evaluation of anti-inflammatory and anti-arthritic

Evaluation of anti-inflammatory and anti-arthritic activities of Juglans

regia L and Trigonella gharuensis Rechf

submitted by

Aisha Mobashar

DPHR02153003

In partial fulfillment for the Degree of doctor of philosophy

in pharmacology

FACULTY OF PHARMACY

THE UNIVERSITY OF LAHORE

LAHORE PAKISTAN

2020

In the name of ALLAH

The Most Beneficial

The Most Merciful

AUTHORrsquoS DECLARATION

I Aisha Mobashar hereby state that my thesis titled ldquoEvaluation of anti-

inflammatory and anti-arthritic activities of Juglans regia L and Trigonella

gharuensis Rechfrdquo is my own work and has not been submitted previously by

me for taking any degree from this university ldquoThe University of Lahorerdquo or

anywhere else in the countryworld

At any time if my statement is found to be incorrect even after my

graduation the university has the right to withdraw my PhD degree

Aisha Mobashar

October 2019

CERTIFICATE OF APPROVAL

This is to certify that research work presented in the thesis entitled ldquoEvaluation of anti-

inflammatory and anti-arthritic activities of Juglans regia L and Trigonella gharuensis

Rechfrdquo was conducted by Ms Aisha Mobashar under the supervision of Dr Arham

Shabbir

No part of this thesis has been submitted anywhere else for any other degree This thesis is

submitted to the university in partial fulfillment of requirements for the degree of Doctor of

Philosophy in the field of Pharmacology Faculty of Pharmacy The University of Lahore

Student Name

Aisha Mobashar Signature

Supervisor Name

Dr Arham Shabbir Signature

Associate Professor

Co-Supervisor Name

Prof Dr Saeed-ul-Hassan Signature ________________________

Professor

Name of Head MPhilPhD Program

Dr Mahtab Ahmad Khan__

Signature Date

Name of HOD

Prof Dr Javed Iqbal_______

Signature Date

i

DEDICATION

I would like to dedicate this thesis to my beloved Teachers and family because whatever

I am today is all due to their prayers and their support

ii

ACKNOWLDGEMENTS

Words are bounded and knowledge is limited to praise ALLAH (SWT) The Most Beneficent

The Merciful Gracious and The Compassionate Whose bounteous blessing and exaltation

flourished my thoughts and thrived my ambition to have the cherished fruit of my modest

efforts in the form of His manuscripts from the blooming spring of blossoming knowledge

Countless blessings upon HOLY PROPHET HAZRAT MUHAMMAND (PBUH) who has

shown the right direction to humanity and thereby enlightening their ways with faith through

his eternal teachings

Itrsquos a great honor and pleasure to pay thanks to my supervisor Dr Arham Shabbir Associate

professor Head of Pharmacy Department The University of Lahore-Gujrat campus for

lending his skilled advice guidance decision devotion support and co-operation His

competence and goodwill encouraged me a lot to complete this important task I consider

myself honored being supervised by my research mentor He is a person with amazing

command on contemporarily research and techniques

My special thanks goes to my co-supervisor Prof Dr Syed Saeed-ul-Hassan The University

of Lahore Pakistan for his invaluable help of constructive comments and suggestions

throughout the thesis work

My sincere gratitude to Prof Dr Javed Iqbal Head of Department The University of Lahore

Pakistan for his unconditional support and encouragement

My sincere appreciation to Dr Muhammad Shahzad Associate Professor University of

Health Sciences Lahore for ensuring provision of lab facilities It wouldnrsquot have been easy

iii

without his countless support and guidance Also my special regards to the professional team

at Department of immunology University of Health Sciences Lahore Pakistan

My in-depth appreciations to Higher Education Commission (HEC) Pakistan for providing

resources and financial assistance in the carrying out of my research in Australia under

International Research Supportive Initiative Program (IRSIP)

I am truly grateful to my husband and parents for being on my side through every thick and

thin during this long journey My extra-ordinary recognition for the support extended by my

colleagues and friends They have always encouraged me to explore my potential and pursue

my dreams

Aisha Mobashar

iv

TABLE OF CONTENTS

DEDICATION i

ACKNOWLDGEMENTS ii

TABLE OF CONTENTS iv

LIST OF TABLES xi

LIST OF FIGURES xiii

LIST OF ABBREVIATIONS xvii

ABSTRACT xix

CHAPTER 1 INTRODUCTION 1

11 Hypothesis 3

111 Null hypothesis 3

112 Alternate hypothesis 3

12 Aim of the study Error Bookmark not defined

13 Objective of study Error Bookmark not defined

CHAPTER 2 LITERATURE REVIEW 4

21 Epidemiology 4

22 Pathophysiology 5

23 Treatments 7

24 Inflammation 10

25 Role of Carrageenan histamine serotonin xylene and dextran 11

26 Plants used 12

CHAPTER 3 MATERIALS AND METHODS 15

31 Plant collection 15

32 Preparation of extracts 15

33 Assessment of anti-arthritic activities 16

331 Arthritis induction 16

v

332 Evaluation of arthritic progression 17

333 Assessment of paw volume 17

334 Histopathological investigations 17

34 Assessment of mRNA expression levels of TNF-α NF-κB IL-6

IL-1β COX-1 COX-2 and IL-4 17

341 RNA extraction 17

3411 Homogenization of blood 18

3412 Phase separation 18

3413 Method of RNA precipitation 18

3414 Washing of RNA 18

3415 Re-suspension of RNA pellets 18

3416 RNA Quantification 18

343 Primer designing 19

344 cDNA synthesis 21

345 Working solution of primers 22

346 Polymerase chain reaction 22

347 Ingredients required for the formulation of TAE reaction

buffer (50X) 22

348 Gel preparation and Gel electrophoresis 23

349 Densitometry 23

3410 Determination of serum PGE2 levels using ELISA 23

34101 Preparation of standard 24

34102 Biotinylated Detection Antibody 24

34103 Concentrated HRP Conjugate 24

34104 Substrate reagent 24

34105 Procedure of ELISA 24

3411 Evaluation of hematological parameters 25

3412 Evaluation of biochemical parameters 25

vi

35 Acute toxicity study 26

36 Evaluation of anti-inflammatory activities 26

361 Experimental design and induction of paw edema using

carrageenan 28

362 Experimental design and paw edema induction using

histamine 28


serotonin 28


dextran 29

365 Experimental design for ear edema induced by xylene 29

366 Experimental design for castor oil-induced diarrhea model 29

37 Phytochemical Evaluation 30

371 Test for Alkaloids 30

3711 Mayerrsquos reagent 30

3712 Wagnerrsquos reagent 31

372 Test for the steroids 31

373 Keller-Kilani for cardiac glycosides 31

374 Tests for terpenoids 31

3741 Salkowski test 31

375 Tests for Phenols 31

376 Test for saponins 32

377 Test for flavonoids 32

38 Gas chromatography-mass spectrometry Analysis 32

39 Statistical analysis 32

CHAPTER 4 RESULTS 33

41 J regia and T gharuensis significantly reduced paw edema in

FCA-induced arthritic model 33

vii

411 Measurement of paw edema at day 8 33




42 J regia and T gharuensis attenuated arthritic Score 39

421 At Day 8 39

422 At Day 15 40

423 At Day 22 41

424 At Day 28 42

43 J regia and T gharuensis attenuated histopatological parameters 45

431 J regia significantly reduced inflammation 45

432 J regia significantly attenuated pannus formation 46

433 J regia significantly reduced erosion of bone 47

44 J regia and T gharuensis downregulated mRNA expression levels

of pro-inflammatory markers and upregulated anti-inflammatory

marker 57

441 J regia and T gharuensis downregulated mRNA expression

levels of TNF-α 57

442 J regia suppressed mRNA expression levels of NF-κB 58

443 J regia and T gharuensis reduced IL-6 mRNA expression

levels 59

444 J regia and T gharuensis decreased the mRNA expression

levels of IL-1β 60

445 J regia and T gharuensis down regulated COX-1 mRNA

expression levels 61

446 J regia and T gharuensis down regulated the mRNA

expression levels of COX-2 62

447 J regia and T gharuensis up regulated the mRNA expression

levels of IL-4 63

viii

448 J regia and T gharuensis significantly reduced PGE2 Levels

64

449 J regia and T gharuensis nearly normalized hematological

parameters 66

4491 J regia and T gharuensis nearly normalized RBC

count 66

4492 J regia and T gharuensis nearly normalized Hb

content 67

4493 J regia and T gharuensis nearly normalized WBC

count 68

4494 J regia and T gharuensis nearly normalized

platelet count 69

4410 J regia and T gharuensis effects on biochemical parameters

71

44101 J regia and T gharuensis effects on urea levels 71

44102 J regia and T gharuensis did not alter creatinine

levels 72

44103 J regia and T gharuensis effects on ALT levels 73

44104 J regia and T gharuensis effects on AST levels 74

44105 J regia and T gharuensis extracts significantly

reduced ALP levels 75

4411 Evaluation of acute Toxicity of extracts of J regia and T

gharuensis 77

45 Pre-treatment with J regia and T gharuensis inhibited paw edema

induced by carrageenan 78

451 At 1st hr 78

452 At 2nd hr 79

453 At 3rd hr 80

454 At 4th hr 81

ix

455 At 5th hr 82

46 Pre-treatment with J regia and T gharuensis inhibited histamine-

induced paw edema 85

461 At 1st hr 85

462 At 2nd hr 86

463 At 3rd hr 87

47 Pretreatment with J regia and T gharuensis prevented serotonin-


471 At 1st hr 90

472 At 2nd hr 91

473 At 3rd hr 92

48 Pretreatment with J regia and T gharuensis inhibited dextran-


481 At 1st hr 95

482 At 2nd hr 96

483 At 3rd hr 97

484 At 4th hr 98

485 At 5th hr 99

49 Pretreatment with J regia and T gharuensis inhibited xylene-


491 Onset time of diarrhea 103

492 Number of wet feces 104

493 Total weight of wet feces 105

494 Total number of feces 106

410 Qualitative analysis of J regia and T gharuensis extracts 108

411 GC-MS analysis of J regia and T gharuensis extracts 109

4111 Assessment of GC-MS analysis of J regia ethanolic extract 109

4112 Assessment of GC-MS of n- hexane extract of J regia 126

x

4113 GC-MS analysis of ethanolic extract of T gharuensis 137

4114 GC-MS analysis of T gharuensis n-hexane extract 148

CHAPTER 5 DISCUSSION 168

CHAPTER 6 CONCLUSION 175

REFERENCES 177

xi

LIST OF TABLES

Page

Table 1 RNA quantification of samples 19

Table 2 Sequences of primers 22

Table 3 Acute inflammatory models 28

Table 4 J regia and T gharuensis extracts significantly reduced paw edema in

FCA induced arthritis model

40

Table 5 J regia and T gharuensis attenuated arthritic development 46

Table 6 J regia and T gharuensis attenuated histopatological parameters 50

Table 7 J regia and T gharuensis attenuated mRNA expression levels of pro-

inflammatory markers and up-regulated anti-inflammatory marker

66

Table 8 J regia and T gharuensis nearly normalized hematological parameters 71

Table 9 J regia and T gharuensis did not alter biochemical parameter 77

Table 10 Acute toxicity study of ethanolic and n-hexane extracts of J regia and

T gharuensis

78

Table 11 The anti-inflammatory properties of the anti-inflammatory

characteristics of J regia and T gharuensis extracts on carrageenan-

induced paw edema

84

xii

Table 12 The anti-inflammatory properties of J regia and T gharuensis extracts

on histamine-induced paw edema model

88


on serotonin-induced paw edema model

92


on dextran-induced Paw edema model

98

Table 15 Pretreatment with J regia and T gharuensis extracts inhibited castor

oil-induced diarrhea

104

Table 16 Qualitative analysis of J regia and T gharuensis extracts 105

Table 17 List of identified constituents of ethanolic extract of J regia 107

Table 18 List of identified constituents of n-hexane extract of J regia 126

Table 19 List of identified constituents of ethanolic extract of T gharuensis 137

Table 20 GC-MS analysis of T gharuensis n-hexane extract 148

xiii

LIST OF FIGURES

Page

Figure 1 Paw edema at day 8 35




Figure 5 Paw edema 39

Figure 6 Arthritic score at day 8 41




Figure 10 Arthritic Score 45

Figure 11 J regia and T gharuensis reduced infiltration of inflammatory cells 47

Figure 12 J regia and T gharuensis reduced pannus formation 48

Figure 13 J regia and T gharuensis reduced bone erosion 49

Figure 14 Histopathology of normal control 51

Figure 15 Histopathology of diseased control 52

Figure 16 Histopathology of piroxicam 53

xiv

Figure 17 Histopathology of EEJR 54

Figure 18 Histopathology of NHJR 55

Figure 19 Histopathology of EETG 56

Figure 20 Histopathology of NHTG 57

Figure 21 J regia and T gharuensis downregulated mRNA expression levels

of TNF-α

58


of NF-κB

59


of IL-6

60


of IL-1β

61


of COX-1

62


of COX-2

63

Figure 27 J regia and T gharuensis upregulated mRNA expression levels of

IL-4

64

Figure 28 J regia and T gharuensis downregulated levels PGE2 65

xv

Figure 29 J regia and T gharuensis nearly normalized RBC count 67

Figure 30 J regia and T gharuensis nearly normalized Hb content 68

Figure 31 J regia and T gharuensis nearly normalized WBC count 69

Figure 32 J regia and T gharuensis nearly normalized Platelet count 70

Figure 33 J regia and T gharuensis effects on urea levels 72

Figure 34 J regia and T gharuensis effects Creatinine level 73

Figure 35 J regia and T gharuensis effects ALT levels 74

Figure 36 J regia and T gharuensis effects AST levels 75

Figure 37 J regia and T gharuensis effects ALP levels 76

Figure 38 Carrageenan At first hour 79

Figure 39 Carrageenan At second hour 80

Figure 40 Carrageenan At third hour 81

Figure 41 Carrageenan At fourth hour 82

Figure 42 Carrageenan At fifth hour 83

Figure 43 Histamine At first hour 85

Figure 44 Histamine At second hour 86

Figure 45 Histamine At third hour 87

xvi

Figure 46 Serotonin At first hour 89

Figure 47 Serotonin At second hour 90

Figure 48 Serotonin At third hour 91

Figure 49 Dextran At first hour 93

Figure 50 Dextran At second hour 94

Figure 51 Dextran At third hour 95

Figure 52 Dextran At fourth hour 96

Figure 53 Dextran At fifth hour 97

Figure 54 Xylene induced ear edema 99

Figure 55 Onset time of diarrhea 100

Figure 56 Number of wet feces 101

Figure 57 Total weight of wet feces 102

Figure 58 Total number of feces 103

xvii

LIST OF ABBREVIATIONS

RA Rheumatoid arthritis

J regia Juglans regia

T gharuensis Trigonella gharuensis

FCA Freundrsquos complete adjuvant

EEJR Ethanolic extract of J regia

NHJR n- hexane extract of j regia

EETG Ethanolic extract of T gharuensis

NHTG n- hexane extract of T gharuensis

AST Aspartate aminotransferase

ALT Alanine transaminase

ALP Alkaline phosphatase

Hb Haemoglobin

RBC Red blood cells

WBC White blood cells

RT-PCR Reverse transcriptase polymerase chain reaction

TNF-α Tumour necrosis factor alpha

NF-κB Nuclear factor kappa B

IL-1β Interleukin 1 beta

IL-6 Interleukin 6

IL-4 Interleukin 4

COX-1 Cyclooxygenase 1


PXM Piroxicam

xviii

IND Indomethacin

DPH Diphenhydramine

Dexa Dexamethasone

LOP Loperamide

ANOVA Analysis of variance

GCMS Gas chromatography mass spectroscopy

I2 Iodine

H2SO4 Sulphuric acid

KI Potassium iodide

NGF Nerve growth factor

DW Distilled water

SM

bw

PO

Synovial membrane

Body weight

Per oral

xix

Evaluation of anti-inflammatory and anti-arthritic activities of

Juglans regia L and Trigonella gharuensis Rechf

ABSTRACT

Background The genus of Juglans and Trigonella have long been used in folk remedy for the

treatment of rheumatoid arthritis and inflammatory disorders The leaves of Juglans regia and

herb of Trigonella gharuensis were collected from Northeast and Southwest areas of Pakistan

respectively

Purpose The study aimed to investigate the immunomodulatory and anti-inflammatory

activities of ethanol and n-hexane extracts of J regia and T gharuensis in different in-vivo

models of acute and chronic inflammation

Methodology Arthritis was induced in rodents using Freundrsquos complete adjuvant induced

model of chronic inflammation Extracts of J regia leaves and T gharuensis were administered

in the doses of 500 mgkg bw po and 400 mgkg bw po respectively Treatment was

started at 8th day of arthritic induction and sustained for 20 days with extracts and piroxicam

as control At 28th day all animals were sacrificed Arthritis was determined using an arthritic

scoring index Blood analysis and biochemical parameters were determined RNA extraction

reverse transcription and polymerase chain reaction (RT-PCR) was performed to determine

expression of pro-inflammatory cytokines such as TNF-α NF-ĸB IL-6 IL-1β COX-1 COX-

