U.S. patent application number 16/093950 was filed with the patent office on 2019-05-02 for applications of butylidenephthalide.
The applicant listed for this patent is NATIONAL DONG HWA UNIVERSITY. Invention is credited to Tzyy-Wen CHIOU, Horng-Jyh HARN, Jeanne HSIEH, Shinn-Zong LIN, Shih-Ping LIU, Kang-Yun LU, Hong-Lin SU.
Application Number | 20190127699 16/093950 |
Document ID | / |
Family ID | 60324827 |
Filed Date | 2019-05-02 |
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United States Patent
Application |
20190127699 |
Kind Code |
A1 |
CHIOU; Tzyy-Wen ; et
al. |
May 2, 2019 |
APPLICATIONS OF BUTYLIDENEPHTHALIDE
Abstract
Applications of butylidenephthalide (BP), comprising the use of
BP in providing a kit for promoting differentiation of stem cells
into brown adipose cells, and the use of BP in preparing a
medicament, wherein the medicament is used for inhibiting the
accumulation of white adipose cells, promoting the conversion of
white adipose cells into brown adipose cells, inhibiting weight
gain and/or reducing the content of triglycerides, glucose, and
total cholesterol in blood.
Inventors: |
CHIOU; Tzyy-Wen; (Hualien
County, TW) ; LIN; Shinn-Zong; (Taichung City,
TW) ; HARN; Horng-Jyh; (New Taipei City, TW) ;
SU; Hong-Lin; (Taichung City, TW) ; LIU;
Shih-Ping; (Taichung City, TW) ; LU; Kang-Yun;
(Taichung City, TW) ; HSIEH; Jeanne; (Hualien
City, Hualien County, TW) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NATIONAL DONG HWA UNIVERSITY |
Hualien County |
|
TW |
|
|
Family ID: |
60324827 |
Appl. No.: |
16/093950 |
Filed: |
May 3, 2017 |
PCT Filed: |
May 3, 2017 |
PCT NO: |
PCT/CN2017/082838 |
371 Date: |
October 15, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62339506 |
May 20, 2016 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2500/76 20130101;
A61K 31/365 20130101; A61K 31/341 20130101; C12N 2501/33 20130101;
A61K 31/522 20130101; C12N 2501/999 20130101; A61K 35/35 20130101;
A61K 31/4439 20130101; A61K 35/28 20130101; A61P 3/04 20180101;
C12N 5/0653 20130101; A61K 31/661 20130101; A61K 31/341 20130101;
A61K 2300/00 20130101; A61K 35/28 20130101; A61K 2300/00 20130101;
A61K 31/365 20130101; A61K 2300/00 20130101; A61K 31/4439 20130101;
A61K 2300/00 20130101; A61K 31/661 20130101; A61K 2300/00 20130101;
A61K 31/522 20130101; A61K 2300/00 20130101 |
International
Class: |
C12N 5/077 20060101
C12N005/077; A61K 31/341 20060101 A61K031/341; A61K 35/28 20060101
A61K035/28; A61K 35/35 20060101 A61K035/35; A61P 3/04 20060101
A61P003/04 |
Claims
1. A kit for promoting the differentiation of a stem cell into a
brown-like adipose cell, comprising the following components: (1) a
conditional medium, comprising a basic medium and an ingredient
capable of inducing the differentiation of a stem cell into an
adipose cell; and (2) butylidenephthalide (BP).
2. The kit as claimed in claim 1, wherein the ingredient is
selected from the group consisting of rosiglitazone, insulin,
3-isobutyl-1-methylxanthine (IBMX), dexamethasone, and combinations
thereof.
3. A method for inhibiting the accumulation of white fat and/or
promoting the conversion of white fat into brown fat, comprising
administering to a subject in need an effective amount of
butylidenephthalide (BP).
4. A method for inhibiting the accumulation of white fat, promoting
the conversion of white fat into brown fat, inhibiting weight gain
and/or reducing the contents of triglycerides, glucose and total
cholesterol in blood, comprising administering to a subject in need
an effective amount of butylidenephthalide (BP), and the
butylidenephthalide (BP) is administered with at least one of a
mesenchymal stem cell and a brown-like adipose cell.
5. The method as claimed in claim 4, which is for anti-obesity
and/or preventing metabolic syndrome associated with obesity.
6. The method as claimed in claim 5, wherein the metabolic syndrome
is at least one of diabetes mellitus, cerebrovascular disease,
cardiovascular disease, hypertension and nephropathy.
7. The method as claimed in claim 4, wherein the mesenchymal stem
cell is an adipose stem cell.
8. The method as claimed in claim 3, wherein the
butylidenephthalide (BP) is administered at an amount ranging from
30 mg (as BP)/kg-body weight to 2000 mg (as BP)/kg-body weight per
day.
9. The method as claimed in claim 4, wherein the
butylidenephthalide (BP) is administered at an amount ranging from
30 mg (as BP)/kg-body weight to 2000 mg (as BP)/kg-body weight per
day.
10. The method as claimed in claim 5, wherein the mesenchymal stem
cell is an adipose stem cell.
11. The method as claimed in claim 6, wherein the mesenchymal stem
cell is an adipose stem cell.
12. The method as claimed in claim 5, wherein the
butylidenephthalide (BP) is administered at an amount ranging from
30 mg (as BP)/kg-body weight to 2000 mg (as BP)/kg-body weight per
day.
13. The method as claimed in claim 6, wherein the
butylidenephthalide (BP) is administered at an amount ranging from
30 mg (as BP)/kg-body weight to 2000 mg (as BP)/kg-body weight per
day.
14. The method as claimed in claim 7, wherein the
butylidenephthalide (BP) is administered at an amount ranging from
30 mg (as BP)/kg-body weight to 2000 mg (as BP)/kg-body weight per
day.
15. The method as claimed in claim 8, wherein the
butylidenephthalide (BP) is administered at an amount ranging from
30 mg (as BP)/kg-body weight to 2000 mg (as BP)/kg-body weight per
day.
16. The method as claimed in claim 9, wherein the
butylidenephthalide (BP) is administered at an amount ranging from
30 mg (as BP)/kg-body weight to 2000 mg (as BP)/kg-body weight per
day.
Description
[0001] This application is a national stage application of
International Patent Application No. PCT/CN2017/082838, filed Mar.
5, 2017, which claims the benefit of U.S. Provisional Application
Ser. No. 62/339506, filed May 20, 2016. The entirety of the
aforementioned applications is incorporated herein by
reference.
FIELD
[0002] The present invention relates to the use of
butylidenephthalide (BP), especially relates to the use of
butylidenephthalide (BP) in promoting the differentiation of a stem
cell into a brown-like adipose cell and the use of
butylidenephthalide (BP) in inhibiting the accumulation of white
fat, promoting the conversion of white fat into brown fat,
inhibiting weight gain and/or reducing the contents of
triglycerides, glucose and total cholesterol in blood. By
administering butylidenephthalide (BP) in combination with a
mesenchymal stem cell and/or a brown-like adipose cell to a
subject, better efficiencies on inhibiting weight gain, reducing
the accumulation of adipose tissue and promoting the conversion of
white fat into brown fat in the subject can be provided.
BACKGROUND
[0003] Fat can be roughly classified into two types: white fat and
brown fat. The appearance of white fat, which consists of white
adipose cells, is of white color; while the appearance of brown
fat, which consists of brown adipose cells, is of brown color.
White adipose cell differentiated from Myf-5 negative myofibroblast
is characterized by containing a single and large adipose droplet
on morphology, and its main function is to store energy. Brown
adipose cell differentiated from Myf-5 positive myofibroblast is
characterized by containing numerous small adipose droplets
dispersed therein and a large number of mitochondria. The brown
adipose cell can also highly express uncoupling protein 1 (UCP1),
and its main function is to metabolize energy.
