ADAPT: Efficacy of a Dietary Supplement During and After Exercise

The Nrf-2 pathway is the major cellular defence against oxidative and electrophilic stresses. Briefly, following dissociation from Keap1 (a scaffolding protein which binds Nrf2 and Cul3 ubiquitin ligase for proteasome degradation), Nrf-2 accumulates in the nucleus where it activates the antioxidant response element (ARE) in the promoter region of many antioxidant genes, including catalase, glutathione peroxidases (GPX), and superoxide dismutase (SOD). As such, it is an attractive target for both dietary and pharmacological interventions aiming to increase resilience to oxidative stress. Most diet-derived bioactive Nrf-2 inducers contain Michael acceptor groups (an α, β-unsaturated carbonyl moiety). Curcumin, for example, contains two Michael acceptor groups and is a known activator of Nrf-2. An additional bioactive with a Michael accepter group is trans-cinnamaldehyde (CA) (a component found in the stem bark of Cinnamomum cassia), which, like curcumin, is an Nrf-2 activator in a dose-dependent manner.

Therefore, the purpose of this study will be to assess the effect of bioactive Nrf-2 inducers, based on the presence of an α, β-unsaturated carbonyl moiety, on resilience markers such as antioxidant enzyme activities and markers of oxidative stress, such as TBARS, to an exercise challenge. The hypothesis is that specific plant bioactive increase the production of antioxidant enzyme activities at baseline, and thereby decrease levels of TBARS upon an exercise challenge. Ultimately, the use of novel resilience markers and signatures could significantly improve the ability to prove the efficacy by which pro-oxidant dietary bioactive improve lifelong health, as well as targeting those with lowest resilience as a way to make best use of limited healthcare resources.

The intervention supplement will consist of a mix of dietary bioactive with an α, β-unsaturated carbonyl moiety (provided in individual capsules), of which the efficacy to beneficially affect platelet activity, with and without a H2O2 (Hydrogen Peroxide) challenge, was recently established: sulforaphane (SUL), green tea extract (epigallocatechin gallate) (EGCG) and cinnamaldehyde (Cin).

It is intended to use a mix of dietary bioactive for the proposed human intervention study, as different compounds could affect multiple pathways and therefore, have a broader efficacy. In addition, bioavailability of compounds may be influenced by presence of other compounds, as has been suggested in the literature (effect of piperine on EGCG bioavailability).

Bioavailability data for each of these compounds were extracted from the literature and these have been used, alongside practical considerations, to decide on the dosing strategy. The desired composition of the supplement mix has been set as follows:

- SUL: ~ 14mg

- EGCG: ~ 400mg

- CIN: ~ 15mg These daily intakes should be sufficient to achieve micromolar quantities in blood plasma for between 1 and 6 hours after ingestion, and perhaps longer. .

Suitable food ingredients which can be used in the study to achieve these intakes have been sourced from reputable suppliers. These ingredients will be used to give desired intakes as follows:

- EnduraCell broccoli sprout powder (with added myrosinase): 1.5g (sufficient to provide ~ 45mg glucoraphanin, the precursor to SUL, which should release approximately ~14mg SUL).

- Bulk Powders Green tea extract: ~ 900mg (containing approximately 400mg EGCG).

- Bulk Powders Cinnamon Bark Extract: 0.75g (containing ~15 - 20mg Cin).

Due to envisaged difficulties with palatability, the ingredient mix will be encapsulated. While one of the ingredients (green tea extract) is available pre-encapsulated from the supplier, it is preferred to obtain all ingredients as free powder, and encapsulate them into size 00 capsules at Science in Sport. This will avoid difficulties with placebo blinding, as the capsules can all be given a similar appearance. Tests indicate that daily doses of cinnamon bark extract and of green tea extract will be represented by 2 capsules, each containing 375mg (cinnamon bark extract) and 450mg (green tea extract), while a daily dose of broccoli sprout powder will be represented by 6 capsules containing 250mg each. Placebo capsules will be prepared using food grade microcrystalline cellulose. Microbial testing will be carried out on all supplements.

For presentation to volunteers, the daily capsules will be divided into two servings, one serving to be taken in the morning with breakfast, and one serving to be taken in the evening with dinner. Each serving containing 5 capsules, as follows:

- EnduraCell broccoli sprout powder: 3 capsules

- Bulk Powders Green tea extract: 1 capsule

- Bulk Powders Cinnamon Bark Extract: 1 capsule

One serving of the placebo intervention contains:

• Placebo (microcrystalline cellulose): 5 capsules

Volunteers will take 5 servings just prior to the exercise challenge tests as follows:

Day -2: two servings (one in the morning and one in the evening) Day -1: two servings (one in the morning and one in the evening) Day 0: one serving (one in the morning with the first meal)

Each serving of capsules will be individually bagged and labelled with a unique identifier. Volunteers will be instructed to take a serving of capsules twice per day, with a small volume of water. They will be asked to record the unique identifier of each capsule serving after consumption, along with the time at which it was consumed. Compliance will be monitored via this daily supplement log, as well as by measuring plasma SUL metabolites.

Volunteers will be encouraged to minimise, as far as is possible, a high intake of products rich in sulforaphane (cruciferous vegetables such as Brussels sprouts, broccoli, cabbage, cauliflower, bok choy, kale, collards, broccoli sprouts, chinese broccoli, broccoli raab, kohlrabi, mustard, turnip, radish, rocket), epigallocatechin gallate (green tea) and cinnamaldehyde (cinnamon), during the 2.5 days prior to the challenge test days.