U.S. patent application number 14/888636 was filed with the patent office on 2016-04-14 for composition containing palmultang extract for promoting proliferation of stem cells derived from bone marrow.
This patent application is currently assigned to COMPREHENSIVE AND INTEGRATIVE MEDICINE INSTITUTE. The applicant listed for this patent is COMPREHENSIVE AND INTEGRATIVE MEDICINE INSTITUTE. Invention is credited to Joon Seok BYUN, Sang Gyung KIM, Seung Mo KIM, Sae Kwang KU, Chang Hyeong LEE, Im Hee SHIN, Ki Cheul SOHN.
Application Number | 20160101156 14/888636 |
Document ID | / |
Family ID | 50145222 |
Filed Date | 2016-04-14 |
United States Patent
Application |
20160101156 |
Kind Code |
A1 |
LEE; Chang Hyeong ; et
al. |
April 14, 2016 |
COMPOSITION CONTAINING PALMULTANG EXTRACT FOR PROMOTING
PROLIFERATION OF STEM CELLS DERIVED FROM BONE MARROW
Abstract
The present invention relates to a composition for promoting the
proliferation of stem cells derived from bone marrow using a
palmultang extract, and more specifically, to a composition for
promoting the proliferation of stern cells derived from bone marrow
by administering a granulocyte colony-stimulating factor to a
subject and then administering the palmultang extract to the
subject. The composition of the present invention remarkably
reduces side effects, such as enlargement of the spleen, which are
caused by the administration of G-CSF alone for proliferation and
differentiation of the stern cells, through administration in
combination with the palmultang extract, thereby further promoting
the proliferation and differentiation of stem cells.
Inventors: |
LEE; Chang Hyeong; (Daegu,
KR) ; KIM; Sang Gyung; (Daegu, KR) ; SHIN; Im
Hee; (Daegu, KR) ; KIM; Seung Mo; (Daegu,
KR) ; BYUN; Joon Seok; (Daegu, KR) ; SOHN; Ki
Cheul; (Daegu, KR) ; KU; Sae Kwang; (Daegu,
KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
COMPREHENSIVE AND INTEGRATIVE MEDICINE INSTITUTE |
Daegu |
|
KR |
|
|
Assignee: |
COMPREHENSIVE AND INTEGRATIVE
MEDICINE INSTITUTE
Daegu
KR
|
Family ID: |
50145222 |
Appl. No.: |
14/888636 |
Filed: |
May 1, 2014 |
PCT Filed: |
May 1, 2014 |
PCT NO: |
PCT/KR2014/003909 |
371 Date: |
November 2, 2015 |
Current U.S.
Class: |
424/85.1 |
Current CPC
Class: |
A61K 36/258 20130101;
A61K 36/07 20130101; A61K 36/232 20130101; A61K 38/193 20130101;
A61K 36/804 20130101; A61K 36/64 20130101; A61K 38/193 20130101;
C12N 5/0647 20130101; A61K 36/258 20130101; A61K 36/076 20130101;
A61K 36/234 20130101; A61K 36/484 20130101; A61K 36/232 20130101;
A61K 36/284 20130101; A61K 36/076 20130101; A61K 2300/00 20130101;
A61K 36/484 20130101; A61K 2300/00 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101; A61K 2300/00 20130101; A61K 36/65 20130101; A61K 2300/00
20130101; A61K 36/65 20130101; C12N 2500/76 20130101; A61K 36/234
20130101; A61K 36/284 20130101; A61K 2300/00 20130101; A61K 36/804
20130101 |
International
Class: |
A61K 38/19 20060101
A61K038/19; A61K 36/284 20060101 A61K036/284; A61K 36/484 20060101
A61K036/484; A61K 36/232 20060101 A61K036/232; A61K 36/64 20060101
A61K036/64; A61K 36/65 20060101 A61K036/65; A61K 36/234 20060101
A61K036/234; A61K 36/258 20060101 A61K036/258; A61K 36/07 20060101
A61K036/07 |
Foreign Application Data
Date |
Code |
Application Number |
May 3, 2013 |
KR |
10-2013-0050323 |
Claims
1. A pharmaceutical composition for promoting proliferation of stem
cells derived from bone marrow to treat a hematologic cancer,
lymphoma, or bone marrow failure, comprising a granulocyte
colony-stimulating factor and a palmultang extract.
2. The pharmaceutical composition of claim 1, wherein the
granulocyte colony-stimulating factor and the palmultang extract
are formulated by being mixed in advance, or are formulated
separately.
3. The pharmaceutical composition of claim 1, wherein the
stimulating factor and the palmultang extract are administered
parenterally, orally, locoregionally, or percutaneously.
4. The pharmaceutical composition of claim 1, wherein
administration of the palmultang extract begins within 30 minutes
after administration of the stimulating factor.
Description
TECHNICAL FIELD
[0001] The present invention relates to a composition for promoting
proliferation of stem cells derived from bone marrow including a
pain hang extract.
BACKGROUND ART
[0002] "Stem cells" generally refers to undifferentiated cells that
can differentiate into various cells constituting biological
tissues and can be obtained from individual tissues of embryos,
fetuses, and adults in a cell stage prior to cell differentiation.
Among various stem cells, blood adult stern cells are stern cells
that are derived from bone marrow and may have totipotency to
differentiate into all type of cells constituting organs and blood
of human bodies.
[0003] Among these, bone-marrow-derived stern cells are considered
as an ultimate tool fir treating diseases such as hematologic
cancers, lymphoma, and bone marrow failure. In recent years,
bone-marrow-derived stem cells have been transplanted for various
purposes. In particular, when it is difficult to transplant organs
into patients suffering from terminal kidney and liver diseases,
attempts have been made to promote regeneration of the liver and
kidneys by autologous transplantation of bone-marrow-derived stern
cells.
[0004] To promote cell differentiation and division after the
autologous transplantation of bone-marrow-derived stem cells, a
human recombinant granulocyte colony-stimulating factor (hG-CSF) is
administered in a full dose. However, the proliferation of stem
cells and the mobilization into blood do not occur in approximately
5 to 30% of patients, and various side effects such as cardiac
infarction, cerebral infarction, pyrexia, ostalgia, splenomegaly,
and ruptures are known to he caused by administration of hG-CSF
(Masood et al,, 2008; Fox et al., 2009). In particular, the use of
hG-CSF is limited. Therefore, a new alternative to enhance an
effect of hG-CSF on proliferation of bone-marrow-derived stem cells
and reduce such side effects is needed.
[0005] Meanwhile, palmultang is a representative qi-strengthening
medicine widely used in the field of Oriental medicine. Literally,
palmultang refers to a recipe including eight medicinal herbs.
