US20160058810A1

US20160058810A1 - Composition containing angelica extract for promoting proliferation of stem cells derived from bone marrow

Info

Publication number: US20160058810A1
Application number: US14/888,682
Authority: US
Inventors: Chang Hyeong LEE; Sang Gyung KIM; Im Hee SHIN; Seung Mo Kim; Joon Seok BYUN; Ki Cheul SOHN; Sae Kwang Ku
Original assignee: COMPREHENSIVE AND INTEGRATIVE MEDICINE INSTITUTE
Current assignee: COMPREHENSIVE AND INTEGRATIVE MEDICINE INSTITUTE
Priority date: 2013-05-03
Filing date: 2014-05-01
Publication date: 2016-03-03
Also published as: KR101373100B1; US20180243358A1; WO2014178676A1; US10342835B2

Abstract

The present invention relates to a composition for promoting proliferation of bone marrow derived stem cells using an angelicae gigantis radix extract, and more particularly, to a composition which administers a granulocyte colony-stimulating factor into an object, and administers the angelicae gigantis radix extract to the object to promote the proliferation of the bone marrow derived stem cells. The composition of the present invention solves side effects such as splenomegaly which were caused by a method of administering only the G-CSF for proliferation and differentiation of the stem cells, thereby significantly mitigating the side effects through co-administration of the angelicae gigantis radix extract, and further promoting the proliferation and the differentiation of the stem cells.

Description

TECHNICAL FIELD

The present invention provides a composition containing an angelicae gigantis radix extract for promoting proliferation of bone marrow derived stem cells.

BACKGROUND ART

“Stem cell” is a generic term for undifferentiated cells that can differentiate into various cells forming biological tissues and obtained from tissue of embryos, fetuses, and adults. Among various stem cells, adult stem cells are cells derived from bone marrow, and are multifunctional stem cells which can be differentiated into any cell forming an organ and blood of a human body.
Among these, bone marrow derived stem cells are considered as an ultimate means of treating diseases such as hematologic cancers, lymphoma, and bone marrow failure. Transplantation of bone marrow derived stem cells is currently being performed for various purposes, and particularly when organ transplantation is difficult in critical kidney and liver patients, a method of promoting regeneration of the liver and kidneys is being attempted by transplanting autologous bone marrow derived stem cells.
After the autologous bone marrow derived stem cells are transplanted, to promote differentiation and division of the stem cells, a recombinant human granulocyte colony-stimulating factor (hG-CSF) is administered in full dose. However, in about 5-30% of patients, proliferation or mobilization toward blood does not occur in the stem cells, and various side effects such as myocardial infarction, cerebral infarction, fever, ostalgia, splenomegaly, splenic rupture, etc. are known to be caused by the administration of hG-CSF (Masood et al., 2008; Fox et al., 2009). Particularly, the use of hG-CSF is being limited due to its high cost. Consequently, a new alternative means which is capable of promoting an effect of hG-CSF in proliferating the bone marrow derived stem cells, or mitigating the side effects of hG-CSF is required.
Angelicae gigantis radix is a dried substance of roots of Angelica gigas NAKAI, A. sinensis DIELS, and A. acutiloba KITAG, which are perennial herbs belonging to the family Umbelliferae, along with other congeneric, closely-related plants, and is known to have effects of replenishing blood to cure lack of blood supply, reducing menstrual cramps, and moisturizing the intestine to relieve constipation. The roots perform a sedative action, an antispasmodic action, a hypotensive action, etc., and aqueous solutions of essential oils of the roots perform an analgesic action and an anti-inflammatory action when used to treat arthritis. In the field of oriental medicine, the roots are mostly used in anemia, gynecopathy, and postpartum recovery, and are also used for serious coughing, boils, etc. Meanwhile, Korean Unexamined Patent Application Publication No. 2012-0092754 has disclosed content related to promoting differentiation of stem cells using Chrysanthemum zawadskii, which is a medicinal herb, but there has been no example of promoting differentiation of “bone marrow derived” stem cells using an angelicae gigantis radix extract.

DISCLOSURE

Technical Problem

The present invention has been devised to resolve the above-mentioned need of the prior art, and is directed to providing a composition for promoting proliferation of bone marrow derived stem cells using an angelicae gigantis radix extract.
However, a technical aspect to be achieved by the present invention is not limited to the above-mentioned aspect, and those of ordinary skill in the art will be able to understand other unmentioned aspects from the description below.

Technical Solution

To achieve the above-mentioned aspect of the present invention, the present invention provides a composition containing a granulocyte colony-stimulating factor and an angelicae gigantis radix extract for promoting proliferation of bone marrow derived stem cells.
According to one embodiment of the present invention, the stimulating factor and the angelicae gigantis radix extract may be mixed in advance and made into a dosage form, or separately made into a dosage form.
According to another embodiment of the present invention, the stimulating factor and the angelicae gigantis radix extract may be administered parenterally, orally, locoregionally, or percutaneously.
According to still another embodiment of the present invention, a period of administering the angelicae gigantis radix extract may begin within thirty minutes after administration of the stimulating factor.

