International Journal of Nutrition, Pharmacology, Neurological Diseases

: 2019  |  Volume : 9  |  Issue : 4  |  Page : 136--145

Neuroprotective Effects of Mitochondria-Targeted Quercetin Against Rotenone-Induced Oxidative Damage in Cerebellum of Mice

Whidul Hasan1, Richa Rajak1, Rajesh Kumar Kori2, Rajesh Singh Yadav2, Deepali Jat1,  
1 Neuroscience Research Lab, Department of Zoology, School of Biological Sciences, Dr. H. S. Gour University, Sagar, MP, India
2 Department of Criminology and Forensic Science, Dr. H. S. Gour University, Sagar, MP, India

Correspondence Address:
Deepali Jat
Assistant Professor Neuroscience Research Lab, Department of Zoology, School of Biological Sciences, Dr. H. S. Gour University, Sagar 470003 (M. P.)


Mitochondrial dysfunction leads to various types of metabolic impairments, including decreased ATP formation, diminished calcium buffering, gathering of metabolic intermediates and amplified production reactive oxygen species and reactive nitrogen species. Rotenone is a strong inhibitor of complex I and causes mitochondrial dysfunction, leads to motor impairment, metabolic disorder and cellular damage. Quercetin is a strong antioxidant and neuroprotective against neurodegenerative diseases, but its efficacy is limited because it does not gather into the mitochondria. Therefore, mitochondrially targeted antioxidants have to be developed by conjugating with lipophilic cation which can freely cross through the mitochondrial membrane and protects mitochondrial injury. This study investigated the protective effect of quercetin and mitochondrial-targeted quercetin (MTQ) in rotenone-induced cerebellar toxicity in mice. Treatment of rotenone (3 mg/kg b.w., p.o for 60 days) in mice significantly increases in the levels of lipid peroxidation, nitric oxide and decrease in the activity of AChE, reduced glutathione, superoxide dismutase and catalase were observed in mice compared to controls. Co-treatment of quercetin and MTQ (30mg/kg b.w., p.o) along with rotenone significantly increased AChE activity and protected against rotenone-induced enhanced oxidative stress. Histological study of cerebellum exhibited necrosis of Purkinje cells as revealed by irregular, damaged cells and perineuronal vacuolation in rotenone-treated mice. Co-treatment of quercetin and MTQ along with rotenone showed protection against rotenone-induced cellular damage in these cells. The results exhibit that both quercetin and MTQ showed a protective effect against rotenone-induced cerebellar toxicity in mice and MTQ is more effectively showed protection than quercetin.

How to cite this article:
Hasan W, Rajak R, Kori RK, Yadav RS, Jat D. Neuroprotective Effects of Mitochondria-Targeted Quercetin Against Rotenone-Induced Oxidative Damage in Cerebellum of Mice.Int J Nutr Pharmacol Neurol Dis 2019;9:136-145

How to cite this URL:
Hasan W, Rajak R, Kori RK, Yadav RS, Jat D. Neuroprotective Effects of Mitochondria-Targeted Quercetin Against Rotenone-Induced Oxidative Damage in Cerebellum of Mice. Int J Nutr Pharmacol Neurol Dis [serial online] 2019 [cited 2019 Dec 6 ];9:136-145
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Full Text


Parkinson’s disease (PD) is the second most common neurodegenerative disorder, characterized by bradykinesia, muscle rigidity, resting tremors and postural abnormalities.[1],[2] The neuropathological signs of PD are the progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) basal ganglia of the mid-brain and reduced dopamine content[3] and the presence of cytoplasmic inclusions named as Lewy bodies.[4],[5] The two main subcortical areas of the brain, cerebellum and basal ganglia, have been stated to disturb various cognitive and motor behaviors.[6] Activation of cerebellar Purkinje cells for a long time was linked with dopaminergic neuronal loss.[7] Inflammation is a major pathogenesis aspect causing age-related neurodegenerative diseases, together with PD.[8] Cerebellum plays a central role in motor learning and motor coordination, and its impairments result in motor learning deficits and cerebellar ataxia [9]. Mounting pieces of evidence suggest that the cerebellum also contributes to non-motor functions,[9] comprising cognitive domains, including executive control,[9] language[10] and working memory.[11] Studies have suggested the involvement of cerebellar dysfunction together with the basal ganglia in mediating many Parkinsonian symptoms.[12]

