Scopoletin Ameliorates Lipopolysaccharide Induced Neuroinflammation, Oxidative Stress and Cognitive Dysfunction in Mice: A Mechanistic Study

Background: Alzheimer’s disease (AD), a progressive neuropsychiatric disorder, is characterized by neuroinflammation, β-amyloid plaques and neurofibrillary tangles. It has been demonstrated that systemic inflammation and neuroinflammation caused by lipopolysaccharide (LPS) injections in rodents leads to elevation in Aβ levels and neuronal cell death, finally resulting in cognitive impairment. Methods: Animals received the experimental compound – Scopoletin (2.5, 5 or 10 mg/kg, i.p) for 31 days. Initially for 14 days, they received only the experimental drug; thereafter for the next 7 days they received LPS (0.25 mg/kg i.p) along with the experimental molecules. Treatment with the experimental molecules was continued for the next 10 days and behavioural tests were performed. Animals were sacrificed on day 32 and biochemical tests were performed on brain tissue homogenate. Original Research Article Gursahani et al.; JPRI, 33(3): 74-90, 2021; Article no.JPRI .65989 75 Results: Scopoletin administration improved memory and cognition as indicated by reduction of escape latency in Morris water maze test and increased number of entries in Y maze test. Malondialdehyde was significantly increased after LPS administration and reduced glutathione, catalyse and superoxide dismutase were significantly reduced in mice brains. Treatment with scopoletin significantly reduced oxidative stress. Inflammatory markers. β-secretase and apoptotic markers were also markedly reduced. Conclusion: At medium and high dose, Scopoletin showed promise response as a prophylactic entity in the LPS induced neuroinflamation and cognitive dysfunction model in mice.


INTRODUCTION
Alzheimer's disease (AD), a progressive neurodegenerative disease associated with hallmarks such as accumulation of β-amyloid plaques, deposition of neurofibrillary tangles composed of hyperposphorylated, and neuroinflammation was described by Alios Alzheimer more than 100 years ago. The autopsy performed by Alios Alzheimer, in 1906, on the brain of late Auguste Deter, a female who presented with abnormal behaviours such as short-term amnestic disorder, disorientation and dysphrasia, and whose symptoms had been studied for many years by Alzheimer, revealed significant cerebral atrophy, abnormality in the regions of the brain that were considered responsible for judgement, language and memory; intraneuronal plaques and tangles in nerve fibres were detected [1][2][3]. The number of cases of AD globally are estimated to increase to 106.8 million by 2050 [4]. According to a systematic analysis conducted by Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) Dementia Collaborators [5], the global burden of dementia was 20.2 million in 1990 and it rose to 43.8 million by 2016; women are affected to a greater extent compared to men. In 2016, dementia was the fifth leading cause of death, claiming 2.4 million lives worldwide [5]. In 2020, the estimated economic burden of AD was $305 billion and it is predicted to rise to more than $1 trillion by 2050 [6]. The interventions that are in clinical practice today, only provide symptomatic relief by counterbalancing the neurotransmitter disturbances; three of these molecules are Acetylcholineterase inhibitors and have been approved for the indication mild to moderate AD, whereas, memantine is an NMDA receptor noncompetitive antagonist that is indicated for moderate to severe AD [7,8]. Extensive research is being carried out to identify disease-modifying drugs that will be able to halt the progression of the disease by impeding an essential characteristic process in the pathophysiology leading to clinical symptoms [9,10].
Compiling evidence suggested that immune responses play a major role in the pathogenesis of AD, and that AD is not a disease that is only restricted to the neuronal system. Understanding the interactions of these two key systems may help us in preventing or delaying the progression of the disease. In AD, neuro inflammation is not merely a beholder that is triggered by deposition of plaques or neurofibrillary tangles, but actually contributes equally, if not more, to the pathogenesis of AD; TREM2 and CD33, genes for immune receptors, have recently been implicated in the development of AD [11][12][13][14]. Recent reports suggest that lipopolysaccharide (LPS), a gram-negative endotoxin has been detected in AD affected brain tissues [15,16]. LPS level in blood of AD patients has been found to be 3-fold the levels in control subjects [17]. Additionally, LPS has been found to be colocalized with amyloid plaques, peri-vascular amyloid, neurons, and oligodendrocytes in AD brains [18]. Zhao et al have shown that LPS injection stimulates microglia via the NF-κB signaling pathway leading to increase in brain Aβ levels and neuronal cell death [19].
Scopoletin (SCP) has been shown to possess anti-amnesic activity comparable to that of donepezil in scopolamine induced amnesia model in mice [20]. It has also been proven to restore the disruption of memory induced by administration of the anticholinergic agent scopolamine, when administered i.c.v., suggesting a potent ameliorating action on the impaired cholinergic transmission [21]. However, its potential activity in AD has not been evaluated. A drug that enhances cholinergic neurotransmission and exerts antioxidative and anti-inflammatory actions [22] would be highly desirable as a therapeutic for neurodegenerative diseases such as Alzheimer's dementia (AD).

