U.S. patent application number 13/208533 was filed with the patent office on 2012-08-30 for chinese herbal aqueous extract having anti-anxiety activities and method of in vitro evaluating the same.
This patent application is currently assigned to National Pingtung University of Science and Technology. Invention is credited to Hso-Chi Chaung, Chih-Cheng Chen, Holi Chen, Hsien-Hsueh Chiu, Tsung-Hui Yang.
Application Number | 20120219645 13/208533 |
Document ID | / |
Family ID | 46719128 |
Filed Date | 2012-08-30 |
United States Patent
Application |
20120219645 |
Kind Code |
A1 |
Chaung; Hso-Chi ; et
al. |
August 30, 2012 |
Chinese Herbal Aqueous Extract Having Anti-Anxiety Activities and
Method of In Vitro Evaluating the Same
Abstract
A Chinese herbal aqueous extract having anti-anxiety activities
and a method of in vitro evaluating the same are disclosed. Neural
cells and/or glial cells are cultured in a medium containing the
Chinese herbal aqueous extract, and then subjected to an electric
pulse treatment to form epileptic cells. The epileptic cells are
applied on evaluation of at least one criterion of anti-anxiety
activities of the Chinese herbal aqueous extract.
Inventors: |
Chaung; Hso-Chi; (Pingtung
County, TW) ; Yang; Tsung-Hui; (Pingtung County,
TW) ; Chen; Holi; (Pingtung County, TW) ;
Chen; Chih-Cheng; (Kaohsiung City, TW) ; Chiu;
Hsien-Hsueh; (Kaohsiung City, TW) |
Assignee: |
National Pingtung University of
Science and Technology
Pingtung County
TW
|
Family ID: |
46719128 |
Appl. No.: |
13/208533 |
Filed: |
August 12, 2011 |
Current U.S.
Class: |
424/728 ;
424/769; 424/770; 435/29; 435/7.1; 435/7.92 |
Current CPC
Class: |
G01N 33/5058 20130101;
G01N 2333/415 20130101; A61K 36/254 20130101; A61P 25/22 20180101;
A61K 36/725 20130101; A23L 33/105 20160801; G01N 2800/301 20130101;
G01N 33/502 20130101; G01N 2500/10 20130101; G01N 2510/00
20130101 |
Class at
Publication: |
424/728 ; 435/29;
435/7.1; 435/7.92; 424/770; 424/769 |
International
Class: |
A61K 36/725 20060101
A61K036/725; A61P 25/22 20060101 A61P025/22; G01N 33/53 20060101
G01N033/53; A61K 36/13 20060101 A61K036/13; C12Q 1/02 20060101
C12Q001/02; A61K 36/254 20060101 A61K036/254 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 25, 2011 |
TW |
100106448 |
Claims
1. A method of in vitro evaluating Chinese herbal aqueous extract
with anti-anxiety activity, comprising: heating a raw material of a
Chinese herbal medicine sample with water and to keep them boiling
for 105 minutes, so as to obtain a Chinese herbal aqueous extract,
wherein the Chinese herbal aqueous extract is a test sample;
culturing a neural cell and/or a glial cell in a testing medium
that includes the test sample or not for 12 hours to 36 hours,
wherein the neural cell and/or the glial cell includes a neural
cell line and/or a glial cell line; applying an electric pulse
treatment on the neural cell and/or the glial cell, allowing the
neural cell and/or the glial cell to be suffered with a
hyper-excitatory electrical potential injury, thereby forming an
epileptic cell; detecting at least one anti-anxiety parameter of
the epileptic cell, the anti-anxiety parameter comprises a cell
viability, a percentage of reactive oxygen species (ROS)--generated
cells and a percentage of apoptotic cells, wherein at least one
test value is defined to the at least one anti-anxiety parameter
obtained from the epileptic cell cultured with the test sample, and
at least one reference value is defined to the at least one
anti-anxiety parameter obtained from the epileptic cell cultivated
without the test sample; and comparing the test value with the
reference value, so as to determine that the test sample has the
anti-anxiety activity based on when the cell viability is more than
the reference value, as well as the percentage of the
ROS--generated cells and the percentage of apoptotic cells are less
than the reference value.
2. The method of claim 1, wherein the neural cell is human
neuroblastoma cell line IMR-32 (deposited at the American Type
Culture Collection under accession No.: ATCC CCL-127), and the
glial cell is human glioblastoma cell line U-87 MG (deposited at
the American Type Culture Collection under accession No.: ATCC
HTB-14).
3. The method of claim 1, wherein the neural cell and/or the glial
cell is cultured in the testing medium for 24 hours.
4. The method of claim 1, wherein the electric pulse treatment is
applied on the neural cell and/or the glial cell by using 5 mV of
pulse voltage and 100 ms of inter-pulse interval for 50 times
continuously.
5. The method of claim 1, wherein the testing medium includes 0.01
g/L to 1 g/L of the test sample.
6. The method of claim 1, wherein the testing medium includes 0.05
g/L to 0.5 g/L of the test sample.
7. The method of claim 1, wherein the testing medium includes 0.1
g/L of the test sample.
8. The method of claim 1, before obtaining the Chinese herbal
aqueous extract, further comprising the step of removing solid
components and water from the raw material of the Chinese herbal
medicine sample.
9. The method of claim 8, wherein the solid components and water
are removed from the raw material of the Chinese herbal medicine
sample through a solid-liquid separation process, a reduced
pressure concentration process or a lyophilization process.
10. The method of claim 1, wherein the Chinese herbal aqueous
extract with anti-anxiety activity is extracted from Poris Cum
(PC), Semen Biotae Orientalis (SBO), Acanthopanacis Cortex (AC) or
Semen Zizyphi Spinosae (SZS).
11. The method of claim 1, wherein a membrane expression of a
Cannabinoid receptor type-1 (CB1) is enhanced in vivo by the
Chinese herbal aqueous extract with anti-anxiety activity.
12. A Chinese herbal aqueous extract with anti-anxiety activity,
comprising the Chinese herbal aqueous extract obtained by using the
method according to any one of claims 1, 2, 10 and 11.
13. A food composition, comprising the Chinese herbal aqueous
extract of claim 12 and at least one food additive.
Description
RELATED APPLICATIONS
[0001] This application claims priority to Taiwan Application
Serial Number 100106448, filed February 2011, which is herein
incorporated by reference.
BACKGROUND
[0002] 1. Field of Invention
[0003] The present invention relates to an active substance having
anti-anxiety activities and a method of evaluating the same. More
particularly, the present invention relates to a Chinese herbal
aqueous extract and a method of in vitro evaluating anti-anxiety
activities of the same.
