|Year : 2011 | Volume
| Issue : 6 | Page : 676-682
Neuroprotective activity of Wedelia calendulacea on cerebral ischemia/reperfusion induced oxidative stress in rats
Tigari Prakash1, Dupadahalli Kotresha2, Rama Rao Nedendla3
1 Department of Pharmacology, Acharya and B.M. Reddy College of Pharmacy, Bangalore - 560 090, Karmataka, India
2 Department of Biochemistry, Indian Institute of Science, Bangalore - 560 012, Karnataka, India
3 Department of Pharmaceutical Chemistry, Chalapathi Institute of Pharmaceutical Science, Guntur - 522 034, Andhra Pradesh, India
|Date of Submission||06-Jul-2010|
|Date of Decision||25-Mar-2011|
|Date of Acceptance||31-Aug-2011|
|Date of Web Publication||14-Nov-2011|
Department of Pharmacology, Acharya and B.M. Reddy College of Pharmacy, Bangalore - 560 090, Karmataka
Source of Support: None, Conflict of Interest: None
Objective : This study was undertaken to evaluate the neuroprotective activity of Wedelia calendulacea against cerebral ischemia/reperfusion induced oxidative stress in the rats.
Materials and Methods : The global cerebral ischemia was induced in male albino Wistar rats by occluding the bilateral carotid arteries for 30 min followed by 1 h and 4 h reperfusion. At various times of reperfusion, the histopathological changes and the levels of malondialdehyde (MDA), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-s-transferase (GST), and hydrogen peroxide (H 2 O 2 ) activity and brain water content were measured.
Results : The ischemic changes were preceded by increase in concentration of MDA, hydrogen peroxide and followed by decreased GPx, GR, and GST activity. Treatment with W. calendulacea significantly attenuated ischemia-induced oxidative stress. W. calendulacea administration markedly reversed and restored to near normal level in the groups pre-treated with methanolic extract (250 and 500 mg/kg, given orally in single and double dose/day for 10 days) in dose-dependent way. Similarly, W. calendulacea reversed the brain water content in the ischemia reperfusion animals. The neurodegenaration also conformed by the histopathological changes in the cerebral-ischemic animals.
Conclusion : The findings from the present investigation reveal that W. calendulacea protects neurons from global cerebral-ischemic injury in rat by attenuating oxidative stress.
Keywords: Brain edema, global cerebral ischemia, histopathology, oxidative stress, Wedelia calendulacea
|How to cite this article:|
Prakash T, Kotresha D, Nedendla RR. Neuroprotective activity of Wedelia calendulacea on cerebral ischemia/reperfusion induced oxidative stress in rats. Indian J Pharmacol 2011;43:676-82
|How to cite this URL:|
Prakash T, Kotresha D, Nedendla RR. Neuroprotective activity of Wedelia calendulacea on cerebral ischemia/reperfusion induced oxidative stress in rats. Indian J Pharmacol [serial online] 2011 [cited 2020 Feb 21];43:676-82. Available from: http://www.ijp-online.com/text.asp?2011/43/6/676/89825
| » Introduction|| |
Stroke is the third leading cause of death after myocardial infarction and cancer, and cerebrovascular diseases are considered second most frequent causes of projected deaths in the year 2020. This is the leading cause of permanent disability and disability-adjusted loss of independent life-years in Western countries. According to the Framingham data, 87% of strokes are caused by atherothrombotic and cardioembolic occlusions, 14% by hemorrhages, and 3% by other or undefined reasons.  Risk factors for stroke include advanced age, hypertension, previous stroke or transient ischemic attack, diabetes, high cholesterol, cigarette smoking, and atrial fibrillation. High blood pressure is the most important modifiable risk factor of stroke. 
Oxidative stress is one of the primary factors that exacerbate damage by cerebral ischemia. Several components of reactive oxygen species (ROS) (superoxide, hydroxyl radical, hydrogen peroxide and peroxynitrite radical) that are generated after ischemia-reperfusion injury play an important role in neuronal loss after cerebral ischemia.  Superoxide and hydroxyl radical are potent in producing destruction of the cell membrane by inducing lipid peroxidation.  Inducible nitric oxide synthase (iNOS) is upregulated after ischemia-reperfusion injury. This results in excessive nitric oxide (NO) production. This excess NO reacts with superoxide to form peroxynitrite, a powerful radical that produces neuronal death after cerebral ischemia. The brain is particularly vulnerable to oxidative stress injury because of its high rate of oxidative metabolic activity, intense production of reactive oxygen species metabolites, and high content of polyunsaturated fatty acids, relatively low antioxidant capacity, low repair mechanism activity, and non-replicating nature of its neuronal cells. 
