Moringa Oleifera Lam Mitigates Oxidative Damage and Brain Infarct Volume in Focal Cerebral Ischemia

Problem statement: At present, the therapeutic outcome of cerebral isc hemia is still not in the satisfaction level. Therefore, the preventive s trategy is considered. Based on the protective effe ct against oxidative damage of Moringa Oleifera Lam. Leaves extract, we hypothesized that this plant extract might protect against cerebral ischemia, on e f the challenge problems nowadays. In order to test this hypothesis, we aimed to determine the pro tective effect of M.oleifera leaves extract in animal model of focal cerebral ischemia induced by permane nt occlusion of right middle cerebral artery. Approach: Male Wistar rats, weighing 300-350 g, were orally g iven the extract once daily at doses of 100, 200 and 400 mg kg −1 BW at a period of 2 weeks, then, they were permane ntly occluded the right Middle Cerebral Artery (MCAO). The animals were ass essed the cerebral infarction volume and oxidative damage markers including MDA level and th e activities of SOD, CAT and GSHPx enzymes at 24 h after occlusion. Results: Rats subjected to M.oleifera extract at all doses used in this study significantly decreased brain infarct volume both a cortical and subcortical structures in accompany with the elevation of SOD activity in both hippocam pus and striatum while only the rats exposed to the extract at doses of 100 and 400 mg kg −1 BW showed the increased GSHPx activity in hippocam pus. No the changes were observed. Therefore, our result s demonstrates the potential benefit of M.oleifera leaves to decrease oxidative stress damage and brai n inf rct volume. Conclusion: This study is the first study to demonstrate the neuroprotective effe ct against focal cerebral ischemia of M.oleifera leaves. It suggests that M.oleifera may be served as natural resource for developing ne uroprotectant against focal cerebral ischemia. However, the preci se underlying mechanism and possible active ingredient are still required further study.


INTRODUCTION
To date, ischemic stroke has been recognized as one of the challenge problems leading to both mortality and disability especially in the developing countries (Feigin, 2007).Despite the advances of technology, the therapeutic outcome of this condition is still not in the satisfaction level.The successful of treatment appears to depend on the treatment within the first 3 hours of symptom onset.Therefore, stroke prevention is considered.
Since oxidative stress damage is one of the earliest mechanisms responsible for tissue demise after cerebral ischemic insult (Warner et al., 2004), antioxidants have been proposed to be the promising agent for treating ischemic stroke (Margaill et al., 2005).Numerous substances possessing antioxidant activity have demonstrated the neuroprotective effect against cerebral ischemia in animal model of focal cerebral ischemia induced by permanent occlusion of right Cerebral Artery (MCAO) (Wattanathorn et al., 2011).
Moringa Oleifera Lam., a plant in a family of Moringaceae, is a multipurpose tree found almost all over the Asian countries including in Thailand.Its leaves are consumed not only as food but also as medicine.It has been shown to have anti-inflammatory, hypotensive (Caceres et al., 1992;Faizi et al., 1995) and nootropics (Mohan et al., 2005) activities.In addition, it also exhibits the protective effect against oxidative damage (Sreelatha and Padma, 2011) and antioxidant effect (Kumar and Pari, 2003).Since M.oleifera leaves are consumed as food, the chance of toxicity is very less.It has been reported that LD 50 of alcoholic extract of M.oleifera leaf is approximate 2.8 g kg −1 BW (Sudha et al., 2010).Therefore, it is quite safe even when consumed in a higher quantity due to its high LD 50.Based on the crucial role of oxidative stress on the pathophysiology of cerebral ischemia and the protective effect against oxidative damage of M.oleifera, the neuroprotective effect of this plant extract is considered.Therefore, this study aimed to determine the effect of M.oleifera leaves extract on the oxidative stress markers including Malondialdehyde (MDA) level and the activities of Superoxide Dismutase (SOD), Catalase (CAT) and glutathione peroxidase (GSHPx) and the brain infarct volume both in cortical and subcortical structures.

MATERIALS AND METHODS
Experimental animals: Healthy male Wistar rats (300-350 gm) were obtained from National Animal Center, Salaya, Nakorn Pathom.They were randomly housed 5 per cage and maintained in 12:12 light: Dark cycle and given access to food and water ad libitum.The experiments were performed to minimize animals suffering and the experiment protocols were approved by the Institutional Animal Care and Unit Committee Khon Kaen University, Thailand.

Plant material:
The fresh Moringa Oleifera Lam (Moringaceae) were harvested during November to December, 2010 from the Khon Kaen province Thailand.The plant specimen was authenticated by Associate Professor Dr. Panee Sirisa-ard, Faculty of Pharmaceutical Sciences, Chiangmai University, Thailand.The voucher specimen was kept at Integrative Complimentary Alternative Medicine Research and Development, Khon Kaen University, Khon Kaen, Thailand.

