Aqueous leaf extract of Phyllanthus amarus protects against oxidative stress and mis�ring of dopaminergic neurons in Paraquat-induced Parkinson’s disease-like model of adult Wistar rats

Background of the study: Phyllanthus amarus has high nutritional value and is bene�cial in managing and treating diverse ailments. This study assessed the role of aqueous leaf extract of Phyllanthus amarus on Paraquat (PQ) induced neurotoxicity in the substantia nigra of Wistar rats. Materials and methods: The role of aqueous leaves extract of Phyllanthus amarus was assessed using an open �eld test (OFT) for motor activity, oxidative stress biomarkers [Catalase (CAT), and Superoxide Dismutase (SOD)], histological examination (H and E stained) for cytoarchitectural changes and immunohistochemical studies using tyrosine hydroxylase (TH) as a marker for dopaminergic neurons. Forty-two (42) rats were categorized into six groups (n = 7); group 1: control was administered 0.5 ml/kg distilled water, group 2: received 10 mg/kg PQ + 10 mg/kg L ‐ dopa as reference drug, group 3; received 10 mg/kg PQ, while group 4: received 10 mg/kg PQ + 200 mg/kg P. amarus, group 5: received 10 mg/kg PQ + 300 mg/kg P. amarus, and group 6: received 10 mg/kg PQ + 400 mg/kg P. amarus respectively, for 14 days. All administrations were done orally; a signi�cant difference was set at p<0.05. Results and discussion: The study's open �eld test (OFT) revealed no motor activity de�cit with Paraquat (PQ) exposure. Also, cytoarchitectural distortions were not observed with Paraquat (PQ) only treatment group compared to the control and other groups pretreated with P. amarus and L-dopa. Moreover, the Paraquat (PQ) only treatment group showed oxidative stress by signi�cantly decreasing the antioxidant enzyme (SOD) compared to the control and L-dopa pretreated group. A signi�cant decrease in tyrosine hydroxylase (TH) expressing dopaminergic neurons was also observed in Paraquat (PQ) only treatment. However, P. amarus treatment showed therapeutic properties by signi�cantly increasing tyrosine hydroxylase (TH) expressing dopaminergic neuron levels relative to control. Conclusion:Aqueous leaf extract of Phyllanthus amarus possesses therapeutic properties against Paraquat (PQ) induced changes in the substantia nigra of Wistar rats.


Introduction
Neurotoxicity is any reversible or irreversible adverse effect on the nervous system's structure, function, or chemistry during development or maturity, produced by physical or chemical causes.(Zahra et al., 2020).
The main mechanisms for neurotoxicity involve the excessive production of reactive oxygen species leading to oxidative stress, the release of cytokines causing neuroin ammation, and dysregulations of apoptosis leading to neuronal death (Teleanu et al., 2018).Parkinson's disease (PD) is a good example of a neurodegenerative disease that is associated with a group of motor disorders in the elderly population (Vaccari et al., 2019) and has affected millions around the world from 1990 to 2015.The neuropathological features of Parkinson's disease (PD) are the loss of dopaminergic neurons in vulnerable brain regions, especially in the substantia nigra (SN) (Liddell and White, 2018; Rai et al., 2019).
Further, the substantia nigra (SN) is a midbrain dopaminergic nucleus critical in modulating motor movement and reward functions as part of the basal ganglia circuitry (Sonne et al., 2019).The substantia nigra (SN) is a dopaminergic nucleus located in the midbrain.It is critical in modulating motor movement and reward functions as part of the basal nucleus circuitry (Sonne et al., 2019).
In the recent past, the awareness of the serious consequences related to the use of herbicides discovered during the second world war on humans has increased among developing nations in Africa and Asia.A very good example of such herbicide is paraquat.Paraquat (PQ) is considered to be among the main herbicide involved in intentional and accidental poisoning despite its use as a weed control.Examples of illness caused by paraquat PQ causes toxicity in vital regions of the brain, including substantial nigra, which plays a critical role in motor coordination, supporting the idea that exposure to this herbicide may contribute to the pathophysiology of neurodegenerative diseases like PD Paraquat (PQ), as a kind of pesticide in agriculture, is a widely used herbicide that was identi ed as a neurotoxicant and is linked to Phyllanthus amarus contains such principal constituents as phyllanthin, hypophyllanthin, corilagin, geraniin, amariin, repandusinic acid, phyllanthusiin D, rutin and quercetin 3-O-glucoside; all of which are reported to potently scavenge free radicals in a range of systems (Londhe et al., 2008).Although the therapeutic activity of Phyllanthus amarus against Lipopolysaccharide-induced neurotoxicity has been reported (Alagan et al., 2019), there needs to be more relevant research evidence to demonstrate a similar therapeutic activity against Paraquat-induced neurotoxicity.This study explores its anti-oxidant and antiin ammatory properties as it concerns the therapeutic role of the aqueous leaf extract of Phyllanthus amarus against Paraquat-induced neurotoxicity in the substantia nigra of Wistar rats.

