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Dr. Gabriella Ambrosio

Dissertation: Ecdysterone Use and Misuse in Sports: Diagnostic Markers in Human Specimen and Determination in Supplements

The focus of this study is ecdysterone, a steroid hormone naturally occurring in plants and invertebrates [19, 20]. Ecdysterone is contained in a wide range of dietary supplements available on internet, and there are many rumors on ecdysterone misuse by athletes. Ecdysterone is not yet included in the list of prohibited substances and methods in sports, even if previous studies reported growth promoting and anabolic effects of ecdysterone in various animal species and in humans [28-45], mediated by its interaction with ERbeta [29, 46, 47]. With the exception of few studies [32, 119, 120], the evaluation of the use of ecdysterone supplementation to improve performances and study on its metabolism, have not yet been extensively investigated in humans. As part of this project, an intervention study of strength training of 46 healthy males has been carried out. The evaluation of a long-term administration of an ecdysterone containing dietary supplement has been made, with a special focus on the enhance of the performance during resistance training. Different doses of a supplement labelled to contain 100 mg of ecdysterone per capsule were administered to volunteers for ten weeks. This study included two training groups, Ec1 and Ec2 , which took respectively two and eight capsules per day, one non-training COG, which took two capsules per day (similar to Ec1), and one PLG, which was training taking a supplement not containing any ecdysterone. This study considered and evaluated anthropometrical and performance parameters after ten weeks of administration of ecdysterone containing supplements, and as results, reports an increase in body weight, muscle mass and upper body strength performances. In addition, the in-vitro study performed, shows a hypertrophy in C2C12 myotubes after 48 hour of treatment with supplement extract. The liver and kidney toxicity of ecdysterone is excluded by evaluation of specific biomarker in serum; changes in the serum concentration of endocrine hormones IGF-1 and T4 are observed. Even if further investigations will be needed, also considering other endocrine hormones, it seems that ecdysterone can antagonize the decrease in IGF-1 levels and that intensive training may have an influence on T4. In serum samples, a dose dependent increase of ecdysterone is reported after a successful development and validation of an UHPLC-MS/MS method. After a complete anti-doping screening, no other prohibited substance has been detected in the post administration urines as well as in the supplements administered to volunteers, demonstrating that the obtained effects on performance are resulting from consumption of ecdysterone. Following evaluation of the steroidal profile, it is proved that ecdysterone cannot be detected by changes in parameters of the steroidal module. This suggests that the anabolic effect of ecdysterone is based on a different mechanism than other AAS. From the analysis of the supplement administered to volunteers results that only 6% of the labelled content of ecdysterone is present in the supplement and there is no presence of other performance enhancing substances. After a single dose administration of pure ecdysterone, its metabolism has been preliminary evaluated. Ecdysterone does not undergo phase II metabolism by conjugation with glucuronic or sulfuric acid. The 14-desoxy-metabolite is identified in post-administration urines through analysis with LC-QTOF-MS and confirmed by comparison with the MS/MS spectra of the synthetized 14-desoxy-ecdysterone. As result, ecdysterone (the most abundant analyte detected in post-administration urines) and the 14-desoxy-ecdysterone can be used for initial testing procedures for monitoring of ecdysterone administration in sport. Even more, excretion profiles of both ecdysterone and the 14-desoxy metabolite have been delineated. Finally, since the use of dietary supplements is increasing and their quality is not always guaranteed to consumers, due to the lack of adequate and uniform regulations, an UHPLC-MS/MS method for quantitation of 12 supplements labelled to contain ecdysterone has been developed and validated. The results report that in the 67% of the analyzed supplements, the labelled content of ecdysterone is highly inaccurate and, for some of them, the composition on the label is imprecise. Thanks to this project, the WADA has included ecdysterone in the 2020 Monitoring Program. Methods to trace back an administration of ecdysterone in urine and serum are made available together with a method for the unambiguous identification and quantitation of ecdysterone in natural preparations.