Note: Descriptions are shown in the official language in which they were submitted.
CA 02857543 2014-07-21
Anti-obesity potential of Calebin A
FIELD OF INVENTION
[0002] The invention in general relates to medicaments for obesity management.
More
specifically, it relates to anti-obesity potential of Calebin A.
DESCRIPTION OF PRIOR ART
[0003] Obesity is the most prevalent nutritional disorder in industrialized
countries and is a
growing problem in developing countries. It is described as a global epidemic
and overweight
and obese individuals (BMI of 25 and above) are at increased risk for various
chronic physical
ailments and psychological problems such as depression, eating disorders and
low self esteem. It
is associated with various diseases like cardiovascular diseases, diabetes
mellitus, osteoarthritis,
obstructive sleep apnea and cancer. WHO considers obesity to be one of the top
10 causes of
preventable death worldwide.
[0004] In obesity, there is an increase in the adipose tissue mass due to the
production of new fat
cells (adipocytes) through the process of adipogenesis and/or the deposition
of increased
amounts of cytoplasmic triglyceride per cell. A fat cell develops as
internally produced lipid
droplets coalesce into a single large mass. Eventually, cellulite results due
to enhanced
adipogenesis and accumulation of chunks of adipocytes under the skin dermis.
[0005] Studies of adipogenesis have proceeded with the hope that manipulation
of this process in
humans might lead to a reduction in the burden of obesity and diabetes. At
molecular level,
several markers have been targeted in treating obesity such as leptin,
adiponectin, TNF-a etc
[0006] Though drugs are available for treating the disorder, there is a
constant need and search
for safe natural approach to help manage obesity and its related socio-
economic consequences.
[0007] Calebin A is known to protect neuronal cells from 0-amy1oid insult
(Park S Y et al, J Nat
Prod. 2002 September; 65(9):1227-31), induce apoptosis and modulate MAPK
family activity in
drug resistant human gastric cancer cells (Li Y et al, Eur J. Pharmacol. 2008
Sep. 4; 591(1-
3):252-8). Zeng Y et al. (Chem Pharm Bull (Tokyo) 2007 June; 55(6):940-3)
discusses two new
calebin derivatives,
4 "-(4 " '-hydroxypheny1-3 " '-methoxy)-2 "-oxo-3 "-buteny1-3 441-
hydroxypheny1)-propenoate and
4 "-(4" '-hydroxypheny1)-2" -oxo-3" -buteny1-3-(4'-
hydroxypheny1-3 '-methoxy)-propeno ate.
[0008] The present invention discloses the potential of Calebin A to prevent
fat accumulation
during the terminal differentiation of adipocytes (fat cells) and applications
thereof in obesity
management. The present invention elucidates the potential of Calebin A to
favorably modulate
biochemical markers associated with obesity. Notable biomodulatory properties
of Calebin A
include inhibiting leptin production, increasing adiponectin expression and
inhibiting local
1
CA 02857543 2014-07-21
(adipocyte) and systemic inflammation caused by pro-inflammatory cytokines
Tumor Necrosis
Factor (TNF-a), Interleukin-6 (IL-6) and Interleukin-1 (IL-113).
[0009] Accordingly, it is the principle objective of the present invention to
disclose anti-obesity
potential of Calebin A.
[0010] The invention fulfills the aforesaid principle objective and provides
further related
advantages
SUMMARY OF THE INVENTION
[0011] The present invention discloses the potential of Calebin A in
inhibiting adipogenesis and
applications thereof in obesity management. The present invention elucidates
the potential of
Calebin A to favorably modulate biochemical markers associated with obesity in
mammals.
Notable biomodulatory properties of Calebin A include inhibiting leptin
production, increasing
adiponectin expression and inhibiting local (adipocyte) and systemic
inflammation caused by
pro-inflammatory cytokines Tumor Necrosis Factor (TNF-a), Interleukin-6 (IL-6)
and
Interleukin-1 (IL-1p).
[0012] Other features and advantages of the present invention will become
apparent from the
following more detailed description, taken in conjunction with the
accompanying drawings,
which illustrate, by way of example, the principle of the invention.
BRIEF DESCRIPTION OF FIGURES
[0013] FIG. 1 shows the graphical representation of the percentage
adipogenesis inhibition
effected by Calebin A at concentrations of 0.5, 1.0 and 2.0 jig/m1 as studied
by the Oil-Red-0-
Staining method.
[0014] FIG. 2 shows the graphical representation of the percentage inhibition
of leptin
production in human adipocytes effected by Calebin A at concentrations of 0.5,
1.0 and 2.0
[tg/ml. P value *:<0.01; **:<0.001.
[0015] FIG. 3 shows the graphical representation of the percentage increase of
adiponectin
expression in human adipocytes effected by Calebin A at concentrations of 0.5,
1.0 and 2.0
pg/ml. P value *:<0.01.
[0016] FIGS. 4 and 5 shows the graphical representation of the percentage
inhibition of TNF-ct
expression (P value *:<0.01; **:<0.001) and IL-6 expression (P value *:<0.01)
respectively, in
human adipocytes effected by Calebin A at concentrations of 0.5, 1.0 and 2.0
g/ml.
[0017] FIG. 6 shows the graphical representation of the effect of multiple
dose of Calebin A on
the expression of TNF-cit and IL-10 in the serum from treated Swiss Albino
mice. No. of
animals=6 per group, P-value: *<0.01; **<0.001 students 't' test.
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[0018] FIG. 7 shows the graphical representation of the effect of multiple
dose of Calebin A on
the expression of IL-6 in the serum from treated Swiss Albino mice. No. of
animals-6 per group,
P-value:*<0.01; **<0.001 students 't' test.
[0019] FIG.8 shows that Calebin A inhibits differentiation and adipogenesis of
3T3-L1
preadipocytes.
[0020] FIGS.9 (A) and (B) respectively show the photographs and graphical
representation of
the effects of diet supplementation on the body weights of experimental groups
of C57BL/6
mice.
[0021] FIGS.10 (A), (B) and (C) show the effect of Calebin A supplementation
on relative
adipose tissue weights in High fat diet (HFD)-fed C57BL/6 mice.
[0022] FIG.11 shows the effect of Calebin A supplementation on leptin
expression in the serum
of C57BL/6 mice.
[0023] FIG.12 shows the effect of Calebin A supplementation on adiponectin
expression in the
serum of C57BL/6 mice.
