Investigation of Neuroprotective Effect of n-Hexane Extract of Cissampelos pareira in Ischemic Stroke-Induced Nerve Damage and Underlying Mechanisms

Abstract

Objective: Cissampelos pareira Linn. (Menispermaceae) has been reported for improving memory and cognitive behavior, and also as a folk remedy, for various neurodegenerative disorders. We investigated Cissampelos pareira for evaluating its neuroprotective effect on rodent model of focal cerebral ischemia. Methods: Male Sprague–Dawley rats (270-300 g) were subjected to permanent middle cerebral artery occlusion (MCAO). The animals were divided into five groups as control, MCAO, and MCAO + Cissampelos pareira (n-hexane, ethyl acetate (EtOAc), and methanol (MeOH) extracts; 50 mg/kg; n = 14). The expression of various inflammatory factors and enzymes including cyclooxygenase (COX-2), c-Jun N-terminal Kinase (p-JNK), and nuclear factor kappa-light-chain-enhancer of activated B cells (p-NF-κB) was detected in response to disease and extracts by immunohistochemistry and enzyme-linked immunosorbent assay. Results: The n-hexane extract (LD₅₀> 5.0 g/kg) reduced the infarction area significantly from in n-hexane extract + MCAO group, reversed neuronal death (P < .01), and relieved the neurobehavioral deficits. The reduced levels of antioxidant enzymes (GST, GSH, CAT, SOD, and GPx), in case of MCAO were significantly elevated in response to n-hexane extract. 1,2-benzenedicarboxylic acid was detected (through gas chromatography/mass spectrometry) as the major component in n-hexane extract. Conclusion: n-hexane extract has the potential to be of therapeutic value in stroke patients, and thus further studies are warranted to elucidate the neuroprotective effects of this extract.

Keywords

neurodegeneration 1 2-Benzenedicarboxylic acid

Introduction

Cissampelos pareira Linn. (Menispermaceae) is a climber of therapeutic repute in the Ayurvedic system of medicine and is a popular folk remedy for many neurodegenerative disorders including epilepsy, convulsions, and as tonic.^1,2 Cissampelos pareira is also reputed for its memory enhancing potential in the Unani system of medicine. Herbal formulations having Cissampelos pareira leaves are reported for their neuroprotective and cognitive effects possibly by anti-acetylcholine esterase and antioxidant potential.³ Moreover, it has demonstrated beneficial effects in various memory impairment models by executing anti-inflammatory and antioxidant actions.⁴ Its immunomodulatory potential has also been recognized.⁵

Ischemic stroke is a common cerebrovascular clinical condition bringing about 5 million deaths and many disabilities annually, making stroke the leading cause of death and disability worldwide.⁶ Cerebral ischemia results from the loss of blood supply to the cerebrovascular region, subsequently activating the pathological cascade involving oxidative stress, glutamate excitotoxicity, calcium overload, and release of pro-inflammatory cytokines. All these events eventually result in neuronal death.⁷ Middle cerebral artery occlusion (MCAO) or ischemic stroke is characterized by permanent or transient obstruction of blood flow to the brain, impeding the delivery of oxygen and essential nutrients to the site. The molecular mechanism of ischemic stroke-induced neurodegeneration is a complex phenomenon, due to the involvement of multiple signaling pathways and varying extent of cellular damage.⁸ Energy failure due to diminished oxygen and glucose supply leads to reduced ATP, ionic imbalance, membrane depolarization and calcium overload. Glutamate over release further facilitates calcium influx and initiates downstream inflammatory and apoptotic mediators. The inflammatory cascade occurs within an hour after ischemic stroke and contributes significantly to the pathophysiology of the stroke.⁹ Brain bombardment with circulating inflammatory cells including granulocytes, neutrophils, lymphocytes, and leukocytes is followed by the rapid activation of astrocytes and microglial cells. Moreover, tissue damage due to the blockage of blood flow also activates the release of interleukin-1 (IL-1β), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6), which are the potential mediators of inflammation. All these events result is slow clinical prognosis and increased infarct volume.¹⁰

