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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
GUJARAT TECHNOLOGICAL UNIVERSITY
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
idate under m
mitted the
eship, Fellow
work done
the thesis rep
e of Supervi
f Supervisor:
hmedabad
k incorporat
xperimental
my supervisi
same resea
wship or oth
by the Rese
presents ind
sor:
Dr. Sunita G
CERT
ted in the the
l condition”
ion/guidance
arch work t
her similar t
earch Schola
ependent res
Goswami
iv
TIFICA
esis "Psycho
” submitted b
e. To the bes
to any othe
titles (ii) the
ar during the
search work
ATE
opharmacol
by Ms Krish
st of my kno
er institutio
e thesis subm
e period of
on the part o
D
logical evalu
hna Shah w
owledge: (i) t
on for any
mitted is a r
study under
of the Resea
Date: ………
uation of he
was carried o
the candidat
degree/dipl
record of ori
r my supervi
arch Scholar
…………
erbal
out by
te has
loma,
iginal
ision,
.
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.
c) There is no fabrication of data or results, which have been compiled / analyzed.
d) There is no falsification by manipulating research materials, equipment or processes, or
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
vii
viii
ix
PhD THESIS Non-Exclusive License to
GUJARAT TECHNOLOGICAL UNIVERSITY
In consideration of being a PhD Research Scholar at GTU and in the interests of the facilitation
of research at GTU and elsewhere, Ms. Krishna Shah having 149997390006 hereby grant a
non-exclusive, royalty free and perpetual license to GTU on the following terms:
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Any abstract submitted with the thesis will be considered to form part of the thesis.
f) I represent that my thesis is my original work, does not infringe any rights of others,
including privacy rights, and that I have the right to make the grant conferred by this non-
exclusive license.
g) If third party copyrighted material was included in my thesis for which, under the terms
of the Copyright Act, written permission from the copyright owners is required, I have
obtained such permission from the copyright owners to do the acts mentioned in
paragraph (a) above for the full term of copyright protection.
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
copyright in my thesis of the rights granted by me to my University in this non-exclusive
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:
xi
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.
xiv
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.
xv
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,
xvi
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
xx
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
xxii
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
xxiv
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)
Effect of polyherbal formulation on ketamine – induced locomotor activity locomotor activity: Day 5
68
FIGURE 4.4.3.1 (c)
Effect of polyherbal formulation on ketamine – induced locomotor activity locomotor activity: Day 14
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)
Effect of polyherbal formulation on ketamine – induced stereotype behaviours on day 5: Head bobbing
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)
Effect of polyherbal formulation on ketamine – induced stereotype behaviours on day 14: Head bobbing
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
Chapter 1 Introduction
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).
Chapter 1 Introduction
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
Chapter 1 Introduction
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
Chapter 2 Review of literature
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).
Chapter 2 Review of literature
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).
Chapter 2 Review of literature
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).
Chapter 2 Review of literature
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
Chapter 2 Review of literature
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-
Chapter 2 Review of literature
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).
Chapter 2 Review of literature
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.
Chapter 2 Review of literature
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
Chapter 2 Review of literature
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.
Chapter 2 Review of literature
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
Chapter 2 Review of literature
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).
Chapter 2 Review of literature
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
Chapter 2 Review of literature
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.
Chapter 3 Materials and methods
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
Chapter 3 Materials and methods
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
Chapter 3 Materials and methods
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.
Chapter 3 Materials and methods
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).
Chapter 3 Materials and methods
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
Food and water deprivation for 4 hours
Overnight illumination
Day 3 Tail pinch for 60 seconds
Cold swimming at temperature 5ºc for 5 minutes
Cage tilting at 45º for 4 hours
Food and water deprivation for 4 hours
Day 4 Housing in mild damp sawdust for 6 hours
Tail pinch for 60 seconds
Cold swimming at temperature 5ºc for 5 minutes
Cage tilting at 45º for 4 hours
Chapter 3 Materials and methods
25
Day 5 Wet sawdust for 4 hours Housing in mild damp sawdust for 6 hours
Tail pinch for 60 seconds
Cold swimming at temperature 5ºc for 5 minutes
Day 6 Overnight illumination Wet sawdust for 4 hours
Housing in mild damp sawdust for 6 hours
Tail pinch for 60 seconds
Day 7 Food and water deprivation for 4 hours
Overnight illumination
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:
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).
Chapter 3 Materials and methods
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
Chapter 3 Materials and methods
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
Chapter 3 Materials and methods
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).
Chapter 3 Materials and methods
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).
3.4.2.2 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 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
Chapter 3 Materials and methods
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).
3.4.2.5 Proinflammatory cytokines estimation:
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:
Estimation of reduced glutathione:
0.1 ml serum was precipitated with 1.0 ml of sulfosalicylic acid (4%). The samples were rest 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-
Chapter 3 Materials and methods
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).
Estimation of lipid peroxidation:
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
Chapter 3 Materials and methods
32
previous studies. Herbal formulation was intraperitoneally administered once daily for 14 days
and ketamine 50 mg/kg for 14 days.
