Anti-depressant activity of Erythrina variegata bark extract and regulation of monoamine oxidase activities in mice

Journal Pre-proof Anti-depressant activity of Erythrina variegata bark extract and regulation of monoamine oxidase activities in mice Jeanette Martins, S. Brijesh PII:

S0378-8741(19)30281-8

DOI:

https://doi.org/10.1016/j.jep.2019.112280

Reference:

JEP 112280

To appear in:

Journal of Ethnopharmacology

Received Date: 21 January 2019 Revised Date:

9 August 2019

Accepted Date: 4 October 2019

Please cite this article as: Martins, J., Brijesh, S., Anti-depressant activity of Erythrina variegata bark extract and regulation of monoamine oxidase activities in mice, Journal of Ethnopharmacology (2019), doi: https://doi.org/10.1016/j.jep.2019.112280. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

Anti-depressant activity of Erythrina variegata bark extract and regulation of monoamine oxidase activities in mice

Jeanette Martinsa, Brijesh Sb

a,b

Sunandan Divatia School of Science, NMIMS (Deemed-to be) University, 3rd Floor, Bhaidas

Sabhagriha Building, Bhaktivedanta Swami Marg, Vile Parle (W), Mumbai 400 056, India.

E-mail address: a

[email protected]

b

[email protected]

b

Corresponding author:

Sunandan Divatia School of Science, NMIMS (Deemed-to-be) University, 3rd Floor, Bhaidas Sabhagriha Building, Bhaktivedanta Swami Marg, Vile Parle (W), Mumbai 400056, India. Tel.: +912242355957; Fax: +912226114512; E-mail address: [email protected].

1

Abstract Ethnopharmacological relevance: Erythrina variegata, commonly referred to as ‘tiger’s claw’ or ‘Indian coral tree’ and ‘Parijata’ in Sanskrit, belongs to the Fabaceae family. It is a plant native to the coast of India, China, Malaysia, East Africa, Northern Australia and distributed in tropical and subtropical regions worldwide. In traditional medicine, ‘Paribhadra’ an Indian preparation, makes use of the leaves and bark of E. variegata to destroy pathogenic parasites and relieve joint pains. E. variegata is known to exhibit anxiolytic and anti-convulsant activities. Folkore medicine also suggests that E. variegata barks act on the central nervous system. However, there is a lack of data demonstrating this. The anti-depressant activity of E. variegata bark has not been reported in literature. Aim of the study: Our study focuses on previously unreported anti-depressant activity of E. variegata bark ethanolic extract (EBE) and determination of its mechanism of action possibly through regulation of monoamine oxidase activity in mouse brain homogenates. Materials and Methods: EBE was characterized using standard protocols for phytochemical analysis, followed by liquid chromatography-mass spectrometry (LC-MS) and gas chromatographymass spectrometry (GC-MS) analysis. Anti-depressant activity of EBE (50, 100, 200 and 500 mg/kg) was evaluated in Swiss white albino mice using acute and chronic forced swim test (FST) models. Furthermore, the potential use of the extract as an adjunct to selective serotonin reuptake inhibitor (SSRI), escitalopram, was evaluated using the chronic unpredictable mild stress test model wherein inhibitory effects on monoamine oxidase (MAO) A and B were assessed by spectrophotometric-chemical analysis in mouse whole brain homogenates. Results: The extract showed significant reduction in immobility time periods in both acute (200 mg/kg) and chronic (100, 200 and 500 mg/kg) FST models. When used as an adjunct with escitalopram (15 mg/kg), the extract (100, 200 and 500 mg/kg) showed significantly greater inhibition of MAO-A and B activities when compared to escitalopram alone (30 mg/kg). Phytochemical analysis of EBE revealed presence of sugars, steroids, glycosides, alkaloids and tannins. LC-MS and GC-MS analysis identified components such as 2-amino-3-methyl-1-butanol, phenylethylamine, eriodictyol, daidzein and pomiferin, N-ethyl arachidonoyl amine, inosine diphosphate, trimipramine, granisetron, 3,4-dihydroxymandelic acid, ethyl ester, tri-TMS and dodecane, previously reported for their anti-depressant activity. Conclusions: The study thus demonstrated potential for use of the E. variegata bark ethanolic extract as an adjunct to currently available SSRI treatment. The study also identified components present in E. variegata bark ethanolic extract that may be responsible for its anti-depressant activity. 2

Furthermore, the study thus confirms the traditional use of E. variegata barks in improving CNS function through its anti-depressant like activity.

Keywords: Depression, Escitalopram, Erythrina variegata L., Forced swim test, Chronic unpredictable mild stress, Monoamine oxidase.

Abbreviations: Erythrina variegata (E. variegata), selective serotonin reuptake inhibitor (SSRI), high-performance thin layer chromatography (HPTLC), liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), forced swim test (FST), chronic unpredictable mild stress (CUMS), monoamine oxidase - A and B (MAO-A and MAO-B).