[0004] 2. Description of Related Art
[0005] Animal central nervous system (CNS) has many kinds of
receptor systems for receiving and responding signals that generate
from body under environmental changes. Some active natural
components, which can keep spirit and mind health, perhaps function
on many CNS receptors including cannabinoid receptor 1 (CB1) and
other receptors. However, the nervous system suffers injury under
oxidative pressure and hyper-excitatory electrical potential.
Therefore, reduction of oxidative pressure and hyper-excitatory
electrical potential is helpful to maintain the cell activity of
the neural cells and the glial cells of the nervous system.
[0006] CB1 receptors are discovered in recent years and mainly
distributed in portions of the CNS. Those portions that include
hippocampus, amygdala, anterior cingulated cortex and prefrontal
cortex are directly associated with emotional management and
behaviors. Physiological ligands of CB1 receptors are arachidonic
acid (AA) derivatives, and endogenous CB1 ligands mainly include
N-arachidonoylethanolamine (anadamide) and 2-arachidonoyl glycerol
(2-AG). The physiological functions of CB1 ligands can reduce pain
and anxiety, increase reward and help for sleepiness. The animal
experiments evidence that the anxiety is induced by blocking of CB1
receptor activation or in CB1 receptor knockout mice. Such anxiety
cannot be effectively improved even through the administration of
anti-anxiety drugs. These evidences indicate that the CB1 receptors
have a significant impact on emotional management.
[0007] Researchers such as di Tomaso E. et al. had published a
report titled as "Brain cannabinoids in chocolate" See di Tomaso E.
et al. Nature 382(6593):677-678 (1996). This report evidenced that
some active constituents of chocolate increased the concentration
of endogenous CB1 ligands of the CNS, thereby producing a transient
feeling of well-being. In addition, human milk also includes high
amount of endogenous CB1 ligand, 2-AG. Other studies demonstrated
that 2-AG is a very important bioactive factor for maintaining
orexis and increasing survival rate of newborn animals.
[0008] However, the existing chemical drugs of anti-anxiety drugs
or sleeping drugs have some disadvantages, such as temporary and
permanent amnesia in long-term usage due to high effect
concentration and most stimulation of the inhibitory potential of
such drugs. As a result, scientists shifted to find naturally
anti-anxiety active substances from plants. AA derivatives are not
existed in plants, whereas more than three hundred kinds of plant
fatty acid N-alkylamides (FAAs) have similar structures and
biosynthesis routes with endogenous CB of animals. Among these
compounds, many plants selected for reducing pain and inflammation
contain active constituents that can bind to CB2 receptors. CB2
receptors are mainly distributed on immune cells, and the active
constituents can affect animal immune cells through the CB2
receptors. Although there are many Chinese herbal medicines for
helping tranquilization and anti-anxiety, the mechanism and related
receptors of these active constituents are not clear. Moreover,
there is no effective, quick and economic in vitro evaluation
method for fast screening natural anti-anxiety active constituents
from numerous raw materials of Chinese herbal medicines.
[0009] Therefore, it is necessary to provide an effective and
precise in vitro evaluating method, thereby finding natural
anti-anxiety active constituents from numerous raw materials of
Chinese herbal medicines.
SUMMARY
[0010] A method of in vitro evaluating Chinese herbal aqueous
extract with anti-anxiety activity is provided. The method
comprises the steps of culturing a neural cell and/or a glial cell
in a testing medium that includes the Chinese herbal aqueous
extract. And then, an electric pulse treatment is applied on the
neural cell and/or the glial cell, so as to form an epileptic cell.
Later, at least one anti-anxiety parameter of the epileptic cell is
detected and compared with respect to the reference value, so as to
evaluate the anti-anxiety activity of the Chinese herbal aqueous
extract. In addition, the animal experimentation has also verified
that the Chinese herbal aqueous extract evaluated in vitro has the
anti-anxiety activity in vivo.
[0011] Moreover, a Chinese herbal aqueous extract with anti-anxiety
activity is provided. The Chinese herbal aqueous extract, which is
evaluated by using the aforementioned method, can enhance a
membrane expression of a Cannabinoid receptor type-1 (CB1).
[0012] Furthermore, a food composition is provided. The food
composition comprises the Chinese herbal aqueous extract of claim
12 and at least one food additive.
[0013] Accordingly, the invention provides a method of in vitro
evaluating Chinese herbal aqueous extract with anti-anxiety
activity is provided. In an embodiment, the method comprises the
steps of extracting a raw material of a Chinese herbal medicine
sample with hot water, so as to obtain a Chinese herbal aqueous
extract, wherein the Chinese herbal aqueous extract is a test
sample. Next, a neural cell and/or a glial cell are cultured in a
testing medium that includes the test sample or not. And then, an
electric pulse treatment is applied on the neural cell and/or the
glial cell to be suffered with a hyper-excitatory electrical
potential injury, thereby forming an epileptic cell. Afterwards, at
least one anti-anxiety parameter of the epileptic cell is detected,
in which the anti-anxiety parameter comprises a cell viability, a
percentage of reactive oxygen species (ROS)--generated cells and a
percentage of apoptotic cells, and at least one test value is
defined to the at least one anti-anxiety parameter obtained from
the epileptic cell cultivated with the test sample, and at least
one reference value is defined to the at least one anti-anxiety
parameter obtained from the epileptic cell cultivated without the
test sample. After that, the test value is compared with the
reference value, so as to determine that the test sample has the
anti-anxiety activity based on when the cell viability is more than
the reference value, as well as the percentage of the
ROS--generated cells and the percentage of apoptotic cells are less
than the reference.
[0014] According to an embodiment of the invention, the
aforementioned neural cell, the glial cell or the combination
thereof includes a neuroblastoma cell line, a glioblastoma cell
line or the combination thereof.
[0015] According to an embodiment of the invention, the
aforementioned electric pulse treatment is applied on the neural
cell and/or the glial cell by using 5 mV of pulse voltage and 100
ms of inter-pulse interval for 50 times continuously.
[0016] According to an embodiment of the invention, the
aforementioned Chinese herbal aqueous extract with anti-anxiety
activity is extracted from Poris Cum (PC), Semen Biotae Orientalis
(SBO), Acanthopanacis Cortex (AC) or Semen Zizyphi Spinosae
(SZS).
[0017] According to an embodiment of the invention, an expression
amount of a Cannabinoid receptor type-1 (CB1) is enhanced in vivo
by the aforementioned Chinese herbal aqueous extract with
anti-anxiety activity.
[0018] Moreover, the invention further provides a Chinese herbal
aqueous extract with anti-anxiety activity, which includes the
Chinese herbal aqueous extract obtained by using the aforementioned
method.
[0019] In addition, the invention further provides a food
composition that includes the aforementioned Chinese herbal aqueous
extract and at least one food additive.
