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ORIGINAL ARTICLE
Improvement in insulin resistance and favourable changes
in plasma inflammatory adipokines after weight loss associated
with two months’ consumption of a combination of bioactive food
ingredients in overweight subjects
Mariangela Rondanelli •Annalisa Opizzi •Simone Perna •Milena Faliva •
Sebastiano Bruno Solerte •Marisa Fioravanti •Catherine Klersy •
Cava Edda •Paolini Maddalena •Scavone Luciano •Ceccarelli Paola •
Castellaneta Emanuela •Savina Claudia •Lorenzo Maria Donini
Received: 31 October 2012 / Accepted: 12 December 2012
ÓThe Author(s) 2012. This article is published with open access at Springerlink.com
Abstract This randomized, double blind, placebo-
controlled, 8 week trial assessed the efficacy on metabolic
changes produced by a consumption of a combination of
bioactive food ingredients (epigallocatechin gallate, capsa-
icins, piperine and L-carnitine) versus a placebo, as part of a
therapeutic ‘lifestyle change’ diet, in 86 overweight subjects.
Forty-one patients (2/14 F/M; age 43.7 ±8.5; BMI
30.3 ±3.5 kg/m
2
) were randomized to the supplemented
group and 45 (29/16; age 40.7 ±10.2; BMI 30.0 ±2.7) to
the control group. We observed that consumption of the
dietary supplement was associated with a significantly
greater decrease in insulin resistance, assessed by homostasis
model assessment (p\0.001), leptin/adiponectin ratio
(p\0.04), respiratory quotient (p\0.008). LDL-choles-
terol levels (p\0.01). Moreover, statistically significant
differences were recorded between the two groups in relation
to urinary norepinephrine levels (p\0.001). Leptin, ghre-
lin, C-reactive protein decreased and resting energy expen-
diture increased significantly in the supplemented group
(p\0.05, 0.03, 0.02 and 0,02 respectively), but not in the
placebo group; adiponectin decreased significantly in the
placebo group (0.001) but not in the supplemented group,
although no statistical significance between the groups was
elicited. BMI, fat mass (assessed by DXA) and vascular
endothelial growth factor significantly decreased, whilst the
resting energy expenditure/free fat mass significantly
increased in both groups. In general, a greater change was
recorded in the supplemented group compared to the pla-
cebo, although no statistically significant difference between
the two groups was recorded. These results suggest that the
combination of bioactive food ingredients studied might be
useful for the treatment of obesity-related inflammatory
metabolic dysfunctions.
Keywords Epigallocatechin gallate Capsaicins
Piperine L-carnitine Dietary supplement Obesity
Inflammation Leptin Adiponectin Insulin resistance
Introduction
The prevalence of individuals who are classified as over-
weight and obese is a primary health concern due to the
M. Rondanelli (&)A. Opizzi S. Perna M. Faliva
Department of Applied Health Sciences, Section of Human
Nutrition and Dietetics, Faculty of Medicine, University of
Pavia, Azienda di Servizi alla Persona di Pavia, Servizio
Endocrino Nutrizionale, Istituto di Riabilitazione ‘‘Santa
Margherita’’, Via Emilia 12, Pavia, Italy
e-mail: mariangela.rondanelli@unipv.it
S. B. Solerte M. Fioravanti
Department of Internal Medicine, Geriatrics and Gerontology
Clinic, University of Pavia, ‘‘Istituto Santa Margherita’’,
Pavia, Italy
C. Klersy
Service of Biometry & Clinical Epidemiology, Fondazione
IRCCS ‘‘Policlinico San Matteo’’, Pavia, Italy
C. Edda L. M. Donini
Experimental Medicine Department, Medical Physiopathology,
Food Science and Endocrinology Section, Food Science and
Human Nutrition Research Unit, Sapienza University of Rome,
Rome, Italy
P. Maddalena S. Luciano C. Paola C. Emanuela
S. Claudia
‘‘Villa delle Querce’’ Clinical Rehabilitation Institute, Rome,
Italy
123
Endocrine
DOI 10.1007/s12020-012-9863-0
relationship between obesity and various cardiovascular
diseases (CVD) [1,2] and associated comorbidities [1].
The scale of this obesity epidemic creates a pressing con-
sumer need for the successful development of a dietary
supplement with a beneficial action on weight control. The
post-absorptive action of a number of ingredients has been
shown to influence satiety [3] and substrate use or ther-
mogenesis [4]; these ingredients include epigallocatechin
gallate (EGCG) from Camellia sinensis, capsaicins from
Capsicum annuum longum, piperine from Piper nigrum
Land L-carnitine. Of these active ingredients, EGCG, a
catechin found in abundance in green tea, is the most
common, and has been found to increase satiety [5,6] and
energy expenditure, as well as increasing markers of
lipolysis [7–12]. Studies have also suggested that EGCG
may alter food digestibility [13,14] and downregulate
stearoyl-CoA desaturase (SCD1) gene expression [13],
which may reduce adiposity, decrease lipid synthesis and
increase liver fatty acid oxidation, as was found in SCD1
knockout mice [13,15]. Furthermore, recent studies have
also shown that EGCG directly inhibits vascular endothe-
lial growth factor (VEGF) expression [16,17]. Moreover,
in humans, green tea consumption has been inversely
correlated with oxidative damage and with the levels of
inflammation markers [18,19].
