Effects of Bee Bread Supplementation on Total Antioxidant Status and F2-Isoprostane Among Athletes

Chee Ping Wong^1*, Chee Keong Chen², Foong Kiew Ooi² & Mahaneem Mohamed³

¹Faculty of Food Science and Nutrition, Universiti Malaysia Sabah, Kota Kinabalu, Sabah, Malaysia
²Exercise and Sports Science Programme, School of Health Sciences, Universiti Sains Malaysia, Kelantan, Malaysia
³Department of Physiology, School of Medical Sciences, Universiti Sains Malaysia, Kelantan, Malaysia

*Correspondence to: Dr. Chee Ping Wong, Faculty of Food Science and Nutrition, Universiti Malaysia Sabah, Kota Kinabalu, Sabah, Malaysia.

Copyright © 2020 Dr. Chee Ping Wong, et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Introduction
Numerous studies reported that oxidative stress are the major causes of cardiovascular diseases [1-2], cancer [3-4], impaired wound healing [5-6], cataracts [7] and gastrointestinal disorders [8-9]. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) such as hydroxyl radical, superoxide, hydrogen peroxide, nitric oxide and peroxynitrite can damage lipids, proteins and DNA in cells [10]. Reactive oxygen species and reactive nitrogen species are also related to the muscle damage, fatigue and decrease exercise performance. Free radicals will increase with increased intensity of exercise and raised body temperature during exercise. Exercise is postulated to generate free radicals by the production of lactic acid that can convert a weakly damaging free radical (superoxide) into a strongly damaging free radical (hydroxyl) and due to inflammatory responses to muscle damage [11-12]. Antioxidants will scavenge the free radical from damaging the cell and reduce oxidative stress.

Bee products have been used thousands of years ago as a healthy supplement in the traditional medicine. Several studies indicated bee products are antioxidant agent [13-15], anti-inflammatory agent [16], antitumor agent [17] and antimicrobial agent [18]. Natural antioxidants can be phenolic, flavonoid and nitrogen compounds such as alkaloids, chlorophyll derivatives, amino acids and amines [19-20]. Bee product contains antioxidant components such as vitamins C and E [21], flavonoid [22] and phenolic compounds [23]. There were several studies reported ergogenic effects of bee products on sports performance [24-26] and may elicit beneficial effects on bone properties [27]. However, there is still scanty information related to the bee products on antioxidant properties at pre and post exercise after short term supplementation. This present study investigated effects of eight weeks supplementation of bee bread on antioxidant properties at pre and post exercise among athletes.

Methodology
Twelve male athletes were recruited in this study. Subjects consumed 20g of bee bread or placebo for 8 weeks. Before exercise trial test, subjects were asked to refrain from exercise for 24 hours before the exercise tests to ensure that they have adequate rest. Their food diary and physical activity diary for the last 72 hours were also collected. Subjects recorded their 3 days food diary prior to the first running trial and repeated the same diet over 3 days before the days of consecutive test to minimize the differences in muscle glycogen between the trials.

Upon arrival at the laboratory, subject’s nude body weight (after emptying their bladder) and height were measured. An indwelling cannula was inserted into a subcutaneous forearm vein and an extension tube was connected to it to facilitate repeated blood withdrawals. Patency of the cannula was maintained with heparinised saline (Pfizer, Australia). During each blood collection, the first 1mL of the blood sample was discarded and this amount was not included in the blood analysis as it was diluted by the heparinised saline. Eight mL of blood was collected in another 10mL sterile syringe. Approximately 1mL of heparinised saline was injected into the extension tube after each blood withdrawal. A standardised breakfast with 500mL of plain water was consumed by the participants who remained in comfortable sitting posture for 30 minutes. These procedures were standardised for all the subjects. During the exercise trial test, subject was asked to warm up for 5 minutes by running at 50% of their VO_2max. Then, the intensity of running was increased to 60% of their respective VO_2max during the 90 minutes running trial and followed by a time trial.

Blood samples were taken at pre supplementation, pre exercise, immediate post exercise and 24 hours post exercise to determine total antioxidant status and F₂-Isoprostane. All the statistical analyses were computed by using the Statistical Programme for the Social Sciences (SPSS) version 26.0 (SPSS Incorp, United States). The level of significance for all analysis was set at p<0.05. All the collected data were expressed in mean and standard deviation (mean±SD). ANOVA with repeated measures was used to determine the differences of the measured total antioxidant status and F₂-Isoprostane parameters over time and between trials. Bonferroni adjustment for multiple comparisons was used to locate the differences when repeated measures analysis of variance revealed a significant main effect of time.