DETAILED DESCRIPTION OF THE INVENTION
[0024] The present invention discloses the potential of Calebin A to prevent
fat accumulation
during the terminal differentiation of adipocytes (fat cells) and applications
thereof in obesity
management. The present invention elucidates the potential of Calebin A to
favorably modulate
biochemical markers associated with obesity. Notable biomodulatory properties
of Calebin A
include inhibiting leptin production, increasing adiponectin expression and
inhibiting local
(adipocyte) and systemic inflammation caused by pro-inflammatory cytokines
Tumor Necrosis
Factor (TNF-a), Interleukin-6 (IL-6) and Interleukin-1 (IL-113).
[0025] In the most preferred embodiment, the present invention relates to a
method of inhibiting
adipogenesis, said method comprising step of bringing into contact the
adipocytes with an
effective amount of Calebin A. In other words, the present invention relates
to a method of
preventing accumulation of fat during the terminal differentiation of
mammalian adipocytes.
(FIGS. 1 and 8).
[0026] In another preferred embodiment, the present invention relates to a
method of inhibiting
leptin expression in adipocytes, said method comprising step of bringing into
contact the
adipocytes with an effective amount of Calebin A (FIG. 2).
[0027] In another preferred embodiment, the present invention relates to a
method of increasing
expression of adiponectin in adipocytes, said method comprising step of
bringing into contact the
adipocytes with an effective amount of Calebin A (FIG. 3).
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[0028] In another preferred embodiment, the present invention relates to a
method of inhibiting
pro-inflammatory cytokine TNF-a expression in adipocytes, said method
comprising step of
bringing into contact the adipocytes with an effective amount of Calebin A
(FIG. 4).
[0029] In yet another preferred embodiment, the present invention relates to a
method of
inhibiting pro-inflammatory cytokine Interleukin-6 expression in adipocytes,
said method
comprising step of bringing into contact the adipocytes with an effective
amount of Calebin A
(FIG. 5).
[0030] In specific embodiment, the adipocytes referred to herein above are
human adipocytes.
[0031] In yet another preferred embodiment, the present invention relates to a
method of
reducing obesity induced systemic expression of pro-inflammatory cytokines in
mammals, said
method comprising step of administering an effective amount of Calebin A to a
subject in need
thereof In specific embodiments, the pro-inflammatory cytokines referred to
herein in this
paragraph include Tumor Necrosis Factor-a (TNF-a), Interleukin-6 (IL-6) and
Interleukin-113
(IL-113) [FIGS. 6 and 71.
[0032] In yet other most preferred embodiments, the present invention relates
to
1. A method of obesity management in mammals with risk of excessive
accumulation of
body fat, said method comprising the step of dietary oral supplementation of
effective
amounts of Calebin A to said mammals to bring about the effect of adipogenesis
inhibition.
2. Use of Calebin A in the management of obesity in mammals with risk of
excessive
accumulation of body fat, said use comprising the step of dietary oral
supplementation of
effective amounts of Calebin A to said mammals to bring about the effect of
adipogenesis
inhibition.
3. A method of inhibiting adipogenesis in mammals with risk of excessive
accumulation of
body fat, said method comprising the step of dietary oral supplementation of
effective
amounts of Calebin A to said mammals.
4. A method of reducing body weight of obese mammals, said method comprising
step of
orally administering effective amounts of Calebin A to said mammals.
5. Use of Calebin A in a method to reduce body weight in obese mammals, said
use
comprising step of orally administering effective amounts of Calebin A to said
mammals.
6. A method of increasing systemic expression of adiponectin in obese mammals,
said
method comprising step of dietary oral supplementation of effective amounts of
Calebin
A to said mammals.
7. A method for aiding in preventing, delaying the onset of and/or slowing the
progression
of diabetes mellitus Type II in an obese mammal, said method comprising step
of orally
administering therapeutically effective amounts of Calebin A to said mammal to
achieve
an increase in systemic adiponectin expression levels.
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8. A method of treating obesity in mammals, said method comprising the step of
dietary
oral supplementation of effective amounts of Calebin A to said mammals to
bring about
the effects of adipogenesis inhibition, reduction in body weight and increased
systemic
expression of adiponectin.
9. A method of promoting lean body mass in a mammal, said method comprising
the step of
dietary oral supplementation of effective amounts of Calebin A to said mammals
to bring
about effect of increase in lean body mass by shifting the proportion between
lean body
mass and adipose tissue in favor of lean body mass.
[0033] In yet another preferred embodiment, the subject is a mammal.
[0034] In yet another preferred embodiment, the subject is a human.
[0035] The potential therapeutic value of Calebin A as an anti-obesity
molecule may be
understood through specific examples elucidated herein below.
Example I
Acute Oral Toxicity of Calebin A
[0036] Table I lists the parameters studied for Acute Oral Toxicity of Calebin
A.
[0037] Results:
[0038] No mortality was observed up to 2000 mg/kg p.o. in mice up to two weeks
of
observation.
TABLE I
Parameters studied for Acute Oral Toxicity of Calebin A
General Behaviour Dermal
Aggression=Nil Blanching=Nil
Fear¨Nil Hyperaemia=Nil
Passive¨Nil Cyanosis=Nil
General Movement=Normal
General Locomotor Activity=Normal
Central Nervous System General Parameters
Excitation= Nil Muscular Weakness=Nil
Motor Activity¨Nil Salivation¨Nil
Tremors=Nil Pilo Erection=Nil
Clonic Convulsions¨Nil Diarrhea=Nil
Tonic Convulsion=Nil
Respiratory System Reflexes
Respiration Rate= Normal Corneal¨No effect
Respiration Depth=Normal Pinnal=No effect
Autonomic Nervous System Food and Water (Intake and Excretion)
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Motor Activity¨Normal Fecal Output=Normal
Atexia¨Nil Urine Output=Normal
Respiration Rate=Normal
Diarrhea=Nil
Example II
Oil Red-O-Staining of Adipogenic Cultures and Estimation of Leptin,
Adiponectin, TNF-a and
IL-6 by ELISA
[0039] Terminal differentiation of adipocytes is accompanied by the
accumulation of great
amounts of lipids in large cytoplasmic vesicles. A common assay to measure
adipocyte
differentiation in cell culture is with the dye Oil Red-0 (ORO). ORO is a
lipid-soluble bright red
dye which is a reliable indicator of adipocyte differentiation (adipogenesis).
