U.S. patent application number 12/084149 was filed with the patent office on 2009-02-12 for extract of the family dioscoreaceae and composition for preventing or treating peripheral neuropathy comprising the same.
Invention is credited to Tong-Ho Kang, Sun-Yeou Kim, Ji-Ho Park.
Application Number | 20090041865 12/084149 |
Document ID | / |
Family ID | 37968005 |
Filed Date | 2009-02-12 |
United States Patent
Application |
20090041865 |
Kind Code |
A1 |
Kim; Sun-Yeou ; et
al. |
February 12, 2009 |
Extract of the Family Dioscoreaceae and Composition for Preventing
or Treating Peripheral Neuropathy Comprising the Same
Abstract
An extract of the family Dioscoreaceae useful for preventing or
treating the peripheral neuropathy; and a pharmaceutical
composition or food composition comprising the extract are
provided. In addition, a pharmaceutical composition or food
composition useful for preventing or treating the peripheral
neuropathy comprising a compound isolated from the extract of the
family Dioscoreaceae is provided.
Inventors: |
Kim; Sun-Yeou; (Seoul,
KR) ; Kang; Tong-Ho; (Seoul, KR) ; Park;
Ji-Ho; (Seoul, KR) |
Correspondence
Address: |
THE NATH LAW GROUP
112 South West Street
Alexandria
VA
22314
US
|
Family ID: |
37968005 |
Appl. No.: |
12/084149 |
Filed: |
October 27, 2006 |
PCT Filed: |
October 27, 2006 |
PCT NO: |
PCT/KR2006/004408 |
371 Date: |
April 25, 2008 |
Current U.S.
Class: |
424/725 ;
514/173; 514/26 |
Current CPC
Class: |
A23V 2002/00 20130101;
A23V 2200/322 20130101; A23V 2250/21 20130101; A23V 2002/00
20130101; A61P 21/00 20180101; A61P 25/02 20180101; A23L 33/105
20160801; A61P 25/00 20180101 |
Class at
Publication: |
424/725 ;
514/173; 514/26 |
International
Class: |
A61K 36/18 20060101
A61K036/18; A61K 31/58 20060101 A61K031/58; A61K 31/7028 20060101
A61K031/7028; A61P 25/02 20060101 A61P025/02 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 28, 2005 |
KR |
10-2005-0102450 |
Claims
1. An extract of a family Dioscoreaceae for preventing or treating
the peripheral neuropathy, the family Dioscoreaceae being at least
one selected from the group consisting of Dioscorea nipponica,
Dioscorea septembloba, Dioscorea quinqueoloba, Dioscorea batatas,
Dioscorea japonica, Dioscorea bulbifera, Dioscorea tokoro, and
Dioscorea tenuipes.
2. The extract of claim 1, wherein the family Dioscoreaceae is
Dioscorea nipponica, Dioscorea quinqueoloba, or Dioscorea
tokoro.
3. The extract of claim 1, wherein the peripheral neuropathy is the
multiple neuropathy.
4. The extract of claim 1, wherein the extract is obtained by
performing an extraction process which comprises extracting a whole
part, root, or aerial part of the family Dioscoreaceae with a first
extraction solvent selected from the group consisting of water,
C.sub.1-C.sub.4 alcohol, and a mixture of water and a
C.sub.1-C.sub.4 alcohol.
5. The extract of claim 4, wherein the extract is obtained by
performing an extraction process which comprises extracting the
root of the family Dioscoreaceae with the first extraction
solvent.
6. The extract of claim 4, wherein the first extraction solvent is
a mixture of water and methanol or a mixture of water and
ethanol.
7. The extract of claim 4, wherein the extraction process further
comprises dispersing the extract obtained by the extraction with
the first extraction solvent in water, and extracting with a
water-saturated C.sub.1-C.sub.4 alcohol (second extraction
solvent).
8. The extract of claim 7, wherein the second extraction solvent is
water-saturated butanol.
9. A pharmaceutical composition for preventing or treating the
peripheral neuropathy, which comprises a therapeutically effective
amount of the extract of claim 1, and a pharmaceutically acceptable
carrier.
10. A food composition for preventing or treating the peripheral
neuropathy, which comprises the extract of claim 1, as an active
ingredient.
11. A pharmaceutical composition for preventing or treating the
peripheral neuropathy, which comprises a therapeutically effective
amount of a compound represented by Formula 1 or salt thereof; and
a pharmaceutically acceptable carrier: ##STR00004## wherein R is a
hydrogen atom, a C.sub.1-C.sub.4 alkyl group, or a saccharide.
12. The pharmaceutical composition of claim 11, wherein R is a
hydrogen atom.
13. The pharmaceutical composition of claim 11, wherein the
saccharide is selected from the group consisting of glucose,
fructose, mannose, galactose, ribose, cellulose, glycogen, sucrose,
maltose, and lactose.
14. A food composition for preventing or treating the peripheral
neuropathy, which comprises a compound represented by Formula 1 or
salt thereof as an active ingredient: ##STR00005## wherein R is a
hydrogen atom, a C.sub.1-C.sub.4 alkyl group, or a saccharide.
15. The food composition of claim 14, wherein R is a hydrogen
atom.
16. The food composition of claim 14, wherein the saccharide is
selected from the group consisting of glucose, fructose, mannose,
galactose, ribose, cellulose, glycogen, sucrose, maltose, and
lactose.
Description
TECHNICAL FIELD
[0001] The present invention relates to an extract of the family
Dioscoreaceae for preventing or treating the peripheral neuropathy;
and a pharmaceutical composition or food composition comprising the
extract or a compound isolated from the extract.
BACKGROUND ART
[0002] Neuropathy is a disease caused by structural or functional
abnormalities of the nervous system. The nervous system is divided
into the central nervous system which is distributed in the brain
and the spinal cord and involved in controlling their functions,
and the peripheral nervous system which is distributed in almost
all organs excluding the brain and the spinal cord and involved in
controlling their functions. The peripheral nervous system is
subdivided into the motor nervous system, the sensory nervous
system, the autonomic nervous system. A peripheral nerve, where
neurites branch out beyond the brain and the spinal cord to the
body, arms, and legs, transmits sensation felt at arms and legs to
the central nerve (brain and spiral cord), and transmits orders of
the central nerve to muscles.