[0004] With factors such as a Western diet culture that is high in
oil, sugar and fat, a lifestyle generally lacking in exercise, and
heredity, obesity has become a worldwide health problem. It is
noticed from researches that characteristics such as the
accumulation of body white fat and the excess contents of
triglycerides, glucose and total cholesterol in blood are often
present in a subject with obesity. The occurrences of metabolic
syndrome such as diabetes mellitus, cardiovascular disease,
cerebrovascular disease, hypertension and nephropathy are also
closely related to high triglycerides, hyperglycemia and high
cholesterol. Though there are many commercially available drugs
being alleged to be able to reduce or control weight, most of those
drugs achieve the alleged purposes by the mechanisms such as
increasing the feeling of satiety, inhibiting appetite and
inhibiting the absorption of fat. Accordingly, the administration
of those drugs is often accompanied by many side effects such as
headache, hypoglycemia, constipation, insomnia, and oily feces, and
may even increase the risk of getting heart disease and stroke.
Therefore, there is still a need in the art for developing a method
or drug that has fewer side effects and is effective in
anti-obesity and preventing metabolic syndrome associated with
obesity.
[0005] Researches have shown that though brown fat is only about
0.1% of the weight of an adult, it burns about 10-20% of the daily
basal metabolic rate and is effective in accelerating the clearance
of triglycerides, ameliorating the hyperactivity of insulin, and
anti-obesity. Therefore, the researches of investigating
anti-obesity and preventing metabolic syndrome associated with
obesity have paid more and more attention to the issue of how to
increasing the proportion of body brown fat.
[0006] It is known that there are two approaches for increasing the
proportion of brown fat in an animal body, one is to promote the
conversion of white fat into brown fat in the animal body (referred
to as "fat browning"), and the other is to inject a cell suspension
of brown adipose cells or brown-like adipose cells to the animal
(referred to as "cell infusion of brown adipose cells" or "cell
infusion of brown-like adipose cells"). The brown-like adipose cell
refers to a cell that has the morphology of a brown adipose cell
characterized by containing numerous small adipose droplets
dispersed therein, containing a large number of mitochondria, and
having highly expressed UCP1 protein, but is different from a brown
adipose cell in the gene expression. It is known that a brown-like
adipose cell can be obtained by inducing and promoting the
differentiation of a stem cell or the conversion of a white adipose
cell. Because brown-like adipose cell is similar to brown adipose
cell on cell morphology, it is deemed as belonging to brown fat in
fat classification.
[0007] However, so far, there is still a lack of effective
technical means for the aforementioned two approaches of increasing
the proportion of body brown fat. Though researches have shown that
exercise or cold environments are effective in stimulating the
occurrence of fat browning in an animal body, many people are
generally lacking in exercise and cold environments are not readily
available. It is impractical to effectively and stably promote the
fat browning in an animal body through exercises or the exposure to
cold environments. In addition, the cell infusion of brown-like
adipose cells/brown adipose cells is not cost-effective because the
source of brown-like adipose cells/brown adipose cells is rare and
the cost for obtaining the cells is high.
[0008] In view of the above issues, there is a necessity and
urgency for developing an effective method for promoting the fat
browning in an animal body and/or solving the problem of the
difficulty of obtaining brown-like adipose cells/brown adipose
cells. Inventors of the present invention discovered that a
conditional medium, which comprises a basic medium and an
ingredient capable of inducing the differentiation of a stem cell
into an adipose cell, when being externally added with
butylidenephthalide (BP), can effectively promote the
differentiation of a stem cell into a brown-like adipose cell. As a
result, the cost for obtaining brown-like adipose cells can be
reduced, and the problem of the difficulty of obtaining brown-like
adipose cells/brown adipose cells can be solved.
[0009] Inventors of the present invention also discovered that the
administration of butylidenephthalide (BP) alone and the
administration of butylidenephthalide (BP) in combination with
mesenchymal stem cells and/or brown-like adipose cells to an animal
can both effectively inhibit the accumulation of white fat, promote
the conversion of white fat into brown fat, inhibit weight gain
and/or reduce the contents of triglycerides, glucose and total
cholesterol in blood. Accordingly, the use of butylidenephthalide
(BP) can promote the tat browning in an animal body so as to
achieve the effects of anti-obesity and prevent metabolic syndrome
associated with obesity, and can avoid the side effects caused by
the commercially available anti-obesity drugs that achieve the
anti-obesity purpose by the mechanisms such as increasing the
feeling of satiety, inhibiting appetite, and inhibiting the
absorption of fat.
SUMMARY
[0010] An objective of the present invention is to provide a kit
for promoting the differentiation of a stem cell to into a
brown-like adipose cell, comprising the following components: (1) a
conditional medium, comprising a basic medium and an ingredient
capable of inducing the differentiation of a stem cell into an
adipose cell; and (2) butylidenephthalide (BP). Preferably, the
ingredient is rosiglitazone, insulin, 3-isobutyl-1-methylxanthine
(IBMX), dexamethasone, and combinations thereof.
[0011] Another objective of the present invention is to provide a
use of butylidenephthalide (BP) in the manufacture of a medicament,
wherein the medicament is used for inhibiting the accumulation of
white fat, promoting the conversion of white fat into brown fat,
inhibiting weight gain and/or reducing the contents of
triglycerides, glucose and total cholesterol in blood.
Particularly, the medicament is used for anti-obesity and/or
preventing metabolic syndrome associated with obesity. Preferably,
the medicament is administered in combination with at least one of
a mesenchymal stem cell and a brown-like adipose cell. More
preferably, the medicament is administered in combination with an
adipose stem cell. The medicament is administered at an amount
ranging from about 30 mg (as BP)/kg-body weight to about 2000 mg
(as BP)14-body weight per day.
[0012] Still another objective of the present invention is to
provide a method for inhibiting the accumulation of white fat,
promoting the conversion of white fat into brown fat, inhibiting
weight gain and/or reducing the contents of triglycerides, glucose
and total cholesterol in blood, comprising administering to a
subject in need an effective amount of butylidenephthalide (BP).
Particularly, the method is for anti-obesity and/or preventing
metabolic syndrome associated with obesity. The metabolic syndrome
is at least one of diabetes mellitus, cerebrovascular disease,
cardiovascular disease, hypertension and nephropathy. Preferably,
the subject in need is administered with an effective amount of
butylidenephthalide (BP) and at least one of a mesenchymal stem
cell and a brown-like adipose cell. More preferably, the subject in
need is administered with an effective amount of
butylidenephthalide (BP) and an adipose stem cell. The amount of
butylidenephthalide (BP) is ranging from about 30 mg (as
BP)/kg-body weight to about 2000 mg (as BP)/kg-body weight per
day.
DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 shows the differentiation process of producing
adipose droplets in a stem cell treated by butylidenephthalide
(BP), wherein from left to right, each of the microscope photos
separately shows the morphology of adipose stem cells, adipose
cells differentiated from adipose stem cells and cells obtained
from cultivating the adipose cells in a conditional medium that was
externally added with butylidenephthalide (BP) in different
magnifications (40-fold, 100-fold and 200-fold);
[0014] FIGS. 2A and 2B shows the contents of adipose droplets of
cells with different treatments, wherein FIG. 2A is a photo of
adipose cells stained by oil red dye by observing with a
microscope, FIG. 2B is a histogram showing the quantified
absorbances of adipose droplets, and FIGS. 2A and 2B both include
the results of adipose stem cells (without cultivation in a
conditional medium; undifferentiated), adipose cells (cultivated in
a conditional medium; differentiated) and adipose cells
(differentiated) separately treated by 2, 10 or 50 .mu.g/mL of
butylidenephthalide (BP);
[0015] FIGS. 3A and 3B show the fat browning effects of
butylidenephthalide (BP) at different concentrations, wherein FIG.
