INTRODUCTION:

Depression is a common mental illness characterised by tenacious sadness, depressed mood, anhedonia or loss of pleasure, low energy or fatigue, restlessness, hopelessness and many more¹. Globally it is a huge contributor for morbidity and large scale of death, under worst environments it may lead to suicide². According to data from the WHO in 2015, more than 300 million individuals (4.3% of the global population) struggle with depression. According to the 2015-16 National Mental

Health Survey in India, roughly fifteen percent of Indian adults require active intervention for one or more mental health issues (WHO 2015). Recent treatment for depression which is available for patient may provide partial relief, it is not consider as ideal. So its immediate requirement to find out more effective, safe and promising drug for the treatment of depression³. Neuroinflamation and oxidative stress are closely associated with depression. Variation in level of oxdative parameters like reduced glutathione (GSH), Malondialdehyde (MDA), and inflammatory mediaters like Interleukins (ILs), Tumor necrosis factor (TNF-α). Numerous research have found links between inflammatory biomarkers and depressive symptoms such as insomnia, tiredness, and cognitive impairment^4,5. Depressive patent observed with elevated level of inflammatory cytokines. Increased in level of inflammatory cytokines activate monoamine neurotransmitters, serotonergic system, nor epinephrine metabolism⁶. Recently, a wide range of medications, including selective serotonin reuptake inhibitors (SSRIs) and serotonin and norepinephrine reuptake inhibitors (SNRIs), have been used to treat depression. Which block the reuptake of both serotonin and norepinephrine, have essentially replaced tricyclic antidepressants and MAO inhibitors as the drugs of choice for treating depression. Most prescribed drugs from SSRIs category were Escitalopram, citalopram, fluoxetine, etc., because of their widespread effect on a variety of psychiatric illnesses⁷. However, these consuming antidepressants remain accompanied with plenty of complexity. These include coma, heart toxicity, and sexual upset include anorgasmia, erectile dysfunction, and less interest in libido^{8, 9}. Pregnancy-related SSRI use has been associated to an increased risk of miscarriages, birth abnormalities, neonatal behavioural disorders and Autism spectrum disorder (ASD), etc¹⁰. Withdrawal symptoms are also seen in this treatment. Hence we must concentrate on alternate approaches. Medicinal plant materials or active phytoconstitutes were effective alternative or synergistic supplements in the treatment of depression. In this article, Gramine (Ga) and Zingeone (Zn), two distinct medications, were the main topics. Numerous studies have shown that the pharmacological effects of green barley extract on the neurological and cardiovascular systems are excellent^{11, 12}. Gramine, is an indole alkaloid with well-known pharmacological actions such as anti-oxidant, anti-proliferation, and anti-inflammation reaction, is the primary component of young barley (Hordeum vulgare L.)^{13, 14}. Gramine's ability to reduce inflammation after spinal cord damage by blocking pro-inflammatory mediators has been researched¹⁵.

Ginger's main ingredient, Zingerone, proved its antioxidant and antiinflamatory properties against stannous chloride, which was responsible for DNA damage by increasing ROS¹⁶. A recent study¹⁷ shown that Zingerone has anti-inflammatory properties against LPS-induced hepatic injury and inflammation in mice. In addition to that, zingerone inhibits mitogen-activated protein kinase and reduces expression of the proinflammatory NF-B gene as people get older¹⁸. Stress build in regular living activity if that was not performed successfully. Failure in day today activity also results depression. Same as that chronic unpredictable mild stress (CUMS) model mimics depression like it. This is the choice of model for this type of depressive activity. This CUMS model increased reactive oxygen species as well as effect on Serotonin and pro- inflammatory factors¹⁹. So on the basis of above literature survey we decided that there is a scope to illustrate action of this phytoconstsiuents against chronic unpredictable mild stress (Depression).

MATERIALS AND METHODS:

Chemicals

Gramine and Zinzeone pure both bioactive constituents bought from Yucca enterprises private limited Mumbai. Ecitalopram (standard), Ellman’s reagent.

Experimental Design

Albino Wistar rats with a sex age of seven–eight weeks and a weight in the range of 130–160 g were obtained from LACSMI Bio Farms, Aundh, Pune, Maharashtra, India. This experimental group of animals was housed in one individual cage with an environment of 27 ± 2 °C, a 12-hour light-dark cycle, and a humidity of 50–70 %. No any restriction or limitation of food and water to animals. Before starting experimental work, animals must be acclimatised in the laboratory for 7 days. The Institutional Animal Ethical Committee (IAEC) of the MET Institute of Pharmacy in Nashik, India, reviewed and approved this study protocol on October 21, 2022. (Approval No. MET-IOP-IAEC/2022-23/11).

