Protective effects of Spinacia oleracea seeds extract ...

Biomedicine & Pharmacotherapy 105 (2018) 1015–1025

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Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha

Protective effects of Spinacia oleracea seeds extract in an experimental model of schizophrenia: Possible behavior, biochemical, neurochemical and cellular alterations

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Monu Yadava, Milind Parlea, Nidhi Sharmaa, Deepak Kumar Jindala, Aryan Bhidhasrab, ⁎ Mamta Sachdeva Dhingrac, Anil Kumarc, Sameer Dhingrad, a

Faculty of Medical Sciences, Department of Pharmaceutical Sciences, Guru Jambheshwar University of Science & Technology, Hisar, 125001, India Department of Veterinary Pharmacology and Toxicology, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar, 125001, India University Institute of Pharmaceutical Sciences, UGC Centre of Advanced Study (UGC-CAS) in Pharmaceutical Sciences, Panjab University, Chandigarh, 160036, India d Faculty of Medical Sciences, School of Pharmacy, The University of the West Indies, St. Augustine, Trinidad and Tobago b c

A R T I C LE I N FO

A B S T R A C T

Keywords: Spinacia oleracea Behavioral Schizophrenia Biochemical Neurochemical Protective

Schizophrenia is one of the psychotic mental disorders characterized by symptoms of thought, behavior, and social problems. Newer biomedicine and pharmacotherapy has been investigated for the treatment of various neuropsychiatric disorders in the past few decades. Spinacia oleracea is one of these, reported to have beneficial effect against several neurodegenerative disorders. The present study was carried to explore the protective effects of Spinacia oleracea seed extract (SOEE) in an experimental model of ketamine-induced schizophrenia in mice. Ketamine (50 mg/kg, i.p.) was used to induce stereotyped psychotic behavioural symptoms in mice. Behavioral studies (locomotor activity, stereotype behaviors, immobility duration and memory retention) were carried out to investigate the protective of SOEE on ketamine-induced psychotic symptoms, followed by biochemical, neurochemical and cellular alterations in the brain. Treatment with SOEE for 15 consecutive days significantly attenuated stereotyped behavioral symptoms in mice. Biochemical estimations revealed that SOEE reduced lipid peroxidation and restored total brain proteins. Furthermore, SOEE remarkably reduced dopamine levels, AChE activity & inflammatory surge (serum TNF-α) and increased the levels of GABA and reduced glutathione in mice. The outcomes of the study suggested that SOEE could ameliorate ketamine-induced psychotic symptoms in mice, indicating a protective effect in the treatment of schizophrenia.

1. Introduction Schizophrenia is an incapacitating ailment, generally characterized by stereotypic behaviour, bizarre behaviours or positive symptoms, depressive or negative symptoms and cognitive symptoms with the global burden around 1% [1]. Dopaminergic deregulation, hypofunction of NMDA receptors and GABAergic activity, diminished cholinergic firing, neuroinflammation and increased oxidative stress has been demonstrated to play a pathophysiological role in schizophrenia [2–5]. Typical (first generation) and atypical (second generation) antipsychotic medicines are currently prescribed for the management of schizophrenia, which are also effective against hallucinations, delusions and thought disorders [6]. First generation antipsychotics are only effective in the management of positive symptoms of schizophrenia and also associated with the extra-pyramidal effects [6], while second generation antipsychotics are beneficial in positive, negative and also in

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Corresponding author. E-mail address: [email protected] (S. Dhingra).

https://doi.org/10.1016/j.biopha.2018.06.043 Received 11 February 2018; Received in revised form 8 June 2018; Accepted 12 June 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.

cognitive symptoms of schizophrenia with lesser extra-pyramidal effects, but may cause diabetes mellitus, cardiovascular disorders and agranulocytosis [7]. Though, antipsychotics have been used clinically for several decades, most of them have incomplete efficacy and also coupled with many adverse effects as well as increased risk of psychotic relapse [7]. Thus, there is a great need of therapeutic interventions for managing psychiatric disorders, which will not only provide symptomatic relief but also halt the progression of disease. Natural medicinal agents are the power house of various bioactive constituents which possess biochemical specificity, chemical diversity and medicinal properties that make them essential remedies in various human disorders including schizophrenia [8]. Various medicinal herbs have been specifically studied against various neuropsychiatric disorders and many of them have shown promising results based on their therapeutic potential and safety profile [8]. Spinacia oleracea Linn. family Chenopodiaceae commonly known as


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2.6. Doses selection

spinach is endowed with a number of medicinal properties [9]. Ethnopharmacological studies proposed that Spinacia oleracea seeds have promising antioxidant, neuroprotective, anti-epileptic, anti-alzheimer and anti-inflammatory effects [10–13]. It is an excellent package of unique phytoconstituents such as ascorbic acid, apigenin, astragalin, caffeic, lutein, β-carotene, ferulic acid, kampeferol, rutin, querecetin, myricetin, luteolin, ortho-coumaric acid, para-coumaric acid, protocatechuic acids, methylenedioxy flavonol, glycoglycerolipids, 20-hydroxyecdysone, spirasaponins, stigmasterol, violaxanthin and vitamins, etc., which are responsible for its biomedicinal and pharmacotherapeutic effects [14–18]. Therefore, the present study was designed to investigate the effects of Spinacia oleracea seeds in ketamine-induced animal models of schizophrenia along with bar test to rule out the cataleptic effect which is commonly associated with antipsychotic agents. Biochemical estimations of GABA, dopamine, reduced glutathione, MDA, TNF-α and AChE activity were carried out to study the mechanism of action of Spinacia oleracea seeds.

Doses of haloperidol (1 mg/kg, i.p), ketamine (50 mg/kg, i.p), olanzapine (5 mg/kg, i.p) and ethanol extract of Spinacia oleracea seeds (SOEE: 50, 100 and 200 mg/kg, p.o.) were selected from the previous studies [19–21]. 2.7. Determination of in vitro antioxidant activity using DPPH scavenging method DPPH (1,1-Diphenyl,2-picryl-hydrazyl) was used to study the free radical scavenging effect (in tripilcate) of SOEE according to the Sanchez-Moreno et al. method [22]. Different concentrations of SOEE (20–100 μg/ml) were prepared in methanol. 0.1 ml of extract samples was added in 3.9 ml of DPPH solution (0.025 g/l in methanol). The mixtures so obtained were incubated in the dark for 30 min and then absorbance was recorded at 517 nm using UV–vis spectrophotometer. Percentage of DPPH∙ remaining was calculated by the calibration curve of DPPH followed by determination of IC50 value [18].

2. Materials and methods 2.1. Plant material

2.8. Determination of in vitro anti-inflammatory activity using the membrane stabilizing method

Dried seeds of Spinacia oleracea Linn.were procured from Chaudhary Charan Singh Haryana Agricultural University, Hisar (Haryana), India. These seeds were identified and authenticated from the National Institute of Science Communication and Information Resources (NISCAIR), New Delhi vide specimen reference number 2015/2894/87.