[0020] With application of the Chinese herbal aqueous extract
having anti-anxiety activities and the method of in vitro
evaluating the same, which use the epileptic cells as an evaluation
platform for screening the Chinese herbal aqueous extract having
anti-anxiety activities. Moreover, the evaluated Chinese herbal
aqueous extract indeed has anti-anxiety activities and enhancement
of CB1 membrane expression evidenced by the animal experimentation,
thereby being applied in food or other compositions.
[0021] It is to be understood that both the foregoing general
description and the following detailed description are by examples,
and are intended to provide further explanation of the invention as
claimed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] The invention can be more fully understood by reading the
following detailed description of the embodiment, with reference
made to the accompanying drawings as follows:
[0023] FIGS. 1A and 1B show schematic diagrams of the
electroporator (FIG. 1A) and its electroporator cuvette (FIG. 1B)
according to an embodiment of the present invention.
[0024] FIGS. 2A and 2B show histograms of the cell viability of the
cell line IMR-32 (FIG. 2A) or U-87 MG (FIG. 2B) through the
electric pulse treatment according to an embodiment of the present
invention.
[0025] FIG. 3 shows a flow cytometric diagram with respect to the
percentage of ROS--generated cells of the cell line IMR-32 or U-87
MG through the electric pulse treatment according to an embodiment
of the present invention.
[0026] FIGS. 4A and 4B depict histograms with respect to the
percentage of ROS--generated cells of the cell line IMR-32 (FIG.
4A) or U-87 MG (FIG. 4B) through the electric pulse treatment
according to an embodiment of the present invention.
[0027] FIG. 5 depicts a two-dimensional dot pot with respect to the
percentage of apoptotic cells of the cell line IMR-32 or U-87 MG
through the electric pulse treatment according to an embodiment of
the present invention.
[0028] FIGS. 6A and 6B depict histograms of the percentage of
apoptotic cells of the cell line IMR-32 (FIG. 6A) or U-87 MG (FIG.
6B) through the electric pulse treatment according to an embodiment
of the present invention.
[0029] FIG. 7A depicts a flow chart of the animal test process
according to an embodiment of the present invention.
[0030] FIG. 7B depicts a stereo diagram of the closed environment
according to an embodiment of the present invention.
[0031] FIG. 7C depicts a stereo diagram of the elevated plus-maze
according to an embodiment of the present invention.
[0032] FIGS. 8A and 8B depict histograms of times of the ICR mouse
entered in open arms (FIG. 8A) or closed arms (FIG. 8B) according
to an embodiment of the present invention.
[0033] FIGS. 9A and 9B depict histograms of the CB1 membrane
expression percentage of neural cells of mice cerebral cortexes
(FIG. 9A) and mice hippocampuses (FIG. 9B) according to an
embodiment of the present invention.
[0034] FIGS. 10A and 10B depict histograms of the cell mortality
percentage of neural cells of mice cerebral cortexes (FIG. 10A) and
mice hippocampuses (FIG. 10B) according to an embodiment of the
present invention.
DETAILED DESCRIPTION
[0035] Reference will now be made in detail to the present
embodiments of the invention, examples of which are illustrated in
the accompanying drawings. Wherever possible, the same reference
numbers are used in the drawings and the description to refer to
the same or like parts.
[0036] As aforementioned, the invention relates to a Chinese herbal
aqueous extract having anti-anxiety activities and a method of in
vitro evaluating the same, which use the epileptic cells as an
evaluation platform for screening the Chinese herbal aqueous
extract having anti-anxiety activities in quick, economical and
precise ways.
In Vitro Evaluating Chinese Herbal Aqueous Extract Having
Anti-Anxiety Activities
[0037] The "Chinese Herbal Aqueous Extract" as discussed
hereinafter is referred to the one obtained by using hot eater
extraction of a raw material of a Chinese herbal medicine sample.
In detail, in an embodiment, the raw material of the Chinese herbal
medicine sample is mixed with sterile water, heated to boiling and
kept heating for about 105 minutes. It is noted that, if the water
temperature is below the boiling point or the heating duration is
insufficient, the bioactive component having anti-anxiety
activities could not be extracted from the raw material of the
Chinese herbal medicine sample. However, if the water temperature
is above the boiling point or the heating duration is more than the
aforementioned heating time, the bioactive component having
anti-anxiety activities in the raw material of the Chinese herbal
medicine sample could be destroyed.
[0038] After the heating step, the solid component (or residual) of
the raw material of the Chinese herbal medicine sample can be
removed by using conventional methods such as filtration,
solid-liquid separation or other methods, followed by removing
water in the raw material of the Chinese herbal medicine sample
through rotary evaporation and/or lyophilization, thereby obtaining
powder of the Chinese herbal aqueous extract. The powder of the
Chinese herbal aqueous extract can be prepared to a concentrated
aqueous solution (e.g. 10 g/L) by rehydrating with water or aqueous
solution. In the subsequent evaluation steps, the Chinese herbal
aqueous extract is a test sample for adding into a testing medium.
The testing medium has an amount of 0.01 g/L to 1 g/L, 0.05 g/L to
0.51 g/L, or about 0.1 g/L.
[0039] Next, in an embodiment, a neural cell and/or a glial cell
can be cultured in testing medium that includes the test sample or
not and incubated in a condition at 37.degree. C. in 5% CO.sub.2.
In an example, the aforementioned neural cell and/or a glial cell
is referred to the neural cell, the glial cell or the combination
thereof, which include any available neuroblastoma cell line,
glioblastoma cell line or the combination thereof. For example, the
neural cell includes but is not limited to human neuroblastoma cell
line IMR-32 (deposited at the American Type Culture Collection
(ATCC) under accession No.: ATCC CCL-127), the glial cell includes
but is not limited to human glioblastoma cell line U-87 MG
(deposited at ATCC under accession No.: ATCC HTB-14), and the two
cells are purchased from the Food Industry Research and Development
Institute (FIRDI), Bioresource Collection and Research Center
(BCRC), Hsinchu, Taiwan). Also, the neural cell and the glial cell
obtained from other sources can be used in the present invention.
In another example, the neural cell and the glial cell cultured in
the medium with the test sample belong to an experimentation group,
and the neural cell and the glial cell cultured in the medium
without the test sample belong to a control group.
[0040] For the purpose of evaluating the protection effect of the
Chinese herbal aqueous extract to the neural cell and/or the glial
cell, the present invention utilizes an electric pulse technology
to establish an epileptic cell as in vitro evaluation platform. The
epileptic cell is generated from human neural cell and/or human
glial cell that are treated with a hyper-excitatory electrical
potential injury. Some physiological activities such as CB1
membrane expression, cell viability, physiological activity and
anti-anxiety activity of the epileptic cell are then detected in
vitro, so as to evaluate the in vitro protection effect of the
Chinese herbal aqueous extract to the neural cell and/or the glial
cell under the condition of oxidative stress or hyper-excitatory
electrical potential injury.
