U.S. patent application number 10/568257 was filed with the patent office on 2007-01-18 for composition comprising notoginseng radix extract for preventing and treating of arthritis as an effective ingredient.
Invention is credited to Dong-Heon Baek, Sunhwa Chang, Youngaim Choi, Dong Sik Jung, Hyung-Gun Kim, Jong-Yeo Kim, Jung-Keun Kim, So-Won Kim, Seon-Yle Ko, Seong-Hee Ko, Byung-Eui Lee, Haejong Woo.
Application Number | 20070014879 10/568257 |
Document ID | / |
Family ID | 36204400 |
Filed Date | 2007-01-18 |
United States Patent
Application |
20070014879 |
Kind Code |
A1 |
Kim; Jung-Keun ; et
al. |
January 18, 2007 |
Composition comprising notoginseng radix extract for preventing and
treating of arthritis as an effective ingredient
Abstract
The present invention relates to a composition comprising
Notoginseng radix extract for preventing and treating arthritis as
an effective ingredient. Notoginseng radix extract of the present
invention inhibits release of tumor necrosis factor-alpha
(TNF-.alpha.) and is the death of activated T-cells only, so that
it can be effectively used for the production of a medicine for
preventing and treating arthritis and health food as well.
Inventors: |
Kim; Jung-Keun; (Kyunggi-do,
KR) ; Kim; So-Won; (Chungcheongnam-do, KR) ;
Kim; Hyung-Gun; (Seoul, KR) ; Ko; Seon-Yle;
(Kongju-si, KR) ; Kim; Jong-Yeo;
(Chungcheongnam-do, KR) ; Chang; Sunhwa;
(Cheonan-si, Chungcheongnam-do, KR) ; Baek;
Dong-Heon; (Seoul, KR) ; Lee; Byung-Eui;
(Daejeon-si, KR) ; Ko; Seong-Hee; (Gangwon-do,
KR) ; Choi; Youngaim; (Seoul, KR) ; Jung; Dong
Sik; (Chungcheongnam-do, KR) ; Woo; Haejong;
(Seoul, KR) |
Correspondence
Address: |
JHK Law
P.O. Box 1078
La Canada
CA
91012-1078
US
|
Family ID: |
36204400 |
Appl. No.: |
10/568257 |
Filed: |
September 6, 2004 |
PCT Filed: |
September 6, 2004 |
PCT NO: |
PCT/KR04/02255 |
371 Date: |
February 13, 2006 |
Current U.S.
Class: |
424/728 |
Current CPC
Class: |
A61P 29/00 20180101;
A61K 36/258 20130101; A61P 19/02 20180101 |
Class at
Publication: |
424/728 |
International
Class: |
A61K 36/254 20060101
A61K036/254 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 6, 2003 |
KR |
10-2003-0062418 |
Claims
1. A composition comprising Notoginseng radix extract for
preventing and treating arthritis as an effective ingredient.
2. The composition for preventing and treating arthritis as set
forth in claim 1, wherein the Notoginseng radix extract is
extracted by water, alcohol or a mixed solvent of water and
alcohol.
3. The composition for preventing and treating arthritis as set
forth in claim 1, wherein the alcohol is ethanol.
4. A pharmaceutical composition for preventing and treating
arthritis comprising the composition of anyone of claim 1 to claim
3.
5. A health food composition for preventing and treating arthritis
comprising the composition of anyone of claim 1 to claim 3.
Description
TECHNICAL FIELD
[0001] The present invention relates to a composition comprising
Notoginseng radix extract for preventing and treating arthritis as
an effective ingredient.
BACKGROUND ART
[0002] Arthritis related diseases are the representative
degenerative intractable diseases, which give 12% of total earth
population pain. And over 2 million people are suffering from such
diseases in Korea.
[0003] Arthritis is the general term for symptoms over all the
musculoskeletal system caused by inflammatory changes in
musculoskeletal and connective tissues. The disease is
characterized by chronic inflammation causing permanent damage in
tissues, deformity, degeneration and troubles by having an effect
on joint, bone, cartilage or the spinal cord (Hofbause, L C,
Heufelder, A E: The role of osteoprotegerin and receptor activator
of nuclear factor kappaB ligand in the pathogenesis and treatment
of rheumatoid arthritis, Arthritis and Rheumatism 44:253-259,
2001).
[0004] Arthritis is classified into degenerative arthritis
(osteoarthritis), rheumatoid arthritis, non-joint rheumatism or
collagen disease.
[0005] Degenerative arthritis, which is the most common of all
arthritis related diseases, is developed by local degeneration by
the worn-out of joint cartilage. The cause of the disease is still
unclear but aging or over-weight might be the reason. Primarily,
degenerative changes appear in joint cartilage. Degeneration first
begins in joint cartilage and kills chondrocytes and then cartilage
matrix is destroyed by cathepsin B, cathepsin D, collagenase, etc.
