METHODS FOR IMPROVING PHYSICAL EXERCISE PERFORMANCE

Abstract

The present invention relates to compositions, uses and methods for improving physical exercise performance, and/or improving adaptation to physical exercise, and/or modulating the concentration of lactate and/or glucose in the blood of a human subject.

Claims

1. (canceled)

2. A method for improving physical exercise performance and/or adaptation to physical exercise in a human subject, comprising the step of treating the human subject with a composition comprising sulforaphane, and/or glucoraphanin and/or myrosinase.

3. The method according to claim 2, wherein the human subject is administered a composition comprising sulforaphane.

4. The method according to claim 2, wherein the human subject is administered a composition comprising glucoraphanin, or glucoraphanin and myrosinase.

5. The method according to claim 2, wherein the composition comprises: broccoli sprouts or an extract thereof; mature broccoli or an extract thereof; Brussels sprouts or an extract thereof; kale sprouts or an extract thereof; kale or an extract thereof; cabbage or an extract thereof; cabbage spouts or an extract thereof; cauliflower or an extract thereof; cauliflower sprouts or an extract thereof; broccoli raab or an extract thereof, broccoli raab sprouts or an extract thereof; red kale or an extract thereof red kale sprouts or an extract thereof; kohlrabi or an extract thereof kohlrabi sprouts or an extract thereof; red mizuna or an extract thereof red mizuna sprouts or an extract thereof; and/or optionally additionally comprises myrosinase.

6. The method according to claim 5, wherein the myrosinase is selected from: brown mustard seeds or an extract thereof; white mustard seeds or an extract thereof; yellow mustard seeds or an extract thereof; rocket or an extract thereof; rocket seeds or an extract thereof; rocket sprouts or an extract thereof; garden cress or an extract thereof; garden cress seeds or an extract thereof; garden cress sprouts or an extract thereof; wasabi or an extract thereof; wasabi seeds or an extract thereof; wasabi sprouts or an extract thereof; daikon or an extract thereof; daikon seeds or an extract thereof; daikon sprouts or an extract thereof; horseradish or an extract thereof; horseradish seeds or an extract thereof; horseradish sprouts or an extract thereof; radish or an extract thereof; radish seeds or an extract thereof; radish sprouts or an extract thereof; or purified or isolated myrosinase.

7. The method according to claim 2, wherein the composition comprises: brown mustard seeds or an extract thereof; white mustard seeds or an extract thereof; yellow mustard seeds or an extract thereof; rocket or an extract thereof; rocket seeds or an extract thereof; rocket sprouts or an extract thereof; garden cress or an extract thereof; garden cress seeds or an extract thereof; garden cress sprouts or an extract thereof; wasabi or an extract thereof; wasabi seeds or an extract thereof; wasabi sprouts or an extract thereof; daikon or an extract thereof; daikon seeds or an extract thereof; daikon sprouts or an extract thereof; horseradish or an extract thereof; horseradish seeds or an extract thereof; horseradish sprouts or an extract thereof; radish or an extract thereof; radish seeds or an extract thereof; radish sprouts or an extract thereof; or purified or isolated myrosinase.

8. The method according to claim 2, wherein the composition comprises broccoli sprouts or an extract thereof and, optionally, mustard seeds or an extract thereof.

9. The method according to claim 2, wherein the composition is for oral administration, and is administered orally to the human subject.

10. The method according to claim 2, wherein the composition comprises sulforaphane and is for intra-peritoneal, transdermal, sublingual or rectal administration or injection, and is administered intra-peritoneally, transdermally, sublingually, rectally or by injection to the human subject.

11. The method according to claim 2, wherein the composition modulates the concentration of lactate in the blood of the human subject.

12. The method according to claim 2, wherein the composition modulates the concentration of glucose in the blood of the human subject, preferably wherein the composition increases the concentration of glucose in the blood of the human subject.

13. The method according to claim 2, wherein the composition is administered once and in a single dose.

14. The method according to claim 13, wherein the human subject is in need, or is desirous, of improved physical exercise performance.

15. The use or method according to claim 13 or 14, wherein the composition comprises between 1 g and 140 g of broccoli sprouts; for example the composition comprises 10 g, 25 g, 50 g, 60 g, 70 g, 80 g, 90 g, 100 g, 110 g, 120 g, 130 g or 140 g of broccoli sprouts.

