Systems pharmacology based approach to investigate the in-vivo therapeutic efficacy of Albizia lebbeck (L.) in experimental model of Parkinson’s disease

Parkinson’s disease (PD) is a chronic age-related neurodegenerative disorder characterized mainly with motor dysfunctions including resting tremors, muscular rigidity, bradykinesia and postural reflex impairments. It has been prevalent in 10 million people around the globe with incidence rate of 219/100000 people in Pakistan [1]. There has been evidence that suggests the oxidative stress, accumulation of misfolded protein and the loss the dopaminergic neurons in substantia nigra pars compacta as the main hallmarks of PD pathogenesis [2]. The neurodegeneration has been accounted for the loss of 80% dopaminergic neurotransmission in striatum that leads to significant neuromuscular dysfunction along with some cognitive deficits at advanced stages [3]. Levodopa is the primary gold standard approach to symptomatically manage the PD but its chronic use has also been associated with development of dyskinesia [4]. Moreover, we have no therapeutic options that provides the neuroprotection or relieve the progression of PD. Therefore, it is the need of time to develop the therapeutic modalities that changes the course of PD progression along with treating it symptomatically.

Plants have been increasingly reported to possess the diverse range of therapeutic compounds in the development of therapeutic options for PD [5]. However, it is costly and tedious to characterize the therapeutic roles of a plant species to the diverse or complex nature of bioactive phytochemicals present in it. Recently, the approach of systems pharmacology has been emerged as a sophisticated tool to dissect the complex pharmacological behavior of phytochemicals within the plant species. This approach integrates the high-dimensional biological data for phytochemicals to construct the computational model to explain their synergism in the development of pharmacological space at the biological systems level. The system pharmacology model extrapolates the phytochemicals from molecular or cellular to organism level in the form of biological network that better explain the mechanism of action and potential drug development from plant species [6]. Albizia lebbeck (L.) Benth, commonly known as Siris, is a large deciduous tree native to tropical southern Asia, widely cultivated in other tropical and subtropical regions. It is a tree with bi-pinnate leaves and white fragrant flowers with a fruit that is a pod containing six to twelve seeds [7]. Traditionally, pods and seeds have been widely used as antiprotozoal, anti-asthmatic, anticancer, antidiabetic, antidiarrheal, aphrodisiac and as a brain tonic [8]. The alcoholic extract of Albizia lebbeck (L.) has been reported to possess antihistaminic property, by neutralizing histamine directly or due to corticotrophic action [9]. Alcoholic extract of the stem bark of Albizia lebbeck (L.) was further corroborated to possess the strong analgesic and moderate anti-inflammatory activities possibly due to the presence of steroids and steroidal glycosides [10]. A study suggested that 70% ethanolic extract of bark of Albizia lebbeck (L.) also possess antioxidant and hepatoprotective effects owing to its principle phenolic components [11]. Moreover, the aqueous extract of Albizia lebbeck (L.) was found to reduce oxidative stress in alloxan-induced diabetic rats [12]. Albizia lebbeck (L.) bark has been found to be involved in disturbed testicular somatic cell function owing to its antifertility potential [13]. Albizia lebbeck is a diverse source of bioactive secondary metabolites including the higher content of saponins, vitamins and polyphenolics or flavonoids owing to its potential antioxidant activity or may be the therapeutic properties [14‐16]. The polyphenolics have also been widely conferred with therapeutic potential to fix the redox balance and induction of endogenous antioxidant defense [17, 18]. There are mounting evidences that addresses the role of oxidative stress to regulate the dopamine metabolism, neuro-inflammation and neuronal loss in Parkinson’s [19]. Moreover, Albizia lebbeck has also been found to modulate the locomotion and motor coordination of experimental convulsive rats [20]. Despite its potent antioxidant and neuro-modulatory capacity, the neuropharmacological studies of Albizia lebbeck (L.) has been limited to anticonvulsant, antidepressant and anxiolytic activities with no direct evidence related to major neurodegenerative disorder i.e. Parkinson’s [20‐22]. These insights provide reasonable justifications to further evaluate the traditional use of Albizia lebbeck in neurodegenerative disorders. Therefore, this study is aimed to employ the In-vivo and system’s pharmacology approach to validate the traditional use and investigate the mechanism of action of the Albizia lebbeck (L.) in Parkinson’s disease.

