s3-ap-southeast-1.amazonaws.com€¦ · iii declaration i declare that the thesis entitled...

Psychopharmacological evaluation of herbal formulation –

an experimental study

A Thesis submitted to Gujarat Technological University

for the Award of

Doctor of Philosophy

In Pharmacy

by

Krishna Mahendrabhai Shah

149997390006

under supervision of

Dr. Sunita Goswami

GUJARAT TECHNOLOGICAL UNIVERSITY

September – 2020

i

Psychopharmacological evaluation of herbal formulation –

an experimental study

A Thesis submitted to Gujarat Technological University

for the Award of

Doctor of Philosophy

In

Pharmacy

by

Krishna Mahendrabhai Shah

149997390006

under supervision of

Dr. Sunita Goswami


Septmember – 2020

ii

© Shah Krishna Mahendrabhai

iii

DECLARATION

I declare that the thesis entitled "Psychopharmacological evaluation of herbal formulation –

an experimental condition” submitted by me for the degree of Doctor of Philosophy is the

record of research work carried out by me during the period from 2014 to 2018 under the

supervision of Dr. Sunita Goswami and this has not formed the basis for the award of any

degree, diploma, associate ship, fellowship, titles in this or any other University or other

institution of higher learning.

I further declare that the material obtained from other sources has been duly acknowledged in the

thesis. I shall be solely responsible for any plagiarism or other irregularities, if noticed in the

thesis.

Signature of the Research Scholar: …………………………… Date:….……………

Name of Research Scholar: Ms. Krishna Mahendrabhai Shah

Place: Ahmedabad

I certify t

formulat

the candi

not subm

Associate

research

and (iii) t

Signature

Name of

Place: Ah

that the work

tion – an ex

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f Supervisor:

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by the Rese

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Date: ………

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v

Course-work Completion Certificate

This is to certify that Ms. Krishna Shah enrollment no. 149997390006 is a PhD scholar enrolled

for PhD program in the branch Pharmacy of Gujarat Technological University, Ahmedabad.

(Please tick the relevant option(s))

He/She has been exempted from the course-work (successfully completed during M.Phil

Course)

He/She has been exempted from Research Methodology Course only (successfully

completed during M.Phil Course)

He/She has successfully completed the PhD course work for the partial requirement for

the award of PhD Degree. His/ Her performance in the course work is as follows-

Grade Obtained in Research Methodology (PH001)

Grade Obtained in Self Study Course (Core Subject) (PH002)

CC AB

Supervisor’s Sign

(Dr. Sunita Goswami)

√

vi

Originality Report Certificate

It is certified that PhD Thesis titled "Psychopharmacological evaluation of herbal

formulation – an experimental condition” by Ms. Krishna Shah has been examined by us.

We undertake the following:

a) Thesis has significant new work / knowledge as compared already published or are under

consideration to be published elsewhere. No sentence, equation, diagram, table,

paragraph or section has been copied verbatim from previous work unless it is placed

under quotation marks and duly referenced.

b) The work presented is original and own work of the author (i.e. there is no plagiarism).

No ideas, processes, results or words of others have been presented as Author own work.

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changing or omitting data or results such that the research is not accurately represented in

the research record.

e) The thesis has been checked using Turnitin software (copy of originality report attached)

and found within limits as per GTU Plagiarism Policy and instructions issued from time

to time (i.e. permitted similarity index <10%).

Signature of the Research Scholar: …………………………… Date: ….………

Name of Research Scholar: Ms. Krishna Shah

Place: Ahmedabad

Signature of Supervisor: ……………………………… Date: ………………

Name of Supervisor: Dr. Sunita Goswami

Place: Ahmedabad

ix

PhD THESIS Non-Exclusive License to


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of research at GTU and elsewhere, Ms. Krishna Shah having 149997390006 hereby grant a

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h) I retain copyright ownership and moral rights in my thesis, and may deal with the

copyright in my thesis, in any way consistent with rights granted by me to my University

in this non-exclusive license.

x

i) I further promise to inform any person to whom I may hereafter assign or license my

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license.

j) I am aware of and agree to accept the conditions and regulations of PhD including all

policy matters related to authorship and plagiarism.

Signature of the Research Scholar: …………………………… Date: ….………

Name of Research Scholar: Ms. Krishna Shah

Place: Ahmedabad

Signature of Supervisor: ……………………………… Date: ………………

Name of Supervisor: Dr. Sunita Goswami

Place: Ahmedabad

Seal:

xii

ABSTRACT

Background: Tensnil syrup is a polyherbal formulation (PHF) containing ingredients such as,

extracts of garmarogor, devdaru, shankhavali, pitapapapdo, brahmi, jatamansi, nagarmoth, kadu,

tagar, himaj, draksha, ashwagandha.

Objective: The objectives for the study were to evaluate toxicity study for the polyherbal

formulation along with ED50 determination. We had also investigated long term effect of this

formulation on brain function by using various animal models such as chronic unpredictable

mild stress mice model, LPS –induced neuroinflammation model and ketamine induced

psychosis model.

Materials and methods: For toxicity study the PHF were administered orally at a therapeutic

dose range (100 - 800 mg/kg/day), for 28 days. All animals were monitored daily for their health

status and signs of abnormalities. The body weight and food intake were measured once weekly.

At the end of the experimental period, various haematological and biochemical parameters were

estimated. For acute study, forced swim test (FST), tail suspension test (TST), elevated plus

maze (EPM) and photoactometer tests were performed at doses of 400 and 800 mg/kg.

Fluoxetine (20 mg/kg, p.o.) was used as standard. In CUMS model mice were subjected to a

series of stressful events for a period of 28 days. Drug treatments were given for a period of 28

days after the induction of disease. Parameters studied included behavioural aspects, sucrose

preference test, brain neurotransmitters (5-HT, nor-adrenaline and dopamine) levels, serum pro-

inflammatory cytokines (TNF-α, IL-1β and IL-1), corticosterone, quinolinic acid and oxidative

markers. In LPS model treatments (PHF (600 mg/kg; p.o.) and fluoxetine (20 mg/kg, p.o.)) were

daily administered for 14 days, and challenged with saline or LPS (0.83 mg/kg, i.p.) on 14th day.

In ketamine – induced psychosis study, the effect of PHF on ketamine (50 mg/kg, i.p.) – induced

behavioral (locomotor activity, stereotype behaviour, memory retention and helplessness

behaviour), biochemical (cytokines and anti – oxidants) and neuroprotective alteration (BDNF -

Brain derived neurotropic factor) in the brain were evaluated. Treatments (PHF (600 mg/kg;

p.o.) and haloperidol (0.25 mg/kg, i.p.))

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Results: Long-term use of PHF did not show any remarkable change in physical, haematological

and biochemical parameters. Further, single dose treatment of PHF for 7 days at the dose of 400

and 800 mg/kg, showed significant antidepressant and anxiolytic activity as evident from

significant reduction of immobility time in FST and TST along with increased locomotor index

and time spent in close arm in EPM in acute study. In CUMS model, treatment with polyherbal

formulation (400 & 800 mg/kg) significantly ameliorated behavioral deficits and reduced (p < 0.001)

anhedonia using sucrose preference test. Significant up regulation of serotonin and other

neurotransmitters along with reduction in oxidative stress was observed in treated animals. Further,

polyherbal formulation also significantly attenuated the stress-induced increase in serum levels of TNF-α,

IL-1β, IL-1, corticosterone and quinolinic acid. Pretreatment with formulation in LPS model significantly

ameliorated the anxiety – like behavior as evident from the results of an elevated plus maze and

locomotor activity. LPS – evoked depressive – like effect produced by forced swim test and learning –

memory deficiency by Morris water maze test were prevented. Pretreatment with formulation also

ameliorated LPS – induced neuroinflammation by attenuating TNF – α, IL- 6, IL - 1β levels along with

decrease in oxidative stress via its potential to increase reduced glutathione concentration and reduction in

lipid peroxidation and nitrite levels. Besides, BDNF (a neuroprotective factor) and quinolinc acid

(neurotoxin) significantly increased and decreased respectively in PHF treated animals.

Conclusion: Formulation could ameliorate anxiogenic, depressive, psychotic symptoms and

biochemical changes in rodents, indicating protective effects in the treatment of neurological disorders

such as depression and psychosis.

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Acknowledgement

Research is to see what everybody has seen but think what nobody has thought. Ph.D. has been

inspiring, often exciting, sometimes challenging, but always interesting experience. “No research

is ever outcome of single individual’s talent or efforts.” Working on a research project needs

guidance, support and encouragement without which it is not possible to easily sail through ups

and down during project work. It provides me pleasure to convey my gratitude to all those who

have directly or indirectly contributed to make this work successfully. Though words are seldom

gives sufficient to express gratitude and feelings, it somehow gives me an opportunity to

acknowledge those who helped me during the tenure of my study. These people include my

parents, teachers, friends, well-wishers, relatives and members of ethics committee.

First, I want to submit my deep pray to god whose blessings remained with me from beginning of

my research work. God is always with us, above us to bless us, below us to support us, before us

to guide us, behind us to protect us, beside us to comfort is and inside us to sustain us. “To dear

God whose eternal blessing and divine presence help us to achieve our goal”.

It gives me an immense pleasure to thank my kind, polite and humble guide Dr. Sunita

Goswami, Associate professor, L.M. College of Pharmacy. Her immense support, guidance and

knowledge have helped me to face boldly the ups and down during my entire project work, she

was willing to help me at any point of time without resistance and hesitation. Apart from guiding

me, her moral support and advice has definitely inspired me a lot. I will remain extremely

indebted to her for shaping me out not only for the project but also for my life. I was fortunate to

work under her guidance.

I am thankful to Ms. Vandana Mody, Vice President, Cadila Pharmaceutical PVT. LTD for

providing me drug samples. My Ph.D. would not have been possible without her kind and gentle

help.

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Knowledge, guidance, innovation, motivation and encouragement are requirement to start any

research work. This is what I have got from Doctorate Progress Committee members: Dr.

Shrikalp Deshpande, Principal and Professor, K.B.I.P.E.R, Gandhinagar and Dr. Ashutosh

Jani, General Manager, Lambda research centre throughout my project work. I take this

opportunity to thank them from bottom of my heart for their never ending helpful hand, precious

guidance, intellectual discussion, strong motivation and friendly and humble nature. They were

always beside me to support and inspire me through their thought and work and made me

confident to do my work. I am really indebted to them for tolerating me and helping me as

without them, my work would not has been a success.

I am thankful to the principal, Dr. M.T. Chhabria, for his inspiration. He was always beside me

to support and inspire me through his thought and provide necessary facilities for my work.

I really want to thank HOD, Dr. Anita Mehta and Dr. Mamta Shah, L.M. College of Pharmacy

for their full support, encouragement and timely suggestion that have made my work go

smoothly. I am also obliged to Dr. Gaurang Shah, pofessor, L.M. College of Pharmacy for his

valuable suggestions.

I would like to express my sincere thanks to Rupaben and Dr. Jayesh Beladiya for their kind

support and help.

I would also like to thank other non teaching members of L. M. College of Pharmacy and

Shivabhai and Malikaka, Department of Pharmacology for their kind support and help during

my work.

I am also indebted to all library and administrative staff of L. M. College of Pharmacy who have

directly or indirectly supported me whenever needed.

My work would not have been completed without the immense support of my friends and it was

most memorable part of my life with them during my Ph. D. course. I thank Vinendra,

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Khushboo, Manthan, Varsha, Disha and Kaushal who were not only friends but gave me a

strong support in all ups and down during my project work.

Lastly, I want to thank the heart of my project, my family without whom I would never have been

able to be as I am now. I am and I will always be indebted infinitely to my beloved parents for

their endless love , ultimate care, whole hearted support and encouragement throughout my life

that made me step out , explore new horizons and become confident in my work. I also thank my

brother Bhaumik for his encouragement and help.

Finally, I offer my endless gratitude to all animals who sacrificed their lives to fulfill the

requirements and contribute to make my project success.

xvii

Table of Content Content Page

No. Chapter 1 Introduction 1 Chapter 2 Review of Literature 5 2.1 Definition and epidemiology 5 2.2 Clinical manifestations 5 2.3 Aetiology 6 2.4 Types of depression 7 2.4.1 Major depression 7 2.4.2 Dysthymic disorder 7 2.4.3 Bipolar disorder 7 2.4.4 Melancholia 7 2.4.5 Cyclothymic disorder 8 2.4.6 Psychotic depression 8 2.4.7 Seasonal affective disorder 8 2.5 Theories of depression 9 2.5.1 Monoaminergic theory 9 2.5.2 Glutamatergic theory 10 2.5.3. Neuroendocrine theory 11 2.5.4 Immunological theory 11 2.5.5 Neurotrophic theory 13 2.6 Diagnosis of depression 13 2.6.1 Hamilton Rating Scale for Depression (HAM-D or HRSD) 13 2.6.2 ICD 10 diagnostic criteria for a depressive episode (WHO 1992) 13 2.7 Treatment of depression 14 2.7.1 Pharmacological therapy 15 2.7.2 Non-pharmacological therapy 16 Chapter 3 Materials and methods 19 Experimental design 21 3.1 Toxicity study 21 3.2 Preliminary screening of activity and ED50 calculation 22 3.3 Acute study 22 3.3.1 Forced swim test 23 3.3.2 Tail suspension test 23 3.3.3 Locomotor activity 23 3.3.4 Elevated plus maze 23 3.4 Chronic studies 24 3.4.1 Chronic mild stress – induced depression in mice model 24 3.4.1.1 Forced swim test 25 3.4.1.2 Tail suspension test 25 3.4.1.3 Locomotor activity 25 3.4.1.4 Elevated plus maze 26

xviii

3.4.1.5 Sucrose preference test 26 3.3.1.6 Proinflammatory cytokines estimation 26 3.4.1.7 Brain neurotransmitters analysis 26 3.4.1.8 Serum corticosterone measurement 27 3.4.1.9 Serum quinolinic acid estimation 27 3.4.1.10 Oxido-nitrosative stress parameters 28 3.4.1.11 Body weight 28 3.4.1.12 Adrenal gland weight 28 3.4.2 LPS – induced neuroinflammation in mice model 29 3.4.2.1 Forced swim test 29 3.4.2.2 Locomotor activity 29 3.4.2.3 Elevated plus maze 29 3.4.2.4 Morrison water maze test 30 3.4.2.5 Proinflammatory cytokines estimation 30 3.4.2.6 Serum corticosterone measurement 30 3.4.2.7 Serum quinolinic acid estimation 30 3.4.2.8 Oxido-nitrosative stress parameters 30 3.4.2.9 Nerve growth factor 31 3.4.3 Ketamine-induced psychosis model 31 3.4.3.1 Locomotor activity 32 3.4.3.2 Stereotype behaviours 32 3.4.3.3 Water maze test 32 3.4.3.4 Catalepsy test – bar test 32 3.4.3.5 Learned helplessness 32 3.4.3.6 Social interaction test 33 3.4.3.7 Proinflammatory cytokines estimation 33 3.4.3.8 Oxido-nitrosative stress parameters 33 3.4.3.9 Nerve growth factor 33 Chapter 4 Results 34 4.1 Toxicity study 34 4.1.1 Effects of polyherbal formulation on body weight and food intake 34 4.1.2 Effect of polyherbal formulation on haematological parameters 35 4.1.3 Effect of polyherbal formulation on the biochemical parameters 35 4.2 Preliminary screening of activity and ED50value determination 36 4.3 Acute study 37

4.3.1 Effect of polyherbal formulation on FST 37 4.3.2 Effect of polyherbal formulation on TST 38 4.3.3 Effect of polyherbal formulation on locomotor activity 39 4.3.4 Effect of polyherbal formulation on EPM 40 4.4 Chronic study 41 4.4.1 Chronic unpredictable mild stress – induced depression in mice model 41 4.4.1.1 Effect of polyherbal formulation on CUMS-induced altered FST 41 4.4.1.2 Effect of polyherbal formulation on CUMS – induced altered TST 43 4.4.1.3 Effect of polyherbal formulation on CUMS – induced altered locomotor 44

xix

activity2 4.4.1.4 Effect of polyherbal formulation on CUMS – induced altered EPM

activity 45

4.4.1.5 Effect of polyherbal formulation on CUMS – induced altered sucrose preference test

46

4.4.1.6 Effect of polyherbal formulation on CUMS – induced altered levels of proinflammatory cytokines

47

4.4.1.7 Effect of polyherbal formulation on CUMS – induced altered levels of neurotransmitters

48

4.4.1.8 Effect of polyherbal formulation on CUMS – induced altered levels of corticosterone

51

4.4.1.9 Effect of polyherbal formulation on CUMS – induced altered levels of quinolinic acid

52

4.4.1.10 Effect of polyherbal formulation on CUMS – induced altered levels of oxido - nitrosative stress parameters

53

4.4.1.11 Effect of polyherbal formulation on CUMS – induced altered adrenal gland weight

55

4.4.2 LPS – induced neuroinflammation in mice model 56 4.4.2.1 Effect of polyherbal formulation on LPS – induced forced swim test 56 4.4.2.2 Effect of polyherbal formulation on LPS – induced locomotor activity 57 4.4.2.3 Effect of polyherbal formulation on LPS – induced elevated plus-maze 58 4.4.2.4 Effect of polyherbal formulation on LPS – induced Morrison water maze

test 59

4.4.2.5 Effect of polyherbal formulation on LPS – induced proinflammatory cytokines

60

4.4.2.6 Effect of polyherbal formulation on LPS – induced serum corticosterone measurement

61

4.4.2.7 Effect of polyherbal formulation on LPS – induced serum quinolinic acid estimation

62

4.4.2.8 Effect of polyherbal formulation on LPS – induced oxido-nitrosative stress parameters

63

4.4.2.9 Effect of polyherbal formulation on LPS – induced altered level of nerve growth factor

66

4.4.3 Ketamine-induced psychosis model 67 4.4.3.1 Effect of polyherbal formulation on ketamine – induced locomotor

activity 67

4.4.3.2 Effect of polyherbal formulation on ketamine – induced stereotype behaviours

70

4.4.3.3 Effect of polyherbal formulation on ketamine – induced water maze test 82 4.4.3.4 Effect of polyherbal formulation on ketamine – induced catalepsy test –

bar test 83

4.4.3.5 Effect of polyherbal formulation on ketamine – induced learned helplessness

84

4.4.3.6 Effect of polyherbal formulation on ketamine – induced social interaction test

85

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4.4.3.7 Effect of polyherbal formulation on ketamine – induced proinflammatory cytokines

86

4.4.3.8 Effect of polyherbal formulation on ketamine – induced oxido-nitrosative stress parameters

89

4.4.3.9 Effect of polyherbal formulation on ketamine – induced altered level of nerve growth factor

90

Chapter 5 Discussion 93 Chapter 6 Conclusion 105 Chapter 7 References 106

xxi

List of Abbreviations

Abbreviation Definition

5-HIAA 5-Hydroxyindoleacetic Acid 5-HT 5-Hydroxy Tryptophan

ACTH Adrenocorticotropic Hormone

ALP Alkaline Phosphatase ALT Alanine Amino Transferase

AMPA Α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid ANOVA Analysis Of Variance

AST Aspartate Amino Transferase ATP Adenosine Triphosphate BCT Bright Light Therapy

BDNF Brain-Derived Neurotrophic Factor BH4 Tetra Hydrobiopterine CMS Chronic Mild Stress CNS Central Nervous System

COMT Catechol-O-Methyltransferase

CPCSEA Committee For The Purpose Of Control And Supervision Of Experiments On

Animals CRF Corticotropin Releasing Factor

CSF Cerebrospinal Fluid CUMS Chronic Unpredictable Mild Stress Model

DA Dopamine DOPAC Noradrenaline DTNB 5, 5′-Dithiobis-(2-Nitrobenzoic) Acid ECT Electroconvulsive Therapy

ED50 Effective Dose 50

EDTA Ethylenediamine tetraacetic Acid ELISA Enzyme-Linked Immunosorbent Assay EPM Elevated Plus Maze FST Forced Swim Test

GABA Gamma-Aminobutyric Acid HAM- D /

HRSD Hamilton Rating Scale For Depression

HCI Hydrochloric Acid HGB Hemoglobin HGH Human Growth Hormone

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HPA Hypothalamic-Pituitary-Adrenal Axis HPT Hypothalamic-Pituitary-Thyroid HRP Horseradish Peroxidase HVA Homovanillic Acid i.p. Intra Peritoneal ICD International Classification Of Diseases IDO Indoleamine 2, 3-Dioxygenase

IL-1β Interleukin - 1 Beta KA Kyneronic Acid KP Kynurenine Pathway

LPO Lipid Peroxidase LPS Lipopolysaccharides

MAO Monoamine Oxidase MCH Mean Corpuscular Hemoglobin

MCHC Mean Corpuscular Hemoglobin Concentration MDA Malondialdehyde

MHPG 3- Methoxy-4-Hydroxyphenylglycol MWM Morrison Water Maze

NA Noradrenaline NE Norepinephrine

NF-kB Nuclear Factor Kappa B NGF Nerve Growth Factor

NMDA N-Methyl-D-Aspartate NO Nitric Oxide

NOAEL No Observed Adverse Effect Level NT-3 Neurotrophin-3 OPT O-Phthalaldehyde

PHF Poly Herbal Formulation PLT Platelet

QUIN Quinolinic Acid rpm Revolutions Per Minute SAD Seasonal Affective Disorder SDS Sodium Dodecyl Sulphate sec Second

SEM Standard Error Of Mean SERT Serotonin Transporter SNRIs Serotonin-Norepinephrine Reuptake Inhibitors SSRIs Selective Serotonin Reuptake Inhibitors TCAs Tricyclic Antidepressants

xxiii

TNF-α Tumor Necrosis Factor TRP Tryptophan TST Tail Suspension Test

WHO World Health Organization

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List of Symbols

Symbol Definition β Beta α Alpha mg Milligram kg Kilogram ml Millilitre % Percentage L Litre cm Centimeter °C Degree Celsius min Minute M Molar pH Potential of Hydrogen nm Nanometer µg Microgram v Volume U Unit nM Nanometer h Hour mM Mill molar w/v Weight by volume fL Facolitre pg Picogram

xxv

List of Figures

No. Description Page No.