[0041] In an embodiment, when the neural cell and/or the glial cell
are cultured after about 12 to about 36 hours, or after about 24
hours, the neural cell and/or the glial cell are subjected to an
electric pulse treatment, allowing the neural cell and/or the glial
cell to be suffered with a hyper-excitatory electrical potential
injury, thereby forming an epileptic cell. In an embodiment, during
the electric pulse treatment, a commercially available
electroporator can apply 5 mV of pulse voltage and 100 ms of
inter-pulse interval on the neural cell and/or the glial cell for
50 times continuously. The neural cell and/or the glial cell are
suffered with the hyper-excitatory electrical potential injury that
decreases 50% cell viability of those cells. If the neural cell
and/or the glial cell were treated with other conditions rather
than using the aforementioned ones during the electric pulse
treatment, the neural cell and the glial cell could not be
qualified as the screening platform due to the following reasons,
for example, the epileptic cells that cannot be simultaneously
formed with 30% to 50% cell injury from the neural cell and the
glial cell, the ROS-positive cells increased in at least 2 times to
10 times, insufficient cell injury, or excessively injured cells
that cannot be self-repaired. Those unqualified cells cannot
provide convincible results in subsequent evaluation process.
[0042] And then, at least one anti-anxiety parameter of the
epileptic cell is detected. In an example, the anti-anxiety
parameter comprises cell viability, percentage of ROS--generated
cell, percentage of apoptotic cells and other distinguishable
characteristics. Among those, at least one test value is defined to
the at least one anti-anxiety parameter obtained from the epileptic
cell cultured with the test sample (i.e. the experimental group),
and at least one reference value is defined to the at least one
anti-anxiety parameter obtained from the epileptic cell cultivated
without the test sample (i.e. the control group).
[0043] Later, the test value is compared with the reference value,
so as to determine that the test sample has the anti-anxiety
activity. In an example, the test sample is determined to have the
anti-anxiety activity based on when the experimental group has more
cell viability, less percentage of the ROS--generated cells and
less percentage of apoptotic cells than the reference value of the
control group. A nervous system of an organism is injured when the
organism is subjected to the oxidative stress or the
hyper-excitatory electrical potential. However, if the Chinese
herbal aqueous extract can reduce the oxidative stress and the
excitatory electrical potential of the epileptic cell, the Chinese
herbal aqueous extract is determined to have the anti-anxiety
activity so that it is beneficial to maintain the cell viability of
the neural cells and the glial cells in the nervous system.
Chinese Herbal Aqueous Extract with Anti-Anxiety Activity
[0044] In an embodiment, the Chinese herbal aqueous extract with
anti-anxiety activity, which has been evaluated by the
aforementioned method, can be extracted from Poris Cum (PC), Semen
Biotae Orientalis (SBO), Acanthopanacis Cortex (AC) or Semen
Zizyphi Spinosae (SZS). In another embodiment, CB1 membrane
expression can be enhanced in vivo by the Chinese herbal aqueous
extract with anti-anxiety activity. In other embodiments, a food
composition includes the evaluated Chinese herbal aqueous extract
alone or mixed with at least one food additive.
[0045] Thereinafter, various applications of the Chinese herbal
aqueous extract having anti-anxiety activities and a method of in
vitro evaluating the same will be described in more details
referring to several exemplary embodiments below, while not
intended to be limiting. Thus, one skilled in the art can easily
ascertain the essential characteristics of the present invention
and, without departing from the spirit and scope thereof, can make
various changes and modifications of the invention to adapt it to
various usages and conditions.
EXAMPLE 1
Establishment of In Vitro Cell Platform
[0046] 1. Cell Culture of Human Neuroblastoma Cell Line IMR-32 and
Human Glioblastoma U-87 MG
[0047] Human neuroblastoma cell line IMR-32 (deposited at ATCC
under accession No.: ATCC CCL-127) and human glioblastoma U-87 MG
(deposited at ATCC under accession No.: ATCC HTB-14) were purchased
from the Food Industry Research and Development Institute (FIRDI),
Bioresource Collection and Research Center (BCRC), Hsinchu,
Taiwan). The cell lines IMR-32 and U-87 MG were cultured in the
minimum essential medium (MEM; Gibco.RTM. BRL, Grand Island, N.Y.)
that contained 2 mM L-glutamine, 1 mM non-essential amino acids,
10% fetal bovine serum (FBS), 0.1 mg/mL streptomycin and 0.5 mg/mL
ampicillin. Those cells were incubated for 48 hours in an incubator
(NUAIR NU4500, USA) with 5% CO.sub.2 humidified atmosphere at
37.degree. C., and changed with the fresh medium every two or three
days. When those cells reached about 90% confluence, the cells were
gently washed in phosphate buffer saline (PBS) and subjected to
trypsinization (0.25% 1 mL trypsin-EDTA/plate) to single cells, so
as to separate several cell plates or to subculture according to
different experimental purposes.
[0048] 2. Preparation of Chinese Herbal Aqueous Extract
[0049] 600 g Poris Cum (PC), 112 g Semen Biotae Orientalis (SBO),
112 g Acanthopanacis Cortex (AC) or 112 g Semen Zizyphi Spinosae
(SZS) was separately heated with 2.25 L sterile water to keep it
boiling for 105 minutes. After cooling down and removing solid
residues, the primary aqueous extract such as 330 mL primary
aqueous extract of PC, 420 mL primary aqueous extract of SBO, 380
mL primary aqueous extract of AC or 460 mL primary aqueous extract
of SZS was obtained, respectively.
[0050] The resulted primary aqueous extracts were concentrated by
using a rotary evporator (BUCHI Rotavapor R-200, Switzerland),
respectively, and then the secondary aqueous extract such as 134 mL
secondary aqueous extract of PC, 53 mL secondary aqueous extract of
SBO, 82 mL secondary aqueous extract of AC or 72 mL secondary
aqueous extract of SZS was obtained, respectively. Later, the
resulted secondary aqueous extracts were lyophilized by using a
freeze drying system (Labconco Freezone dry system, USA), and then
the Chinese herbal aqueous extract such as 14.17 g PC aqueous
extract, 9.74 g SBO aqueous extract, 15.93 g AC aqueous extract or
12.75 g SZS aqueous extract was obtained, respectively.
[0051] 100 mg of PC, SBO, AC or SZS aqueous extract was prepared to
10 mg/mL PC, SBO, AC or SZS aqueous extract solution,
respectively.
[0052] 3. Epileptic Cell Platform
[0053] After the cell line IMR-32 or U-87 MG is subjected to
trypsinization (trypsin-EDTA/plate), the single cell solution was
adjusted to cell density of 1.times.10.sup.6 cells/mL, seeded into
96-well microplate with 100 .mu.L cell solution per well (i.e.