C-Jun N-terminal kinases (JNKs) are important members of the mitogen-activated protein kinases (MAPK) family which are involved in apoptotic and inflammation.¹¹ JNK (as p-JNK) activates apoptotic cell death by transcriptional and post-transcriptional modifications. JNKs are critical in death receptor-initiated extrinsic and mitochondrial elicited intrinsic apoptotic pathways. P-JNK is involved in both adaptive and innate immune responses as it can be activated by various pro-inflammatory cytokines like TNF-α, IL-1β, and Toll-like receptor ligands. Moreover, a feedback mechanism exists between inflammatory cytokines and activated p-JNK, suggesting critical role of p-JNK in inflammation.^12,13 This process is followed by the proteasome-mediated degradation of Iκ-B (IkappaB) kinase, activation and nuclear translocation of p-NF-κB, where it induces transcription of ROS generation through inducible nitric oxide synthase (iNOS) and COX-2.¹⁴ Activation of p-NF-κB encodes the bulk of inflammatory mediators that exacerbate ischemic brain injury.

The only available U.S. Food and Drug Administration (FDA) approved therapy for treating ischemic stroke is thrombolysis with tissue-type plasminogen activator (tPA).¹⁵ However, a short time window is a major limitation, as treatment with tPA has to be started within 3 to 5 h after an ischemic attack.¹⁶ This highlights the urgent need to develop effective therapeutic agents in this context. Mechanistic studies reveal that searching for ideal candidates targeting both oxidative stress and neuronal necrosis/apoptosis in post-ischemic reperfusion can be an effective and valid option. The number of plant extracts and natural products reported for the therapeutic interventions in the ischemic injury-related cascade of events is consistently increasing.¹⁷ The reported anti-inflammatory, immunomodulatory, and antioxidant indications motivated us to further extend research on Cissampelos pareira in the MCAO model of ischemic stroke in experimental rats. The selected biomarkers including COX-2, p-JNK, and p-NF-κB have direct implications in inflammation, cellular stress, apoptosis, cell proliferation, necrosis, and immune regulation.

Results and Discussion

Neuroprotective Effect of Cissampelos pareira on MCAO Induced Nerve Damage

The number of survived animals in n-hexane, EtOAc, and MeOH extracts of Cissampelos pareira (n = 14/group) was recorded (Figure 1a) following 72 h of ischemic injury. Moreover, a variable mortality rate was observed for all the three extracts. The maximum survival rate (60% or 8 out of 14 rats) was recorded in case of n-hexane extract. No animal could survive in the case of EtOAc while 2 animals survived in MCAO, 3 in MeOH extract, 8 in n-hexane extract and all in the sham group (n = 14). The animals could not show any toxicity or death at 5000 mg/kg (LD₅₀> 5.0 g/kg) of n-hexane extract.

Figure 1.

(A) The number of survived animals in each group; sham, MCAO, EtOAc, MeOH, and n-hexane extracts of leaves of Cissampelos pariera; (B) neurological composite scoring recorded in each group (n = 14/group); (C) analysis of the infarct area in all studied group. Symbol ## represent P < .01 and *** indicates P < .001. Symbols # shows a significant difference relative to MCAO while * shows a significant difference relative to sham; (D) Nissl stained brain coronal sections, which differentiates between non-ischemic and ischemic areas. The white area represents infarction; (E) and (F) Effect of Cissampelos pareira on neuronal cell death. H&E staining (E) and Nissl staining (F) was used to determine the effect of Cissampelos pareira on the extent of surviving neurons in the striatum and frontal cortex at a magnification of 40× with n = 8/group and scale bar 40 μm. Data were expressed as mean ± SEM. Symbol ## indicates P < .01 and *** indicates P < .001. Symbols # shows a significant difference relative to the MCAO group, while * shows a significant difference relative to the sham group. Data were examined by ANOVA (two-way) pursued by multiple comparison test (post-hoc Bonferroni). The bold arrow indicates vacuole formation while the thin arrow indicated a necrotic neuron (E). Likely arrowhead indicated scalloped neuronal structure (F).