Materials And Methods
This study was performed in line with the principles of the Declaration of Helsinki, as revised in 2013.1999).The open eld utilized in this study was a square wooden arena (100cm x 100cm x 38cm) with lines on its oor dividing it into 18cm by 18cm square.The open eld apparatus was cleaned with alcohol between each rat to avoid irritability due to odor.The rats were conveyed to the test room in their cages, and each rat was tested individually once for ve (5) minutes in an open eld apparatus, and behaviors were scored.The behaviors scored included: line crossing, rearing, walling, and center square activity (Frequency of entry into a central square and Duration spent in the center square).

Biochemical studies
Brains were weighed using a digital weighing scale (Acculab Vicon VIC-511 Precision Balance/Scale, USA, 0.001 g) and mechanically homogenized in 0.1 M phosphate buffer (pH 7.4) (1 g tissue/4 ml) according to already established protocols.Homogenate was analyzed for oxidative stress biomarkers superoxide dismutase, [SOD], and catalase (CAT).Biochemical analysis was conducted at the Department of Human Anatomy, ABU, Zaria.Enzymatic antioxidant activity was estimated by using appropriate enzyme lysate immunosorbent assay kits.

Histological studies using H & E techniques
The recommended procedure of Alagan et al., 2019 was adopted.Histological para n sections were processed and stained with Hematoxylin and Eosin (H and E) stains for demonstration of the cytoarchitecture of SN in the Histology Unit of the Department of Human Anatomy, FUTA, Ondo.
Microscopy (using Digital Microscopic Camera, MA 500 AmScope®, USA) was conducted in the Microscopy Research Laboratory of FUTA, Akure.

Immunohistochemical Staining with Tyrosine Hydroxylase (TH) Procedures
Brain samples were taken to the Department of Anatomy FUTA, where tissues were processed for immunohistochemical studies.The sections were depara nized in xylene and taken to water with descending grades of alcohol.Antigen retrieval was performed.The slides were washed in phosphatebuffered saline (PBS) for about 2 minutes.Endogenous peroxidase blocking was performed using 0.3% hydrogen peroxide in phosphate-buffered saline (PBS) for 10 minutes.The slides were washed with phosphate-buffered saline (PBS).The sections were blocked in 2.5% normal animal serum for 20 minutes.The sections were incubated in primary antibody (anti-tyrosine hydroxylase) at 1:7500 for 3 hours at room temperature.The sections were then washed in PBS for 5 minutes.Sections were incubated in ImmPRESS (peroxidase) Polymer Anti-Rabbit IgG Reagent, made in horse for 30 minutes.
The sections were washed twice, 5 minutes at a time.The color was developed with a DAB peroxidase (HRP) Substrate kit (Vector®).Sections were then rinsed well in tap water and Counter-stained in hematoxylin, dehydrated using graded alcohols, cleared using xylene, mounted, and coverslipped using Distyrene Plasticizer Xylene (DPX).Slides were subjected to quality control assessment and stored at room temperature before photomicrography.The processed tissues were viewed under a Digital Light microscope, and digital photomicrographs were taken by an attached camera at x400 magni cation using OMAX software.Using the cell counter plugin Field, NIH-sponsored ImageJ software was used to digitally analyze photomicrographs (Edobor et al., 2021).

