U.S. patent application number 10/569844 was filed with the patent office on 2006-12-14 for extract of nelumbins semen for the treatment of depression.
Invention is credited to Hyeon-Su Bae, Moo-Chang Hong, Moon-Kyu Kang, Jung-Wan Oh, Min-Kyu Shin, Jeong-Hwan Yoon.
Application Number | 20060280822 10/569844 |
Document ID | / |
Family ID | 34567608 |
Filed Date | 2006-12-14 |
United States Patent
Application |
20060280822 |
Kind Code |
A1 |
Shin; Min-Kyu ; et
al. |
December 14, 2006 |
Extract of nelumbins semen for the treatment of depression
Abstract
Disclosed are a Nelumbinis Semen extract for treating
depression, method of preparing the extract, and a pharmaceutical
composition and a health food, comprising the extract. The
Nelumbinis Semen extract is prepared by extracting Nelumbinis Semen
with an alcohol or an alcohol solution. The Nelumbinis Semen
extract has been demostrated using animal behavioral, bio-chemical
and molecular biological methods to have strong antidepressive
activity, as well as inhibiting immune suppression caused by
depression, leading to normal immune responses, and reducing side
effects of conventional antidepressants, thereby ensuring safety.
Thus, The Nelumbinis Semen extract is useful for preparing a
composition or health food for treating depression.
Inventors: |
Shin; Min-Kyu; (Seoul,
KR) ; Hong; Moo-Chang; (Seoul, KR) ; Bae;
Hyeon-Su; (Seoul, KR) ; Yoon; Jeong-Hwan;
(Gyeonggi-do, KR) ; Kang; Moon-Kyu; (Gyeonggi-do,
KR) ; Oh; Jung-Wan; (Gyeonggi-do, KR) |
Correspondence
Address: |
ROBERT E. BUSHNELL
1522 K STREET NW
SUITE 300
WASHINGTON
DC
20005-1202
US
|
Family ID: |
34567608 |
Appl. No.: |
10/569844 |
Filed: |
August 28, 2003 |
PCT Filed: |
August 28, 2003 |
PCT NO: |
PCT/KR03/01743 |
371 Date: |
February 28, 2006 |
Current U.S.
Class: |
424/778 |
Current CPC
Class: |
A23V 2002/00 20130101;
A61K 36/62 20130101; A23V 2002/00 20130101; A61P 25/24 20180101;
A23V 2002/00 20130101; A23G 9/42 20130101; A23L 33/105 20160801;
A61P 25/18 20180101; A23L 13/65 20160801; A23V 2250/21 20130101;
A23V 2200/322 20130101; A23V 2200/324 20130101; A23V 2250/21
20130101; A61K 36/185 20130101; A23G 4/068 20130101 |
Class at
Publication: |
424/778 |
International
Class: |
A61K 36/185 20060101
A61K036/185 |
Claims
1. A Nelumbinis Semen extract having antidepressive activity, which
is prepared by extracting Nelumbinis Semen with an alcohol or an
alcohol solution.
2. The Nelumbinis Semen extract according to claim 1, wherein the
alcohol or alcohol solution is selected from the group consisting
of 10-100% ethyl alcohol and 10-100% methyl alcohol.
3. The Nelumbinis Semen extract according to claim 2, wherein the
alcohol or alcohol solution is 70-100% ethyl alcohol.
4. A method of preparing a Nelumbinis Semen extract comprising: 1)
extracting Nelumbinis Semen with an alcohol or an alcohol solution;
and 2) filtering and concentrating a resulting extract.
5. The method according to claim 4, wherein the extraction of
Nelumbinis Semen is selected from the group consisting of cold
extraction (maceration), reflux extraction and ultrasonic
extraction.
6. The method according to claim 5, wherein the extraction is
ultrasonic extraction.
7. A pharmaceutical composition for treating depression, comprising
the Nelumbinis Semen extract of claim 1 as an effective
component.
8. A health food for treating depression, comprising the Nelumbinis
Semen extract of claim 1 as an effective component.
9. The method according to claim 4, further comprising
freeze-drying a resulting concentrate.
10. A Nelumbinis Semen extract prepared by the method of claim 4.
Description
TECHNICAL FIELD
[0001] The present invention relates, in general, to an extract of
Nelumbinis Semen (Nelumbo nucifera) having a therapeutic effect on
depression, a method of preparing the extract, and a pharmaceutical
composition and a health food comprising the extract. More
particularly, the present invention relates to a Nelumbinis Semen
extract obtained by extracting Nelumbinis Semen with an alcohol or
an alcohol solution and concentrating and drying the resulting
extract, a method of preparing the Nelumbinis Semen extract, and a
pharmaceutical composition and a health food which comprise the
Nelumbinis Semen extract as an effective component.
BACKGROUND ART
[0002] Mental damage occurring in the complicated modern society
is, contrary to in the past, mostly caused by weak but prolonged
and repeated stress from usual activities rather than large
psychological impact or stimuli. Such stress is difficult to be
recognized by patients and easily overlooked during hospital visits
by patients, and thus accumulates, causing individuals to suffer
from depression.
[0003] Depression is an emotional pathological phenomenon occurring
regardless of objective situations. Emotional symptoms of
depression include depressed behavior during all activities,
anhedonia (loss of interest or pleasure), diminished mental
capacity, pessimism, poor self-esteem, and suicidal thoughts that
occasionally lead to suicide attempts. Physical symptoms of
depression include decreased appetite, insomnia, constipation,
diminished sexual desire, reduced immune functions, and patients'
susceptibility to diseases due to the reduced immune function.
