Anti-Parkinson’s evaluation of Brassica juncea leaf extract and underlying mechanism of its phytochemicals

Department of Pharmacology, Faculty of Pharmaceutical Sciences, Government College University, 38000 Faisalabad, Pakistan, Yunnan Herbal Laboratory, College of Ecology and Environmental Sciences, Yunnan University, 650091 Kunming, Yunnan, China, International Joint Research Center for Sustainable Utilization of CordycepsBioresouces in China and South-east Asia, Yunnan University, 650091 Kunming, Yunnan, China, Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Government College University, 38000 Faisalabad, Pakistan, Riphah Institute of Pharmaceutical Sciences, Riphah International University, 54000 Lahore, Pakistan, Department of Botany, Faculty of Biological and Health Sciences, Hazara University, 21120 Mansehra, Pakistan, Department of Biosciences, Faculty of Sciences, COMSATS University Islamabad, 57000 Sahiwal, Pakistan, Department of Chemistry, Federal Urdu University of Arts, Science & Technology, 75300 Karachi, Pakistan, Dr. Muhammad Ajmal Khan Institute of Sustainable Halophyte Utilization, University of Karachi, 75270 Karachi, Pakistan, Department of Pharmaceutical Chemistry, College of Pharmacy, Taif University, 21944 Taif, Saudi Arabia, Department of Clinical Pharmacy, College of Pharmacy, Taif University, 21944 Taif, Saudi Arabia, Department of Physiology and Biochemistry, Faculty of Medicine and Surgery, University of Malta, MSD2080 Msida, Malta, Centre for Molecular Medicine and Biobanking, University of Malta, MSD2080 Msida, Malta, Department of Pharmacology, Bromatology, Toxicology, Faculty of Pharmacy and Biochemistry, Universidad Nacional Mayor de San Marcos, Jr. Puno 1002, 15001 Lima, Peru, E-Health Research Center, Universidad de Ciencias y Humanidades, 15001 Lima, Peru, Department of Biology, Faculty of Applied Science, Umm Al-Qura University, 21961 Makkah, Saudi Arabia


Introduction
Parkinson's disease (PD) is a severe neurologic dysfunction described by compromised motor coordination (postural imbalance, shaking, stiffness, loss of autonomic movements, and bradykinesia) and nonmotor symptoms (coherent defacement, disorganization, melancholy, reduced ability to detect the smell, hypersalivation, chewing and swallowing problems) [1][2][3]. It is considered the second most severe abnormality of the brain after Alzheimer's disease (AD). Patients with PD are reported about 2-3% after 65 years of age. Pathologically, it is a multifaceted disease with ambiguous etiology. Though gene mutation, environmental factors certain toxins (herbicide and pesticides), age and sex impart conclusive contribution in the disease etiology. Men are more prone towards this syndrome than women. It is ascribed to incessant dopaminergic neuronal loss in substantia nigra, lodgment of alphasynuclein protein (Lewy bodies), neuronal inflammation, the damage of mitochondria along with oxidative stress. However, the cause for the deterioration of dopaminergic neurons is not well documented [4][5][6]. Glycation of αsynuclein is the main factor that leads to aggregation and formation of Lewy bodies, thus cause neuronal cell death in PD.
Up to date there is no curative therapy for PD. The available therapeutic options including levodopa, monoamine oxidase B inhibitors (MAO-B), beta-blockers, amantadine, and anticholinergics is not enough to treat the PD and associated organ demages [7,8]. Moreover, there are a lot of adverse effects associated with the available therapies so that putative drugs and alternative costeffective therapies are required with less or no adverse effects.
Several plant species and their secondary metabolites have been identified to possess excellent therapeutic potential against neurodegenerative disorders [9,10]. Experimental and molecular mechanisms of natural PD inhibitors isolated from plants by experimental in vitro and in vivo analyses have been highlighted in several studies [10][11][12]. These plants have been found to exert a diverse range of protective effects that soothe devastating neurodegeneration. Generally, plant species with antioxidant properties have been widely recognized to ameliorate the disease process. For example, Salvia Officinalis possess rosmarinic acid, a potent free antioxidant that has been attributed to protecting the neurons from free radicals associated with cellular degeneration in AD and PD [13]. Certain herb families were found to regulate synaptic neurotransmission such as Ginseng [14,15]. However, there is a long list of isolated bioactive compounds from medicinal plants that have been found to ameliorate neurodegenerative disorders [16][17][18].
Brassica juncea belongs to the family Cruciferae also called Indian mustard, mustard green, yellow mustard, black mustard [19]. The main planting nations are found in Asian countries. It is considered as a principal weed in Canada, Argentina, and Australia. Indian mustard was broadly dispersed also cultivated in subtropics regions and pleasant weather [20]. Previous studies suggested that B. juncea leaves are traditionally used as stimulants, diuretics, and expectorants and also as a food [21,22]. Its seeds lubricant is mostly used as a traditional spice, for arthritis, foot ache, and rheumatism [23,24]. B. juncea is an economically important edible plant [25]. The mustard plant is famous for its important components such as vitamin A, vitamin C, iron, calcium, selenium, chromium, and zinc supplements [26]. B. juncea contains a high proportion of alkenyl glucosinolates (85-96%) with the majority of sinigrin contents [27]. Phenyl isothiocyanate, phenolic compounds, fatty acids, glycosides, flavonoid, and proteins contents are present in this plant [21]. This plant contains phenolic and flavonoid compounds which have antioxidant and free radical scavenging activity [21]. Previous literature has highlighted the importance of B. juncea leaves for various diseases, but the purpose of this study was to examine the therapeutic potential of B. juncea leaves and their secondary metabolites by the experimental evaluation with respect to high performance liquid chromatography (HPLC), physicochemical, quantitative, in vitro and in vivo studies. Moreover, molecular docking simulations assisted to validate the binding interactions of the selected metabolites within the catalytic cleft of enzyme acetyl cholinesterase (AChE ) whose binding site was already determined and studied for drug design and development study. Significant binding energies in terms of Kcal/mol and binding interactions of the functional residues of AChE target active site with atoms of the selected metabolites could lead towards the novel drug development for the treatment of PD, and that might be significant for the treatment of several other brain diseases because AChE is a renowned molecular target for neurodegenerative diseases. In silico pharmacokinetics and toxicity prediction are the prerequisite of drug design studies and could be helpful for the initial screening of drug-like parameters of the selected metabolites, which could highlight major issues of the drug to be chemically improved in future before moving towards the drug development clinical studies. Findings of this experimental and computational analysis will add knowledge, and motivates the neuroscience researcher towards the advanced methodologies in future for the novel PD drug development.