After the acclimatisation period, the 54 animals were allocated into nine groups (n = 6). The CUMS technique described by Gupta and Joneth Fernandes (2019) was modified somewhat²⁰.

Group 1: Control group (non- stressor + free food and water access)

Group 2: CUMS group (Stressed by CUMS test + Vehicle)

Group 3: Stressed by CUMS test + Zn (75mg/kg)

Group 4: Stressed by CUMS test + Zn (125mg/kg)

Group 5: Stressed by CUMS test + Zn (250mg/kg)

Group 6: Stressed by CUMS test + Ga (13mg/kg)

Group 7: Stressed by CUMS test + Ga (27.5 mg/kg)

Group 8: Stressed by CUMS test + Ga (55mg/kg)

Group 9: Stressed by CUMS test + Ecitalopram (15mg/kg) (Standard)

Treated animals (Group 3-8) given test drugs and standard drug (Group-9) given orally with respective dose prior induction of CUMS stress.

Induction of chronic unpredictable mild stress (CUMS)

The CUMS paradigm included seven distinct types of stressors, and the rat participants were subjected to one form of stress per day for a period of 21 days (timing 8am to 4pm). The types of stressors included in follows:-

Evaluating Parameters

Animals were exposed to behavioural, biochemical, and neurochemical examinations on the 22nd day.

Behavioural tests analysis:

Sucrose preference test (SP):-

Sucrose preference tests have been carried out before and after the 21-day evaluation as part of the experiment paradigm. 8 hr. before start this SP test, rats were restricted for water and food consumption. Next to that give the free access for 12 hours of two bottles in which first contain 1% sucrose solution and other contain normal tap water. The places of bottles were switched after 6 hours to avoid the effects of side preference on drinking familiarity. The consumption of both waters was determined by weighting their respective bottles. The sucrose preference (SP) was calculated as a percentage of sucrose solution ingested relative to total liquid drank²¹. The Sucrose Preference Test was used in this study for recognising anhedonia, one of the main symptoms associated with serious depression.

Sucrose Solution Drunk (g)

SP = ---------------------------------------------- x 100

Sucrose Solution Drunk (g)+Tap water Drunk (g)

Force swim test (FST):-

FST is an excellent behavioural model for screening depression in rats and mice. Individually, the animals were obligated to swim in a cylinder-shaped plastic object (46 cm hgt. x 20 cm inside diameter) filled to a height of 30 cm with water at (23±2°C), and a duration of immobility was recorded. During the initial time period of FST, animals are hyperactive, but over a gradually increasing time period, their movements become slowed or they are observed immobilised and trying to float on the water surface. The position of floating on water is like posture: slightly slumped but upright, nose above water, termed "immobile. There were two sessions performed: the first was a pre-test for 15 minutes, followed by a 5-minute 'test' after 24 hr.^22,
23

Brain sampling for biochemical analysis

Last day of study all animals were euthanized after completing behavioural study, brain of animals isolated, rinsed in ice-cold normal saline solution then instantly collected midbrain tissue samples were washed with cold 0.1M Phosphates buffer solution (_PH 7.4) and weight it properly. Homogenised brain with this 9 volumes of ice cold 0.1M Phosphates buffer solution. Furthermore this homogenate centrifuged at 10 thousand rpm for 15 min at 4°C, finally supernatant utilised for next biochemical estimation.

UV analysis of brain tissue homogenate to measure the MDA level

The amount of malondialdehyde (MDA) in brain tissue samples was measured using thiobarbituric acid reactive substances (TBARS). 0.5 ml of post-mitochondrial supernatant and 0.5 ml of Tris-HCl were incubated at 37°C for 2 hours with 10% tricholoroacetic acid, 0.67% thiobarbituric acid, and 10 minutes of immersion in scalding water. After chilling, 1 ml of double-distilled water was added and 532 nm absorbance was determined (coefficient for calculation 1.56 x 105 M-1 cm1)²⁴.

Solution preparation methods

10% tricholoroacetic acid = dissolve 2.2g of TCA in 1 ml of water to get a 100% (w/v) solution. After that, formulate a 10% solution by mixing 0.9 ml of water with 0.1 ml of 100% TCA.

(Abs.)