Extensive research over the past two decades has revealed
that obesity is a proinflammatory disease [20], and increasing
evidence identifies inflammation as the potential link between
adipose tissue expansion and cardiometabolic complications
[21]. In fact, obesity is now considered to be a condition that
facilitates the development of a low-grade inflammatory state
characterized by increased plasma levels of pro-inflammatory
cytokines and cytokine-like proteins known as adipokines
[22]. One of the consequences of this state of inflammation is
the development of insulin resistance [23].
Several spices have been shown to exhibit activity
against obesity through their antioxidant and anti-inflammatory
mechanisms [24]. Of these, in addition to EGCG, capsaicin
has been investigated most extensively as an antioxidant
agent [25]. The use of capsaicin has therefore been
investigated as a treatment for obesity and obesity-related
metabolic diseases, as well as for its thermogenic activity
[26–30] and its satiating effect [31,32].
Piperine is an active component of black pepper that can
effectively suppress lipid peroxidation [33,34] and
enhance the bioavailability of curcumin and other sub-
stances through the inhibition of drug-metabolizing
enzymes in the liver [35].
Carnitine is an integral component in the transportation
of long-chain fatty acids within the mitochondria for oxi-
dation [36]. Supplemental carnitine has been shown to
increase weight loss in animals [37] and to increase the
oxidation of long-chain fatty acids in adults [38,39].
Allium sativa is a highly appropriate ingredient for
inclusion within a dietary supplement for the treatment of
obesity-related pathologies, due to its wide range of sig-
nificant biological activities (hypocholesterolemic, hypo-
lipidemic, anti-hypertensive, anti-diabetic, anti-thrombotic
and anti-hyperhomocysteinemia effects, which are typical
of thiosulfinates and other organosulfur compounds) [40].
Studies have shown that the diet typically consumed by
the Italian population is insufficient to prevent sub-clinical
iodine deficiency. In fact, physicians often advise people to
use iodine salt. Fucus vesiculosus can present a valid
alternative, especially for those consuming low-sodium
diets. The use of iodine to treat obesity can ensure that the
total daily requirement is consumed and, consequently, can
ensure optimal thyroid function [41].
Therefore, the purpose of this study was to examine the
changes produced by ingesting a combination of bioactive
food ingredients (EGCG, capsaicin, piperine, carnitine,
Allium sativa and Fucus vescicolosus) for two months on:
(1) body composition, assessed using dual energy X-ray
absorptiometry (DXA) evaluation, as the primary end-
point; (2) satiety control, calculated using a visual ano-
logue scale and ghrelin assessment; (3) thermogenesis,
calculated using resting energy expenditure (REE) and
respiratory quotient (RQ) assessment; 4) serum markers of
lipolysis [free fatty acids (FFA) and glycerol]; (5) adipo-
kine release (leptin, adiponectin); (6) indices of inflam-
mation [C-reactive protein (CRP) -PCR-] and insulin
resistance [homostasis model assessment (HOMA)]; (7)
angiogenesis marker release (-VEGF-); and (8) urinary
noradrenalin. Moreover, the effect on health-related quality
of life, evaluated using the Short-Form 36-Item Health
Survey, was also assessed. The decision to use a combi-
nation of compounds was prompted by the concept of
putting together interesting compounds with different
activities on the various causes of obesity, in order to act on
the multiple issues involved.
Methods
Participants
The study was carried out following approval by the Ethics
Committee of the Department of Internal Medicine and
Medical Therapy at the University of Pavia (Italy). Sub-
jects provided their written consent to participate in the
study. Healthy males and females ages 25–45 years, with a
body mass index greater than 25 kg/m
2
and less than
35 kg/m
2
, were eligible for the study. All subjects had to
provide complete medical histories, and all underwent a
physical examination, anthropometric assessment and
routine laboratory tests. Individuals suffering from any
Endocrine
123
hepatic or renal disease, diabetes, unstable CVD, uncon-
trolled hypertension, an eating disorder (diagnosed buli-
mia), active cancer or who had undergone surgery for
weight loss were all excluded from the study. Moreover,
patients were excluded from the study if they met the
Diagnostic and Statistical Manual-IV (DSM-IV) criteria for
a current diagnosis of major depressive disorder as deter-
mined by the Structured Clinical Interview for DSM-IV
Axis 1 Disorders (SCID-1) [42].
Subjects were also excluded if they were using any type of
medication for weight loss, or were pregnant or lactating, or
if they had entered menopause. All participants agreed to
refrain from participating in any other weight-loss pro-
gramme. Alcohol intake, smoking habits and physical
activity were recorded. Sedentary and non-smoking subjects,
who did not drink more than 6 glasses of wine a week and did
not drink hard liquor, were admitted to the study.
Procedures and study design
After 12 h of fasting and abstinence from water since
midnight, the subjects arrived at around 8:00 am, using
motorised transportation, at the Endocrinology and Clinical
Nutrition Unit of Azienda di Servizi alla Persona di Pavia
at the University of Pavia (Italy) and at the Dietetic and
Metabolic Unit, ‘Villa delle Querce’ Clinical Rehabilita-
tion Institute in Rome, Italy.
Blood samples were taken for the routine analysis
measurements and to measure leptin, adiponectin, ghrelin,
insulin, glycerol and FFAs. The assessment of REE and RQ
by indirect calorimetry, and the assessment of body com-
position by DXA and anthropometry were carried out in
the fasting state at baseline.
