a) title of the thesis abstract€¦ · a) title of the thesis psychopharmacological evaluation of...

a) Title of the thesis

Psychopharmacological evaluation of herbal formulation – an experimental study

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. Evaluation of long term effect of this formulation on brain function by using

different models such as chronic unpredictable mild stress mice model, LPS –induced

neuroinflammation model and ketamine - induced psychosis model.

Materials and methods: Therapeutic dose range 100 to 800 mg/kg/day were orally administered

for 28 days and animals were monitored for signs of abnormalities. Physical, hematological and

biochemical parameters were evaluated. Seven days acute study was performed using various

behavioral models such as FST (forced swim test), TST (tail suspension test), EPM (elevated

plus maze), PAM (photoactometer) at the doses of 400 and 800 mg/kg. 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 were daily administered for 14 days, and

challenged with saline or LPS (0.83 mg/kg, i.p.) on day 14th

. In ketamine – induced psychosis

study, the effect of PHF on ketamine (50 mg/kg, i.p.) – induced behavioral (locomotor activity,

stereotype behavior, memory retention and helplessness behavior, biochemical (cytokines and

anti – oxidants) and neuroprotective alteration (BDNF - Brain derived neurotropic factor) in the

brain were evaluated.

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

and biochemical parameters. In acute study, dose of 400 and 800 mg/kg, showed significant

antidepressant and anxiolytic activity as evident from significant reduction of immobility time in

along with increased locomotor index and time spent in close arm. 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. 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. In ketamine – induced model, treatment with

formulation significantly attenuated behavioral symptoms in mice. Biochemical estimations

revealed that PHF ameliorated the lipid peroxidation and nitrite level and restored the reduce

glutathione level. Furthermore, PHF remarkably reduced the inflammatory surge (TNF – α, IL –

6 and IL - 1β), and increased BDNF in mice.

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

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

disorders such as depression and psychosis.

b) Art of research topic:

Depression - the greatest decrement in personal health and highest cost of care. Functional Link

between Stress – inflammation and depression. Any type of stress activates HPA (hypothalamus

pituitary axis) system and there by release of cortisol from the adrenal gland. This released

cortisol is again responsible for the activation of macrophages and down regulates immune

system. It also produces inflammation, where it increases pro-inflammatory cytokines, adhesion

molecules and chemokines. These markers are highly lipophillic in nature and ability to cross

BBB (Blood brain barrier). There they produce monoamines depletion, excitotoxicity and

depletion of trophic factor [1].

c) Definition of the problem:

Clinical evidence has been found indicating that antidepressant drugs are less efficacious in

recurrent depression and in preventing relapse [2]. They have been described inducing adverse

events such as withdrawal symptoms at discontinuation, onset of tolerance and resistance

phenomena , unfavorable long-term outcomes and paradoxical effects and increase vulnerability

to relapse [3].

d) Objectives and scope of the work

Ayurveda is one of the traditional medicinal systems of Indian. Traditional pharmacognosy

isolates single active principles which may be self-defeating because overall biological effects

rely on synergistic interactions between plant components. As it may modify the absorption,

distribution, metabolism and excretion of bioactive constituents, or reduce the side-effects [4].

Poly herbal formulation (PHF) possesses some advantages such as to reduction in dose,

convenience, ease of administration and multi target responses obtained. [4-7].

The objectives were to evaluate toxicity study for the polyherbal formulation along with ED50

determination along with 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.

e) Original contribution by thesis

The current study provided scientific data regarding potential use of polyherbal formulation for

the treatment of neurological and psychological disorders as well as mechanisms responsible for

ameliorative effects of this formulation in consequences associated with neurological disorders.

f) Methodology of Research, Results / Comparisons

a. Methods

i. Experimental animals and ethics approval

Mice were housed and acclimatized for two weeks under controlled environment (Temperature

- 22 ± 1◦C and 12 h light/dark cycles) prior to experimentation. Standard laboratory animal

feed and water was provided ad libitum. All the experiments were conducted between 9.00and

17.00 h, in accordance with the Institutional Animal Ethics Committee (IAEC)

[(LMCP/COLOGY/16/09), (LMCP/Pharmacology/Ph.D/17/15)].

ii. Study plan

Part 1: Sub - acute toxicity study (28 days toxicity study)

Selection of dose: The therapeutic doses of Tensnil syrup for mice were selected as 100, 200,

400, 600, 800 mg/kg by calculating from the human clinical dose (1000 - 1500 mg/day/70 kg)

and was calculated based on the total body surface area [9]. The animals were divided into six

groups, each having three animals.

Toxicity study: PHF 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 control group.

Part 2: Preliminary screening

Part 2A: ED50 determination using forced swim test

The animals were divided into six groups, each having six animals. PHF 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 control group. ED50 doses of

formulation was calculated by using graphical method.

Part 2B: Preliminary behavior screening for various CNS activities

Forced swim test [8]: The mice were taken to a separate room and were immediately 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 the duration of 5 minutes.

Tail suspension test [9]: TST was implemented based on the previous method that the mouse

was hung 25 cm above the floor by the tip of the tail (1 cm) tied up to the level. The

immobility time was counted during a test period of 6 min (prior 1 min to adapt and recorded

the last 5 min).

Locomotor activity[10]: Each mouse was placed in a closed square (30 cm) area equipped with

infrared light-sensitive photocells using a digital photoactometer. The mice was observed for

a period of 5 min and the values were expressed as counts per 5 min.

Elevated plus maze[11]: Elevated plus maze (EPM) assesses unconditioned anxiety like

behavior in mice. EPM consisted of two open arms (30×5 cm), two enclosed arms (30×5 cm),

and a connecting central platform (5×5 cm). The maze was elevated 38.5 cm above the ground.

At the beginning of the 5-min session.

