Hylocereus polyrhizus effect on catalase and malondialdehyde in rats with oxidative stress

The excessive production of free radicals causes oxidative damage and degenerative diseases. Whereas, catalase is a macromolecule with a specific activity, but Malondialdehyde (MDA) is a small molecule with no activity. The peels of red dragon fruit (H. polyrhizus) are potential to be natural exogenous antioxidants. This research aimed to determine the effects of methanol fraction from H. polyrhizus peels on catalase activity and MDA level. The peel was macerated using chloroform and fractionated using methanol. The stress condition was done through five-day fasting and ten-minute swimming per day. The activity of catalase and MDA was measured using UV-Vis spectrophotometer. Thirty five rats were divided into 7 groups: normal, negative, dose I (5 mg/200gBW), dose II (10 mg/200gBW), dose III (20 mg/200gBW), positive control (quercetin) (4 mg/200gBW) and vitamin E (17.64 mg/kg). The activity of catalase in the normal, negative, vitamin E, and quercetin, dose I, II, and II respectively were: 28.37; 7.8; 24.85; 34.33; 59.92; 28.18; and 70.85 Unit/mL. The average MDA level in the groups respectively were: 0.042; 0.051; 0.034; 0.042; 0.037; 0.033; 0.030 μg/mL. The result of One Way ANOVA test showed that the catalase activity in dose III was significantly different than normal group; the MDA levels in all groups were significantly different than normal group. This research revealed that methanol fraction from H. polyrhizus peels is a potent antioxidant for its capacity to increase the activity of catalase and reduce the MDA level in rats with oxidative stress.


INTRODUCTION
The demand for fulfilling the life needs was inevitable so that people had to work hard, and it often led to irregular meal and rest time. The hard work without rest would finally make body organs bear burdens and trigger free radicals. Free radicals could be produced from endogenous process as a normal response to either intracel or extracel biochemical process and extracel and exogenous process as a result of pollution, food and skin injection and absorption. The production of free radicals in body was able to increase oxidative stress condition. In a normal condition, the produced free radicals would be neutralized by antioxidants in the body. The high concentration of free radicals made endogenous antioxidants unable to neutralize free radicals, and this resulted in oxidative stress, an imbalance between free radicals and antioxidants [1]. The indicators used to determine oxidative stress in human body were catalase and malondialdehyde (MDA). Catalase was an enzyme containing heme that catalyzes hydrogen peroxide dismutation to water and oxygen; MDA was lipid peroxidation resulted from free radicals. The oxidative stress induced with swimming and fasting could reduce the activity of catalase in rats' liver, muscle, heart and brain and increase the MDA concentration in rats' liver [2,3], thus antioxidants from outside the body was needed.
The peels of red dragon fruit (H. polyrhizus) contained beta carotene and betacyanin which have antioxidant activity [4,5]. Based on antioxidant activity test using DPPH radical scavenging activity method, methanol extracts from H. polyrhizus peels had higher antioxidant activity than the extracts from white dragon fruit (H. Undatus) peels and from H. polyrhizus flesh [6,7]. Methanol extracts, ethyl acetate soluble fractions, and ethyl acetate insoluble fractions from red dragon fruit peels had antioxidant activity with IC50 241.19 μg/mL; 8.34 μg/mL and 46.84 μg/mL respectively [8]. The antioxidant activity of the fruit peel isolates was 2.952,14 μg/mL [9]. Those researches revealed that the antioxidant activity was tested *Corresponding Author: ekakartika@pharm.untan.ac.id through in vitro method. Therefore, a research to investigate the increase on catalase concentration and measure MDA concentration in male Wistar rats with oxidative stress after injection of methanol fraction from H. polyhizus peels is required.

Collection and Process of Fruit Peels
The fruit was obtained from Petani Mekar Sari Plantation, Segedong Sub-district, Pontianak District, West Kalimantan Province. The fruit peels were washed with flowing water, chopped, and wet sorted. Furthermore, they were dried and put in a blender to generate simplicia passing 40 mesh strainer.

Extraction and Fractionation
The extraction method applied in this research was maceration. The simplicia of H. polyrhizus peels was put into a maceration vessel and added with chloroform until all samples got submerged in the solvent; the tube was then closed. The maceration took 7 days. The maceration results were filtered to separate their extract and residue. The generated extract was collected and concentrated with evaporator at 40°C to evaporate the solvent and obtain thick chloroform extract.
It was fractionated in liquid form with methanol. This process resulted in two phases of separation based on the polarity index of methanol fraction and residue fraction. Methanol fraction found on the upper layer was collected and concentrated with waterbath at the temperature to obtain thick methanol fraction from the peels.

