Mechanistic Insight Into Diosmin-Induced Neuroprotection And Memory Improvement in Intracerebroventricular-Quinolinic Acid Rat Model: Resurrection Of Mitochondrial Functions And Antioxidants Part 4

2.12.4. Superoxide Dismutase Activity. .e rate of SOD (EC 1.15.1.1) action (units per mg protein) was considered by the procedure of Kakkar et al. [33]. .e reaction concoction involved 0.3 ml homogenate, 100 µl 5-methylphenazinium methyl sulfate (197 μM), and 1.3 ml sodium diphosphate tetrabasic (0.066 mM, pH 7.2). .e reaction was commenced using 200 µl of β-nicotinamide adenine dinucleotide (DPNH) (780 μM) and halted 60 seconds later using 1 ml glacial CH3COOH in this blend. The chromogen quantity generated was computed by noting the color strength at λmax� 560 nm. 

Superoxide dismutase (SOD) is an important enzyme substance that can play an antioxidant role in the body. More and more studies have shown that there is a close relationship between SOD and memory.

First, the SOD enzyme can help the body remove free radicals and reduce the damage of oxidative stress to neurons, thereby protecting the normal function of the nervous system. The normal function of the nervous system is the key to human memory, learning, and other advanced thinking activities. Therefore, a sufficient SOD level can help us maintain a good state of memory, cognition, and learning.

Secondly, SOD can also help the human body replenish enough oxygen. With age and changes in the environment, the supply of oxygen in the body will gradually decrease. Oxygen is one of the key substances for human cell metabolism and is also very important for maintaining a good memory. SOD can help the body maintain oxygen levels and ensure the normal basic metabolic functions of the human body, thereby providing conditions for the generation and maintenance of memory.

In daily life, it is also very easy to maintain a sufficient SOD level. We can increase the body's SOD content through diet, such as consuming vegetables rich in SOD such as green vegetables, cabbage, and carrots. In addition, maintaining good living habits can also help us maintain a healthy SOD level, such as moderate exercise, adequate sleep, etc.

In short, there is a close relationship between SOD enzyme and memory. Increasing the body's SOD content can help us maintain a healthy nervous system and promote the normal progress of advanced thinking activities. We should help the body maintain sufficient SOD levels through scientific diet, living habits, and physical exercise, to better protect our memory. It can be seen that we need to improve memory, and Cistanche can significantly improve memory because Cistanche has antioxidant, anti-inflammatory, and anti-aging effects, which can help reduce oxidation and inflammatory reactions in the brain, thereby protecting the health of the nervous system. In addition, Cistanche can also promote the growth and repair of nerve cells, thereby enhancing the connectivity and function of neural networks. These effects can help improve memory, learning ability, and thinking speed, and can also prevent the occurrence of cognitive dysfunction and neurodegenerative diseases.

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2.12.5. Catalase Activity. To assess the rate of catalase (EC 1.11.1.6) action, the O.D. discrepancy (λmax � 240 nm) of the analyzed concoction (3.0 ml) comprising 50 µl investigating sample, 1.22 ml H2O2 (0.03 M) in Na+ -K+ [PO4] 2- buffer (pH 7.91, 0.06 M), and 1.63 ml of 0.06 M Na+ -K+ [PO4] 2- buffer (pH 7.1) was recorded. 

Catalase activity (µmol H2O2 decayed per minute per mg protein of brain) was computed by applying ε � 43.6/M/cm [34]. 2.12.6. Whole Nitrites Level. .e technique of Sastry et al. [35] was implemented to evaluate entire brain nitrites (μmol per mg of brain protein). 

In test tubes encompassing 100 µl investigative sample, 145 mg amalgam of copper–cadmium, 500 µl H2CO3 buffer (pH 8.89), 0.44 M 100 µl NaOH, and 119.8 mM 400 µl ZnSO4 were centrifugated at 4,500 × g power for 10 minutes, and the superfluous upper liquid (supernatant) was secluded. 

Griess chemical (50 µl) was included in 100 µl of s superfluous liquid. After 60 minutes of incubation, O.D. (λmax � 548 nm wavelength) was noted employing a twin-beam UV1700 spectrophotometer (Shimadzu). A typical curve of nitrous acid sodium (0.02–0.2 mM) was designed, and the entire nitrite was equated. 