Part 3: Chronic models

Part 3A: Chronic unpredictable mice model[12]

The mice were randomly divided into five groups (6 mice in each): control, CUMS exposed,

CUMS + Fluoxetine 20 mg/kg, CUMS + PHF 400 mg/kg, CUMS + PHF 800 mg/kg. Mice

were exposed to a random pattern of mild stressors daily for 28 days which scheduled for

period of 1 week and repeated throughout experiment. Stressors included cage tilting at 450,

cold swimming, tail pinch, housing in mild damp saw dust, wet saw dust, overnight

illumination, and food and water deprivation. Behavior 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 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. The brain was dissected out on an ice plate for analysis of brain

neurotransmitters namely 5-hydroxy tryptamine, noradrenaline, and dopamine.

Part 3B: LPS-induced model[13]

Animals were randomly divided into four experimental groups (n = 6) for behavioral and

biochemical assessment. The group I - vehicle control group, group II - LPS control group, (

treated with saline (p.o.) for 14 days and then challenged with LPS (0.83 mg/kg, i.p.)) group -

III and IV treated with fluoxetine 20 mg/kg and PHF – 600 mg/kg respectively, for 14 days and

then challenged with LPS. Anxiety-like behavior was assessed by elevated plus maze and

photoactometer. Depressive-like behavior and memory function were assessed during forced

swim test and morrison water maze test. Blood samples were collected from the retro orbital

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. The brain was

dissected out on an ice plate for analysis of nerve growth factor (BDNF – Brain Derived

Neurotropic Factor).

Part 3C: Ketamine – induced psychosis model[14]

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

biochemical assessment. The group I - vehicle control group while group II - ketamine control

group, group III and IV were treated orally with haloperidol 0.25 mg/kg and PHF – 600 mg/kg

respectively along with intraperitoneally 50 mg/kg ketamine. Motor activity assessed by

photoactometer and cataleptic behavior with the use of bar test. Memory task was performed

by Morrison water maze test and psychosis was analyzed via stereotype behavior and learned

helplessness model. Blood samples were collected from the retro orbital for the estimation of

serum proinflammatory cytokines (TNF-α, IL-1β and IL-6), and levels of oxidative and anti-

oxidant enzymes. Then, the mice were killed by decapitation.The brain was dissected out on an

ice plate for analysis of BDNF.

b. Results

Part 1: Sub - acute toxicity study (28 days toxicity study)

1.1 Effects of Poly herbal formulation (PHF) on body weight and food intake

Body weight and food intake of all the animals were measured every week throughout the

study as shown in table 1.1(a) and 1.1(b) respectively. There was no remarkable changes were

observed.

Table 1.1(a) Effect of Poly herbal formulation (PHF) on body weight (g)

Study

day

Body weight (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

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 1.1(b) Effect of Poly herbal formulation (PHF) on food intake (g)

Study

Group

Food Intake

(g)

Control 3.1 ± 0.07

100 mg/kg 2.9 ± 0.08

200 mg/kg 3.1 ± 0.06

400 mg/kg 3.0 ± 0.09

600 mg/kg 3.3 ± 0.05

800 mg/kg 3.2 ± 0.09


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


1.2 Effects of Poly herbal formulation (PHF) 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 1.2.

Table 1.2 Effect of Poly herbal formulation (PHF) on the haematological parameters in mice


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

Lympho(%) 18.03 ± 0.95 26.53 ± 1.82 23.23 ± 1.72 20.60 ± 2.26 17.80 ± 0.48 24.70 ± 2.67

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

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

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

Day 28 29.33 ±

0.42

28.33 ±

0.42

3083 ±

1.51

31.17 ±

1.28

29.83 ±

0.75

31.00 ±

0.68

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

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

MCHC 53.28 ± 1.06 53.63 ± 1.03 54.33 ± 1.22 53.18 ± 0.52 53.10 ± 0.76 53.12 ± 0.44

PLT

(*109/L)

17.12 ± 0.41 17.20 ± 0.61 17.25 ± 0.45 17.17 ± 0.49 16.13 ± 1.25 16.88 ± 0.56

HGB (%) 32.17 ± 0.64 32.05 ± 0.81 31.77 ± 0.87 31.68 ± 1.12 30.28 ± 2.37 31.78 ± 0.92




1.3 Effects of Poly herbal formulation (PHF) 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 (table 1.3).

Table 1.3 Effect of Poly herbal formulation (PHF) on the biochemical parameters in mice


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




Part 2: Preliminary screening

Part 2A: ED50 determination using forced swim test

The ED50value of different doses of the formulation obtained from the FST was 600 mg/kg p.o.,

in mice (table 2).

Table 2A Effect of Poly herbal formulation (PHF) 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

Part 2B: Preliminary behavior screening for various CNS activities

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

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

2B.1 Forced swim test (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 and PHF 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 2B.1).

Figure 2B.1 Effect of Poly herbal formulation (PHF) on FST

Forced Swim Test

Nor

mal

Con

trol

Disea

se C

ontr

ol

Fluox

etin

e (2

0 m

g/kg)

PHF (4

00 m

g/kg)

PHF (8

00 m

g/kg)

Nor

mal

Con

trol

Disea

se C

ontr

ol

Fluox

etin

e (2

0 m

g/kg)

PHF (4

00 m

g/kg)

PHF (8

00 m

g/kg)

Nor

mal

Con

trol

Disea

se C

ontr

ol

Fluox

etin

e (2

0 m

g/kg)

PHF (4

00 m

g/kg)

PHF (8

00 m

g/kg)

0

50

100

150

200

Day 0

Day 7

Day 14

Day 0 Day 7 Day 14

* indicates p < 0.001 when compared with normal control; # indicates p < 0.001 when compared with disease control

*

#

* * *

#

#

Groups

Du

rati

on

of

Imm

ob

ilit

y (

sec.)

2B.2 Tail suspension test (TST):

There was significant reduction in 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 2B.2).