Preparation of Experimental Animals
Male Wistar rats were used in this research. They were at the age of 2-3 months with average weight of 100-200g. The animals were adapted to the experimental cage for 1 week and classified into 7 experimental groups. Each group consist of 3 rats. Group 1 was normal rats without treatment; group 2 was positive oxidative stress; group 3 was stress oxidative treatment with methanol fraction 5mg/200gBW; group 4 was stress oxidative treatment with methanol fraction 10mg/200gBW; group 5 was stress oxidative treatment with methanol fraction 20mg/200gBW; group 6 was stress oxidative treatment with quercetin 4mg/200grBW; group 7 was stress oxidative treatment with Vitamin E 17.64 mg/kgBW. The oxidative stress treatment was performed through fasting, without food but with ad libitum water. In addition, the rats were made to swim for 10 minutes per day for 5 days. Methanol fraction, quercetin, and Vitamin E were given orally using sonde. After 5 days of treatment, the rats were terminated by cervical dislocation, after anesthetized with chloroform. The rats were dissected for their livers. The organs were washed with phosphate buffered saline (PBS).

Preparation of Rat Liver Homogenate
Rat livers (1.25g), in 5mL PBS containing 11.5g/L KCL, were chopped and centrifuged with microcentrifuge at 4000 rpm for 10 minutes at 4°C to obtain clear supernatants (homogenate). These were used to analyze the activity of antioxidant enzymes including catalase [10].

Measurement of Catalase Activity
The clear supernatants of rat livers (0.5 mL) were added with 2.0 mL potassium phosphate buffer (50 mM (pH 7.0)) containing 10 mM H2O2. The changing absorptions were measured with UV-Vis spectrophotometer at λ 240 nm, recorded every 15 seconds for 1 minute.
The catalase activity was measured bassed on the hydrogen peroxide reduction; it was calculated using absorption slope curve of the sample solvent and blank solvent with the formula [11].

Measurement of Liver MDA
The clear supernatants of rat livers (0.5 mL) were added with 2.0 mL HCL (0.25 N) containing 15% TCA, 0.38% TBA and 0.5% BHT. The mixture was heated at 80ᵒC for 1 hour. Its supernatants were collected and measured for their absorbance with spectrophotometer at λ 532 nm. Tetramethoxypropane (TMP) was used as the standard solvent [11].

Statistical Analysis
One-way ANOVA and Least Significant Difference (LSD) test were applied to analyze the data.