This study was aimed to employ the experimental and system’s pharmacology approach to validate the traditional use and investigate the mechanism of action of Albizia lebbeck (L.) in Parkinson’s disease (PD). In this study, haloperidol (1 mg/kg) was intraperitoneally administered for 21 days to induce the cataleptic feature of PD. Haloperidol is a neuroleptic drug that acts primarily through dopamine receptor subtype D2 and produce the catalepsy as a extrapyramidal side effect [37]. The haloperidol induced catalepsy serve as a robust model to investigate the anti-Parkinsonian activity [38]. The catalepsy is referred to a state where animals are unable to correct their imposed postures due to motor dysfunctions [39]. The HPD group was found to be highly cataleptic as demonstrated with higher duration of catalepsy at all intervals that was consistent with literature [23]. However, the co-treatment with ALE 100, 200 and 300 significantly reduced the catalepsy in a dose dependent manner that was comparable STD. Moreover, the motor coordination and muscular strength was further assessed with the narrow beam and hang test. The HPD group revealed the lowest time latency in hang test and highest time latency to walk through the beam. However, the ALE – coadministration improved the globular muscle strength and coordination thereby reversing the motor dysfunction of Parkinson’s. In addition to this, locomotor activity and exploratory activity of experimental rats was also assessed in open field test to address the cognitive and motor dysfunction associated with the Parkinson’s. This test works on the principle that rats with lower cognition, skeleton muscles strength and endurance shows lower locomotor activity and exploratory behavior [27]. The HPD group revealed the marked reduction in horizontal exploration and locomotor activity as compare to STD groups. However, the ALE co-administration improved the locomotor and exploratory activity suggesting its role in improving the motor dysfunction of Parkinson’s. Together, these behavior studies suggested the overall improvements in motor function of experimental groups treated with ALE.

In addition to its cataleptic function, the haloperidol has also been implicated as potential source of oxidative stress [40]. Interestingly, dopamine can also be metabolized to free radicals by the monoamine oxidase (MAO) or auto-oxidation in PD [41]. These reactive metabolites of dopamine can form the cysteinyl adducts at substantia nigra of PD patient thus induces the neurodegeneration [42]. Moreover, the susceptibility of α-synuclein to reactive metabolites can also activates the microglia that further fuel the neurotoxicity [43]. In our study, the endogenous antioxidant enzymes and GSH content in the brain tissue of HPD group was severely compromised that showed acute oxidative stress consistent with previous studies [23]. Superoxidse dismutase (SOD) is a key endogenous antioxidant enzyme that has been found to be depressed in the PD and results into the neuronal load of O₂• free radicals [44]. Moreover, the activity of catalase (CAT) was also found to be compromised in PD that suggests the production of OH• free radicals by Fenton type reaction in neurons thus contributing to the neurodegeneration in PD. Additionally, the content of GSH is depleted along the saturation of antioxidant enzyme [45]. However, the ALE co-administration significantly recovered the GSH content and activity of these enzymes thereby supported the therapeutic potential of ALE to mitigate the oxidative stress in PD. These results were further corroborated with histological studies that revealed the ALE potential to ameliorate the lipid peroxidation and neurodegeneration while reversing the tissue architecture of PD. Therefore, together these biochemical studies supported the functional outcomes of behavioral studies and advocated the therapeutic potential of ALE in the treatment of PD.

