AIP Conference Proceedings 2230, 020013 (2020); https://doi.org/10.1063/5.0006534 2230, 020013
© 2020 Author(s).
Effect of Hibiscus sabdariffa Linn. on oxidative stress in cardiac tissue of
overtrained rat: Study on malonildialdehid (MDA), superoxide dismutase (SOD),
glutathione (GSH), and NADPH oxidase (Nox2)
Cite as: AIP Conference Proceedings 2230, 020013 (2020); https://doi.org/10.1063/5.0006534 Published Online: 04 May 2020
Nurul Paramita, Imma Fatayati, Syarifah Dewi, et al.
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Effect of Hibiscus sabdariffa Linn. on Oxidative Stress in Cardiac Tissue of Overtrained Rat: Study on
Malonildialdehid (MDA), Superoxide Dismutase (SOD), Glutathione (GSH), and NADPH Oxidase (Nox2)
Nurul Paramita
1, a), Imma Fatayati
2, Syarifah Dewi
3, Dewi Irawati Soeria Santoso
1, Trinovita Andraini
1, Neng Tine Kartinah
1, Ermita Isfandiary Ibrahim Ilyas
1,
Mariyal Qibtiyah
2, Sri Yunita
21Department of Medical Physiology, Faculty of Medicine Universitas Indonesia, Jakarta, Indonesia.
2Graduate Student of Magister Program in Biomedical Sciences, Faculty of Medicine Universitas Indonesia, Jakarta, Indonesia.
3Department of Biochemistriy and Molecular Biology, Faculty of Medicine Universitas Indonesia
a) Corresponding author:[email protected]
Abstract. Accumulation of overtraining/OT volume, in the long run can lead to decreased performance called overtraining syndrome (OTS). Oxidative stress, an imbalance state of endogenous antioxidants and free radicals, is closely related to pathophysiology of OTS and in the long run can cause cardiovascular disorders. Some research shows that oxidative stress can be inhibited through antioxidants supplementation. Hibiscus Sabdariffa Linn. (H. sabdariffa) is a plant that contains high antioxidants. This study aims to explore the effect of OT on oxidative stress level in rat’s cardiac tissue and the effect of H. Sabdariffa supplementation to the level of oxidative stress in OT rats. The study was an experimental study using 25 adult Wistar rats, 8–10 weeks, 290–390 gr, randomized into five groups: Control (C), Control + Hibiscus (C-Hib), Aerobic Exercise (A), Overtraining (OT) and Overtraining + Hibiscus (OT-Hib). Dosage of H. sabdariffa given: 500 mg / kg / day. Aerobic physical exercise was given 2 x/wk, 10 min, 12 m/min, lasted for 11 wks.
Overtraining protocols was given 5x/wk with increasing frequency and decreasing recovery time between sessions for 11 wks. Calculated levels of MDA, SOD and GSH using spectrophotometry and Nox2 using ELISA at the end of Week 11.
The result showed that overtraining induces an oxidative stress condition in the rat’s cardiac tissue characterized by higher MDA levels than aerobic and control rats, and lower GSH levels than aerobic rats. The administration of H.
sabdariffa may induce oxidative stress reduction in overtraining rat’s cardiac tissue, characterized by lower MDA levels and higher GSH levels in overtraining rats given H. sabdariffa compared to overtraining rats.
INTRODUCTION
Accumulation of excessive training volume in the long run can cause a decrease in performance called Overtraining Syndrome (OTS). Decreased performance occurs due to the imbalance between excessive workout, time of rest, and recovery.1
During overtraining, Reactive Oxygen Species (ROS) will be released 10-20 times higher than during resting condition. ROS is a type of free radical that is highly reactive. If the total amount of endogenous antioxidants cannot compensate for the amount of ROS, this condition is called oxidative stress wich can cause lipids, proteins and DNA damage.2, 3
At the physiological level, ROS acts as a signalling molecule to change proteins or lipid species inside mitochondria and cardiomyocyte so that they can carry out their functions. Nevertheless, too much ROS can cause
as atherosclerosis, ischemic injury with chronic ischemic heart disease, cardiomyopathy, congestive heart failure and arrhythmias. Myocardial damage that occurs in athletes with OTS can lead to sudden death. 4, 5, 6
In overtraining condition, angiotensin II will continue to be produced, to regulate fluid volume, blood pressure, and cardiac remodelling. Angiotensin II can stimulate the release of dinucleotide adenine phosphate oxidase (Nox), one of the main source for ROS production. Nox2 is the type of nox that plays a part in the cardiac tissue.7,8 The most common site of damage caused by free radicals is in the cell membrane, through a lipid peroxidation mechanism. Lipid peroxidation process can produce Malonildialdehyde (MDA), therefore MDA widely used as an indicator of lipid peroxidation.3,6 Determination of MDA in blood plasma or tissue homogenates is one of the useful methods to predict the oxidative stress levels.9
First antioxidant which combat free radicals are Superoxide Dismutase (SOD). It degrades superoxide anion into oxygen and hydrogen peroxide. Further, Catalase (CAT) and glutathione peroxidase (GPx) mediated by glutathione (GSH) cofactors will break down hydrogen peroxide into oxygen and water.10
Additional antioxidants obtained from food ingredients or supplements can reduce the risk of oxidative stress.