[0004] There has been so far no theory that perfectly explains the
mechanism causing depression and the action mechanism of
antidepressants for treating depression. However, for many years,
the prevailing hypothesis is that depression is caused by an
absolute or relative deficiency of monoamine neurotransmitters in
synapses of the central nervous system, such as serotonin,
norepinephrin and dopamine. In this regard, all antidepressants
have pharmaceutical action to increase concentrations of
neurotransmitters in central serotonin or noradrenaline
synapses.
[0005] Antidepressants are divided into three major types according
to the mechanism involving increasing the neurotransmitter levels:
tricyclic antidepressants (TCA); monoamine oxidase inhibitors
(MAOI); and selective serotonin reuptake inhibitors (SSRI).
[0006] Monoamine oxidase inhibitors, such as phenelzine developed a
relatively long time ago, have a severe adverse effect of inducing
heart diseases, and thus, have not been widely used recently.
Tricyclic antidepressants such as imipramine also have
anticholinergic, sedative, and other side effects related to the
cardiovascular system. Thus, recent research focuses on the
development of therapeutic agents against depression using
selective serotonin (5-HT) reuptake inhibitors (hereinafter,
referred to simply as "SSRI") as antidepressants with fewer side
effects. Representative examples include fluoxetine (brand name:
Prozac), paroxetine (brand name: Seroxate), and sertraline (brand
name: Zoloft), which are widely approved due to their clinical
efficacy. However, the aforementioned drugs also have side effects
such as whole-body fatigue, sexual dysfunction and insomnia.
Administration of antidepressants was reported to typically
activate a serotonin receptor by increasing serotonin levels,
leading to an activation of PKA that is downstream of the serotonin
receptor and eventually increases in protein levels of CREB,
brain-derived neurotrophic factor (BDNF) and its receptor, trkB.
These increased protein levels are considered to indicate effective
actions of antidepressants in molecular levels (J. of Psychosomatic
Research 53, 687-697 (2002)). In addition, the administration of
antidepressants restores to normal levels decreased concentrations
of cortisol and IL-2 and decreased cell numbers of WBC and
lymphocytes, all of which are representative responses of
individuals with depression, thereby providing a normal immune
system (Ann N Y Acad Sci. 917, 478-487 (2000)). These effects may
be another therapeutic effect of antidepressants.
[0007] Recently, in the Western countries, medicinal herbal
extracts have been recognized for their therapeutic effects and
studied. With regard to depression, extracts of Hypericum
perforatum (known also as St. John's wort) have been studied
(Neuropharmacology, 1999, 21(2), 247-257; Cochrane Database Syst
Rev, 2000, (2), CD000448; Drugs Aging, 2000, 16(3), 189-197).
[0008] According to a report that compared a Hypericum perforatum
extract with imipramine for therapeutic efficacy on depression, the
Hypericum perforatum extract has similar efficacy to imipramine in
treating depression and has fewer side effects (BMJ, 2000, 321,
536-539). Also, the Hypericum perforatun extract has the potential
to inhibit the activities of human cytochrom P450 enzymes (J
Pharmacol Exp Ther, 2000, 294(1), 88-95).
[0009] The Hypericum perforatum extract contains a large number of
structurally different compounds that directly or indirectly affect
the central nervous system (CNS). That is, the Hypericum perforatum
extract contains bioactive compounds, such as hypericin and
hyperforin, and dimeric flavors, which are known to have
antidepressive and apprehension-removing effects in animals and
humans.
[0010] The action mechanisms of the constituents of Hypericum
perforatum are as follows. Hypericin is proved to have the
antidepressive effect in the presence of dimeric procyanidines
contained in the Hypericum perforatum extract (Regensburg, Germany,
V. Butterwecke et.al., 45th Annual Congress of the Society for
Medicinal Plant Research, 1997, Abstract No. 011). Hyperforin
increases 5-HT (serotonin) levels in the hypothalamus and
hippocampus, indicating that the antidepressive effect of
hyperforin is associated with the serotonergic system (J Pharm
Pharmacol, 2001, 53(5), 583-600; Pharmacopsychiatry, 2000, 33(2),
60-65). However, about 20% of depression patients are not treated
with conventional antidepressants, and recently developed
antidepressants such as SSRI have fewer side effects than other
antidepressants, but they are still not negligible.
[0011] On the other hand, various depression animal models have
been tried in the development process of antidepressants for
treating depression. Strong stimuli such as intense foot-shock,
cold water immersion and 48 h food/water deprivation were initially
preferred, but, recently, preferred methods are to use weak
repetitive stresses better capable of mimicking usual activities of
modern people experiencing weak prolonged chronic stresses
(Psychopharmacology, 1984, 83, 1-16). Among the recent methods, a
chronic mild stress (hereinafter, referred to simply as "CMS")
model, suggested by Willner et al., has been approved as an
excellent animal model of depression having reliability and
validity (Neuroscience and Biobehavial Review, 1981, 5, 231-246;
TIPS, 1991, 12, 131-136).
[0012] "Mildly stressed rats" means that, when CMS-induced
behavioral changes are observed for a prolonged administration
period of weeks, the behavioral changes do not occur habitually, or
habitual changes occur within a constant limitation
(Psychopharmacology, 1997, 134, 319-320). In general experiments, a
variety of chronic weak stressors, such as overnight illumination,
periods of food and/or water deprivation, cage tilt and change of
cage mate, are used (Psychopharmacology, 1997, 134, 319-320).
Repeated exposure of white rats to such stressors results in a
significant decrease in consumption of a sucrose solution, which is
comparable to anhedonia, the representative symptom of depression
of white rats. Upon no appropriate treatment, such decrease in
consumption of a sucrose solution is known to last for several
weeks after withdrawal of a CMS procedure. Many antidepressants
have been approved that they have effects of recovering the reduced
sucrose intake induced by the CMS procedure to an original level
(Psychopharmacology 1992, 109, 433-438).