Changes in antioxidant status in nervous tissue have been implicated in normal ageing and the pathologies of neurodegenerative disorders, such as amyotrophic lateral sclerosis, Parkinson's disease, and Alzheimer's disease. Not only oxidative stress implicated in chronic neuro-pathologies, similar mechanisms have been suggested for acute injuries such as ischemic stroke.  The polyphenolics including flavonoids, which are found in many herbal extracts, have been shown to be strong ROS scavengers, antioxidants, and protectors of neurons from lethal damage in in vitro. 
In addition, the neuroprotective effects of flavonoids in herbal extracts and their physiologically relevant conjugates against ischemia/reperfusion (I/R)-induced oxidative damage have also been reported. , Phenolic antioxidants from medicinal plants have also been evaluated in vivo as neuroprotective agents in animal models of I/R (ischemia/reperfusion) induced oxidative stress. Coumestan derivative wedelolactone and norwedelo-lactone are the main active constituents of the Wedelia calendulacea and it was reported that wedelolactone inhibits lipopolysaccharide (LPS)-induced caspase-11 expression in cultured cells by inhibiting NF-Jb-mediated transcription.  Caspase-11 is a key regulator of proinflammatory cytokine IL-1b maturation and pathological apoptosis. Apart from this, constituents in plant include coumestans, flavonoids, steroids, triterpenoids, ployaetylenes, and thiophene derivatives.
It is reported to have stimulatory effect on liver cell regeneration, constituents exhibited anti-hepatotoxic activity in rat hepatocytes. In addition, wedelolactone is one of the most powerful inhibitors of 5-lipoxygenase isolated from plants.  Present study was undertaken to evaluate the neuroprotective potential of methanolic extract of W. calendulacea in bilateral common carotid artery (BCA) occlusion induced global cerebral ischemia model in rats.
| » Materials and Methods|| |
Chemicals and Drugs
Glutathione (oxidized and reduced), nicotinamide adenine dinucleotide phosphate reduced (NADPH), 1-chloro-2,4-dinitrobenzene (CDNB), thiobarbituric acid (TBA), ethylenediaminetetraacetic acid (EDTA), and nitroblue tetrazoleum chloride (NBT), were purchased from Sigma Aldrich (St. Louis, MO, USA), SRL, Bombay and other chemicals were AR grade.
Male Wistar albino rats (250-300 g) were obtained from the National Institute of Mental Health and Neuro Science (NIMHANS), Bangalore. Rats were housed in polypropylene cages in air-conditioned room. Standard rat chow pellets and water was allowed ad libitum. The experimentations on animals (Ref No: 13/P'cology/2006-2009) were approved by the Institutional Animal Ethical Committee (IAEC) under the regulation of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), New Delhi.
The stem part of W. calendulacea was collected from Indian Institute of Science, Bangalore and authenticated by Department of Botany, Bangalore University, Bangalore. A voucher specimen (No: 001/2007) has been deposited in the Department of Pharmacology.
Fresh stem part of W. calendulacea was successively extracted with petroleum ether, chloroform and methanol. Petroleum ether and chloroform extract was discarded. Subsequently, the residue was extracted with methanol (yield: 8.9 g) in a Soxhlet apparatus for 48 h. The methanol solvent was removed under reduced pressure in a rotary vacuum evaporator.
Experimental Protocol for Global Ischemia
The protocol was divided into two main groups of 1 h and 4 h reperfusion models. Each main group again divided into six groups containing of six Wistar male rats, fed with methanolic extract or vehicle for 10 days prior to the experiment and treated as follows:
- Group I: Normal saline (10 ml/kg, orally), no ischemia.
- Group II: Normal saline (10 ml/kg, orally), bilateral carotid artery occlusion (BCAO) for 30 min and followed by 1 h and 4 h reperfusion individually (ischemic control).
