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The effect of Cissus
quadrangularis (CQR-300)
and a Cissus
formulation (CORE) on
obesity and
obesity-induced
oxidative
stress
Abstract
Aim: Obesity is
generally linked to
complications in lipid
metabolism and oxidative
stress. The aim
of this study was to
compare the effect of a
proprietary extract of
Cissus quadrangularis
(CQR-300)
to that of a proprietary
formulation containing
CQR-300 (CORE) on
weight, blood lipids,
and
oxidative stress in
overweight and obese
people.
Methods: The first part
of the study
investigated the in
vitro antioxidant
properties of CQR-300
and CORE using 3
different methods, while
the second part of the
study was a double-blind
placebo
controlled design,
involving initially 168
overweight and obese
persons (38.7% males;
61.3%
females; ages 19–54), of
whom 153 completed the
study. All participants
received two daily doses
of CQR-300, CORE, or
placebo and were
encouraged to maintain
their normal levels of
physical
activity. Anthropometric
measurements and blood
sampling were done at
the beginning and end of
the study period.
Results: CQR-300 as well
as CORE exhibited
antioxidant properties
in vitro. They also
acted as in
vivo antioxidants,
bringing about
significant (p < 0.001)
reductions in plasma
TBARS and carbonyls.
Both CQR-300 and CORE
also brought about
significant reductions
in weight, body fat,
total
cholesterol,
LDL-cholesterol,
triglycerides, and
fasting blood glucose
levels over the
respective
study periods. These
changes were accompanied
by a significant
increase in
HDL-cholesterol levels,
plasma 5-HT, and
creatinine.
Conclusion: CQR-300 (300
mg daily) and CORE (1028
mg daily) brought about
significant
reductions in weight and
blood glucose levels,
while decreasing serum
lipids thus improving
cardiovascular risk
factors. The increase in
plasma 5-HT and
creatinine for both
groups
hypothesizes a mechanism
of controlling appetite
and promoting the
increase of lean muscle
mass
by Cissus
quadrangularis, thereby
supporting the clinical
data for weight loss and
improving
cardiovascular health.
Background
The incidence of obesity
in adults as well as
children is on
an increase globally.
Once considered a
problem of developed
countries, this global
epidemic also affects
developing
countries. Coupled to
this epidemic are
obesityrelated
complications such as
cardiovascular disease,
stroke, depression, and
Type-2 diabetes, which
are spreading
rapidly across poor and
middle-income countries,
where infectious
diseases and
malnutrition have
previously
overshadowed such
illnesses [1]. Obesity
is a principal
causative factor of
metabolic syndrome
[2-6]. The
coexistence of these
metabolic syndromes
(hyperglycemia,
dyslipidemia, and
hypertension in the same
individual)
is a growing medical
problem in
industrialized
countries [6-9]. It has
been reported that
obesity may
induce systemic
oxidative stress and
that increased oxidative
stress in accumulated
fat is associated with
dysregulation
of adipocytokines and
development of metabolic
syndrome [6]. Oxidative
stress has been shown to
be
involved in the process
of atherogenesis [10],
ischemic
heart disease [11],
obesity [6], metabolic
syndrome or
syndrome X, diabetes
[12,13], as well as in
immunodeficiency
[14]. The stability of
tissues against
oxidative stress
is however enhanced by
antioxidant compounds,
which
could be present in the
diet [15]. Antioxidants
have also
been shown to be very
effective in inhibiting
the oxidation
of LDL and scavenging of
free radicals and
reactive oxygen
species in vitro
[16-20]. These compounds
are present in
fruits and vegetables,
which contain natural
antioxidants,
and are known to delay
the onset of
atherogenesis.
Cissus quadrangularis
(Linn) has been used by
common
folk in India for
promoting the fracture
healing process. It
has been prescribed in
Ayurveda as an
alterative,
anthelmintic, dyspeptic,
digestive, tonic,
analgesic in eye
and ear diseases, and in
the treatment of
irregular menstruation
and asthma. In Cameroon,
the whole plant is
used in oral
re-hydration, while the
leaf, stem, and root
extracts of this plant
are important in the
management of
various ailments.
Earlier works on Cissus
quadrangularis
report its effectiveness
on the management of
obesity and
complications associated
with metabolic syndrome
[21],
as well as its
antioxidant and free
radical scavenging
activity
in vitro [22,23].