Hibiscus sabdariffa Linn or rosella which can be found in Indonesia contains potent antioxidants like polyphenols, flavonoids, anthocyanins, and vitamin C.11,12 Zuraida, et al showed that supplementation of rosella flower extract can reduce the serum level of MDA and increase activity of endogenous antioxidant, one of them was catalase.13 Herdiani, et al also concluded that the rosella flower extract may help prevent oxidative stress due to increased activity of serum SOD.14 Ilyas’ research showed that overtraining rats with H. sabdariffa administration had lower serum MDA levels and higher serum GPx levels than those in overtraining rats without H. sabdariffa.15 This study aim to explore the effect of overtraining on oxidative stress levels in rat’s cardiac tissue and the effect of H.
Sabdariffa administration to oxidative stress in overtraining rats.
MATERIALS AND METHODS Experimental Animals
This was an experimental study. Twenty five adult male Wistar rats (Rattus norvegicus), aged 8-10 weeks, initial weight 290-390 g were used. Before and during the length of the study, all the animals were in good and healthy condition. Animals were housed under controlled temperature conditions (230C), with an alternate of 12 hours light/12 hours darkness condition and ad libitum feeding and drinking. The animals were appropriately maintained in accordance with the code of animal handling commission for the use of experimental animals. Prior to intervention, the animals were introduced to the research enviroment, treadmill and other study condition for two weeks. The total number of animals used was determined using the Federer formula: (t-1) (n-ZKHUHWLVWKH number of treatment groups. Animals were randomly and evenly assigned into 5 groups: control group treated with placebo (C), control group with supplementation of Hibiscus sabdariffa Linn. (C-Hib), aerobic exercise group (A), overtraining group (OT), and overtraining with supplementation of Hibiscus sabdariffa Linn. (OT-Hib).
Supplementation of Hibiscus Sabdarifa Linn.
Hibiscus sabdarifa Linn.was given at a dose of 500 mg/kgbodyweight/day16 initiated after the completion of acclimatization period, 2 days/week for 11 weeks, given before training. Methanol extract of Hibiscus sabdarifa Linn. obtained from the Laboratory of Medicinal Study Center - Bogor Agricultural University.
Exercise Training Protocols
Group A were given aerobic exercise using rodent treadmill. Treadmill running was performed 2 days/week for 11 weeks, consisted of 10 minutes running. The running speed was maintained at 12 m/minute. In each training session, animal performed a 5 minute warm-up and 5 minute cool down period at the speed of 5 m/minute.17
Group OT and OT-Hib were given overtraining exercise using rodent treadmill. Treadmill running was performed 5 days/week for 11 weeks, consisted of several phases with gradual increase in exercise duration and speed. Table 1 shows the overtraining exercise protocols performed in this study.17
TABLE 1. Overtraining protocols Research time
(Week) Phase of exercises Speed
(m / Min) Duration (Min) Frequency of exercise / day
Inter-session break time
(Hours)
Acclimatization - - - -
1 A-1 15 20 1 24
2 A-1 20 30 1 24
3 A-1 22.5 45 1 24
4 A-1 25 60 1 24
5 A-2 25 60 1 24
6 A-2 25 60 1 24
7 A-2 25 60 1 24
8 A-2 25 60 1 24
9 T-2X 25 60 2 4 10 T-3X 25 60 3 3 11 T-4X 25 60 4 2 A-1: Adaptation 1; A-2: Adaptation 2; T-2X: treadmill exercise 2 twice a day; T-3x: treadmill exercise thrice a day; T-4X:
Treadmill exercise 4 times a day.
Tissue Preparation
After 11 weeks, all animals were sacrificed and cardiac organ were removed from the chest cavity and stored in a freezer (temperature of -80qC) until measurement of MDA, SOD, GSH and Nox2.
