Study protocol
This observational pilot study was designed in agreement with the Declaration of Helsinki and approved by the local Ethical Committee. All subjects volunteered to the study and gave their informed consent.
The enrolled subjects were asked to fill out an anonymous questionnaire in order to obtain information about their knowledge and/or personal experience with plant-derived nutritional supplements. Those who declared to consume any of these products were included in the study as “users” who were asked to provide a blood sample for laboratory analysis.
Subjects
Over a period of 6 months, 740 trained subjects (420 body builders, 70 cyclists, and 250 fitness athletes) were enrolled in the study.
All subjects have been training regularly for at least 1 year, 1–2 hours per day, 3–6 days per week and most of them had practiced the same, or other, sports in the past. All subjects, through the compilation of the anonymous questionnaire, denied the consumption of any prohibited substances.
Athletes were instructed to abstain from caffeine, alcohol and drug consumption and to refrain from any strenuous physical activity for 24 hours before the examination that consisted of a blood sampling in the morning (08:00 h, after an overnight fast) and a medical evaluation which included a detailed familiar, medical and sportive personal history and a complete physical examination.
Laboratory analysis
Of the 740 athletes who completed the questionnaire, 26 declared to use plant-derived supplements and 23 of them gave their consent for the blood sample collection. Subjects n° 1, 3, 4 and 9 in Figure 1 and subjects n° 2, 3 and 5 in Figure 2 consumed, for at least 6 months 1,5 gr/die of a commercially available product containing Caffein, Citrus A., Zingiber, Guggul, Cacao, Naringina and Bioperine. Subjects n° 2, 5,and 6 in Figure 1 and subjects 1, 4, 9 and 12 in Figure 2 consumed, for at least 1 year, 3 gr/die of a commercially available product: 5-Methyl-7Methoxyisoflavone, 7-Isopropoxyisoflavone, 20-Hydroxyecdysone, Secretagogues, Triboxybol, Saw Palmetto extract, Beta Sitosterol, Pygeum extract, Guarana extract and Cordyceps extract. Subjects n° 7 and 8 in Figure 1 and subjects n° 6 and 8 in Figure 2 consumed, for at least 1 year and at different dosages, a commercially available product containing Rhaponticum Carthamoides extract (in 1 case, subject 6 in Figure 2, associated with another commercially available product containing Ajuga Turkestanica and Rhaponticum Carthamoides root extract). The remaining subjects consumed high doses of soy derived proteins (2–2.5 gr/kg/die for at least 1 year in some cases associated with Muira Puama and/or Gotu Kola extracts). All subjects also consumed daily different proportions of vitamins, proteins and branched-chain amino acids.
In addition, 30 subjects matched for age, gender, sport discipline, body mass index (BMI) and training volume were recruited as controls among those who denied the use of any nutritional supplements were enrolled as controls.
Blood samples were collected in SST II tubes with serum separator gel, immediately frozen and analyzed within the same day. Testosterone, Dehydroepiandrosterone (DHEA), Estrogens, Progesterone, luteinizing hormone (LH), follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), FT3, FT4 and Cortisol were analyzed by the immunometric method (Axym abbott Diagnostic Laboratories, Abbott Park, Illinois, USA). Urea, creatinine, aspartate aminotransferase (GOT), alanine aminotransferase GPT), lactate dehydrogenase (LDH), creatine kinase (CK), gamma glutamyl transpeptidase (GGT), alkaline phosphatise (APH), total and partial bilirubin, were measured spectrophotometrically by clinical-chemistry analyzer Integra 800 (Roche).
GC/MS analysis
Since androgenic anabolic hormones have been frequently detected in steroid-free nutritional supplements [15, 16], three natural products, used by 20 out of the 23 users, (Animal Stuck, EnerZona soy protein and TestostroGrow) were analyzed at the World Anti-Doping Agency (WADA) accredited Laboratory of Rome, following the appropriate authorization, to test their eventual contamination by steroid hormones for a total of 20 different hormones.
Sample Preparation: 1 g of powder was dissolved in carbonate buffer (PH:9), 50μL of internal standard (17 α-methyl-testosterone, final concentration 500 ng/mL) were added and the extraction was performed with 10 mL of pentane in a multimixer for 5 minutes. The organic layer was separated and evaporated under nitrogen at 70 °C. The dry residue was derivatized using 50μL of TMSJ at 75° C for 30 minutes. 2 μL of the derivatized layer were injected into a gas cromatograph connected to a mass spectrometer.
Instrumental Conditions: GC/MS was performed on an HP 6890 mass selective detector (Agilent Technologies, Tokio, Japan) connected with a 5973 quadruple mass spectrometry, with ionization energy modality, at 70 eV and SIM acquisition. The fused-silica capillary column used was HP1 with 0.20 mm diameter and 0.11 μm film thickness). Helium was used as a carrier gas (flow rate: 1 mL/min, split ratio 1:10).
Statistical analysis
Database management and all statistical analyses were performed using the Statistica 6 for Windows software package (Statsoft Inc., Tulsa, OK). Normality of data was assessed by the Wilk-Shapiro’s test. Differences were analysed by means of the two-tailed Student’s t test. If a significant difference was present, a Dunn’s post hoc test was used to locate the difference. Levels of statistical significance were set to p < 0.05.