On the same morning, the patients took the 24-h urine test for
noradrenalin assessment. The same evaluations were assessed
after two months’ ingestion of 2 capsules per day of the dietary
supplement or the placebo. The subjects were randomly
assigned to one of the 2 groups in a double-blind parallel study.
Rating of satiety
Visual analogue scales (VAS) were used to assess appetite
sensations. The scale was dotted with phrases describing
the various degrees of hunger or satiety, but subjects were
free to choose any point along the scale; the point chosen
was defined as the Haber score [43]. In order to calculate
the values of the area under the curve (AUC) in response to
the treatment (with respect to ground), the VAS measure-
ments for each day, over the entire study period, were
considered in this calculation using the trapezoid method,
after rescaling the score from 0 to 20. The test was per-
formed every day before lunch time by all the subjects
included in the study.
Body composition measurement
Bone mineral density and body composition was measured
at baseline and at 12 months by DXA, using a Lunar
Prodigy DEXA (GE Medical Systems, Waukesha, WI)
[44].
Assessment of REE
Respiratory exchange measurements using indirect calo-
rimetry (Deltatrac Monitor II MBM-200, Datex Engstrom
Division, Instruments Corp. Helsinki, Finland) were used
to estimate REE, adhering to the recommended measure-
ment conditions [45]. REE was calculated from O
2
and
CO
2
volumes—as well as from urine excretion nitrogen
values—using the Weir formula, and expressed as kcal/day
[46].
Anthropometry, weight-loss programme and food
intake
Nutritional status was assessed using anthropometric
measurements at baseline and after 2 months in both
groups. Body weight and height were measured and the
body mass index (BMI) was calculated (kg/m
2
). Skinfold
thicknesses (biceps, triceps, suprailiac, subscapular) were
measured twice using a Harpenden skinfold caliper at
5 min intervals at each site, following a standardized
technique [47]. Sagittal abdominal diameter was measured
at the L
4–5
level in the supine position and waist girth was
also measured. Anthropometric variables were measured
by a single investigator.
Body weight reduction was induced by a low-energy
mixed diet (55 % carbohydrates, 30 % lipids and 15 %
proteins) providing 600 kcal less than individually estimated
energy requirements based on the measured REE. The
energy content and macronutrient composition of the diets
adhered to the nutritional recommendations of the American
Diabetes Association [48,49]. These diets were designed to
achieve weight losses of 0.5–1 kg per week; this type of diet
is considered to be a low-risk intervention [50]. Individual
diet plans were drawn up for each subject by the research
dietitian. To optimize compliance, dietary instructions were
reinforced each week by the same research dietician. Each
consultation included a nutritional assessment and weighing.
A 3-day weighed-food record of 2 weekdays and 1 weekend
day was performed before the study and during the last week
of intervention. One-day weighed-food records were com-
pleted in weeks 2, 5 and 7. Low-energy diets and dietary
records were analyzed using a food-nutrient database
(Rational Diet, Milan, Italy). In order to assess compliance to
the weight-reduction programme, a 24-h dietary summary
was assessed by the nutritionist at the end of the study.
Endocrine
123
Assessment of depressive symptoms and health-related
quality of life
Depressive symptoms, assessed by the Beck depression
inventory (BDI) [51] and health-related quality of life, assessed
by the Short-Form 36-Item Health Survey (SF-36) [52], were
evaluated at baseline and after 2 months in both groups.
Biochemical analyses
Fasting venous blood samples were drawn between 08.00
and 10.00 a.m. with the subjects in a sitting position.
Clinical Chemistry parameters were detected on the Roche
Cobas Integra 400 plus analyzer (Roche Diagnostics,
Basel, Switzerland), using specially-designed commercial
kits provided by the manufacturer. In particular, total
serum cholesterol, triacylglycerol, HDL-cholesterol, total
proteins, total bilirubin, iron, glucose, uric acid, creatinine,
transaminase alanine aminotransferase, aspartate amino-
transferase and gamma glutamyl transferase were mea-
sured using enzymatic-colorimetric methods. LDL
cholesterol was calculated according to the Friedewald
formula [53] for those specimens with triacylglycerol lev-
els less than 400 mg/dl (\4.5 mmol/l). The CRP was
determined by Nephelometric High-Sensitivity CRP (Dade
Behring, Marburg, Germany). Hemochrome was measured
using a Coulter automated cell counter MAX-M (Beckman
Coulter, Inc., Fullerton, USA). Serum insulin levels were
measured on a Roche Elecsys 2010 analyzer (Roche
Diagnostics, Basel, Switzerland) using dedicated com-
mercial electrochemiluminescent immunoassays. Insulin
resistance was evaluated using the HOMA [54] and
Quantitative insulin-sensitivity check index (QUICKI)
[55]. Serum concentrations of FFA and glycerol were
determined by a quantitative colorimetric assay (BioAssay
Systems, Hayward, CA). Plasma acylated and unacylated
ghrelin levels were measured using an enzyme immuno-
metric assay based on a double-antibody sandwich tech-
nique (BioVendor, Brno, CZECH REPUBLIC). Serum
adiponectin levels were measured using an enzyme-linked
immunosorbent assay (ELISA) (R&D Systems, Inc., Min-
neapolis, MN, USA). Serum leptin levels were measured
using an ELISA (R&D Systems, Inc., Minneapolis, MN,
USA). Serum VEGF (VEGF) levels were measured using
an ELISA (R&D Systems, Inc., Minneapolis, MN, USA).