Sagunjatang is known as a qi-strengthening medicine and a samultang
is known as a blood-nourishing medicine, and palmultang is a recipe
obtained by combination of these two medicines. Sagunjatang
includes the four medicinal herbs Panax ginseng, Atractylodes
ovata, Glycyrrhiza uralensis, and Wolfiporia extensa, and samultang
includes the four medicinal herbs Rehmannia glutinous, Paeonia
lactiflora, Cnidium officinale, and Angelica gigas. Sagunjatang is
known to promote metabolic actions, enhance immune functions, and
improve blood-increasing activities and digestive absorption
functions, and samultang is known to be applied to weakness from a
disease.
DISCLOSURE
Technical Problem
[0006] Therefore, the present invention is designed to solve the
problems of the prior art, and therefore it is an object of the
present invention to provide a composition for promoting
proliferation of bone-marrow-derived stem cells using a palmultang
extract.
[0007] However, the technical objects of the present invention are
not limited thereto, and other objects of the present invention
which are not disclosed herein will become more apparent to those
of ordinary skill in the art by describing in detail exemplary
embodiments thereof
Technical Solution
[0008] To solve the above problems, according to an aspect of the
present invention, there is provided a composition for promoting
proliferation of bone-marrow-derived stem cells, which includes a
granulocyte colony-stimulating factor and a palmultang extract.
According to one exemplary embodiment of the present invention, the
stimulating factor and the palmultang extract may be formulated by
being mixed in advance, or may be formulated separately.
[0009] According to another exemplary embodiment of the present
invention, the stimulating factor and the palmultang extract may be
administered parenterally, orally, locoregionally, or
percutaneously.
[0010] According to still another exemplar, embodiment of the
present invention, administration of the palmultang extract may
begin within 30 minutes after administration of the stimulating
factor.
Advantageous Effects
[0011] The con position for promoting proliferation of stem cells,
which includes the palmultang extract provided in the present
invention as an active ingredient, includes a human recombinant
granulocyte colony-stimulating factor (hG-CSF) and the palmultang
extract, and thus can be useful in promoting proliferation and
differentiation of stem cells when the composition is administered
prior to transplantation of stem cells.
[0012] Also, the composition according to one exemplary embodiment
of the present invention can be used as a new alternative to solve
the technical problems of the prior art, for example, side effects
such as cardiac infarction, cerebral infarction, pyrexia, ostalgia,
splenomegaly, and rupture, when proliferation of stem cells is
induced by administering hG-CSF to promote proliferation and
differentiation of the stem cells.
DESCRIPTION OF DRAWINGS
[0013] FIG. 1 is a diagram showing a structure of hG-CSF used in
the present invention.
[0014] FIG. 2 is a diagram showing changes in body weights and
weight gains of mice when hG-CSF or a natural-substance-derived
candidate compound is administered.
[0015] FIG. 3 is a diagram showing the results obtained by
determining the sizes of spleens from the mice into which the
hG-CSF or the natural-substance-derived candidate compound is
administered (A: untreated, B: hG-CSF, C: palmultang+hG-CST,
samchulgeonbitang (SCGBT)+hG-CSF, E: Astragalus membranaceus Bunge
var. membranaceus+hG-CST, F: Capreolus capreolus ochracea
Thomas+hG-CSF, and G: Angelica gigas+hG-CSF).
[0016] FIGS. 4 and 5 are diagrams showing the results obtained by
observing CD34+ cells of the mice into which the hG-CSF or the
natural-substance-derived candidate compound is administered using
a fluorescence-activated cell sorter (FACS) (A: untreated, B:
hG-CSF, C: palmultang+hG-CSF, D: SCGBT+hG-CSF, E: Astragalus
membranaceus Bunge var. membranaceus+hG-CST, F: Capreolus capreolus
ochracea Thomas+hG-CST, and G: Angelica gigas+hG-CSF),
[0017] FIGS. 6 and 7 are diagrams showing the results obtained by
observing CD45+ cells of the mice into which the hG-CSF or the
natural-substance-derived candidate compound is administered using
a FACS (A: untreated, B: hG-CSF, C: palmultang+hG-CSF, D:
SCCBT+bG-CSF, E: Astragalus membranaceus Bunge var.
membranaceus+hG-GSF, F: Capreolus capreolus ochracea Thomas+hG-CSF,
and G: Angelica gigas+hG-CSF).
[0018] FIG. 8 is a diagram showing the results obtained by
determining the total thicknesses of spleens of the mice into which
the hG-CSF or the natural-substance-derived candidate compound is
administered, and nuclear cells in red and white pulp of the mice
per unit area (A to C: untreated, D to F: hG-CSF, G to I:
palmultang+hG-CSF, J to L: SCGBT+hG-CSF, M to O: Astragalus
membranaceus Bunge var. membranaceus+hG-CSF, P to R: Capreolus
capreolus ochracea Thomas+hG-CSF, and S to U: Angelica
gigas+hG-CST).
[0019] FIG. 9 is a diagram showing the results obtained by
observing increases in the numbers of CD34 immunoreactive cells in
the spleens and bone marrow of the mice into which the hG-CSF or
the natural-substance-derived candidate compound is administered (A
to B: untreated, C to): hG-CSF, F to F: palmultang+hG-CSF, G to H:
SCGBT+hG-CSF, I to J: Astragalus membranaceus Bunge var.
membranaceus+hG-CSF, K to L: Capreolus capreolus ochracea
Thomas+hG-CSF, and M to N: Angelica gigas+hG-CSF).
[0020] FIG. 10 is a diagram showing the results obtained by
observing increases in the numbers of CD45 immunoreactive cells in
the spleens and bone marrow of the mice into which the hG-CSF or
the natural-substance-derived candidate compound is administered (A
to D: untreated, E to H: hG-CSF, I to L: palmultang+hG-CSF, M to P:
SCGBT+hG-CSF, Q to T: Astragalus membranaceus Bunge var.
membranaceus+hG-CSF, U to X: Capreolus capreolus ochracea
Thomas+hG-CST, and Y to AB: Angelica gigas+hG-CSF).
BEST MODE
[0021] The present inventors have focused on one medicinal herb in
order to develop a composition capable of reducing various side
effects caused when a human recombinant granulocyte
colony-stimulating factor is administered to promote cell
differentiation and division after transplantation of
bone-marrow-derived stern cells and increasing production of the
bone-marrow-derived stem cells, and found that the medicinal herb
has an excellent effect of promoting proliferation and
differentiation of stem cells when the medicinal herb is added to
palmultang. Therefore, the present invention has been completed
based on these facts.
[0022] Therefore, according to an aspect of the present invention,
there is provided a composition for promoting proliferation of
bone-marrow-derived stem cells, which included a granulocyte
colony-stimulating factor and a palmultang extract.