2 and anti-inflammatory cytokine IL-4 Prostaglandin E2 level (PGE2) was evaluated using

ELISA Paw edemas were measured using digital water plethysmometer

For evaluation of anti-inflammatory activities acute inflammatory paw edema models

(carrageenan- histamine- serotonin- and dextran-induced paw edema) ear edema model

xx

(xylene-induced ear edema) and castor oil-induced diarrhea model were used Qualitative

analysis and gas chromatography-mass spectrometry (GC-MS) analysis were performed for

the identification of potential anti-inflammatory phytochemicals

Results Treatment with J regia and T gharuensis extracts inhibited arthritic development and

significantly attenuated paw edema All evaluated histopathological parameters were found

significantly reduced after treatment with plant extracts The RT-PCR analysis showed

significant attenuation of pro-inflammatory TNF-α NF-κB IL-6 and IL-1β levels and

potentiation of IL-4 levels COX-2 expression level was also found reduced in extracts treated

groups ELISA demonstrated suppression of PGE2 levels in all extracts treated groups The

results of all extracts were comparable to the effects of piroxicam The treated groups restored

levels of Hb and RBC Moreover levels of WBCs and platelets were found to be nearly

normalized in treated groups The non-significant difference in the levels of creatinine urea

ALT and AST were observed Both plants extracts significantly reduced paw edema induced

by carrageenan in first (early) and second (late) acute inflammation phases indicating the

possible inhibition of autacoids as one of the mechanisms of anti-inflammatory activity The

extracts also significantly prevented histamine- and serotonin-induced paw edema which

complemented inhibition of carrageenan-induced inflammation in the first acute phase

Suppression of edema by plant extracts in dextran- and xylene-induced inflammatory models

supported inhibition of autacoids Attenuation of castor oil-induced diarrhea by both extracts

supported inhibition of carrageenan-induced inflammation via prostaglandin inhibition in the

second acute inflammation phase Qualitative phytochemical screening and gas

chromatography-mass spectrometry analysis indicated constituents with anti-inflammatory

activity for example fatty acid esters and flavonoids in both extracts

xxi

Conclusion Extracts of J regia leaves and T gharuensis herb ameliorated experimentally

induced arthritis which may be ascribed to their immunomodulatory effects Moreover

attenuation of inflammatory edema in different acute models not only validated anti-

inflammatory potential but also suggested possible inhibition of autacoids as one of the

mechanisms along with immunomodulatory effects of both plants

1

CHAPTER 1 INTRODUCTION

Rheumatoid arthritis (RA) is a chronic and autoimmune disease having worldwide prevalence

of 1 in industrialized populations (1 2) The symptoms of RA are characterized by stiffness

pain synovitis of diarthrodial joints resulting into articular destruction bone erosion and

pannus formation (3) TNF-α (tumor necrosis factor-α) stimulates IL-6 (interleukin-6)

production which causes deterioration of proteoglycans and collagen causing joint destruction

(4) It is also stimulator of B and T cells (5) IL-1β (interleukin-1 beta) is present in inflamed

rheumatoid synovial tissue and stimulates bone erosion and pannus formation NF-κB (Nuclear

factor-κB) stimulates recruitment of pro-inflammatory cytokines such as TNF-α IL-1β and IL-

6 (6) Activation of cyclo-oxygenase-2 (COX-2) by NF-κB exacerbates the inflammation

COX-2 activation also leads to the production of prostaglandin-E2 (PGE2) which in turn

causes bone erosion and cartilage destruction All these cytokines contribute towards

progression of RA (7 8)

FCA was used to induce arthritis in rat model Induction of arthritis through FCA in rat paw is

a well-recognized model of arthritis The features of this chronic poly-arthritic model closely

resemble with human arthritic model This model has been extensively used to evaluate anti-

inflammatory and immunomodulatory properties of plant extracts and therapeutic agents (9)

Inflammation is regarded as cardinal answer to injury and pathogens caused by body It is

manifested by augmented vascular permeability recruitment of leukocytes release of

cytokines and chemotaxis The two types of inflammatory conditions acute and chronic are

based on time duration and underlying cascades of events that lead to physical symptoms The

acute phase can be further classified into first (early) and second (late) phases (10) The early

acute phase is caused by the release of histamine and serotonin followed by the release of

2

bradykinin prostaglandins leukotrienes and activation of polymorphonuclear cells in the late

acute phase Increased vascular permeability leads to edema and inflammation (11 12)

Although inflammation is a shielding phenomenon of the body to injurious stimuli but its

pathophysiological implications are often undesirable Steroids opiates and Non-steroidal

anti-inflammatory drugs (NSAIDs) are preferred remedies to control the negative outcomes of

inflammation (13) Various disease-modifying anti-rheumatic drugs (DMARD) such as

methotrexate TNF-α and IL-1β antagonists have been used to treat autoimmune disorders but

their use is limited due to higher vulnerability of patients to infections (14 15) NSAIDs may

induce gastric perforation erosion and bleeding due to inhibition of prostaglandins (16)

Tolerance and dependence are associated with opiates (3) Peptic ulcer precipitation of

diabetes osteoporosis and increased susceptibility to infections are associated with

corticosteroids (17)

DMARDs are used as the mainstream for the management of inflammation and pain in arthritic

patients in addition to NSAIDs However the adverse effects of synthetic drugs suggest a need

to focus on medicinal plants due to the easy availability of plants and lower cost (18) The

undesirable adverse effects of above mentioned drugs specify worth of traditional medicinal

plants for the cure of inflammation and arthritis (19)

Juglans regia L (Enaglish name walnut local name Akhrot) belongs to the family

Juglandaceae These plants are distributed all over the world in temperate regions in particular

Asia United States Western South America and Europe (20) J regia leaves have been

extensively used in the pharmaceutical and cosmetic industry due to its easy accessibility (21)

Traditionally J regia leaves have been used to treat rheumatic pains (22) Its decoction has

also been used for rheumatic pain and inflammatory disorders (7) The plant is known to

possess antinociceptive (21) antidiabetic antimicrobial antioxidant hepatoprotective (20)

3

anti-atherogenic osteoblastic (23) aphicidal (21) antihyperlipidemic (24) antiproliferative

(25) antitumour immunoregulatory (26) and antimycobacterial (27) activities

Trigonella gharuensis Rech f is collected from the hilly areas of Quetta It is perennial herb

suberect 6-9 mm broad 50cm tall 14 cm long Petiole 1 cm long leaflets (28) Its distribution

is worldwide (29) It belongs to family Fabeacae the second largest family of flowering plants

(30) Traditionally member of genus Trigonella were used to treat inflammatory diseases

Different plants of Trigonella genus such as Trigonella stellate and Trigonella foenum

graceum possess known pharmacological anti-inflammatory properties (18 31) T gharuensis

is also known for possessing medicinally important active ingredients (32) We did not find out

any literature regarding its anti-inflammatory prospective So prime focus of this study was to

explore its anti-arthritic and anti-inflammatory potential

In order to evaluate antirheumatic properties of J regia and T gharuensis using FCA induced

chronic model of inflammation this current study was performed Furthermore this study also

investigated anti-inflammatory mechanisms of both plant extracts in various acute

inflammatory models using mice

11 Hypothesis

111 Null hypothesis

Extracts of J regia and T gharuensis do not possess anti-arthritic and anti-inflammatory

properties

112 Alternate hypothesis

Extracts of J regia and T gharuensis possess anti-arthritic and anti-inflammatory properties

4

CHAPTER 2 LITERATURE REVIEW

Rheumatoid arthritis (RA) is progressive as well as reversible when diagnosed at early stages

Joint inflammation redness cartilage damage and bone erosion are prominent clinical

manifestations of RA Laboratory findings showed leukocytosis anemia fever weight loss

and anorexia Joint inflammation is absent in early rheumatoid arthritis However when disease

progresses joint swelling appears with fusiform appearance RA can be classified into four

stages First stage does not include any destructive changes Second stage includes moderate

destruction of subchondral bone and cartilage damage Joint deformities are absent at this stage

although mobility of joint is compromised Stage 3 is crucial and it causes muscle atrophy

Stage 4 involves lesion of soft tissues of extra-articular organs (33) Extra articular involvement

affects other organs such as eyes lungs renal nervous system and various gastrointestinal

system It usually associated with mortality Usually 40 patients get affected with extra

articular disease when they encountered with this disease and during the course of disease (34)

21 Epidemiology

Rheumatoid arthritis is occurring throughout world Majority of population suffering with this

disease and its estimated prevalence is about 05-1 in Northern Europe and North American

and mean annual incidence is 002ndash005 Patient survival is decreased in 3 to 10 years It is a

multifactorial disease involving genetic as well as environmental factors The major risk factors

are sex age smoking infectious agents These factors contribute towards severity of disease

Females have higher risk of developing arthritis as compared to male due to estrogen which

has stimulatory effect on immune system (35)

Cytokines control inflammatory processes which participate in RA progression Imbalance is

well understood between anti-inflammatory and pro-inflammatory roles of cytokines This

5

imbalance causes the stimulation of chronic inflammatory mediators as a result of which ankle

joint damage occurs Therefore cytokines are the new targets for the treatment of arthritis (36)

22 Pathophysiology

Immunologic mechanisms RA gave a wide knowledge about the pathogenesis and etiology of

rheumatoid arthritis However the exact immune pathways for understanding pathogenesis and

etiology of RA is still insufficient There are some clinical and pathological manifestations that

are obtained from the activation of various interrelated immune pathways Modern concepts of

RA that is supported by laboratory studies animal models and clinical observation have

revised and reestablished some of the opinions Early study emphasized on the importance of

immune complexes and auto-antibodies in the initiation of RA It suggested a function of T

cells in initiating inflammatory response in RA It was recognized that these interrelated

mechanisms suggestively contribute in pathogenesis of RA T cells also play function in

initiating and perpetuation of disease Immune pathways play role in defining the implication

of possible treatment because their modulation directed towards specific targets gives benefits

to the patients suffering with RA (36)

RA is a progressive autoimmune disease having systemic effects The precise pathogenesis of

this disease is unidentified but there are some environmental as well as genetic factors which

play contributory role in this disease The interaction between pro-inflammatory cytokines as

well as B- and T cells promotes progression of RA The destruction of joint starts from synovial

membrane Activation of mononuclear cells and angiogenesis leads to synovitis in joints

Pannus is a part of the synovial membrane that is rich with osteoclast cells Pannus destroys

bones and the enzymes which are released by chondrocytes and synoviocytes degrade the

cartilage Antigen-activated CD4 T cells strengthen the immune response of body by activating

osteoclast mononuclear cells chondrocytes and also synovial fibroblasts The release of

6

cytokines from these cells especially the release of TNF-α IL-1 and IL-6 lead to inflammation

of synovial membrane In addition to articular effects of cytokines the pro-inflammatory

cytokines stimulate the systemic effects which include acute-phase proteins production

cardiovascular diseases anemia and osteoporosis (37)

Synovial fibroblast is a part of composite cellular network that plays a significant function in

pathogenesis of RA Lymphocytes and macrophages secreted factors that play role in the

activation of fibroblasts This activation pattern is further characterized by the changes in

regulating signaling cascades and genes expression and alterations in these pathways lead to

apoptosis (38) IL-1 along with TNF-α play role in dys-regulation of cartilage and bone

remodeling characteristics of RA In case of bone remodeling both cytokines stimulate the

formation of NF-κB ligand receptor activator that plays role in stimulating bone resorption

caused by osteoclastic activity TNF-α increases the formation of mature osteoclasts IL-1

cytokines show its effect directly on osteoclast to stimulate both cells which stimulate bone-

resorbing ability TNFα and IL-1 both cytokines aggravate the remodeling of cartilage but IL-

1 cytokine is more effective than another This cytokine stimulates the production of factors

and also increase degradation of cartilage matrix The improved understanding of pathways

mechanism that underlays the processes of destruction of the joint will enable for more specific

and selective application of pharmacological and therapeutic agents which target these pro-

inflammatory cytokines (39)

Study was conducted to determine different types of arthritis that was expressed by both

biologically and histo-chemically active nerve growth factor in synovium A model of arthritis

induced by carrageenan was used to assess joint inflammation on the level of NGF The study

determined that in the rats experimentally induced arthritis is caused by stimulation in nerve

growth factor in peri-vascular area in synovium It was determined that nerve growth factor

7

injection in synovium did not lead to any inflammation and destruction of the peripheral

sympathetic innervations considerably reduced both level of nerve growth factor and

inflammation following carrageenan injection

A histomorphometric method was used to determine the association between ostopenia and

arthritis that emphasized total changes in the composition of bone Carrageenan injection was

injected into the knee of mature rabbit to check the trabecular bone volume and its turnover

Histomorphometry method was used to study trabecular bone volume of femoral condyles and

was established by the special technique photodensitometry on the standard macroradiographs

Calcein fluorochrome is a newly developed technique that was used to study osteogenesis by

using histological sections which were not decalcified A major defeat of the cancellous bone

occurred within 49 days in lateral and medial femoral condyle in the arthritis group The net

Osteogenesis was emphasized within four hold It was considered that rise in the total bone

resorption leads to negative bone balance The study also showed that periarticular bone loss

of the RA is also directly linked with increased bone turnover When quantitative study on the

kinetics of bone remodeling in RA was done it provides the foundation for therapeutic attempts

to reverse or prevent the therapeutic attempts Osteopenia also increases the risk of fracture in

inflammatory arthritis (40)

23 Treatments

NSAIDs are extensively used drugs all over the world The adverse effects which restrain its

uses is GIT bleeding Endoscopic studies showed that 15 to 30 of patients usually

encountered with gastric or duodenal ulcer The major side effects of NSAIDs are bleeding and

more than 100000 patients are hospitalized and among which 16500 patients die as a result of

gastrointestinal events in the United States NSAIDS inhibited cyclooxygenase which is

involved in the synthesis of prostaglandin COX-I is overexpressed constitutively and it

8

protects gastric mucosa While COX-2 increases prostaglandin production which mediates

inflammation COX-2 inhibition prevented harmful effects of prostaglandin which is

inflammation and pain while COX-I inhibition is associated with harmful effects in the GIT

wall and inhibition of platelets aggregation Agents that inhibit COX-2 enymes showed similar

effects such as reduction of inflammation and pain similar to non-selective NSAIDs (41)

NSAIDs was invented in 1972 and they was introduced in market in 1982 by Pfizer Piroxicam

has long half-life which is 45 hours in human being It possesses analgesic and antipyretic It

has been used in treatment of RA Like others NSAIDs its mechanism of action is inhibition of

cyclooxygenase (42)

Cyclooxygenase act on arachidonic acid to form prostaglandins While isomers of these

enzymes transform the arachidonic acid to prostaglandins COX-1 a constitutive enzyme form

is present in the human body and play a role in the homeostatic function for example influence

blood flow and maintain normal gastric mucosa The COX-2 on another hand acts as an

inducer and is expressed to physiological stimuli and is also concerned in prostaglandins

production which arbitrate the pain as well as inflammation Non-specifically the Nonsteroidal

anti-inflammatory drugs (NSAIDs) reduce the production of both forms The beneficial effects

of NSAIDs through minimizing inflammation and producing analgesic effects occur by

decreasing synthesis of COX-2 but with some unwanted effects like mild bleeding and

gastrointestinal adverse effects occur due to concurrent inhibition of important COX-1 The

agents which selectively decrease the COX-2 synthesis while sparing the COX-1 regarded as

an highly interesting and beneficial therapeutically and can also characterize as a chief progress

in the treatment of RA as well as osteoarthritis (43)