Principle:
[0040] Oil Red-0 (Solvent Red 27, Sudan Red 5B, C.I. 26125, and C26H24N40) is
a
lysochrome (fat-soluble dye) diazo dye used for staining of neutral
triglycerides and lipids on
frozen sections and some lipoproteins on paraffin sections. It has the
appearance of a red powder
with maximum absorption at 518(359) nm. Oil Red-0 is one of the dyes used for
Sudan staining.
Similar dyes include Sudan III, Sudan IV, and Sudan Black B. The staining has
to be performed
on fresh samples, as alcohol fixation removes the lipids. Oil Red 0 largely
replaced Sudan III
and Sudan IV, as it provides much deeper red color and the stains are
therefore much easier to
see.
[0041] Oil Red-0 is an oil soluble dye. Oil soluble dyes exhibit greater
solubility of the dye in
lipid substances in the tissues/cells, than in the usual hydro alcoholic dye
solvents. Hence, it will
deeply stain the cells.
Methodology:
[0042] 3T3-L1 cells approximately 60x104 cells are seeded for 48-72 hrs to get
70-80%
confluence. After 48 hrs 200 IA of AIM (Adipogenesis induction medium) freshly
prepared is
added. 72 hrs later 200 I APM (Adipogenesis progression medium) with the test
compounds in
different concentrations is added to the wells. The cells are incubated for 48
hrs in a humidified
atmosphere (37 C) of 5% CO2 and 95% air. The supernatant is collected and
stored for the
estimation of leptin, adiponectin, IL-6 and TNF-a by ELISA. Cells are fixed by
adding 100 1 of
10% formalin and ORO staining is done. OD is read at 492 nm in microplate
reader. The results
are expressed as IC50 values using Graphpad prism software.
[0043] The percentage of inhibition of adipogenesis is calculated as follows,
% inhibition=C-T/T * 100
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Where C-absorbance of Oil red 0 in differentiating/undifferentiated cells
T-absorbance of Oil Red-0 in sample treated differentiating/undifferentiated
cells. The
estimation of leptin, adiponectin, IL-6 and TNF-a is done according to user's
manual from R&D
Systems.
REFERENCES
[0044] 1. Wu Z, Xie Y, Morrison R F, Bucher NLR, Farmer SR 1998. PPAR 7
induces the
Insulin-dependent Glucose Transporter GLUT4 in the absence of C/EBPo during
the conversion
of 3T3 fibroblasts into adipocytes. J Clin Invest. 101:22-32.
[0045] 2. A pre-adipose 3T3 cell variant highly sensitive to adipogenic
factors & to human
growth hormone. L A Salazar-Olivo, F Castro-Munozledo & W Kuri-Harcuch.
Department of
Cell Biology, Centro de Investigation y de Estudios Avanzados del I.P.N.,
Mexico D.F., Mexico.
Journal of Cell Science, 1995.Vol 108, Issue 5 2101-2107.
[0046] 3. A Nuclear Receptor Atlas: 3T3-L1 Adipogenesis. Mingui Fu, Tingwan
Sun, Angie L.
Bookout, Micheal Downes, Ruth T. Yu, Ronald M. Evans and David J. Mangelsdorf.
Molecular
Endocrinology, 2005. 19 (10): 2437-2450.
[0047] 4. " Expression of a Constitutively Active Akt Ser/Thr Kinase in 3T3-L1
Adipocytes
Stimulates Glucose Uptake and Glucose Transporter 4 Translocation, Aimee D
Kohn et al, J.
Biol. Chem. 1996, 271:31372-31378.
Result:
[0048] FIG. 1 shows percentage adipogenesis inhibition of 32.43%, 38.59% and
35.8%
respectively effected by Calebin A at concentrations of 0.5, 1.0 and 2.0
,g/m1 studied by the Oil-
Red-O-Staining method.
[0049] FIG. 2 shows percentage inhibition of leptin production (34.92%, 41.04%
and 39.48%
respectively) in human adipocytes by Calebin A at concentrations of 0.5, 1.0
and 2.0 tg/ml. The
importance of the effects of Calebin A in inhibiting leptin production in
human adipocytes and
correlation thereof to obesity management stems from the following facts
(Notes on
Pathophysiology of the Endocrine System, Colorado State University).
[0050] Leptin is a protein hormone expressed predominantly in adipocytes. It
has important
effects in regulating body weight, metabolism and reproductive function.
Encoded by the obese
(ob) gene, the protein is approximately -16 kDa in mass. At normal
concentrations, leptin's
biological function is predominantly vested in its effects on hypothalamic
centers of the brain
that control hunger, appetite, regulation of body temperature and energy
metabolism. Thus
leptin, in a non-obese individual could result in weight loss by two important
mechanisms. (i)
Decrease in hunger and food consumption most probably through the inhibition
of neuropeptide
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Y that controls feeding behavior and (ii) increase in energy expenditure
through increased body
temperature, oxygen consumption and loss of adipose tissue mass. However,
excessive secretion
of leptin as in case of obesity or experimental models of induced obesity
leads to disrupted
functions of hypothalamic centers that an obese subject fails to attain
satiations and tends to go
on a over feeding mode. Hence it becomes imperative to bring about effective
reduction of the
over excessive levels of leptin in obesity and Calebin A shows promise in this
area as indicated
in FIG. 2.
[0051] FIG. 3 shows percentage enhancement of adiponectin expression (27.12%,
34.06% and
32.8% respectively) in human adipocytes by Calebin A at concentrations of 0.5,
1.0 and 2.0
1.tg/m1. Adiponectin is a cytokine produced almost exclusively by adipocytes
and is expressed in
very high levels by lean and healthy individuals. Obese individuals on the
other hand express
reduced levels of this adipokine and are prone to coronary heart disease
(CAD), diabetes mellitus
and hypertension.
REFERENCES
[0052] 1. Tamar. R. Aprahamian and Flora Sam, "Adiponectin in Cardiovascular
Inflammation
and Obesity, Int J. Inflam. 2011; 2011: 376909;
[0053] 2. Hotta K, Funahashi T, Arita Y, et al. Plasma concentrations of a
novel, adipose-
specific protein, adiponectin, in type 2 diabetic patients. Arteriosclerosis,
Thrombosis and
Vascular Biology. 2000; 20(6):1595-1599;
[0054] 3. Iwashima Y, Katsuya T, Ishikawa K, et al. Hypoadiponectinemia is an
independent
risk factor for hypertension. Hypertension. 2004; 43(6):1318-1323;
[0055] 4. Kumada M, Kihara S, Sumitsuji S, et al. Association of
hypoadiponectinemia with
coronary artery disease in men. Arteriosclerosis, Thrombosis and Vascular
Biology. 2003;
23(1):85-89 and
[0056] 5. Lindsay R S, Funahashi T, Hanson R L, et al. Adiponectin and
development of type 2
diabetes in the Pima Indian population. The Lancet. 2002; 360(9326):57-58.