[0023] The term "palmultang extract" used in the present invention
refers to an extract obtained by extracting eight medicinal herbs.
The eight medicinal herbs are Panax ginseng, Atractylodes ovata,
Glycyrrhiza uralensis, Woltiporia extensa, Rehmannia glutinosa,
Paeonia lactiflora, Cnidium officinale, and Angelica gigas.
[0024] According to one exemplary embodiment of the present
invention, the stimulating factor and the palmultang extract may be
formulated by being mixed in advance, or may be formulated
separately.
[0025] The palmultang extract may be administered within 30
minutes, preferably 15 minutes, and most preferably 5 minutes after
administration of the granulocyte colony-stimulating factor, but
the present invention is not limited thereto.
[0026] The granulocyte colony-stimulating factor used in the
present invention is characterized in that it is a human
recombinant granulocyte colony-stimulating factor, but the present
invention is not limited thereto.
[0027] The granulocyte colony-stimulating factor and the palmultang
extract may be administered parenterally, orally, locoregionally,
or percutaneously. Preferably, the palmultang extract may be
administered orally, but a route of administration may be properly
chosen by those skilled in the related art according to a condition
and body weight of a patient, the severity of a disease,
administration duration, etc.
[0028] In the present invention, the term `subject` refers to a
target requiring treatment of a disease, and, more particularly, to
a mammal such as a human or a non-human primate, a mouse, a rat, a
dog, a cat, a horse, and cattle.
[0029] According to another aspect of the present invention, there
is provided a pharmaceutical composition for enhancing production
of bone-marrow-derived stem cells, which includes a palmultang
extract.
[0030] The pharmaceutical composition according to one exemplary
embodiment of the present invention may include a pharmaceutically
acceptable carrier. The pharmaceutically acceptable carrier may
include a physiological saline solution, polyethylene glycol,
ethanol, vegetable oil, and isopropyl myristate, but the present
invention is not limited thereto.
[0031] According to one exemplary embodiment of the present
invention, a preferred dosage of the pharmaceutical composition may
vary according to a condition and body weight of a patient, the
severity of a disease, the form of a drug, and a route and time of
administration, but may be properly chosen by those skilled in the
related art. However, the pharmaceutical composition may be
preferably administered at a dose of 0.001 to 300 mg/kg of body
weight, and more preferably 0.01 to 200 mg/kg of body weight a
day.
[0032] The pharmaceutical composition according to one exemplary
embodiment of the present invention may be administered to a mammal
such as a rat, a mouse, livestock, and a human through various
routes of administration. Methods of administration are not
particularly limited. For example, the composition may be
administered orally, rectally, or by intravenous, intramuscular,
subcutaneous, cervical epidural, or intra-cerebroventricular
injection.
[0033] In the present invention, when the palmultang extract was
administered orally after administration of hG-CSF, it was revealed
that splenomegaly was relieved when the palmultang extract was
orally co-administered within 5 minutes (see Example 2), an
increase in weight of the spleen and splenomegaly findings by
proliferation of nuclear cells in red pulp was inhibited
significantly (p<0.01), no effect on overall proliferation of
granulocytes was observed (see Example 3), and proliferation and
mobilization of the bone-marrow-derived stem cells by hG-CSF were
significantly enhanced (p<0.01) (see Example 4).
[0034] Therefore, the composition for promoting proliferation of
bone-marrow-derived stem cells, which includes the palmultang
extract according to one exemplary embodiment of the present
invention, has effects of enhancing production of the
bone-marrow-derived stem cells and simultaneously reducing various
side effects caused when the hG-CSF is administered alone as known
in the related art.
MODE FOR INVENTION
[0035] Hereinafter, preferred embodiments are provided to aid in
understanding the present invention. However, it should be
understood that detailed description provided herein is merely
intended to provide a better understanding of the present
invention, but is not intended to limit the scope of the present
invention.
EXAMPLES
[0036] In these Examples, effects of palmultang, samchulgeonbitang
(also referred to as SCGBT), Astragalus membranaceus Bunge var.
membranaceus, capreolus capreolus ochracea Thomas, and Angelica
gigas on medicinal effects and side effects of hG-CSF, particularly
on mobilization and splenomegaly of stem cells, were evaluated
using Balb/c mice which are often used to mobilize stem cells by
means of hG-CSF.
Example 1
Preparation for experiment
[0037] 1-1. Preparation of Materials
[0038] hG-CSF was purchased from Life Technologies (Carlsbad,
Calif., USA to be used, and the structure of the hG-CSF is shown in
FIG. 1
[0039] Palmultang (also referred to as `PMT`) was purchased from
HanZung Pharmaceutical Co. Ltd. (Daejeon, Korea) to be used, and
the components and their amounts in the palmultang are listed in
the following Table 1.
TABLE-US-00001 TABLE 1 Herbs Scientific Names/Produce Region Amount
(g) Angelicae Gigantis Angelica gigas N. 2.46 Radix Atractylodis
Rhizoma Atractylodes ovata (Thunb.) DC. 2.46 Cnidii Rhizoma Cnidium
officinale Makino 2.26 Ginseng Radix Alba Panax ginseng C. A.
Meyer. 1.6 Glycyrrhizae Radix Glycyrrhiza uralensis Fisch 1.8
Hoelen Poria cocos Wolf 0.13 Paeoniae Radix Paeonia lactiflora
Pall. 1.8 Rehmanniae Radix Rehmannia glutinosa Liboschitz ex 3.33
Preparata Steudel Total 8 types 15.84
[0040] For comparison, SCGBT, Astragalus membranaceus Bunge var.
membranaceus, Capreolus capreolus ochracea Thomas, and Angelica
gigas were chosen as medicinal rials to compare their effects with
the effects of the palmultang. The components and amounts of the
medicinal herbs in the SCGBT are listed in the following Table
2.
TABLE-US-00002 TABLE 2 Scientific Names/ Amounts Herbs Produce
Region (g) Amomi Fructus Amomum xanthioides Wallich 0.16
Atractylodis Rhizoma Atractylodes ovata (Thunb.) DC. 0.98 Alba
Citri Unshii, Pericarpium Citrus unshiu S. Marcov. 0.85 Ginseng
Radix Alba Panax ginseng C. A. Meyer. 0.64 Glycyrrhizae Radix et
Glycyrrhiza uralensis Fisch 0.36 Rhizoma Hawthorn Fruit (Crataegi
Crataegus pinnatifida Bunge 0.93 Fructus) var. typica Schneider
Hordei Fructus Hordeum vulgare Linn. 0.34 Germiniatus Zizyphi
Fructus Zizyphus jujuba var. inermis 4.17 (Bunge) Rehder Magnoliae
Cortex Magnolia officinalis Rehder 0.21 et Wilson Massa Medicata
Triticum aestivum L. 0.67 Fermentata Paeoniae Radix Paeonia
lactiflora Pall. 0.54 Ponciri Fructus Poncirus trifoliata 0.58
Hoelen Poria cocos Wolf 0.05 Zingiberis Rhizoma Zingiber officinale
Roscoe 0.26 Crudus Total 14 types 10.74
[0041] 1-2. Preparation of Laboratory Animals
[0042] In this Example, Balb/c CrSlc mice (6-week-old females, SLC,
Shizuoka, Japan) were selected and used as laboratory animals.