[0003] The peripheral nerve can be injured by various causes, which
is called collectively as the peripheral neuropathy. The
mono-neuropathy refers to a case in which a single peripheral nerve
is injured, and the multiple neuropathy refers to a case in which
many peripheral nerves are injured to a similar level. The
mono-neuropathy usually occurs when a single peripheral nerve is
abnormally pressed or traumatically injured during extending to
ends of arms and legs. The mono-neuropathy can be treated with
operations.
[0004] The multiple neuropathy can be induced by various causes,
such as metabolic diseases (e.g., diabetes, renal failure,
hypothyroidism), drugs (e.g., antitumor agents, antituberculosis
drugs) or toxic substance intoxication (e.g., Pb, organic
solvents), malnutrition (e.g., vitamin deficiency, alcoholism),
connective tissue disorders (e.g., rheumatoid arthritis, systemic
lupus erythematosus), inflammatory diseases (Guillain-Barre
syndrome), or genetically determined neuropathy. In addition, the
multiple neuropathy can be caused by cancers.
[0005] Until now, the neuropathy has been treated with drugs used
as a symptomatic therapy that improves symptoms only and there are
almost no fundamental remedies for the neuropathy. Only epalrestat,
an aldose reductase inhibitor, was approved by US Food and Drugs
Administration (FDA), with respect to diabetic peripheral
neuropathy, one of the multiple neuropathies, but epalrestat is not
used due to its low therapeutic effects (Foster D W., Harrison's
Principles of Internal Medicine 13, p 1979, 1999; Stephen L D,
Applied Therapeutics: the clinical use of drugs. 6, p 48.1-48.62,
1996).
[0006] Meanwhile, a protein that affects the growth,
differentiation, and survival of neurons in the central nervous
system (CNS) and the peripheral nervous system (PNS) is called
collectively as a neurotrophic factor (NF), which is one of neuron
control factors that regulate the growth, differentiation, and
death of neurons. Examples of the NF include a brain-induced
neurotrophic factor (BDNF), neurotrophin-3 (NT-3), NT-4, and NT-5.
These NFs are synthesized at different areas, and have different
differentiation, different expression, and different target
regions.
[0007] A nerve growth factor (NGF), one of the NFs, inhibits the
degeneration and death of neurons so as to prevent a decrease in
the number of neurons, to protect neurons from damages, and to
retain mature neuron sources (Hefti F., J. Neurosci., 6(8), pp
2155-2162, 1986; and Levi-Montalcini R., et al., Proc. Natl. Acad.
Sci. USA, 46, pp 384-391, 1960). It is known that when the nervous
system normally develops, about 50% of neurons on growth are
removed by cell death (Raff M C., et al., Science, 262(5134), pp
695-70, 1993), and that NGFs secreted by a target cell determine
survival of neurons. In order for neurons to survive, grow, and
differentiate in a normal state, a growth factor, such as the NGF,
is necessarily required. Such feasibility of the NGF has led
development of a recombinant human nerve growth factor in order to
treat a diabetic neuropathy, one of the multiple neuropathies.
However, the recombinant human nerve growth factor is still
unsatisfactory in safety and efficacy (Apfel S C et al., Journal of
American Medical Association 284(17), pp 2215-2221, 2000).
DISCLOSURE OF INVENTION
Technical Problem
[0008] The present invention provides herbal extracts and compounds
isolated therefrom useful for preventing or treating the peripheral
neuropathy induced by various causes.
[0009] That is, the present invention provides an extract of the
family Dioscoreaceae for preventing or treating the peripheral
neuropathy.
[0010] The present invention also provides a pharmaceutical
composition or food composition comprising the extract or a
compound isolated from the extract as an active ingredient.
Technical Solution
[0011] According to an aspect of the present invention, there is
provided an extract of a family Dioscoreaceae for preventing or
treating the peripheral neuropathy, the family Dioscoreaceae being
at least one selected from the group consisting of Dioscorea
nipponica, Dioscorea septembloba, Dioscorea quinqueoloba, Dioscorea
batatas, Dioscorea japonica, Dioscorea bulbifera, Dioscorea tokoro,
and Dioscorea tenuipes.
[0012] According to another aspect of the present invention, there
is provided a pharmaceutical composition for preventing or treating
the peripheral neuropathy, which comprises a therapeutically
effective amount of the extract; and a pharmaceutically acceptable
carrier.
[0013] According to still another aspect of the present invention,
there is provided a food composition for preventing or treating the
peripheral neuropathy, which comprises the extract as an active
ingredient.
[0014] According to still another aspect of the present invention,
there is provided a pharmaceutical composition for preventing or
treating the peripheral neuropathy, which comprises a
therapeutically effective amount of a compound represented by
Formula 1 or salt thereof; and a pharmaceutically acceptable
carrier:
##STR00001##
[0015] wherein R is a hydrogen atom, a C.sub.1-C.sub.4 alkyl group,
or a saccharide.
[0016] According to still another aspect of the present invention,
there is provided a food composition for preventing or treating the
peripheral neuropathy, which comprises the compound represented by
Formula 1 or salt thereof as an active ingredient.
Advantageous Effects
[0017] An extract of the family Dioscoreaceae and/or a compound
isolated from the extract induces an endogenous nerve growth factor
in an organism so that it can be used in a wide range of
applications for preventing or treating peripheral neuropathy.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 and FIG. 2 shows the effect of the compound isolated
from an extract according to an embodiment of the present invention
on neurite outgrowth.
[0019] FIG. 3 is a graph illustrating a change in the level of a
nerve growth factor (NGF) in normal mouse serum resulting from
administration of a compound isolated from an extract according to
an embodiment of the present invention.
[0020] FIG. 4 is a graph illustrating a change in the level of a
NGF in the serum of a diabetes-induced mouse resulting from
administration of an extract according to an embodiment of the
present invention and a compound isolated from the extract.
