1. Introduction
The Endoplasmic Reticulum (ER) is a key subcellular organelle involved in the synthesis, folding, modification, and transport of proteins. Chronic ER stress impairs cognitive functions and causes weaknesses in learning and memory (Sindi, Kareholt et al., 2017), depression (Mahar et al., 2014), and anxiety (Herbison et al., 2017). Overstimulation of the hypothalamic-pituitary-adrenal axis harms the central nervous system (Marin et al., 2011). Zhang et al. (2014) proposed that ER stress due to restraining causes hippocampal apoptosis and cognitive impairments. 
ER is the main site for steroids, cholesterol, and other lipids. Chronic restraint stress can cause depressive-like behavior by causing neuroinflammation and oxidative stress. Many factors are involved in this process, like pro-inflammatory cytokines and reactive oxygen species generation. The release of Ca2+ from ER enters the mitochondria, where they release reactive oxygen or nitrogen species (ROS/RNS). This reaction further enhances ER stress leading to apoptosis, neuroinflammation, and neurotoxicity. ER stress-induced apoptosis is involved in the stimulation of ER-resident caspase-12, which accordingly initiates caspase-3 (Nakagawa & Yuan, 2000; Nakagawa et al., 2000). Scheper, & Hoozemans, (2015) reported that unfolded protein response up-regulates genes encoding ER chaperones, decreases translation, or increases ER-associated degradation of aggregated proteins. Bettigole, & Glimcher (2015) reported that protein kinase R-like ER kinase, inositol-requiring enzyme 1α, and Activating Transcription Factor 6 (ATF6) are crucial transmembrane proteins that start unfolded protein response.
Imipramine, a tricyclic antidepressant, reduces sadness and lethargy and improves mood and overall body tone. The drug has successively been used in various neurodegenerative disorders like anxiety or depression.
Zanthoxylum alatum (ZA) Roxb. (Rutaceae) is an important medicinal xerophyte, tree, or shrub, that grows up to 6 m with dense foliage and armed branched flattened prickles. It comprises about 150 genera (Nasir, 1979). The dried fruit of ZA contains an aroma, which is present in the pericarp shell of the brown fruit wall (Latika et al., 2013). In India, the plant Z. alatum (Rutaceae) is found in the fierier valleys of the Himalayas from Jammu and Kashmir to Assam and Khasi hills, in the Eastern Ghats in Orissa and Andhra Pradesh, and the lesser Himalayan areas in the North-Eastern states of India, including Naga Hills, Meghalaya, Mizoram, and Manipur (Kala et al., 2005). Z. alatum is known for its curative properties as a traditional remedy for various ailments. It is a carminative, stomachic, and anthelmintic drug (Singh, & Singh, 2011). Its fruit and seeds are used for curing fever and dyspepsia as an aromatic tonic. Fruits extract is beneficial in roundworm infestation. It is also used in treating cold and cough, tonsillitis, headache, fever, vertigo, diarrhea, and dysentery (Geweli et al., 2008). Ethanolic extract of ZA possesses antioxidant (Batool et al., 2000) and anti-inflammatory activity (Sati et al., 2011). The essential oil of ZA has antispasmodic, antimicrobial, cytotoxic, and phytotoxic properties (Barkatullah et al., 2013). Essential oils of ZA seeds have various chemicals, including alkaloids, flavonoids, flavonol glycosides, lignins, phenolics, sterols, terpenoids, fatty acids, alkanoic acids, and amino acids (Kalia et al.,1999). In our previous studies, we reported its anti-depression (Barua et al., 2018), anticholinergic, antihistaminic, antiserotonergic activity (Saikia et al., 2017), and memory-enhancing property (Saikia et al., 2018). However, the seeds have a strong aroma and contain essential oil. Thus, we aimed to explore the modulation of genes involved in ER stress at a molecular level in the chronic restraint stress model in mice. The 78-kDa Glucose-Regulated Protein (GRP78) and 94-kDa glucose-regulated protein (GRP94) genes are crucial to keep the ER functions. Sharma et al. (2018) reported that inhibition of Protein Kinase RNA (PKR)- like ER kinase expression in the hippocampus could improve hippocampal-dependent memory and undo memory decline in mice. The GRP78, GRP94, ATF6, XBP1, ATF4, and CHOPgene expression increased in the hippocampus of rats with learned helplessness (Timberlake, & Dwivedi, 2016). To correlate its behavioral parameters, a few important stress markers, namely CHOP, GRP78, GRP94, and Caspase-12, were also investigated. We theorized that Z. alatum treatment could ameliorate the chronic restrain stress-induced depressive-like behavior and cognitive impairment.