Advantageous Effects

A composition containing an angelicae gigantis radix extract as an effective component for promoting proliferation of stem cells which is provided in the present invention includes a recombinant human granulocyte colony-stimulating factor (hG-CSF) and the angelicae gigantis radix extract, and is administered before transplanting stem cells, thereby promoting proliferation and differentiation of stem cells.
In addition, when the hG-CSF is administered to promote proliferation and differentiation of stem cells and the proliferation of the stem cells is induced, the composition of the present invention may be used as a new alternative means which is able to solve the problem of the prior art which had side effects such as myocardial infarction, cerebral infarction, fever, ostalgia, splenomegaly, splenic rupture, etc.

DESCRIPTION OF DRAWINGS

FIG. 1 is a view illustrating a structure of an hG-CSF used in the present invention.

FIG. 2 is a view illustrating results of changes in body weights and body weight gain of mice due to administration of the hG-CSF or a natural product derived candidate substance.

FIG. 3 is a view illustrating results of checking sizes of spleens of the mice to which the hG-CSF or the natural product derived candidate substance was administered (A: untreated, B: hG-CSF, C: palmultang+hG-CSF, D: samchulgeonbitang+hG-CSF, E: astragali radix+hG-CSF, F: cervi cornu parvum+hG-CSF, and G: angelicae gigantis radix+hG-CSF).

FIGS. 4 and 5 are views illustrating results of observing a fluorescence-activated cell sorter (FACS) of CD34+ cells of the mice to which the hG-CSF or the natural product derived candidate substance was administered (A: untreated, B: hG-CSF, C: palmultang+hG-CSF, D: samchulgeonbitang+hG-CSF, E: astragali radix+hG-CSF, F: cervi cornu parvum+hG-CSF, and G: angelicae gigantis radix+hG-CSF).

FIGS. 6 and 7 are views illustrating results of observing a FACS of CD45+ cells of the mice to which the hG-CSF or the natural product derived candidate substance was administered (A: untreated, B: hG-CSF, C: palmultang+hG-CSF, D: samchulgeonbitang+hG-CSF, E: astragali radix+hG-CSF, F: cervi cornu parvum+hG-CSF, and G: angelicae gigantis radix+hG-CSF).

FIG. 8 is a view illustrating observations of total thicknesses of spleens and nuclear cells per unit area of red pulp and white pulp of the spleens of the mice to which the hG-CSF or the natural product derived candidate substance was administered (A-C: untreated, D-F: hG-CSF, G-I: palmultang+hG-CSF, J-L: samchulgeonbitang+hG-CSF, M-O: astragali radix+hG-CSF, P-R: cervi cornu parvum+hG-CSF, and S-U: angelicae gigantis radix+hG-CSF).

FIG. 9 is a view illustrating observations of a numerical increase in CD34 immunoreactive cells in spleens and bone marrow of the mice to which the hG-CSF or the natural product derived candidate substance was administered (A-B: untreated, C-D: hG-CSF, E-F: palmultang+hG-CSF, G-H: samchulgeonbitang+hG-CSF, I-J: astragali radix+hG-CSF, K-L: cervi cornu parvum+hG-CSF, and M-N: angelicae gigantis radix+hG-CSF).

FIG. 10 is a view illustrating observations of a numerical increase in CD45 immunoreactive cells in the spleens and the bone marrow of the mice to which the hG-CSF or the natural product derived candidate substance was administered (A-D: untreated, E-H: hG-CSF, I-L: palmultang+hG-CSF, M-P: samchulgeonbitang+hG-CSF, Q-T: astragali radix+hG-CSF, U-X: cervi cornu parvum+hG-CSF, and Y-AB: angelicae gigantis radix+hG-CSF).