3.4.3.1 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(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
Chapter 3 Materials and methods
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).
3.4.3.7 Proinflammatory cytokines estimation:
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:
Estimation of reduced glutathione:
0.1 ml serum was precipitated with 1.0 ml of sulfosalicylic acid (4%). The samples were rest 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-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).
Estimation of lipid peroxidation:
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
Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple
comparison test. Results are expressed as mean ± SEM, n = 6, p < 0.05 as compared to control
group, where no significant difference observed.
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
Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple
comparison test. Results are expressed as mean ± SEM, n = 6, p < 0.05 as compared to control
group, where no significant difference observed.
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
Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple
comparison test. Results are expressed as mean ± SEM, n = 6, p < 0.05 as compared to control
group, where no significant difference observed.
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
Statistical analysis was performed by one-way ANOVA followed by Bartlett’s test. Results are
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
Statistical analysis was performed by one-way ANOVA followed by Bartlett’s test. Results are
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
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 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
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 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
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 as compared to the
normal control group. # p < 0.001 as compared to the disease control group.
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
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 as compared to the
normal control group. # p < 0.001 as compared to the disease control group
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
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 as compared to the
normal control group. # p < 0.001 as compared to the disease control group
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
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 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
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 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
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
)
FIGURE 4.4.1.7 Effect of polyherbal formulation on CUMS – induced altered levels of
neurotransmitters: 5-HT
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 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
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)
FIGURE 4.4.1.8 Effect of polyherbal formulation on CUMS – induced altered levels of
corticosterone
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 as compared to the
normal control group. # p < 0.001 as compared to the disease control group.
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)
FIGURE 4.4.1.9 Effect of polyherbal formulation on CUMS – induced altered levels of
quinolinic acid
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 as compared to the
normal control group. # p < 0.001 as compared to the disease control group. $ p < 0.05 as
compared with the standard (fluoxetine (20 mg/kg)) group.
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
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 as compared to the
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
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 as compared to the
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
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 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
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
Statistical analysis was performed by two way ANOVA followed by Bonferroni multiple
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
Statistical analysis was performed by two way ANOVA followed by Bonferroni multiple
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
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 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):
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
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
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 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.
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
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 as compared to the
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
Serum reduced glutathione levels (µM/ml)
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
Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple
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
Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple
comparison test. Results are expressed as mean ± SEM, n = 6. * indicates p < 0.001 as compared
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.
Serum lipid peroxidase levels(nM/ml)
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
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 as compared 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 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
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 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
Locomotor Activity: Day 5
0 min 30 min 60 min0
50
100
150
200
250
Normal Control
Disease Control
Haloperidol - 0.25 mg/kg
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
locomotor activity: Day 5
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
69
Locomotor Activity: Day 14
0 min 30 min 60 min0
50
100
150
200
250
Normal Control
Disease Control
Haloperidol - 0.25 mg/kg
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
locomotor activity: Day 14
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
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
Haloperidol - 0.25 mg/kg
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
Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple
comparison test. Results are expressed as mean ± SEM, n = 6.p < 0.05 as compared to control
group, where no significant difference observed.
Chapter 4 Results
71
Effect on stereotype behaviour on day 0
0 min 30 min 60 min0
2
4
6
8
Normal Control
Disease Control
Haloperidol - 0.25 mg/kg
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
Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple
comparison test. Results are expressed as mean ± SEM, n = 6.p < 0.05 as compared to control
group, where no significant difference observed.
Chapter 4 Results
72
Effect on stereotype behaviour on day 0
0 min 30 min 60 min0.0
0.5
1.0
1.5
2.0
2.5
Normal Control
Disease Control
Haloperidol - 0.25 mg/kg
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
Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple
comparison test. Results are expressed as mean ± SEM, n = 6.p < 0.05 as compared to control
group, where no significant difference observed.
Chapter 4 Results
73
Effect on stereotype behaviour on day 0
0 min 30 min 60 min0
5
10
15
Normal Control
Disease Control
Haloperidol - 0.25 mg/kg
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
behaviours on day 0: Falling
Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple
comparison test. Results are expressed as mean ± SEM, n = 6.p < 0.05 as compared to control
group, where no significant difference observed.