16. The method according to claim 13, wherein the composition comprises between 0.055 mg and 500 mg of glucoraphanin (optionally between 1.75 mg and 500 mg of glucoraphanin); for example 0.0055 mg, 0.01 mg, 0.025 mg, 0.05 mg, 0.075 mg, 0.1 mg, 0.25 mg, 0.5 mg, 0.75 mg, 1 mg, 1.75 mg, 17.5 mg, 43.75 mg, 87.5 mg, 105 mg, 122.5 mg, 131.25 mg, 140 mg, 157.5 mg, 175 mg, 192.5 mg, 210 mg, 227.5 mg, 245 mg, 300 mg, 400 mg or 500 mg.

17. The method according to claim 13, wherein the composition comprises between 5.9 ?g and 100 mg of sulforaphane (optionally between 0.18768 mg and 100 mg of sulforaphane); for example 5.9 ?g, 10 ?g, 25 ?g, 50 ?g, 75 ?g, 0.1 mg, 0.15 mg, 0.188 mg, 1.188 mg, 4.962 mg, 9.384 mg, 11.261 mg, 13.138 mg, 15.014 mg, 16.891 mg, 18.768 mg, 20.6448 mg, 22.5216 mg, 24.3984 mg, 26.2752 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg or 100 mg.

18. The method according to claim 13, wherein the composition comprises between 0.5 g and 10 g of mustard seeds, for example 0.5 g, 0.75 g, 1 g, 1.5 g, 2 g, 3 g, 4 g, 5 g, 6 g, 7 g, 8 g, 9 g, 10 g of mustard seeds; optionally wherein the mustard seeds are brown mustard seeds.

19. The method according to claim 13, wherein the composition reduces the concentration of lactate in the blood of the human subject.

20. The method according to claim 13, wherein the composition is administered to the human subject before physical exercise, and preferably: less than six hours before; or less than five hours before; or less than three hours before; or less than two hours before; or less than one hour before physical exercise.

21. The method according to claim 13, wherein the composition improves physical exercise performance in the human subject.

22. The method according to claim 13, wherein the composition improves the ability of the human subject to endure oxidative stress resulting from exercise.

23. The method according to claim 13, wherein the composition improves the physical endurance of the human subject during exercise.

24. The method according to claim 13, wherein the human subject is suffering from lactic acidosis.

25. The method according to claim 19 wherein the composition is used in the prevention or treatment of medical conditions associated with increased lactic acidosis and/or fatigue, for example; mitochondrial myopathies (MELAS syndrome), biotin deficiency, bacterial infection in the bloodstream or body tissues (sepsis), glycogen storage diseases, Reye syndrome, short-bowel syndrome, liver failure, hypoxia (for example hypoxia caused by a defect in the heart or blood vessels), bacterial meningitis, thiamine deficiency (especially during TPN), impaired delivery of oxygen to cells in tissues (e.g. from impaired blood flow (hypoperfusion)), bleeding, polymyositis, ethanol toxicity, shock, advanced liver disease, diabetic ketosis, excessive exercise (overtraining), regional hypoperfusion (e.g. bowel ischemia or marked cellulitis), cancers such as Non-Hodgkin's and Burkitt lymphomas, pheochromocytoma, and/or tumour lysis syndrome.

26. The method according to claim 19, wherein the composition is used to reduce lactic acidosis caused by metformin or acetaminophen.

27. The method according to claim 19, wherein the composition is used to reduce increased blood lactate levels caused by Linezolid, Isoniazid, Propofol, Epinephrine, Propylene glycol, Nucleoside reverse-transcriptase inhibitors (for example Abacavir/dolutegravir/lamivudine), Emtricitabine/tenofovir, Potassium cyanide (cyanide poisoning) and/or Fialuridine.

28. The method according to claim 2, wherein the composition is administered daily, for a period of at least two days; preferably for a period of three days, or four days, or five days, or six days, or seven days, or two weeks, or three weeks, or four weeks, or one month, or two months, or three months, or four months, or five months, or six months, or one year, or more.

29. The method according to claim 28, wherein the human subject is in need, or is desirous, of improved adaptation to physical exercise and/or improved physical exercise performance.

30. The method according to claim 28, wherein the composition comprises more than 120 g of broccoli sprouts; preferably more than 150 g of broccoli spouts; for example 200 g of broccoli sprouts.

31. The method according to claim 28, wherein the total amount of broccoli sprouts consumed within a 16-hour period of each day is between 120 g and 200 g; preferably between 150 g and 200 g, for example wherein the human subject consumes two doses of 75 g of broccoli sprouts within a 16-hour period of each day.

32. The method according to claim 28, wherein the composition increases the concentration of lactate in the blood of the human subject; optionally by at least 10%, at least 15%, at least 20%, or at least 24%, compared to the normal blood lactate concentration in the human subject.

33. The method according to claim 28, wherein the composition results in an increase in blood glucose in the human subject.

34. The method according to claim 28, wherein the composition reduces the amount of time the human subject spends in hypoglycaemia.