Anti-inflammatory activity of SOEE was evaluated by hypotonic solution induced erythrocyte haemolysis [23]. The blood of swiss albino mouse was collected in a container containing anticoagulant. Washings were done thrice with normal saline and then centrifuged at 3000 rpm for 10 min. Normal saline was used for washing of packed cells and using hypotonic solution (sodium phosphate buffered saline, composed of 154 mM NaCl in 10 mM Sodium Phosphate Buffer at pH 7.4), 40% v/v suspension was made, collectively used as RBC suspension or stock erythrocyte. The test sample contained 0.030 ml of stock erythrocyte with 5 ml hypotonic solution containing different concentrations of extract. The control sample composed of 0.030 ml stock erythrocyte with a hypotonic buffered solution only. Diclofenac sodium used as a reference drug at a concentration of 100 μg/ml. These mixtures were incubated for 10 min at room temperature, further centrifuged at 3000 rpm for 10 min and then absorbance of supernatant was measured at 540 nm using spectrophotometer. The percentage inhibition of haemolysis was calculated using the equation-

2.2. Extraction The coarsely powdered Spinacia oleracea Linn. seeds (800 g) were defatted using petroleum ether followed by successive extraction with ethanol using soxhlet extraction. The prepared extract was concentrated by distilling off the solvent followed by evaporation on a water bath to obtain the dried extract and stored in a desiccator for further evaluation. 2.3. Experimental animals Swiss albino mice (3 months old, 25–30 g weight, either sex) were purchased from Disease Free Small Animal House, Lala Lajpat Rai University of Veterinary and Animal Sciences (LUVAS), Hisar (HaryanaIndia). Six mice in a group were housed in a cage under standard laboratory conditions and acclimatized for at least seven days before the start of the experiments. The experiments were conducted between 9.00 a.m. to 5.00 pm. The animal protocol was approved by the Institutional Animals Ethics Committee (IAEC) and the animal care was taken as per the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forests, Government of India (Registration no. 0436).

%Inhibition of haemolysis = [(Ac − At)/ Ac]× 100 Where: Ac = Absorbance of control sample At = Absorbance of test/reference sample 2.9. Acute toxicity studies An Acute oral toxicity study was done as per OECD-423 guidelines [24]. Swiss albino mice of either sex (n = 3) were randomly selected and their body weight was measured on a daily basis during the SOEE administration. The animals were fasted for 4 h. The SOEE was administered orally at a concentration of 50 mg/kg initially and mortality was observed for 3 days. If the mortality was found in 2/3 or 3/3 animals, then the concentration administered was considered as toxic dose. If, mortality was seen only in one animal out of three animals then the same concentration was repeated again to confirm the toxic dose. If mortality was not seen, the procedure was then repeated with higher doses.

2.4. Drugs protocol Haloperidol (RPG Science Pharmaceutical Pvt. Ltd., India), ketamine (Neon Pharmaceutical Pvt. Ltd., India) and olanzapine (Ranbaxy Laboratories, India) were used for this study. ELISA kits for TNF-α were procured from Research and Development Systems, USA. Analytical grade chemicals were used for biochemical estimations. 2.5. Drug solutions

2.10. Experimental design Haloperidol, ketamine and olanzapine were separately diluted in normal saline. The ethanol extract of Spinacia oleracea Linn. seeds were suspended in 2% v/v Tween 80. All drug solutions were freshly prepared and administered to animals.

The animals were divided into 14 groups with 6 mice in each: Group I: Control, mice received vehicle only Group II: Negative control, mice received ketamine (50 mg/kg, i.p.) 1016


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free zone, if they remain on the platform for 60 s. After 24 h, memory was tested in a similar manner, except the shocks were not given and SDL was observed with a cutoff time of 5 min [28].

only Group III, IV: Positive control, mice received haloperidol (1 mg/kg, i.p.) or olanzapine (5 mg/kg i.p.) 30 min before ketamine (50 mg/kg, i.p.) Group V, VI, VII: Test sample, mice received SOEE (50 mg/kg, p.o.), SOEE (100 mg/kg, p.o.) or SOEE (200 mg/kg, p.o.) 30 min before ketamine (50 mg/kg, i.p) Animals in group VIII to XIV were treated in similar manner as in group I to VII. The drugs were administered daily for 15 days.

2.11.5. Bar test for catalepsy This test is used for assessment of catalepsy, a side effect of typical antipsychotic drugs. The mouse was placed with its front paws on the horizontal bar, which was 4.5 cm high and parallel to the base. When the mouse removed its either paw from the bar, time was recorded. The maximum cutoff time for bar test was 180 s [29].

2.11. Behavioral tests 2.12. Biochemical estimations

Psychotic symptoms were divided into stereotypic behaviours (turning, falling, head bobbing and weaving), bizarre or positive, depressive or negative and cognitive symptoms. Some behavioral models have potential relevance to signs and symptoms of schizophrenia [5–7]. On 7th day of drug administration, locomotor activity was measured using actophotometer (hyperlocomotor activity represents positive symptoms of schizophrenia). Number of falls and turns (stereotypic behaviours) were counted just after the administration of ketamine on the 10thday. Further, anhedonia, a negative symptom of schizophrenia were tested by recording immobility duration of mice using force swim test on the 12th day of drugs administration. Step down latency was measured to assess the effect of drug on learning and memory on passive avoidance test on 13th and 14th day of the experiment. On the 15th day, bar test was performed to investigate the cataleptic effect of SOEE and thereafter, blood was collected and serum separated by centrifugation which was used for analysing TNF-α level. Then, animals were sacrificed by decapitation method and brain was isolated for the estimation of biochemical parameters. Timeline depicting the treatment and the different tests

2.12.1. Collection of blood samples On the 15th day of the protocol, blood (0.5–0.8 ml) was removed from the retro-orbital plexus of mice in groups I to VII. Blood were centrifuged for 15 min at 3000 rpm using cooling centrifuge (Remi Instruments, Mumbai, India) to separate the serum, which was used for estimation of TNF-α levels. 2.12.2. Estimation of serum TNF-α level The reagents and standard dilutions were prepared as per the manufacturer’s instructions. 50 μL of assay diluents was added into each well of the precoated microtitre plate. Then, 50 μL of sample or standard was added per well and mixed. The plates were covered with the adhesive strip and incubated at room temperature for two hours. Each well was washed with wash buffer five times. The plate was inverted and blotted against a clean paper towel. Then, 100 μL of mouse TNFConjugate was added to each well and covered with adhesive strip. The plate was again incubated for two hours at room temperature. Repeated aspiration and washes were done. Then, 100 μL of substrate solution was added into each well and incubated at room temperature for 30 min, protected from light. Finally, 100 μL of stop solution was added to each well and the absorbance of wells was read in ELISA reader at 450 nm [30].

2.11.1. Locomotor activity Acute administration of ketamine produced hyperlocomotor activity in mice, which was measured using actophotometer (INCO, Ambala, India). The mouse was placed in the activity chamber for five minutes before assessing actual locomotor counts. Total number of locomotor counts were noted for further 5 min [25].