Animals
Swiss albino mice weighing 25 procured from the National Institute of Biosciences, Pune. Mice were hous polypropylene cages in a temperature and humidity-controlled environment under a 12 hours light-dark cycle. Food and water were provided ad libitum. Experimentation was s after a 7-days acclimatisation period. Behavioural experiments were carried out between 10 a.m. and 4 p.m.

Experimental Design Administration
A total of 30 mice were divided into 5 groups (each with n = 6). They received saline or SCP (2.5, 5 or 10 mg/kg i.p) totally for 31 days. Initially for 14 days, they received the experimental drug alone; thereafter for the next 7 days, they received LPS (0.25 mg/kg i.p) along with the SCP. Treatment with the SCP was continued for the next 10 days and behavioural tests were performed. The experimental scheme has been described in Fig. 1.

Morris water maze (MWM) test
The morris water maze test, which is a commonly used test to evaluate AD dysfunction was used to study hippocampal spatial memory impairment [24,25]. This test is

MATERIALS AND METHODS
Swiss albino mice weighing 25-35 g were procured from the National Institute of Biosciences, Pune. Mice were housed in temperature and controlled environment under a 12 dark cycle. Food and water were . Experimentation was started days acclimatisation period. Behavioural experiments were carried out between 10 a.m.

Design and Drug
A total of 30 mice were divided into 5 groups 6). They received saline or SCP kg i.p) totally for 31 days. Initially for 14 days, they received the experimental drug alone; thereafter for the next 7 days, they mg/kg i.p) along with the SCP. Treatment with the SCP was continued for s and behavioural tests were performed. The experimental scheme has been

Morris water maze (MWM) test
water maze test, which is a commonly used test to evaluate AD-like dysfunction was used to study hippocampal spatial memory impairment [24,25]. This test is designed in such a way that the mice swim in a circular pool containing an opaque fluid until they find the hidden platform, which is the only safe place in the pool. The apparatus consisted of a circular pool (122 cm in diameter, 50 cm in height, and filled to a depth of 30 cm with opaque water [prepared by dissolving milk powder in water]) and a hidden platform (10 ×10 cm placed in the pool such that its surface was 1 cm below the surface of opaque water. Bright coloured clues were placed around the pool and the placing was such that they would be visible to the mouse from the pool. This was provide spatial clues to the mice, so that the mice will be able to locate the platform easily when the mice would be exposed to the same challenge the next time. The experimental setup was maintained exactly identical during the training and evaluation challenges; the environmental conditions and spatial clues were uniformly maintained. Two perpendicular threads were secured firmly on the circumference of the pool in order to divide the pool into 4 equal quadrants, such that the threads would form pool would be divided; this was done so that the animal could be placed in a random quadrant during training and evaluation challenges. The hidden platform was placed in the third quadrant throughout the training and evaluation process. During the training phase, mice were placed randomly in one of the other three quadrants with their face towards the wall of the circular pool. The mice were to swim through the pool and identify the safe place i.e. the hidden platform. The maximum time allowed for finding the hidden platform was 120s. If the mouse failed to find the platform, he was gently guided towards the platform. The mouse was allowed to stay on the platform for 30s in order to explore the spatial clues. Four consecutive training sessions carried out at an interval of 30 min between training sessions for four days. Escape latency (time taken to locate the hidden platform) was recorded subsequently.