Rotenone is a natural pesticide that is extremely lipophilic, crosses blood-brain barrier and also inhibiting the mitochondrial complex I of the respiratory chain.[5] It induces the production of free radicals that leads to oxidative stress and neuroinflammation. The oxidative stress causes selective neurodegeneration, which has been directly related to the onset of PD.[2],[5],[13] Oxidative stress caused by rotenone plays a major role in pathogenesis of PD by increasing the mitochondrial oxidative stress.[13] Mitochondrial dysfunction leads to various types of metabolic impairments, including decreased ATP formation, diminished calcium buffering, gathering of metabolic intermediates and amplified production of reactive oxygen species (ROS) and reactive nitrogen species (RNS).[14]

Numerous forms of antioxidants have been verified for their neuroprotective role in neurological disorders. Studies have been observed that natural antioxidant such as vitamin C, α-tocopherol and vitamin E and plant extracts such as Ginkgo biloba extract, curcumin, quercetin reduced oxidative stress in the mouse brain,[15],[16],[17],[18] suggesting that these antioxidants may be neuroprotective against PD. Epidemiological studies recommend that the combination of high dose of vitamin C and vitamin E administration decreased progression of PD.[19] But their efficacy is limited because they do not gather into the mitochondria. Therefore, be resolve this issue mitochondrially targeted antioxidants to be developed which can freely cross through the mitochondrial membrane and protects mitochondrial injury. In numerous studies, lipophilic triphenylphosphonium ion (TPP) is used as a positively charged cation to target mitochondria.[19] TPP works as a perfect vehicle to transport antioxidants to mitochondria because of its lipophilic nature, it quickly permeates into the mitochondrial membrane and accumulates inside it.[19] TPP linked accumulation of antioxidants within mitochondria increases the effectiveness of antioxidants against oxidative damage.[20] Quercetin (3,3′,4′,5,7-pentahydroxyflavone) is a plant flavonoid present in various types of fruits and vegetables. Various studies have reported its protective effects on cancers, inflammatory processes, infections, gastrointestinal tract function, cardiovascular diseases, diabetes and numerous nervous disorders.[21],[22],[23] Recently, neuroprotective role of quercetin in radiation-induced brain injury in rats has been reported.[24] In view of this, this study has been carried out to investigate the protective efficacy of quercetin and mitochondria-targeted quercetin (MTQ) on oxidative damage induced by rotenone in the cerebellum of mice.

 Materials and Methods

Chemicals: Dimethyl formamide (DMF), 1-bromo-4-chlorobutane, Sodium iodide (NaI), Potassium carbonate (K2CO3), Ethyl acetate (EtOAc), Triphenylphosphine ion, quercetin, Haematoxylin and eosin stain, thiobarbituric acid (TBA), reduced glutathione (GSH) and bovine serum albumin (BSA) were purchased from Himedia, India. Hydrochloric acid (HCl), Dichloromethane (CH2Cl2), Magnesium sulphate (MgSO4), acetone and hydrogen peroxide (H2O2) and toluene were purchased from CDH, India. Acetyl chloride, n-hexane, diethyl ether, pre-coated silica gel GF aluminum plate were purchased from Merck, India. Superoxide dismutase assay kit (Abcam, ab65354), Acetylthiocholine Iodide and Griess reagent were purchased from Sigma Aldrich.

Synthesis of targeted antioxidant

MTQ was synthesized by covalent linkage of quercetin with lipophilic TPP as given procedure described.[25] The charge of the lipophillic cation evenly distributes over a large hydrophobic surface area by which activation energy for their movement across the mitochondrial membrane lowers down thus they easily cross through mitochondrial membrane.[26] To synthesize targeted derivative of quercetin, a solution of TPP (lipophilic cation) and the quercetin was refluxed then solvent was eliminated under reduced pressure below 100°C to obtain MTQ.