2.12.3. Preparation of brain homogenate for antioxidant parameters and AChE estimations After behavioural tests on the 15th day, animals in group I to VII were sacrificed using decapitation and their brain were isolated from the skull. The isolated brain was weighed and washed with buffer (pH 7.4) and divided into two parts: one part of the brain was weighed and homogenized in 0.1 M phosphate buffer (pH 7.4) and the resultant homogenate was used for estimation of reduced glutathione (GSH) and malondialdehyde (MDA). The other part was weighed and homogenized in 10 volumes of phosphate buffer (pH 8) followed by centrifugation for 10 min at 3000 rpm using cooling centrifuge and supernatant was used for the estimation of acetylcholinesterase (AChE) activity.

2.11.2. Stereotypic behaviours Acute administration of ketamine induced stereotypic behaviours in mice, which was assessed by counting total number of falling (number of falls) and turning (turn around) [26]. 2.11.3. Force swim test (FST) Chronic administration of ketamine induced immobility in mice, a sign of anhedonia, negative symptom of schizophrenia, was assessed using force swim test. The mouse was forced to swim in a water container of 25 × 12 × 25 cm, which was filled with water (23 ± 2 °C) up to 15 cm height. After an initial period of vigorous activity, each animal attained a typical immobile posture. The duration of immobility was measured during the 4 min period. The animal was considered to be immobile when it ceased to struggle, and the limbs seldom moved to keep the head above the water [27].

2.12.4. Estimation of brain reduced glutathione Ellman, 1959 method [30] was used for the estimation of reduced glutathione (GSH) level in brain tissue. 4% trichloroacetic acid (TCA) was used to precipitate the brain homogenate. This mixture was centrifuged at 5000 rpm for 10 min using cooling centrifuge (Remi Instruments, Mumbai, India). Then, 2 ml of 0.3 M disodium hydrogen phosphate buffer (pH 8.4) and 0.4 ml of double distilled water were added in 0.5 ml of resultant supernatant. 0.25 ml of 0.001 M DTNB [5,5′-dithiobis(2-nitrobenzoic acid)] was dissolved in 1% w/v sodium citrate and added into the above reaction mixture. The reaction mixture was incubated at room temperature for 10 min. The yellow colour developed was read in UV–vis Spectrophotometer at 412 nm. GSH level was calculated using a molar extinction coefficient of (1.36 × 104 M−1cm−1) and expressed as micromole per milligram protein.

2.11.4. Passive avoidance test The apparatus consisted of a box having three wood walls, one plexiglas wall, one grid floor with wooden platforms. A 15 W bulb was used to illuminate the box and shock (20 V AC) was given to the floor. Two training sessions were carried out. The animal was placed individually on the platform. When the mouse stepped down with its all paws on the floor, shocks were given for 15 s. Animals showing SDL between 2–15 second during first training were used for second training and further for memory test. The second training was given after 90 min of first training. When mouse stepped down before 60 s, shocks were given for 15 s. In the second training, mouse was removed from shock 1017


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2.12.5. Estimation of brain malondialdehyde Malondialdehyde (MDA), a parameter of lipid peroxidation, was estimated as thiobarbituric acid reactive substances (TBA) using Wills, 1965 method [31]. By mixing homogenate with tris−HCl (pH 7.4) in a ratio of 1:1 the reaction mixture was prepared and incubated for 2 h at 37 °C. After incubation, 1 ml of 10% ice cold TCA was added to the reaction mixture and centrifuged for 10 min at 1000 rpm using a refrigerated centrifuge. Then, 1 ml of the resultant supernatant was mixed with 1 ml of 0.67% TBA and kept the tubes for 10 min in boiling water bath. After cooling, 1 ml of double distilled water was added and absorbance was measured at 532 nm using UV–vis Spectrophotometer. Thiobarbituric acid reactive substances were calculated using an extinction coefficient of 1.56 × 105M−1cm−1 and expressed as nanomole of malondialdehyde per milligram of brain protein [31].

before iodine). Internal reagent standards were prepared by adding 2.5 ml HCl-Butanol and 0.125 ml distilled water to 500 ng of dopamine standard. The internal reagent blank was prepared by adding 0.125 ml water to 2.5 ml HCl-Butanol.

2.12.6. Estimation of brain acetylcholinesterase (AChE) activity Brain AChE activity was estimated according to the method described in Ellman, 1961 [32]. 0.4 ml of supernatant was added into test tube having 2.6 ml of phosphate buffer. Further, 0.1 ml DTNB (5, 5dithiobis-2-nitrobenzoic acid) reagent was added and absorbance was taken at 412 nm using UV–vis Spectrophotometer. 0.02 ml of acetylthiocholine iodide solution was added and absorbance was again recorded after 15 min. Then, a change in absorbance per minute was determined. The rate of hydrolysis of substrate was estimated by using the given formula.

2.13. Statistical analysis

2.12.9. Estimation of brain GABA level Gamma amino butyric acid (GABA) level was estimated as per the method described by Lowe (1958) [34]. 0.1 ml of the homogenate was added in 0.2 ml of 0.14 M ninhydrin solution in 0.5 M carbonate bicarbonate buffer (pH 9.95), kept for 30 min on water bath at 60 °C, then, cooled and treated with 5 ml of copper tartrate reagent (0.16% disodium carbonate, 0.03% copper sulphate and 0.0329% tartaric acid). After 10 min, samples were read at 377/455 nm in a spectrofluorometer.

The data were interpreted by One-way analysis of variance (ANOVA) followed by Tukey’s test using GraphPad Instat (San Digeco, USA) software. The results are expressed as mean ± standard error of mean and p < 0.05 was considered as statistically significant. 3. Results 3.1. DPPH scavenging activity of SOEE

R = 5.74(10−4) ΔA/Co

The DPPH free radical scavenging activity of SOEE was determined and compared with the quercetin. SOEE showed a tendency to scavenge the free radicals as indicated by decrease in DPPH∙concentration representing good antioxidant potential (Table 1).

R = rate of hydrolysis of acetylthiocholine iodide in mmol/min/g of tissue ΔA = change in absorbance per min Co = weight of tissue homogenate in mg/ml

3.2. Membrane stabilizing effect of SOEE 2.12.7. Preparation of brain homogenate for neurotransmitter level estimation On the 15th day, after behavioural tests, animals of Group VIII to XIV were sacrificed using decapitation method and their brain was isolated and weighed. The isolated brain was divided into two equal parts: one part was used for the estimation of dopamine level and another one for GABA level estimation. The first part of brain tissue wasweighed and homogenized in 3 ml HCl-Butanol (0.1 M HCl in butanol) in an ice cool environment and used for estimation of dopamine level. Second part of the brain was weighed and homogenized in 5 ml of 0.01 M hydrochloric acid and used for the estimation of GABA level.

in vitro antiinflammatory activity of SOEE was evaluated by a membrane stabilizing method in terms of percentage inhibition of haemolysis. SOEE has shown concentration dependent significant stabilization of erythrocyte membrane. The percentage inhibition by SOEE at different concentrations and their comparison with the standard drug (diclofenac sodium) has been shown in Table 2. 3.3. Acute toxicity studies An ethanol extract of SOEE did not produce any mortality and no change in body weight was observed even up to the dose of 2000 mg/ kg, p.o.