Plant material preparation:
The fresh leaves were immediately cleaned, than cut into small pieces and dried at the temperature less than 50°C.The dried plant material was ground into fine coarse powder and extracted with 50% alcoholic.After that evaporation of solvent in rotary evaporator affords a crude extract of the soluble components and filtrate was lyophilized.The percent yield of extract was 17.49%.The extract contained total phenolic compounds at concentration of 86.73-93.6±0.51 mg of GAE.g −1 extract.The extracts were stored at-25°C in a dark bottle until used.The crude extract was suspended in 1% CMC (Sodium carboxymethylcellulose).
Focal cerebral ischemic induction: Animals were induced food deprivation at a period of 12 hours before surgery whereas water was allowed to assess.Then, they were anesthesized by injecting thiopental sodium at dose of 60 mg kg −1 body weight via intraperitoneal route.After the animals were anesthesized, the focal cerebral ischemic induction was performed.In brief, through a ventral midline incision, the bifurcation of right common carotid artery was exposed.The internal carotid artery and external carotid artery were distally dissected free from the adjacent tissues and ligated.The silicone-coated nylon monofilament was gently advanced passed up through the lumen of the internal carotid artery via arteriectomy in common carotid artery approximate 17 mm from the carotid bifurcation and advanced to the Circle of Willis to occlude the origin of the right middle cerebral artery.Then, the distal end of monofilament was tied up and the wound was suture.

Determination of infarction volume:
Animals were sacrificed 24 h after right middle cerebral artery occlusion, the brain was immediately removed and prepared as coronal sections at 2 mm thick and immersed in 2% 2, 3, 5-Triphenyltetrazolium Chloride (TTC) solution at a 15 min period.Morphometric measurement was performed using Image analyzer (Image Pro Plus 3).Total infarct volume for each brain was calculated by summation of the infracted area of all brain slices (area of infarct in square millimetersxthickness (2 mm)) from the same hemisphere.

Determination of malondialdehyde:
The brain regions including cortex, hippocampus and striatum were isolated and prepared as brain homogenate with 1.15% KCL.The Malondialdehyde (MDA) level in brain homogenate sample was determined by using thiobarbituric acid reaction (Tong-Un et al., 2010).Briefly, the following reagents including 100 µL of 8.1% sodium dodecyl sulfate, 750 µL of 20% acetic acid (pH 3.5), 750 µL of 0.8% thiobarbituric acid and 300 µL of distilled water were added to 100 µL of brain homogenate sample in glass tube.The mixture was mixed and heated in water bath at 95°C for 1 h.After cooling, 500 µL of distilled water and the mixture of nbutanol: Pyridine (15:1) were added.The test tubes were centrifuged at 4,000 g for 10 min and the organic layer was taken to measure the absorbance at 532 nm with spectrophotometer (Pharmacia LKB-Biochrom 4060).1, 1, 3, 3-Tetramethoxy Propane (TMP) at concentrations of ranging between 2-20 nmol were used as standard calibration curve.The results were normalized with protein concentration which determined and expressed as nmol mg −1 protein (Lowry et al., 1951).

Determination of superoxide dismutase (SOD):
The activity of SOD in brain homogenate sample was estimated by using the xanthine/xanthine oxidase system for generating superoxide anion and measuring the reduction of cytochrom c (colorimetric reaction) as a scavenging activity of SOD via reader (Bio Rad model 680) at 550 nm (Kakkar et al.,1984).The activity of SOD was expressed as units per milligram protein.

Determination of glutathione peroxidase (GSHPx):
The activity of GSHPx in brain homogenate was determined indirectly by measuring the yellow colored 5-thio-2-nitrobenzoic acid which produced from the reaction of glutathione (GSH) and DTNB (Eyer and Podhradský, 1986).Standard GSHPx concentrations ranging between 1-100 units ml −1 were used as calibration curve.The activity was normalized with protein concentration and expressed as units per milligram protein.

Determination of Catalase (CAT):
Catalase activity was measured by recording the rate of H 2 O 2 reduction (Goldblith and Proctor, 1950) The brain homogenate sample or standard CAT concentrations ranging between 0-100 units.ml−1 were allowed to react with 0.01 N H 2 O 2 and stopped the reaction with 5 N sulfuric acid solution.5 mM Potassium permanganate was added to perform the reaction with excess peroxide and the excess permanganate from the reaction with peroxide was determined photometrically at 515 nm.The standard curve was plotted as the A515 nm against the catalase activity.The data was expressed as units of catalase per mg protein.
Experimental protocols: Animals were divided into 6 groups; each group consisted of 8 animals as following: • Group I: Sham operation group, animals were orally administered with 1% carboxymethylcellulose and received sham operation which used to determine the effect of surgery • Group II: Vehicle treated group, animals were orally administered with 1% carboxymethylcellulose and served as control group • Group III: Vitamin C, a well known antioxidant which was used as positive control was orally administered to the animals at dose of 250 mg kg −1 body weight • Group IV-VI: M. oleifera extract treated groups which were orally given the leaves extract at doses of 100, 200 and 400 mg kg −1 body weight All animals were orally given the assigned substances for 14 days period, then, the right middle cerebral artery were performed in animals of group II-VI.24 h after the induction, animals were sacrificed for determining the infarction volume (n = 4/group) and biochemical assays (n = 4/group).
Statistical analysis: All data were presented as mean ± SEM value.Statistical analysis was operated using SPSS® (v.17.0 for Window®).Statistical significant of data were performed using one way Analysis Of Variance (ANOVA) followed by LSD post hoc test for multiple comparison.The statistical significant level was set at p-value <0.05.