3A is a curve diagram showing the effect of different
concentrations of butylidenephthalide (BP) on the oxygen
consumption rate of adipose cells, FIG. 3B is a histogram showing
the effect of different concentrations of butylidenephthalide (BP)
on the spare respiratory capacity of adipose cells, and FIGS. 3A
and 3B both include the results of undifferentiated adipose stem
cells (represented as "A"), differentiated adipose cells
(represented as "0"), adipose cells cultivated in a conditional
medium that was externally added with butylidenephthalide (BP) (to
a final concentration of 2 .mu.g/mL) for 7 days (represented as
"2"), adipose cells cultivated in a conditional medium that was
externally added with butylidenephthalide (BP) (to a final
concentration of 10 .mu.g/mL) for 7 days (represented as "10"), and
adipose cells cultivated in a conditional medium that was
externally added with butylidenephthalide (BP) (to a final
concentration of 50 .mu.g/mL) for 7 days (represented as "50");
[0016] FIGS. 4A and 4B show the results of the expression of UCP1
gene in the adipose cell determined by reverse transcription
polymerase chain reaction (RT-PCR) and the expression of UCP1
protein in the adipose cell determined by Western blot, wherein
FIG. 4A is a photo showing the expression levels of UCP1, PARP4
(the internal control of adipose cells) and Actin (the internal
control of all cells) genes, FIG. 4B is a photo showing the
expression levels of UCP1, FABP4 and Actin proteins, and FIGS. 4A
and 4B both include the results of control group (cultivated in a
conditional medium for 14 days; represented as "Control group"),
butylidenephthalide (BP)-treated group (cultivated in a conditional
medium for 7 days, and then cultivated in a conditional medium that
was externally added with BP for another 7 days; represented as
"BP"), tofacitinib-treated group (cultivated in a conditional
medium for 7 days, and then cultivated in a conditional medium that
was externally added with tofacitinib for another 7 days;
represented as "Tofacitinib"), adenosine-treated group (cultivated
in a conditional medium for 7 days, and then cultivated in a
conditional medium that was externally added with adenosine for
another 7 days; represented as "Adenosine"),
proanthocyanidin-treated group (cultivated in a conditional medium
for 7 days, and then cultivated in a conditional medium that was
externally added with proanthocyanidin for another 7 days;
represented as "Proanthocyanidin");
[0017] FIGS. 5A and 5B show the effect of butylidenephthalide (BP)
and cell infusion on the morphology of mouse fat, wherein FIG. 5A
is a photo showing the result of IHC staining of mouse subcutaneous
fat slice and FIG. 5B is a photo showing the result of IHC staining
of mouse visceral fat slice; the mice in "ND group" were only fed
with a normal diet, those in "HFD group" were only fed with a high
fat diet, those in "HFD+BP group" were fed with a high fat diet and
administered with butylidenephthalide (BP), those in "HFD+ADSC
group" were fed with a high fat diet and administered with adipose
stem cells, those in "HFD+BP+ADSC group" were fed with a high fat
diet and administered with butylidenephthalide (BP) and adipose
stem cells, those in "HFD+BLC group" were fed with a high fat diet
and administered with brown-like adipose cells, and those in
"HFD+BP+BLC group" were fed with a high fat diet and administered
with butylidenephthalide (BP) and brown-like adipose cells;
[0018] FIGS. 6A to 6C show the effect of butylidenephthalide (BP)
and cell infusion on the accumulation of mouse body fat, wherein
FIG. 6A is a photo showing the appearances of the heart, liver,
kidney, subcutaneous adipose tissue and visceral adipose tissue,
FIG. 6B is a histogram showing the weight of mouse subcutaneous
adipose tissue, FIG. 6C is a histogram showing the weight of mouse
visceral adipose tissue, and FIGS. 6A, 6B and 6C all include the
results of "ND group", "HFD group", "HFD+BP group", "HFD+ADSC
group", "HFD+BP+ADSC group", "HFD+BLC group" and "HFD+BP+BLC
group";
[0019] FIG. 7 is a curve diagram showing the effect of
butylidenephthalide (BP) and cell infusion on mouse weight by
showing the change of mouse weight during 9 weeks to 18 weeks old,
including the results of "ND group", "HFD group", "HFD+BP group",
"HFD+ADSC group", "HFD+BP+ADSC group", "HFD+BLC group" and
"HFD+BP+BLC group"; and
[0020] FIGS. 8A to 8C show the effect of butylidenephthalide (BP)
and cell infusion on the mouse serum biochemical values, wherein
FIG. 8A is a histogram showing the content of glucose in blood,
FIG. 8B is a histogram showing the content of triglycerides in
blood, FIG. 8C is a histogram showing the content of total
cholesterol in blood, and FIGS. 8A, 8B and 8C all include the
results of "ND group", "HFD group", "HFD+BP group", "HFD+ADSC
group", "HFD+BP+ADSC group", "HFD+BLC group" and "HFD+BP+BLC
group".
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0021] The following paragraphs will describe some of the
embodiments of the present invention in detail. However, without
departing from the spirit of the present invention, the present
invention may be embodied in various embodiments and should not be
limited to the embodiments described in the specification and the
appended claims. Unless otherwise indicated herein, the expression
"a," "an," "the," or the like recited in the specification of the
present invention (especially in the claims) are intended to
include both the singular and plural forms.
[0022] According to the standard of Health Promotion
Administration, Ministry of Health and Welfare of Taiwan, the
definition of obesity is BMI>24, and the definition of metabolic
syndrome is BMI>24, high-density lipoprotein (HDL) cholesterol
<50 mg/dL, fasting blood glucose>100 mg/dL and
triglycerides>150 mg/dL. In the standard of World Health
Organization (WHO), the definition of obesity of European and
American races is BMI>30. According to National Cholesterol
Education Program Adult Treatment Panel-III (NCEP ATP III) of
United State, the definition of metabolic syndrome is a subject
having at least three indices of blood pressure>130/85 mmHg,
triglycerides>150 mg/dL, HDL cholesterol<40 mg/dL (male) or
<50 mg/dL (female), waistline>102 cm (male) or >88 cm
(female), and fasting blood glucose>110 mg/dL. Therefore, in the
research, high cholesterol, hyperglycemia and high triglycerides
are generally used as the indices of obesity.
[0023] It is known that the occurrence of metabolic syndrome such
as diabetes mellitus, cardiovascular disease, cerebrovascular
disease, hypertension and nephropathy is closely associated with
obesity. These can be seen in "Cardiac Abnormalities in Youth with
Obesity and Type 2 Diabetes, Curr Diab Rep. 2016 July; 16(7):62",
"Impact of obesity on cardiovascular health. Best Pract Res Clin
Endocrinol Metab. 2013 April; 27(2):147-156", "Obesity and risk of
vascular disease: importance of endothelium-dependent
vasoconstriction. Br Pharmacol. 2012 February; 165(3):591-602", and
"Obesity-Related Chronic Kidney Disease-The Role of Lipid
Metabolism, Metabolites. 2015 Dec. 11; 5(4):720-732", which are
entirely incorporated hereinto by reference.
[0024] As described above, the researches of investigating
anti-obesity and preventing metabolic syndrome associated with
obesity have paid more and more attention to the issue of how to
increase the proportion of body brown fat. It has been confirmed by
researches that thermogenesis in brown fat can be driven by
uncoupling the respiratory chain of mitochondria in cells, wherein
with the use of the PET/CT image, it is discovered in adult body
that the thermogenesis activity of adipose tissue is positively
related to the expression level of UCP1. Specifically, the cells
highly expressing UCP1 (including brown adipose cells and
brown-like adipose cells) can conduct oxidation through the
electron transport chain in uncoupling the respiratory chain of
mitochondria, but the cells cannot conduct phosphorylation, and
thus ATP will not be produced. This process can promote the
consumption of nutrition and oxygen, and thus, can be used for
achieving the purpose of weight loss. These can be seen in "Brown
and beige fat: development, function and therapeutic potential. Nat
Med. 2013 October; 19(10):1252-1263, which is entirely incorporated
hereinto by reference.
[0025] It is known that the approaches of increasing the proportion
of body brown fat include promoting the fat browning in an animal
body and infusing, brown adipose cells and/or brown-like adipose
cells. However, as described above, there is neither an effective
method for promoting the fat browning in an animal body nor an
effective mean for solving the problem of the difficulty of
obtaining, brown adipose cells and brown-like adipose cells. Though
prior researches have shown that drugs such as tofacitinib,
adenosine and proanthocyanidin can be used for promoting the
differentiation of a stem cell into a brown-like adipose cell, the
effect thereof is limited.