MODES OF THE INVENTION

To develop a composition which is capable of mitigating various side effects that occur when the recombinant hG-CSF is administered to promote differentiation and division of bone marrow derived stem cells after the bone marrow derived stem cells are transplanted, and further increasing production of the bone marrow derived stem cells, the present inventors have focused on medicinal herbs, and found that, among the medicinal herbs, angelicae gigantis radix has an outstanding effect in promoting proliferation and differentiation of the stem cells, thus completing the present invention.
Consequently, the present invention is directed to providing a composition containing a G-CSF and an angelicae gigantis radix extract for promoting proliferation of bone marrow derived stem cells.
According to one embodiment of the present invention, the G-CSF and the angelicae gigantis radix extract may be mixed in advance and made into a dosage form, or separately made into a dosage form.
A period of administering the angelicae gigantis radix extract may begin within thirty minutes after administration of the G-CSF, preferably within fifteen minutes, and most preferably within five minutes, but the administration period is not limited thereto.
The G-CSF used in the present invention is the recombinant hG-CSF, but is not limited thereto.
The G-CSF and the angelicae gigantis radix extract may be administered parenterally, orally, locoregionally, or percutaneously. Although it is preferable that the angelicae gigantis radix extract be administered orally, the administration method may be properly selected by those of ordinary skill in the art in accordance with a condition and body weight of a patient, an extent of a disease, a period, etc.
In the present invention, an “individual” refers to an object which requires a treatment of a disease, and more specifically, refers to mammals such as humans or non-human primates, mice, rats, dogs, cats, horses, cattle, etc.
In addition, the present invention may provide a pharmaceutical composition containing the angelicae gigantis radix extract for increasing the production of the bone marrow derived stem cells.
In addition, the composition of the present invention may contain a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may include normal saline, polyethylene glycol, ethanol, vegetable oil, isopropyl myristate, etc., but is not limited thereto.
According to an embodiment of the present invention, a preferred administration amount of the pharmaceutical composition varies in accordance with a condition and body weight of a patient, an extent of a disease, a form of a drug, an administration route, and a period, but may be properly selected by those of ordinary skill in the art. However, preferably, the pharmaceutical composition is administered in 0.001 to 300 mg/kg of body weight per day, and more preferably in 0.01 to 200 mg/kg of body weight per day.
The pharmaceutical composition of the present invention may be administered to mammals such as rats, mice, livestock, humans, etc. in various routes. The administration methods are not limited, and for example, the pharmaceutical composition of the present invention may be administered orally, rectally, or by intravenous, intramuscular, subcutaneous, intrauterine, or intra cerebroventricular injection.
In the present invention, as a result of orally administering the angelicae gigantis radix extract after administering the hG-CSF, it was confirmed that splenomegaly can be mitigated by orally co-administering the angelicae gigantis radix extract within five minutes after administering the hG-CSF (refer to example 2), spleen weight increase and features of splenomegaly caused by proliferation of nuclear cells in red pulp were significantly (p<0.01) suppressed, overall proliferation of granulocytes was unaffected (refer to example 3), and proliferation and mobilization of bone marrow derived stem cells by hG-CSF were significantly (p<0.01) increased (refer to example 4).
Consequently, the composition containing the angelicae gigantis radix extract for promoting proliferation of the bone marrow derived stem cells of the present invention is able to increase production of the bone marrow derived stem cells, and mitigate various side effects that were caused before when only a G-CSF was administered.
Hereinafter, preferred embodiments will be provided to assist in understanding of the present invention. However, the following embodiments are provided only to enable easier understanding of the present invention, and the content of the present invention is not limited by the following embodiments.

EMBODIMENTS

In the present embodiments, effects of palmultang, samchulgeonbitang, astragali radix, cervi cornu parvum, and angelicae gigantis radix on medicinal effects and side effects of the hG-CSF, particularly on mobilization of stem cells and splenomegaly, were evaluated using Balb/c mice which are commonly used in the mobilization of stem cells by hG-CSF.

Example 1

Experiment Preparation

1-1. Material Preparation
The hG-CSF was purchased from Life Technologies (Carlsbad, Calif., USA) and used, and a structure thereof is illustrated in FIG. 1.
Palmultang (PMT) was purchased from Hanzung Pharmaceutical Co. Ltd. (Daejeon, Korea) and used, and components thereof and amounts of the components are shown in Table 1 below.

TABLE 1

Herbs	Scientific Names/Produce Region	Amounts (g)

Angelicae Gigantis	Angelica gigas N.	2.46
Radix
Atractylodis	Atractylodes ovata (Thunb.) DC.	2.46
Rhizoma
Cnidii Rhizoma	Cnidium officinale Makino	2.26
Ginseng Radix	Panax ginseng C. A. Meyer.	1.6
Alba
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

As medicinal herbs to be compared with an effect of angelicae gigantis radix, PMT, samchulgeonbitang (SCGBT), astragali radix, and cervi cornu parvum were selected and compared. Among the above medicinal herbs, components of SCGBT and amounts of the components are illustrated in Table 2.

TABLE 2

Herbs	Scientific Names/Produce Region	Amounts (g)

Amomi Fructus	Amomum xanthiodes Wallich	0.16
Atractylodis	Atractylodes ovata (Thunb.) DC.	0.98
Rhizoma Alba
Citri Unshii	Citrus unshiu S. Marcov.	0.85
Pericarpium
Ginseng Radix	Panax ginseng C. A. Meyer.	0.64
Alba
Glycyrrhizae	Glycyrrhiza uralensis Fisch	0.36
Radix et
Rhizoma
Hawthorn	Crataegus pinnatifida Bunge var.	0.93
Fruit(Crataegi	typica Schneider
Fructus)
Hordei Fructus	Hordeum vulgare Linn.	0.34
Germiniatus
Zizyphi Fructus	Zizyphus jujuba var. inermis	4.17
	(Bunge) Rehder
Magnoliae	Magnolia officinalis Rehder et	0.21
Cortex	Wilson
Massa Medicata	Triticum aestivum L.	0.67
Fermentata
Paeoniae Radix	Paeonia lactiflora Pall.	0.54
Ponciri Fructus	Poncinus trifoliata	0.58
Hoelen	Poria cocus Wolf	0.05
Zingiberis	Zingiber officinale Roscoe	0.26
Rhizona Crudus
Total	14 types	10.74

1-2. Experimental Animal Preparation
In the present example, Balb/c CrSlc mice (6-week age, female, SLC, Shizuoka, Japan) were selected and used as experimental animals. Seven healthy SPF Balb/c mice were obtained and refined for 34 days. Then, ten experimental animals which had constant body weights were selected for each group, and the experimental animals were divided into seven groups as illustrated in Table 3 below, and used in the experiment. All experimental animals were fasted for eighteen hours overnight before the start date of administering hG-CSF and the drug and a final autopsy date (drinking water was freely provided), and the objects were identified using picric acid.

TABLE 3

		Test substances and dose
Group	Inducer	(mg/kg/day)	Animal No.

