Effect of Hypericum perforatum extract on behavioural studies in experimentally induced neurodegenerative disease

Chandran Satheesh kumar1,2, Srinivasagam Raja Sankar*3, Kaliyaperumal Prabu4 1Department of Research, Bharath Institute of Higher Education and Research, Chennai-600073. Tamil Nadu, India 2Department of Anatomy, Melmaruvathur Adhiparasakthi Institute of Medical Sciences and Research, Tamil Nadu 600073, India 3Department of Anatomy, Velammal Medical College, Madurai-625009, Tamil Nadu, India 4Department of Anatomy, Sree Balaji Medical College, Chennai-600044, Tamil Nadu, India

Behavioral studies, Hypericum perforatum, MPTP, Parkinson's disease A Parkinson's disease (PD) is a common neurodegenerative movement disease affecting a large number of people worldwide. In animals, the intracranial or systemical application of the neurotoxin 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) can result in severe injury to the nigrostriatal dopaminergic system. Hypericum perforatum is a plant generally used as an antidepressant that also has anti-in lammatory and antioxidant properties. Hypericum perforatum has been used to lessen the mild to moderate symptoms of depression. The present study was done to ind the effect of Hypericum perforatum methanolic extract pretreatment and post-treatment on behavioral studies in MPTP induced Parkinson's disease model. The behavioral assessments were performed with a forced swim test, pole test, tail suspension test, and catalepsy test. Hypericum perforatum extract ameliorated depressive-like behavior better in post-treatment as compared with pretreatment group in MPTP model of Parkinson's disease. It can be said that Hypericum perforatum methanolic extract might be a therapeutic agent for the treatment of PD, but further clinical studies are required.

INTRODUCTION
PD is a multisystem neurodegenerative disease in which progressive loss of midbrain dopamine neurons, with resulting dopaminergic deafferentation of the basal ganglia, excites characteristic motor instabilities that comprise muscular rigidity, resting tremor and slowing of movement. It affects 1% of the elderly population (Deng et al., 2018). Clinically PD is characterized by postural tremor, bradykinesia, rigidity and instability along with psychiatric, autonomic and cognitive problems. Many neurotoxins used for PD model induction that leads to nigral cell degeneration with accentuating oxidative stress (Pablo-Fernández et al., 2019).
The neurotoxin "MPTP" became a priceless tool to make experimental parkinsonism, which is used as a model of PD. In individuals, MPTP generates dopaminergic nigrostriatal neurodegeneration of the basal ganglia, which is predominantly af licted in Parkinson's disease (Kasahara et al., 2017). This neurotoxin excites virtually all symptoms of the idiopathic disorder, comprising rigidity, akinesia, gait/posture abnormalities and tremor (Yang et al., 2017). In medical society, the treatment for Parkinsonism without adverse effects is required more for the people suffering from this disease.
Hypericum perforatum (Saint John's wort) was considered a traditional folk medicine used topically for treating wounds, burns, abrasions, sunburns and in lammatory skin disorders. Its use in wound healing could be justi ied with its antimicrobial, anti-in lammatory, and astringent effects. Hypericum oil can be used topically for treating haemorrhoids and burns, to decrease in lammation, and as an antiviral and anesthetic. Hypericum perforatum extracts are becoming one of the standard treatment for antidepressant therapy (Ng et al., 2017). H. perforatum has various therapeutic potential, including anti-in lammatory and anxiolytic effects. H. perforatum extract has a bene iciary effect on chronic stress model in Hypericum extract has many components with well documented biological activity including naphthodianthrones (pseudohypericin and hypericin), a wide range of lavonoids (hyperoside, quercetin, and isoquercitrin) and the phloroglucinols (pseudohyperforin and hyperforin) (Cao et al., 2017). With this evidence this plant was selected for research work to ind out the ef icacy of Hypericum perforatum on degenerative neuro diseases. Their present study was done to ind the effectiveness of Hypericum perforatum methanolic extract (HPE) on behavioural studies in MPTP induced Parkinson disease.

Animals
Three months old C57BL/6 male mice weighing 23g -28g were used to carry out the experiments. The experiments were approved by IAEC, Adhiprasakthi College of Pharmacy (APCP/IAEC/2015-2016/4) and performed out in accordance with standard operating procedures in "guidelines on the regulation of scienti ic experiments on animals" (CPCSEA guidelines) by the ministry of environment and forest, Government of India. The animals were kept in polycarbonate cages in the standard day-night cycle and the temperature kept at 22±2 0 C. The mice were fed with Amruth Rat Feed, provided by Pranav Agro Industries (Pune, India) and had unrestricted access to water ad libitum.

