (hES).
Example 4
Preparation of Sequoia (Sequoiadendron giganteum) Callus Extract
Containing Shikimic Acid
[0165] 20 mg of the sequoia callus powder of Example 1 was
dissolved in 1 mL of a DMSO solvent. Similarly, 1 g of the sequoia
callus powder was dissolved in 10 mL of a mixture solvent of BG and
EtOH to prepare a sequoia callus extract containing shikimic acid.
The prepared extracts were used as samples in the following test
example.
Test Example 1
Compositional Analysis of Sequoia Callus Extract
[0166] Of the sequoia extracts prepared in Example 4, the sequoia
callus extract prepared using the mixture solvent of BG and EtOH
was subjected to compositional analysis by HPLC.
[0167] The compositional analysis of the extract was performed
using the Waters 1525.mu. Binary HPLC pump and the Gemini 5u C18
110 A column (5 .mu.m, 4.6 mm.times.250 nm, Phenomenex). Solvent A
(water, containing 0.1% TFA) and solvent B (acetonitrile,
containing 0.1% TFA) were used as mobile phases and a 230-nm UV
lamp was used. Measurement was carried out with a flow rate of 1
mL/min, a run time of 46 minutes and an extract injection volume of
20 .mu.L.
[0168] The LC spectrum of the sequoia extract is shown in FIG. 8
and the analysis result is given in Table 1. In Table 1, % Area
denotes the percentage (%, w/v) of the substance contained the
sequoia extract. It can be seen that the sequoia callus extract
contains shikimic acid in a larger amount than any other substance.
It can be also seen that the sequoia callus extract contains, in
addition to the shikimic acid, caffeic acid and ferulic acid.
TABLE-US-00001 TABLE 1 RT Area % Area Height 1 3.589 2413779 7.91
122548 2 (shikimic acid) 4.248 9990137 32.75 633894 3 17.115
2660895 8.72 146243 4 (caffeic acid) 18.666 7252283 23.78 363051 5
(ferulic acid) 21.672 5225473 17.13 320196 6 22.411 2775302 9.10
159198 7 28.044 183049 0.60 15075
Test Example 2-1
Increase of Oct 3/4 Expression
[0169] 5.times.10.sup.5 neonatal human dermal fibroblasts
(NHDF-Neonatal) (CC-2509, Lonza, USA) were treated with a
permeabilization buffer and 10 .mu.g/mL digitonin and then with the
20 .mu.g/mL sequoia callus extract (mixture solvent of BG and EtOH)
of Example 4 or 5 .mu.g/mL shikimic acid, respectively. Untreated
NHDF-Neonatal were used as a negative control group. On day 3 after
the treatment, the cells (5,000 cells) were attached on a 4-chamber
slide. Next day, i.e., on day 4, the cells were fixed with 3.8%
formaldehyde in PBS (diluted from 38% paraformaldehyde) and kept at
room temperature for 10 minutes. Then, 400.times. images were
obtained using the Carl Zeiss Confocal microscope LSM510 by
immunocytochemistry using Oct3/4 antibody (Genetex, GTX100622,
.times.200) and Alexa Fluor.RTM. 488 goat anti-rabbit IgG. The
obtained images are shown in FIG. 10. The ratio of Oct3/4-positive
cells was determined by analyzing the Carl Zeiss Confocal
microscopic images obtained from the Oct3/4 ICC experiment, for 4
or more images per sample, 5 on average. The result is shown in
Table 2.
TABLE-US-00002 TABLE 2 Number of Oct3/4-positive Number of total
Ratio of Oct3/4- Ratio of Oct3/4- Treated cells (per image) cells
(per image) positive cells positive cells (%) compound #1 #2 #3 #4
#5 #1 #2 #3 #4 #5 #1 #2 #3 #4 #5 Mean SD Negative control 0 0 0 0 0
11 6 17 20 13 0.0 0.0 0.0 0.0 0.0 0% 0% 20 .mu.g/mL sequoia 0 3 4 6
3 6 6 10 13 11 0.0 0.5 0.4 0.5 0.3 33% 5% calluss extract (BG +
EtOH) 5 .mu.g/mL shikimic 2 4 1 5 12 14 7 9 0.2 0.3 0.1 0.6 29% 5%
acid (water)
[0170] As can be seen from FIG. 10, the HDF treated with the
sequoia callus extract of Example 4 showed increased expression of
the Oct3/4 gene as compared to the normal HDF (negative control),
which was higher than those treated with shikimic acid.