GCSF-2012-PD: Effects on the hG-CSF-treated mice

Control	Saline	Distilled water oral 10 ml/kg	M01~M10
	10 ml/kg	[Intact vehicle]
Control	hG-CSF	Distilled water oral 10 ml/kg	M11~M20
	250 μg/kg	[hG-CSF]
Active	hG-CSF	PMT oral (200 mg/kg) [PMT]	M21~M30
	250 μg/kg
Active	hG-CSF	SCGBT oral (200 mg/kg) [SCGBT]	M31~M40
	250 μg/kg
Active	hG-CSF	AR oral (200 mg/kg) [AR]	M41~M50
	250 μg/kg
Active	hG-CSF	CCP oral (200 mg/kg) [CCP]	M51~M60
	250 μg/kg
Active	hG-CSF	AGR oral (200 mg/kg) [AGR]	M61~M70
	250 μg/kg

1-3. Administration Method
Administration of hG-CSF and Candidate Drugs
In accordance with previous methods (Verma et al., 1997; Levesque et al., 2003), 250 μg/kg of the hG-CSF (Life Technologies, Carlsbad, Calif., USA) was subcutaneously administered once per day for six consecutive days to promote proliferation of leukocytes and mobilization of bone marrow derived stem cells, and within five minutes of administering the hG-CSF, 200 mg/kg of PMT, SCGBT, astragali radix, cervi cornu parvum, and angelicae gigantis radix extracts were orally administered once per day for the six consecutive days. All five types of natural product derived extracts were dissolved in sterilized distilled water and forcibly orally administered in a volume of 10 mL per kg of animal body weight using a 1 mL syringe to which a metallic zonde was attached, and the hG-CSF was dissolved in normal saline and subcutaneously administered in a volume of 10 ml/kg in dorsal subcutaneous portions. Only sterilized distilled water of the same volume was administered in an hG-CSF control group instead of the natural product derived extracts, and normal saline and sterilized distilled water of the same volume were administered at five minute intervals in a normal media control group instead of the hG-CSF and the natural product derived extracts. The administration amount of the natural product derived extracts used in the present experiment, i.e., 200 mg/kg, was selected based on results of individual animal experiments.
1-4. Observations
Together with changes in body weights and spleen weights, and total number of bone marrow nuclear cells and blood leukocytes, numbers of CD34+ and CD45+ cells, which are representative markers of bone marrow derived stem cells, were observed in bone marrow and blood by a method of using the fluorescence activated cell sorter (FACs). In addition, the numbers of CD34+ and CD45+ cells per unit area in tissue samples of spleens and bone marrow were observed using an area histochemical method, and total thicknesses of spleens, amounts and diameters of white pulp, and amounts of red pulp in spleens and nuclear cells in bone marrow per unit area were also evaluated using an automatic image analyzer (iSolution FL ver 9.1, IMT i-solution Inc., Quebec, Canada). To observe clearer changes, a % change between the normal media control group and the hG-CSF control group, and % changes between each of the groups to which natural product derived extracts were administered and the hG-CSF control group were calculated and compared.

Example 2

Confirmation of Changes in Weights and Weight Gains

As a result of the observation, changes in weights and weight gains were measured and are illustrated in Table 4 and FIG. 2. As can be seen in FIG. 2, compared with the normal media control group, significant changes in weights and weight gains related to administration of the hG-CSF or the natural product derived candidate substances were not seen in the entire period of the experiment.

	TABLE 4

	Body weight(g)	Body weight gains

	At first		(g) during
Groups	treatment [A]	Sacrifice [B]	treatment [B − A]

Controls

Intact	16.24 ± 0.63	17.19 ± 0.56	0.95 ± 0.44
vehicle
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 mean ± S.D. of 10 mice

However, the hG-CSF control group exhibited a 20.00% change in the weight gain amount compared to the normal media control group during the six days of the entire experiment period, and the groups to which 200 mg/kg of PMT, SCGBT, astragali radix, cervi cornu parvum, and angelicae gigantis radix extracts were administered exhibited changes of −11.40, 0.00, −17.54, −3.51, and 4.39%, respectively, compared to the hG-CSF control group.

Example 3

Confirmation of Changes in Weights of Spleens

Amounts of changes in the weights of spleens observed in the present example are illustrated in Table 5 below. Significant (p<0.01) increase in relative weights with respect to spleens and body weights due to noticeable splenomegaly was seen in the hG-CSF control group compared to the normal media control group, and significant (p<0.01) decrease in weights of spleens were seen in the groups to which PMT and angelicae gigantis radix extracts were administered compared to the hG-CSF control group.