Various formulations now
contain
extracts of Cissus
quadrangularis in
combination with other
compounds, used for the
purpose of management of
overweight
and obesity, as well as
complications resulting
from these conditions,
notably metabolic
syndrome (syndrome
X). Phytochemical
analyses of Cissus
quadrangularis
revealed high contents
of ascorbic acid,
carotene, anabolic
steroidal substances,
and calcium. The stem
contains two
asymmetric tetracyclic
triterpenoids, and two
steroidal
principles. The presence
of β-sitosterol,
δ-amyrin, δ-amyrone,
and flavanoids
(quercetin) has also
been reported
[24,25], all these
components having
potentially different
metabolic and
physiologic effects.
Although different uses
of Cissus quadrangularis
have been
investigated, [26,27]
the antioxidant
potential of various
Cissus quadrangularis
formulations have not
been evaluated
in the modulation of
obesity-induced
oxidative
stress. It was for this
reason that the present
study was
designed, using a Cissus
quadrangularis
formulation
(CORE) and CQR-300 (a
standardized extract of
Cissus
quadrangularis, 2.5 %
keto-steroids, and 15%
soluble
plant fiber).
Methods
The study was approved
by the Cameroon National
Ethics
Board. The purpose,
nature, and potential
risks of the
study were explained to
all participants, who
gave their
written informed consent
before participation.
The study
was conducted in
accordance with the
Helsinki Declaration
(1983 version).
Sample
CORE (Table 1) was
obtained from Soy Labs,
LLC., Fairfield,
California, USA;.
CQR-300, a standardized
extract of
Cissus quadrangularis
containing 2.5%
keto-steroids and
15% soluble plant fiber
(Gateway Health
Alliances, Inc,
Fairfield, California,
USA).
In vitro antioxidant
potential of the CORE
formulation
and CQR-300
Both CORE and CQR-300
were dissolved in
acidified
methanol prepared as
previously described by
Agbor et al.
[19] for the in vitro
antioxidant study. Three
methods were
used for the
determination of
antioxidant potential:
Folin
(polyphenol content),
ferric reducing
antioxidant power
(FRAP, antioxidant
power),
1,1-Diphenyl-2-picrilhydrazyl
(DPPH, redical
scavenging potential).
Polyphenol content
The Folin Ciocalteu
reagent (Sigma Chemical
Co., St
Louis, MO, USA) was used
to determine the
concentration
of polyphenol as a
measure of antioxidant
potential of
CORE and CRQ-300. The
reagent was diluted 10
times
before used as described
by Singleton et al.
[28]. The
absorbance was measured
at 750 nm (Genesys
Spectronic
20).
Ferric Reducing
Antioxidant Power (FRAP)
was measured
as earlier described by
Benzie and Strain. [29].
In brief,
2000 μl of freshly
prepared FRAP reagent
(10 parts of 300
mM acetate buffer (pH
3.6), 1 part of 10 mM
TPTZ
(Sigma, in 400 mM HCL),
and 1 part of 20 mM
ferric
chloride). After an
initial incubation for
15 minutes at
37°C, the absorbance was
read at 593 nm.
Scavenging potential
against
1,1-Diphenyl-2-picrilhydrazyl
(DPPH) measured the
ability of the extracts
to scavenge
free radicals. 20 μl of
extract was introduced
into 2
ml methanolic solution
of DPPH (0.3 mM) and
kept in
the dark for 30 minutes.
The extract was replaced
by methanol
for the control, and
catechin was used as the
standard.
The absorbance was read
at 517 nm, and the
antioxidant content and
percentage inhibition of
the
extract calculated as
earlier described by Yen
and Duh,
[30].
In all three methods
mentioned above,
measurements
were done in
triplicates.
In vivo study
Participants
A total of 168
overweight, obese, and
normal weight
participants
aged between 19 and 50
years were selected from
a group responding to a
radio and poster advert.
The BMIs
of participants ranged
from 25.0 to 48.7, and
their
weights ranged from 70.6
to 142 kg. After
physical examination,
which included
measurement of blood
pressure,
participants with
unusually elevated
fasting blood glucose
levels, those who were
pregnant or lactating,
as well as
those on any form of
weight-reducing
medication were
excluded from the study.
Also excluded were
participants
who were involved in
intense exercise
programs, had
medical conditions known
to affect serum lipids,
or had a
history of drug or
alcohol abuse. A total
of 153 participants
completed the study,
while 15 participants
dropped
out of the study for
personal reasons (7
moved out of
town or had to travel
during study period; 4
participants
thought they had lost
enough weight; while 4
participants
were on malaria
treatment and were
excluded before the
end of the study
period).