[0171] Also, as can be seen from Table 2, the percentage of the
cells positive for the Oct3/4 gene was the highest as 33% for the
sequoia callus extract extracted with a mixture solvent of BG
(butylene glycol) and EtOH, which was higher than that for shikimic
acid.
Test Example 2-2
Increase of Oct 3/4 Expression
[0172] 1.times.10.sup.6 neonatal human dermal fibroblasts
(NHDF-Neonatal) (CC-2509, Lonza, USA) were treated with 10 .mu.M or
10 mM shikimic acid, respectively. Untreated NHDF-Neonatal were
used as a negative control group. On day 5 after the treatment, the
cells (5,000 cells) were attached on a 4-chamber slide. 3 days
later, i.e., on day 8, the cells were fixed with 3.8% formaldehyde
in PBS (diluted from 38% paraformaldehyde) and kept at room
temperature for 10 minutes. Then, 400.times. images were obtained
using the Carl Zeiss Confocal microscope LSM510 by
immunocytochemistry using Oct3/4 antibody (Genetex, GTX100622,
.times.200) and Alexa Fluor.RTM. 488 goat anti-rabbit IgG. The
obtained images are shown in FIG. 11. The ratio of Oct3/4-positive
cells was determined by analyzing the Carl Zeiss Confocal
microscopic images obtained from the Oct3/4 ICC experiment, for 4
or more images per sample, 5 on average. The result is shown in
Table 3.
TABLE-US-00003 TABLE 3 Number of Oct3/4-positive Number of total
Ratio of Oct3/4- Ratio of Oct3/4- cells (per image) cells (per
image) positive cells positive cells (%) #1 #2 #3 #4 #5 #1 #2 #3 #4
#5 #1 #2 #3 #4 #5 Mean SD Negative control 0 0 0 0 0 11 6 17 20 13
0.0 0.0 0.0 0.0 0.0 0% 0% Shikimic 10 .mu.M 18 4 10 14 24 54 27 33
53 60 0.33 0.15 0.30 0.26 0.40 29% 2% acid 10 mM 11 11 12 0 18 33
32 59 36 39 0.33 0.34 0.20 0.00 0.46 27% 4% (water)
[0173] As can be seen from FIG. 11, the HDF treated with shikimic
acid showed increased expression of the Oct3/4 gene as compared to
the untreated normal HDF. The Oct3/4 gene expression increased with
the concentration of shikimic acid.
[0174] Also, as can be seen from Table 3, the percentage of the
cells positive for the Oct3/4 gene was 27-29% on average when
treated with the shikimic acid in water. The value was
significantly higher as compared to the untreated group.
[0175] From the above results, it was confirmed that the shikimic
acid which is the main ingredient of the sequoia callus extract is
effective in increasing the expression of the Oct3/4 gene which
plays a critical role in inducing induced pluripotent stem cells.
Accordingly, by injecting the shikimic acid or the extract
containing the same according to the present disclosure into
somatic cells, the induction of induced pluripotent stem cells can
be induced through expression of the Oct3/4 gene.
Test Example 3-1
Increase of Expression of Stem Cell Marker Alkaline Phosphatase
(ALP)
[0176] 5.times.10.sup.5 NHDF-Neonatal (CC-2509, Lonza, USA) were
treated with a permeabilization buffer and 10 .mu.g/mL digitonin
and then with the 20 .mu.g/mL sequoia callus extract (mixture
solvent of BG and EtOH) of Example 4 or 5 .mu.g/mL shikimic acid,
respectively. Untreated NHDF-Neonatal were used as a negative
control group. On day 6 after the treatment, the cells (5,000
cells) were attached on a 12-well plate. 5 days later, i.e., on day
11, the cells were fixed with 3.8% formaldehyde in PBS and kept at
room temperature for 15 minutes. Then, the cells were treated with
200 .mu.L of the NBT/BCIP.RTM. ALP substrate solution diluted in 10
mL of ALP buffer, with 0.5 mL each time. 20 hours later, the cells
were observed under the Olympus CKX41 optical microscope at
40.times. magnification. The result is shown in FIG. 12.