[0013] On the other hand, Nelumbinis Semen is the skinned ripe seed
of lotus (Nelumbo nuncifera), which has a green core. Nelumbinis
Semen has no smell and a sweet, fresh and slightly astringent
taste.
[0014] Nelumbinis Semen contains a large quantity of starch and
raffinose sugar, and is known to have the therapeutic effects of
strengthening the spleen and stomach, alleviating insomnia,
whitening the skin, relieving inflammation and healing wounds in
the skin. However, to date, there is no report of its ability to
alleviate depression symptoms.
DISCLOSURE
[0015] Therefore, the present invention aims to provide a
Nelumbinis Semen extract having antidepressive activity, a method
of preparing the Nelumbinis Semen extract, and a pharmaceutical
composition and a health food comprising the Nelumbinis Semen
extract as an effective component.
[0016] Based on the fact that Nelumbinis Semen is used as a Chinese
traditional herbal medicine, the intensive and thorough animal
behavioral research into the therapeutic effects of an extract of
Nelumbinis Semen, conducted by the present inventors, resulted in
the finding that the Nelumbinis Semen extract is superior in
treating depression to conventional antidepressants, Hypericum
perforatum extract and fluoxetine (brand name: Prozac) one of SSRI,
the recently most commonly used antidepressants, thereby providing
a pharmaceutical composition for treating depression comprising the
Nelumbinis Semen extract of the present invention.
[0017] In addition, the molecular biological and biochemical
research revealed the mechanism of the antidepressive action of the
Nelumbinis Semen extract of the present invention, and resulted in
the finding that the Nelumbinis Semen extract has another effect of
normalizing immune functions, thereby providing a pharmaceutical
composition for treating depression comprising the Nelumbinis Semen
extract of the present invention.
[0018] Further, the animal behavioral research resulted in the
finding that the Nelumbinis Semen extract of the present invention
does not have the side effects that are observed upon application
of conventional antidepressants.
[0019] Thus, in one aspect, the present invention provides a
Nelumbinis Semen extract having antidepressive activity.
[0020] In another aspect, the present invention provides a method
of preparing the Nelumbinis Semen extract.
[0021] In a further aspect, the present invention provides a
pharmaceutical composition for treating depression, comprising the
Nelumbinis Semen extract as an effective component.
[0022] In yet another aspect, the present invention provides a
health food for treating depression, comprising the Nelumbinis
Semen extract as an effective component.
[0023] Hereinafter, the present invention will be described in
detail.
[0024] The present invention provides a Nelumbinis Semen extract
having antidepressive activity.
[0025] The Nelumbinis Semen extract of the present invention is
prepared by a process including 1) extracting Nelumbinis Semen with
an alcohol or an alcohol solution; 2) filtering and concentrating
the resulting extract; and 3) freeze-drying the resulting
concentrate.
[0026] The alcohol or alcohol solution may be selected from the
group consisting of 10-100% ethyl alcohol and 10-100% methyl
alcohol. Preferred is 70-100% ethyl alcohol.
[0027] The extraction is carried out by cold extraction
(maceration), under reflux conditions or by ultrasonic treatment.
The ultrasonic extraction is preferred.
[0028] The present inventors investigated that the Nelumbinis Semen
extract of the present invention has antidepressive activity. After
the Nelumbinis Semen extract of the present invention was
administered, experimental animals were stressed by being exposed
to bright light for a period of 48 hrs before a forced swim test.
During forced swimming, the following behaviors were evaluated:
struggling time (time spent struggling, defined as strongly moving
all four limbs), first latency (latency until first floating) and
first rest duration (length of the first floating). As a result,
the Nelumbinis Semen extract of the present invention displayed
antidepressive activity and had a higher antidepressive effect than
a comparative group, Hypericum perforatum extract.
[0029] In addition, the present inventors compared the Nelumbinis
Semen extract of the present invention with other Chinese medical
herbs. The Chinese medical herbs as comparative subjects included
Rehmanniae Radix Preparat, Lycii Fructus and Corni Fructus, which
strengthen body functions, and Pinelliae Rhizoma that has an
expectorating effect (Table 1). TABLE-US-00001 TABLE 1 Chinese
medical herbs and their amount used Chinese medical herb
(Pharmaceutical name) Amount Rehmanniae Radix Preparat 500 g Corni
Fructus 500 g Lycii Fructus 500 g Pinelliae Rhizoma 500 g
Nelumbinis Semen 500 g Hypericum perforatum 500 g
[0030] As a result, the Nelumbinis Semen extract of the present
invention was found to have a higher antidepressive effect than
extracts of Rehmanniae Radix Preparat, Lycii Fructus, Corni Fructus
and Pinelliae Rhizoma.
[0031] In addition, the Nelumbinis Semen extract of the present
invention was tested for its antidepressive activity and for
overcoming sexual dysfunction, which is a representative side
effect of conventional antidepressants. In this test using the
aforementioned CMS model of depression in rats, being applicable to
practical situations, where rats were exposed to CMS to induce
depression, the rats were administered with conventional
antidepressants, Prozac and a Hypericum perforatum extract, and the
Nelumbinis Semen extract of the present invention, antidepressive
effects and side effects of the administered drugs were evaluated
by objective comparison between test groups for behavioral changes
including changes in weight, sucrose intake and physical activity
in an open place. Also, a reduction in sexual behavior, which is a
representative side effect of the SSRI depressants, was
investigated by comparison between test groups for mating behavior
according to the above drugs. As a result, the Nelumbinis Semen
extract of the present invention was found to have a higher
antidepressive effect than the comparative drugs, Hypericum
perforatum extract and Prozac, while not displaying reduced sexual
behavior that is a side effect of the above conventional
antidepressive drugs, thereby indicating that the present
Nelumbinis Semen extract does not have the side effects found upon
the application of conventional antidepressive drugs.