[0057] Calebin A is shown (FIG. 3) to effectively increase levels of
adiponectin in human
adipocytes and thus show promise in the area of obesity management.
[0058] FIGS. 4 and 5 show the percentage inhibition of TNF-a (36.03%, 40.81%
and 45.47%
respectively) and IL-6 (21.31%, 32.37% and 31.7% respectively) by Calebin A at
concentrations
of 0.5, 1.0 and 2.0 ilg/ml. Bastard JP et al, "Recent Advances in the
relationship between obesity,
inflammation and insulin resistance", Eur Cytokine Netw. 2006 March; 17(1):4-
12 cite that
obesity is associated with low-grade inflammation of the white adipose tissue
(WAT). The
authors also remark that in obesity, WAT is characterized by increased
expression of pro-
inflammatory molecules like TNF-a and IL-6 which not only exert effects on WAT
but also on
8
CA 02857543 2014-07-21
other systemic organs of the body. FIGS. 4 and 5 demonstrate that Calebin A is
effective in
reducing TNF-a and IL-6 expression in adipocytes and would be a useful agent
to modulate
effects of local and systemic inflammation in obesity.
Example III
Modulation of Systemic Inflammation by Calebin A
[0058] The present inventors also adduce extra evidence to support the ability
of Calebin A to
suppress intracellular TNF and extracellular IL-10 in murine neutrophil
systems (Table II, Table
III). Neutrophils are isolated by histopaque gradient method tested for their
ability to produce in
vitro TNF-a following stimulation with Lipopolysaccharide (LPS). The cells
were incubated
with phycoerythrin (PE)-labeled anti-mouse TNF-a. in the dark, and after being
washed with
sterile PBS, samples were resuspended in PBS (pH 7.4) and acquired directly on
the flow
cytometer (BDLSR; Becton Dickinson). A fluorescence trigger was set on the PE
(FL1)
parameter of the gated neutrophil populations (10,000 events). Rolipram at 100
ps/m1 was used
as standard inhibitor of TNF-a in this study. Fluorescence compensation, data
analysis, and data
presentation were performed using Cell Quest Pro software (Becton Dickinson).
REFERENCES
[0060] 1. Clara, B., R. C. Arancha, G. M. Andre's, P. Atanasio, A. Julia, and
O. Alberto. 2003. A
new method for detecting TNF-a-secreting cells using direct immunofluorescence
surface
membrane stainings. J. Immuno. Methods 264:77-87.
[0061] 2. Khurshid A. Bhat, Bhahwal A. Shah, Kuldeep K. Gupta, Anjali Pandey,
Sarang Bani,
Subhash C. Taneja. Semi-synthetic analogs of pinitol as potential inhibitors
of TNF-a cytokine
expression in human neutrophils. Bioorganic & Medicinal Chemistry Letters 19
2009, 1939-
1943.
TABLE II
Serial No Sample Concentration Expression of % Activity
( ,g/m1) TNF-a
Mean + S E
1 LPS Control 2.62 0.01
2 Calebin A 0.5 1.87 0.04* 28.62%1
3 Calebin A 1.0 1.70 0.02** 35.11%1
4 Calebin A 2.0 1.59 0.05** 39.31%1
Rolipram 100 0.73 0.09** 72.13%1
%1: indicates suppression of TNF-a expression
No. of observations = 3
9
CA 02857543 2014-07-21
P-value: *<0.01; **<0.001 students`e test
TABLE III
Samples Treatment Concentration (pg/ml) = % Activity
LPS Control 51.80 2.18
Calebin A
0.5 tg/m1 41.24 1.16* 20.38%1
1.0 ug/m1 39.26 2.52* 24.20%1
2.0 ug/m1 37.16 2.11** 28.26%1
Rolipram Standard
100 ug/m1 22.52 1.60** 56.52%1
%1: indicates suppression of IL-1 (3 expression
No. of observations = 3
P-value: *<0.01; **í0001 students`f test
[0062] The present inventors also adduce study data on the ability of Calebin
A to reduce
expression of Extracellular TNF-a, IL-1 beta [FIG. 6] and IL-6 [FIG. 7] in
serum from treated
mice (in-vivo models). Swiss albino male mice aged 6-8 weeks were maintained
at 22 2 C.
under 12/12 h light dark cycle. Mice received oral treatment of test drugs at
graded doses (w/v)
for 6 days, followed by intravenous injection of 1 mg/kg of LPS according to
the method
described by Brieva A, Guerrero A, Alonso-Lebrero J L and Pivel J P. 2001.
Immunoferon, a
glycoconjugate of natural origin, inhibits LPS-induced TNF-a production and
inflammatory
responses. International Immunopharmacology 1, 1979-1987. Six mice were
employed in each
group and experiments were performed in triplicates. TNF-a, IL-1 beta and IL-6
production was
evaluated by a commercial ELISA kits (R&D Systems) in serum from treated mice,
90 min after
LPS injection. Rolipram at 30 mg/kg was used as standard drug.
[0063] FIGS. 6 and 7 demonstrate that Calebin A is effective in reducing TNF-
a, IL-1 beta and
IL-6 thus indicating that the compound is a useful agent to modulate effects
of local and systemic
inflammation in obesity.
Example IV
Adipogenesis Inhibition by Calebin A
[0064] Cell culture and adipocyte differentiation
[0065] Mouse 3T3-L1 pre-adipocytes purchased from the American Type Culture
Collection
(Rockville, MD) were grown in Dulbecco's Modified Eagle's Medium (DMEM)
supplemented
CA 02857543 2014-07-21
with 2 mM glutamine (GIBCO BRL), 1% penicillin/streptomycin (10000 units of
penicillin/mL
and 10 mg streptomycin/mL) and 10% fetal calf serum (FCS) at 37 C under a
humidified 5%
CO2 atmosphere. Briefly, cells were seeded into 24-well (2 x104/mL) or 10 cm
dish with DMEM
containing 10% fetal bovine serum (FBS) to full confluence. Two days after
confluence (defined
as day 0), cells were incubated in differentiation medium (MDI) containing 1.7
M insulin, 0.5
mM 3-isobutylmethylxanthine (IBMX) and 12.7 M dexamethasone (DEX) in DMEM
containing 10% FBS for 2 days. The medium was then replaced by DMEM containing
10% FBS
and insulin (1.7 M) with or without Calebin A which was replaced every 2
days. The final
concentrations of dimethyl sulfoxide (DMSO) in the culture medium were <
0.05%. The cells
were harvested after 8 days (at day 10) for Oil Red 0 staining.
