Centella asiatica attenuates the neurobehavioral, neurochemical and ...

Neurol Sci (2013) 34:925–933 DOI 10.1007/s10072-012-1163-1

ORIGINAL ARTICLE

Centella asiatica attenuates the neurobehavioral, neurochemical and histological changes in transient focal middle cerebral artery occlusion rats Rizwana Tabassum • Kumar Vaibhav • Pallavi Shrivastava • Andleeb Khan Md. Ejaz Ahmed • Hayate Javed • Farah Islam • Sayeed Ahmad • M. Saeed Siddiqui • Mohammed M. Safhi • Fakhrul Islam

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Received: 18 March 2012 / Accepted: 11 July 2012 / Published online: 4 August 2012 Ó Springer-Verlag 2012

Abstract Centella asiatica has been used as psychoactive and antioxidant herbal medicine since ancient time. The present study was design to evaluate the preventive role of ethanolic extract of C. asiatica in middle cerebral artery occlusion (MCAO) in rats. Male Wistar rats were gavaged orally with C. asiatica extract (100, 200 and 300 mg/kg body weight once daily) for 21 days and thereafter subjected to right MCAO for 2 h followed by 22-h

Electronic supplementary material The online version of this article (doi:10.1007/s10072-012-1163-1) contains supplementary material, which is available to authorized users. R. Tabassum  K. Vaibhav  P. Shrivastava  A. Khan  Md. Ejaz Ahmed  H. Javed  M. Saeed Siddiqui  F. Islam (&) Neurotoxicology Laboratory, Department of Medical Elementology and Toxicology (DST-FIST and UGC-SAP-BSR funded department), Jamia Hamdard (Hamdard University), Hamdard Nagar, New Delhi 110062, India e-mail: [email protected] Present Address: P. Shrivastava Department Neurology, Robert Wood Johanson Medical School, UMDNJ, New Jersey, USA F. Islam Department of Biotechnology, Faculty of Pharmacy, Jamia Hamdard (Hamdard University), Hamdard Nagar, New Delhi 110062, India S. Ahmad Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Jamia Hamdard (Hamdard University), Hamdard Nagar, New Delhi 110062, India Present Address: M. M. Safhi  F. Islam Neuroscience and Toxicology Unit, Faculty of Pharmacy, Jazan University, Jazan, Kingdom of Saudi Arabia

reperfusion. Brain injury was evaluated by 2,3,5-triphenyltetrazolium chloride and hematoxylin and eosin staining. Behavioural outcomes as neurological deficit, rota rod test, and grip strength were assessed. In addition, lipid peroxidation, enzymatic and non enzymatic antioxidants were analyzed to assess the oxidative stress. Our results revealed that C. asiatica administration greatly improved neurobehavioral activity and diminished infarction volume along with the restored histological morphology of brain in MCAO rats. Furthermore, supplementation with this extract to MCAO group has reduced the level of thiobarbituric acid reactive species, restored glutathione content and augmented the activities of antioxidant enzymes— catalase, glutathione peroxidase, glutathione reductase, glutathione-S-transferase and superoxide dismutase in a dose-dependent manner in ischemic rats. The remarkable antioxidant activity of C. asiatica may be attributed to its bioactive triterpenes, asiatic acid, asiaticoside, madecassic acid and madecosside and may be translated to clinical level for prevention of ischemic stroke. Keywords Asiatic acid  Centella asiatica  Ischemia–reperfusion  Neurobehavior  Oxidative stress

Introduction Transient focal cerebral ischemia is a disruption of cerebral blood supply to the part of the brain for a certain duration. Ischemia–reperfusion (I/R) injuries aggravate the pathological process by generation of free radicals including superoxide anions (O2 ) and hydroxyl radicals (OH) [1]. The brain tissue is more prone to oxidative damage due to its high content of unsaturated fatty acids, rapid oxidative metabolic activity, and comparatively little endogenous