1

INTRODUCTION

Depression is a syndrome that is generally comprised of loss of interest, anxiety, disturbance in sleep, loss of appetite, lack of energy and suicidal thoughts, which can be recurrent. It is a leading cause for morbidity and mortality, and under worst circumstances can lead to suicide (Jones et al., 2001). The WHO reports that more than 300 million (4.4%) people suffer from depression globally (WHO, 2017). Pharmacological treatments for depression involve use of several classes of anti-depressant drugs. The tricyclic antidepressants and monoamine oxidase (MAO) inhibitors which were the first choice for treatment have increasingly been replaced by the selective serotonin reuptake inhibitors (SSRIs) and the serotonin and norepinephrine reuptake inhibitors (SNRIs). Major SSRIs such as escitalopram, citalopram, fluoxetine, etc., are considered as primary medications due to their broad effect on various psychiatric illnesses (Joseph and Lieberman, 2003). However, SSRIs have a number of side effects including coma, seizures, cardiac toxicity, sexual dysfunctions such as anorgasmia, erectile dysfunction and diminished libido (Coleman, 2011; Isbister et al., 2004). Use of SSRIs during pregnancy is associated with increased rate of miscarriages, birth defects, newborn behavioural syndrome, autism, etc. (Croen et al., 2011; Domar et al., 2012). Discontinuations of SSRIs have also been reported to produce disturbing withdrawal symptoms (Broekhoven et al., 2002). Hence, there is a need to explore alternative approaches that can either reduce the negative profiles of the SSRIs or help reduce their dosage thereby decreasing their side effects. Medicinal plants, which are cost effective and more readily available, may serve to fulfill this niche. They may also show synergistic effects similar to dual acting drugs like SNRIs, and hence may also act as adjuncts in SSRI treatment. 3

E. variegata is a rapidly growing, deciduous tree, 50–60 feet tall with prickly stems and branches, green to yellow triangular leaflets and large coral red flowers. It is commonly known as the “Indian Coral Tree”. It is cultivated throughout the tropics, particularly as an ornamental tree and as a shade and soil improvement tree for other tree crops such as coffee and cacao. Erythrina belongs to the Fabaceae family which is represented by 290 species. Erythrina is derived from the Greek word “erythros” which means “red” and refers to the distinct colour of its flowers. In folk medicine, plants of the genus Erythrina have long been utilized for a wide variety of human diseases. The bark and leaves of E. variegata are largely used in traditional medicine in India, China and Southeast Asia as a febrifuge, anti-bilious, anti-helminthic, anti-asthmatic and antiepileptic (Hegde, 1993). In India, the juice from the leaves is mixed with honey and ingested to treat parasitic conditions. This juice is ingested by women also to stimulate lactation, menstruation and to relieve dysentery and joint pains. The bark is used as a laxative, diuretic and an expectorant (Kumar et al., 2011). E. variegata is a rich source of alkaloids such as: 3-demethoxyerythratidinone, erythraline, erythramine, erythrinine, erythratidinone, erysonine, erysotine, erysodine, erysovine, 11-hydroxyepi-erythratidine, erythratidine, epierythratidine, erysodienone, erysotrine, erysopitine and 11-β hydroxyerysotrine, isolated from various parts of the plant and scoulerine, coreximine, Lreticuline and erybidine have been isolated from the leaves of E. variegata (Rastogi and Mehrotra, 2006). Isoflavonoids such as: erythrinins A, B and C, osajin and alpinum isoflavone, in addition to the

styrene

oxyresveratrol

and

dihydrostilbene

dihydroxyresveratrol,

are

also

major

phytoconstituents present in E. variegata (Rahman et al., 2007). From the roots of E. variegata, diphenylpropan-1,2-diols: eryvarinols A and B, isoflavonoids: eryvarins M-O, 2-arylbenzofurans: eryvarins P and Q and a 3-aryl 2,3-dihydrobenzofuran: eryvarin

R, have been isolated and

structurally elucidated (Tanaka et al., 2004). Fractionation of the bark extract of E. variegata has resulted in the isolation of newer isoflavones together with seven known compounds: euchrenone b10, isoerysenegalensein E, wighteone, laburnetin, lupiwighteone, erythrodiol and oleanolic acid (Xiaoli et al., 2006). As reported in Indian Materia Medica, E. variegata barks have been traditionally known to act on the central nervous system (CNS) so as to diminish or abolish its functions (Nadkarni et al., 1976) and has been used in India as a nervine sedative (Ghosal et al., 1972). Studies have also confirmed on CNS improving properties of E. variegata, through its anxiolytic and anti-convulsant activities (Bhattacharya et al., 1972; Pitchaiah et al., 2008; Sangale et al., 2015). E. variegata barks may thus be clinically useful in possessing muscle relaxant and anti-depressant like properties. However, there is a lack of data demonstrating this and scientific evidence on the anti-depressant activity and mechanism of action of E. variegata bark in models of depression such as: forced swim 4

test and chronic mild stress model, has not been studied or reported in literature to the best of our knowledge. Hence, this study was designed to evaluate the potential anti-depressant activity of E. variegata bark ethanolic extract (EBE) in acute and chronic forced swim test models and to explore its mechanism of action and potential for use as an adjunct to current SSRI treatment using the chronic mild stress model of depression in mice.

2

MATERIALS AND METHODS

2.1 Collection and extraction of plant material The bark of E. variegata was collected and authenticated by Dr. P. Tetali, Department of Botany, Regional Ayurveda Institute for Fundamental Research (RAIFR), Pune, Maharashtra, India. A voucher specimen (No. 763, dated 28-02-2018) has been deposited at the Naoroji Godrej Centre for Plant Research (NGCPR), Shirwal, Maharashtra, India. The name of the plant was confirmed with http://www.theplantlist.org. The bark of E. variegata was air dried under shade at room temperature and powdered using a grinder. Thirty grams of the powdered bark was extracted to exhaustion successively with petroleum ether (60–80 °C), chloroform (purity ≥ 99.5 %) and ethanol (purity ≥ 99.9 %), obtained from Sigma Aldrich, using a Soxhlet apparatus. The extracts were evaporated to dryness using a vacuum oven and stored at 4 °C until used for further studies. The present article focuses on further studies carried out using the ethanolic extract (EBE).