Experimental induction of MPTP
The experimental induction of Parkinsonism to mice was done by giving an intraperitoneal (i.p) injection of MPTP hydrochloride (30 mg/kg b.w), dissolved in physiological saline for ive consecutive days. Safety protections for the use of MPTP in chemical preparation and animal injections were taken by following the method of (Kasahara et al., 2017).

Plant material and preparation
Hypericum perforatum plant was purchased from JK medicinal plants introduction center (JKMPIC), Srinagar, Jammu and Kashmir with authentication (no: JKMPIC-(K) R&D 20119). The air-dried sample was powdered inely using an auto-mix blender and kept in a deep freezer till use. The methanolic extract made using Soxhlet apparatus was concentrated by rotary evaporator at 40 • C and kept in a cool place. These samples were evaporated to dryness and were dissolved in water for further behavioral studies.

Experimental grouping
The animals were randomly classi ied into 5 groups Group 1-Control group, the mice received 1ml of distilled water orally.
Group 2-Mice were treated with Hypericum perforatum methanolic extract (HPE) 1ml orally as a single dose.
Group 3-Mice were administered with MPTP (30mg/kg, i.p) as a single dose for 5 consecutive days.
Group 4-Mice were administered with MPTP (30mg/kg, i.p) as a single dose for 5 consecutive days and post-treated orally with HPE (300mg/kg b.w) as a single dose.
Group 5-Mice pretreated orally with HPE (300mg/kg b.w) as a single dose and then injected with MPTP (30mg/kg, i.p) as a single dose for 5 successive days.
After the treatment period, behavioral assessments were performed with a swim test, tail suspension test, pole test and catalepsy test.

Forced swimming test
Forced swimming was carried out following the method of (Cao et al., 2017). Brie ly, a transparent acrylate cylinder (60 cm high; 24 cm diameter) was illed using 25 • C water (25 cm deep). The mice swim for 5 consecutive days for 10 min daily and 4 weeks later on day 33. The total time period of the FST was ixed at 4 minutes (240 seconds). During behavioral analysis, the time each mice expend mobile is evaluated and noted. The complete mobility time is reduced from 240 seconds. This shows the immobility of time. This method was taken as it is better to note down the movements. Any movement other than those essential for stabilizing the body and keep the head above the water was measured as mobility.

Tail suspension test
The tail suspension test was done according to the method of (Pałucha-Poniewiera et al., 2017). The tail (2 cm from the end of the tail) of the animal was hanged upward down using adhesive tape. An animal was arbitrated to be immobile when it stopped moving limbs and body, making only movements allowing to breathe and the total period of immobility in seconds was measured.

Pole Test
Animals were kept head-up on top of a vertical wooden pole. The pole base is kept in a cage having a bed and when kept on the pole, mice orient themselves downward and incline the length of the pole back into the cage. Animals received ive test trials after two days of training. The times to orient downward and total duration to incline are measured (Yao and Zhao, 2018).

Catalepsy test
The hind limbs of mice were kept on a wooden block (3 cm high), and mice latency in moving to the smooth surface was examined (Bhattacharjee et al., 2016).

Statistical analysis
All the results were measured as Mean ± SEM of a number of experiments. The statistical variation was observed by one-way analysis of variance (ANOVA) with the help of SPSS version 11.5 (SPSS, Cary, NC, USA) and the intercomparison between the groups was attained by Duncan's Multiple Range Test (DMRT). A P value less than 0.05 was measured to indicate a signi icant variation between groups.