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antioxidant potential and insufficient neuronal cell repair capacity [2]. Therefore, the oxidative stress after I/R ultimately mediates the induction of deleterious events, like mitochondrial dysfunction that may lead to apoptosis, inflammation and exitotoxicity [3]. Extensive evidences have reported that various antioxidants ameliorate oxidative stress and salvage neurons from I/R-induced injury [4, 5]. Even, a number of extracts of aqueous garlic extract [6] Crocus sativus [7] and Nardostachys jatamansi [8] have been proved as neuroprotective in I/R injury. Therefore, traditional system of medicines is getting more approachable for their effectual and extensively pharmacological treatments nowadays. Centella asiatica is a psychoactive therapeutic plant belonging to the family Apiaceae that has been used for several years as a Medhya Rasayana in Indian Ayurvedic medicine [9]. In addition, it has potent antioxidant [10] and anti-inflammatory properties [11]. The medicinal importance of this extract is credited to its bioactive triterpenes content namely asiatic acid, asiaticoside, madecassic acid and madecosside [12]. Recent reports also discovered its protective role in ameliorating the amyloid beta levels in Alzheimer’s disease animal model [13] and in experimentally induced Parkinsonism in aged Sprague–Dawley rat [14]. Therefore, keeping emphasis on its properties, the present work was planned to study the effects of ethanolic extract of C. asiatica (C) on the prevention of cerebral ischemia in rats.

Materials and methods Ethanolic extract of C. asiatica (C) was purchased from Saiba Industries, Mumbai, India. Oxidized glutathione (GSSG), reduced glutathione (GSH), (-)epinephrine, 1-chloro-2,4dinitrobenzene (CDNB), 5,50 -dithiobis-2-nitrobenzoic acid (DTNB), glutathione reductase (GR), nicotinamide adenine dinucleotidephosphate reduced (NADPH), thiobarbituric acid (TBA), and 2,3,5-triphenyltetrazolium chloride (TTC) were purchased from Sigma-Aldrich Chemicals Pvt. Ltd., India. Other chemicals were of analytical reagent grade. Animals Male Wistar rats (260–280) were obtained from the Central Animal House Facility, Jamia Hamdard, and New Delhi, India. The animals were used in accordance with the procedure approved by the Animal Ethics Committee of Jamia Hamdard. Experimental plan The animals were randomly divided into six groups representing (1) sham (S): saline administered control, (2)

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MCAO: saline administered ischemic group, (3) C100 ? MCAO: ischemic group pretreated with C (100 mg/kg body weight once daily for 21 days), (4) C200 ? MCAO: ischemic group pretreated with C (200 mg/kg body weight once daily for 21 days), (5) C300 ? MCAO: ischemic group pretreated with C (300 mg/kg body weight once daily for 21 days), (6) C300 ? S: pretreatment with C (300 mg/kg body weight of once a day for 21 days) in sham group. Induction of ischemia Ischemia was induced using a MCAO model as described by Ahmad et al. [4]. Briefly, rats were anaesthetized with intraperitoneal injection of chloral hydrate 400 mg/kg body weight. A silicon-coated suture (4 0-033REPK0, DOCCOL, USA) was inserted into the right external carotid artery (ECA) and pushed through the internal carotid artery (ICA) up to the origin of middle cerebral artery (MCA). After 2 h of MCAO, the suture was retracted carefully and animals were allowed to recover from anesthesia. After 22-h reperfusion, animals were evaluated for behavioral deficits and then sacrificed to collect brains. Neurological deficits Neurological deficits were evaluated by flexion test (FT) and spontaneous motor activity (SMA). After 22-h reperfusion, FT was assessed by placing the animals in their normal environment (cage) for 5 min on four point scoring scale. 0: No neurological deficit; 1: contralateral forelimb flexion and shoulder adduction; 2: reduced resistance to lateral push; and 3: circling movements towards the paretic side [15]. SMA was evaluated on four point scales, 0: no neurological deficit, 1: unable to extend left forepaw fully, 2: circling to the left, 3: falling to the left [16]. Grip strength test Grip strength was analyzed on six scale system: 0: falls off; 1: hangs onto string by two forepaws; 2: as for 1 but attempts to climb on string; 3: as for 1 plus one or both hind limbs; 4: as for 1 plus tail wrapped around string and 5: escape. The highest reading of three successive trials was taken for each animal [17]. Muscular coordination Rats were subjected to the rota rod task as described by Sommer et al. [18]. The time each rat remained on the rotating bar was recorded for a maximum period of 180 s per trial. The speed was set at 10 rpm. Data were presented as mean time on the rotating bar over the five test trials.