2.2 Physicochemical evaluation Physicochemical evaluation of EBE was carried out, wherein, loss on drying, total ash, acid insoluble ash and sulphated ash values were determined based on standard analytical procedures (Mukherjee, 2008).

2.3 Phytochemical analysis EBE was tested for the presence of bioactive compounds using established standardized methods (Khandelwal and Sethi, 2014). The presence of various phytoconstituents such as carbohydrates, sugars, proteins, amino acids, steroids, cardiac glycosides, anthraquinone glycosides, saponins, flavonoids, alkaloids and tannins was evaluated.

2.4 High performance thin layer chromatography (HPTLC) fingerprint analysis

5

The HPTLC fingerprint analysis of EBE was carried out on GF254 silica gel plates (Merck KGaA, Darmstadt, Germany). Briefly, 2 µl of sample was spotted on the plate and the chromatogram was developed using the solvent system consisting of toluene, chloroform and ethanol in the ratio 8:8:2. The chromatogram was derivatized with anisaldehyde sulphuric acid. The HPTLC bands were visualised at 366 nm and photodocumented using a CAMAG visualizer. Densitometric scanning (slit dimensions: 5 × 0.45) was performed at 366 nm using CAMAG TLC Scanner 4 operated in a reflectance–absorbance mode.

2.5 Liquid chromatography-mass spectrometry (LC-MS) analysis LC-MS analysis of EBE was performed using Dual AJS ESI (Agilent, USA). The mobile phase consisted of 100 % water (A containing 0.1 % formic acid in water) and 100 % acetonitrile (B containing 90 % acetonitrile + 10 % water + 0.1 % formic acid). Initial conditions were solvent A 95 %: B 5 %; 0–20 min, changed to solvent A 5 %: B 95 %; 20–26 min and went back to solvent A 95 %: B 5 % 26–30 min. The flow rate was set to 0.4 ml/min, the injection volume was 3 μl and the column used was C 18 (Zorbax Eclipse).

2.6 Gas chromatography-mass spectrometry (GC-MS) analysis GC-MS analysis of EBE was carried out on 1300 GC (Thermo Trace, USA) connected to TSQ 8000 MS (Thermo Fischer, USA) employing the following conditions: column TG 5 MS (30 m × 0.25 mm × 0.25 μm), inert gas helium was used as a carrier gas at a constant flow rate of 1 ml/min. Mass transfer line and injector temperatures were set at 280 and 250 °C, respectively. The oven temperature was initially set at 80 °C for 2 min and later roused to 260 °C for 10 min. The total run time was 28.10 min. The compounds in the extract were identified and characterized by employing the National Institute of Standards and Technology (NIST) library.

2.7 Experimental animals Male swiss albino mice weighing around 25–30 g were purchased from Bombay Veterinary College, Parel and from National Institute of Biosciences, Pune. The animals were housed at SVKM’s animal house facility (1830/PO/Re/S/15/CPCSEA). ‘Principles of laboratory animal care’ (NIH publication no. 82-23, revised 1985) guidelines were followed. The animals were housed in perspex cages (six mice per cage) under standard conditions of temperature (22–24 °C) and humidity (50–60 %) with alternating light and dark cycle of 12 hours each. They had free access to 6

food (dry pellets, Nutri Vet Life Sciences) and water (ad libitum). The animals were acclimatized for at least seven days before behavioural experiments were conducted. The study was carried out between 10:00 am and 5:00 pm. The study was approved (CPCSEA/IAEC/SOS/P-61/2017) by the Institutional Animal Ethics Committee (IAEC) and all experiments were carried out in compliance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Social Justice and Empowerment, Government of India.

2.8 Forced swim test (FST) The FST, which is routinely used as a model to assess behavioural despair in rodents, was used for testing the anti-depressant activity of the extract. The method was as previously described by Porsolt et al. (1977). The study consisted of a glass tank (21 cm height and 12 cm diameter) filled to 10 cm with water maintained at 24–25 °C. For inducing depression, the mice were individually placed into the tank forcing them to swim. The immobility period (time when the animal remains passive or inactive or made small limb movements necessary for floating) was estimated for the last 4 min out of the total time of 6 min. Mobility time period was noted as the time when the animal was swimming or when the limbs were active. After each experiment, the mouse was cleaned with a hand towel before returning to its cage. Male swiss albino mice were randomly distributed into six experimental groups comprising of six animals each. Group 1 served as the stress control group. Group 2 was orally administered with escitalopram (Lundbeck Pharmaceuticals, Copenhagen, Denmark) (30 mg/kg b.w.) using a gavage feeding needle, whereas Groups 3–6 were treated with EBE at doses of 50, 100, 200 and 500 mg/kg b.w. The test was carried out for assessing the antidepressant effects of the extract in both acute and chronic FST models. For the acute model, FST was performed 1 h after single dose of saline/SSRI/extract administered orally to the animals. For the chronic model, the animals were orally administered daily with saline/SSRI/extract for a period of 14 days and FST was performed 1 h after oral administration on the 14th day.