2.12.7. Determination of Total Proteins. .e overall protein level (mg/ml of homogenate) was computed using a typical curvature graph of bovine serum albumin with the solution strength ranging from 0.3 to 3.8 mg/ml. .e examination combination was organized with 250 µl homogenate, 5.1 ml Lowry's reagent, Na+ -K+ [PO4] 2- buffer (900 µl), and 1.1 N 500 µl FCR. .e discrepancy of O.D. was observed at λmax � 650 nm [36]. 

2.13. Brain Sections Histopathology. Using a gravity-fed diffusion setup, rats were intracardially (via left ventricle) diffused with 10% neutral buffered formaldehyde (10% NBF) solution and acutely anesthetized. The hippocampus and cortical sectors are immersed in a fixative (10 :1 fixative: tissue proportion), namely 10% NBF for one week (4°C), accompanied by 0.04% natriumazid (pH 7.4). 

Ethyl alcohol (70%) was employed as a packing solution for fixed tissue portions kept at 4°C. A microtome cutter (rotation type) was employed to acquire thin portions (8.0 μm), which were then tinted with colorant hematoxylin and eosin (H&E). .e slides were made permanent using DPX-resin, which were later cover-slipped and inspected through an optical microscope (binocular) at ×40 magnifications. 

2.14. Statistical Analysis. A skilled experimenter blinded to miscellaneous drug regimens given to animal cohorts scrutinized and evaluated the data. Outliers were not pragmatic (Grubb's test) in the data, and the Kolmogorov–Smirnov test and Levene's test confirmed the normal distribution of variables and homogeneity of variance (HOV p > 0.05, Levene's test), respectively. 

Otherwise, in case of unequal variance (HOV p < 0.05, Levene's test), Welch's ANOVA (p < 0.05, F′-statistic), and Games–Howell post hoc tests can be applied. .e means of normally distributed variables were scrutinized and related by one-way or repeated measures of two-way analysis of variance (ANOVA). In the case of ANOVA, outcomes are significant (p < 0.05) in F-statistics, multiple comparison tests, namely Tukey's HSD (honest significant difference) or Bonferroni, were applied. Statistical significance was deemed at p < 0.05, and the results were stated as mean- ± Standard Error of Mean (SEM).

3. Results

3.1. Outcomes of DSM on Body Mass (g), Diet, And Water Ingestion of Rats Administered QA-ICV. Body weights, feed, and water ingestion were analyzed weekly, starting from day 1. 

A significant reduction (p < 0.001) in body mass (g), feed, and water intake on days 7, 14, and 21 was pragmatic in rats subjected to QA-ICV injection on day 1 when compared to sham counterparts (Figure 2). DSM (100 mg/kg) dosing caused a significant increase in the body weight (day 7 p < 0.05, day 14 p < 0.01, day 21 p < 0.001), feed (day 7 p < 0.05, day 14 p < 0.01, day 21 p < 0.001), and water intake (day 7 p < 0.01, day 14 p < 0.05) in rats against QA-ICV. DSM (50 mg/kg) also significantly attenuated QA-ICV-triggered decrease in the body weight (day 21 p < 0.05) and feed intake (day 7 p < 0.01) of rats relative to rats that have lone QA-ICV injections. QA-ICV- and DNP-treated rats disclosed a significant escalation in body mass (g) (day 7 p < 0.05, day 14 p < 0.001, day 21 p < 0.001), feed (day 7 p < 0.001, day 14 p < 0.01, day 21 p < 0.001), and water intake (day 7, 14, 21 p < 0.01) relative to rats that remained exposed to lone QA-ICV. 

3.2. Effect of DSM on Locomotion, Motor Coordination, and Gait of Rats against QA-ICV. In this study, the locomotor activities of animals were not affected by QA-ICV or drug treatments. 

QA group depicted no significant change in the mean counts per 5 minutes in the actinometer apparatus in comparison to the sham group (Figure 3(a)). Rotarod and footprint analysis were used to evaluate the sensorimotor performance and gait of rats. 

Results showed that QA-ICV significantly hampered (p < 0.001) motor coordination (Figure 3(b)) and gait (Figure 3(c)) of rats, reflected by a decrease in latency to fall from the revolving shaft and the stride length of rats in footprint analysis in comparison to sham counterparts. 

QA + DSM50 and QA + DSM100 groups portrayed a noteworthy increase in the falling latency (p < 0.05, p < 0.01) and stride length (p < 0.05, p < 0.001) relative to the QA group. 