Measurement of MDA, SOD, GSH, and Nox2
Homogenization was performed on the rat’s cardiac tissue. Will’s method was used to measure MDA level.
Thiobarbituric acid (TBA) was reacted with MDA, which produce a coloured solution. Using spectrophotometry, the absorbance was measured at 532 nm.18 Randox kit was used to measure SOD level. SOD is reacted with Xanthin Oxidase and using spectrophotometry, the absorbance was measured at 505 nm after 30 second and 3 minutes.19 Ellman’s methods was used to measure GSH level. -SH compound was reacted with S-S compound namely diiobisnitrobenzoic acid (DNTB). A yellow solution was form from the reaction. The absorbance was measured at 412 nm wavelength, using spectrophotometry.20 Measurement of Nox2 levels using the ELISA technique. The ELISA kit used from Bioassay Technology Laboratory. The study was conducted in Histology animal house laboratory and Biochemistry laboratory, Faculty of Medicine, Universitas Indonesia and laboratory of Faculty of Dentistry Medicine, Universitas Indonesia.
Data Analysis
Data normality was confirmed using Shapiro Wilk test. Kruskal Wallis test was used to test the mean differences between each group, because the data was not distributed normally,. Statistical analyses were performed with SPSS version 20. Significant difference was determined if 3
RESULTS Body Weight
Body weight (BW) measurement data was presented in Figure 1. The measurement was done in Week 1 before treatment and in Week 11. The homogeneity and normality tests were carried out on each of the mean BW on Week 1 and Week 11. Based on the analysis, we found that data in Week 1 were homogenous and distributed normally
(340±10.8070 g), A (350.8±8.9032 g), OT (319.72±8.1126 g), OT-Hib (342.4±17.1117 g). At the end of the study we found that there was an increase in BW in C, C-Hib, A, and OT group, but the difference in the increase BW in the OT group is not as high as that of C, C-Hib, and A group. There was a decrease BW in OT-Hib group.
FIGURE 1. Comparison of rat’s mean body weight in Week 1 and Week 11. Description: C (control); C-Hib (control and Hibiscus sabdariffa Linn.); A (aerobic); OT (overtraining); OT-Hib (overtraining and Hibiscus sabdariffa Linn.). (Data
represent mean ± SEM; n = 5 rats/group.)
Specific SOD activity in the cardiac tissue were presented in Figure 2. The result of data significant test were p=0.372 (p>0.05). However, it can be seen from the number that the highest SOD level were obtained in group C (0.1187±0.0257 U/g protein), followed by group A (0.0822±0.0062 U/g protein), group C-Hib (0.0795±0.001326 U/g protein), group OT-Hib (0.0776±0.0077 U/g protein) and the lowest in the group OT (0.0637±0.01173 U/g protein).
FIGURE 2 SOD specific activity in cardiac tissue (U/mg protein) of each treatment group. Description: C (control); C-Hib (control and Hibiscus sabdariffa Linn.); A (aerobic); OT (overtraining); OT-Hib (overtraining and Hibiscus sabdariffa Linn.).
(Data represent mean ± SEM; n = 5 rats/group. Kruskal-Wallis).
MDA level
MDA levels in the cardiac tissue are presented in Figure 3. Data significant test result showed that p=0.008 (p<0.05). The lowest MDA levels were in group A (0.05931±0.1785 nmol/mL) and the highest level in group OT group (3.3701±2.3028 nmol/mL). In the group OT-Hib, MDA levels were lower (1.3425±0.4955 nmol/mL) than in the group OT (3.3701±2.3028 nmol/mL), although it was not lower than in group C (1.2482±0.6441 nmol/mL) and group C-Hib (1.108±0.3504 nmol/mL).
FIGURE 3. Mean MDA levels (μl/mL) in each treatment group. Description: C (control); C-Hib (control and Hibiscus sabdariffa Linn.); A (aerobic); OT (overtraining); OT-Hib (overtraining and Hibiscus sabdariffa Linn.). (Data represent mean ± SEM; n = 5
rats/group. **p<0.001*p<0.005, Kruskall-Wallis and Mann-Whitney post hoc).
GSH Level
GSH levels in cardiac tissue are presented in Figure 4. Data significant test result showed that p=0.001 (p<0.05).
The lowest GSH level were in C group (0.0096±6.9947 μg/mL) and the highest level in group A (0.0108±0.0001 μg/mL). Significant differences were noticed between group C and C-Hib (p = 0.0079), C and A (p = 0.0079), group C-Hib and OT-Hib (p = 0.0079), group C-Hib and OT (0.0079), group A and OT (p = 0.0079), group OT and OT-Hib (p = 0.0079), and group A and OT-Hib (p = 0.0317).