RESULTS AND DISCUSSION
The reduced activity of catalase that occurred in normal group was significantly different form that in negative control (p<0.05). This indicated that swimming treatment for 10 minutes and fasting could cause oxidative stress. This was in accordance with the research by Misra, et.al stating that swimming treatment for rats resulted in oxidative stress, and it could significantly reduce the activity of catalase [2]. Oxidative stress was a serious imbalanced between the production of reactive oxygen species (ROS) and the defence of antioxidant. It was able to cause tissue injuries, damage of all molecule targets, DNA, protein, and lipid (lipid peroxidation) [12]. Figure 1 shows the catalase activity of rats with some treatments.
The principle of MDA measurement was the reaction of one MDA molecule with two Thiobarbituric acid (TBA) molecules which generated pink color measured using spectrophotometer at 532 nm of wavelength. The standard used to measure MDA concentrations was tetramethoxypropane (TMP). In acid condition, TMP could be hydrolyzed resulting in hemiacetal and methanol. The produced hemiacetal was then decomposed into methanol and aldehyde which were able to react with TBA [13]. The linear regression equation generated from the standard curve measurement was y = 8.17993x -0.09969 with r = 0.99268. Figure 3 shows the graph of relation between absorbance and standard curve of TMP. The result of ANOVA analysis showed that the MDA concentration of negative control was significantly different from those of the other groups (p<0.05). This explained that 10-minute swimming and fasting implemented to Wistar rats resulted in oxidative stress due to the higher increased MDA concentration than that in normal condition. The rats induced with swimming and 5-day fasting in negative control group had higher MDA concentration than the normal group did [3,13]. In accordance, this research showed that the negative control group had higher MDA concentration than the normal 28 group did. Figure 4 shows the graph of average concentration for each group. The extraction method applied was maceration. The method was selected because it was simple, did not require complex tools, and was able to avoid component damage of compounds which could not bear heat as the method was without heating process. The chloroform extracts from H. polyrhizus peels were fractionated with methanol. Accordingly based on preliminary study the chloroform extract generate strong antioxidant activity. Extraction in liquid form was an extraction method based on solubility of the component target and in two separated solvent materials [14]. Enzymatic antioxidants found in body including catalase had an important role to protect body against reactive oxygen species (ROS). It was a term of oxidant groups involving free radicals and molecules able to produce free radicals. ROS included superoxide anion (O2), hydrogen peroxide (H2O2), peroxyl radical (ROO), and hydroxyl radical (OH) [15].
Catalase would catalyze the decomposition of hydrogen peroxide (H2O2) into water and oxygen to protect cells from oxidative damage [2]. The first reaction explained that one molecule of hydrogen peroxide (H2O2) oxidized heme found in catalase in resting-state form (Enz (Por-FeIII)) into oxyferryl. One oxidation was equal to the reduction of iron (Fe) and one of porphyrin (Por) ring to produce compound I (Cpd I) that was porphyrin cation radical (Por+-FeIV=O). Moreover, one molecule of hydrogen peroxide in the second reaction was used as reductor of compound I to regenerate resting-state enzyme (Enz (Por-FeIII)), water (H2O), and oxygen (O2) [12]. The reactions were: Enz (Por-FeIII) +H2O2 → Cpd I (Por+-FeIV=O)+H2O (2) Cpd I(Por+-FeIV=O)+H202 → Enz (Por-FeIII)+H2O+O2 (3) During the physical activities such as swimming, the oxygen consumption of body increased. Rats treated with physical activity in the form of swimming, will be easily suffer from oxidative stress. Under physiologic condition, free radicals, as the result of metabolism, can be overcome by endogenous antioxidant. Increase activity or oxidant will lead to higher need of the antioxidant. Under streesfull condition, catalase will be lessersince this enzyme component as a complex protein has been damaged by free radicals. The excessive radicals would result in oxidative stress condition since endogenous antioxidants were unable to absorb the free radicals [16].
In addition to swimming, rats were fasting but with ad libitum water. Fasting was a condition that could cause oxidative stress. The neutral fats were catabolized into fatty acid and glycerol; fatty acid was the main source of energy production. In a normal condition, the catabolism of fatty acid occurred in the mitochondria through ß-oxidation process. Whereas, in a hunger condition, ß-oxidation process increased in peroxisome that was normally a minor line of the process [17]. The increased activity of ß-oxidation increased number of free radicals, additional result of metabolism process. The addition of methanol extracts (5mg/mL, 10mg/mL and 20mg/mL) from the peels of red dragon fruits could increase the activity of catalase more significantly than that of the negative control group (p<0.05). The rats given dose of 5mg/mL and10mg/mL had catalase activity which was not significantly different from that of the normal rats (p>0.05). It was better than that of the normal group after the addition of 20mg/mL methanol fractions from the fruit peels.
This was predicted as the effect of secondary metabolites in the peels of red dragon fruit. Kim stated that the peels of red dragon fruit contained polyphenols and flavonoids [6]. The