Figure 2B.2 Effect of Poly herbal formulation (PHF) on TST

Tail Suspension Test

Nor

mal

Con

trol

Disea

se C

ontr

ol

Fluox

etin

e (2

0 m

g/kg)

PHF (4

00 m

g/kg)

PHF (8

00 m

g/kg)

Nor

mal

Con

trol

Disea

se C

ontr

ol

Fluox

etin

e (2

0 m

g/kg)

PHF (4

00 m

g/kg)

PHF (8

00 m

g/kg)

Nor

mal

Con

trol

Disea

se C

ontr

ol

Fluox

etin

e (2

0 m

g/kg)

PHF (4

00 m

g/kg)

PHF (8

00 m

g/kg)

0

50

100

150

200

250

Day 0

Day 7

Day 14

Day 0 Day 7 Day 14

* indicates p < 0.001 when compared with normal control; $ indicates p < 0.001 when compared with

disease control

*

#

* * *

#

#

Groups

Du

rati

on

of

Imm

ob

ilit

y (

sec.)

2B.3 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 2B.3).

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

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.

Figure 2B.3 Effect of Poly herbal formulation (PHF) on locomotor activity

Photoactometer

Nor

mal

Con

trol

Disea

se C

ontr

ol

Fluox

etin

e (2

0 m

g/kg)

PHF (4

00 m

g/kg)

PHF (8

00 m

g/kg)

Nor

mal

Con

trol

Disea

se C

ontr

ol

Fluox

etin

e (2

0 m

g/kg)

PHF (4

00 m

g/kg)

PHF (8

00 m

g/kg)

Nor

mal

Con

trol

Disea

se C

ontr

ol

Fluox

etin

e (2

0 m

g/kg)

PHF (4

00 m

g/kg)

PHF (8

00 m

g/kg)

0

50

100

150

200

Day 0

Day 0 Day 7 Day 14

* indicates p < 0.01, when compared with normal control; ** indicates p < 0.001, when

compared with normal control; # indicates p < 0.001, when compared with disease control

Day 7

Day 14

#

*** ** *

#

#

Groups

Lo

co

mo

tor

ind

ex (

co

un

ts/5

min

.)

2B.4 Elevated plus maze (EPM):

Time spent in open 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 2B.4 Effect of Poly herbal formulation (PHF) on Elevated plus maze

Elevated Plus Maze

Nor

mal

Con

trol

Disea

se C

ontr

ol

Fluox

etin

e (2

0 m

g/kg)

PHF (4

00 m

g/kg)

PHF (8

00 m

g/kg)

Nor

mal

Con

trol

Disea

se C

ontr

ol

Fluox

etin

e (2

0 m

g/kg)

PHF (4

00 m

g/kg)

PHF (8

00 m

g/kg)

Nor

mal

Con

trol

Disea

se C

ontr

ol

Fluox

etin

e (2

0 m

g/kg)

PHF (4

00 m

g/kg)

PHF (8

00 m

g/kg)

0

20

40

60

80

Day 0

Day 7

Day 14

Day 0 Day 7Day 14

* indicates p < 0.05 when compared with normal control; # indicates p < 0.01 when compared with

disease control

* * **

# #

Groups

Tim

e s

pen

t in

op

en

a

rm (

sec.)

Part 3: Chronic models

Part 3A: Chronic unpredictable mice model

3A.1 Effect of treatments on CUMS-induced altered forced swim test:

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

the duration of immobility time of the. Treatment with Fluoxetine (reference standard, 20 mg/kg)

and PHF (400 mg/kg &800 mg/kg) after 4th

week, significantly reduced the immobility time in

comparison to the disease control group.



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.



expressed as mean ± SEM, n = 6.* p< 0.05 as

compared to the normal control; # p < 0.01 as

compared to the disease control

Figure 3A.1 Effect of treatments on CUMS-induced altered forced swim test

Forced Swim Test

Nor

mal

Con

trol

Disea

se C

ontr

ol

Fluox

etin

e (2

0 m

g/kg)

PHF (4

00 m

g/kg)

PHF (8

00 m

g/kg)

0

50

100

150

200

*

##

#

Groups

Du

rati

on

of

Imm

ob

ilii

ty (

sec)

3A.2 Effect of treatments on CUMS-induced altered tail suspension test:

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

control group (P < 0.001, Fig. 2). 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.

Figure 3A.2 Effect of treatments on CUMS-induced altered forced swim test

Tail Suspension Test

Nor

mal

Con

trol

Disea

se C

ontr

ol

Fluox

etin

e (2

0 m

g/kg)

PHF (4

00 m

g/kg)

PHF (8

00 m

g/kg)

0

50

100

150

200

250*

# # #

Groups

Du

rati

on

of

Imm

ob

ilii

ty (

sec)

3A.3 Effect of treatments 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 significant (p < 0.001) raised

locomotor index.

Figure 3A.3 Effect of treatments on CUMS-induced altered locomotor activity

Locomotor activity

Nor

mal

Con

trol

Disea

se C

ontr

ol

Fluox

etin

e (2

0 m

g/kg)

PHF (4

00 m

g/kg)

PHF (8

00 m

g/kg)

0

50

100

150

200

*

# # #

Groups

Loco

moto

r in

dex

(co

un

ts/5

min

.)

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


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.


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.

3A.4 Effect of treatments on CUMS-induced altered elevated plus maze test:

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.

Figure 3A.4 Effect of treatments on CUMS-induced altered elevated plus maze test

Elevated Plus Maze (EPM)

Nor

mal

Con

trol

Dise

ase

Con

trol

Fluox

etin

e (2

0 m

g/kg)

PHF (4

00 m

g/kg)

PHF (8

00 m

g/kg)

0

50

100

150

200

250

*

# ##

Groups

Tim

e sp

ent

in o

pen

arm

(se

c)

3A.5 Effect of treatments on CUMS-induced altered sucrose preference test:

There was 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) or PHF (400 & 800 mg/kg) for 28 successive days (figure 3A.5).