Seven healthy SPF Balb/c mice were purchased, and acclimatized for
34 days. Thereafter, only the laboratory animals having uniform
body weights were chosen and divided into seven groups of ten mice,
and used for this experiment as listed in the following Table 3.
All the laboratory animals were fasted overnight for 18 hours, a
period of which spanned from a start date on which hG-CSF and
medicines were administered up to a final date of autopsy (drinking
water was freely supplied), and the subjects were identified using
picric acid.
TABLE-US-00003 TABLE 3 Test substances and dose Group Inducers
(mg/kg/day) Animal No. GCSF-2012-PD: Effects on hG-CSF-treated mice
Control Saline 10 ml/kg Distilled water oral 10 M01 to M10 ml/kg
[intact vehicle] Control hG-CSF 250 .mu.g/kg Distilled water oral
M11 to M20 10 ml/kg [hG-CSF] Active hG-CSF 250 .mu.g/kg PMT oral
(200 mg/kg) M21 to M30 [PMT] Active hG-CSF 250 .mu.g/kg SCGBT oral
(200 mg/kg) M31 to M40 [SCGBT] Active hG-CSF 250 .mu.g/kg AR oral
(200 mg/kg) [AR] M41 to M50 Active hG-CSF 250 .mu.g/kg CCP oral
(200 mg/kg) M51 to M60 [CCP] Active hG-CSF 250 .mu.g/kg AGR oral
(200 mg/kg) M61 to M70 [AGR]
[0043] 1-3. Methods of Administration
[0044] Administration of hG-CSF and Candidate Drugs
[0045] 250 .mu.g/kg of hG-CSF (Life Technologies, Carlsbad, Calif.,
USA) was continuously subcutaneously administered once a day for 6
days according to the previous methods (Verma et al., 1997;
Levesque et al., 2003) to promote proliferation of leukocytes and
mobilization of bone-marrow-derived stem cells. Thereafter, 200
mg/kg of each of palmultang, SCGBT, Astragalus membranaceus Bunge
var. membranaceus, Capreolus capreolus ochracea Thomas and Angelica
gigas extracts was orally administered within 5 minutes after
administration of hG-CSF. In this case, the administration was
performed once a day for 6 days. All the five
natural-substance-derived extracts were dissolved in sterile
distilled water, and then forcibly orally administered to the mice
at a dose of 10 ml/kg of body weight using a 1 mL syringe with a
metallic zoned needle, and 11G-CSF was dissolved in a physiological
saline solution, and then subcutaneously administered into
subcutaneous regions of the backs of the mice at a dose of 10
ml/kg, in the hG-CSF control, only an equivalent dose of sterile
distilled water was administered instead of the
natural-substance-derived extracts, and, in the normal medium
control, only equivalent doses of the physiological saline solution
and sterile water were subcutaneously and orally administered
instead of the hG-CSF and the natural-substance-derived extract at
intervals of 5 minutes. The dose used in this experiment, that is,
200 mg/kg of the natural-substance-derived extract, was chosen
based on the results obtained from each animal experience.
[0046] 1-4, Observation Items
[0047] Changes in body weights and spleen weights, and the number
of CD34+ and CD45+ cells as representative markers for labeling
bone-marrow-derived stem cells as well as the total number of bone
marrow nuclear cells and blood leukocytes in the bone marrow and
blood were determined using a fluorescence-activated cell sorting
(FACS) method. Also, the number of CD34+ and CD45+ cells per unit
area in tissue samples from the spleen and bone marrow was
determined using an immunohistochemical method, and the total
thickness of the spleen, the amount and diameters of white pulp,
and the number of nuclear cells in the spleen red pulp and bone
marrow per unit area were also evaluated using an automated image
analyzer (iSolution FL ver 9.1, IMT i-solution Inc,, Quebec,
Canada), To observe clearer changes, the changes (%) in the normal
medium control and the hG-CSF control, and the respective changes
(%) in the groups to which the natural-substance-derived extract
was administered and the hG-CSF control were calculated and
compared.
Example 2
Determination of Changes in both Weight and Weight Gain
[0048] Based on the observation results, changes in body weight and
weight gain were measured, and are listed in Table 4 and shown in
FIG. 2. As shown in FIG. 2, it could be seen that the changes in
body weight and weight gain associated with administration of the
hG-CSF or the natural-substance-derived candidate compound compared
to the normal medium control were not shown to be significant for
the entire experimental period.
TABLE-US-00004 TABLE 4 Body weight (g) Body weight gain At first
treatment (g) during treatment Groups [A] Sacrifice [B] [B - A]
Controls Intact vehicle 16.24 .+-. 0.63 17.19 .+-. 0.56 0.95 .+-.
0.44 hG-CSF 16.10 .+-. 0.68 17.24 .+-. 0.79 1.14 .+-. 0.36 Natural
extract orally co-administered PMT 16.19 .+-. 0.51 17.20 .+-. 1.06
1.01 .+-. 0.76 SCGBT 16.27 .+-. 0.93 17.41 .+-. 0.78 1.14 .+-. 0.55
AR 16.37 .+-. 0.89 17.31 .+-. 0.79 0.94 .+-. 0.51 CCP 16.10 .+-.
0.71 17.20 .+-. 1.16 1.10 .+-. 0.51 AGR 16.48 .+-. 1.15 17.67 .+-.
1.27 1.19 .+-. 0.57 Values are expressed as mean .+-. standard
deviation (S.D.) for 10 mice
[0049] However, it was revealed that the weight gain for the entire
experimental period of 6 days changed by 20.00% in the hG-CSF
control, compared to the normal medium control, and that the weight
gains changed by -11.40, 0.00, -17.54-3.51, and 4.39% in the groups
to which the palmultang, SCGBT, Astragalus membranaceus Bunge var.
membranaceus, Capreolus capreolus ochracea Thomas, and Angelica
gigas extracts (200 mg/kg) were administered, respectively,
compared to the hG-CSF control.