Animals and Treatment

Adult male Swiss albino mice (28±2g) were procured from the Veterinary Science and Animal Husbandry, Mhow, India and acclimatized for ten days before starting the experiment. Mice were housed in polypropylene cages under standard hygiene condition at a temperature of 25±2°C with a 12-hour light/dark schedule and were provided standard food and water ad libitum. The animals were randomly divided into four groups and four animals in each group. In group I, mice were treated with vehicle and served as controls; in group II, mice were treated with rotenone (3 mg/kg, b.w., p.o. dissolved in corn oil) for 60 days; in group III they were co-treated with quercetin (30mg/kg, b.w., p.o. dissolved in corn oil) and rotenone for 60 days and in group IV, mice were co-treated with MTQ (30mg/kg, b.w., p.o. dissolved in corn oil) and rotenone for 60 days. For biochemical studies, mice were sacrificed by cervical decapitation around 24 h after the last day of treatment. Brains were taken out quickly, washed in ice-cold saline and cerebellum was dissected following the standard procedure.[27] The isolated cerebellum samples were processed immediately for the assay of oxidative stress parameters. For the histological study, brain from each treatment group was isolated from the perfused mice and processed within four to five days following the standard protocol. All procedures were done in accordance with Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and the study was approved by the Institutional Animal Ethical Committee (IAEC No. AIPS/2015/2459/IAEC-04), Adina College of Pharmacy, Sagar (MP), India.

Biochemical analysis

Assay of acetylcholinesterase activity: Activity of acetylcholinesterase (AChE) was determined in cerebellum following the method of Ellman et al.,[28] using acetylthiocholine iodide as a substrate and 5, 5’-dithiobis-2 nitrobenzoic acid (DTNB) as the colouring agent. To the reaction mixture containing 1 ml phosphate buffer (0.1 M, pH 7.4), 5mM DTNB, post-mitochondrial fraction of the cerebellum containing protein, and acetylthiocholine iodide were added and the change in absorbance was monitored at 412 nm. The enzyme activities were expressed as μmole/min/mg protein.

Nitric oxide assay: Nitric oxide measured as a nitrite was estimated using Griess reagent according to standard protocol.[29] Formation of nitric oxide from L-arginine is catalysed by nitric oxide synthase that quickly reacts with oxygen to yield nitrite. Nitrite ions react with the Griess reagent to form a pink diazo dye by a diazonium coupling reaction which is measured at 540 nm. To calculate the concentration of nitrite, a standard curve plot of sodium nitrite was used and values were expressed in μmole/mg protein.

Assay of lipid peroxidation: Lipid peroxidation was determined by measuring the level of malondialdehyde (MDA) in the cerebellum. MDA was determined by measuring thiobarbituric acid reactive substance (TBARS), yielded a pink coloured compound according to the method of Ohkawa et al.[30] The pink colour product was determined by measuring the absorbance at 548 nm. The result was expressed as nmol MDA/mg protein.

Assay of reduced glutathione: The reduced glutathione content was determined in the supernatants of cerebellum homogenate as described by Hasan and Haider,[31] involving the spectrophotometric assessment of the formation of 5-thio-2-nitrobenzoate from DTNB in the presence of NADPH and glutathione reductase. In parallel, a calibration curve was prepared using glutathione (2-10 µg) as standard. The values were expressed in terms of µg GSH/mg protein.

Assay of superoxide dismutase activity: Activity of superoxide dismutase (SOD) was carried out in cerebellum by colorimetric assay kit (Abcam, ab65354). The assay utilizes a tetrazolium salt WST-1 that produces a water-soluble formazan dye upon reduction with superoxide anion that can be detected at 450 nm. The rate of WST-1 reduction is linearly related to the inhibition activity of xanthine oxidase (XO) by SOD. SOD catalyses the dismutation of the superoxide anion into H2O2 and molecular oxygen, resulting in a decrease of WST-1 reduction. The activity of SOD is expressed in inhibition rate %.

Assay of catalase activity: The activity of catalase was assayed following the method of Aebi,[32] spectrophotometrically in a post-mitochondrial fraction using H2O2 as substrate. The activity of the catalase was expressed in µmole/min/mg protein.