Based on these observations, only the n-hexane extract was selected for further study and was subjected to column chromatography for further fractionation. The obtained column fractions (1-cp, 2-cp, 3-cp, and 4-cp) on evaluation for the same effect resulted in the death of all tested animals (data not shown). Therefore, we could not continue further isolation of n-hexane extract, and carried our further study on n-hexane crude extract. Dose-dependent effects were noted in case of the n-hexane extract at 50, 200, and 400 mg/kg. A significant improvement in neuroscore was recorded at 200 (P < .05, F = 2.7) and 400 mg/kg (P < .01, F = 3.8, Figure 1b) compared to the MCAO group. No behavioral alterations could be observed in the sham-operated control group (26.57 ± 0.06). A highly significant increase in neuro-function score was recorded at 400 mg/kg of n-hexane extract. Moreover, 2, 3, 5-triphenyl tetrazolium chloride (TTC) staining demonstrated that treatment with n-hexane extract of Cissampelos pareira alleviated the infarct area relative to the MCAO operated group (Figure 1c) at P < .01 (F = 2.6). The infarct area was 5.86 ± 0.46, 41.57 ± 0.57, and 22.57 ± 0.37% in sham, MCAO and n-hexane extract of Cissampelos pareira + MCAO respectively (Figure 1c).

To further validate, we used hematoxylin and eosin (H&E) and Nissl staining to determine the rate and extent of surviving neurons (Figure 1e and f). The frontal cortex and striatum regions were analyzed for morphological analysis as these are the most vulnerable to ischemic attack. No morphological alteration was noticed in sham-operated animals while MCAO operated animals demonstrated significant alterations at the cellular level including vacuoles formation in the cytoplasm, scalloped shaped neuron along with shrunken organelle with dried up dendrites. Cissampelos pareira treatment attenuated these MCAO induced damages, resulted in decreased vacuoles and less compressed and pyknotic nuclei (Figure 1e and f).

Effect of n-Hexane Extract of Cissampelos pareira on Neuroinflammatory Biomarkers

The n-hexane extract of Cissampelos pareira significantly suppressed the MCAO induced activation and expression levels of p-NF-κB (P < .05, F = 4.8) and p-JNK (P < .05, F = 2.6) in cortical homogenate (Figure 2a and b). To further validate these findings, we performed immunohistochemical analysis. Immunohistochemical analysis revealed significantly higher expression of p-NF-κB, p-JNK, and COX-2 in the MCAO group compared to the sham group (Figure 2c-e). Moreover, Cissampelos pareira treatment significantly decreased this hyper expression of p-NF-κB, p-JNK, and COX-2 in the cortex and striatal regions (P < .05).

Figure 2.

Cissampelos pareira treatment decreased the levels of p-NF-κB (A) and p-JNK (B) in the cortex as detected through ELISA. All data were expressed as mean ± SEM. # P < .05, and P < .001 and n = 6/group. (C) Immunohistochemistry results of p-NF-κB; (D) p-JNK and (E) COX-2 in the frontal cortex and striatum are shown at a magnification of 40×, scale bar 40 μm. Data were presented as mean ± SEM. Symbol # indicates P < .05, ## indicates P < .01 and *** indicates P < .001. Symbol # shows a considerable difference compared to the MCAO group, while * shows a considerable difference compared to the sham group. Data were examined by ANOVA (two-way) pursued by multiple comparison test (post-hoc Bonferroni).

