INTRODUCTION:

Anxiety and depression disorders related with most of the clinical conditions like cardiovascular, thyroid and Neuro disorders such as parkinson’s diseases which is characterized by an increase in Monoamine oxidase (MAO), this may induce neuronal degeneration in brain due to oxidative stress¹.

MAO involves in generations of reactive oxygen species (ROS) which contributes to various disorders such as cancer, liver diseases, atherosclerosis, respiratory diseases², tissue damage, parkinson’s, Alzheimer’s, hydrogen peroxide and lipid Peroxidation in specific areas of brain which are toxic to dopamine cells and leads to reduction of dopamine levels and its metabolites. Long term usage of synthetic drugs results in adverse effects, In view of this, there is an urgent need of a drug that can overcome these symptoms. Therefore, the plant based products that can be identified to possess both antianxiety and antidepressant effects might prove useful as a therapeutic agent in these disorders³,⁴Justicia gendarussa belongs to family Acanthaceae and commonly known as Nili-Nirgundi which is wild or cultivated in Indonesia, India, China, Malaysia, Sri Lanka, and Bangladesh⁵. Talakona, Tirumala in Andhra Pradesh, India. This plant is found to have a broad spectrum of activities due to the presence of phytochemical active constituents like alkaloids, flavonoids, phenolic compounds, steroids, carbohydrates, carotenoids and terpenoids, proteins and aromatic amines⁶. Leaves of JG contains flavonoids like Vitexin and Apigenin besides lignans, triterpenoid stigmasterol, lupeol and 16-hydroxy lupeol⁷. JG with high Naringenin and kaempferol contents were successfully screened⁵. Justicia gendarussa has been utilized to treat hemiplegia, facial paralysis, muscle pain, headache, antiseptic, fever, antioxidant, analgesic and anti-inflammatory^8,9,10. Present study is aimed to investigate the behavioral activity of the various extracts of Justicia gendarussa leaves in MPTP induced mice, a plant known for its various therapeutic properties in Indian traditional medicine.

MATERIALS AND METHODS:

Animals:

Male Swiss albino mice weighing between 25-30 gm were obtained from Sri Venkateswara enterprises, Bangalore and used for the experiments. Animal studies were conducted as per IAEC (1677/PO/RE/S/2012/ CPCSEA/IAEC/05) Dated 23-02-2019. MPTP and L-dopa used in this work was Sigma Aldrich and all other chemicals and solvents used were Merck and analytical grade.

Collection and Preparation of Justicia jendarussa leaf extracts:

Leaves of Justicia gendarussa (JG) were collected from Tirupati rural region in month of September and authenticated by Dr. Madhavachetty, Botanist, Dept. of Botany, SVU, Tirupati, specimen deposited in herbarium with number 1610. Collected leaves were dried, Coarsely powdered and extracted with solvents of increasing polarity n-hexane, ethyl acetate, ethanol and aqueous by Soxhlation for 6hrs¹¹. After each extraction, the solvent was distilled off and extract was concentrated to dryness at 45–50^oC to obtain dried extracts, percentage yield was calculated and used for further studies.

Preliminary Phytochemical Studies:

Phytochemical analysis was performed for the presence of different secondary metabolites present in various extract of JG¹².

Experimental protocol:

Acute toxicity and Gross Behavioral Changes:

All the plant extracts used in this study were subjected to acute toxicity studies. Animals were grouped into 4 containing 3 animals each. Group-1 control and received 2% v/v tween 80/kg b.w orally. Groups 2-4 animals received 500, 1000 and 2000mg/kg of extracts suspended in 2% w/v tween 80. The animals were observed continuously for 2 hours and then intermittently at gap of one hour till 6, 12, 24 and 48hrs for behavioral and neurological effects. The mice were observed for mortality at the end of 48hrs to calculate LD 50(OECD guidelines, 420)¹³.

Evaluation of Neuropharmacological Activity:

Test for Spontaneous motor activity:

The locomotor activity was measured by using Actophotometer¹⁴. It consists of cage which is 30 x 30 x 30cm and has a wire mesh at the bottom with six lights and 6 photo cells placed in the outer periphery of the bottom in such a way that a single mice blocks only one beam. Photocell is activated when the rays of light falls on photocells, the beam of light is cut as and when animal crosses the light beam, number of cut offs were recorded for 10 minutes.