Example 3
Determination of Change in Weight of Spleen
[0050] Changes in weights of spleens observed in this Example are
listed in the following Table 5. In the hG-CSF control,increases in
weight of the spleen and relative weight with respect to the body
weight caused by splenomegaly were significant (p<0.01),
compared to the normal medium control. In the groups to which the
palmultang and Angelica gigas extracts were administered, a
decrease in weight of the spleen was shown to be significant
(p<0.01), compared to the hG-CSF control.
TABLE-US-00005 TABLE 5 Spleen weight Groups Absolute (g) Relative
(% of body weight) Controls Intact vehicle 0.066 .+-. 0.005 0.384
.+-. 0.018 hG-CSF 0.132 .+-. 0.012.sup.a 0.768 .+-. 0.090.sup.a
Natural extract orally co-administered PMT 0.104 .+-. 0.008.sup.ab
0.608 .+-. 0.064.sup.ab SCGBT 0.139 .+-. 0.019.sup.a 0.803 .+-.
0.128.sup.a AR 0.125 .+-. 0.011.sup.a 0.721 .+-. 0.050.sup.a CCP
0.150 .+-. 0.018.sup.ac 0.871 .+-. 0.085.sup.ac AGR 0.109 .+-.
0.008.sup.ab 0.618 .+-. 0.061.sup.ab
[0051] As shown in FIG. 3, it was also revealed that the size of
the spleen decreased in the groups to which the palmultang and
Angelica gigas extracts were administered when observed with naked
eye, compared to the normal medium control.
[0052] Meanwhile, in the group to which the Capreolus capreolus
ochracea Thomas extract was administered, increases in absolute and
relative weights of the spleen were shown to be significant
(p<0.05), compared to the hG-CSF control. In the groups to which
the SCGBT and Astragalus membranaceus Bunge var. membranaceus
extracts were administered, changes in absolute and relative
weights of the spleen were not shown to be significant, compared to
the hG-CSF control.
[0053] In the hG-CSF control, the absolute weight of the spleen
changed by 99.55%, compared to the normal medium control. In the
groups to which the palmultang, SCGBT, Astragalus membranaceus
Bunge var. membranaceus, Capreolus capreolus ochracea Thomas, and
Angelica gigas extracts (200 mg/kg) were administered, the absolute
weights of the spleens changed by -21,08, 5,61, -5.38, 13.65, and
-17,51% respectively, compared to the hG-CSF control.
[0054] In the hG-CSF control, the relative weight of the spleen
changed by 99.81%, compared to the normal medium control. In the
groups to which the palmultang, SCGBT, Astragalus membranaceus
Bunge par. membranaceus, Capreolus capreolus ochracea Thomas, and
Angelica gigas extracts (200 mg/kg) were administered, the relative
weights of the spleens changed by -20.85, 4.57, -6.13, 1146, and
-19.46%, compared to tle hG-CSF control.
[0055] Based on the experimental results, it was revealed that the
side effects (e.g., splenomegaly) of hG-CSF were relieved when the
hG-CSF and the palmultang extract were co-administered.
Example 4
Determination of Number of Blood Leukocytes and Bone Marrow Nuclear
Cells
[0056] In this Example, changes in numbers of blood leukocytes and
bone marrow nuclear cells were observed. The results are listed in
the following Table 6.
TABLE-US-00006 TABLE 6 Total cell counts Blood leukocytes Bone
marrow nuclear cells Groups (.times.10.sup.3 cells/.mu.l)
(.times.10.sup.4 cells/.mu.l) Controls Intact vehicle 5.06 .+-.
2.00 47.10 .+-. 20.93 hG-CSF 53.40 .+-. 22.63.sup.a 457.90 .+-.
129.50.sup.c Natural extract orally co-administered PMT 55.50 .+-.
17.92.sup.a 440.80 .+-. 115.78.sup.c SCGBT 47.70 .+-. 13.34.sup.a
444.10 .+-. 105.38.sup.c AR 52.50 .+-. 11.74.sup.a 431.90 .+-.
127.46.sup.c CCP 73.00 .+-. 12.94.sup.ab 590.10 .+-. 134.67.sup.c
AGR 57.10 .+-. 16.93.sup.a 444.80 .+-. 92.76.sup.c
[0057] In the hG-CSF control,increases in total numbers of blood
leukocytes and bone marrow nuclear cells shown to be significant
(p<0,01), compared to the normal medium control However, the
increases in the total numbers of blood leukocytes and bone marrow
nuclear cells were shown to be significant in the hG-CSF/Capreolus
capreolus ochracea Thomas co-administered group, but changes in the
total number of blood. leukocytes and the bone n arrow nuclear
cells were not shown to be significant in any of the natural
substance extract co-administered groups.
[0058] In the hG-CSF control, the total number of blood leukocytes
changed by 955.34%, compared to the normal medium control. In the
groups to which the palmultang, SCGBT, Astragalus membranaceus
Bunge var. membranaceus, Capreolus capreolus ochracea Thomas, and
Angelica gigas extracts (200 mg/kg) were administered, the total
numbers of blood leukocytes changed by 3.93, -10.67, -1.69, 36.70,
and 6.93%, respectively, compared to the hG-CSF control.
[0059] In the hG-CSF control, the total number of bone marrow
nuclear cells changed by 872.19%, compared to the normal medium
control. In the groups to which the palmultang, SCGBT, Astragalus
membranaceus Bunge var. membranaceus Capreolus capreolus ochracea
Thomas, and Angelica gigas extracts (200 mg/ g) were administered,
the total numbers of bone marrow nuclear cells changed by -3.73,
-3.01 -5.68, 28.87, and -2.86%, respectively, compared to the
hG-CSF control.
[0060] Therefore, it was revealed that the total numbers of blood
leukocytes and bone marrow nuclear cells did not increase but
remained at similar levels when the palmultang extract was
administered, compared to the hG-CSF control,
Example 5
FACS results: Determination of Changes in Number of CD34+ and CD45+
Cells in Blood and Bone Marrow
[0061] Changes in the numbers of CD34+ and CD45+ cells in the blood
and bone marrow were observed using a FACS method. The results are
listed in the following Table 7.
TABLE-US-00007 TABLE 7 Blood leukocytes Bone marrow nuclear cells
CD34+ cells CD45+ cells CD34+ cells CD45+ cells Groups
(.times.10.sup.2 cells/.mu.l) (.times.10.sup.2 cells/.mu.l)
(.times.10.sup.2 cells/.mu.l) (.times.10.sup.2 cells/.mu.l)
Controls Intact vehicle 7.93 .+-. 1.05 10.41 .+-. 1.47 16.60 .+-.
3.76 13.16 .+-. 2.32 hG-CSF 26.81 .+-. 6.02.sup.a 42.94 .+-.