Effect of n-Hexane Extract of Cissampelos pareira on Antioxidant Biomarkers and Enzymes

The levels of glutathione (GSH), glutathione S-transferase (GST) (antioxidant markers), catalase (CAT), superoxide dismutase (SOD), and GPx (antioxidant enzymes) were significantly reduced to 10.5 ± 1.41, 27.66 ± 2.11, 29.75 ± 2.19, 99.68 ± 2.15, and 26.69 ± 2.12 respectively in the MCAO-treated group compared with the control group (sham). The values for GSH, GST, CAT, SOD, and GPx in sham group were 24.9 ± 1.13, 53.25 ± 1.63, 78.25 ± 1.59, 161.25 ± 1.57, and 71.25 ± 1.52 respectively (Figure 3). Treatment with n-hexane extract of Cissampelos pareira at 400 mg/kg significantly increased GSH, GST, CAT, SOD, and GPx to 16.64 ± 0.45, 36.9 ± 2.55, 62.9 ± 2.49, 125.9 ± 2.38, and 52.9 ± 2.49 respectively in the MCAO group.

Figure 3.

Illustration of the data obtained from MCAO-induced antioxidant biomarkers and antioxidant enzymes. MCAO reduced the levels of antioxidant biomarkers (GST and GSH) and antioxidant enzymes (CAT, SOD, and GPx) in the cortex (** P < .01, n = 6/group). The administration of Cissampelos pareira extract resulted in significant recovery of down-regulated antioxidant biomarkers and antioxidant enzymes.

GC-MS Analysis of the n-Hexane Extract

Multiple constituents were identified in the n-hexane extract of Cissampelos pareira. The retention times and percentage presence of the identified phytoconstituents is given in Table 1. 1,2-benzenedicarboxylic acid ditridecyl ester (20.48%), 5-methyl octadecane (13.41%), (Z)-7-hexadecenal (11.57%), 17-pentatriacontene (8.52%), and 2-(tetradecyloxy)-ethanol (9.77%) were among major constituents. Among these, the largest peak belonged to 1,2-benzene dicarboxylic acid ditridecyl ester (20.48%).

Table 1.

GC-MS Analysis Profile of the n-Hexane Extract of Leaves of Cissampelos pareira.

S. No.	Name of the compound	Molecular formula	Molecular weight	Retention time (min.)	Retention index	Peak area (%)
1	1,2-Benzenedicarboxylic acid, ditridecyl ester	C34H58O4	530	22.609	3498	20.48
2	5-methyl-octadecane	C19H40	268	20.816	1854	13.41
3	(Z)-7-Hexadecenal	C16H30O	238	18.880	1798	11.57
4	2-(tetradecyloxy)- Ethanol	C16H34O2	258	21.643	2031	9.77
5	1,1′-oxybis- Decane	C20H42O	298	19.940	2060	9.15
6	17-Pentatriacontene	C35H70	490	23.236	3484	8.52
7	Nonadecane	C19H40	268	17.067	1867	4.27
8	2-Hexyl-1-octanol	C14H30O	214	18.335	2069	3.22
9	1-chloro- Tetradecane	C14H29Cl	232	13.638	1668	2.54
10	1-Pentanol, 4-methyl-2-propyl	C9H20O	144	11.189	1363	2.02
11	Hexadecane	C16H34	226	14.965	268.29	1.70
12	Dodecane, 2,6,10-trimethyl-	C15H32	212	16.258	1374	1.64
13	Hexane	C6H14	86	8.444	564.4	1.64
14	1-chloro-decane,	C10H21Cl	176	13.219	1275	0.83
15	2,3-dimethyl-Pentane	C7H16	100	10.716	668.2	0.79
16	Decane	C10H22	142	11.766	159.66	0.17
17	2-butyl-1-Octanol	C12H26O	186	15.082	1277	0.15

Discussion

Accumulation of toxic radicals and oxidative stress is closely related to neuro-inflammatory activation, which further exacerbates ischemic pathogenesis.¹⁸ Cissampelos pareira treatment, in this study, resulted in strong protective effects through diminishing neuro-inflammatory mediators through by down-regulating ROS generation. The clinical interventions with neuro-protective agents, targeted at a particular single event rather than interconnected steps of pathological process, could not show considerable success.¹⁹ A neuro-protective agent acting at different stages and targeting multiple players, may be more advantageous to intervene in the vicious cycles in MCAO. Cissampelos pareira in this study ameliorated the detrimental effects of MCAO by targeting multiple aspects of the stroke pathogenesis, including neuro-inflammation, oxidative stress, and neuronal necrosis (Figure 1 to 3).