FIGURE 2.1 Theories of depression 9 FIGURE 2.2 Mechanisms of dopamine synthesis and release 10 FIGURE 2.3 Immunological alterations during depression 11 FIGURE 3.2 The protocol diagram for animal groups for sub-acute toxicity

study 21

FIGURE 4.3.1 Effect of polyherbal formulation on FST 37 FIGURE 4.3.2 Effect of polyherbal formulation on TST 38 FIGURE 4.3.3 Effect of polyherbal formulation on locomotor activity 39 FIGURE 4.3.4 Effect of polyherbal formulation on EPM 40 FIGURE 4.4.1.1 Effect of polyherbal formulation on CUMS – induced altered FST 42 FIGURE 4.4.1.2 Effect of polyherbal formulation on CUMS – induced altered TST 43 FIGURE 4.4.1.3 Effect of polyherbal formulation on CUMS – induced altered

locomotor activity 44

FIGURE 4.4.1.4 Effect of polyherbal formulation on CUMS – induced EPM activity

45

FIGURE 4.4.1.5 Effect of polyherbal formulation on CUMS – induced altered sucrose preference test

46

FIGURE 4.4.1.6 Effect of polyherbal formulation on CUMS – induced altered levels of proinflammatory cytokines

48

FIGURE 4.4.1.7 (a)

Effect of polyherbal formulation on CUMS – induced altered levels of neurotransmitters: NA

48

FIGURE 4.4.1.7 (b)

Effect of polyherbal formulation on CUMS – induced altered levels of neurotransmitters: DA

49

FIGURE 4.4.1.7 (c)

Effect of polyherbal formulation on CUMS – induced altered levels of neurotransmitters: 5-HT

50

FIGURE 4.4.1.8 Effect of polyherbal formulation on CUMS – induced altered levels of corticosterone

51

FIGURE 4.4.1.9 Effect of polyherbal formulation on CUMS – induced altered levels of quinolinic acid

52

FIGURE 4.4.1.10 (a)

Effect of polyherbal formulation on CUMS – induced altered levels of oxido-nitrosative stress parameters: reduced glutathione

53

FIGURE 4.4.1.10 (b)

Effect of polyherbal formulation on CUMS – induced altered levels of oxido-nitrosative stress parameters: lipid peroxidase

54

FIGURE 4.4.1.11 Effect of polyherbal formulation on CUMS – induced altered adrenal gland weight

55

FIGURE 4.4.2.1 Effect of polyherbal formulation on LPS – induced forced swim test

56

FIGURE 4.4.2.2 Effect of polyherbal formulation on LPS – induced locomotor activity

57

FIGURE 4.4.2.3 Effect of polyherbal formulation on LPS – induced elevated plus maze

58

xxvi

FIGURE 4.4.2.4 Effect of polyherbal formulation on LPS – induced Morrison water maze test

59

FIGURE 4.4.2.5 Effect of polyherbal formulation on LPS – induced proinflammatory cytokines

60

FIGURE 4.4.2.6 Effect of polyherbal formulation on LPS – induced serum corticosterone measurement

61

FIGURE 4.4.2.7 Effect of polyherbal formulation on LPS – induced serum quinolinic acid estimation

62

FIGURE 4.4.2.8 (a)

Effect of polyherbal formulation on LPS – induced oxido-nitrosative stress parameter: reduced glutathione

63

FIGURE 4.4.2.8 (b)

Effect of polyherbal formulation on LPS – induced oxido-nitrosative stress parameter: lipid peroxidase level

64

FIGURE 4.4.2.8 (c)

Effect of polyherbal formulation on LPS – induced oxido-nitrosative stress parameter: nitrite level

65

FIGURE 4.4.2.9 Effect of polyherbal formulation on LPS – induced altered level of nerve growth factor

66

FIGURE 4.4.3.1 (a)

Effect of polyherbal formulation on ketamine – induced locomotor activity locomotor activity: Day 0

67

FIGURE 4.4.3.1 (b)


68

FIGURE 4.4.3.1 (c)


69

FIGURE 4.4.3.2 (a)

Effect of polyherbal formulation on ketamine – induced stereotype behaviours on day 0: Falling

70

FIGURE 4.4.3.2 (b)

Effect of polyherbal formulation on ketamine – induced stereotype behaviours on day 0: Head turning

71

FIGURE 4.4.3.2 (c)

Effect of polyherbal formulation on ketamine – induced stereotype behaviours on day 0: Head bobbing

72

FIGURE 4.4.3.2 (d)

Effect of polyherbal formulation on ketamine – induced stereotype behaviours on day 0: Sniffing

73

FIGURE 4.4.3.2 (e)

Effect of polyherbal formulation on ketamine – induced stereotype behaviours on day 5: Falling

74

FIGURE 4.4.3.2 (f) Effect of polyherbal formulation on ketamine – induced stereotype behaviours on day 5: Head turning

75

FIGURE 4.4.3.2 (g)


76

FIGURE 4.4.3.2 (h)

Effect of polyherbal formulation on ketamine – induced stereotype behaviours on day 14: Sniffing

77

FIGURE 4.4.3.2 (i) Effect of polyherbal formulation on ketamine – induced stereotype behaviours on day 14: Falling

78

FIGURE 4.4.3.2 (j) Effect of polyherbal formulation on ketamine – induced stereotype behaviours on day 14: Head turning

79

FIGURE 4.4.3.2 (k)


80

FIGURE 4.4.3.2 (l) Effect of polyherbal formulation on ketamine – induced stereotype behaviours on day 14: Sniffing

81

xxvii

FIGURE 4.4.3.3 Effect of polyherbal formulation on ketamine – induced water maze test

82

FIGURE 4.4.3.4 Effect of polyherbal formulation on ketamine – induced catalepsy test – bar test

83

FIGURE 4.4.3.5 Effect of polyherbal formulation on ketamine – induced learned helplessness

84

FIGURE 4.4.3.6 Effect of polyherbal formulation on ketamine – induced social interaction test

85

FIGURE 4.4.3.7 (a)

Effect of polyherbal formulation on ketamine – induced proinflammatory cytokines: TNF – α

86

FIGURE 4.4.3.7 (b)

Effect of polyherbal formulation on ketamine – induced proinflammatory cytokines: IL – 6

87

FIGURE 4.4.3.7 (c)

Effect of polyherbal formulation on ketamine – induced proinflammatory cytokines: IL-1β

88

FIGURE 4.4.3.8 (a)

Effect of polyherbal formulation on ketamine – induced oxido-nitrosative stress parameters: reduced glutathione

89

FIGURE 4.4.3.8 (b)

Effect of polyherbal formulation on ketamine – induced oxido-nitrosative stress parameters: nitrite level

90

FIGURE 4.4.3.8 (c)

Effect of polyherbal formulation on ketamine – induced oxido-nitrosative stress parameters: LPO

91

FIGURE 4.4.3.9 Effect of polyherbal formulation on ketamine – induced altered level of nerve growth factor

92

xxviii

List of Tables

No. Description Page No.

TABLE 2.1 Plants for the herbal formulation 16 TABLE 2.2 Plants and their reported activity 17 TABLE 3.1 Composition of Tensnil syrup 20 TABLE 3.4.1 Chronic mild stress (CMS) procedure 24 TABLE 4.1.1(a) Effect of polyherbal formulation on body weight (g) 34 TABLE 4.1.1(b) Effect of polyherbal formulation on food intake (g) 34 TABLE 4.1.2 Effect of polyherbal formulation on the haematological parameters

in mice 35

TABLE 4.1.3 Effect of polyherbal formulation on the biochemical parameters in mice

36

TABLE 4.2 Effect of polyherbal formulation on % inhibition of immobility using forced swim test (FST)

36

xxix

List of Appendices

Appendix A: IEC certificate

Appendix B: List of publications

Appendix C: Images for pharmacological methods

Chapter 1 Introduction

1

CHAPTER 1

Introduction

According to WHO, around 450 million public affected by mental illness out of which 10–20

million commit suicide every year universally. In India, the frequency of such illness is around

24.4% and 18.5%, correspondingly, and co-morbidity of anxiety with depression is high about

87%(1). Depression is a mental confusion, which encompasses emotion, cognition, and physical

symptoms with significant morbidity and mortality (2, 3). It was commonly elicited by diverse

factors, including psychological, social, environmental, genetic and metabolic factors (4, 5).

WHO forecasts that depression will be the 2nd highest illness to threaten human’s health (6).

Clinical depression is described by low mood, anhedonia, reduced cognition, low or impair

psychomotor action and sleep trouble(7). Depression produces the greatest decrement in personal

health when compared with chronic physical diseases such as angina, arthritis, asthma and

diabetes (8). The link between stress and depression is not novel and there is a functional link

with stress exposure and depression (9). Stress is one of the best-studied mediators by which

genetic vulnerabilities are translated into mood disorder pathology through the process of

neuroprogression (10).

Stress is a conversion in an environmental situation that upset the normal physiological stability

and connected to various neurological illnesses (11, 12). According to a recent investigation link

between inflammation and the immune system, deregulation has been established in the

pathophysiology of depression (13, 14). The two chief areas response system in both humans and

other animals are (1) a part of the nervous system called the sympathetic nervous structure and

(2) a hormone system called the hypothalamic-pituitary-adrenal (HPA) axis. Both systems allow

the brain to correspond with the rest of the body. Commencement of the sympathetic nervous

system produces several physiological responses within seconds, such as an accelerated heart

rate, augmented respiration, and blood flow redistribution from the skin to the skeletal muscle.

Their response assists the “fight or flight” behavioural reaction. Activation of the HPA axis

induces glucocorticoid discharge, which in spin affect a wide area of physiological response,

such as change in blood sugar level, and blood pressure, fat relocation, muscle collapse, and


2

immune system inflexion (15). Activation of the HPA axis controls the emission of

glucocorticoid hormones from the adrenal gland into general movement. Glucocorticoid

hormones played an important role in the whole body, together with the central nervous

organization (i.e., the brain and spinal cord). Cortisol’s ability to affect many body systems

allows this hormone to be an effective mediator of a generalized stress response. At the same

time, however, the wide range of cortisol’s effect necessitates tight regulation of the hormone’s

levels. This control is achieved largely through a negative feedback mechanism (16).

Pathogenesis also includes abnormal neurotransmitters metabolism, distorted neuroendocrine

functions and partial neural plasticity (17, 18). Improved level of proinflammatory cytokines

such as tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1 beta (IL-1β)

in serum have been reported (18, 19). They activate IDO (Indoleamine 2, 3-dioxygenase) and up-

regulate tryptophan degradation via the kynurenine pathway, and produces neuroactive

metabolite quinolinic acid (QUIN) (20). This QUIN is reported for dysfunctioning of neurons

and neuronal death, thereby inducing permanent damage (21, 22). These cytokines also lead to

hyper activation of the HPA axis (19) which releases a surplus of corticosterone in the body (23)

and generates oxido-nitrosative stress which concerned in the pathophysiology of depression and

anxiety. The experimental effects consist of lipid peroxidation, reduced glutathione level and

reduction in the level of antioxidant enzymes. Therefore, targeting these multiple targets can be a

beneficial approach to provide protection against depression (24).

Clinical facts have been found indicating that even though antidepressant drugs are effective in

treating depressive episodes, they are less efficacious in recurrent depression and in preventing

relapse(25). In some cases, antidepressants have been described inducing adverse events such as

withdrawal symptoms at discontinuation, onset of tolerance and resistance phenomena, switch,

and cycle acceleration in bipolar patients. Unfavorable long-term outcomes and paradoxical

effects (depression inducing and symptomatic worsening) have also been reported. All these

phenomena may be explained based on the oppositional model of tolerance. Continued drug

treatment may recruit processes that oppose the initial acute effect of a drug. When drug

treatment ends, these processes may operate unopposed, at least for some time and increase

vulnerability to relapse (26).


3

Ayurveda is one of the traditional therapeutic systems of Indian. The philosophy behind

Ayurveda is preventing redundant suffering and living a long healthy life. Ayurveda involves the

use of natural essentials to eliminate the root cause of the disease by restoring balance, at the

same time create a healthy life-style to prevent the recurrence of imbalance. In India, about

15,000 medicinal plants have been recorded, in which the communities used 7,000-7,500 plants

for curing diverse diseases. In Ayurveda, single or multiple herbs (polyherbal) are used for the

treatment. Traditional pharmacognosy extracts single active principles, which may be self-

defeating as overall biological property relies on synergistic interactions between plant

components. An extract may contain compounds which do not in a straight line affect the

pathophysiological processes but may change the absorption, distribution, metabolism, and

excretion of bioactive constituents, or reduce the side-effects (27). Polyherbal formulation (PHF)

possesses a littleadvantage such as decline in dose, convenience, and ease of administration. (27-

29). The multitarget impacts of herbal drugs are established to be favourable in chronic

conditions and so forth, and also in restoring the health status (30).

Thus, there is a scope for the improvement of such treatment which works not only behavioural

defects of the depression and anxiety but also helpful for the elimination of toxins from the brain

and produces a calming effect. Poly Herbal Formulation (PHF) contains extracts of garmarogor,

devdaru, shankhavali, pitapapapdo, brahmi, jatamansi, nagarmoth, kadu, tagar, himaj, draksha,

ashwagandha. These plants have been reported to be used in nervous system disorders as they

calm down the brain, produce quality sleep (31), and remove toxins from the brain (32).

Therefore, they can be used in anxiety and sleep disorders. Cassia Fistula appears anti-oxidant,

anti-inflammatory, antibacterial, antidiabetic, antifertility, hepatoprotective, antitumor, antifungal

properties (33). Cedrus deodara has anti-oxidant, anti-inflammatory, antibacterial, antidiabetic,

antifertility, hepatoprotective, antitumor, antifungal properties (34).Evolves Alsinoids is one of

the prime medhya plants of Ayurveda, which may be useful for neural regeneration and synaptic

plasticity. Pre-clinical (in vivo and vitro) investigations have demonstrated anti-amnesic,

antistress (adaptogenic), anxiolytic, cognitive enhancing, antimicrobial and gastroprotective

activity (35). Fumaria Parviflora is an excellent drug that has CNS stimulant, anti-depressant

(36, 37). Hydrocotyl Asiaticain ayurveda is known as cognitive enhancing, anxiolytic and anti-

depressant activity and used for sleep disorders (38). Nardostachys Jatamansi appears to be an


4

excellent candidate for tranquillizing, anti-oxidant, neuroprotective, anticonvulsant activity,

antiparkinson’s activity, hepatoprotective, hypotensive, anti-diabetic (39).Cyperus Rotundushas

cytoprotective, anti-oxidant activity, stimulant,anti-inflammatory, antidiabetic, antidiarrhoeal,

antimutagenic, antimicrobial, antibacterial, and apoptotic, antipyretic and analgesic activities

(40). Picrorrhiza Kurroa is well known for its anti-oxidant, anti-inflammatory antioxidant and

immunomodulatory activities (41).Valeriana Walichii is another most valuable plant for

anxiolytic and anti-oxidant activities and it is beneficial in treating insomnia, nervous

problems(42). Terminalia Chebula is the best herb that has anti-oxidant, anti-inflammatory, and

protective effects on various vital organs (43).Vitis vinifera the herb which has neuroprotective,

anti-oxidant activity, anti-inflammatory, and antimicrobial, cardioprotective, hepatoprotective

activities (44). Withania Somnifera is another important anti-ageing plant along with anti-stress,

adaptogenic (45). Although the active phytochemical constituent of individual plants have been

well conventional, they usually present in small quantity and at all times, they are inadequate to

attain the desired therapeutic property. For this, scientific studies have discovered that these

plants of varying effectiveness when collective may hypothetically create a superior result, as

compared to individual use of the plant and also the sum of their individual effect. This fact of

positive herb-herb relations is known as synergism. Desired therapeutic actions can only be

achieved when different plants confined together having individual potential. In the present

study, the objectives are to perform toxicity study for the polyherbal formulation used under the

study and to determine ED50 value. In addition, long term effect of this formulation is studied on

brain function by using various animal models such as chronic unpredictable mild stress mice

model, LPS –induced neuroinflammation model and ketamine induced psychosis model.

Chapter 2 Review of literature

5

CHAPTER 2

Review of Literature

2.1 DEFINITION AND EPIDEMIOLOGY

Major depressive disorder is a mood disease in which the human being experiences one or more

major depressive episode without a history of manic, mixed, or hypomanic episodes (46). There

are two diverse types of depressive disease, namely unipolar depression, and bipolar affective

disorder. The disease can be additional categorized as bipolar I, where developed episodes of

mania occur, and bipolar II, where depressive episode are intersperse with less severe hypomanic

episodes(47).

The duration risk of rising a bipolar I disorder is said to be about 1% (0.3–1.5%). A correct

estimation for the more generally distinct bipolar II disorder is more difficult and it may be much

more frequent, with studies suggesting a lifetime prevalence of between 0.2% and 10.9%. The

frequency of bipolar I is normally reported to be the same for both man and woman, where some

studies propose that bipolar II may be somewhat more ordinary in woman. Chances of

occurrence of depression usually falls in mid -20s.Although some studies found the incidence

and peak of occurrence of depression in women at the age of 35–45 years (48, 49).

2.2 CLINICAL MANIFESTATIONS(50)

The symptom of depression include emotional and biological components.

Emotional symptoms:

Misery, apathy and pessimism

Low self-esteem: Feelings of guilt, inadequacy and ugliness

Indecisiveness, loss of motivation


6

Physiological symptoms:

Slowing down of thought process

Reduction of sexual activity

Incomplete sleep and food intake

2.3 AETIOLOGY

The aetiology of depressive disease is too compound to be totally explained by a single social,

developmental, or biologic hypothesis. A number of factors emerge to work together to cause or

precipitate depressive disorders. Various factors such as genetic, hormonal, biochemical,

environmental and social all have same role in developing the disease.

Genetic causes

An instant family (parents, children, or siblings) includes e person with depression; the familial

frequency is 1.5 to 3 times higher. When one identical twin develops depression, the view that

the other identical twin will also develop depression is 25 to 93%. Animal models of depression

have drawn in ETP-binding type sub-family B constituent 1, histone deacetylase, e promoter

region related to serotonin transporter gene transcription, neuritin, and disrupted in schizophrenia

linked with depressive episodes (51).

Environmental factors

The life dealings described as ‘threatening’ are more possible to be connected with depression.

Employment, higher socioeconomic class and the reality of a close and confiding association

gives safety for overcoming attack of episode(52).

Biochemical factors

This theory deals with the shortage of neurotransmitter amines in the brain namely noradrenaline

(norepinephrine), serotonin (5-hydroxytryptamine) and dopamine (53).


7

Endocrine factors

Two basic systems mainly hypothalamic-pituitary-adrenal (HPA) axis and the hypothalamic-

pituitary-thyroid (HPT) axis involved in the disease development. Elevated levels of cortisol

have been found and linked to dysfunction within the HPA axis (54).

2.4 TYPES OF DEPRESSION:

Depressive disorder has been recognized as diverse. The major categories are discussed below.

2.4.1 Major depression

It includes low mood, loss of interest along with other symptoms. It may be called as unipolar

depression (55, 56).