DNP treatment significantly attenuated QA-ICV and triggered a decrease in latency to fall (p < 0.001) and stride length (p < 0.001) when compared to rats that were given QA-ICV alone. Furthermore, DSM (100 mg/kg) treatment displayed a significant improvement (p < 0.01) in gait relative to DSM (50 mg/kg) in rats subjected to QA-ICV. 

3.3. Effect of DSM on Working Memory and Spatial Long-Term Memory of Rats against QA-ICV. A decrease in the discrimination index (%) in NORT corroborated a decrease in the working type memory. 

A gradual increase in ELT over 4 days of training trials and a decrease in TSTQ in the retrieval trials (conducted 24 hours after the last training trial) in MWM denoted long-term memory loss in rats. 

In this study, rats that were exposed to QA-ICV treatment alone indicated a noteworthy decline (p < 0.001) in the discrimination index (%) relative to sham (Figure 4(a)). 

In the MWM test, day 17 training trials revealed no significant alteration in ELT among different groups, however, on day 18, a marked change in ELT was noted. QA cohort displayed a substantial (p < 0.001) increase in ELT (day 18–20) (figure 4(b)) and a decrease in TSTQ (day 21) (Figure 4(c)) relative to sham counterparts. 

Treatment with DSM (100 mg/kg)- attenuated QA-ICV prompted diminution in discrimination index (%) (p < 0.001), increase in ELT (day 18 p < 0.05, day 19 p < 0.01, day 20 p < 0.001), and decrease in TSTQ (p < 0.001) when compared with the rats that have undergone QA-ICV injection alone. 

QA + DSM50 cohort exhibited a substantial upsurge in discrimination index (%) (p < 0.001), a decrease in day 20 ELT (p < 0.01), and an increase in TSTQ (p < 0.001) relative to the QA group. 

DNP treatment enhanced the discrimination index (%) (p < 0.001), decreased ELT (day 18 p < 0.01, day 19 p < 0.001, day 20 p < 0.001), and increased TSTQ (p < 0.001) in rats that were administered QA-ICV in comparison to vehicle-treated QA-ICV rats. Furthermore, DSM (100 mg/ kg)-repeated injections disclosed a substantial improvement in memory functions relative to DSM (50 mg/kg) in rats subjected to QA-ICV.

3.4. Outcomes of DSM on Brain Mitochondrial Complex in QA-ICV Injected Rats. 

Mitochondrial activity in the whole-brain homogenate was evaluated after behavioral trials. Results showed a significant decline (p < 0.001) in the complex I/II rates in the mitochondrial fraction of the brain homogenate by QA-ICV relative to sham (Figure 5). .is decrease in complex I/II activity by QA-ICV treatment was attenuated (complex I p < 0.05, p < 0.001; complex II p < 0.01, p < 0.001) by DSM (50 and 100 mg/kg) given for 21 successive days in comparison to QA-ICVadministered rats that were given drug vehicle treatment alone. DNP significantly enhanced (p < 0.001) the activity of complex I/II in contrast to the vehicle in QA-ICV injected rats. 

3.5. Effect of DSM on Brain Mitochondrial Oxidative Stress in QA-ICV Injected Rats. Results exhibited a noteworthy increase (p < 0.001) in the TBARS and total nitrites and a decline in GSH, GPx, SOD, and catalase activities in the mitochondrial fraction of the brain homogenate by QA-ICV relative to sham (Figure 6). .is augmentation in the brain TBARS (p < 0.05, p < 0.001) and total nitrites (p < 0.05, p < 0.01) and decline in GSH (p < 0.05, p < 0.001), GPx (p < 0.05, p < 0.001), SOD (p < 0.01, p < 0.001), and catalase (p < 0.05, p < 0.001) activities by QA-ICV treatment was attenuated by DSM (50 and 100 mg/kg) given for 21 uninterrupted days in comparison to QA-ICV-administered rats that were given drug vehicle treatment only. DNP significantly depriciated (p < 0.001) brain TBARS and total nitrites accumulation and enhanced (p < 0.001) the activity of GSH, GPx, SOD, and catalase in comparison to the vehicle in QA-ICV-treated rats. 