35. The method according to claim 28, wherein the composition reduces hypoglycaemia associated with intense physical exercise in the human subject.

36. The method according to claim 28, wherein the composition improves adaptation to physical exercise in the human subject.

37. The method according to claim 36, wherein the improved adaptation to physical exercise comprises: increased number and/or density and/or activity of mitochondria in cells of the human subject; and/or increased number of capillaries in the body of the human subject, for example resulting from activation of mitochondrial biogenesis pathways.

38. The method according to claim 28, wherein the composition improves the physical endurance of the human subject during physical exercise and/or improves tolerance to oxidative stress.

39. The method according to claim 36, wherein the improved adaptation to physical exercise results from the body of the human subject adapting to stress resulting from lactate levels.

40. The method according to claim 28, wherein after administration for a period of at least two days, physical exercise performance is improved in the human subject.

41. The method according to claim 32, wherein the composition is used in the prevention or treatment of McArdle's disease, LDH-deficiency, Fructose 1,6-bisphosphatase deficiency, Glucose-6-phosphatase deficiency, GRACILE syndrome, pyruvate dehydrogenase deficiency, pyruvate carboxylase deficiency and/or Leigh syndrome.

42. The method according to claim 2, wherein the human subject is male or is female.

43. The method according to claim 2, wherein the human subject is an athlete.

44. The method according to claim 2, wherein the human subject is desirous, and/or in need, of improved athletic performance.

45. The method according to claim 2, wherein improved physical exercise performance in the human subject comprises one or more of: increased physical output; increased workload; increased maximal oxygen uptake; increased maximal effort; reduced exhaustion; reduced muscle fatigue; reduced hypoglycaemia; maintenance of normal blood glucose; reduced lactic acidosis; a decrease in the time to exhaustion at a constant workload; and/or a decrease in the time taken to complete a specific work (e.g. to run, swim or cycle a specific distance).

46. The method according to claim 2, wherein improving adaptation to physical exercise in the human subject comprises one or more of: maintained maximal heart rate; increased mitochondrial density; increased mitochondrial respiration; increased level of muscular lactate transport; and/or reduced hypoglycaemia.

47. The method according to claim 2, wherein the composition comprises broccoli sprouts (or an extract thereof) and/or mustard seeds (or an extract thereof), wherein the composition has a broccoli sprout (or extract thereof) to mustard seed (or extract thereof) ratio between 100:0 to 1:10; for example a broccoli sprout (or extract thereof) to mustard seed (or extract thereof) ratio of 100:1.

48. The method according to claim 2, wherein the composition comprises 50 g of broccoli sprouts (or an extract thereof) and 0.5 g mustard seeds (or an extract thereof).

49. The method according to claim 2, wherein the composition is administered between two and ten times per day.

50. (canceled)

51. A method for modulating the concentration of lactate and/or increasing the concentration of glucose in the blood of a human subject, comprising the step of treating the human subject with sulforaphane, or with glucoraphanin and/or myrosinase.

52-54. (canceled)

Description

LIST OF FIGURES

[0251] FIG. 1: Effect of acute administration of BSJ on blood lactate concentration after submaximal exercise.

[0252] FIG. 2: Effect of chronic administration of BSJ on blood lactate levels at submaximal exercise.

[0253] FIG. 3: Effect of chronic administration of BSJ on maximal attainable workload during incremental exercise.

[0254] FIG. 4: Effect of chronic administration of BSJ on blood lactate levels in subjects undertaking exercise training

[0255] FIG. 5A to 5C: Effect of chronic administration of BSJ in subjects undertaking exercise training on (A) maximal power output, (B) total time to exhaustion, and (C) maximal oxygen uptake.

[0256] FIG. 6: Effect of chronic administration of BSJ on mitochondrial respiration in subjects undertaking exercise training.

[0257] FIG. 7: Effect of chronic administration of BSJ on levels of NRF2 protein present in skeletal muscles in subjects undertaking exercise training.

[0258] FIG. 8: Effect of chronic administration of BSJ on the time spend in hypoglycaemia in subjects undertaking exercise training.

[0259] FIG. 9: Effect of chronic administration of BSJ on maximal heart rate during maximum work in subjects undertaking exercise training.

[0260] FIG. 10: Overview of conversion of glucosinolates by myrosinase, including the conversion of glucoraphanin to sulforaphane. The glucosinolates glucoraphanin and glucoerucin can be hydrolysed by myrosinase enzymes to give glucose (Glc) and unstable aglycones. These unstable aglycones may form isothiocyanates (e.g. sulforaphane and erucin) and their corresponding nitriles depending on the exact reaction conditions. Furthermore, erucin and sulforaphane can be interconverted (Dinkova-Kostova et al. 2017).