Experimental groups and study design
; Article no.JPRI .65989 designed in such a way that the mice swim in a circular pool containing an opaque fluid until they find the hidden platform, which is the only safe place in the pool. The apparatus consisted of a circular pool (122 cm in diameter, 50 cm in height, and filled to a depth of 30 cm with opaque water [prepared by dissolving milk powder in en platform (10 ×10 cm 2 ) was placed in the pool such that its surface was 1 cm below the surface of opaque water. Bright coloured clues were placed around the pool and the placing was such that they would be visible to the mouse from the pool. This was done to provide spatial clues to the mice, so that the mice will be able to locate the platform easily when the mice would be exposed to the same challenge the next time. The experimental setup was maintained exactly identical during the training ation challenges; the environmental conditions and spatial clues were uniformly maintained. Two perpendicular threads were secured firmly on the circumference of the pool in order to divide the pool into 4 equal quadrants, such that the threads would form a '+' and the pool would be divided; this was done so that the animal could be placed in a random quadrant during training and evaluation challenges. The hidden platform was placed in the third quadrant throughout the training and evaluation process.
g the training phase, mice were placed randomly in one of the other three quadrants with their face towards the wall of the circular pool. The mice were to swim through the pool and identify the safe place i.e. the hidden platform.
for finding the hidden platform was 120s. If the mouse failed to find the platform, he was gently guided towards the platform. The mouse was allowed to stay on the platform for 30s in order to explore the spatial clues. Four consecutive training sessions were carried out at an interval of 30 min between training sessions for four days. Escape latency (time taken to locate the hidden platform) was

Y maze task [26]
The apparatus consisted of three identical arms of equal size-the first arm, the familiar arm and the novel arm. The animal was always placed in the first arm which was always open. The familiar arm was also always open for the animal to explore. The novel arm was blocked during the training trail but was open during the evaluation phase. The training trial was carried out and then after an hour, the evaluation trial was carried out. In the training trial, the novel arm was blocked, and the animal was allowed to explore the start arm and the familiar arm for a period of 10 min. After an hour, the novel arm was unblocked, and the animal was placed in the start arm. The animal was free to explore all the three arms for 5 min. The number of entries in the novel arm was recorded. A novel arm 'entry' was counted when all four limbs of the animal were in the novel arm.

Brain tissue processing for biochemical evaluation
Mice were euthanized by CO 2 overdose on the 32 nd day and perfused with ice-cold 0.1 M phosphate buffer (pH 7.4). The brains were isolated and weighed, and 10% tissue homogenates were prepared in 0.1 M phosphate buffer (pH 7.4). The homogenates were centrifuged at 4000 rpm for 10 min and the supernatant was stored at -80ᵒC until further use.

Determination of lipid peroxidation
Free radical attacks on polyunsaturated fatty acids leads to formation of reactive electrophilic aldehydes such as malondialdehyde (MDA). This process of lipid peroxidation leads to neuronal damage. The content of MDA in the supernatant was assayed in the form of Thiobarbituric acid reactive substance (TBARS) by the method described by Niehaus and Samuelsson, 1968. Reaction of thiobarbituric acid (TBA) with MDA produces a red complex which has a peak absorbance of 535 nm. 0.1 ml of the supernatant was treated with 2 ml of TBA-TCA-HCl (1:1:1) reagent (0.37% thiobarbituric acid, 0.25 N HCl and 15% TCA) and heated on a boiling water bath for 15 min. It was cooled and centrifuged at 4000 rpm for 10 min at room temperature and the absorbance of the clear supernatant was measured at 535 nm using a spectrophotometer [27]. The MDA content was determined by extrapolating from the standard curve.

Determination of Reduced glutathione (GSH) concentration
GSH is a potent antioxidant that scavenges free radicals and protects the cells from their damaging effects. The method described by Moron et al. 1979 was followed [28]. 0.4 ml of the supernatant was precipitated with an equal quantity of 20% TCA. This mixture was then centrifuged at 10,000 rpm at 4ᴼC for 20 min. From the resultant supernatant 0.25 ml was mixed with 2 ml of 0.6 mM DTNB reagent and the final volume was made up to 3ml with 0.2 M phosphate buffer. The absorbance of the yellow product that was formed in-situ was measured at 412 nm in a spectrophotometer.

Determination of Catalase (CAT) activity
According to the method described by Aebi, 1984 [29], decomposition of H2O2 was followed by measuring the change in absorbance at 240 nm. In this assay, 0.1 ml of the supernatant was mixed with 0.8 ml of phosphate buffer (50 mM, pH 7.0) and 0.1 ml of 0.02% Triton X-100. This mixture was incubated for 10 min at room temperature. This mixture was added to 2.0 ml of 0.03M H 2 O 2 and the change in absorbance was monitored every minute for five minutes at 240 nm. The activity was expressed as micromole H 2 O 2 decomposed/min/mg protein.