Results and Discussion
Twelve male athletes were recruited in this study. The mean maximum oxygen consumption (VO_2max) reflected that the subjects had good cardio-respiratory fitness. Mean body mass index of the subjects was categorised as normal and healthy (Table 1).

Figure 1 showed mean values of total antioxidant status in two different trials. Total antioxidant status increased significantly after 8 weeks supplementation in the bee bread trial but not in the placebo trial (p<0.01). Total antioxidant status was significantly higher at pre exercise, immediate post exercise and 24 hours post exercise in bee bread trial compared to the placebo trial (p<0.05). Total antioxidant status was significantly higher at pre exercise, immediate post exercise and 24 hours post exercise compared to the pre supplementation value in the bee bread trial (p<0.01).

* denotes significant difference from corresponding value of placebo trial at p<0.05
#, ## denotes significant difference from respective pre supplementation values at p<0.05 and p<0.01 respectively.
++ denotes significant difference from respective pre exercise values p<0.01.

* denotes significant difference from corresponding value of placebo trial at p<0.05.
#, ## denotes significant difference from respective pre supplementation values at p<0.05 and p<0.01 respectively.
+, ++ denotes significant difference from respective pre exercise values at p<0.05 and p<0.01 respectively.

Figure 2 showed mean values of F₂-Isoprostane in two different trials. F₂-Isoprostane decreased significantly after 8 weeks supplementation in the bee bread trial but not in the placebo trial (p<0.05). F₂-Isoprostane concentration was significantly lower at pre exercise, immediate post exercise and 24 hours post exercise in bee bread trial compared to placebo trial (p<0.05). F₂-Isoprostane concentration was significantly higher at immediate post exercise compared to the respective pre exercise values in both trials (p<0.01).

Total antioxidant status increased significantly at immediate post exercise compared to the resting value in both of the trials. Increased antioxidant status during exercise is due to increased activities of antioxidant enzymes response of prolonged exercise. It is assumed that exercise may increase antioxidant defence and the magnitude increased of antioxidant defence is dependent on the antioxidant status of the athletes. Total antioxidant status concentration was significantly higher in the bee bread trial in comparison with placebo trial at immediate post exercise. This present findings indicated that eight weeks supplementation of bee bread was able to increase antioxidant status compared to the placebo. It is speculated that higher total antioxidant status in the bee bread trial compared to the placebo trial during exercise may have scavenged the free radicals and may beneficial to the exercise performance. Free radicals such as reactive oxygen species may limit the exercise performance because they are causing damage to the nucleic acids cell and promote muscular soreness and fatigue.

F₂-Isoprostane is a prostaglandin-like compound formed from the free radical catalysed peroxidation of arachidonic acid [36]. Quantification of F₂-isoprostanes has proven to be an important indicator to measure free radicals and to assess oxidative stress [37-38]. A lower of F₂-Isoprostane in the bee bread trial reflects that there was less oxidative damage compared to the placebo trial due to antioxidant properties of bee bread. F₂-Isoprostane increased significantly at immediate post exercise compared to the resting value in both of the trials. This finding was similar with the previous studies also reported that F₂-Isoprostane increased significantly after exercise [39-40]. It is speculated that F₂-Isoprostane increased during exercise due to the oxygen flux increased to the exercising skeletal muscle [41]. Exercise increases the oxygen uptake 10 to 20 fold and promotes generation of reactive oxygen species [42]. This present finding found F₂-Isoprostane in the bee bread trial was lower compared to the placebo trial at immediate and 24-hour post exercise. This observation reflected that bee bread supplementation reduced oxidative stress productions during exercise compared to the placebo and a lower oxidative stress means less damage to biological macromolecules especially deoxyribonucleic acid, polyunsaturated fatty acids, amino acids and active proteins of the skeletal muscle cells [41]. This finding indicated oxidative stress was significantly reduced at rest and exercise in the bee bread trial compared to the placebo trial. This reduction in oxidative stress could have contributed to a better exercise performance due to the speculated less oxidative damage to the muscle cells. This present finding found that eight weeks supplementation of bee bread was able to increase total antioxidant status and decrease F₂-Isoprostane at pre and post exercise among athletes.

Conclusion
Supplementation of bee bread showed positives impact on antioxidant status among athletes.