Figure 3A.5 Effect of treatments on CUMS-induced altered sucrose preference test

Sucrose Preferance Test

Nor

mal

Con

trol

Disea

se C

ontr

ol

Fluox

etin

e (2

0 m

g/kg)

PHF (4

00 m

g/kg)

PHF (8

00 m

g/kg)

0

20

40

60

80

100

###

*

Groups

% S

ucr

ose

pre

fera

nce

3A.6 Effect of treatments on CUMS – induced altered levels of proinflammatory cytokines (TNF

– α, IL – 6, IL-1β):

CUMS animals showed significantly (p < 0.001) increase in the levels of neuroinflammation

markersas compared to the disease group. PHF (400 &800 mg/kg) treatment significantly (p

<0.001) attenuated the increased levels of markers when compared with the CUMS-induced

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

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 < 00.001 as compared to the disease control group

disease control group (Fig. 3A.6). Further, comparison between PHF treated group and

fluoxetine, PHF treated group significantly (p < 0.05) lowered levels of cytokines.

Figure 3A.6 Effect of treatments on CUMS – induced altered levels of proinflammatory

cytokines (TNF – α, IL – 6, IL-1β)

Serum TNF- estimation (pg/ml)

Nor

mal

Con

trol

Disea

se C

ontr

ol

Fluox

etin

e (2

0 m

g/kg)

PHF (4

00 m

g/kg)

PHF (8

00 m

g/kg)

0

50

100

150

*

## #

$

Groups

TN

F-

(p

g/m

l)

Serum IL - 6 concentration (pg/ml)

Nor

mal

Con

trol

Disea

se C

ontr

ol

Fluox

etin

e (2

0 m

g/kg)

PHF (4

00 m

g/kg)

PHF (8

00 m

g/kg)

0

20

40

60

80

100

*

# ##

#$

Groups

IL -

6 (

pg

/ml)

Serum IL-1 concentration (pg/ml)

Nor

mal

Con

trol

Disea

se C

ontr

ol

Fluox

etin

e (2

0 m

g/kg)

PHF (4

00 m

g/kg)

PHF (8

00 m

g/kg)

0

20

40

60

80

100

*

##

#$

Groups

IL-1

(p

g/m

l)

3A.7 Effect of treatments on CUMS – induced altered levels of neurotransmitters (NA, DA, 5-

HT):

All three neurotansmitters namely were significantly (p < 0.001) reduced in the disease control

group as compared to normal control. Fluoxetine and PHF showed significantly (p < 0.001)

reversal effect. 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).

Figure 3A.7 Effect of treatments on CUMS – induced altered levels of neurotransmitters

Brain Noradrenaline level

Nor

mal

Con

trol

Disea

se C

ontr

ol

Fluox

etin

e (2

0 m

g/kg)

PHF (4

00 m

g/kg)

PHF (8

00 m

g/kg)

0

100

200

300

400

500

*

#

#

#$ $

Groups

Nora

dre

nali

ne

(ng

/mg

wei

gh

t of

bra

in)

Brain Dopamine level

Nor

mal

Con

trol

Disea

se C

ontr

ol

Fluox

etin

e (2

0 m

g/kg)

PHF (4

00 m

g/kg)

PHF (8

00 m

g/kg)

0.0

0.1

0.2

0.3

0.4

*

#

#

#$

Groups

Dop

am

ine

(ng

/mg

wt

of

bra

in)

Brain 5 - Hydroxytryptamine level

Nor

mal

Con

trol

Disea

se C

ontr

ol

Fluox

etin

e (2

0 m

g/kg)

PHF (4

00 m

g/kg)

PHF (8

00 m

g/kg)

0.00

0.05

0.10

0.15

0.20

*

##

#

Groups

5 -

HT

(n

g/m

l w

eig

ht

of

bra

in)


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 < 0.01 as

compared to the disease control group. $ p < 0.05 when compared with the standard (fluoxetine

(20 mg/kg)) group.


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.

3A.8 Effect of treatments on CUMS-induced altered levels of serum 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.

Figure 3A.8 Effect of treatments on CUMS-induced altered levels of serum corticosterone

Serum Corticosterone (ng/ml)

Nor

mal

Con

trol

Disea

se C

ontr

ol

Fluox

etin

e (2

0 m

g/kg)

PHF (4

00 m

g/kg)

PHF (8

00 m

g/kg)

0

50

100

150

*

##

#

Groups

Cort

icost

eron

e (n

g/m

l)

3A.9 Effect of treatments on CUMS – induced altered level 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.

Figure 3A.9 Effect of treatments on CUMS – induced altered level of quinolinic acid

Serum Quinolinic Acid (pg/ml)

Nor

mal

Con

trol

Disea

se C

ontr

ol

Fluox

etin

e (2

0 m

g/kg)

PHF (4

00 m

g/kg)

PHF (8

00 m

g/kg)

0

1

2

3

4

*

##

#$

Groups

Qu

inol

inic

Aci

d (

pg

/ml)

3A.10Effect of treatments on CUMS – induced altered levels of oxido - nitrosative stress

parameters (reduced glutathione and lipid peroxidase):

CUMS produced significant increase in oxidative stress in the disease control group when

compared with 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



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.



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


disease control group respectively. A higher lipid peroxidase level was observed in the disease

control group. Further, poly herbal formulation significantly (p < 0.05) attenuated the lipid

peroxidase level as compared to fluoxetine treated animals.

Figure 3A.10 Effect of treatments on CUMS – induced altered levels of oxido-nitrosative stress

parameters (reduced glutathione and lipid peroxidase)

Lipid peroxidase levels

(nM/ml)

Nor

mal

Con

trol

Disea

se C

ontr

ol

Fluox

etin

e (2

0 m

g/kg)

PHF (4

00 m

g/kg)

PHF (8

00 m

g/kg)

0

5

10

15

*

## #

$

Groups

LP

O (

nM

/ml)

3A.11 Effect of treatments on CUMS – induced altered adrenal gland weight:

Statistical analysis of the relative adrenal gland weight revealed a significant main effect of

CUMS. CUMS increased (p < 0.001) the 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.

Figure 3A.11Effect of treatments on CUMS – induced altered adrenal gland weight

Adrenal gland weight

Nor

mal

Con

trol

Disea

se C

ontr

ol

Fluox

etin

e (2

0 m

g/kg)

PHF (4

00 m

g/kg)

PHF (8

00 m

g/kg)

0

2

4

6

8

#

#$

#$

*

Groups

wei

gh

t (m

g)

Part 3B: LPS-induced model

3B.1 Effect of treatments on LPS - induced altered 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, which indicated depressive-like behavior. Fluoxetine

& PHF (600 mg/kg) significantly (P < 0.05) alleviated the LPS-induced depressive behavior as

evident from reduced immobility time in FST paradigms.