FIGURE 4. Mean GSH levels (μg/mL) in each treatment group. Description: C (control); C-Hib (control and Hibiscus sabdariffa Linn.); A (aerobic); OT (overtraining); OT-Hib (overtraining and Hibiscus sabdariffa Linn.). (Data represent mean ± SEM; n = 5
rats/group. **p<0.00,*p<0.005, Kruskall-Wallis and Mann-Whitney post hoc).
Nox2 Level
Nox2 levels in the cardiac tissue are presented in Figure 5. Data significant test result showed that p=0.01 (p<0.05). The lowest Nox2 level were in group C-+LEȡOPJDQGWKHORZHVWLQgroup C (0.0091 ± ȡOPJ1R[OHYHOVLQ group 27ȡOmg) were lower than group OT-Hib (0.0151 ± 0.0067 ȡOPJDQGLQgroup A ȡOPJ but they did not show a significant difference. There was significant differences between group C and C-Hib (p = 0.0079), group C with A (p = 0.0317), and group C-Hib with A (p = 0.0079).
FIGURE 5. Mean Nox2 levels (Ul/mg) in each treatment group. Description: C (control); C-Hib (control and Hibiscus sabdariffa Linn.); A (aerobic); OT (overtraining); OT-Hib (overtraining and Hibiscus sabdariffa Linn.). (Data represent mean ±
SEM.**p<0.001, *p<0.005, Kruskall-Wallis and Mann-Whitney post hoc).
DISCUSSION
The result of the study found that increase body weight (BW) in overtraining group was lower compared to group control. Overtraining can cause hypermetabolism and proteolysis which inhibits increase of BW. Therefore hindrance of increase BW is an indication and marker of overtraining.17 This is in accordance with the results of the study by Ilyas, et al. which found that the overtraining group has a body weight 50% lower than control.1
There was no significant specific SOD activity difference between each group. This result is not in accordance with Saragih et.al that shows excessive physical training can decrease level of SOD.21 However this result is in accordance with the study by Azizbeige, et al. that showed no significant differences in SOD specific activity in various groups given different physical exercises. This could be because measurement of SOD in this study was not specific for the type of SOD which acts to neutralize ROS during physical exercise. During physical exercise ROS is produced in cells, therefore the type of SOD that plays a major role during physical exercise is MnSOD which is located in the mitochondria.22 Nevertheless, the specific SOD activity in control (0.1187±0.0257 U/g protein) was higher compare to overtraining (0.0637±0.01173 U/g protein). This shows overtraining can cause interruption of the repair process that leads to adaptation failure. One of the possible mechanisms is because overtraining can disturb the capacity of endogenous antioxidants. Reduced antioxidant activity (which responsible to weakened oxidants) will lead to oxidative stress, in which oxidant levels are higher than antioxidant activity.23
From this study we found that the highest MDA level in cardiac tissue was in the overtraining group (3.3701 ± 2.3028 nmol / mL), and it was significantly higher than control group (1.2482 ± 0.6441 nmol / mL), C-Hib group (1.108±0.3504 nmol/mL) and A group (0.05931 ± 0.1785 nmol / mL). The high levels of oxidants in overtraining can react with cells, especially in the lipid layer and result in higher production of MDA.24 Our result is in accordance with the result of study by Saragih et.al that shows increase activity of lipid peroxidation in overtraining.21
GSH level was highest in A group (0.0108 ± 0.0001 μg / mL), and significantly different compared to control (0.0096 ± 6.9947 μg / mL). This is possibly due to the protection effect of the antioxidant GSH against free radicals produced during physical exercise.25 Aerobic endurance training can promote physiological and beneficial adaptation to the heart tissue, causing increase antioxidant levels to provide a protective effect on the heart.26 This result is in accordance with the study result of Power, et.al that shows aerobic exercise can increase the antioxidant activity of SOD in the myocardium, which is important for cardioprotection.26 GSH levels in the overtraining (0.0098 ± 3.2482 μg / mL) was significantly lower compared with A group (0.0109 ± 0.0001 μg / mL). This shows that free radicals formed during overtraining are much higher, and consequently more antioxidants are needed, therefore GSH levels are lower. This is in accordance with the result of Marpaung study that shows higher levels of hippocampal GSH in the aerobic group than in control.27 The results of MDA and SOD levels also supported this result. MDA levels in aerobic group were significantly lower compared to OT group. Although the difference was not significant, SOD levels in aerobic group were higher than OT group. Lower levels of GSH in OT group were not significantly different than control group presumably due to the presence of other antioxidants.
