THE EFFECT OF HONEY SUPPLEMENTATION BEFORE PHYSICAL ACTIVITY TOWARDS THE PLASMA MALONDIALDEHYDE LEVEL IN MALE WISTAR RATS (RATTUS NORVEGICUS)

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1 443 THE EFFECT OF HONEY SUPPLEMENTATION BEFORE PHYSICAL ACTIVITY TOWARDS THE PLASMA MALONDIALDEHYDE LEVEL IN MALE WISTAR RATS (RATTUS NORVEGICUS) Krisnanda DA Faculty of Sports Science, Yogyakarta State University Abstract Objectives : An exercise has positive and negative impacts. The benefits of exercise are improving the fitness and health of a body, improving performance, and preventing several diseases. One of the negative effects of exercise is the formation of oxidant compounds. The oxidant compounds can trigger oxidative stress, a condition caused by the imbalance between the production of free radicals and the antioxidants. Malondialdehyde (MDA) is one of the biological markers of oxidative stress in organism. Oxidative stress can be prevented by giving exogenous antioxidants. Honey is a rich source of antioxidants. The objective of this study aims to determine the effect of honey supplementation before physical activity towards reduction levels on plasma malondialdehyde (MDA) in male rats. Methods :The study was an experimental laboratory with the posttest only control group design and which was conducted in 7 days. The samples of this study were using 20 male white strain Wistar rats (Rattus norvegicus). The rats were divided into 2 groups randomly and each group consists of 10 rats. The two groups are: K 0 = control group (mean of weight 170,89±12,12, physical activity 148,44±11,28 and MDA plasma 12.02±1,85), K 1 = supplemented honey 5 g / kg body weight (mean of weight 170,89±12,12, physical activity 154,22±11,60 and MDA plasma 7,75±1,61). Swimming 70% of maximum capacity was used as physical activity with the additional weights of 6% from rat body weight. Honey supplementation given once daily for 7 days by oral feeding via sonde. The data were analyzed using statistical program SPSS 16. Results:The results showed that the mean levels of plasma MDA in group K 0 was 12.02±1.85 nmol/ml and 7.75±1.61 nmol/ml in group K 1. Independent sample t-test showed a significant difference p = (p <0.05). Reduction levels of plasma MDA was 35.52%. Conclusions: Independent sample t test showed a significant differences among the groups (p<0.05). Supplemented honey with 5 g / kg gave the significant decrease in MDA levels. The results can be used as a basic knowledge for further research to determine its use in human being. Keywords : honey, physical activity, plasma MDA, wistar rats. INTRODUCTION Healthy is a lifestyle, by exercises we can keep our body health. Regular and measurable exercise can improve the various components of physical fitness, improve performance for athletes and reduce the risk of disease. Exercise can improve health and maintain pulmonary heart fitness (Hottenrott et al., 2012). Exercise also has a negative effect, causing an imbalance between reactive oxygen species (ROS) and antioxidants that lead to fatigue (Alessio, 2000). Exercise increases the formation of oxidant compounds followed by the occurrence of oxidative stress (Harjanto, 2003). Oxidative stress occurs due

