Investigating the Efficacy of Low or High Dose Tart Cherry on Recovery From Exercise Induced Muscle Damage and Establishing Its Molecular Mechanisms of Action

Study Overview
=================
Brief Summary
-----------------
To determine the dose-response effects of 10-day tart cherry product consumption (0, 30 ml, and 60 ml) on knee extensor isometric strength 24-h and 48-h after muscle damaging exercise and to elucidate the mechanisms of action for TC supplementation.

Detailed Description
-----------------
Montmorency cherry supplementation has been shown to exert antioxidant and anti-inflammatory effects that can be beneficial for improving recovery from exercise. However, the investigator is currently not aware of how tart cherry polyphenol supplementation produces these effects. This study aims to determine the dose-response effects of 10-day tart cherry product consumption (placebo, 30 ml, and 60 ml Montmorency tart cherry concentrate in a 500 ml beverage) on knee extensor isometric strength 24-h and 48-h after muscle damaging exercise on supplementation Day 8; on enhancing recovery of other measures of muscle function (single leg eccentric and concentric force development and single leg jump height) and muscle soreness; on reducing markers of oxidative stress and inflammation in plasma, muscle and urine; on inducing signaling in muscle via the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway to upregulate endogenous antioxidant enzymes in muscle; on inhibiting muscle cyclo-oxygenase (COX-1 and COX-2). This study also aims to identify and establish the molecular mechanisms of action through which tart cherry polyphenols exert antioxidant and anti-inflammatory effects. Oxidative stress and inflammatory signaling in primary human myogenic cells will be assessed by incubating primary human myogenic cells (commercial cell line) in sera derived from 6 participants consuming 8-day placebo vs. 60 ml/day Montmorency tart cherry concentrate supplement. Subjects are permitted to participate in both the molecular mechanism of action part of the study (Part A, no exercise component) and the damaging exercise muscle recovery part of the study (Part B). For 60 ml doses in Parts A and B, global proteomics analysis of the muscle tissue will be conducted generate more insight into the mechanisms of action. This would identify the specific pathways that are influenced by cherry supplementation and allow identification of the full range of mechanisms involved, rather than assume antioxidant/anti-inflammatory effects alone.

Official Title
-----------------
Investigating the Efficacy of Low or High Dose Tart Cherry on Recovery From Exercise Induced Muscle Damage and Establishing Its Molecular Mechanisms of Action

Conditions
-----------------
Muscle Recovery

Intervention / Treatment
-----------------
* Other: 500 ml base solution
* Other: 500 ml containing 30 ml tart cherry concentrate diluted with water and base solution
* Other: 500 ml containing 60 ml tart cherry concentrate diluted with water only


Participation Criteria
=================
Eligibility Criteria
-----------------
Inclusion Criteria: Male in good health 18-40y BMI ≤ 29.9 Recreationally active but not highly trained (Participating in sport or physical activity at least once per week for the last 6 months and lower limb resistance training ≤ 1/week) (Part B only) No lower limb musculoskeletal injury that prevented exercise in the last 3 months (Part B only) Understanding of the procedures to be undertaken as part of the study Willing to participate in the exercise protocols (Part B only) and follow supplementation guidelines and other instructions provided by the experimenter Willing to abstain from caffeine, alcohol and strenuous exercise for two 5 day periods (from 48h prior to muscle damaging exercise and throughout the follow up period; days 6-10 of the trial) for Part B, and abstain from caffeine and alcohol for two 3-day periods for Part A Willing to keep food diaries for the duration of each arm of the trial and avoid multiple servings per day of foods that have a high polyphenol content from a list provided. Willing to construct their diet for the first arm of the trial from a list of recipes provided, and to then replicate this diet during the second arm of the trial. Willing to wear a triaxial accelerometer throughout each arm of the trial, and during the second arm of the trial to replicate their physical activity pattern completed in the first arm of the trial. Willing to abstain from consumption of anti-inflammatory, analgesic medications or those with anti-coagulant effects especially NSAIDs and aspirin for 48h prior to and throughout each arm of the trial. (Part B only) Willing to abstain from icing or applying compression bandages to the exercised/biopsied leg (Part B only) Ability to provide voluntary written informed consent to participate in the study. Exclusion Criteria: Female <18y or >40y BMI ≥ 30 Inactive or well-trained (see inclusion criteria) (Part B only) Lower limb musculoskeletal injury in the last 3 months (Part B only) Under the care of a physician Known health condition that could interfere with any element of the data collection protocol. Any known pulmonary, gastrointestinal (e.g., irritable bowel syndrome, Crohn's disease), kidney, cardiovascular, or metabolic diseases, blood-borne infections, or coagulopathies including deep vein thrombosis (DVT) Current regular consumption of nutritional/dietary supplements or previous use of any dietary supplements in the past 6 months known to have a lasting effect. Current use of prescription medication; including the use of an inhaler Non-steroidal anti-inflammatory drugs, aspirin or other analgesic medication or any anti-coagulants are prohibited during the study and for 48h prior to commencing supplementation Allergy/sensitivity/intolerance to cherries, any cherry derived product, or any of the additional ingredients found within the TC test products, including sensitivity to sorbitol. Allergy to lidocaine or other local anaesthetics. (Part B only) Active drug or alcohol dependency Current or active usage of nicotine or tobacco related products Participation in a clinical trial within the past 30 days or concurrently during this trial Participation in any PepsiCo trial within past 6 months, except that Part A participants may take part in Part B after a >=14 day washout from Part A Inability to provide written informed consent