Reduced Glutathione

Nor

mal

Con

trol

Disea

se C

ontr

ol

Fluox

etin

e (2

0 m

g/kg)

PHF (4

00 m

g/kg)

PHF (8

00 m

g/kg)

0.00

0.05

0.10

0.15

#

# # #

# # #

*

Groups

seru

m r

edu

ced

glu

tath

ion

e µ

M/m

l 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

< 0.05as compared to the disease control

group. $ p < 0.05 when compared with the




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.

Figure 3B.1 Effect of treatments on LPS - induced altered forced swim test

3B.2 Effect of treatments on LPS - induced altered locomotor activity:

LPS treated rats showed 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 3B.2 Effect of treatments on LPS - induced altered locomotor activity

3B.3 Effect of treatments on LPS - induced altered morris water maze test (MWM):

Time spent in target quadrant was measured in the MWM to evaluate the LPS –induced memory

status in mice. Time spent in target quadrant of LPS challenged group was significantly reduced

than that of control group. Drugs pre – treatments significantly increased time spent in target

quadrant as compared to disease group. After drugs treatment, time of Fluoxetine and PHF

groups was significantly increased as compared to disease group (Fig: 3B.2).

Figure 3B.3 Effect of treatments on LPS - induced altered morris 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, ** indicates p < 0.01 as

compared to normal control group on day 15. # p < 0.05 as

compared to disease control group on day 15.



Mean ± SEM with n=6.* p < 0.001, ** indicates p < 0.01 as

compared to normal control group on day 15. $ p < 0.05 as

compared to disease control group on day 15.

Forced swim test

Nor

mal

Con

trol

Disea

se C

ontr

ol

Fluox

etin

e - 2

0 m

g/kg

PHF -

600

mg/

kg0

50

100

150

200

Day 0 Day 15

** *#

* *#

Du

rati

on

of

imm

ob

ilit

y (

sec.

)

Morris water maze test

Nor

mal

Con

trol

Disea

se C

ontr

ol

Fluox

etin

e - 2

0 m

g/kg

PHF -

600

mg/

kg0

50

100

150

Day 0 Day 15

*

* *$

* *$ $

Tim

e sp

ent

in t

arg

et q

urd

ent

(sec

.)

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, ** indicates p < 0.01 as compared to

normal control group on day 14. $ p < 0.05 as compared to disease

control group on day 14.

Locomotor activity

Nor

mal

Con

trol

Disea

se C

ontr

ol

Fluox

etin

e - 2

0 m

g/kg

PHF -

600

mg/

kg0

50

100

150

Day 0 Day 14

*

# #

Loco

moto

r in

dex

(co

un

ts/5

min

.)

3B.4 Effect of treatments on LPS - induced altered elevated plus maze test:

LPS treatment induced an anxiogenic effect that was evident by reduction in the open arm time

(P < 0.001) in EPM test when compared with vehicle-treated control group. Fluoxetine &PHF

(600 mg/kg) pretreated rats showed significant increase in time spent (P < 0.01) in the open arm

as compared to LPS - treated group (Fig: 3B.4).

Figure3B.4 Effect of treatments on LPS - induced altered elevated plus maze test

3B.5Effect of treatments on LPS - induced altered levels of serum corticosterone

LPS treatment significantly 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 3B.5 Effect of treatments on LPS - induced altered levels of serum corticosterone

3B.6 Effect of treatments on LPS - induced altered levels of cytokines

LPS treated animals showed significant (p < 0.001) rise in neuroinflammation markers, namely

TNF – α, IL - 1β, IL -6 as compared to the normal group. PHF (600 mg/kg) treatment

significantly (p <0.001) attenuated the increased levels of TNF – α, IL - 1β, IL -6 when

compared with the LPS-induced disease control group.

Figure 3B.6 Effect of treatments on LPS - induced altered levels of cytokines (TNF – α)

Elevated Plus Maze

Nor

mal

Con

trol

Disea

se C

ontr

ol

Fluox

etin

e - 2

0 m

g/kg

PHF -

600

mg/

kg0

20

40

60

80

100

Day 0 Day 14

*

* *

#

* *

#

Tim

e sp

ent

in o

pen

arm

(se

c.)

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, ** indicates p < 0.05 as compared to normal

control group on day 14. # p < 0.05 as compared to

disease control group on day 14.

Serum Corticosterone (ng/ml)

Nor

mal

Con

trol

Disea

se C

ontr

ol

Fluox

etin

e - 2

0 m

g/kg

PHF -

600

mg/

kg

0

50

100

150

200

*

*# *

#$

Groups

Cort

icost

eron

e (n

g/m

l)


followed by tukey’s multiple comparison test. Results are

presented as Mean ± SEM with n=6.* p < 0.001 as compared

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

control group. $ p < 0.05 as compared to fluoxetine treated

group.

TNF- (pg/ml)

Nor

mal

Con

trol

Disea

se C

ontr

ol

Fluox

etin

e - 2

0 m

g/kg

PHF- 6

00 m

g/kg

0

50

100

150

*

* *#

* *#

Groups

TN

F-

(p

g/m

l)

IL-6 (pg/ml)

Nor

mal

Con

trol

Disea

se C

ontro

l

FLuoxe

tine - 2

0 m

g/kg

PHF- 6

00 m

g/kg

0

10

20

30

40

50

*

* * *

# #

Groups

IL-6

(pg/m

l)

IL-1 (pg/ml)

Nor

mal

Con

trol

Dise

ase

Contro

l

FLuoxe

tine

- 20

mg/

kg

PHF- 6

00 m

g/kg

0

20

40

60

*

* * *

# #

Groups

IL-1

(pg

/ml)

3B.7 Effect of treatments on LPS - induced altered levels of neurotoxin (Quinolinic Acid)

LPS treatment significant rise in serum quinolic 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 markers. Moreover, serum concentration of

quinolinic acid in PHF - 600 mg/kg treated animals lowered significantly (p < 0.05) as compared

to the standard treatment (fluoxetine)

Figure 3B.7 Effect of treatments on LPS - induced altered levels of neurotoxin (Quinolinic

Acid)

3B.8 Effect of treatments on LPS - induced altered levels of anti-oxidants

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 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 levels and

nitrite levels as compared to LPS treated animals

Figure 3B.8 Effect of treatments on LPS - induced altered levels of anti-oxidants

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 <0.01 as compared to

disease control group.