[0066] Oil Red-0 staining
[0067] At the end of differentiation, cells were washed twice with phosphate-
buffered saline
(PBS), fixed with 10% formalin for 60 min, stained with 0.5% Oil Red 0 in
isopropanol for 1 h
at room temperature. Excess Oil Red-0 dye was washed twice with distilled
water and then
dried. The stained lipid droplets within cells were visualized by light
microscope and
photographed with a digital camera at 100x magnification. To quantify lipid
accumulation, the
stained lipid droplets were dissolved in isopropanol and the absorbance was
measured at 520 nm.
[0068] FIG.8 shows that Calebin A inhibits differentiation and adipogenesis of
3T3-L1
preadipocytes. Differentiation of 3T3-L1 preadipocytes stained with Oil Red 0
and
photographed (upper and middle). 3T3-L1 preadipocytes were incubated with MDI
(DMEM
with IBMX, DEX, and insulin) for 2 days and then replaced with DMEM containing
insulin with
or without Calebin A (0, 5, 10, 15, 20, 25 and 30 M), respectively, for 8
days. Lipid content
was extracted from Oil Red 0 stained cells by 2-propanol and quantified by
spectrophotometric
analysis at 520 nm.
[0069] Animal Experiment-STUDY I
[0070] Male C57BL/6J mice at 5 weeks of age were purchased from the BioLASCO
Experimental Animal Center (Taiwan Co., Ltd., Taipei, Taiwan) and housed in a
controlled
atmosphere (25 1 C at 50% relative humidity) and with a 12-h light/12-h dark
cycle. After 1
week of acclimation, animals were randomly distributed into four groups of 8
animals each as
follows: normal diet (ND, 15% energy as fat), high fat diet (HFD; 40% energy
as fat), and HFD
supplemented with 0.25% or 1% Calebin A (2.5 g or 10 g Calebin A /kg diet),
respectively, for
12 weeks (Table V). The experimental diets were modified from the Purina 5001
diet (LabDiet,
PMI Nutrition International) and the composition is listed in Table IV.
Animals had free access
to food and water at all times. Food cups were replenished with fresh diet
daily. The diet intake
of animals was monitored every day and the body weight was recorded weekly.
All animal
experimental protocol used in this study was approved by Institutional Animal
Care and Use
Committee of the National Kaohsiung Marine University (IACUC, NKMU). At the
end of the
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CA 02857543 2014-07-21
study, all animals were fasted overnight and sacrificed by CO2 asphyxiation.
Blood samples were
collected from the heart for biochemical analysis. Liver, spleen, kidney and
fat pads
(perigonadal, retroperitoneal and mesenteric fat) were immediately removed,
weighed (TABLE
VI) and photographed. FIG.9 (A) shows the representative photographs of each
group at the end
of week 12. Body weight was monitored weekly, and the average body weight of
each group was
expressed as the mean SE. Statistical analysis was done by Student's t test.
(*) P < 0.01,
compared with ND group; (#) P < 0.01, compared with HFD group. ND, normal diet
and HFD,
high-fat diet (FIG.9 (B)).
[0071] FIGS. 10 (A), (B) and (C) show the photographs of perigonadal fat,
retroperitoneal and
mesenteric fat and also the graphical representation of the % relative
perigonadal, retroperitoneal
and mesenteric fat weights.
TABLE IV
Composition of Experimental diets
Composition/Ingredient ND HFD HFD+0.25`)/0 HFD+1
Calebin A Calebin A
Macronutrient Composition
Protein % of Energy 20.0 14.0 14.0 14.0
Carbohydrate % of Energy 65.0 46.0 46.0 46.0
Fat % of Energy 15.0 40.0 40.0 40.0
Ingredient g/kg
Lard 150.0 150.0 150.0
.
Soybean oil 15.0 15.0 15.0
Cholesterol- 1.0 1.0 1.0
Calebin A. - 2.5 10.0
Energy Content kJ/g 35.8 50.7 50.7 50.7
TABLE V
Effect of Calebin A on Body Weight Gain and Food Intake in Mice Fed HFDa
Weight ND HFD HFD + 0.25% HFD
+ 1%
Parameters Calebin A
Calebin A
Initial wt (g) 21.06 0.99 22.13 1.13 22.27 0.49
21.88 0.91
Final wt (g) 28.05 1.08 38.63 3.92*** 35.28
2.38 31.99 2.504
wt gain (g) 6.99 0.48 16.50 2.90* 13.02 2.52
10.12 2.48"
Food intake
4.35 0.63 3.77 0.52 3.57 0.47 3.57 0.40
(g/mouse/day)
12
CA 02857543 2014-07-21
[0072] 'Mice were fed diet for 12 weeks as described under Materials and
Methods, and the body
weights were monitored twice weekly. The average body weight of each group is
expressed as
the mean SE (n = 8 per group), and statistical analysis was done by
Student's t test. ND,
normal diet; HFD, high-fat diet. *, P< 0.01, and ***, P< 0.0001 compared with
ND group. #, P <
0.01,and ##P< 0.001 compared with HFD group.
TABLE VI
Effects of Calebin A on relative organ weights in mice fed with HFDa
Organ ND HFD HFD + 0.25% HFD
+ 1%
Calebin A
Calebin A
liver (%) 3.86 0.36 4.74 0.66 4.05 0.28 4.00
0.25
Kidney (%) 1.30 0.07 1.29 0.23 1.22 0.12 1.30
0.15
Spleen (%) 0.20 0.07 0.22 0.09 0.16 0.04 0.15
0.04
[0073] a Mice were fed HFD supplemented with or without Calebin A (0.25 and
1%) for 12
weeks. Mice of each group were sacrificed at the end of week 12; the liver,
spleen, and kidney
were removed, photographed, weighed, and recorded. Data are presented as the
mean SE (n =
8 per group). The relative organ weight is expressed as a percentage of body
weight (liver
weight/body weight x 100). ND, normal diet and HFD, high-fat diet.