Inhibitors of synthesis of PGE2 nonsteroidal anti-inflammatory drugs and some other

prostanoids can be used in the treatment the RA To understand mechanism by which

prostaglandin plays role in pathogenesis of this disease experiment was conducted on rats

9

Rats which lack each of the four known prostaglandin receptor E2 receptors were used

Homozygous deletion of all receptors (E1 E2 and E3) did not influence the arthritis

development while disease progress was found in mice which have low expression of above-

mentioned receptor These rats also showed the reduction in the inflammation when assessed

with low circulating serum amyloid A and IL-6 levels Therefore antagonists of these receptors

give new agents for treatment of RA (44)

Cyclooxygenase-2 is stimulated in inflamed joint tissues and is involved in the production of

prostaglandin While the exact phenomenon of cyclooxygenase expression is regulated stays

to be elucidated Various study suggested that prostaglandin play role in inflammation

angiogenesis and apoptosis These events are responsible of possible structural changes which

characterize the arthritic disease NSAIDs are prescribed for the treatment of RA and give

effective relief from the symptoms of RA while serious GIT complications occur with their

use The mechanism by which NSAIDS can reduce pain and inflammation in patients of

rheumatoid arthritis is that they inhibit the production of cyclooxygenase -2 while toxicity is

related to inhibition of COX-1 Selective C0X-2 inhibitors show better results when compared

with placebo and are effective for the management of RA symptoms They also improve the

GIT safety and tolerability Various studies also shows that there is still some complication of

NSAIDs with the selective COX-2 inhibitors The major feature is the rheumatoid arthritis is

overexpression of -2 protein in articular tissues While the exact molecular mechanisms are

still unclear By understanding more clear mechanism of COX-2 it will become effective to

treat rheumatoid arthritis easily

When anti-tumur necrosis factor α antibody (infliximab) and methotrexate are administered in

a combination for the treatment of RA it shows a positive effect Though some patients did

not show any response to this treatment The study was conducted on a patients with

10

rheumatoid arthritis by administering repeated high dose of intra-articular infliximab and

infliximab infusions into an inflamed knee When the patient was assessed it was determined

that that there were not any clinical effects When knee arthroscopic biopsy was done before

injection it was demonstrated that TNFα staining was present there Moreover

immunohistochemistry report revealed presence of lymphotoxin α Thus it was suggested that

the resistance to TNF receptor blockage might be due to insufficient amount of TNFα The

study also suggested that LTα may also have a significant role in RA

RA synovium is associated with increased levels of COX-2 as well as angiogenesis Both

phenomena were investigated and it was found that selective COX-2 inhibitor such as rofecoxib

effects on human dermal and reduce ability of angiogenesis and chemotactic activity The

pretreatment with selective COX-2 inhibitor (rofecoxib) considerably decreased angiogenesis

When RA was firstly treated with selective COX-2 inhibitor (rofecoxib) for at least 14 hours

followed by stimulation with the IL-1β less angiogenesis was observed The COX-2 induced

angiogenic activity is the important active mechanism in RA synovium hence these are

preferred drug therapies in the treatment of RA

24 Inflammation

Inflammation helps the body in healing wounds infections or damage and without

inflammatory response it is impossible to heal wounds Inflammation is very beneficial for the

body but can also be harmful if it is present in more quantity and can persist in body for long

time than usual When a baby is born various defences are naturally present in the body of

human which is known as innate immunity Inflammation is of two types In acute

inflammation onset of action in acute inflammation is very rapid however time period of

action is short The symptoms of chronic and acute inflammation vary depending upon nature

of disease The causes of acute inflammation are chemical agents (tissue necrosis reducing

11

agents alkalis and corrosive acids) physical agents (trauma vibration heat cold ionizing

agents) hypersensitivity reactions and microbial infections Chronic inflammation has slow

onset of action but duration of action is long

25 Role of Carrageenan histamine serotonin xylene and dextran

Carrageenan is commonly used chemical that play role in releasing inflammatory and

histamine prostaglandins leukotrienes TNF-α as well as bradykinin Acute inflammation

route of carrageenan is a biphasic and when carageenan is injected serotonin kinins and

histamine is released in first phase while second phase is responsible of release of prostaglandin

(45)

Histamine play crucial part in early inflammatory response Stored histamine present in white

blood cells eg basophils eosinophils and mast cells Mast cells are present in large quantity

at the site of potential injury such as feet mouth internal body surfaces nose and blood vessels

Non-mast cells histamine is also found in various tissues such as in brain where it functions as

neurotransmitter Another significant site where histamine is stored and released is ECL of

stomach The important function of histamine release from mast cells and basophils is

immunologic (46)

Basophilocytes from the patients (patients with rheumatoid arthritis) respond to the leukocyte

nuclei from normal people with the release of histamine A similar release of histamine induced

by nuclear components of DNA and RNA has been demonstrated in normal people Patients

with RA were treated with anti-histamines (H1 and H2) and results showed that there was a

direct effect of histamine in RA (47)

12

Serotonin has two functions related to inflammation that it increases vascular permeability and

causes vasodilation It is also a vasoactive amine just like histamine It also plays role in the

dilation of capillaries and in contraction of non-vascular smooth muscles (48)

Xylene is responsible for severe vasodilation and can cause edematous changes of skin that is

sign of acute inflammation Xylene is also responsible for histopathological changes such as it

increase the thickness of skin tissues when it is applied topically (49)

Dextran is responsible for infilteration of mononuclear cells as well as regenerative changes in

epithelium The exact mechanism through which dextran play role in inflammation is no clear

but studies show that it induces B cells T cells macrophages and polymorphonuclear changes

26 Plants used

There are eight genera of plant Juglandaccac family while the best-known species of the family

is J regia because it produces timber that can used for many purposes and edible nuts It is

cultivated in all over the world especially in South of Asia West Indies South Eastern Eurpoe

Japan and in Eastern and Southern region of US The height of this plant is 25-35m while the

diameter of its trunk is 2m and grow well in full light of moon It is mostly present in mountain

region of Central Asia for the purpose of making medicaments J regia leaves has been

extensively use in the pharmaceutical and cosmetic industry due to its easy accessibility (21)



possess antinociceptive (21) antidiabetic antimicrobial antioxidant and hepatoprotective (20)

anti-atherogenic and osteoblastic (23) aphicidal (21) antihyperlipidemic (24)

antiproliferative (25) antitumour immunoregulatory (26) and anti-mycobacterial (27)

activities Walnut is made up of many chemical compounds such as flavonoids caffeic acid

quercetin and ascorbic acid and due to presence of these compounds it has anti-oxidant activity

13

(50) The roots of Juglans are use to treat diabetes and its related drugs The leaves of plant

have anti-inflammatory anthelmintic depurative and astringent activity Thus they are used

to treat chronic cough dairrhoea asthma dyspepsia and constipation They are also used to

treat stramous sores skin ailments and to purify the blood The rind of plant has astringent and

anodyne activity They are used to treat anemia and diarrhea The seeds of plant are stimulant

and diuretic The oil of seed is anthelmintic It is also used to treat dry skin and menstrual

problems The cotyledons are used to treat cancer because this plant has anti-cancer activity

(51)


suberect 6-9 mm broad 50cm tall 14 cm long Petiole and 1 cm long leaflets (28) Its

distribution is worldwide (29) It belongs to family Fabeacae the second largest family of

flowering plants It has 650 genus and 18000 species It contains perennial and annual herbs

with trifoliate leaves It is most widely present in Iran and almost 48 taxa of genus are present

in Iran (30) Traditionally member of genus Trigonella were considered as best treatment for

inflammatory disorders Different plants of Trigonella genus such as Trigonella stellate and

Trigonella foenum graceum possess known pharmacological anti-inflammatory properties T

gharuensis is also known for possessing medicinally important active ingredients (32) T

gharuensis is used as anti-cancer drug It is also used as flavoring agent The literature

determined that it has as anti-diabetic activity anti-fungal activity anti-bacterial activity anti-

carcinogenic anti-lipidemic and anti-carcinogenic activities It has little side-effects

therefore widely used in pharmaceutical nutritional and therapeutic potential (52)

14

Aim of the study

To evaluate anti-arthritic and anti-inflammatory potentials of J regia and T gharuensis

extracts using different experimental models of acute and chronic inflammation

Objective of study

Arthritic score and paw edema were assessed through macroscopic criteria and plethysmometer

in FCA induced arthritic model respectively Histopathological parameters such as pannus

formation bone erosion and cartilage damage were evaluated using H amp E staining method

Effects of extracts were checked on expression levels of IL-4 TNF-α IL1β IL-6 NF-кB

COX-1 and COX-2 using reverse transcription PCR The levels of prostaglandin E2 were

measured through ELISA Effects of extracts were evaluated on hematological parameters

(WBC platelets and RBC counts and Hb content) liver function tests (AST and ALT) and

renal function tests (urea and creatinine) Paw edema models such as Carrageenan- histamine-

serotonin- and dextran- induced models were used to evaluate anti-inflammatory effects of

extracts Xylene induced ear edema and castor oil-induce diarrhea models were also used to

assess anti-inflammatory effects of extracts Qualitative analysis was performed on all types of

extracts Identification of T gharuensis and J regia extracts constituents were carried out using

GC-MS analysis

15

CHAPTER 3 MATERIALS AND METHODS

31 Plant collection

J regia leaves were gathered from Chitral in the northeast area of Pakistan The plants were

identified by a botonist namely Dr Abdul Rehman Niazi The Department of Botany

University of the Punjab Lahore and a token sample (LAH 7261) was deposited on 02-05-

2018 in the herbarium wwwtheplantlistorg at 18-07-2018 was the website from where plant

name was checked

The herb T gharuensis was acquired from district Quetta Balochistan Pakistan Prof Rasool

Baksh Tareen identified this herb from University of Balochistan (UOB) Quetta Pakistan The

plant was kept in university herbarium and it was allotted voucher specimen no TG- RBT-

05

32 Preparation of extracts

Leaves of J regia were dried under shade and pounded by using electrical grinder The powder

(100 g) was soaked for seven days in ethanol (500 ml) or n-hexane (500 ml) and incubated at

25 C (room temperature) with occasional trembling The mixtures were strained through

muslin fabric followed by filter paper Filtrates were then concentrated at 37 ˚C in rotary

evaporator (IKA Germany) Extracts were dried in an incubator at 40 ˚C The percentage yield

was calculated as 15 for ethanolic extract and 7 for n-hexane extract

Herb of T gharuensis was dried under shade and it was chopped into small pieces Crushing

was performed by using electrical grinder The powdered herb (1kg) underwent cold

maceration using 3 liters of ethanol and n-hexane separately for seven days Mixtures were

filtered using muslin cloth Then the whole mixtures were filtered Filtrate was subjected to

evaporation in a rotary evaporator maintaining conditions of constant temperature at 37 ˚C

under reduced pressure Semi solid crude extracts was obtained and it was placed into

16

incubator at 40 ˚C for further drying (53) The extract was dissolved in 1 Tween 80 (54 55)

The yield of ethanolic extract of T gharuensis was 18 while yield of n-hexane extract of T

gharuensis was 10 The doses of extracts of J regia leaves and T gharuensis used in the

experiments were picked on the basis of previous literature of anti-inflammatory studies (56

57)

33 Housing of Animals

Sprague-Dawley rats 6ndash8 weeks old of both genders weighing 200ndash250 g were allowed to

familiarize with environment for one week before the start of experiment Natural day and night

cycles (12 h) were maintained Temperature as well as moisture were kept at 25˚C plusmn 2 and 60-

70 respectively Rats were provided with free access to food and water To determine anti-

inflammatory activities 6-8 weeks-old BALBc mice weighing 28-33 grams were used with

same above-mentioned conditions Experiments were conducted after getting permission from

the Institutional Research Ethics Committee The University of Lahore (IREC-2017-23) (12)

34 Assessment of anti-arthritic activities

341 Arthritis induction

Thirty rats were segregated into five groups with each having 6 animals Group-I (vehicle

control) and group-II (arthritic control) were given vehicle ie 1 Tween 80 in water (54)

Group-III was considered as reference group and received piroxicam (10 mgkg bw po)

(58) Group-IV (EEJR) and group-V (NHJR) were treated with the dose of 500 mgkg bw

po respectively While group-VI (EETG) and group-VII (NHTG) were administered 400

mgkg bw po respectively (56) FCA (015 ml) was injected into left paws of all the animals

(sub plantar region) except vehicle control group at 0 day Animals were treated with extracts

and piroxicam starting from day 8 till day 28 (12)

17

342 Evaluation of arthritic progression

Inflammation redness and erythema were observed on day 8 15 22 and 28 Arthritic scoring

was done on the basis of macroscopic observation of paw inflammation redness and swelling

Score 0 (normal) 1 (minimal) 2 (mild) 3 (moderate) and 4 (severe ) were used to evaluate

redness erythema and inflammation of paws (12)

343 Assessment of paw volume

Arthritis was induced at 0 day and then paw edema was evaluated using digital water

displacement plethysmometer at day eight fifteen twenty two and twenty eight after injecting

FCA (59)

344 Histopathological investigations

Rats were sacrificed on day 28 and ankle joints were separated Ankle joints were transected

longitudinally to obtain equal halves and immersed in 10 formalin for fixation The samples

were decalcified using formic acid The tissue slides were prepared by first embedding in

paraffin blocks and then slicing into thin sections (5microm) Slides were treated with H amp E

(hematoxylin and eosin) Blinded histopathologist evaluated slides for different parameters

such as pannus formation as well as bone erosion Scoring criteria was used as mentioned in

342 (12)

35 Assessment of mRNA expression levels of TNF-α NF-κB IL-6 IL-1β COX-1 COX-

2 and IL-4

351 RNA extraction

Total RNA was extracted from blood of rats using TRIzol method

18

3511 Homogenization of blood

Blood (200 micro) and TRIzol LS reagent (600 microL) was mixed in an Eppendorf tube It was

homogenized using vortex mixer After that it was incubated for five minutes

3512 Phase separation

200 microL of chilled chloroform was taken and it was added in each tube followed by vigorous

mixing for 15-30 seconds Vortex mixer was used for this purpose Mixture was placed at 15-

30degC for 2 mins Samples were centrifuged using 12000 rpm for fifteen minutes at controlled

temperature of 4degC The resultant upper transparent layer containing RNA was carefully

separated

3513 Method of RNA precipitation

Aqueous phase was transferred in tube Isopropanol was mixed with it and amount of

isopropanol was same as of aqueous phase Then these tubes were kept at room temperature

for ten minutes Tubes were placed in centrifuged machine for ten minutes whose rpm was

12000 at 4degC RNA pellet was precipitated

3514 Washing of RNA

For washing of RNA pellet 75 ethanol (1 ml in each tube) was used Tubes were placed in

centrifugal machine After that excess ethanol was removed and air dried to get RNA pellets

3515 Re-suspension of RNA pellets

RNA pellet was dissolved in 20 microL of RNAase free water Finally RNA was stored at -80degC

3516 RNA Quantification

Nanodrop spectrophotometric technique was used to quantify the total RNA

19

352 Primer designing

Sequence of primers for example IL-6 IL-1β NF-κB IL-4 COX-1 and COX-2 were picked

from previously published studies (58 60) Steps of manually designing of TNF-α and GAPDH

primers are given below

Ensemble Genome browser was consulted to find out gene sequences of TNF-α and

GAPDH

Both Forward as well as reverse primers were selected from gene exons having 20 base

pairs sequence

Physical characteristics of all primers were checked using online oligocalc calculator

Annealing temperatures of all primers were kept at 55-60oC

GC content of primers were selected between 50-55

Product size of primers were kept between 190-300 bp

In-silico PCR was performed

Hairpin formation and self-annealing were absent

In end specificity of primers was checked using nucleotide blast

20

Table 1 Sequences of primers

Genes Forward primer Reverse primer Product size Reference

IL-4

5´- CACCTTGCTGTCACCCTGTT -3´

231 (58)