- Group III: W. calendulacea (250 mg/kg, single dose/day, orally), BCAO for 30 min and followed by 1 h and 4 h reperfusion individually.
- Group IV: W. calendulacea (250 mg/kg, double dose/day, orally), BCAO for 30 min and followed by 1 h and 4 h reperfusion individually.
- Group V: W. calendulacea (500 mg/kg, single dose/day, orally), BCAO for 30 min and followed by 1 h and 4 h reperfusion individually.
- Group VI: W. calendulacea (500 mg/kg, double dose/day, orally), BCAO for 30 min and followed by 1 h and 4 h reperfusion individually.
Induction of Global Cerebral Ischemia and Reperfusion (I/R)
Group of animals were subjected to bilateral carotid artery occlusion. Rats were anesthetized with thiopentone sodium (40 mg/kg, i.p.). Animals were placed on the back; a midline ventral incision was made in neck. Trachea of animal was exposed followed by the right and left common carotid arteries were located. Both carotid arteries were exposed with special attention paid to separating and preserving the vagus nerve fibers. A cotton thread was passed below each carotid artery and a surgical knot was put on both arteries for 30 min induced ischemia. After 30 min of global cerebral ischemia, the thread was removed to allow the reflow of blood through carotid arteries (reperfusion) for 1 h and 4 h individually. Body temperature of rats was maintained around 37 ± 0.5°C throughout the surgical procedure by heated surgical platform. Sham control animals received the same surgical procedures except BCA were not occluded. After the completion of reperfusion period, the animals were assessed for their neuroprotective activity and were sacrificed thereafter. The brains were dissected out for determination of biochemical parameter, brain weight; histopathology study and assessment of cerebral infract size.
Preparation of Post-Mitochondrial Supernatant
After producing BCAO, the animals were sacrificed immediately by decapitation. Their brains were taken out, washed in pre-cooled 0.9% saline and frozen at −20°C for 5 min. The brain was then subsequently blotted on filter paper, then weighed and homogenized in chilled sodium phosphate buffer (0.1 M, pH 7.4) using a REMI tissue homogenizer. Homogenate was centrifuged at 10,000 g for 20 min at 4°C and post-mitochondrial supernatant (PMS) obtained from 10% (w/v) brain tissue was stored at -10°C for further assays.
To a sample of 0.2 ml of PMS (10% w/v), 0.2 ml of 8.1% sodium dodecyl sulphate, 1.5 ml of 20% acetic acid solution, and 1.5 ml of 0.8% aqueous solution of TBA were added. The mixture was made up to 5 ml with distilled water and then heated in an oil bath at 95°C for 60 min using a glass ball as a condenser. After cooling with tap water, 5 ml of the mixture of n-butanol and pyridine (15:1 v/v) was added and shaken vigorously. After centrifugation at 4000 g for 10 min, the organic layer was taken and its absorbance was measured at 532 nm. The tissue MDA levels were measured from the standard graph of known concentrations of MDA and expressed as nmol/g tissue.
Glutathione peroxidase activity
The reaction mixture consisted of 0.4 ml of phosphate buffer (0.4 M, pH 7.0), 0.2 ml of EDTA (0.8 mM), 0.1 ml of sodium azide (10 mM), 0.1 ml of reduced glutathione (4 mM), 0.1 ml of H 2 O 2 (30 mM), and 0.2 ml of PMS (10%, w/v). The mixture was incubated at 37°C for 10 min. Stored the tubes at room temperature and added 0.5 ml of 10% TCA and centrifuged at 200 g for 10 min, supernatant 0.1 ml of DTNB (0.04% in 1% trisodium citrate solution) solution was added. The optical density was read at 420 against blank (i.e. without homogenate).The enzyme activity was calculated as nM of glutathione oxidized/min/mg protein by using molar extinction coefficient 6.22×10 3 M−1 cm−1 .
Glutathione reductase activity
The assay system consisted of 1.65 ml phosphate buffer (0.1 M, pH 7.6), 0.1 ml NADPH (0.1 mM), 0.1 ml EDTA (0.5 mM), 0.05 ml oxidized glutathione (1 mM), and 0.1 ml PMS (10%, w/v) in a total volume of 2.0 ml. The enzyme activity was quantitated at room temperature by measuring the disappearance of NADPH at 340 nm and was calculated as nM NADPH oxidized/min/mg protein by using molar extinction coefficient of 6.22×10−3 M−1 cm−1 .