Plants are the rich source of diverse bioactive chemical species that determines the medicinal properties of them. The multi-component system and synergism between them makes the plants challenging source to probe the potential bioactive compounds responsible for their pharmacological activities. Interestingly, the virtue of synergism is complementing the emerging approach of “multi-drugs and multi-targets” with the fall of “one-drug and one target” approach due to dynamic biological systems [46]. In this context, an integrate approach of system’s pharmacology has been emerged that considers the synergism of phytochemicals to approximate their pharmacological properties with potential targets in a dynamic biological system [47]. The holistic approach of systems pharmacology to integrate the genomics, proteomics, metabolomics and bioinformatics provides the exceptional platform to study the essence of synergism among the phytochemicals to treat complex diseases. Therefore, we adopted this approach to delineate the Albizia lebbeck (L.) mechanism of action in PD. In the present study, the 86% of total 30 compounds were filtered by the Lipinski rule of five for druglikeness in SwissADME. Lipinski rule of five describe the relationship between physicochemical properties and pharmacokinetics by defining the physicochemical benchmarks to predict the orally active compounds [32]. These compounds were further mapped to predict the 132 targets by reverse pharmacophore modelling in Swiss Target Prediction tool. The reverse pharmacophore approach computes the 2D and 3D pharmacophore of targets with known ligands and predict them by the measure of structural similarity [33]. The class of these targets or proteins was distinguished and relevance to biological functions was determined by PANTHER classification system. These targets were predominantly classified as oxidoreductase, receptors (G-Protein coupled), hydrolases, and nucleic acid binding proteins, transporter, transcription factors and transferases. The relevance in variety of cellular, metabolic, biological regulation, multicellular organismal, response to stimulus, developmental, immune system and localization processes suggested the vitality of these proteins in biological system. The compound-target (C-T) network explained that most of the compounds actively involved in the synergistic pattern of interaction with targets. This network also indicated that many of the compounds were interacting with more than one target and vice versa. Therefore, synergistic modulation of series of the targets by number of compounds suggested the “multi-drugs and multi-targets” as a essence to the mechanism of action of Albizia lebbeck (L.). It is generally accepted that compounds or targets with the higher degree of interaction are more pharmacologically crucial [48]. Degree is a basic topological parameter in network analysis that determine the importance of compounds and targets [49]. Betweenness centrality is another important topological parameter that determine the significance of node location within the network [50]. In our network, we found the correlation between the degree and betweenness centrality as compounds, such as kaemferol, phyosterol and okanin, demonstrated both the higher degree of interaction and betweenness centrality thus represented as key players of Albizia lebbeck (L.). The tyrosyl – DNA phosphodiesterase 1 (TDP1) and microtubule-associated protein tau (MAPT) were the central nodes amongst the targets with highest degree of interaction thus represented as the targets modulated by majority of Albizia lebbeck (L.). TDP1 is an DNA repair enzyme that has been found to catalyze breakage of the covalently linked oxidative stress induced DNA adducts at 3′- phosphate end and implicated as a target of various anticancer drugs [51]. It has been found to repair the oxidative stress induced single strand breaks (SSB) in astrocytes and loss of TDP1 function has also been implicated in development of progressive age-related cerebellar atrophy that highlighted its involvement in neural homeostasis [52]. MAPT encodes the tau protein that has been implicated to regulate the axonal and micro tubular function. Moreover, single nucleotide polymorphism (SNP) at MAPT has been associated with tauopathies in development of neurodegenerative disease like Alzheimer’s and Parkinsonism [53]. There are also evidences that suggest the risk associated with MAPT haplotypes in the pathogenesis of PD [54, 55]. The higher centrality or degree of interaction of these target nodes in C-T network of Albizia lebbeck (L.) represent them the key player of its mechanism of action in PD. Furthermore, the functional association of the targets in STRING analysis enriched the 48 KEGG pathways. The neuroactive ligand receptor pathway was found to be in top ten overrepresented pathway in the genome-wide association studies (GWAS) of PD [56]. The experimental evidence had suggested the association of neuroactive ligand receptor pathway in α-synuclein toxicity in the pathogenesis of PD [57]. The serotoninergic synapses has been implicated as increase post-synaptic density of serotonin receptors (5HT_1A, 5HT_2A) in neocortex of PD patients [58]. A study has suggested the degeneration of serotonergic terminals is an early process in the pathogenesis of PD and with preferential lower rate of degeneration as compare of dopaminergic neurons [59]. The aberrant spread of serotonergic terminals in putamen, non-physiologically converts the exogenous levodopa and results into higher swings of dopamine that develops the levodopa associated dyskinesia [60]. The cholinergic and dopaminergic neurons are highly conserved in the striatum and extensively interact bidirectionally to regulate the motor coordination, cognitive function and rewards [61]. Cholinergic synapse has been found to induce glutamatergic and GABAergic plasticity and increase the Ach at striatum that induce the levodopa associated dyskinesia in PD [62]. Various lines of evidences have also suggested the MAPK signaling in regulation of autophagy and neurodegeneration in PD [63]. Therefore, these pathways further highlighted the functional association of interactome of Albizia lebbeck (L.) targets to the PD. However, all of the KEGG pathways were further indexed to KEGG disease database and Pathway – Disease (P-D) network was constructed to explore the therapeutic space of Albizia lebbeck (L.). The global view of network revealed that these pathways mainly interacted with the metabolic, endocrine, cancer, cardiovascular, nervous system diseased nodes which was consistent with the majority of Albizia lebbeck (L.) traditional uses [8]. Among all the disease nodes, we found that nervous system diseases node shared the highest degree of interaction with 21 pathways after the node of congenital malformation which interacted with 24 pathways. Therefore, these insights of Albizia lebbeck (L.) systems pharmacology were found to be consistent with in-vivo experimental results of anti-Parkinsonian activity and provided the deep understanding regarding its mechanism of action in PD.

The in-vivo experiment for anti-Parkinson’s activity of Albizia lebbeck (L.) concluded that Albizia lebbeck (L.) improved the motor functions and reversed the biochemical damages in brain tissue of PD. The systems pharmacology approach in this study investigated the mechanism of action of the Albizia lebbeck (L.) in PD and clearly suggested the synergistic effect of its phytochemicals in the development of its therapeutic effect. This approach supported the theory of “multi-drug and multi target” and indicated the therapeutic efficiency of Albizia lebbeck (L.) for nervous system diseases. However, the networks also highlighted the therapeutic efficiency for other disease system that should also be deciphered experimentally. Therefore, the present work served as the alternative strategy to provide the deep understanding of therapeutic behavior and sophistically validates the traditional uses of Albizia lebbeck (L.) to improve the efficiency of drug discovery.