[0026] Inventors of the present invention discovered that a
conditional medium, which comprises a basic medium and an
ingredient capable of inducing the differentiation of a stem cell
into an adipose cell, being externally added with
butylidenephthalide (BP), can effectively promote the
differentiation of a stem cell into a brown-like adipose cell.
[0027] Therefore, the present invention relates to a kit for
promoting the differentiation of a stem cell into a brown-like
adipose cell, comprising the following components: (1) a
conditional medium, comprising a basic medium and an ingredient
capable of inducing the differentiation of a stem cell into an
adipose cell, and (2) butylidenephthalide (BP). The use of the kit
in accordance with the present invention can effectively promote
the differentiation of a stem cell into a brown-like adipose cell
and stably provide a large amount of brown-like adipose cells in a
cheaper way. The provided brown-like adipose cells not only can be
used in the cell infusion of brown-like adipose cells, but also can
be used in the investigation related to obesity or energy
metabolism. The application range of the brown-like adipose cell is
wide.
[0028] In the kit provided in accordance with the present
invention, any suitable ingredient capable of inducing the
differentiation of a stern cell into an adipose cell can be used in
component (1) (i.e., a conditional medium) of the kit. For example,
the ingredient can be selected from the group consisting of
rosiglitazone, insulin, 3-isobutyl-1-methylxanthine (IBMX),
dexamethasone, and combinations thereof, but is not limited
thereby.
[0029] In component (1) of the kit (i.e., a conditional medium)
provided in accordance with the present invention, the basic medium
comprises the essential ingredient capable of providing nutrition
and condition (e.g., pH value and humidity) for stem cell growth,
which is generally adjusted depending on the need of the cultivated
stem cell. In general, examples of the adoptable basic media
include DMEM medium (Dulbecco's Modified Eagle's Medium), MEM
medium (Minimum Essential Medium), .alpha.-MEM medium, BME medium
(Basal Media Eagle), MEM/F12 medium, Ham's F10 medium, Ham's F12
medium, and RPMI medium (Rosewell Park Memorial Institute), but is
not limited thereby. In some embodiments of the present invention,
DMEM medium was used in the kit.
[0030] In the kit in accordance with the present invention,
component (1) (i.e., a conditional medium) and component (2) (i.e.,
butylidenephthalide (BP)) are normally independently packaged and
stored, and can be transported or sold separately or in a set.
Optionally, the subcomponents in component (1) could be
independently packaged and stored. In addition, the kit can further
comprise an instruction manual, which provides the procedures and
program for the user to mix the components on-site for culturing,
treating and using the cells.
[0031] For example, when the subcomponents of component (1) and
component (2) are independently packaged and stored and transported
or sold separately, the ingredient(s) capable of inducing the
differentiation of a stem cell into an adipose cell (e.g.,
rosiglitazone, insulin, 3-isobutyl-1-methylxanthine (IBMX) and
dexamethasone) and butylidenephthalide (BP) could be kept in a dark
environment at a temperature of less than 4.degree. C., and the
basic medium could be kept in an environment at a temperature of
-20.degree. C. Also, when the components in the kit in accordance
with the present invention are transported and sold in a set,
butylidenephthalide (BP) and the ingredient(s) capable of inducing
the differentiation of a stem cell into an adipose cell could be
kept in a container with an interior temperature of less than
4.degree. C. and the basic medium could be kept in a container with
an interior temperature of -20.degree. C. (e.g., an ice box). There
is no particular limitation on the shape and size of the
containers, as long as the containers can serve the desired
insulation function to ensure that the storage temperatures of
components will not affect each other when the components are
transported and sold in a set.
[0032] When using the kit in accordance with the present invention,
there is no particular limitation for the order to formulate and
mix each component. For example, when the subcomponents of the
conditional medium are packaged separately, the conditional medium
could be formulated, and then mixed with butylidenephthalide (BP).
Also, butylidenephthalide (BP) could be mixed with the basic
medium, and then mixed with other subcomponents; or each
subcomponent of the conditional medium could be mixed with
butylidenephthalide (BP) simultaneously. In addition,
butylidenephthalide (BP) can be directly mixed with the conditional
medium or basic medium; or butylidenephthalide (BP) can be
dissolved in a solvent to provide a butylidenephthalide (BP)
solution, and then the butylidenephthalide (BP) solution is mixed
with the conditional medium or basic medium. Examples of the
solvents capable of dissolving butylidenephthalide (BP) include
dimethyl sulfoxide (DMSO), polyoxyethylene castor oil
(Kolliphor.RTM. EL), ethanol, vegetable oil and animal oil, but is
not limited thereby.
[0033] It is discovered that when using the kit in accordance with
the present invention, butylidenephthalide (BP) at an overly low
concentration cannot effectively promote the differentiation of a
stem cell into a brown-like adipose cell; however,
butylidenephthalide (BP) at an overly high concentration may cause
cell damage. Therefore, the used concentration generally ranges
from about 1 .mu.g to about 100 .mu.g per mL of a conditional
medium, preferably ranges from about 2 .mu.g to about 50 .mu.g per
mL of a conditional medium, and more preferably ranges from about 5
.mu.g to about 20 .mu.g per mL of a conditional medium. For
example, as shown in the appended examples, butylidenephthalide
(BP) can effectively promote the differentiation of a stem cell
into a brown-like adipose cell at a concentration of about 10 .mu.g
per mL of a conditional medium.
[0034] Inventors of the present invention also discovered that the
administration of butylidenephthalide (BP) alone and the
administration of butylidenephthalide (BP) in combination with
mesenchymal stem cells and/or brown-like adipose cells to an animal
can both achieve the effects on inhibiting the accumulation of
white fat, promoting the conversion of white fat into brown fat,
inhibiting weight gain and/or reducing the contents of
triglycerides, glucose and total cholesterol in blood.
[0035] Therefore, the present invention also relates to a use of
butylidenephthalide (BP) in the manufacture of a medicament,
wherein the medicament is used for inhibiting the accumulation of
white fat, promoting the conversion of white fat into brown fat,
inhibiting weight gain and/or reducing the contents of
triglycerides, glucose and total cholesterol in blood. The
medicament provided in accordance with the present invention
especially can be used for anti-obesity and/or preventing metabolic
syndrome associated with obesity. The metabolic syndrome associated
with obesity is at least one of diabetes mellitus, cerebrovascular
disease, cardiovascular disease, hypertension and nephropathy.
Optionally, the medicament could be administered in combination
with mesenchymal stem cells and/or brown-like adipose cells.
[0036] Depending on the desired purpose, the medicament of the
present invention could be provided in any suitable form without
specific limitations. For example, the medicament could be
administered to a subject in need by an oral or parenteral (such as
subcutaneous injection, intravenous injection, muscular injection,
peritoneal injection, transdermal, subcutaneous implantation or
interstitial implantation) route, but is not limited thereby.
Particularly, the oral administration can be easily taken by
patients themselves on time. Depending on the form and purpose,
suitable carriers could be chosen and used to provide the
medicament, as long as the carriers do not adversely affect the
desired effects of butylidenephthalide (BP). Examples of the
carriers include excipients, diluents, auxiliaries, stabilizers,
absorbent retarders, disintegrating agent, hydrotropic agents,
emulsifiers, antioxidants, adhesives, binders, tackifiers,
dispersants, suspending agents, lubricants, hygroscopic agents,
etc.
[0037] As for a dosage form for oral administration, examples of
suitable carriers include, but are not limited to, water, saline,
dextrose, glycerol, ethanol or its analogs, cellulose, starch,
sugar bentonite, and combinations thereof. The medicament could be
provided in any suitable form for oral administration in any
suitable way, such as in a solid form of a tablet, a pill, a
capsule, granules, a pulvis, or in a liquid form of an oral liquid,
a syrup, a spirit, an elixir, a tincture, etc., but is not limited
thereby.