2.9 Chronic unpredictable mild stress (CUMS) test The CUMS model of depression in mice was used to assess the potential of EBE as an adjunct to SSRIs. The CUMS procedure was performed as described by Chhillar and Dhingra (2013) with minor modifications. The mice were divided into seven groups comprising of five animals each. Group 1 served as the non-stress control group, which was provided with food and water ad libitum. Group 2 served as the stress control group. Groups 3 and 4 were orally administered with escitalopram (15 and 30 mg/kg b.w., respectively) using a gavage feeding needle and groups 5–7 7

were treated with a combination of the standard drug escitalopram (15 mg/kg b.w.) and varying concentrations (50, 100 and 200 mg/kg b.w.) of the extract. The mice in groups 2–7 were subjected to a CUMS paradigm over a period of three weeks accompanied by daily dosing of escitalopram and/or the extract orally, one hour prior to exposure to stress. The CUMS paradigm consisted of six different kinds of stressors wherein the mice were exposed to one type of stress each day. The types of stressors included tail pinch (60 s), cold water swim at 12°C (6 min), food deprivation (24 h), water deprivation (10 h), tilting of cage (45 min) and soiled cages with rat excreta (12 h). The schedule of stress exposure was as described in table 1.

2.10 Collection of brain homogenate for analysis On the 22nd day of CUMS, FST was performed for all the groups after which the mice were sacrificed and brain samples were collected immediately on an ice plate. The procedure of isolation and preparation of mouse brain homogenate was performed as described by Dhingra and Kumar (2007). Briefly, the collected brain samples were washed with cold 0.25 M sucrose- 0.1 M tris- 0.02 M EDTA buffer (pH 7.4), obtained from Sigma Aldrich and weighed. The brain samples were then homogenized in 9 volumes of cold sucrose- tris- EDTA buffer and centrifuged twice at 800 g for 10 min at 4 °C in a cooling centrifuge (Remi instruments, Mumbai). The pellets were discarded and the supernatant was again centrifuged at 12000 g for 20 min at 4 °C. The precipitates were washed twice with 10 ml of sucrose- tris- EDTA buffer and suspended in 9 volumes of cold sodium phosphate buffer (10 mM, pH 7.4, containing 320 mM sucrose), (purity ≥ 96 %) obtained from Sigma Aldrich and mingled well at 4 °C for 20 min. The mixture was then centrifuged at 15000 g for 30 min and the pellets were re-suspended in nine volumes of cold sodium phosphate buffer. The protein concentrations of the mitochondrial fractions of the brain homogenates thus obtained were estimated by the Folin-Lowry method using bovine serum albumin (BSA), (purity ≥ 96 %) obtained from Sigma Aldrich as the standard.

2.11 Effect of EBE on monoamine oxidase (MAO)-A and MAO-B activities The effect of EBE on MAO-A and MAO-B activities in the brain homogenate when used as an adjunct to escitalopram was evaluated using a method described by Yu et al. (2002). Briefly, for estimating MAO-A activity, the assay mixture (1 ml) consisted of 100 µl of 4 mM 5-hydroxy tryptamine (5-HT), (purity ≥ 98 %) purchased from Sigma Aldrich, 250 µl of the mitochondrial fraction of brain homogenate and 650 µl of sodium phosphate buffer (100 mM, pH 7.4), (purity ≥ 96 %) obtained from Sigma Aldrich. The mixture was incubated at 37 °C for 20 min. The reaction 8

was stopped by adding 200 µl of 1 N HCl, (purity ≥ 36.5–38 %) obtained from Sigma Aldrich. The reaction product was extracted with 5 ml butyl acetate, (purity ≥ 99 %) obtained from Sigma Aldrich and the absorbance of the organic phase was measured at 280 nm using UV/Vis spectrophotometer (Jasco V – 550). For estimating MAO-B activity, the assay mixture (1 ml) consisted of 100 µl of 2 mM β-phenylethylamine (β-PEA), (purity ≥ 99 %) obtained from Sigma Aldrich, 250 µl of the mitochondrial fraction of brain homogenate and 650 µl of sodium phosphate buffer (100 mM, pH 7.4). The mixture was incubated at 37 °C for 20 min. The reaction was stopped by adding 200 µl of 1 N HCl. The reaction product was extracted with 5 ml cyclohexane, (purity ≥ 99.9 %) obtained from Sigma Aldrich and the absorbance of the organic phase was measured at 242 nm using UV/Vis spectrophotometer. Blank samples were prepared by adding 1 N HCl prior to the enzyme substrate reaction and were worked up subsequently in the same manner. Percent inhibition of MAO-A and MAO-B activity by the extract was then calculated.

2.12 Statistical analysis The values obtained are expressed as mean + SD. The data were analyzed using one-way analysis of variance (ANOVA) followed by the Dunnett’s post-test and Tukey’s multiple comparison test using GraphPad Prism 5.0 (GraphPad software, Inc., USA). P-value less than 0.05 (p < 0.05) was considered to be significant.

3

RESULTS

3.1 Extraction of plant material Successive extraction of powdered bark of E. variegata using soxhlet apparatus resulted in higher yields in ethanolic (1.05 + 0.18 %, w/w) and chloroform (0.98 + 0.23 %, w/w) extracts compared to petroleum ether extract (0.45 + 0.01 %, w/w).