[0021] FIG. 5 and FIG. 6 illustrates the effect of a compound
isolated from an extract according to an embodiment of the present
invention on the transmission speed of the motor nerve and sensory
nerve in a sciatic nerve of diabetes-induced mice (FIG. 5: DG
treatment for one-month, FIG. 6: DG treatment for two-month)
[0022] FIG. 7 is a graph illustrating a change in the level of a
NGF in the sciatic nerve of a diabetes-induced mouse resulting from
administration of a compound isolated from an extract according to
an embodiment of the present invention.
[0023] FIG. 8 is a graph illustrating a change in the level of
sorbitol in the sciatic nerve of a diabetes-induced mouse resulting
from administration of a compound isolated from an extract
according to an embodiment of the present invention.
[0024] FIG. 9 is images illustrating histological changes in the
sciatic nerve of a diabetes-induced mouse resulting from
administration of an extract according to an embodiment of the
present invention and a compound isolated from the extract.
BEST MODE FOR CARRYING OUT THE INVENTION
[0025] In the present specification, the term "peripheral
neuropathy" refers to a condition of peripheral nerves (motor
nerves, sensory nerves, and autonomic verves) injured by various
causes. The peripheral neuropathy may be subdivided into the
mono-neuropathy and the poly-neuropathy (also called as multiple
neuropathy). The multiple neuropathy includes any neuropathy caused
by, metabolic diseases (e.g., diabetes, renal failure,
hypothyroidism), drugs (e.g., antitumor agents, antituberculosis
drugs) or toxic substance intoxication (e.g., Pb, organic
solvents), malnutrition (e.g., vitamin deficiency, alcoholism),
connective tissue disorders (e.g., rheumatoid arthritis, systemic
lupus erythematosus), inflammatory diseases (Guillain-Barre
syndrome), or genetically determined neuropathy. In addition, the
multiple neuropathy may include a neuropathy caused by genetic
factors and cancers.
[0026] An extract of the family Dioscoreaceae according to an
embodiment of the present invention or a compound isolated from the
extract derives neurite outgrowth and increases the amount of an
endogenous nerve growth factor secreted, so that nerves of the
peripheral nervous system can be effectively differentiated,
protected, and reinnervated. In particular, the extract and/or the
compound allow oral administration, which improve patients
medication compliance.
[0027] The present invention provides an extract of the family
Dioscoreaceae for preventing or treating the peripheral
neuropathy.
[0028] The family Dioscoreaceae is at least one selected from
Dioscorea nipponica, Dioscorea septembloba, Dioscorea quinqueoloba,
Dioscorea batatas, Dioscorea japonica, Dioscorea bulbifera,
Dioscorea tokoro, and Dioscorea tenuipes. Preferably, the family
Dioscoreaceae is Dioscorea nipponica, Dioscorea quinqueoloba,
and/or Dioscorea tokoro. More preferably, the family Dioscoreaceae
is Dioscorea nipponica.
[0029] The extract according to the present invention can be
obtained through an extraction process which includes extracting a
whole part, root, or aerial part (for example, leave or stem) of
the family Dioscoreaceae with an extraction solvent (first
extraction solvent) selected from the group consisting of water,
C.sub.1-C.sub.4 alcohol, and a mixture of water and a
C.sub.1-C.sub.4 alcohol. For example, the extract of the family
Dioscoreaceae can be obtained by extracting the root of the family
Dioscoreaceae with the first extraction solvent. The first
extraction solvent may be a mixture solvent of water and methanol
or a mixture solvent of water and ethanol.
[0030] In the extraction process, the whole part, root, or aerial
part, preferably, the root of the family Dioscoreaceae is cut into
small sections, and then extracted with the first extraction
solvent. At this time, the amount of the first extraction solvent
may be 1 to 20 times, preferably about 3 to 10 times, greater than
that of the family Dioscoreaceae.
[0031] The first extract solvent may be a mixture solvent of water
and methanol (for example, about 85% methanol solution) or a
mixture solvent of water and ethanol (for example, about 85%
ethanol solution). The extraction is not affected by temperature,
and can be performed at various temperature ranges, such as a
temperature of 15.degree. C. to 100.degree. C. The extraction can
be performed by cold extraction, hot extraction, superfluid
extraction, centrifugal extraction, ultrasonic extraction, or
reflux cooling extraction. The extraction time may vary according
to the extraction method. For example, the extraction can be
performed once or multiple times for about 1 hour to 10 days.
Preferably, the extraction can be performed twice or three times at
room temperature for about 2 days using the first extraction. The
extract obtained by extraction with the first extraction solvent
can be a liquid form in which impurities in the extract are removed
using a conventional method, e.g., filtration, or a powder form
obtained by concentrating under reduced pressure or drying the
liquid extract using a conventional method.
[0032] In addition, when needed, the extraction process may further
include obtaining a fraction having higher contents of active
ingredients. That is, the extraction process further includes:
dispersing the extract obtained by the extraction with the first
extraction solvent in water; and extracting the resultant solution
with water saturated C.sub.1-C.sub.4 alcohol (second extraction
solvent), thereby increasing the contents of active ingredients in
the obtained extract.
[0033] When the extract obtained by extracting with the first
extraction solvent is dispersed in water, a liquid form per se
obtained by the extraction with the first extraction solvent may be
dispersed in water, or a powder form obtained by concentrating the
liquid extract under reduced pressure and/or drying the liquid
extract using a conventional method may be dispersed in water.
[0034] The water saturated C.sub.1-C.sub.4 alcohol (second
extraction solvent) may be water saturated butanol.
[0035] The present invention includes, within its scope, a
composition comprising a compound isolated from the extract, i.e.,
a steroidal saponin or steroidal sapogenin. That is, the present
invention includes a pharmaceutical composition for preventing or
treating the peripheral neuropathy, which comprises a
therapeutically effective amount of a compound represented by
Formula 1 or salt thereof; and a pharmaceutically acceptable
carrier:
##STR00002##
[0036] wherein R is a hydrogen atom, a C.sub.1-C.sub.4 alkyl group,
or a saccharide.
[0037] In the compound of Formula 1, R may be hydrogen or methyl,
preferably hydrogen. That is, the compound of Formula 1 can be
3-.beta.,25R-spirost-5-en-3-ol.