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Cerebral infarct assessment

Catalase (CAT)

The brain was sliced into 3.0 mm coronal section in thickness with the help of brain matrix (Activational Systems, MI, and USA) and incubated in 0.1 % TTC for 30 min at 37 °C [5]. After incubation, color images of these sections were taken. The infarct area is measured by Image-J 1.37v, NIH, USA Analysis software. Infarct volumes were presented as percentage of infarct volume of ipsilateral hemisphere.

CAT activity was measured by the procedure of Claiborne et al. [22]. Catalase activity was calculated in terms of nmol H2O2 consumed/min/mg protein using molar extinction coefficient of 43.6 9 103 M-1 cm-1.

Histopathology After 22-h reperfusion, the animals were anesthetized with chloral hydrate (400 mg/kg, i.p.) and perfused as described previously by Nakayama et al. [19] with the mixture of formaldehyde (40 %), glacial acetic acid and methanol (1:1:8, v/v). The brain was embedded in paraffin and cut to give 5-lm thick coronal sections. Every ten sections of the hippocampus and cortex region was mounted on glass slides, and processed for hematoxylin and eosin staining. The images were analyzed using Olympus BX50 microscope.

Biochemical studies Sample preparation

Glutathione peroxidise (GPx) GPx activity was recorded as described by Mohandas et al. [23] as disappearance of NADPH at 340 nm at room temperature and expressed as nmol NADPH oxidized/min/mg protein using molar extinction coefficient 6.22 9 103 M-1 cm-1. Glutathione reductase (GR) GR activity was measured at room temperature by measuring the disappearance of NADPH at 340 nm and was calculated as nmol NADPH oxidized/min/mg protein using molar extinction coefficient 6.22 9 103 M-1 cm-1 [23]. Glutathione-S-transferase (GST) GST activity was measured as the change in absorbance recorded at 340 nm and expressed as nmol of CDNB conjugate formed/min/mg protein using molar extinction coefficient 9.6 9 103 M-1 cm-1 [24].

Ipsilateral hippocampus and frontal cortex were dissected to prepare 5 % (w/v) homogenate (10 mM phosphate buffer (pH 7.4) having 10 ll/ml protease inhibitor). The homogenate were centrifuged at 8259g for 5 min at 4 °C to separate nuclear debris. The supernatant was used for the assay of lipid peroxidation and the remaining supernatant was further centrifuged at 10,5009g for 15 min at 4 °C to obtain post-mitochondrial supernatant (PMS) which was used for the estimation of GSH and antioxidant enzymes.

Superoxide dismutase (SOD)

Lipid peroxidation (LPO)

Protein was estimated by the method of Lowry et al. [26] using bovine serum albumin as standard.

LPO was assayed by measuring thiobarbituric acid reactive substance (TBARS) at 535 nm as described by Islam et al. [20]. The rate of LPO was expressed as nmol TBARS formed/h/mg protein using a molar extinction coefficient of 1.56 9 105 M-1 cm-1.

SOD activity was assayed by monitoring the auto oxidation of (-) epinephrine at pH 10.4 for 3 min at 480 nm and calculated as nmol (-) epinephrine protected from oxidation/min/mg protein using molar extinction coefficient 4.02 9 103 M-1 cm-1 [25]. Protein

Statistical analysis Results are expressed as mean ± SEM. The result was analyzed by one-way ANOVA followed by Tukey’s test. The p value \0.05 was considered significant.