3.2 Physicochemical evaluation Physicochemical evaluation of plant based drugs is important for detecting any possible adulteration or improper handling during collection, drying or extraction processes. The moisture content of the dry powder of EBE was 3.43 + 0.25 % which was sufficiently low to prevent any bacterial or fungal growth. The ash value was determined in the form of total ash, acid insoluble ash and sulphated ash. The total ash helps in determining the purity of drugs with respect to foreign inorganic materials such as metallic salts or silica. Acid insoluble ash determines the amount of silica present, 9

especially sand. Sulphated ash determines the amount of residual substance not volatilized from the sample when ignited in the presence of sulphuric acid. The total ash of the dry powder of the extract was 7.38 + 0.09 %, acid insoluble ash was 0.32 + 0.01 % and sulphated ash was 9.79 + 0.51 %. All the parameters were within the limits as per the Ayurvedic pharmacopoeia indicating that the content of inorganic matter and silica in the extract were within acceptable limits (Ministry of AYUSH, 2016).

3.3 Phytochemical analysis The results of the qualitative phytochemical analysis of EBE are shown in table 2. The extract showed presence of phytoconstituents such as hexose sugars, steroids, cardiac glycosides, alkaloids and tannins.

3.4 HPTLC fingerprint analysis The HPTLC fingerprint obtained for EBE is shown in fig.1. The chromatogram developed using the solvent system consisting of toluene, chloroform and ethanol (in the ratio 8:8:2) shows the bands obtained following derivatization with anisaldehyde sulphuric acid (fig. 1a). The chromatogram was visualized at 366 nm using a CAMAG visualizer. The densitogram (fig 1b) shows the corresponding retention factors and percent areas for each of the bands obtained.

3.5 LC-MS analysis Compounds identified in EBE by LC-MS analysis are shown in table 3. LC-MS analysis revealed the presence of phytoconstituents such as 2-amino-3-methyl-1-butanol, phenylethylamine, eriodictyol, daidzein, inosine diphosphate, trimipramine, pomiferin, n-ethyl arachidonoyl amine, and granisetron (fig. 2) that have been previously reported for their anti-depressant activity (Aithal et al., 2014; Avraham et al., 2013; Bahramsoltani et al., 2015; DeMorais et al., 2016; Hirose et al., 2016; Maj et al., 1998; Messina and Gleason, 2016; Muto et al., 2014; Sabelli et al., 1996).

3.6 GC-MS analysis Compounds identified in EBE by GC-MS analysis are shown in table 4. GC-MS analysis revealed the presence of phytoconstituents such as 3,4-dihydroxymandelic acid and dodecane (fig. 3) that have been previously reported for their anti-depressant activity (Smith et al., 2009; Sule et al., 2017). 10

3.7 Forced swim test The effect of EBE on immobility time period in FST study is shown in fig.4. In the acute FST study, the control animals showed immobility time period of (144.17 + 26.23 sec). When treated with the extract, significant reduction (47.57 + 9.94 %) in immobility time period (84.00 + 21.60 sec) was observed at the dose of 200 mg/kg b.w. The standard drug escitalopram (30 mg/kg b.w.) also resulted in significant reduction (59.90 + 13.97 %) in the immobility time period (55.25 + 28.35 sec). In the chronic FST study, the control animals showed immobility time period of (153.17 + 24.49 sec). When treated with the extract, significant reduction in immobility time period was observed at 100, 200 and 500 mg/kg b.w. doses with maximum reduction (41.35 + 5.94 %) in the immobility time period (94.40 + 20.03 sec) at 200 mg/kg b.w. The standard drug escitalopram (30 mg/kg b.w.) also resulted in significant reduction (57.12 + 12.01 %) in the immobility time period (68.00 + 29.18 sec).

3.8 Effect of EBE on MAO-A and MAO-B activities Fig. 5 shows the effect of EBE on MAO-A and MAO-B activities in mouse brain following the CUMS when used as an adjunct to the SSRI, escitalopram. Following exposure to the CUMS, significant increase in MAO-A (43.34 + 3.11 %) and MAO-B (36.09 + 11 %) activities was observed in the brain of the animals in the stress control group (Group 2) compared to the nonstress control group (Group 1). The standard drug escitalopram showed 7.61 + 1.79 % and 19.45 + 1.84 % decrease in MAO-A activity at 15 and 30 mg/kg b.w., respectively, and 10.21 + 2.11 % and 22.12 + 6.61 % decrease in MAO-B activity, respectively. When administered in combination with the extract, escitalopram (15 mg/kg b.w.) showed significantly greater decrease in MAO-A and MAO-B activities at all the combinations compared to escitalopram alone at both 15 and 30 mg/kg b.w. The results indicated synergistic activity between escitalopram and EBE suggesting potential use of the extract as an adjunct to standard SSRIs.

4

DISCUSSION

In the present study, FST was used as a model to assess the anti-depressant activity of EBE in swiss albino mice. The FST model of depression is a widely accepted model that provides a rapid and reliable behaviour screening test for anti-depressant drugs (Porsolt et al., 1977). The characteristic behaviour evaluated in these tests, termed as “immobility” is a state of lowered mood or helplessness, which the animals experience when they are subjected to a stressful situation like FST 11