The destruction outpaces the generation of proteoglycan and
collagen, and adaptability of cartilage to outside force becomes
weaker, resulting in microfractures in subchondral bone tissues. As
the disease progresses, the hardening of subchondral bone,
over-ossification around joint, joint deformation, etc. are
observed. Then, the surface of cartilage becomes rough and
inflammation in joint cavity enveloped by joint capsule repeats,
resulting in constant pain, ankylosis and gradual motor disturbance
in joint.
[0006] Rheumatoid arthritis is a chronic inflammatory disease over
the whole body and its symptoms occur symmetrically to movable
joints. The disease is also known as an autoimmune disease caused
by malfunction of immune system. However, the cause of the disease
is still in question. Rheumatoid arthritis is characterized by
continuous inflammatory synovitis causing the destruction of
cartilage and bone erosion, resulting in deformity of joint
structure. Symptoms of rheumatoid arthritis are joint edema, joint
tenderness, inflammation, morning stiffness and acute pain with
bending. As the disease progresses, structural damage can be found
such as bone erosion and joint destruction (Firestein, G S:
Evolving concept of rheumatoid arthritis. Nature 423:356-361,
2003). In addition, a patient with rheumatoid arthritis might
suffer from other symptoms by additional organ damage, for example
damage of skin, kidney, heart, lung, central nervous system and
eye, which is resulted from vasculitis related to autoimmune
process.
[0007] Arthritis related symptoms include acceleration of
erythrocyte sedimentation rate and increase of the concentration of
serum C-reactive protein (CRP) or soluble IL-2 receptor (IL-2r).
The acceleration of erythrocyte sedimentation rate is detected in
almost every active rheumatoid arthritis patients. The
concentration of serum C-reactive protein also increases in those
patients. It is related to the activation of the disease and the
possibility of progressive joint damage. The concentration of
soluble IL-2r, a product of T-cell activation, increases in serum
and synovial fluid of active rheumatoid arthritis patients, too
(Udagawa, N., Kotake, S., Kamatani, N., Takahashi, N., and Suda, T:
The molecular mechanism of osteoclastogenesis in rheumatoid
arthritis. Arthritis Research 4:281-289, 2002).
[0008] It is generally believed that Th1 type CD4+ T cells play an
important role in the progress and continuation of rheumatoid
arthritis. That is, CD4+ T lymphocytes stimulate macrophages and
synovial cells to have inflammatory cytokines (TNF-.alpha.,
IFN-.gamma., GM-CSF, IL-2, IL-6) and matrix metalloproteinase
secreted, for which signals were transmitted by soluble materials
such as interferon-gamma (IFN-.gamma.) and IL-17 and by cell
surface component such as CD69. The secreted cytokines stimulate
the proliferation of synovial membrane to form a pannus and destroy
cartilage in cooperation with matrix metalloproteinase. The
activated CD4+ T cells induce the activation of B cells through the
contact with them on cell surface by CD40L, CD28, and a1b2
integrin, leading to the production of antibody containing
rheumatoid factors. When CD4+ T cells are activated,
osteoprotegerin ligand is expressed on the surface, which
stimulates osteoclastogenesis, an important factor for bone
destruction (Kong Y Y, Feige U, Sarosi I., et al.: Activated T
cells regulate bone loss and joint destruction in adjuvant
arthritis through osteoprotegerin ligand. Nature 402, 304-309,
1999). The activated macrophages and fibroblasts accelerate
angiogenesis by secreting VEGF, FGF, etc. The activated vascular
endothelial cells in synovial membrane make an amplified cycle of
inflammation by secreting chemokine such as IL-8, inducing the
expression of adhesion molecule and speeding up the infiltration of
inflammatory cells. Rheumatoid arthritis is also believed to be a
T-cell mediated autoimmune disease, which is related to the
antigen-nonspecific intracellular interaction between T-lymphocytes
and antigen-presenting cells. The reaction size of T-cells is
determined by simultaneous stimuli induced by the interaction
between a T-cell surface molecule and its' ligand. A major
simultaneous stimulus signal is given by the interaction between
T-cell surface receptors, CD28 and CTLA4, and their ligands such as
B7-related molecules on antigen-presenting cells, that is CD80
(B7-1) and CD86 (B7-2) (Linsley, P. and Ledbetter, J.: The role of
the CD28 receptor during T cell responses to antigen. Ann. Rev.
Immunol. 11:191-212, 1993). T-cell activation without simultaneous
stimuli results in anergic T-cell response, indicating that immune
system does not response to a stimulus [Schwartz, R. H.:
Costimulation of T lymphocytes: the role of CD28, CTLA-4, and
B7/BB1 in interleukin-2 production and immunotherapy. Cell
71:1065-1068, 1992].
[0009] Fundamental treatment of arthritis to cure the cause is
still far, and all the medicines developed so far are just for
relieving a pain, inhibiting inflammation or keeping the function
as it is. Such medicines are supposed to be administered for a long
time, but long-term administration of those drugs cause side
effects in gastrointestinal system, central nervous system,
hematopoietic organ, kidney, liver, etc. (Langenegger T, Michel B
A.: Drug treatment for rheumatoid arthritis. Clin. Orthop.