Histological study

Histological studies were carried out according to the standard procedure.[33] Briefly, mice were anesthetized with diethyl ether vapours inside a glass desiccator with closed lid and then perfused through transcardial puncture, initially with phosphate-buffered saline (PBS, 0.01 M, pH 7.4) followed by ice-cold 4% para-formaldehyde in 0.1 M PBS for fixation of tissues. The brains were removed, post fixed in 10% para-formaldehyde solution for overnight and processed for paraffin sectioning. After that tissues were thoroughly washed with water and dehydrated with an increasing series of ethyl alcohol (30–100%). Then, the tissues were cleared in toluene and kept in paraplast prepared at 50–55°C for proper impregnation of wax and tissue block. The tissue was cut in sagittal sections of cerebellum at a thickness of 8 µm using microtome and the sections were collected on gelatin-coated slides. To observe the histological changes, tissue sections were stained with haematoxylin and eosin stain.

Protein estimation: Protein concentration was determined by Folin-phenol reaction assay as described by Lowry et al.[34] using BSA as a standard.

Statistical Analysis

All the statistical analysis was performed using statistical software Microsoft Office Excel 2007 version and Sigma plot 12.0. Values were expressed as mean ± standard error of the mean (SEM). The comparison of groups was performed by one-way analysis of variance (ANOVA), following by Tukey’s multiple comparison as posthoc test. The level of significance was set at p≤0.05.


Effect on AChE activity: The activity of AChE was significantly decreased in the cerebellum (60 %, p≤0.01) in rotenone-treated mice as compared to controls. Co-treatment of quercetin and MTQ along with rotenone in mice caused a significant increase in the activity of AChE in cerebellum (41%, p>0.05; 52%, p≤0.05), respectively as compared to those treated with rotenone alone. The MTQ-treated group showed greater protection against rotenone-induced cerebellar toxicity as compared to quercetin [Figure 1].{Figure 1}

Effect on nitric oxide levels: Mice treated with rotenone exhibited significantly marked elevation in nitric oxide contents in cerebellum (37%, p≤0.001) as compared to controls. Co-treatment of quercetin and MTQ along with rotenone showed significantly decreased levels of nitric oxide (16%, p>0.05; 28% p≤0.01), respectively, in cerebellum as compared to those treated with rotenone alone. The treatment of MTQ was more effective in reducing the level of nitric oxide (14%, p>0.05) against rotenone-induced toxicity as compared to quercetin [Figure 2].{Figure 2}

Effect on lipid peroxidation: A significant increase in the level of lipid peroxidation in cerebellum (38%, p≤0.01) was observed in rotenone-treated mice as compared to controls. Co-treatment of quercetin and MTQ along with rotenone reduced the levels of lipid peroxidation in cerebellum (21%, p≤0.05; 30%, p≤0.01), respectively, as compared to those treated with rotenone alone. MTQ was found to be more effective in reducing the level of lipid peroxidation (11%, p>0.05) against rotenone-induced toxicity as compared to quercetin [Figure 3].{Figure 3}

Effect on reduced glutathione levels: GSH serves as a major antioxidant and detoxifying enzyme and maintains the appropriate mitochondrial redox environment to avoid or repair oxidative modifications. As shown in [Figure 4], the rotenone-treated mice caused a significant decrease in GSH levels in cerebellum (31%, p≤0.01) as compared to control. Co-treatment of quercetin and MTQ along with rotenone maintains the GSH level (25%, p≤0.05; 31%, p≤0.01), respectively, in cerebellum as compared to those treated with rotenone alone. The MTQ was found more effective in preserving the GSH level in cerebellum (8%, p>0.05) in rotenone-induced toxicity as compared to quercetin.{Figure 4}

Effect on superoxide dismutase: The SOD activity was found to be significantly lower in the rotenone-treated group in cerebellum (50%, p≤0.001) as compared to the control group. SOD activity was significantly enhanced in cerebellum (19%, p>0.05; 36%, p≤0.01), co-treatment of quercetin and MTQ along with rotenone as compared to rotenone-treated groups. MTQ-treated group was found to show potentially enhanced SOD activity in cerebellum (20%, ≤0.05) than quercetin [Figure 5].{Figure 5}

Effect on catalase activities: The activity of catalase was significantly diminished in rotenone-treated group in cerebellum (56%, p≤0.01) as compared to control. Co-treatment of quercetin and MTQ along with rotenone significantly increased the catalase activity in cerebellum (23%, p>0.05; 52%, p≤0.05), respectively, as compared to those treated with rotenone alone. Catalase activity prevented more in MTQ-treated group in cerebellum (37%, p>0.05) against rotenone-induced toxicity as compared to quercetin [Figure 6].{Figure 6}