Determination of Superoxide dismutase (SOD) activity
SOD activity was measured based on the method described by Marklund and Marklund, 1974 [30]. The principle of this method was based on the ability of SOD to prevent the autooxidation of pyrogallol. In this assay, 10 μl of tissue homogenate, 180 μl of phosphate buffer, and 10 μl of pyrogallol were added to each well of a 96-well microplate. The absorbance was read at 325 nm for 5 min. For control solution, the tissue homogenate was replaced with distilled water. One-unit activity of SOD activity was defined as 50% inhibition of autoxidation of pyrogallol.

Determination of Acetylcholine esterase (AChE) inhibitory activity
The assay method described by Ellman

Determination of inflammatory cytokines (TNF-alpha and IL-6) by ELISA
Content of TNF-alpha in the brain tissue supernatants was determined by using commercially available ELISA kit (Krishgen biosystems). Results were expressed as pg/mg protein.

Determination of Caspase-3 by ELISA
Caspase-3 activation has been demonstrated as an early neurodegenerative event in the progression of AD; Caspase-3 is the primary executioner of apoptosis. Numerous studies have presented elevated level of caspase-3 in AD. Caspase-3 level in the brain homogenate were estimated using commercially available ELISA kit (Bioassay Technology Laboratory)

Determination of NF-ĸB by ELISA
To determine the mechanistic pathway by which LPS induces pathological changes and to study the mechanism of action of the experimental molecules, NF-kB levels in the brain homogenate were estimated using commercially available ELISA kit (Bioassay Technology Laboratory)

Total protein
Protein estimation was carried out by using Bovine serum albumin (BSA) as the standard and extrapolating the values from the standard curve.

Statistical analysis
The data obtained was analyzed statistically using analysis of variance (ANOVA) followed by Tukey's Multiple Comparison Test. P<0.05 is considered as significant. The analysis was performed using Graph Pad Prism 5.0. U.S.A.

Effect of SCP on Escape Latency in Morris Water Maze Test
MWM task is a standard behavioral tool designed to access spatial learning and memory. Over the course of training, animals learned to find the hidden platform; animals in the disease control group took significantly longer to find the hidden platform (escape latency) than animals in the vehicle control group. Administration of SCP showed dose-dependent effect on escape latency and animals could find the platform in a shorter span of time (Fig. 2).

Effect of SCP on Number of Entries in Novel Arm in Y Maze Test
The number of entries in the novel arm significantly reduced in the disease control group; this effect was significantly (p<0.001) reversed by administration of high dose of SCP (Fig. 3).

Effect of SCP on Lipid Peroxidation
Administration of LPS significantly increased (p<0.001) MDA level, a lipid peroxidation biomarker, in the brain of mice. Treatment with SCP reduced MDA level in a dose dependant manner (Fig. 4).

Effect of SCP on GSH Content
GSH, a major free radical scavenger in the brain, was reduced significantly after LPS administration. SCP significantly improved (p<0.01) GSH level in the brain at the highest dose administered (Fig. 5).

Fig. 2. Effect of SCP on escape latency in MWM test on day 25, 26, 27, and 28
Results are represented as Mean± S.D (n = 6). Disease control group has been compared with the vehicle control group; all treatment groups have been compared with disease control. *** p< 0.001

Fig. 3. Effect of SCP on number of entries in novel arm in Y test
Results are represented as Mean± S.D (n = 6). Disease control group has been compared with the vehicle control group; all treatment groups have been compared with disease control. *** p< 0.001, ns=not significant

Effect of SCP on CAT Activity
Catalase, an enzyme involved in preventing cellular oxidant damage by metabolizing hydrogen peroxide to water and oxygen, was significantly reduced after LPS administration. There was a significant (***p<0.001, **p<0.01) dose-dependent increase in catalase activity following the administration of SCP (Fig. 6).

Effect of SCP on SOD Activity
The activity of SOD, the first detoxification enzyme and most powerful antioxidant in the cell, was significantly reduced in the brain of mice post the administration of LPS. All the three doses of SCP significantly (p<0.001) improved the SOD activity all the tested dose levels (Fig.  7).

Effect of SCP on AChE Activity
Acetylcholinesterase, the enzyme responsible for the breakdown of acetylcholine, was significantly inhibited (p<0.001) by SCP at all the administered doses (Fig. 8).