ANOVA followed by tukey’s multiple comparison

test. Results are presented as Mean ± SEM with n=6.

*p <0.001 , ** p < 0.01 when compared with normal

control group, #p <0.001 when compared with the

disease control group, $p <0.05 when compared with

the standard (fluoxetine (20 mg/kg)) group

Serum Quinolinic Acid (pg/ml)

Nor

mal

Con

trol

Disea

se C

ontr

ol

Fluox

etin

e - 2

0 m

g/kg

PHF -

600

mg/

kg

0

5

10

15

*

*#

* *#$

Groups

Qu

inoli

nic

Aci

d (

pg

/ml)

Reduced Glutathione

Nor

mal

Con

trol

Disea

se C

ontr

ol

Fluox

etin

e - 2

0 m

g/kg

PHF -

600

mg/

kg

0.00

0.05

0.10

0.15

0.20

*

*# #

*#$

Groups

Ser

um

red

uce

d g

luta

thio

ne

(µM

/ml)


(nM/ml)

Nor

mal

Con

trol

Disea

se C

ontr

ol

Fluox

etin

e - 2

0 m

g/kg

PHF -

600

mg/

kg

0

5

10

15

*

* *#

* * *#

Groups

LP

O (

nM

/ml)

Nitrite levels (M/ml)

Nor

mal

Con

trol

Disea

se C

ontro

l

Fluox

etin

e - 2

0 m

g/kg

PHF -

600

mg/

kg

0

2

4

6

*

* *#

* * *#

Groups

Nit

rite

(

M/m

l)

3B.9 Effect of treatments on LPS - induced altered levels of nerve growth factor (BDNF)

Furthermore, BDNF levels were 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 disease

control group.

Figure 3B.9 Effect of treatments on LPS - induced altered levels of nerve growth factor (BDNF)

Part 3C: Ketamine – induced psychosis model

3C.1 Effect of treatments on ketamine - induced stereotype behavior

Ketamine (50 mg/kg, i.p.) induced stereotype behavior including head turning, bobbing, head

falling and sniffing in mice as compared to control animals (p < 0.001). Treatment with PHF

significantly decreased stereotype behavior.

Figure 3C.1.1 Effect of treatments on ketamine - induced stereotype behavior: Day 0


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

Fal

lin

g (

cou

nts

/10

min

s.)


0 min 30 min 60 min0

2

4

6

8

Normal Control

Disease Control


PHF (600 mg/kg)

Disease Control - 2

Head

tu

rnin

g (

co

un

ts/1

0 m

ins.

)



0.5

1.0

1.5

2.0

2.5

Normal Control

Disease Control


PHF (600 mg/kg)

Disease Control - 2

Hea

d b

ob

bin

g (

cou

nts

/10

min

s.)



5

10

15

Normal Control

Disease Control


PHF (600 mg/kg)

Disease Control - 2

Sn

iffi

ng (

cou

nts

/10

min

s.)



0.5

1.0

1.5

2.0

Normal Control

Disease Control


PHF (600 mg/kg)

^

^

^

Disease Control - 2

Fal

lin

g (

cou

nts

/10

min

s.)



2

4

6

8

10

Normal Control

Disease Control


PHF (600 mg/kg)

^ ^

^ ^ ^

^ ^ ^

*** ****

@ @ @

# #

Disease Control - 2

Hea

d t

urn

ing

(co

un

ts/1

0 m

ins.

)



5

10

15

Normal Control

Disease Control


PHF (600 mg/kg)

^ ^ ^ * * *

@ @

@ @ @

Disease Control - 2

* * * * * *@ @ @H

ead

bo

bb

ing

(co

un

ts/1

0 m

ins.

)



10

20

30

Normal Control

Disease Control


PHF (600 mg/kg)

^ ^ ^

* * *

Disease Control - 2

^ ^ ^ ^ ^ ^

* * * * * *

Sni

ffin

g (c

ount

s/10

min

s.)


comparison test. Results are presented as Mean ± SEM with n=6. *p < 0.001, ** p < 0.01, ***

p < 0.05 when compared with the normal control group. # p < 0.001, ## p <0.01 when

compared with the disease control group.


ANOVA followed by tukey’s multiple comparison

test. Results are presented as Mean ± SEM with n=6.

*p < 0.001, ** p < 0.01 when compared with normal

control group. # p < 0.01, ## p < 0.001 when

compared with the disease control group.

Brain Derived Neurotrophic Factor

Nor

mal

Con

trol

Disea

se C

ontr

ol

Fluox

etin

e - 2

0 m

g/kg

PHF -

600

mg/

kg

0

100

200

300

*

* *# # #

Groups

BD

NF

(p

g/g

wt

of

tiss

ue)

3C.1.3 Effect of treatments on ketamine - induced stereotype behavior: Day 14


3C.2 Effect of treatments on ketamine - induced altered water maze test

In water maze test, Ketamine (50 mg/kg, i.p.) significantly decreased the time spent in target

quadrant as compared to control animals showing memory impairment. Whereas treatments

notably decreased, the time spent in target quadrant.

Figure 3C.2 Effect of treatments on ketamine - induced altered water maze test



0.5

1.0

1.5

2.0

Normal Control

Disease Control


PHF (600 mg/kg)

^ ^ ^

! ! !

Disease Control - 2

Fal

lin

g (

cou

nts

/10

min

s.)