5´-TCACCGAGAACCCCAGACTT-3´

IL-6

5-CCCACCAAGAACGATAGTCA-3

247 (5)

5-CTCCGACTTGTGAAGTGGTA-3

TNF-α

5prime-AGTCCGGGCAGGTCTACTTT-3prime

202 NM_0126753

5prime-GGAAATTCTGAGCCCGGAGT -3prime

NF-КB

5prime-TGAGATCCATGCCATTGGCC-3prime

207 (58)

5ʹ-AGCTGAGCATGAAGGTGGATG-3ʹ

COX-1

5prime -CTGCCCTCTGTACCCAAAGA-3prime

200 (59)

5ʹ-GGACCCATCTTTCCAGAGGT-3 ʹ

COX-2

5´-CCAGATGGCCAGAGGACTCA-3ʹ

237 (58)

5´-TGTGAGTCCCGAGGGAATAGA-3

IL-1β

5-GCTGTCCAGATGAGAGCATC-3

293 (58 60)

5-GTCAGACAGCACGAGGCATT-3

GAPDH

5´-GTCATCAACGGGAAACCCAT-3ʹ

197 NM_0170084

5´ATCACAAACATGGGGGCATC-3ʹ

21

353 cDNA synthesis

cDNA synthesis kit (Thermo Scientific America) was used to synthesize cDNA by

using reverse transcription technique

Sterile tube was placed in ice box and following order was adopted for cDNA synthesis

Total RNA (1000 ng) was used for reverse transcription in each sample

1 microL of oligo dt primer was added in each tube containing sample

Nuclease free water (qs) was added to adjust volume up to 12 microL

Then mixture was placed in centrifuge machine for a short time period

Mixture was incubated in thermal cycler at 65 oC for five minutes in thermal cycler model

no 9902 (Applied Bio-Rad system Veriti)

The mixture was chilled on ice immediately

After that remaining components of kits were added in the following sequence

4 microL of reaction Buffer (5X) (20 mM of magnesium chloride 250 mM of Tris-HCl

250 mM of KCl (pH=83) and 50 mM of DTT) was added

1 microL of Ribolock RNAse inhibitor (20 UmicroL) was added

2 microL of dNTP mix (10 mM) was mixed

1 microL of M-MuLV enzyme (200 UmicroL) was added

20 microL mixture of each sample was prepared and it was centrifuged for a short time period

Then all sample tubes were kept at 40 ordmC for 60 min in thermal cycler followed by

reaction termination by heating at 70 ordmC for 5 min

The synthesized cDNA was stored at -20 ordmC

22

354 Working solution of primers

Stock solution was prepared by taking 25 nm primers were diluted with 250 microL nuclease free

water Concentration of stock solutions was100 microM15 microL of stock solution was diluted with

nuclease free water (135 microL) The concentration of stock solution was 10 microM

355 Polymerase chain reaction

cDNA was synthesized by taking cDNA (2 microL) of each sample in each separate tube then

primer (1 microL) followed by mixing of PCR Master Mix (6 microL) and nuclease free water (3 microL)

Then vials were placed in thermal cycler For denaturation 95oC temperature was set for 10

seconds For annealing 58-62 oC temperature was adjusted for 20 seconds and extension was

performed at 72 oC for 30 seconds 35 cycles were adjusted

356 Ingredients required for the formulation of TAE reaction buffer (50X)

Each one litre

Tris base 242 g

Glacial acetic acid 571 ml

05M EDTA 100 ml

Trisaminomethane base was dissolved in distilled water (qs) using magnetic stirrer up-to

600ml

Anhydrous acetic acid was mixed with DW

EDTA was added in above mixture

Total volume was adjusted up-to 1 litre

23

357 Gel preparation and Gel electrophoresis

Following steps were used

11 gram of agarose gel was taken in beaker and it was mixed with 55 ml of TAE (1Χ)

buffer

Then flask was heated in an oven to dissolve all solid particles After that flask was

immediately cooled using tap water

Subsequently ethidium bromide (3 microL) was added in above flask for visualization Gel

caster was taken and liquefied gel present in flask was transferred into gel caster

Gel electrophoresis apparatus was filled with TAE buffer (1Χ) All samples were loaded

with blue dye (2 microL)

DNA ladder having 100 bp (5 microL) was added in first well while rest of wells were loaded

with samples (5 microL)

Gel was run at 120 volts up to forty-five minutes Gel documentation system was

applied to visualize bands Resultant bands were calculated against standard ladder

358 Densitometry

Finally result of all samples were finalized using image J software Sample results were semi-

quantified through densitometry

359 Determination of serum PGE2 levels using ELISA

ELISA kit protocols of Elab Science (E-EL-0034 96T) were followed for the evaluation of

serum PGE2 levels Following were the reagents which were prepared before initiation of

experiment

24

3591 Preparation of standard

Standard was prepared 15 min before the start of experiment Standard reference vial was

centrifuged at 10000 g for one min It was reconstituted by taking 10 ml of reference diluent

with 1 ml of sample diluent Vial was tightly closed and the contents were mixed thoroughly

This stock solution had concentration of 2000 pgml Then serial dilutions of 1000 500 250

125 625 3125 and 0 pgml were prepared

3592 Biotinylated Detection Antibody

Stock tube was centrifuged and concentrated biotinylated detection antibody was diluted to

working solution 1100 ratio 200 microL excess amount was prepared

3593 Concentrated HRP Conjugate

Stock tube was centrifuged and concentrated HRP Conjugate was diluted to working solution

1100 ratio 200 microL excess amount was prepared Total amount of 6000 microL was prepared

3594 Substrate reagent

Substrate reagent is light sensitive so it was added into wells immediately after opening of

bottle due to its light sensitivity Sterilized tips were used to aspirate required dose of this

reagent and unused reagent was discarded after its use

3595 Procedure of ELISA

50 microL standard (duplicate) and sample were added in the each well of ELISA plate

carefully After that concentrated biotinylated antibiotic was added to each well

immediately

The plate was incubated for 45 min in an incubator

All samples present in wells were aspirated and then plate was kept in automated

washer for washing of plate This procedure was repeated for three times

25

After washing of plate 100 microL of HRP conjugate was added to each well Then the

plate was sealed and it was incubated at 37 C for 15 min

Again the sample from all wells were aspirated and plate was subjected to washing for

five times

90 microL of substrate reagent was added to each well The plate was sealed and it was

incubated for 15 min at 37 C in an incubator

After that 50 microL of stop solution was added into each well The analysis of samples

was performed twice and OD (optical density) was determined by ELISA reader

(BioTek ELx-800) while wavelength was set at 450 nm

3510 Evaluation of hematological parameters

Through cardiac puncture blood samples were collected in vacutainer (Lab Vac) containing

EDTA (ethylenediaminetetraacetic acid) Hb contents red blood cells (RBC) white blood cells

(WBC) and platelets were counted using an automated hematology analyzer (Sysmex XT-

1800i)

3511 Evaluation of biochemical parameters

Firstly blood was collected (intracardiac puncture) in BD Vacutainer containing gel Then

serum was separated Different commercially available kits were used to measure urea and

creatinine for evaluation of renal function while ALT AST and ALP for assessing liver

function by using chemistry analyzer which was automated (Humalyzer 3500) and protocols

of kit manufacturers were followed

26

36 Acute toxicity study

Nine groups of N=6 BALBc mice were used Group I was administered with vehicle only

Doses of EEJR and NHJR ie 050 gkg 1 gkg 15 kg and 2 gkg were administered orally

to Groups II III IV and V and Doses of EETG and NHTG 040 gkg 8 gkg 12 kg and

16 gkg were given to Groups VI VII VIII and IX respectively After the extract

administration food was withheld for two hours The extracts were given by oral gavage (po)

All groups were kept under close observation over 72 h and then for 14 days for signs of acute

toxicity Different effects including restlessness respiratory distress diarrhea motor activity

posture and mortality if any were determined (61)

37 Evaluation of anti-inflammatory activities

Table 2 Acute inflammatory models

Models Groups

Carrageenan-induced paw edema 01 mL 1 wv in distilled water

VC 3 mlkg (1 Tween 80)

EEJR (500 mgkg)

NHJR (500 mgkg )

EETG (400 mgkg)

NHTG (400 mgkg)

Piroxicam (10 mgkg )

Histamine-induced paw edema 01mL 1 wv in normal saline


EEJR (500 mgkg)

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)

Indomethacin (10 mgkg)

Serotonin-induced paw edema VC 3 mlkg (1 Tween 80)

EEJR (500 mgkg)

27

01thinspmL 10-3thinspmgmL dissolved in distilled

water

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)


Xylene-induced ear edema 03 ml of xylene


EEJR (500 mgkg)

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)

Dexamethasone (1 mgkg)

Dextran-induced paw edema 01 ml of 15 wv in distilled water


EEJR (500 mgkg)

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)

Diphenhydramine (60 mgkg)

Castor oil-induced diarrhea 03 ml of castor oil

VC 3 mlkg bw (1 Tween 80)

EEJR (500 mgkg)

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)

Loperamide (5 mgkg)

VC = vehicle control EEJR = Ethanolic extract of J regia NHJR = n-hexane extract of J

regia EETG = Ethanolic extract of T gharuensis NHTG = n-hexane extract of T gharuensisa

bw = body weight

28

371 Experimental design and induction of paw edema using carrageenan

Mice were allocated into different four groups (n=6) Vehicle and piroxicam were given to

group I and group VI while extracts were administered orally to group II (EEJR) group III

(NHJR) group IV (EETG) and group V (NHTG) Edema was induced by injecting

carrageenan (01mL 1 wv in normal saline) into the sub plantar tissue of the right hind paw

after one hour of extracts administration Measurement of paw edema was done at one hour

intervals for five hours by plethysmometer after administration of carrageenan (12) The

percentage inhibition was calculated using the formula given by Uroos et al (62)

372 Experimental design and paw edema induction using histamine

Mice were allocated into different four groups (n=6) Vehicle and Indomethacin were given to


(NHJR) group IV (EETG) and group V (NHTG) Edema was induced by injecting histamine

(01mL 1 wv in normal saline) into the sub plantar tissue of the right hind paw after one

hour of extracts administration Measurement of paw edema was done at one hour intervals for

three hours by plethysmometer after administration of histamine (12) The percentage

inhibition was calculated using the formula given by Uroos et al (62)

373 Experimental design and paw edema induction using serotonin

Mice were allocated into different four groups (n=6) Vehicle and Indomethacin orally were

given to group I and group VI while extracts were administered orally to group II (EEJR)

group III (NHJR) group IV (EETG) and group V (NHTG) Edema was induced by injecting

serotonin (01thinspmL 10-3thinspmgmL dissolved in distilled water) into the sub plantar tissue of the

right hind paw after one hour of extracts administration Measurement of paw edema was done

29

at one hour intervals for three hours by plethysmometer after administration of serotonin (12)

The percentage inhibition was calculated using the formula given by Uroos et al (62)

374 Experimental design and induction of paw edema using dextran

Mice were allocated into different four groups (n=6) Vehicle and Diphenhydramine were



dextran (01 ml of 15 wv in distilled water) into the sub plantar tissue of the right hind paw


intervals for five hours by plethysmometer after administration of dextran (12 63)

375 Experimental design for ear edema induced by xylene

Mice were allocated into four sets of mice containing (N = 6) as given in table 1 Xylene drop

was applied on the inner surface of the right ear of all groups The other ear (left) was

considered as untreated control Vehicle (group I) EEJR (group II) NHJR (group III) EETG

(group IV) and NHTG (group V) and dexamethasone (group VI) was administered one hour

before application of xylene After 15 minutes mice were sacrificed Both ears of mice were

cut and weighed using established methods (10)

376 Experimental design for castor oil-induced diarrhea model

Diarrhea was induced by castor oil using established methods (64) and detail of grouping was

mentioned in table1 Pretreatment with extracts and loperamide were conducted thirty minutes

30

before induction of diarrhea Diarrhea was induced using 06 ml of castor oil Four parameters

were evaluated for four hours time of onset of first diarrhea (min) total number of drywet

feces and total weight of wet feces (g) (65 66)

38 Phytochemical Evaluation

The initial screening of extracts were performed for different chemical constituents for

example alkaloids steroids terpenoids glycosides tannins phenols saponins and flavonoids

using previously published methods (67-70)

381 Test for Alkaloids

Mayerrsquos reagent and Wagnerrsquos reagent were extensively used to assess the presence of

alkaloids Crude extracts of EEJR NHJR EETG and NHTG were slightly warmed with dilute

HCl for 2 min in separate test tubes Filter paper was used for filtration After that solution was

divided into two parts and poured into two different tubes 2 to 3 drops of Mayerrsquos reagent or

Wagnerrsquos reagent was added in test tube White creamy precipitation was obtained which

showed positive result with Mayerrsquos reagent While reddish-brown precipitate with Wagnerrsquos

reagent confirmed the presence of alkaloids in the extract (71)

3811 Mayerrsquos reagent

5 grams of KI was mixed with DW Then more distilled water (qs) was added to make up its

volume up to 20 ml (Solution A) 136 g of mercuric iodide was mixed in 60 ml DW (Solution

B) After mixing both solutions their final volume was adjusted up to 01 litre (71)

31

3812 Wagnerrsquos reagent

Two grams of KI and 127 grams of I2 soln were taken in test tube Then DW (qs) was added

in tube up to 10ml (71)

382 Test for the steroids

Crude extracts of EEJR NHJR EETG and NHTG extracts of plant were mixed with 2 ml of

chloroform Conc H2S04 was mixed alongside wall of test tube After that acetic acid (2ml)

was added The formation of Green color confirmed that steroids are present in the extracts

(68)

383 Keller-Kilani for cardiac glycosides

Crude extracts of EEJR NHJR EETG and NHTG were mixed with glacial CH₃COOH (2ml)

having one drop of FeCl3 solution Then 1 ml of conc Sulphuric acid was poured with the wall

of test tube Ultimately dark brown ring appeared that indicates the existence of cardiac

glycosides (70)

384 Tests for terpenoids

3841 Salkowski test

Crude extracts of EEJR NHJR EETG and NHTG and chloroform (2 ml) were mixed

Subsequently 1-2 ml of conc H2S04 was poured into side wall of test tube A dark brown ring

appeared where acid and extracts interact to each other (70)

385 Tests for Phenols

EEJR NHJR EETG and NHTG extracts were mixed in 2 to 3 ml of water in each test tubes

After that dilute I2 solution was added Phenols were confirmed due to production of transient

red colour (71)

32

386 Test for saponins

500 mg of crude extract of EEJR NHJR EETG and NHTG were mixed with 5 ml of DW in

each test tube After thoroughly shaking persistent froth was observed Then three olive oil

drops were added in each tube and mixtures were shaken again vigorously to notice the

emulsion formation (70)

387 Test for flavonoids

Crude extracts of EEJR NHJR EETG and NHTG were taken in separate tubes and they were

treated with of 1 aluminum solution (few drops) Appearance of yellow color confirmed

presence of flavonoids in extracts (72)

39 Gas chromatography-mass spectrometry Analysis

GC-MS analysis was conducted using a previously published protocol with minor

modifications (62) GC-MS analysis was performed using capillary column (025 μm 30 m x

025 mm) Following were the settings Carrier gas Helium column velocity flow 10 mlmin

mode split less injection volume 05 microl Conditions of mass spectrometer were as follows

initial temperature 110 ˚C for 2 min raised at 10 ˚C per min until reached to 200 ˚C then rate

decreased to 5 ˚C per min until 280 ˚C ionizing voltage 70 eV mz range 20-800 (62)

310 Statistical analysis

Graph Pad Prism (V 60) was selected for interpretation of data Data was presented as mean plusmn

SEM (standard error of mean) was used Data of pharmacological activities were analyzed by

using one or two way ANOVA (one way analysis of variance) which is followed byTukeyrsquos

post-hoc test for comparison Significance level was observed as p lt 005

33

CHAPTER 4 RESULTS

41 J regia and T gharuensis significantly reduced paw edema in FCA-induced arthritic

model

411 Measurement of paw edema at day 8

The piroxicam (0970 plusmn 0006) EEJR (0958 plusmn 0007) NHJR (0943 plusmn 0007) EETG (0936 plusmn