2.4.2 Dysthymic disorder

Similar symptoms with less severity to that of the major depression has been observed. Although

symptoms last longer (57, 58).

2.4.3 Bipolar disorder

It’s a 'manic depression' since the person’s mood swings from depression to mania. Mania is like

the reverse of depression including feeling great, lots of power, racing judgment, slight need for

sleep, talking rapidly, obscurity concentrating on tasks, and showing irritated and touchy

behavior (59).

2.4.4 Melancholia

Melancholic depression is psychomotor alteration (usually retardation) and is more frequent in

bipolar depression (bipolar I) than in major depressive disorder. Slow movement of the patient is

the core identification symptoms. It was found that psychomotor agitation is more common in

bipolar II depression (60, 61).


8

2.4.5 Cyclothymic disorder

It’s a milder form of bipolar disorder, where a person experiences chronic unpredictable moods

over at least two years, involving periods of hypomania (a mild to moderate level of mania) and

periods of depressive symptoms, with very short periods of normality between. The period of the

symptoms are shorter, less rigorous and not as usual(62).

2.4.6 Psychotic depression

Sometimes people with a depressive disorder can lose contact with actuality and experience

psychosis. This can involve hallucinations or delusions, such as believing they are bad or evil, or

that they are being watched or followed (63).

2.4.7 Seasonal affective disorder (SAD)

SAD is a mood disorder that has a seasonal blueprint. The cause of the disorder is indistinct, but

it's characterized by mood aggressive (either periods of depression or mania) that begin and end

in a particular season (64).


9

2.5 THEORIES OF DEPRESSION

There are five theories of depression which include monoaminergic, neurotrophic theory, HPA-

axis, immunological and glutamatergic theories of depression.

FIGURE 2.1 Theories of depression(65)

2.5.1 Monoaminergic Theory

Serotonin (5-HT, 5-hydroxy tryptophan) is a monoamine neurotransmitter involved in mood and

appetite regulation. Metabolic studies showed lower level of 5-HIAA in CSF in hospitalized

depressives and associated with an increased risk for suicide (66-69). The 5HT1Aautoreceptor

controls release of serotonin from the presynaptic neuron. Increased 5HT1ABmax (binding sites)

has been reported in suicide victims. An increase in binding sites (B-max) has been stated in

depressed and suicidal patients (70-72).

Dopamine (DA) is a precursor for norepinephrine. CSF levels of homovanillic acid (HVA),

urinary DOPAC levels are decreased in depressives compared with controls. Although not


10

pictured, extreme cytokine-induced release of glutamate and quinolinic acid may also add to

augmented oxidative stress and excitotoxicity (73-75).

FIGURE2.2 Mechanisms of dopamine synthesis and release(76)

Norepinephrine (NE) is a catecholamine is synthesized from the amino acid tyrosine; NE is

degraded by the enzymes catechol-o-methyltransferase (COMT) and monoamine oxidase. A

metabolite 3- methoxy-4-hydroxyphenylglycol (MHPG) which is derived from the brain has

20% to 30% concentration. Urinary MHPG reported significantly lower levels in depressed

patients than healthy controls and exposed that low urinary MHPG levels were seen particularly

in bipolar depressives and a subgroup of unipolar patients (77, 78).

2.5.2 Glutamatergic Theory

Glutamate is an excitatory neurotransmitter which acts by NMDA and AMPA type of receptors.

Up-regulation of NMDA receptor function and consequent cell death has been observed (79). IL-

1β increases production of nitric oxide and hence an increase in glutamate release (80). TNF-α

leads to production of AMPA receptors lacking the GluR2 subunit and facilitate calcium influx

into the neuron. This predisposes the neuron to glutamate-induced excitotoxicity (81). High

concentrations of these compounds are thought to contribute to excitotoxicity and calcium-


11

mediated cell death (82). GABA is a chief inhibitory neurotransmitter in the brain and regulates

seizure threshold as well as norepinephrine and dopamine turnover. GABA levels have been

reported to be decreased in the plasma as well as CSF of depressed patients in a few studies (83,

84).

2.5.3 Neuroendocrine Theory

Three axes, hypothalamic– pituitary–adrenal (HPA), hypothalamic-pituitary-thyroid (HPT),

human growth hormone (HGH), in particular, have been studied in major depression.HPA axis

dysregulation may be a result of distressed physiology of the hypothalamic and limbic

system centers that control the secretion of corticotropin-releasing factor (CRF)

and adrenocorticotropic hormone (ACTH). On the other hand, abnormal neurophysiology

and central nervous system function may cause the depressed state and HPA axis over activity

(15, 16).

2.5.4 Immunological Theory

The central release of corticotrophin-releasing hormone in depressed persons activates the

hypothalamic-pituitary-adrenal axis and altered with evidence of immune suppression (e.g.,

decreases in lymphocyte responses), as well as inflammation. Cytokine to brain communication

occurs when proinflammatory cytokines that bind to cytokine receptors throughout the brain

(86). The proinflammatory cytokines in the peripheral blood go through the weak region of the

blood-brain barrier and exhibit higher circulating levels of several proinflammatory cytokines

(87).

https://www.sciencedirect.com/topics/medicine-and-dentistry/limbic-system



https://www.sciencedirect.com/topics/medicine-and-dentistry/corticotropin-releasing-factor

https://www.sciencedirect.com/topics/medicine-and-dentistry/corticotropin

https://www.sciencedirect.com/topics/medicine-and-dentistry/central-nervous-system-function


12

FIGURE 2.3 Immunological alterations during depression(88)

The main types of interleukins or pro-inflammatory cytokines implicated in depression are IL -

1, IL - 2, IL - 6 and TNF – α (89, 90).

a. Interleukin 1: IL - 1 has significant effects on the brain. The IL-1 receptors are found in

hypothalamus, hippocampus, raphe nucleus and locus coeruleus which are the main structure of

the brain. IL - 1 manages most of the body's major neurotransmitters and hormones.

b. Interleukin 2: IL - 2 has powerful effects growth and survival of nerve growth, nerve impulses

andaction of neurotransmitter. The brain and has IL - 2 molecules and IL- 2 receptors all over

and they can also cross the blood-brain barrier.

c. Interleukin6: The production of IL - 6 increases in the body with the age, in contrast to most

cytokines which decline with age. This has a vital impact on degenerative brain disorders

Alzheimer's disease and like Parkinson's disease.

d. TNF - α: They are mainly secreted from macrophages, and they affect different cells to

produce fever, chemotaxis, fibroblast activation, endothelial regulations and leukocyte

adherence.


13

2.5.5 Neurotrophic Theory

Neurogenesis has emerged as an important process in the development of depression and the

activity of antidepressant medications (91, 92). The cytokines theory reveals that stress-induced

decrease in neurogenesis as well as the expression of relevant nerve growth factors, including

BDNF. In vitro, studies specify that the inhibitory effect of IL-1 on neurogenesis is mediated by

the activation of NF-kB (93).

2.6 DIAGNOSIS OF DEPRESSION

Various rating scale has been developed which may help the psychiatrist assess the severity of

the disorder. These rating scales are described below:

2.6.1 Hamilton Rating Scale for Depression (HAM-D or HRSD):

This is one of the earliest scales to be developed for depression. The original HAM- D included

21 items, but Hamilton pointed out that the last four items diurnal variation, depersonalization,

paranoid symptoms, and obsessive-compulsive symptoms should not be counted toward the total

score because these symptoms are either uncommon or do not reflect depression severity. Thus,

the 17-item version of the HAM-D has become the standard for clinical trials and over the years,

the most widely used scale for controlled clinical trials in depression. The scale is broadly used

in clinical trials and in clinical practice, and usually, it is carried out weekly. Qualified

interviewer or clinician use this scale and take care that all information must be filled with proper

observation of symptoms. The variables are measured either on five-point or three-point scales

(94, 95).

2.6.2 ICD 10 diagnostic criteria for a depressive episode (WHO 1992): (96)

In the UK, International Classification of Diseases (ICD 10) has developed for diagnosis of

depression.

Usual Symptoms: Depressed mood, loss of interest and enjoyment, and reduced energy

leading fatigue and diminished activity.

Common Symptoms: Reduced concentration and attention, reduced self-esteem and

self- confidence, Ideas of guilt and unworthiness (even in a mild type of episode),Bleak


14

and pessimistic views of the future, Ideas or acts of self-harm or suicide, Disturbed sleep,

diminished appetite

Mild depressive episode: For at least 2 weeks, at least two of the usual symptoms of a

depressive episode and two of the common symptoms listed above.

Moderate depressive episode: For at least 2 weeks, at least two or three of the usual

symptoms of a depressive episode plus at least three (preferably four) of the common

symptoms listed above.

Severe Depressive episode: For at least 2 weeks, at least all three of the usual symptoms

of a depressive episode plus at least four of the common symptoms listed above, some of

which should be of severe intensity.

The other scales are used for diagnosis of depression are The Beck Depression Inventory,

Inventory of Depressive Symptomatology (IDS or QIDS), and DSM-IV- TR criteria for major

depressive episode (94).

2.7 TREATMENT OF DEPRESSION:

The goals of treatment are to decrease the symptoms of acute depression, help the patient’s

return to the level of functioning. There are 3 phases of treatment to consider when treating

patients with major depressive disorder (97, 98).

1. The acute phase lasting from 6 to 10 weeks in which the goal is remission

2. The continuation phase listing 4 to 9 months after remission is achieved, in which the

goal is to eliminate residual symptoms or prevent relapse

3. Maintenance phase lasting at least 12 to 36 months in which the goal is to prevent

recurrence (i.e, separate episode of depression). The risk of reappearance increase as the

number of pest episodes increases.


15

2.7.1 Pharmacological Therapy:

Antidepressants can be classified in several ways(99).

1) Monoamine oxidase inhibitors (MAO) Inhibitors

2) Tricyclic antidepressants (TCAs)

• NA (Nor-adrenaline) + 5-HT (5-Hydroxytryptamine) reuptake inhibitors:

• Predominantly NA reuptake inhibitors

3) Selective Serotonin reuptake inhibitors (SSRIs)

4) Serotonin-norepinephrine reuptake inhibitors (SNRIs)

5) Atypical antidepressant: Bupropion, Nefazodone, Trazodone, Mirtazapine

MAO Inhibitors(100):

Phenelzine, Isocarboxazid, Tranylcypromine, Selegeline, Moclobemide, Clorgyline

MAOIs inhibit the metabolism of the neurotransmitters via oxidative deamination of

monoamines. MAO is of two types: MAO-A and MAO-B. All monoamines are primarily

deaminated by MAO-A but phenethylamine and benzylamine are deaminatedby MAO-B. MAO-

A’s activity is predominant in peripheral tissues, whereas, MAO-B isin the brain. The most

common early side effects of MAOIs include orthostatic hypotension, dizziness, drowsiness,

insomnia, and nausea. Others are weight gain, oedema, muscle pains, myoclonus, paresthesias,

and sexual dysfunction.

Tricyclic antidepressants (101, 102):

Imipramine, Amitriptyline, Trimipramine, Doxepin, Dothiepin, Clomipramine

Desipramine, Nortriptyline, Amoxapine, Reboxetine

TCAs compete for the binding site of the amine transporter. Most TCAs inhibit noradrenaline

and 5-HT uptake by brain synaptosomes. TCAs acts on the histaminergic or acetylcholinergic

systems, leading to sedation, hypotension, blurred vision, dry mouth, and other unwanted effects.

SSRIs (103):

Fluoxetine, Fluvoxamine, Paroxetine, Sertaline, Citalopram, Escitalopram


16

The primary mechanism of action of SSRIs is selective inhibition of the serotonin transporter

(SERT). SSRI blocks the serotonin reuptake pump there by increases somatodendritic serotonin

concentration desensitized the somatodendritic 5-HT1A autoreceptors, disinhibited neuronal

impulse flow, and increased release of serotonin from terminal presynaptic membrane region; the

final step is the desensitization of both the terminal presynaptic 5-HT1B autoreceptors and the

postsynaptic serotonin receptors. Disinhibition of the serotonergic pathway from brainstem to

hypothalamus, which mediates aspects of appetite and eating behaviours, is responsible for the

reduced appetite, nausea, and even weight loss associated with SSRIs administration.

SNRI(104):

Venlafaxine, Desvenlafaxine, Duloxetine

Specific serotonin and norepinephrine reuptake inhibitors act on both neuroamines of depression:

norepinephrine and serotonin. They are active on depressive symptoms, as well as on certain

comorbid symptoms. They are active significant in rate of remission, decreasing the risk of

relapse and recurrence. They are the drug of choice for long-term treatment and in high doses in

refractory depression or with strong potential of relapse.

2.7.2 Non-Pharmacological Therapy: Electroconvulsive Therapy:

Electroconvulsive therapy (ECT) is a safe and efficient treatment, including major depressive

disorder as well as other selected psychiatric illnesses. Patients with depression are candidates

for ECT when risks of other treatments outweigh potential benefits. ECT, in humans, involves

stimulus from side to side electrodes, with the patient lightly anaesthetized, paralyzed with a

short-acting neuromuscular-blocking drug (e.g. succinylcholine), so as to avoid physical injury,

and artificially ventilated (105, 106).

Bright Light Therapy is another nonpharmacologic treatment for depression. Consequently,

anyone undergoing light therapy should receive baseline and periodic eye examinations. The

combination of bright light therapy and an antidepressant may provide additional benefit beyond

either approach alone(107, 108).


17

TABLE 2.1 Plants for the herbal formulation:

Botanical Name English Name Traditional Name Part used Cassia fistula Golden shower Garmarogor Pulp Cedrus deodara Deodar Devdaru Bark Evolvulus alsinoides Shankhpushpi Shankhavali Hall herb Fumaria parviflora Fine-leaved Fumitory Pitapapapdo Seed Hydrocotyl asiatica Brahmi Brahmi Hall herb Nardostachys Jatamansi Spikenard Jatamansi Rhizome Cyperus rotundus Nutgrass Nagarmoth Rhizome Picrorhiza kurroa Picrorrhiza Kadu Root Valeriana wallichii Valerian Tagar Rhizome Terminalia chebula Chebulic myrobalan Himaj Fruit Vitis vinifera Common grape wine Draksha Fruit Withania somnifera Indian ginseng Ashwagandha Stem

TABLE 2.2 Plants and their reported activity:

Plants Reported activity

Cassia fistula Anti-oxidant, anti-inflammatory, anti-aging(109), antibacterial, antidiabetic, antifertility, hypatoprotective, antitumor, antifungal properties (33, 110)

Cedrusdeodara Anti-oxidant, anti-inflammatory, antibacterial, antidiabetic, antifertility, hepatoprotective, antitumor, antifungal properties (34)

Evolvulus alsinoides Neural regeneration and synaptic plasticity. Pre-clinical (in vivo and vitro) investigations have demonstrated anti-amnesic, antistress (adaptogenic), anxiolytic, cognitive enhancing, antimicrobial and gastroprotective activity (35)

Fumaria parviflora CNS stimulant, anti-depressant (36, 37) Hydrocotyl easiatica Cognitive enhancing, anxiolytic and anti-depressant activity and used for

sleep disorders (38) Nardostachys jatamansi

Tranquillizing, anti-oxidant, neuroprotective, anticonvulsant activity, antiparkinson’s activity, hepatoprotective, hypotensive, anti-diabetic (39)

Cyperus rotundus Cytoprotective, anti-oxidant activity, stimulant, anti-inflammatory, antidiabetic, antidiarrhoeal, antimutagenic, antimicrobial, antibacterial, and apoptotic, antipyretic and analgesic activities (40)

Picrorhiza kurroa anti-oxidant, anti-inflammatory and immunomodulatory activities (41) Valeriana wallichii Anxiolytic and anti-oxidant activities and it is beneficial in treating insomnia,

nervous problem (42)

Terminalia chebula Anti-oxidant, anti-inflammatory, and protective effects on various vital


18

organs (43)

Vitis vinifera Neuroprotective, anti-oxidant activity, anti-inflammatory, and antimicrobial, cardio protective, hepatoprotective activities (44)

Withania somnifera Anti-ageing plant along with anti-stress, adaptogenic (45)

Chapter 3 Materials and methods

19

CHAPTER 3

Materials and Methods

Animal husbandry and feeds

Swiss albino mice (20-30g) of either sex were housed in a room maintained at 22 ± 1°C with a

relative humidity of 55 ± 5% and a 12 h light-dark cycle. Animals had free access to standard

pellet diet and filtered tap water. All experiments were carried out with strict adherence to ethical

guidelines and were conducted as per protocol (LMCP/COLOGY/16/09),

(LMCP/Pharmacology/Ph.D./17/15) approved by the Institutional Animal Ethics Committee

(IAEC) and as per Indian norms laid down by the Committee for the Purpose of Control and

Supervision of Experiments on Animals (CPCSEA), New Delhi. Throughout the entire study

period, the animals were monitored for growth, health status, and food intake capacity to be

certain that they were healthy.

Drugs and Chemicals

Polyherbal formulation was supplied by the manufacturer, Cadila Pharmaceutical Private

Limited and fluoxetine powder was gifted by pharmACE laboratory. Horse redox peroxidase

was purchased from Sigma-Aldrich, USA. Sodium dodecyl sulphate, thiobarbituric acid,

quinolinic acid were purchased from himedia, India. Dopamine, nor-adrenaline, serotonin,

heptane, O- Phthalaldehyde, Diethyl ether (Rankem, New Delhi, India)

Kits for triglycerides, total protein, uric acid, albumin, glucose, creatinine, urea, total bilirubin,

direct bilirubin, aspartate amino - transferase (AST), alkaline phosphatase (ALP), alanine amino

- transferase (ALT) and cholesterol were purchased from span diagnostics (Gujarat, India).

ELISA kits for TNF-α, IL-6, IL-1β were purchased from Krishgen Biosystem, CA, USA. ELISA

kit for BDNF was purchased from Booster bioscience, CA, USA.


20

Composition of TENSNIL syrup

TABLE 3.1 Composition of TENSNIL syrup

Botanical Name English Name Traditional Name

Part used Each 10 ml contain Extract derived from powders of

Cassia fistula Golden shower Garmaro gor Pulp 40 mg Cedrus deodara Deodar Devdaru Bark 40 mg Evolvulus alsinoides

Shankhpushpi Shankhavali Hall herb 40 mg

Fumaria parviflora

Fine-leaved Fumitory

Pitapapapdo Seed 40 mg

Hydrocotyle asiatica

Brahmi Brahmi Hall herb 40 mg

Nardostachys jatamansi

Spikenard Jatamansi Rhizome 40 mg

Cyperus rotundus

Nut grass Nagarmoth Rhizome 40 mg

Picrorhiza kurroa Picrorrhiza Kadu Root 40 mg Valeriana wallichii

Valerian Tagar Rhizome 40 mg

Terminalia chebula

Chebulic myrobalan

Himaj Fruit 40 mg

Vitis vinifera Common grape wine

Draksha Fruit 40 mg

Withania somnifera

Indian ginseng Ashwagandha stem 40 mg

Flavored syrup base:

Q.S

FIGURE 3.1Images of the herbs incorporate in the formulation


21

Experimental design

3.1 Toxicity study

Selection of dose

The human clinical dose of Tensnil syrup is 10 ml for two to three times/day. The mice

therapeutic doses of Tensnil syrup selected under the study were, 100, 200, 400, 600, 800 mg/kg

by calculating from the human clinical dose (1000 - 1500 mg/day/70 kg). It was calculated based

on the total body surface area of the mice, using 0.0026 as the conversion factor (111). The drug

was administered in a volume of 2, 4, 8, 12, 16 ml/kg.

Toxicity study

The animals were divided into six groups, each having six animals. Tensnil Syrup was

administered orally at five dose levels i.e. 100 mg/kg, 200 mg/kg, 400 mg/kg, 600 mg/kg and

800 mg/kg body weight for twenty eight days. Normal saline was administered to the animals of

the control group.

FIGURE 3.2The protocol diagram for animal groups for sub-acute toxicity study


22

Physical Parameters

Physical parameters (body weight and food intake), and local injury were studied throughout the

treatment. Mortality if any, in all the groups, during the course of treatment was also recorded.

At the and of treatment hematological and biochemical studied.

Biochemical Parameters

Serum biochemical parameters include triglycerides, total protein, uric acid, albumin, glucose,

creatinine, urea, total bilirubin, direct bilirubin, aspartate amino - transferase (AST), alkaline

phosphatase (ALP), alanine amino - transferase (ALT) and cholesterol.

Haematological Parameters

RBC, WBC, lymphocytes (%), monocytes (%), eosinophils (%), basophils (%), MCV (Mean

Corpuscular Volume) (%), MCH (Mean Corpuscular Hemoglobin) (%), MCHC (Mean

Corpuscular Hemoglobin Concentration), PLT (Platelet) (*109/L), and HGB (Hemoglobin) (%)

were estimated.