2 444 to an imbalance between oxidant production and antioxidants (Leeuwenburgh & Heinecke, 2001). Aerobic exercise can increase oxygen consumption times in the body and times in skeletal muscle (Revan & Erol, 2011). Increased oxygen consumption during exercise adds to ROS formation and triggers oxidative stress. The degree of oxidative stress can be determined by measuring the levels of malondialdehyde (MDA) plasma. The body has an endogenous antioxidant defense system and exogenous antioxidants to counteract free radicals. Antioxidants are substances capable of delaying, preventing or eliminating free radicals (Erejuwa et al., 2012). Honey has benefits including antibacterial, anti-inflammatory and antioxidant (Natalia et al., 2014). The antioxidant content of honey include: vitamin E, vitamin C, vitamin A, phenolic acid, flavonoids, zinc and selenium minerals. The honey supplementation is expected to prevent fatigue and maintain subsequent performance. The honey supplementation is expected to increase levels of antioxidants and reduce levels of free radicals in the body. The effect of honey supplementation on MDA levels as a marker of increased free radical production in mice treated with physical activity was not known certainty. Based on that idea the honey supplementation before physical activity towards the level of malondialdehid plasma still need to be investigated further. METHOD This study was a true experimental design using posttest control group design (Notoatmojo, 2012). This research was conducted in Faculty of Veterinary Medicine of Airlangga University, place of maintenance and treatment of animal try. The study was conducted in June The population in this study was male rats (Rattus norvegicus). The sample was 10 rats of each group with body weight gram, 3-4 months old with healthy physical condition. The sample was divided into two treatment groups. The sample used fulfilled the criteria of healthy physical inclusion characterized by clear eyes, shiny fur, active movement agile, good / not soft feces, weight did not down from 10% during acclimatization. Samples will be excluded from research if ill. Sampling and group division was done by simple random alocation (Asami, 2013). The dependent variable in this research is plasma MDA level, while the independent variable is honey. Honey kelengkeng perhutani given in this research, obtained from the office honey perhutani, road Ahmad Yani no. 276 Bandung, given at a dose 5 g / kg body weight, was administered once daily for 7 days (Hardianty, 2011) via sonde. Acclimatization was done for one week with the same conditions in order to adapt to the new environment. The experimental group of animals was: K0 = control group, K1 = honey treatment group dose 5 g / kg body weight. Maximum ability measurement was done by swimming mice by adding weight of 6% of body weight. The maximum swimming capacity was calculated if the rats were drowned and release large air bubbles. Moderate exercise was being done after 1 hour of placebo and honey (Schramm et al., 2003), rats were rejuvenated into a water bath for 70% of the maximum mouse swimming time with 6% weight. The collection of blood specimens originated from the heart of mice, where anesthesia was previously performed using ketamine HCl doses of mg / kg body weight in intra kardia. After the eyes fade and the body did not move, then the skin of the chest abdomen skinned with a scalpel and after seeing his heart then with a 5 ml syringe of blood taken from the heart as much as 3 ml (Kusumawati, 2004). Blood specimens in all groups were left in the syringe then given identity in accordance with the order of specimens in the group and immediately taken to the Biochemistry Laboratory of the Faculty of Medicine, University of Airlangga to measure the levels of malondialdehyde (MDA). Research data was tabulated and analyzed using Statistical Product And Service Solution for

3 445 Windows XP (SPSS) program which includes statistical analysis as follows: descriptive analysis, normality test and independent samples t test. RESULT AND DISCUSSION This study used 20 white rats (Rattus norvegicus) Wistar strains, males, healthy, adults 2-3 months old, gram weight. The sample was divided into 2 (two) groups: K0 = control group, K1 = group of honey dose 5 g / kg body weight. The data were analyzed based on the minimum number of samples that was 9 rats of each group, because there was one dead rat (K1) on the 7th day. Moderate exercise may increase plasma MDA levels. The honey supplementation as an antioxidant is expected to reduce plasma MDA levels. Descriptive analysis was used to calculate mean value and standard deviation of weight variable before treatment, physical activity (70%) and plasma MDA level. The result of descriptive analysis between groups can be seen in table 1. Table 1. Descriptive Analysis Results Variable Body weight (gram) exercise 70% (second) MDA plasma level mean K 0 (n=9) 170,89±12,12 148,44±11, ±1,85 group mean K 1 (n=9) 170,89±12,12 154,22±11,60 7,75±1,61 The normality test results show all p values> 0.05, so the data is normally distributed. Data can be seen in table 2 below: Body weight Exercise 70% MDA level Table 2. The Normality Test in the Group variable n sig , Testing homogeneity with Levene test showed value p = 0,273 (p> 0,05). So it can be concluded that plasma MDA levels have a homogeneous variant. Post hoc test result showed p value (significance) between group p <0,05, so there was significant difference between group. Results of post hoc test analysis can be seen in table 3. Table 3. Independent samples t-test on plasma MDA levels Variable Sig. MDA The data above shows that elevated plasma MDA levels occur after the treatment of physical activity. This is due to heavy activity that exceed the limit of fatigue can lead to the formation of free radicals, as proposed by Arsana (2014) that the exercise with the intensity of 70% of maximum activity leads to the production of high free radicals. Exercise to exceed the limit of fatigue increases oxygen consumption in the body times or more and in muscle fibers that contract the use of oxygen can be increased times above normal needs (Sauza, 2005). Increased oxygen consumption triggers the release of free radicals that is superoxide radicals (Cooper, 2001).

