An Investigation into the Authenticity of Citrus Aurantium-Listing Pre-Workout Supplements and Functional Evaluations of their Trace Amines on the Vasculature.

  • Andy Koh

Student thesis: Doctoral Thesis


Pre-workout supplements (PWS) are stimulatory multi-ingredient dietary supplements used to improve athletic performance. The stimulatory effects of these supplements typically arise from ingredients such as caffeine and phenethylamine alkaloids, commonly derived from a variety of plant extracts such as Citrus aurantium L. (Bitter orange). It contains natural phenethylamines such as synephrine, octopamine, and tyramine, which are structurally related to ephedrine. Additionally, these amines exist in trace amounts in the mammalian system and are called trace amines. The safety of PWS has come into question following reports of cardiovascular adverse events associated with PWS consumption, such as arrhythmias, myocardial infarctions, and aortic dissections. While there is some evidence that the inclusion of C. aurantium extracts is beneficial for weight loss and exercise, the distinct mechanisms of action of each trace amines on different components of the mammalian vascular system remain poorly understood.

PWS are categorised and regulated as complementary medicines in Australia. Manufacturers are not bound to provide evidence for their efficacy and have much less stringent labelling requirements than pharmaceutical drugs. Additionally, manufacturers of PWS are not required to reveal the quantities of stimulatory ingredients on the label when part of a proprietary blend, which makes it difficult to determine, which ingredient contributes to an ergogenic effect. However, trace amines in C. aurantium generally exhibit a unique ratio of amines and a predominance of a particular enantiomer, which can be useful in differentiating from other non-natural sources. Hence, two main aims were developed to answer a set of questions raised from these observations.

The first aim was to investigate the comparative vascular effects of trace amines (synephrine, octopamine and tyramine) found in C. aurantium-listing PWS on three main arteries (mesenteric, coronary, and renal) that contribute to blood pressure. In vivo models have been previously explored but have failed to yield a clear explanation of the mechanisms of action of the trace amines on the vascular smooth muscle tissue. Therefore, the scope of the study was limited to the determination of vascular effects on isolated porcine tissues and elucidated their respective mechanisms of action with functional assays involving various antagonists, including the recently developed TAAR1 antagonist EPPTB. These studies concluded that the three trace amines elicited their contractile effects from complex mechanisms of action that was different across the three selected arteries investigated. Interestingly, the contractile responses to synephrine were affected by TAAR1 blockade, suggesting that synephrine may mediate its effects via this pathway in addition to the direct α1-adrenoceptor activity.

The next aim of the study was to determine the levels of trace amines found in C. aurantium-listing PWS and whether they correlate with their labels and are from an authentic natural source. Two quantitative analytical methods for assessing the authenticity of C. aurantium-containing PWS were developed and validated using HPLC-UV-MS. The study first compared the ratios of the trace amines in PWS to that found in C. aurantium. Secondly, the compared enantiomeric ratio of synephrine in the PWS sample set to that found in C. aurantium standardised reference materials. These studies showed that only three out of twelve evaluated C.aurantium PWS contained authentic sources of plant material. Additionally, thevalues of the amines were not consistent with their labelled amounts.

In conclusion, this thesis showed that the combined results of total synephrine quantified in C. aurantium PWS could elicit vascular responses, whereas there were negligible amounts of octopamine and tyramine. Therefore, some of the observed adverse cardiovascular events may be attributed to synephrine in C. aurantium PWS, but further vascular effects from other stimulants such as caffeine or synthetic sympathomimetics need further exploration. The variability of stimulant levels in PWS carries a risk to consumers, particularly those with cardiovascular comorbidities such as obesity.
Date of Award15 Jun 2022
Original languageEnglish
SupervisorAnna Lohning (Supervisor) & Russ Chess-Williams (Supervisor)

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