2.12.8. Estimation of brain dopamine level Schlumpf (1974) method [33] with slight modifications was used for the estimation of dopamine. HCl-Butanol brain homogenate was centrifuged for 10 min at 2000 rpm using cooling centrifuge. Then, 0.8 ml of supernatant was removed and added into an Eppendorf reagent tube having 2 ml of heptane and 0.25 ml 0.1 M HCl. After 10 min of shaking, the sample was again centrifuged and the aqueous phase was used for dopamine estimation. To 1 ml of HCl phase, 0.25 ml of 0.4 M HCl and 0.5 ml of EDTA/sodium acetate buffer (pH 6.9) were added, followed by 0.5 ml iodine solution (0.1 M in ethanol) for oxidation. This reaction was stopped after 2 min with the addition of 0.5 ml sodium thiosulphate (5 M in sodium hydroxide). After 90 s, 0.5 ml of 10 M acetic acid was added. Then, the above solution was heated at 100 °C for 6 min. When the samples were again reached to room temperature, excitation and emission spectra were recorded (330–375 nm) in photofluorometer (Systronic, Model 152, Ahmedabad, Gujarat). Then, the tissue values (fluorescence of tissue extract minus fluorescence of tissue blank) were compared with the internal reagent standard (fluorescence of internal reagent standard minus fluorescence of internal reagent blank). The tissue blanks were prepared by adding the reagents of the oxidation step in reverse order (sodium thiosulphate

3.4. Behavioural observations 3.4.1. Effect of SOEE on locomotor activity in actophotometer As shown in Table 3, treatment of SOEE (50, 100 and 200 mg/kg, p.o.) for seven successive days produced significant (p < 0.05, p < 0.001 and p < 0.001, respectively) decrease in the total number Table 1 Effect of ethanolic extract of Spinacia oleracea seeds (SOEE) on antioxidant activity. S. No.

Conc. (μg/ml)

Quercetin (% DPPH∙remaining)

SOEE (% DPPH∙remaining)

1. 2. 3. 4. 5.

20 40 60 80 100 IC50

44.33+0.62 34.33+0.94 23.36+0.31 11.71+0.74 2.06+0.49 9.9

88+0.61 78.32+0.52 68.88+0.26 47.85+0.47 33.99+0.21 80

Values are expressed as Mean ± SEM (n = 6). 1018


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Table 2 Percentage inhibition of haemolysis by SOEE.

Table 4 Effect of SOEE on immobility duration.

S. No.

Conc. (μg/ml)

Diclofenac sodium

SOEE

Treatments

Immobility duration (sec)

1. 2. 3. 4. 5.

100 200 300 400 500

63.82+0.17 67.49+0.43 74.63+0.25 79.16+0.31 87.43+0.63

47.36+0.24 54.28+0.26 57.62+0.58 63.25+0.43 71.48+0.30

Vehicle Keta (50 mg/kg) Keta+Halo (1 mg/kg) Keta + Olz (5 mg/kg) Keta + SOEE (50 mg/kg) Keta + SOEE (100 mg/kg) Keta + SOEE (200 mg/kg)

77.83 ± 1.35 140.00 ± 2.87* 132.66 ± 1.20 83.66 ± 1.33a 138.00 ± 1.39 119.66 ± 1.95c 106.66 ± 2.91b

Values are expressed as Mean ± SEM (n = 6).

Values are Mean ± SEM (n = 6). One way ANOVA followed by Tukey’s test. ‘*’ p < 0.001 vs vehicle treated group. ‘a’ p < 0.001, ‘b’ p < 0.01, ‘c’ p < 0.05 vs ketamine treated group. Keta: ketamine, Halo: Haloperidol, Olz: olanzapine.

Table 3 Effect of SOEE on locomotor activity and stereotypic behaviour. Treatment

Locomotor counts

Number of falls

Number of turns

Vehicle Keta (50 mg/kg) Keta+Halo (1 mg/kg) Keta + Olz (5 mg/kg) Keta + SOEE (50 mg/ kg) Keta + SOEE (100 mg/ kg) Keta + SOEE (200 mg/ kg)

288.5 ± 4.90 508.21 ± 2.63* 293.66 ± 3.18 a 285.16 ± 4.52a 401.66 ± 3.09 c

0.00 ± 0.00 12.50 ± 0.28* 3.33 ± 0.1 a 3.16 ± 0.12 a 9.53 ± 0.29c

0.00 ± 0.00 11.33 ± 0.55* 3.33 ± 0.21 a 2.33 ± 0.33a 7.00 ± 0.36b

374.66 ± 3.81a

7.83 ± 0.22b

5 ± 0.51a

362.66 ± 3.19

a

5.16 ± 0.30

a

4.33 ± 0.42

immobility duration (Table 4) 3.4.4. Effect of SOEE on step down latency in passive avoidance test Administration of SOEE (100 and 200 mg/kg, p.o.) significantly (p < 0.01 and p < 0.001, respectively) reversed the ketamine-induced cognitive deficit in mice by increasing the step down latency. Olanzapine (5 mg/kg, i.p.) was also potential (p < 0.001) to increase the step down latency in passive avoidance test (Table 5).

a

Values are shown as Mean ± SEM (n = 6). One way ANOVA followed by Tukey’s test. ‘*’ p < 0.001 vs vehicle treated group. ‘a’ p < 0.001, ‘b’ p < 0.01, ‘c’ p < 0.05 vs ketamine treated group. Keta: ketamine, Halo: Haloperidol, Olz: olanzapine, SOEE: Spinacea oleracea seedsethanol extract.

3.4.5. Cataleptic effect of SOEE in bar test SOEE was also studied for extra-pyramidal side effect using bar test. As displayed in Table 6, SOEE (50, 100 and 200 mg/kg) did not show catalepsy on the 15th day of treatment.

of locomotor counts as compared to ketamine treated animals. 3.5. Biochemical, neurochemical and cellular observations 3.4.2. Effect of SOEE on stereotypic behaviors in mice Pre-treatment with SOEE (50, 100 and 200 mg/kg, p.o.) significantly decreased ketamine-induced stereotypic behaviours. Moreover, haloperidol (1 mg/kg, i.p.) and olanzapine (5 mg/kg, i.p.) were also remarkable (p < 0.001 and p < 0.001, respectively) to diminish the ketamine induced stereotypic behaviours (Table 3) (Fig. 1).

3.5.1. Effect of SOEE on reduced glutathione level SOEE (100 and 200 mg/kg, p.o.) was found to reinforce antioxidant mechanism by increasing reduced GSH levels (p < 0.001 and p < 0.001, respectively) in mice brain. Furthermore, haloperidol (1 mg/kg, i.p.) and olanzapine (5 mg/kg, i.p.) was also found to decrease ketamine-induced oxidative stress (Fig. 2).

3.4.3. Effect of SOEE on immobility duration in FST In order to confirm the effect of SOEE against ketamine-induced negative symptoms in mice, forced swim test was used. Administration of SOEE (100 and 200 mg/kg, p.o.) was significant (p < 0.05 and p < 0.01, respectively) to reduce ketamine-induced immobility duration in FST. Olanzapine (5 mg/kg, i.p.) was also effective (p < 0.001) to decrease the ketamine induced negative symptom by decreasing the

3.5.2. Effect of SOEE on lipid peroxidation level SOEE (100 and 200 mg/kg, p.o.) was found to reduce oxidative stress by decreasing lipid peroxidation (decreased MDA level) (p < 0.01 and p < 0.001, respectively) in mice brain. Moreoever, haloperidol (1 mg/kg, i.p.) and olanzapine (5 mg/kg, i.p.) was also effective to decrease ketamine-induced oxidative stress (Fig. 3).