HPLC, physiochemical analysis, and quantitative phytochemical analysis of leaves powder
The physicochemical analysis of B. juncea leaves powder revealed that the loss on drying was 2.60% thus moisture content was found to be within the hygroscopicity range limit. It contained total ash contents of 4.55%, water-soluble ash 1.05%, acid insoluble ash 3.60%, alcohol soluble contents 9.42%, water-soluble contents 1.05%. The alcohol and water contents of the plant revealed an aqueousalcoholic cosolvent with advanced extractive capacity and purity while sulfated ash was found to be 5.1% contents.
Alkaloid contents of B. juncea extract were estimated by the linear regression equation y = 0.0009x (R 2 = 0.9748) which was obtained by drawing a graph between concentration versus absorbance. The extract of B. juncea contained 42.66 µg atropine equivalent/mg of alkaloids. The phenolic contents were determined by the mean of linear regression equation y = 0.0014x (R 2 = 0.9764) which was obtained by drawing a graph between concentration versus absorbance. The extract contained 107 µg gallic acid equivalent/mg of contents. The contents of flavonoids were estimated by the linear regression equation y = 0.0009x (R 2 = 0.9711) which was obtained by drawing a graph between concentration versus absorbance. The plant extract con-tained 4 µg quercetin standard equivalent/mg of contents. In Fig. 1, the standard curves were demonstrated.
Eleven compounds were identified and quantified in the HPLC. The compounds in the increasing order of quantity (mg/g extract) are catechol (25.34

In vitro antioxidant potential
The 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging assay was used to assess the antioxidant potential of plant extract at various concentrations. The percentage radical scavenging activity of the plant extract was increased dose-dependently. The maximum % age scavenging was presented by plant extract at 1000 µg/mL (70%) which was notably varied from ascorbic acid (56%). The IC 50 value of extract and ascorbic acid were calculated using linear regression equation was y = 0.0.0253x (R 2 = 0.9232) and y = 0.0.0338x (R 2 = 0.9272) respectively. The IC 50 value of extract was 137.54 µg/mL while the IC 50 value of ascorbic acid was 159.85 µg/mL.

Behavioral studies
The catalepsy in haloperidol-induced PD rats was determined by employing block, hang and triple horizontal bar tests. The catalepsy timing was significantly (p < 0.001) rose in the DCG as compared to the NCG in the block test (Fig. 3A). The plant extract at 200, 400, and 600 mg/kg dosage had expressed a significant (p < 0.001) reduction in the level of catalepsy on the 7th, 14th, and 21st day of study as compared to the DCG on respective days. Hang test and horizontal bar timing were significantly (p < 0.001) decreased in the DCG in comparison to the NCG (Fig. 3B,C). The diseased rats treated with the plant extract at 200, and 400 mg/kg showed a significant (p < 0.001) rise  in the timing in hang as well as horizontal bar test on the 7th, 14th, and 21st day of study as compared to that of DCG on a respective day. Horizontal bar and hang bar timing was increased in a dose-dependent manner as the dose increased the time to stay in the horizontal bar test was also increased. The SOD, CAT, and GSH levels were significantly decreased with an increase in the MDA levels in the DCG as compared to the NCG. SOD, CAT, and GSH levels were increased significantly in the groups treated with the B. juncea extract. The extract showed maximum effect at 600 mg/kg ( Table 2).

Effect of B. juncea extract treatments on dopamine levels
As shown in Fig. 4, the level of dopamine was significantly (p < 0.001) raised in the animals treated with the B. Juncea extract at different dose levels when compared with the disease control. A dose-dependent increase in the levels of dopamine was observed in the animals treated with B. juncea.

Effect of B. juncea extract treatments on MAO-B levels
As shown in Fig. 5, B. juncea at 600 mg/kg dose level significantly reduced the levels of MAO-B in the brain of the treated animals when compared with the disease control. However, other doses also showed a decrease but it was non-significant when compared with disease control.

Histopathological examination of brain tissue
The histopathological study of brain tissue of DCG revealed an increase in vacuolated cytoplasm (i.e., lipid peroxidation) and depletion of dopaminergic neurons. The NCG brain slide showed no vacuolated cytoplasm and as well as no depletion in dopaminergic neurons. The standard group revealed significant recovery from cytoplasmic vacuolation and dopaminergic neurons (Fig. 6C). However, the extract 200 mg/kg treated rat's brain showed moderate recovery from vacuolation and dopaminergic neurons while extract-treated groups at 400 and 600 mg/kg exhib- The values are expressed as mean ± SEM, (n = 6). ###p < 0.001 vs normal control. ***p < 0.001 vs disease control group. ited better recovery from haloperidol-induced brain damage as shown in Fig. 6.