[0261] FIG. 11: Conversion of glucoraphanin to sulforaphane in the absence of externally added mustard seeds. Whole sprouts were homogenised in a water solution and kept at room temperature for 14 days. Samples of the homogenate were collected before and after the incubation period and were frozen in liquid nitrogen for later analysis of sulforaphane and glucoraphanin.

EXAMPLES

[0262] Study I: Acute Administration of Broccoli Sprout Juice

[0263] The aim of study I was to assess blood concentrations of lactate after a single dose i.e. acute administration.

[0264] Three healthy male human subjects were enrolled on this study. Subjects were provided compositions of broccoli sprout juice (BSJ) containing broccoli sprouts mixed with mustard seeds and water (2 ml per gram of broccoli sprouts), in the amounts outlined in the table below. A minimum of 48 hours wash-out period was used between administering the different doses within a subject. Five different doses of broccoli sprouts were administered; 0, 10 g, 50 g, 75 g and 150 g.

[0265] The total amount of glucosinolates in the broccoli sprouts was assessed according to the methods set out in Meitinger & Kreis, 2018 and was found to be 46 ?mol per gram fresh weight. The amount of glucoraphanin and glucoiberin were analyzed using a UPLC-mass spectrophotometric method and found to be 2.3 and 0.15 ?g/gram tissue weight, respectively. Peak detection showed particularly high amounts of gluconapin were present in the broccoli sprouts and suggested that gluconapin was the most abundant glucosinolate in the sprouts. The same type of broccoli sprouts were used in studies I, II and II.

TABLE-US-00001 Powdered brown Composition Broccoli sprouts (g) mustard seeds (g) Water (ml) A 0 0 B 10 0.1 20 C 50 0.5 100 D 75 0.75 150 E 150 1.5 300

[0266] Before the consumption of the BSJ, a pre-test was conducted where the subjects cycled on a stationary cycle ergometer at 2 work rates for 5 minutes per work rate at an intensity corresponding to 65 and 80% of maximal oxygen consumption. A capillary blood sample was taken from a punctured fingertip and assessed for lactate. Immediately following the pre-test, the BSJ was consumed by the subject and an identical exercise test was performed 3-3.5 hours after.

[0267] The blood lactate was measured both in blood from the capillaries of a punctured finger-tip. The lactate was measured using an enzymatic method using a Biosen C-line lactate analyser.

[0268] As shown in FIG. 1, BSJ had an acute effect on blood lactate concentration after submaximal exercise. Surprisingly, the dose-response curve was U-shaped with 10, 50 and 75 grams of BSJ significantly reducing blood lactate concentrations, while 150 grams of BSJ significantly increased blood lactate concentrations (1-way ANOVA). The reduction in blood lactate at the 10-gram dose was 9.6%, at the 50-gram dose was 22%, at the 75-gram dose it was 18% lower, while it was 28% higher at the 150-gram dose. The changes in blood lactate levels are relative to the blood lactate level measured during the pre-test phase.

[0269] The distinct U-shaped dose response curve suggests a bimodal use of BSJ during exercise training. A lower blood lactate concentration during submaximal work is a classic positive adaptation to a period of exercise training indicative of enhanced mitochondrial oxidation of pyruvate. The magnitude of decrease in blood lactate at the 50 and 75 gram doses are similar to what can be expected after several weeks of endurance training (Mayes et al. 1987) and are expected to be associated with an improved physical performance. Decreasing lactate accumulation with the pharmacological agent dichloroacetate acutely improves performance (Ludvik et al, 1993).

[0270] The 150 grams dose of BSJ significantly increased blood lactate levels during submaximal exercise. This indicates a lowering of mitochondrial oxidation of pyruvate and should be negative for exercise capacity acutely. However, lactate has also been shown to have signalling properties during exercise and elevated lactate levels have been associated with positive long-term adaptations to exercise training (Hashimoto et al 2007).

[0271] Study IIChronic Administration of Broccoli Sprout Juice or a Placebo for 7 Days

[0272] Six healthy human subjects (4 females, 2 males) were enrolled to participate in a double-blind study into the effects of chronic administration of broccoli sprout juice. Four of the subjects were given broccoli sprout juice (BSJ) and the other two received juice containing an equal amount of juice containing alfalfa sprouts, which contained no glucoraphanin or sulforaphane. The subjects taking alfalfa juice can be used as a negative control.

[0273] The juices were administered twice a day (a total dose of 150 g of sprouts daily) for 7 days, with each dose containing 75 g of broccoli or alfalfa sprouts and 150 ml of water. To each juice, 0.75 g of powdered brown mustard seeds were added before consumption to enhance the myrosinase activity and thereby sulforaphane content. The last dose was taken 90 minutes before the exercise test.