Reduced glutathione (GSH) Estimation of asiatic acid The GSH was determined by the method of Jollow et al. [21]. The yellow color developed was read immediately at 412 nm in spectrophotometer. The GSH level was expressed as lmol GSH/mg protein using a molar extinction coefficient of 13.6 9 103 M-1 cm-1.

Asiatic acid, an active component in C. asiatica extract was estimated using CAMAG HPTLC Scanner III apparatus with the help of Wincats software. The standard solution (4 ll) of asiatic acid and sample (6 ll) was applied on

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Fig. 1 Behavioral deficits. The FT and SMA were severe in MCAO group as compared to sham group. C administration has protected the severity of deficits in C200 ? MCAO and C300 ? MCAO groups (a). Muscular strength (b) and motor coordination skills (c) were decreased considerably in MCAO group, as compared to sham group.

A significant improvement was observed in C200 ? MCAO and C300 ? MCAO groups. C100 ? MCAO (vs. MCAO) and C300 ? S (vs. Sham) groups have shown no significant change. Values are expressed as mean ± SEM. #p \ 0.01; versus sham; *p \ 0.05 and **p \ 0.01; versus MCAO

Fig. 2 Infarct volume and histological alterations. a Representative photograph is TTC (0.1 %) stained coronal sections of the brain. b Degree of injuries expressed as percentage of infarct volume. Pretreatment with C has shown significant recovery in infarct volume of C200 ? MCAO (*p \ 0.01) and C300 ? MCAO groups (*p \ 0.01) as compared to MCAO group. c H&E staining of hippocampus and cortex of sham group shows neurons with well-characterized round

nuclei (black arrow) (I and VII). MCAO caused neuronal injury associated with injured neurons with triangular and pycnotic nuclei (white arrow) (II and VIII). C-pretreated group exhibited a significant protection of neuronal injury in terms of restoration of hippocampal and cortical neurons with more intact neurons (III–V and IX–XI). Furthermore, histology of higher dose (C300 ? S) did not presented any toxicity (VI and XII)

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314.74 ± 13.66 313.34 ± 12.97 758.80 ± 20.35 310.38 ± 5.80

GPX (nmol NADPH oxidized/min/mg protein)

GR (nmol NADPH oxidized/min/mg protein)

GST (nmol CDNB conjugate formed/min/mg protein)

SOD (nmol epinephrine protected/min/mg protein)

#

1.33 ± 0.05 (5.55%) 18.01 ± 1.70 (14.13%) 175.06 ± 10.71 (4.99%) 168.72 ± 11.11 (8.71%) 446.63 ± 27.79 (14.28%) 175.58 ± 5.34 (11.28%)

15.85 ± 2.14# (-48.32%) 167.25 ± 7.18# (-46.86%) 155.20 ± 10.71# (-50.46%) 390.92 ± 14.79# (-48.48%) 157.77 ± 6.51# (-49.16%)

8.85 ±0.5 (-3.38%)

C100 ? MCAO

1.26 ± 0.03# (-33.33%)

9.16 ± 0.41 (94.89%)

MCAO

203.22 ± 1.69* (28.80%)

537.32 ± 26.06* (37.45%)

193.53 ± 9.13* (24.69%)

211.28 ± 5.34* (26.32%)

22.45 ± 2.25* (1.64%)

1.42 ± 0.08* (12.69%)

7.03 ± 0.46* (-23.25%)

C200 ? MCAO

241.76 ± 3.25** (53.23%)

594.55 ± 9.99** (52.08%)

243.79 ± 10.60** (57.08%)

226.69 ± 7.63** (35.53%)

26.56 ± 1.50** (67.57%)

1.57 ± 0.03** (24.60%)

5.92 ± 0.29** (-35.37%)

C300 ? MCAO

303.09 ± 8.50 (-2.34%)

768.79 ± 54.58 (?1.31%)

290.44 ± 11.34 (-7.30%)

290.44 ± 11.34 (-1.35%)