Molecular docking analysis
Molecular Operating Environment (MOE) software package 2016.10 was used for molecular docking of AchE protein with selected 12 phytochemicals. Molecular docking analysis explains the functional residues involved in the inhibitory activity of AChE protein to design a potential drug against PD by using the dataset of 12 phytochemicals (Table 3).
Molecular docking interpretations show that the binding energies of best-bounded confirmation of the selected 12 phytochemicals were in the range of -7.8793 to -4.4390 Kcal/mol. As the molecule that has presented the lowest binding energy was considered the most significant AChE inhibitor. So that there are four ligands (sinapic acid, rutin, ferulic acid, and caffeic acid), that were considered to be the best AChE inhibitors based on their bestbounded confirmation, lowest binding affinities, and excellent binding interactions within the catalytic pocket of enzyme AChE (PDB ID: 4EY7) (Fig. 7). Other 7 phytochemicals have also presented significant interactions in terms of Vader Waals or hydrophobic interactions, hydrogen bonds, Pi-lone pair, Pi-Pi T-shaped, and Pi-alkyl interactions, but their binding energies were not up to the significant threshold value (Table 4).
For the entry into AChE, target protein's through the active binding site, there is a narrow cleft linked with the pattern of aromatic amino acid residues, hence allow the substrate to enter into the catalytic triad of HIS, SER, and GLU amino acid residues. It is known as the hydrophobic amino acid residues are linked with the aromatic amino acids supervision that revealed the highest degree of flexibility and permits the ligands of significant size. Though, the opening and closing posture of the active site is associated with a fluctuating cleft, e.g., ligand atoms interacting with TYR337 residue [28]. The conformational investigation exposed that sinapic acid, ferulic acid, and caffeic acid, penetrated the active site cleft, and interrupted the aromatic amino acids, and inhibit the substrate-binding (Fig. 7). Moreover, these ligands accept the challenge of blocking the fluctuating cleft and overturned near the catalytic triad to inhibit the target protein activity [29].
The docking results of sinapic acid, ferulic acid, and caffeic acid are excellent in terms of binding interactions with functional residues and their docked complex binding energies are above 7 Kcal/mol and shown the strongest efficacy towards the AChE target protein among the selected 12 phytochemicals. Moreover, the other contents of Brassica juncea extracts, caffeic acid, and sinigrin has exhibited binding energies of less than 7 Kcal/mol which is less than the good threshold values, and shown moderate protein-ligand interaction results and energy values in the range of 6 to 7 Kcal/mol. The other six phyto-chemicals; Pyrogallol, gallic acid, catechol, catechin, hydroxybenzoic acid, chlorogenic acid, coumaric acid, and rutin have presented binding energies less than 6 Kcal/mol that is less than the threshold values. It means the docking results show less potential of these six compounds towards inhibition of AChE target protein.

Pharmacokinetics and toxicity risk assessment
Calculation of in silico pharmacokinetic parameters is significant to narrow down the large dataset to highly significant drugs with the minimum risk of rejection in the early drug development procedures [29]. Four bestscored phytochemicals were subjected for the estimation of in silico pharmacokinetic properties by Swiss ADME and toxicity estimations by Osiris molecular property explorer. Hence, three phytochemicals, sinapic acid, ferulic acid, and caffeic acid showed the maximum results in the acceptable range shown in Tables 4 and 5.
The drug or compound we selected should be metabolized by the body or not, it is required to be understood because the only pharmacokinetics properties won't be enough to understand the membrane permeability of selected phytochemicals. After all, the high logP and molecular weights of the compounds from natural sources could not accept the drug-likeness parameters, although in several studies in silico drug-like calculations have been performed [30] while in this study three phytochemicals have shown that they have accepted the rules of drug-likeness, and only one compound rutin has violated the rule due to its large chemical compound size.
Additionally, cytochromes P450 were calculated to support the pharmacokinetics of the selected phyto-chemicals, it is reported that the essential isoforms, e.g., CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4 are important for drugs activity within the body. The mechanism of isoforms of the cytochrome P450 superfamily is becoming a challenge for the natural chemicals concerning the metabolism and excretion of drugs through the liver [31]. Chemicals that could inhibit the cytochromes P450 assist the interactions of selected natural compounds and are attributable to drug administration that may not be able to encompass the metabolism-related activity and accumulate as toxic substantials inside the body.
Lipophilicity (LogP) values are also calculated for the four putative phytochemicals because it is important to explain the bioavailability of the drug, some minor violations were observed in three selected phytochemicals other than rutin, these drug-like parameters assist the oral administration of chemicals, such as poor lipophilic drugs corresponding to poor gastrointestinal absorption and owns the lowest BBB permeability, so it's critical to understand the protein-ligand interactions intricated in the disease of the central nervous system [32]. The water solubility (LogS) of the phytochemical was calculated to describe the absorption and distribution, all four chemicals have presented very good solubility and all these properties were explained well in Tables 4 and 5.
The undesired or unnecessary structural issues known as Pan assay interference compounds (PAINS) and Brenk alerts were also studied in the four selected phytochemicals, it is thought to be eliminated before moving towards the early drug development procedures [28]. All four phytochemicals have displayed PAINS and Brenk alerts, which could be a preliminary hypothesis and could be helpful for the improvement of structural issues. Osiris molecular property explorer demonstrated that four phytochemicals results are positive, there are no harmful effects on four major grounds of tumorigenic, mutagenic, irritant, and reproductive properties, Moreover, drug-likeness and drug scores are important to understand the ADMET profile of a chemical, that is also very good and acceptable result are shown in Table 5 and Fig. 8. Hence, it is demonstrated that the ADMET profile of the selected phytochemicals along with the protein-ligand interaction results is most acceptable, so our proposed chemical compounds could be potential inhibitors of the AChE protein target and treat the PD well [31].