366:22-30, 1999).
[0010] As explained hereinbefore, arthritis related diseases are
considered to be chronic inflammatory diseases and T-cell medicated
immune system disorders, so that it is an urgent need, for the
treatment of such diseases, to develop a medicine to inhibit
release of cytokine and to destroy activated T cells
selectively.
[0011] The present inventors have made every effort to find out a
material from herb medicines that can inhibit release of cytokine
and destroy activated T-cells only. And the present inventors have
completed this invention by confirming that Notoginseng radix
extract can inhibit separation of cytokine and destroy activated
T-cells only.
DISCLOSURE OF INVENTION
Technical Solution
[0012] It is an object of the present invention to provide a
composition comprising Notoginseng radix extract for preventing and
treating arthritis as an effective ingredient.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] The application of the preferred embodiments of the present
invention is best understood with reference to the accompanying
drawings, wherein:
[0014] FIG. 1 is a schematic diagram showing the method for
extracting and separating Notoginseng radix extract of the present
invention,
[0015] FIG. 2 is a graph showing the effect of Notoginseng radix
extract of the present invention on release of tumor necrosis
factor-alpha (TNF-.alpha.),
[0016] FIG. 3 is a graph showing that Notoginseng radix extract of
the present invention destroys activated T-cells selectively,
[0017] FIG. 4 is a graph showing the inhibiting effect of
Notoginseng radix extract of the present invention on the arthritis
progress tested by using animals with type 2 collagen induced
arthritis, which is presented by arthritis index,
[0018] FIG. 5 is a set of photographs showing the inhibiting effect
of Notoginseng radix extract of the present invention on the
arthritis progress tested by animals with type 2 collagen induced
arthritis.
BEST MODE FOR CARRYING OUT THE INVENTION
[0019] In order to achieve the above object, the present invention
provides a composition comprising Notoginseng radix extract for
preventing and treating arthritis as an effective ingredient.
[0020] The composition of the present invention includes a
pharmaceutical composition for preventing and treating arthritis
and a composition for health food
[0021] Notoginseng radix extract of the present invention inhibits
release of tumor necrosis factor-alpha (TNF-.alpha.) and is the
death of activated T-cells selectively, so that it can be
effectively used for the production of improved health food or the
development of a medicine for preventing and treating
arthritis.
[0022] Notoginseng radix is a root of a perennial herb belonging to
Panax notoginseng (Burk.) F. H. Chen. It is smaller than a ginseng
and has 7 pieces of leaves. Its' root is in a small thread drum
shape and it is raised widely in Yunnan and Sichuan, southern
China. Since the plant has 7 leaves on three branches, it has been
called `Samchil (three-seven)` and often called `Samchil ginseng`
owing to its similar appearance to Korean ginseng. The root has
3-8% saponin and its' major components are ginsenoside Rb1, Rg1 and
Re, and notoginsenoside R1, R2, Fa and Fc, but small amount of
ginsenoside R2, b2, d, e, c are also included. R0 is not contained
or if it is, it must be least. Essential oil composition is fewer
in Notoginseng radix than in Panax ginseng. Notoginseng radix
additionally includes oleanolic acid. Its' root has hemostatic and
cardiotonic activities. It was confirmed from animal tests that the
root has efficacy of increasing blood flow of coronary artery,
decreasing oxygen consumption of cardiac muscle and lowering the
levels of lipid and cholesterol in blood. Notoginseng radix also
has functions of anti-inflammation, analgesia and hemostasis, so
that it is very useful for the treatment of not only inflammatory
diseases including hepatitis but also bleeding from trauma, cut,
etc., and internal hemorrhage. Applying to a wound or oral
administration give the same effects.
[0023] Notoginseng radix extract of the present invention is
extracted by using water, alcohol or a mixed solvent of water and
alcohol. At this time, alcohol is preferred to be ethanol.
[0024] Conventional extraction methods including cold
precipitation, hot precipitation, heating, etc., using the solvent
mentioned above are used.
[0025] Notoginseng radix extract of the present invention inhibits
release of tumor necrosis factor-alpha (TNF-.alpha.), so that it
can be used for the production of health food or a medicine for
preventing and treating arthritis.
[0026] In order to investigate how Notoginseng radix extract of the
present invention worked to inhibit release of tumor necrosis
factor-alpha (TNF-.alpha.), THP-1 cells, a human monocytic cell
line, were treated with lipopolysaccharide (LPS) and Notoginseng
radix extract of the present invention at the concentration of 2 or
10 .mu.l/ml. Then, the amount of released tumor necrosis
factor-alpha (TNF-.alpha.) in cell culture medium was measured by
ELISA. As a result, the amount of released tumor necrosis
factor-alpha (TNF-.alpha.) was remarkably decreased by the
treatment of 10 .mu.l/ml of Notoginseng radix extract of the
present invention (see Experimental Example 1).