[0032] Further, the mechanism of the antidepressive action of the
Nelumbinis Semen extract of the present invention was assessed
according to the following molecular biological and biochemical
methods.
[0033] First, according to a molecular biological method, after a
depression-induced experimental animal was administered with an
effective amount of the Nelumbinis Semen extract of the present
invention and the conventional antidepressive drugs, the
experimental animal with improved depression was incised at the
cerebral frontal cortex. Total RNA was isolated from the cerebral
frontal cortex, and double-stranded cDNA was synthesized using the
isolated RNA and an oligo(dT)24-T7 primer. cRNA was synthesized by
in vitro transcription using the synthesized cDNA, and was
biotin-labelled and applied to an oligonucleotide microarray to
determine gene expression patterns in each test group. As a result,
the Nelumbinis Semen extract of the present invention was found,
like the Hypericum perforatum extract and Prozac used as
comparative drugs, to significantly increase expression levels of
CREB, BDNF and trkB genes that are representative in vivo markers
of antidepression.
[0034] Also, according to a biochemical method, the therapeutic
efficacy of a candidate drug was primarily examined by measuring
changes in 5-HT and norepinephrine. (NE) levels in a chronic CMS
model by microdialysis and HPLC-ECD. Catecholamine content was
measured using an HPLC system equipped with an electrochemical
detector. A mobile phase containing 0.05 M monobasic sodium
phosphate, 0.1 N sodium acetic acetate and 1% methanol was adjusted
to pH 4.4 with a phosphate buffer for HPLC. DA was composed of a
Supelcosil LC-8-DB 3-.mu.m column (150.times.4.6 mm, Supelco,
Bellefonte, Pa.) protected by an LC-18 guard column. As a result,
the Nelumbinis Semen extract of the present invention was found,
like the Hypericum perforatum extract and Prozac used as
comparative drugs, to significantly increase levels of the 5-HT and
NE neurotransmitters.
[0035] Secondarily, antidepressive effects were evaluated by an
increase or decrease in receptor binding of a serotonin 2A receptor
agonist, [.sup.3H]spiperone, in the rat brain frontal cortex, as
follows. A behavioral test in rats was carried out for a period of
three weeks, and the rats were administered with each drug. The
brain frontal cortex was excised from the rats and frozen in liquid
nitrogen. The frontal cortex was cryo-sectioned into a horizontal
form using a cryostat microtome, and each section was incubated in
a [.sup.3H]spiperone-containing Tris-HCl buffer for two hours,
dried, placed in an autoradiography cassette and exposed to an
autoradiography film for four weeks in a refrigerator. Then, the
film was developed and scanned with a densitometer to measure light
and dark intensity that corresponds to binding strengtHypericum
After calibrated values were calculated using a standard scale bar,
binding strength of each cryo-section was expressed as Ci/mg
tissue. When test groups were compared with a control group for an
increase or decrease in receptor binding as an indication of
antidepressive action, the Nelumbinis Semen extract of the present
invention was found, like the Hypericum perforatum extract and
Prozac used as comparative drugs, to significantly increase the
binding of the serotonin 2A receptor agonist,
[.sup.3H]spiperone.
[0036] Thirdly, an increase in the antidepressive markers, CREB,
BDNF and trkB proteins, was examined in the rat cerebral frontal
cortex by Western blotting, as follows. A behavioral test in rats
was carried out for a period of three weeks, and the rats were
administered with each drug. The brain frontal cortex was excised
and centrifuged, and the supernatant was recovered. Each sample was
mixed with a SDS-PAGE sample buffer to reduce proteins and boiled
to denature proteins. Samples were loaded onto a SDS-PAGE (sodium
dodesyl sulfate poly acrylamide gel electrophoresis) gel and run at
100-200 V for about 1-2 hrs. The SDS-PAGE gel was transferred onto
a PVDF or nitrocellulose membrane by electro-transferation. The
membrane was incubated with a primary antibody for a target
protein, washed and incubated with a secondary antibody. Then, the
target protein was detected by an enhanced chemiiluminascent
method. As a result, the Nelumbinis Semen extract of the present
invention was found, like the Hypericum perforatum extract and
Prozac used as comparative drugs, to significantly increase protein
levels of CREB, BDNF and trkB.
[0037] Fourthly, an increase in the antidepressive markers, CREB,
BDNF and trkB proteins, was examined in the rat cerebral frontal
cortex by 2-DE, as follows. A behavioral test in rats was carried
out for a period of three weeks, and the rats were administered
with each drug. The brain frontal cortex was excised and
centrifuged, and the supernatant was recovered. Each sample was
mixed with an IEF sample buffer and loaded onto an IEF gel.
Proteins were separated according to isoelectric point by
isoelectric focusing (IEF) in the first dimension and according to
molecular weight by SDS-PAGE at 100-200 V for about 1-2 hrs in the
second dimension. The gel was stained by a Gel-Code Blue staining
method and evaluated for elevated or newly emerged proteins by the
antidepressive substances. The elevated or newly emerged proteins
by the antidepressive substances were subjected to mass
spectrometry. As a result, the Nelumbinis Semen extract of the
present invention was found, like the Hypericum perforatum extract
and Prozac used as comparative drugs, to significantly increase
protein levels of CREB, BDNF and trkB.
[0038] In addition, the Nelumbinis Semen extract of the present
invention was evaluated to determine its ability to help overcome
immune suppression caused by depression, by the following
biochemical method.