[0038] As for the form of injection or drip suitable for
subcutaneous, intravenous, muscular, or peritoneal administration,
the medicament provided in accordance with the present invention
could comprise one or more ingredient(s), such as an isotonic
solution, a salt-buffered saline (e.g., phosphate-buffered saline
or citrate-buffered saline), a hydrotropic agent, an emulsifier, a
5% sugar solution, and other carriers to provide the medicament as
an intravenous infusion, an emulsified intravenous infusion, a
powder for injection, a suspension for injection, or a powder
suspension for injection, etc. Alternatively, the medicament could
be prepared as a pre-injection solid. The pre-injection solid could
be provided in a form which is soluble in other solutions or
suspensions, or in an emulsifiable form. A desired infection is
provided by dissolving the pre-injection solid in other solutions
or suspensions or emulsifying it prior to being administered to a
subject in need. In addition, as for the external dosage form for
transdermal administration, the medicament could be provided in a
form of such as a liniment (e.g., an emulsion, a cream, a gel, a
dispersing paste, an ointment), a spray, a patch, or a solution
(e.g., a cleaning liquid, a suspension), etc.
[0039] As for a dosage form suitable for subcutaneous implantation
or interstitial implantation, the medicament provided in accordance
with the present invention could further comprise one or more
ingredients, such as an excipient, a stabilizer, a buffer, other
carriers, etc., to provide the medicament in a form of such as a
wafer, a tablet, a pill, a capsule, and the likes. Therefore, the
medicament could be implanted into a subject to slowly and
continuously release the butylidenephthalide (BP) contained therein
to the tissues surrounding the implanted site, and thus, could
achieve a locally stable high dose of medicament for inhibiting the
accumulation of white fat, promoting the conversion of white fat
into brown fat, inhibiting weight gain and/or reducing the contents
of triglycerides, glucose and total cholesterol in blood. For
example, the medicament provided in accordance with the present
invention could be mixed with p(CPP-SA) copolymer to provide a
mixture; the mixture is then dissolved in methylene dichloride and
dried to provide a powder; thereafter, the powder is put in a mold
and compressed under a slight pressure to provide a wafer for
subcutaneous implantation or interstitial implantation, but is not
limited thereby.
[0040] Optionally, the medicament provided in accordance with the
present invention could further comprise a suitable amount of
additives, such as a flavoring agent, a toner, or a coloring agent
for enhancing the palatability and the visual perception of the
medicament, and a buffer, a conservative, a preservative, an
antibacterial agent, or an antifungal agent for improving the
stability and storability of the medicament. In addition, the
medicament could optionally further comprise one or more other
active ingredients such as .beta.-adrenaline receptor agonists
(e.g., CL316,243), PPAR-.gamma. agonists (e.g., rosiglitazone),
metabolic promotion related factors (e.g., adiponectin),
immunomodulators, etc., or be used in combination with a medicament
comprising one or more other active ingredients, to further enhance
the effect of the medicament, or to increase the application
flexibility and adaptability of the preparation thus provided, as
long as the other active ingredients do not adversely affect the
desired effects of butylidenephthalide (BP).
[0041] Depending on the need, age, body weight, and health
conditions of the subject, the medicament provided in accordance
with the present invention could be dosed at various administration
frequencies, such as once a day, multiple times a day, or once
every few days, etc. For example, when the medicament provided in
accordance with the present invention is administered by oral
administration to a subject to inhibit the accumulation of white
fat, promote the conversion of white fat into brown fat, inhibit
weight gain and/or reduce the contents of triglycerides, glucose,
total cholesterol in blood, the medicament is administered at an
amount ranging from about 30 mg (as BP)/kg-body weight to about
2000 mg (as BP)/kg-body weight per day, preferably ranging from
about 100 mg (as BP)/kg-body weight to about 1,000 mg (as
BP)/kg-body weight per day, and more preferably ranging from about
200 mg (as BP)/kg,-body weight to about 500 mg (as BP)/kg-body
weight per day, wherein the unit "mg/kg-body weight" refers to the
amount required for per kg-body weight of the subject. In some
embodiments of the present invention, the medicament provided in
accordance with the present invention is used for anti-obesity
(i.e., inhibiting the accumulation of white fat, promoting the
conversion of white fat into brown fat, inhibiting weight gain
and/or reducing the contents of triglycerides, glucose and total
cholesterol in blood), wherein the medicament is administered at an
amount of about 250 mg (as BP)/kg-body weight per day.
[0042] Besides the administration of the medicament provided in
accordance with the present invention, additional mesenchymal stem
cells and/or brown-like adipose cells could be optionally
administered to the subject in need, and the mesenchymal stem cells
and/or brown-like adipose cells could be administered in
combination with the medicament simtaneously or separately.
Depending on the need, age, body weight and health conditions of
the subject, the mesenchymal stem cells and/or brown-like adipose
cells could be administered at various frequencies, such as once a
day, multiple times a day, once every few days, or once every few
weeks, etc. For example, when the cells are administered to the
subject by cell infusion, the cells are administered at an amount
ranging from about 4.times.10.sup.4 cells to about 4.times.10.sup.6
cells every two weeks, based on the total amount of mesenchymal
stem cells and brown-like adipose cells. In some embodiments of the
present invention, besides administering the medicament provided in
accordance with the present invention to a subject in need, the
subject could further be administered with about 4.times.10.sup.5
adipose stem cells or about 4.times.10.sup.5 brown-like adipose
cells by cell infusion every two weeks.
[0043] The present invention also relates to a method for
inhibiting the accumulation of white fat, promoting the conversion
of white fat into brown fat, inhibiting weight gain and/or reducing
the contents of triglycerides, glucose and total cholesterol in
blood, comprising administering to a subject in need an effective
amount of butylidenephthalide (BP). The method is especially for
anti-obesity and/or preventing metabolic syndrome associated with
obesity. The applied route, applied type, applied amount, applied
form in combination and uses in related applications of
butylidenephthalide (BP) are all in line with the above
descriptions.
[0044] The present invention will be further illustrated in detail
with specific examples as follows. However, the following examples
are provided only for illustrating the present invention. The scope
of the present invention is not limited thereby and will be
indicated in the appended claims.
EXAMPLES
[0045] [Cellular Experiment]
[0046] A1. Preparation of a Conditional Medium
[0047] A DMEM medium (brand name: GIBCO; product number: 11965092)
was used as a basic medium, and rosiglitazone (concentration; 0.5
.mu.M, solvent: dimethyl sulfoxide (DMSO)), insulin (concentration:
170 nM, solvent: culture medium), 3-isobutyl-1-methylxanthine
(IBMX; concentration: 0.5 .mu.M, solvent: DMSO) and dexamethasone
(concentration: 1 .mu.M, solvent: DMSO) were externally added
therein to provide a conditional medium.
[0048] B1. Cell Cultivation
[0049] B1-1. Pre-Cultivation of Adipose Stem Cells
[0050] The adipose stem cells (ADSCs) were cultivated in a DMEM/F12
medium at 37.degree. C. 5% CO.sub.2 for 5 days. The morphology of
aforementioned pre cultivated cells was observed at different
magnifications (including 40fold, 100-fold and 200-fold) by a
microscope and taken photos. The results are shown in FIG. 1 (i.e.,
adipose stem cells (undifferentiated)).
[0051] B1-2. Differentiation of Adipose Stem Cells
[0052] The aforementioned pre-cultivated adipose stem cells
provided by Example B1-1 were cultivated in a conditional medium
provided by Example A1 at 37.degree. C., 5% CO.sub.2 for 7 days to
differentiate into adipose cells. The morphology of the cells thus
obtained was observed at different magnifications (including
40-fold, 100-fold and 200-fold) by a microscope and taken photos.
The results are also shown in FIG. 1 (i,e., adipose cells
(differentiated)).