Furthermore, DSM (100 mg/kg) treatment displayed a noteworthy depreciation in lipid peroxidation (p < 0.001) and intensification in endogenous antioxidants, such as GSH (p < 0.001), GPx (p < 0.01), SOD (p < 0.05), and catalase (p < 0.05), relative to DSM (50 mg/ kg) in rats subjected to QA-ICV. 3.6. Effect of DSM on Brain Histopathology in Rats against QAICV. 

In histopathology analysis, major changes in the cellular architecture were observed in the QA group. Sham animals showed no signs of neurodegeneration. QA-ICV treatment caused marked changes highlighted by pyknosis and the blebbing of the plasma membrane in the cortical and hippocampus (CA 1 and (2) neurons. Treatment of QAICV rats with DSM or DNP attenuated the pathological signs of neurodegeneration (Figure 7).

4. Discussion

QA (2,3-pyridine dicarboxylic acid) is an excitotoxin similar to glutamate and is capable of evoking neurodegeneration as its concentration amplifies with age [5]. .e antagonists of NMDARs and amino phosphonates can prohibit the neurodegenerative excitotoxicity of QA, which suggests that QA acts through NMDARs in the brain [9]. BBB acts as a protective barrier and limits the neurotoxicity of QA. 

However, the pieces of evidence indicate enhanced pathological accumulation of QA in diverse neurodegenerative disorders. 

In the brain, quinolinate phosphoribosyltransferase (QPRT) catabolizes QA to NAD+ and carbon dioxide. .e activity of QPRT is maximum in the olfactory bulb and lowermost in the cortex, hippocampus, and striatum, where QA may exert neurotoxic action to a great extent in these brain regions. These brain areas are adversely affected by numerous neurodegenerative disorders, such as AD, PD, HD, and schizophrenia that lead to severe cognitive decline [9, 10]. 

In this study, QA was administered directly through the ICV route to overcome the BBB constraint in the adult rats. DSM is a bioactive natural flavonoid that has shown therapeutic effects against traumatic brain injury [16], scopolamine-induced amnesia [17], apomorphine- and ketamine-induced psychosis [18], and chronic unpredictable mild stress [19]. 

DSM is capable of enhancing glucose metabolism, insulin signaling, and diabetic complications, and it may prevent energy depletion and the ensuing adverse consequences implicated in neurodegenerative disorders [14, 15]. 

QA prohibits mitochondrial function and energy-producing aerobic respiratory pathways in the brain regions adversely affected by neurodegenerative disorders [5]. 

Hence, in the present study, rats administered with QA-ICV on day one were exposed to DSM treatment for 21 consecutive days, and biochemical parameters and behavioral functions were assessed.

idonitrosative stress and depreciation of endogenous antioxidant levels by QA-ICV in the mitochondrial fraction of the whole-brain homogenate. Previous studies also indicate NMDAR-dependent and NMDAR-independent increases in free radicals and inflammatory deterioration by QA in experimental animals [5, 9, 10]. 

In current experiments, QA enhanced lipid peroxidation and total nitrites in the brain. Free radicals and the ensuing modifications in cellular biomolecules, such as lipids, proteins, and DNA, underlie major pathogenic changes in neurodegenerative disorders. 

Lipid peroxides, such as malondialdehyde (MDA), 4-hydroxy 2-nominal (4-HNE), isoprostanes, and acrolein are highly toxic aldehydes that readily accumulate in the form of bio-adducts and are resistant to autophagic and other mechanisms of removal [37, 38]. 

A pathogenic rise in these insoluble adducts breaches the integrity of the cell, which leads to a loss of internal homeostasis, leakage of internal components, and cell death [38]. 

Furthermore, an increase in nitrates directly correlates with the level of nitric oxide release in the brains of rats. .e administration of QA-ICV on the first day instigated a noteworthy escalation in nitrates in the brains of rats. Nitric oxide is a gaseous neurotransmitter that participates in synaptic modulation and long-term potentiation by acting in a rearward manner through NMDARs [39, 40]. 

Nitric oxide is biosynthesized by nitric oxide synthase (NOS-neuronal) in response to the activation of postsynaptic NMDARs. An influx of calcium ions through postsynaptic NMDARs activates neuronal NOS, leading to the generation of nitric oxide that stimulates presynaptic glutamate release in the synapse. 

However, excessive NMDAR activation and the resulting overt intracellular influx of calcium ions and nitric oxide biosynthesis leads to a rise in the reactive oxygen species (ROS), such as alkoxyl (RO_ ), superoxide (O•− 2), peroxyl (RO2·), hydroxyl radicals (OH• ), and hydrogen peroxide (H2O2), and reactive nitrogen species (RNS), such as nitrous anhydride (N2O3), peroxynitrite (ONOO− ), and nitrogen dioxide (•NO2) [41–43]. 