1.times.10.sup.5 cells/100 .mu.L/well), and incubated for 24 hours
in the incubator (NUAIR NU4500, USA) with 5% CO.sub.2 humidified
atmosphere at 37.degree. C., and added with 1 .mu.L PC, SBO, AC or
SZS aqueous extract solution (i.e. 10 .mu.g Chinese herbal aqueous
extract/well), respectively. After being incubated for 24 hours in
the incubator (NUAIR NU4500, USA) with 5% CO.sub.2 humidified
atmosphere at 37.degree. C., the cells in each well were subjected
to trypsinization (0.25% 20 .mu.L trypsin-EDTA/well), and the
trypsinized cells of each well were loaded into each electroporator
cuvette (shown in FIG. 1B) of an electroporator (shown in FIG. 1A;
BTX ECM830, USA) for being subjected to the electric pulse
treatment.
[0054] Reference is made to FIGS. 1A and 1B, which show schematic
diagrams of the electroporator (FIG. 1A) and its electroporator
cuvette (FIG. 1B) according to an embodiment of the present
invention. The electroporator cuvette 110 has a cuvette body 111
and a cap 113. The cuvette body 111 receives the being-treated cell
solution 115 therein. The cuvette body 111 further has an electrode
plate (unshown). In this EXAMPLE, a main body 101 of the
electroporator (FIG. 1A) applied 5 mV of pulse voltage and 100 ms
of inter-pulse interval on the cells (for example, the cell
solution 115) in the electroporator cuvette 110 (FIGS. 1A and 1B)
for 50 times continuously through connection lines and related
equipments, thereby forming epileptic cells. After the electric
pulse treatment was completed, the cell solution was seeded into
96-well plates, incubated for about 60 minutes and washed with PBS.
Subsequently, cell viability, percentage of ROS--generated cells,
percentage of apoptotic cells and other distinguishable
characteristics were analyzed by using MTT test or other
methods.
EXAMPLE 2
In Vitro Evaluating Chinese Herbal Aqueous Extract
[0055] This EXAMEPLE was directed to evaluate the anti-anxiety
activities of the Chinese herbal aqueous extracts of EXAMPLE 1 in
vitro by using the epileptic cell platform established by EXAMPLE
1.
[0056] 1. Evaluation of Cell Viability
[0057] In this EXAMPLE, cell viability of EXAMPLE 1 was detected by
using MTT test. At first, methylthiazoletetrazolium (MTT; Sigma,
St. Louis, Mo., USA) solution was prepared by adding 5 mg MTT into
1 mL PBS. Next, each well (i.e. 1.times.10.sup.5 cells/well) of
96-well microplate was added with 10 .mu.L MTT solution, incubated
for 24 hours in the incubator (NUAIR NU4500, USA) in the dark with
5% CO.sub.2 humidified atmosphere at 37.degree. C., and then
centrifuged for 10 minutes in a rotation speed of 1,000.times.g at
4.degree. C. The supernatant was discarded and 100 .mu.L dimethyl
sulfoxide (DMSO; Merck, Darmstadt, Germany) solution is added. The
microplate was vibrated gently for 10 minutes. Absorbance at 550 nm
(OD.sub.550 nm) of each well was detected by ELISA reader (Thermo,
USA) for evaluating cell viability. The result was shown in FIGS.
2A and 2B.
[0058] Reference is made to FIGS. 2A and 2B, which show histograms
of the cell viability of the cell line IMR-32 (FIG. 2A) or U-87 MG
(FIG. 2B) through the electric pulse treatment according to an
embodiment of the present invention, in which the symbol "*" is
referred to the statically significant difference of the cell
viability values in the experimental group as compared with the
control group (i.e. "Epileptic cell") (P<0.05).
[0059] Based on the results of FIGS. 2A and 2B, as compared with
the normal cell (i.e. "Normal cell") that was untreated with the
electric pulse treatment, the control group (i.e. "Epileptic cell")
including the cell line IMR-32 (FIG. 2A) or U-87 MG (FIG. 2B) had
significantly less cell viability. However, as compared with the
control group (i.e. "Epileptic cell"), the epileptic cell line
IMR-32 (FIG. 2A) or U-87 MG (FIG. 2B) treated with the PC, SBO, AC
or SZS aqueous extract solution had significantly increased cell
viability.
[0060] 2. Evaluation of Percentage of ROS--Generated Cells
[0061] In this EXAMPLE, 10 mM/.mu.L 2,7-dichlorofluorescein
diacetate (H2DCF-DA; Invitrogen, USA) solution was prepared by
adding H2DCF-DA into DMSO anhydrous. Next, the cell line IMR-32 or
U-87 MG of EXAMPLE 1 was washed with PBS, added into PBS (including
10 .mu.M H2DCF-DA) and incubated for 30 minutes in the incubator at
37.degree. C. After being washed with PBS, the cells were recovered
in phenol red-free MEM for 5 minutes in the incubator at 37.degree.
C. The cells were collected, and then the DCF (the fluorescent
product of H2DCF-DA) fluorescence intensity at the emission
wavelength of 526 nm (FL1) of each group was measured by using a
FACScan flow cytometry (Beckman Coulter Epics XL-MCL, USA) at the
excitation wavelength of 488 nm, so as to analyze the percentage of
ROS--generated cells (%). The flow cytometric result was shown in
FIG. 3.
[0062] Reference is made to FIG. 3, which shows a flow cytometric
diagram with respect to the percentage of ROS--generated cells of
the cell line IMR-32 or U-87 MG through the electric pulse
treatment according to an embodiment of the present invention, in
which the open peak is referred to ROS-negative cells (i.e. the
cells without generating ROS), the shaded peak is referred to
ROS-positive cells (i.e. the cells with generating ROS), and the
percentage presented in the region D is referred to the percentage
of ROS--generated cells.
[0063] Reference is also made to FIGS. 4A and 4B, which depict
histograms with respect to the percentage of ROS--generated cells
of the cell line IMR-32 (FIG. 4A) or U-87 MG (FIG. 4B) through the
electric pulse treatment according to an embodiment of the present
invention, in which the symbol "*" is referred to the statically
significant difference of the percentage of ROS--generated cells in
the experimental group as compared with the control group (i.e.
"Epileptic cell") (P<0.05).
[0064] Based on the results of FIGS. 4A and 4B, as compared with
the normal cell (i.e. "Normal cell") that was untreated with the
electric pulse treatment, the control group (i.e. "Epileptic cell")
including the cell line IMR-32 (FIG. 4A) or U-87 MG (FIG. 4B) had
significantly increased percentage of ROS--generated cells.
However, as compared with the control group (i.e. "Epileptic
cell"), the epileptic cell line IMR-32 (FIG. 4A) or U-87 MG (FIG.
4B) treated with the PC, SBO, AC or SZS aqueous extract solution
had significantly reduced percentage of ROS--generated cells from
the epileptic cells.