Twenty-four hour urinary excretion of noradrenalin was
also determined at baseline and after 8 weeks, using the
chromatographic-colorimetric method.
Dietary supplement
The subjects received two capsules per day of a dietary
supplement or an identical placebo. The product was
manufactured by Medestea Research & Production S.p.a.,
(Torino)—Italy. The composition of the dietary supple-
ment is listed in Table 1. Capsicum extract contains a
pungent active ingredient, capsaicin. In this particular
dietary supplement, the capsicum extract is micro-encap-
sulated to improve its tolerability. This innovative method
of micro-encapsulation enables the administration of one
total dose to be subdivided into hundreds of micro-doses.
These micro-doses are spread out evenly over a very
extensive area of the intestinal mucosa, thus enabling
uniform absorption of the active ingredient, over a pre-
established timeframe (6 h). The resulting concentration of
the active ingredient in each micro-dose is minimal, thus
decreasing the risk of local irritation. Moreover, the tablets
of this product are gastro-resistant, in order to avoid any
kind of irritation in the oral and gastric areas. Identical
capsules for each treatment group were assigned to a
subject number according to a coded (AB) block random-
ization table prepared by an independent statistician.
Investigators were blinded to the randomization table, to
the code assignments and to the procedure. Compliance to
the supplementation regimen was defined as the number of
tablets actually taken by each subject, divided by the
number of tablets that should have been taken over the
course of the study. Adverse events were based on spon-
taneous reporting by subjects as well as open-ended
enquiries by members of the research staff.
Sample size calculation
The sample size calculation is based on the primary end-
point and on information retrieved from scientific literature
[56]. We hypothesized a decrease in free fat mass of
0.5 ±2.5 kg in the control group and of 2.5 ±2.5 kg in
the experimental group, corresponding to a between-group
difference of 0.8 standard deviations (effect size). To show
such a difference, with a 2-sided type I error of 5 % and a
power of 90 %, 50 patients per group are required. The
power would be reduced to 82 % in the presence of a
Table 1 Characteristics of the dietary supplement
Botanical extracts mg/cpr mg/die
Active ingredients
Camellia sinensis decaffeinated dried extract,
mixed with soya phospholipids
150 300
Microencapsulated oleoresin of Capsicum
annum
7.5 15
L-Carnitine 150 300
Fucus vesiculosus dry extract 56.5 113
Allium sativum dried extract 2.5 5
Microencapsulated mint essential oil 2.5 5
Piper nigrum dry extract 3 6
Endocrine
123
dropout rate of about 20 %. nQuery 4 (Statistical Solutions,
Cox, IRL) was used to perform the calculations.
Statistical analysis
Data were described as mean and standard deviation if
continuous, and as counts and percentage if categorical.
Pre-post comparisons were carried out within each treat-
ment group using the paired Student ttest; mean changes
over time and 95 % confidence intervals (95 %CI) were
calculated. Finally, the changes between the treatment
groups were compared using a general linear regression
model, adjusting for baseline values. Huber White robust
standard errors were calculated. The mean difference
between the changes and 95 % CI was recorded. Stata 12
(StataCorp, College Station, TX, USA) was used for these
calculations. A 2-sided pvalue \0.05 was considered to
be statistically significant.
Results
One hundred and three overweight subjects were inclu-
ded in the study, out of one hundred and seven eligible
participants (Fig. 1). Forty-one patients (26 women; 15
men; aged 43.7 ±8.5 years with BMI 30.3 ±3.5 kg/m
2
)
were randomized to the supplemented group and 45
(29 women; 16 men; age 40.7 ±10.2 years and BMI
30.0 ±2.7 kg/m
2
) to the control group. The baseline
characteristics of the supplemented group and the control
group are summarized in Table 2, with no relevant dif-
ferences observed in any of the baseline variables. In
addition, there was no significant difference in the
baseline characteristics between study completers and
drop-outs. The dietary supplement was well tolerated,
and there were no reports of any serious adverse events
as a result of administration. This finding supports and is
consistent with the safety profile that is already widely
reported in scientific literature regarding the use of cap-
saicin and the other plant extracts contained in the for-
mulation [57]. Compliance was completed in both groups
and there were no comments from any of the studied
patients regarding the content of the supplement he/she
was taking, or regarding their perception of having been
included in one of the two groups. Changes from
694
695
696
697
698