Inflammation worsens the clinical prognosis of the stroke and compromises its therapeutic outcome. The release of inflammatory mediators substantiates ischemia-induced neuronal death by several mechanisms. Activation of TLR-4 on glial cells stimulates stress-related kinases, including JNK and p38-MAPK, which triggers the mitochondrial apoptotic pathway.²⁰ The activated microglia, astrocytes, and neurons along with the activated kinases result in cytokine storm within the first hour of ischemic injury.²¹ Activated nuclear transcriptional machinery like p-NF-κB ultimately elicits the production of pro-inflammatory factors, such as TNF-α, IL-1β, and nitric oxide, which further aggravate the damage already done. The higher expression of p-NF-κB and p-JNK in our study (Figure 2) was in agreement with previous reports where p-JNK played cytotoxic roles in ischemic brain injury and resulted in increased infarction area.^22,23

It has been reported that p-NFκB activation triggers iNOS and COX-2 production.¹⁴ Both COX-2 and iNOS are toxic mediators of inflammatory cascade which can be down-regulated by inhibiting p-NFκB.²⁴ The observed downregulation of COX-2 in this study seems to be the manipulation of inhibition of NF-kB (Figure 2a and e). In accordance with other results, the reduced expression of antioxidant enzymes GST and GSH in ischemic tissue was significantly restored by Cissampelos pareira, showing the antioxidant effect of Cissampelos pareira (Figure 3). In literature, correlation between inflammation and oxidative stress is evident in both neuronal and non-neuronal models.²⁵

Another very interesting aspect of this study is the significant difference in the rate of survival of MCAO operated animals in response to different extracts. The survival rate of n-hexane extract administered group encouraged us to further sub-fractionate the extract and follow the bioactivity guided isolation till single constituents (Figure 4d). However, we could not go further due to the death of all the animals in response to these sub-fractions administration (data not shown). This should not be surprising. A multitude of actions resulting from a mixture of particular constituents in specific proportions can potentiate a particular pharmacological activity or healing process. Here the example of antibacterial activity of berberine, isolated from Berberis fremontii (Berberidaceae) can be rightly given.²⁶ The growth inhibitory effect of berberine against Staphylococcus aureus was potentiated by two other constituents, yet neither could show any activity alone.

Figure 4.

(A) Wild growing Cissampelos pareira; (B) Dried leaves of Cissampelos pareira; (C) Extracts in n-hexane, EtOAc and MeOH; (D) Fractions of n-hexane extract obtained through column chromatography.

In our study, the GC-MS analysis of the n-hexane extract of leaves of Cissampelos pareira detected 1,2-benzenedicarboxylic acid di-tri-decyl-ester (20.48%), as major constituent (Table 1). There are several reports from literature which makes us to confident to conclude that the ameliorative role of n-hexane might be due to the 1,2-benzenedicarboxylic acid. This compound reduced Aβ-induced neurotoxicity, possibly by reducing oxidative stress in another report from literature.²⁷ The antioxidant and anti-inflammatory properties of 1,2-benzendicarboxylic acid are also reported.²⁸

So we can conclude that n-hexane extract attenuated MCAO-induced oxidative stress and inflammatory cascade, possibly by modulating the JNK/NFκB/COX2 pathway, eventually accounting for its neuro-protective effects against neuronal apoptosis. Our evidence suggest that the multiple constituents might have worked synergistically to manifest this outcome, with 1,2-benzenedicarboxylic acid di-tri-decyl-ester (20.48%), being the major constituent. However, we could not isolate the individual constituent/s. The study could not involve human study, either. So we recommend future clinical studies, to further validate the findings.