	TABLE 5

	Spleen weights

		Relative (% of
Groups	Absolute (g)	body weight)

Controls

Intact	0.066 ± 0.005	0.384 ± 0.018
vehicle
hG-CSF	0.132 ± 0.012^a	0.768 ± 0.090^a

Natural extract orally co-administered

PMT	0.104 ± 0.008^ab	0.608 ± 0.064^ab
SCGBT	0.139 ± 0.019^a	0.803 ± 0.128^a
AR	0.125 ± 0.011^a	0.721 ± 0.050^a
CCP	0.150 ± 0.018^ac	0.871 ± 0.085^ac
AGR	0.109 ± 0.008^ab	0.618 ± 0.061^ab

In addition, as illustrated in FIG. 3, as a result of checking the sizes of the spleens with the naked eye, it was confirmed that the sizes of spleens of the groups to which PMT and angelicae gigantis radix extracts were administered decreased compared to the normal media control group.
Meanwhile, significant (p<0.05) increase in absolute and relative weights of spleens was seen in the cervi cornu parvum administration group compared to the hG-CSF control group, and significant changes in the weights of the spleens were not seen in the groups to which SCGBT and astragali radix extracts were administered compared to the hG-CSF control group.
The hG-CSF control group exhibited a 99.55% change in the absolute weights of spleens compared to the normal media control group, and the groups to which 200 mg/kg of PMT, SCGBT, astragali radix, cervi cornu parvum, and angelicae gigantis radix extracts were administered exhibited changes of −21.08, 5.61, −5.38, 13.65, and −17.51%, respectively, compared to the hG-CSF control group.
The hG-CSF control group exhibited a 99.81% change in the relative weights of spleens compared to the normal media control group, and the groups to which 200 mg/kg of PMT, SCGBT, astragali radix, cervi cornu parvum, and angelicae gigantis radix extracts were administered exhibited changes of −20.85, 4.57, −6.13, 13.46, and −19.46%, respectively, compared to the hG-CSF control group.
From the above results, it was confirmed that co-administration of the G-CSF and the angelicae gigantis radix extract can mitigate splenomegaly, which is a side effect of the G-CSF.

Example 4

Numerical Confirmation of Blood Leukocytes and Nuclear Cells in Bone Marrow

In the present experiment, numerical changes in blood leukocytes and bone marrow nuclear cells were observed and the results thereof are illustrated in Table 6.

	TABLE 6

	Total Cell Counts

	Blood leukocytes	Bone marrow nuclear cells
Groups	(×10³cells/μl)	(×10³cells/μl)

Controls

Intact	5.06 ± 2.00	47.10 ± 20.93
vehicle
hG-CSF	53.40 ± 22.63^a	457.90 ± 129.50^c

Natural extract orally co-administered

PMT	55.50 ± 17.92^a	440.80 ± 115.78^c
SCGBT	47.70 ± 13.34^a	444.10 ± 105.38^c
AR	52.50 ± 11.74^a	431.90 ± 127.46^c
CCP	73.00 ± 12.94^ab	590.10 ± 134.67^c
AGR	57.10 ± 16.93^a	444.80 ± 92.76^c

Although significant (p<0.01) increases in total numbers of blood leukocytes and bone marrow nuclear cells were seen in the hG-CSF control group compared to the normal media control group, significant changes in the total numbers of blood leukocytes and bone marrow nuclear cells were not seen in any of the natural product extract co-administration groups except that noticeable increases in the total numbers of blood leukocytes and bone marrow nuclear cells were seen in the cervi cornu parvum co-administration group.
The hG-CSF control group exhibited a 955.34% change in the total number of blood leukocytes compared to the normal media control group, and the groups to which 200 mg/kg of PMT, SCGBT, astragali radix, cervi cornu parvum, and angelicae gigantis radix extracts were administered exhibited changes of 3.93, −10.67, −1.69, 36.70, and 6.93%, respectively, compared to the hG-CSF control group.
The hG-CSF control group exhibited a 872.19% change in the total number of bone marrow nuclear cells compared to the normal media control group, and the groups to which 200 mg/kg of PMT, SCGBT, astragali radix, cervi cornu parvum, and angelicae gigantis radix extracts were administered exhibited changes of −3.73, −3.01, −5.68, 28.87, and −2.86%, respectively, compared to the hG-CSF control group.
Consequently, it was confirmed that the total numbers of blood leukocytes and bone marrow nuclear cells were not increased compared to the hG-CSF control group when the angelicae gigantis radix extract was administered, but remained at a similar increasing level.

Example 5

Results of FACs: Confirmation of Numerical Chances in CD34+ and CD45+ Cells in Blood and Bone Marrow

Numerical changes in CD34+ and CD45+ cells in blood and bone marrow were observed by a method using the FACs, and the results thereof are illustrated in Table 7.

	TABLE 7

	Blood leukocytes	Bone marrow nuclear cells

	CD34+ cells	CD45+ cells	CD34+ cells	CD45+ cells
	(×10²	(×10²	(×10²	(×10²
Groups	cells/μl)	cells/μl)	cells/μl)	cells/μl)

Controls

Intact	7.93 ± 1.05	10.41 ± 1.47	16.60 ± 3.76	13.16 ± 2.32
vehicle
hG-CSF	26.81 ± 6.02^a	42.94 ± 8.62^a	30.41 ± 6.69^a	24.63 ± 3.82^a

Natural extract orally co-administered

PMT	43.40 ± 5.46^ab	61.99 ± 3.32^ab	45.09 ± 5.83^ab	32.35 ± 1.77^ab
SCGBT	22.14 ± 5.46^a	41.76 ± 6.09^a	31.21 ± 1.60^a	25.37 ± 2.47^a
AR	25.10 ± 6.36^a	45.28 ± 8.58^a	32.42 ± 3.31^a	23.97 ± 2.46^a
CCP	40.66 ± 2.59^ab	56.79 ± 6.14^ab	40.44 ± 1.13^ab	28.44 ± 4.73^a
AGR	41.25 ± 6.00^a	61.28 ± 7.68^ab	44.34 ± 3.59^ab	31.17 ± 2.59^ab