3.2 Preliminary screening of activity and ED50 calculation

The animals were divided into six groups, each having six animals. Formulation was

administered orally at five dose levels i.e. 100 mg/kg, 200 mg/kg, 400 mg/kg, 600 mg/kg and

800 mg/kg body weight. Normal saline was administered to the animals of the control group.

ED50 doses of formulation was calculated in forced swim test (FST) using dose-response curve

with different doses in geometrical progression versus immobility time in seconds.

3.3 Acute study

Mice were grouped into 5 groups having 6 animals in each group. Groups 1 to 5 were normal

control, disease control, fluoxetine treated and PHF-400 & 800 mg/kg doses respectively.

Animals were forced to swim for duration of 10 min. into a closed container for continuously 7

days. On day 7 behavioural parameters were measured. On day 7, 14 animals were treated with

their treatment. Again on day 14, behavioural parameters were evaluated.


23

3.3.1 Forced swim test:

The mice were taken to the isolated room end placed in e cylinder (45 cm high, 20 cm diameter)

filled to 30 cm depth and maintained et 25 ± 1°C. Mice were examined for the duration of 5

minutes. They were dried end returned to their respective home cages later. The oral treatments

in the various groups were carried out 1 hour prior to the forced swim test in the second session.

The cylinder used had been freshly cleaned end disinfected prior to the forced swim test. Clean

water was used for each behavioural trial (112).

3.3.2 Tail suspension test:

TST was performed based on the earlier method (113) that the mouse was hung 25 cm over the

floor by the tip of the tail (1 cm) tied up to the level end immobility time was counted for 6 min

(prior 1 min to adept end recorded the lest 5 min). End only when the mouse hung passively end

completely motionless, it could be noted as immobile.

3.3.3 Locomotor activity:

Each mouse was placed in a closed square (30 cm) area prepared with infrared light-sensitive

photocells using a digital photoactometer and the values were expressed as counts per 5 min. The

apparatus was placed in a darkened, light- and sound- and ventilated test room (114).

3.3.4 Elevated plus maze:

Elevated plus maze (APM) assesses anxiety-like behaviour in mice. It consisted of two open

arms (30×5 cm), two enclosed arms (30×5 cm), and a connecting central platform (5×5 cm) and

was elevated 38.5 cm above the ground. At the beginning of the 5-min session, each mouse was

placed in the middle natural zone, facing one of the closed arms. Percentage time in the open and

central arms were recorded in situ by two blind experimenters. An arm entry was defined as a

mouse having entered an arm of the maze with all four lags (115).


24

3.4 Chronic studies

3.4.1 Chronic mild stress-induced depression in mice model

Experimental Design

Mice were exposed to an unsystematic pattern of mild stressors (116) daily for 28 days. These

stressors were randomly planed for a period of 1 week and repeated during the experiment.

Stressors incorporated cage tilting at 450 for 4 hours, cold swimming at temperature 50c for 5

minutes, tail pinch for 60 seconds, housing in mild damp sawdust for 6 hours, wet sawdust for 4

hours, overnight illumination, and food and water deficiency for 4 hours. The whole

experimentation lasted for 8 weeks (56 days). Behaviour tests including forced swim test, tail

suspension test, locomotor activity using photoactometer, elevated plus maze and sucrose

preference test were performed at the end of every week. Blood samples were collected from the

retro orbital vein at the end of the study for the estimation of serum proinflammatory cytokines

(TNF-α, IL-1β and IL-6), corticosterone, quinolinic acid and levels of oxidative and anti-oxidant

enzymes. Then, the mice were sacrificed by decapitation. The skull was opened, and the brain

was taken out on an ice plate for analysis of brain neurotransmitters that is 5-hydroxy tryptamine,

noradrenaline, and dopamine.

TABLE 3.4.1 Chronic mild stress (CMS) procedure

Days/weeks Week 1 Week 2 Week 3 Week 4

Day 1 Cage tilting at 45º for 4 hours

Food and water deprivation for 4 hours

Overnight illumination

Wet sawdust for 4 hours

Day 2 Cold swimming at temperature 5ºc for 5 minutes

Cage tilting at 45º for 4 hours



Day 3 Tail pinch for 60 seconds

Cold swimming at temperature 5ºc for 5 minutes



Day 4 Housing in mild damp sawdust for 6 hours

Tail pinch for 60 seconds




25

Day 5 Wet sawdust for 4 hours Housing in mild damp sawdust for 6 hours



Day 6 Overnight illumination Wet sawdust for 4 hours

Housing in mild damp sawdust for 6 hours


Day 7 Food and water deprivation for 4 hours


Wet sawdust for 4 hours

Housing in mild damp sawdust for 6 hours

3.4.1.1 Forced swim test:

The mice were taken to the isolated room and placed in the cylinder (45 cm high, 20 cm

diameter) filled to 30 cm depth and maintained et 25 ± 1°C. Mice were examined for the duration

of 5 minutes. They were dried and returned to their respective home cages later. The oral

treatments in the various groups were carried out 1 hour prior to the forced swim test in the

second session. The cylinder used had been freshly cleaned end disinfected prior to the forced

swim test. Clean water was used for each behavioural trial(112).

3.4.1.2 Tail suspension test:

TST was performed based on the earlier method (113) that the mouse was hung 25 cm over the

floor by the tip of the tail (1 cm) tied up to the level and immobility time was counted for 6 min

(prior 1 min to adept end recorded the least 5 min). End only when the mouse hung passively and

completely motionless, it could be noted as immobile.

3.4.1.3 Locomotor activity:



apparatus was placed in a darkened, light- and sound- and ventilated test room (114).


26

3.4.1.4 Elevated plus maze:

Elevated plus maze (APM) assesses anxiety-like behaviour in mice. It consisted of two open

arms (30×5 cm), two enclosed arms (30×5 cm), and a connecting central platform (5×5 cm) and

was elevated 38.5 cm above the ground. At the beginning of the 5-min session, each mouse was

placed in the middle netural zone, facing one of the closed arms. Percentage time in the open and

central arms were recorded in situ by two blind experimenters. An arm entry was defined as a

mouse having entered an arm of the maze with all four lags (115).

3.4.1.5 Sucrose preference test:

The sucrose preference test was performed as documented previously with minor modifications

(117) at the end of the week of the study. Mice were first trained to drink 1% sucrose solution

before starting of CMS procedure for 1 hour. Three days later, mice received sucrose preference

test. Each group provided simultaneously with both sucrose (1%) and water. Sucrose intake was

calculated by measuring the bottle at 60 min (118).

Sucrose solution intake (g)

Sucrose preference = -----------------------------------------------------------------------

Sucrose solution intake (g) + water intake (g)

3.4.1.6 Proinflammatory cytokines estimation:

ELISA kits for TNF-α, IL-6, IL-1β (Krishgen Biosystem, CA, USA) were used for estimation of

proinflammatory cytokines.

3.4.1.7 Brain neurotransmitters analysis:

The evaluation of serotonin, noradrenaline and dopamine in mice brain was carried out according

to the fluorometric technique(119, 120). Brain tissue sample was homogenized in 10 volumes of

cold acidified N-butanol using a glass homogenizer for 10 min at 2000 rpm. An aliquot

supernatant segment (1 ml) was removed and further to centrifuge tube containing 2.5 ml

heptane and 0.31 ml HCl of 0.1 M. After 10 min of vigorous shaking, the tube was centrifuged

under the same environment as above (10 min at 2000 rpm) in order to separate the two phases,

and the overlaying organic phase was discarded. The aqueous phase (0.2 ml) was then in use


27

either for 5-HT or NA and DA assay. Whole procedure was carried out at 0° C. To the 0.2 ml

of aqueous phase, 0.05 ml 0.4 M HCl and 0.1 ml of eDTA / Sodium acetate buffer (pH 6. 9)

were added, followed by 0.1 ml iodine solution (0.1 M in ethanol) for oxidation. The reaction

was stopped after 2 min by adding of 0.1 ml Na2SO3 solution. 0.1 ml acetic acid is added after

1.5 min. The solution was then heated to 100°C for 6 min as soon as the sample again reached

room temperature, excitation and emission spectra were read from the spectrofluorimeter. The

observations were taken at 330-375 nm for dopamine and 395-485 nm for nor-adrenaline. For 5-

HT estimation 0.2 ml aqueous extract 0.25 ml of OPT reagent was added. The fluorophore was

developed by heating to 100°C for 10 min. Once the samples reached equilibrium with the

ambient temperature, readings were taken at 360 - 470 nm in the spectrofluorimeter. Tissue

blanks for Dopamine and nor-adrenaline were prepared by adding the reagents of the oxidation

step in reversed order (sodium sulphite before iodine). For serotonin tissue blank, 0.25 ml HCI

without OPT was added. Internal Standard: 500 µg/ml each of noradrenaline, dopamine and

serotonin are prepared in distilled water: HCl - butanol in 1:2 ratio (121).

3.4.1.8 Serum corticosterone measurement:

Estimation of plasma level of corticosterone was done by spectrophotometer according to the

method of Katyara and Pandya. 0.1 ml of serum was treated with 0.2 ml newly prepared

chloroform: methanol mixture (2:1, v/v), 3 ml of chloroform. The samples were vortexed for 30

sec and centrifuged at 2,000 rpm (for 10 min). The chloroform layer was carefully taken. The

chloroform extract than treated with 0.1 N NaOH by vortexing quickly and NaOH layer was

rapidly removed. The sample was reacted with 30 N H2SO4 by vortexing vigorously. After phase

separation, the chloroform layer on top was discarded. The tubes containing H2SO4 was kept in

away from light for 30–60 min and afterwards fluorescence measurements carried out in

fluorescence spectrophotometer (RF-5301 pc, Shimadzu) with excitation and emission

wavelength sat at 472 and 523.2 nm respectively(122).

3.4.1.9 Serum quinolinic acid estimation:

HRP (Horseradish Peroxidase) Method for Determining QA: - HRP solution (1.0ml, 10U/ml)

was mixed with serum (1.0ml, 1.0 – 5.0 nM of QA), 0.5M H solution (1.0 ml), 0.1M lactate

buffer solution (3.0 ml, pH 5.0). The mixture was incubated at 30˚C for 90 min without contact


28

to light. The fluorescence intensity of the solution was measured with excitation and emission

wavelength at 328 and 377 nm in spectrophotometer (RF-5301 pc, Shimadzu) respectively. The

fluorescence concentration of the blank solution was similarly measured under the same

conditions.

3.4.1.10 Oxido-nitrosative stress parameters :

Estimation of reduced glutathione:

0.1 ml serum was precipitated with 1.0 ml of sulfosalicylic acid (4%). The samples wererested at

4˚C for at least 1 h and then subjected to centrifugation (1200 rpm for 15 min). The assay

mixture contained 0.1 ml supernatant, 2.7 ml phosphate buffer (0.1 M, pH 7.4), and 0.2 ml 5, 50-

dithiobis-(2-nitro benzoic acid) (Ellman’s reagent, 0.1 mM, pH 8.0) in 3 ml total volume. The

yellow color was developed and read immediately at 412 nm (123).

Estimation of lipid peroxidation:

Malondialdehyde (MDA) content was measured quantitatively by performing the method of

Ohkawa et al. Briefly, where 0.1 ml of sample was added to 0.1 ml of 8.1 % sodium dodecyl

sulphate (SDS), 0.75 ml of 20 % acetic acid solution (pH 3.4), and 0.75 ml of 0.8 %

thiobarbituric acid. Final volume was made up to 3 ml with distilled water. The final mixture was

then heated on a water bath at 95 °C for 60 min, cooled, and then centrifuged at 10,000 rpm for

10 min. Supernatant was collected, and the absorbance was taken at 532 nm (124).

3.4.1.11 Bodyweight:

The bodyweight of all animals was measured at the end of every week i.e. day 0, 7, 14, 21, 28,

35, 42, 49, 56 and percentage change in body weight recorded at the end of experiment.

3.4.1.12 Adrenal gland weight:

The animals were killed using the CO2 chamber. The adrenal gland was taken out from animal

and cleaned using saline. It was soaked on filter paper and their weight was taken using digital

weighing balance. The adrenal gland weight was used in this study as an indirect parameter of

the HPA axis activation (125).


29

3.4.2 LPS – induced neuroinflammation in mice model

Experimental Design

Animals were alienated into four groups (n=6/group). On the day of administration, fresh

solutions of LPS was prepared from 1 mg/ml stock solutions. The doses of herbal formulation

(600 mg/kg) were selected. Herbal formulation was orally administered once daily for 15 days

prior to, and on the same day of LPS injection. LPS was dissolved in sterile, endotoxin-free

normal saline (0.9% w/v NaCl) and injected intraperitoneally at the dose of 0.83 mg/kg of body

weight (126). Both LPS and drug were administered at the dose level of 10 ml/kg.

Drug treatments

On the day of administration fresh solutions of LPS and fluoxetine were prepared from 1 mg/ml

stock solutions. The dose of PHF – 600 mg/kg was selected based on previous studies. Herbal

formulation was orally administered once daily for 14 days prior to, and on the same day of LPS

injection. LPS was dissolved in sterile, endotoxin-free normal saline (0.9% w/v NaCl) and

injected intraperitoneally at the dose of 0.83 mg/kg of body weight.

3.4.2.1 Forced swim test:

The mice were taken to an isolated room and placed in a cylinder (45 cm high, 20 cm diameter)

filled to 30 cm depth and maintained at 25 ± 1°C. Mice were examined for a duration of 5

minutes. The oral treatments in the different groups were carried out 1 hour before a forced swim

test in the second session(112).




apparatus was positioned in a darkened, light- and sound-attenuated and ventilated test

room(127).

3.4.2.3 Elevated plus maze:

Elevated plus maze (EPM) assesses anxiety-like behaviour in mice. It consisted of two open

arms (30×5 cm), two enclosed arms (30×5 cm), and a linking middle platform (5×5 cm) and was


30

high 38.5 cm above the floor. At the beginning of the 5-min session, each mouse was placed in

the centre unbiased zone, facing one of the closed arms. Percentage time in the open and central

arms were recorded by two blind experimenters. An arm entry was defined as a mouse having

entered an arm of the maze with all four legs(115).

3.4.2.4 Morrison water maze test:

Mice were lifted by the base of the tail and gently placed into the water, facing the edge of the

pool. If the mouse found the stage before the 60-sec cut-off, allowed the mouse to stay on the

stage for 5 seconds then return it to its home cage. If the mouse did not find the stage, placed the

mouse on the stage and allowed it to stay there for 20 sec before returning it to its home cage.

Whole trial was repeated for all mice. Each trial was begun with a different stage location and

starting direction. For each day and each mouse, average the 5 trials were given and single-path

length and escape latency and time spent in the stage quadrant for each subject was tested(128).


Proinflammatory cytokines TNF-α, IL-6, IL-1β (Krishgen Biosystem, CA, USA) were estimated

using ELISA kits (129).

3.4.2.6 Serum corticosterone measurement:

Serum corticosterone level was estimated by spectrophotometer according to the method of

Katyare and Pandya (122).

3.4.2.7 Serum quinolinic acid estimation

Estimation of quinolinic acid was performed according to the method of Junichi, Masahiko and

Akihito using fluorometric method (130).

3.4.2.8 Oxido-nitrosative stress parameters:


0.1 ml serum was precipitated with 1.0 ml of sulfosalicylic acid (4%). The samples were rest at




31

dithiobis-(2-nitrobenzoic acid) (Ellman’s reagent, 0.1 mM, pH 8.0) in 3 ml total volume. The

yellow color was developed and read immediately at 412 nm(131).


The lipid peroxidation was measured according to the method of Wills. The amount of MDA

was measured by reacting it with thiobarbituric acid and measured at 532 nm (132).

Estimation of Nitrite level:

Plasma nitrite levels were measured by using thegoal of Graan at al (133).

3.4.2.9Nerve growth factor:

BDNF levels were measured using commercial enzyme-linked immunosorbent assay (ELISA)

kits (BOSTER Immunoleader, Boster Biological Technology Co., Ltd., CA, USA.)(134).

3.4.3 ketamine-induced psychosis model

Experimental Design

Animals were randomly divided into five experimental groups (n = 6) for behavioural and

biochemical assessment. The group I served as a normal control group while group II served as a

disease control group and treated with ketamine for 14 days. Group III and IV were treated orally

with haloperidol 0.25 mg/kg and PHF – 600 mg/kg respectively, for 14 days and along with

intraperitoneally 50 mg/kg (i.p.) ketamine. Group V was disease control group treated with

ketamine for 5 consecutive days only. Motor activity assessed by photoactometer and cataleptic

behaviour with the use of bar test. Memory task was performed using Morrison water maze test

and psychosis was analyzed via stereotype behaviour and learned helplessness model.

Blood samples were collected from the retroorbital for the analysis of serum proinflammatory

cytokines (TNF-α, IL-1β and IL-6), and levels of oxidative and anti-oxidant enzymes. Then, the

mice were sacrificed by decapitation and the brain was dissected out on an ice plate for

analysisof nerve growth factor (BDNF – Brain Derived Neurotropic Factor).

Drug treatments

On the day of administration, fresh solutions of Haloparidol was prepared from 1 mg/ml stock

solutions. The dose of PHF (Polyherbal formulation) – 600 mg/kg was selected based on


32

previous studies. Herbal formulation was intraperitoneally administered once daily for 14 days

and ketamine 50 mg/kg for 14 days.




apparatus was placed in a darkened, light- and sound- and ventilated test room(127).

3.4.3.2 Stereotype behaviours:

Ketamine (50 mg/kg, i.p.) was injected for 14 days to produce stereotype behaviours (falling,

turning, head bobbing and sniffing) in mice, measured by total number of falls (falling),

turnaround (turning), neck wave right, left, up and down (head bobbing) and frequent rearing and

grooming behaviours (sniffing) for 10 min over 60 min at an interval of 10 min(135).

3.4.3.3 Water maze test:

Mice were lifted by the base of the tail and gently placed into the water, facing the edge of the

pool. If the mouse found the platform before the 60-sac cut-off, allowed the mouse to stay on the

platform for 5 seconds then return it to its home cage. If the mouse did not find the platform,

placed the mouse on the platform and allowed it to stay there for 20 sec before returning it to its

home cage. Whole trial was repeated for all mice. Each trial was begun with a different platform

location and starting direction. For each day and each mouse, average the 5 trials were given and

single-path length and escape latency and time spent in the platform quadrant for each subject

was tested (128).

3.4.3.4 Catalepsy test – bar test:

Catalepsy is a side effect usually observed with first generation antipsychotic drugs. Animals

were placed individually with its forepaws on horizontal bar, 9 cm at height. When the animal

withdraws its either paw from the bar, the time was noted with the cut off time of 3 min (136).

3.4.3.5 Learned helplessness:

Mice were submitted to inescapable foot shocks with a mean interval of 5–10s for 1 min. The

mice in the control group were placed into the same chamber for 60 min but no shock was


33

delivered during this time. A single climbing from the electrified compartment to the platform

made within this latter period was called an escape response. If no escape response occurred,

tone and shock were turned off, and this was recorded as an escape failure (learned helplessness

behaviour). During the test session, the number of escape failures was recorded (137).

3.4.3.6 Social interaction test:

Three chambered test was performed for this activity evaluation. Time spent by test mice with

probe mice was measured(138).


Proinflammatory cytokines TNF-α, IL-6, IL-1β (Krishgen Biosystem, CA, USA) were estimated

using ELISA kits (129).

3.4.3.8 Oxido-nitrosative stress parameters:


0.1 ml serum was precipitated with 1.0 ml of sulfosalicylic acid (4%). The samples were rest at



dithiobis-(2-nitrobenzoic acid) (Ellman’s reagent, 0.1 mM, pH 8.0) in 3 ml total volume. The

yellow color was developed and read immediately at 412 nm (131).


The lipid peroxidation was measured according to the method of Wills. The amount of MDA

was measured by reacting it with thiobarbituric acid and measured at 532 nm (132).

Estimation of Nitrite level:

Plasma nitrite levels were measured by using the method of Graan at al (133).

3.4.3.9 Nerve growth factor:

BDNF levels were measured using commercial enzyme-linked immunosorbent assay (ELISA)

kits (BOSTER Immunoleader, Boster Biological Technology Co., Ltd., CA, USA) (134).

Chapter 4 Results

34

CHAPTER 4

Results

4.1 Toxicity study

4.1.1 Effects of polyherbal formulation on body weight and food intake

Bodyweight and food intake of all the animals were measured every week throughout the study

as shown in table 4.1.1(a) and 4.1.1(b) respectively. There was no remarkable change in body

weight and food intake of the mice in any of the study groups.