[0274] Before and after the supplementation period comprehensive physiological tests (outlined below) were performed with metabolic measurements of oxygen uptake, substrate oxidation and assessment of blood lactate formation during incremental submaximal and maximal exercise. Subjects were instructed to maintain their regular lifestyle and did not participate in any regular exercise training. The pre-tests were performed before the supplementation period started and the post-test was done 90 minutes after the last dose (75 grams) of juice was taken.

[0275] Physiological Testing

[0276] The following physiological tests were performed on the human subjects. For inclusion and assessment of baseline physiological characteristics during cycling, a pre-test was performed on a SRM ergometer (Schoberer Rad Messtechnik, SRM, J?lich, Germany). For assessment of the physiological response to submaximal work rate a standard protocol was used which comprised of a series of five minutes long intervals separated with one minutes of rest where capillary blood lactate and glucose were taken and analyzed using a Biosen C-Line Clinic (EKF-diagnostics, Barleben, Germany). The work rate was set individually at 80-100 W at the first stage and thereafter increased with 15-30 W per stage until a substantial blood lactate accumulation was evident. After a short rest, an incremental maximal exercise test was initiated to determine VO.sub.2max. The test started at the work rate of the previous stage, and increased with 20-30 W.Math.min.sup.?1 until fatigue. VO.sub.2max were expressed as the average of the four consecutive highest 10-second long periods. Breath by breath sampling of gas exchange were performed during the entire session using an Oxycon Pro device (Erich Jaeger GmbH, Hoechberg, Germany). Heart rate was measured continuously (Polar Electro OY, Kempele, Finland) and the subjects rated their perceived exertion using the BORG scale (Borg, 1982) at every stage and at exhaustion. The equipment was calibrated according to manufactures instructions.

[0277] As shown in FIG. 2, blood lactate concentrations were higher at submaximal work in BSJ group but not in the placebo (alfalfa) group. Despite the higher blood lactate concentration during submaximal exercise, the maximal attainable workload during incremental exercise to exhaustion was unchanged between pre and post testing (see FIG. 3), indicating that positive adaptations to the increased lactate levels had occurred.

[0278] Lactate has been shown to have important signalling properties during exercise and elevated lactate levels have been associated with positive long-term adaptations to exercise training (Hashimoto et al. 2007).

[0279] Study III: Chronic Administration of Broccoli Sprout Juice or a Placebo in Combination with Exercise Training

[0280] In this study, nine healthy recreationally active subjects (6 females, 3 males, age 25+/?4 years) were enrolled. The study was a double-blinded, randomized, placebo controlled, cross-over study with 4 weeks wash-out between the interventions. A battery of physiological tests (outlined below) similar to those used in study II were performed prior to the supplementation period.

[0281] Similarly to study II, subjects received 2?75 g of BSJ (active) or alfalfa sprouts (placebo) daily, i.e. a total daily dose of 150 g of sprouts, for 10 days. A dose was taken 90 minutes before the subjects donated the muscle biopsy and the last dose was taken 90 minutes before the exercise test on the 10th day. In parallel with the supplementation, subjects performed supervised daily high-intensity interval training at >90% of maximal oxygen consumption for 7 days (outlined below). On day 8, 9 and 10 the subjects continued the supplementation but rested from the training.

[0282] Physiological Testing

[0283] For inclusion and assessment of baseline physiological characteristics during cycling, a pre-test was performed on an SRM ergometer (Schoberer Rad Messtechnik, SRM, J?lich, Germany). For assessment of the physiological response to submaximal work rate a standard protocol was used which comprised of a series of five minutes long intervals separated with one minutes of rest where capillary blood lactate and glucose were taken and analyzed using a Biosen C-Line Clinic (EKF-diagnostics, Barleben, Germany). The work rate was set individually at 80-100 W at the first stage and thereafter increased with 15-30 W per stage until a substantial blood lactate accumulation was evident. After a short rest, an incremental maximal exercise test was initiated to determine VO.sub.2max. The test started at the work rate of the previous stage, and increased with 20-30 W.Math.min.sup.?1 until fatigue. VO.sub.2max were expressed as the average of the four consecutive highest 10-second long periods. Breath by breath sampling of gas exchange were performed during the entire session using an Oxycon Pro device (Erich Jaeger GmbH, Hoechberg, Germany). Heart rate was measured continuously (Polar Electro OY, Kempele, Finland) and the subjects rated their perceived exertion using the BORG scale (Borg, 1982) at every stage and at exhaustion. The equipment was calibrated according to manufactures instructions.