Test for Motor Co-ordination (Rota Rod Test):

Motor Co-ordination test was conducted using a Rota Rod apparatus¹⁵. The animals were placed on the moving rod prior to the treatment and the mice stayed on the rod without falling for 120 seconds were chosen for the study. The time of animals taken for falling from the rotating rod was noted before and after extract treatment.

Hole Board test for alertness:

The Hole Board consisted of a 0.5m³wooden board with 16 holes (3cm in diameter). The mice was placed at the corner of the board and allowed to move freely. First two minutes were allowed for adaptation and the number of head dipping in next 4 min was counted¹⁵

Tail Suspension Test (TST):

Immobility induced by tail suspension was measured. Mice were suspended on the edge of a table 50cm above the floor by the adhesive tape placed approximately 1cm from the tip of the tail. The total duration of immobility was recorded during the next 4 min of a total 6 min test¹⁶.

Forced swimming test (FST):

Mice were forced to swim individually in a glass jar (25 x 12 x 25cm³) containing fresh water of 15cm height and maintained at 25°C (±3°C). The total duration of immobility was recorded during the last 4 min of a total 6min test¹⁶.

Experimental design:

Treatment protocol:

The animals were divided into 11 groups each containing six mice. Group-I: control (2% Tween 80 p.o), Group-II: 20mg/kg i.p MPTP four injections at 2h intervals. Group-III: L-dopa, Standard (100mg/kg, i.p.) Group-IV and XI received various extract of JG like ethanol extract (EEJG), ethyl acetate extract (EAEJG), aqueous extract (AEJG), n-Hexane extract (HEJG) at 200 and 400mg dose orally for seven days along with 20mg/kg MPTP on day 1. Behavioral studies were conducted after MPTP induced animals on 1^st ,4^th and 7^th day of treatment.

Statistical analysis:

The results were analyzed using Graph pad prism version 8.1.5. All values are expresses as Mean±SEM. Behavioral studies were analyzed using one-way (ANOVA) test for multiple comparison followed by Tukey-Krammer test. In all the tests, the criteria for statistical significance was P<0.05.

RESULTS:

The percentage yield of Ethanol, ethyl acetate, Aqueous and n- hexane extracts were found to be 9.4, 7.2, 7.9 and 8.1%(w/w). Preliminary phytochemical analysis of all the extracts showed presence of flavonoids, polyphenols, amino acids, glycosides, proteins, carbohydrates, alkaloids, steroids, terpenoids and tannins.

Spontaneous motor activity:

Spontaneous motor activity score was significantly (P<0.001) decreased in MPTP induced group as compared to normal control and L-Dopa groups. Locomotor activity was significantly and dose dependently increased with all the extracts of JG with 200, 400 dose on 4^th and 7^th day of treatment as correlated with control, L-dopa and MPTP groups, 7^th day of treatment values are given in (Table 1, Figure 1).

Table 1: Effect of Various extracts of JG leaves on neuropharmacological activity in MPTP induced mice on 7^th day of treatment.