Trial protocol
The study was
double-blind placebo
controlled, with the
168 overweight, obese,
or normal weight
participants of
both sexes (between 19
and 50 years)
distributed as outlined
in Table 2. The
participants who were
either on their
normal diet or on an
energy restricted (2100
Kcal/day)
diet, received two daily
doses in the form of
capsules of
CORE for 8 weeks, and
CQR-300 or placebo for 6
weeks.
The capsules were
identical in shape,
color, and appearance,
with neither the
participants nor
researchers knowing
what capsule they
received. Side effects
were solicited
on each weekly visit.
Body weight and
percentage body fat
were determined in
12-hour fasted
participants using a
Tanita™ scale. Height
was measured with a
stadiometer to
the nearest 0.5 cm.
Blood samples were
obtained at the
start and end of the
trial period after a
12-hour overnight
fast, into heparinized
tubes, for the
measurement of LDL
oxidation (TBARS) [31]
and protein carbonyl
[32] content,
total cholesterol,
triacylglycerol,
HDL-cholesterol,
LDL-cholesterol, and
glucose (cholesterol
Infinity, triglyc-eride
Infinity, EZ HDL™
cholesterol, EZ LDL™
cholesterol,
Glucose Trinder) from
SIGMA Diagnostics.
Serotonin was
measured using an enzyme
immunoassay method
(Serotonin
EIA kit, BioSource
Europe S.A, Belgium),
creatinine
by a modification of the
method of Bartels et al.
[33],
while MDA was measured
by a standard
established
method [34].
Statistical analyses
Statistical Package for
the Social Sciences
(SPSS) [35] software
was used for all
statistical analysis.
The data were presented
as means ± SD. The
statistical difference
between
samples was assessed by
a Student's t-test for
normal distribution
or the Mann-Whitney test
for non-normal
distribution,
after ANOVA testing of
all the groups showed
that
significant differences
existed. Paired
Student's t-test was
carried out on the start
and end values of all
the groups.
Results
In vitro fantioxidant
potential of CORE and
CQR-300
The in vitro antioxidant
capacity of CORE was
significantly
(p < 0.01) higher than
that of CQR-300
irrespective of the
method of analysis used.
Considering the
composition of
CORE (Table 1), it is
likely that the other
components
present act
synergistically with the
Cissus quadrangularis
extract present (Table
3).
Table 4 presents the
effect of CORE and
CQR-300 on the
oxidative stress
parameter (TBARS and
carbonyls). CORE
was more effective in
reducing oxidative
stress than CQR-
300, the reduction being
more obvious in the
dietrestricted
group. It significantly
(p < 0.01) reduced the
formation
of TBARS and carbonyls
compared to CQR-300.
Effect of CORE and
CQR-300 on body weight
Obese participants, who
received CQR-300 (300 mg
daily), had
significantly (p < 0.05)
greater reduction in
body weight compared to
those on placebo (Table
5). This
reduction in body weight
corresponded to a 5.4 %
reduction
in BMI. CORE had a more
significant (p < 0.01)
effect
(8.5% reduction for
participants on an
energy restricted
diet) on the weight of
participants compared to
CQR-300.
There was no significant
net change in weight in
participants
on placebo during the
study period.
Table 6 presents the
effect of CORE and
CQR-300 on
blood lipids and fasting
blood glucose levels.
For participants
on a restricted diet,
six weeks use of CQR-300
reduced plasma total
cholesterol by 18.0%,
LDL-cholesterol
by 29.0%,
triacylglycerol by
21.7%, and fasting
blood glucose by 14.6%.
This treatment also
increased the
concentration of
HDL-cholesterol by
21.1%. On the other
hand, CORE (group 3)
reduced the
concentration of
plasma total cholesterol
by 26.0%,
LDL-cholesterol by
32.4%, triacylglycerol
28.0%, and fasting blood
sugar
16.1%. The CORE
formulation also
increased HDL
cholesterol
by 43.0%. The above
mentioned changes were
less
obvious in participants
whose diets were not
restricted.
The effect of CORE and
CQR-300 on
malondialdehyde
(MDA), serum serotonin,
and creatinine levels
are presented
in Table 7. CQR-300
significantly (p < 0.05)
reduced the
concentration of plasma
MDA. This effect was
accompanied by a slight
increase in the urinary
concentration
of MDA though not
significant. CQR-300
also significantly
(p < 0.05) increased the
concentration of plasma
5-HT by 53.3% and plasma
creatinine levels by
23.5%. An
increase in 5-HT of
17.0% was also observed
in the placebo
group, while CORE (group
3) showed a significant
increase of 39.1%. As
such results for 5-HT
and creatinine
were significantly (p <
0.05) lower for CORE
than CQR-
300.