[0065] 3. Evaluation of Percentage of Apoptotic Cells
[0066] In this EXAMPLE, the cell line IMR-32 or U-87 MG of EXAMPLE
1 was washed with PBS, collected into 1.5 mL eppendorf vials,
respectively. Each vial is added with 100 .mu.L annexin V-binding
buffer of Vybrant.RTM. Apoptosis Assay Kit (Invitrogen, USA), 5
.mu.L annexin V and 1 .mu.L 0.15 mM propidium Iodide (PI) dye, and
mixed well, and reacted at room temperature. Later, each vial is
added with 400 .mu.L annexin V-binding buffer and put on ice.
Afterward, the fluorescence intensity at the wavelength of 488 nm
and 595 nm was measured by using a FACScan flow cytometry (Beckman
Coulter Epics XL-MCL, USA), in which the cells bound with annexin V
(annexin V.sup.+) were referred to apoptotic cells, and the cells
stained with PI dye were referred to necrotic cells, so as to
evaluate the percentage of apoptotic cells. The result of the
percentage of apoptotic cells was shown in FIG. 5.
[0067] Reference is also made to FIG. 5, which depicts a
two-dimensional dot pot with respect to the percentage of apoptotic
cells of the cell line IMR-32 or U-87 MG through the electric pulse
treatment according to an embodiment of the present invention, in
which the block D1 included Pl.sup.+ annexin V.sup.- cells (i.e.
the necrotic cells), the block D3 included PI.sup.- annexin V.sup.-
cells (i.e. the viable cells), the blocks D2 and D4 included
annexin V.sup.+ cells (i.e. the apoptotic cells).
[0068] Moreover, reference is made to FIGS. 6A and 6B, which depict
histograms of the percentage of apoptotic cells of the cell line
IMR-32 (FIG. 6A) or U-87 MG (FIG. 6B) through the electric pulse
treatment according to an embodiment of the present invention, in
which the symbol "*" is referred to the statically significant
difference of the percentage of apoptotic cells in the experimental
group as compared with the control group (i.e. "Epileptic cell")
(P<0 .05).
[0069] Based on the results of FIGS. 6A and 6B, as compared with
the normal cell (i.e. "Normal cell") that was untreated with the
electric pulse treatment, the control group (i.e. "Epileptic cell")
including the cell line IMR-32 (FIG. 6A) or U-87 MG (FIG. 6B) had
significantly increased percentage of apoptotic cells. However, as
compared with the control group (i.e. "Epileptic cell"), the
epileptic cell line IMR-32 (FIG. 6A) or U-87 MG (FIG. 6B) treated
with the PC, SBO, AC or SZS aqueous extract solution had
significantly reduced percentage of apoptotic cells from the
epileptic cells.
EXAMPLE 3
In Vivo Evaluating Anti-Anxiety Activities of Chinese Herbal
Aqueous Extract by Using Experimental Animal Model
[0070] This EXAMEPLE was directed to evaluate the anti-anxiety
activities of the Chinese herbal aqueous extracts of EXAMPLE 1 in
vivo by using an animal model.
[0071] 1. Establishment of Experimental Animals
[0072] 1.1 Care of Experimental Animals
[0073] In this EXAMPLE, fifty six-week-old specific pathogen-free
(SPF) imprinting-control-region (ICR) mice (purchased from BioLASCO
Taiwan Co., Ltd., Yi-Lan, Taiwan) were randomly allocated into four
experimental groups (i.e. PO group, SBO group, AC group and SZS
group) and one control (PBS) group, each group of which had ten
mice.
[0074] All ICR mice were bred in a SPF environment with positive
air pressure. Ambient temperature was controlled at 24.degree. C.,
relative humidity at 65.+-.5. In addition, the animals were
maintained on a reverse 12 h light-dark cycle. Mice were provided
with standard laboratory chow and water ad libitum. All
experimental procedures were performed according to the NIH Guide
for the Care and Use of Laboratory Animals.
[0075] 1.2 Feeding with Chinese Herbal Aqueous Extract
[0076] Chinese herbal aqueous extracts of EXAMPLE 1 were prepared
to solutions by adding the Chinese herbal aqueous extract into
sterile water. The ICR mice of the four experimental groups were
given 100 mg of Chinese herbal aqueous extract of EXAMPLE 1 per kg
body weight once per day by oral administration (for example,
orogastric intubation) for continuing 35 days. The ICR mice of the
control group were orally administrated with distilled water or PBS
once per day. The whole flow chart of the animal test process was
shown in FIG. 7A.
[0077] 2. Evaluation of Anti-Anxiety Behaviors of Experimental
Animals
[0078] 2.1 Establishment of Closed Environments
[0079] This EXAMPLE is designed to direct the ICR mouse into a
closed environment, inducing them to generate anxiety state.
Reference is made to FIG. 7B, which depicts a stereo diagram of the
closed environment according to an embodiment of the present
invention. The ICR mouse 725 was firstly entered into the closed
environment 720 of FIG. 7B before carrying the subsequent plus-maze
test. In this EXAMPLE, the volume of the closed environment 720 was
about 110 cm.sup.3, and several air outlets 723 (for example, six
air outlets with diameter of about 1 mm) are installed on the
plastic plug. The closed duration is one hour, so as to make the
ICR mouse 725 to generate anxiety state.
[0080] 2.2 Elevated Plus-Maze
[0081] After the ICR mouse generated anxiety state, the ICR mouse
was moved to an elevated plus-maze for one hour, allowing the mouse
to adapt this environment.
[0082] Reference is made to FIG. 7C, which depicts a stereo diagram
of the elevated plus-maze according to an embodiment of the present
invention. The elevated plus-maze 730 is widely applied in an
animal behavior test for measuring anxiety of an experimental
animal. Anxiety associated behavior of the ICR mouse 735 was
determined by counting the times that the ICR mouse 735 entered or
stayed in open arms 731 or closed arms 733 as well as the duration
time that the ICR mouse 735 spends in each arm. The open arms 731
were heightened and openly, and the closed arms 733 were sheltered
by black plates. The elevated plus-maze 730 was elevated about 50
cm from the floor and lit it by dim light, the ICR mouse 735 must
have been not subjected to such a test.
[0083] The ICR mouse having anxiety state was placed in the center
737 of the elevated plus-maze 730 and the test was started. The ICR
mouse behavior was recorded by an animal behavior tracking system
(for example, EthoVision XT system) and monitored by a camera (for
example, DSP CCD CAMERA). The test was spent 5 minutes for counting
the times and the duration time that the ICR mouse 735 freely
entered or stayed in open arms 731 or closed arms 733. The anxiety
of the ICR mouse 735 was compared by measuring the proportion of
the duration time that the ICR mouse 735 spent in open arms 731
(i.e. time in open arms/total duration time), The times of the ICR
mouse 735 entered into open arms 733 were counted for evaluating
the ICR mouse 735 activity. However, it should be supplemented
that, the anxiety of the comparison must be based on the condition
that the ICR mouse 735 had little difference of activity.