Chapter 4 Results
74
Effect on stereotype behaviour on day 5
0 min 30 min 60 min0.0
0.5
1.0
1.5
2.0
Normal Control
Disease Control
Haloperidol - 0.25 mg/kg
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
behaviours on day 5: Falling
Statistical analysis was performed by two way ANOVA followed by bonferroni multiple
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
Effect on stereotype behaviour on day 5
0 min 30 min 60 min0
2
4
6
8
10
Normal Control
Disease Control
Haloperidol - 0.25 mg/kg
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
Statistical analysis was performed by two way ANOVA followed by bonferroni multiple
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
76
Effect on stereotype behaviour on day 5
0 min 30 min 60 min0
5
10
15
Normal Control
Disease Control
Haloperidol - 0.25 mg/kg
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
Statistical analysis was performed by two way ANOVA followed by bonferroni multiple
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
77
Effect on stereotype behaviour on day 5
0 min 30 min 60 min0
10
20
30
Normal Control
Disease Control
Haloperidol - 0.25 mg/kg
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
Statistical analysis was performed by two way ANOVA followed by bonferroni multiple
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
78
Effect on stereotype behaviour on day 14
0 min 30 min 60 min0.0
0.5
1.0
1.5
2.0
Normal Control
Disease Control
Haloperidol - 0.25 mg/kg
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
Statistical analysis was performed by two way ANOVA followed by bonferroni multiple
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 < 0.05, !! 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
Effect on stereotype behaviour on day 14
0 min 30 min 60 min0
2
4
6
8
Normal Control
Disease Control
Haloperidol - 0.25 mg/kg
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
Statistical analysis was performed by two way ANOVA followed by bonferroni multiple
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 < 0.05, !! 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
80
Effect on stereotype behaviour on day 14
0 min 30 min 60 min0
2
4
6
8
Normal Control
Disease Control
Haloperidol - 0.25 mg/kg
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
Statistical analysis was performed by two way ANOVA followed by bonferroni multiple
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 < 0.05, !! 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
81
Effect on stereotype behaviour on day 14
0 min 30 min 60 min0
5
10
15
20
25
Normal Control
Disease Control
Haloperidol - 0.25 mg/kg
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
Statistical analysis was performed by two way ANOVA followed by bonferroni multiple
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 < 0.05, !! 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
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
Haloperidol - 0.25 mg/kg
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
Statistical analysis was performed by two way ANOVA followed by bonferroni multiple
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
Haloperidol - 0.25 mg/kg
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
Statistical analysis was performed by two way ANOVA followed by Bonferroni multiple
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
Haloperidol - 0.25 mg/kg
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
Statistical analysis was performed by two way ANOVA followed by bonferroni multiple
comparison test. Results are presented as Mean ± SEM with n=6. ** p < 0.01 as compared to
normal control group on day 14. $$ p < 0.01 as compared to disease control group on day 14.
Chapter 4 Results
85
4.4.3.6 Effect of polyherbal formulation on ketamine – induced social interaction test:
Effect of PHF on the exploratory behaviour (i.e.) the time spent in the probe chamber decreased
significantly (P < 0.01) in all groups a compare to normal control group on day 5. Treatments
significantly ameliorate this behaviour and showed via more time spent in the probe chamber on
day 14.
Social Interaction test
Day 0 Day 5 Day 140
100
200
300
400
500
Normal Control
Disease Control
Haloperidol - 0.25 mg/kg
PHF (600 mg/kg)
^ ^
Disease Control - 2
^ ^ ^ ^ ^ ^ * *
Tim
e sp
ent i
n pr
obe
cham
ber (
sec)
FIGURE 4.4.3.6 Effect of polyherbal formulation on ketamine – induced social interaction test:
Statistical analysis was performed by two way ANOVA followed by Bonferroni multiple
comparison test. Results are presented as Mean ± SEM with n=6 ** p < 0.01 as compared to the
normal control group on day 14. ^^ p < 0.01 as compared to normal control group on day 5.
Chapter 4 Results
86
4.4.3.7 Effect of polyherbal formulation on ketamine – induced proinflammatory cytokines (TNF
– α, IL – 6, IL-1β):
Ketamine treated animals showed significant (p < 0.001) rise in TNF – α as compared to the
normal group while IL – 6, IL – 1 levels were not affected. PHF (600 mg/kg) treatment
significantly (p <0.001) attenuated the increased levels of TNF – α when compared with the
ketamine-induced disease control group.
Serum TNF-α levels (pg/ml)
Normal C
ontrol
Disease
Control
Haloperi
dol - 0.25 mg/kg
PHF- 600 mg/kg
Disease
Control -
20
50
100
150
*
*$$$
* *$
$
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
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
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
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
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.
Serum reduced glutathione levels (µM/ml)
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
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
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
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.
Chapter 4 Results
91
Serum lipid peroxidase levels(nM/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 L
PO le
vels
(nM
/ml)
FIGURE 4.4.3.8 (c) Effect of polyherbal formulation on ketamine – induced oxido-nitrosative
stress parameters: LPO
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.
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)
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, 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).
Chapter 5 Discussion
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
Chapter 5 Discussion
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
Chapter 5 Discussion
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
Chapter 5 Discussion
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
Chapter 5 Discussion
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
Chapter 5 Discussion
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.
Chapter 5 Discussion
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
Chapter 5 Discussion
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
Chapter 5 Discussion
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.
Chapter 5 Discussion
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
Chapter 5 Discussion
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
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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
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Images for pharmacological methods
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Forced swim test
Tail suspension test
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Locomotor activity
Elevated plus maze
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Sucrose preference test
Morris water maze test
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Catalepsy test – bar test
Learned helplessness
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Social interaction test