8.62.sup.a 30.41 .+-. 6.69.sup.a 24.63 .+-. 3.82.sup.a Natural
extract orally co-administered PMT 43.40 .+-. 5.46.sup.ab 61.99
.+-. 3.32.sup.ab 45.09 .+-. 5.83.sup.ab 32.35 .+-. 1.77.sup.ab
SCGBT 22.14 .+-. 5.46.sup.a 41.76 .+-. 6.09.sup.a 31.21 .+-.
1.60.sup.a 25.37 .+-. 2.47.sup.a AR 25.10 .+-. 6.36.sup.a 45.28
.+-. 8.58.sup.a 32.42 .+-. 3.31.sup.a 23.97 .+-. 2.46.sup.a CCP
40.66 .+-. 2.59.sup.ab 56.79 .+-. 6.14.sup.ab 40.44 .+-.
1.13.sup.ab 28.44 .+-. 4.73.sup.a AGR 41.25 .+-. 6.00.sup.a 61.28
.+-. 7.68.sup.ab 44.34 .+-. 3.59.sup.ab 31.17 .+-. 7.59.sup.ab
Values are expressed as mean .+-. S.D. for 5 mice
[0062] 5.1. Changes in Numbers of CD34+ Cells in Blood and Bone
Marrow
[0063] The FACS results of CD34+ cells are shown in FIGS. 4 and 5.
Based on the FACS results listed in Table 7, increases in the
numbers of CD34+ cells in the blood and bone marrow were shown to
be significant (p<0,01) in the hG-CSF control, compared to the
normal medium control. In the groups to which the palmultang,
Capreolus capreolus ochracea Thomas, and Angelica gigas extracts
were administered, increases in the numbers of CD34+ cells in the
blood and bone marrow were also shown to be significant
(p<0.01), compared to the hG-CSF control. On the other hand, in
the groups to which the SCGBT and Astragalus membranaceus Bunge
var. membranaceus extracts were administered, changes in the
numbers of CD34+ cells in the blood and bone marrow were not shown
to be significant, compared to the hG-CSF control.
[0064] In the hG-CSF control, the number of CD34+ cells in the
blood changed by 238.08%, compared to the normal medium control. In
the groups to which the palmultang, SCGBT, Astragalus membranaceus
Bunge var. membranaceus, Capreolus capreolus ochracea Thomas, and
Angelica gigas extracts (200 mg/kg) were administered, the numbers
of CD34+ cells in the blood changed by 61.89, -17.41, -6.39, 51.65,
and 53.85%, respectively, compared to the hG-CSF control.
[0065] In the hG-CSF control, the number of CD34+ cells in the bone
marrow changed by 83.14%, compared to the normal medium control. In
the groups to which the palmultang, SCGBT, Astragalus membranaceus
Bunge var. membranaceus, Capreolus capreolus ochracea Thomas, and
Angelica gigas extracts (200 mg/kg) were administered, the numbers
of CD34+ cells in the bone marrow changed by 48.29, 2.64, 6.62,
33.00, and 45.80%, respectively, compared to the hG-CSF
control.
[0066] Based on the experimental results, it was revealed that the
numbers of CD34+ cells in the blood and bone marrow significantly
increased when the hG-CSF and palmultang extract were
co-administered, compared to when the hG-CSF was administered
alone.
[0067] 5.2. Changes in Numbers of CD45+ Cells in Blood and Bone
Marrow
[0068] The FACS results of CD45+ cells are shown in FIGS. 6 and 7.
Based on the FACS results listed in Table 7, increases in the
numbers of CD45+ cells in the blood and bone marrow were shown to
be significant (p<0.01) in the hG-CSF control, compared to the
normal medium control. In the groups to which the palmultang,
Capreolus capreolus ochracea Thomas, and Angelica gigas extracts
were administered, increases in the numbers of CD45+ cells in the
blood and bone marrow were shown to be significant, compared to the
hG-CSF control. On the other hand, in the groups to which the SCGBT
and Astragalus membranaceus Bunge var. membranaceus extracts were
administered, changes in the numbers of CD45+ cells in the blood
and bone marrow were not shown to be significant, compared to the
hG-CSF control.
[0069] In the hG-CSF control, the number of CD45+ cells in the
blood changed by 312.39%, compared to the normal medium control. In
the groups to which the palmultang, SCGBT, Astragalus membranaceus
Bunge var. membranaceus, Capreolus capreolus ochracea Thomas, and
Angelica gigas extracts (200 mg/kg) were administered, the numbers
of CD45+ cells in the blood changed by 44.38, -2.75, 5.45, 32.26,
and 42.72%, respectively, compared to the hG-CSF control.
[0070] In the hG-CSF control, the number of CD45+ cells in the bone
marrow changed by 87,18%, compared to the normal medium control. In
the groups to which the palmultang, SCGBT, Astragalus membranaceus
Bunge var. membranaceus, Capreolus capreolus ochracea Thomas, and
Angelica gigas extracts (200 mg/kg) were administered, the number
of CD45+ cells in the bone marrow changed by 31.35, 3.02, -2.68,
15.47, and 26.58%, respectively, compared to the hG-CSF
control.
[0071] Based on the experimental results, it was revealed that the
numbers of CD45+ cells in the blood and bone marrow significantly
increased when the hG-CSF and palmultang extract were
co-administered, compared to when the hG-CST was administered
alone.
[0072] Therefore, based on the results of Examples 5.1 and 5.2, it
could be seen that the co administration of the hG-CSF and
palmultang extract caused a significant increase in proliferation
of the bone-marrow-derived stem cells.
Example 6
Determination of Histopathologic Changes
[0073] Histopathologic changes of spleens and femoral bone marrow
in eight groups were observed. The results are listed in the
following Table 8.
TABLE-US-00008 TABLE 8 Spleen Bone marrow Total Amount of Mean
diameters Mean number Mean thickness white pulp of white pulp of
red pulp number of (mm/central (white (.mu.m/white nuclear cells
nuclear cells Groups regions) pulp/mm.sup.2) pulp) (.times.10.sup.2
cells/mm.sup.2) (.times.10.sup.2 cells/mm.sup.2) Controls Intact
1.58 .+-. 1.16 14.50 .+-. 2.37 459.22 .+-. 98.51 3.34 .+-. 0.80
16.30 .+-. 2.26 vehicle hG-CSF 1.86 .+-. 0.12a 14.60 .+-. 1.84
478.38 .+-. 70.00 27.18 .+-. 5.19a 74.51 .+-. 18.28a Natural
extract orally co-administered PMT 1.63 .+-. 0.07b 14.10 .+-. 2.33
493.19 .+-. 68.93 19.09 .+-. 2.44ab 72.46 .+-. 11.54a SCGBT 2.06
.+-. 0.16ab 14.00 .+-. 2.98 484.79 .+-. 84.57 28.92 .+-. 5.91a
70.77 .+-. 11.22a AR 1.85 .+-. 0.18a 14.80 .+-. 1.75 482.04 .+-.