[0038] The saccharide can be monosaccharide, disaccharide, or
polysaccharide, such as glucose, fructose, mannose, galactose,
ribose, cellulose, glycogen, sucrose, maltose, and lactose.
[0039] The salt of the compound of Formula 1 can be a conventional
inorganic acid and/or organic acid addition salt prepared from
steroidal saponin or sapogenin compounds. Examples of the salt of
the compound of Formula 1 include salts disclosed in International
Laid-open Patent Publication No. WO2003/082893. These salts can be
prepared in situ during final separating and purifying processes of
a compound. In particular, an acid addition salt can be prepared by
reacting a refined compound in a free base form with a suitable
organic or inorganic acid and then separating the produced salt
(see S. M. Berge, et al., Pharmaceutical Salts, J. Pharm. Sci., 66:
p. 1-19(1977)). International Laid-open Patent Publication No.
WO2003/082893 and the journal of. M. Berge, et al. are used as a
reference in the present invention. A base addition salt can be
prepared by reacting a refined compound in an acid form with a
suitable organic or inorganic base and separating the produced
salt. The base addition salt can be a pharmaceutically acceptable
metal or amine salt. The acid addition salt can be a salt prepared
from an acid selected from hydrochloric acid, sulfuric acid,
phosphoric acid, and nitric acid. The base addition salt can be a
salt prepared from a base selected from sodium hydroxide, potassium
hydroxide, and ammonium hydroxide.
[0040] The compound of Formula 1 can be isolated from the extract
according to an embodiment of the present invention, synthesized
using a known method (see Herbert O. House, Modern Synthetic
Reactions, The Benjamin Cummings Publishing Company, 1972), or
commercially obtained (Sigma Co., USA.).
[0041] The method for isolating the compound of Formula 1 from the
extract may include an acid hydrolysis process and an
recrystallization process using the extract obtained according to
an embodiment of the present invention (for example, an extract
obtained using the first extraction solvent and the second
extraction solvent.) For example, the method for isolating the
compound of Formula 1 from the extract may include: hydrolyzing
with an acid, such as 2.5N hydrochloric acid, at a temperature of
50 to 150.degree. C., preferably at about 94.degree. C., for 30
minutes to 3 days, preferably for about 4 hours; solvent extracting
the obtained hydrolysate (that is, aglycone sapogenin) with an
organic solvent, such as chloroform, acetone, benzene, or xylene,
for 1 to 60 minutes, preferably for about 15 minutes, and then
separating the organic layer; when needed, concentrating the
separated organic layer at a temperature of 10 to 100.degree. C.,
preferably 30 to 35.degree. C.; recrystallizing the organic layer
or the concentrated solution of the organic layer with
C.sub.1-C.sub.4 alcohol or C.sub.1-C.sub.4 alcoholic solution (for
example, 95% ethanol solution); and, washing the obtained
precipitate with water when needed, and recrystallizing with
acetone.
[0042] The present invention provides a pharmaceutical composition
for preventing or treating the peripheral neuropathy, which
comprises a therapeutically effective amount of an extract of the
family Dioscoreaceae or a compound of Formula 1 or salt thereof; or
a pharmaceutically acceptable carrier.
[0043] The pharmaceutical composition according to the present
invention includes a pharmaceutically acceptable carrier, and can
be formulated into oral dosage form, external dosage form,
suppository, and sterile injection solution, such as powders,
granules, tablets, capsules, suspensions, emulsions, syrups, or
aerosols. The pharmaceutically acceptable carrier can be lactose,
dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol,
maltitol, starch, acacia rubber, alginate, gelatin, calcium
phosphate, calcium silicate, cellulose, methyl cellulose,
microcrystalline cellulose, polyvinyl pyrrolidone, water,
methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium
stearate, or mineral oil. The pharmaceutical composition may
further include a diluent or an excipient, such as filler,
expander, binder, humectant, disintegrant, or surfactant. A solid
oral formulation can be a tablet, a pill, a powder, a granule, or a
capsule. Such solid formulations may include at least one excipient
selected from, for example, starch, calcium carbonate, sucrose,
lactose, and gelatin. In addition, such solid formulations may
further include a lubricant, such as magnesium stearate or talc. A
liquid oral formulation can be a suspension, a solution, an
emulsion, or syrup. In addition, the liquid oral formulation may
include a diluent, such as water, liquid paraffine; humectant;
sweetening agent; odorant; or preservative. A parenteral
formulation can be a sterile aqueous solution, a non-aqueous
solution, a suspension, an emulsion, a lyophilized formulation, or
a suppository. Non-aqueous solvents or suspending agents can be
propylene glycol, polyethylene glycol, natural oil, such as olive
oil, or injectable esters, such as ethylolate. Vehicles for
suppository can be witepsol, macrogol, Tween 61, cacao butter,
Laurin, or glycerogelatine.
[0044] In the pharmaceutical composition according to the present
invention, a dose of the extract of the family Dioscoreaceae or the
compound of Formula 1 may vary depending on patient's state or body
weight, seriousness of disease, dosage forms, administration
routes, and the period of administration, and can be appropriately
determined by a person having ordinary skill in the art. For
example, the extract of the family Dioscoreaceae or the compound of
Formula 1 can be administered in an amount of 0.0001 to 1000 mg/kg,
preferably 0.001 to 1000 mg/kg, per day. The administration can be
completed once or through several times per day. In the
pharmaceutical composition according to the present invention, the
amount of the extract of the family Dioscoreaceae or the compound
of Formula 1 may be in the range of 0.001 to 50% by weight based on
100% by weight of the pharmaceutical composition.
[0045] The pharmaceutical composition can be administered to
mammals, such as rats, mouse, livestock, or human beings, through
various routs, e.g., orally, rectally, intravenously,
intramuscularly, subcutaneously, through intrauterine dura mater
injection, or through intracerebroventricular injection.
[0046] The present invention includes, within its scope, a food
composition for preventing or treating the peripheral neuropathy,
which comprises an extract of the family Dioscoreaceae or the
compound of Formula 1 as an active ingredient.