[0074] Animal Experiment-STUDY 2-DEMONSTRATION OF BODY WEIGHT LOSS IN
OBESE MAMMALIAN MODELS
[0075] Test System details
Animal species Mice
Strain C57
Source In-House
Body weight range Males ¨ 22.1 - 25.8 g
Females ¨ 20.3 ¨ 23.9 g
Age at treatment 8-10 weeks
Number of Groups 5 groups ( One Control, One High fat
diet
control and three treatment groups)
Number of animals / group Each group consists of 10 animals (5
Males + 5
Females). Female animals used were
nulliparous and non-pregnant
Total number of animals 50
Identification Cage cards and individual animal ear
notching
method
[0076] Test Performance
13
CA 02857543 2014-07-21
[0077] A. Husbandry
[0078] a. Conditions: The animals were housed under standard laboratory
conditions, air-
conditioned with adequate fresh air supply (Air changes 12-15 per hour), room
temperature 22
3oC, relative humidity 30-70 %, with 12 hours light and 12 hours dark cycle.
The temperature
and relative humidity are recorded once daily.
[0079] b. Housing: Individual animals were housed in a standard polypropylene
cage (Size: L
290 x B 140 x H 140 mm) with stainless steel mesh top grill having facilities
for holding pellet
feed and drinking water in water bottle fitted with stainless steel sipper
tube. Clean sterilized
paddy husk is provided as bedding material.
[0080] c. Acclimatization: The animals were acclimatized for 5 days to
laboratory conditions and
were observed for clinical signs daily.
[0081] d. Diet: The animals were fed ad libitum with AMRUT Laboratory Animal
Feed
manufactured by Pranav Agro Industries Limited, Sangli, Maharastra throughout
the
acclimatization.Open Source Diet D12450B diet (with 10 kcal% Fat) and Open
Source Diet
D12492 High fat diet (with 60 kcal% Fat) manufactured by Research Diet Inc,
USA procured
from Indus Marketing, Hyderabad, Andhra Pradesh, INDIA was used for induction
of obesity
and the main study.
[0082] e. Water: Clean drinking water was provided ad libitum throughout the
acclimatization
and obesity induction period. Deep bore-well water passed through reverse
osmosis unit was
provided in plastic water bottles with stainless steel sipper tubes.
[0083] B. Grouping
[0084] Grouping of animals was done on the last day of acclimatization by body
weight
randomization and stratification method. Grouping of animals was done such
that body weight
variation of animals used does not exceed 20% of the mean body weight of each
group.
[0085] C. Study Design
[0086] The animals were divided into 5 groups viz., Group 1, 2, 3, 4 and 5
consisting of 10
animals (5 male and 5 female) each. The group details, doses and number/sex of
animals per
group are presented in Table VII.
TABLE VII
Dose Number. of
Group TreatmentAnimal numbers
Animals
(mg/kg
14
CA 02857543 2014-07-21
Bwt) Male Female Male Female
G1 Control (with 10 kcal% Fat) - 5 5 1-5
26-30
High fat diet Control
G2 - 5 5 6-10 31-35
(with 60 kcal /o Fat)
Calebin A 5 mg/kg +
03 5 5 5 11-15 36-40
High fat diet (with 60 kcal% Fat)
Calebin A 10 mg/kg +
G4 10 5 5 16-20 41-45
High fat diet (with 60 kcal% Fat)
Calebin A 20 mg/kg +
G5 20 5 5 21-25 46-50
High fat diet (with 60 kcal% Fat)
Total: 25 25 -
-
Total number of animals: 50
[0087] D. Animal treatment
[0088] a. Dose Volume: Dose volume/animal=10 ml/kg body weight for all animals
throughout
the study period
[0089] b. Obesity induction: The G1 Control group animals were fed with normal
control diet
feed D12450B containing 10 kcal % fat and the G2 to G5 group animals were fed
with high fat
diet feed D12492 containing 60 kcal % fat during the induction of obesity and
during main
study.
[0090] c. Main Study: The main study was started after the induction of
obesity. The 3 doses of
Calebin A was administered to animals from Day 29 daily consecutively for a
period of 28 days.
The feeding of diets continued in the main study in a similar way as performed
in induction of
obesity. The G1 Control and G2 High fat diet control group animals were
administered with
0.5% CMC (Carboxy Methyl Cellulose) while other group animals received test
item from Day
29 to Day 56 of the study period. The dose volume of administration was
maintained according
to the weekly body weight of individual animals. The total duration of the
study was 61 days (5
days Acclimatization period + 28 days Induction of obesity + 28 days Main
study).
CA 02857543 2014-07-21
[0091] Statistical analysis: The raw data obtained from the present study were
subjected to
computer statistical processing. The computer printout of the data (in the
form of appendix) was
verified with the original raw data. After verification, the data was
subjected to One-way
ANOVA (Analysis of Variance) with Dunnett's post test for the data on body
weights,
hematology and clinical chemistry parameters, organ weights using GraphPad
Prism version
5.01, GraphPad Software. All analyses and comparisons was evaluated at the 95%
level of
confidence (P<0.05), indicated by the designated by the superscripts of a
where G1 is compared
to G3, G4, G5, and G6 and b where G2 is compared to G3, G4, G5, and G6
throughout the
report as stated below: *: Statistically significant (P(0.05) wherever
applicable.
The data were subjected to One way ¨ ANOVA statistical analysis by comparing
the following:
G1 group {Control group (with 10 kcal% Fat)} to G3 group { Calebin A 5 mg/kg +
High fat diet
(with 60 kcal% Fat)}, G4 group { Calebin A -10 mg/kg + High fat diet (with 60
kcal% Fat)} and
G5 group { Calebin A 20 mg/kg + High fat diet (with 60 kcal% Fat)} as
represented below:
G3 group
Calebin A 5 mg/kg +
High fat diet (with 60 kcal% Fat)
G1 group
G4 group
Control group
Calebin A -10 mg/kg + High fat
(with 10 kcal% Fat) diet (with 60 kcal% Fat)
G5 group
Calebin A 20 mg/kg + High fat
diet (with 60 kcal% Fat)
G2 - High fat diet Control (with 60 kcal% Fat) to G3 group { Calebin A 5 mg/kg
+ High fat diet
(with 60 kcal% Fat)}, G4 group { Calebin A -10 mg/kg + High fat diet (with 60
kcal% Fat)} and
G5 group { Calebin A 20 mg/kg + High fat diet (with 60 kcal% Fat)} as
represented below:
G2 group G3 group
High fat diet Control Calebin A 5 mg/kg +
16
CA 02857543 2014-07-21
(with 60 kcal% Fat) High fat diet (with 60 kcal% Fat)
G4 group
Calebin A -10 mg/kg + High fat diet
(with 60 kcal% Fat)
G5 group
Calebin A 20 mg/kg + High fat diet
(with 60 kcal% Fat)
[0092] Results
[0093] Body Weight: Individual animal body weights were recorded on the day of
receipt on
Day 1 and weekly ( 1 day) thereafter during the study period.