Experimental Section

Preparation of the Extracts and Fractions

All experiments were performed at Riphah Institute of Pharmaceutical Sciences, Riphah International University, Islamabad, Pakistan. Leaves of Cissampelos pareira were collected in September 2018 from Islamabad, Pakistan. All leaves, irrespective of age, were used in this experiment. Dr Mushtaq Ahmad (Associate Professor at Department of Plant Sciences, Quaid-i-Azam University, Islamabad, Pakistan) identified the plant (voucher specimen having number 130282). In brief, the dried leaves were ground to form a powder which were extracted by the maceration process (using n-hexane, EtOAc and MeOH [500 g/5000 mL, w/v]), at room temperature for 14 days (Figure 4a-d). All these extracts were then filtered and dried using rotary evaporator. Most potential (n-hexane) extract (22 g) was further fractionated through column chromatography (Figure 4d). Elution was initiated with 100% n-hexane and increased progressively in polarity with a 5% increase in more polar EtOAc until 50:50 n-hexane and EtOAc ratio was reached. All fractions were collected continuously. These fractions were further dried with the help of the rotary evaporator. Fraction number 1-2, fractions 3-5, fractions 6-8, and fraction number 9-11 were combined as fractions 1-cp, 2-cp, 3-cp, and 4-cp respectively, based on TLC (Silica 60 F₂₅₄:Merck; n-hexane : EtOAc (7:3)) spots under UV light as shown in Figure 4d (Spectroline, E-Series, ENF-240C/FE, Ultraviolet Hand Lamps, USA).

Animal Grouping and Drug Treatment

Adult male Sprague–Dawley rats, weighing 270 to 300 g, were purchased from the National Institute of Health (NIH), Islamabad, Pakistan. The experimental animals were kept in an animal house at Riphah Institute of Pharmaceutical Sciences, under a 12 h dark/light cycle at 18 °C to 22 °C. The animals had free access to water and diet throughout the study. All experimental procedures were set as per the guidelines of the Riphah Ethical Committee (REC), Riphah Institute of Pharmaceutical Sciences (Ref. No. REC/RIPS/2019/20). Rats were divided into three experimental groups as follows: Sham/vehicle administered control group (n = 14); MCAO (rats undergoing permanent MCAO surgery) (n = 14); Cissampelos pareira + MCAO (n = 14). All three dried extracts including n-hexane, EtOAc, and MeOH (50 mg/kg) of Cissampelos pareira were dissolved in distilled water, DMSO (0.1%) and tween 80 (0.1%). Intra-peritoneal administration started from 30 min before MCAO, and then repeated after every 6 h (for 3 days). Based on the initial findings (survival rate), only the n-hexane extract was selected for further study.

MCAO Surgery

MCAO surgery was conducted following the previously established protocol in our lab.²⁹ A mixture of ketamine and xylazine (3.2:1) was used intra-peritoneally to anesthetize the rats. The common carotid artery, external carotid artery, and internal carotid artery were exposed over and a midline cervical incision was done. The external carotid artery was tied, and a (3/0) nylon filament with a blunted rounded tip around 30 mm in length was introduced from the external carotid artery into the internal carotid artery. It was proceeded further into the middle cerebral artery where some resistance to the nylon movement occurred. Sham-operated animals were exposed to the same procedures as the MCAO group except for filament insertion.

Neurobehavioral Test

Neuroscore (28 points) was used to assess the sensorimotor deficits based upon several tests comprising (1) circling, (2) motility, (3) general condition, (4) righting reflex when placed on its back, (5) paw placement on the table top, (6) ability to pull itself up on a horizontal bar, (7) climbing on an inclined platform, (8) grip strength, (9) contralateral reflex, (10) visual forepaw reaching, and (11) contralateral rotation when held by the base of its tail. A collective score of 28 points indicated less or no impairment while a score of (0) showed severe neurological impairment.³⁰

TTC Staining

After occlusion, the rats were decapitated under anesthesia at the end of 3 days protocol. Brains were carefully separated and washed with cold phosphate buffer saline (PBS, pH 7.4). Around 3 to 4 mm thick slices were cut from the frontal lobe by using a sharp knife blade. These slices were incubated in 2% TTC (the solution was made in PBS) for 10 to 20 min until a clear demarcation was witnessed for the lateral and contralateral brain for MCAO and Cissampelos pareira + MCAO operated rats, while sham-operated rats were stained as deep red. The sections were fixed in 4% paraformaldehyde and then photographed. The percent infarct area was measured using Image J (a computer-based program). The corrected brain infarction was evaluated as follows: corrected infarct area = left hemisphere area − (right hemisphere area − infarct area). These coronal sections were then embedded in paraffin and 4 μm thin coronal sections were cut by a rotary microtome and were proceeded for the further morphological analysis.