TABLE 4.1.1(a) Effect of polyherbal formulation on body weight (g)

Study day

Bodyweight (g) Control 100 mg/kg 200 mg/kg 400 mg/kg 600 mg/kg 800 mg/kg

Day 0 28.33 ± 0.84 25.83 ± 0.65 30.00 ± 1.91 30.67 ± 2.01 30.83 ± 1.90 32.50 ± 2.11 Day 7 28.00 ± 0.45 26.50 ± 0.43 29.83 ± 1.58 30.00 ± 1.39 28.50 ± 0.89 30.00 ± 1.03 Day 14 28.67 ± 0.56 27.33 ± 0.42 30.33 ± 1.58 30.17 ± 1.45 29.17 ± 0.79 31.00 ± 1.03 Day 21 29.17 ± 0.48 27.83 ± 0.54 30.83 ± 1.47 30.83 ± 1.45 29.50 ± 0.89 30.67 ± 0.84 Day 28 29.33 ± 0.42 28.33 ± 0.42 30.83 ± 1.51 31.17 ± 1.28 29.83 ± 0.75 31.00 ± 0.68

Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple

comparison tests. Results are expressed as mean ± SEM, n = 6, p < 0.05 as compared to control

group, where no significant difference observed.

TABLE 4.1.1(b) Effect of polyherbal formulation on food intake (g)

Study Group Food Intake (g) Control 3.1 ± 0.07 100 mg/kg 2.9 ± 0.08 200 mg/kg 3.1 ± 0.06 400 mg/kg 3.0 ± 0.09 600 mg/kg 3.3 ± 0.05 800 mg/kg 3.2 ± 0.09


comparison test. Results are expressed as mean ± SEM, n = 6, p < 0.05 as compared to control


Chapter 4 Results

35

4.1.2 Effect of polyherbal formulation on hematological parameters

Our observations of the study for the period of 28 days did not reveal any significant change in

any of the haematological parameters as shown in table 4.1.2. The formulation was found safe up

to 800 mg/kg dose level.

TABLE 4.1.2 Effect of polyherbal formulation on the haematological parameters in mice

Normal Control 100 mg/kg 200 mg/kg 400 mg/kg 600 mg/kg 800 mg/kg

RBC 10.90 ± 2.22 11.84 ± 1.59 12.55 ± 2.04 12.01 ± 2.17 12.01 ± 1.86 11.45 ± 1.55

WBC 6.95 ± 0.54 10.19 ± 1.08 8.33 ± 0.50 8.35 ± 0.99 7.83 ± 0.36 9.24 ± 1.70

Lymphocytes(%) 81.82 ± 2.78 76.63 ± 4.12 76.05 ± 3.78 76.30 ± 4.53 79.27 ± 2.72 80.05 ± 2.14

Monocytes (%) 2.27 ± 0.44 2.25 ± 0.20 2.50 ± 1.08 1.45 ± 0.33 2.10 ± 0.38 2.73 ± 0.57

Eosinophils (%) 1.67 ± 0.35 1.83 ± 0.49 1.87 ± 0.40 1.60 ± 0.47 1.97 ± 0.41 2.02 ± 0.37

Basophils (%) 0.12 ± 0.05 0.08 ± 0.04 0.08 ± 0.04 0.10 ± 0.05 0.07 ± 0.03 0.07 ± 0.03

HCT (%) 43.37 ± 0.40 43.80 ± 0.51 42.40 ± 0.95 41.13 ± 1.17 40.77 ± 0.50 44.40 ± 0.59

MCV(fL) 53.28 ± 1.06 53.63 ± 1.03 54.33 ± 1.22 53.18 ± 0.52 53.10 ± 0.76 53.12 ± 0.44

MCH (pg) 17.12 ± 0.41 17.20 ± 0.61 17.25 ± 0.45 17.17 ± 0.49 16.13 ± 1.25 16.88 ± 0.56

MCHC 32.17 ± 0.64 32.05 ± 0.81 31.77 ± 0.87 31.68 ± 1.12 30.28 ± 2.37 31.78 ± 0.92

PLT (*109/L) 1570.00 ± 570.16

1123.17 ± 38.86

1193.00 ± 75.50

1026.50 ± 106.76

1027.83 ± 90.81

1227.33 ± 71.38

Haemoglobin (%) 12.95 ± 1.04 14.57 ± 0.29 13.92 ± 0.23 13.25 ± 0.72 13.58 ± 1.02 13.90 ± 0.26




4.1.3 Effect of polyherbal formulation on the biochemical parameters

The repeated oral dose treatment for 28 days did not show any significant changes in hepatic

functional transaminases viz. ALT, AST and ALP levels. The renal function was evaluated by

measuring serum urea and creatinine. Other biochemical parameters like triglyceride, total

protein, uric acid, albumin, glucose, total bilirubin, direct bilirubin, globulin, cholesterol levels

also did not change remarkably.

Chapter 4 Results

36

TABLE 4.1.3 Effect of polyherbal formulation on the biochemical parameters in mice

Control 100 mg/kg 200 mg/kg 400 mg/kg 600 mg/kg 800 mg/kg

TG 178.07 ± 9.83 179.32 ± 11.64 178.07 ± 12.37 171.15 ± 10.55 170.13 ± 14.12 170.52 ± 12.14 Total Protein 6.01 ± 0.66 5.78 ± 0.41 6.18 ± 0.58 6.68 ± 0.91 6.27 ± 0.74 6.18 ± 0.57

Uric Acid 2.13 ± 0.13 2.35 ± 0.21 2.30 ± 0.17 2.43 ± 0.23 2.48 ± 0.18 2.31 ± 0.20 Albumin 3.54 ± 0.17 3.78 ± 0.11 3.42 ± 0.06 3.26 ± 0.18 3.39 ± 0.09 3.40 ± 0.15

Glucose 112.53 ± 4.71 103.79 ± 5.34 102.52 ± 3.99 97.15 ± 5.90 95.48 ± 3.96 100.86 ± 6.08 Creatinine 0.39 ± 0.01 0.43 ± 0.01 0.43 ± 0.01 0.36 ± 0.01 0.43 ± 0.03 0.41 ± 0.01

Urea 40.94 ± 3.41 38.35 ± 4.30 45.05 ± 5.94 47.20 ± 8.22 45.92 ± 7.69 46.87 ± 7.09 Total Bilirubin 0.50 ± 0.08 0.64 ± 0.11 0.60 ± 0.09 0.50 ± 0.08 0.64 ± 0.11 0.60 ± 0.09 Direct Bilirubin 0.12 ± 0.02 0.11 ± 0.03 0.12 ± 0.03 0.14 ± 0.03 0.08 ± 0.02 0.14 ± 0.03

Globulin 2.47 ± 0.64 2.00 ± 0.51 2.77 ± 0.56 3.42 ± 0.97 2.88 ± 0.82 2.78 ± 0.65

AST 106.82 ± 3.21 103.28 ± 2.66 105.05 ± 4.78 106.37 ± 4.44 106.08 ± 2.83 99.89 ± 7.26

ALP 87.69 ± 2.84 90.40 ± 2.44 84.75 ± 2.64 87.91 ± 2.41 89.72 ± 1.13 90.85 ± 2.55 ALT 43.91 ± 2.12 42.73 ± 1.53 42.14 ± 1.58 42.28 ± 2.11 40.37 ± 2.45 41.11 ± 1.53

Cholesterol 106.03 ± 4.44 104.65 ± 3.61 101.32 ± 5.54 103.23 ± 5.38 99.57 ± 6.30 99.27 ± 4.23




4.2 Preliminary screening of activity and ED50value determination

The ED50 value of different doses of the formulation obtained from the FST was 600 mg/kg p.o.,

in mice.

TABLE 4.2 Effect of polyherbal formulation on % inhibition of immobility using forced swim

test (FST)

% Inhibition of duration of immobility time using FST

Groups Male mice Female mice Control 100 100

PHF(100 mg/kg) 99.853 93.282 PHF(200 mg/kg) 74.444 71.543 PHF(400 mg/kg) 57.206 53.761 PHF(600 mg/kg) 48.888 45.143 PHF(800 mg/kg) 44.577 42.27

Chapter 4 Results

37

4.3 Acute study

This study was performed by using various behavioural parameters, which included forced swim

test, tail suspension test, locomotor activity, elevated plus mazes tests.

4.3.1 Effect of polyherbal formulation on FST:

In the forced swim test, the duration of immobility was significantly (p < 0.001) increased in the

disease control group without any treatment on day 7 when compared with the results of duration

of immobility day 0. Fluoxetine (reference standard) and PHF – 400 mg/kg and 800 mg/kg

treatment from day 7 to day 14 resulted in significant reduction in duration of immobility time as

compared to data of disease control group on day 7 (figure 4.3.1).

Forced Swim Test

Normal

Contro

l

Disease

Contro

l

Fluoxe

tine (

20 m

g/kg)

PHF (400

mg/k

g)

PHF (800

mg/k

g)

Normal

Contro

l

Disease

Contro

l

Fluoxe

tine (

20 m

g/kg)

PHF (400

mg/k

g)

PHF (800

mg/k

g)

Normal

Contro

l

Disease

Contro

l

Fluoxe

tine (

20 m

g/kg)

PHF (400

mg/k

g)

PHF (800

mg/k

g)

0

50

100

150

200

Day 0

Day 7

Day 14

Day 0 Day 7 Day 14

*

#

* * *

#

#

Groups

Dur

atio

n of

Imm

obili

ty (s

ec)

FIGURE 4.3.1 Effect of polyherbal formulation on FST

Statistical analysis was performed by one-way ANOVA followed by bartlett's test. Results are

expressed as mean ± SEM, n = 6, * p < 0.001 as compared to the normal control group. # p<

0.001 as compared to the disease control group.

Chapter 4 Results

38

4.3.2 Effect of polyherbal formulation on TST:

Marked decline was observed in the duration of immobility in fluoxetine and PHF (400 and 800

mg/kg) treated mice as compared with the disease control group on day 14 (figure 4.3.2).

Tail Suspension Test

Normal

Contro

l

Disease

Contro

l

Fluoxe

tine (

20 m

g/kg)

PHF (400

mg/k

g)

PHF (800

mg/k

g)

Normal

Contro

l

Disease

Contro

l

Fluoxe

tine (

20 m

g/kg)

PHF (400

mg/k

g)

PHF (800

mg/k

g)

Normal

Contro

l

Disease

Contro

l

Fluoxe

tine (

20 m

g/kg)

PHF (400

mg/k

g)

PHF (800

mg/k

g)

0

50

100

150

200

250

Day 0

Day 7Day 14

Day 0 Day 7 Day 14

*

#

* * *

##

Groups

Dur

atio

n of

Imm

obilit

y (s

ec)

FIGURE 4.3.2 Effect of polyherbal formulation on TST

Statistical analysis was performed by one-way ANOVA followed by Bartlett’s test. Results are

expressed as mean ± SEM, n = 6, * p < 0.001 as compared to the normal control group. # p<

0.001 as compared to the disease control group.

Chapter 4 Results

39

4.3.3 Effect of polyherbal formulation on locomotor activity:

The locomotor activity observed using photoactometer was significantly (p < 0.05) reduced in

the disease control group as compared to the normal control group on day 7. Treatment with

fluoxetine and PHF from day 7 to day 14 significantly increased locomotor activity as matched

to the disease control group (figure 4.3.3).

Photoactometer

Normal

Contro

l

Disease

Contro

l

Fluoxe

tine (

20 m

g/kg)

PHF (400

mg/k

g)

PHF (800

mg/k

g)

Normal

Contro

l

Disease

Contro

l

Fluoxe

tine (

20 m

g/kg)

PHF (400

mg/k

g)

PHF (800

mg/k

g)

Normal

Contro

l

Disease

Contro

l

Fluoxe

tine (

20 m

g/kg)

PHF (400

mg/k

g)

PHF (800

mg/k

g)

0

50

100

150

200

Day 0

Day 0 Day 7 Day 14

Day 7Day 14

#

*** ** *

#

#

Groups

Loco

mot

or in

dex

(cou

nts/

5 m

in)

FIGURE 4.3.3 Effect of polyherbal formulation on locomotor activity


expressed as mean ± SEM, n = 6.* p< 0.01 and ** p < 0.001, as compared to the normal control

group; # p < 0.001, as compared to the disease control group.

Chapter 4 Results

40

4.3.4 Effect of polyherbal formulation on EPM:

Time spent in close arm for fluoxetine and PHF groups (400, 800 mg/kg) was found statistically

significant (p < 0.05) as compared to the disease control group on day 14 (figure 4.3.4).

FIGURE 4.3.4 Effect of polyherbal formulation on EPM


expressed as mean ± SEM, n = 6.* p< 0.05 as compared to the normal control; # p < 0.01 as

compared to the disease control

Elevated Plus Maze

Normal

Contro

l

Disease

Contro

l

Fluoxe

tine (

20 m

g/kg)

PHF (400

mg/k

g)

PHF (800

mg/k

g)

Normal

Contro

l

Disease

Contro

l

Fluoxe

tine (

20 m

g/kg)

PHF (400

mg/k

g)

PHF (800

mg/k

g)

Normal

Contro

l

Disease

Contro

l

Fluoxe

tine (

20 m

g/kg)

PHF (400

mg/k

g)

PHF (800

mg/k

g)

0

20

40

60

80

Day 0

Day 7Day 14

Day 0 Day 7Day 14

* * **

# #

Groups

Tim

e sp

ent i

n cl

ose

arm

(sec

)

Chapter 4 Results

41

4.4 Chronic study

Chronic study was performed by using different animal models namely, chronic unpredictable

mild stress model – including chronic unpredictable mild stress model (CUMS),

lipopolysaccharide-induced depression model and ketamine-induced antipsychotic model. Each

chronic study included two components in the study namely behavioural test and biochemical

analysis.

4.4.1 Chronic unpredictable mild stress – induced depression in mice model

4.4.1.1 Effect of polyherbal formulation on CUMS-induced altered FST:

Exposure to CUMS for 4 weeks resulted in depressive-like behaviour as it significantly increased

the duration of immobility time of the FST. Treatment with Fluoxetine and PHF (400 mg/kg&

800 mg/kg) after 4th week, showed reduction in the immobility time in comparison to the disease

control group (61.59%) (p < 0.001).

Chapter 4 Results

42

Forced Swim Test

Normal

Contro

l

Disease

Contro

l

Fluoxe

tine (

20 m

g/kg)

PHF (400

mg/k

g)

PHF (800

mg/k

g)0

50

100

150

200

*

# # #

Groups

Dur

atio

n of

Imm

obili

ity (s

ec)

FIGURE 4.4.1.1 Effect of polyherbal formulation on CUMS – induced altered FST


comparison test. Results are expressed as mean ± SEM, n = 6. *p < 0.001 as compared to the

normal control group. # p < 0.001 as compared to the disease control group

Chapter 4 Results

43

4.4.1.2 Effect of polyherbal formulation on CUMS – induced altered TST:

The duration of immobility was measured in the TST to evaluate the stress-related despairing

status in mice. The duration of immobility of CUMS group was significantly longer than that of

the control group (P < 0.001). After drugs treatment, the immobility time of Fluoxetine and PHF

groups was significantly reduced as compared to the disease group (P < 0.001), suggesting that

PHF (400 & 800 mg/kg) could reverse despairing status in the CUMS-induced mice.

Tail Suspension Test

Normal

Contro

l

Disease

Contro

l

Fluoxe

tine (

20 m

g/kg)

PHF (400

mg/k

g)

PHF (800

mg/k

g)0

50

100

150

200

250*

# # #

Groups

Dur

atio

n of

Imm

obili

ity (s

ec)

FIGURE 4.4.1.2 Effect of polyherbal formulation on CUMS – induced altered TST


comparison test. Results are expressed as mean ± SEM, n = 6. * p < 0.001 as compared to the

normal control group. # p < 0.001 as compared to the disease control group.

Chapter 4 Results

44

4.4.1.3 Effect of polyherbal formulation on CUMS – induced altered locomotor activity:

The locomotor activity using photoactometer was significantly (p < 0.001) reduced in the disease

control group treated with CUMS. Fluoxetine and PHF (400 & 800 mg/kg) treated groups were

compared with CUMS-induced disease control group, showed a significant (p < 0.001) raised

locomotor index.

Locomotor activity

Normal

Contro

l

Disease

Contro

l

Fluoxe

tine (

20 m

g/kg)

PHF (400

mg/k

g)

PHF (800

mg/k

g)0

50

100

150

200

*

# # #

Groups

Loco

mot

or in

dex

(cou

nts/

5 m

in)

FIGURE 4.4.1.3 Effect of polyherbal formulation on CUMS – induced altered locomotor activity




Chapter 4 Results

45

4.4.1.4 Effect of polyherbal formulation on CUMS – induced altered EPM activity:

CUMS-induced an anxiogenic effect in diseased group and significantly (p < 0.001) increased

the time spent in open arm in plus-maze. Both the treatments including fluoxetine and PHF

significantly (p < 0.001) reversed the time spent in open arm when compared with the disease

control group.

Elevated Plus Maze (EPM)

Normal

Contro

l

Disease

Contro

l

Fluoxe

tine (

20 m

g/kg)

PHF (400

mg/k

g)

PHF (800

mg/k

g)0

50

100

150

200

250

*

# # #

Groups

Tim

e sp

ent i

n op

en a

rm (s

ec)

FIGURE 4.4.1.4 Effect of polyherbal formulation on CUMS – induced EPM activity




Chapter 4 Results

46

4.4.1.5 Effect of polyherbal formulation on CUMS – induced altered sucrose preference test:

Results showed no significant difference observed in sucrose preference (%) among all the

groups in the baseline test. Exposure of the mice to stress for 28 successive days significantly

decreased sucrose preference (%) in stressed mice as compared to control group. Reduced

sucrose preference (%) in stressed mice was significantly restored by the administration of

fluoxetine (20 mg/kg (96.62%)) or PHF (400 (96.08%) & 800 mg/kg (99.39%)) for 28

successive days.

FIGURE 4.4.1.5 Effect of polyherbal formulation on CUMS – induced altered sucrose

preference test




Sucrose Preference Test

Normal C

ontrol

Disease

Control

Fluoxetine (

20 mg/kg)

PHF (400 m

g/kg)

PHF (800 m

g/kg)0

20

40

60

80

100

###

*

Groups

% S

ucro

se pr

eferen

ce

Chapter 4 Results

47

4.4.1.6 Effect of polyherbal formulation on CUMS – induced altered levels of proinflammatory

cytokines (TNF – α, IL – 6, IL-1β):

CUMS treated animals showed significant (p < 0.001) increase in the levels of

neuroinflammation markers, TNF – α, IL – 6, IL – 1 as compared to the disease group. PHF (400

& 800 mg/kg) treatment significantly (p < 0.001) attenuated the increased levels of TNF – α, IL –

6, IL – 1 when compared with the CUMS - induced disease control group (Fig. 5). Further,

comparison between PHF 800 mg/kg treated group and fluoxetine (20 mg/kg), PHF treated

group significantly (p < 0.05) lowered TNF – α, IL – 6, IL - 1βlevels.

Cytokines-induced neuroinflammationin CUMS model

Normal

Contro

l

Disease

Contro

l

Fluoxe

tine (

20 m

g/kg)

PHF (400

mg/k

g)

PHF (800

mg/k

g)

Normal

Contro

l

Disease

Contro

l

Fluoxe

tine (

20 m

g/kg)

PHF (400

mg/k

g)

PHF (800

mg/k

g)

Normal

Contro

l

Disease

Contro

l

Fluoxe

tine (

20 m

g/kg)

PHF (400

mg/k

g)

PHF (800

mg/k

g)

0

50

100

150

TNF - αIL - 6IL - 1β

*

* *$ * * *

$

@

*

$ $$

@

*

* *$ * *

$$@

TNF - α

IL - 6

IL - 1β

Groups

Seru

m c

ytok

ines

leve

ls (p

g/m

l)

FIGURE 4.4.1.6 Effect of polyherbal formulation on CUMS – induced altered levels of

proinflammatory cytokines (TNF – α, IL – 6, IL-1β)

Statistical analysis was performed by one-way ANOVA followed by tukey’s multiple

comparison test. Results are expressed as mean ± SEM, n = 6. * p < 0.001, ** p < 0.01, *** p <

0.05 as compared to the normal control group. $ p < 0.001, $$ p < 0.01, $$$ p < 0.05 as

compared to the disease control group. @ p < 0.05, @@ p< 0.01 as compared to the fluoxetine

treated group.