RNS modifies proteins, leading to protein nitrosylation and the formation of S-glutathiols and nitrosothiols. Nitric oxide participates in vascular damage (e.g., BBB and ischemia injury) and inflammatory response by the activation of macrophages, astrocytes, matrix metalloproteinases (MMPs), and adhesion molecules [44, 45]. 

Peroxynitrites cause the nitration of guanine nucleotides, resulting in DNA single-strand rupture, triggering the PARP pathway, and also prohibiting DNA repair enzymes [46]. 

Nitric oxide inhibits cytochrome c oxidase and thereby suppresses mitochondrial ATP production [39]. Hence, excess nitrite accumulation can cause an energy-deficient state in the brain that further ensures the dysfunction of ATP-dependent ion pumps, accumulation of sodium ions (causing cell swelling), increased calcium influx, and hyperexcitability [47]. .e augmentation of cytoplasmic calcium levels is the primary mechanism of ROS and RNS output and the activation of calcium-dependent cell demise pathways through the activation of proteases and calpains [48]. 

In neurodegenerative diseases, free radicals, calcium, lipid peroxidation, and DNA mutilation are at the core of the pathogenic progression of diseases. In previous studies, the analysis of the biomarkers of oxidative stress revealed the amplification of MDA, 4- HNE, and 8-hydroxy-2' -deoxyguanosine (8-OHdG) in the cerebrospinal fluid, brain, and blood samples [49, 50]. 

In the current experiments, DSM (50 and 100 mg/kg) significantly abrogated QA-ICV-triggered intensification in lipid peroxidation and total nitrites in the mitochondrial portion of the brain. 

Earlier reports also substantiate the free radical attenuating and anti-inflammatory deeds of DSM in the entire brain of experimental animals [16–20]. .e standard drug, DNP, also attenuated lipid peroxidation (TBARS) and total nitrites in the brains of rats exposed to QA-ICV.

.e mitochondria, peroxisomes, endoplasmic reticulum, and plasma membranes are the chief locations of ROS and RNS biosynthesis [51]. Cellular respiration in the mitochondria is the chief generator of ROS, such as superoxide and hydroxyl radicals. Peroxisomes are the central hub for hydrogen peroxide generation. 

Superoxide anion acquires an electron from molecular oxygen and dismutases to hydrogen peroxide via the Fenton reaction. Subsequently, this hydrogen peroxide is metabolized by catalase to water and oxygen, or it may generate hydroxyl radicals. 

H2O2 can also generate toxic hydroxyl radicals through the Haber-Weiss reaction. Superoxide anion, by reacting with nitric oxide, can form peroxynitrites [52, 53]. .ese free radicals trigger mitochondrial permeability transition pores (mPTP), leading to the leakage of cytochrome c, mitochondrial degeneration, and cell demise. 

Mitochondrial complex I is the gateway for electrons' entry from NADH into the respiratory chain. Complex I and II can generate superoxide anions in surplus in response to a higher NADH/NAD+ ratio, leading to an abridged FMN (flavin mononucleotide) site on complex I, and electron contribution to the succinate dehydrogenase (SDH)-reduced coenzyme Q is associated with a high proton motive force, leading to reverse electron transport [54]. QA is a well-recognized inhibitor of mitochondrial functions [5]. 

In this study, QA-ICV caused the inhibition of the brain mitofragment complex I and II rates in rats. .e QA-induced aberrations in the mitochondrial electron transport chain functions might be the primary cause of oxidative mutilation in the brains of rats. However, DSM (50 and 100 mg/kg) resurrected the brain complex I and II activities in QA-ICV-challenged rats. 

DNP treatment also showed significant improvement in complex I and II functions in the brain mitochondria against QA-ICV cytotoxicity. .e analysis of antioxidant levels revealed that DSM treatment for 21 consecutive days attenuated QA-ICV-induced decline in endogenous antioxidants, such as GSH, GPx, SOD, and catalase. 