0008) and NHTG (0931 plusmn 0013) inhibited paw edema when compared to positive control

group (0940 plusmn 0010) at day 8

Figure 1 Paw edema at day 8

Figure 1 indicates increase in paw edema in comparison to vehicle control group at day 8

shows the comparison between vehicle control group and arthritic control group

34



0013) and NHTG (0863 plusmn 0006) reasonably decreased (p lt 0001) paw edema in comparison

to positive control group (1144 plusmn 0017) at day 15


Figure 2 shows significant attenuation of paw edema in Piroxicam EEJR NHJR EETG and

NHTG p lt 0001 as compared to arthritic group indicates difference between vehicle

control and arthritic control group

35


Similarly at day 22 significant inhibition (p lt 0001) was determined while treating edema

with piroxicam (0718 plusmn 0007) EEJR (0753 plusmn 0011) NHJR (0744 plusmn 0011) EETG (0737

plusmn 0009) and NHTG (0727 plusmn 0010) as compared to arthritic control group (1242 plusmn 0012)



NHTG groups p lt 0001 as compared with arthritic group indicates difference

between two control groups

36


Significant inhibition (p lt 0001) was observed with piroxicam (0627 plusmn 0008) EEJR (0647

plusmn 0010) NHJR (0688 plusmn 0018) EETG (0630 plusmn 0007) and NHTG (0670 plusmn 0017) treated

groups in contrast to arthritic control group (1366 plusmn 0009) at day 28


Figure 4 denotes significantly attenuation of paw edema in Piroxicam EEJR NHJR EETG

and NHTG treated groups p lt 0001 as compared with arthritic control group indicates

difference between two control groups

37

Figure 5 Paw edema

Figure 5 shows that treated groups significantly reduced inflammation at day 8 15 22 and 28

p lt 001 and p lt 0001 in contrast with arthritic control group

38

Table 1J regia and T gharuensis extracts significantly reduced paw edema in FCA induced arthritis model

Days Arthritic Control Piroxicam EEJR NHJR EETG NHTG

Mean plusmn SEM (ml)

Day 8 0942 plusmn 0013 0960 plusmn0013 0950 plusmn 0004 0943 plusmn0008 0937 plusmn 0008 0932 plusmn 0014

Day 15 1144 plusmn 0017 0783 plusmn 0008 0788 plusmn 0010 0883 plusmn 0006 0770 plusmn 0013 0863plusmn0006

Day 22 1242 plusmn 0012 0718 plusmn 0007 0753 plusmn 0011 0744 plusmn 0011 0737 plusmn 0009 0727 plusmn 0010


EEJR Ethanolic extract of Juglans regia NHJR n-hexane extract of Juglans regia EETG Ethanolic extract of Trigonella gharuensis NHTG

n-hexane extract of Trigonella gharuensis p lt 0001 in contrast to arthritic control group

39

42 J regia and T gharuensis attenuated arthritic Score

421 At Day 8

The development of arthritis was observed on day 8 in piroxicam (3083 plusmn 0083) EEJR (3083

plusmn 0083) NHJR (300 plusmn 0129) EETG (3083 plusmn 0083) and NHTG (3083 plusmn 0083) in

comparison with arthritic group (3083 plusmn 0083)

Figure 6 Arthritic score at day 8

Figure 6 shows increase in arthritic score at day 8 There is no difference between treatment

groups and arthritic control group Bar chart of vehicle group is invisible because the values of

the normal group are represented as 0 in these figures

40

422 At Day 15

Attenuation in arthritic development was found with piroxicam (2250 plusmn 0111 p lt 0001)

EEJR (2750 plusmn 0111 p lt 001) NHJR (2750 plusmn 0111 p lt 001) EETG (2833 plusmn 0105 p lt

001) and NHTG (2833 plusmn 0106 p lt 001) treated groups in comparison to arthritic control

group (3417 plusmn 0083) on day 15


Figure 7 shows attenuation of arthritic development with Piroxicam EEJR NHJR EETG and

NHTG Bar chart of vehicle group is invisible because the values of the normal group are

represented as 0 in these figures p lt 001 and p lt 0001 in contrast to arthritic control

group

41

423 At Day 22

Similarly at day 22 significant inhibition was observed with piroxicam (2500 plusmn 0182 p lt

0001) EEJR (2583 plusmn 0153 p lt 0001) NHJR (2750 plusmn 0111 p lt 001) EETG (2667 plusmn

0105 p lt 001) and NHTG (2917 plusmn 0153 p lt 005) treated groups in comparison to arthritic

control group (3500 plusmn 0129)




represented as 0 in these figures p lt 005 p lt 001 and p lt 0001 in contrast to arthritic

control group

42

424 At Day 28

The trend continued on 28th day as we observed significant inhibition with (p lt 0001) with

piroxicam (2083 plusmn 0083) EEJR (2583 plusmn 0153) NHJR (2583 plusmn0083) EETG (2533 plusmn

0105) and NHTG (2500 plusmn 0129) treated groups as compared to diseased group (3583 plusmn

0153)




represented as 0 in these figures p lt 0001 in contrast to arthritic control group

43

Figure 10 Arthritic Score

Figure 10 shows attenuation of arthritic development with Piroxicam EEJR NHJR EETG

and NHTG Bar charts of vehicle group are invisible because the values of the normal group

are represented as 0 in these figures p lt 0001 in contrast to arthritic control group

44

Table 2 J regia and T gharuensis attenuated arthritic development


n-hexane extract of Trigonella gharuensis p lt 005 p lt 001 and p lt 0001 in contrast to arthritic control group

Days

Arthritic Control Piroxicam EEJR NHJR EETG NHTG


Day 8 3083 plusmn 0083 3083 plusmn 0083 3000 plusmn 0129 3000plusmn 0129 3083 plusmn 0083 3083 plusmn 0083

Day 15 3417 plusmn 0083 2250 plusmn 0111 2750plusmn 0111 2750 plusmn 0111 2833 plusmn 0105 2833 plusmn 0166

Day 22 3500 plusmn0129 2500 plusmn0182 2583plusmn 0153 2750 plusmn 0111 2667 plusmn 0105 2917 plusmn 0153

Day 28 3583plusmn 0153 2083 plusmn0083 2580 plusmn 0153 2583 plusmn 0083 2533 plusmn 0105 2500 plusmn 0129

45

43 J regia and T gharuensis attenuated histopathological parameters

431 J regia significantly reduced inflammation

Significant reduction in inflammation was observed with piroxicam (1583 plusmn 0083 p lt 0001)


0001) NHTG (2083 plusmn 0083 p lt 005) and piroxicam treated groups in contrast to arthritic


Figure 11 J regia and T gharuensis reduced infiltration of inflammatory cells

Figure 11 shows reduction of infiltration of inflammatory cells Bar chart of vehicle group is

invisible because normal group values are represented as 0 in these figures p lt 005 and p

lt 0001 in contrast to arthritic control group

46

432 J regia significantly attenuated pannus formation

Reduction (p lt 0001) in pannus formation was observed with piroxicam (2333 plusmn 0105) EEJR

(2583 plusmn 0083) NHJR (2667 plusmn 0105) EETG (2583 plusmn 0083) and NHTG (2583 plusmn 0083) in

comparison to arthritic group (3417 plusmn 0083)

Figure 12 J regia and T gharuensis reduced pannus formation

Figure 12 explains reduction of pannus formation Bar chart of vehicle group is invisible

because normal group values are represented as 0 in these figures p lt 0001 in contrast to

arthritic control group

47

433 J regia significantly reduced erosion of bone

Significant elevation of bone destruction was observed in arthritic control group (2583 plusmn

0083) in contrast to vehicle group A significant attenuation of bone erosion was observed

with piroxicam (2083 plusmn 0083 p lt 001) EEJR (2083 plusmn 0083 p lt 001) NHJR (2167 plusmn

0105 p lt 005) EETG (2083 plusmn 0083 p lt 001) and NHTG (2167 plusmn 0105 p lt 005) in

contrast to arthritic control group

Figure 13 J regia and T gharuensis reduced bone erosion

Figure 13 indicates that Piroxicam EEJR NHJR EETG and NHTG significantly attenuated

bone erosion Bar chart of vehicle group is invisible because normal group values are

represented as 0 in these figures p lt 005 and p lt 001 in contrast to arthritic control group

48

Table 3J regia and T gharuensis attenuated histopatological parameters

Parameters

Arthritic control

Piroxicam

EEJR

NHJR

EETG

NHTG

Mean plusmn SEM

Infiltration of

inflammatory cells 2583 plusmn 0083 1583 plusmn 0083 1833 plusmn 0105 2083 plusmn 0083 1917 plusmn 0154 2083 plusmn 0083

Pannus formation 3417 plusmn 0083 2333plusmn 0105 2583 plusmn 0083 2667 plusmn 0105 2583 plusmn 0083 2583 plusmn 0083

Bone erosion 2583 plusmn 0083 2083 plusmn 0083 2083 plusmn 0083 2167 plusmn 00105 2083 plusmn 0083 2167 plusmn 0105



49

Figure 14 Histopathology of normal control

Normal Control Thin synovial membrane layer surrounded by flattened epithelium of synovial

lining Arrows was used to specify it The oblong circle spans sub-intimal (supportive) layer of

synovial membrane which consists of adipose tissue as well as fibrous tissue in addition to

blood vessels No cell infiltrate of inflammatory cells was seen in the synovial membrane No

pannus formation is present (Fig 9)

50

Figure 15 Histopathology of diseased control

Arthritic control Diffuse and extensive infiltrate of inflammatory cell (I) surrounding synovial

membrane (SM) large plaques of fibrovascular tissue as well as granulation tissue involving

central as well as peripheral synovial membrane Focus of bone erosion (BE) is also shown

(Fig 15)

51

Figure 16 Histopathology of Piroxicam

Piroxicam synovial membrane (SM) is surrounded by focal aggregates of inflammatory cell

infiltrate Central part of SM is marked by circle which indicates inflammation of pauci area

Line spans the large plaques of fibro vascular tissue as well as granulation tissue (Figure 16)

52

Figure 17 Histopathology of EEJR

EEJR Arrow indicates thin layer of SM whereas oblong circles point out sub-intimal layer of

SM This layer consists of blood vessels fibrous as well as tissues Moreover inflammatory

cells and pannus formation are absent (Figure 17)

53

Figure 18 Histopathology of NHJR

NHJR Arrow indicates thin layer of SM whereas rectangle points out sub-intimal layer of SM

This layer consists of blood vessels fibrous as well as tissues Moreover inflammatory cells

and pannus formation are absent Figure 18

54

Figure 19 Histopathology of EETG

TE synovial membrane (SM) is surrounded by focal aggregates of inflammatory cell infiltrate

Most of the areas of SM are pauci-inflamed which are denoted by oblong circles The inlet on

right lower corner depicts a closer look at the above-mentioned findings (Figure 19)

55

Figure 20 Histopathology of NHTG

NHTG Focal scattered inflammatory cells around SM Most of the areas of SM are pauci-

inflamed which are denoted by oblong circles The inlet on right lower corner depicts a closer

look at the above-mentioned findings with no focus on pannus formation (Figure 20)

56

This picture shows reduction of inflammation of pannus formation in experimental groups as

compared to diseased control group which shows bone erosion

57

44 J regia and T gharuensis downregulated mRNA expression levels of pro-

inflammatory markers and upregulated anti-inflammatory marker

441 J regia and T gharuensis downregulated mRNA expression levels of TNF-α

An increase (p lt 0001) in expression levels were detected in arthritic control group (5019 plusmn

1983) in contrast with the vehicle group (3342 plusmn 1787) Treatment with Piroxicam (3292 plusmn

1043) EEJR (3396 plusmn 1639) NHJR (3588 plusmn 0792) EETG (3429 plusmn 1538) and NHTG

(3538 plusmn 0571) significantly down regulated (p lt 0001) the levels of TNF α

Figure 21 J regia and T gharuensis downregulated mRNA expression levels of TNF-α

Figure 21 shows that J regia and T gharuensis downregulated mRNA expression levels p

lt 0001 in contrast with diseased arthritic control group indicates difference between two

control groups

58

442 J regia suppressed mRNA expression levels of NF-κB

An upregulation (p lt 0001) in levels of NF-κB were observed in arthritic control group (5369

plusmn 0684) in contrast with vehicle control group (3403 plusmn 1541) Significant attenuation (p lt

0001) of NF-κB expression levels were seen with piroxicam (3592 plusmn 1236) EEJR (3892 plusmn

1100) NHJR (4290 plusmn 1333) EETG (3908 plusmn 1365) and NHTG (4125 plusmn 0690) treated

groups

Figure 22 J regia and T gharuensis downregulated mRNA expression levels of NF-κB

Figure 22 shows that J regia and T gharuensis suppressed mRNA expression levels of NF-

κB p lt 0001 in contrast with arthritic control group indicates difference between two

control groups

59

443 J regia and T gharuensis reduced IL-6 mRNA expression levels

Significant downregulation of IL-6 was observed with piroxicam (3209 plusmn 1043 p lt 0001)



group (4069 plusmn 1188)

Figure 23 J regia and T gharuensis downregulated mRNA expression levels of IL-6

Figure 23 shows treatment with piroxicam EEJR NHJR EETG and NHTG attenuated IL-6

levels p lt 001 and p lt 0001 as compared with arthritic control group indicates


60

444 J regia and T gharuensis decreased the mRNA expression levels of IL-1β

IL-1β expression levels were upregulated (p lt 0001) in arthritic control group (5269 plusmn 1445)

in comparison with vehicle group (3409 plusmn 1472) Significant inhibition (p lt 0001) was

determined with piroxicam (3225 plusmn 1236) EEJR (3409 plusmn 1682) NHJR (3692 plusmn 0835)

EETG (3446 plusmn 1239) and NHTG (3583 plusmn 0649) treated groups

Figure 24 J regia and T gharuensis downregulated mRNA expression levels of IL-1β

Figure 24 shows that treatment with piroxicam EEJR NHJR EETG and NHTG attenuated

levels of IL-1β p lt 0001 as compared with arthritic control group indicates difference


61

J regia and T gharuensis down regulated COX-1 mRNA expression levels

COX-1 expression levels were upregulated (p lt 0001) in arthritic control group (5163 plusmn

1484) as compared to the vehicle control group (3387 plusmn 1036) Significant attenuation (p lt

0001) in COX-1 levels were observed with piroxicam (3287 plusmn 1036) EEJR (3337 plusmn 1747)

NHJR (3655 plusmn 0715) EETG (3298 plusmn 1757) and NHTG (3678 plusmn 863) treated groups

Figure 25 J regia and T gharuensis downregulated mRNA expression levels of COX-1

Figure 25 shows that treatment with piroxicam EEJR NHJR EETG and NHTG significantly

attenuated mRNA expression levels of COX-1 p lt 0001 as compared to arthritic control

group indicates difference between two control groups

62

445 J regia and T gharuensis down regulated the mRNA expression levels of COX-2

COX-2 expression levels were increased significantly (p lt 0001) in arthritic control group

(5506 plusmn 1223) as compared to vehicle control group (3342 plusmn 1787) COX-2 levels were

significantly reduced (p lt 0001) in piroxicam (3317 plusmn 0888) EEJR (3317 plusmn 0888) NHJR

(3469 plusmn 0650) EETG (3106 plusmn 0791) and NHTG (3473 plusmn 0499) treated groups


Figure 26 represents that treatment with piroxicam EEJR NHJR EETG and NHTG

significantly attenuated mRNA expression levels of COX-2 p lt 0001 as compared with

arthritic control group indicates difference between two control groups

63

446 J regia and T gharuensis up regulated the mRNA expression levels of IL-4

IL-4 expression levels were decreased (p lt 0001) in arthritic control group (2085 plusmn 1757) as

compared to the vehicle control group (2348 plusmn 0516) Significant up-regulation (p lt 0001)

in IL-4 was determined in piroxicam (2892 plusmn 0734) EEJR (3064 plusmn 0650) NHJR (3247 plusmn

0760) EETG (3182 plusmn 0551) and NHTG (3316 plusmn 0427) treated groups

Figure 27 J regia and T gharuensis upregulated mRNA expression levels of IL-4


upregulated mRNA expression levels of IL-4 p lt 0001 as compared with arthritic control


