2.1. Study Design and Participants
A randomized, double-blind, placebo-controlled study was conducted at the Health Sciences Department of Universidad Católica San Antonio de Murcia (UCAM), in Murcia, Spain. Participants were mainly recruited by advertising the study through social networks and mass media. Inclusion criteria were as follows: Caucasian men and women; age between 18 and 65 years; not belonging to the group of people considered as priorities by the Third Joint Task Force for the prevention of CVD due to high risk [22
]; serum cholesterol levels >200 mg/dL and/or LDL-C levels >130 mg/dL without pharmacological treatment; cardiovascular risk according to the Systematic Risk Evaluation (SCORE) risk chart [23
] of less than 5% for an ischemic event in a 10-year period; not receiving any pharmacological or nutraceutical treatment for any cardiovascular risk factor (hypertension, diabetes, hyperlipidemia); absence of any treatment affecting weight or appetite; and absence of thyroid gland diseases, heart diseases, renal dysfunction, liver dysfunction, lung diseases, or neurological disorders. Exclusion criteria were as follows: the use of any dietary supplement in the last 3 months, willingness to follow a diet during the study, alcohol abuse or consumption of more than three glasses of wine/beer per day, chronic terminal illness, and ineligibility as judged by the investigators.
The study protocol was approved by the Ethics Committee of Universidad Católica San Antonio de Murcia (code F-PR-AC05-01-05, approval date 23 February 2007) (Murcia, Spain) and was registered in the ClinicalTrials.gov (NCT04330937). Written informed consent was obtained from all participants.
2.2. Intervention and Study Variables
Participants were randomly assigned to the intervention group (dietary intervention with the nutritional supplement) or to the control group (supplementation with placebo) using a computer-generated table of random numbers but ensuring homogeneity of the study groups regarding age, sex, assessment of cardiovascular risk, and serum total cholesterol levels. The active product was a commercially available nutritional supplement (Citrolive™, iff-Murcia Natural Ingredients, Site Plant Nutrafur, Alcantarilla, Murcia, Spain) based on the combination of two hydroethanolic extracts, one from bitter orange (Citrus aurantium L.), flavanones (naringin, neohesperidin, neoeriocitrin, and hesperidin) and flavones (luteolin-7-glucoside, apigenin-7-glucoside, diosmetin-7-glucoside, luteolin, and diosmetin), and one hydroethanolic extract from olive leaf (Olea europaea
L., olive secoiridoids, oleuropein family, hydroxytyrosol, etc.). The quantitative composition and absolute content (% w/w
, according to the corresponding standards) of the main bioactive ingredients have been previously reported [20
Citrolive™ and each individual ingredient were classified according to the Global Harmonized System (GHS) into category 5 (unclassified substance or of very low toxicity). Subjects in the intervention group were instructed to take two capsules a day (2 × 500 mg each), 12 h apart, for 90 days. Subjects in the control group received identical-appearing placebo capsules (maltodextrin) and followed the same regimen. All patients were advised to maintain their usual diet and the level of physical activity during the study.
Participants were visited at baseline and at 90 days (final visit). At baseline, written informed consent was obtained, fulfilment of the inclusion criteria was checked, and the study product was provided. Clinical assessments included detailed medical history, comorbidity, physical activity, complete physical examination, and cineanthropometric study based on the model of De Rose and Guimaraes [24
] using a scale with a height rod (Seca 220; sensitivity ±5 mm height, ±100 g weight), Holtain skinfold caliper and a Holtain pachymeter, and an inextensible tape measure. All anthropometric measurements were performed in compliance with the International Society of Advancement of Kinanthropometry protocol [25
]. Anthropometric variables were as follows: weight, height, body mass index (BMI), waist-to-hip ratio, sum of skinfold thicknesses (subscapular, triceps, suprailiac, abdominal, anterior thigh, medial leg), fat mass (using the Carter, Yuhasz and Faulkner equations for women and men, respectively), skeletal mass (through Von Döbeln equation modified by Rocha), residual mass (through Würch equation), and muscle mass (as the difference between total mass and the sum of fat, skeletal and residual mass). Air displacement plethysmography (BOD POD model 2000A, Life Measurements Instruments, Concord, CA, USA) was used to measure body fat. Participants also completed a 3-day food record including 2 weekdays and either a Saturday or Sunday. All these variables were recorded at the end of the study and at the final visit at 90 days.
Venous blood samples were taken after 12 h of fasting at each of the visits (baseline and 90-day visit). Laboratory studies included blood cell count, cholesterol, triglycerides, LDL-C, HDL-C, glucose, creatinine, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyl transpeptidase (GGT) using the clinical chemistry analyzer ILAB 600 (Instrumentation Laboratory). Serum apolipoproteins A1 (apo A1) and B (apo B) were determined using an immunoturbidimetric assay based on the measurement of immunoprecipitation at a wavelength of 340 nm as described by Riepponen et al. [26
]. Oxidized LDL (ox-LDL) was measured by Human Oxidized LDL ELISA kit (Elabscience Biotechnology Inc., Houston, TX, USA), and paraoxonase/arylesterase 1 (PON1) by a spectrophotometric technique according to the method described by Ferré et al. [27
2.3. Statistical Analysis
The sample size was calculated according to the ox-LDL as the main variable of the study. Considering a standard deviation of ox-LDL levels of 4 U/L reported in a similar population [28
], for a precision of 4 U/L with an alpha risk of 5% and statistical power of 80%, 13 subjects in each group were needed, increasing to 15 subjects per group assuming a 15% loss to follow-up.
The per-protocol (PP) data set of all participants who completed the 90-day study period was analyzed. Categorical variables are expressed as frequencies and percentages and continuous variables as mean and ± standard deviation (SD). Data analysis included the chi-square (χ2) test or the Fisher’s exact test for comparison of categorical variables between the study groups, and the analysis of variance (ANOVA) for repeated measures, with time (baseline and final) as the within-subject factor and intervention (active nutritional supplement and placebo) as between-subject factor. Statistical significance was set at p < 0.05. SPSS version 21.0 (IMB Corp., Armonk, NY, USA) was used for statistical analysis.