Effect on histopathology in the cerebellum: The photomicrographs for histological study were taken at 4X and 40X resolution from the high-resolution microscope (Zeiss, P95, M37/52 × 0.75). Sagittal section of the cerebellum with haematoxylin and eosin-stained sections shows histological changes in rotenone-treated mice [Figure 7]. The most conspicuous damage was in loss of Purkinje neurons in the foliated tree-like structure in mice treated with rotenone as compared to controls, where there was a marked reduction in the number of cells. Co-administration of quercetin and MTQ with rotenone ameliorated the cerebellum from damage in cortical neural in the Purkinje layer induced by rotenone [Figure 7].{Figure 7}


Mitochondrial impairment and oxidative stress are the primary factors leading to neuronal death and the characteristics of neurodegenerative diseases.[3],[4],[35] Rotenone has been suggested as a prime risk factor in the progression of PD through the ROS production. Numerous studies have also been reported that many of the pathological features namely dopaminergic neurodegeneration, α-synucleinopathy, motor dysfunction[5],[37] and the neurotoxicity induced by rotenone involve oxidative stress. Increased lipid peroxidation and mitochondria dysfunctions in the cerebellum after intrastriatal injection of rotenone in the rat model have been demonstrated.[8],[36],[37] Rotenone exposure has been found to be associated with elevated oxidative stress in different brain regions.[38] AChE, an enzyme involved in the degradation of acetylcholine, has been reported to play an important role in the neurotransmission process. It is a very sensitive enzyme that can be inhibited via environmental toxicant due to increased generation of free radicals. The inhibited activity of AChE has also been observed in the cerebellum of rotenone-treated rats[36],[39] suggesting cognitive impairments. In this study, significant decreases in the activity of AChE following exposure to rotenone have been observed which was in agreement with the earlier studies, suggesting that the rotenone induced oxidative stress that leads to inhibition of AChE activity. In addition, quercetin and MTQ co-treated with rotenone for 60 days showed restoration of the activity of AChE.

Role of nitric oxide in the formation of RNS and to cause neurodegeneration is well established.[40],[41] Nitric oxide interacts with superoxide anion (O2–) and forms peroxynitrite (ONOO–), a strong oxidant and nitrating agent that damage the lipids, proteins, nucleic acids and is associated with neurodegeneration.[42] The reduced form of glutathione mainly participates in protecting cells from oxidative stress and maintaining intracellular reducing environment.[43] The elevated oxidative stress was also demonstrated by decreased levels of GSH, suggesting consumption of this scavenger molecule by excess free radical. Increased nitric oxide and decreased activity of SOD, catalase and GSH concentrations have been reported in the cerebellum and other brain regions of rotenone-treated rats.[39],[44] The level of nitric oxide was increased in rotenone-treated mice, and most likely due to increased nitric oxide production due to induction of the inducible form of nitric oxide synthase (iNOS).[44],[45] It increases protein carbonyl levels in rodent midbrain culture, indicative of oxidative damage to proteins.[46] In this study, there are increased levels of nitric oxide, lipid peroxidation and decreased activity of SOD, catalase and GSH in the cerebellum, suggesting oxidative stress and mitochondrial dysfunction in rotenone-exposed mice. These results are consistent with the previous studies indicating increased generation of reactive oxygen metabolites by the rotenone.

In cerebellum, the function of neurotransmitters has been shown to involve in several behavioural functions, including motor, cognition and emotional behaviour.[47] Alterations in the neurotransmitter levels following exposure to rotenone have been found to cause a histopathological modification in the cerebellum associated with the motor functions in rats.[36] Pathological alterations in the cerebellum following dopaminergic degeneration were also observed in patients with PD and animal models and a consequent imbalance between dopaminergic and cholinergic modulation of striatal output.[12] Pathological abnormalities in the cerebellum of mice following exposure to rotenone have been observed in this study suggesting rotenone-induced neurodegeneration.