116.86 30.65 .+-. 6.14a 67.22 .+-. 10.57a CCP 2.00 .+-. 0.34a 14.90
.+-. 2.60 487.78 .+-. 65.78 26.76 .+-. 3.43a 80.03 .+-. 12.18a AGR
1.68 .+-. 0.14b 14.60 .+-. 2.01 472.34 .+-. 65.60 17.61 .+-. 3.18ab
67.30 .+-. 11.77a Values are expressed as mean .+-. S.D. for 10
mice
[0074] 6.1. Histopathologic Change of Spleen
[0075] As listed in Table 8, it was revealed that the splenomegaly
findings caused by infiltration of nuclear cells in spleen red pulp
were significant in the hG-CSF control. Also, it was revealed that
increases in the total thickness of the spleens and the number of
nuclear cells in the red pulp per unit area were significant
(p<0.01), compared to the normal medium control, but the amount
and diameters of the white pulp were observed to be similar to the
normal medium control.
[0076] FIG. 8 is an image showing the total thicknesses of the
spleens and the number of nuclear cells in the red and white pulp
per unit area. As shown in FIG. 8, it was revealed that decreases
in the total thickness of the spleens and the number of nuclear
cells in the red pulp per unit area were significant (p<0.01) in
the groups to which the palmultang and Angelica gigas extracts were
administered, compared to the hG-CSF control, but an increase in
the total thickness of the spleens was significant (p<0.01) in
the SCGBT-administered group, compared to the hG-CSF control, and
the histopathologic changes of the spleens were not significant in
the groups to which the Astragalus membranaceus Bunge var.
membranaceus and Capreolus capreolus ochracea Thomas extracts were
administered, compared to the hG-CSF control.
[0077] In the hG-CSF control, the total thickness of the spleen
changed by 17.47%, compared to the normal medium control. In the
groups to which the palmultang, SCGBT, Astragalus membranaceus
Bunge var. membranaceus, Capreolus capreolus ochracea Thomas, and
Angelica gigas extracts (200 mg/kg) were administered, the total
thicknesses of the spleens changed by -12.02, 10.72, -0.48, 7.81,
and -9.75%, respectively, compared to the hG-CSF control.
[0078] In the hG-CSF control, the amount of spleen white pulp
changed by 0.69%, compared to the normal medium control. In the
groups to which the palmultang, SCGBT, Astragalus membranaceus
Bunge Var. membranaceus, Capreolus capreolus ochracea Thomas, and
Angelica gigas extracts (200 mg/kg) were administered, the amounts
of spleen white pulp changed by -3.42, -4.11, 1.37, 2.05, and
0.00%, respectively, compared to the hG-CSF control.
[0079] In the hG-CSF control, the mean diameter of the spleen white
pulp changed by 4.17%, compared to the normal medium control. In
the groups to which the palmultang, SCGBT, Astragalus membranaceus
Bunge var. membranaceus, Capreolus capreolus ochracea Thomas, and
Angelica gigas extracts (200 mg/kg) were administered, the mean
diameters of the spleen white pulp changed by 3.09, 1.34, 0.77,
1.96, and -1.26%, respectively, compared to the hG-CSF control.
[0080] In the hG-CSF control, the amount of spleen ed pulp per unit
area changed by 714.75%, compared to the normal medium control, In
the groups to which the palmultang, SCGBT, Astragalus membranaceus
Bunge var. membranaceus Capreolus capreolus ochracea Thomas, and
Angelica gigas extracts (200 mg/kg) were administered, the amounts
of spleen red pulp per unit area changed by -29.76, 6.39, 12.76,
-1.55 and -35.21%, respectively, compared to the hG-CSF
control.
[0081] 6.2. Histopathologic Change of Femoral Bone Marrow
[0082] As listed in Table 8, it was revealed that the proliferation
of the granulocytes was significant in the hG-CSF control As shown
its FIG. 9, it was revealed that an increase in the number of
nuclear cells in the bone marrow per unit area was consequently
significant (p<0.01), compared to the normal medium control, but
the histopathologic changes of the femoral bone marrow were not
significant in any of the groups to which the
natural-substance-derived extracts were administered, compared to
the hG-CSF control.
[0083] In the hG-CSF control, the number of nuclear cells in the
femoral bone marrow per unit area changed by 357.02%, compared to
the normal medium control. In the groups to which the palmultang,
SCGBT, Astragalus membranaceus Bunge var. membranaceus, Capreolus
capreolus ochracea Thomas and Angelica gigas extracts (200 mg/kg)
were administered, the numbers of nuclear cells in the femoral bone
marrow per unit area changed by -2.75, -5.02, -9.79, 7.40, and
-9.68%, respectively, pa ed to the hG-CSF control.
[0084] Based on the results of Examples 5.1 and 5.2, it could be
seen that the side effects (e.g., splenomegaly) of hG-CSF were
prevented and the number of leukocytes also increased when the
hG-CSF and the palmultang extract were co-administered, compared to
when the hG-CSF was administered alone.
Example 7
Determination of Immunohistochemical Changes
[0085] The numbers of CD34 and CD45 immunoreactive cells in the
spleens and bone marrow in seven groups were observed. The results
are listed in the following Table 9.
TABLE-US-00009 TABLE 9 Number of spleen Number of bone marrow
immunoreactive cells (cells/mm.sup.2) immunoreactive cells
(cells/mm.sup.2) Groups CD34+ CD45+ CD34+ CD45+ Controls Intact
vehicle 16.40 .+-. 2.72 44.90 .+-. 15.04 11.40 .+-. 3.92 35.80 .+-.
11.86 hG-CSF 133.40 .+-. 20.30.sup.c 303.50 .+-. 79.69.sup.c 46.20
.+-. 9.74.sup.c 325.40 .+-. 98.79.sup.a Natural extract orally
co-administered PMT 174.60 .+-. 18.35.sup.cd 405.20 .+-.
64.22.sup.cd 74.20 .+-. 10.88.sup.cd 490.10 .+-. 112.39.sup.ab
SCGBT 137.70 .+-. 20.82.sup.c 300.50 .+-. 70.36.sup.c 41.30 .+-.
6.13.sup.c 350.00 .+-. 70.41.sup.a AR 146.80 .+-. 14.77.sup.c
327.50 .+-. 46.42.sup.c 49.40 .+-. 8.83.sup.c 364.90 .+-.
116.30.sup.a CCP 125.50 .+-. 13.74.sup.c 478.80 .+-. 111.65.sup.cd
42.20 .+-. 6.98.sup.c 344.40 .+-. 102.18.sup.a AGR 160.60 .+-.