Values are expressed mean ± S.D. of 5 mice

5.1. Numerical Changes in CD34+ Cells in Blood and Bone Marrow
FACS observation results of CD34+ cells are illustrated in FIGS. 4 and 5. When the results are viewed in conjunction with Table 7, significant (p<0.01) numerical increase in CD34+ cells in blood and bone marrow was seen in the hG-CSF control group compared to the normal media control group, and significant (p<0.01) numerical increases in CD34+ cells in blood and bone marrow were seen in the groups to which PMT, cervi cornu parvum, and angelicae gigantis radix extracts were administered even when compared to the hG-CSF control group. Meanwhile, significant numerical increases in CD34+ cells in blood and bone marrow were not seen in the groups to which SCGBT and astragali radix extracts were administered compared to the hG-CSF control group.
The hG-CSF control group exhibited a 238.08% change in the number of CD34+ cells in blood compared to the normal media control group, and the groups to which 200 mg/kg of PMT, SCGBT, astragali radix, cervi cornu parvum, and angelicae gigantis radix extracts were administered exhibited changes of 61.89, −17.41, −6.39, 51.65, and 53.85%, respectively, compared to the hG-CSF control group.
The hG-CSF control group exhibited a 83.14% change in the number of CD34+ cells in bone marrow compared to the normal media control group, and the groups to which 200 mg/kg of PMT, SCGBT, astragali radix, cervi cornu parvum, and angelicae gigantis radix extracts were administered exhibited changes of 48.29, 2.64, 6.62, 33.00, and 45.80%, respectively, compared to the hG-CSF control group.
By the above results, compared with a case only G-CSF is administered, it was confirmed that the numbers of CD34+ cells in the blood and the bone marrow significantly increase when the angelicae gigantis radix extract is co-administered with the G-CSF.
5.2. Numerical Changes in CD45+ Cells in Blood and Bone Marrow
FACs observation results of CD45+ cells are illustrated in FIGS. 6 and 7. When the results are viewed in conjunction with Table 7, significant (p<0.01) numerical increase in CD45+ cells in blood and bone marrow was seen in the hG-CSF control group compared to the normal media control group, and significant (p<0.01) numerical increases in CD45+ cells in blood and bone marrow were seen in the groups to which PMT, cervi cornu parvum, and angelicae gigantis radix extracts were administered even when compared to the hG-CSF control group. Meanwhile, significant numerical increases in CD45+ cells in blood and bone marrow were not seen in the groups to which SCGBT and astragali radix extracts were administered compared to the hG-CSF control group.
The hG-CSF control group exhibited a 312.39% change in the number of CD45+ cells in blood compared to the normal media control group, and the groups to which 200 mg/kg of PMT, SCGBT, astragali radix, cervi cornu parvum, and angelicae gigantis radix extracts were administered exhibited changes of 44.38, −2.75, 5.45, 32.26, and 42.72%, respectively, compared to the hG-CSF control group.
The hG-CSF control group exhibited a 87.18% change in the number of CD45+ cells in bone marrow compared to the normal media control group, and the groups to which 200 mg/kg of PMT, SCGBT, astragali radix, cervi cornu parvum, and angelicae gigantis radix extracts were administered exhibited changes of 31.35, 3.02, −2.68, 15.47, and 26.58%, respectively, compared to the hG-CSF control group.
By comparing the above results with a case in which only G-CSF is administered, it was confirmed that the numbers of CD45+ cells in the blood and the bone marrow significantly increase when the angelicae gigantis radix extract is co-administered with the G-CSF.
Consequently, referring to the results of examples 5.1 and 5.2, it can be confirmed that the co-administration of the angelicae gigantis radix extract significantly increases proliferation of the bone marrow derived stem cells.

Example 6

Confirmation of Histopathological Chances

Histopathological changes of spleens and femoral bone marrow were observed in eight groups and values thereof are illustrated in Table 8 below.

	TABLE 8

	Spleen

			Mean	Mean number	Bone marrow
	Total	Number of	diameters of	of red pulp	Mean number
	thickness	white pulp	white pulp	nuclear	of nuclear
	(mm/central	(white	(μm/white	cells(×10²	cells (×10²
Groups	regions)	pulps/mm²)	pulp)	cells/mm2)	cells/mm²)