[0039] First, changes in concentrations of cortisol, which is a
representative marker to determine whether the immune suppression
has been overcome, according to administration of the Nelumbinis
Semen extract were examined, as follows. After urine samples were
collected from rats,
[0040] (1) 1.0 ml of urine was placed into a tube with a cap, 2.0
ml dichloromethane was added to the tube, and the tube was covered
with the cap with caution;
[0041] (2) the mixture was vortexed for 5-10 min;
[0042] (3) the mixture was centrifuged at 1500.times.g (rpm), the
upper layer was aspirated, and 50 .mu.l of the lower layer were
aliquotted into coated tubes;
[0043] (4) samples were evaporated for dryness.
[0044] Urinary cortisol concentrations were measured as
follows.
[0045] (1) The coated tubes were individually labeled with NSB, Std
(A-F), a control (CON6 No. 5) and sample numbers (for NSB, a green
tube was used).
[0046] (2) 25 .mu.l of each of NSB, Std (A-F), a control (CON6 No.
5) and samples was added to the corresponding tube (free cortisol
was added to the completely dried coated tube, and 25 .mu.l of Std.
A was added to the NSB tube).
[0047] (3) 1.0 ml of .sup.125I-cortisol was added to the tubes,
followed by mixing.
[0048] (4) The tubes were incubated in a water bath at 37.degree.
C. for 45 min and aspirated.
[0049] (5) After the content of the tubes was completely aspirated,
radioactivity was measured for at least 1 min using a
Y-counter.
[0050] As a result, the Nelumbinis Semen extract of the present
invention was found, like the Hypericum perforatum extract and
Prozac used as comparative drugs, to restore cortisol
concentrations to levels similar to a normal group.
[0051] Secondarily, changes in concentrations of IL-2, which is a
representative marker to determine whether the immune suppression
has been overcome, according to administration of the Nelumbinis
Semen extract was examined, as follows. After blood samples were
collected from rats,
[0052] (1) 100 .mu.l of assay diluent QD6-23 were aliquotted onto a
microplate strip;
[0053] (2) 50 .mu.l of each sample and a standard were aliquotted
onto the strip;
[0054] (3) the strip was incubated with shaking at room temperature
for 2 hrs;
[0055] (4) the strip was washed four times;
[0056] (5) 200 .mu.l of an IL-2 conjugate was aliquotted onto the
strip, and the strip was incubated with shaking at room temperature
for 3 hrs;
[0057] (6) 200 .mu.l of a substrate (luminol+hydrogen peroxide) was
aliquotted onto the strip, and the strip was incubated at room
temperature for 20-40 min;
[0058] (7) emitted light was measured using a luminometer; and
[0059] (8) IL-2 concentrations were determined using a standard
quantitative curve.
[0060] As a result, the Nelumbinis Semen extract of the present
invention was found, like the Hypericum perforatum extract and
Prozac used as comparative drugs, to restore
[0061] IL-2 concentrations to levels similar to a normal group.
[0062] Thirdly, changes in WBC cell number, which is a
representative marker to determine whether the immune suppression
has been overcome, according to administration of the Nelumbinis
Semen extract, was examined, as follows. After blood samples were
collected from rats, cell volume, conductivity and light scattering
were measured.
1) Cell Volume
[0063] Cell volume was measured according to the Coulter Principle,
which is universally recognized as a reference method for sizing of
volumes. An aperture between two electrodes is placed in a cell
flow, and direct current flows between the electrodes. When WBC
maintained at nearly natural states passes through the aperture,
electrical resistance to the direct current flow increases, thus
generating a voltage pulse being in proportion to cell volumes. The
size of the pulse is one of the distinct features allowing
determination of the type of WBC. However, to distinguish between
two types of cells with similar size, a different phenotype
allowing the distinction of two types of cells should be measured.
For example, mature basophils and small lymphocytes are about 9-12
.mu.m in diameter, and immature prolymphocytes and mature
neutrophils are 12-14 .mu.m in diameter. Different types of cells
with similar size are difficult to determine based on only their
size, and thus, in this case, conductivity and light scattering are
simultaneously measured.
2) Conductivity
[0064] Conductivity measurement is based on measuring cell contents
using a high-frequency electromagnetic field. This technology is a
method of measuring cell contents, which started to be developed in
the 1960s by Doctor Wallace Coulter, the founder of the Coulter
Electronics Company, and was patented in USA in 1970. Doctor
Coulter demonstrated that a high-frequency current is able to
penetrate the cell membrane. The current that has penetrated cells
exists in specific forms according to the composition of the
nucleus and granules and the internal chemical composition of
cells. The application of this high-frequency electromagnetic field
creates a novel practical method capable of providing information
about contents of cells. Doctor Coulter suggested a very important,
new measurement item called "opacity", which has information about
composition of the cytoplasm and the nucleus in an electromagnetic
field where a high-frequency current penetrates cells. "Opacity" is
a conductivity signal reflecting the internal composition of cells,
which is not influenced by cell size. "Opacity" measurement
provided only by the Coulter Company is the most accurate and
reliable technology for measuring cell contents. Conductivity is
useful for separating cells of similar size, but different internal
composition. Only volume measurement does not distinguish basophils
from small lymphocytes. However, since conductivity measurement is
carried out by measuring the difference between cell types in the
ratio of the nucleus to the cytoplasm, granularity, etc., it is
very useful for separating cell types.
3) Light Scattering
[0065] In addition to volume and conductivity data, light
scattering characteristics on the cell surface distinctly differ
between cell types. Homogeneous light emitted from a laser is
collected using a lens and converted to a voltage pulse. Thus,
light scattering is very useful for sorting cells according to the
morphology and amount of granules.