29.21 ± 1.39 (-4.76%)

1.85617 ± 0.08 (2.11%)

4.73±0.33 (-0.67%)

C300 ? S

226.29 ± 14.14 260.23 ± 11.21 699.72 ± 28.48 314.87 ± 4.26

GPx (nmol NADPH oxidized/min/mg protein)

GR (nmol NADPH oxidized/min/mg protein)

GST (nmol CDNB conjugate formed/min/mg protein)

SOD (nmol epinephrin protected/min/mg protein)

C100 ? MCAO 7.78 ± 0.74 (-5.46%) 1.21 ± 0.06 (?5.21%) 16.87 ± 1.19 (8.69%) 131.37 ± 28.55 (16.38%) 154.83 ± 15.71 (9.71%) 391.71 ± 25.64 (7.75%) 176.23 ± 7.27 (7.17%)

MCAO 8.23 ± 0.37# (?68.30%) 1.15 ± 0.08# (36.46%) 15.52 ± 1.16# (-45.26%) 121.23 ± 12.21# (-46.48%) 141.13 ± 14.51# (45.76%) 363.21 ± 14.74# (-48.03%) 164.43 ± 8.66# (-47.77%)

209.29 ± 4.33* (27.28%)

522.38 ± 22.58* (43.65%)

180.28 ± 5.70* (27.74%)

141.09 ± 12.77* (16.38%)

20.50 ± 0.96* (32.08%)

1.41 ± 0.07* (22.60%)

7.01 ± 0.26* (-14.82%)

C200 ? MCAO

253.68 ± 4.06** (54.27%)

589.48 ± 11.88** (54.42%)

212.32 ± 10.30** (50.44%)

174.90 ± 8.23** (44.27%)

24.18 ± 1.14** (55.79%)

1.60 ± 0.05** (39.13%)

5.93 ± 0.22** (-27.93%)

C300 ? MCAO

296.05 ± 40.71 (-5.97%)

726.28 ± 42.31 (3.79%)

255.70 ± 11.99 (-1.74%)

207.43 ± 3.14 (-8.33%)

27.87 ± 1.68 (-1.69%)

1.85 ± 0.18 (2.21%)

4.20 ± 0.42 (-14.11%)

C300 ? S

Values are represented as mean ± SEM. Values in parentheses show the percentage increase or decrease with respect to their control (#p \ 0.01 vs. S group and *p \ 0.05, **p \ 0.01 vs. MCAO)

28.35 ± 0.62

1.81 ± 0.07

GSH (lmol glutathione conjugate formed/mg protein)

CAT (nmol H2O2 consumed/min/mg protein)

4.89 ± 0.20

Sham (S)

LPO (nmol TBARS formed/h/mg protein)

Cortex

Table 2 Protective effect of Centella asiatica on lipid peroxidation (LPO), reduced glutathione (GSH), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glutathioneS-transferase (GST), and superoxide dismutase (SOD) in cortex of focal cerebral ischemic rat

Values are represented as mean ± SEM. Values in parentheses show the percentage increase or decrease with respect to their control (#p \ 0.01 vs. S group and *p \ 0.05, **p \ 0.01 vs. MCAO)

30.67 ± 1.68

1.89 ± 0.06

GSH (lmol glutathione conjugate formed/mg protein)

CAT (nmol H2O2 consumed/min/mg protein)

4.70 ± 0.36

Sham (S)

LPO (nmol TBARS formed/h/mg protein)

Hippocampus

Table 1 Protective effect of Centella asiatica on lipid peroxidation (LPO), reduced glutathione (GSH) content and the activity of catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-S-transferase (GST) and superoxide dismutase (SOD) in hippocampus of focal cerebral ischemic rat

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5 9 10 cm precoated silica plate in duplicate. The solvent system, toluene: ethyl acetate: formic acid (5:4:1, v/v/v) was used as mobile phase. The plate was developed by spraying anisaldehyde–sulfuric acid reagent and kept at 110 °C for 10 min and scanned at 600 nm.