[0284] Training Intervention

[0285] All physiological tests and HIIT sessions were carefully supervised by highly experienced personnel with extensive background in physiological and performance testing. Subjects were blinded to their performance during HIIT sessions throughout the intervention and were instructed to perform all sessions with the ambition to produce the highest possible mean power output. The intervention period consisted of daily HIIT-sessions (5?4 min at 95% VO2max or 5?8 min at 90% VO2max 4 of the HIIT-sessions were ended with up to 4 30 sec sprints at all-out effort) for 7 days in a row at under close surveillance and careful monitoring of power output and heart rate. The subjects were blinded to power output, cadence and heart rate during all HIIT sessions. All HIIT sessions started with a submaximal warm up of 10 minutes at 100 W and 70 rpm. The subjects were instructed to perform all HIIT sessions to achieve the highest possible mean power output over all intervals. In order to help the subjects with a recommended (standardized) pacing strategy they were paced at the first interval at a power output corresponding to their previously highest measured mean power during a HIIT session.

[0286] Nutritional Intervention

[0287] After each HIIT session a recovery drink containing 1 g kg.sup.?1 bw of carbohydrates and 0.25 g kg.sup.?1 bw of protein was ingested by the subjects. After the last HIIT session, subjects were supplied with an evening meal consisting of 74 g carbohydrates, 38 g protein and 26 g fat that was consumed two hours post exercise. They thereafter remained fasted and reported to the laboratory in early morning for donation of a muscle biopsy.

[0288] On day 8, 9 and 10 the subjects continued the supplementation but rested from the training. On the morning of the 8th day a skeletal muscle biopsy was donated and on the 10th day a battery with physiological tests identical to the pre-tests was performed.

[0289] Muscle Biopsies

[0290] All biopsies were collected in fasted state in early morning following 14 hour of rest after the last HIIT-session. Biopsies were taken from the vastus lateralis. First, local anestethsia (2% Carbocain, AstraZeneca, Sodertalje, Sweden) was injected at the biopsy site. A small incision was made and approximately 150 mg of wet tissue was removed with a Weil-Blakesley chonchotome or a 4 mm Bergstrom needle with manually applied suction. The muscle samples were blotted and dissected clean from visual fat, connective tissue and blood and divided in to three portions; ?50 mg to ice cold ISO-medium for respirometrics and two portions of 50-100 mg to liquid nitrogen and thereafter stored in ?80? C. for later analysis.

[0291] Mitochondrial Isolation and Respirometry

[0292] Mitochondria were isolated from ?50 mg of musclein isolation medium (Sucrose 100 mM, KCl 100 mM, Tris-HCl 50 mM, KH.sub.2PO.sub.4 1 mM, EGTA 100 ?M, BSA 0.1%; pH 7.4 as described by Gnaiger and Kuznetsov, 2002). Muscle was kept on ice and homogenized first by scissors and thereafter after addition of 0.2 mg ml.sup.?1 bacterial protease, further homogenization was performed in a water-cooled glass homogenizer. The homogenate was centrifuged at 700 rcf at 4? C. for 10 min in a 15 ml tube and the supernatant was transferred to new 1.5 ml tubes and centrifuged at 10000 g at 4? C. The pellets were resolved and transferred to a single 1.5 ml tube and centrifuged at 7000 rcf at 4? C. for 5 min giving a resultant pellet that was liquated in 0.6 ?l per initial mg wet weight of muscle in preservation medium (EGTA 0.5 mM, MgCl2 6H2O 3 mM, K-lactobionate 60 mM, Taurine 20 mM, KH.sub.2PO.sub.4 10 mM, HEPES 20 mM, Sucrose 110 mM, BSA 1 g L.sup.?1 Histidine 20 mM, Vitamin E succinate 20 ?M, Glutathione 3 mM, Leupeptine 1 ?M, Glutamate 2 mM, Malate 2 mM, Mg-ATP 2 mM).

[0293] Mitochondrial respiration were measured using a two-channel high-resolution respirometer (Oxygraph-2k, Oroboros Instruments Corporation, Innsbruck, Austria). 5 ul of isolated mitocondria were added to two 2 ml wells containing respiration medium MIR05 (EGTA 0.5 mM, MgCl2.Math.6H2O 3 mM, K-lactobionate 60 mM, Taurine 20 mM, KH2PO4 10 mM, HEPES 20 mM, Sucrose 110 mM, BSA 1 g L-1). All experiments were performed at 37? C. and 02 calibration calibration was performed according to the manufactures' instructions. All measurements were performed and analyzed in DatLab 5.2 software (Oroboros, Paar, Graz, Austria). Mitochondrial respiration was related to the wet weight of the initial piece of muscle before mitochondrial isolation.