[0066] WBC counting using the above methods resulted in the finding
that the Nelumbinis Semen extract of the present invention was
found, like the Hypericum perforatum extract and Prozac used as
comparative drugs, to restore the cell number of WBC to levels
similar to a normal group.
[0067] Fourthly, changes in cell number of lymphocytes, which is a
representative marker to determine whether the immune suppression
has been overcome, according to administration of the Nelumbinis
Semen extract, was examined as follows. After blood samples were
collected from rats, changes in lymphocyte cell number were
examined by the following method.
[0068] 1. Assay Principle
[0069] When diluted blood corpuscles are suspended in an
electrolyte solution (Isoton sol.) that is a separate insulator,
the electrolyte suspension of particles is suctioned for 4 sec
through an aperture with a predetermined size. The aperture is
placed between an internal electrode and an external electrode, and
a current flows between the electrodes. When particles pass through
the aperture between the two electrodes, resistance increases while
voltage decreases. The difference between decreased voltage and
ground voltage is expressed as height by a threshold circuit. The
number of pulses indicates the number of particles, and the
amplitude of pulses is in proportion to the volume of
corpulscles.
[0070] 2. Assay Method: Automatic Analyzer
[0071] 3. Machine Used: ADVIA120, Bayer, USA
[0072] 4. Reagents Used:
[0073] 1) Isoton III, Beckman Coulter, USA
[0074] 2) Coulter clenz, Beckman Coulter, USA
[0075] 3) Lyse S III, Beckman Coulter, USA
[0076] 4) 4% sod. hypochloride-solution, Beckman Coulter, USA
[0077] 5) 4C-plus, Beckman Coulter, USA
[0078] 6) Scatter Pak, Beckman Coulter, USA
[0079] As a result, the Nelumbinis Semen extract of the present
invention was found, like the Hypericum perforatum extract and
Prozac used as comparative drugs, to restore the cell number of
lymphocytes to levels similar to a normal group.
[0080] In addition, the present invention provides a pharmaceutical
composition for treating depression, comprising the Nelumbinis
Semen extract as an effective component.
[0081] The present pharmaceutical composition for treating
depression includes the Nelumbinis Semen extract as an effective
component. The pharmaceutical composition may be administered
orally or parenterally and may be formulated into typical
pharmaceutical preparations.
[0082] That is, the Nelumbinis Semen extract of the present
invention may be formulated into various formulations for oral and
parenteral administration upon clinical application. In the
formulation, diluents or excipients may be used, which are
exemplified by fillers, thickeners, binders, humectants,
disintegrators and surfactants.
[0083] Examples of solid formulations for oral administration
include tablets, pills, powders, granules and capsules. The solid
formulations may include, in addition to the Nelumbinis Semen
extract, at least one excipient selected from among starch, calcium
carbonate, sucrose, lactose, gelatin, etc. Also, the solid
formulations may include, in addition to a simple excipient, a
lubricant such as magnesium stearate or talc.
[0084] Examples of liquid formulations for oral administration
include suspensions, internal solutions, emulsions and syrups. The
liquid formulations may include, in addition to commonly used
simple diluents such as water and liquid paraffin, various
excipients which are exemplified by humectants, sweeteners,
aromatics and preservatives.
[0085] Examples of preparations for parenteral administration
include sterile aqueous solutions, non-aqueous solutions,
suspensions, emulsions, freeze-dried preparations and
suppositories. In the formulation into non-aqueous solutions and
suspensions, propylene glycol, polyethylene glycol, vegetable oils
such as olive oil, and injectable esters such as ethyl oleate may
be used. As a base of suppositories, witepsol, macrogol, Tween 61,
cacao fat, lanolin fat, glycerol and gelatin may be used.
[0086] The unit dose, may, for example, occurs one, two, three or
four times, or a half, third or quarter of an individual dose. The
individual dose preferably contains the amount of an effective
drugs which is given in one administration and usually corresponds
to a whole daily dose or a half, third or quarter of the daily
dose.
[0087] In the pharmaceutical composition for treating depression,
an effective amount of the Nelumbinis Semen extract ranges from 30
to 700 mg/kg, and preferably 100 to 500 mg/kg, and may be
administered once to six times daily. The dosage for a specific
patient may vary according to the patient's weight, age, sex,
health state and diet, administration duration, administration
routes, excretion rates and severity of the illness.
[0088] When the Nelumbinis Semen extract of the present invention
was orally, intraperitoneally and subcutaneously administered to
white rats to evaluate its toxicity, 50% lethal dose (LD50) of the
Nelumbinis Semen extract upon the intraperitoneal administration
was higher than 20 g/kg. This result demonstrates that the
Nelumbinis Semen extract is safe.
[0089] In addition, the present invention provides a health food
for treating depression, comprising the Nelumbinis Semen extract as
an effective component.
[0090] In the case of using the present extract as a food, the
present extract may be added as it exists or in combination with
other food or food ingredients, and may be used suitably according
to general methods. Mixed amounts of effective components may be
suitably determined according to the intended use (preventive,
health or therapeutic purposes). Typically, the present extract may
be added in an amount of 0.01 to 1 wt %, and preferably 0.1 to 1 wt
%, based on the total weight of raw materials used in preparing a
food or drink. An effective amount of the present extract may be
determined based on an effective amount of the pharmaceutical
composition. When consumed for a long period of time for health and
sanitary purposes or health control, the present extract may be
used in an amount lower than the range. Also, it is apparent that
the present extract can be used in an amount higher than the range
because the effective component carries no safety risk.