Results Effect of C on behavioral deficits High neurological deficits were observed in flexion test (92.83 %) and spontaneous motor activity (93.20 %) in MCAO group (p \ 0.01) as compared to sham that was alleviated in C200 ? MCAO (-42.91 and -40.00 %; p [ 0.05) and C300 ? MCAO (-71.67 and -66.40 %; p \ 0.01) (Fig. 1a). Further, significant weak grip strength and muscular co-ordination were observed in MCAO group (-64.96 and -63.92 %; p \ 0.01). C has improved the grip strength and motor co-ordination significantly in C200 ? MCAO (61.58 and 37.04 %; p \ 0.05) and C300 ? MCAO group (89.63 and 95.69 %; p \ 0.01) in a dose-dependent manner (Fig. 1b, c). The lower dose C100 ? MCAO did not show any significant changes versus MCAO group. Further, no significant difference was observed between C300 ? S and sham group. Effect of C on infarct volume and histological alterations TTC staining of the coronal sections of the brain presented a marked white infarct in ischemic brains (Fig. 2a). C200 ? MCAO group (-54.12 %; p \ 0.05) and C300 ? MCAO (-70.59 %; p \ 0.01) group significantly decreased infarct volume as compared to MCAO group where as C100 ?MCAO group did not exhibit significant decrement in infarction (Fig. 2b). In addition, H&E staining of brain sections revealed densely arranged healthy neurons with well-characterized round nuclei in CA1 region of hippocampus and penumbral cortex of sham group while the MCAO group showed severe neuronal degeneration associated with vacuolated tissue having scantly arranged neurons and pyknotic nuclei which has been protected by C administration (Fig. 2c). Effect of C on oxidative stress The MCAO group exhibited a significant increased level of TBARS in hippocampus and cortex (?94.89 and ?68.30 %; p \ 0.01) in comparison to sham group. The C200 ? MCAO group showed a significant amelioration of TBARS in hippocampus and frontal cortex (-23.25 and -13.6 %; p \ 0.05), whereas the C300 ?MCAO group

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attenuated more TBARS in hippocampus and frontal cortex (-35.37 and -27.94 %; p \ 0.01). GSH content and the activity of various antioxidant enzymes (GPx, GR, SOD, GST and CAT) were depleted significantly in hippocampus (p \ 0.01) and frontal cortex (p \ 0.01) of MCAO group as compared to sham group. The pre-treatment with C has protected their activity in hippocampus and frontal cortex in C200 ? MCAO group (p \ 0.05) and C300 ? MCAO group (p \ 0.01) in a dose reliant way as compared to MCAO group, whereas C100 ? MCAO group has not shown significant changes as compared to MCAO (Tables 1, 2). Quantification of asiatic acid HPTLC fingerprint shows a moderate amount of asiatic acid (0.069 mg/g of extract) present in C that may be attributed for its antioxidant activities (Fig. 3). The fingerprint, however, shows a number of significant peaks (2–4 and 6–16) indicating the presence of other functional components Therefore, the role of other components like asiaticoside, madecassic acid and madecosside cannot be ignored in the protection against I/R injury.

Discussion It is well established that I/R insult initiates free-radical generation that induce oxidation of building macromolecules viz. lipid [27], protein [5] and DNA [28] resulting the impaired behavioral, neurochemical and histological outcomes in rat brain [4, 5, 29]. In the present study, ischemic group rats exhibited the higher score of neurological deficits with considerable weak grip strength and loss of muscular coordination. C has ameliorated the behavioral alterations significantly in both doses except the lower dose group (C100 ? MCAO). The reason for this may be the lower dose could not maintain the optimum level in blood to counter free radicals. The I/R injury results in impaired energy metabolism leading to mitochondrial injury [30, 31]. The administration of C prior to MCAO has significantly regressed neuronal injury as shown by small infarct in TTC-stained brain sections. TTC a colorless compound gets reduced to red formazan by mitochondrial dehydrogenase enzymes of viable cells [32]. The present outcome of C administration is also attested by previous studies [3, 4, 33]. Many of the histological alterations due to cerebral ischemic injury were successfully reverted in C-pretreated groups. As reported earlier, C is a remarkable antioxidant and has been proved to scavenge free radicals [10]. Thus, it may facilitate in retaining the biological antioxidant, and able to successful amelioration of neuronal injury.