H&E Staining

Tissue sections on simple slides were rinsed thrice in xylene and hydrated with graded ethyl alcohol series from 100% to 70%. Segments were stained with Harris’ hematoxylin solution (Sigma-Aldrich, St. Louis, MO, USA) for 15 min and Eosin Y (Sigma-Aldrich) for 10 min. Segments were washed with water, dehydrated with graded ethyl alcohol series, mounted with a paramount mounting solution (Thermo Fisher Scientific, Waltham, MA, USA), and photographed using an Olympus microscope (Olympus, Tokyo, Japan).

Nissl Staining

Tissue slides were rinsed with distilled water and 0.01 M PBS, stained with 0.5% crystal violet solution, rinsed with distilled water and dehydrated with graded ethyl alcohol (70, 95% and 100%). Brain slides were cleared with xylene and mounted with a glass coverslip. The stained images were taken with a light microscope (Olympus, Japan), saved as TIF files, and analyzed by Image J computer-based program.

Immunohistochemistry

Following de-paraffinization and hydration, the tissue sections on coated slides were treated with protein kinase for antigen retrieval, followed by treatment with 3% hydrogen peroxidase for blocking the peroxidase activity.³¹ The slides were washed and subsequently blocked with 5% serum depending upon the origin of secondary antibodies used. The slides were incubated over night with COX-2, p-NF-κB, and p-JNK (Santa Cruz Biotechnology) antibodies. The next morning, slides were incubated with biotinylated secondary antibodies for 2 h and then successively with avidin-biotin complex (ABC) reagents (Standard Vectastain ABC Elite Kit; Vector Laboratories, Burlingame, CA, United States) for 1 h at room temperature. The slides were washed with PBS and stained with di-amino-benzidine-tetra-hydrochloride solutions; then washed with distilled water, dehydrated in graded ethanol (70%, 80%, 90%, 95%, and 100%), fixed in xylene, and cover-slipped by a mounting medium. Immunohistochemistry results were examined by a light microscope (Olympus, Japan), which was connected to a digital photomicroscope camera. For quantitative determination, a computer-based software Image J, was used to measure hyper-activated COX-2, p-NF-κB, and p-JNK in the striatum and frontal cortex by optimizing the background of images according to the threshold intensity. COX-2, p-NF-κB and p-JNK positive cells were analyzed at the same threshold intensity for all groups. The intensity is demonstrated as the relative density of the value of the samples relative to the sham (control).

Measurement of p-NF-κB and p-JNK Levels Through ELISA

Enzyme-linked immunosorbent assay (ELISA) was performed to determine the levels of p-NF-κB and p-JNK, according to the instructions of the manufacturer (Shanghai Yuchun Biotechnology, China). Briefly, the brain tissues were stored at −80 °C and were then homogenized and the supernatant was collected after centrifugation (at 13 500 × g for 1 h). The supernatant was then analyzed for p-NF-κB and p-JNK quantification through rat anti-p-NF-κB (Cat. No.SU-B28069, Shanghai Yuchun Biotechnology, China) and anti-p-JNK antibodies (Cat. No. SU-B30586, Shanghai Yuchun Biotechnology, China) respectively.