Discussion
The role of antioxidants
from natural products in
degenerative
disease has attracted
more interest on natural
products research. In
this study, we evaluated
the antioxidant
potential of CORE and
CQR-300 on
obesity-induced
oxidative stress. The
parent plant in these
two formulations
is Cissus
quandrangularis. The
formulation had a
higher in vitro
antioxidant potential
than CQR-300
irrespective
of the method used for
the assay. The high
antioxidant
potential of CORE may be
due to its composition.
It contains some tea
polyphenols and selenium
that are
potential antioxidants
and thus complement the
antioxidant
potential of the parent
plant in this
formulation (Cissus
quandrangularis). On the
other hand, CQR-300 is a
standardized extract of
Cissus quandrangularis
and no antioxidant
was added to it. Obesity
may induce systemic
oxidative stress, and
increased oxidative
stress in accumulated
fat is one of the
underlying causes of
dysregulation of
adipocytokines and
development of metabolic
syndrome [6]. Oxidative
stress
plays critical roles in
the pathogenesis of
various diseases
[36]. In order to
investigate if oxidative
stress was
increased in the obese
participants, we
measured lipid
peroxidation (which
represent the plasma
TBARS) and the
carbonyl compounds as
markers of oxidative
injury,
which correlates with
the BMI. The high plasma
concentration
of TBARS and carbonyl
compounds was an
indication
of oxidative stress in
the obese and overweight
participants. These
concentrations were
significantly (p <
0.01) reduced after
treatment, with CORE
being more
effective than CQR-300.
These samples may
function
through two mechanisms:
either by scavenging
free radicals
to reduce oxidative
stress, or by clearing
the plasma of
the products that are
themselves potential
oxidants. These
activities may be
attributed to the
polyphenols present in
the different
formulations.
The use of CORE and
CQR-300 during the study
period
brought about a
significant reduction in
the weight and
BMI of obese patients.
This loss in weight was
comparable
to that observed with
cissus studies [21],
sibutramine for
one year [37], and
orlistat for 6 months or
1 year [38,39].
This reduction in BMI
was accompanied by an
increase in
HDL-cholesterol, and
corroborates earlier
work that
showed an inverse
relation between BMI and
HDL-cholesterol,
the latter imparting
possible health benefits
in overweight
and obese people
[40,41]. The increase in
the
concentration of
HDL-cholesterol and a
decrease in the
concentration of
LDL-cholesterol could
lead to a lowering
of the atherogenicity
and therefore a
significant reduction
in the potential
incidence of coronary
heart disease [42]
(54% reduction of risk
for a 0.6 mmol/L
reduction of
serum cholesterol) [43].
A reduction of fasting
blood glucose
levels as well as MDA
levels have been
previously
reported to accompany
weight loss in obese
subjects [39].
The above observation
could be linked to an
increase in
circulating creatinine
and serotonin over the
eight-week
trial period. Serotonin
is known to have a
positive effect
on mood and to reduce
binge eating, which is
common in
obese people. Several
previous studies [44,45]
have
shown a direct link
between serotonin levels
and weight
loss. On the other hand,
an increase in
creatinine
concentrations
parallels an increase in
lean muscle mass and a
probable reduction in
body fat.
Furthermore, in vitro
studies (submitted in a
different
publication) show the
ability of Cissus
quadrangularis
extracts to inhibit
pancreatic lipase by
approximately
60%, alpha-amylase by
approximately 90%, as
well as
alpha-glucosidase by
approximately 39%, all
of which
could contribute to
weight reduction in
obesity.
Conclusion
The CORE and CQR-300
(300 mg daily) brought
about a
significantly greater
weight loss than placebo
during the
study period in obese
individuals. This was
accompanied
by a significant
improvement in the lipid
profiles, blood
sugar profiles, and
serotonin profiles of
study participants.
They could have
additional properties as
antioxidants
against oxidative stress
in obese individuals.
Thus,
CQR-300 as well as CORE
possesses antioxidant
and free
radical scavenging
properties that could
have applications
in metabolic as well as
other physiological
complications
in which there is an
increase in oxidative
stress.
These new findings
warrant further
exploration into the
active phytonutrients of
Cissus quadrangularis
and the
potential of its newly
discovered weight loss
and cardiovascular
health benefits.
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