Discussion
PD is a chronic brain disorder. It is slowly progressive and characterized by tremors, rigidity, stiffness, and difficulty in walking [6]. Non-motor symptoms are usually associated with cognitive decline and behavioral abnormalities and worsen in the advanced stages of this disease [33]. The motor symptoms of PD are mainly induced by dopaminergic neuronal apoptosis in substantia     nigra pars compacta and brought about dopamine deficiency [34]. Until now, no cure for this disease is available. Although, the conventional treatment with levodopa, monoamine oxidase inhibitors, and dopamine agonists are effective only in the early stages. These therapeutic agents are ineffective and associated with severe side effects in advanced stages such as involuntary muscular movements [35]. Oxidative stress, mitochondrial dysfunction, neuronal apoptosis, neuronal excitotoxicity, and accumulation of misfolded proteins like alpha-synuclein are major pathological hallmarks. Due to the existence of a higher amount of dopamine and its metabolites, the substantia nigra of the middle brain is more vulnerable to oxidative stress [36].
Most of the medicinal plants enriched with alkaloids, flavonoids, and polyphenols have been reported with neuroprotective and antioxidant potential against neurodegenerative disorders [37]. In the current study, HPLC analysis of B. juncea revealed the existence of a favorable amount of polyphenolic antioxidant compounds. Catechol and hydroxyl benzoic acid existed in higher concentrations (mg/g of plant extract) compared to other compounds in ethanol extract of B. juncea. Congruent to previous study catechol polyphenolic compounds and lipids compounds are associated with neuroprotective, anti-inflammatory, and antioxidant potential in oxidative stress-induced neurotoxicity in rat brain and modulated dopamine neurotransmission [38,39]. Hydroxy benzoic acid in B. juncea is associated with antioxidant and neuroprotective effects in the PD model similar to reported literature in which this compound remarkably mitigated the pathological hallmarks of neurodegeneration through modulation of antioxidant and anti-neuro-inflammatory signaling pathways [40,41]. A favorable amount of antioxidant rutin is detected in ethanol extract of B. juncea, which is associated with therapeutic potential to reverse the neurodegeneration via modulation of brain-derived neurotrophic factor, nerve growth factor, and through inhibition of neuronal apoptosis in rat brain in previous studies [42]. In the repeated cerebral ischemic rat model of impaired spatial memory, rutin ameliorated neuronal death and spatial memory impairment [42,43]. Pyrogallol [44,45], gallic acid [46][47][48], catechin [49], chlorogenic acid [50], caffeic acid [51], sinapic acid [52], coumaric acid [53], and ferulic acid [54] found in ethanol extract of B. juncea are responsible for neuroprotective effect against haloperidol-induced neurotoxicity in corroboration to previously reported literature through modulation of Nrf2/OH-1 -1 pathway and other antioxidant signaling pathways, neuronal cell death, and mitochondrial dysfunctions. In this study detection of antioxidant compounds through Hplc analysis is supported by DPPH scavenging assay [55]. B. juncea as a source of antioxidants exhibited free radical scavenging/inhibiting potential, therefore could be effective in the treatment of numerous oxidative stress associated with human ailments [37].
The neuroprotective role of B. juncea was evaluated in haloperidol-induced PD in the rat. Haloperidolinduced catalepsy is widely accepted in an animal model of PD through the non-selective blockade of D 2 receptors leading to dopamine exhaustion at nerve endings. So that haloperidol (1 mg/kg) induced catalepsy was remarkably recovered by B. juncea ethanol extract in a dose-dependent manner demonstrating the neuroprotective role of plant extract through modulation of dopamine neurotransmission [56].
Hang test revealed that B. juncea extracts improved neuromuscular coordination and muscular strength impaired by haloperidol injection and showed a correlation with the findings of our previous research published in 2020 [36]. While the results concluded from this study have presented a similar horizontal bar test as mentioned in our other published research work [57] indicated that administration of B. juncea extract mitigated the haloperidol-induced motor dysfunctions and recovered the locomotion abnormalities.
Haloperidol-induced reversible blockade of D 2 receptors results in the generation of free radicals. Free radicals perpetuated mitochondrial dysfunctions and neuronal death. Oxidative stress was measured in this study through the estimation of catalase activity, superoxide dismutase, malondialdehyde, and reduced glutathione level in brain homogenates [58].
Haloperidol-induced neurotoxicity through the production of free radicals and hydrogen peroxide due to its inhibitory potential on mitochondrial complex I and IV in the electron transport chain. These free radicals induced lipid peroxidation, protein denaturation in brain tissues of PD patients [59]. Lipid peroxidation was assessed through estimation of the thiobarbituric acid level in brain homogenate as a sensitive biomarker of oxidative stress [60]. Unsaturated fatty acids and arachidonic acids are vulnerable to radical attack and produce lipid peroxyl radicals, perpetuating further initiation of free radicals chain reaction and attack on the double bond of unsaturated fatty acids [61]. Lipid peroxidation induced impaired structural and functional integrity of biological membrane leading to deactivation of membrane-bound enzymes [62]. The raised level of lipid peroxidation and products are found in substantia nigra PD brain tissues. Lipid peroxidation marker, Malnodialdehyde level was increased in brain tissues after haloperidol treatment compared to control similar to previous findings. B. juncea extract ameliorated the raised level of Malnodialdehyde and decreased lipid peroxidation due to its antioxidant potential. Hydrogen peroxide produced in brain tissues is neutralized by catalase into the water and molecular oxygen and inhibited the accumulation of precursors for the biosynthesis of free radicals. The decreased level of catalase is reported in PD brain tissues congruent to the current study. B. juncea extract recovered the reduced level of catalases in brain tissues similar to previous find-ings [35]. The dismutation of superoxide reaction is catalyzed by superoxide dismutase as a first-line antioxidant defence system in most of the cells. Haloperidol decreased the level of superoxide dismutase in this study parallel to our previous findings. However, B. juncea extracts improved the decreased level of superoxide dismutase. The dopaminergic neuronal loss resulted due to decreased level of reduced glutathione level in the PD brain [57].
Reduced levels of reduced glutathione impaired the neuron to detoxify hydrogen peroxide and raised oxidative stress. It was evident from the present study that haloperidol declined the level of reduced glutathione similarly as reported earlier [57]. Though, B. juncea extract decreased the oxidative stress by retrieving the level of reduced glutathione in treatment groups. Therefore, it is evident from biochemical analysis that B. juncea extract has antioxidant potential, and findings are also supported by histopathological analysis. In the current study histopathological findings showed a protective effect of B. juncea extract on neuronal necrosis, neuronal density, intracellular spaces, and neutrophil infiltration in the striatum region of the brain [58]. Therefore, it is concluded from the present study that B. juncea extract has neuroprotective potential against neurotoxicity caused by haloperidol through modulation of behavioral and biochemical parameters and will be considered as a lead compound in the future neurotherapeutic to treat cureless neurodegeneration.
All 12 phytochemicals extracted from B. Juncea have also presented significant interactions in terms of Van der Waals' or hydrophobic interactions, Pi-alkyl, hydrogen bonds, Pi-lone pair, Pi-Pi T-shaped interactions within the native active binding site of the target protein (Table 4). Computational analysis of the proposed results of the extracted compounds has explained that four phytochemicals have presented very good results in terms of protein-ligand binding interactions as well as ADMET profile estimations (Figs. 7 and 8). Following isolated selected compounds evaluated in the integrated experimental and computational study have shown antioxidant potential and it is also highlighted in previous literature, hence providing support to our concluded results [17,63,64]. Sinapic acid, ferulic acid, and caffeic acid are excellent AChE inhibitors with binding energies above 7 Kcal/mol. While caffeic acid and sinigrin have exhibited moderate AChE inhibitory potential with binding energies in the range of 6 to 7 Kcal/mol. The other six phytochemicals; Pyrogallol, gallic acid, catechol, catechin, hydroxybenzoic acid, chlorogenic acid, coumaric acid, and rutin are weak AChE inhibitors. Tables 4 and 5 enlisted ADMET properties of the four putative phytochemicals, and seems are very much acceptable, only rutin have presented more violations because of its large chemical size, moreover, Fig. 8 shows that all four phytochemicals druglikeness and drug scores are in the acceptable range, and sinapic acid is showing the remarkable ADMET properties as well as drug-interactions within the active binding site of AChE target protein. Sinapic acid has been evaluated for brain diseases previously and has shown remarkable results [65,66]. In vitro and in vivo studies also highlighted the efficacy of the natural components of the B. juncea leaves for PD and neuroprotective disorders.