2. Materials and Methods 
Chemicals
Imipramine hydrochloride was procured from Sigma-Aldrich Corporation (St. Louis, USA). Ethanol, ether, and acetonitrile were procured from Merck (M) and 2-thiobarbituric acid from “HiMedia.” 

Laboratory animals
We obtained 30 healthy male Swiss albino mice (30±5 g) from the animal house of the Department of Pharmacology and Toxicology, College of Veterinary Science, Khanapara, Assam. The Institutional Animal Ethics Committee (IAEC) of the College of Veterinary Sciences, Assam Agricultural University, Khanapara, permitted the study protocol (No.770/ac/CPCSEA/FVSc, AAU/IAEC/15-16/367). The animals were familiarized with the Lab condition for two weeks before conducting the experiment. The mice were kept in polypropylene cages, and food and clean drinking water were provided ad libitum. They were maintained in a standard laboratory condition (12:12 h light/dark cycle at an ambient temperature of 22°C-25°C and 30% relative humidity) according to the National Institutes of Health (NIH) guidelines for the Care and Use of Laboratory Animals.

Plant material and preparation of extract
Seeds of Z. alatum were obtained from Arunachal Pradesh from July to August. The seeds were identified by Dr. I.C. Barua, Principal Scientist, Department of Agronomy, Assam Agricultural University. The voucher specimen was kept in the herbaria (5109, dated-25.09.2014) for future reference. The seeds were cleaned and dried in the shade for a week and then grinded in an electric grinder and powdered. Next, 250 g of powder was soaked in 1000 mL of hydroalcoholic solution (ethanol and water in the ratio of 70:30) for 72 h in a beaker; the mixture was stirred with a sterile glass rod till it became colorless. The filtrate was evaporated using a rotary evaporator (Buchi R-210, BÜCHI Labortechnik AG, Switzerland) to remove the solvent. The recovery percentage was 19.71% w/v. Phytochemical screening of Z. alatum disclosed the presence of steroids, glycosides, alkaloids, diterpenes, and triterpenes (Kalia et al., 1999). 

Identification of active compound
We used an ultrahigh-performance liquid chromatography UHPLC system with an ESI OrbitraPMS/MS to spot the phytoconstituents in the hydroethanolic extract of Z. alatum (Kumar et al., 2016). The mobile phase of solvent A: water with formic acid (0.01%) and solvent B: 100% acetonitrile were used with a steady flow rate of 0.3 mL/min by subsequent gradient method. It began with 95% A for 2 min, then gradually reduced to 5% A in 6 mins and hold at 5% A for 1 min, then to the starting conditions, 95% A for 1 min. Samples (5 μL) were injected onto a Hypersil GOLD C18 column (150 x 3.00 mm, Thermo, USA). For the identification, by simultaneous screening at 275 nm, 366 nm, and 200-400 nm, we used a photodiode array detector. Also, we used it for the analysis of the full mass peak and fragmentation pattern of the phytoconstituents mass spectrometer. The observed mass-to-charge ratio of the sample was compared with the literature and mass databases that were the primary tool for the characterization of the phytoconstituents (Figures 1, 2, and Table 2).