RESULTS AND DISCUSSION
The novel therapeutic strategies able to modify the course of neurodegenerative disorders are cur-   rently one of the major goals for the researchers of this area. Developing novel pharmaceutical compounds having antioxidant properties will remarkably increase our understanding of their functions besides radical species in biological systems. The forced swim test (FST) is more appropriate for animal models with bilateral lesions and with partial dopamine diminution. Such animal models are appropriate for drug discovery studies, and thus, FST can serve as a valuable tool for assessing motor behavioural aberration in neuroprotective studies (Chonpathompikunlert et al., 2018).The FST was used for evaluating depressive-like behavior, as described previously (Chen et al., 2015). In the present study, HP alone treated mice showed non-signi icant results when compared with control mice. Rodents administered with MPTP showed a signi icant increase in swim test scores when compared to the control group. The swim test scores were found decreased in the pre and post Hypericum perforatum extract (HPE) treated group when compared with MPTP administered mice (Figure 1). * * * p<0.001 statistically signi icant from the control group; @@ p<0.01, @@@ p<0.001 statistically signi icant as compared with MPTP treated group.
Similar to the FST, in the tail suspension test, mice were kept in an inescapable but mild stressful situation. The lack of associated escape behavior is measured as immobility (Ramalhete et al., 2016). In the tail suspension test, MPTP administered mice showed a signi icant increase in tail suspension test scores when compared to the control group. Reduced tail suspension scores were found in HPE pre and post-treated mice when compared with the MPTP group of mice (Figure 2). * * * p<0.001 statistically signi icant from the control group; @@ p<0.01, @@@ p<0.001 statistically signi icant as compared with MPTP treated group.
This immobility might be associated with a difference in stress-induced behavioral depression and can be linked with the psychological construct of frame-up seen in clinical depression (Kiasalari et al., 2016). As a pointer of the antidepressant effect, it was observed that the immobility time of animals in the tail-suspension tests was shorter, i.e. the effect of the animals was greater in HPE treated mice.
The results of this administration of H. perforatum plant extract using forced swim test model display antidepressant effects several times lower than those in post-treatment experiments. Hesperidin, an active component present in HPE, was observed to be effective in reducing cognitive and depressive de icits by controlling neurotransmitter systems (Zirak et al., 2019). It has been shown recently that a lavonoid fraction obtained from a crude extract of Hypericum perforatum was remarkably active in the forced swimming test (Kordjazy et al., 2016). Future trials are required to further characterize the mechanism of the antidepressant action of H. perforatum, particularly of single compounds of the extract, as the only natural drug with antidepressant action. The tail suspension tests are acute trials of antidepressant effect that depends on behav-ioral despair or immobility to detect depressive-like behavior of mice.
The pole test is generally used for measuring basal ganglia related mobility diseases (Pałucha-Poniewiera et al., 2017).
MPTP-administered mice are proposed as a useful tool for measuring the capacity of pharmacological agents to hinder recognition de icits in PD. Rodents administered with MPTP showed a signi icant increase in pole test scores when compared to the control group. Decreased pole test scores were found in the posttest group when compared with MPTP administered mice (Figure 3). * * * p<0.001 statistically signi icant from the control group; @@ p<0.01, @@@ p<0.001 statistically signi icant as compared with MPTP treated group.
Catalepsy (immobility re lex, tonic immobility, and animal hypnosis) is considered by muscular rigidity resulting in prolonged immobility and an inability to correct an externally imposed awkward posture. Excessive catalepsy-like dyskinesia in human is a pathological symptom happening in mood disorders (e.g. depression), schizophrenia, and Parkinson's' disease (Poleszak et al., 2019).Cataleptic behavior in the PD mice has been hired as a standard model of bradykinesia and rigidity in human Parkinson's disease. The characteristic catalepsy test involves keeping mice into an unusual posture and recording the time taken to come back to the normal posture. Catalepsy, the mobility impairment, is an extrapyramidal dysfunction called a prominent motor symptom of PD, which is associated with striatal dopamine reduction (Vajdi-Hokmabad et al., 2017). The MPTP administered rats showed a signi icant increase in catalepsy test scores when compared to the control group. The decreased catalepsy scores were found both in the HPE pre and post-treated group in comparison with MPTP administered mice (Figure 4). * * * p<0.001 statistically signi icant from the control group; @@ p<0.01, @@@ p<0.001 statistically signi icant as compared with MPTP treated group.
The results of this pretreatment study on mice after H. perforatum plant extract therapy proved antidepressant effects several times lower than those in pretreatment experiments. Hesperidin, an active component present in HPE, was observed to be effective in reducing depressive and cognitive de icits in mice via modulating neurotransmitter systems (German-Ponciano and Rosas-Sánchez, 2018). It has been proved recently that a lavonoid content taken from Hypericum perforatum extract was remarkably active in the FST (Ben-Eliezer and Yechiam, 2016). Future trials are required to further characterize the mechanism of the antidepressant action of H. perforatum.

CONCLUSION
Hypericum perforatum extract ameliorated depressive-like behavior better in post treatment as compared with pretreatment group in MPTP model of Parkinson's disease. It can be said that methanolic extract of Hypericum perforatum can be a therapeutic agent for Parkinson's treatment, however further clinical studies are required.