[0294] Immunoblotting

[0295] Homogenization was performed in freeze dried samples of ?2 mg muscle. The protocol for homogenization is extensively described earlier (Samuelsson et al., 2016). Briefly, 100 ?l/mg dry WT of homogenization buffer consisting of 2 mM HEPES (pH 7.4), 1 mM EDTA, 5 mM EGTA, 10 mM MgCl2, 50 mM ?-glycerophosphate, 1% Triton X-100, 1 mM Na.sub.3VO.sub.4, 2 mM dithiothreitol, 1% phosphatase inhibitor cocktail (Sigma P-2850) and 1% (vol/vol) Halt Protease Inhibitor Cocktail (Thermo Scientific, Rockford, IL) was added to each sample and processed in a bullet blender until homogenized. After rotation for 30 min at 4? C. and centrifugation at 10,000 g for 10 min at 4? C. the supernatant was analysed for protein content and diluted with homogenization buffer and Laemmli buffer (Bio-Rad, Richmond, CA) obtaining a protein concentration of 1 ?g/?l. All samples were denatured at 95? C. for five minutes and stored at ?80? C.

[0296] Samples of muscle homogenates 20, 16 or 14 ?g of protein were loaded to 26-well Criterion TGX gradient gels (4-20% acrylamide; Bio-Rad). Electrophoresis (300 V for 32 minutes kept on ice) were performed in transfer buffer containing 25 mM Tris base, 192 mM glycine, and 10% methanol and the proteins were then transferred to a polyvinylidine fluoride membranes (Bio-Rad) at a constant current of 300 mA for 3 h kept on ice. The membranes were stained with MemCode Reversible Protein Stain Kit (Thermo Scientific) as a loading control. The membranes were then destained and blocked with Tris-buffered saline (TBS; 20 mM Tris base, 137 mM NaCl, pH 7.6) containing 5% non-fat dry milk for 1 h at room temperature. Thereafter, incubation overnight followed with antibodies diluted with TBS buffer with 2.5% non-fat dry milk and 0.1% Tween. The antibody used was from Cell Signaling Technology; Nrf2 (D1Z9C). After incubation, the membranes were washed and incubated with secondary antibodies conjugated with horseradish peroxidase 1 h at room temperature. The membranes were washed again and Super Signal West Femto Chemiluminescent Substrate (Thermo Scientific) was added and target protein visualized and quantified in Molecular Imager ChemiDoc XRS system with Quantity One software (version 4.6.3; Bio-Rad) and in ChemiDoc MP with Quantity One software (version 6.0.1; Bio-Rad).

[0297] Glucose Monitoring

[0298] Furthermore, the subjects wore a continuous glucose monitor (a skin-mounted sensor (FreeStyle Libre, Abbot)) throughout the supplementation period that measured interstitial glucose concentrations every 15 minutes. The sensor was fitted laterally on the subject's deltoideus and automatically stored measurements for a maximum of eight hours before it had to be emptied using a portable reader. If the sensor failed to measure, or if the subject did not export data from the sensor to the reader within eight hour from the last export, the sensor overwrites previously stored data and leave a gap in the exported text fil. The first 12 hours of measurements were also excluded from all subjects due to differences in time before readings appeared stable.

[0299] After 4 weeks of wash-out the procedure was repeated but the subjects that first received BSJ now received placebo and vice versa.

[0300] As shown in FIG. 4, blood lactate concentrations at submaximal workloads (50-75% of VO2max) increased significantly after training in the placebo group and decreased significantly in the BSJ condition. The classical adaptation to endurance exercise training is a decreased lactate concentration at submaximal workloads due to enhanced mitochondrial respiration and better aerobic capacity. The increased lactate concentrations in the placebo condition may be interpreted as a maladaptation to the intense training load, whereas in the BSJ condition the subjects' ability to adapt to the workload was enhanced.

[0301] Subjects performed an incremental exercise protocol to exhaustion to determine maximal power output, time to exhaustion and VO.sub.2max. As shown in FIG. 5A, the maximum power output at exhaustion was significantly enhanced in the BSJ condition only and all subjects experienced an increase in maximal power output. The response in the placebo condition was not significant with 3 subjects showing adverse responses to the training stimuli (FIG. 5A).

[0302] As shown in FIG. 5B, the time to exhaustion during the same incremental exercise protocol was significantly increased in the BSJ condition only, with all subjects experiencing an increase in the amount of time taken to exhaustion. The response in the placebo condition was not significant with some subjects experiencing a decrease in their time to exhaustion (FIG. 5B).