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.



2

4

6

8

Normal Control

Disease Control


PHF (600 mg/kg)

^ ^ ^

^^

* * *

!% % %

@ @ @

# #

# # #

Disease Control - 2

% % %

Hea

d t

urn

ing

(co

un

ts/1

0 m

ins.

)



2

4

6

8

Normal Control

Disease Control


PHF (600 mg/kg)

^ ^ ^

!

!!

* * *

% % % @ @

@

Disease Control - 2

% % %@ @

Hea

d b

ob

bin

g (

cou

nts

/10

min

s.)



5

10

15

20

25

Normal Control

Disease Control


PHF (600 mg/kg)

^ ^ ^

! !! ! !

* * *

% % %

Disease Control - 2

% % %

Sn

iffi

ng (

cou

nts

/10

min

s.)

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.

Morris water maze test

Day 0 Day 5 Day 140

50

100

150

Normal Control

Disease Control


PHF (600 mg/kg)

* ** ** @@@

#$

Disease Control - 2

* @@

Tim

e sp

ent

in t

arg

et q

ua

dra

nt

(sec

.) Statistical analysis was performed by two way ANOVA followed


Mean ± SEM with n=6. * p < 0.05, ** p < 0.001 as compared to

normal control group on day 5. @ p < 0.001, @@ p < 0.01 as

compared to normal control group on day 14. # p < 0.001 as

compared to disease control group on day 14. $ p < 0.01 as

compared to Haloperidol treated group on day 14.

3C.5 Effect of treatments on ketamine - induced altered 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.

Figure 3C.5 Effect of treatments on ketamine - induced altered catalepsy test – bar test

3C.6 Effect of treatments on ketamine - induced altered learned helplessness model

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.

Figure 3C.6 Effect of treatments on ketamine - induced altered learned helplessness model

3C.7 Effect of treatments on ketamine - induced altered social interaction test

In social interaction test treatments significantly ameliorate this behavior and showed via more

time spent in probe chamber on day 14.

Figure 3C.7 Effect of treatments on ketamine - induced altered social interaction test

Catalepsy

Day 0 Day 5 Day 140

1

2

3

4

Normal Control

Disease Control


PHF (600 mg/kg)

@ # $

Disease Control - 2

Des

cen

t L

ate

ncy

(se

c.)

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.



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.

Learned helplessness model

Day 0 Day 5 Day 140

2

4

6

8

Normal Control

Disease Control


PHF (600 mg/kg)

@

#

@

#

Disease Control - 2

@

#

Nu

mb

er o

f fa

ilu

res

Social Interaction test

Day 0 Day 5 Day 140

100

200

300

400

500

Normal Control

Disease Control


PHF (600 mg/kg)

*

Disease Control - 2

* * * @

Tim

e sp

ent

in p

rob

e ch

am

ber

(se

c.) 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 5. @ p < 0.01 as

compared to normal control group on day 14.

3C.8 Effect of treatments on ketamine - induced altered locomotor activity

For locomotor activity haloperidol (0.25 mg/kg) &PHF (600 mg/kg) pretreatment produced a

significant reduction in the locomotor index (P < 0.01) on day 14 at 0, 30, 60 min. time points.

Figure 3C.8 Effect of treatments on ketamine - induced altered locomotor activity

3C.9 Effect of treatments on ketamine - induced altered levels of cytokines

Figure 3C.9 Effect of treatments on ketamine - induced altered levels of cytokines

IL-1 (pg/ml)

Nor

mal

Con

trol

Disea

se C

ontr

ol

Hal

oper

idol

-0.2

5 m

g/kg

PHF -

600

mg/

kg

Disea

se C

ontr

ol -

2

0

5

10

15

Groups

IL-1

(p

g/m

l)

TNF- (pg/ml)

Nor

mal

Con

trol

Disea

se C

ontr

ol

Hal

oper

idol

- 0.

25 m

g/kg

PHF- 6

00 m

g/kg

Disea

se C

ontr

ol -

2

0

50

100

150

Groups

TN

F-

(p

g/m

l)

*

*# #

* *#

#

IL - 6 (pg/ml)

Nor

mal

Con

trol

Disea

se C

ontr

ol

Hal

oper

idol

-0.2

5 m

g/kg

PHF -

600

mg/

kg

Disea

se C

ontr

ol -

2

0

5

10

15

Groups

IL -

6 (

pg

/ml)

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 <0.01

as compared to disease control group.

Locomotor Activity: Day 5


50

100

150

200

250

Normal Control

Disease Control


PHF -600 mg/kg

* *

^

Disease Control - 2

!

!

^ ^ ^!

!

* *

Lo

com

oto

r in

dex

(co

un

ts/5

min

.)



50

100

150

Normal Control

Disease Control


PHF -600 mg/kg

Disease Control - 2

Lo

com

oto

r in

dex

(co

un

ts/5

min

.)



50

100

150

200

250

Normal Control

Disease Control


PHF -600 mg/kg

@

?

#

Disease Control - 2

$

[ ]"" "

[ ] [ ]?

?

Lo

com

oto

r in

dex

(co

un

ts/5

min

.)

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 < 0.001, ! P < 0.001 as compared to normal control group on day 0 at 0,

30, 60 minutes respectively.

% P < 0.01 as compared to normal control group on day 5 at 60 minutes.

@ p < 0.001, # p < 0.001, $ P < 0.001 as compared to normal control group on day

14 at 0, 30, 60 minutes respectively.

? p < 0.001, “ p < 0.001, [ ] P < 0.001 as compared to normal control group on day

14 at 0, 30, 60 minutes respectively.

3C.10 Effect of treatments on ketamine - induced altered levels of anti-oxidants

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 disease

control group. Further, higher levels of lipid peroxidase and nitrite content were noted in the

disease control group that was also attenuated significantly (p < 0.05) in treatment.