[0177] As can be seen from FIG. 12, the HDF treated with the
sequoia callus extract showed a larger stained area than the normal
HDF, suggesting that the expression of ALP was increased
remarkably. Also, it can be seen that the cells treated with the
sequoia callus extract showed higher ALP expression than those
treated with the shikimic acid.
[0178] During the process in which fibroblasts are converted to
induced pluripotent stem cells, ALP is begin to be expressed 3 day
after the genes such as Oct4 are expressed in the cells. It is a
marker known to play an important role in the early stage of
formation of dedifferentiated stem cells. Therefore, when the
sequoia callus extract according to the present disclosure is
injected into somatic cells, the expression of ALP increases
remarkably and the Oct4 gene is expressed. Accordingly, induced
pluripotent stem cells can be induced.
[0179] From this experimental result, it was confirmed that the
sequoia callus extract according to the present disclosure exhibits
a remarkable effect of activating stem cells by promoting the
expression of the stem cell marker ALP.
Test Example 3-2
Increase of Expression of Stem Cell Marker Alkaline Phosphatase
(ALP)
[0180] 1.times.10.sup.6 NHDF-Neonatal (CC-2509, Lonza, USA) were
treated with 10 .mu.M or 10 mM shikimic acid, respectively.
Untreated NHDF-Neonatal were used as a negative control group. On
day 5 after the treatment, the cells (100,000 cells) were attached
on a 6-well plate. 7 days later, i.e., on day 12, the cells were
fixed with 3.8% formaldehyde in PBS and kept at room temperature
for 15 minutes. Then, the cells were treated with 200 .mu.L of the
NBT/BCIP.RTM. ALP substrate solution diluted in 10 mL of ALP
buffer, with 0.5 mL each time. 20 hours later, the cells were
observed under the Olympus CKX41 optical microscope at 40.times.
magnification. The result is shown in FIG. 13.
[0181] As can be seen from FIG. 13, the HDF treated with the
shikimic acid showed a larger area stained deep blue than the
normal HDF, suggesting that the expression of ALP was increased
remarkably. Also, it can be seen that the cells treated with the
shikimic acid showed higher ALP expression than those treated with
the shikimic acid.
[0182] During the process in which fibroblasts are converted to
induced pluripotent stem cells, ALP is begin to be expressed 3 day
after the genes such as Oct4 are expressed in the cells. It is a
marker known to play an important role in the early stage of
formation of dedifferentiated stem cells. Therefore, when the
shikimic acid and a plant extract containing the same according to
the present disclosure is injected into somatic cells, the
expression of ALP increases remarkably and the Oct4 gene is
expressed. Accordingly, induced pluripotent stem cells can be
induced.
[0183] From this experimental result, it was confirmed that the
shikimic acid and a plant extract containing the same according to
the present disclosure exhibits a remarkable effect of activating
stem cells by promoting the expression of the stem cell marker
ALP.
Test Example 4-1
Increase of Colony-Forming Ability of Dermal Cells
[0184] 5.times.10.sup.5 NHDF-Neonatal (CC-2509, Lonza, USA) were
treated with a permeabilization buffer and 10 .mu.g/mL digitonin
and then with the 100 ppm or 200 ppm sequoia callus extract
(mixture solvent of BG and EtOH) of Example 4, which had been
filtered through a 0.4-.mu.m filter, and a 20 ppm DMSO solution.
Untreated NHDF-Neonatal were used as a negative control group. On
day 10 after subculturing, the cells (200 cells) were attached on a
60-mm plate. On day 23, the cells were washed with ice-cold PBS and
then fixed with ice-cold methanol kept at -20.degree. C. for 10
minutes. The cells were stained by treating for 5-10 minutes with a
working solution, which had been prepared by diluting 1% crystal
violet in an ethanol stock solution 1/10 with PBS, washed 4 times
with PBS and then imaged. For quantitative analysis, the cells were
eluted by treating with an elution buffer consisting of 50%
ethanol, 40% DW, and 10% acetic acid for 5 minutes. Then,
absorbance was measured at 580 nm after transferring 200 .mu.L of
the cells to a 96-well plate. The obtained images are shown in FIG.