Nox2 levels in aerobic group were significantly higher than control group. Nox2 levels in overtraining group were higher than control group, although the difference was not significant. This result was in line with MDA levels as the end result of lipid peroxide process caused by ROS, which is also higher in OT group than in control group. It can be seen that Nox2 has some contribution in increasing ROS levels and is thought to also induce cardiac hypertrophy in overtraining. These results are consistent with Looi, et al. research showing mice conditioned without Nox2 had lower levels of atrial natriuretic factor (hypertrophy marker) and lower levels of 3-nitrotyrosine protein (markers of oxidative stress in the myocardium) than controls. This can mean that Nox2 involved in the remodeling of the heart to the pathologic direction such as hypertrophy, and contractility dysfunction.28
MDA levels in cardiac tissue of overtraining group given H. sabdariffa (OT-Hib) was significantly lower than overtraining (OT) group. GSH levels in cardiac tissue of OT-Hib group were significantly higher than OT group.
SOD specific activity in OT-Hib group was higher than in OT group even though the difference was not significant.
These results indicate that the administration of H. sabdariffa can prevent the occurrence of oxidative stress in the overtraining rat’s cardiac tissue, and has a potential cardioprotective effect in overtraining conditions. These results are consistent with the study of Ilyas, et.al., that showed lower serum MDA levels and higher serum GPx antioxidant levels in OT-Hib group when compared to OT group.15 The results of the Marpaung study, et.al., showed greater hippocampal GSH levels in OT-Hib group compared to OT group.27 The results of the study of Zainalabidin, et al.
concluded that the administration of H. sabdariffa could reduce MDA as an oxidative stress marker and stimulate an increase in enzymatic antioxidants such as SOD, CAT and GSH.29 Accordance with Tsao, et al. research, polyphenols can induce the formation of enzymatic antioxidants such as SOD, CAT, GPx which can decompose free radicals and inhibit the expression of xanthinoxidase enzymes.30 Furthermore Singh, et al. concluded anthocyanin in H. sabdariffa stabilizes and neutralizes free radicals through electron donors or H+ atoms. Thus reducing the level of oxidation.12
The C-Hib group had significantly higher Nox2 level than the control group, as was the SOD activity, although the difference was not significant. This is consistent with study result of Poljsak et.al, which concluded that vitamin C can serve as an antioxidant when the cells is in oxidative stress situation but can become prooxidant if the cells do not undergo an oxidative stress situation.31 Therefore, further research is still needed to determine the appropriate indication for administration of H. sabdariffa.
In addition to C-Hib group, Nox2 was also higher in OT-Hib group compared to control, although the difference was not significant. This is probably due to active substance contained in the H. sabdariffa. One research showed the presence of lectins in H. sabdariffa seeds.32 Study conducted by Gorudko et.al., showed that certain lectins from several types of plants have different affinities in binding to gp91phox glycans to activate Nox. Lectin is a Nox plasma membrane stimulator in human neutrophil cells. Lectins can bind to the glycoreceptor on the cell membrane and trigger a signal that causes phosphorylation and translocation phox protein in cells. This mechanism will activate Nox to produce ROS. In addition, lectins can also bind to GP91phox glycans, which is one of the subunits of Nox, the formation of the lectin complex with GP91phox will activate Nox, which also produce ROS.33 Research conducted by Karlsson, shows that Wheat germ agglutinin (WGA) which is a lectin compound in wheat mediates the release of ROS both intra and extra neutrophil cells. Activation of Nox by lectins can also occur through transduction of formyl-methionyl-leucyl-phenylalanine (fMLP) receptor that bind to G protein. Activated fMLP receptor will cause phospholipase C activation and trigger the release of Ca2+ and activation of protein kinase C. The activity of Nox through this pathway occurs in extracellular space. Intracellular Nox activation occurs through an increase in Ca2+ by ionophore ionomycin activity, resulting in activation of protein kinase C.34 Therefore further
CONCLUSION
Overtraining can lead to oxidative stress in rat’s cardiac tissue characterized by higher MDA levels in the overtraining group compared to aerobic and control, and lower GSH levels in the overtraining group than the aerobic group.
The administration of H. sabdariffa has the posibility to reduce oxidative stress levels in cardiac tissue of overtraining rats, characterized by lower MDA levels and higher GSH levels in the overtraining group given H.
sabdariffa compared with the overtraining group without H. sabdariffa administration.
ACKNOWLEDGMENTS
This study was funded by Hibah PITTA Universitas Indonesia 2018.We would like to thank everyone who support the preparation of this paper.
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