MDA Conc.= -------------------------------

(0.156 ×gm of tissue in homogenate)

UV analysis of brain tissue homogenate to measure GSH level

The Ellman reaction was used to determine the quantity of reduced glutathione (GSH) in a brain sample. The post-mitochondrial supernatant was precipitated with sulphosalicylic acid and centrifuged at 4°C. 0.1 ml of supernatant, 2.7 ml of phosphate buffer, and 0.2 ml of 5.5'-dithiobis (2-nitrobenzoic acid) were the components in the test mixture. The yellow hue was measured using a Shimadzu UV-visible spectrophotometer at a wavelength of λ 412 nm.

Solution preparation methods

0.1 M phosphate buffer solution pH 8.0: Mix 0.523g of KH2PO4 and 16.73g of dipotassium (dibasic) hydrogen phosphate in water and dilute this mixture up to 1000 ml with H₂O.

Ellman’s reagent: 0.039g dissolved in 10 ml phosphate buffer.

GSH Conc.=(Abs.Sample-Abs.Blank )×Dilution factor/Ꜫ (14.15)×Vol. of sample in ml

The determination of the neurotransmitter levels (5-HT or Serotonin)

As per given instructions from the manufacturer, KRISHGEN Bio Systems provides a kit (Cat. No. KLR0866 Ver. 2.2), which is used to determine the neurotransmitter serotonin with the support of an enzyme-linked immunosorbent assay (ELISA). Before starting experimental process tissue sample was prepared. Blood which is attached to tissue was clean with help of PBS of pH 7.4 before homogenisation tissue must be weight. This homogenate centrifuge at temperature 2-8 °C at 2000-3000 RPM for 20 min, separate Supernatant cautiously. Then next steps were performed accurately following the instructions of the manufacturer. The quantitative sandwich enzyme immunoassay method is used in this assay. Serotonin-specific antibodies were previously coated on a microtiter plate. The use of duplicates or triple copies for all Standards and Samples is highly advised. 50 ul of Standard Diluent should be added to each blank well. 50 ul of the prepared Standards should be added to each sample well. 40 ul of the sample should be added to each sample well. 10 ul of biotinylated serotonin antibody should be pipetted into each sample well. 50 ul of Streptavidin: HRP pipette Apply to all wells. Mix thoroughly. After sealing the plate, incubate it at 37°C for 60 min. Plate must have to be aspirated, cleaned with diluted Wash Buffer (1X) four times, and any excess buffer needs to be blotted by vigorously pressing the plate with absorbent paper. Remove any liquid that may have collected on the bottom outside of the microtiter wells because it could interfere with the reading process. 100 ul TMB in a pipette Add substance to each well. The plate should be incubated for 10 minutes at 37 °C. DO NOT SHAKE as this could lead to greater backgrounds and less precise results. Positive wells should change colour to a bluish hue. Stop Solution 100 ul added to all wells with the help pipette. The colour of the wells should change from blue to yellow. After adding the Stop solution, measure the absorbance at 450 nm with a microplate within 15 to 20 minutes²⁵. Instrument used for procedure is 96 well Microplate reader (EPOCH Version) Make by Bio Tek-Agilent, USA

The determination of the Pro- Inflammatory Cytokines (IL-6) and (TNF- α)

The GENLISATM ELISA kits (Cat. Nos. KB3068 Ver 5.3) and (Cat. Nos. KB3145 Ver 6.1) provided by KRISHGEN Bio Systems were used to measure (IL-6) and (TNF- α) respectively. Following the manufacturer's protocols. The levels of IL‑6 and TNF- α were expressed as pg/ml. The similar devices mentioned above was employed for this procedure also.

Statistical analysis.

Statistical analysis were perfumed with support of Graph Pad Prism 5.01. Values are expressed as the mean ± standard error mean as indicated. Significance was analysed using one‑way method, next comparisons by Dunnett’s test. Significance was set at p<0.05; least significant difference between the control and treatment groups.

RESULT:

Effect of Zn and Ga in Sucrose preference test (%)

In this Learning process, anhedonia (a lack of interest in routine or day today activities) was identified using a sucrose preference test. Existing study sucrose preference test was performed before induction of CUMS and after 21days treatment with doses of Zingerone (75, 125, 250 mg/kg) and Gramine (13, 27.5,55 mg/kg). Before start of study SP test observed normal in all groups of animals (Figure. 1). After completing 21 days exposure of CUMS stress we observed that preferences to the consumption of Sucrose water get decrease significantly and simultaneously treatment with Zingirone dose 125mg/kg and 250mg/k, Gramine (13, 27.5,55 mg/kg) all doses and Ecitalopram (Std.) drugs shows significantly increase in consumption or preference to Sucrose water when compare with normal control group, significant changes in SP (where, (*p < 0.05)). (Figure. 2).