50 allocated
to the supplement group
All 50 received the allocated
intervention
41 followed up at
week 8
53 allocated
to placebo group
All 53 received the
allocated intervention
45 followed up at
week 8
41 analysed 45 analysed
107 eligible patients
103 randomized
8 drop-outs, all for personal
reasons: 5 due to family
problems, 2 already
scheduled for gynecological
surgery, 1 hospitalized for
appendicitis
9 drop-outs, all for
personal reasons:
1 due to
translocation, 5 due
to family problems,
1 admitted for renal
colic, 2 due to
bereavement, 1 due
to a car accident
4subjects
decided not to
participate
Fig. 1 Flow diagram
illustrating a clinical trial of a
food supplement versus a
placebo in the treatment of
healthy overweight subjects
Table 2 Basal characteristics and biochemical parameters of subjects
studied
Variable Supplemented Placebo
No. of subjects studied 50 53
No. of females/males 37/13 37/16
No. of drop-outs 9 8
Age (y) 43.7 (±8.5) 40.8 (±10.2)
BMI (kg/m
2
) 30.4 (±3.6) 30.0 (±2.8)
Waist (cm) 98.7 (±11.8) 97.9 (±7.4)
Hips (cm) 109.7 (±8.3) 106.8 (±6.6)
Fat mass (kg) 34.4 (±6.8) 33.3 (±7.3)
Free fat mass (kg) 46.2 (±10.1) 45.5 (±9.7)
Resting energy expenditure
(kcal/die)
1483 (±328) 1504 (±295)
Resting energy expenditure/
free fat mass
32.5 (±5.7) 35.0 (±1.7)
RQ 0.82 (±0.06) 0.82 (±0.06)
FFAs (mM/L) 0.41 (±0.2) 0.41 (±0.19)
Glycerol (mM/L) 0.15 (±0.05) 0.14 (±0.04)
Adiponectin (ng/mL) 83.63 (±46.26) 83.6 (±53.02)
Leptin (pg/mL) 214.16 (±140.5) 222.68 (±187.5)
Leptin/Adiponectin 3.5 (±3.11) 3.3 (±3.15)
VEGF (pg/mL) 304.75 (±190.42) 332.84 (±228.63)
Ghrelin (pg/mL) 414.58 (±235.25) 395.61 (±275.51)
Urinary norepinephrine
(nM/24 h)
40.58 (±24.27) 41.53 (±23.99)
Values are means (±SD) (All randomized patients are included)
Endocrine
123
baseline were compared between the two treatment arms;
these results are summarized in Table 3(for biochemical
and urinary parameters), Table 4(for metabolic, body
composition and QoL parameters) and Table 5(for
anthropometric parameters). A significant difference
between treatment groups was observed for the following
parameters: insulin levels (p=0.003), insulin resistance
(assessed by HOMA (p=0.002) and QUIKI
(p=0.007)), leptin/adiponectin ratio (p=0.040), RQ
(p=0.008), LDL-cholesterol levels (p=0.031) and
urinary norepinephrine levels (p\0.001). No significant
differences between the treatment arms were shown for
leptin, ghrelin, PCR, REE and adiponectin. Nonetheless,
there was a significant decrease in leptin, ghrelin and
PCR and an increase in REE (by 120 kcal/die) in the
supplemented group but not in the placebo group, whilst
adiponectin levels decreased significantly in the placebo
group, but not in the supplemented group. Similarly, no
significant differences were observed between treatment
groups for BMI, fat mass, REE/free fat mass and VEGF.
Once again, there was a significant decrease in BMI, fat
mass and VEGF and a significant increase in the REE/
free fat mass ratio in both groups, with a greater change
recorded overall in the supplemented group in compari-
son to the placebo group. No significant differences were
observed neither for any blood chemistry values nor for
any of the other anthropometric parameters studied
(which all showed a significant decrease from baseline).
Finally, no significant differences between the treatment
groups were apparent in relation to the sense of satiety,
BDI-II and the 2 subscales of the 36-item Short-Form
Survey. In the supplemented group, 14 patients (34 %)
lost at least 5 % of their body weight, whilst just 10
patients (22 %) from the placebo group fell into this
category [Risk difference (IC95 %): 12 % (-7 to 31 %),
p=0.24]. Table 6shows the results of a 3-day weighed-
food record of 2 weekdays and 1 weekend day, carried
out by all patients from both groups during the first week
and the last week of intervention. No significant differ-
ences were apparent between the two treatment groups.
Table 3 Comparison of biochemical and urinary parameters between the supplemented and placebo groups
Variable DDietary supplement group
mean change (95 % CI)
a
Placebo group mean
change (95 % CI)
a
Treatment effect mean difference (95 %
CI) [adjusted for baseline]
pvalue
Insulin (pmol/L) 1.8 (0.72 to 2.9) 0.11 (-0.88 to 1.1) 17 910
-4
(6 910
-4
to 27 910
-4
) 0.003
Homeostatic metabolic
assessment index (HOMA)
0.42 (0.14 to 0.71) 0.02 (-0.21 to 0.26) 4 910
-4
(1 910
-4
to 7 910
-4
) 0.007
Quantitative Insulin-sensitivity
check index (QUICKI)
-0.01 (-0.02 to -0.01) 0.00 (-0.01 to 0.01) -12 910
-6
(-20 910
-6
to -
5910
-6
)
0.002
Leptin/Adiponectin 0.46 (-0.09 to 1.0) -0.54 (-1.4 to 0.30) 88 910
-5
(3 910
-5
to 173 910
-5
) 0.04
LDL-cholesterol (mmol/L) -4.9 (-10.8 to 0.93) 6.6 (-1.4 to 14.6) -0.01 (-0.02 to 0.00) 0.013
Urinary norepinephrine
(nM/24 h)
-24.6 (-39.6 to -9.6) 5.0 (-3.4 to 13.4) -0.03 (-0.04 to -0.01) \0.001