14. And, the result of absorbance measurement relative to the
negative control group is shown in FIG. 16.
[0185] As can be seen from FIG. 14, the cells treated with the
sequoia callus extract showed a larger stained area than the
negative control group NHDF-Neonatal, suggesting that the dermal
cells differentiated actively and formed a large colony.
[0186] Also, as can be seen from FIG. 16, the cells treated with
the sequoia callus extract showed about 2.6 times increased
colony-forming ability as compared to the negative control group,
which is statistically significant. The cells treated with the
shikimic acid showed increase of about 3.8 times as compared to the
negative control group.
[0187] Accordingly, it was confirmed that the sequoia callus
extract according to the present disclosure remarkably promotes the
proliferation of fibroblasts.
[0188] Also, it was confirmed that the sequoia extract according to
the present disclosure promotes skin regeneration by remarkably
promoting the proliferation of fibroblasts.
Test Example 4-2
Increase of Colony-Forming Ability of Dermal Cells
[0189] 1.times.10.sup.6 NHDF-Neonatal (CC-2509, Lonza, USA) were
treated with 10 .mu.M, 50 .mu.M, 100 .mu.M, 1 mM or 10 mM shikimic
acid, respectively. Untreated NHDF-Neonatal were used as a negative
control group. On day 5 after subculturing, the cells (200 cells)
were attached on a 60-mm plate. On day 17, the cells were washed
with ice-cold PBS and then fixed with ice-cold methanol kept at
-20.degree. C. for 10 minutes. The cells were stained by treating
for 5-10 minutes with a working solution, which had been prepared
by diluting 1% crystal violet in an ethanol stock solution 1/10
with PBS, washed 4 times with PBS and then imaged. For quantitative
analysis, the cells were eluted by treating with an elution buffer
consisting of 50% ethanol, 40% DW, and 10% acetic acid for 5
minutes. Then, absorbance was measured at 580 nm after transferring
200 .mu.L of the cells to a 96-well plate. The obtained images are
shown in FIG. 15. And, the result of absorbance measurement
relative to the negative control group is shown in FIG. 17.
[0190] As can be seen from FIG. 15, the cells treated with the
shikimic acid showed a larger stained area than the negative
control group NHDF-Neonatal, suggesting that the dermal cells
differentiated actively and formed a large colony.
[0191] Also, as can be seen from FIG. 17, the cells treated with
the shikimic acid showed about 1.27-5.06 times increased
colony-forming ability as compared to the negative control group,
which is statistically significant. In particular, the cells
treated with 1 mM shikimic acid showed the most increase in
colony-forming ability. Therefore, it can be seen that treatment
with 1 mM shikimic acid leads to the most promotion of cellular
proliferation.
[0192] Accordingly, it was confirmed that the shikimic acid
according to the present disclosure remarkably promotes the
proliferation of fibroblasts.
[0193] Also, it was confirmed that the shikimic acid according to
the present disclosure promotes skin regeneration by remarkably
promoting the proliferation of fibroblasts.
Test Example 5
Increase of Proliferating Ability of Dermal Cells
[0194] 2,000 NHDF-Neonatal (CC-2509, Lonza, USA) were attached on a
96-well plate. The next day, the cells were treated with the 20
.mu.g/mL sequoia callus extract (mixture solvent of BG and EtOH) of
Example 4 and 5 .mu.g/mL shikimic acid. Untreated NHDF-Neonatal
were used as a negative control group. Then, we were subculturing
it for 7 days (as confluency becomes to 90% or below on 7.sup.th
day). After 6 days later, 10 .mu.L of WST-1 was treated and
absorbance was measured at 450 nm. The result relative to the
negative control group is shown in FIG. 18.
[0195] As can be seen from FIG. 18, treatment with the sequoia
callus extract according to the present disclosure resulted in
about 1.5 times or more increased cell division as compared to the
negative control group, which is statistically significant. And,
the shikimic acid resulted in 2 times increased cell division as
compared to the normal control group.
[0196] Accordingly, it was confirmed that the sequoia callus
extract according to the present disclosure promotes the cell
division of fibroblasts remarkably.