The reaction mixture consisted of 1.425 ml phosphate buffer (0.1 M, pH 6.5), 0.2 ml reduced glutathione (1 mM), 0.025 ml CDNB (1 mM), and 0.3 ml PMS (10%, w/v) in a total volume of 2.0 ml. The changes in absorbance were recorded at 340 nm and the enzyme activity was calculated as nM CDNB conjugate formed/min/mg protein using a molar extinction coefficient of 9.6×10 3 M−1 cm−1 .
Hydrogen peroxide was estimated by added 0.2 ml of PMS to 0.5 ml of 10 mM potassium phosphate buffer (pH 7.0), and 1.0 ml of 1M of potassium iodide. The absorbance of the reaction mixture was measured at 390 nm. The rate of H 2 O 2 production was calculated using a standard graph of H 2 O 2 and expressed as μM/g tissue.
Protein concentration in all PMS 10% (w/v) samples was determined by the method of Lowry et al.  by using span diagnostic kit.
Brain weight and water content
In another study involving the same protocol and treatment, brains from twelve groups of animals were removed and immediately weighed in pre-weighed glass vials and the wet weights recorded.  The percentage water content of each brain was calculated.
Similar protocol was placed for studying the histopathology of brain. Twelve groups of six animals each and were given similar treatment and induced the BCAO for 30 min and followed by 1 h and 4 h reperfusion individually. Coronal brain sections from control and experimental groups of global ischemia were removed, rinsed with cold normal saline and were fixed with a mixture of formaldehyde (40%), glacial acetic acid and methanol (1:1:8, v/v). Brain slices cut into 4-5 mm thickness and embedded in paraffin blocks. Brain sections of 4-6 μm thickness were cut and stained with hematoxylin and eosin.
The results were expressed as mean ± S.E.M. Statistical difference between mean were determined by one-way analysis of variance (ANOVA), followed by Dunnett t-test. The diagrammatic representation of the data was performed by using; Microcal™ Origin® Version 6.0 (Origin 6.0 AddOn, Data analysis and Technical graphics) software was used for all statistical calculations. Differences were considered significant at P<0.05.
| » Results|| |
Effect of W. calendulacea on Biochemical Analysis
The biochemical results are showed in [Figure 1],[Figure 2],[Figure 3],[Figure 4],[Figure 5]. The results showed that the cerebral ischemia and reperfusion significantly decreased antioxidative activities (GPx, GR, and GST) and increased the level of lipid peroxidation (malondialdehyde content, an index of lipid peroxidation) and hydrogen peroxide (H 2 O 2 ) in the injured brain tissue of rats as compared with the sham control group. However, the pretreatment of rats with W. calendulacea (250, 500 mg/kg, single and double dose/day) was markedly increased GPx, GR, and GST activity. In contrast, MDA and H 2 O 2 content in the injured brain tissue of rats decreased significantly (P<0.01) in W. calendulacea extract treated group compared to ischemic control group. However, the accumulation of MDA and H 2 O 2 content was significantly lower in cerebral ischemia in extract treated animals. Interestingly, the double dose/day treated group (250 and 500 mg/kg) had significantly alters enzyme activities than the single dose/day treated group with methanolic extract of W. calendulacea in cerebral ischemia-subjected rats.