[0047] The food composition according to the present invention can
be used as a health functional food. According to Article 6727 of
Korean Health Functional Food law, the "health functional food"
refers to a food which is produced and processed using a source or
component that carries out good functions on the human body. The
"function" refers to an intake purporting to attain good health
effects, that is, a nutrient control with respect to the structure
and function of the human body or a physiological operation.
[0048] The food composition according to the present invention can
include a conventional food additive. The conformity of the "food
additive" is determined, as long as there are no other regulations,
in consideration with the standard and criteria of the
corresponding item according to the general rule of the food
additives codex and general tests approved by Korea Food & Drug
Administration.
[0049] The items listed on the "food additives codex" include a
chemically synthesized substance, such as ketone, glycine,
potassium citrate, nicotinic acid, or cinnamic acid; natural
additives, such as persimmon color, an extract of licorice,
crystalline cellulose, caoliang color, or guar gum; or mixed
formulation, such as L-sodium glutamate formulation, alkali
additives for noodles, preservatives, or tar color formulation.
[0050] The food composition according to the present invention may
include the extract of the family Dioscoreaceae or the compound of
Formula 1 in an amount of 0.01 to 95% by weight, preferably 1 to
80% by weight, based on 100% by weight of the food composition, in
order to prevent and/or treat the peripheral neuropathy. In
addition, in order to prevent and/or treat the peripheral
neuropathy, the food composition can be produced and processed into
tablets, capsules, powder, granule, liquid phase, or pills.
[0051] For example, in order to produce a health functional food in
a tablet form, a mixture of the extract of the family Dioscoreaceae
or the compound of Formula 1, an excipient, a binder, a
disintegrant, and other additives can be granulated using a
conventional method, and then compression molding process is
preformed with a lubricant. Alternatively, the mixture can be
directly subjected to the compression molding process. In addition,
when needed, the health formulated food in a tablet form may
include sweetening agents, and when needed, the health formulated
food in a tablet form can be coated with coating materials.
[0052] Among health functional foods in a capsule form, a hard
capsule formulation can be produced by filling a conventional hard
capsule with a mixture of the extract of family Dioscoreaceae or
the compound of Formula 1 and an additive, such as an excipient, or
granules of the mixture, or coated granules of the mixture; and a
soft capsule formulation can be produced by filling a capsule
support of gelatin with a mixture of the extract of family
Dioscoreaceae or the compound of Formula 1 and an additive, such as
an excipient. When needed, the soft capsule formulation can include
plasticizer, such as glycerin or sorbitol, a coloring agent, and a
preservative.
[0053] A health functional food in a pill form can be produced by
molding a mixture of the extract of family Dioscoreaceae or the
compound of Formula 1, an excipient, a binder, and a disintegrant
using a suitable method. When needed, the health functional food in
a pill form can be coated with white sugar or other coating
materials, or can be covered with starch, talc, or other
materials.
[0054] A health functional food in a granule form can be produced
by granulating a mixture of the extract of family Dioscoreaceae or
the compound of Formula 1, an excipient, a binder, and a
disintegrant using a suitable method. When needed, the health
functional food in a granule form can include a flavoring agent and
a sweetening agent.
[0055] The excipient, the binder, the disintegrant, the lubricant,
the sweetening agent, and the flavoring agent used in the present
invention can be defined as corresponding materials having the same
or similar functions disclosed in references known in the art (The
Korean pharmacopoeia review, Moonsungsa Publication Co., Korea
Pharmaceutical University Association, Fifth edition, p 33-48,
1989).
MODE FOR THE INVENTION
[0056] The present invention will be described in further detail
with reference to the following examples. These examples are for
illustrative purposes only and are not intended to limit the scope
of the present invention.
Example 1
Preparation of Extract
[0057] Dioscorea Nipponica was dried and the root thereof was cut
into small sections. The 500 g of the sample was added to 10 l of
85% methanol solution and then extracted three times (each for 2
hours) at room temperature. Such an extraction process was repeated
twice. The resultant supernatants were collected and concentrated
under reduced pressure, thereby obtaining 74 g of a crude
extract.
[0058] The 74 g of the crude extract was suspended in 1 l of
distilled water, 1 l of water-saturated butanol was added thereto,
and then, the generated organic layer was separated, which was
repeated five times. The obtained organic layers were collected
altogether and dried under reduced pressure. As a result, 17 g of
the extract of Dioscorea Nipponica was obtained.
Example 2
Preparation of Extract
[0059] Dioscorea quinqueoloba was dried and the root thereof was
cut into small sections. The 500 g of the sample was added to 5 l
of 85% ethanol solution and then extracted three times (each for 2
hours) at room temperature. Such an extraction process was repeated
twice. The resultant supernatants were collected and concentrated
under reduced pressure, thereby obtaining 90 g of a crude
extract.
[0060] The 90 g of the crude extract was suspended in 1 l of
distilled water, 1 l of water-saturated butanol was added thereto,
and then, the generated organic layer was separated, which was
repeated five times. The obtained organic layers were collected
altogether and dried under reduced pressure. As a result, 26 g of
the extract of Dioscorea quinqueoloba was obtained.
Example 3
Preparation of Extract
[0061] Dioscorea tokoro was dried and the root thereof was cut into
small sections. The 300 g of the sample was added to 3 l of 85%
ethanol solution and then extracted three times (each for 2 hours)
at room temperature. Such an extraction process was repeated twice.
The resultant supernatants were collected and concentrated under
reduced pressure, thereby obtaining 35 g of a crude extract.
[0062] The 35 g of the crude extract was suspended in 0.5 l of
distilled water, 0.5 l of water-saturated butanol was added
thereto, and then, the generated organic layer was separated, which
was repeated five times. The obtained organic layers were collected
altogether and dried under reduced pressure. As a result, 11 g of
the extract of Dioscorea tokoro was obtained.
Example 4
Separation of Active Compound
[0063] 1 g of the extract of Dioscorea nipponica obtained in
Example 1 was hydrolyzed at 94.degree. C. for four hours by adding
10 ml of 2.5N HCl 10 ml thereto. Then, the resultant hydrolysate
was extracted with 10 ml of chloroform for 15 minutes. The
chloroform layer was separated, filtered, and then concentrated
under reduced pressure at a temperature of 30-35.degree. C. The
obtained residue was recrystallized at 4.degree. C. using 5 ml of
95% ethanol solution. The recrystallized precipitate was filtered,
washed with water, recrystallized at 4.degree. C. using 3 ml of
acetone, and then filtered to obtain about 100 mg of the
precipitate. The precipitate was identified as 3beta,
25R-spirost-5-en-3-ol represented by Formula 2.