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 mean ± S.D. of 10 mice

6.1. Histopathological Change in Spleens
As can be seen in Table 8 above, the hG-CSF control group showed a feature of splenomegaly caused by significant infiltration of nuclear cells in red pulp of spleens. Although significant (p<0.01) increases in the total thicknesses of the spleens and nuclear cells per unit area of the red pulp were seen compared to the normal media control group, the amount and the diameter of white pulp was observed to be similar with the normal media control group.
FIG. 8 is a picture illustrating total thicknesses of the spleens and the numbers of nuclear cells per unit area of the red pulp and the white pulp of the spleens. As can be seen in FIG. 8, although the groups to which PMT and angelicae gigantis radix extracts were administered showed significant (p<0.01) decreases in the total thicknesses of the spleens and the numbers of nuclear cells per unit area of the red pulp of the spleens compared to the hG-CSF control group, the SCGBT administration group showed a significant (p<0.01) increase in the total thickness of the spleen compared to the hG-CSF control group, and the groups to which the astragali radix and cervi cornu parvum extracts were administered showed no significant histopathological changes in the spleens compared to the hG-CSF control group.
The hG-CSF control group exhibited a 17.47% change in the total thickness of the spleen compared to the normal media control group, and the groups to which 200 mg/kg of PMT, SCGBT, astragali radix, cervi cornu parvum, and angelicae gigantis radix extracts were administered exhibited changes of −12.02, 10.72, −0.48, 7.81, and −9.75%, respectively, compared to the hG-CSF control group.
The hG-CSF control group exhibited a 0.69% change in the amount of white pulp in the spleen compared to the normal media control group, and the groups to which 200 mg/kg of PMT, SCGBT, astragali radix, cervi cornu parvum, and angelicae gigantis radix extracts were administered exhibited changes of −3.42, −4.11, 1.37, 2.05, and 0.00%, respectively, compared to the hG-CSF control group.
The hG-CSF control group exhibited a 4.17% change in the mean diameter of the white pulp of the spleen compared to the normal media control group, and the groups to which 200 mg/kg of PMT, SCGBT, astragali radix, cervi cornu parvum, and angelicae gigantis radix extracts were administered exhibited changes of 3.09, 1.34, 0.77, 1.96, and −1.26%, respectively, compared to the hG-CSF control group.
The hG-CSF control group exhibited a 714.75% change in the number of nuclear cells per unit area of the red pulp of the spleen compared to the normal media control group, and the groups to which 200 mg/kg of PMT, SCGBT, astragali radix, cervi cornu parvum, and angelicae gigantis radix extracts were administered exhibited changes of −29.76, 6.39, 12.76, −1.55, and −35.21%, respectively, compared to the hG-CSF control group.
6.2. Histopathological Change in Femoral Bone Marrow
As can be seen in Table 8 above, the hG-CSF control group showed significant proliferation of granulocytes. As illustrated in FIG. 9, although a significant (p<0.01) increase in nuclear cells per unit area of bone marrow was seen in comparison to the normal media control group, no significant histopathological changes in femoral bone marrow were seen in any of the groups to which natural product derived extracts were administered compared with the hG-CSF control group.
The hG-CSF control group exhibited a 357.02% change in the number of nuclear cells per unit area of the femoral bone marrow compared to the normal media control group, and the groups to which 200 mg/kg of PMT, SCGBT, astragali radix, cervi cornu parvum, and angelicae gigantis radix extracts were administered exhibited changes of −2.75, −5.02, −9.79, 7.40, and −9.68%, respectively, compared to the hG-CSF control group.
From examples 6.1 and 6.2, it can be confirmed that the co-administration of the angelicae gigantis radix can prevent the side effects such as splenomegaly caused by administration of only the G-CSF, and increase leukocytes at the same time.

Example 7

Confirmation of Area Histochemical Chance

Numbers of CD34 and CD45 immunoreactive cells in spleens and bone marrow with respect to seven groups are illustrated in Table 9 below.

	TABLE 9

	Spleen immunoreactive cell	Bone marrow immunoreactive cell
	numbers (cells/mm²)	numbers (cells/mm²)

Groups	CD34+	CD45+	CD34+	CD45+

Controls

Intact	16.40 ± 2.72	44.90 ± 15.04	11.40 ± 3.92	35.80 ± 11.86
vehicle
hG-CSF	133.40 ± 20.30^c	303.50 ± 79.69^c	46.20 ± 9.74^c	325.40 ± 98.79^a

Natural extract orally co-administered

PMT	174.60 ± 18.35^cd	405.21 ± 64.22^cd	74.20 ± 10.88^cd	490.10 ± 112.39^ab
SCGBT	137.70 ± 20.82^c	300.50 ± 70.36^c	41.30 ± 6.13^c	350.00 ± 70.41^a
AR	146.80 ± 14.77^c	327.50 ± 46.42^c	49.40 ± 8.83^c	364.90 ± 116.30^a
CCP	125.50 ± 13.74^c	478.80 ± 111.65^cd	42.20 ± 6.98^c	344.40 ± 102.18^a
AGR	160.60 ± 14.21^cd	462.40 ± 80.42^cd	81.00 ± 19.10^cd	536.70 ± 82.72^ab