[0197] Also, it was confirmed that the sequoia extract according to
the present disclosure promotes skin regeneration by remarkably
promoting the cell division of fibroblasts.
[0198] The foregoing description of the present disclosure is for
the purpose of illustration only and those of ordinary skill in the
art to which the present disclosure belongs will understand that
the changes can be easily made thereto without departing from the
technical spirit and scope of this disclosure. Therefore, the
above-described examples should be interpreted as being
exemplary.
[0199] Hereinafter, the formulation examples of the composition
will be described. However, the following formulation examples are
for illustrative purposes only.
Formulation Example 1
Soft Capsule
[0200] 40 .mu.g of shikimic acid or the sequoia callus extract of
Example 4, 9 mg of vitamin E, 9 mg of vitamin C, 2 mg of palm oil,
8 mg of hydrogenated vegetable oil, 4 mg of yellow beeswax and 9 mg
of lecithin were mixed according to a commonly employed method, and
400 mg of the mixture was filled per capsule. Separately from this,
a soft capsule sheet was prepared from 66 parts by weight of
gelatin, 24 parts by weight of glycerin and 10 parts by weight of a
sorbitol solution and the mixture was filled therein to prepare a
soft capsule in which 400 mg of the composition according to the
present disclosure is contained.
Formulation Example 2
Tablet
[0201] 40 .mu.g of shikimic acid or the sequoia callus extract of
Example 4, 9 mg of vitamin E, 9 mg of vitamin C, 200 mg of
galactooligosaccharide, 60 mg of lactose and 140 mg of maltose were
mixed and granulated using a fluidized-bed dryer. After adding 6 mg
of sugar ester, 500 mg of the resulting composition was prepared
into a tablet according to a commonly employed method.
Formulation Example 3
Drink
[0202] 40 .mu.g of shikimic acid or the sequoia callus extract of
Example 4, 9 mg of vitamin E, 9 mg of vitamin C, 10 g of glucose,
0.6 g of citric acid and 25 g of oligosaccharide syrup were mixed.
After adding 300 mL of purified water, 200 mL of the mixture was
filled per bottle and sterilized at 130.degree. C. for 4-5
seconds.
Formulation Example 4
Granule
[0203] 40 .mu.g of shikimic acid or the sequoia callus extract of
Example 4, 9 mg of vitamin E, 9 mg of vitamin C, 250 mg of
anhydrous crystalline glucose and 550 mg of starch were mixed,
granulated using a fluidized-bed granulator and then filled in a
pouch.
Formulation Example 5
Injection
[0204] An injection (for 2-mL ampoule) was prepared according to a
commonly employed method as described in Table 4.
TABLE-US-00004 TABLE 4 Ingredients Contents Shikimic acid or
sequoia callus extract of Example 4 40 .mu.g Sterile distilled
water for injection adequate pH control agent adequate
Formulation Example 6
Softening Lotion (Skin Lotion)
[0205] A softening lotion was prepared according to a commonly
employed method as described in Table 5.
TABLE-US-00005 TABLE 5 Ingredients Contents (wt %) Shikimic acid or
sequoia callus extract of Example 4 0.2 Glycerin 3.0 Butylene
glycol 2.0 Propylene glycol 2.0 Carboxyvinyl polymer 0.1 PEG-12
nonyl phenyl ether 0.2 Polysorbate 80 0.4 Ethanol 10.0
Triethanolamine 0.1 Preservative, pigment and fragrance adequate
Purified water balance
Formulation Example 7
Nourishing Lotion (Milk Lotion)
[0206] A nourishing lotion was prepared according to a commonly
employed method as described in Table 6.
TABLE-US-00006 TABLE 6 Ingredients Contents (wt %) Shikimic acid or
sequoia callus extract of Example 4 1.0 Glycerin 3.0 Butylene
glycol 3.0 Propylene glycol 3.0 Carboxyvinyl polymer 0.1 Beeswax
4.0 Polysorbate 60 1.5 Caprylic/capric triglyceride 5.0 Squalane
5.0 Sorbitan sesquioleate 1.5 Liquid paraffin 0.5 Cetearyl alcohol
1.0 Triethanolamine 0.2 Preservative, pigment and fragrance
adequate Purified water balance
Formulation Example 8
Nourishing Cream
[0207] A nourishing cream was prepared according to a commonly
employed method as described in Table 7.