SOD, catalase, and thiol-dependent antioxidants, such as GSH and GPx, are the 1st line of defense against oxidative mutilation. SOD and catalase detoxify superoxide anions and H2O2, respectively, and GPx and GSH are involved in the removal of H2O2 and breakdown of lipid peroxides (MDA, 4-HNE, etc.) to their respective alcohols, particularly in the mitochondria and cytoplasm [55]. In the current study, DSM (50 and 100 mg/kg) or DNP regimens boosted the antioxidant actions in the brains of rats that were rendered toward QA-ICV neurotoxicity. 

Neuroprotection by DSM and DNP was evident in the H&E staining analysis of the hippocampus and cortical regions of rat brains. QA-ICV treatment caused marked changes in the cellular architecture highlighted by pyknosis, cell swelling, and blebbing of the plasma membrane. These pathogenic changes were attenuated by DSM and DNP treatments in separate groups of rats. In the current protocol, DSM (100 mg/kg) showed significant improvement in biochemical parameters against QA-ICV and also attenuated pathological cell mutilation evident in histological analysis in comparison to DSM (50 mg/kg). Hence, these findings depicted the dose-dependent effects of DSM in the QA-ICV rat model of neurodegeneration. 

In the weekly analysis, a significant decline in mean body mass (g), feed, and water ingestion was pragmatic in QAICV-treated rats. Motor coordination (rotarod test) and gait (footprint analysis) were also adversely affected in rats treated with QA-ICV alone. 

Locomotor activity was not affected by diverse drug treatments in the current set of experiments. However, DSM or DNP-treated rats showed significant improvement in motor coordination and gait against QA-ICV toxicity. Body bulk (g), diet, and water consumption were also enhanced by DSM or DNP in separate groups of QA-ICV-treated rats. 

Memory parameters were assessed using NORT (day 16) and MWM (days 17–21) paradigms. A decline in the discrimination ability of QAICV-treated rats in NORT supported the loss of working memory. 

In MWM trials, QA-ICV caused an increase in ELT during four days of training trials and a decrease in TSTQ in retrieval trials on the 5th day. These findings showed the depreciation of long-term spatial memory of rats by QAICV treatment. DSM or DNP treatments attenuated the decline of discrimination index, TSTQ, and increase in ELT in rats that were challenged with QA-ICV on day 1. .e contemporary findings are in harmony with the former reports substantiating the memory improvement activity of DSM in experimental animals [16–20]. 

DSM (100 mg/kg) showed dose-dependent improvement in working memory and long-term memory in comparison to DSM (50 mg/kg) against QA-ICV. .e findings showed that DSM could ameliorate brain functions such as memory, motor coordination, and gait in rats in the QA-ICV model. Diosmin is a flavonoid glycoside possessing a sugar moiety (rutinoside disaccharide) and aglycone group diosmetin. In several preclinical studies, diosmin is administered through the parenteral route (i.p.) [17, 56], and there is an impending urgency to find a suitable formulation to better translate the pre-clinical findings in clinical settings. 

Particle size reduction and increasing the surface area can be utilized to enhance its transport across the biological barriers [57]. In this context, a micronized diosmin formulation [58] was attempted that showed greater bioavailability and pharmacokinetic characteristics, however, the targeted brain-specific delivery of diosmin is still a huge challenge.

5. Conclusions

In the current therapeutic scenario where no such drug is available that can revive the pathogenic evolution of neurodegenerative conditions, such as AD and HD, a shift toward natural remedies is pragmatic. Current therapeutic strategies focus on symptomatic improvement only, and none can reverse the sequence of the disease development. We observed that diosmin resurrected cognitive functions (working and long-term spatial memory) in the QA-ICV rat model of neurodegeneration. Diosmin improved the sensorimotor performance and the gait of rats against QA-ICV. .e observed improvement of behavioral functions in QA-ICV rats treated with diosmin is primarily because of the attenuation of mitochondrial dysfunctions and oxidative mutilation of the brain. Hence, diosmin might be used as an alternative therapeutic agent against mitochondrial dysfunction origin neurodegenerative disorders. However, additional investigations are mandatory to envisage its neuroprotective mechanism and applications in clinical settings.

Data Availability

.e data of this study are accessible upon an appropriate demand from the corresponding author.

Ethical Approval
.e entire set of animal experimentations was approved by the IAEC (Approval no. ASCB/IAEC/14/20/145) and was executed by implementing the ethical guidelines on animal testing issued by the "Committee for Control and Supervision of Experiments on Animals (CPCSEA), GOI, New Delhi."

Conflicts of Interest

The authors declare that there are no conflicts of interest.

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