Adiponectin (ng/mL) 4.6 (-1.7 to 10.9) 11.5 (5.8 to 17.2) -0.01 (-0.01 to 0.00) 0.08
Total cholesterol (mmol/L) -1.8 (-10.1 to 6.5) 7.2 (-0.58 to 15.1) -0.01 (-0.02 to 0.00) 0.09
Leptin (pg/mL) 32.6 (9.9 to 55.3) 5.2 (-24.2 to 34.6) 0.03 (0.00 to 0.06) 0.10
Ghrelin (pg/mL) 83.3 (4.8 to 161.8) 0.45 (-85.4 to 86.3) 0.07 (-0.02 to 0.16) 0.14
Apolipoprotein B (g/L) -8.3 (-16.7 to 0.17) -1.8 (-8.5 to 4.8) -0.01 (-0.01 to 0.00) 0.16
FFAs (mM/L) 0.04 (-0.02 to 0.10) -0.02 (-0.08 to 0.03) 5 910
-5
(-2910
-5
to 11 910
-5
) 0.16
CRP (mg/dL) 0.14 (0.02 to 0.27) 0.08 (-0.04 to 0.19) 5 910
-5
(-3910
-5
to 13 910
-5
) 0.20
Glycaemia (mmol/L) 1.7 (-1.8 to 5.2) 0.42 (-1.7 to 2.5) 1 910
-3
(-2910
-3
to 4 910
-3
) 0.36
Triacylglycerol (mmol/L) 6.8 (-13.5 to 27.1) -11.6 (-48.2 to 25.0) 0.02 (-0.02 to 0.05) 0.39
HDL-cholesterol (mmol/L) 1.8 (-0.77 to 4.3) 3.0 (0.64 to 5.3) -1910
-3
(-4910
-3
to 2 910
-3
) 0.42
Apolipoprotein A1 (g/L) -1.1 (-11.6 to 9.4) -3.2 (-13.0 to 6.7) 0.00 (-0.01 to 0.01) 0.42
VEGF (pg/mL) 94.9 (43.3 to 146.5) 86.9 (43.0 to 130.8) 0.02 (-0.03 to 0.07) 0.49
Glycerol (mM/L) 0.00 (-0.02 to 0.01) -0.01 (-0.02 to 0.01) -1910
-6
(-17 910
-6
to
14 910
-6
)
0.87
Tot Chol/HDL-Chol -0.14 (-0.29 to 0.01) -0.12 (-0.33 to 0.08) -1910-5(-23 910-5to
21 910-5)
0.90
a
Within-group changes are calculated as a baseline-final value. So a plus sign (?) corresponds to higher values at baseline (or a decrease during
follow-up) and a minus sign (-) to lower values at baseline (therefore indicating an increase during follow-up)
(Variables with statistically significant differences are presented first; the other variables are listed according to the calculated pvalue)
Endocrine
123
Discussion
In this study, which was carried out on overweight subjects,
we observed that the consumption of this combination of
bioactive food ingredients was associated with a decrease in
insulin resistance and in inflammatory adipokines. In addi-
tion, the difference in body composition was greater in the
active treatment arm, particularly in relation to the primary
endpoint (fat mass), although no statistically significant
results were produced. Obesity is now considered to be a
condition that facilitates the development of a low-grade
inflammatory state characterized by increased plasma levels
of pro-inflammatory cytokines and cytokine-like proteins
known as adipokines [22], and that one of the consequences
of this state of inflammation is the development of insulin
resistance [23]. Adipose tissue secretes adipokines, which
may be the missing link between insulin resistance and
obesity, influencing body weight, glucose and lipid metab-
olism [58]. Adiponectin has anti-atherogenic, anti-diabetic
and anti-inflammatory properties [59]. Increased adiponectin
is associated with a lower risk of impaired glucose tolerance
and a decrease in the risk of myocardial infarction, and has
Table 5 Comparison of the anthropometric parameters studied between the supplemented and placebo groups
Variable DDietary supplement group mean
change (95 % CI)
a
Placebo group mean
change (95 % CI)
a
Treatment effect mean difference (95 %
CI) [adjusted for baseline]
pvalue
Weight (kg) 3.1 (2.2 to 4.0) 2.0 (1.1 to 2.8) 9 910
-4
(-1910
-4
to 20 910
-4
) 0.08
BMI (kg/m
2
) 1.1 (0.78 to 1.4) 0.71 (0.39 to 1.0) 33 910
-5
(4 910
-5
to 70 910
-5
) 0.08
Waist hip ratio
(WHR)
0.00 (0.00 to 0.01) 0.01 (0.00 to 0.03) -1910
-5
(-2910
-5
to 15 910
-5
) 0.09
Arm
circumference
(cm)
0.60 (0.28 to 0.92) 0.59 (0.12 to 1.1) 1 910
-4
(-4910
-4
to 6 910
-4
) 0.65
a
Within-group changes are calculated as the baseline-final value. Therefore a plus sign (?) corresponds to a higher value at baseline (or a
decrease during follow-up) and a minus sign (-) to a lower value at baseline (thus indicating an increase during follow-up)
(Variables with statistically significant differences are presented first; the other variables are listed according to the calculated pvalue)
Table 4 Comparison of the metabolic, body composition and health-related quality of life parameters studied between the supplemented and
placebo groups
Variable DDietary supplement group
mean change (95 % CI)
Placebo group mean
change (95 % CI)
Treatment effect mean difference
(95 % CI) [adjusted for baseline]
pvalue
RQ 0.04 (-0.01 to 0.09) -0.04 (-0.09 to 0.01) 8 910
-5
(2 910
-5
to
13 910
-5
)
0.008
Resting energy expenditure (kcal) -120.6 (-220.9 to -20.2) 8.6 (-86.8 to 103.9) -0.10 (-0.21 to 0.02) 0.10
Fat free mass (kg) 0.36 (-0.03 to 0.76) -0.13 (-0.66 to 0.40) 4 910
-4
(-1910
-4
to
10 910
-4
)
0.14
Resting energy expenditure/Free fat
mass
-3.0 (-5.5 to -0.58) 2.1 (-2.3 to 6.4) -2910
-3
(-4910
-3
to
1910
-3
)
0.22
Fat mass (kg) 2.5 (1.6 to 3.3) 1.9 (1.1 to 2.7) 5 910
-4
(-4910
-4
to
14 910
-4
)
0.24
36-item short-form survey. Mental
component subscale (SF-36 MCS)