[0027] Notoginseng radix extract of the present invention can be
used for the production of health food or a medicine for preventing
and treating arthritis owing to its ability to death activated
T-cells selectively.
[0028] In order to investigate whether or not Notoginseng radix
extract of the present invention was able to death activated
T-cells only, a lymph node of a 5-week-old female mouse was taken
and single cells were prepared. The cells were cultured, during
which T cells were activated The apoptosis of activated
T-lymphocytes was investigated. As a result, when cells were
treated with over 5 .mu.l/ml of Notoginseng radix extract of the
present invention, only activated T-cells were killed (inactivated
T-cells were still alive) (see Experimental Example 2).
[0029] Notoginseng radix extract of the present invention also
inhibits the progress of the disease in animals having type 2
collagen induced arthritis.
[0030] In order to investigate the treatment effect on arthritis of
Notoginseng radix extract of the present invention, collagen
suspension was intra-dermally injected in tail head of a mouse to
induce arthritis. Notoginseng radix extract of the present
invention was orally administered to the mouse with arthritis,
which was then observed. As a result, the progress of arthritis was
remarkably inhibited from the 9.sup.th day after oral
administration of the extract (see Experimental Example 3).
[0031] A composition of the present invention can additionally
include, in addition to Notoginseng radix extract, one or more
effective ingredients having a similar to or the same function as
Notoginseng radix extract.
[0032] A composition of the present invention can additionally
include, in addition to Notoginseng radix extract, one or more
effective ingredients having a different function from that of
Notoginseng radix extract.
[0033] A composition of the present invention can contain at least
one of pharmaceutically acceptable carriers, in addition to the
above effective ingredients, for the convenience of the
administration. Pharmaceutically acceptable carriers can be
selected from a group consisting of saline, sterile water, Ringer's
solution, buffered saline, dextrose solution, maltodextrin
solution, glycerol, ethanol and a mixture of them (one or more
components). If necessary, other additives such as anti-oxidants,
buffers, fungistats, etc., can be included. A composition of the
present invention can also be prepared in the forms of pills,
capsules, granules, tablets and injectable solutions such as
acqueous solutions, suspensions, emulsions, etc., produced by being
mixed with generally used diluents, disintegrating agents,
surfactants, binders and lubricants. Besides, a composition of the
present invention can be prepared in different forms considering a
disease and included ingredients by general method well-known to
the people in this field or the method described in Remington's
Pharmaceutical science (Newest edition), Mack Publishing Company,
Easton Pa. Calcium or vitamin D.sub.3 can be added to a composition
of the present invention to enhance its medicinal effect of
preventing and treating arthritis.
[0034] The administration method of a composition of the present
invention varies from the purpose of the treatment; either oral
administration or parenteral administration (for example,
intravenous, intradermal, intraperitoneal or local injection) is
fine. And the dosage of the composition is determined awarding to
weight, age, gender, health condition of a patient, diet,
administration times and method, excretion rate, and severity of a
disease. The effective usage of Notoginseng radix extract of the
present invention is 0.1.about.10 mg/kg, and 0.1.about.3 mg/kg is
more preferable. The administration times can be once a day or
preferably several times a day.
[0035] The acute toxicity test in mice via oral administration was
performed to see if the Notoginseng radix extract of the present
invention has acute toxicity in mice. As a result, its estimated
LD.sub.50 values are much greater than 2 g/kg in mice, indicating
that this extract is evaluated to be a safe substance.
[0036] A composition of the present invention can be treated for
preventing and treating arthritis either independently or in
combination with surgical operation, radiotherapy, hormone therapy,
chemotherapy and other biological response regulators.
[0037] A composition of the present invention can be added to
health food to improve arthritis related diseases. Notoginseng
radix extract of the present invention can be added to food as it
is or together with other food or food ingredients by general
method for food process. The mixing ratio of effective ingredients
is determined by the purpose of use (for prevention, for promoting
health, or for treatment of a disease). In general, Notoginseng
radix extract of the present invention is added to food or
beverages under 100 weight %, preferably under 50 weight %.
However, in the case of long-term administration for the purpose of
health and sanitation or health control, the amount of a
composition added to food or beverages might be less than the
above, but since the composition is safe for human, it could be
added more than the above.
[0038] There is no limitation in food category applicable to the
extract of the present invention. So, the extract can be added to
meat, sausages, bread, chocolate, candies, snacks, cookies, pizza,
ramyun, noodles, gums, dairy product including ice cream, soups,
beverages, tea, drinks, alcoholic drinks and vitamin complex, etc.
and other ordinary health food.
[0039] A composition for health promoting beverages can
additionally include various flavors or natural carbohydrates, like
any other ordinary beverages. Natural carbohydrates are exemplified
by monosaccharides such as glucose and fructose, disaccharides such
as maltose and sucrose, polysaccharides such as dextrin,
cyclodextrin, and sugar alcohols such as xilytole, sorbitol and
erythritol. As a sweetening agent, natural sweeteners such as
thaumatin and stevia extract, and synthetic sweeteners such as
saccharin and aspartame can be used. It is preferred to add natural
carbohydrates by 0.1.about.20 g per 100 ml of a composition of the
present invention, and is more preferred to add 1.about.10 g of
natural carbohydrates to 100 ml of the composition.