Plant extract	Doses	Spontaneous motor activity scores	Alertness (No. of head dippings)	Rota rod test (Grip strength)	Tail suspension test(immobility time in sec)	Forced swim test(immobility time in sec)
	Control	405.2±8.87	62.17±2.91	123.5±1.25	123.7±2.26	112.8±2.97
	MPTP	115.2±13.69⁺⁺⁺	24±1.39⁺⁺⁺	60.33±2.34⁺⁺⁺	145.0±1.86⁺⁺⁺	141.5±3.31⁺⁺⁺
	L-Dopa	410.7±6.60^***	68.83±3.38^***	131.7±1.38^***	57.50±3.57^+++,***	67.17±0.94^+++, ^***
Ethanol extract of JG	200mg/kg	391.5±2.82^***	54.83±0.87^***,##	110.5±4.07^+, ^***,###	75.33±0.88^+++,***,###	68.17±1.37^+++, ^***
Ethanol extract of JG	400mg/kg	409.5±5.03^***	69±3.20^***	133.7±1.90^*^	63.17±1.37^+++,***	62.67±0.71^+++, ^***
Ethyl acetate extract of JG	200mg/kg	348.5±3.19^+++, ^***,###	48±1.39^++,***, ^###	103.0±1.21^+++,***,###	84.00±0.85^+++,***,###	103.7±1.22^+,***,###
Ethyl acetate extract of JG	400mg/kg	387.7±0.84^***	60.67±0.71^***	113.5±2.68^***,###	72.33±0.84^+++, ^***,###	85.50±0.76^+++, ^***,###
Aqueous extract of JG	200mg/kg	337.5±3.58^+++, ^***,###	46.67±1.05^++, ^***,###	96.67±3.04^++, ^***,###	82.83±0.79^+++, ^***,###	103.5±0.99^+,***,###
Aqueous extract of JG	400mg/kg	387.3±1.70^***	53.83±0.79^***,##	113.5±2.82^***,###	72.67±1.22^+++, ^***,###	84.50±1.54^+++, ^***,###
n-hexane extract of JG	200mg/kg	212.8±1.24^+++, ^***,###	36.5±2.99^+++,*,###	70.33±2.88^++, ^####	132.5±0.76^++,***,###	132.2±0.94^+++,*,###
n-hexane extract of JG	400mg/kg	226.8±1.078⁺⁺⁺ ^***,###	45.67±3.91^+++,***,###	80.67±1.82^+++,***,###	123.8±1.27^***,###	113.8±1.95^*** ^,###

Values are expressed in Mean±SEM (n=6); +++(P<0.001), ++(P<0.01),+(P<0.05) Vs Control; ***(P<0.001), *(P<0.05) Vs MPTP; ###(P<0.001), ##(P<0.01) Vs L-Dopa

Fig. 1: Effect of ethanol extract of Justicia gendarussa on Spontaneous motor activity scores in MPTP induced mice.

Values are expressed as Mean ± SEM (n = 6); +++(P< 0.001) Vs Control group; ***(P< 0.001) Vs MPTP group.

Rota rod test:

The retention time or grip strength on rota rod was decreased significantly (P<0.001) in MPTP induced group as correlated to control and L-Dopa treated groups, it was drastically improved in a significantly (P<0.001) and dose dependent manner on 4^thand 7^thday of treatment with all extract of JG leaves with 200, 400 mg doses when compared to control, L-dopa and MPTP groups, values are given in (Table 1, Figure 2).

Fig.2: Effect of ethanol extract of Justicia gendarussa on Grip strength in MPTP induced mice

Values are expressed as Mean ± SEM (n = 6); +++(P< 0.001) Vs Control; *(P< 0.05), ***(P< 0.001) Vs MPTP.

Hole board test:

Hole board test revealed that, significant (P<0.001) decrease in the number of head dipping in MPTP induced mice as compared to normal (62.17±2.91) and L-Dopa groups Number of head dipping was elevated significantly (P<0.001) and dose dependently with all the extracts of JG with dose of 200,400mg/kg on 4^th and 7^th day of treatment when compared to MPTP, control and L-dopa treated groups, results were given in(Table 1,Figure 3).

Fig. 3: Effect of ethanol extract of Justicia gendarussa on Alertness in MPTP induced mice

Values are expressed as Mean ± SEM (n = 6);+++(P< 0.001) Vs Control;**(P< 0.01), ***(P< 0.001) Vs MPTP; #(P< 0.05) Vs L-Dopa group.

Immobility time in TST and FST:

Immobility time in TST and FST was significantly (P<0.001) increased in MPTP induced animals. Immobility time was significantly (P<0.001) and dose dependently decreased with various extracts of JG at all the doses on 4^th and 7^th day of treatment. Among all the extract, The ethanol extract of JG at 400mg on 7^th day exhibited more effect and n-hexane extract showed moderate effect as compared to MPTP and other treated groups. it was observed that, these evidences suggest that JG leaf extracts, could contain CNS stimulant active constituents and JG leaf extracts may considered as antidepressant evidenced by decreased immobility time in TST and FST (Table 1, Figure 4,5)

Fig. 4: Effect of ethanol extract of Justicia gendarussa on immobility time in Tail suspension test in MPTP induced mice

Values are expressed as Mean ± SEM (n = 6); +++(P< 0.001) Vs Control; ***(P< 0.001) Vs MPTP group.