4.0 (0.68 to 7.4) 1.4 (-2.6 to 5.4) 2 910
-3
(-2910
-3
to
6910
-3
)
0.24
Beck depression inventory (BDI-II) 3.3 (1.4 to 5.2) 2.3 (0.79 to 3.7) 9 910
-4
(-7910
-4
to
25 910
-4
)
0.26
AUC for satiety VAS – – 0.02 (-0.02 to 0.06) 0.36
Android fat (%) 1.8 (1.1 to 2.5) 1.4 (0.59 to 2.2) 4 910
-4
(-5910
-4
to
13 910
-4
)
0.36
36-item short-form survey. Physical
component subscale (SF-36 PCS)
5.1 (-0.59 to 10.8) 3.1 (-1.4 to 7.6) 2 910
-3
(-4910
-3
to
8910
-3
)
0.42
Gynoid fat (%) 1.6 (0.92 to 2.2) 1.4 (0.71 to 2.1) 1 910
-4
(-6910
-4
to
9910
-4
)
0.70
a
Within-group changes are calculated as a baseline-final value. Thus, a plus sign (?) corresponds to a higher value at baseline (or a decrease
during follow-up) and a minus sign (-) to a lower value at baseline (thus indicating an increase during follow-up)
(Variables with statistically significant differences are presented first; the other variables are listed according to the calculated pvalue)
Endocrine
123
been proposed as a biomarker of early atherosclerosis [60].
Leptin, a cytokine-like molecule secreted by adipose tissue,
regulates adipose mass and body weight by inhibiting food
intake and stimulating energy expenditure [61,62].
Numerous publications suggest leptin as a biomarker for
obesity, insulin resistance and Metabolic Syndrome in adults
[61,62]. The leptin-to-adiponectin ratio was recently pro-
posed as a biomarker with the benefits of both indices [63–
65]. Although weight and BMI are the best indicators for the
patients, a positive outcome must be measured by an
improvement in their overall well-being, not just by weight
loss alone. An improvement in the patient’s inflammatory
state therefore represents a significant positive outcome. The
novelty of our study is the fact that overweight subjects
taking the dietary supplement obtained a positive metabolic
outcome at the end of the study. In fact, when comparing the
effect of the dietary supplement with that of the placebo in
our study, we observed that consumption of the dietary
supplement was associated with a significant decrease in
insulin resistance (assessed by HOMA and QUIKI), leptin/
adiponectin ratio, RQ and LDL-cholesterol levels. Leptin,
ghrelin and CRP significantly decreased in the supplemented
group but not in the placebo group, whilst adiponectin levels
significantly decreased in the placebo group but not in the
supplemented group, although no statistically significant
difference between the groups was recorded. This frame-
work constitutes a significant improvement in the metabolic
status of the supplemented patients, especially in light of the
fact that weight loss does not always lead to an improvement
in a patient’s inflammatory state and insulin resistance, as
demonstrated by numerous studies [66,67]. These positive
results on metabolic status are probably due to the synergistic
action of the bioactive compounds of this dietary supple-
ment, in particular EGCG and capsaicins. Capsaicin, a bio-
logically active compound found in red pepper, has anti-
inflammatory activities [68–70] and demonstrates potential
benefits for treating obesity and insulin resistance, both in
animal models and in clinical studies [71–74]. Our results
therefore concur with previous studies in this area [27–29,32,
72]. Interestingly, the supplemented subjects experienced a
significant reduction in RQ. It has also been suggested that
the RQ, as a reflection of carbohydrate and fat oxidation, may
be a metabolic index which predicts subsequent weight gain
[75]. In one study, a higher 24-h RQ (reflecting greater car-
bohydrate and less fat oxidation) was found to correlate with
a greater risk of weight gain, independently of low-energy
expenditure [76]. The findings of the Baltimore Longitudinal
Study supported this, in that a higher resting RQ correlated
with subsequent weight gain, at least amongst lean men [77].
Another reflection of this pattern of energy usage lies in the
fact that insulin sensitivity—and, hence, a tendency to
greater carbohydrate use—has been reported to produce a
greater weight gain in Pima Indians [75] and in individuals
who are already obese [78], but not in lean individuals [78].
Another important result of the study is the significant
increase in urinary norepinephrine levels, which was
observed only in the supplemented group. This urinary
parameter was evaluated to produce an indirect assessment
of the absorption and effectiveness of the dietary supple-
ment. EGCG can inhibit catechol O-methyltransferase, an
enzyme involved in the degradation of norepinephrine [79].
Consequently, once released, norepinephrine remains in the
synaptic cleft for longer, and provides prolonged stimulation
of the adrenergic receptors. Interestingly, a number of
compounds extracted from plants—such as capsaicin from
pungent spices—can modulate catecholamine release and
activity [80].