Drug treatments and experimental design
Acute toxicity study
The acute toxicity study was conducted following the protocol of Organization for Economic Cooperation and Development guidelines for testing chemicals (OECD 423). The extract was fed orally at 2000 mg/kg to 3 mice, and the percentage of mortality, if any, was noted. They were observed for the next 14 d for mortality or gross abnormality with the given doses. Based on the acute toxicity study, 100 and 200 mg/kg oral doses were selected for the present study. 

Experimental Design
Five groups (n=6) of experimental animals were restrained for six hours every day for 28 days in 50 mL polystyrene tubes. They were divided into the following groups: group I, or normal control, which received vehicle (Tween-80 and saline PO); group II, negative control, or restraint group; group III, or standard group, which received imipramine 10 mg/kg IP; group IV, or ZAHA 100 mg/kg PO; and group V, or ZAHA 200 mg/kg PO. 

Stress protocol 
The extract was given per os from the 22nd day of restraint till the 28th day (Chiba et al., , 2012). Administration of drugs was done for 7 days 45 min prior to stress. Imipramine (10 mg/kg, IP) was administered to group III. A forced swim test was performed for the behavioral study. After that, the mice were sacrificed by cervical decapitation immediately after the last day of restrain. Their hippocampus was dissected carefully for further biochemical and molecular analysis and kept at -80°C till further estimation.

Behavioral studies 
To evaluate the despair behavior of mice, we performed the forced swim test (Porsolt et al., 1977) 24 h after the last day of restrain. We kept each animal in an unpreventable chamber of measurement 10 cm in diameter filled up with water (25°C) up to 15 cm for a complete time span of 6 min; an initial 2 min were considered as the acclimatization period, and perception for the last 4 min was considered to conduct the stressful behavior. The immobility duration of mice was recorded using Any Maze apparatus (Stoelting Co., USA).

Body weight 
To study the effect of stress on food intake, the body weight of the animals was taken at the beginning and the end of the experiment.

Oxidative stress analysis
Lipid peroxidation and enzyme assays 
The lipid peroxide in the brain homogenate was assessed by the thiobarbituric acid reactive substances (Ohkawa et al., 1979) method at 532 nm (Multiskan GO, Themofisher Scientific). The method of Bradford (1976) was employed to measure the total protein. Reduced glutathione (GSH) was measured with the slight modification of the Moron et al. (1979) method and superoxide dismutase (SOD) by Marklund and Marklund’s (1974) method.

Quantitative real-time PCR
The mRNA expression levels of GRP94, GRP78, CHOP, and Caspase-12 genes were assayed by real-time PCR (Applied Biosystems). Messenger RNA was isolated from the hippocampus using TRIzol (Ambion), followed by 1 µg of mRNA as reverse transcribed using the RevertAid First Strand cDNA synthesis kit (Thermo Fisher Scientific India Pvt). The resultant cDNA was amplified separately with specific primers for GRP94, GRP78, CHOP, and Caspase-12 using a standard protocol (Applied Biosystems 7500 Real-Time PCR system). Table 1 lists the primers (ILS primers, India) . 

Statistical analysis
The results are expressed as Mean±Standard Error of the Mean. Statistical analysis was performed by 1-way analysis of variance followed by Dunnett’s post hoc test in Graph Pad Prism software version 5.0 (version 5.0, Graph Pad Software Inc., San Diego, CA, USA). All results were considered statistically significant when P<0.05.

3. Results
UHPLC-ESI OrbitraPMS/MS analysis to identify phenolic compounds
The gradient method was used to identify the phytoconstituents in Z. alatum extract. The chromatogram was recorded at 365 nm. Figure 1 shows the chromatographic representation.

The peaks were identified by comparing the retention time (RT), λ max, and mass spectra of the Z. alatum extract from the literature and database. Peaks with RT (min) of 1.70, 2.08, 9.31, and 9.53 (peaks 1-4) were recognized as hesperidin, magnoflorine, melicopine, and sesamin (Figure 2).