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Fig. 3 Asiatic acid content. Track 1 HPTLC developed silica plate represents asiatic acid standard while track 2 and 3 represents C extract (a). Peak 4 corresponds to Rf value 0.43 in finger print of track 1 of silica plate at 600 nm, showed maximum response for asiatic

acid standard (b). In the fingerprint of track 3 at 600 nm, peak 5 corresponded to Rf value of asiatic acid and concentration was found to be 0.069 mg/g extract (c)

Oxidation of polyunsaturated fatty acids of the cell membrane of the ischemic brain manifests alteration in membrane structure, fluidity and permeability [34]. In this study, C administration depleted TBARS in MCAO group that is also in accord with the prior in vivo study [34]. Increased ROS by I/R injury may cause the exhaustion in an endogenous antioxidant GSH, which is the first line of neuronal injury [35]. In the current study, depletion of reduced glutathione may be attributed to scavenge the ROS in MCAO group [35]. C treatment has restored this GSH content significantly as compared to MCAO group in a dose-dependent manner by virtue of its ROS scavenging ability. It has been reported that inadequate CAT and GPx activity leads to the increment of H2O2, consequently transformed into more reactive hydroxyl ions in MCAO group [36]. In the detoxifying reaction of H2O2 by GPx,

reduced glutathione is converted to oxidized glutathione (GSSG) [37]. Hence, the role of GR is also a crucial in regeneration of GSH from its oxidized form GSSG for protecting cells against ROS. It was also proved in earlier reports that GST, a glutathione-dependent enzyme, is detoxifier of oxidized metabolites of catecholamine (o-quinone) mediating as antioxidant agent by refraining degenerative cellular process [38]. It has previously reported that SOD proved as protective as it scavenges the superoxide produced in I/R injuries and may limit the consequent DNA damage in rat model of cerebral ischemia [39]. Protection of the activities of these antioxidant enzymes by administration of C is indicative of strong ROS scavenging ability of the extract. It can be argued that the anti-ischemic activity of C may be attributed to its antioxidant compounds.

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In the present study, the extract was characterized by HPTLC method for one of its bioactive compounds, asiatic acid to establish the identification of the extract and HPTLC fingerprint has been shown in the manuscript. The amount of asiatic acid in C. asiatica extract was found to be 0.069 mg/g which might have contributed to the protection of brain after ischemia reperfusion (I/R) injury. Since, ethanolic extract of C. asiatica is enriched with a number of bioactive components such as asiatic acid, madecassic acid, asiaticoside and madecosside, etc. [12] which being lipophilic in nature, may cross the blood brain barrier and may possess augmented protective role by ameliorating oxidative stress through robust antioxidant and free-radical scavenging potential [14, 40]. Therefore, it can be inferred that protection afforded by C. asiatica extract is the combined effect of various active compounds present in the extract [14, 41–44] and has been observed as different peaks in the fingerprint (Fig. 3c). In conclusion, C prevented neuronal injury induced by MCAO by its antioxidant and free-radical scavenging potentials and that successfully improved the neurobehavioral, neurochemical outcomes and histology of ischemic brain. These findings also strongly put C as a candidate for pharmacological investigation for further understanding the mechanism behind the neuroprotection afforded by this. However, the role of individual component in acute ischemic injury remains to be evaluated. Therefore, further studies may be obligatory to reveal the precise molecular mechanism of different bioactive component of C. asiatica against cerebral ischemic injury. Acknowledgments The author (R. Tabassum) is highly thankful to UGC, Govt. of India for financial help. Conflict of interest Authors declare that there is no conflict of interest involved with this work.

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