[0091] The type of the food is not particularly limited. Examples
of foods to which the present extract can be added include meats,
sausages, breads, chocolates, candies, snacks, confectionary,
pizza, instant noodles, other noodles, gums, dairy products
including ice creams, various soups, beverages, teas, drinks,
alcoholic beverages and vitamin complexes, as well as traditional
therapeutic preparations for use as an antianemic, a body
function-strengthening agent, a skin whitening agent, and the like.
In addition, the present invention may be used in various
prescriptions of Chinese medical decoctions, such as Reu Do Han
Shao Tang, Quing Sin Shan Yao Tang and Tai Yin Tiao Wei Tang.
[0092] A better understanding of the present invention may be
obtained, in conjunction with the accompanying drawings, through
the following examples and experimental examples which are set
forth to illustrate, but are not to be construed as the limit of
the present invention.
DESCRIPTION OF DRAWINGS
[0093] FIG. 1 is a graph showing the struggling time measured in a
forced swim test using white rats, in which, for struggling time, a
test group administered with a Nelumbinis Semen extract according
to the present invention is compared to a control group and other
test groups individually administered with a Rehmanniae Radix
Preparat extract, a Corni Fructus extract, a Lycii Fructus extract;
a Pinelliae Rhizoma extract and a Hypericum perforatum extract;
[0094] FIG. 2 is a graph showing the first latency time measured in
a forced swim test using white rats, in which, for first latency
time, a test group administered with a Nelumbinis Semen extract
according to the present invention is compared to a control group
and other test groups individually administered with a Rehmanniae
Radix Preparat extract, a Corni Fructus extract, a Lycii Fructus
extract, a Pinelliae Rhizoma extract and a Hypericum perforatum
extract; and
[0095] FIG. 3 is a graph showing the first rest duration measured
in a forced swim test using white rats, in which, for first rest
duration, a test group administered with a Nelumbinis Semen extract
according to the present invention is compared to a control group
and other test groups individually administered with a Rehmanniae
Radix Preparat extract, a Corni Fructus extract, a Lycii Fructus
extract, a Pinelliae Rhizoma extract and a Hypericum perforatum
extract.
MODE FOR INVENTION
EXAMPLE 1
Preparation of Nelumbinis Semen Extract
[0096] 500 g of Nelumbinis Semen dried powder was placed into a
flask containing 1 L of 70% ethyl alcohol and subjected to
ultrasonic extraction (Branson Co., USA) at room temperature for 10
min, and the supernatant was recovered. The pellet was further
extracted with 85% and 100% ethylalcohol according to the same
method as described above. The supernantants were pooled and
filtered through a gauze. The filtrate was concentrated using a
vacuum filter (Eyela, Japan) and freeze-dried, thus yielding 95 g
of a Nelumbinis Semen extract according to the present
invention.
EXPERIMENTAL EXAMPLE 1
Evaluation of Antidepressive Activity of the Nelumbinis Semen
Extract
[0097] The Nelumbinis Semen extract of the present invention was
orally administered to postnatal 85-95-day Sprague-Dawley male
rats. A comparative group was orally administered with a Hypericum
perforatum extract. For 48 hrs before a forced swim test, the rats
were stressed by being exposed to bright light (300 Lux).
[0098] A forced swim test was carried out as follows. On day 1, the
white rats were placed into a cylindrical water bath (22 cm in
diameter; 30 cm water depth) and forced to swim for 10 min. On day
2, the rats were forced to swim for 5 min, and during this forced
swimming, struggling time was measured.
[0099] As a result, during the forced swimming, the comparative
group administered with the Hypericum perforatum extract showed a
non-significant increase in struggling time by 25.2% in comparison
with a control group. In contrast, the Nelumbinis Semen extract
significantly increased the struggling time by 43.9% (FIG. 1).
[0100] In addition, compared to the control group, administration
of the Hypericum perforatum extract resulted in a non-significant
increase in first latency time by 75.8%. In contrast, the
Nelumbinis Semen extract significantly increased the first latency
time by 90.2% (FIG. 2).
[0101] Further, compared to the control group, in the comparative
group administered with the HYPERICUM perforatum extract, no change
was observed in first rest duration. In contrast, in the group
administered with the Nelumbinis Semen extract, the first rest
duration decreased by 59.0% (FIG. 3).
[0102] These results indicate that the Nelumbinis Semen extract of
the present invention has antidepressive activity and is superior
to the Hypericum perforatum extract used as a comparative group in
counteracting depression.
COMPARATIVE EXAMPLE
Comparison of the Nelumbinis Semen Extract with Other Chinese
Medicinal Herbal Extracts for Antidepressive Activity
[0103] The antidepressive effect of the Nelumbinis Semen extract
was examined according to the same method as in Experimental
Example 1 and compared with other Chinese medicinal herbal extracts
of Rehmanniae Radix Preparat, Corni Fructus, Lycii Fructus and
Pinelliae Rhizoma.
[0104] As a result, during the forced swimming, in comparison with
a control group, administration of the Rehmanniae Radix Preparat
and Lycii Fructus extracts resulted in a non-significant increase
in struggling time by 15.2% and 4.9%, respectively, while
administration of the Corni Fructus and Pinelliae Rhizoma extracts
resulted in a reduction of 3.9% and 1.1%, respectively. In
contrast, the Nelumbinis Semen extract significantly increased the
struggling time by 43.9% (FIG. 1).
[0105] In addition, compared to the control group, administration
of the Rehmanniae Radix Preparat, Corni Fructus and Pinelliae
Rhizoma extracts resulted in a non-significant increase in first
latency time by 38.4%, 29.2% and 65.5%, respectively, while
administration of the Lycii Fructus extract resulted in a reduction
of 21.4%. In contrast, the Nelumbinis Semen extract significantly
increased the first latency time by 90.2% (FIG. 2).