|Figure 1: Effect of W. calendulacea on MDA in rats subjected to global cerebral ischemia followed by reperfusion. I: Sham control, no occlusion, II: ischemic control (normal saline, 10 ml/ kg, p.o.), III: W. calendulacea (250 mg/kg, single dose/day, p.o.) + ischemia, IV: W. calendulacea (250 mg/kg, double dose/day, p.o.) + ischemia, V: W. calendulacea (500 mg/kg, single dose/day, p.o.) + ischemia, VI: W. calendulacea (500 mg/kg, double dose/day, p.o.) + ischemia. Values are expressed as mean ± S.E.M., (n = 6). a = P<0.01 vs Sham control, b = P<0.01 vs ischemic control, by one-way analysis of variance (ANOVA), followed by Dunnett t-test|
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|Figure 2: Glutathione peroxidase (GPx) (a = P<0.01 and b = P<0.001 vs Sham control, c = P<0.01 and d = P<0.001 vs ischemic control (see the legends in Fig. 1)|
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|Figure 3: Glutathione reductase (GR) (a = P<0.001 vs Sham control, b = P<0.01 vs ischemic control (see the legends in Fig. 1)|
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|Figure 4: Glutathione-S-transferase (GST) (a = P<0.001 vs Sham control, b = P<0.01 vs ischemic control (see the legends in Fig. 1)|
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|Figure 5: Hydrogen peroxide (H2O2) (a = P<0.001 vs Sham control, b = P<0.01 vs Sham control, c = P<0.01 vs ischemic control (see the legends in Fig. 1)|
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Effect of W. calendulacea on Brain Weight and Water Content
The content of cerebral water (edema) was increased significantly in the ischemic/reperfusion control group. Pretreatment with W. calendulacea (250, 500 mg/kg) produced significant reduction (P<0.01) in water content and more than two-fold decrease the water content was observed in treated rat as compared to ischemic control group. However, there was significant decrease of brain weight observed in treated groups [Figure 6].
|Figure 6: Effect of methanolic extract of W. calendulacea on water content in brain in rats subjected to global cerebral ischemia followed by reperfusion. a = P<0.001 vs Sham control, b = P<0.01 vs ischemic control (see the legends in Fig 1)|
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Effect of W. calendulacea on Histopathology
The histopathology of the brain of ischemia-reperfusion and extract treated groups are showed in [Figure 7] and [Figure 8]. From the histopathological study, it was observed that section of brain tissue showing swollen neurons, dilated blood vessels with neuronal loss occurred in brain regions of I/R rats induced by BCAO for 30 min followed by 1 h and 4 h reperfusion in ischemic control group [Figure 7]b and [Figure 8]b. While no apparent morphological changes in sham control and brain section showing normal structure [Figure 7]a and [Figure 8]a. W. calendulacea (250 and 500 mg/kg) treated group of 1 h reperfusion brain section showed significantly prevented the neuron loss by compared with ischemic control group and in the other hand no significant difference between the doses of 250 mg/kg of W. calendulacea on 4 h reperfusion ischemic treated groups.
|Figure 7: Histopathological photographs of coronal sections of brain after 30 min of occlusion and 1 h of reperfusion in bilateral common carotid arteries occluded rats. (A) Sham control, no ischemia (normal saline, 10 ml/kg, p.o.); (B) ischemic control (normal saline, 10 ml/kg, p.o.); (C) W. calendulacea (250 mg/kg, single dose/day, p.o.) + ischemia; (D) W. calendulacea (250 mg/kg, double dose/day, p.o.) + ischemia; (E) W. calendulacea (500 mg/kg, single doses/day, p.o.) + ischemia; (F) W. calendulacea (500 mg/kg, double doses/day, p.o.) + ischemia|
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|Figure 8: Histopathological photographs of coronal sections of brain after 30 min of occlusion and 4 h of reperfusion in bilateral common carotid arteries occluded rats (see the legends in Fig. 7)|
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| » Discussion|| |
In a previous study, our group described the neuroprotective effect of the methanolic and aqueous extract of W. calendulacea.  The main compounds found in W. calendulacea include coumestan derivative, such as: wedelolactone, norwedelo-lactone, flavonoids, steroids, triterpenoids, ployaetylenes, and thiophene derivatives. This study was therefore carried out to evaluate the cerebroprotective property of the W. calendulacea.
In the present study, we observed therapeutic potential of W. calendulacea on global model of ischemia for the study of stroke in rats. Here, we selected two different reperfusion (1 h and 4 h reperfusion) models because the extension of damage produced may differ significantly. Furthermore, our aim was to evaluate the brain damage caused by both reperfusion models and then subsequently observe the protective action of methanolic extract of W. calendulacea pretreatment.