Chapter 4 Results

48

4.4.1.7 Effect of polyherbal formulation on CUMS – induced altered levels of neurotransmitters

(NA, DA, 5-HT):

All three neurotransmitters namely noradrenaline (NA), dopamine (DA) and 5-hydroxy

tryptamine (5 – HT) were significantly (p < 0.001) reduced in the disease control group as

compared to normal control. Fluoxetine and PHF (400 & 800 mg/kg) showed significantly (p <

0.001) reversal effect, i.e. increased in the levels of all three neurotransmitters after treatments

for 28 days with. Also treatment with 800 mg/kg PHF showed significance rise into levels of

noradrenaline (p < 0.01) and dopamine (p < 0.05) when compared with the standard treatment of

fluoxetine (20 mg/kg).

Brain Noradrenaline levels

Normal

Contro

l

Disease

Contro

l

Fluoxe

tine (

20 m

g/kg)

PHF (400

mg/k

g)

PHF (800

mg/k

g)0

100

200

300

400

500

*

#

#

#$ $

Groups

Nor

adre

nalin

e le

vels

(ng/

mg

wt o

f bra

in)

FIGURE 4.4.1.7 (a) Effect of polyherbal formulation on CUMS – induced altered levels of

neurotransmitters: NA


comparison test. Results are expressed as mean ± SEM, n = 6.* p < 0.001 as compared to the

normal control group. # p < 0.001 as compared to the disease control group. $ p < 0.05 when

compared with the standard (fluoxetine (20 mg/kg)) group.

Chapter 4 Results

49

Brain Dopamine levels

Normal

Contro

l

Disease

Contro

l

Fluoxe

tine (

20 m

g/kg)

PHF (400

mg/k

g)

PHF (800

mg/k

g)0.0

0.1

0.2

0.3

0.4

*

##

#$

Groups

Dop

amin

e le

vels

(ng/

mg

wt o

f bra

in)

FIGURE 4.4.1.7 (b) Effect of polyherbal formulation on CUMS – induced altered levels of

neurotransmitters: DA





Chapter 4 Results

50

Brain 5 - Hydroxytryptamine levels

Normal

Contro

l

Disease

Contro

l

Fluoxe

tine (

20 m

g/kg)

PHF (400

mg/k

g)

PHF (800

mg/k

g)0.00

0.05

0.10

0.15

0.20

*

# #

#

Groups

5 -H

T le

vels

(ng/

ml w

t of b

rain

)


neurotransmitters: 5-HT





Chapter 4 Results

51

4.4.1.8 Effect of polyherbal formulation on CUMS – induced altered levels of corticosterone:

CUMS-induced significant increased the levels of serum corticosterone in the disease control

group as compared to the normal control group. Treatment with the fluoxetine 20 mg/kg and

PHF - 400 & 800 mg/kg showed significantly reduced levels of corticosterone.

Serum corticosterone levels(ng/ml)

Normal

Contro

l

Disease

Contro

l

Fluoxe

tine (

20 m

g/kg)

PHF (400

mg/k

g)

PHF (800

mg/k

g)0

50

100

150

*

# #

#

Groups

Seru

m c

ortic

oste

rone

leve

ls (n

g/m

l)


corticosterone




Chapter 4 Results

52

4.4.1.9 Effect of polyherbal formulation on CUMS – induced altered levels of quinolinic acid:

CUMS-induced significant increased the levels of serum quinolinic acid in the disease control

group as compared to the normal control group. Treatment with the fluoxetine 20 mg/kg and

PHF-400 & 800 mg/kg showed significantly reduced levels of this neurotoxin (quinolinic acid).

Moreover, serum concentration of quinolinic acid in PHF-800 mg/kg treated animals was found

significantly (p < 0.05) lowered as compared to the standard treatment with fluoxetine indicating

better safety of the test drug under the study.

Serum quinolinic acid (pg/ml)

Normal Control

Disease C

ontrol

Fluoxetine (2

0 mg/kg)

PHF (400 mg/kg)

PHF (800 mg/kg)0

1

2

3

4

*

# ##$

Groups

Serum

quino

linic

acid l

evels

(pg/m

l)


quinolinic acid



normal control group. # p < 0.001 as compared to the disease control group. $ p < 0.05 as


Chapter 4 Results

53

4.4.1.10 Effect of polyherbal formulation on CUMS – induced altered levels of oxido -

nitrosative stress parameters (reduced glutathione and lipid peroxidase):

CUMS produced a significant increase in oxidative stress in the disease control group when

compared with the normal group. Treatments with fluoxetine and PHF-400 &800 mg/kg

significantly (p < 0.05, 0.05 and 0.001) ameliorated the level of reduced glutathione as to that of

disease control group respectively. A higher lipid peroxidase level was observed in the disease

control group. Further, polyherbal formulation significantly (p < 0.05) attenuated the lipid

peroxidase level as compared to fluoxetine treated animals.

Serum reduced glutathione levels (µM/ml)

Normal Control

Disease C

ontrol

Fluoxetine (2

0 mg/kg)

PHF (400 mg/kg)

PHF (800 mg/kg)0.00

0.05

0.10

0.15

#

# # ## # #

*

Groups

serum

redu

ced gl

utathi

one le

vels (

µM/m

l)

FIGURE 4.4.1.10 (a) Effect of polyherbal formulation on CUMS – induced altered levels of

oxido-nitrosative stress parameters: reduced glutathione



normal control group. # p < 0.001 as compared to the disease control group. # # # p < 0.05 as

compared to the disease control group. $ p < 0.05 as compared with the standard (fluoxetine (20

mg/kg)) group.

Chapter 4 Results

54

Serum lipid peroxidase levels(nM/ml)

Normal

Contro

l

Disease

Contro

l

Fluoxe

tine (

20 m

g/kg)

PHF (400

mg/k

g)

PHF (800

mg/k

g)0

5

10

15

*

## #

$

Groups

Seru

m L

PO le

vels

(nM

/ml)

FIGURE 4.4.1.10 (b) Effect of polyherbal formulation on CUMS – induced altered levels of

oxido-nitrosative stress parameters: lipid peroxidase



normal control group. # p < 0.001 as compared to the disease control group. # # # p < 0.05 as

compared to the disease control group. $ p < 0.05 as compared with the standard (fluoxetine (20

mg/kg)) group.

Chapter 4 Results

55

4.4.1.11 Effect of polyherbal formulation on CUMS – induced altered adrenal gland weight:

Applied variable stressors showed prominent effect on the relative adrenal gland weight revealed

a significant main effect of CUMS. As shown in Fig. 10, stressed animals showed higher (p <

0.001) relative weight of adrenal gland when compared with that of controlled mice. PHF at both

the doses was found significantly (p < 0.001) effective against the increase of the relative adrenal

gland weight produced by CUMS.

Adrenal gland weight

Normal C

ontrol

Disease

Control

Fluoxetine (2

0 mg/kg)

PHF (400 mg/kg)

PHF (800 mg/kg)

0

2

4

6

8

##$

#$

*

Groups

Adren

al gla

nd w

t (mg

)

FIGURE 4.4.1.11 Effect of polyherbal formulation on CUMS – induced altered adrenal gland

weight





Chapter 4 Results

56

4.4.2 LPS – induced neuroinflammation in mice model

4.4.2.1 Effect of polyherbal formulation on LPS – induced forced swim test:

LPS-challenged mice exhibited a marked increase (P < 0.001) in immobility time in FST as

compared to vehicle-treated control group that indicated depressive-like behaviour. Fluoxetine &

PHF (600 mg/kg) significantly (P < 0.05) alleviated the LPS-induced depressive behaviour as

evident from reduced immobility time in FST paradigms.

Forced swim test

Normal C

ontrol

Disease

Control

Fluoxetine -

20 mg/kg

PHF - 600 mg/kg

0

50

100

150

200

Day 0 Day 15

* * *#

* *#

Durat

ion of

immo

bility

(sec

)

FIGURE 4.4.2.1 Effect of polyherbal formulation on LPS – induced forced swim test

Statistical analysis was performed by two way ANOVA followed by Bonferroni multiple

comparison test. Results are expressed as Mean ± SEM with n=6. * p < 0.001 as compared to

normal control group on day 15. ** p < 0.01 as compared to normal control group on day 15.

*** p < 0.05 as compared to normal control group on day 14. # p < 0.05 as compared to the

disease control group on day 15.

Chapter 4 Results

57

4.4.2.2 Effect of polyherbal formulation on LPS – induced locomotor activity:

LPS treated mice showed a significant reduction in locomotor index (P < 0.001). Fluoxetine

&PHF – 600 mg/kg pretreatment produced a significant increase in the locomotor index (P <

0.01).

FIGURE 4.4.2.2 Effect of polyherbal formulation on LPS – induced locomotor activity


comparison test. Results are presented as Mean ± SEM with n=6.* p < 0.001 as compared to the

normal control group on day 14. # p < 0.01 as compared to the disease control group on day 14.

Locomotor activity

Normal C

ontrol

Disease

Control

Fluoxetine -

20 mg/kg

PHF - 600 mg/kg

0

50

100

150

Day 0 Day 14

*

# #

Loco

motor

inde

x (co

unts/

5 min)

Chapter 4 Results

58

4.4.2.3 Effect of polyherbal formulation on LPS – induced elevated plus-maze:

LPS treatment induced an anxiogenic effect that was evident by a reduction in the closed arm

time (P < 0.001) in EPM test when compared with the vehicle-treated control group. Fluoxetine

&PHF (600 mg/kg) pretreated rats showed a significant increase in time spent (P < 0.01) in the

closed arm as compared to LPS - treated group.

Elevated Plus Maze

Normal C

ontrol

Disease

Control

Fluoxetine -

20 mg/kg

PHF - 600 m

g/kg0

20

40

60

80

100

Day 0 Day 14

*

* *#

* *#

Time

spen

t in c

lose

d arm

(sec

)

FIGURE 4.4.2.3 Effect of polyherbal formulation on LPS – induced elevated plus maze


comparison test. Results are presented as Mean ± SEM with n=6. * p < 0.001 as compared to

normal control group on day 14. ** p < 0.05 as compared to the normal control group on day 14.

# p < 0.05 as compared to the disease control group on day 14.

Chapter 4 Results

59

4.4.2.4 Effect of polyherbal formulation on LPS – induced Morrison water maze test:

Time spent in the target quadrant was measured in the MWM to evaluate the LPS –induced

memory status in mice. Time spent in the target quadrant of LPS challenged group was

significantly reduced than that of the control group (P < 0.001). Drugs pre – treatments

significantly increased time spent in the target quadrant as compared to disease group (P < 0.05).

After drug treatment, the time of Fluoxetine (81.76%) and PHF (85.57%) groups was

significantly increased as compared to the disease group (65.51%) (P < 0.05).

Morrison water maze test

Normal C

ontrol

Disease

Control

Fluoxetine -

20 mg/kg

PHF - 600 mg/kg

0

50

100

150

Day 0 Day 15

*

* *$

* *$ $

Time s

pent

in tar

get q

uadra

nt (se

c)

FIGURE 4.4.2.4 Effect of polyherbal formulation on LPS – induced Morrison water maze test


comparison test. Results are expressed as Mean ± SEM with n=6. * p < 0.001 as compared to

normal control group on day 15. ** p < 0.01 as compared to normal control group on day 15. $ p

< 0.05 as compared to disease control group on day 15.

Chapter 4 Results

60

4.4.2.5 Effect of polyherbal formulation on LPS – induced proinflammatory cytokines (TNF – α,

IL – 6, IL-1β):

LPS treated animals showed significant (p < 0.001) rise in neuroinflammation markers, namely

TNF – α, IL – 6, IL – 1 as compared to the normal group. PHF (600 mg/kg) treatment

significantly (p < 0.001) attenuated the increased levels of TNF – α, IL – 6, IL – 1β when

compared with the LPS-induced disease control group.

Cytokines-induced neuroinflammationin LPS model

Normal C

ontrol

Disease

Control

Fluoxetine (2

0 mg/kg)

PHF (600 mg/kg)

Normal C

ontrol

Disease

Control

Fluoxetine (2

0 mg/kg)

PHF (600 mg/kg)

Normal C

ontrol

Disease

Control

Fluoxetine (2

0 mg/kg)

PHF (600 mg/kg)

0

50

100

150

TNF - αIL - 6IL - 1β

*

* *$

$*

* * *$ $

*

* * *$ $

TNF - α

IL - 6

IL - 1β

Groups

Serum

cytok

ines l

evels

(pg/m

l)

FIGURE 4.4.2.5 Effect of polyherbal formulation on LPS – induced proinflammatory cytokines

(TNF – α, IL – 6, IL-1β):


comparison test. Results are expressed as mean ± SEM, n = 6. * p < 0.001, ** p< 0.01, *** p <


compared to the disease control group. @ p < 0.05, @@ p < 0.01 as compared to the fluoxetine

treated group.

Chapter 4 Results

61

4.4.2.6 Effect of polyherbal formulation on LPS – induced serum corticosterone measurement:

LPS treatment significant rise in serum corticosterone levels in the disease control group as

compared to the normal control group. Treatment with fluoxetine (20 mg/kg) and PHF (600

mg/kg) showed significantly lowered levels of the marker.

FIGURE 4.4.2.6 Effect of polyherbal formulation on LPS – induced serum corticosterone

measurement



normal control group. # p < 0.001 as compared to the disease control group. $ p < 0.05 as

compared to the standard (fluoxetine (20 mg/kg)) group.

Serum corticosterone levels (ng/ml)

Normal C

ontrol

Disease

Control

Fluoxetine -

20 mg/kg

PHF - 600 m

g/kg0

50

100

150

200

*

*# *

#$

Groups

Seru

m co

rtico

stero

ne le

vels

(ng/m

l)

Chapter 4 Results

62

4.4.2.7 Effect of polyherbal formulation on LPS – induced serum quinolinic acid estimation:

LPS treatment significant rise in serum quinolinic acid levels in the disease control group as

compared to the normal control group. Treatment with fluoxetine (20 mg/kg) and PHF (600

mg/kg) showed significantly lowered levels of the marker. Moreover, the serum concentration of

quinolinic acid in PHF - 600 mg/kg treated animals lowered significantly (p < 0.05) as compared

to the standard treatment (fluoxetine).

Serum quinolinic acid levels (pg/ml)

Normal Control

Disease C

ontrol

Fluoxetine -

20 mg/kg

PHF - 600 mg/kg

0

5

10

15

*

*#

* *#$

Groups

Serum

quino

linic

acid l

evels

(pg/m

l)

FIGURE 4.4.2.7 Effect of polyherbal formulation on LPS – induced serum quinolinic acid

estimation



normal control group. ** p < 0.01 as compared to the normal control group. # p < 0.001 as

compared to the disease control group. $ p < 0.05 as compared to the standard (fluoxetine (20

mg/kg)) group.

Chapter 4 Results

63

4.4.2.8 Effect of polyherbal formulation on LPS – induced oxido-nitrosative stress parameters

(reduced glutathione, LPO, nitrite level):

LPS treatment significant rise in oxidative stress in the disease control group when compared

with the normal group. Treatments with fluoxetine (20 mg/kg) and PHF (600 mg/kg)

significantly ameliorated the level of reduced glutathione as compared to that of the disease

control group. Further higher level of lipid peroxidase and nitrite content were observed in the

disease control group that was also attenuated significantly (p < 0.05) the lipid peroxidase level

and nitrite level as compared to LPS treated animals


Normal Control

Disease C

ontrol

Fluoxetine -

20 mg/kg

PHF - 600 mg/kg

0.00

0.05

0.10

0.15

0.20

*

*# #

*#$

Groups

Serum

redu

ced gl

utathi

one le

vels (

µM/m

l)

FIGURE 4.4.2.8 (a) Effect of polyherbal formulation on LPS – induced oxido-nitrosative stress

parameter: reduced glutathione


comparison test. Results are expressed as mean ± SEM, n = 6. * indicates p < 0.001 as compared

to the normal control group. # indicates p < 0.01 as compared to the disease control. ## indicates

p < 0.001 as compared to the disease control group. $ indicates p < 0.05 as compared to the

standard (fluoxetine (20 mg/kg)) group.

Chapter 4 Results

64

FIGURE 4.4.2.8 (b) Effect of polyherbal formulation on LPS – induced oxido-nitrosative stress

parameter: lipid peroxidase level



to the normal control group. ** p < 0.01 as compared to the normal control group. *** p < 0.05

as compared to the normal control group. # p < 0.001 as compared to the disease control.


Norm

al Con

trol

Diseas

e Con

trol

Fluox

etine -

20 m

g/kg

PHF - 60

0 mg/k

g0

5

10

15

*

* *# * * *

#

Groups

Seru

m L

PO le

vels

(nM

/ml)

Chapter 4 Results

65

Normal

Contro

l

Disease

Contro

l

Fluoxe

tine -

20 m

g/kg

PHF - 600

mg/k

g0

2

4

6

*

* *#

* * *#

Serum nitrite levels (µM/ml)

Groups

Seru

m n

itrite

leve

ls (µ

M/m

l)

FIGURE 4.4.2.8 (c) Effect of polyherbal formulation on LPS – induced oxido-nitrosative stress

parameter: nitrite level



normal control group. ** p < 0.01 as compared to the normal control group. *** p < 0.05 as

compared to the normal control group. # p < 0.001 as compared to the disease control group.

Chapter 4 Results

66

4.4.2.9 Effect of polyherbal formulation on LPS – induced altered level of nerve growth factor (BDNF – Brain-Derived Neurotrophic Factor - BDNF):

Furthermore, BDNF level was significantly reduced (P < 0.001) after 24 h of LPS administration

as compared to the normal control group as shown (Fig. 13). PHF - 600 mg/kg (P < 0.001)

significantly prevented the LPS-induced BDNF depletion as compared to the disease control

group.

Brain Derived Neurotrophic Factor

Normal Control

Disease C

ontrol

Fluoxetine -

20 mg/kg

PHF - 600 mg/kg

0

100

200

300

*

* *# # #

Groups

BDNF

levels

(pg/g

wt o

f tissu

e)

FIGURE 4.4.2.9 Effect of polyherbal formulation on LPS – induced altered level of nerve

growth factor (BDNF – Brain-Derived Neurotrophic Factor - BDNF)

Statistical analysis was performed by one way ANOVA followed by Tukey’s multiple


to the normal control group. ** indicates p < 0.01 as compared to the normal control group. #

indicates p < 0.01 as compared to the disease control group. ## indicates p < 0.001 as compared

to the disease control group.

Chapter 4 Results

67

4.4.3 Ketamine-induced psychosis model

4.4.3.1 Effect of polyherbal formulation on ketamine – induced locomotor activity:

Ketamine treated animals showed a significant increase in locomotor index (P < 0.001) on day 5

at 0, 30 and 60 min. Haloperidol (0.25 mg/kg) (92.77%))&PHF (600 mg/kg) (98.89%)

pretreatment produced a significant reduction in the locomotor index (P < 0.01) on day 14 at 0,

30, 60 min. time points.

Locomotor Activity: Day 0

0 min 30 min 60 min0

50

100

150

Normal Control

Disease Control

Haloperidol - 0.25 mg/kg

PHF -600 mg/kg

Disease Control - 2

Loco

motor

inde

x (co

unts/

5 min)

FIGURE 4.4.3.1 (a) Effect of polyherbal formulation on ketamine – induced locomotor activity

locomotor activity: Day 0

Statistical analysis was performed by two way ANOVA followed by bonferroni multiple

comparison test. Results are expressed as mean ± SEM, n = 6. * p < 0.001, $ p < 0.001, # p <

0.001 as compared to the normal control group at 0, 30, 60 min respectively on day 5. ^ p <

0.001, @ p < 0.001, % p < 0.001 as compared to the normal control group at 0, 30, 60 min

respectively on day 14. ψ p < 0.001, Ω p < 0.001, ! p < 0.001 as compared to disease control group

at 0, 30, 60 min. respectively on day 14.

Chapter 4 Results

68



50

100

150

200

250

Normal Control

Disease Control


PHF -600 mg/kg

* *

$

Disease Control - 2

#

* *

$ $ $

# ##

Loco

mot

or in

dex

(cou

nts/5

min

)

FIGURE 4.4.3.1 (b) Effect of polyherbal formulation on ketamine – induced locomotor activity








Chapter 4 Results

69



50

100

150

200

250

Normal Control

Disease Control


PHF -600 mg/kg

^

Ψ

@

Disease Control - 2

%

!Ω! !Ψ

Ψ

Ω Ω

Loco

mot

or in

dex (

coun

ts/5 m

in)

FIGURE 4.4.3.1 (c) Effect of polyherbal formulation on ketamine – induced locomotor activity








Chapter 4 Results

70

4.4.3.2 Effect of polyherbal formulation on ketamine – induced stereotype behaviours:

Ketamine (50 mg/kg, i.p.) induced stereotype behaviour including head-turning, bobbing, head

falling and sniffing in mice as compared to control animals (p < 0.001). Treatment with PHF

significantly decreased stereotyped behaviours.