[0053] B1-3. Conversion of Adipose Cells
[0054] The adipose cells provided by Example B1-2 were divided into
seven groups and independently subjected to the following
treatments:
[0055] (1) Control group: cells were cultivated in a conditional
medium provided by A1 for 7 days;
[0056] (2) BP-2 group: cells were cultivated in a conditional
medium that was externally added with butylidenephthalide (BP) (to
a final concentration of 2 .mu.g/mL) for 7 days
(butylidenephthalide (BP) was purchased from ECHO CHEMICAL Co.,
Ltd. (Taiwan); product number: A10353);
[0057] (3) BP-10 group: cells were cultivated in a conditional
medium that was externally added with butylidenephthalide (BP) (to
a final concentration of 10 .mu.g/mL) for 7 days;
[0058] (4) BP-50 group: cells were cultivated in a conditional
medium that was externally added with butylidenephthalide (BP) (to
a final concentration of 50 .mu.g/mL) for 7 days;
[0059] (5) Tofacitinib group: cells were cultivated in a
conditional medium that was externally added with tofacitinib (to a
final concentration of 2 .mu.M) for 7 days (tofacitinib was
purchased from UNI-ONWARD Corp. (Taiwan); product number:
PZ0017);
[0060] (6) Adenosine group: cells were cultivated in a conditional
medium that was externally added with adenosine (to a final
concentration of 1 .mu.M) for 7 days (adenosine was purchased from
UNI-ONWARD Corp. (Taiwan); product number: A4036); and
[0061] (7) Proanthocyanidin group: cells were cultivated in a
conditional medium that was externally added with proanthocyanidin
(to a final concentration of 10 .mu.M) for 7 days (adenosine was
purchased from Hong Jing Co., Ltd. (Taiwan); product number:
sc-344976).
[0062] Then, the morphology of cells in each group was observed at
different magnifications (including 40-fold, 100-fold and 200-fold)
by a microscope and taken photos. The results of "BP-10 group" are
also shown in FIG. 1.
Example 1
Effect of Butylidenephthalide (BP) on Promoting the Differentiation
of a Stem Cell into a Brown-Like Adipose Cell
[0063] As described above, different from white adipose cells, the
morphology or brown-like adipose cells/brown adipose cells is
characterized by (i) containing numerous adipose droplets, (ii)
containing a large number of mitochondria and (iii) having highly
expressed uncoupling protein 1 (UCP1). Because of containing a
large number of mitochondria, brown-like adipose cells/brown
adipose cells possess higher oxygen consumption rate and spare
respiratory capacity (wherein the spare respiratory capacity refers
to the difference between the maximum oxygen consumption rate and
the basic oxygen consumption rate, representing the oxygen
consumption that is not used for producing ATP), as compared to
white adipose cells. Therefore, the number and proportion of
brown-like adipose cells/brown adipose cells can be conceived by
observing the number of adipose droplets in the cells, the oxygen
consumption rate of adipose cells and the gene and protein
expressions of UCP1.
[0064] (1-1) Observation of the Production Efficiency of Adipose
Droplets in Cells
[0065] First, the cell morphology of each group in photos taken in
Examples B1-1, B1-2 and B1-3 was observed. As shown in FIG. 1, as
compared to the adipose stem cells (undifferentiated) provided by
Example B1-1 and adipose cells (differentiated) provided by Example
B1-2, the adipose cells in "BP-10 group" provided by Example B1-3
contained more adipose droplets. The results indicate that the use
of BP can effectively promote the differentiation of an adipose
stem cell into a brown-like adipose cell.
[0066] Thereafter, the adipose droplets of adipose stem cells
(undifferentiated) provided by Example B1-1, adipose cells
(differentiated) provided by Example B1-2 and adipose cells in
"BP-2 group", "BP-10 group" and "BP-50 group" provided by Example
B1-3 were stained by oil red dye, and the morphology thereof was
taken photos and recorded. The results are shown in FIG. 2A. Then,
the number of adipose droplets of adipose cells in each group was
quantified. The results are shown in FIG. 2B. As shown in FIGS. 2A
and 2B, as compared to the adipose cells that were not treated by
butylidenephthalide (BP), the adipose cells that were treated by
butylidenephthalide (BP) in "BP-2 group", "BP-10 group" and "BP-50
group" contained more adipose droplets, wherein the adipose cells
in "BP-10 group" had the highest content of adipose droplets. The
results indicate that the use of BP can effectively promote the
differentiation of an adipose stem cell into a brown-like adipose
cell.
[0067] (1-2) Observation of the Efficiency of Enhancing Oxygen
Consumption Rate
[0068] The adipose stem cells (undifferentiated) provided by
Example B1-1, adipose cells (differentiated) provided by Example
B1-2 and adipose cells in "BP-2 group", "BP-10 group" and "BP-50
group" provided by Example B1-3 were separately seeded in a XF
24-well cell culture plate (purchased from Seahorse Bioscience)
with a density of 1.65.times.10.sup.4 cells/well and cultivated in
a DMEM medium free of buffer ingredients (i.e., the medium
containing 2 mM (GlutaMAX, 1 mM sodium pyruvate, 1.85 g/L NaCl, 25
mM glucose) at 37.degree. C. for 24 hours. After a medium was
replaced with a fresh medium, the cells were continuously
cultivated at 37.degree. C. for 1 hour. Then, the mitochondrial
biosynthesis of cells in each group was separately interfered with
25 .mu.M oligomycin, 0.5 .mu.M FCCP and 5 .mu.M rotenone/antimycin.
A sequentially, and was continuously detected by a XF24
extracellular flux analyser (purchased from Seahorse Bioscience)
prior to and during conduction of the aforementioned interference.
The oxygen tension and acidification degree in the culture medium
were detected by an adjusted probe to further calculate the oxygen
consumption rate and spare respiratory capacity. The results of
oxygen consumption rate are shown in FIG. 3A, and the results of
basic oxygen consumption rate and spare respiratory capacity are
shown in FIG. 3B.
[0069] As shown in FIGS. 3A and 3B, as compared to the adipose
cells (differentiated) that were not treated by butylidenephthalide
(BP), the cells that were treated by butylidenephthalide (BP) in
"BP-2 group", "BP-10 group" and "BP-50 group" had higher oxygen
consumption rate (FIG. 3A) and spare respiratory capacity (FIG.
3B). The results indicate that the use of BP can effectively
promote the differentiation of an adipose stem cell into a
brown-like adipose cell.
[0070] (1-3) Analysis of UCP1 Expression
[0071] The cells in each group provided by Example B1-3 were
seprately collected, and then the total RNA and protein of the
cells were individually extracted for the following
experimentation.
[0072] The total RNA was subjected to reverse transcription to
provide cDNA, and then the expression of UCP1 gene of the cells in
each group was analyzed by reverse transcription polymerase chain
reaction (RT-PCR). The results are shown in FIG. 4A, wherein FAB4
gene was a biomarker of mature adipose cells, and thus, it was used
as an internal control of adipose cells in this experimentation.
The actin gene was used as an internal control of all the cells.
Furthermore, the expression of UCP1 protein of the cells in each
group was analyzed by Western blot. The results are shown in FIG.
4B, wherein FABP4 protein was used as an internal control of
adipose cells. The actin was used as an internal control of all the
cells.
[0073] As shown in FIGS. 4A and 4B, as compared to that of control
group, the expressions of UCP1 gene and UCP1 protein of "BP-10
group" both significantly increased, and the increments of UCP 1
gene and UCP1 protein were higher than that of tofacitinib group,
adenosine group and proanthocyanidin group. The results indicate
that the use of BP can effectively promote the differentiation of
an adipose stem cell into a brown-like adipose cell.
[0074] As shown by the results of aforementioned Examples (1-1) to
(1-3), BP can effectively promote the differentiation of a stem
cell into a brown-like adipose cell, and the effect is better than
that of the known drugs capable of promoting the differentiation of
a stem cell into a brown-like adipose cell, including tofacitinib,
adenosine and proanthocyanidin.