The m/z of the hesperidin (C28H34O15) was 610.565 [M-H]+ (calculated: 610.565), of the magnoflorine (C20H24NO4) was 341.09201 [M-H]+ (calculated: 342.415), of the melicopine (C17H15NO5) was 314.19891 [M+H]+ (calculated: 313.309), and of the sesamin (C20H18O6) was 353.09232 [M-H]+ (calculated: 354.358) (Bhatt, Sharma, Kumar, Sharma, & Singh, 2017; Kumar et al., 2014). 

Effect of zanthoxylum alatum on the duration of immobility in the forced swim test
A significant increase in the immobility time (128±2.66 s, P<0.001) was observed compared to the normal control group (49.50 ±1.36 s). However, ZAHA pretreatment at 100 and 200 mg/kg reduced stress, as shown by the immobility time (92.53±2.92 s and 75.99±2.56 s, P<0.001). A similar result was observed with imipramine (Figure 3).

Effect of zanthoxylum alatum on the body weight
The mice’s mean body weight decreased (-3.94±0.11 g, P<0.001) in the restraint group compared to the normal control group (2.61±0.62 g). Imipramine (10 mg/kg, IP) caused significant gain (0.49±0.11 g, P<0.001) in the body weight. Also, ZAHA pretreatment at both doses significantly increased (-2.46±0.21 g, P<0.05) the body weight compared to the chronic restraint group (-1.16±0.07 g, P<0.001) (Figure 4).

Effect of Zanthoxylum alatum on lipid peroxidation
We found a significant increase (7.78±0.64 nM/mg protein, P<0.01) in the malondialdehyde (MDA) level in the chronic restraint group compared to the normal control group (3.99±0.52 ƞM/mg protein). Imipramine showed significant decline (4.17±0.38 nM/mg protein, P<0.01) in MDA level. ZAHA at both doses significantly reduced the MDA level (5.13±0.88 ƞM/mg protein, P<0.05; 4.51±0.73 nM/mg protein, P<0.01) compared to the chronic restraint group (Figure 5a).

Effect of Zanthoxylum alatum on oxidative enzymes 
Here we discuss the effect of pretreatment on reduced glutathione (GSH) and superoxide dismutase (SOD).

Reduced glutathione
There was a significant reduction in the GSH level (0.92±0.29 µg/mg protein, P<0.01) in the stress group when compared to a normal control group (3.20±0.22 µg/mg protein). Imipramine significantly increased GSH level (2.90±0.16 µg/mg protein, P<0.01). Pretreatment with ZAHA at both doses, significantly increased GSH level (2.38±0.36 µg/mg protein, P<0.05) compared to the chronic restraint stress group (2.56±0.66 µg/mg protein, P<0.01) (Figure 5b).

Superoxide dismutase
A significant decrease in the SOD level (1.01±0.32 U/mg protein; P<0.001) was recorded in the stress group compared to the normal control group. Imipramine as a standard drug increased SOD level (2.95±0.36 U/mg protein, P<0.01). Pretreatment with ZAHA (100 and 200 mg/kg) significantly increased SOD (2.38±0.28 U/mg protein, P<0.05) compared to the stress group (2.71±0.20 U/mg protein, P<0.01) (Figure 5c).

Effect of Zanthoxylum alatum on mrna expression by real-time polymerase chain reaction
In the restraint group, significant upregulation of the hippocampal genes, i.e., GRP94 (P<0.001), GRP78 (P<0.001), CHOP(P<0.001), and Caspase-12 mRNA (P<0.001) compared to the vehicle control group were observed. The ZAHA treatment significantly down-regulated the expression of the GRP94, GRP78, CHOP, and Caspase-12 mRNA compared to the control group (P<0.001, P<0.05, P<0.001, and P<0.001 for 100 and 200 mg/kg of ZAHA), but (P<0.01) for 100 mg/kg of ZAHA in the CHOPgene expression (Figure 6a-d).

GRP94 gene expression
GRP94 gene expression in the restraint group was upregulated significantly (P<0.001) compared to the normal control group. On the contrary, a significant down-regulation (P<0.001) in the expression of the gene was observed in the imipramine, ZAHA (200 mg/kg and 100 mg/kg) treated groups (Figure 6a).