Data analysis
Data obtained from this study were expressed as mean ± standard error of the mean (SEM).One-way analysis of variance was used to compare the mean difference between groups, followed by Tukey's Posthoc test, two-way analysis of variance, and Bonferroni's multiple comparisons tests.GraphPad Prism version 8.0.2(263) was used for statistical analysis.A signi cant difference was set at p < 0.05.

Discussion
In this study, the role of the aqueous leaves extract of Phyllanthus amarus on paraquat-induced neurotoxicity in the substantia nigra of Wistar rats was studied using neurobehavioral, biochemical, histological, and immunohistochemical assessments.
The open eld test is widely used to evaluate motor function in Parkinson's disease animal models (Crist ´ova˜o et al., 2020), and epidemiologic studies link paraquat exposure to Parkinson's disease (PD) development (Brouwer et al., 2017).Line crossing, rearing, walling, frequency, and duration in the center square are usually used as measures of locomotor activity as well as exploration and anxiety in open eld tests, with a higher frequency of these behaviors indicating increased locomotion and exploration and low anxiety (Walsh and Cummins, 1976).This study assessed an open eld test for de cit or improved motor function.A comparison of open eld tests across the study period revealed no motor function de cit with the paraquat-only treatment group.This nding is at variance with reports on the paraquat-induced motor de cit.Following paraquat exposure, Fernandes et al. and Ait-Bali et al. reported signi cant di culties, reduced motor activities, and modi ed motor coordination, attributing these adverse observations to paraquat neurotoxicity.Variance in ndings with other reports could be attributed to differences in paraquat dosage administered, duration, and routes of administration adopted in this study.Besides, Rojo et al. demonstrated that paraquat administered at different intranasal doses for four weeks (28 days) did not cause motor de cits in rats which could be similar to the ndings of this study.However, L-dopa and P. amarus (400 kg/mg) pretreated group showed improvement in motor coordination across the period of this study, as shown in Fig. 4.7, Fig. 4.8, and Fig. 4.12.This suggests the therapeutic properties of these treatments against paraquat-induced motor de cits in rodents.
Furthermore, antioxidant agents have been reported to improve motor coordination functions in paraquatinduced motor de cits in rodents (Ateş et al., 2019;Mirshekar et al., 2020).An established L-dopa action mechanism is the biological system's down-regulation of reactive oxygen species (ROS) (Olanow, 2015).P. amarus also contains bene cial compounds with strong antioxidant activities, such as phyllanthin and hypophyllanthin, which are reported to potentially scavenge free radicals in various systems (Londhe et al., 2008).Oxidative stress is the imbalance in intracellular biochemical redox activity, ultimately impairing cellular integrity and function.The central nervous system is especially sensitive to oxidative stress because of the relatively high oxygen utilization, high polyunsaturated fat, and low antioxidant level (Carvalho et al., 2014), and paraquat increases the formation of free radicals and oxidative stress (Ranjbar et al., 2018).Superoxide dismutase (SOD) and catalase (CAT) are critical to the antioxidant system.Superoxide dismutase (SOD) breaks down highly reactive superoxide into less reactive hydrogen peroxide.Hydrogen peroxide is further disintegrated into water molecules and oxygen by catalase (CAT).
Exacerbating the level of superoxide and hydrogen peroxide will eventually downplay the activities of superoxide dismutase (SOD) and catalase (CAT), respectively, thereby resulting in oxidative stress (Omotoso et al., 2018).
Widdowson et al. demonstrated that the administration of paraquat at a dose of 5 mg/kg/day for four weeks (28 days) does not cause biochemical changes, which partially reinforces the nding of this study where no signi cant difference was observed in catalase (CAT) activity in paraquat, only treatment group, compared to the control group as shown in Fig. 4.16.except for superoxide dismutase (SOD) activity, where a signi cant reduction in superoxide dismutase (SOD) activity was observed in paraquat only treatment group when compared to the control group and was alleviated by L-dopa pretreatment as shown in Fig. 4.17.This agrees with a previous report on L-dopa therapeutic activity against oxidative stress-triggered neurotoxicity (Olanow et al., 2015).
In addition, Edobor et al. reported cytoarchitectural distortions in the substantia nigra of rats following paraquat exposure for four weeks (28 days).This is at variance with this present study in which cytoarchitectural distortions were not observed in the paraquat (PQ) only treatment group compared to the control and other groups pretreated with P. amarus L-dopa as shown in Fahim et al. reported a remarkably decreased number of dopaminergic neurons in the substantia nigra exposed to paraquat.They attributed the loss of neurons to paraquat-triggered oxidative stress, which is in agreement with this study, where the immunohistochemical assessments showed a signi cant decrease in the number of tyrosine hydroxylase (TH) expressing dopaminergic neurons in paraquat only treatment group compared to the control group, P. amarus (300 mg/kg), P. amarus (400 mg/kg), and Ldopa pretreated groups as shown in Fig. 4.24.This suggests that the reduction in the number of dopaminergic neurons in the substantia nigra induced by paraquat (PQ) (10 mg/kg) was alleviated by P.
amarus (300 mg/kg), and P. amarus (400 mg/kg), as well as L-dopa.Previous reports have shown that phyllanthin, a major lignan in Phyllanthus amarus leaves, is a potent neuroprotective agent (Tao et al., 2020;Yuan et al., 2021).In agreement with these reports, the observed therapeutic activity of Phyllanthus amarus leaves extract against paraquat-triggered pathological changes in the substantia nigra of Wistar rats in this study may be attributable to phyllanthin.Thus, P. amarus, especially at 300 mg/kg and 400 mg/kg doses, possesses therapeutic properties comparable to the reference drug, L-dopa.