Effect on stereotype behaviour on day 0

0 min 30 min 60 min0.0

0.2

0.4

0.6

0.8

Normal Control

Disease Control


PHF (600 mg/kg)

Disease Control - 2

Falli

ng (c

ount

s/10

min

)

FIGURE 4.4.3.2 (a) Effect of polyherbal formulation on ketamine – induced stereotype

behaviours on day 0: Falling


comparison test. Results are expressed as mean ± SEM, n = 6.p < 0.05 as compared to control


Chapter 4 Results

71



2

4

6

8

Normal Control

Disease Control


PHF (600 mg/kg)

Disease Control - 2

Hea

d tu

rnin

g (c

ount

s/10

min

)

FIGURE 4.4.3.2 (b) Effect of polyherbal formulation on ketamine – induced stereotype

behaviours on day 0: Head turning




Chapter 4 Results

72



0.5

1.0

1.5

2.0

2.5

Normal Control

Disease Control


PHF (600 mg/kg)

Disease Control - 2

Hea

d bo

bbin

g (c

ount

s/10

min

)

FIGURE 4.4.3.2 (c) Effect of polyherbal formulation on ketamine – induced stereotype

behaviours on day 0: Head bobbing




Chapter 4 Results

73



5

10

15

Normal Control

Disease Control


PHF (600 mg/kg)

Disease Control - 2

Sniff

ing

(cou

nts/

10 m

in)

FIGURE 4.4.3.2 (d) Effect of polyherbal formulation on ketamine – induced stereotype





Chapter 4 Results

74



0.5

1.0

1.5

2.0

Normal Control

Disease Control


PHF (600 mg/kg)

^

^

^

Disease Control - 2

^

Falli

ng (c

ount

s/10

min

)

FIGURE 4.4.3.2 (e) Effect of polyherbal formulation on ketamine – induced stereotype



comparison test. Results are presented as Mean ± SEM with n=6. ^ p < 0.05, ^^ p < 0.01, ^^^ p <

0.001 as compared to NC group at 0 min. * p < 0.01, ** p < 0.001 as compared to NC group at 30

min. @ p < 0.05, @@ p < 0.01, @@@ p < 0.001 as compared to NC group at 60 min.

Chapter 4 Results

75



2

4

6

8

10

Normal Control

Disease Control


PHF (600 mg/kg)

^ ^

^ ^ ^

^ ^ ^

*** ****

@ @ @

# #

Disease Control - 2

^ ^ ^

Hea

d tu

rnin

g (c

ount

s/10

min

)

FIGURE 4.4.3.2 (f) Effect of polyherbal formulation on ketamine – induced stereotype

behaviours on day 5: Head turning





Chapter 4 Results

76



5

10

15

Normal Control

Disease Control


PHF (600 mg/kg)

^ ^ ^ * * *@ @

@ @ @

Disease Control - 2

* * * * * *@ @ @H

ead

bobb

ing

(cou

nts/

10 m

in)

FIGURE 4.4.3.2 (g) Effect of polyherbal formulation on ketamine – induced stereotype

behaviours on day 5: Head bobbing





Chapter 4 Results

77



10

20

30

Normal Control

Disease Control


PHF (600 mg/kg)

^ ^ ^

* * *

Disease Control - 2

^ ^ ^ ^ ^ ^

* * * * * *

Sniff

ing

(cou

nts/

10 m

in)

FIGURE 4.4.3.2 (h) Effect of polyherbal formulation on ketamine – induced stereotype

behaviours on day 5: Sniffing





Chapter 4 Results

78



0.5

1.0

1.5

2.0

Normal Control

Disease Control


PHF (600 mg/kg)

^ ^ ^

! ! !

Disease Control - 2

Falli

ng (c

ount

s/10

min

)

FIGURE 4.4.3.2 (i) Effect of polyherbal formulation on ketamine – induced stereotype behaviors

on day 14: Falling



0.001 as compared to NC group at 0 min.! p < 0.05, !! p < 0.01, !!! p < 0.001 as compared to DC

group at 0 min. *p < 0.05, ** p < 0.01, *** P < 0.001 as compared to NC group at 30 min. % p <

0.01, %%%%%p < 0.001 as compared to DC group at 30 min. @ p < 0.05, @@ p < 0.01, @@@ p <

0.001 as compared to NC group at 60 min. # p < 0.05, ## p < 0.01, ### p < 0.001 as compared to

DC group at 60 min.

Chapter 4 Results

79



2

4

6

8

Normal Control

Disease Control


PHF (600 mg/kg)

^ ^ ^

^^

* * *

! % % %

@ @ @

# #

# # #

Disease Control - 2

% % %

Hea

d tu

rnin

g (c

ount

s/10

min

)

FIGURE 4.4.3.2 (j) Effect of polyherbal formulation on ketamine – induced stereotype behaviors

on day 14: Head turning







DC group at 60 min.

Chapter 4 Results

80



2

4

6

8

Normal Control

Disease Control


PHF (600 mg/kg)

^ ^ ^

!!!

* * *

% % % @ @@

Disease Control - 2

% % % @ @

Hea

d bo

bbin

g (c

ount

s/10

min

)

FIGURE 4.4.3.2 (k) Effect of polyherbal formulation on ketamine – induced stereotype

behaviors on day 14: Head bobbing







DC group at 60 min.

Chapter 4 Results

81



5

10

15

20

25

Normal Control

Disease Control


PHF (600 mg/kg)

^ ^ ^

! !! ! !

* * *

% % %

Disease Control - 2

% % %

Sniff

ing

(cou

nts/

10 m

in)

FIGURE 4.4.3.2 (l) Effect of polyherbal formulation on ketamine – induced stereotype behaviors

on day 14: Sniffing







DC group at 60 min.

Chapter 4 Results

82

4.4.3.3 Effect of polyherbal formulation on ketamine – induced water maze test:

Ketamine (50 mg/kg, i.p.) significantly (77.80%) decreased the time spent in target quadrant as

compared to control animals (100.00%) showing memory impairment. Whereas treatments

(PHF: 100.39%, haloperidol: 80.69%) notably decreased, the time spent in target quadrant.

Morris water maze test

Day 0 Day 5 Day 140

50

100

150

Normal Control

Disease Control


PHF (600 mg/kg)

^ ^ ^ ** *

$@ @

Disease Control - 2

^ * *

Tim

e spe

nt in

targ

et qu

adra

nt (s

ec)

FIGURE 4.4.3.3 Effect of polyherbal formulation on ketamine – induced water maze test


comparison test. Results are presented as Mean ± SEM with n=6. $ p < 0.001, $$ p < 0.01, $$$ p <

0.05 as compared to the disease control group on day 14. @ p < 0.05, @@ p < 0.01, @@@ p < 0.001

as compared to the Haloperidol group on day 14. ^ p < 0.05, ^^ p < 0.01, ^^^ p < 0.001 as

compared to normal control group on day 5.

Chapter 4 Results

83

4.4.3.4 Effect of polyherbal formulation on ketamine – induced catalepsy test – bar test:

Cataleptic symptoms were observed in mice treated with haloperidol (0.25 mg/kg, i.p.) (p <

0.001) (14th day) as compared to control animals. While treatment with PHF did not show any

cataleptic symptoms.

Catalepsy

Day 0 Day 5 Day 140

1

2

3

4

Normal Control

Disease Control


PHF (600 mg/kg)

*

Disease Control - 2

Desc

ent L

atenc

y (se

c)

FIGURE 4.4.3.4 Effect of polyherbal formulation on ketamine – induced catalepsy test – bar test


comparison test. Results are presented as Mean ± SEM with n=6. * p < 0.001 as compared to

normal control, disease control and PHF – 600 mg/kg treated groups with Haloperidol group on

day 14.

Chapter 4 Results

84

4.4.3.5 Effect of polyherbal formulation on ketamine – induced learned helplessness:

Administration of PHF (600 mg/kg, p.o) significantly (p < 0.01) inhibited the helplessness

response in mice as indicated by decreased in number of failure. Haloperidol (0.25 mg/kg; i.p.)

remarkably (p < 0.01) reduced the helplessness response in mice as indicated by decreased in

number of failure.

Learned helplessness model

Day 0 Day 5 Day 140

2

4

6

8

Normal Control

Disease Control


PHF (600 mg/kg)

* *$ $

* *$ $

Disease Control - 2

* *$ $

Numb

er of

failu

res

FIGURE 4.4.3.5 Effect of polyherbal formulation on ketamine – induced learned helplessness


comparison test. Results are presented as Mean ± SEM with n=6. ** p < 0.01 as compared to

normal control group on day 14. $$ p < 0.01 as compared to disease control group on day 14.

Chapter 4 Results

85

4.4.3.6 Effect of polyherbal formulation on ketamine – induced social interaction test:

Effect of PHF on the exploratory behaviour (i.e.) the time spent in the probe chamber decreased

significantly (P < 0.01) in all groups a compare to normal control group on day 5. Treatments

significantly ameliorate this behaviour and showed via more time spent in the probe chamber on

day 14.

Social Interaction test

Day 0 Day 5 Day 140

100

200

300

400

500

Normal Control

Disease Control


PHF (600 mg/kg)

^ ^

Disease Control - 2

^ ^ ^ ^ ^ ^ * *

Tim

e sp

ent i

n pr

obe

cham

ber (

sec)

FIGURE 4.4.3.6 Effect of polyherbal formulation on ketamine – induced social interaction test:


comparison test. Results are presented as Mean ± SEM with n=6 ** p < 0.01 as compared to the

normal control group on day 14. ^^ p < 0.01 as compared to normal control group on day 5.

Chapter 4 Results

86

4.4.3.7 Effect of polyherbal formulation on ketamine – induced proinflammatory cytokines (TNF

– α, IL – 6, IL-1β):

Ketamine treated animals showed significant (p < 0.001) rise in TNF – α as compared to the

normal group while IL – 6, IL – 1 levels were not affected. PHF (600 mg/kg) treatment

significantly (p <0.001) attenuated the increased levels of TNF – α when compared with the

ketamine-induced disease control group.

Serum TNF-α levels (pg/ml)

Normal C

ontrol

Disease

Control

Haloperi

dol - 0.25 mg/kg

PHF- 600 mg/kg

Disease

Control -

20

50

100

150

*

*$$$

* *$

$

Groups

Seru

m TN

F-α

levels

(pg/m

l)

FIGURE 4.4.3.7 (a) Effect of polyherbal formulation on ketamine – induced proinflammatory

cytokines: TNF – α

Statistical analysis was performed by one-way ANOVA followed by Tukey's multiple

comparison test. Results are presented as Mean ± SEM with n=6. * p < 0.001, ** p < 0.01 as

compared to normal control group. @ p < 0.001, @@@ P < 0.05 as compared to disease control

group.

Chapter 4 Results

87

Serum IL - 6 levels (pg/ml)

Normal

Contro

l

Disease

Contro

l

Halope

ridol

-0.25

mg/k

g

PHF - 600

mg/k

g

Disease

Contro

l - 2

0

5

10

15

Groups

Ser

um

IL

- 6

leve

ls (

pg/

ml)

FIGURE 4.4.3.7 (b) Effect of polyherbal formulation on ketamine – induced proinflammatory

cytokines: IL – 6




group.

Chapter 4 Results

88

Serum IL-1β levals(pg/ml)

Normal

Contro

l

Disease

Contro

l

Halope

ridol

-0.25

mg/k

g

PHF - 600

mg/k

g

Disease

Contro

l - 2

0

5

10

15

Groups

Seru

m IL

-1β

leve

ls (p

g/m

l)

FIGURE 4.4.3.7 (c) Effect of polyherbal formulation on ketamine – induced proinflammatory

cytokines: IL-1β




group.

Chapter 4 Results

89

4.4.3.8 Effect of polyherbal formulation on ketamine – induced oxido-nitrosative stress

parameters (reduced glutathione, LPO, nitrite level):

Ketamine treatment significant rise in oxidative stress in the disease control group when

compared with the normal group. Treatments with haloperidol (0.25 mg/kg) and PHF (600

mg/kg) significantly ameliorated the level of reduced glutathione as compared to that of the

disease control group. Further higher level of lipid peroxidase and nitrite content were observed

in the disease control group that was also attenuated significantly (p < 0.05) the lipid peroxidase

level and nitrite level as compared to ketamine treated animals.


Normal C

ontrol

Disease

Control

Haloperid

ol -0.25 mg/kg

PHF - 600 mg/kg

Disease

Control -

20.00

0.05

0.10

0.15

0.20

*

* *$

* * *$

* * *$

Groups

Serum

redu

ced g

lutath

ione l

evels

(µM

/ml)

FIGURE 4.4.3.8 (a) Effect of polyherbal formulation on ketamine – induced oxido-nitrosative

stress parameters: reduced glutathione

Statistical analysis was performed by one way ANOVA followed by tukey’s multiple



compared to the disease control group.

Chapter 4 Results

90

Serum nitrite levels (µM/ml)

Normal

Contro

l

Disease

Contro

l

Halope

ridol

-0.25

mg/k

g

PHF - 600

mg/k

g

Disease

Contro

l - 2

0

2

4

6

*

* *$ * * *

$

*$

Groups

Seru

m n

itrite

leve

ls (µ

M/m

l)

FIGURE 4.4.3.8 (b) Effect of polyherbal formulation on ketamine – induced oxido-nitrosative

stress parameters: nitrite level




compared to the disease control group.

Chapter 4 Results

91


Normal

Contro

l

Disease

Contro

l

Halope

ridol

-0.25

mg/k

g

PHF - 600

mg/k

g

Disease

Contro

l - 2

0

5

10

15

*

* *

$

*$ *

$

Groups

Seru

m L

PO le

vels

(nM

/ml)

FIGURE 4.4.3.8 (c) Effect of polyherbal formulation on ketamine – induced oxido-nitrosative

stress parameters: LPO


comparison test. Results are expressed as mean ± SEM, n = 6. * p < 0.001, ** p< 0.01, *** p<

0.05 as compared to the normal control group. $ p < 0.001, $$ p< 0.01, $$$ p< 0.05 as compared

to the disease control group.

Chapter 4 Results

92

4.4.3.9 Effect of polyherbal formulation on ketamine – induced altered level of nerve growth

factor (BDNF – Brain-Derived Neurotrophic Factor - BDNF):

BDNF level was significantly reduced (P < 0.001) after ketamine administration as compared to

the normal control group as shown. PHF - 600 mg/kg (P < 0.001) significantly prevented the

ketamine-induced BDNF depletion as compared to the disease control group.

Brain Derived Neurotrophic Factor

Normal

Contro

l

Disease

Contro

l

Haloper

idol -0

.25 m

g/kg

PHF - 600

mg/k

g

Disease

Contro

l - 2

0

100

200

300

*

* $

* *$ *

$

Groups

BDNF

leve

ls (p

g/g w

t of t

issue

)

FIGURE 4.4.3.9 Effect of polyherbal formulation on ketamine – induced altered level of nerve

growth factor (BDNF – Brain-Derived Neurotrophic Factor - BDNF)


comparison test. Results are expressed as mean ± SEM, n = 6. * p < 0.001, ** p < 0.01, as

compared to the normal control group. $ p < 0.001, , $$$ p < 0.05 as compared to the disease

control group.

Chapter 5 Discussion

93

CHAPTER 5

Discussion

Psychiatric illnesses account for 22.8% of the worldwide burden of diseases. Depression is one

of the chief origins of illness universal (139). Depression which has substantially increased since

1990, largely driven by population advance and ageing. There are many executor interacting

pathways that are concerned in the pathogenesis of depression due to its intricacy and

heterogeneity (140). Several studies have described the link between inflammation and

depression (87, 141). Depressive illness is closely linked with chronic inflammatory path, which

is manifested by augmented levels of proinflammatory cytokines, chemokines, and adhesion

molecules in the periphery and central nervous system response (141-144).

Even though numerous antidepressant drugs are available now, yet their efficacy and usefulness

are highly uncertain especially because of their side effects. As herbal medications are generally

related with favorable safety outlines, therefore they have the likely potential to deliver effective

alternates to presently existing synthetic antidepressants (145-147). Overall, biological effect

relies on synergistic relations between plant components although single active principles of

plant extract can be self-defeating for given cause. Therefore, polyherbal formulation is used in

current practice. Polyherbal formulation (PHF) possesses some advantages such as to decrease in

dose, ease of administration (27-29). The multitarget responses of herbal drugs are confirmed to

be beneficial in chronic conditions and so forth, and as well in restoring the health status (30).

So, there is a range for the development of such treatment which works not only by behavioural

defects of the depression and anxiety but also useful for the elimination of toxins from the brain

and produces a calming effect. Tensnil Syrup, a PHF (Poly Herbal Formulation), contains

extracts of Garmarogor (109), devdaru, shankhavali, pitapapapdo, brahmi, jatamansi, nagarmoth,

kadu, tagar, himaj, draksha, ashwagandha. These plants have been reported to be used in nervous

system disorders as they calm down the brain, produce quality sleep (31), and remove toxins

from the brain (32).


94

So based on the above reports, the present study was divided into three parts in which, part 1 was

toxicity study. In part two, ED50 determinations were performed by using forced swim test and

preliminary screening of the formulation was done using a forced swim test, tail suspension test,

locomotor activity elevated plus-maze test. Based on the results obtained from part 2 study,

chronic studies were planned in part three wherein various animal models were used namely

chronic unpredictable mild stress, lipopolysaccharide-induced depressive behavioural model and

ketamine-induced experimental psychosis in mice.

In part one study, Tensnil syrup was administered orally at five different dose levels i.e. 100

mg/kg, 200 mg/kg, 400 mg/kg, 600 mg/kg and 800 mg/kg body weight for twenty eight days. No

mortality or abnormal behaviour was seen in the animals treated with Tensnil syrup, up to the

dose of 800 mg/kg. The formulation did not have any significant impact on the body weight and

food intake indicating that treatment with Tensnil syrup did not affect the common health status

of the animals.

The hematopoietic system is one of the most susceptible targets for toxic compounds and an

important index of physiological and pathological status in man and animal (148). The treatment

with Tensnil syrup did not have a significant impact on the hematological study. The levels of

glucose, cholesterol, and triglyceride remained unaffected indicating that the formulation did not

interfere with the carbohydrate and lipid metabolism in mice (149). Treatment with Tensnil

syrup in mice did not alter the hepatic and renal function, as identified from the hepatic enzyme

AST, ALT levels, and renal serum biomarkers of creatinine. It further confirmed the normal

functioning of hepatocytes and nephrons during treatment period. Based on these findings, the

safety of the formulation is confirmed at the therapeutic dose level under the study. In addition to

this, no observed adverse effect level (NOAEL) of Tensnil syrup was observed up to the dose of

800 mg/kg.

In part two, ED50 was derived to 600 mg/kg dose, for this PHF using a dose range 100 - 800

mg/kg in the study. Mice treated with acute stress produced an increase in immobility time in

FST and TST along with decreased locomotor index in photoactometer and reduced the time

spent in close arm. Our study showed parallel results with those of previous studies in which


95

exposure to stress augmented immobility time (150). In FST, mice were forced to swim in a

constrained space, which induced a typical behaviour of immobility. In TST, mice were hanging

by their tip of the tail from a metal rod which in addition induced a state of immobility in

animals like that in FST. This immobility reflects a state of despair in animals and is claimed to

reproduce a condition similar to depression in humans. PHF produced a marked decrease in the

duration of immobility when compared with the disease control group and thereby produced

anti-depressant activity. Locomotor activity is measured as an index of alertness and a decrease

in it is investigative of sedative effect. However, none of the doses of PHF were found to have

any sedative effect activity. The Elevated plus maze was used to evaluate the anxiety state in

animals. It is a simple and less time-consuming procedure wherein acquisition can be considered

as transfer latency on the first-day trials and the retention/consolidation later. The animals treated

with PHF (400, 800mg/kg) showed a significant increase in time spent in closed arm indicating

anxiolytic activity of the drug under investigation.

Part 3 study was divided into chronic study with different animal models for evaluating the

effects of long term use of the formulation. The first model was the CUMS model, in which mice

were exposed to a unsystematic pattern of mild stressors daily for 28 days which were scheduled

for a period of one week and repeated throughout the experiment. Stressors incorporated cage

tilting at 450, cold swimming, tail pinch, housing in mild damp sawdust, wet sawdust, overnight

illumination, and food and water deprivation. The entire experimentation lasted for 8 weeks (56

days). Behaviour tests together with forced swim test, tail suspension test, locomotor activity

using photoactometer, elevated plus maze and sucrose preference test were performed at the end

of every week. Blood analysis was performed at the end of the experiment for the estimation of

serum proinflammatory cytokines (TNF-α, IL-1β and IL-6), corticosterone, quinolinic acid and

levels of oxidative and anti-oxidant enzymes. Then, after the mice were killed and skull was

opened, and the brain was dissected out on an ice plate for analysis of brain neurotransmitters

namely 5-hydroxy tryptamine, noradrenaline, and dopamine.