[0040] In addition to the above, a composition of the present
invention can also include various nutrients, vitamins,
electrolytes, flavoring agents, coloring agents, pectic acid and
its salts, alginic acid and its salts, organic acids, protective
colloidal thickeners, pH regulators, stabilizers, antiseptics,
glycerin, alcohol, carbonating agents used in carbonated beverages,
etc. The composition of the present invention can further include
sarcocarps to produce natural fruit juices, fruit beverages and
vegetable beverages. Each ingredient is used either independently
or in combination with others. At this time, the mixing rate is not
so important but in general, 0.05.about.50 parts of weight per 100
parts of weight of the composition of the present invention is
preferred.
MODE FOR THE INVENTION
EXAMPLES
[0041] Practical and presently preferred embodiments of the present
invention are illustrative as shown in the following Examples.
[0042] However, it will be appreciated that those skilled in the
art, on consideration of this disclosure, may make modifications
and improvements within the spirit and scope of the present
invention.
Example 1
Preparation of Notoginseng radix Extract
[0043] Cultivated Notoginseng radix was purchased from a wholesale
dried medicinal herb store.
[0044] <1-1> Preparation of Notoginseng radix Crude
Extract
[0045] <1-1-1> Crude Alcohol Extract of Notoginseng radix
[0046] Notoginseng radix was cut into 1.about.2 cm fragments. The
fragments were washed with running water to eliminate impurities.
The fragments were pulverized. 200 g of the Notoginseng radix
powder was put in a 3 l flask, which was stirred at reflux at
78.5.degree. C. using 2,000 ml of ethanol. Extraction by heating
was repeated three times for 4 hours. The extract was filtered and
vacuum-concentrated under reduced pressure by using vacuum rotary
evaporator under 40.degree. C., resulting in Notoginseng radix
crude extract containing 2.7 g of Notoginseng radix powder (RF1M)
(yield: 1.35%).
[0047] <1-1-2> Crude Water Extract of Notoginseng radix
[0048] Notoginseng radix crude extract was extracted by the same
method as described in the above <1-1-1> and the only
difference in the procedure was that water was used instead of
ethanol as an extraction solvent.
[0049] <1-1-3> Crude Mixed Solvent Extract of Notoginseng
radix
[0050] Notoginseng radix crude extract was extracted by the same
method as described in the above <1-1-1> and the only
difference in the procedure was that a mixed solvent of water (25%)
and ethanol (75%) was used instead of ethanol as an extraction
solvent.
[0051] <1-2> Separation of Notoginseng radix Crude
Extract
[0052] A fraction (RF1MB) was obtained from the crude extract
(FF1M) prepared in the above <1-1-1> at room temperature by
using 500 ml of normal butanol (n-butanol) as a solvent, for which
a fraction funnel was used and solvent fractionation was repeated
three times.
[0053] RF1MB4 fraction was separated from the RF1MB fraction by
column chromatography. Column chromatography was performed again
with the RF1MB4 fraction, resulting in the final fraction of
Notoginseng radix extract (RF1MB4b).
[0054] Extraction and separation method of Notoginseng radix
extract of the present invention is described in FIG. 1.
[0055] In experimental examples of the invention, the final
extraction of Notoginseng radix extract (RF1MB4b) was concentrated
and then freeze-dried. The dried fraction was diluted with water
and used for in vitro and animal tests.
Experimental Example 1
Inhibition of the Release of TNF-.alpha. by Notoginseng radix
Extract of the Present Invention
[0056] Following experiments were performed to investigate whether
or not Notoginseng radix extract of the present invention inhibited
the release of TNF-.alpha., a cytokine separated from human
monocytic cell line `THP-1 cell`.
[0057] <1-1> Cell Selection and Culture
[0058] The below cell line was used to investigate the effect of
Notoginseng radix extract of the present invention on the release
of TNF-.alpha..
[0059] Human originated cell line THP-1 (ATCC No. TIB-202) was
purchased from ATCC (Rockville, USA) and cultured in RPMI 1640
(Gibco, BRL, USA) medium supplemented with 10% FBS (fetal bovine
serum).
[0060] <1-2> Quantification of released TNF-.alpha.
[0061] In order to investigate the effect of Notoginseng radix
extract of the present invention on the release of TNF-.alpha., the
amount of released TNF-.alpha. was measured by ELISA using cells
prepared in the above <1-1>.
[0062] Cells were plated into a 96-well plate by 5.times.10.sup.5
cells/ml and lipopolysaccharide (LPS) was added in order to
activate cells for the release of TNF-.alpha..