from which they cannot escape. This is thought to reflect either a failure to persist in escape directed behaviour after persistent stress or the development of passive behaviour that disengages the animal from active forms of coping with stressful stimuli (Lucki, 1997). Effective anti-depressant drugs decrease the immobility time period in FST. The results obtained in the present study showed significant reduction in immobility time period when treated with EBE in both the acute and chronic FST models. The study thus demonstrated the anti-depressant activity of E. variegata. CUMS was used as a model to assess the potential application of EBE as an adjunct to SSRI drugs. CUMS procedure is a commonly used stress paradigm as a model for depression. It consists of exposing animals to stressful situations which usually last for a period of three weeks. During this period, different kinds of stressors are introduced to the animals such as water and food deprivation, etc. A wide variety of drugs can be tested for their anti-depressant activity using this model (Mahar et al., 2014). EBE was administered in combination with the standard SSRI, escitalopram and its effect on MAO-A and MAO-B activities in the brain following exposure of the animals to the CUMS model was assessed. MAO-A and MAO-B are a class of enzymes that bring about the oxidative deamination of monoamines in the brain. Inhibition of these enzymes by various drugs and plant compounds can arrest the neurotransmitter breakdown process, leading to increase in their concentrations in the brain, and thus bringing about the anti-depressant activity (Kalgutkar et al., 2001). The results showed that EBE, when used in combination with escitalopram (15 mg/kg b.w.), demonstrated significantly greater inhibition of MAO-A and MAO-B activities in the brain compared to the standard SSRI alone (30 mg/kg b.w.). The study therefore demonstrated the potential of EBE for use as an adjunct to currently used standard SSRIs. Phytochemical analysis of EBE showed presence of the following classes of compounds: hexose sugars,

steroids,

cardiac

glycosides

(deoxysugars),

alkaloids,

tannins

and

phenolics.

Phytocompounds such as flavonoids, phenolic acids, lignanes, coumarins, alkaloids, terpenes, terpenoids, saponins, sapogenins, amines and carbohydrates from various plants have been previously reported to possess anti-depressant activity (Bahramsoltani et al., 2015). Further, LC-MS and GC-MS analyses identified the following phytoconstituents previously reported for their antidepressant activity: 2-amino-3-methyl-1-butanol (amino alcohol), phenylethylamine (monoamine alkaloid), eriodictyol (flavanone), daidzein and pomiferin (isoflavones), N-ethyl arachidonoyl amine (fatty acid neurotransmitter), inosine diphosphate (purine nucleoside), trimipramine (tricyclic anti-depressant), granisetron (serotonin receptor antagonist), 3,4-dihydroxymandelic acid, ethyl ester, tri-TMS (norepinephrine metabolite), and dodecane (alkane hydrocarbon molecule). The phytochemical studies thus identified the possible phytoconstituents that may be responsible for the 12

anti-depressant activities observed in both FST and CUMS models, possibly via inhibition of MAO-A and MAO-B activities. The present study demonstrated the anti-depressant activity of EBE in both acute and chronic FST models of depression. The anti-depressant activity of the extract may be due to the presence of major phytocompounds such as 2-amino-3-methyl-1-butanol, phenylethylamine, eriodictyol, daidzein, pomiferin, n-ethyl arachidonoyl amine, inosine diphosphate, trimipramine, granisetron, 3,4-dihydroxymandelic acid and dodecane. Further, the extract showed increased inhibition of the MAO-A and MAO-B activities in brain when administered in combination with escitalopram in the CUMS model. The study therefore demonstrated the potential of EBE for use as an adjunct to standard SSRIs which could help in decreasing the amount of usage of these drugs in clinical practice and thus help in reducing the toxic profile of these drugs.

ACKNOWLEDGEMENTS The authors would like to thank and acknowledge Dr. P. Tetali and the Department of Botany at Regional Ayurveda Institute for Fundamental Research (RAIFR), Pune, for helping with the collection and authentication of E. variegata bark; Bombay Veterinary College, Parel and National Institute of Biosciences, Pune for supplying us with the animals needed to perform the studies.

FUNDING SOURCES None.

CONFLICT OF INTEREST The authors declare no conflict of interest.

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17

LEGENDS TO TABLES

Table 1: CUMS schedule for the stress experimental groups. Table 2: Phytochemical analysis of EBE. Table 3: The components present in EBE identified by LC-MS analysis. Table 4: The components present in EBE identified by GC-MS analysis.

LEGENDS TO FIGURES

Figure

1:

High

performance

thin

layer

chromatography

fingerprinting

analysis

of

phytoconstituents present in EBE observed at 366 nm after derivatization with anisaldehyde sulphuric acid. A) Chromatogram showing bands of phytoconstituents. B) Densitogram showing retention factors and percent areas of phytoconstituents.

Figure 2: The chromatogram obtained from LC-MS analysis of EBE showing phytoconstituents previously

reported

for

anti-depressant

activity,

viz.,

2-amino-3-methyl-1-butanol,

phenylethylamine, eriodictyol, daidzein, inosine diphosphate, trimipramine, pomiferin, n-ethyl arachidonoyl amine and granisetron.

Figure 3: The chromatogram obtained from GC-MS analysis of EBE showing phytoconstituents previously reported for anti-depressant activity, viz., 3,4-dihydroxymandelic acid and dodecane.

Figure 4: Mean immobility time period of mice when treated with single and repeated doses of EBE in acute (A) and chronic (B) FST model of depression. 1: Control; 2: Escitalopram (30 mg/kg b.w.); 3: EBE (50 mg/kg b.w.); 4: EBE (100 mg/kg b.w.); 5: EBE (200 mg/kg b.w.); 6: EBE (500 mg/kg b.w.). The values are expressed as mean ± SD. p < 0.05 was considered to be statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001 when compared to the control group (Group 1).