64


There were increased (p lt 0001) in PGE2 levels in arthritic control group (0986 plusmn 0027) as

compared to the vehicle control group (0486 plusmn 0043) We observed significant reduction (p lt

0001) in PGE2 levels with piroxicam (0628 plusmn 0021) EEJR (0759 plusmn 0035) NHJR (0780 plusmn

0035) EETG (0747 plusmn 0026) and NHTG (0837plusmn 0017) treated groups

Figure 28 J regia and T gharuensis reduced levels of PGE2

Figure 28 shows treatment with piroxicam EEJR NHJR EETG and NHTG significantly

down-regulated prostaglandin E2 levels p lt 0001 as compared with arthritic control group

indicates difference between two control groups

65

Table 4 J regia and T gharuensis attenuated mRNA expression levels of pro-inflammatory markers and up-regulated anti-inflammatory

marker

Markers

Vehicle

Control

Arthritic

Control Piroxicam

EEJR

NHJR

EETG

NHTG

Mean plusmn SEM

TNF-α 3342 plusmn 1787 5019 plusmn 1983 3292 plusmn 1043 3396 plusmn 1639 3588 plusmn 0792 3429 plusmn 1538 3538 plusmn 0571

NF-κB

3403 plusmn 1541 5369 plusmn 0684 3592 plusmn 1236 3892 plusmn 1100 4290 plusmn 1333 3908plusmn 1365 4125plusmn 0690

IL-6

3375 plusmn 1751 4069 plusmn 1188 3209 plusmn 1043 3209plusmn 1639 3392 plusmn 0901 3386 plusmn 0850 3361plusmn 1195

IL-1β

3409 plusmn 1472 5269 plusmn 1445 3225 plusmn 1236 3409 plusmn 1682 3692 plusmn 0835 3446 plusmn 1239 3583 plusmn 0649

COX-1

3387 plusmn 1036 5163 plusmn 1484 3287 plusmn 1036 3337 plusmn 1747 3655 plusmn 0715 3298 plusmn1757 3678 plusmn 0863

COX-2 3342 plusmn 1787 5506 plusmn 1223 3317 plusmn 0888 3317 plusmn 0888 3469 plusmn 0650 3106 plusmn0791 3473 plusmn 0499

IL-4 3475 plusmn 1004 2348 plusmn 0516 2892 plusmn 0734 3064 plusmn 0650 3247 plusmn 0760 3182 plusmn 0551 3316 plusmn 0427

EEJR Ethanolic extract of Juglans regia NHJR n-hexane extract of Juglans regia EETG Ethanolic extract of Trigonella gharuensis NHTG n-

hexane extract of Trigonella gharuensis p lt 001 and p lt 0001 as compared with arthritic control group presents comparison


66

448 J regia and T gharuensis nearly normalized hematological parameters

4481 J regia and T gharuensis nearly normalized RBC count

Suppression (p lt 0001) of RBC count in the arthritic control group (5642 plusmn 0336) was noticed

in comparison to vehicle control group (8298 plusmn 0315) Significant elevation (p lt 001) in RBC

counts were observed with piroxicam (6807 plusmn 0372) EEJR (7280 plusmn 0200) NHJR (7465 plusmn

0183) EETG (736 plusmn 0365) and NHTG (7337 plusmn 0304) as compared to arthritic control

group

Figure 29 J regia and T gharuensis nearly normalized RBC count

Figure shows that treatment with piroxicam EEJR NHJR EETG and NHTG nearly

normalized RBC levels p lt 001 as compared with arthritic control group indicates


67

4482 J regia and T gharuensis nearly normalized Hb content

Suppression (p lt 0001) of Hb content in the arthritic control group (1173 plusmn 0237) was

observed in comparison to vehicle control group (1475 plusmn 0131) Statistically significant

elevation of Hb content was observed with piroxicam (1367 plusmn 0353 p lt 001) EEJR (1397

plusmn 0458 p lt 0001) NHJR (1343 plusmn 0145 p lt 001) EETG (1402 plusmn 0425 p lt 0001) and

NHTG (1367 plusmn 0353 p lt 001) in comparison to arthritic control group

Figure 30 J regia and T gharuensis nearly normalized Hb content


normalized Hb content p lt001 and p lt 0001 as compared with arthritic control group


68

4483 J regia and T gharuensis nearly normalized WBC count

The data showed reduction in WBC count with piroxicam (1291 plusmn 0239 p lt 0001) EEJR

(1288 plusmn 0209 p lt 0001) NHJR (12580 plusmn 0416 p lt 0001) EETG (1288 plusmn 0209 p lt

0001) and NHTG (1325 plusmn 0324 p lt 001) treatment in comparison to arthritic control group

(1512 plusmn 0256)

Figure 31 J regia and T gharuensis nearly normalized WBC count


reduced WBC count as compared to arthritic control p lt 001 and p lt 0001 as compared

with arthritic control group indicates difference between two control groups

69

4484 J regia and T gharuensis nearly normalized platelet count

Elevated platelet count (1444 plusmn 1844) was found in arthritic control group when compared

with vehicle control group (7987 plusmn 2782) Reduction (p lt 0001) in platelet count was

observed with piroxicam (1044 plusmn 3237) EEJR (9372 plusmn 1330) NHJR (1163 plusmn 4131) EETG

(9108 plusmn1584) and NHTG (1099 plusmn 3413) as compared to arthritic control group

Figure 32 J regia and T gharuensis nearly normalized Platelet count

Figure 32 displays treatment with piroxicam EEJR NHJR EETG and NHTG significantly

decreased platelets counts p lt 0001 as compared with arthritic control group indicates


70

Table 5 J regia and T gharuensis nearly normalized hematological parameters

Parameters

Control Arthritic control Piroxicam EEJR NHJR EETG NHTG

Mean plusmn SEM

RBC

(106Ul) 8298 plusmn 0315 5642 plusmn 0366 7473 plusmn 0348 7280 plusmn 0200 7465 plusmn 0183 7360 plusmn 0365 7337 plusmn 0304

Hb

(gdl)


WBC

(103Ul)


Platelets

(103Ul)



hexane extract of Trigonella gharuensis p lt 001 and p lt 0001 as compared with arthritic control group indicates difference between

two control groups

71


4491 J regia and T gharuensis effects on urea levels

No differences in urea levels were observed with piroxicam (2617 plusmn 0401) EEJR (2733 plusmn

0333) NHJR (2633 plusmn 0210) EETG (2767 plusmn 0421) and NHTG (2617 plusmn 0477) when

compared with arthritic control group (2633 plusmn 0614)

Figure 33 J regia and T gharuensis effects on urea levels

Figure 33 shows treatment with piroxicam EEJR NHJR EETG and NHTG did not produce

any sign of nephrotoxicity as indicated by non-significant difference among the levels of urea

in all groups

72

4492 J regia and T gharuensis did not alter creatinine levels

No differences in creatinine levels were observed with piroxicam (0850 plusmn 0022) EEJR (0800

plusmn 0025) NHJR (0800 plusmn 0025) EETG (0866 plusmn 0021) and NHTG (0858 plusmn 0020) as

compared to arthritic control group (0833 plusmn 0210)

Figure 34 J regia and T gharuensis effects on creatinine level

Figure 34 represents treatment with piroxicam EEJR NHJR EETG and NHTG did not alter

creatinine levels

73

4493 J regia and T gharuensis effects on ALT levels

Insignificant change was observed in ALT levels when vehicle control (3033 plusmn 0557) arthritic

control (3183 plusmn 0477) piroxicam (3367 plusmn 0421) EEJR (3367 plusmn 0872) NHJR (3383plusmn

0792) EETG (3317 plusmn 0654) and NHTG (3317 plusmn0792) groups were compared with each

other

Figure 35 J regia and T gharuensis effects on ALT levels

Figure 35 shows that treatment with piroxicam EEJR NHJR EETG and NHTG did not alter

creatinine levels

74

4494 J regia and T gharuensis effects on AST levels

Insignificant differences were observed in AST levels among vehicle control (9671 plusmn 1714)

arthritic control (9886 plusmn 1519) piroxicam (9471 plusmn 0190) EEJR (96 plusmn 1852) NHJR (9557

plusmn 1212) EETG (9800 plusmn 0617) and NHTG (9671 plusmn134) groups when compared with each

other

Figure 36 J regia and T gharuensis effects on AST levels

Figure 36 shows treatment with piroxicam EEJR NHJR EETG and NHTG did not alter AST

levels

75

4495 J regia and T gharuensis extracts significantly reduced ALP levels

The comparison between arthritic control group (1567 plusmn 2472) and normal control group

(1308 plusmn 1621) indicates a significant enhancement in ALP levels between both groups

Comparison of arthritic control group with piroxicam (131 plusmn 1633) EEJR (1313 plusmn 1333)

NHJR (1322 plusmn 1493) EETG (131 plusmn 1065) and NHTG (1322 plusmn 1167) indicates reduction

(p lt 0001) of raised ALP levels

Figure 37 J regia and T gharuensis extracts significantly reduced ALP levels

Figure 37 presents J regia and T gharuensis extracts significantly attenuated ALP levels near

to control group p lt 0001 as compared with arthritic control group indicates


76

Table 6 J regia and Tgharuensis did not alter biochemical parameter

Biochemical

Parameters

Vehicle


Mean plusmn SEM

Urea

(mgdl) 2633plusmn 0614 28plusmn 0856 2617plusmn 0401 2733plusmn 0333 2633plusmn 0210 2767plusmn 0421 2617 plusmn0477

Creatinine

(mgdl) 0833plusmn 0210 0883plusmn 0016 0850plusmn 0022 0800plusmn 0025 0800plusmn 0025 0866plusmn 0021 0858plusmn 0020

AST

(IUL) 9671plusmn 1714 9886plusmn 1519 9471plusmn 1190 96plusmn 1852 9557plusmn 1212 98plusmn0617 9671plusmn 134

ALT

(IUL) 3033plusmn 0557 3183plusmn 0477 3367plusmn 0421 3367plusmn 0872 3383plusmn 0792 3317plusmn 0654 3317plusmn 0792


hexane extract of Trigonella gharuensis Whereas p lt 005 was considered significant

77

4410 Evaluation of acute Toxicity of extracts of J regia and T gharuensis

Acute toxicity studies revealed safety of ethanolic and n- hexane extracts of J regia and T

gharuensis extracts up to maximum dose of 2000 mgkg Different doses of J regia extracts

(500 mgkg 1000 mgkg 1500 mgkg and 2000 mgkg) and T gharuensis extracts (400 mgkg

800 mgkg 1200 mgkg and 1600 mgkg) were given orally and they did not produce any signs

of toxicity such as restlessness respiratory distress diarrhea motor activity posture

abnormality and mortality during 72 hours as presented in Table 10

Table 7 Acute toxicity study of ethanolic and n-hexane extracts of J regia and T

gharuensis

Groups Restless Respiratory

distress Diarrhea

Motor

activity

Posture

abnormality Mortality

Group-1 - - - - - -

Group-1I - - - - - -

Group-1II - - - - - -

Group-1V - - - - - -

Group-V - - - - - -

Group-VI - - - - - -

Group-VII - - - - - -

Group-

VIII - - - - - -

GROUP-

IX - - - - - -

78

45 Pre-treatment with J regia and T gharuensis inhibited paw edema induced by

carrageenan

451 At 1st hr

Substantial edema inhibition (p lt 0001) was observed with EEJR (0210 plusmn0003) NHJR

(0235 plusmn 0002) EETG (0226 plusmn0004) NHTG (0255 plusmn 0003) and piroxicam (0208 plusmn 0007)

in contrast with control group (0288 plusmn 0004) The percentage inhibition in these groups were

found to be 2708 1840 2139 1145 and 2777 respectively At first hour the

maximum inhibition was shown by EEJR

Figure 38 Carrageenan At first hour

Figure 38 depicts inhibition of paw edema as a consequence of pre-treatment p lt 0001 in

contrast with control group

79

452 At 2nd hr

At second hour pretreatment with piroxicam (0213 plusmn 0004) EEJR (0195 plusmn 0002) NHJR

(0218 plusmn 0003) EETG (0236 plusmn 0005) and NHTG (0250 plusmn 0002) demonstrated significant

inhibition (p lt 0001) in contrast with control group (0290 plusmn 0003) The percentage inhibition

in these groups were found to be 3275 2482 1866 and 1379 and 2655

respectively The maximum inhibition was shown by EEJR at second hour

Figure 39 Carrageenan At second hour

Figure 39 depicts significant inhibition of paw edema as a consequence of pre-treatment

p lt 0001 in contrast with control group

80

453 At 3rd hr

Consistent prevention was noticed in treatment groups for instance EEJR (0172 plusmn 0003 P lt 0001)

NHJR (0206 plusmn 0003 P lt 0001) EETG (0231 plusmn 0006 P lt 0001) NHTG (0280 plusmn 0010 P lt 001)

and piroxicam (0210 plusmn 0003 P lt 0001) in contrast to control group (0318 plusmn 0007) The percentage

inhibition in these groups were found to be 4591 3522 2735 1194 and 3396

respectively The maximum inhibition was shown by EEJR at third hour

Figure 40 Carrageenan At third hour

Figure 40 depicts suppression of paw edema as a consequence of pre-treatment p lt 001

and P lt 0001 in contrast with control group

81

454 At 4th hr

Considerable decrease (p lt 0001) remained the hallmark of pretreatment with EEJR (0156 plusmn 0003)

NHJR (0193 plusmn 0003) EETG (0210 plusmn 0006) NHTG (0260 plusmn 0010) and piroxicam (0183 plusmn 0004)

in comparison with control group (0340 plusmn 0007) The percentage inhibition in these groups were

found to be 5411 4323 3823 2352 and 4617 respectively The maximum

inhibition was shown by EEJR at fourth hour

Figure 41 Carrageenan At fourth hour


in contrast with control group

82

455 At 5th hr

Notable (P lt 0001) anti-inflammatory activity of EEJR (0125 plusmn 0003) NHJR (0175 plusmn 0003) EETG

(0195 plusmn 0008) NHTG (0236 plusmn 001) and piroxicam (0165 plusmn 0003) were detected in contrast with

control group (0358 plusmn 0008) The percentage inhibition in these groups were found to be 6508

5111 4553 3407 and 5391 respectively The maximum inhibition was shown

by EEJR at fifth hour

Figure 42 Carrageenan At fifth hour



83

EEJR Ethanolic extract of Juglans regia NHJR n-hexane extract of Juglans regia EETG Ethanolic extract of Trigonella gharuensis n-NHTG

hexane extract of Trigonella gharuensis

84

Table 8 The anti-inflammatory characteristics and percentage inhibition of paw edema caused by J regia and T gharuensis extracts on

carrageenan-induced paw edema

Groups

1st hr 2nd hr 3rd hr 4th hr 5th hr

Mean plusmn SEM ( paw edema inhibition)

Control 0288 plusmn 0004 0290 plusmn 0003 0318 plusmn 0007 0340 plusmn 0007 0358 plusmn 0008

EEJR

(500 mgkg)

0210 plusmn0003

(2708)

0195 plusmn 0002

(3275)

0172 plusmn0003

(4591)

0156 plusmn0004

(5411)

0125 plusmn 0003

(6508)

NHJR

(500 mgkg)

0235 plusmn 0002

(1840)

0218 plusmn 0003

(2482)

0206 plusmn0003

(3522)

0193 plusmn0003

(4323)

0175 plusmn 0003

(5111)

EETG

(400 mgkg)

0226 plusmn0004

(21387)

0236 plusmn 0005

(1866)

0231 plusmn0006

(2735)

0210 plusmn0006

(3823)

0195 plusmn 0008

(4553)

NHTG

(400 mgkg)

0255 plusmn0003

(1145)

0250 plusmn 0002

(1379)

0280 plusmn0010

(1194)

0260 plusmn0010

(2352)

0236 plusmn 0011

(3407)

Piroxicam

(10 mgkg)

0208 plusmn0007

(2777)

0213 plusmn 0004

(2655)

0210 plusmn0003

(3396)

0183 plusmn0004

(4617)

0165 plusmn 0003

(5391)


hexane extract of Trigonella gharuensis p lt 001 and p lt 0001 as compared to control group

85

46 Pre-treatment with J regia and T gharuensis inhibited histamine-induced paw

edema

461 At 1st hr

Treatment with EEJR (0235 plusmn 0002) NHJR (0251 plusmn 0003) EETG (0240 plusmn 0004) NHTG (0256 plusmn