Plant collection and identification
The B. juncea leaves were collected in the winter season from Toba Tek Singh district, Pakistan. The herbarium of B. juncea plant was prepared and identified from the Department of Botany, Government College University Faisalabad by Taxonomist Dr. Qasim Ali under authentication number Z245-BOT-19.

Physiochemical analysis and quantitative phytochemical analysis
The physicochemical analysis of B. juncea leaves powder including loss on drying, total ash, acid-insoluble, water-insoluble and sulfated ash contents, alcohol and water-soluble contents were performed by following United States Pharmacopoeia-National Formulary (2003) methods [67]. Das et al. [68] method was adopted for the quantification of alkaloids and Saleem et al. [69] methods were followed for the flavonoids and phenolic contents determination.

Loss on drying
Two-gram of plant powdered was dried in the oven for 30 min at 105 • C by putting it in the tarred china dish. After cooling, the dried plant was weighed. Moisture content was calculated as a loss on drying by dividing the dried weight with actual powdered plant weight.

Total ash
2 gram of plant powder was incinerated in the electric furnace at 675 ± 25 • C until it got free from carbon. This carbon-free ash was cooled in the desiccator and weighed. Total ash content was calculated by dividing the ash weight with the initial powdered weight.

Water-insoluble ash
Total ash obtained in the previous ash test was boiled with 25 mL of distilled water for five minutes and then filtered through ashless filter paper. The filtrate contained the soluble material of the plant and residue on the filter paper was an insoluble matter that was washed with warm distilled water to extract any soluble material in the residue. Then filter paper was dried and ignited in the china dish (that was pre-tarred) till the carbon-free ash was obtained. After cooling this ash, it was weighed and waterinsoluble ash was calculated by dividing this ash weight with the initial total ash weight.

Acid-insoluble ash
Total ash obtained in the previous ash test was boiled with 25 mL of HCl for five minutes and then filtered through ashless filter paper. The filtrate contained the soluble material of the plant and residue on the filter paper was an insoluble matter that was washed with warm distilled water to extract any soluble material in the residue. Then filter paper was dried and ignited in the china dish (that was pretarred) till the carbon-free ash was obtained. After cooling this ash, it was weighed and acid-insoluble ash was calculated by dividing this ash weight with the initial total ash weight.

Sulfated ash
Two grams of plant powder were put in the china dish and sulphuric acid was added to it to make a pastelike material. This china dish was put on the burner, white fumes started to originate and ignition was done till the white fumes ended to arise. Then it was cooled and ash content was weighed.