Bibliography

1. Hertog, M. G., Feskens, E. J. & Hollman, P. C. (1993). Dietary antioxidant flavonoids and risk of coronary heart disease: the zutphen elderly study. J Lancet., 342(8878), 1007-1011.
2. Rakha, M. K., Nabil, Z. I. & Hussien, A. A. (2008). Cardioactive and vasoactive effects of natural wild honey against cardiac malperformance induced by hyperadrenergic activity. J Med Food., 11(1), 91-98.
3. Ginter, E. (1995). The role of antioxidants in the prevention of tumours. Bratisl Lek Listy., 96, 195-209.
4. Dragan, S., Nicola, T. & Ilina, R. (2007). Role of multi-component functional foods in the complex treatment of patients with advanced breast cancer. Rev Medic Chir., 111(4), 877-884.
5. Subrahmanyam, M. (1991). Topical application of honey in the treatment of burns. Br J Surg., 78(4), 497-498.
6. Wana, A. (1997). Oxygen free radicals impair wound healing in ischemic rats. Ann Plast Surg., 39(5), 516-523.
7. Gerster, H. (1989). Antioxidant vitamins in cataract prevention. Z Ernahrungswis., 28, 56-75.
8. Smirnov, D. A. (1994). Acute pancreatitis and biological antioxidants. J Khirurgiia., 3, 30-32.
9. Ali, A. T. (1995). Natural honey exerts its protective effects against ethanol-induced gastric lesions in rats by preventing depletion of glandular nonprotein sulfhydryls. Scand J Gastroenterol., 16(1), 18-26.
10. Orrenius, S., Gogvadze, V. & Zhivotovsky, B. (2007). Mitochondrial oxidative stress: implications for cell death. Annual Rev Pharma Toxico., 47, 143-183.
11. Sen, A. (1995). Oxidants and antioxidants in exercise. J Appl Physiol., 79(3), 675-686.
12. Dekkers, J. C., Van Doornen, L. J. & Kemper, H. C. (1996). The role of antioxidant vitamins and enzymes in the prevention of exercise-induced muscle damage. Sports Med., 21(3), 213-238.
13. Nagai, T., Sakai, M., Inoue, R., Inoue, H. & Suzuku, N. (2001). Antioxidative activities of some commercially honeys, royal jelly and propolis. Food Chem., 75(2), 237-240.
14. Nagai, T., Nagashima, T., Suzuku, N. & Innoue, R. (2005). Antioxidant activity and angiotensin I-converting enzyme inhibition by enzymatic hydrolysates from bee bread. Z Naturforsch C., 60(2), 133-138.
15. Nakajima, Y., Tsuruma, K., Shimazawa, M., Mishima, S. & Hara, H. (2009). Comparison of bee product based on assays of antioxidant capacities. BMC complement Alter Med., 9(4), 1-9.
16. Maruyama, H., Sakarmoto, T., Araki. & Hara, H. (2010). Anti-inflammatory effects of bee pollen ethanol extract from Cistus sp. of Spanish on carrageenan-induced rat hind paw edema. J Med., 10(30), 1-11.
17. Yang, X. P., Guo, D., Zhang, J. M. & Wu, M. C. (2007). Characterization and antitumor activity of pollen polysaccharide. Int Immunopharma., 7(3), 401-408.
18. Abouda, Z., Zerdani, I., Kalalou, I., Faid, M. & Ahami, M. T. (2011). The antibacterial activity of Moroccan bee bread and bee-pollen (fresh and dried) against pathogenic bacteria. Res J of Microbiol., 6(4), 376-384.
19. Hall, C. A. & Cuppet, S. L. (1997). Structure- activities of natural antioxidants. In: Antioxidant methodology in vivo and in vitro Concepts. United States of America: Champaign.
20. Larson, R. A. (1988). The antioxidants of higher plants. Phytochemistry., 27(4), 4-8.
21. Crane, E. (1975). Honey: a comprehensive survey. New York, USA: Crane Russak.
22. Ferreres, F., Ortiz, A., Silva, C. & Garcia-Viguera, C. (1992). Flavonoids of ‘‘La Alcarria’’ Honey. A study of their botanical origin. Z Lebensm Unters Forsch., 194, 139-143.
23. Al-Mamary, M., Al-Meeri, A. & Al-Habori, M. (2002). Antioxidant activities and total phenolic of different types of honey. Nutr Res., 22(9), 1041-1047.
24. Earnest, C. P., Lancaster, S. L., Rasmussen, C. J., Kerkisck, C. M., Lucia, A., Greenwood, M. C., Almada, A. L., et al. (2004). Low versus high glycemic index carbohydrate gel ingestion during stimulated 64 km cycling time trial performance. J Strenght Cond Res., 18(3), 466-472.