Ages Eligible for Study
-----------------
Minimum Age: 18 Years
Maximum Age: 40 Years

Sexes Eligible for Study
-----------------
Male

Accepts Healthy Volunteers
-----------------
Accepts Healthy Volunteers

Study Plan
=================
How is the study designed?
-----------------

Design Details

Primary Purpose: Basic Science
Allocation: Randomized
Intervention Model: Crossover Assignment
Interventional Model Description: Repeated measures, double-blind, counter-balanced
Masking: Quadruple


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Placebo Comparator: 0 ml tart cherry concentrate<br>Still cherry-flavored beverage, similar in appearance, taste, aroma and calories to the 30 ml and 60 ml tart cherry concentrate beverages | Other: 500 ml base solution<br>* Color, taste, aroma, and calorie matched water base<br>|
| Experimental: 30 ml tart cherry concentrate<br>Low dose tart cherry concentrate beverage | Other: 500 ml containing 30 ml tart cherry concentrate diluted with water and base solution<br>* Montmorency cherry concentrate<br>|
| Experimental: 60 ml tart cherry concentrate<br>High dose tart cherry concentrate beverage | Other: 500 ml containing 60 ml tart cherry concentrate diluted with water only<br>* Montmorency cherry concentrate<br>|

What is the study measuring?
-----------------
Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Isometric knee extensor strength | Maximum voluntary contraction (MVC) as a measure of functional recovery from muscle damage. Unit of measurement is Newtons. Stronger is better. | Changes from immediately before and immediately after muscle damaging exercise on day 8 of tart cherry supplementation to 24 hours and 48 hours after muscle damaging exercise (days 9 and 10 of supplementation). | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Single leg eccentric and concentric isokinetic force production | Dynamic muscle strength measured by isokinetic contraction (IKC, 60 deg.s-1). More forceful is better. | Changes from immediately before and immediately after muscle damaging exercise on day 8 of tart cherry supplementation to 24 hours and 48 hours after muscle damaging exercise (days 9 and 10 of supplementation). | 
| Single leg vertical jump height (SLVJ) | Dynamic muscle strength measured by jump height (cm). Higher is better. | Changes from immediately before and immediately after muscle damaging exercise on day 8 of tart cherry supplementation to 24 hours and 48 hours after muscle damaging exercise (days 9 and 10 of supplementation). | 
| Leg muscle soreness by Pressure Pain tolerance (PPT) | Pressure pain tolerance (PPT) measured by algometer. Higher tolerance is better. | Changes from immediately before and immediately after muscle damaging exercise on day 8 of tart cherry supplementation to 24 hours and 48 hours after muscle damaging exercise (days 9 and 10 of supplementation). | 
| Leg muscle soreness by Visual Analogue Scale (VAS) | Measured using a visual analogue pain scale from 0 mm (no pain) to 100 mm (extremely painful). Lower score is better. | Changes from immediately before and immediately after muscle damaging exercise on day 8 of tart cherry supplementation to 24 hours and 48 hours after muscle damaging exercise (days 9 and 10 of supplementation). | 
| Leg muscle biopsy for nuclear factor k-B (NFκB) | Inflammation marker expression activity of the NFκB pathway will be measured by immunoblotting. Lower is better. | Changes from pre and post exercise on day 8 of tart cherry supplementation to pre-exercise on days 9 and 10 of supplementation. Biopsies are done after blood samples and before muscle soreness measures. | 
| Leg muscle biopsy for cyclo-oxygenase COX1 protein | Inflammation marker of cyclo-oxygenase measured by total protein carbonylation (immunoblotting). A reduction (inhibiting) is better. | Changes from pre and post exercise on day 8 of tart cherry supplementation to pre-exercise on days 9 and 10 of supplementation. Biopsies are done after blood samples and before muscle soreness measures. | 