Figure 1. Sucrose water consumption preferences (%) given by animals before induction of CUMS.

Figure 2. Sucrose water consumption preferences (%) consider after treatment of 21days with Zingerone and Gramine, induction of CUMS. Mean ±SEM; N=6. Significant alteration (*p < 0.05)

Effect of Zn and Ga in Forced Swim test

21 days of continuous exposure to CUMS stressor procedures significantly increased immobility time in the force swim test observed in group 2. After treatment with Zingerone, we observed that comparatively, 75 and 125 mg/kg have less significance (reduction in mobility) than 250 mg/kg when compared with the CUMS group. Gramine doses of 13 and 27.5 mg/kg show significant effects, but doses of 55 mg/kg and Ecitalopram (standard) show more significance (maximum reduction in immobility) when compared with the CUMS group (Figurer 3).

Figure: - 3 Effects of Zn and Ga treatment on the immobility time in the FST after the CUMS paradigm. Mean ±SEM. N=6. Significant alteration (*p < 0.05)

Effect of Zn and Ga on oxidative parameter MDA/ LPO

When comparing CUMS stressor group 2 with the normal control group, lipid peroxidation levels in the rat brain were found to be significantly greater. Furthermore, zingirone-treated groups 75, 125, and 250 mg/kg were found to be less significant when compared to CUMS group 2, while gramine-treated groups 13, 27.5, and 55 mg/kg, as well as Ecitalopram (standard), have been demonstrated to be more significant, to CUMS group 2 (Figure 4).

Figure: - 4 Effect on oxidative parameter MDA/ LPO activity in rat brain. Mean ±SEM. N=6. Significant alteration (*p < 0.05)

Effect of Zn and Ga in oxidative parameter GSH

When comparing CUMS stressor group 2 with the normal control group, reduced glutathione levels in the rat brain were found to be significantly decreased. This reduced glutathione level in Zingerone group only dose of 250 mg/kg significantly increased, and not only Gramine groups doses of 27.5 and 55 mg/kg but also Ecitalopram (standard) showed a significant increased level of reduced glutathione when compared with CUMS stressor group 2 (Figure. 5).

Figure: - 5 Effect on oxidative parameter GSH activity in rat brain. Mean± SEM. N=6. Significant alteration (*p < 0.05)

Effect of Zn and Ga in neurotransmitter levels (5-HT or Serotonin)

The neurotransmitter serotonin level was significantly decreased in CUMS stressor rats when compared with the control group. After treatment with Zn, doses of 250 mg/kg showed a significant rise in the level of 5HT, but doses of 75 mg/kg and 125 mg/kg were less significant. Groups treated with gramine and ecitalopram (standard) showed a significant elevation in serotonin levels when compared with CUMS stressor rats in group 2. (*p < 0.05) Figure. 6).

Figure. 6 Effect on Serotonin (5HT) activities in rat brain. Mean± SEM. N=6. Significant alteration (*p < 0.05)

Effect of Zn and Ga in Pro-Inflammatory Cytokines (IL-6) and (TNF- α)

The levels of cytokines were significantly higher in group 2 compared with the control. In particular, when Zn and Ga were administered to all treated groups, including the standard group, elevated levels of IL-6 were significantly reduced. Likewise, TNF-α level in stressed rats treated with Zn group 75 mg/kg was observed as not significant, 125 mg/kg was less significant, and 250 mg/kg showed a significant effect. In a similar fashion, Ga administration-treated groups (13 mg/kg not significant, 27.5 mg/kg less significant, 55 mg/kg, and Ecitalopram (standard)) showed a significant decrease in level of TNF-α when compared to the CUMS stressor group. (*p < 0.05) (Figures. 7 and 8).

Figure. 7 and 8 Effect Interlukin-6 (IL-6) and TNF-α activity in rat brain. Mean± SEM. N=6. Significant alteration (*p < 0.05)

DISCUSSION:

The chronic unpredictable mild stress model in rodents demonstrated an ameliorating connection with several progressive mental diseases and types of depression. As a result, the CUMS stressor model is the most appropriate and compatible model for studying a wide range of depression problems and mental illnesses^{26, 27}. Animals exposed to the CUMS model displayed a variety of symptoms that were comparable to those experienced by patients with depression^{28, 29}. If we divide this model into three parts, we might recognise numerous biological mechanisms of direct antidepressant medicines as well as elaborate pathological mechanisms underlying depression. They are as follows: Construction (Observation of anhedonia as a basic symptom), Prediction (effective medications were reversed observation of CUMS), and Validation (Comparison of biochemical values with humans). For these reasons³⁰, we have included the CUMS model in our investigation of Zingerone and Gramine.