25. Rahim, M., Ooi, F. K. & Hamid, W. Z. A. (2011). Effects of combined aerobic dance exercise and honey supplementation on bone metabolism and immune functions in Women. 9th International Sports Science Conference, Kota Bharu, Kelantan, Malaysia.
26. Shukri, N., Ooi, F. K., Chen, C. K. & Sirajudeen, K. N. S. (2011). Effects of acacia honey drink supplementation compared to sports drink on blood glucose and running performance in the heat. Proceeding of 16th National conference on Medical and Health Sciences, Kelantan, Malaysia.
27. Tavafzadeh, S. S., Ooi, F. K., Krasilshchikov, O. & Sulaiman, S. A. (2011). Effect of a combination of jumping exercise and honey supplementation on the mass, strength and physical dimensions of bones in young female rats. J Api Product Api Medic Sci., 3(1), 26-32.
28. Chen, L., Mehta, A., Berenvaum, M., Zangerl, A. R. & Engeseth, J. (2000). Honeys from different floral sources as inhibitors of enzymatic browning in fruit and vegetable homogenates. J Agric Food Chem., 48(10), 4997-5000.
29. Aljadi , A. M. & Kamaruddin, M. Y. (2004). Evaluation of the phenolic contents and antioxidant capacities of two Malaysian floral honeys. Food Chem., 85, 513-518.
30. Stanciu, O. G., Marghitas, L. A. & Dezmirean, D. (2009). Macro- and oligo-mineral elements from honeybee-collected pollen and beebread harvested from transylvania (Romania). Anim Sci Biotechnol., 66(1-2), 1-5.
31. Vinson, J. A., Dabbagh, Y. A., Serry, M. M. & Jang, J. (1995). Plant flavonoids, especially tea flavonols, are powerful antioxidants using an in vitro oxidation model for heart disease. J Agri Food Chem., 43(11), 2800-2802.
32. Cao, G., Sofic, E. & Prior, R. L. (1997). Antioxidant and prooxidant behaviour of flavonoids: structure-activity relationships. Free Radical Bio Med., 22, 749-760.
33. Gulcin, I. M. E., Buyukokuroglu, M. O. & Kufrevioglu, O. (2003). Antioxidant and analgesic activities of turpentine of Pinus nigra Arn.subsp. pallsiana (Lamb.) Holmboe. J. Ethnophar., 86(1), 51-58.
34. Halliwell, B. (1990). How to characterize a biological antioxidant. Free Radic Res Commun., 9(1), 1-32.
35. Aruoma, O. I. (1994). Nutrition and health aspects of free radicals and antioxidants. Food Chem Toxicol., 32, 671-683.
36. Morrow, J. D., Minton, T. A., Mukundans, C. R., Campbell, M. D., Zackers, W. E., Daniel, V. C., Badri, K. F., et al. (1994). Free Radical-induced generation of Isoprostanes in vivo: Evidence for the formation of D-ring and E-ring Isoprostane. J Bio Chem., 269(6), 4317-4326.
37. Morroe, J. D., Hill, K. E., Burk, R. F., Nammour, T. M., Badr, K. F. & Roberts, L. J. (1990). A series of prostaglandin F2-like compounds are produced in vivo in humans by a non-cyclooxygenase, free radical-catalyzed mechanism. Proc Natl Acad Sci., 87(23), 9383-9387.
38. Erik, S. M. & Jason, D. M. (2005). F₂-Isoprosatne as markers of oxidant stress: an overview. Curr Protoc Toxicol., 17(5), 1-10.
39. McAnulty, S., McAnulty, L., Pascoe, D., Gropper, S., Keith, R., Morrow, J. & Gladden, L. (2005). Hyperthermia increases exercise-induced oxidative stress. Int J Sports Medic., 26(3), 188-192.
40. Davison, G., Callister, R., Williamson, G., Cooper, K. A. & Gleeson, M. (2011). The effect of acute pre-exercise dark chocolate consumption on plasma antioxidant status, oxidative stress and immune endocrine responses to prolonged exercise. Eur J Nutr., 51(1), 69-79.
41. Kelkar, G., Subhadra, K. & Chengappa, R. K. (2008). Effect of antioxidant sup-plementation on haematological parameters, oxidative stress and performance of Indian athletes. J Hum Ecol., 24(3), 209-213.
42. Lambertucci, R. H., Levada-Pires, A. C., Rossoni, L. V., Curi, R. & Pithon-Curi, T. C. (2007). Effects of aerobic exercise training on antioxidant enzyme activities and mRNA levels in soleus muscle from young and aged rats. Mech Ageing Dev., 128(3), 267-275.