| Leg muscle biopsy for cyclo-oxygenase COX1 activity | Inflammation marker of cyclo-oxygenase activity measured by commercially available colorimetric assay. A reduction (inhibiting) is better. | Changes from pre and post exercise on day 8 of tart cherry supplementation to pre-exercise on days 9 and 10 of supplementation. Biopsies are done after blood samples and before muscle soreness measures. | 
| Leg muscle biopsy for nuclear factor erythroid 2-related factor 2 (Nrf2), absolute expression, nuclear translocation | Oxidative damage marker Nrf2 pathway intended to upregulate endogenous antioxidant enzymes in muscle. Induction of signaling is better. | Changes from pre and post muscle damaging exercise on day 8 of tart cherry supplementation to pre-exercise on days 9 and 10 of supplementation. Biopsies are done after blood samples and before muscle soreness measures. | 
| Leg muscle biopsy for global proteomics analysis of muscle tissue samples for the 60 ml dose only | Tissue analysis to identify pathways influenced by cherry supplementation and identify mechanisms of action. | Changes from pre and post muscle damaging exercise on day 8 of tart cherry supplementation to pre-exercise on days 9 and 10 of supplementation. Biopsies are done after blood samples and before muscle soreness measures. | 
| Leg muscle biopsy for endogenous antioxidant enzyme expression | Enzymes (superoxide dismutase 1, superoxide dismutase 2, glutathione peroxidase 1 and catalase) protein expression quantified by immunoblotting. Higher values (upregulation by Nrf2) is better. | Changes from pre and post muscle damaging exercise on day 8 of tart cherry supplementation to pre-exercise on days 9 and 10 of supplementation. Biopsies are done after blood samples and before muscle soreness measures. | 
| Leg muscle biopsy for immune cell infiltration | Inflammation marker response measured by immunohistochemistry. Less infiltration is better. | Changes from pre and post muscle damaging exercise on day 8 of tart cherry supplementation to pre-exercise on days 9 and 10 of supplementation. Biopsies are done after blood samples and before muscle soreness measures. | 
| Plasma interleukin-6 (IL-6) | Plasma inflammatory stress response measured by ELISA. A reduction is better. | Changes from pre and post muscle damaging exercise on day 8 of tart cherry supplementation to pre-exercise on days 9 and 10 of supplementation. Pre exercise blood sampling is done after urine collection. Post exercise blood sampling done before biopsy. | 
| Plasma protein carbonyls | Oxidative stress response and antioxidant status measured by ELISA. A reduction is better. | Changes from pre and post muscle damaging exercise on day 8 of tart cherry supplementation to pre-exercise on days 9 and 10 of supplementation. Pre exercise blood sampling is done after urine collection. Post exercise blood sampling done before biopsy. | 
| Plasma phenolic concentration | LC-MS-MS metabolite analysis to evaluate plasma levels associated with tart cherry supplementation at 0, 30 and 60 ml/day. | Changes from pre and post muscle damaging exercise on day 8 of tart cherry supplementation to pre-exercise on days 9 and 10 of supplementation. Pre exercise blood sampling is done after urine collection. Post exercise blood sampling done before biopsy. | 
| Urine oxidation-reduction potential (ORP) | Urinary oxidation-reduction potential to measure oxidative damage and antioxidant status measured by probe. Less oxidative stress/free radical production is better. | Change from pre-exercise value day 8 to days 9 and 10 of tart cherry supplementation. Collected upon arrival to the study site. | 

 Terms related to the study
=================
Keywords Provided by Centre Hospitalier Valida
-----------------
cherry, tart cherry, Montmorency cherry, beverage, exercise, muscle recovery, muscle soreness, muscle damage, muscle function, muscle strength, muscle biopsy, mechanism, oxidative stress, inflammation, anthocyanin, polyphenol, antioxidant, cyclo-oxygenase, myotube, mygenic culture