[0084] Reference is made to FIGS. 8A and 8B, which depict
histograms of times of the ICR mouse entered in open arms (FIG. 8A)
or closed arms (FIG. 8B) according to an embodiment of the present
invention, in which the symbol "*" is referred to the statically
significant difference of the averaged times of the experimental
group as compared with the control group (i.e. "Epileptic cell")
(P<0 .05).
[0085] Based on the results of FIGS. 8A and 8B, as compared with
the mice of the control group that were fed without the Chinese
herbal aqueous extracts, the mice of the experimental groups (i.e.
PC, SBO, AC and SZS groups) had significantly increased times of
the ICR mouse entered in open arms after the mice were in anxiety
state. Similarly, the mice of the experimental groups (i.e. PC,
SBO, AC and SZS groups) had significantly reduced times of the ICR
mouse entered in closed arms after the mice were in anxiety state
as compared with the mice of the control group. Therefore, with
respect to the anxiety induced by the closed environment, the mice
fed with the Chinese herbal aqueous extracts can significantly
increased the times of the mice entered in open arms and reduced
the times of the mice entered in closed arms. The results showed
that the PC, SBO, AC and SZS aqueous extracts of the Chinese herbal
aqueous extracts had physiologically active component of
anti-anxiety.
[0086] 3. Detection of Anti-Anxiety-Related Activities of Central
Nervous System of Experimental Animals
[0087] 3.1 Excision of Brain Tissue
[0088] All mice were sacrificed after the whole process. The mice
brains were excised and cleaned with MEM medium (including 3% FBS,
Gibco.RTM. BRL, Grand Island, N.Y.). And then, the left and right
hippocampuses and the cerebral cortex of each mouse were separated,
respectively.
[0089] 3.2 Primary Cell Culture
[0090] The tissue specimens of the mice hippocampuses and cerebral
cortexes were homogenized in a tissue culture plate (for example,
the plate with diameter of 9 cm). The homogenized tissues were
filtrated through 100 .mu.m cell filter and collected in 50 mL
centrifuge tube, followed by centrifuging at a centrifugal force of
400.times.g for 10 minutes by using a centrifuge (BECKMAN Cs-6R,
USA). After centrifuging, the supernatant was discarded, and the
remained cell layer are added with PBS and adjusted to a cell
density of 1.times.10.sup.7/mL, and every 100 .mu.L aliquot
(1.times.10.sup.6/100 .mu.L) of which was added into a 1.5 mL
eppendorf vial.
[0091] 3.3 Detection of CB1 Membrane Expression by Using
Fluorescence Antibodies
[0092] The eppendorf vials including 100 .mu.L neural cell solution
(1.times.10.sup.6/100 .mu.L) were put on ice. Next, a primary
antibodies were added, which included 1 .mu.L rabbit anti-mouse
neuron specific enolase (rabbit anti-mouse NSE) antibody (Abcam,
Cambridge, UK) for labeling neural cells and 1 .mu.L goat
anti-mouse cannabinoid receptor I (CB1) antibody (Abcam, Cambridge,
UK) for labeling CBI receptor, so as to detecting the CB1 membrane
expression. After mixing the primary antibodies well, the reaction
mixture was reacted in the dark for 30 minutes. Afterward, the
unbound primary antibodies were washed by thrice repetition of
resuspending cells with PBS, centrifuging at a centrifugal force of
400.times.g for 10 minutes and discarding the supernatant, for
completely removing the unbound primary antibodies.
[0093] The cell pellets were resuspended with 100 .mu.L PBS and
added with secondary antibodies that included 2 .mu.L goat anti
rabbit IgG R-phycoerythrin conjugated antibody (Abcam, Cambridge,
UK) and 2 .mu.L donkey anti-goat IgG FITC conjugated antibody
(Abcam, Cambridge, UK). After mixing the secondary antibodies well,
the reaction mixture was reacted in the dark for 30 minutes.
Afterward, the unbound secondary antibodies were washed out by
thrice repetition of resuspending cells with PBS, centrifuging at a
centrifugal force of 400.times.g for 10 minutes and discarding the
supernatant, for completely removing the unbound secondary
antibodies. The cell pellets were resuspended and fixed with 500
.mu.L 4% formalin. The CB1 membrane expression of the neural cells
was measured by using a FACScan flow cytometry (Beckman Coulter
Epics XL-MCL, USA). The flow cytometric results were shown in FIGS.
9A and 9B.
[0094] Reference is made to FIGS. 9A and 9B, which depict
histograms of the CB1 membrane expression percentage of neural
cells of mice cerebral cortexes (FIG. 9A) and mice hippocampuses
(FIG. 9B) according to an embodiment of the present invention, in
which the control group is referred to the mice fed without Chinese
herbal aqueous extracts of EXAMPLE 1, and the symbol "*" is
referred to the statically significant difference of the averaged
percentage of the experimental group as compared with the control
group (i.e. "Control") (P<0.05).
[0095] Based on the results of FIGS. 9A and 9B, as compared with
the mice of the control group that were fed without the Chinese
herbal aqueous extracts, the mice of the experimental groups (i.e.
PC, SBO, AC and SZS groups) had significantly increased CB1
membrane expression percentage of neural cells of mice cerebral
cortexes (FIG. 9A) and mice hippocampuses (FIG. 9B). The results
showed that the PC, SBO, AC and SZS aqueous extracts of the Chinese
herbal aqueous extracts had physiologically active component of
anti-anxiety.
[0096] 3.4 Detection of Neural Cell Mortality by Using Fluorescence
Antibodies
[0097] The eppendorf vials including 100pL neural cell solution
(1.times.10.sup.6/100 .mu.L) were put on ice. Next, 2 .mu.L PI dye
was added, mixed well and reacted in the dark for 10 minutes.
Afterward, the cells were washed by thrice repetition of
resuspending cells with PBS, centrifuging at a centrifugal force of
400.times.g for 10 minutes and discarding the supernatant. The cell
pellets were resuspended with 100 .mu.L PBS and added with a
primary antibody, which included 1 .mu.L rabbit anti-mouse neuron
specific enolase (rabbit anti-mouse NSE) antibody (Abcam,
Cambridge, UK) for labeling neural cells. After mixing the primary
antibody well, the reaction mixture was reacted in the dark for 30
minutes. Afterward, the unbound primary antibody was washed by
thrice repetition of resuspending cells with PBS, centrifuging at a
centrifugal force of 400.times.g for 10 minutes and discarding the
supernatant, for completely removing the unbound primary
antibody.