Finally, another important result of this study was the
effect of the dietary supplement on REE; this increased
significantly in the supplemented group but not in the
placebo group. Studies examining changes in energy
requirements following moderate weight loss have shown
Table 6 A 3-day weighed-food record of 2 weekdays and 1 weekend day, performed during the first week and the final week of intervention
First week of intervention
(dietary supplement)
Final week of intervention
(dietary supplement)
First week of
intervention (placebo)
Final week of
intervention (placebo)
Energy (kJ) 6761 (65.2) 6792 (64.5) 6652 (59.9) 6766 (67.2)
Protein (g); (% energy) 70.8 (4.1); 17.5 (0.9) 70.8 (5.0); 17.5 (1.0) 63.0 (6.3); 15.9 (1.1) 69.3 (5.9); 17.2 (1.0)
Fat (g); (% energy) 48.7 (3.3); 27.2 (2.1) 47.1 (2.9); 26.1 (1.9) 46.9 (4.2); 26.6 (2.0) 52.5 (5.3); 29.2 (2.1)
Saturated fatty acids
(g); (% energy)
12.2 (1.5); 6.8 (0.7) 12.8 (2.1); 7.1 (1.0) 12.0 (2.4); 6.8 (0.8) 14.9 (3.0); 8.3 (0.9)
Carbohydrate (g); (%) 238.2 (6.1); 55.3 (1.6) 243.8 (5.7); 56.4 (1.9) 243.8 (7.4); 57.5 (2.0) 231.0 (8.0); 53.6 (2.1)
Complex (g); (%) 152.3 (20.4); 35.3 (3.1) 167.1 (21.8); 38.7 (2.9) 181.7 (22.3); 42.9 (3.0) 165.7 (23.2); 38.5 (4.0)
Simple (g); (%) 85.9 (10.2); 20 (2.5) 76.7 (11.1); 17.7 (2.7) 62.1 (12.5); 14.6 (2.9) 65.2 (9.9); 15.1 (2.4)
Dietary fibre (g) 26.3 (2.9) 26.5 (3.6) 25.8 (4.2) 24.1 (4.3)
Cholesterol (mg) 128.9 (31.7) 178.6 (30.5) 159.2 (32.4) 204.1 (33.0)
Mean (SD); Nutritional evaluation carried out by Carnovale E, Marletta L: ‘‘Food composition tables’’, Italian National Insitute of Nutrition,
Rome 1997 (‘‘Tabelle di composizione degli alimenti’’, Istituto Nazionale della Nutrizione, Roma 1997)
Endocrine
123
that active weight loss is associated with a decrease in
REE, and that this decrease is disproportionately large in
relation to the loss of body tissues [81–83]. Our results
show that the dietary supplement, in combination with a
low-calorie diet, can counteract this decrease in REE. The
results of the study demonstrated that REE increases only
in the treated group. This increase in REE is significantly
advantageous from a metabolic perspective. Our results
concur with the available literature: there is a substantial
quantity of data available to substantiate the metabolism-
enhancing properties of pepper and green tea [4,26–30].
As regards appetite, the results of this study confirm that
EGCG and capsaicin inhibit appetite effectively [6,32].
The dietary supplement used in this study is certainly an
innovation. We consider that the majority of the results
produced by this study can be attributed to the synergistic
action of the components. Compared to the results pub-
lished in previous scientific literature, this supplement is
more effective in increasing the basal metabolic rate,
improving insulin resistance and inducing favourable
changes in plasma inflammatory adipokines.
Our study does have some limitations, the first of which
is the relatively short duration of the observation period,
although this is similar to the duration of other previous
studies carried out on the same substances [6,8,27,37]. A
further limitation lies in the fact that we did not test the
efficacy of the individual components of the dietary sup-
plement used in the trial, although there is a substantial
quantity of scientific literature available relating to the
activities of the individual components within this
combination of bioactive food ingredients [5–14,25–38].
Another problem stems from the fact that the personalized
weight-loss programme was relatively short, and therefore
its efficacy could have overlapped at least partially with the
effects of the tested dietary supplement. However, the
study had a placebo arm and the placebo subjects also
received a personalized weight-loss programme. Changes
in biochemical indices and body composition are separate
endpoints and were therefore evaluated as such, as pro-
jected in the protocol. We might hypothesize that some of
the effect seen in one endpoint may be explained by the
other. Post-hoc sub-analyses are planned in the future in
order to address this, along with other issues.
In conclusion, the results of this study indicate that this
combination of bioactive food ingredients could produce
significant beneficial actions in the management of over-
weight-related inflammation, by reducing insulin resistance
and inflammatory adipokines.
Acknowledgments A. Opizzi, S. Perna, M. Faliva, E. Cava,
M. Paolini, L. Scavone, P. Ceccarelli, E, Castellaneta and C. Savina
recruited the subjects and conducted the intervention study at the
clinic. M. Rondanelli and L. Donini were the investigators who
designed and wrote the study, as well as providing supervision.
M. Fioravanti and S.B. Solerte were responsible for collecting the
samples of biochemical markers, and also provided supervision.
C. Klersy was responsible for statistical issues and provided advice on
interpreting the results.
Conflict of interest All authors deny any financial and personal
relationships with other people or organisations that could inappro-
priately influence this study.
Open Access This article is distributed under the terms of the
Creative Commons Attribution License which permits any use, dis-
tribution, and reproduction in any medium, provided the original
author(s) and the source are credited.
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