TABLE-US-00007 TABLE 7 Ingredients Contents (wt %) Shikimic acid or
sequoia callus extract of Example 4 2.0 Glycerin 3.0 Butylene
glycol 3.0 Liquid paraffin 7.0 Beta-glucan 7.0 Carbomer 0.1
Caprylic/capric triglyceride 3.0 Squalane 5.0 Cetearyl glucoside
1.5 Sorbitan stearate 0.4 Polysorbate 60 1.2 Triethanolamine 0.1
Preservative, pigment and fragrance adequate Purified water
balance
Formulation Example 9
Massage Cream
[0208] A massage cream was prepared according to a commonly
employed method as described in Table 8.
TABLE-US-00008 TABLE 8 Ingredients Contents (wt %) Shikimic acid or
sequoia callus extract of Example 4 2.0 Glycerin 8.0 Butylene
glycol 4.0 Liquid paraffin 45.0 Beta-glucan 7.0 Carbomer 0.1
Caprylic/capric triglyceride 3.0 Beeswax 4.0 Cetearyl glucoside 1.5
Sorbitan sesquioleate 0.9 Vaseline 3.0 Paraffin 1.5 Preservative,
pigment and fragrance adequate Purified water balance
Formulation Example 10
Pack
[0209] A pack was prepared according to a commonly employed method
as described in Table 9.
TABLE-US-00009 TABLE 9 Ingredients Contents (wt %) Shikimic acid or
sequoia callus extract of Example 4 0.2 Glycerin 4.0 Polyvinyl
alcohol 15.0 Hyaluronic acid extract 5.0 Beta-glucan 7.0 Allantoin
0.1 Nonyl phenyl ether 0.4 Polysorbate 60 1.2 Ethanol 6.0
Preservative, pigment and fragrance adequate Purified water
balance
Formulation Example 11
Health Food
[0210] A health food was prepared according to a commonly employed
method as described in Table 10.
TABLE-US-00010 TABLE 10 Ingredients Contents Shikimic acid or
sequoia callus extract of Example 4 20 .mu.g Vitamin A acetate 70
.mu.g Vitamin E 1.0 mg Vitamin B.sub.1 0.13 mg Vitamin B.sub.2 0.15
mg Vitamin B.sub.6 0.5 mg Vitamin B.sub.12 0.2 .mu.g Vitamin C 10
mg Biotin 10 .mu.g Nicotinamide 1.7 mg Folic acid 50 .mu.g Calcium
pantothenate 0.5 mg Ferrous sulfate 1.75 mg Zinc oxide 0.82 mg
Magnesium carbonate 25.3 mg Monobasic potassium phosphate 15 mg
Dibasic calcium phosphate 55 mg Potassium citrate 90 mg Calcium
carbonate 100 mg Magnesium chloride 24.8 mg
[0211] The above-described composition of the mixture of vitamins
and minerals is only exemplary and may be changed as desired.
Formulation Example 12
Health Drink
[0212] A health drink was prepared according to a commonly employed
method as described in Table 11.
TABLE-US-00011 TABLE 11 Ingredients Contents Shikimic acid or
sequoia callus extract of Example 4 20 .mu.g Citric acid 1000 mg
Oligosaccharide 100 g Taurine 1 g Purified water balance
[0213] According to a commonly employed health drink preparation
method, the above ingredients were mixed and heated at 85.degree.
C. for about 1 hour under stirring. The resulting solution was
filtered and sterilized.
Formulation Example 13
Injection Containing Pluripotent Stem Cells
[0214] An injection containing pluripotent stem cells was prepared
according to a commonly employed method as described in Table
12.
TABLE-US-00012 TABLE 12 Ingredients Contents Pluripotent stem cells
of Example 2 40 .mu.g Sterile distilled water for injection
adequate pH control agent adequate
[0215] While the exemplary embodiments have been shown and
described, it will be understood by those skilled in the art that
various changes in form and details may be made thereto without
departing from the spirit and scope of this disclosure as defined
by the appended claims.
* * * * *