0004) and indomethacin (0238 plusmn 0003) produced anti-inflammatory effects (P lt 0001) in treatment

groups in contrast with control group (0301 plusmn 0003) The percentage inhibition in these groups

were found to be 2192 1860 2093 1993 and 2093 respectively The

maximum inhibition was shown by EEJR at first hour

Figure 43 Histamine At first hour

Figure 43 depicts significant inhibition of paw edema as a consequence of pre-treatment P

lt 0001 in contrast with control group

86

462 At 2nd hr

Extracts of EEJR (0220 plusmn 0003) NHJR (0240 plusmn 0002) EETG (0228 plusmn 0005) NHTG (0246 plusmn

0002) and indomethacin (0223 plusmn 0002) depicted remarkable reduction (P lt 0001) in comparison to

control group (0318 plusmn 0001) at second hour The percentage inhibition in these groups were found

to be 3081 2452 2830 2264 and 2087 respectively The maximum inhibition

was shown by EEJR at second hour

Figure 44 Histamine At second hour



87

463 At 3rd hr

A significant inhibition (p lt 0001) was observed with EEJR (0186 plusmn 0004) NHJR (0225 plusmn 0008)

EETG (0225 plusmn 0005) NHTG (0236 plusmn 0002) and indomethacin (0203 plusmn 0004) in contrast with

control group (0321 plusmn 0004) at third hour The percentage inhibition in these groups were found


was shown by EEJR at third hour

Figure 45 Histamine At third hour



88


hexane extract of Trigonella gharuensis Ind Indomethacin

89

Table 9The anti-inflammatory properties and percentage inhibition of J regia and T gharuensis extracts on histamine-induced paw

edema model

Groups

1sthr 2ndhr 3rdhr

(Mean plusmnSEM)

(ml)

Inhibition

(Mean plusmnSEM)

(ml)

Inhibition

(Mean plusmnSEM)

(ml)

Inhibition

Control 0301 plusmn 0003

- 0318 plusmn 0001 - 0321 plusmn 0004 -

EEJR

( 500 mgkg)

0235 plusmn 0002

2192

0220 plusmn 0003

3081

0186 plusmn 0004

4205

NHJR

( 500 mgkg)

0251 plusmn 0003

1860

0240 plusmn 0002

2452

0225 plusmn 0008

2990

EETG

( 400 mgkg) 0240 plusmn 0004 2093 0228 plusmn 0005 2830 0225 plusmn 0005 2990

NHTG


Indomethacin

(10 mgkg)

0238 plusmn 0003

2093

0223 plusmn 0002

2987

0203 plusmn 0004

3676



90

47 Pretreatment with J regia and T gharuensis prevented serotonin-induced paw edema

471 At 1st hr

Significant inhibition (p lt 0001) was observed with EEJR (0236 plusmn 0005) NHJR (0258 plusmn 0004)




by EEJR at first hour

Figure 46 Serotonin At first hour



91

472 At 2nd hr

Significant inhibition (p lt 0001) was noticed with EEJR (0223 plusmn 0004) NHJR (0236 plusmn 0004) EETG

(0238 plusmn 0003) NHTG (0253 plusmn 0003) and indomethacin (0230 plusmn 0005) in contrast to control group

(0331 plusmn 0004) The percentage inhibition in these groups were found to be 3262 2870

2809 2356 and 2051 respectively The maximum inhibition was shown by EEJR at

second hour

Figure 47 Serotonin At second hour



92

473 At 3rd hr

Substantial inhibition (p lt 0001) of edema was observed with EEJR (0206 plusmn 0003) NHJR (0225 plusmn

0005) EETG (0183 plusmn 0004) NHTG (0241 plusmn 0001) and indomethacin (0195 plusmn 0003) in contrast

to control group (0340 plusmn 0004) The percentage inhibition in these groups were found to be 3941


by EEJR at third hour

Figure 48 Serotonin At third hour


in contrast to control group

93

This picture shows inhibition of paw edema induced by serotonin with experimental groups as compared to control group EEJR Ethanolic extract

of Juglans regia NHJR n-hexane extract of Juglans regia EETG Ethanolic extract of Trigonella gharuensis n-NHTG hexane extract of

Trigonella gharuensis Ind Indomethacin plt 0001 as compared to control group

94

Table 10 The anti-inflammatory properties of J regia and T gharuensis extracts on serotonin-induced paw edema model

Groups

1sthr 2ndhr 3rdhr

(Mean plusmn SEM)

(ml) Inhibition

(Mean plusmn SEM)

(ml) Inhibition

(Mean plusmn SEM)

(ml) Inhibition


- 0331 plusmn 0004 - 0340 plusmn 0004 -

EEJR

( 500 mgkg)

0236 plusmn 0005

2411

0223 plusmn 0004

3262

0206 plusmn 0003

3941

NHJR

( 500 mgkg)

0258 plusmn 0004

1704

0236 plusmn 0004

2870

0225 plusmn 0005

3382

EETG

( 400 mgkg) 0238plusmn 0006 2347 0238 plusmn 0003 2809 0218 plusmn 0004 3588

NHTG

( 400 mgkg) 0261 plusmn 0003 1607 0253 plusmn 0003 2356 0241plusmn 0001 2911

Indomethacin ( 10 mgkg) 0238 plusmn 0003

2347

0230 plusmn 0005

2051

0195 plusmn 0003

4264

EEJR Ethanolic extract of Juglans regia NHJR n-hexane extract of Juglans regia EETG Ethanolic extract of Trigonella gharuensis n-hexane

extract of Trigonella gharuensis p lt 001 and p lt 0001 as compared to control group

95

48 Pretreatment with J regia and T gharuensis inhibited dextran-induced paw edema

481 At 1st hr

EEJR (0216 plusmn 0004 p lt 0001) NHJR (0238 plusmn 0006 p lt 0001) EETG (0243 plusmn 0001 p lt 0001)

NHTG (0266 plusmn 0006 p lt 001) and diphenhydramine (0193 plusmn 0004 p lt 0001) extracts presented

anti-inflammatory activity in contrast to control group (0300 plusmn 0005) The percentage inhibition in

these groups were found to be 28 2066 19 and 1133 and 2667 respectively

The maximum inhibition was shown by EEJR at first hour

Figure 49 Dextran At first hour

Figure 49 depicts suppression of paw edema as a consequence of pre-treatment p lt 001 and

P lt 0001 in contrast with control group

96

482 At 2nd hr

An inhibition (p lt 0001) was detected with EEJR (0206 plusmn 0004) NHJR (0225 plusmn 0002) EETG (0226

plusmn 0004) NHTG (0245 plusmn 0008) and diphenhydramine (0195 plusmn 0004) in comparison with control

group (0316 plusmn 0003) The percentage inhibition in these groups were found to be 4801 2879

2848 2246 and 3829 respectively The maximum inhibition was shown by EEJR

at second hour

Figure 50 Dextran At second hour



97

483 At 3rd hr

EEJR (0193 plusmn 0004) NHJR (0205 plusmn 0006) EETG (0213plusmn 0004) NHTG (0223 plusmn 0006) and

diphenhydramine (0176 plusmn 0003) produced significant inhibitory effects (p lt 0001) in contrast with




Figure 51 Dextran At third hour



98

484 At 4th hr

EEJR (0175 plusmn 0002) NHJR (0193 plusmn 0004) EETG (0195 plusmn 0002) NHTG (0218 plusmn 0005) and

diphenhydramine (0168 plusmn 0003) prevented (p lt 0001) paw edema development in contrast with


4405 4347 3681 and 5130 The maximum inhibition was shown by EEJR at

fourth hour

Figure 52 Dextran At fourth hour

Figure 52 represents suppression of paw edema as a consequence of pre-treatment p lt 0001


99

485 At 5th hr


diphenhydramine (0161 plusmn 0006) caused significant prevention (p lt 0001) of edema in contrast with




Figure 53 Dextran At fifth hour



100


extract of Trigonella gharuensis DPH Diphenhydramine

101

Table 11 The anti-inflammatory properties of J regia and T gharuensis extracts on dextran-induced Paw edema model

Groups

1sth

2ndh

3rdh

4thh

5thh



0316 plusmn 0003 0336 plusmn 0005 0345 plusmn 0004 0353 plusmn 0006

EEJR

(500 mgkg)

0216 plusmn 0004

(28)

0206 plusmn 0004

(4801)

0193 plusmn 0004

(4255)

0175 plusmn 0002

(4927)

0173 plusmn 0004

(5099)

NHJR

(500 mgkg)

0238 plusmn 0006

(2066)

0225 plusmn 0002

(2879)

0205 plusmn 0006

(3898)

0193 plusmn 0004

(4405)

0183 plusmn 0009

(4815)

EETG

(400 mgkg)

0243 plusmn 0005

(19)

0226 plusmn 0004

(2848)

0213 plusmn 0004

(3660)

0195 plusmn 0002

(4347)

0183 plusmn 004

(4815)

NHTG

(400 mgkg)

0266 plusmn 0005

(1133)

0245 plusmn0005

(2246)

0223 plusmn 0006

(3363)

0218 plusmn 0005

(3681)

0205 plusmn 0006

(4192)

DPH

(60 mgkg)

0220 plusmn 0004

(2667)

0195 plusmn 0004

(3829)

0176 plusmn 0003

(4761)

0168 plusmn 0003

(5130)

0161 plusmn0 006

(5443)


hexane extract of Trigonella gharuensis DPH Diphenhydramine p lt 001 and p lt 0001 as compared to control group

102

49 Pretreatment with J regia and T gharuensis inhibited xylene-induced paw edema

Contrary to control group (0035 plusmn 0002) EEJR (0213 plusmn 0005 p lt 0001) NHJR (0026 plusmn

0006 p lt 0001) EETG (0033 plusmn 0001 p lt 0001) NHTG (0037 plusmn 0006 p lt 001) and

dexamethasone (0012 plusmn 0001 p lt 0001) showed substantial inhibition of ear edema

induced by xylene

Figure 54 Xylene induced ear edema

Figure 54 depicts significant suppression of ear edema as a consequence of pre-treatment p

lt 001 and p lt 0001 as compared with control group

103

413 Pretreatment with J regia and T gharuensis prevented castor oil- induced diarrhea

491 Onset time of diarrhea

Onset of diarrhea in groups pre-treated with EEJR (1922 plusmn 2136) NHJR (1652 plusmn 1302)

EETG (1947 plusmn 1838) NHTG (1742 plusmn 1537) and loperamide (218 plusmn 3559) was significantly

delayed (p lt 0001) as compared to control group (1095 plusmn 1335) Pretreatment with both

extracts significantly prolong onset time of diarrhea

Figure 55 Onset time of diarrhea

Figure 55 depicts that time of onset of diarrhea was prolonged as a consequence of pre-

treatment p lt 0001 as compared with control group

104

492 Number of wet feces

As compared to control group (9833 plusmn 0307) number of wet feces was significantly reduced

(p lt 0001) due to pretreatment with EEJR (4167 plusmn 0307) NHJR (5833 plusmn 0401) EETG

(5833 plusmn 0401) NHTG (7500 plusmn 0223) and loperamide (5333 plusmn 0210)

Figure 56 Number of wet feces

Figure 56 depicts that as a consequence of pretreatment number of feces were reduced p lt

0001 as compared with control group

105

493 Total weight of wet feces

Total weight of wet feces was significantly reduced (p lt 0001) with EEJR (2045 plusmn 1335)

NHJR (2575 plusmn 1118) EETG (210 plusmn 1693) NHTG (2608 plusmn 2330) and loperamide (1865 plusmn

2473) as compared to control group (4057 plusmn 1542)

Figure 57 Total weight of wet feces

Figure 57 depicts significant attenuation of total weight of feces as a consequence of pre-


106

494 Total number of feces

Total number of feces was significantly reduced (p lt 0001) with EEJR (4167 plusmn 0307) NHJR

(7500 plusmn 0341) EETG (6167 plusmn 0307) NHTG (9500 plusmn 0341) and loperamide (4500 plusmn

0223) in comparison to control group (1333 plusmn 0614)

Figure 58 Total number of feces

Figure 58 depicts reduction of total number of feces because of pre-treatment with extracts and

loperamide p lt 0001 as compared with control group

107

Table 12 Pretreatment with J regia and T gharuensis extracts inhibited castor oil-induced diarrhea

Parameters

Control group EEJR NHJR EETG NHTG Piroxicam

Mean plusmn SEM

Time of onset of

diarrhea (mins) 1095 plusmn 1335 1922 plusmn 2136 1652 plusmn 1302 1947 plusmn 1838 1742 plusmn 1537 218 plusmn 3559

Number of wet feces 9833 plusmn 0307 4167 plusmn 0307 5833 plusmn 0401 5833 plusmn 0401 75 plusmn 0223 5333 plusmn 0210

Total weight of feces

(grams) 4057 plusmn 1542 2045 plusmn 1335 2575 plusmn 1118 210 plusmn 1693 2608 plusmn 2330 1865 plusmn 2473

Total number of feces 1333 plusmn 0614 4167 plusmn 0307 7500 plusmn 0341 6167 plusmn 0307 9500 plusmn 0341 4500 plusmn 0223



108

410 Qualitative analysis of J regia and T gharuensis extracts

Qualitative analysis confirmed presence of many biological compounds such as alkaloids

saponins phenols terpenoids flavonoids and tannins in both extracts while it revealed the

absence of glycosides saponins and steroids as presented in Table 16

Table 13 Qualitative analysis of J regia and T gharuensis extracts

Tests EEJR NHJR EETG NHTG

Alkaloids + + + +

Glycosides _ _ _ _

Saponins _ _ _

Phenols + + + +

Terpenoids + + + +

Steroids _ _ _ _

Flavonoids + + + +

Tannins + _ + +

109

411 GC-MS analysis of J regia and T gharuensis extracts

Detail of GC-MS analysis of J regia and T gharuensis extracts was elucidated below

4111 Assessment of GC-MS analysis of J regia ethanolic extract

GC-MS analysis of EEJR revealed the presence of thirty compounds All compound names

retention times molecular formulae molecular weights and their chemical names are

presented in Table 17 Benzene (1-butylheptyl) Benzene (1-pentylheptyl) and Benzene (1-

pentyloctyl) were found in the highest concentration (12704 8340 and 6753

respectively) in EEJR These compounds belong to different classifications alkyl halides (1-

chlorododecane) aliphatic compounds (decane undecane dodecane and heptacosane)

aromatic compounds (Benzene (1-butylhexyl)- Benzene (1-butylheptyl)- Benzene (1-

pentylheptyl)- and Benzene(1-pentyloctyl) saturated fatty acid esters (methyl palmitate and

ethyl palmitate) saturated fatty acid esters (methyl palmitate ethyl palmitate 16-

Methylheptadecanoic acid methyl ester) plasticizer compound such as 12-

benzenedicarboxylic acid mono (2-ethylhexyl) ester and triterpene (squalene)

110

Table 14 List of identified constituents of ethanolic extract of J regia

Sr

No

Retention

Time

(Minute)

Total

Percentage

Name of Identified

Compound

Molecular

Formula

Molecular

Weight

(gmol)