Fig. 1. Timeline depicting the treatment and the different tests. 1019


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3.5.5. Effect of SOEE on dopamine level Pre-treatment with SOEE (100 and 200 mg/kg p.o.) significantly (p < 0.01 and p < 0.001, respectively) downregulated the ketamineinduced dopamine hyperactivation. Haloperidol (1 mg/kg, i.p.) and olanzapine (5 mg/kg, i.p.) were also effective in decreasing the dopamine level (Fig. 6).

Table 5 Effect of SOEE on SDL in passive avoidance test. Treatments

SDL (sec) duration learning session

SDL (sec) during memory session

Vehicle Keta (50 mg/kg) Keta+Halo (1 mg/kg) Keta + Olz (5 mg/kg) Keta + SOEE (50 mg/ kg) Keta + SOEE (100 mg/kg) Keta + SOEE (200 mg/kg)

83.83 72.00 75.83 77.50 73.00

1.97 1.75 2.05 2.34 1.33

248.00 ± 3.39 87.00 ± 1.46* 90.16 ± 1.99 186.00 ± 2.22a 89.66 ± 1.26

75.50 ± 2.59

113.66 ± 1.94b

76.16 ± 1.30

148.33 ± 1.40a

± ± ± ± ±

3.5.6. Effect of SOEE on GABA level Pre-treatment with SOEE (50, 100 and 200 mg/kg p.o.) was effective (p < 0.01, p < 0.001 and p < 0.001, respectively) to increase the GABA level in psychotic mice. Moreover, haloperidol (1 mg/kg, i.p.) and olanzapine (5 mg/kg, i.p.) were also found to increase the GABA level (Fig. 7).

Values are Mean ± SEM (n = 6). One way ANOVA followed by Tukey’s test. ‘*’ p < 0.001 vs vehicle treated group. ‘a’ p < 0.001, ‘b’ p < 0.01 vs ketamine treated group.Keta: ketamine, Halo: Haloperidol, Olz: olanzapine.

4. Discussion Oxidative stress is produced from the imbalance between reactive oxygen species, free radicals and antioxidant systems. These oxidative species reacts proteins, lipids and DNA and cause anomalous neuronal degradation and cell death [35]. Excessive oxidative damage can also be induced from chronic inflammation and mitochondrial abnormalities. These abnormalities have been involved in the development of psychotic symptoms [36]. Antioxidants help to decrease such changes produced by free radicals through different mechanisms like decreasing the free radical formation as well as scavenging them, resulting in retardation of the lipid peroxidation process [37,38]. Exogenous administration of phytoconstituents in the form of herbal medicines has been linked with reduced risk, delayed progression and enhanced recovery from various chronic neuropsychiatric disorders [39,40]. Various medicinal plants contain lutein, β-carotene, hesperidin, stigmasterol, kampeferol, apigenin, rutin, quercetin, myricetin, luteolin, berberine, astragalin, para-coumaric acid, ortho-coumaric acid, ascorbic acid, violaxanthin, methylenedioxy flavonol, glycoglycerolipids, caffeic acid and protocatechuic acids have been shown to exert neuroprotective, antioxidant and anti-inflammatory effects [26], which might be supportive for managing positive, negative and cognitive symptoms of schizophrenia with minimal side effects in comparison to synthetic compounds. Therefore, we have selected the commonly consumed plant worldwide viz. Spinacia oleracea because of its tremendous medicinal properties with promising neuroprotective bioactive constituents. in vitro assay using DPPH has shown free radical scavenging efficiency of SOEE representing the antioxidant potential of SOEE. Further, the concentration dependent inhibition of haemolysis shown by SOEE indicates anti-inflammatory activity of SOEE. Our outcomes are in line with the literature reported, suggesting its effectiveness in

Table 6 Effect of SOEE on catalepsy in bar test. Treatments

15th day

Vehicle Keta (50 mg/kg) Keta+Halo (1 mg/kg) Keta + Olz (5 mg/kg) Keta + SOEE (50 mg/kg) Keta + SOEE (100 mg/kg) Keta + SOEE (200 mg/kg)

5.16 ± 0.30 4.50 ± 0.22 19.16 ± 0.79* 5.00 ± 0.36 4.50 ± 0.22 7.33 ± 0.49 7.60 ± 0.22

Values are Mean ± SEM (n = 6). One way ANOVA followed by Tukey’s test. ‘*’ p < 0.001 vs vehicle treated group. Keta: ketamine, Halo: Haloperidol, Olz: olanzapine.

3.5.3. Effect of SOEE on serum TNF-α level SOEE (50, 100 and 200 mg/kg p.o.) was found to attenuate (p < 0.05, p < 0.01 and p < 0.001, respectively) ketamine-induced neuroinflammation as shown by reducing serum TNF-α level. Furthermore, haloperidol (1 mg/kg, i.p.) and olanzapine (5 mg/kg, i.p.) also decreased the inflammation by decreasing the TNF-α level (Fig. 4).

3.5.4. Effect of SOEE on acetylcholinesterase activity SOEE (100 and 200 mg/kg p.o.) was effective (p < 0.01 and p < 0.01, respectively) to attenuate ketamine-induced acetylcholinesterase activity. Also, haloperidol (1 mg/kg, i.p.) and olanzapine (5 mg/kg, i.p.) was significant to decrease AChE activity (Fig. 5).

Fig. 2. Effect of SOEE on reduced glutathione (GSH) level. Values are Mean ± SEM (n = 6). One way ANOVA followed by Tukey’s test. ‘*’ p < 0.001 vs vehicle treated group. ‘a’ p < 0.001 vs Ketamine treated group. 1020


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Fig. 3. Effect of SOEE on malondialdehyde (MDA) level. Values are Mean ± SEM (n = 6). One way ANOVA followed by Tukey’s test. ‘*’ p < 0.001 vs vehicle treated group. ‘a’ p < 0.001, ‘b’ p < 0.01 vs Ketamine treated group.