Figure 5: Inhibition of MAO-A and MAO-B activities by EBE in the CUMS model. 1: Stress control; 2: Escitalopram (15 mg/kg b.w.); 3: Escitalopram (30 mg/kg b.w.); 4: Escitalopram (15 mg/kg b.w.) + EBE (500 mg/kg b.w.); 5: Escitalopram (15 mg/kg b.w.) + EBE (100 mg/kg b.w.); 6: Escitalopram (15 mg/kg b.w.) + EBE (200 mg/kg b.w.). 18

The values are expressed as mean ± SD. p < 0.05 was considered to be statistically significant. ***p < 0.001 when compared to the stress control group (Group 1).

###

p < 0.001 when compared

to the Escitalopram (30 mg/kg b.w.) group (Group 3).

19

TABLES

Table 1. CUMS schedule for the stress experimental groups Weeks Monday

Tuesday

Wednesday

Thursday

Friday

Saturday

Sunday

1

T

F

W

CT

SC

C

F

2

W

T

CT

SC

C

F

W

3

CT

C

SC

F

T

W

SC

Key: C: Cold water swim; T: Tail pinch; F: Food deprivation; W: Water deprivation; CT: Cage tilt; SC: Soiled cage with rat excreta.

20

Table 2. Phytochemical analysis of EBE extract Phytoconstituent

Test

Result

Carbohydrates

Molisch’s test

–

Reducing sugars

Fehling’s test

–

Benedict’s test

–

Hexose sugars

Cobalt chloride test

+

Non - reducing sugars

Iodine test

–

Proteins (General test)

Biuret’s test

–

Millon’s test

–

Proteins (containing tyrosine and tryptophan) Xanthoprotein test

–

Amino acids

Ninhydrin test

–

Steroids

Salkowski reaction

+

Cardiac glycosides (deoxysugars)

Keller-Killiani test

+

Anthraquinone glycosides

Borntrager’s test

–

Saponin glycosides

Foam test

–

Flavonoids

Shinoda test

–

Sulphuric acid test

–

Dragendorff’s test

+

Mayer’s test

+

Wagner’s test

+

Hager’s test

+

Ferric chloride test

+

Lead acetate test

+

Alkaloids

Tannins

(+ : present, – : absent)

21

Table 3. The components present in EBE extract identified by LC-MS analysis Retention