Fig. 5: Effect of ethanol extract of Justicia gendarussa on immobility time in Forced swimming test in MPTP induced mice

Values are expressed as Mean ± SEM (n = 6); +++(P< 0.001) Vs Control; ***(P< 0.001), **(P< 0.01), Vs MPTP group.

DISCUSSION:

Anxiety and depressive are the global burden diseases increasing day to day life which are identified as recurring psychiatric conditions that effects individuals mood and behavoir¹⁷. Currently benzodiazepines are most widely prescribed medication for anxiety disorders but due their distributing side effects there is need to develop a novel pharmacological agent from plant source. In finding of new curative therapeutic products for the treatment of various Neuropharmacological disorders, research has progressed constantly towards natural plants. In folk medicine, Justicia gendarussa (JG), is known to have anxiolytic action¹⁸. Due to cognitive deficits in motor functioning and co-ordination are the cordial signs of PD¹⁹. In our present study, evaluated effect of various extracts of JG on motor impairment with four Behavioral parameters against MPTP induced mice model. Actophotometer is used for screening the locomotor and anti-anxiety activity, among animal groups induced with MPTP showed lethargic movement in the Actophotometer, lethargic movement was reversed with various extracts of JG significant (P<0.001) and dose dependent manner as compared to MPTP, Standard and control treated groups. This could be due to presence of CNS stimulant constituents²⁰. Impairment of motor coordination and muscular coordination in MPTP induced mice could be due to muscle relaxant effect. Treatment with various extracts of JG with 200 and 400 mg/kg doses improved the retention time due to increased grip strength on the rota rod it might be due to presence of L-dopa like substances and polyphenols and flavonoids, Naringenin and isoflavone. Hole board test is used to screen anxiolytic activity based on number of head dipping represents alertness. Number of head dipping were deliberately increased with various extract of JG on 7^th day of treatment, present findings of anxiolytic effect are similar to previous findings^16,21,22,26. Immobility time indicates antidepressant effect in TST and FST, immobility time period in MPTP treated animals were increased, it might be due to depressant effect produced by inducer. In our study various extracts of JG at two doses 400 and 200mg/kg produced significant reduction (P<0.001) in the immobility time period in both the parameters, it could be due to presence of antidepressant phytoconstituents in JG extract and findings correlated with previous results done by another methods and It might be due to presence of phytoconstituents like L-dopa, polyphenols and flavonoids, Due to presence of L-dopa in Justicia gendarussa, thus showing anti-depressant activity^23,24. Earlier reports reveled that presence of probable flavanoids like Naringenin has been proved for its anti-oxidant potential and able to cross Blood Brain Barrier (BBB)²⁵. These evidence showed that Naringenin possess neuroprotective effect in various animal models of neurological diseases like epilepsy, stroke, alzheimer's disease. Previous findings showed that Naringenin improved motor function in MPTP induced experimental mouse model Further, it exerts neuroprotection by decreasingα-synuclein accumulation and neuroinflammation and improved neuronal activity by increasing tyrosine hydroxyl levels in the MPTP mouse model²¹.

CONCLUSION:

Justicia gendarussa extracts exhibited significant improvements in motor performance, coordination and spontaneous activity enhancing learning and memory performance in mice, probably as a result of reductive oxidative stress by antioxidant defense mechanisms. This effect of Justicia concerned modulation of cholinergic neurotransmission, may be utilized as a potential agent in treatment of neurodegenerative diseases such as Alzheimer’s disease, Parkinsonism and other type of dementia and memory deterioration.

ACKNOWLEDGEMENT:

The Principal Investigator is thankful to the SERB, New Delhi for Granting research fund under EMEQ scheme to carry out the present research project.

CONFLICT OF INTEREST:

Author has no conflict of interest to publish this research paper.

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Received on 12.01.2020 Modified on 06.03.2020

Research J. Pharm. and Tech. 2020; 13(12):5793-5798.

DOI: 10.5958/0974-360X.2020.01010.0