14.21.sup.cd 462.40 .+-. 80.42.sup.cd 81.00 .+-. 19.10.sup.cd
536.70 .+-. 82.72.sup.ab Values are expressed as mean .+-. S.D. for
10 mice
[0086] 7.1. Changes in Numbers of CD34 Immunoreactive Cells in
Spleen and Bone Marrow
[0087] As listed in Table 9 and shown in FIG. 9, it was revealed
that increases in it e numbers of CD34 immunoreactive cells in the
spleen and bone marrow were significant (p<0.01) in the hG-CSF
control, compared to the normal medium control, and that increases
in the numbers of CD34 immunoreactive cells in the spleens and bone
marrow were significant (p<0.01) in the groups to which the
palmultang and Angelica gigas extracts were administered, compared
to the hG-CSF control. On the other hand, it was revealed that
changes in the numbers of CD34 immunoreactive cells in the spleen
and bone marrow per unit area were not significant in the groups to
which the SCGBT, Capreolus capreolus ochracea Thomas, and
Astragalus membranaceus Bunge var. membranaceus extracts were
administered, compared to the hG-CSF control.
[0088] In the hG-CSF control, the number of CD34 immunoreactive
cells in the spleen changed by 713.41%, compared to the normal
medium control. In the groups to which the palmultang, SCGBT,
Astragalus membranaceus Bunge var. membranaceus, Capreolus
capreolus ochracea Thomas, and Angelica gigas extracts (200 mg/kg)
were administered, the numbers of CD34 immunoreactive cells in the
spleen changed by 30.88, 3.22, 10.04, -5.92, and 20.39%,
respectively, compared to the h.G-CSF control.
[0089] In the hG-CSF control, the number of CD34 immunoreactive
cells in the bone marrow changed by 305.26%, compared to the normal
medium control. In the groups to which the palmultang, SCGBT,
Astragalus membranaceus Bunge var. membranaceus, Capreolus
capreolus ochracea Thomas and Angelica gigas extracts (200 mg/kg)
were administered, the numbers of CD34 immunoreactive cells in the
bone marrow changed by 60.61, -10.61 6.93, -8.87, and 75.32%,
respectively,compared to the hG-CSF control.
[0090] 7.2. Changes in Numbers of CD45 Immunoreactive Cells in
Spleen and Bone Marrow
[0091] As listed in Table 9 and shown in FIG. 10, it was revealed
that increases in the numbers of CD45 immunoreactive cells in the
spleen and bone mars were significant (p<0.01) in the hG-CSF
control, compared to the normal medium control, and that increases
in the numbers of CD45 immunoreactive cells in the spleens and bone
marrow were significant (p<0.01) in the groups to which the
palmultang and Angelica gigas extracts were administered, compared
to the hG-CSF control. On the other hand, it was revealed that an
increase in the number of CD45 immunoreactive cells in the spleen
was also significant (p<0.01) in the group to which the
Capreolus capreolus ochracea Thomas extract was administered,
compared to the hG-CSF control, but the number of CD45
immunoreactive cells in the bone marrow was observed to be similar
to the hG-CSF control, and changes in the numbers of CD45+ cells in
the blood and bone marrow were not significant in the groups to
which the SCGBT and Astragalus membranaceus Bunge var. membranaceus
extracts were administered, compared to the hG-CSF control.
[0092] In the hG-CSF control, the number of CD45 immunoreactive
cells in the spleen changed by 575.95%, compared to the normal
medium control. In the groups to which the palmultang, SCGBT,
Astragalus membranaceus Bunge var. membranaceus, capreolus
capreolus ochracea Thomas, and Angelica gigas extracts (200 mg/kg)
were administered, the numbers of CD45 immunoreactive cells in the
spleens changed by 33.51, -0.99, 7.91, 57.76, and 52.36%,
respectively, compared to the hG-CSF control.
[0093] In the hG-CSF control, the number of CD45 immunoreactive
cells in the bone marrow changed by 808.94%, compared to the normal
medium control. in the groups to which the palmultang, SCGBT,
Astragalus membranaceus Bunge var. membranaceus, capreolus
capreolus ochracea Thomas, and Angelica gigas extracts (200 mg/kg)
were administered, the numbers of CD45 immunoreactive cells in the
spleens changed by 50.61, 7.56, 12.14, 5.84, and 64.94%,
respectively, compared to the hG-CSF control.
[0094] Based on the experimental results, it was confirmed that the
number of immunoreactive cells significantly increased when the
hG-CSF and the palmultang extract were co-administered, compared to
when the hG-CSF was administered alone.
[0095] In summary, the experimental results of Examples 2 to 7
showed that the increases in the weights of the spleens and the
total numbers of nuclear cells in the blood and bone marrow by
administration of the hG-CSF and the increases in the numbers of
CD34+ and CD45+ cells in the blood and bone marrow were
significant, and that the increases in the total thicknesses of the
spleens and the numbers of nuclear cells in the red pulp and
femoral bone marrow per unit area were significant in a
histopathologic aspect, and the increases in the numbers of CD34
and CD45 immunoreactive cells in the spleens and bone marrow were
significant in an immunohistochemical aspect.
[0096] Therefore, it was revealed that the proliferation and
mobilization of the bone-marrow-derived stem cells by the hG-CSF
significantly increased (p<0.01) when the palmultang extract was
orally co-administered within 5 minutes, and the increases in the
weight of the spleen and splenomegaly findings by proliferation of
the nuclear cells in the red pulp were significantly inhibited
(p<0.01), but the administration of the hG-CSF had no influence
on the overall proliferation of the granulocytes.
[0097] Meanwhile, it was revealed that the proliferation and
mobilization of the bone-marrow-derived stem cells were also
significant in the group to which the Capreolus capreolus ochracea
Thomas extract was administered, but the splenomegaly findings
actually worsened, and that the co-administration of the SCGBT and
Astragalus membranaceus Bunge var. membranaceus extracts had no
influence on the effect of the hG-CSF on the proliferation and
mobilization of the granulocytes and bone-marrow-derived stem
cells, and had no influence on the side effects such as
splenomegaly either.
[0098] Therefore, the palmultang extract is expected to provide
anew combined medical system of Oriental and Western medicine which
is very useful in improving an effect of the hG-CSF on mobilization
of the bone-marrow-derived stem cells and reducing side effects
such as splenomegaly.
[0099] The present invention has been described in detail. However,
it should be understood that the detailed description and specific
examples, while indicating preferred embodiments of the invention,
are given by way of illustration only, and various changes and
modifications within the spirit and scope of the invention will
become apparent to those skilled in the art from this detailed
description.
* * * * *