Compound

time

Molecular formula

0.206

Isoamyl nitrite

C5H11NO2

0.803

2-Amino-3-methyl-1-butanol

C5H13NO

0.859

Triparanol

C7H13NO2

1.316

Indospicine

C7H15N3O2

4.669

Rimantadine

C12H21N

5.155

Octopine

C9H18N4O4

5.157

2-amino-4'-hydroxy-Propiophenone

C9H11NO2

5.266

Isoacitretin

C21H26O3

5.312

Fendiline

C23H25N

5.343

Pro Ser Pro

C13H21N3O5

5.432

Lys Ser Lys

C15H31N5O5

5.662

2-Propylbenzimidazole

C10H12N2

6.007

Phenylethylamine

C8H11N

6.064

Gln Arg Arg

C17H34N10O5

6.263

26,26,26,27,27,27-hexafluoro-1-alpha,24 dihydroxy vitamin

C27H38F6O3

D3 6.268

1alpha,25-dihydroxy-26,27-dimethyl-20,21,22,22,23,23-

C30H44O3

hexadehydro-24 ahomo vitamin D3 6.441

Protoporphyrinogen IX

C34H40N4O4

7.13

Pro Trp Ly

C22H31N5O4

7.91

Arg Gly Val

C13H26N6O4

8.396

Ursinic acid

C15H16O5

8.466

Eriodictyol

C15H12O6

8.72

Daidzein

C15H10O4

8.88

Leu Arg Thr

C16H32N6O5

9.321

1-[[2-(2,3-dihydro-2-oxo-1Hindol-4-yl) ethyl] propyl

C20H26N2O9

carbamate] glucuronide 9.815

2-alpha-Fluoro-19-nor-22-oxa-1 alpha, 25-dihydroxy vitamin

C25H41FO4

D3 9.834

6-(sulfooxy)-2-naphthaleneacetic acid

C12H10O6S 22

9.88

Pteryxin

C21H22O7

9.998

Deoxysappanone B trimethyl ether

C19H20O5

10.012

Lys Cys His

C15H26N6O4S

10.09

Phenyl glucuronide

C12H14O7

10.091

Penicillamine cysteine disulfide

C8H16N2O4S2

10.545

Dihydrodeoxystreptomycin

C21H41N7O11

10.613

Praziquantel

C19H24N2O2

10.614

Ala Trp Asp

C18H22N4O6

10.739

Dipyridamole

C24H40N8O4

11.185

Sappanone A 7-methyl ether

C17H14O5

11.214

10-Hydroxyloganin

C17H26O11

11.218

Desmethylondansetron

C17H17N3O

11.297

Trp Asp Val

C20H26N4O6

11.346

Inosine diphosphate

C10H14N4O11P2

12.55

Phytosphingosine

C18H39NO3

12.586

Trimipramine

C20H26N2

12.668

Pomiferin

C25H24O6

12.779

Pseudouridine

C9H12N2O6

13.027

Acetyl tyrosine ethyl ester

C13H17NO4

13.892

Dihydroceramide C2

C20H41NO3

13.968

2-Isoprenylemodin

C20H18O5

13.969

Khellin

C14H12O5

14.626

Ala Ile Arg

C15H30N6O4

14.626

N-ethyl arachidonoyl amine

C22H37NO

14.707

Benzylbutylphthalate

C19H20O4

15.611

Lys Cys Cys

C12H24N4O4S2

15.719

Granisetron

C18H24N4O

16.249

Desmethyldehydronimodipine

C20H22N2O7

16.804

Artesunate

C19H28O8

16.893

Deuteroporphyrin IX

C30H30N4O4

17.058

Desmethylmianserin glucuronide

C23H26N2O6

17.214

2,3-Dinor-8-iso-Prostaglandin F2alpha

C18H30O5

17.267

Thr Arg Met

C15H30N6O5S 23

17.366

Phthalic acid mono-2-ethylhexyl ester

C16H22O4

17.39

6'-Hydroxysiphonaxanthin

C40H56O5

17.503

4-Keto-4'-hydroxyalloxanthin

C40H50O4

17.837

2R-hydroperoxy-9Z,12Z,15Z-octadecatrienoic acid

C18H30O5

17.859

GPSer(17:0/14:1(9Z))

C37H73N2O10P

18.116

GPEtnNMe(O-14:0/O-14:0)

C34H72NO6P

18.373

Vitamin D3 glucosiduronate

C33H52O7

18.612

Phylloquinone (Vitamin K1)

C31H46O2

18.706

Lys Arg

C12H26N6O3

19.111

Idebenone Metabolite (1,4-Benzenediol, 2-(10-hydroxydecyl)-

C19H32O5

5,6-dimethoxy-3-methyl-) 19.52

18-acetoxy-1alpha,25-dihydroxyvitamin D3 / 18-acetoxy-

C29H46O5

1alpha,25-dihydroxycholecalciferol 19.585

Desmethylnimodipine

C20H24N2O7

20.46

(±)5-HETrE

C20H34O3

24.209

3beta-hydroxy-11-oxo-5betacholan-24-oic acid

C24H38O4

24

Table 4. The components present in EBE extract identified by GC-MS analysis Retention Compound

Molecular

time

formula

4.03

Tetrahydro-3-methyl-5-oxo-2-furancarboxylic acid

C6H8O4

4.03

Ethyl 4-(ethyloxy)-2-oxobut-3-enoate

C8H12O4

4.03

3-Methyl-isoxazol-5(4H)-one

C4H5NO2

5.36

1,1,3,3,5,5,7,7-Octamethyl-7-(2methylpropoxy)tetra siloxan-1-ol

C12H34O5Si4

5.36

Cyclotetrasiloxane, octamethyl-

C8H24O4Si4

5.36

3-Butoxy-1,1,1,5,5,5-hexamethyl-3-(trimethylsiloxy) trisiloxane

C13H36O4Si4

6.37

2-Hexynyl aldehyde diethyl acetal

C10H18O2

6.37

2,4-Hexadiene,1,1-diethoxy-

C10H18O2

6.37

2,4-Hexadiene,1,1-diethoxy-,(E,E)-

C10H18O2

7.82

Cyclopentasiloxane, decamethyl-

C10H30O5Si5

7.82

Benzoic acid, 2,6-bis[(trimethylsilyl)oxy]-, trimethylsilyl ester

C16H30O4Si3

7.82

3,4-Dihydroxymandelic acid, ethyl ester, tri-TMS

C19H36O5Si3

8.40

Dodecane

C12H26

8.40

Tetradecane

C14H30

8.40

Pentadecane

C15H32

10.37

Cyclohexasiloxane, dodecamethyl-

C12H36O6Si6

10.37

Heptasiloxane, 1,1,3,3,5,5,7,7,9,9,11,11,13,13-tetradecamethyl-

C14H44O6Si7

10.37

Octasiloxane, 1,1,3,3,5,5,7,7,9,9,11,11,13,13,15,15-

C16H50O7Si8

hexadecamethyl11.19

Dodecane, 2,5-dimethyl-

C14H30

12.65

Cycloheptasiloxane, tetradecamethyl-

C14H42O7Si7

12.65

3-Isopropoxy-1,1,1,7,7,7-hexamethyl-3,5,5-tris(trimethylsiloxy)

C18H52O7Si7

tetrasiloxane 12.65

Octasiloxane, 1,1,3,3,5,5,7,7,9,9,11,11,13,13,15,15-

C16H50O7Si8

hexadecamethyl17.19

Dibutyl phthalate

C16H22O4

17.19

Phthalic acid, butyl hept-4-yl ester

C19H28O4

17.19

Phthalic acid, butyl hept-3-yl ester

C19H28O4

17.69

Hexadecanoic acid, ethyl ester

C18H36O2

17.69

Hexadecanoic acid, 2-methyl-, methyl ester

C18H36O2 25

17.69

Ethyl 13-methyl-tetradecanoate

C17H34O2

19.23

9,12-Octadecadienoic acid, ethyl ester

C20H36O2

19.23

n-propyl 9,12-octadecadienoate

C21H38O2

19.23

Butyl 9,12-octadecadienoate

C22H40O2

19.54

Heptadecanoic acid, 15-methyl-, ethyl ester

C20H40O2

19.54

Octadecanoic acid, ethyl ester

C20H40O2

19.54

Ethyl 13-methyl-tetradecanoate

C17H34O2

22.37

Diisooctyl phthalate

C24H38O4

22.37

Phthalic acid, di(2-propylpentyl) ester

C24H38O4

22.37

Bis(2-ethylhexyl) phthalate

C24H38O4

24.58

13-Docosenamide, (Z)-

C22H43NO

24.58

trans-13-Docosenamide

C22H43NO

24.58

Bis(cis-13-docosenamido) methane

C45H86N2O2

26