[0098] The cell pellets were resuspended with 100 .mu.L PBS and
added with a secondary antibody that included 2 .mu.L goat anti
rabbit IgG R-phycoerythrin conjugated antibody (Abcam, Cambridge,
UK). After mixing the secondary antibody well, the reaction mixture
was reacted in the dark for 30 minutes. Afterward, the unbound
secondary antibody was washed out by thrice repetition of
resuspending cells with PBS, centrifuging at a centrifugal force of
400.times.g for 10 minutes and discarding the supernatant, for
completely removing the unbound secondary antibody. The cell
pellets were resuspended and fixed with 500 .mu.L 4% formalin. The
fluorescence intensity of the neural cells was measured by using a
FACScan flow cytometry (Beckman Coulter Epics XL-MCL, USA), so as
to obtain the cell mortality percentages of the neural cells by
measuring the proportion of PI stained cells in NSE labeled cells.
The NSE labeled cells are referred to neural cells, and the NSE
labeled and PI stained cells are referred to necrotic neural cells.
The results were shown in FIGS. 10A and 10B.
[0099] Reference is made to FIGS. 10A and 10B, which depict
histograms of the cell mortality percentage of neural cells of mice
cerebral cortexes (FIG. 10A) and mice hippocampuses (FIG. 10B)
according to an embodiment of the present invention, in which the
control group is referred to the mice fed without Chinese herbal
aqueous extracts of EXAMPLE 1, and the symbol " * " is referred to
the statically significant difference of the averaged percentage of
the experimental group as compared with the control group(i.e.
"Control") (P<0.05).
[0100] Based on the results of FIGS. 10A and 10B, as compared with
the mice of the control group that were fed without the Chinese
herbal aqueous extracts, the mice of the experimental groups (i.e.
PC, SBO, AC and SZS groups) had significantly increased cell
viability percentage of neural cells of mice cerebral cortexes
(FIG. 10A) and mice hippocampuses (FIG. 10B). The results showed
that the PC, SBO, AC and SZS aqueous extracts of the Chinese herbal
aqueous extracts had physiologically active component of
anti-anxiety.
[0101] Although the present invention has been described in
considerable detail with reference to certain embodiments thereof,
other embodiments are possible. It is necessarily supplemented
that, specific Chinese herbal aqueous extracts, specific cells,
specific extraction process, specific electric pulse treatment,
specific experimental animals, specific analysis methods or
specific apparatuses are employed as exemplary embodiments for
clarifying the Chinese herbal aqueous extract having anti-anxiety
activities and method of in vitro evaluating the same of the
present invention. However, as is understood by a person skilled in
the art, other Chinese herbal aqueous extracts, other cells, other
extraction process, other electric pulse treatment, other
experimental animals, other analysis methods or other apparatuses
can be also employed in the Chinese herbal aqueous extract having
anti-anxiety activities and method of in vitro evaluating the same
of the present invention, rather than being limited thereto.
[0102] In addition, ICR mice further evaluated the biosafety test
of aforementioned Chinese herbal aqueous extracts. Sixty-five
four-week-old ICR mice were randomly allocated into thirteen groups
that included one control (water) group and twelve experimental
groups. The experimental groups included four Chinese herbal
aqueous extracts, each of which had three different dosages. The
dosages of Chinese herbal aqueous extracts in the biosafety test
included 0.1 time of recommended intake (0.1.times.), one time of
recommended intake (1.times.) and ten times of recommended intake
(10.times.), in which one time of recommended intake was defined to
100 mg intake per kg body weight (100 mg/ kg body weight). The
biosafety test was carried out for 28 days. After the biosafety
test was completed, the bloods of all groups were obtained, and
biochemical parameters in the bloods were analyzed. The biochemical
parameters in the bloods included blood glutamic-pyruvic
transaminase (GPT), blood glutamic-oxaloacetic transaminase (GOT),
blood urea nitrogen (BUN) and serum creatinine level, for example.
The biochemical parameters in the bloods were measured by
conventional methods rather than being described in detail. There
was no statically significant difference among all groups
(P>0.05).
[0103] Reference is made to TABLE 1, which lists the result of the
biosafety test of Chinese herbal aqueous extracts according to an
embodiment of the present invention.
TABLE-US-00001 TABLE 1 Creatinine GOT (U/L) GPT (U/L) BUN (mg/dL)
(mg/dL) Control 106 .+-. 17 62 .+-. 15 21.23 .+-. 3.67 0.83 .+-.
0.17 10X PC 110 .+-. 16 67 .+-. 14 22.16 .+-. 2.60 0.86 .+-. 0.16
1X PC 104 .+-. 23 63 .+-. 16 21.85 .+-. 2.30 0.85 .+-. 0.16 0.1X PC
99 .+-. 22 50 .+-. 25 21.75 .+-. 2.71 0.82 .+-. 0.06 10X SBO 123
.+-. 13 77 .+-. 13 22.58 .+-. 2.41 0.86 .+-. 0.08 1X SBO 114 .+-.
22 69 .+-. 18 21.64 .+-. 1.78 0.85 .+-. 0.16 0.1X SBO 111 .+-. 5 54
.+-. 11 20.92 .+-. 1.97 0.84 .+-. 0.06 10X AC 130 .+-. 19 77 .+-.
12 21.44 .+-. 4.50 0.85 .+-. 0.04 1X AC 128 .+-. 13 54 .+-. 15
20.71 .+-. 2.75 0.86 .+-. 0.14 0.1X AC 102 .+-. 18 50 .+-. 24 21.54
.+-. 1.06 0.84 .+-. 0.18 10X SZS 122 .+-. 4 79 .+-. 14 22.58 .+-.
1.92 0.83 .+-. 0.07 1X SZS 123 .+-. 19 73 .+-. 7 21.44 .+-. 1.94
0.82 .+-. 0.15 0.1X SZS 105 .+-. 9 53 .+-. 8 21.64 .+-. 2.15 0.82
.+-. 0.13
[0104] Based on the result of TABLE 1, the evaluated Chinese herbal
aqueous extracts had nontoxic to livers and kidneys of the mice.
Therefore, a food composition can include the evaluated Chinese
herbal aqueous extract alone or mixed with at least one food
additive. The kinds and dosage of the food additives are well known
by the artisan in the art rather than being described in
detail.
[0105] According to the embodiments of the present invention, the
aforementioned Chinese herbal aqueous extract having anti-anxiety
activities and method of in vitro evaluating the same
advantageously use the epileptic cells as an evaluation platform
for screening the Chinese herbal aqueous extract having
anti-anxiety activities. Moreover, the evaluated Chinese herbal
aqueous extract indeed has anti-anxiety activities and enhancement
of CB1 membrane expression evidenced by the animal experimentation,
thereby being applied in food or other compositions.
[0106] It will be apparent to those skilled in the art that various
modifications and variations can be made to the structure of the
present invention without departing from the scope or spirit of the
invention. In view of the foregoing, it is intended that the
present invention cover modifications and variations of this
invention provided they fall within the scope of the following
claims.
* * * * *