[0063] An experimental group was treated with Notoginseng radix
extract (RF1MB4b) at the concentration of 2 or 10 .mu.l/ml together
with LPS. After the treatment, the released TNF-.alpha. in culture
supernatant was quantified by ELISA.
[0064] The results are presented in FIG. 2.
[0065] As shown in FIG. 2, when an experimental group was treated
with low concentration (2 .mu.l/ml) of Notoginseng radix extract
(RF1MB4b), the amount of released TNF-.alpha. of the experimental
group was just a little different from that of a control group not
treated with the extract. But, when the extract was provided with
high concentration (10 .mu.l/ml), the amount of released
TNF-.alpha. in the experimental group was greatly decreased,
comparing to a control group.
[0066] Thus, the above results indicate that Notoginseng radix
extract of the present invention inhibits the release of
TNF-.alpha..
Experimental Example 2
Selective Apoptosis of Activated T-Cells by Notoginseng radix
Extract of the Present Invention
[0067] In order to confirm whether or not Notoginseng radix extract
of the present invention could destroy activated T-cells only,
following experiments were performed.
[0068] <2-1> Separation and Activation of T-Cells
[0069] A lymph node of a 5-week-old female mouse was taken out and
mashed by the back tip of a sterilized syringe to extract cells.
The cells were filtered by a cell-filter (Falcon, NJ USA) and
washed with PBS, then put in a culture medium at the concentration
of 2.times.10.sup.6cells/ml. As a culture medium, RPMI 1640 (Gibco,
BRL, USA) supplemented with 10% FBS (fetal bovine serum) was
used.
[0070] In order to activate T-cells only, concanavalin A was added
by 5 .mu.g/ml to the medium, followed by culture for 48 hours.
After 48 hours of culture, 10 mg/ml of
methyl-.alpha.-D-mannopyranoside (sigma, Germany) was put in the
medium, followed by further culture for 30 minutes. Then, the cells
were washed with PBS three times and put in a culture medium
supplemented with 100 units/ml of human interleukine-2 (hIL-2,
R&D, MN, USA), followed by further culture for 48 hours and
cell density was maintained as 2.times.10.sup.6 cells/mg during the
culture (Lenardo M J. et al.: Interleukin-2 programs mouse alpha
beta T lymphocytes for apoptosis. Nature. 353(6347):858-61.
1991).
[0071] <2-2> Investigation of Selective Apoptosis of
Activated T-Cells
[0072] The concentration of activated T-cells was adjusted to
1.times.10.sup.6 cells/ml, then they were put in a 96-well plate
(Falcon, USA) by 200 .mu.l/well. At that time, 100 units/ml of
human interleukine-2 (hIL-2) was added to each well.
[0073] While a control group was not treated with Notoginseng radix
extract, an experimental group was treated with the final fraction
(RF1MB4b) of Notoginseng radix extract prepared in the above
example at different concentrations (5 .mu.g/ml, 10 .mu.g/ml, 20
.mu.g/ml) before being cultured for 24 hours.
[0074] As a control, inactivated cells were prepared as
follows.
[0075] Single cells were collected from spleen and cell density was
adjusted to 2.times.10.sup.6 cells/ml, which were distributed to a
96 well plate by 200 .mu.l/well. Notoginseng radix extract was
added thereto, followed by culture for 24 hours. After 24 hours of
culture, the cells were transferred to a flow tube, to which
propidium iodide (PI) was added. Then, live cells were counted for
20 seconds by using CellQuest program of FACSCaliver (Becton
Dickinson, France).
[0076] Apoptosis was calculated as follows: (1-F extract treated
cells/untreated cells).times.100. All candidate drugs were examined
by that math formula to choose a drug to induce high apoptosis of
activated T-cells but low apoptosis of naive T-cells (Sabapathy K,
Hu Y, Kallunki T, Schreiber M, David J P, Jochum W, Wagner E F,
Karin M.: JNK2 is required for efficient T-cell activation and
apoptosis but not for normal lymphocyte development. Curr. Biol.
11;9(3): 116-25. 1999).
[0077] The results are presented in FIG. 3.
[0078] As shown in FIG. 3, when Notoginseng radix extract of the
present invention was treated with high concentration over 5
.mu.l/ml, activated T-cells were selectively destroyed while
inactivated T-cells still remained.
[0079] Thus, it was confirmed that Notoginseng radix extract of the
present invention destroys activated T-cells selectively and the
apoptosis effect was concentration-dependent.
Experimental Example 3
Inhibition of the Progress of Arthritis in Test Animals with Type 2
Collagen Induced Arthritis by Notoginseng radix Extract of the
Present Invention
[0080] In order to investigate whether or not Notoginseng radix
extract of the present invention could inhibit the progress of
arthritis in test animals having type 2 collagen induced arthritis,
following experiments were performed.
[0081] <3-1> Inducement of Arthritis in Test Animals
[0082] In order to prepare test animals having type 2 collagen
induced arthritis, 5-6 week old male DBA1 mice were purchased from
SCI company, Japan, and the mice were raised at 21.degree. C. with
40% humidity.