[0075] [Animal Experiment]
[0076] A2. Preparation of Experimental Animal Mouse Model
[0077] The C57BL/6 mice (purchased from National Laboratory Animal
Center, Taiwan) were bred in Laboratory Animal Center of China
Medical University, Taiwan (conditions: 3 mice were bred per cage;
the temperature of the breeding room ranged from 20.degree. C. to
24.degree. C., and the relative humidity ranged from 50% to 70%;
the light cycle was 12 hours; sufficient water and normal diet were
supplied daily) until they were 4 weeks old. Then, the mice were
randomly divided into seven groups (six mice per group), and
individually subjected to the following treatment and
experimentation:
[0078] (1) Normal diet group (i.e., "ND" group): the mice were
continuously fed with a normal diet until they were 18 weeks
old.
[0079] (2) High fat diet group (i.e., "HFD" group): the mice were
continuously fed with a high fat diet until they were 18 weeks old,
and no additional treatment was conducted.
[0080] (3) Oral administration of butylidenephthalide (BP) group
(i.e., "HFD+BP" group): the mice were continuously fed with a high
fat diet, and then simultaneously administered with
butylidenephthalide (BP) by oral route at the beginning of the
ninth week (dosage amount: every day, 250 mg/kg-body weight) until
they were 18 weeks old.
[0081] (4) Cell infusion of adipose stem cells group (i.e.,
"HFD+ADSC" group): the mice were continuously fed with a high fat
diet, and then simultaneously administered with adipose stem cells
by intravenous injection at the beginning of the eleventh week
(dosage amount: every two weeks, 4.times.10.sup.5 cells suspended
in 100.about.150 .mu.L of normal saline) until they were 18 weeks
old (four injections in total).
[0082] (5) Oral administration of butylidenephthalide (BP) in
combination with cell infusion of adipose stem cells group (i.e.,
"HFD+BP+ADSC" group): the mice were continuously fed with a high
fat diet, then simultaneously administered with butylidenephthalide
(BP) by oral route at the beginning of the ninth week (dosage
amount: every day, 250 mg/kg-body weight), and further
simultaneously administered with adipose stem cells by intravenous
injection at the beginning of the eleventh week (dosage amount:
every two weeks, 4.times.10.sup.5 cells suspended in 100.about.150
.mu.L of normal saline) until they were 18 weeks old (four
injections in total).
[0083] (6) Cell infusion of brown-like adipose cells group (i.e.
"HFD+BLC" group): the mice were fed with a high fat diet, and then
simultaneously administered with brown-like adipose cells provided
by Example 1 by intravenous injection at the beginning of the
eleventh week (dosage amount: every two weeks, 4.times.10.sup.5
cells suspended in 100.about.150 .mu.L of normal saline) until they
were 18 weeks old (four injections in total).
[0084] (7) Oral administration of butylidenephthalide (BP) in
combination with cell infusion of brown-like adipose cells group
(i.e., "HFD+BP+BLC" group): the mice were continuously fed with a
high fat diet, then simultaneously administered with
butylidenephthalide (BP) by oral route (dosage amount: every day,
250 mg/kg-body weight) at the beginning of the ninth week, and
further simultaneously administered with brown-like adipose cells
provided by Example 1 by intravenous injection at the beginning of
the eleventh week (dosage amount: every two weeks, 4.times.10.sup.5
cells suspended in 100.about.150 .mu.L of normal saline) until they
were 18 weeks old (four injections in total).
[0085] The standard diet (1326, Altromin, Lage, Germany; purchasing
broker: ejoy2 Co., Ltd.) is a granulated diet with the granule size
of 13 mm, containing 19% of crude protein, 4% of crude fat, 6% of
crude fiber, 13.5% of water, 7% of gray matter and 50.5% of
non-nitrogen compound therein. The high fat diet (TD.06415, Harlan,
Frederick, State of Maryland, U.S.A; purchasing broker:ejoy2. Co.,
Ltd.) is a special formulated diet mixing with lard and soybean
oil, containing 22.7% of fat (as weight) and 22.8% of sucrose (as
weight) therein, and the amount of beat is 4.6 kcal/g wherein 45%
of calories are from fat.
[0086] B2. Observation, Recordation and Sample Collection of Mouse
Model
[0087] B2-1.In the preparation process of above Example A2, the
weight change of mice was recorded every week until they were 18
weeks old.
[0088] B2-.2. After Example B2-1 was accomplished, the mice was
subjected to fast for 12 hours, and then the blood of mice was
collected in a serum tube by cardic blood collection. The collected
blood was stand for 30 minutes, and then subjected to centrifuge at
3000 rpm, 4.degree. C. for 10 minutes. The supernatant thus
provided was collected (i.e., blood sample) and kept at a
temperature of 4.degree. C. for following experimentation and
analysis.
[0089] B2-3. After Example: B2-2 was accomplished, the mice were
sacrificed, and the liver, heart, kidney, subcutaneous adipose
tissue and visceral adipose tissue thereof were taken out to take
photos. The weight of the subcutaneous adipose tissue and visceral
adipose tissue of mice were measured and recorded, and the
collected organs were kept at a temperature of -80.degree. C. for
following experimentation and analysis,
Example 2
Observation of the Condition of Mouse Fat Browning
[0090] It is known that there are numerous small adipose droplets
in brown adipose cells. To understand whether butylidenephthalide
(BP) is effective in promoting the occurrence of the fat browning
in an animal body or not, the subcutaneous adipose tissue and
visceral adipose tissue provided by Example B2-3 were fixed by
formalin, mounted by wax, and then sliced. The aforementioned
slices were stained by hematoxylin-eosin stain (H&E stain) to
observe the cell morphology of adipose tissue of mice in each
group. The results are shown in FIGS. 5A (subcutaneous fat) and 5B
(visceral fat).
[0091] As shown in FIGS. 5A and 5B, the subcutaneous adipose tissue
and visceral adipose tissue of mice in "HFD" group both consist of
single and large white adipose cells, and those in "HFD+BP" group,
"HFD+ADSC" group, "HFD+BP+ADSC" group, "HFD+BLC" group and
"HFD+BP+BLC" group contained the brown adipose cells with numerous
small adipose droplets. In addition, as compared to the "HFD+BP"
group or "HFD+ADSC" group, mice in "HFD+BP+ADSC" group had
significantly larger number of brown adipose cells with numerous
small adipose droplets. On the other hand, as compared to the
"HFD+BP" group or "HFD+BLC" group, mice in "HFD+BP+BLC" group had
significantly larger number of brown adipose cells with numerous
small adipose droplets.
[0092] The results indicate that the administration of
butylidenephthalide (BP) alone and the administration of
butylidenephthalide (BP) in combination with cell infusion can both
effectively convert the white fat of mice induced by a high fat
diet into brown adipose cells with numerous small adipose droplets.
That is, butylidenephthalide (BP) and a combination of BP and cell
infusion both are effective in promoting the fat browning in an
animal body, wherein the effect of a combination of BP and cell
infusion is better.
Example 3
Appearances of the Heart, Liver, Kidney, Subcutaneous Adipose
Tissue and Visceral Adipose Tissue of Mice
[0093] To understand whether butylidenephthalide (BP) can alleviate
the accumulation of white fat or not, the photos of appearances of
the liver, heart, kidney, subcutaneous adipose tissue and visceral
adipose tissue of mice in each group recorded in Example B2-3 are
shown in FIG. 6A. The weight of subcutaneous adipose tissue and
visceral adipose tissue of mice in each group recorded in Example
B2-3 were respectively averaged. The results are shown in Table 1,
FIGS. 6B and 6C.