It was observed that W. calendulacea attenuated the impaired neurological deficit and sensory motor functions of the ischemic rats. The activity of W. calendulacea appears to restore the altered antioxidant enzymes as well as decreased the production of MDA in brain regions induced by BCA occlusion. There was a considerable evidence supported the role of ROS in the pathogenesis of I/R induced oxidative stress in brain.  The endogenous antioxidant enzyme activity of the brain impaired by I/R is particularly important and measurement of those antioxidant enzymes after reperfusion can assess the vulnerability of the particular areas of the brain.  Therefore, the potent radical scavenging properties or lipid proxidation inhibiting ability of polyphenolic natural compounds protecting the neurons from oxidative stress may provide useful therapeutic agent for the treatment of neurodegenerative diseases such as I/R induced oxidative stress. Peroxidation of lipid bi-layer was reported after cerebral IR injury.  So that lipids are most susceptible macromolecules to oxidative stress. From results of our work reveals that the production of MDA was significantly elevated in ischemic brain regions of the rats after 1 h and 4 h of reperfusion period. The results demonstrated that pretreatment with W. calendulacea had markedly reduced the MDA level and inhibits the neuronal injuries from propagating chain reaction of lipid peroxidation.
The presence of many antioxidant enzymes such as, GPx, GR, and GST in the brain prevents these tissues from the oxidative damage by free radical formation.  GPx is another important enzyme contributing to hydrogen peroxide scavenging. Dysfunction of GPx may result in loss of protective activity exerted by these enzymes and manifested as increased infarction in many studies.  Antioxidant treatment should prevent the loss of GPx activity by effectively scavenging the excess ROS. GPx are involved in the detoxification of H 2 O 2 , both at high and low concentrations. In this study, reduction in the GPx activity was significantly prevented by W. calendulacea administration.
Glutathione reductase an important enzyme for the maintenance of intracellular concentration of reduced glutathione, has a crucial role as a free radical scavenger.  A decline in glutathione reductase could both increase/reflect oxidative stress. The level of brain glutathione reductase was significantly reduced after ischemic injury.  In our study also, it was significantly decreased in I/R control group compared to sham control group. This depletion was directly associated with elevation in brain lipid peroxidation. Dysfunction of the glutathione system has been implicated in a number of neurodegenerative diseases.  Therefore, further extends the support to our idea for observing the glutathione levels in the present study. Glutathione-S-transferase catalyses the detoxification of oxidized metabolites of catecholamines (o-quinone) and may serve as antioxidant, preventing degenerative processes. 
Accumulation of hydrogen peroxide was reported to impair the mitochondrial function. H 2 O 2 is a longer-lasting reactive species electrically neutral and is able to pass through cell membranes. Hydrogen peroxide is reported to be more stable than superoxide anion, hydroxyl free radical, and other ROS.  Therefore, hydrogen peroxide may persist for longer time after reperfusion to produce neuronal injury. Level of H 2 O 2 in I/R rats brain were markedly reversed and restored to near normal levels in the groups' pre-treated with W. calendulacea. Due to it increases in endogenous antioxidant defense enzymes and may prevent the neuronal injury produce by hydrogen peroxide.
Brain edema has also been studied extensively in ischemia to assess the impact of brain damage. , Cerebral edema occurs as a result of ionic imbalance (and hence the osmotic pressure) across the cellular membrane. A simple pathophysiological mechanism is that energy failure results in neuron depolarization, which causes activation of glutamate receptors, which in turn alters ionic gradients of Na + , Ca ++ , Cl - and K + . As glutamate increases in the extracellular space, peri-infarct depolarization occurs. Then, as water shifts occur, cells swell with resulting cerebral edema. W. calendulacea also reversed effects on brain water content in the ischemia reperfusion animals as compared to the ischemic control group. These results are pointing to its potential therapeutic value in cerebrovascular diseases including stroke. The observed results leading to neurodegeneration is also confirmed by the histopathological differences between treatment and ischemic control groups. There was reversal of the brain damage observed in extract administered twice a day and it was prevented the neuron loss by approximately 40%. It is suggested that pretreatment of W. calendulacea could exert a neuroprotective effect on the brain subjected to I/R.
| » Conclusion|| |
Methanolic extract of W. calendulacea could reduce neuronal loss of the ischemic brain tissue. W. calendulacea showed antioxidant activity in reperfusion induced oxidative stress.
| » Acknowledgment|| |
The authors would like to express their gratitude to Mr. Premanath Reddy, Chairman, Acharya Institute, and Dr. Divakar Goli, Principal, Acharya and BM Reddy College of Pharmacy, Bangalore, India, for providing the necessary facilities and support to carry out the research work.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]