Several pharmacological and herbal agents, including quercetin, resevratrol, curcumin and gallic acid, have been found to protect against oxidative damage. They reduce lipid peroxidation, protein carbonylation and increase the GSH level, SOD, catalase activity.[15],[16],[17],[18] Anti-inflammatory, antiviral, anti-allergy, antioxidant, anti-asthmatic and anti-tumor activities of quercetin were found to prevent the propagation of ROS production in tissues and inhibit the neurodegeneration.[15],[49] Mitochondria are the main source of oxidative stress and environmental neurotoxicant primarily act on it to cause mitochondrial dysfunctions. The mitochondria-targeted antioxidant is necessarily designed to accumulate within the mitochondrial matrix, and it is useful to reduce mitochondrial oxidative stress.[50] Therefore, researchers developed several antioxidants targeted to mitochondria such as MitoQ, MitoVitE, Mito TEMPO, Mitocurcuminoids, and mitochondriotropic derivative of quercetin by attaching lipophilic cation TPP can be used to inhibit neurodegeneration from oxidative damage.[24],[51] The protective efficacy of quercetin is enhanced many folds when it enters into the mitochondria as compared to quercetin present outside the mitochondria. MitoQ is an anti-oxidant that is selectively targeted to mitochondria accumulated inside it at a very high concentration in a trans-membrane potential-driven process. It has been shown to prevent ischemia/reperfusion-induced cardiac dysfunction[52] and also protects against endothelial dysfunction in vivo and in vitro models.[53] Another mitochondria-targeted antioxidant, vitamin E derivative prevents hepatic oxidative stress and inhibits fat deposition in mice.[54] Mounting evidence has shown the protective efficacy of quercetin against rotenone-induced oxidative stress by modulating antioxidative enzyme defence in the brain.[22],[55] The effects of rotenone on oxidative stress in the brain were substantially reduced by co-administration of quercetin. Thus, quercetin decreases lipid peroxidation levels, nitric oxide concentrations, restored the activity of SOD, catalase and reduced glutathione, suggesting that co-administration of quercetin and MTQ with rotenone decreased oxidative and nitrosative stress. Our findings are in agreement with previous findings and further highlight the prophylactic protective efficacy of quercetin in ameliorating the toxicity of neurotoxin such as rotenone.In cerebellar vermis, Purkinje neurons regulate and coordinate body movement and through the release of GABA neurotransmitter.[47] Large number of GABAergic inputs from the cerebellar cortex to brainstem project through Purkinje cells[56] and therefore controlling the output of these nuclei in the motor function.[57] Pathological modifications in the cerebellum might be generated by dopaminergic neurodegeneration, abnormal drives from the dopaminergic treatment.[58] Pathological alterations in the cerebellum following dopaminergic degeneration were observed in PD patients and animal models.[12] Several studies on in-vitro experimental animals and humans have given supportive evidence for the neuroprotective effects of quercetin against neurotoxic chemicals. In rotenone-treated mice, the reduction in Purkinje cells in cerebellum vermis has been reported in rodents.[35],[36]

Recent research on a chronic parkinsonian model of monkey found that continuous hyper-activation of Purkinje cells linked with the level of dopaminergic leads to neuronal loss in the substantia nigra.[59] In this study, rotenone-induced loss of Purkinje neurons in cerebellum indicated that rotenone-induced neuronal death through oxidative damage causes histopathological damage in the cerebellum. In contrast, co-treatment with quercetin and MTQ exquisitely protects from oxidative-induced loss of Purkinje neurons in the cerebellar vermis.


The findings in this study indicate a protective action of quercetin and MTQ against rotenone-induced neurotoxicity. Quercetin and its mitochondria-targeted derivative exerted an antioxidant action, decreasing brain lipid peroxidation, nitric oxide and increasing activity of AChE and antioxidant enzymes induced by rotenone in the cerebellum of mice brain. It is also observed that supplementation of quercetin and MTQ can attenuate Purkinje neuronal death from oxidative damage in the cerebellum evoked by rotenone. The quercetin targeted to mitochondria was more effective than untargeted quercetin in preventing rotenone-induced oxidative stress and histological damage in the cerebellum. Our data therefore suggest that targeting mitochondria with these synthesized antioxidants may enhance the bioavailability and could be the preferred approach as a novel therapeutic agent for neurodegenerative diseases induced by oxidative stress.


The authors are thankful to the CIL, Dr. Harisingh Gour Vishwavidyalaya (A Central University), Sagar (MP), India, for providing instrumentation.

Financial support and sponsorship

The authors are thanful to UGC, New-Delhi, India, for providing financial support.

Conflicts of interest

The authors declare no conflict of interest.


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