positive symptoms of schizophrenia [26,42]. In the present study, administration of SOEE at the doses of 50, 100 and 200 mg/kg, p.o. significantly decreased ketamine-induced locomotor counts and stereotypic behaviours in mice. Interestingly, neurochemical estimations suggest that SOEE was effective to attenuate dopamine levels and increased GABA levels in the brain. This effect may be due to the presence of flavonoids and polyphenols in SOEE, which are known to possess GABA agonist effect. This observation is further supported by previous reports on GABA agonist agents resulting in increased GABA levels, which may prevent or delay the progression of schizophrenia [26,43]. Moreover, it has been suggested in a study that flavonoids improves NMDA receptor antagonists induced hyperlocomotor activity [43], which supports the present study. Further, SOEE was also screened for extra-pyramidal effects using bar test for catalepsy shown by typical antipsychotic drugs. SOEE treated animals did not show any cataleptic effect in bar test. Immobility enhancement after treatment with ketamine has been used as a screening model for negative symptoms (flattening of affect and avolition) of schizophrenia [26]. It has been found that treatment with SOEE decreased the immobility period in force swim test. The

inflammation and oxidant stress which is helpful in the treatment of schizophrenia [26,41]. By virtue of antioxidant and anti-inflammatory effects, we have screened SOEE against ketamine-induced schizophrenia in mice. Haloperidol (typical antipsychotic: effective in stereotypic behaviours and positive symptom) and olanzapine (atypical antipsychotic: effective in stereotypic behaviours, positive, negative and cognitive symptoms) were selected as standard drugs to understand the similarity of test drug with standard drugs. Some pharmacological studies have represented the etiological role of N-methyl-D-aspartate (NMDA) receptors in psychosis [3–6]. NMDA receptor antagonists induce psychotic symptoms, including stereotypic behaviours, positive symptoms, negative symptoms and cognitive symptoms in small laboratory animals as well as in healthy human beings [3–5]. Ketamine antagonises the NMDA receptors on GABAergic efferent neurons inhibiting the release of GABA [26]. GABA is an inhibitory neurotransmitter, which further regulates the dopamine release. The inhibitory action of the GABAergic neurons has been reduced by ketamine, this in turns enhances the dopamine release which stimulates the stereotypic behaviors, locomotor activity representing

Fig. 4. Effect of SOEE on TNF-α level. Values are Mean ± SEM (n = 6). One way ANOVA followed by Tukey’s test. ‘*’ p < 0.001 vs vehicle treated group. ‘a’ p < 0.001, ‘b’ p < 0.01, ‘c’ p < 0.05 vs Ketamine treated group. 1021


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Fig. 5. Effect of SOEE on acetylcholinesterase levels (AChE). Values are Mean ± SEM (n = 6). One way ANOVA followed by Tukey’s test. ‘*’ s p < 0.001 vs vehicle treated group. ‘A’ p < 0.001, ‘b’ p < 0.01 vs Ketamine treated group.

Fig. 6. Effect of SOEE on dopamine level. Values are Mean ± SEM (n = 6). One way ANOVA followed by Tukey’s test. ‘*’ p < 0.001 vs vehicle treated group. ‘a’ p < 0.001, ‘b’ p < 0.01 vs Ketamine treated group.

Fig. 7. Effect of SOEE on GABA level. Values are Mean ± SEM (n = 6). One way ANOVA followed by Tukey’s test. ‘*’ p < 0.001 vs vehicle treated group. ‘a’ p < 0.001, ‘b’ p < 0.01 vs Ketamine treated group.

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Fig. 8. Possible mechanisms involved in the protective effect of Spinacea oleracea seeds.

which further abnormally increase glutamate neurotransmission that can lead to excitotoxic damage seen in schizophrenia [6]. In the present investigation, ketamine administration significantly increased TNF-α level, which was confirmed by increased serum TNF-α level in mice. On the other hand, treatment with SOEE has reduced TNF-α level due to anti-inflammatory and antioxidant properties which acts via inhibition of microglial cell activation. Cognitive impairments, including deficits in attention, executive function, working memory and spatial memory are associated with schizophrenia [3–6]. Acetylcholine, a neurotransmitter involved in learning and memory process, is degraded by the enzyme AChE which also plays a putative role in neurodegenerative diseases [55]. Various earlier reports demonstrate that acetylcholine is suppressed in hippocampus with the administration of the ketamine via nicotinic acetylcholine receptor (nAChR) blockade and also enhances AChE activity leading to memory impairment [6,42,56]. Passive avoidance test has already been used for screening of antipsychotic agents, e.g., haloperidol and olanzapine on cognitive symptoms in animals [26]. Our findings displayed the remarkable effect of SOEE in the treatment of cognitive symptoms of schizophrenia as shown by increased time to step down latency in passive avoidance test. In addition to behavioural parameter, biochemical estimation reflected that the AChE activity was markedly increased in ketamine treated mice, which may lead to decrease in acetylcholine levels while reduction in AChE activity was observed with the SOEE pre-treatment. These results point out the favourable effect of SOEE in cognitive symptoms of schizophrenia by reducing AChE activity, which can enhance cholinergic function that increases glutamate binding on NMDA receptor binding in the brain and thus indirectly may enhance glutamatergic neurotransmission. Other studies reported the effect of dietary flavonoids on brain, including reduction in AChE activity, suppression of neuroinflammation, neuroprotection against neurotoxins and potential to increase memory, learning and cognitive function [57–59]. Thus, the effect of SOEE on cognitive symptoms of schizophrenia is probably due to the presence of flavonoids as well as its antioxidant and anti-inflammatory properties. SOEE decreased stereotypic behaviours, positive, negative and cognitive symptoms of schizophrenia and also free from extra-pyramidal side

effectiveness of SOEE in negative symptoms has been attributed to its anti-inflammatory, antioxidant and neuroprotective properties and hence we examined antioxidant and anti-inflammatory biomarkers. Ketamine has been reported to produce oxidative stress in small animals [26,43]. Metabolism of dopamine may generate a large amount of hydrogen peroxide (H2O2) and superoxide radical (O2−) which can damage DNA, lipids and proteins and finally cellular dysfunction led to psychiatric disorders [41,44]. Glutathione is an endogenous antioxidant biomarker; it acts by reducing inactive disulfide linkage of enzymes to the active sulfhydryl group. The sulfhydryl group of glutathione gets oxidized by donating an electron to the free radicals to neutralize them and prevents cellular oxidation. Thus, glutathione plays a significant role in protecting membrane peroxidation and also diminishes hydrogen peroxide [45,46]. Lipid peroxidation is oxidative degradation of polyunsaturated fatty acids initiated by hydrogen addition or abstraction of oxygen radical which causes the impaired membrane function and inactivates membrane bound enzymes [47]. The degree of lipid peroxidation can be measured by estimating the level of MDA, a lipid peroxidation product. Therefore, the deficiency of activity of these enzymes leads to an imbalance between antioxidant defense mechanism and free oxidant radicals that induces neuropsychiatric disorders [48,49]. It has been reported that antipsychotic drugs reduce the oxidative damage by potentiating the enzymatic activity of endogenous antioxidants [50,51]. In our study, SOEE significantly alleviated ketamine-induced oxidative stress by reversal of above mentioned antioxidant parameters. Our results on oxidative stress biomarkers are in concordance with previous studies [52,53]. Chemically, Spinacia oleracea seeds are reported as an excellent source of polyphenols, phytoecdysteroids and flavonoids, which are identified to have potent antioxidant and anti-inflammatory [11,13,14]. Neuroinflammation plays a vital role in the pathophysiology of schizophrenia. Brain injury, oxidative stress, infection and NMDA antagonist can activate microglial cells, resident phagocytes in the brain [3–5]. Microglia are a primary reservoir of inflammatory cytokines such as TNF-α, which are responsible for various pathological findings in psychiatric patients [54]. TNF-α can increase the surface expression of αamino-3-hydroxy-5-methyl-4-isoxazolepropionic acid AMPA receptors 1023


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effects similar to olanzapine (atypical antipsychotic).