CUMS for 28 days, significantly activate HPA axis, which is indicated by high levels of

proinflammatory cytokines, chemokines, and adhesion molecules in the periphery and central

nervous system and caused production of reactive oxidative stress markers (14, 151-153). In this


96

study, CUMS experience induced a depressive status in mice as it resulted in increased

immobility time in the FST and TST. The forced swim test has been used to detect helpless

behaviour as measured by immobility time in the chronic mild stress model in mice. Our data

showed that stressed mice exhibited a significant persistence of immobility time in the FST/TST

over the end of the last week of CUMS, compared to control and this is also supported by an

previous study (154). PHF (400 & 800 mg/kg) treatment significantly reduced the duration of

immobility in the FST/TST, suggesting that the polyherbal formulation reversed the depression-

like symptoms of CUMS exposed mice, thus showed significant antidepressant-like effect.

Anxiety is thought to be a negative sentiment caused by many kinds of stress. In this ground, the

EPM task has become one of the most accepted animal paradigms used in our study. In this test,

the anxiety-like behaviour (i.e., decreased time spent in the closed arms) is potentiated by

previous exposure to a variety of stressors (155), as confirmed by CUMS procedure. Our study

has confirmed decreased the anxiety-like effects of stress in mice after the CUMS protocol by

the pretreatment of orally administered polyherbal formulation. The locomotor activity is

considered as an index of alertness and a decrease in it is indicative of anxiety-like activity. The

effect of stress on locomotor activity is still controversial (156, 157). However, both of the doses

of PHF under the study have shown an anti-anxiety effect against photoactometer.

SPT signifies the anhedonia-like behavioural change, a core indicator of depressive disorder

(158). With this test, reduced utilization of sucrose solutions reflecting a decrease in

responsiveness to rewards that are interpreted as an indication of anhedonia. In our study, mice

experienced to CUMS procedures consumed less sucrose solution as compared to the control

group, while treatment with PHF significantly reversed this change of behaviour, enlightening

antidepressant effect. Taken together, the behavioural finding reveals that, PHF treatment exerts

antidepressant-like effects in the CUMS-induced mice.

Further, depression is accompanied by altering immune function and beginning of the

inflammatory response in central and peripheral nervous system (159). In animals, a range of

stressors increases the concentration of proinflammatory cytokines, mainly TNF-α, IL-6 and IL-

1β (129,160, 161) that is also observed in our model. Repetitive administration with the


97

fluoxetine and polyherbal formulation significantly reduced an increase in the levels of pro-

inflammatory cytokines in CUMS exposed mice. Further, herbal treatment was found

significantly superior to Fluoxetine treatment. These results suggest that the antidepressant-like

effect of formulation might be associated with a decrease in stress-induced anxiety.

It has been documented that the HPA axis could be activated by an inflammatory cytokines (162,

163) which leads to unusually high glucocorticoid (corticosterone in rodents or cortisol in

primates) levels in blood (164) in that way plays an important role in the pathophysiology of

depression (165, 166). Cortisol is identified to regulate neuronal endurance, neuronal

excitability, and neurogenesis and memory acquirement. Higher levels of cortisol may thus

contribute to the demonstration of depressive symptoms by impairing these brain function (167).

CUMS-induced hyperactivity of HPA axis led to an increase in plasma corticosterone levels and

an increase in adrenal gland weight, in one study (168). There is a report of reduced HPA

activity in anti-depressant response in rodents (169). Treatment with PHF also reduced CUMS-

induced hyperactivity of HPA axis in mice that is evident from significant lessening of plasma

corticosterone and adrenal gland weights of treated mice.

The typical monoamine hypothesis of depression still is one of the proposed theories regarding

the aetiology of depression (170). Once cytokine signals get to the brain, they have the capability

to manipulate the synthesis, release, and reuptake of mood-relevant neurotransmitters including

the monoamines (171). The breakdown of TRP (tryptophan) is believed to contribute to reduced

serotonin accessibility (172). Cytokines also have been shown to influence the synthesis of DA

(dopamine). Activation of microglia is associated with increased NO (nitric oxide) production

(173), suggesting an influence of cytokines on BH4 (tetrahydrobiopterine) via NO may be a

general mechanism by which cytokines reduce DA accessibility in related brain regions (174).

Deficiency of 5-HT, NE, and DA in the brain are commonly observed in both animals and

patients experiencing stress and depression (170). Fluoxetine, a classic antidepressant, plays an

antidepressant role by efficiently raising the level of 5-HT and improving serotonergic

transmission. In addition, fluoxetine uniquely increases extracellular levels of DA and NE as

well as 5-HT. Consistent with this result, our data showed a decrease in noradrenaline,

dopamine, and 5-hydroxytryptamine levels in CUMS- treated mice. However, PHF treatment


98

restored the concentration of these neurotransmitters, and thereby amelioration of depressive

behaviours after PHF treatment.

Besides, kynurenine pathway (KP) of tryptophan metabolism has appeared in current ages as an

important controller of the production of both neuroprotective (e.g. Kynurenic and picolinic

acid, and the essential cofactor NAD+) and neurotoxic metabolites (e.g. quinolinic acid,3-

hydroxykynurenine) (175). KA (Kyneronic Acid) inhibits the discharge of glutamate, while

QUIN (quinolinic acid) promote glutamate release through activation of N-methyl-D-aspartate

(NMDA) receptors (176). QUIN also activates and/or kills astrocytes and this amplifies the

inflammatory response in the brain. Our data also showed raised QUIN levels in the CUMS

treated mice, while the anti-depressant like effects of PHF was accompanied with the decrease of

serum QUIN levels. Additionally, this formulation seems to exert a more distinct antidepressant-

like effect as compared to fluoxetine.

Another major mechanism of QUIN - induced neurotoxicity is through the lipid peroxidation in

grouping with glutamate release causative CNS excitotoxicity (177, 178). Studies also have

revealed that QUIN forms a complex with iron and electron transfer from this complex to

oxygen consequences in the formation of reactive oxygen species which then arbitrate lipid

peroxidation (179). Oxygen-free radicals can gather in the brain and have a powerful function in

neurodegeneration linked with depression (180). Oxidative stress is major cause of neuronal

dysfunction and depression (181). In our study, we reported a significantly increase in oxidative

harm that is reflected from increased lipid peroxidation, and reduction of reduced glutathione

levels thus strengthening the theory of oxidative stress-induced depressive illness. PHF treatment

for four consecutive weeks significantly reversed these stress-related parameters. Thus the anti-

oxidant activity of the PHF is very well established in the present study.

Another chronic model under the study was LPS-induced depressive and anxiety-like behaviour

in mice. LPS is a very well-known endotoxin that can cause anxiety and depression-like

behaviour in rodents after central or peripheral administration (182). LPS also elicits the

production of proinflammatory cytokines (IL-β, IL-6, TNF-α) and reactive oxygen species

(ROS) that eventually leads to peripheral as well as systemic inflammation (143). Oxidative


99

stress and inflammatory mediators generate a vicious cycle that further depletes the neurotrophic

factor such as BDNF, nerve growth factor (NGF), and neurotrophin-3 (NT-3) levels in the cortex

and hippocampus. Exaggerated oxidative stress, neuroinflammation, and the resulted depletion

of neurotrophic factors in the brain eventually manifest the development of anxiety and

depressive-like behaviour (183). Our results are in line with the previous studies wherein

oxidative stress, neuroinflammation, and BDNF depletion play a key role in the pathogenesis of

anxiety and depressive illness.

Results of behavioural tests of LPS model showed that LPS-challenged mice exhibit anxiety and

depressive-like behaviour at 3 - and 24 - h post - LPS administration, respectively. Elevated plus

maze test and photoactometer test is the behavioural paradigms frequently used to assess the

anxiety behaviour in rodents. We found a significant reduction in the closed arm time and

locomotor index in the photoactometer test showing anxiety behaviour in LPS-challenged mice.

Depressive behaviour in the LPS-treated group is evident from a marked reduction in the

immobility time in FST as compared to the control group. These behavioural test results are in

concordance with the previous experimental studies (184, 185). The reported experiments also

explored the effects of peripheral LPS administration on learning and memory processes as

measured by the Morrison water maze test by evaluating time spent in target quadrant. The

results of this study also confirm that LPS disrupts learning and memory processes in accordance

with the previous study (186). PHF treatment significantly restored up the learning and memory

task. The observed anxiety and depressive behaviour in behavioural paradigms is possibly

accompanied by elevation of corticosterone, quinolinic acid, cytokines, oxidative stress as well

as BDNF depletion by LPS treatment.

Further, it is reported that LPS - treated mice show significant increase in TNF - α, IL - 6, and IL

- 1β that might be responsible for the neuroinflammation, and neurobehavioral changes(187).

The current findings of the study are parallel with the earlier reports wherein LPS induces

neuroinflammation which is responsible for behavioural alterations (184, 188). Further, PHF

(600 mg/kg) pre-treatment also significantly attenuated an increased level of TNF - α, IL - 6, and

IL - 1β probably via inhibition of pro-inflammatory cytokines.


100

It has been reported that the hypothalamic-pituitary-adrenal (HPA) axis could be activated by

inflammatory cytokines which leads to abnormally high glucocorticoid levels in blood and plays

an important role in the pathophysiology of depression. LPS-induced hyperactivity of HPA axis

led to an increase in plasma corticosterone levels which is supported by observations from other

studies (189, 190). Treatment with PHF reduced hyperactivity of HPA axis in mice, as evident

from significant reduction of plasma corticosterone levels in LPS challenged mice. Also, there

was a reduction of QUIN levels in drug-treated mice indicating the neuroprotective effect of

PHF.

Numerous studies illustrate the role of oxidative stress and neuroinflammation in the

pathogenesis of depression. LPS induces pro-inflammatory cytokines release and activates

microglia causing a marked increase in the production of reactive oxygen species, nitrites and

peroxides, which may further lead to inflammation, lower antioxidant status, and consequently

cause neurobehavioral alterations (191, 192). In the current study, marked oxidative damage in

LPS - treated mice after 24 h was significantly attenuated by pre-treatment of PHF. The results

are in line with the previous findings that prevent oxidative stress by restoring antioxidant

enzyme activity namely reduced glutathione along with reduction of the levels of nitrite and lipid

peroxidase. (193). A previous report demonstrated that peripheral administration of BDNF

exerted anti-anxiety and antidepressant action in mice. Moreover, there are many reports that

have shown therapeutic actions of antidepressants mediated via the presence of BDNF We

observed a significant reduction in neurotrophin, levels (BDNF) after 24 h of LPS – challenged

to mice. Thus, treatments significantly restored up this and thereby showed neuroprotective

potential of the formulation.

The phase 3 of the study includes a chronic model of chronic activity of schizophrenia.

Schizophrenia is a heterogeneous neuropsychiatric disorder characterized by distorted or non-

existent sense of reality (194). Schizophrenia is characterized by positive (e.g., hallucinations),

negative (e.g., social isolation) and cognitive (e.g., executive and memory dysfunction)

symptoms (195). The positive symptoms results from hyperdopaminergic activity in the

mesolimbic pathways, but the negative and cognitive deficits produced from hypodopaminergic

system of the prefrontal cortex (196). The antagonists of the N-methyl-D-aspartate glutamate


101

receptor (NMDAR), such as phencyclidine (PCP) and ketamine, transiently induce symptoms of

acute schizophrenia and led to a paradigm shift from dopaminergic to glutamatergic dysfunction

in pharmacological models of schizophrenia. Ketamine inhibits the release of GABA through

NMDA receptor inhibition located on GABAergic efferent neurons in the brain. GABA, an

inhibitory neurotransmitter, is known to play an important role to control the release of

dopamine. Thus, release of GABA is reduced with ketamine administration leading to increased

dopamine release which further stimulates stereotyped behaviors and locomotor activity (positive

symptoms of psychosis) (197, 198). In our study, treatment with PHF was found effective to

attenuate falling, turning, sniffing and head bobbing behaviours induced by ketamine. PHF was

found effective against ketamine-induced positive symptoms of psychosis as it significantly

attenuates locomotor activity. The above outcomes are also supported by other researchers (199).

This dopamine dysfunctioning is also responsible for social withdrawal a negative symptoms of

psychosis (200). The results are in line with the available literature in which treatments improve

those negative symptoms.

Cognition is defined as recording the events, information or sensory stimuli and its retention for

shorter or longer periods of time (201, 202). Acetylcholine plays a promising role in the

cognition process, get degraded by the enzyme acetylcholinesterase (203). It has been reported

that ketamine suppresses acetylcholine inputs in the hippocampus with the nAChR blockade and

increases the acetylcholinesterase activity (204). Thus, cholinergic deficits contribute to the

cognitive symptoms of psychosis. Learned helplessness test is used widely to screen the effect of

antipsychotics on memory (205). In our study, PHF treatment increased the time spent in the

target quadrant in the Morrison water maze test and reduced number of failure in the learned

helplessness model. Extrapyramidal side effects are the most common side effects observed with

the antipsychotic medicines (206). Bar test has been used to assess the probable side effects (e.g.,

catalepsy) of PHF on mice. In our study, no cataleptic effect was observed in PHF treated group.

These findings indicate that PHF might be a promising molecule for treatment of psychosis,

devoid of extra-pyramidal side effects.

Metabolism of dopamine may create a large amount of hydrogen peroxide (H2O2) and

superoxide radical (O2−) which can impair DNA, a lipids and proteins and finally cellular


102

dysfunction leading to psychiatric disorders. Glutathione plays a role as an endogenous

antioxidant and it reduces inactive disulfide linkage of enzymes to the active sulfhydryl group

and thereby plays an important role in shielding membrane peroxidation with reduction of

hydrogen peroxide. The amount of lipid peroxidation can be measured by estimating the level of

MDA, a lipid peroxidation product. Therefore, the deficiency of action of these enzymes leads to

an inequity between antioxidant protection mechanism and free oxidant radicals that encourage

neuropsychiatric disorders (207). Ketamine promotes oxidative stress by generating free radicals

with demolition of the antioxidant defense mechanism of the brain and subsequently causes

negative as well as cognitive symptoms (208-210). Free radicals accumulate in brain tissue and

stimulate neuropsychiatric disorders associated with memory loss and depression (211-213). In

the current investigation, oxidative stress observed with ketamine was evident from reduction of

reduced glutathione levels and increase of lipid peroxidation and nitrite content, thus

strengthening the theory of oxidative damage induced depressive and cognitive symptoms of

psychosis with ketamine. Oxidative stress is also well known to produce neuroinflammation and

vice versa (214, 215). In addition, oxidative stress causes microglia cell activation that increases

the release of inflammatory cytokines, which further reinforce the oxidative stress leading to

neuronal toxicity and progression of psychosis (216, 217). Ketamine has shown a significant

increase in the serum cytokines levels that is reversed by PHF treatment, which might be due to

its antioxidant and anti-inflammatory effect. Thus, this result when taken collectively reflects

that PHF has free radical scavenging effect, which further reduces neuroinflammation associated

with depressive symptoms of psychosis.

BDNF is a neurotrophin and highly expressed in the mammalian brain that plays a prominent

role in neurogenesis, neural regeneration, synaptic transmission, and synaptic plasticity (200).

Numerous preclinical and clinical studies have shown the key role of BDNF in the

pathophysiology of anxiety and depression. Moreover, there are numerous reports that show the

therapeutic actions of antidepressants mediated through the BDNF (218). In the present study,

we have observed a significant reduction in the BDNF level this neurotrophin after ketamine –

challenged to mice. Drug treatments significantly restored up this and thereby showed

neuroprotective potential of the formulation.


103

In summary, the preliminary data from the toxicity study suggest that there was no observable

finding of serious signs and no significant changes in the physical, hematological, and

biochemical parameters of 28 - day’s administration of Tensnil syrup treated mice. Therefore,

Tensnil syrup reflected the innocuous nature of this formulation on hepatic, renal and

hematopoietic system even at a high dose level of daily administration, indicating the safety of

the formulation and devoid of any neurotoxicity effect. -Further, psychopharmacological

findings revealed significant improvement in depression and anxiety in mice. These findings

have scientifically validated the traditional claim and suggested the valuable role of PHF in the

treatment of neurological disorders. As this study is based on the behavioural models without

any associated neurochemical estimations, it becomes necessary to carry out specific binding

studies and estimations of the neurotransmitter levels in the brain, to understand the exact

mechanism of action and extend these results further.

In the CUMS model study, PHF treatment could significantly mitigate behavioural deficits and

showed considerable up-regulation of serotonin and other neurotransmitters alongside with

lessening in the oxidative stress. Besides, PHF treatment also significantly attenuated the stress-

induced raise in serum levels of TNF-α, IL-1β, IL-6, corticosterone, quinolinic acid. In addition,

PHF pretreatment significantly attenuated the LPS - induced increase in serum level of TNF - α,

IL - 1β, IL - 6, corticosterone, quinolinic acid. In the ketamine – induced psychosis model, PHF

treatment could significantly mitigate behavioural deficits, elicited by ketamine administration. It

also showed up-regulation of BDNF levels.PHF treatment also significantly attenuated the

ketamine-induced raise in serum levels of TNF-α, IL-1β, IL-6. The mechanism of action of PHF

under the study is attributed to, reduction into the levels of corticosterone, proinflammatory

Cytokines like TNF-α, IL-6, IL-1β; oxidative stress and quinolinic acid along with increase in

the levels of neurotransmitters namely 5-HT, DA and NA. Thus, studies provide new insight into

the anti-depressant and anti-psychotic actions of this polyherbal formulation with multiple

targets of depression and helpful novel therapeutic strategies for depression.

Psychopharmacological animal models have founded on the current understanding of the effects

of drugs as they rely on the observation that certain drugs induce behaviors that mimic or predict

symptoms of the diseases in humans. Our studies have several limitations that need to be


104

addressed in future studies. Few of them were significantly impacted including the small size and

diversity of the ‘less reliable’ sample of labs, severity of different stress regimes, the serotype,

route of administration. As a whole, behavioral measurements are limited in their ability to

translate animal model measurements to humans. Nevertheless, these models might shed

mechanistic insights related to signaling in systemic neuroinflammation, responsible immune

pathways participating in inflammatory events leading to neurodegeneration, gene expression

using blood samples. Further studies with different models of neuroinflammation are an

important tool for deciphering pathological mechanisms involved in neurodegeneration as well

as for testing potential therapeutic molecules.

Chapter 6 Conclusion

106

CHAPTER 6

Conclusion

In the present study, our data from the toxicity study suggest that Tensnil syrup has an innocuous

nature on hepatic, renal and hematopoietic system even at high dose level of daily administration

and indicating safety of the formulation and devoid of any neurotoxicity effect. In addition,

neuropsychological findings with the help of CUMS model, LPS – induced neuroinflammation

model and ketamine – induced psychosis model revealed significant improvement in depression

and anxiety in mice. These findings have scientifically validated the traditional claim via

attenuation of the stress-induced increase in serum levels of TNF-α, IL-1β, IL-1, lipid

peroxidation, nitrite, corticosterone, quinolinic acid and up regulation of reduced glutathione and

BDNF level. Also the levels of three major neurotransmitters involved in the aetiology of

depression namely noradrenaline, dopamine and 5-Hydroxytryptamine were restored and thereby

amelioration of depressive behaviours after PHF treatment.

Formulation could ameliorate anxiogenic, depressive, psychotic symptoms and biochemical

changes in rodents, indicating protective effects in the treatment of neurological disorders such

as depression and psychosis.

Chapter 7 References

106

CHAPTER 7

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IEC certificate

128

List of Publications

1. Shah Krishna M., Mody Vandana and Goswami Sunita S. Preliminary screening of

psychopharmacological effects and toxicity testing of tensnil syrup in swiss albino mice.

WJPR,6(8) : 2265-2277

2. Shah Krishna M., Mody Vandana and Goswami Sunita S. Reversal of

neuroinflammation and oxidative stress by polyherbal formulation in an animal model of

chronic unpredictable mice model. Asian Journal of Pharmacy and Pharmacology 2019;

5(5):991-999

151

Images for pharmacological methods

152

Forced swim test

Tail suspension test

153

Locomotor activity

Elevated plus maze

154

Sucrose preference test

Morris water maze test

155

Catalepsy test – bar test

Learned helplessness

156

Social interaction test

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