According to the current study, there was successful induction of depression by the CUMS model based on animal behaviour studies. Sucrose preference test and force swim test models are most validated models used in this CUMS study. The sucrose preference test demonstrated that less consumption of sucrose water or a significant decrease in sucrose preference (%) when compared before beginning the study and after completing 21 days of treatment without drugs showed anhedonia. Furthermore, a considerable increase in immobility time in the Force swim test when compare with control group which demonstrated the successful induction of CUMS depression in rats³¹.

Now next to the induction process, treatment with Zingerone and Gramine demonstrated good reversal anti-depressant activity, which also indicated by increased consumption of sucrose water. After 21 days daily administration with Zn, Except dose 75mg/kg dose other doses of Zn 125, 250 mg/kg, Ga (13, 27.5, 55 mg/kg) and Standard Ecitalopram showed significantly restored in percentage of sucrose water consumption when compare with CUMS group. The nerve cells in the brain reward system are damaged, according to the chronic unpredictable stress study. The condition is thought to be linked to the serotonergic (5-HT) systems, leading a loss of happiness or pleasure. ^{32, 33}.

According to this study's force swim test, Gramine drugs had better reversal anti-depressant efficacy than Zingerone, exhibited by the rats' improved immobility time and decreased struggle time ³⁴.

Changes in reactive oxygen species and variations in oxidative stress are key factors in the aetiology of depression³⁵. In this study, we observed that the CUMS stressor group increased levels of lipid peroxidation or malondialdehyde and declined levels of reduced glutathione when compared with the normal control group, which ultimately shows a decrease in antioxidant activity in rats. After treatment with Zn and Ga, we observed that higher doses of both drugs effectively decreased MDA levels and increased GSH levels; hence, it proved that both drugs have potential antioxidant activity.

According to recent research, persistent stress is a crucial role in the advancement of depression related with brain function. Neuroinflammation and its contributors^36,37. Cytokinine induced behavioural abnormalities, where seratonine level was varied³⁸. That is why we have included serotonin in our study. In this study, we found that there was a decrease in serotonin levels in the CUMS group when compared to the control group after 21 days of regular treatment with Zn and Ga. We concluded that Gramine elevates or regulates serotonin levels properly, but Zingerone was less effective on serotonin.

Pro-inflammatory cytokines are a particular target for depression as well. In this study, IL-6 and TNF-α were taken into consideration due to research into these pro-inflammatory cytokines, which are excellent mimics of CUMS-induced depression^{39, 40}. In the existing study, we inspected whether the CUMS-induced depression group observed a remarkable increase in levels of IL-6 as well as TNF-α. After regular treatment with Zn and Ga at all doses, a remarkable reduction in IL-6 was observed. While TNF-α levels slightly change at lower doses, there is a remarkable decrease at high doses of Zn and Ga.

CONCLUSION:

After considering all factors, primary we report that Zingerone and Gramine having potential antidepressant activity when used against CUMS stressor model. Our finding suggested that comparatively Gramine possess best inhibition on IL-6 and Serotonin factors affected by CUMS. Antioxidant property of both drugs was observed remarkable. Further studies are needed to explore molecular mechanism of Zingerone and Gramine effects in CUMS-induced depression.

CONFLICT OF INTEREST:

The authors’ Rahul R. Sable, Dr. Ghanshyam B. Jadhav, declare no conflict of interest, financially or otherwise.

ACKNOWLEDGEMENT:

I thank my mentor Dr. Ghanshyam B. Jadhav for their assistance and I would like to thank Respected Principal Dr. D.V. Derle, MVPs Institute of Pharmacy Nashik and Respected Principal Dr. S.J. Kshirsagar, MET’s Institute of Pharmacy, Adgaon, Nashik for their Support. Special thanks to dear Colleague Dr. Pwan Udawant for their technical assistance.

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Received on 16.10.2022 Modified on 28.03.2023

Research J. Pharm. and Tech 2023; 16(7):3067-3074.

DOI: 10.52711/0974-360X.2023.00504

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