TABLE-US-00001 TABLE 1 Subcutaneous Visceral adipose tissue (g)
adipose tissue (g) "ND" group 0.22 0.59 "HFD" group 0.42 1.0 "HFD +
BP" group 0.24 0.57 "HFD + ADSC" group 0.31 0.62 "HFD + BP + ADSC"
group 0.28 0.64 "HFD + BLC" group 0.21 0.54 "HFD + BP + BLC" group
0.20 0.46
[0094] As shown in FIG. 6A, as compared to that of "ND" group, the
visceral of mice is "HFD" group contained a large amount of
accumulated white fat, wherein the accumulation condition
surrounding kidney was most serious. However, as compared to that
of "HFD" group, the accumulation condition of white fat in visceral
of mice in "HFD+BP" group, "HFD+ADSC" group, "HFD+BP+ADSC" group,
"HFD+BLC" group and "HFD+BP+BLC" group significantly alleviated. On
the other hand, as compared to that of "HFD+BP" group or "HFD+ADSC"
group, the condition of mice in "HFD+BP+ADSC" group was alleviated
more significantly. In addition, as compared to that of "HFD+BP"
group or "HFD+BLC" group, the condition of mice in "HFD+BP+BLC"
group was alleviated more significantly.
[0095] As shown in Table 1, FIGS. 6B and 6C, as compared to that of
"ND" group, the weights of subcutaneous fat and visceral fat in
"HFD" group were both significantly higher. However, as compared to
that of "HFD" group, the weights of subcutaneous fat and visceral
fat in "HFD+BP" group, "HFD+ADSC" group, "HFD+BP+ADSC" group,
"HFD+BLC" group and "HFD+BP+BLC" group were all significantly
lower.
[0096] The results indicate that the administration of
butylidenephthalide (BP) alone and the administration of
butylidenephthalide (BP) in combination with cell infusion can both
effectively reduce the accumulation of subcutaneous white fat and
visceral white fat of mice induced by a high fat diet, wherein the
effect of a combination of butylidenephthalide (BP) and cell
infusion is better.
Example 4
Observation of the Changes of Mouse Physiological Parameters
[0097] 4-1. Changes of Mouse Weight
[0098] To understand whether butylidenephthalide (BP) is effective
in inhibiting weight gain or not, the average value of the recorded
weights of mice in each group of Example B2-1 was obtained weekly.
The results are shown in FIG. 7. As shown in FIG. 7, as compared to
that of "HFD" group, the weights of mice in "HFD+BP" group,
"HFD+ADSC" group, "HFD+BP+ADSC" group, "HFD+BLC" group and
"HFD+BP+BLC" group increased significantly less. On the other hand,
as compared to that of "HFD+BP" group or "HFD+ADSC" group, the
weights of mice in "HFD+BP+ADSC" group increased significantly
less. In addition, as compared to that of "HFD+BP" group or
"HFD+BLC" group, the weights of mice in "HFD+BP+BLC" group
increased significantly less.
[0099] The results indicate that the administration of
butylidenephthalide (BP) alone and the administration of
butylidenephthalide (BP) in combination with cell infusion can both
effectively inhibit weight gain of mice induced by a high fat diet,
wherein the effect of a combination of butylidenephthalide (BP) and
cell infusion on inhibiting weight gain is better.
[0100] 4-2. Contents of Glucose, Triglycerides and Total
Cholesterol in Blood
[0101] To understand whether butylidenephthalide (BP) is effective
in reducing the contents of glucose, triglycerides and total
cholesterol in blood or not, the blood sample of mice in each group
provided by Example B2-2 was detected by a SYSMEX K-1000 and
TOSHIBA TBA200FA automatic blood analyzer to analyze the contents
of glucose, triglycerides and total cholesterol in blood of mice.
The results of each group were averaged. The results are shown in
Table 2 and FIGS. 8A to 8C. Based on the result of "HFD" group, the
relative contents of glucose, triglycerides and total cholesterol
of each group were calculated. The results are also shown in Table
2.
TABLE-US-00002 TABLE 2 Serum biochemical values of mice in groups
fed by a normal diet or a high fat diet HFD + HFD + HFD + HFD + HFD
+ ND HFD BP ADSC BP + ADSC BLC BP + BLC Glucose (mg/dL) 229.67 .+-.
484.50 .+-. 411.50 .+-. 362.50 .+-. 333.00 .+-. 327.00 .+-. 91.50
.+-. 27.48 70.50 43.50 46.50 41.00 91.00 77.50 Relative content --
100 85 75 69 67 60 of glucose Triglycerides 149.00 .+-. 253.00 .+-.
213.50 .+-. 234.50 .+-. 157.00 .+-. 109.50 .+-. 219.50 .+-. (mg/dL)
8.16 33.00 50.50 24.50 14.00 44.50 15.50 Relative content -- 100 84
93 62 43 87 of triglycerides Total cholesterol 55.00 .+-. 197.00
.+-. 157.50 .+-. 105.50 .+-. 123.00 .+-. 165.00 .+-. 149.50 .+-.
(mg/dL) 0.00 34.00 1.50 4.50 14.00 35.00 39.50 Relative content --
100 80 54 62 84 76 of total cholesterol
[0102] As shown in Table 2 and FIG. 8A, as compared to that of "ND"
group, the content of glucose in blood of mice in "HFD" group was
significantly higher. However, as compared to that of "HFD" group,
the contents of glucose in blood of mice in "HFD+BP" group,
"HFD+ADSC" group, "HFD+BP+ADSC" group, "HFD+BLC" group and
"HFD+BP+BLC" group were all significantly lower. On the other hand,
as compared to that of "HFD+BP" group or "HFD+ADSC" group, the
content of glucose in blood of mice in "HFD+BP+ADSC" group was
lower. In addition, as compared to that of "HFD+BP" group or
"HFD+BLC" group, the content of glucose in blood of mice in
"HFD+BP+BLC" group was lower.
[0103] As shown in Table 2 and FIG. 8B, as compared to that of "ND"
group, the content of triglycerides in blood of mice in "HFD" group
was significantly higher. However, as compared to that of "HFD"
group, the contents of triglycerides in blood of mice in "HFD+BP"
group, "HFD+ADSC" group, "HFD+BP+ADSC" group, "HFD+BLC" group and
"HFD+BP+BLC" group were all significantly lower. On the other hand,
as compared to that of "HFD+BP" group or "HFD+ADSC" group, the
content of triglycerides in blood of mice in "HFD+BP+ADSC" group
was lower.
[0104] As shown in Table 2 and FIG. 8C, as compared to that of "ND"
group, the content of total cholesterol in blood of mice in "HFD"
group was significantly higher. However, as compared to that of
"HFD" group, the contents of triglycerides in blood of mice in
"HFD+BP" group, "HFD+ADSC" group, "HFD+BP+ADSC" group, "HFD+BLC"
group and "HFD+BP+BLC" group were all significantly lower. On the
other hand, as compared to that of "HFD+BP" group, the content of
triglycerides in blood of mice in "HFD+BP+ADSC" group was lower. In
addition, as compared to that of "HFD+BP" group or "HFD+BLC" group,
the content of triglycerides in blood of mice in "HFD+BP+BLC" group
was lower.
[0105] The results indicate that the administration of
butylidenephthalide (BP) alone and the administration of
butylidenephthalide (BP) in combination with cell infusion can both
effectively reduce the contents of glucose, triglycerides and total
cholesterol in blood of mice induced by a high fat diet, wherein
the effect of a combination of butylidenephthalide (BP) and cell
infusion is better.
[0106] As shown by the above results of cellular and animal
experiments, butylidenephthalide (BP) is capable of promoting the
differentiation of a stem cell into a brown-like adipose cell,
inhibiting the accumulation of white fat, promoting the conversion
of white fat into brown fat, inhibiting weight gain and/or reducing
the contents of triglycerides, glucose and total cholesterol in
blood, and thus, can be administered to an animal to anti-obesity
and/or prevent metabolic syndrome associated with obesity.
Particularly, the metabolic syndrome associated with obesity is at
least one of diabetes mellitus, cerebrovascular disease,
cardiovascular disease, hypertension and nephropathy. In addition,
administration of butylidenephthalide (BP) with mesenchymal stem
cells and/or brown-like adipose cells can further enhance the
aforementioned effects.
[0107] The above embodiments are only used to illustrate the
principle and function of the present invention, and the scope of
the present invention is hot limited thereby. Without departing
from the principle and spirit of the present invention, people
skilled in the art can modify and change the above embodiments. The
scope of the present invention is indicated in the appended
claims
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