##STR00003## [0064] (1) Formula: C.sub.27H.sub.42O.sub.3 [0065] (2)
Molecular Weight: 414.62 [0066] (3) Melting Point: 204-207.degree.
C. [0067] (4) [.alpha.].sup.25.sub.D-129 [0068] (5) NMR Data: refer
to Table 1
TABLE-US-00001 [0068] TABLE 1 .sup.13C Chemical shift
(.delta.).sup.a .sup.1H Chemical shift (.delta.).sup.a C-1
37.6-37.7 H-1 1.60-1.70; 0.87-0.91 (m, o) C-2 30.3-30.4 H-2
2.00-2.03; 1.75-1.79 (m, o) C-3 78.1-78.7 H-3 3.81-3.90 (m, J =
4.7-5.6 Hz) C-4 38.9-39.5 H-4 2.57-2.29 (m, J = 13-14 Hz; J = 5.0;
12.0 Hz) C-5 140.9-141.1 C-6 121.9-122.0 H-6 5.23-5.26 (d, J =
4.6-5.4 Hz) C-7 32.4-32.5 H-7 1.77-1.82; 1.41-1.46 (m, o) C-8
31.8-31.9 H-8 1.44-1.48 (m, o) C-9 50.4-50.5 H-9 0.80-0.83 (m) C-10
37.2-37.3 C-11 ~21.3 H-11 1.35-1.38 (m, o) C-12 40.0-40.1 H-12
1.60-1.62 (m, o) C-13 40.6-40.7 C-14 56.8-56.8 H-14 1.01 (m, o)
C-15 32.4-32.5 H-15 1.95-1.98 (m, J = 5.9-6.1 Hz); 1.31-1.35 (m, o)
C-16 81.3-81.3 H-16 4.45-4.48 (m, J = 7.0-7.4 Hz) C-17 63.0-63.1
H-17 1.72-1.75 (m, o) C-18 16.5-16.6 H-18 0.73-0.76 (s) C-19
19.6-19.6 H-19 0.80-1.00 (s) C-20 42.1-42.2 H-20 1.88 (m) C-21
15.2-15.2 H-21 1.05-1.07 (d, J = 6.8-7.2 Hz) C-22 109.4-109.5 C-23
32.0-32.0 H-23 1.57-1.63 (m, o) C-24 29.4-29.5 H-24 1.48-1.52 (m,
o) C-25 30.8-30.8 H-25 1.50 (m, o) C-26 67.0-67.1 H-26 3.50-3.40
(m, J = 10.5; 3.0; 10.5 Hz) C-27 17.5-17.5 H-27 0.60-0.63 (d, J =
4.7-5.8 Hz)
Experimental Example 1
Measurement of Neurite Outgrowth
[0069] In an incubator with conditions including 5% CO.sub.2 and a
temperature of 37.degree. C., PC 12 cell (pheochromocytoma, ATCC
Number: CRL-1721) was cultured in a RPMI 1640 medium supplemented
with horse serum (10%, v/v), fetal bovine serum (5%, v/v), and 1%
penicillin-streptomycin
[0070] In order to find the effect of the compound of Formula 2 on
the neurite outgrowth, the mediums supplemented with 2% horse
serum, 1% fetal bovine serum, and 1% penicillin-streptomycin were
added to each 6-well plate coated with poly-d-lysine and then the
PC12 cells were inoculated in 5.times.10.sup.4 cells each well.
After 24 hours, these wells were treated with 10 .quadrature./ml of
ethanol, 10 .quadrature./.quadrature. of the compound of Formula 2,
and 50 ng/.quadrature. of a nerve growth factor (R&D system,
USA), respectively. Then, after 48 hours, the length of the neurite
was measured using an inverted image contrast microscope (CK-2,
Olympus, USA) (See FIG. 1 and FIG. 2)
[0071] Referring to FIGS. 1 and 2, the neurite outgrowth was not
observed in the ethanol-injected group (control), but the neurite
outgrowth was induced both in the compound of Formula 2-treated
group (DG) and in the nerve growth factor-treated group (NGF).
Accordingly, it was found that the compound of Formula 2 induced
differentiation of the PC 12 cell by inducing the neurite
outgrowth.
Experimental Example 2
Measurement of Level of Nerve Growth Factor in Mouse Serum
[0072] The compound of Formula 2 was dissolved in 0.2 ml of a
solution of dimethylsulfoxide:ethanol (3:1), and then orally
administered once to 7-week old male ICR mice (n=7) in an amount of
10 mg/kg. After 24 hours, the amount of an endogenous nerve growth
factor was measured by ELISA. As a control group, an ICR mouse was
orally administered once with 0.2 ml of the
dimethylsulfoxide:ethanol (3:1). Then, the amount of a nerve growth
factor in the control group was measured in the same manner as
described above.
[0073] Referring to FIG. 3, the compound of Formula 2-administered
group (DG 10 mg/kg P.O) showed a nerve growth factor in the serum
about 2.5 times greater than the control group. Such results show
that the compound of Formula 2 can treat the neuropathy by
suppressing the degeneration and death of neurons in a mouse and
thus preventing a decrease in the number of neurons.
Experimental Example 3
Measurement of Level of Nerve Growth Factor in Serum of
Diabetes-Induced Mouse
[0074] An alloxan-induced diabetic mouse was prepared as an animal
model having diabetic neuropathy, one of the multiple neuropathies.