[0106] Further, compared to the control group, administration of
the Rehmanniae Radix Preparat, Corni Fructus, Lycii Fructus and
Pinelliae Rhizoma extracts resulted in a reduction of 63.1%, 31.6%,
12.4% and 62.4%, respectively, in first rest duration. In contrast,
administration of the Nelumbinis Semen extract resulted in a
reduction of 59.0% in first rest duration (FIG. 3).
[0107] These results indicate that the Nelumbinis Semen extract of
the present invention has higher antidepressive activity than the
Rehmanniae Radix Preparat extract.
EXPERIMENTAL EXAMPLE 2
Evaluation of Acute Toxicity of the Nelumbinis Semen Extract Upon
Oral Administration to Rats
[0108] An acute toxicity test was carried out using 6-week specific
pathogen-free (SPF) SD rats. The Nelumbinis Semen extract of the
present invention was suspended in a 0.5% methylcellulose solution
and orally administered to groups each consisting of five rats in a
single dose of 5 g/kg, 10 g/kg and 20 g/kg. After administration of
the extract, death, clinical symptoms and weight change were
observed, and a hematological test and hematobiochemical analysis
were performed. Upon autopsy, abnormality in abdominal organs and
chest organs was visually observed.
[0109] As a result, all rats administered with the extract
exhibited no particular clinical symptoms, no death, no change in
weight and no toxicity upon the hematological assay,
hematobiochemical analysis and autopsy. As a result, the Nelumbinis
Semen extract of the present invention exhibited no toxicity even
at a dose of 10 g/kg in all rats, and thus had a 50% lethal dose
(LD50) higher than 20 g/kg upon oral administration. This result
demonstrates that the Nelumbinis Semen extract is safe.
FORMULATION EXAMPLE 1
Preparation of Soft Capsules
[0110] Soft capsules were prepared according to a soft capsule
preparation method described in General Rules for Preparation in a
guidebook, Korean Pharmacopoeia, using 100.0 mg per capsule of the
Nelumbinis Semen extract prepared in Example 1, 175.0 mg of soybean
oil, 45.0 mg of cera flava, 127.5 mg of hydrogenated palm oil, 21.0
mg of soybean phospholipids, 212.0 mg of gelatin, 50.0 mg of
glycerin (gravity: 1.24), 76.0 mg of di-sorbitol, 0.54 mg of
methyl-paraoxybenzoate, 0.90 mg of propyl-paraoxybenzoate, 0.56 mg
of methylvanillin, and a proper amount of yellow no. 203.
FORMULATION EXAMPLE 2
Preparation of Tablets
[0111] 100.0 mg of the Nelumbinis Semen extract prepared in Example
1, 90.0 mg of corn starch, 175.0 mg of lactose, 15.0 mg of
L-hydroxypropylcellulose, 5.0 mg of polyvinylpyrolidone 90 and a
proper amount of ethanol were homogeneously mixed, granulated by
wet granulation, mixed with 1.8 mg of magnesium stearic acid, and
forced into 400 mg tablets.
FORMULATION EXAMPLE 3
Preparation of Capsules
[0112] 100.0 mg of the Nelumbinis Semen extract prepared in Example
1, 83.2 mg of corn starch, 175.0 mg of lactose and 1.8 mg of
magnesium stearic acid were homogeneously mixed, and filled into
capsule shells at 360 mg per capsule.
FORMULATION EXAMPLE 4
Preparation of Food and Beverage
[0113] The present inventors prepared food and a beverage
comprising the Nelumbinis Semen extract as an effective component,
as follows.
[0114] <4-1> Preparation of Chewing Gum
[0115] Chewing gum was prepared according to a general method using
0.24-0.64% of the Nelumbinis Semen extract prepared in Example 1,
20% of gum base, 76.36-76.76% of sugar, 1% of a fruit aromatic and
2% of water.
[0116] <4-2> Preparation of Ice Cream
[0117] Ice cream was prepared according to a general method using
0.24-0.64% of the Nelumbinis Semen extract prepared in Example 1,
10.0% of milk fat, 10.8% of SNF (Solids Not Fat), 12.0% of sugar,
3.0% of starch syrup, 0.5% of an emulsion stabilizer (span), 0.15%
of an aromatic (strawberry) and 63.31-62.91% of water.
[0118] <4-3> Preparation of Beverage
[0119] A beverage was prepared according to a general method using
0.48-1.28 mg of the Nelumbinis Semen extract prepared in Example 1,
522 mg of honey, 5 mg of thioctic acid amide, 10 mg of nicotinic
acid amide, 3 mg of riboflavin hydrochloride sodium, 2 mg of
pyridoxine hydrochloride, 30 mg of inositol, 50 mg of orotic acid
and 200 ml of water.
[0120] <4-4> Preparation of Sausage
[0121] Sausage was prepared according to a general method using
0.24-0.64% of the Nelumbinis Semen extract prepared in Example 1,
63.6% of pork, 27.5% of chicken, 3.5% starch, 1.7% of soybean
proteins, 1.62% of edible salt, 0.5% of glucose and 0.94-1.34% of
another additive (glycerin).
INDUSTRIAL APPLICABILITY
[0122] As described hereinbefore, the Nelumbinis Semen extract of
the present invention has very strong antidepressive activity, and
Nelumbinis Semen, as the raw material of the Nelumbinis Semen
extract, is a natural raw material used in Chinese medicine that is
not harmful to the body and is well absorbed by the body when used
as a pharmaceutical composition for treating depression. Therefore,
the Nelumbinis Semen extract is very useful for treating and
preventing depression and various related diseases.
* * * * *