Effect of SCP on TNF-α and IL-6 level
TNF-α, the multifunctional cytokine, was significantly elevated after LPS administration; this elevation was dose dependently alleviated (*** p<0.001) by administration of SCP at medium and high dose. Brain level of the proinflammatory cytokine IL-6 was significantly increased after LPS administration. SCP administration significantly reduced (*** p<0.001, *p<0.05) IL-6 level at all studied dose levels ( Fig.  9).

Effect of SCP on Caspase-3 Level
Caspase-3, a critical executioner of apoptosis, was significantly elevated in the disease control group. SCP administration caused a significant (*** p<0.001, *p<0.05) reduction in the apoptotic marker level (Fig. 10).

Effect of CSP on NF-ĸB Level
Brain level of NF-ĸB, a mediator of proinflammatory gene induction, was significantly increased after LPS injection. SCP administration reduced NF-ĸB level in a doses dependant manner at all three dose levels (Fig. 11).
The pathophysiology of AD has been shown to encompass immunological responses, and brains of AD patients have shown chronic inflammation. It has been hypothesised that contribution of inflammation in AD pathophysiology starts decades before the clinical onset and cognitive decline. Also, there is a strong association between Aβ accumulation, tau hyperphosphorylation and neuroinflammation [33][34][35][36][37][38][39][40][41]. In this study, we examined the pharmacological effect of SCP at three dose levels in LPS induced neuroinflammation and cognitive impairment in mice. Administration of LPS resulted in increase in escape latency in MWM task, whereas it lead to a significant reduction in number of entries in novel arm in Y maze test. MWM task is a standard behavioral tool designed to access spatial learning and memory. Over the course of training, animals learned to find the hidden platform; animals in the disease control group took significantly longer to find the hidden platform (escape latency) than animals in the vehicle control group. This finding was consistent with the previously reported effect of LPS on learning and memory [42]. Administration of SCP showed dose-dependent effect on escape latency and animals could find the platform in a shorter span of time. The Y maze test novel arm preference test is a widely-accepted test to study hippocampus-dependant spatial memory; mice with special deficits tend to enter the novel arm less frequently in comparison to normal mice. We observed that mice receiving the highest dose of SCP entered the novel arm more frequently that the mice in the disease control group, thus indicating that SCP has a positive effect on spatial memory [43][44][45]. The brain is extremely sensitive to oxidative stress since it utilizes 20-30% of the inspired oxygen. Previous studies have demonstrated that i.p. injection of LPS caused significant changes in oxidative stress markers in the brain of mice [46]. Moreover, the brain contains high levels of redox transition metals and polyunsaturated fatty acids (PUFA). Lipid peroxidation, an early event in the progression of AD, is a major source of freeradical mediated injury that leads to neuronal membrane damage and yields a number of secondary products that were also in turn responsible for extensive cellular damage [47]. Free radical attack to PUFAs leads to the formation of highly reactive electrophilic aldehydes such as malondialdehyde (MDA) which is used as a marker of oxidative stress [48]. SCP administration (medium and high dose) significantly lowered brain MDA level. Diminished GSH levels make cells more vulnerable to oxidative stress. Post-mortem studies of AD brains have shown diminished GSH levels and increased glutathione disulphide (GSSG) content in patients with mild cognitive impairment, as mild/moderate and severe AD patients compared to controls. Similarly in proton magnetic resonance spectroscopy studies, reduction in in vivo GSH levels have been observed in AD patients; the reduction has mainly been observed in frontal cortex and hippocampus i.e. regions of the brain that show pathological changes in AD. LPS administration diminished GSH level in disease control mice brains. This effect was significantly reversed in mice receiving high dose of SCP [49][50][51]. Similiarly, the activity of SOD, the first detoxification enzyme and most powerful antioxidant in the cell, was significantly reduced in the brain of mice post the administration of LPS; SOD levels were significantly rejuvenated after administration of each of the SCP doses. Clinically, it has been observed that in postmortem AD brains, catalase level is significantly reduced [52]. Administration of LPS significantly reduced catalse level, and this effect was reversed by prophylactic administration of SCP at all three doses. The pathogenesis of AD has been associated with reduced level of the brain neurotransmitter acetylcholine. Subsequently, acetylcholinesterase inhibitors (AChEIs) were introduced for the symptomatic treatment of AD and are the mainstay of symptomatic AD management. The