7-week old male ICR mice underwent alimentary abstinence for 18
hours, and then alloxan dissolved in a physiological saline was
once injected to them by intraperitoneal injection in an amount of
160 mg/kg to induce diabetes. Mice that maintained their fasting
blood sugar to 200 mg/dl or more for one week, that is,
diabetes-induced mice were selected and then divided into a control
group (n=10), an extract-administered group (n=10), and a
compound-administered group (n=10). The control group was
administered with 0.2 ml of a solution of
dimethylsulfoxide:ethanol(3:1), the extract-administered group was
administered with the extract obtained according to Example 1
dissolved in a solution of dimethylsulfoxide:ethanol (3:1) in an
amount of 100 mg/kg, and the compound-administered group was
administered with the compound of Formula 2 dissolved in a solution
of dimethylsulfoxide:ethanol (3:1) in an amount of 10 mg/kg. The
all groups were orally administered three times per one week, and
the entire administration period was one month. The amount of an
endogenous nerve growth factor in the serum was measured by
ELISA.
[0075] Referring to FIG. 4, the amounts of the endogenous nerve
growth factor in the serum of both the extract-administered group
(DN 100 mg/kg p.o) and the compound-administered group (DG 10 mg/kg
p.o) were three and four times higher than the control group to
which only a vehicle was injected, respectively. Such results show
that the extract and compound according to the present invention
can treat the diabetic neuropathy by suppressing the degeneration
and death of neurons in nerve-injured diseases caused by diabetes
and thus preventing a decrease in the number of neurons.
Experimental Example 4
Measurement of Transmission Speed of Motor
[0076] Nerve and Sensory Nerve of Sciatic Nerve in Diabetes-induced
Mouse The therapeutic effect of the compound according to the
present invention on the diabetic neuropathy, one of the multiple
neuropathies, was identified by measuring the effect of the
compound according to the present invention on the transmission
speed of the motor nerve and sensory nerve in the sciatic
nerve.
[0077] 7-week old male ICR mice underwent alimentary abstinence for
18 hours, and then alloxan dissolved in a physiological saline was
once injected to them by intraperitoneal injection in an amount of
160 mg/kg to induce diabetes. Mice that maintained their fasting
blood sugar to 200 mg/dl or more for one week, that is,
diabetes-induced mice were selected and then divided into a control
group (n=7), and a compound-administered group (n=7). The control
group was administered with 0.2 ml of a solution of
dimethylsulfoxide:ethanol (3:1), and the compound-administered
group was administered with the compound of Formula 2 dissolved in
a solution of dimethylsulfoxide:ethanol (3:1) in an amount of 10
mg/kg. 7-week old male ICR mice in which diabetes was not induced
were grouped as a normal group (n=7), and the normal group was
administered with 0.2 ml of a solution of
dimethylsulfoxide:ethanol(3:1). The all groups were orally
administered three times per one week, and the entire
administration period was 2 months. Administration-completed mice
were sacrificed by cervical dislocation, and then the skin and
muscle in the femoral region were quickly removed. Then, left and
right sciatic nerves were respectively separated in a length of 20
mm or more and stored in a physiological saline while air flows
through the physiological saline. The separated sciatic nerves were
placed on a 20 mm round measurement plate. Then, a sensor and a
stimulating probe were connected to respective neuroterminals and
the electrical conductivity was measured using a digital storage
oscilloscope to assess the nerve transmission speed (see FIGS. 5
and 6.)
[0078] In general, the myelin is a phospholipid membrane
surrounding axons with several layers, and also called as myelin
sheath. Like the plastic coating of an electric wire, the myelin,
through a white lipid material, prevents the electrical signals
transmitted by neurons from leaking or dispersing. The myelin is
regularly spaced between nodes of ranvier (a portion that forms
nodes of myelin) at which the myelin is not formed, and surrounds
axons. The electrical signal is transmitted along the space,
impulses are quickly transmitted along neurons, and the myelin
increases the electrical impulse speed. Accordingly, when the
myelin is destructed by nerve injury due to the neuropathy induced
by diabetes, axons stops their function and the nerve transmission
speed decreases.
[0079] During one month of a diabetes-induced period, the
compound-administered group (DM-DG) showed a transmission speed of
the sensory nerve 25% higher than the diabetes-induced control
group (DM) to which only the vehicle was administered (see FIG. 5).
During two months of a diabetes-induced period, the
compound-administered group (DM-DG) showed a transmission speed of
the sensory nerve 45% higher than the control group, and showed a
transmission speed of the motor nerve 40% higher than the control
group (see FIG. 6). Accordingly, it was found that the compound
according to the present invention has a therapeutic effect on
nerve injury due to the diabetic neuropathy by increasing the
transmission speed of the sensory nerve and motor nerve of a
diabetes-induced mouse.
Experimental Example 5
Measurement of Level of Nerve Growth Factor in Sciatic Nerve in
Diabetes-induced Mouse
[0080] 7-week old male ICR mice underwent alimentary abstinence for
18 hours, and then alloxan dissolved in a physiological saline was
once injected to them by intraperitoneal injection in an amount of
160 mg/kg to induce diabetes. Mice that maintained their fasting
blood sugar to 200 mg/dl or more for one week, that is,
diabetes-induced mice were selected and then divided into a control
group (n=5), and a compound-administered group (n=5). The control
group was administered with 0.2 ml of a solution of
dimethylsulfoxide:ethanol (3:1), and the compound-administered
group was administered with the compound of Formula 2 dissolved in
a solution of dimethylsulfoxide:ethanol (3:1) in an amount of 10
mg/kg. The all groups were orally administered three times per one
week, and the entire administration period was one month. The
amount of an endogenous nerve growth factor in the sciatic nerve
was measured by ELISA (see FIG. 7)
[0081] Referring to FIG. 7, it was found that the nerve growth
factor in the sciatic nerve of the compound-administered group
(DM-DG 10 mg/kg P.O) was about 30% higher than that of the control
group to which only the vehicle was injected. Accordingly, it is
considered that the results shown in FIGS. 5 and 6 result from the
nerve protecting function of the nerve growth factor.
Experimental Example 6
Measurement of Change in Amount of Sorbitol in Sciatic Nerve of
Diabetes-Induced Mouse
[0082] The therapeutic effect of the compound according to the
present invention on the neuropathy was identified by measuring a
change in the amount of sorbitol in the sciatic nerve.