[0083] Bovine type 2 collagen (Condrex Co., Japan) was dissolved in
0.05% acetic acid, making the concentration 2 mg/ml. Then the type
2 collagen was mixed with the same amount of complete adjuvant
(Condrex Co., Japan). While cooling down with ice, the mixture
became homogeneous suspension by using T-connector linked to 3 ml
syringe. After confirming the suspension was prepared rightly, tail
head of a mouse was sterilized with alcohol cotton and 100 .mu.l of
collagen suspension was injected under the skin of the tail
head.
[0084] <3-2> Oral Administration of Notoginseng radix Extract
(RF1MB4b) of the Present Invention
[0085] Notoginseng radix extract (RF1MB4b) prepared in the above
example was dissolved in water, resulting in 2.5 mg/ml solution.
The solution was filtered by 0.25 uM filter.
[0086] The filtered solution was diluted to 0.2 mg/ml and was
administered to the mouth of a mouse through sonde linked to a 1 ml
syringe, once a day and by 0.05 mg/250 .mu.l/mouse.
[0087] <3-3> Progress of Arthritis: Naked Eye Observation and
Diagnosis
[0088] In order to investigate arthritis treating effect of
Notoginseng radix extract (RF1MB4b) of the present invention, the
Notoginseng radix extract (RF1MB4b) prepared in the above example
was administered by the same method as described in the above
<3-2> to test animals having arthritis induced by the
injection of collagen suspension.
[0089] Arthritis was developed 30 days after collagen suspension
was injected to a mouse. Naked eye observation on lesion of
arthritis was performed by using following scores based on
literature cited.
[0090] 0: No swelling or flair, 1: Light swelling and flair in
joint, 2: Clear swelling and flair in joint, 3: Severe swelling and
flair in joint including knuckle joint, 4: Severe swelling in all
over the joint.
[0091] Therefore, the highest score of lesion of arthritis is 16
per mouse, which sums up scores of forelegs and hind legs, and the
highest score per one leg is 4 (Courtenay J S, Dallman M J, Dayan A
D, et al.: Immunization against heterologous type II collagen
induces arthritis in mice. Nature 283: 666-668. 1980).
[0092] FIG. 4 and FIG. 5 present the results of investigation,
after oral administration of the extract, of arthritis progress
inhibiting effect of Notoginseng radix extract of the present
invention in test animals with type 2 collagen induced
arthritis.
[0093] In FIG. 4, the arthritis progress inhibiting effect of
Notoginseng radix extract of the present invention in test animals
with type 2 collagen induced arthritis was presented as arthritis
index, and FIG. 5 is a set of photographs showing the arthritis
progress inhibiting effect of Notoginseng radix extract of the
present invention in animals having type 2 collagen induced
arthritis.
[0094] As shown in FIG. 4, when Notoginseng radix extract of the
present invention was orally administered into a mouse having type
2 collagen induced arthritis, the progress of the disease was
obviously inhibited from the 9.sup.th day of administration,
comparing to a control group.
[0095] As shown in FIG. 5, both a control medicine without
Notoginseng radix extract and an experimental medicine including
the extract were orally administered respectively to mice having
type 2 collagen induced arthritis. Big difference between the two
was observed after 21 days from the administration. A mouse treated
with a control medicine showed very severe swelling all over the
joints but a mouse administered with an experimental medicine just
showed light flair and swelling in joints.
[0096] Therefore, it was confirmed that Notoginseng radix extract
of the present invention effectively inhibits the progress of
arthritis.
Example 4
Acute Toxicity Test with Notoginseng radix Extract of the Present
Invention
[0097] Notoginseng radix extract of the present invention is
classified into a food material, indicating that it is safe. But,
for the use as a treatment medicine, acute toxicity of the extract
had to be investigated as follows.
[0098] 6-week old SPF mice were used in the tests for acute
toxicity. Notoginseng radix extract (RF1MB4b) prepared in the above
example was suspended in distilled water and orally administered
once to 5 mice per group at the dosage of 2, 1, and 0.5 g/kg.
[0099] Death, clinical symptoms, and weight change in mice were
observed, hematological tests and biochemical tests of blood were
performed, and any abnormal signs in the gastrointestinal organs of
chest and abdomen were checked with eyes during autopsy.
[0100] The results showed that Notoginseng radix extract of the
present invention did not cause any specific clinical symptoms,
weight change, or death in mice. No change was observed in
hematological tests, biochemical tests of blood, and autopsy.
[0101] Notoginseng radix extract (RF1MB4b) of the present invention
used in this experiment is evaluated to be safe substance since it
does not cause any toxic change in mice up to the level of 2 g/kg
and its estimated LD.sub.50 values are much greater than 2 g/kg in
mice.
Manufacturing Example 1
Preparation of Pharmaceutical Formulations
[0102] <1-1> Preparation of Powders
[0103] Notoginseng radix extract 2 g
[0104] Lactose 1 g
[0105] Powders were prepared by mixing all the above components and
filled airtight bag with them.