Effect of M. oleifera leaves extract on brain infarction:
We had determined the brain infarct volume in rats subjected to the right middle cerebral artery occlusion.Our results in Fig. 1 clearly demonstrated that rats which receive sham operation showed no infarction volume whereas rats which received the occlusion of right middle cerebral artery showed the significant increase in infarction volume of both cortex and subcortex (p-value<0.001all; compared with vehicle plus sham operation).Rats which subjected vitamin C significantly decreased the infarction volume both in cortex and subcortex (p-value<0.01 all; compared with vehicle plus MCAO).Interestingly, it was found that M.oleifera leaves extract at doses of 100, 200 and 400 mg kg −1 BW could decrease brain infarction volume in cortex (p-value<0.01,0.05 and 01.05 respectively; compared with vehicle plus MCAO) and subcortex (p-value<0.01,0.05 and 0.01 respectively; compared with vehicle plus MCAO).

Effect of M. oleifera leaves extract on the alteration of malondialdehyde level:
The current data in Fig. 2 revealed that rats which were induced MCAO significantly increased MDA level in cortex, hippocampus and striatum (p-value<0.01,0.01 and 0.05 respectively; compared with vehicle plus sham operation).Vitamin C treated group reversed the elevation of MDA induced by MCAO in cerebral cortex, hippocampus and striatum (p-value<0.01,0.01 and 0.05 respectively; compared with vehicle plus MCAO).

DISCUSSION
Our study have demonstrated the neuroprotective potential of hydroalcoholic extract of M.oleifera leaves extract against ischemia induced oxidative stress as well as histopathological alteration.
It is well documented that focal MCAO gives rise to neurological and histopathological abnormalities in some brain areas.These abnormalities have been reported to be associated with Reactive Oxygen Species (ROS), which react with cellular macromolecules such as lipids, proteins and nucleic acids resulting in oxidative damage of neurons (Negishi et al., 2001).In this study, our results have demonstrated the reduction of SOD and CAT in cerebral cortex, hippocampus and striatum whereas the reduction of GSHPx was observed only in cerebral cortex and hippocampus of cerebral ischemic rats.The decreased enzymes activities in turn enhanced the excess reactive oxygen species resulting in the increased oxidative damage reflecting by the elevation of MDA in cerebral cortex, hippocampus and striatum leading to neurodegeneration and brain infarction.It was found that M.oleifera leaves extract markedly decreased MDA level in all areas mentioned earlier and decreased brain infarction.The extract could enhance the activity of SOD both in hippocampus and striatum whereas the elevation of GSHPx enzyme activity was observed only in hippocampus.Unfortunately no changes of any enzymes just mentioned were observed in cerebral cortex.Therefore, the reduction of MDA level in this area might occur via the reduction of oxidative stress formation.
Although the determination of active ingredient responsible for the neuroprotective effect of this plant extract is beyond the scope of this study, we do suggest that the polyphenolic compound in the extract might play a vital role.It has been reported that plant polyphenols provide protection against neurodegenerative changes associated with cerebral ischemia (Simonyi et al., 2005).Moreover, this compound could also reduce the infarct volume, prevented motor impairment and inhibited lipid peroxidation (Sinha et al., 2002).Based on the effect of polyphenolic compounds just mentioned, it was also possible that the neuroprotective effect of the plant extract was associated with these compounds.Though our results suggested that the neuroprotective effect of M.oleifera leaves extract occurred partly via the decreased oxidative stress, other mechanisms such as the decreased apoptosis and intracellular calcium which also played the crucial roles on neurodgeneration and brain damage in cerebral ischemia still could not be cut off.

CONCLUSION
In conclusion, this study is the first study to demonstrate the neuroprotective effect against focal cerebral ischemia of M.oleifera leaves.It suggests that M.oleifera may be served as natural resource for developing neuroprotectant against focal cerebral ischemia.However, the precise underlying mechanism and possible active ingredient are still required further study.

Fig. 1 :
Fig. 1: The effect of Vitamin C, M. oleifera extract at the doses of 100, 200 and 400 mg kg −1 BW on the brain infarction volume.The coronal brain sections were determined using TTC staining.The data value are expressed as mean ± S.E.M. a : p-value<.05,aa : p-value<0.01,aaa : p-value<0.001;compared with vehicle plus sham operation.*: p-value<0.05,**: p-value<0.01;compared with vehicle plus MCAO