SUPPLEMENTARY MATERIAL
Clinical evaluation of Moro (Citrus sinensis (L.) Osbeck) orange juice supplementation
for the weight management
Venera Cardilea, Adriana Carol Eleonora Grazianoa, Alessandro Vendittib,c*
a Department
of Bio-medical Sciences, Section of Physiology, University of Catania, Viale Andrea
Doria 6, 95125 Catania, Italy.
b Dipartimento
di Chimica, Sapienza Università di Roma, Piazzale Aldo Moro 5, 00185 Roma, Italy;
c
Dipartimento di Biologia Ambientale, “Sapienza” Università di Roma, Piazzale Aldo Moro 5, 00185
Roma, Italy
*E-mail: alessandro.venditti@uniroma1.it
* Corresponding author. Tel. +39 0649913229; fax: +39 0649913841; E-mail address:
alessandro.venditti@uniroma1.it (A. Venditti).
Abstract
In the last years, several studies has recently evaluated beneficial effect of red orange juice (Citrus
sinensis (L.) Osbeck) and its active components in weight management and obesity. Moro orange
is a cultivar of red orange, particularly rich in active compounds such as anthocyanins,
hydroxycinnamic acids, flavone glycosides and ascorbic acid, that showed to possess anti-obesity
effects in vitro and in vivo studies. In the present clinical study, the effect of a Moro juice extract
(Morosil®, 400 mg/die) supplementation was evaluated in overweight healthy human volunteers
for 12 weeks. Results showed that Moro juice extract intake was able to induce a significant
reduction of BMI (Body Mass Index) after 4 weeks of treatment (p<0.05). Moreover, in subjects
treated with Moro extract, body weight, BMI, waist and hip circumference were significant
different to the placebo group (p<0.05). In conclusion, it could be suggested that the active
compounds contained in Moro juice possess a synergistic effects on fat accumulation in humans
and Moro juice extract can be used in the weight management and in the prevention of human
obesity.
Key words: red orange; Moro juice; weight management; obesity
3 - Experimental
3.1 Reagents and test material
The powder extract of Moro red orange variety (Morosil® batch 02201306-10) used in this study
was supplied by Bionap srl (Catania, Italy). It is a standardized solid extract obtained from Moro
orange fruits, containing the following active substances: anthocyanins (cyanidin-3-O-glucoside)
0.8-0.9%, hydroxycinnamic acids (caffeic, cumaric, ferulic, sinapic acid) 2-2.2%, flavone glycosides
(narirutin, hesperidin) 0.8-1% and ascorbic acid 4.3-4.5%. All the standard compounds and the
reagents for analytical evaluation were supplied from Extrasynthese (Lyon, France) and SigmaAldrich (Milan, Italy). Tablets containing 400 mg of Morosil® extract or 400 mg of maltodextrin
were prepared for the in vivo study.
3.2 Polyphenols analysis of Moro orange extract
Active compounds contained in Moro orange extract were analysed using a Shimadzu HPLC
system (Kyoto, Japan) equipped with an SCL-10Avp controller, two LC-10ADvp pumps, a DGU-14A
degasser, and an SPD-M10vp diode array detector. The column used was an Ascentis C18 (25cm x
4.6 mm x 5 µm) column purchased from Sigma-Aldrich. Hydroxycinnamic acids were analysed by
the HPLC method reported by Rapisarda et al (Rapisarda, 1998). Chromatograms were recorded
with a 300 nm UV light and isocratic elution was performed with a solvent mixture of 18%
tetrahydrofuran and 82% water-acetic acid solvent system. Caffeic, cumaric, ferulic and sinapic
acid obtained from Extrasynthese (France) were used as external standards. For flavone glycosides
contained in the extract a solvent system contained water/acetonitrile/acetic acid 79.5:0.5:20
(v/v/v) was used for a run time of 25 minutes at 280 nm UV-light. External standards (narirutin,
hesperidin) purchased from Extrasynthese (France) were used as external standard. Finally, an
isocratic solvent system contained methanol 0.1% orthophosphoric acid and at 245 nm UV-light
was used to detect ascorbic acid contained in the extract. Anthocyanins class compounds were
identify using a UV-spectrophotometric method (UV-1700, PharmaSpec, Shimadzu). Moro extract
was solubilized in a 1% HCl methanol solution and, afterwards, Moro extract solution was diluted
with a pH=3 buffer solution (citric acid/sodium phosphate dibasic). Anthocyanins were detected at
520 nm and identified by using cyanidin-3-glucoside chloride (Extrasynthese, France) as external
standard.
3.3 Human clinical study
3.3.1 Subjects
A randomized, placebo, double blinded clinical trial was performed on 60 healthy volunteers (aged
21-50 years old), recruited after medical screening including the completion of a health
questionnaire followed by physical examination. Inclusion criteria was: a body mass index (BMI)
between 25-35 Kg/m2, not taking any drugs or dietary food supplement during the
experimentation. Moreover, volunteers maintained their previous daily physical exercise and
eating habits (1500-2000 cal/day) without any particular changes or dietetic program during the
clinical study. Excluding criteria was: pregnancy, smokers, having a history of thyroid disease,
cardiovascular disease, diabetes, using weight loss medications, laxative or diuretic, recent
unexplained weight loss or gain, taking medications during the previous months (including vitamin
and antioxidant supplement) or any significant dysfunctions. After pre-inclusion, volunteers were
fully informed of the nature of the study, substances and procedures involved, they gave their
written informed consent before participation.
3.3.2 Treatment protocol
The volunteers were assigned by randomization into two groups of 30 peoples. The placebo group
received a dietary supplement of one tablet per day, between meals, containing 400 mg of
maltodextrin while the MoroEx group received one tablet per day, between meals, containing 400
mg of Moro juice extract. During the clinical trial, subjects were evaluated at the beginning of the
study (T0), after 2 weeks (T1), 4 weeks (T2), 8 weeks (T3) and 12 weeks (T4) of treatment. At each
time point, subjects were monitored for several anthropomorphic parameters such as body weight
and body mass index (BMI); waist circumference and hip circumference were monitoring at the
beginning and the end-point of the study. Vital signs such as blood pressure were measured to
monitored the general health of the subjects and any side effects observed were recorded during
each visit.
3.3.4 Statistical analysis
All data obtained were submitted to a statistical analysis. All statistical comparisons in data were
evaluated using Kruskal-Wallis test and difference between groups were tested using the MannWhitney U test. A p value of less than 0.05 was considered significant.
Reference.
Rapisarda P, Carollo G, Fallico B, Tomaselli F, Maccarone E. 1998. Hydroxycinnamic Acids as
Markers of Italian Blood Orange Juices. J. Agric. Food Chem. 46: 464-470.