[0303] As shown in FIG. 5C, maximal oxygen uptake was significantly increased in the BSJ condition. Furthermore, there were a greater number of adverse responders in the placebo condition.

[0304] The results in FIGS. 5A to 5C suggest that positive adaptations to physical exercise have taken place in those subjections consuming BSJ and experiencing the decreased lactate concentration.

[0305] A muscle biopsy was taken pre and post each supplementation condition. The expected response after a training period is that mitochondrial respiration should increase. No effect on mitochondrial respiration in the placebo group but a tendency (p=0.067) towards an improvement was observed in the BSJ condition (FIG. 6).

[0306] Nrf2 protein abundance in the skeletal muscle biopsy was assessed using western blotting technique. We found that nrf2 was significantly increased in the BSJ condition but not in the placebo condition. Induction of nrf2 by sulforaphane or broccoli sprouts in skeletal muscle is a novel finding (FIG. 7).

[0307] Additionally, the inventors found that nrf2 indeed was increased after consumption of compositions comprising sulforaphane and/or myrosinase and/or glucoraphanin (e.g. broccoli sprout juice) but not after the placebo condition. nrf2 can also be activated by exercise (Done & Traustad?ttir, 2016) and this is an important part of the muscle cell's ability to withstand higher oxidative load and adapt to a higher metabolic requirement, i.e. improve physical performance. There are indications that the activation of nrf2 from sulforaphane and physical exercise occurs through different mechanisms, which consequently there may be synergistic effects between sulforaphane intake and exercise.

[0308] Intensive physical exercise releases a large amount of free oxygen radicals (ROS) that needs to be effectively scavenged by the muscle cell. Furthermore, oxidative stress has also been shown to be linked to the aging process and several pathological conditions (Lu et al, 2004). However, in the context of exercise, this oxidative stress has been shown to be necessary for the initiation of the body's adaptation processes and will ultimately lead to a better physical performance (Ristow et al 2009). Conversely, we have recently shown that excessive physical exercise leads to a ROS load that the cell's antioxidant defenses cannot handle, ultimately leading to inhibited mitochondrial function and oxidative damage at the cellular level (Larsen et al, 2016). It is clearly conceivable that supplementation with broccoli sprouts can stimulate this antioxidant defence by activating nrf2 in skeletal muscle and ultimately lead to better performance and better resistance to intensive exercise that has been partially found in animal studies (Malaguti et al, 2009).

[0309] Periods of very intense exercise training are typically associated with adverse responses such as an increase in nocturnal hypoglycaemic episodes and a reduction in maximal heart rate. To assess whether BSJ could protect against these adversities, blood glucose concentrations were monitored using continuous glucose monitors (CGMs) during the supplementation period. The time spent in the hypoglycaemic range was significantly reduced in the BSJ condition as compared to the placebo condition (FIG. 8). During the placebo condition the subjects spent 22% of their time (more than 5 hours per day) in the hypoglycaemic range while during the broccoli condition this was reduced to only 9%.

[0310] BSJ enhances the resistance to a period of very hard training, presumably through activation of nrf2 and modulation of lactate levels during exercise. The clear dose-response effect on blood lactate levels during submaximal exercise is both novel and surprising. The inventors are not aware of any other nutritional supplement that has this effect. A decrease in lactate levels during exercise is the hallmark of positive training adaptations to an endurance training program. In athletes, lactate levels are more sensitive to changes in fitness level than VO2max that usually remains fairly constant over time in already highly trained subjects.

[0311] Furthermore, maximum heart rate during maximal work was significantly reduced in the placebo condition but was maintained in the BSJ condition (FIG. 9).

[0312] Study IV: Effect of Mustard Seeds on Sulforaphane Formation from Glucoraphanin

[0313] The inventors conducted a study to determine whether any physiological effects might be produced by BSJ in the absence of myrosinase provided in the form of mustard seeds.

[0314] Frozen sprouts were thawed, homogenised and left in room temperature (20 degrees Celsius) to assess the formation of sulforaphane without adding myrosinase-rich mustard seeds. Glucoraphanin and sulforaphane were assessed before and after 14 days incubation at 20 degrees Celsius. During incubation glucoraphanin concentrations declined from 118.1 to 6.6 g/l, while sulforaphane increased from 0.09 to 18.6 g/I indicating abundant myrosinase activity in the sprout homogenate. Glucoraphanin and sulforaphane were analysed using UPLC mass spectrometry with both positive and negative ionization.

[0315] Sulforaphane levels generated from the conversion of glucoraphanin in the absence of myrosinase from mustard seeds were measured (FIG. 11). The myrosinase activity in frozen BSJ is enough to hydrolyse all glucoraphanin into sulforaphane.

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