Hylocereus Polyrhizus Effect On Catalase And Malondialdehyde In Rats With Oxidative Stress
(Sri Wahdaningsih, Eka Kartika Untari) ___________________________________________________________________________________________________ fruit peels contained beta carotenes and betalains [4]. Polyphenols in the peels were considered to increase the activity of catalase since the compounds could be antioxidant. The addition of polyphenolic compounds from Halimeda opuntia (Linnaeus) Lamouroux could increase the activity of catalase in rat liver induced with carbon tetrachloride [18]. It was considered as the effect of polyphenols that could influence the transcription reaction in expression process of catalase gene [18]. Moreover, another antioxidant compound that could increase the catalase activity was flavonoid. The increase of catalase activity in hepatic cells of human after the addition of flavonoids was daidzein [18]. The compunds were considered able to induce the transcription of catalase gene through interaction with the part of the gene promotor [19].
Another compound functioning as antioxidant in methanol fractions from the red dragon peels was betalain included in secondary metabolite of phenolics. The extracts of Cyclea peltata containing alkaloid tetrandrine (Tet), fangchinoline, and coclaurine had capability to increase the activity of antioxidant enzymes such as catalase, superoxide dismutase (SOD), and glutathione (GSH) in rats induced with carbon tetrachloride (CC14) [20]. Metals such as copper and iron found in biological system would interact with H2O2 through enton reaction to produce hydroxyl radical (OH-). It was able to induce the damaged DNA chain and cells. The alkaloids found in methanol fraction from the peels of red dragon fruit functioned as antioxidant; they played a role as scavangers of hydroxyl radical (OH-) and ion chelators [21,22].
Beta carotene found in methanol fractions from the red dragon peels also functioned as antioxidant. The addition of beta caroteneswas able to increase the activity of catalase in rats induced with acetaminophen [23]. The compound was considered as antioxidant since it could increase gene expression of antioxidant enzymes which were catalase and superoxide dismutase (SOD) by enhancing the number of mRNA [24].
The positive control used was quercetin. It was considered able to detoxify H2O2 and inhibit the ROS production through fenton reaction to generate hydroxyl radical with ion chelators of transition metals such as Cu2+ [25]. Antioxidant activity of quercetin showed that the compound was able to inhibit DNA damage and alkaline oxidation of pyrimidine in cells induced with hydrogen peroxide, free radical [26].
Furthermore, Vitamin E was also used as the positive control. The catalase activity in Vitamin E group was significantly different from those of the other groups (p<0.05). Vitamin E or alpha-tocopherol was used to inhibit oxidative damage by stopping the propagation process of oxidation of doublebond unsaturated fatty acid [27]. Vitamin E was considered able to control hydrogen peroxide production directly in mitochondria, and the excessive production of ROS in mitochondria was the main cause of the injured tissues in rats deficient in Vitamin E [28].
The ability of methanol fractions from the peels of red dragon fruit to increase the activity of catalase in rats with oxidative stress was not as good as that of positive control, Vitamin E. this was due to the unidentified exact number of secondary metabolite found in methanol fraction functioning to increase the catalase activity. Nevertheless, the fraction remained potential to be antioxidant, indicated by the increased catalase activity in rats given methanol fraction compared with the goups of negative control, normal control, and positive control (quercetin).
Moreover, the data analysis of positive control group (Vitamin E) showed that the MDA concentration in this group was significantly different form that of the negative control (p<0.05). The average of MDA concentrations was 0.034 μg/ml. Vitamin E functioned as chain breaking antioxidants inhibiting the propagation stage by one hydrogen ion donor from 6-hydroxyl group of chroman ring able to change peroxyl radicals, result of oxidized lipids, into less reactive tocopherol radicals which was unable to break the fatty acid chain [29]. Tocopherol scavenged peroxyl radicals faster than peroxyl radicals to react with lipid substrate did. This compound would be stable free radicals (tocopheroxyl radical). The reaction of tocopheroxyl radicals with peroxyl radicals would generate two stable products which were α-tocopherylquinone and epoxyquinone.
The reduced MDA concentration in quercetin group was significantly different from that of the negative control group (p<0.05). The MDA concentration of the first group was 0.042 μg/ ml. Quercetin had antioxidant effects so that it could reduce the MDA concentration in rats with oxidative stress. In adiition, it was able to reduce the MDA concentration in Wistar rat gastric induced with ethanol.
The Least Significant Difference (LSD) test, dose I, II, and III groups had reduced MDA concentration significantly different from that of negative control (p<0.05). The reduced MDA concentration, however, was not significantly different from those of normal control, positive control Vitamin E, and quarcetin. This indicated that the three doses of methanol fractions from the furit peels had antioxidant effects. H. polyrhizus contained secondary metabolites which were beta carotenes and betacyanins. Kim showed that methanol extracts from the peels of H. polyrhizus contained secondary metabolites which were polyphenols and flavonoids [6]. The metabolites were considered responsible for the antioxidant activities. The peels of H. polyrhizus contained beta carotenes, terpenoids types of carotenoids which had antioxidant activities [4]. Carotenoids were antioxidant with a role as free radical breaker. It reacted with peroxyl radicals, result of oxidized lipids, and resulted in less reactive antioxidant radicals to restart the propagation process of free radicals. The formation of free radicals could be terminated when it rected with other radicals by forming stable products to break the free radical chains.

CONCLUSION
The methanol fractions from the peels of H. polyrhizus with doses of 5mg/200gBW, 10mg/200gBW and 20mg/200gBW were able to increase the activities of catalase in rats with oxidative stress and reduce the MDA concentrations.