Alcohol and water-soluble extractives
In the tarred flask, 5 g of powdered plant material was placed and macerated with 100 mL of 95% ethanol (in case of alcohol-soluble extractive) and 100 mL distilled water in case of water-soluble extractive for 24 h. Then extract was filtered and in the china dish, 25 mL of filtrate was evaporated till residue remained at the bottom. This residue was further dried in the oven at 105 • C and weighed. The percentage of the extractives was calculated regarding the weight of the powdered material used in the test.

Quantification of alkaloid content
One gram extract was mixed with dimethyl sulfoxide in the test tube and 1 mL of 2 N HCl was added to this test tube. The solution was filtered using Whatman no. 1 filter paper and 5 mL bromocresol green solution along with 5 mL phosphate buffer was added to it. The mixture was shaken thoroughly. After that, trichloromethane was added at different quantities as 1-, 2-, 3-, and 4 mL. Then the mixture was added to a 10 mL volumetric flask and dilution was made with trichloroacetic acid. Standard atropine solution at different concentrations 10-, 20-, 30-, 40-, 50-and 60 µg/mL were made similarly to that of extract (test sample). The absorbance of a test sample and reference sample was measured by an ultraviolet spectrophotometer at 470 nanometers. A blank sample was used for error correction. The presence of total alkaloids in the extract was measured as milligrams of atropine equivalent (AEq/g) of extract.

Quantification of phenolic content
The phenolic content of the extract was calculated by using Folin-Ciocalten's reagent method. In this method, 1 mL of sample and 1 mL of standard solution were mixed in a test tube then phenolic reagent 0.2 mL was added to it and mixed thoroughly. After 4 min, 1 mL of a sodium carbonate solution was added and stored in the darkness. Various concentrations (10-, 20-, 30-, 40-, 50-and 60 µg/mL) of the reference sample were prepared similarly to that of the test sample (extract) to make a standard curve. The absorbance of the test sample and reference sample was recorded at 750 nm. The percentage of total phenolic content was calculated using the following equation: Here, T = total phenolic content (mg/g), C = gallic acid equivalent (µg/mL), V = volume of extract (mL), M = weight of the extract (g).

Quantification of flavonoid content
The total flavonoid content of the extract was calculated by using the AlCl 3 colorimetric method. Extract 0.3 mL was added to the test tube. Then 3.4 mL of methanol 30%, 0.15 mL of 0.5 M sodium nitrite, and 0.15 mL of aluminum trichloride hexahydrate (0.3 M) were added to the test tube having a sample in it and after few minutes, the 1 mL solution of sodium hydroxide (1 M) was added. Quercetin was used as a reference sample. Reference solution was made similarly to that of sample extract. The standard curve was constructed by drawing the graph between various concentrations (10-, 20-, 30-, 40-, 50-and 60 µg/mL) of quercetin vs respective absorbance. Blank was prepared identically to that of the sample but it was without extract/reference sample. The mixture was shaken well and absorbance was measured against the blank at 506 nm. Total flavonoid content was determined as mg of quercetin equivalents/g of ethanol extract.

High-performance liquid chromatography analysis
The methanolic Plant extract was filtered through a 0.2 µm membrane filter (Millex-HV) and injected into HPLC. The HPLC system (Shimadzu LC-20AT) was used. Mobile phase composed of (A) 0.1% acetic acid and (B) methanol and the composition gradient was: 25% (B) for 10 min; 40% (B) for 15 min; then 50%, 60%, 80% and 100% (B) for every 10 min. The flow rate was 0.8 mL/min. Phenolic moieties were acknowledged with the spectra of standard compounds at 280 nm [70].

Extract preparation by microwave-assisted extraction method
B. juncea leaves were washed thoroughly with tap water and the leaves were dried in a hot air oven at 40 • C temperature. Then, the leaves were crumbled to fine powder by utilizing an electronic blender. The powder was passed over a sieve and deposited in an airtight container. The 100 g powder of B. juncea was weighed and taken in each of 3 beakers that contain 750 mL ethanol each. All three beakers were placed in a microwave oven already adjusted at 9000 watt, and then the oven heated up for 2 min, stopped the oven, and opened it for 30 sec. This specific method was repeated 5 times. In the end, the supernatant was filtered through filter paper. In the 2nd cycle again 500 mL ethanol was added in each beaker containing residue. Again all the beakers were placed in a microwave oven. The oven was heated up for 2 min and then stopped for 30 sec. The same method was repeated 5 times. The supernatant solution was filtered through filter paper. In the 3rd cycle again 500 mL ethanol was added to each beaker and repeated the same method 5 time. Filtrates were pooled in the same reservoir. Excess solvent was removed from the filtrate/extract using a rotary evaporator at 40 • C and semisolid ethanolic leaves extract (ELE) was obtained. Lipids were removed by washing the extract with n-hexane. It was stored in a refrigerator at 4 • C till further use.

Determination of antioxidant potential
The in vitro antioxidant properties of B. juncea ethanol extract were determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay by the earlier procedure [68]. The extract solution at different concentrations (25-, 50-, 100-, 150-and 200 µg/mL) was prepared in methanol by two-fold dilution method while ascorbic acid solution (1-100 µg/mL) that was prepared in methanol, used as standard. For the test, 500 µL DPPH solution (0.1 mM) was added in 250 µL of extract solution and 250 µL of methanol. The solution was allowed to stand at 25 • C for half an hour in a dark place and absorbance was measured at 517 nm using an ultraviolet-visible (UV-Vis) spectrophotometer. The sample was kept for. The percentage for DPPH-radical scavenging activity of the extract was determined by the following formula: Percentage Inhibition (%) = Blank absorbance − Sample absorbance Blank absorbance * 100 Extract IC 50 was measured using linear regression equation y = 0.253x (R 2 = 0.9232) by drawing graph between absorbance verses concentration.