4 446 In this study all groups of Wistar rats were given moderate intensity activity (70%). According to Bompa (1994), the intensity of 70% of maximal ability is classified into intermediate to medium intensity. Exercise with the intensity of 70% of maximum activity leads to the production of high free radicals (Arsana, 2014). Castro et al. (2009) states that the effectiveness of the antioxidant system in offsetting the free radical production reaches saturation conditions in physical activity with an intensity of 70% of the maximum heart rate. Oztasan et al. (2004) mentioned that the high Thiobarbituric Acid Reactive Substance (TBARS) level was caused by shorter training duration. During physical activity, ROS is formed as a by-product of the phosphorylated oxidation reaction to form energy (ATP) in the electron transport chain in the mitochondria. The phosphorylated oxidation process requires O 2 to bind to hydrogen to form water, but not all of the consumed O 2 (about 4% -5%) turns into ROS (Marciniak et al., 2009). Physical activity increases the body's metabolism. Increased body temperature can trigger the formation of oxidant compounds. An elevating 1 C will increase 12% of body metabolic activity (Guyton, 1996). Physical activity increases the secretion of adrenal hormones that can increase the formation of oxidant compounds. Treatment group in this study was given honey with dose 5 g / kg body weight for 7 days. Determination time of 7 days based on research that has been done by Hardianty (2011). Giving honey as an antioxidant can lower levels of MDA (Yao et al., 2011). Another study conducted by Fajrilah (2013) showed a decrease in plasma MDA levels along with the addition of doses of honey. The supplementation of honey in the treatment group is due to: 1) Honey is a rich source of antioxidants (Erejuwa et al., 2012) that work synergistically (Aljadi, 2004), 2) Honey contains various antioxidant components, including: polyphenols, vitamin E, vitamin C, enzymes (catalase, peroxidase and glucose oxidase), phenolic, carotenoid (vitamin A) (Gheldof, 2002), 3) Other results suggest that honey components, especially flavonoids and phenolic acids, have been shown to contribute significantly to antioxidant capacity (Moussa, 2012), 4) Honey contains mineral selenium (glutathione peroxidase enzyme component) and zinc (one of the co-factors of SOD) that acts as an antioxidant (Bogdanov et al., 2008), 5) The main nutritional component in honey is carbohydrates with elements of glucose and fructose monosaccharides (National Honey Board, 2015) and 6) Honey provides a good source of energy for athletes (Williams, 2007). Various antioxidant content in honey plays a role in inhibiting the formation of free radicals. Vitamin E acts as a hydrogen donor that can convert peroxyl radicals into less reactive tocopherol radicals that are unable to attack fatty acids (Stampfer et al., 1993). Vitamin C becomes the first defense against ROS that can counteract the hydroxyl radical and act as a hydrogen donor for a radical change of tocopherol into alpha tocopherol. Vitamin A can clean the oxygen singlet and also react with the peroxyl radical compounds (Deddy, 2013). Flavonoids have the ability to donate hydrogen atoms, churning metal ions, cleaning hydroxyl radicals, superoxide anion radicals and peroxy lipids (Saikat and Raja, 2011). Selenium is a micro mineral that has strong antioxidant power and is a component of the enzyme glutathione peroxidase (Deddy, 2013). Zinc is one of the co-factors of SOD. SOD activity can run when zinc minerals are available in sufficient quantities (Pande and Gayatri, 2010). Glucose and fructose are the main nutritional components in honey. High carbohydrate content in honey has provided clinical evidence that honey can act as an energy supplier to exercise (Mayhew, 2007). Short-duration exercise leads to the use of large amounts of glucose and muscle glycogen. The main mechanism of increased use of glucose from the blood (glucose uptake) into the muscles during exercise is through GLUT 4 translocation (Garrett, 2000).