Figure 3C.10 Effect of treatments on ketamine - induced altered levels of anti-oxidants

3C.12 Effect of treatments on ketamine - induced altered levels of BDNF brain derived

neurotrophic factor)

BDNF level was significantly reduced (P < 0.001) after ketamine administration as compared to

the normal control group as shown in figure 25. PHF - 600 mg/kg (P < 0.001) significantly

prevented the ketamine - induced BDNF depletion as compared to disease control group.

Figure 3C.12 Effect of treatments on ketamine - induced altered levels of BDNF brain derived

neurotrophic factor)

Brain Derived Neurotrophic Factor

Nor

mal

Con

trol

Disea

se C

ontr

ol

Hal

oper

idol

-0.2

5 m

g/kg

PHF -

600

mg/

kg

Disea

se C

ontr

ol -

2

0

100

200

300

*

* *#

#

*

Groups

BD

NF

(p

g/g

wt

of

tiss

ue)


followed by tukey’s multiple comparison test. Results are

presented as Mean ± SEM with n=6. * p < 0.001, ** p < 0.001

as compared to normal control group. # p < 0.01, ## p < 0.001

as compared to disease control group.

Reduced glutathione

Nor

mal

Con

trol

Disea

se C

ontro

l

Hal

oper

idol

-0.2

5 m

g/kg

PHF -

600

mg/

kg

Disea

se C

ontro

l - 2

0.00

0.05

0.10

0.15

0.20

*

* *#

* * *#

* * *#

Groups

Ser

um

red

uce

d g

luta

thio

ne

(µM

/ml)

Nitrite levels (M/ml)

Nor

mal

Con

trol

Disea

se C

ontro

l

Hal

oper

idol

-0.2

5 m

g/kg

PHF -

600

mg/

kg

Disea

se C

ontro

l - 2

0

2

4

6

*

* *#

* * *#

*

Groups

Nit

rite

(

M/m

l)


(nM/ml)

Nor

mal

Con

trol

Disea

se C

ontro

l

Hal

oper

idol

-0.2

5 m

g/kg

PHF -

600

mg/

kg

Disea

se C

ontro

l - 2

0

5

10

15

*

* *#

$ $

*# *

#

Groups

LP

O (

nM

/ml)


comparison test. Results are presented as Mean ± SEM with n=6. * p < 0.001, ** p < 0.01,

*** p <0.05 as compared to normal control group. # p < 0.001 as compared to the disease

control group. $$ p < 0.01 as compared to Haloperidol treated group.

g) Achievements with respect to objectives

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 level, corticosterone, quinolinic acid and upregulation of reduced

glutathione level and BDNF level.

h) Conclusion

Formulation could ameliorate anxiogenic, depressive, psychotic symptoms and biochemical

changes in rodents, indicating protective effects in the treatment neurological disorders such as

depression and psychosis.

i) Papers

1) Shah Krishna M., Mody Vandana and Goswami Sunita S. 2017. Preliminary screening of

psychopharmacological effects and toxicity testing of tensnil syrup in swiss albino mice.

World Journal of Pharmaceutical Research. Issue 8. Volume 6. Page Number: 2265-77

2) Shah Krishna M., Mody Vandana and Goswami Sunita S. 2019. Reversal of neuro-

inflammation and oxidative stress by polyherbal formulation in an animal model of

chronic unpredictable mild stress. Asian Journal of Pharmacy and Pharmacology.

(Accepted but Under publication)

j) References

1. Slavich, G.M. and M.R. Irwin, From stress to inflammation and major depressive

disorder: a social signal transduction theory of depression. Psychol Bull, 2014. 140(3):

p. 774-815.

2. Ionescu, D. and G. Papakostas, Experimental medication treatment approaches for

depression. Transl Psychiatry., 2017. 7(3): p. e1068. doi 10.1038/t 2017.33.

3. Fava, G.A. and E. Offidani, The mechanisms of tolerance in antidepressant action. Prog

Neuropsychopharmacol Biol Psychiatry, 2011. 35(7): p. 1593-602.

4. Modak, M., et al., Indian herbs and herbal drugs used for the treatment of diabetes. J

Clin Biochem Nutr, 2007. 40(3): p. 163-73.

5. Little, C.V., Simply because it works better: exploring motives for the use of medical

herbalism in contemporary U.K. health care. Complement Ther Med, 2009. 17(5-6): p.

300-8.

6. Bhushan, B., S. Sardana, and G. Bansal, Acute and sub-acute toxicity study of

Clerodendrum inerme, Jasminum mesnyi Hance and Callistemon citrinus. Journal of

Acute Disease, 2014. 3(4): p. 324-327.

7. Morphy, R., C. Kay, and Z. Rankovic, From magic bullets to designed multiple ligands.

Drug Discov Today, 2004. 9(15): p. 641-51.

8. Porsolt, R.D., et al., Behavioural despair in rats: a new model sensitive to antidepressant

treatments. Eur J Pharmacol, 1978. 47(4): p. 379-91.

9. Steru, L., et al., The tail suspension test: a new method for screening antidepressants in

mice. Psychopharmacology, 1985. 85(3): p. 367-70.

10. Bhosale, et al., Study of central nervous system depressant and behavioral activity of an

ethanol extract of Achyranthes aspera (Agadha) in different animal models.(Indexed with

Pubmed/medline). Vol. 1. 2011. 104-8.

11. Lister, R.G., The use of a plus-maze to measure anxiety in the mouse.

Psychopharmacology, 1987. 92(2): p. 180-5.

12. Murua, V.S., et al., Shuttle-box deficits induced by chronic variable stress: Reversal by

imipramine administration. Pharmacology Biochemistry and Behavior, 1991. 38(1): p.

125-130.

13. Jangra, A., et al., Protective effect of mangiferin against lipopolysaccharide-induced

depressive and anxiety-like behaviour in mice. Eur J Pharmacol, 2014. 740: p. 337-45.

14. Yadav, M., et al., Protective effect of gallic acid in experimental model of ketamine-

induced psychosis: possible behaviour, biochemical, neurochemical and cellular

alterations. Inflammopharmacology, 2018. 26(2): p. 413-424.

a) title of the thesis abstract€¦ · a) title of the thesis psychopharmacological evaluation of...

Documents