[0083] 7-week old male ICR mice underwent alimentary abstinence for
18 hours, and then alloxan dissolved in a physiological saline was
once injected to them by intraperitoneal injection in an amount of
160 mg/kg to induce diabetes. Mice that maintained their fasting
blood sugar to 200 mg/dl or more for one week, that is,
diabetes-induced mice were selected and then divided into a control
group (n=3), and a compound-administered group (n=3). The control
group was administered with 0.2 ml of a solution of
dimethylsulfoxide:ethanol (3:1), and the compound-administered
group was administered with the compound of Formula 2 dissolved in
a solution of dimethylsulfoxide:ethanol (3:1) in an amount of 10
mg/kg. 7-week old male ICR mice in which diabetes was not induced
were grouped as a normal group (n=3), and the normal group was
administered with 0.2 ml of a solution of dimethylsulfoxide:ethanol
(3:1). The normal group, the control group, and the
compound-administered group were orally administered three times
per one week, and the entire administration period was 2 weeks.
Administration-completed mice were sacrificed by cervical
dislocation, and then the amount of the sorbitol in the sciatic
nerve was measured by HPLC (see FIG. 8).
[0084] A polyol pathway refers to a process in which glucose is
converted to sorbitol by aldose reductase and the sorbitol is
changed into fructose by sorbitol dehydrogenase. In a hyperglycemic
state, such as diabetes, excess glucose enters cells and sorbitol
is generated and accumulated by the polyol pathway. At this time,
nerves can be injured by the osmotic operation drawing water into
cells. Accordingly, as a result of measuring the effect of the
compound according to the present invention on the sorbitol
accumulation in the sciatic nerve of the diabetes-induced mouse, as
shown in FIG. 8, it was found that the sorbitol in the sciatic
nerve of the diabetes-induced control group (DM-CON) was about 40%
higher than that of the normal group, but the sorbitol in the
sciatic nerve of the compound-administered group (DM-DG) was 10%
lower than that of the diabetes-induced control group (DM-CON).
Such results show that the compound according to the present
invention can partially decrease aggressive factors causing nerve
injury.
Experimental Example 7
Histological Comparison of Sciatic Nerve of Diabetes-Induced
Mouse
[0085] The therapeutic effect of the compound according to the
present invention on the neuropathy was identified by measuring a
histological change of the sciatic nerve.
[0086] 7-week old male ICR mice underwent alimentary abstinence for
18 hours, and then alloxan dissolved in a physiological saline was
once injected to them by intraperitoneal injection in an amount of
160 mg/kg to induce diabetes. Mice that maintained their fasting
blood sugar to 200 mg/dl or more for one week, that is,
diabetes-induced mice were selected and then divided into a control
group (n=5), an extract-administered group (n=5), and a
compound-administered group (n=3). The control group was injected
with 0.2 ml of a solution of dimethylsulfoxide:ethanol (3:1), the
extract-administered group was administered with the extract
obtained according to Example 1 dissolved in a solution of
dimethylsulfoxide:ethanol (3:1) in an amount of 100 mg/kg, and the
compound-administered group was administered with the compound of
Formula 2 dissolved in a solution of dimethylsulfoxide:ethanol
(3:1) in an amount of 10 mg/kg. All groups were treated three times
by oral administration per one week, and the entire administration
period was 2 months. Administration-completed mice were sacrificed
by cervical dislocation, and then the sciatic nerve was separated,
dyed according to the following conditions, and then observed using
a 600.times., 1200.times. confocal microscope. [0087] (1)
prefixing: 2.5% glutaraldehyde, 12 or more hours [0088] (2)
washing: pH 7.4, 0.1M phosphate buffer solution, 15 minutes/twice
[0089] (3) postfixing: 1% OsO.sub.4 osmium tetroxide, 60 minutes
[0090] (4) washing: pH 7.4, 0.1M phosphoate buffer solution, 5
minutes/twice [0091] (5) dehydrating: [0092] 50, 70, 80, 90%
alcohol: 10 minutes/each [0093] 100% alcohol: 15 minutes/twice
[0094] (6) substituting: prophylene oxide, 15 minutes/twice [0095]
(7) permeating: propylene oxide (1), EPOK 812 (2), 12 or more hours
[0096] (8) embedding: refined EPOK 812 (80.degree. C.
polymerization): 12 or more hours [0097] (9) sectioning: semi-thin
section, 35-95 [0098] (10) dyeing: 1% toluidine blue
[0099] FIGS. 9A and 9B show the results obtained with 600
magnification (9A) and 1200 magnification (9B), respectively. In
the diabetes-induced group (DM), the axon and the myelin in the
central part of the sciatic nerve were significantly destructed.
However, in the compound-administered group (DM-DG) and the
extract-administered group (DM-DN), the axon and the myelin in the
central part of the sciatic nerve were clearly observed. Such
results show that the extract or compound according to the present
invention can protect and treat nerves injured by the
neuropathy.
[0100] The compound according to the present invention was
formulated into the following forms. However, these formulation
examples are for illustrative purposes only and are not intended to
limit the scope of the present invention.
Formulation Example 1
Tablet Formation
TABLE-US-00002 [0101] Compound of Formula 2 200 Lactose 100 Starch
100 Magnesium stearate proper These components were mixed and
compressed into tablets according to a conventional tablet
formulating method.
Formulation Example 2
Liquid Formulation
TABLE-US-00003 [0102] Compound of Formula 2 1000 CMC-Na 20 g
Isomerized sugar 20 g Lemon flavor proper
[0103] Purified water was added so that the volume of the entire
solution was 1000. These components were mixed according to a
conventional liquid formulation method, filled into a brown bottle,
and sterilized, thereby producing the liquid formulation.
Formulation Example 3
Capsule Formulation
TABLE-US-00004 [0104] Compound of Formula 2 300 Crystalline
cellulose 3 Lactose 14.8 Magnesium stearate 0.2
[0105] These components were mixed according to a conventional
capsule formulating method, and then filled to a gelatin capsule,
thereby producing the capsule formulation.
Formulation Example 4
Injection Formulation
TABLE-US-00005 [0106] Compound of Formula 2 300 Mannitol 180
Injectable sterile distilled water 2974 Na.sub.2HPO.sub.412H.sub.2O
26
[0107] An injection containing the components having amounts
described above per one ample (2 was manufactured according to a
conventional injection manufacturing process.
* * * * *