Values are expressed mean ± S.D. of 10 mice

7.1. Numerical Changes in CD34 Immunoreactive Cells in Spleens and Bone Marrow
As can be seen in Table 9 and FIG. 9, the hG-CSF control group showed a significant (p<0.01) numerical increase in CD34 immunoreactive cells in spleens and bone marrow compared to the normal media control group, and the groups to which PMT and angelicae gigantis radix extracts were administered showed significant (p<0.01) numerical increases in CD34 immunoreactive cells in the spleens and the bone marrow even when compared to the hG-CSF control group. Meanwhile, no significant numerical changes in CD34 immunoreactive cells per unit area of the spleens and the bone marrow were seen in the groups to which SCGBT, cervi cornu parvum, and astragali radix extracts were administered compared to the hG-CSF control group.
The hG-CSF control group exhibited a 713.41% change in the number of CD34 immunoreactive cells in the spleen compared to the normal media control group, and the groups to which 200 mg/kg of PMT, SCGBT, astragali radix, cervi cornu parvum, and angelicae gigantis radix extracts were administered exhibited changes of 30.88, 3.22, 10.04, −5.92, and 20.39%, respectively, compared to the hG-CSF control group.
The hG-CSF control group exhibited a 305.26% change in the number of CD34 immunoreactive cells in the bone marrow compared to the normal media control group, and the groups to which 200 mg/kg of PMT, SCGBT, astragali radix, cervi cornu parvum, and angelicae gigantis radix extracts were administered exhibited changes of 60.61, −10.61, 6.93, —8.87, and 75.32%, respectively, compared to the hG-CSF control group.
7.2. Numerical Changes in CD45 Immunoreactive Cells in Spleens and Bone Marrow
As can be seen in Table 9 and FIG. 10, the hG-CSF control group showed a significant (p<0.01) numerical increase in CD45 immunoreactive cells in spleens and bone marrow compared to the normal media control group, and the groups to which PMT and angelicae gigantis radix extracts were administered showed significant (p<0.01) numerical increases in CD45 immunoreactive cells in the spleens and the bone marrow even when compared to the hG-CSF control group. Meanwhile, although a significant (p<0.01) numerical increase in the CD45 immunoreactive cells in the spleen was seen in the cervi cornu parvum extract administration group compared to the hG-CSF control group, the CD45 immunoreactive cells in the bone marrow were observed to be similar with the hG-CSF control group, and no significant numerical changes in CD45 immunoreactive cells per unit area of the spleens and the bone marrow were seen in the groups to which SCGBT and astragali radix extracts were administered compared to the hG-CSF control group.
The hG-CSF control group exhibited a 575.95% change in the number of CD45 immunoreactive cells in the spleen compared to the normal media control group, and the groups to which 200 mg/kg of PMT, SCGBT, astragali radix, cervi cornu parvum, and angelicae gigantis radix extracts were administered exhibited changes of 33.51, −0.99, 7.91, 57.26, and 52.36%, respectively, compared to the hG-CSF control group.
The hG-CSF control group exhibited a 808.94% change in the number of CD45 immunoreactive cells in the bone marrow compared to the normal media control group, and the groups to which 200 mg/kg of PMT, SCGBT, astragali radix, cervi cornu parvum, and angelicae gigantis radix extracts were administered exhibited changes of 50.61, 7.56, 12.14, 5.84, and 64.94%, respectively, compared to the hG-CSF control group.
From the above results, it was confirmed that the co-administration of the angelicae gigantis radix extract can significantly increase the number of immunoreactive cells compared to the case in which only the G-CSF is administered.
To summarize the results of examples 2 to 7, a significant increase in the weight of spleens, an increase in total numbers of nuclear cells in blood and bone marrow, and numerical increases in CD34+ and CD45+ cells in the blood and the bone marrow were seen, increases in total thicknesses of spleens and numerical increases in nuclear cells per unit area of red pulp and femoral bone marrow were seen in a histopathological aspect, and numerical increases in CD34 and CD45 immunoreactive cells in the spleens and the bone marrow were seen in an immunohistochemical aspect.
Consequently, by the oral co-administration of the angelicae gigantis radix extract within five minutes of administration of the hG-CSF, proliferation and mobilization of bone marrow derived stem cells by the hG-CSF were significantly (p<0.01) increased, an increase in the weights of spleens and features of splenomegaly due to proliferation of nuclear cells in red pulp were significantly (p<0.01) suppressed, and proliferation of overall granulocytes was not affected.
Meanwhile, although significant proliferation and mobilization of the bone marrow derived stem cells were also seen in the cervi cornu parvum extract administration group, the features of splenomegaly actually worsened. Administering the SCGBT and the astragali radix extracts was observed not to affect granulocytes of hG-CSF and proliferation and mobilization of the bone marrow derived stem cells, and was also observed not to affect side effects such as splenomegaly.
Consequently, the angelicae gigantis radix extract is expected to provide a new method of fusing western and oriental medicine which is very useful in enhancing an effect of hG-CSF in mobilizing the bone marrow derived stem cells and mitigating side effects such as splenomegaly.
The above description of the present invention is for illustrative purposes, and those of ordinary skill in the art will be able to understand that the examples of the present invention may be easily modified in other specific forms without changing the technical spirit or essential characteristics of the present invention. Therefore, the above-described examples should be construed as being illustrative in every aspect, not limitative.

Claims

1. A pharmaceutical composition for promoting proliferation of bone marrow derived stem cells, the composition comprising:

a granulocyte colony-stimulating factor; and

an angelicae gigantis radix extract,

wherein the stem cells are CD34+ cells.

2. The composition according to claim 1, wherein the stimulating factor and the angelicae gigantis radix extract are mixed in advance and made into a dosage form, or separately made into a dosage form.

3. The composition according to claim 1, wherein the stimulating factor and the angelicae gigantis radix extract are administered parenterally, orally, locoregionally, or percutaneously.

4. The composition according to claim 1, wherein a period of administering the angelicae gigantis radix extract begins within thirty minutes after administration of the stimulating factor.