Experimental animals
Wistar albino rats weighing 180-200 g were purchased from the Animal House of the University of Agriculture, Faisalabad. They were kept in an animal house (under standard laboratory conditions; room temperature: 25 • C, 12 h light and dark cycle, humidity 60%) of Govt. College University, Faisalabad for one week before the start of the experiment to acclimatize the animals. They had free access to food and water.

Ethical approval for animal studies
The in vivo study was executed after getting ethical approval from Institutional Review Board (Reference No.

Study design
Thirty-six healthy rats of both sexes were divided into 6 groups (n = 6). Group I was normal to control (NCG), to whom the vehicle was given orally. Group II served as disease control (DCG), receiving haloperidol 1 mg/kg intraperitoneally [71,72]. Group III was the standard group, received L-Dopa 100 mg/kg + carbidopa 25 mg/kg orally. Groups IV-VI treatment groups received extract at 200, 400, and 600 mg/kg dose levels respectively via the oral route. All the groups were given their relevant treatments for 21 days using a gavage needle.
Behavioral evaluation of the NCG was started from day one whereas in other groups behavioral observations were started from the eighteenth day of the study to evaluate the anti-PD effect of the extract.
On the 22nd day, all the animals were humanely killed by cervical dislocation under mild chloroform anesthesia. Brains were excised from all the animals for biochemical and histopathological analysis to examine the anti-PD effect of the plant extract. For the preparation of tissue homogenate (10% w/v) and biochemical assays brain was isolated and the homogenate was prepared with phosphate buffer.

Behavioral studies
To observe the effect of plant extract on motor functions of rats with haloperidol-induced Parkinsonism, various types of behavioral tests were employed.

Catalepsy test
The catalepsy test was performed to evaluate the muscle's rigidity. The bar has 1 cm diameter and 3 cm highest from the floor was used in the experiment. Rat's forelimbs were placed on the bar and time was recorded in which rats step down from the bar. The time at which the animals fall was considered as cataleptic time (sec). This test was carried out on the 7th, 14th and 21st day of the study [73,74].

Hang test
In the grid hang test neuromuscular strength was determined. This test was carried out on the 7th, 14th, and 21st days during the study. Rats were picked up from their tails and steadily put down on a straight gauze and hold up until they snatched the gauze with their front and hind paws. The gauze was then upturned so that the rats were allowed to hang upside down. The gauze was ascended 20 cm overhead a solid surface, to depress the falling but not become the cause of any injury if the rat falls. The reading was taken on a stopwatch after 30 sec when the rats fell [74].

Horizontal bar test
Muscle coordination was checked in this test. The apparatus comprised of a bar (brass material) 38 cm in length that was held with wooden support up to 49 cm high from the surface. Bars of three diameters (2, 4, and 6 mm) were used to assess coordination. This test was carried out on the 7th, 14th, and 21st days. Rats were held by their tails and put down on the bench in front of the apparatus. They were slipped quickly backward at least 20 cm when rats gripped the bar with their forepaw, their tails were left. Time was noted with the stopwatch to record how long a rat takes to fall on the ground. The utmost break-off time was 30 seconds [75].

Biochemical analysis of brain
Brains tissue homogenate (10% w/v) was prepared in 0.1 M (Ph 7.4) phosphate buffer using automated tissue homogenizer for estimation of SOD, catalase, lipid peroxidation, and GSH to sort out the anti-PD effect of the extract on these organs.
The total protein contents were estimated by adopting the previously followed method. Briefly, the absorbance of the reaction mixture was taken at 660 nm using a UV-Vis spectrophotometer. Standard curve of bovine serum was used for the expression analysis of the protein levels were expressed in mg/mL [5]. 5.9.2.1 Estimation of Superoxide dismutase (SOD) activity 0.1 M potassium phosphate buffer (pH 7.4) (2.8 mL) and 0.1 mL tissue homogenate and pyrogallol solution (500 mg pyrogallol in 2 mL water + 12 g potassium hydroxide in 8 mL water) were mixed in a test tube. Absorbance was taken at 325 nm [5].

Estimation of catalase (CAT) Levels
For CAT activity, the reaction mixture was contained 0.05 mL of sample, 1.95 mL of phosphate buffer (50 Mm; pH 7), and 1mL of H 2 O 2 (30 mM) solution. The absorbance was measured at 240 nm [76]. Micromoles of H 2 O 2 per min per mg was used as an expression of CAT levels. The cat activity was calculated by using the following formula: CAT activity = OD/E × vol of sample × mg of protein

Estimation of Malondialdehyde (MDA) level
For determining lipid peroxidation in tissue homogenate of treated animals, malondialdehyde level was estimated which is an indirect method by following earlier procedure. In this test, 1 mL of the sample and 3 mL thiobarbituric acid (TBA; 0.38% w/w) was taken in a test tube, mixed properly followed by holding on an ice bath for 15 min. Afterward, centrifugation at 3000 rpm was done for 10 min. The absorbance of the upper layer was taken at 532 nm. The concentration of MDA was calculated by using the following formula.

Estimation of reduced glutathione (GSH) level
In this assay, 1 mL of tissue homogenate (10% w/v) was precipitated with 1 mL of 10% trichloroacetic acid (TCA). The aliquot of the supernatant was added with 4 mL of phosphate solution and 0.5 mL of DTNB (5,5-dithiobis-2-nitrobenzoic acid) reagent. The sulfhydryl group of glutathione reacts with DTNB and produces a yellow-colored 5-thio-2-nitrobenzoic acid (TNB). The absorbance of the solution was measured at 412 nm. GSH is determined by using the following formula.