[0094] The summary of weekly body weight of male and female animals is
presented in Tables
VIII (a) / VIII (b) and IX (a) / IX (b) respectively.
TABLE VIII (a)
BODY WEIGHT (grams)
GROUP TREATMENT DAYS
1 8 15 22
G1 a Control 25.30 1.49 26.12 1.83 26.60 2.03
26.86 2.01
(with 10 kcal%
Fat)
G2b High fat diet 25.20 0.89 26.30 1.44 28.22 1.10
30.10 1.44
Control
(with 60 kcal%
Fat)
G3 Calebin A 25.02 1.45 25.78 1.14 27.68 0.95
29.98**a
mg/kg 1.03
+
High fat diet
G4 Calebin A 25.28 0.88 25.82 0.91 27.58
0.65 29.92**a
mg/kg 0.97
+
High fat diet
(with 60 kcal%
Fat)
G5 Calebin A 25.88 1.25 26.66 1.73 28.94*a
30.64**a
mg/kg 1.59 1.16
+
17
CA 02857543 2014-07-21
High fat diet
(with 60 kcal%
Fat)
n=5; Values are Mean + Standard Deviation; * - Significant difference, P>0.05
TABLE VIII (b)
BODY WEIGHT (grams)
GROUP TREATMENT DAYS
29 36 43 50
56
Gla Control 27.26 28.42 28.64 28.90
29.26
(with 10 kcal% Fat) +2.45 3.09 3.16 +3.35
3.49
G2b High fat diet 31.72 32.50 33.90
34.94 35.96
Control 1.66 +1.47 +1.52 +1.19
0.90
(with 60 kcal% Fat)
G3 Calebin A 30.92**a 31.70*a 30.84***b 30.04***b
29.98***b
mg/kg +1.37 +1.09 +1.49 2.13 1.93
+
High fat diet
G4 Calebin A 31.02**a 31.06*b 29.80***b 28.98***b
28.62***b
mg/kg 0.70 +1.55 +1.48 +1.10 +1.18
+
High fat diet
(with 60 kcal% Fat)
G5 Calebin A 31.86***a 31.48*b 29.12***b 27.74***b
27.54***b
mg/kg +1.14 +1.21 +1.34 1.28 1.87
+
High fat diet
(with 60 kcal% Fat)
n=5; Values are Mean Standard Deviation; * - Significant difference, P>0.05
TABLE IX (a)
BODY WEIGHT (grams)
GROUP TREATMENT DAYS
1 8 15
22
Gla Control 23.18 + 0.91 24.12 1.21 24.68 + 0.90 25.36 +
0.80
(with 10 kcal% Fat)
G2b High fat diet 22.86 1.05 23.82 + 0.94 25.18 1.00
26.48 1 0.55
Control
(with 60 kcal% Fat)
G3 Calebin A 22.36 0.65 23.18 0.79 24.36 0.87 25.66 0.48
5 mg/kg
+
High fat diet
G4 Calebin A 23.16 0.99 24.16 1.29
25.62 1.26 26.76 1.27
18
CA 02857543 2014-07-21
mg/kg
+
High fat diet
(with 60 kcal% Fat)
G5 Calebin A 23.52 1.10 24.66 1.24 26.26* a
27.26* a
mg/kg 1.05 1.25
+
High fat diet
(with 60 kcal% Fat)
n=5; Values are Mean Standard Deviation; * - Significant difference, P>0.05
TABLE IX (b)
BODY WEIGHT (grams)
GROUP TREATMENT _ DAYS
29 36 43 50 56
G1 a Control 25.68 26.28 26.88 27.52 28.08
(with 10 kcal% Fat) 0.79 0.86 0.83 0.70 0.73
G2b High fat diet 28.10 29.14 30.26 32.00 33.16
Control 0.42 0.36 0.63 0.54 0.36
(with 60 kcal% Fat)
G3 Calebin A 27.40* a 28.18**a 28.46* 28.20***b
28.12***b
5 mg/kg 0.60 0.68 a **b 0.45 0.70
,
+ 0.71
High fat diet
G4 Calebin A 28.64***a 28.16* 27.54***b 27.04***b
10 mg/kg 28.46*** a 0.62 a ***b 0.38 0.47
+ 1.10 0.36
High fat diet
(with 60 kcal% Fat)
G5 Calebin A 28.74***a 28.40**a 27.80***b 27.44***b 27.10***b
20 mg/kg 0.93 1.03 0.99 0.95 0.93
+
High fat diet
(with 60 kcal% Fat)
n=5; Values are Mean Standard Deviation; * - Significant difference, P>0.05
[0095] In male animals, there was statistically significant increase in mean
weekly body weight
values on Day 15 in G5 group { Calebin A - 20 mg/kg + High fat diet (with 60
kcal% Fat)}
compared to G1 group {Control group (with 10 kcal% Fat)}. These changes were
considered to
be related to difference in fat content of the feed.
[0096] In male animals, there was statistically significant increase in mean
weekly body weight
values on Day 22 and Day 29 in G3 group { Calebin A - 5 mg/kg + High fat diet
(with 60 kcal%
Fat)}, G4 group { Calebin A - 10 mg/kg + High fat diet (with 60 kcal% Fat)},
G5 group {
Calebin A - 20 mg/kg + High fat diet (with 60 kcal% Fat)} compared to G1 group
{Control
19
CA 02857543 2014-07-21
group (with 10 kcal% Fat)). These changes were considered to be due to
difference in fat content
of the feed.
[0097] In male animals, there was statistical significant increase in mean
weekly body weight
values on Day 36 in G3 group { Calebin A - 5 mg/kg + High fat diet (with 60
kcal% Fat)}
compared to G1 group {Control group (with 10 kcal% Fat)}. These changes were
considered to
be due to difference in fat content of the feed.