[20] N.K. Jain, A.K. Singhai, Ameliorative effects of Spinacia oleracea L. eeds on carbon tetrachloride (CCl4)-induced hepatotoxicity: in vitro and in vivo studies, Asian Pac. J. Trop. Biomed. 2 (2012) 232–237. [21] R. Kadian, M. Parle, Evaluation of Terminalia bellerica for its antipsychotic potential, Int. J. Pharm. Sci. Rev. Res. 30 (2015) 247–252. [22] C. Sánchez-Moreno, J.A. Larrauri, F. Saura-Calixto, Free radical scavenging capacity and inhibition of lipid oxidation of wines,grape juices and related polyphenolic constituents, Food Res. Int. 32 (1999) 407–412. [23] V.R. Ravikumar, M. Dhanamani, T. Sudhamani, In-vitro anti-inflammatory activity of aqueous extract of leaves of Plectranthus amboinicus (Lour.), Spreng. Anc. Sci. Life 28 (2009) 7–9. [24] OECD Test Guideline-423, Acute Oral Toxicity - Acute Toxic Class Method, (2010). [25] L. de Oliveira, C.M. Spiazzi, T. Bortolin, L. Canever, F. Petronilho, F.G. Mina, F. DalPizzol, J. Quevedo, A.I. Zugno, Different sub-anesthetic doses of ketamine increase oxidative stress in the brain of rats, Prog. Neuropsychopharmacol. Biol. Psychiatry 33 (2009) 1003–1008. [26] M. Yadav, D.K. Jindal, M.S. Dhingra, A. Kumar, M. Parle, S. Dhingra, Protective effect of gallic acid in experimental model of ketamine-induced psychosis: possible behaviour, biochemical, neurochemical and cellular alterations, Inflammopharmacology 26 (2018) 413–24. [27] R.D. Porsolt, A. Bertin, M. Jalfre, Behavioral despair in mice: a primary screening test for antidepressants, Arch. Int. Pharmacodyn. Ther. 229 (1977) 327–336. [28] H. Joshi, M. Parle, Evaluation of nootropic potential of Ocimum sanctum Linn. in mice, Ind. J. Expt. Biol. 44 (2006) 133–136. [29] D.C. Hoffman, H. Donovan, Catalepsy as a rodent model for detecting antipsychotic drugs with extrapyramidal side effect liability, Psychopharmacology 120 (1995) 128–133. [30] G.L. Ellman, Tissue sulphydryl groups, Arch. Biochem. Biophys. 82 (1959) 70–77. [31] E.D. Wills, The effect of inorganic iron on the thiobarbituric acid method for the determination of lipid peroxides, Biochim. Biophys. Acta 184 (1964) 475–477. [32] G.L. Ellman, K.D. Courtney, V. Andres, R.M. Featherstone, A new and rapid colorimetric determination of acetylcholinesterase activity, Biochem. Pharmacol. 7 (1961) 88–95. [33] M. Schlumpf, W. Lichtensteiger, H. Langemann, P.G. Waser, F. Hefti, A fluorimetric micro method for the simultaneous determination of serotonin, noradrenaline and dopamine in milligram amount of brain tissue, Biochem. Pharmacol. 23 (1974) 2337–2346. [34] I.P. Lowe, E. Robins, G.S. Eyerman, The fluorimetric measurement of glutamic, decarboxylase measurement and its distributionin brain, J. Neuro. Chem. 3 (1958) 8–18. [35] A. Ciobica, M. Padurariu, I. Dobrin, C. Stefanescu, R. Dobrin, Oxidative stress in schizophrenia - focusing on the main markers, Psychiatr. Danub. 23 (2011) 237–245. [36] E. Asevedo, A. Gadelha, C. Noto, R.B. Mansur, A. Zugman, S.I. Belangero, A.A. Berberian, B.S. Scarpato, E. Leclerc, A.L. Teixeira, C.S. Gama, Impact of peripheral levels of chemokines, BDNF and oxidative markers on cognition in individuals with schizophrenia, J. Psychiatr. Res. 47 (2013) 1376–1382. [37] J. Davis, S. Moylan, B.H. Harvey, M. Maes, M. Berk, Neuroprogression in schizophrenia: pathways underpinning clinical staging and therapeutic corollaries, Aust. N. Z. J. Psychiatry 48 (2014) 512–529. [38] F.E. Emiliani, T.W. Sedlak, A. Sawa, Oxidative stress and schizophrenia: recent breakthroughs from an old story, Curr. Opin. Psychiatry 27 (2014) 185–190. [39] Z.J. Zhang, Therapeutic effects of herbal extracts and constituents in animal models of psychiatric disorders, Life Sci. 75 (2004) 1659–1699. [40] V.K. Gupta, S.K. Sharma, Plants as natural antioxidants, Nat. Prod. Radiance 5 (2006) 326–334. [41] G.E. Hardingham, K.Q. Do, Linking early-life NMDAR hypofunction and oxidative stress in schizophrenia pathogenesis, Nat. Rev. Neurosci. 17 (2016) 125–134. [42] M. Chatterjee, R. Verma, S. Ganguly, G. Palit, Neurochemical and molecular characterization of ketamine-induced experimental psychosis model in mice, Neuropharmacol. 63 (2012) 1161-71. [43] S. Okuyama, T. Fukata, Y. Nishigawa, Y. Amakura, M. Yoshimura, T. Yoshida, M. Nakajima, Y. Furukawa, Citrus flavonoid improves MK-801-induced locomotive hyperactivity: possible relevance to schizophrenia, J. Funct. Foods 5 (2013) 2002–2006. [44] Y.D. Kim, S.M. Lantz-McPeak, S.F. Ali, M.T. Kleinman, Y.S. Choi, H. Kim, Effects of ultrafine diesel exhaust particles on oxidative stress generation and dopamine metabolism in PC-12 cells, Environ. Toxicol. Pharmacol. 37 (2014) 954–959. [45] R. Dringen, Metabolism and functions of glutathione in brain, progress in neurobiology, Prog. Neurobiol. 62 (2000) 649–671. [46] O.M. Dean, M.V. den Buuse, A.I. Bush, D.L. Copolov, F. Ng, S. Dodd, M. Berk, A role for glutathione in the pathophysiology of bipolar disorder and schizophrenia? Animal models and relevance to clinical practice, Curr. Med. Chem. 16 (2009) 2965–2976. [47] A. Dietrich‐Muszalska, B. Kontek, Lipid peroxidation in patients with schizophrenia, Psychiatry Clin. Neurosci. 64 (2010) 469–475. [48] S.P. Mahadik, S. Mukherjee, R. Scheffer, E.E. Correnti, J.S. Mahadik, Elevated plasma lipid peroxides at the onset of nonaffective psychosis, Biol. Psychiatry 43 (1998) 674–679. [49] Y. Gong, R. Zhao, B. Yang, Superoxide dismutase activity and malondialdehyde levels in patients with travel-induced psychosis, Shanghai Arch. Psychiatry 24 (2012) 155–161. [50] M. Kuloglu, B. Ustundag, M. Atmaca, H. Canatan, A.E. Tezcan, N. Cinkilinc, Lipid peroxidation and antioxidant enzyme levels in patients with schizophrenia and bipolar disorder, Cell. Biochem. Funct. 20 (2002) 171–175. [51] S.P. Mahadik, S. Mukherjee, Free radical pathology and antioxidant defense in