[0106] <1-2> Preparation of Tablets
[0107] Notoginseng radix extract 100 mg
[0108] Corn starch 100 mg
[0109] Lactose 100 mg
[0110] Magnesium stearate 2 mg
[0111] Tablets were prepared by mixing all the above components by
the conventional method for preparing tablets.
[0112] <1-3> Preparation of Capsules
[0113] Notoginseng radix extract 100 mg
[0114] Corn starch 100 mg
[0115] Lactose 100 mg
[0116] Magnesium stearate 2 mg
[0117] Capsules were prepared by mixing the components above and
filled gelatin capsules with them according to the conventional
method for capsules.
Manufacturing Example 2
Preparation of Food
[0118] Foodstuff containing Notoginseng radix extract of the
present invention was prepared as follows.
[0119] <2-1> Preparation of Cooking Spices
[0120] Health improving spices and condiments containing
Notoginseng radix extract of the present invention by 20-95 weight
% were prepared.
[0121] <2-2> Preparation of Tomato Ketchup and Sauce
[0122] Health improving tomato ketchup or sauce was prepared by
adding Notoginseng radix extract of the present invention by
0.2-1.0 weight % to original tomato ketchup or sauce.
[0123] <2-3> Preparation of Flour Food
[0124] Health improving flour food was prepared by adding
Notoginseng radix extract of the present invention by 0.5-5.0
weight % to wheat flour and then making the flour into bread,
cakes, cookies, crackers and noodles.
[0125] <2-4> Preparation of Soups and Gravies
[0126] Notoginseng radix extract of the present invention was added
by 0.1-5.0 weight % to soups and gravies to produce health
improving processed meats, noodle soups and gravies.
[0127] <2-5> Preparation of Ground Beef
[0128] Notoginseng radix extract of the present invention was added
by 10 weight % to ground beef to prepare health improving ground
beef.
[0129] <2-6> Preparation of Dairy Products
[0130] Notoginseng radix extract of the present invention was added
by 5-10 weight % to milk to prepare health improving dairy products
such as butter, ice cream, etc.
[0131] <2-7> Preparation of Sunsik
[0132] Brown rice, barley, glutinous rice and coix (job's tear)
were gelatinizated by the conventional method, followed by drying.
The dried mixture was distributed and pulverized, resulting in
60-mesh grain size of powders.
[0133] Black bean, black sesame and perilla were steamed and dried
by the conventional method. The dried mixture was distributed and
pulverized, resulting in 60-mesh grain size of powders.
[0134] Notoginseng radix extract of the present invention was
vacuum-concentrated under reduced pressure using a vacuum
concentrator, which was then spray-dried with a hot-air drier. The
dried material was pulverized by a grinder, resulting in 60-mesh
grain size of powders.
[0135] The prepared grain, seeds, and dried Notoginseng radix
extract powders were all mixed at the following ratio.
[0136] Grain (brown rice 30 weight %, coix 15 weight %, barley 20
weight %),
[0137] Seeds (perilla 7 weight %, black bean 8 weight %, black
sesame 7 weight %),
[0138] Dried powder of Notoginseng radix extract (3 weight %),
[0139] Ganoderma lucidum (0.5 weight %),
[0140] Rehmannia glutinosa (0.5 weight %)
Manufacturing Example 3
Preparation of Beverages
[0141] <1-1> Preparation of Carbonated Beverages
[0142] Sugar (5-10%), citric acid (0.05-0.3%), caramel
(0.005-0.02%) and vitamin C (0.1-1%) were mixed, to which purified
water (79-94%) was added to make syrup. The prepared syrup was
sterilized at 85-98.degree. C. for 20-180 seconds, then mixed with
cooling water at the ratio of 1:4. Then, carbon dioxide gas
(0.5-0.82%) was given to the mixture to prepare carbonated
beverages containing Notoginseng radix extract of the present
invention.
[0143] <1-2> Preparation of Health Beverages
[0144] Acid fructose (0.5%), oligosaccharide (2%), sugar (2%), salt
(0.5%) and water (75%) were all mixed with Notoginseng radix
extract evenly, followed by sterilization. The mixture was put in a
small container such as a glass bottle or pat bottle, resulting in
health beverages.
[0145] <1-3> Preparation of Vegetable Juice
[0146] 5 g of Notoginseng radix extract of the present invention
was added to 1,000 ml of tomato or carrot juice to prepare health
vegetable juice.
[0147] <1-4> Preparation of Fruit Juice
[0148] 1 g of Notoginseng radix extract of the present invention
was added to 1,000 ml of apple or grape juice to produce health
fruit juice.
INDUSTRIAL APPLICABILITY
[0149] As explained hereinbefore, Notoginseng radix extract of the
present invention has activities of inhibiting TNF-.alpha. release
and destroying activated T-cells selectively.
[0150] Therefore, Notoginseng radix extract of the present
invention can be effectively used for the production of health food
or a medicine for preventing and treating arthritis.
* * * * *