5 447 Physical activity of 50-60% intensity of VO 2 max or more increases the use of muscle glycogen. The process of gluconeogenesis is not fast enough to replace lost glycogen, so more blood glucose is used. Blood glucose is known to inhibit adrenaline secretion (Pedersen, 2000) that can trigger the formation of oxidant compounds. The use of glucose in honey as a source of energy, inhibits adrenaline secretion in the process of gluconeogenesis. Gluconeogenesis is the process of glucose formation from sources other than carbohydrates, for example in the liver and muscles. Honey contains various components that are known to play the role of antioxidants, so that honey is used as a natural source to ward off free radicals (Wilczyńska, 2010). Exercise is known to increase the formation of oxidant compounds followed by the occurrence of oxidative stress events (Harjanto, 2003). Exercise can cause an imbalance between reactive oxygen species (ROS) and antioxidants that lead to fatigue (Alessio, 2000). The honey supplementation before physical activity can reduce plasma MDA levels, so that cell damage due to physical activity can be prevented. If muscle damage can be inhibited then this will provide benefits in improving performance and can perform subsequent performance after exercise. This is in accordance with the results of research that can be seen in the diagram below: MDA Plasma Level placebo honey 5 g/kg Group Treatment Figure 1. MDA Plasma Bar Chart CONCLUSION AND SUGGESTION The honey supplementation before physical activity can significantly decrease plasma MDA levels of male wistar rat (Rattus norvegicus). This suggests that honey does have a high antioxidant potential in an effort to prevent oxidative stress as one cause of fatigue due to physical activity,

6 448 ACKNOWLEDGMENT The authors would like to thank all lecturers especially Dr. Gadis Meinar Sari, dr., M.Kes., AIFO and Prof. Dr.Nancy Margarita Rehatta, dr., Sp.An., KIC., KNA, master students and staff of Department of Physiology, Faculty of Medicine, Airlangga University who contributed to this study. REFERENCES Alessio HM, Hagerman AE, Fukerson BK, Ambrose J, Rice RE, Wiley RL Generation of reactive oxygen species after exhautive aerobic and isometric exercise. Med Sci Sports Exerc. 32 (9): Aljadi AM, Kamaruddin MY Evaluation of the phenolic contents and antioxidant capacities of two Malaysian floral honeys. Food Chem 85: Arsana, I Nyoman Ekstrak Kulit Buah Manggis (Garcinia mangostana L.) dan Pelatihan Fisik Menurunkan Stres Oksidatif pada Tikus Wistar (Rattus norvegicus) selama Aktivitas Fisik Maksimal. Disertasi. Denpasar: Fakultas Kedokteran, Program Pascasarjana Universitas Udayana. Asami RK Pengaruh Ekstrak Gingseng Jawa {Talinum Paniculatum (JACQ.) GAERTN.} pada Kebugaran Tikus Putih (Rattus norvegicus) Jantan. Tesis tidak diterbitkan. Surabaya: Fakultas Kedokteran, Universitas Airlangga. Bogdanov, Stefan., Jurendic, Tomislav., Sieber, Robert., Peter, Gallmann Honey for Nutrition and Health: a Review. American Journal of the College of Nutrition 27: Bompa, Tudor O Theory and Methodology of Training: The Key to Athletic Performance. Kendall/Hunt Publishing Company, IOWA. USA. Hal: 2-3. Cooper, K.H Sehat Tanpa Obat, Empat Langkah Revolusi Antioksidan yang Mengubah Hidup Anda. Cetakan ke-1. Bandung : Penerbit Kaifa. Castro, M. A. C. de., Neto, F. F. C., Lima, L. M. C., Silva, F.M. da., Oleiveira, R. J. de., dan Zanesco Production of Free Radical and Catalase Activity During Acute Exercise Training in Young Men. Biology of Sport. 26 (2): Deddy Muhtadi Antioksidan & Kiat Sehat di Usia Produktif. Bandung: CV.Alfabeta. Erejuwa,O.O., Sulaiman, S.A., Wahab, M.S.A Honey: A Novel Antioxidant. Molecules 17. Pp Fajrilah, Bela Risqiyani., Indrayani, Ulfah Dian, Djam an, Qathrunnada Pengaruh Pemberian Madu terhadap Kadar Malondialdehyde (MDA) Plasma Darah pada Tikus yang Diinduksi Alloxan Studi Experimental pada Tikus Putih Jantan Galur Wistar. Sains Medika 5(2): Garrett WE, Kirkendal DT Exercise and Sport Science. Philadelpia: Lippincott Williams&Wilkins. Gheldof N, Engeseth NJ Antioxidant capacity of honeys from various floral sources based on the determination of oxygen radical absorbance capacity and inhibition of in vitro lipoprotein oxidation in human serum samples. J Agric Food Chem 50: Guyton AC, Hall JE Text Book of Medical Physiology, 9 th ed. Philadelpia: WB Saunders Company.

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