Conclusion
In conclusion, the aqueous leaf extract of Phyllanthus amarus possesses therapeutic properties against paraquat-triggered pathological changes in the substantia nigra of Wistar rats.Therapeutic properties could be attributed to bioactive compounds in aqueous leaves extract of Phyllanthus amarus with potent antioxidant activities against reactive oxygen species-associated paraquat-triggered pathologies.

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Fig. 4 .Fig. 4 . 20 .
Fig.4.20.Variance in ndings could be attributed to differences in paraquat dosage administered and the duration adopted in this study.The gold standard marker used to identify a dopaminergic neuron is the presence of tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of dopamine, catalyzing the conversion of L-tyrosine to L3, 4-dihydroxyphenylalanine (L-DOPA) and broadly expressed in noradrenergic and dopaminergic neurons in the central nervous system (White and Thomas, 2012;Levitt et al., 1965).
Fresh leaves of Phyllanthus amarus were collected in bulk in the swampy areas of Federal Housing Estate Farm, Igba, Ondo City, Ondo State.Collected plant material was identi ed (herbarium voucher label UNIMED/P.B.T.H/013) in the Department of Biology Biotechnology Department, University of Medical Sciences, Ondo City, Ondo State, Nigeria.
Anatomy, Faculty of Basic Medical Sciences, University of Medical Sciences, Ondo City, Ondo State, Nigeria.This test measured the experimental animals' locomotion, exploration, and anxiety (Brown et al., in the PQ (10mg/kg) + P. amarus (400mg/kg) group on Day 15 compared to PQ (10mg/kg) + P. amarus (400mg/kg) group on day 8 (p < 0.05), n = 3.All other comparisons of experimental groups between days 8 and 15 are not signi cantly different (ns) p > 0.05.graph C bar graph shows a comparison between day eight and day 15 of the frequency of walling made by the experimental rats.There was a signi cant decrease in the frequency of walling in the control group on day 15 compared to the control group on day 8 (p < 0.05), n = 3.All other comparisons of experimental groups between days 8 Figure 1:bar graphs comparing days eight and 15 of the Open eld test.Graph A represents the frequency of line crossing made by the experimental rats in all groups over horizontal and vertical lines and shows no signi cant difference between day eight and day 15 in the frequency of line crossing across all experimental groups (p > 0.05).Graph B bar graph shows a comparison between day eight and day 15 of the frequency of rearing made by the experimental rats.There was a signi cant increase in the frequency of rearing Figure 2:bar graphs showing the level of oxidative stress markers among treatment groups.Graph A shows no signi cant difference in the level of catalase across all experimental groups (p > 0.05); control [31.00 ± 0.5774], PQ (10mg/kg) + l-dopa [32.43 ± 0.8647], PQ (10mg/kg) [27.00 ± 1.155], PQ (10mg/