Oxidative Stress Analysis

Oxidative stress markers including GST and GSH were determined to evaluate the effect of the n-hexane extract following ischemic stroke. The determination of GSH was done as reported earlier with some modifications.³² Freshly prepared 5′, 5′-dithio-bis (2-nitrobenzoic acid) (DTNB) solution, phosphate buffer solution, and plant extract were added simultaneously and color change was observed at 412 nm with the help of a spectrophotometer. DTNB solution was used as control whereas phosphate buffer was used as blank. The increased value of absorbance of the mixture indicated the presence of GSH in the plant extract. By subtracting the absorbance of control from that of the sample, real absorbance was calculated. The resulted GSH values are expressed in µmoles of GSH/mg of sample. For GST, the earlier protocol was repeated.³² 1-Chloro-2, 4-dinitrobenzene (CDNB) and GSH were mixed with the plant extract and the optical density was recorded at 340 nm through spectrophotometer. The assay mixture without plant extract served as control. By using the extinction coefficient of the product formed, GST activity was calculated and expressed as µmoles of CDNB conjugated/min/mg of protein. The levels of key antioxidant enzymes, including CAT, SOD, and GPx, were also determined. CAT activity was measured by mixing 3000 µL of H₂O₂ and 50 µL of tissue supernatant. The absorbance was measured at 240 nm against a blank containing only 3000 µL of PBS. The absorbance is proportional to the H₂O₂ level, which is decreased by catalase as it degrades the H₂O₂. This is the measure of H₂O₂ breakdown and hence is expressed as μmol H₂O₂ decomposed per mg of protein/min. SOD activity was carried out by mixing supernatant of tissue homogenate in xanthine oxidase and xanthine solution, followed by incubation at 37 °C. The reaction mixture was then allowed to react quantitatively with 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (iodo-nitrotetrazolium) to generate superoxide anion radicals (red formazan crystals). Absorbance was measured at 505 nm within 3 min after the reaction. The activity is expressed as U/mg protein. For GPx detection, the assay mixture used consisted of 50 mM phosphate buffer (pH 7), 1 mM EDTA, 1 mM NaN₃, 3 U glutathione reductase, 1 mM GSH, 0.2 mM NADPH, 0.25 mM H₂O₂, and 20 to 30 μg tissue homogenate as an enzyme source in a final volume of 1.5 mL. Oxidation of NADPH was recorded at 340 nm at 15 s intervals for 2 min and the enzyme activity was expressed as nmol NADPH oxidized/min/mg protein using a molar extinction coefficient of 6.22 × 10³/M/cm.

Gas Chromatography Mass Spectrometry (GCMS) Analysis

A Shimadzu GCMS-QP 5050A gas chromatograph-mass spectrometer connected to a Shimadzu GC-17A gas chromatograph, equipped with an injector and a capillary column of D8-5 fused-silica (30 m × 0.25 mm; 0.25 μm film thickness), was used to perform GC-MS analysis. Helium was used as carrier gas at 1 mL/min flow rate. At 250 °C, maintain injection port, and 94 was the split ratio. Programming of oven temperature at 10 °C/min was ended from 60 °C to 300 °C, and it remained for 5 min at 300 °C. The temperature was retained at 250 °C interface. Electron impact ionization was the ionization mode and from 35 to 580 m/z was the scanning range. At 0.5 s interval, mass spectra were acquired. NIST library (NIST27.LIB) standard mass spectra were used as standards.

Statistical Analysis

Image J software was used to evaluate morphological data and neurological deficit scores. Immunohistochemical data were examined by ANOVA (two-way) pursued by multiple comparison test (post-hoc Bonferroni). Moreover, TTC, Nissl staining and ELISA were analyzed by one-way analysis of variance followed by post-hoc Bonferroni multiple comparison tests (GraphPad Prism 6). Symbols # or * indicates P < .05, ## or ** indicates P < .01 and ### or *** indicates P < .001. Symbols # show a significant difference compared to the MCAO group, while * shows a significant difference compared to the sham group.

Footnotes

Acknowledgments

The authors thank Prof. Dr Arif-ullah Khan, Dean Riphah Institute of Pharmaceutical Sciences, for providing free access to all the facilities.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Ethical Approval

All experimental procedures were set as per the guidelines of the Riphah Ethical Committee (REC), Riphah Institute of Pharmaceutical Sciences (Ref. No. REC/RIPS/2019/20).

ORCID iD

Muhammad Sohaib Khalid

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