GRP78 gene expression
The level of expression GRP78 was upregulated significantly (P<0.001) in the restraint group compared to the normal control group. On the other hand, a significant down-regulation of the gene was observed in the standard drug (imipramine) treated group (P<0.001), ZAHA (100 mg/kg) (P<0.05) and ZAHA (200 mg/kg) (P<0.05) compared to chronic restraint stress-induced group (Figure 6b).

CHOPgene Expression
CHOPgene expression in the restraint group also upregulated significantly (P<0.001) compared to the normal control group. Conversely, a significant down-regulation (P<0.001) in the expression of the gene could be observed in the imipramine, ZAHA (200 mg/kg and 100 mg/kg) (P<0.01) treated groups compared to the restraint group (Figure 6c).

Caspase-12 gene expression
The level of expression of Caspase-12 was upregulated significantly (P<0.001) in the restraint group, compared to the normal control group, whereas a significant down-regulation of the gene could be observed in the imipramine treated group (P<0.001), ZAHA (100 mg/kg) (P<0.001) and ZAHA (200 mg/kg) (P<0.001) compared to the restraint group (Figure 6d).

4. Discussion
In this study, we evaluated the effect of Zanthoxylum alatum on depression-like behavior induced by chronic restraint stress. We took imipramine as a reference drug to compare the effects of Zanthoxylum alatum because it has a strong antidepressant potential shown by previous studies (Han et al., 2011). The forced swim test is a physiological model for interpreting chronic to restraint stress-induced depressive-like behavior. In the present study, the forced swim test results showed increased immobility time with chronic restraint stress, which reflects behavioral despair and depressive-like behavior in mice. Since the reduction in immobility time is considered beneficial in assessing antidepressant agents, imipramine, a tricyclic antidepressant and conventional drug for depression, was found to be effective in reducing the immobility time. ZAHA also showed a similar effect but with less potency than imipramine. 
Loss of body weight in the chronic restraint group was also observed compared to the normal control group, which might be due to reluctance in eating due to restraint stress for 28 days continuously. Imipramine and ZAHA (slightly) ameliorated the loss in body weight. 
Antioxidant enzymes can stabilize or deactivate free radicals and inhibit oxidative damage. This phenomenon is depicted by reduced SOD and GSH levels in the hippocampus, one of the possible mechanisms in depression pathophysiology (Ahmad et al., 1993). These free radicals react with membrane lipids rich in polyunsaturated fatty acid and form lipid peroxidation, generating Malondialdehyde (MDA) that causes cell membrane damage. 
In our study, we found that imipramine and ZAHA significantly reduced oxidative stress and malondialdehyde formation. The results are very promising in this model, and the test compound showed potential in counteracting chronic stress, and the resultant depressive behavior is also taken care of. This effect is due to their diverse phytochemicals, which were identified in our study.
According to previous studies, up-regulation of ER stress-related genes in depression could compensate for the harmful effects of prolonged stress, glucocorticoid release from vulnerable brain regions such as the hippocampus, and so on (Bown et al., 2000; Ishisaka et al., 2011; Barua et al., 2018). Studies also show ER stress is responsible for memory impairment under different pathophysiological situations (Zhang et al., 2014; Jangra et al., 2016; Barua et al., 2018).
The phytochemical study reveals hydroalcoholic extract contains alkaloids, glycoside, triterpenes, tannic acid, etc. Its chemical analysis was reported by various other researchers (Akhtar et al., 2009; Ranawat et al., 2010). We identified magnoflorine, melicopine, sesamin, and hesperidin in our study. Hesperidin possesses antioxidant, anti-inflammatory, neuroprotective effects, and anti-carcinogenic activities (Cho, 2006; Roohbakhsh et al., 2015). Magnoflorine has cytotoxic, antiviral (Mohamed et al., 2010), antioxidant (Li, & Wang, 2014), and anti-amnesic properties (Koch et al.,2017). Melicopine is an acridone alkaloid with cytotoxic and antimalarial activity (Wang et al., 2014). Sesamin has antinociceptive, anti-inflammatory (Monteiro et al., 2014), and anti-chronic stress activity (Zhao et al., 2016). 
CHOP, GRP78, GRP94, and Caspase12, the standard indicators for stress, were upregulated following ER stress in the experimental animals. However, following pretreatment with ZAHA, the above genes were down-regulated, indicating that they counteracted ER stress. The C/EBPhomologous protein (CHOP) is a transcription factor that activates at different levels during ER. It is activated by the p38 kinase (Wang, & Ron, 1996). Deregulated CHOPmovement compromises cell viability (Zhan et al., 1994), and cells without CHOPare essentially shielded from the deadly outcomes of ER stress (Oyadomari et al., 2002). This condition was observed in our study. 
GRP78 is a key regulator of the ER stress response. Overexpression of GRP78 inhibits the up-regulation of CHOP, which induces apoptosis. ZAHA treatment downregulated the expression of this deleterious gene and inhibited apoptosis.
Caspase-12 shows resistance to ER stress-mediated apoptosis (Nakagawa et al., 2000; Rao et al., 2001). Activated caspase-12 can activate caspase-9, which cleaves procaspase-3. Activated procaspase-3 would lead to the apoptosis of cells (Yingying et al., 2008). In our study, expression of Caspase-12 was also down-regulated following treatment with imipramine and ZAHA.
Glucose-regulated protein 94 is the HSP90-like protein in the lumen of the endoplasmic reticulum. This protein functions in the development and physiology of organisms as antigen-presenting cells, producing pro-inflammatory cytokines and priming the adaptive immune response (Yang et al., 2007). Thus, the amplified expression of GRP94 indicates ER-stressed cell priming for inflammatory interactions was significant in our study in the restraint group. Davide et al. (2010) reported that many recent genetic, biochemical, and cell biological studies have shed light on the functions of GRP94. 
Our investigation revealed that ZAHA could restrain GRP94 and Caspase-12 expression by recovering ER stress. Treatment with methanolic extract of Entada phaseoloides seeds weakened ER stress in chronic stress in mice (Barua et al. 2018). Jangra et al. (2016) reported that Honokiol degenerates ER stress by down-regulating GRP78 and CHOPexpressions. Barua et al. (2017) reported that Elsholtzia communis counteracts stress by modulating the expression of hsp14, CHOP, Nrf2, Caspase-3, and brain-derived neurotrophic factor in rat hippocampus.