[0098] In male animals, there was statistical significant decrease in mean
weekly body weight
values on Day 36 in G3 group { Calebin A - 5 mg/kg + High fat diet (with 60
kcal% Fat)}, G4
group { Calebin A - 10 mg/kg + High fat diet (with 60 kcal% Fat)} compared to
G2 group {High
fat diet Control (with 60 kcal% Fat)}. These changes were considered to be
related to the effect
of administration of test item Calebin A.
[0099] In male animals, there was statistical significant decrease in mean
weekly body weight
values on Day 43, 50 and Day 56 in G3 group { Calebin A - 5 mg/kg + High fat
diet (with 60
kcal% Fat)}, G4 group { Calebin A - 10 mg/kg + High fat diet (with 60 kcal%
Fat)}, G5 group {
Calebin A - 20 mg/kg + High fat diet (with 60 kcal% Fat)} compared to G2 group
{High fat diet
Control (with 60 kcal% Fat)}. These changes were considered to be due to
administration of test
item Calebin A.
[0100] In female animals, there was statistically significant increase in mean
weekly body weight
values on Day 15 and Day 22 in G5 group { Calebin A - 20 mg/kg + High fat diet
(with 60
kcal% Fat)} compared to G1 group {Control group (with 10 kcal% Fat)}. These
changes were
considered to be due to difference in fat content of the feed.
[0101] In female animals, there was statistically significant increase in mean
weekly body weight
values on Day 29 and Day 36 in G3 group { Calebin A - 5 mg/kg + High fat diet
(with 60 kcal%
Fat)}, G4 group { Calebin A - 10 mg/kg + High fat diet (with 60 kcal% Fat)},
G5 group {
Calebin A - 20 mg/kg + High fat diet (with 60 kcal% Fat)} compared to G1 group
{Control
group (with 10 kcal% Fat)}. These changes were considered to be due to
difference in fat content
of the feed.
[0102] In female animals, there was statistically significant increase in mean
weekly body weight
values on Day 43 in G3 group { Calebin A - 5 mg/kg + High fat diet (with 60
kcal% Fat)}, G4
group { Calebin A - 10 mg/kg + High fat diet (with 60 kcal% Fat)}, compared to
G1 group
{Control group (with 10 kcal% Fat)}. These changes were considered to be due
to difference in
fat content of the feed.
[0103] In female animals, there was statistically significant decrease in mean
weekly body
weight values on Day 43, 50 and Day 56 in G3 group { Calebin A - 5 mg/kg +
High fat diet
(with 60 kcal% Fat)}, G4 group { Calebin A - 10 mg/kg + High fat diet (with 60
kcal% Fat)}, G5
group { Calebin A - 20 mg/kg + High fat diet (with 60 kcal% Fat)} compared to
G2 group
CA 02857543 2014-07-21
{High fat diet Control(with 60 kcal% Fat)} . These changes were considered to
be due to
administration of test item Calebin A.
[0104] It could thus be concluded that Calebin A had an effect in decreasing
the body weights of
high fat diet induced obese male and female C57 animals at test concentrations
of 5, 10 and 20
mg/kg body weight.
[0105] Further, after completion of the study period (Day 57) the animals were
humanely
sacrificed by exposure to excess CO2 in gas chamber and organ weights were
noted. The brain,
thymus, liver, adrenals, kidneys (paired), spleen, heart and ovaries/testes
(paired) from all
animals were trimmed off any adherent tissue, as appropriate and weighed wet
as soon as
possible to avoid drying. While in general, there was no statistically
significant difference in
organ weights in males and females, organ specific improvements in weight,
example for liver in
the male group was observable (See TABLE X). This result corroborates with
those in TABLE
VI for liver. It may be noted that Behnke, A. R. 1953. Lean body mass. A.M.A.
Arch. Int. Med.
91, 585 indicates liver as an index of lean body mass promotion and
H.F.Kraybill et al, J ANIM
SCI 1954, 13:548-555 indicate that other visceral organs may also be equally
predictive of lean
body mass promotion. It is quite possible that statistical significance in
terms of sustainable
increase in organ weights without indications of toxicity may be achieved with
a larger sample
size (more number of tested animals) over extended testing periods. The
results of Table VI and
Table X may be interpreted as a preliminary indication of Calebin A's
potential to not only
inhibit adipogenesis and reduce body weight, but also promote to the lean body
mass
TABLE X
GROUP TREATMENT
LIVER WEIGHT (g)
Gla Control (with 10 kcal% Fat) 1.47
0.36
G2b High fat diet Control (with 60 kcal% Fat) 1.60
0.35
G3 Calebin A 5 mg/kg + High fat diet (with 60 kcal%
Fat) 1.41
0.17
G4 Calebin A 10 mg/kg + High fat diet (with 60 kcal%
Fat) 1.25
0.17
G5 Calebin A 20 mg/kg + High fat diet (with 60 kcal% 1.79
0.19
Fat)
[0106] Further, on completion of the study period blood samples were collected
from all the
animals in tubes containing potassium ethylene di-amide tetra acetic acid (K2-
EDTA)
anticoagulant for estimation of systemic expression of leptin and adiponectin.
Blood samples
were collected humanely from retro-orbital plexus puncture method under mild
ether anesthesia
with the help of a fine capillary tube. The blood samples collected in tubes
without anticoagulant
were centrifuged at 3000 rpm for 10 minutes to obtain serum which was
subjected to ELISA
technique for the estimation of leptin and adiponectin. The import of leptin
and adiponectin
21
CA 02857543 2014-07-21
expression as biomarkers in obesity has been discussed well in Paragraphs 0050
and 0051.
Calebin A showed an insignificant effect on the inhibition of leptin
expression in the serum of
obese animals (Fig.11) and showed a significant effect in enhancing the
adiponectin expression
in the serum levels of obese animals (Fig.12). Low systemic adiponectin levels
have been cited
as predictive factors in the progression of disease states like Type II
Diabetes mellitus
(Chamukuttan Snehalatha et al, "Plasma Adiponectin Is an Independent Predictor
of Type 2
Diabetes in Asian Indians", Diabetes Care December 2003 vol. 26 no. 12 3226-
3229). The
ability of Calebin A to significantly enhance the levels of systemic
adiponectin in mammalian
models of obesity indicates its ability to aid in preventing the onset of
diabetes mellitus Type II
in said mammals.
[0107] The scope of the claims should not be limited by the preferred
embodiments set forth in
the examples, but should be given the broadest interpretation consistent with
the description as a
whole.
22