Estimation of dopamine levels
For the estimation of dopamine, HCI-butanol was used for the preparation of brain tissue homogenate. The reaction mixture was then centrifuged at 2000 rpm for 10 min. In 1 mL of supernatant, 2.5 mL heptane and 0.3 mL (0.1M HCI) were added and centrifuged at 2000 rpm for 10 min. Remove the aqueous layer and in a 0.2 mL of layer, 0.05 mL (0.4 M HCI) and 0.1 mL of EDTA were added. For the start of oxidation, 0.1 mL of iodine solution was added. Sodium sulphite (0.1 mL) and 0.1 mL of acetic acid were added to the reaction mixture to stop the oxidation reaction. The reaction mixture was heated at 100 • C for 6 min. The absorbance of the reaction mixture was measured at 350 nm. The regression line of dopamine was used for the estimation of dopamine levels in brain tissue [78].

Estimation of MAO-B levels
In a 250 µL of serotonin and buffer, brain tissue homogenate (250 µL) was added and incubated the mixture at 37 • C for 20 min. 1M HCI (200 µL) was added to arrest the reaction then 5 mL cyclohexane was added and the organic phase was removed and absorbance was taken out at 242 nm. In a blank 1 M HCI was added before the serotonin and buffer [33]. Levels of MAO-B were calculated by using the following formula: X = Sample (abs) − Blank(abs)× Conc.of Standard ÷ Standard (abs) − Blank (abs)

Histopathology analysis
The isolated brains were preserved in 10% formalin solution and the slides were prepared for microscopic evaluation. Digital images were captured using an optical microscope under the polarized light.

Molecular docking analysis
Molecular docking assists to understand and visualize the binding interactions of extracted phytochemicals with the enzyme acetylcholine-esterase, which is one of the remarkable drug targets for PD [79]. For the analysis of protein-ligand interactions, preliminary requirements were the three-dimensional structure of protein and ligand dataset, X-rays crystallographic structure of AChE protein with PDB ID: 4EY7 with the resolution of 2.35 Å was used for molecular docking interpretations [80]. in three-dimensional conformations were downloaded from PubChem database [35]. Molecular Operating Environment (MOE) software package (version 2016.10 Chemical Chemical Computing Group Montreal, Quebec, Canada) was used for molecular docking of AchE protein with selected 12 phytochemicals. The chemical structures were directed to the system to prepare and optimize the files before the docking procedure. The integral issues were found in the co-crystallized macromolecule and corrected, missing residues were added, the irrelevant molecular moieties such as water elements, co-factor, ions were removed. Polar hydrogen atoms were added. The protonate function was applied for the optimization. By using the Dock module of MOE, molecular docking was performed, the native binding site was identified from the co-crystallized protein structure. The energy minimization was done by the Amber: 10EHT force field. The triangular matcher placement method was selected with the London dG scoring function. The initially docked poses were refined by the induced-fit docking protocols and selected the GBVI/WSA dG scoring function. Docked complex with the lowest binding energies/highest binding affinities was subjected for interaction analysis.

Pharmacokinetics and toxicity risk assessment
In silico pharmacokinetic parameters were evaluated by the SwissADME web tool (http://www.swissadme. ch/), major properties calculated were medicinal chemistryrelated parameters such as pharmacokinetics, lipophilicity, water-solubility, drug-likeness, identification of undesirable structural moieties, and toxicity alerts of the selected phytochemicals. Osiris property explorer (https://www.or ganic-chemistry.org/prog/peo/) was used to estimate the toxicity on the four major grounds such as tumorigenic, irritant, mutagenic, and reproductive properties. It also calculated the drug-likeness and drug score, which explains the ADMET (absorption, distribution, metabolism, elimination, and toxicity) profile of the extracted phytochemicals as explained in our previous study [32].

Statistical analysis
Results were expressed as the mean ± S.E.M. the data was analyzed by the application of two-way ANOVA followed by the Bonferroni test by the GraphPad prism (version 5.0, GraphPad Software, San Diego, California USA), the value of p < 0.05 was set as the statistical significant parameter value.

Conclusions
PD is the most frequent neurodegenerative disease in an older population. Plants have been the backbone of the development of treatment options for acute to chronic diseases. In this study, we have studied and established the therapeutic potential of B. juncea in PD. Molecular docking analysis explains the protein-ligand interactions of the selected 12 phytochemicals and concluded with the four best results of sinapic acid, rutin, ferulic acid, and caffeic acid within the active binding site of AChE target protein, and ADMET profile estimations also supported the selection of potential compounds. B. juncea improved motor functions and enhanced the antioxidant enzymes in brain tissues. B. juncea at 600 mg/kg dose level highly significantly improved the locomotion and neurotransmitters levels. B. juncea at the same dose significantly reduced the MAO-B levels in the brain that means the breakdown of dopamine is inhibited significantly. Hence, it is estimated that B. Juncea and its extracted metabolites could be efficacious for the treatment of PD and further development in this regard may provide us with effective and potential treatment.

Ethics approval and consent to participate
The experimental protocol involving the caring, handling, and treatment of rats for the experiment was approved by the Government College University Faisalabad, Pakistan Institutional Review Board (GCUF/ERC/2035).

Acknowledgment
The authors would like to extend their sincere appreciation to Taif University Researchers Supporting Project number (TURSP-2020/309), Taif University, Taif, Saudi Arabia.

Conflict of interest
The authors declare no conflict of interest.