5. Conclusion
The seed extracts of Z. alatum have many customary medicinal properties, including their use as nerve tonic and stimulant in debilitated patients. Its positive effect has been shown in reverting ER stress in our study. It thwarts the restraint of stress-induced depressive-like behavior in mice under its antioxidant property, down-regulating the stress markers like GRP78, GRP90, CHOP, and Caspase-12 due to an assortment of phytoconstituents present therein. 

Ethical Considerations
Compliance with ethical guidelines
Mice were used according to the NIH Guide for the Care and Use of Laboratory Animals, and the experiments were performed following approval of the protocol by the Ethics Committee of the College of Veterinary Sciences, Assam Agricultural University (No.770/ac/CPCSEA/FVSc, AAU/IAEC/15-16/367) to minimize the suffering.

Funding
This work was financially supported by the Life Science Research Board, Defense Research and Development Organization, and the Government of India (Grant No.: 81/48222/ LSRB-286/EPB/2014 dt 17.11.14).

Authors' contributions
All authors equally contributed to preparing this article.

Conflict of interest
The authors declared no conflict of interest.

Acknowledgments
We sincerely thank the Director of Research (Vety), AAU, Khanapara, for providing the necessary facilities to carry out this work. Also, we thank taxonomist Dr. Iswar Chandra Barua, Principal Scientist, Department of Agronomy, AAU, Jorhat, for identifying the plant. 

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