current opinion is that the efficacy of AChE inhibitors is due to the improvement of acetylcholine-medicated neuron to neuron transmission. However, AChE inhibitors also protect cells from free radical toxicity and β-amyloid-induced injury and increased production of antioxidants. It has also been shown that AChE inhibitors directly inhibit the release of cytokines from microglia and monocytes. These observations indicate a role of acetylcholine in suppression of cytokine release through a 'cholinergic anti-inflammatory pathway' [53]. Significant ACHE inhibitory activity of SCP was observed in vivo. The exact pathophysiology of AD is still unknown; however, mounting evidence suggests the role of inflammation in neurodegenerative diseases, specially AD [54][55][56]. It has been suggested that accumulation of Aβ leads to the activation of caspases and signal-dependent transcription factors (NF-kB and AP-1) which cause regulation of inflammation amplifiers (e.g., IL-1β, TNF-α, IL-6); these proinflammatory cytokines may directly act on neurons to cause apoptosis [57,58] or activate astrocytes, and these factors released from astrocytes possess the capacity to further activate microglia [59]. Studies have demonstrated that TNF-α triggers cytotoxic cascades and apoptotic cell death; Aβ causes neuronal cell death via TNF-α-mediated signalling. Moreover, it also effects learning and memory processes by disrupting synaptic plasticity and has detrimental effects on synaptic transmission and plasticity [60,61]. There was a significant reduction in expression of proinflammatory cytokines in SCP-administered mice brain as compared to disease control.
Caspase-3 is a principal cell death protease involved in neuronal apoptosis during physiological development and under pathological conditions. It has been shown that wild-type Amyloid precursor protein (APP) is an intrinsic activator of caspase-3-mediated death machinery in postmitotic neurons [62]. SCP administration diminished caspase-3 level at all three test doses. APP, presenilin (a component of γ secretase) and BACE1 (β-secretase) have NF-kB sites in their promoters, and thus their expression is governed by NF-kB; Sastre et al have demonstrated that proinflammatory cytokines upregulate the expression of these factors in neurons [63]. Inflammatory mediators acting on neurons increase production of Aβ, further activating microglia-mediated inflammation.
Thus, neurons and glia communication may amplify the production of hallmark AD pathological factors. Guzman-Martinez et al have hypothesized that during the course of AD, damage signals activate glial cells leading to NF-ĸB activation and synthesis and release of proinflammatory cytokines that affect neuronal receptors with an overexpression of protein kinases [64]. Furthermore, NF-kB, a pleiotropic transcription factor, regulates several other genes that are involved in inflammatory responses and stimulates numerous cellular signalling pathways, which leads to the increased production of inflammatory cytokines. The LPSinduced NF-kB activation in glia has been reported to upregulate the expression of inflammatory mediators [65,66]. SCP administration significantly lowered brain NF-ĸB level. BACE1, the β-secretase enzyme, cleaves the APP and releases a C99 fragment which gives rise to various species of Aβ peptide upon further processing by γ-secretase. BACE inhibition will result in an upstream interference with the amyloid cascade. Several studies report that BACE1 inhibitors hold great potential as a target strategy to reduce Aβ brain concentrations, thus preventing the progression of AD [67,68]. High dose of SCP demonstrated significant BACE-1 inhibitory effect.

CONCLUSION
In summary, intraperitoneal injection of LPS lead to cognitive and memory impairment in behavioral tests, and an increase in proinflammatory cytokines (TNF-α and IL-6) via the activation of NF-ĸB signaling pathway, oxidative stress (increase in MDA; reduction in SOD, GSH, and CAT), upregulation of AChE, cell death (caspase-3), and BACE-1 activity. Our findings revealed that SCP attenuated oxidative stress, inflammation and degeneration in a dosedependent manner. Further, it is important to study the activity of SCP in other chemically induced animal models. Catalase, an enzyme involved in preventing cellular oxidant damage by metabolizing hydrogen peroxide to water and oxygen, was significantly reduced after LPS administration.

FUNDING
Authors would like to thank University Grants Commision, New Delhi, India for funding this research work

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by University Grants Commission.

CONSENT
It's not applicable.

ETHICAL APPROVAL
Approval for animal experimentation was obtained from Institutional Animal Ethics Committee of T. N. Medical College and BYL Nair Charitable Hospital, Mumbai (Protocol No. IAEC/4A dated 29 th February 2016) and all the experimentation was carried out in accordance with the Committee for Control and Supervision of Experimentation on Animals (CPCSEA) guidelines.