5. Conclusion The present investigation shows that SOEE was effective against stereotypic behaviours, positive, negative and cognitive symptoms of schizophrenia induced by ketamine in mice. The extract did not show extra-pyramidal side effects. Hence, SOEE may provide an imperative lead in the invention of newer antipsychotic agent, which may be further studied clinically in patients with predominant psychotic symptoms. Moreover, its protective effect in schizophrenia may be associated with its regulating effect on dopamine, GABA, AChE, GSH and MDA levels as illustrated in Fig. 8. Conflict of interest The authors report no conflict of interest. Acknowledgements Authors are grateful to Guru Jambheshwar University of Science & Technology, Hisar for financial assistance and also thankful to Dr. M. K. Rana, Department of Vegetable Science, CCS Haryana Agricultural University, Hisar, Haryana (India) for providing Spinacia oleracea seeds for research work. References [1] J. McGrath, S. Saha, D. Chant, J. Welham, Schizophrenia: a concise overview of incidence, prevalence, and mortality, Epidemiol. Rev. 30 (2008) 67–76. [2] A.M. Fineberg, L.M. Ellman, Inflammatory cytokines and neurological and neurocognitive alterations in the course of schizophrenia, Biol. Psychiatry 73 (2013) 951–966. [3] J.T. Coyle, NMDA receptor and schizophrenia: a brief history, Schizophrenia Bull. 38 (2012) 920–6. [4] D.C. Javitt, J.T. Coyle, Decoding schizophrenia, Sci. Am. 290 (2004) 48–55. [5] J. Frohlich, J.D. Van Horn, Reviewing the ketamine model for schizophrenia, J. Psychopharmacol. 28 (2014) 287–302. [6] A. Kumar, M. Yadav, P. Parle, D.K. Dhull, S. Dhingra, Potential drug targets and treatment of schizophrenia, Inflammopharmacology 25 (2017) 277–292. [7] J. Horacek, V.B. Valesova, M. Kopecek, T. Palenicek, C. Dockery, P. Mohr, C. Hoschl, Mechanism of action of atypical antipsychotic drugs and the neurobiology of schizophrenia, CNS Drugs 20 (2006) 389–409. [8] P. Garcia-Garcia, F. Lopez-Munoz, G. Rubio, B. Martin-Agueda, C. Alamo, Phytotherapy and psychiatry: bibliometric study of the scientific literature from the last 20 years, Phytomedicine 15 (2008) 566–76. [9] M. Yadav, M. Parle, M. Kadian, K. Sharma, A review on psychosis and anti-psychotic plants, Asian. J. Pharm. Clin. Res. 8 (2015) 24–28. [10] G.R. Kumar, Hypolipidemic activity of Spinacia oleracea L. in atherogenic diet induced hyperlipidemic rats, J. Bio. Pharm. Res. 1 (2012) 39–43. [11] R.K. Verma, R. Sisodia, A.L. Bhatia, Role of Spinacia oleracea as antioxidant: a biochemical study on mice brain after exposure of gamma radiation, Asian. J. Exp. Biol. Sci. 17 (2003) 51–57. [12] Y. Wang, C.F. Chang, J. Chou, H.L. Chen, X. Deng, B.K. Harvey, J.L. Cadet, P.C. Bickford, Dietary supplementation with blue berries, spinach or spirulina reduces ischemic brain damage, Exp. Neurol. 193 (2005) 75–84. [13] R. Sultana, M. Perluigi, D.A. Butterfield, Protein oxidation and lipid peroxidation in brain of subjects with Alzheimer’s disease: insights into mechanism of neurodegeneration from redox proteomics, Antioxid. Redox Signal. 8 (2006) 2021–2037. [14] N. Sharma, M. Kapoor, B. Nehru, Spinacia oleracea L. extract protects against LPS induced oxidative stress, inflammatory response and ensuing biochemical, neurochemical and neurobehavioral impairment in mice, Int. J. Pharm. Pharm. Sci. 6 (2014) 203–210. [15] F. Ferreres, M. Castaner, F.A. Tomas-Barberan, Acylated flavonol glycoside from spinach leaves (Spinacia oleracea), Phytochemistry 45 (1997) 1701–1705. [16] A. Bunea, M. Andjelkovic, C. Socaciu, O. Bobis, M. Neacsu, R. Verhe, J. Van-Camp, Total and individual carotenoids and phenolic acids content in fresh, refrigerated and processed spinach (Spinacia oleracea L.), Food. Chem. 108 (2008) 649–656. [17] P.S. Gaikwad, R.V. Shete, K.V. Otari, Spinacia oleracea Linn: a pharmacognostic and pharmacological overview, Int. J. Res. Ayu. Phar. 1 (2010) 78–84. [18] A. Sathya, P. Siddhuraju, Role of phenolics as antioxidants, biomolecule protectors and as anti–diabetic factors-evaluation on bark and empty pods of Acacia auriculiformis, Asian Pac, J. Trop. Med. 51 (2012) 757–765. [19] M. Yadav, M. Parle, N. Sharma, S. Dhingra, N. Raina, J.D. Kumar, Brain targeted oral delivery of doxycycline hydrochloride encapsulated tween 80 coated chitosan nanoparticles against ketamine induced psychosis: behavioural, biochemical, neurochemical and histological alterations in mice, Drug. Deliv. 24 (2017) 1429–1440.

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M. Yadav et al.

but still living relationship, Behav. Brain. Res. 221 (2011) 424–429. [56] M.E. Hasselmo, The role of acetylcholine in learning and memory, Curr. Opin. Neurobiol. 16 (2006) 710–715. [57] C. Gutierrez-Merino, C. Lopez-Sanchez, R.K. Lagoa, A. Samhan-Arias, C. Bueno, V. Garcia-Martinez, Neuroprotective actions of flavonoids, Curr. Med. Chem. 18 (2011) 1195–1212. [58] I. Uriarte-Pueyo, M. Calvo, Flavonoids as acetylcholinesterase inhibitors, Curr. Med. Chem. 18 (2011) 5289–5302. [59] C. Grosso, P. Valentao, F.B. Ferreres, P. Andrade, The use of flavonoids in central nervous system disorders, Curr. Med. Chem. 20 (2013) 4694–4719.

schizophrenia: a review, Schizophr. Res. 19 (1996) 1–17. [52] F.F. Brinholi, C.C. de Farias, K.L. Bonifacio, L. Higachi, R. Casagrande, E.G. Moreira, D.S. Barbosa, Clozapine and olanzapine are better antioxidants than haloperidol, quetiapine, risperidone and ziprasidone in in vitro models, Biomed. Pharmacother. 81 (2016) 411–415. [53] I. Sadowska-Bartosz, S. Galiniak, G. Bartosz, M. Zuberek, A. Grzelak, A. DietrichMuszalska, Antioxidant properties of atypical antipsychotic drugs used in the treatment of schizophrenia, Schizophr. Res. 176 (2016) 245–251. [54] A. Monji, T. Kato, S. Kanba, Cytokines and schizophrenia: microglia hypothesis of schizophrenia, Psychiatry Clin. Neurosci. 6 (2009) 257–265. [55] J. Micheau, A. Marighetto, Acetylcholine and memory: a long, complex and chaotic

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