Structures

1 4880 1062 1-chlorododecane C12H25Cl 204

2 5350 3956 Decane C10H22 142

3 6255 1169 (E)-3(10)-Caren-4-ol C10H16O 152

OH

CH2

CH3

CH3

4 6795 2182 Undecane C11H24 156

CH3

CH3

CH3 Cl

CH3

CH3

111

5 8311 0859 Dodecane C12H26 170

CH3

CH3

6 14886 3267 Benzene (1-butylhexyl)- C16H26 218

CH3

CH3

7 15077 2691 Benzene (1-propylheptyl)- C16H26 218

CH3

CH3

8 15461 2345 Benzene (1-ethyloctyl)- C16H26 218

CH3CH3

9 16236 2542 Benzene (1-methylnonyl)- C16H26 218 CH3

CH3

112

10 17045 12704 Benzene (1-butylheptyl)- C17H28 232

CH3

CH3

11 17246 6128 Benzene (1-propyloctyl)- C17H28 232

CH3

CH3

12 17664 5710 Benzene (1-ethylnonyl)- C17H28 232

CH3

CH3

13 18430 4841 Benzene (1-methyldecyl)- C17H28 232

CH3

CH3

14 18988 8340 Benzene (1-pentylheptyl)- C18H30 246

CH3

CH3

113

15 19101 4175 Benzene (1-butyoctyl)- C18H30 246

CH3

CH3

16 19327 5055 Benzene (1-propylnonyl)- C18H30 246

CH3

CH3

17 19745 4279 Benzene (1-ethyldecyl)- C18H30 246

CH3

CH3

18 20512 3754

Benzene (1-

methylundecyl)-

C18H30 246

CH3

CH3

19 20947 6753 Benzene (1-pentyloctyl)- C19H32 260

CH3

CH3

114

20 21086 4226 Benzene (1-butylnonyl)- C19H32 260

CH3

CH3

21 21313 3038 Benzene (1-propyldecyl)- C19H32 260

CH3 CH3

22 21748 2501 Benzene (1-ethylundecyl)- C19H32 260

CH3

CH3

23 22506 2311

Benzene (1-

methyldodecyl)-

C19H32 260

CH3 CH3

24 22758 1105 Methyl palmitate C17H34O2 270

CH3

O

O CH3

25 25475 0713 Methyl linoleate C19H34O2 294

CH3

O

O

CH3

115

26 25580 1165 Methyl linolenate C19H32O2 292

O

O

CH3

CH3

27 25885 0319 Methyl stearate C19H38O2 298 CH3

O

O

CH3

28 30178 0212

12-Benzenedicarboxylic

acid mono(2-ethylhexyl)

ester

C16H22O4 278

O

OH

O

O

CH3

CH3

29 32303 1309 Squalene C30H50 410

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

30 32738 0380 Heptacosane C27H56 380

CH3

CH3

116

117

1 Dodecane 1-chloro-

2 Decane

30( E )-3 (10)-Caren-4-ol

118

4 Undecane

5 Dodecane

6 Benzene (1-propylheptyl)

119

7 Benzene (1ethyloctyl)-

8 Benzene (1-methylonyl)-

9 Benzene (1-butylheptyl)-

120

10 Benzene (1-propyloctyl)-

11 Benzene (1-methyldecyl)-

12 Benzene (1-pentylheptyl)-

121

13 Benzene (1-butyloctyl)-

14 Benzene (1-propylonyl)-

15 Benzene (1-ethyldecyl)-

122

16 Benzene (1-methylundecyl)-

17 Benzene (1-pentyloctyl)-

18 Benzene (1-Butylonyl)-

123

19 Benzene (1-propyldecyl)

20

Benzene (1-ehtylundecyl)-

21Benzene (1-methyldodecyl)-

124

22 Hexadecanoic acid methyl ester

23 912-Octadecadienoic acid (ZZ)- methyl ester

24 91215 -Octadecadienoic acid methyl ester (ZZZ)-

125

25 Octadecanoic acid methyl ester

2612-Benzenedicarboxylic acid mono(2-ethylhexyl) ester

27 Heptacosane

126

4112 Assessment of GC-MS of n- hexane extract of J regia

GC-MS analysis of NHJR revealed presence of fifteen compounds in NHJR Ethyl palmitate

(16955) ethyl linolenate (1533) squalene (15138) ethyl linolelaidate (7868)

heptacosane (7554) vitamin E (6222) and ethyl stearate (5535) were found in the

highest concentration These compounds belong to different chemical classifications aromatic

compounds (Benzene (1-butylheptyl) Benzene (1-methylundecyl)- Benzene(1-pentylheptyl)

aliphatic compounds octadecane heptacosane octadecane and 3-ethyl-5-(2-ethylbutyl)

triterpene (squalene) naphthalone derivative (388-Trimethoxy-3-piperidyl-22-

binaphthalene-1144-tetrone) sesquiterpene lactone (224-trimethyl-1233a68a-

hexahydroazulene-57-dicarbaldehyde also known as costunolide) unsaturated fatty acid

esters (methyl lineoleate and linolenic acid methyl ester) saturated fatty acid esters (ethyl

palmitate) diterpene alcohol (phytol) and tocopherol (vitamin E) (Table 18)

127

Table 15 List of identified constituents of n-hexane extract of J regia

Sr

No

Retention

Time

(Minute)

Total

Percentage

Name of Identified

Compound

Molecular

Formula

Molecular

Weight

(gmol)

Structures

1 16845 2133 Benzene (1-butylheptyl) C17H28 232

CH3

CH3

2 17498 4905

Velleral

C15H20O2 232 CH3

CH3

CH3O

O

3 20372 2361

Benzene (1-

methylundecyl)-

C18H30 246

CH3

CH3

128

4 20773 2105

Benzene (1-

pentylheptyl)

C18H30 246

5 20930 2199

ZZZ-1469-

Nonadecatetraene

C19H32 260

CH3 CH2

6 23934 16955 Ethyl palmitate C18H36O2 284

CH3

O

O

CH3

7 25737 4597 Phytol C20H40O 296

OH CH3

CH3

CH3

CH3

129

8 26311 7868 Ethyl linolelaidate C20H36O2 308 O

CH3

O

CH3

9 26407 15553 Ethyl linolenate C20H34O2 306 O CH3

O

CH3

10 26686 5535 Ethyl stearate C20H40O2 312 O

CH3

O

CH3

11 30178 2740

388-Trimethoxy-3-

piperidyl-22-

binaphthalene-1144-

tetrone

C28H25NO7 487

N

O

O

O

O

O

O

CH3CH3

O

CH3

130

12 32294 15138 Squalene C30H50 410

CH3

CH3

CH3

CH3CH3

CH3

CH3

CH3

13 32730 4184

Octadecane3-ethyl-5-(2-

ethylbutyl)-

C26H54 366 CH3

CH3

14 34193 7504 Heptacosane C27H56 380 CH3

CH3

15 34759 6222 Vitamin E C29H50O2 430 O

CH3

CH3

OH

CH3

CH3

CH3

CH3

CH3

131

132


2 56-Azulenedicarboxaldehyde 123a8a-Hexahydro-228-trimethyl-


133


5 ZZZ ndash 1469-Nonadecatetraene

6 Hexadecanoic acid ethyl ester

134

7 Phytol

8 912-Octadecadienoic acid ethyl ester

9 91215-Octadecatrienoic acid ethyl ester (ZZZ)-

135

10 Octadecanoic acid ethyl ester

11 3rsquo88rsquoTrimethoxy-3-piperidyl22rsquobinaphthalene-11rsquo44rsquo-tetrone

6 Squalene

136

7 Octadecane 3-ethyl-5-(2-ethylbutyl)-

8 Heptacosane

9 Vitamin E

137

4113 GC-MS analysis of ethanolic extract of T gharuensis

Assessment of GC-MS analysis of ethanolic extract of T gharuensis revealed presence of

fifteen compounds 2H-1-Benzopyran-2-one (53050) 1H-2-Benzopyran-1-one34-dihydro

(15848) and ethyl palmitate (6588) were found in the highest concentrations These

compounds belong to different classifications the unsaturated fatty acids esters (ethyl

linolenate and ethyl linoelaidic acid) saturated fatty acid esters (ethyl myristate ethyl laurate

ethyl stearate and ethyl palmitate) diterpene (phytol) coumarins (2H-1-Benzopyran-2-one

and 1H-2-Benzopyran-1-one34-dihydro) aromatic compound (2-methylbenzaldehyde) and

aliphatic compounds (Nonacosane 3-Ethyl-5-(2-ethylbutyl)octadecane and hexatriacontane)

(Table 19)

138

Table 16 List of identified constituents of ethanolic extract of T gharuensis

SrNo Retention

Time (mins)

Total

(age)

Name of Identified

Compound Mol Formula

MW

(gmol) Structures

1 8398 0430 Benzaldehyde 2-methyl- C8H8O 120

O

CH3

2 12186 15848 1H-2-Benzopyran-1-

one34-dihydro C9H8O2 148

OO

3 13588 53050 2H-1-Benzopyran-2-one C9H6O2 146 OO

4 15983 1342 Dodecanoic acid ethyl

ester C14H28O2 228

5 16175 1580

Tetracyclo

[33101(39)]decan-10-

one

C10H12O 148 OO

139

6 20093 0951 Tetradecanoic acid ethyl

ester C16H32O2 256

CH3

O

CH3

O

7 23803 1281 n-Hexadecanoic acid C16H32O2 256 CH3

OH O


CH3

O

O

CH3

9 25737 0614 Phytol C20H40O 296 OH CH3

CH3

CH3

CH3

10 26320 2475 912-Octadecadienoic acid

ethyl ester C20H36O2 308

O

CH3

O

CH3

140

11 26425 4714 91215-Octadecatrienoic

acid ethyl ester C20H34O2 306

O CH3

O

CH3


CH3

O

CH3

13 32764 6519 Nonacosane C29H60 408 CH3

CH3

14 33435 0459 Octadecane3-ethyl-5-(2-

ethylbutyl)- C26H54 366 CH3

CH3

15 34210 3039 Hexatriacontane C36H74 506 CH3

CH3

141

142

1 Benzaldehyde 2-methyl-

2 1H-2-Benzopyran-1-one 34-dihydro-


143

4 2H-1-Benzopyran-2-one

5 Dodecanoic acid ethyl ester

6 Tetracyclo[33101(39)]decan-10-one

144

7 Tetradecanoic acid ethyl ester

8 n-Hexadecanoic acid


145

10 Phytol



146


14 Nonacosane


147

16 Hexatriacontane

148

4114 GC-MS analysis of T gharuensis n-hexane extract

Assessment of T gharuensis extract revealed presence of thirty-three compounds Benzene

(1-butylheptyl)- (11968) Benzene (1 pentylheptyl)- (8653) and Benzene (1-

pentyloctyl)- (6996) were found in the highest concentration The unsaturated fatty acids

esters (Ethyl linolenate Ethyl linolelaidate Ethyl stearate and Linolenic acid methyl ester)

saturated fatty acid esters (tetradecanoic acid ethyl ester Ethyl palmitate) diterpene (phytol)

triterpene (squalene) aromatic compounds (benzene-1-ethyl-3-methyl) decan-5-ylbenzene

Dodecan-6-ylbenzene benzene-1-pentyloctyl) flavonoids (2H-1-Benzopyran-2-one)

aliphatic compounds (decane undecane dodecane nonacosane Octadecane 3-ethyl-5-2-

ethylbutyl tetratriacontane and hexatriacontane) naphthalone derivative (388-Trimethoxy-3-

piperidyl-22-binaphthalene-1144-tetrone) Tetracyclo [33101(39)]decan-10-one (Table

20)

149

Table 17 GC-MS analysis of T gharuensis n-hexane extract

Sr

No

Retention

Time

(mins)

Total

(age) Name of Identified Compound

Mol

Formula

MW

(gmol) Structures

1 4914 1538 Benzene 1-ethyl-3-methyl- C9H12 120

CH3

CH3

2 5358 3731 Decane C10H22 142 CH3

CH3

3 5663 0376 Decane 4-methyl- C11H24 156 CH3

CH3

CH3

4 6264 1048 (E)-3(10)-Caren-4-ol C10H16O 152

OH

CH2

CH3

CH3

150

5 6804 1990 Undecane C11H24 156 CH3

CH3

6 8311 0750 Dodecane C12H26 170 CH3

CH3



CH3

CH3


CH3

CH3

10 15486 2170 Benzene (1-ethyloctyl)- C16H26 218 CH3

CH3

151

11 16262 2355 Benzene (1-methylnonyl)- C16 H26 218 CH3

CH3


CH3

CH3


CH3

CH3

14 17698 5073 Benzene (1-ethylnonyl)- C17H28 232 CH3

CH3

15 18465 4620 Benzene (1-methyldecyl)- C17H28 232 CH3

CH3

152

16 19048 8653 Benzene (1 pentylheptyl)- C18H30 246

CH3

CH3


CH3

CH3

18 19789 4412 Benzene (1-ethyldecyl) C18H30 246

CH3

CH3

19 20555 3701 Benzene (1-methylundecyl) C18H30 246 CH3

CH3


CH3

CH3

21 21138 4273 Benzene (1-butylnonyl) C19H32 260

CH3

CH3

153

22 21365 3206 Benzene (1-propyldecyl) C19H32 260

CH3 CH3

23 21792 2679 Benzene (1-ethylundecyl) C19H32 260

CH3

CH3

24 22540 2403 Benzene (1-methyldodecyl)- C19H32 260 CH3 CH3

25 22749 0590 Hexadecanoic acid methyl ester C17H34O2 270 CH3O

O

CH3

26 23986 1301 Hexadecanoic acid ethyl ester C18H36O2 284

CH3

O

O

CH3

27 25562 0356 91215- Octadecatrienoic acid

methyl ester (ZZZ)- C19H32O2 292

O

O

CH3

CH3

154

28 26320 0252 912- Octadecatrienoic acid ethyl

ester C20H36O2 308

O

CH3

O

CH3


ethyl ester (ZZZ)- C20H34O2 306

O CH3

O

CH3

30 26694 0214 Octadecanoic acid ethyl ester C20H40O2 312 O

CH3

O

CH3

31 30187 0535 12- Benzenedicarboxylic acid

mono(2-ethylhexyl) ester C16H22O4 278

O

OH

O

O

CH3

CH3

155

32 32799 2525 Nonacosane C29H60 408

CH3

CH3

33 34227 0957 Tetratriacontane C34H70 478 CH3

CH3

156

157

1 Decane

2 Decane 4-methyl-

3 (E)-3(10)-caren-4-ol

158

3 Undecane

4 Dodecane


159

7 Benzene (1-butylhexyl)-

8 Benzene (1-propylheptyl)-

9 Benzene (1-ethyloctyl)-

160

10 Benzene (1-methylnonyl)-


12 Benzene (1-propyloctyl

161

13 Benzene (1-ethylnonyl)-



162

17 Benzene (1-propylnonyl)-


18 Benzene (1-methylundecyl)

163


20 Benzene (1-butylnonyl)-

21 Benzene (1-propyldecyl

164

22 Benzene (1-ethylundecyl)-

23 Benzene (1-methyldodecyl)-


165


26 9 12 15-Octadecatrienoic acid methyl ester (ZZZ)-

27 912-Octadecadienoic acid ethyl ester28

166

28 9 12 15-Octadecatrienoic acid ethyl ester (ZZZ)-



167

31 Nonacosane

32 Tetratriacontane

33 Benzene 1-ethyl-3-methyl

168

CHAPTER 5 DISCUSSION

The roles of anti-inflammatory and immunomodulatory substances in treating chronic

inflammatory conditions are well established RA is characterized by over production of TNF-

α NF-ĸB IL-6 IL-1β and PGE2 and reduced levels of anti-inflammatory IL-4 Substances

which down regulate pro-inflammatory cytokines have gained importance in the treatment of

inflammatory disorders (58 60)

J regia and T gharuensis have been frequently used for the treatment of arthritis in folk

medicine (56) FCA-induced model of rheumatoid arthritis is preferred due to its similarities

with human arthritic disorder characterized by synovial hyperplasia vascular formation

cartilage destruction and bone erosion (12) The pannus formation is a hallmark of RA which

is mainly composed of macrophages and fibroblasts like synoviocytes (73) It is believed that

erosion observed during rheumatic diseases is mainly due to TNF-α IL-6 and IL-1β (74) J

regia and T gharuensis extracts significantly attenuated bone erosion and pannus formation in

joints which was ascribed to attenuation of pro-inflammatory cytokines levels

The key focus in the treatment of RA is to reduce inflammation caused by pro-inflammatory

cytokines TNF-α is considered as main cytokine involved in the inflammatory disorders It

activates IL-6 in both paracrine and autocrine manner Activation of fibroblasts by TNF-α tends

to destroy cartilage by producing matrix degrading enzyme (58) NF-ĸB is associated with

resorption of bone by differentiation besides activation of osteoclasts These events also lead

to the development of Th1 response It is a transcription factor which activates other cytokines

IL-6 TNF-α in addition IL-1β and causes recruitment neutrophils as well as activation COX-

2 is activated by NF-ĸB thus amplifying prostaglandin production and inflammatory response

(5) This study showed that J regia and T gharuensis significantly decreased TNF-α and NF-

169

ĸB mRNA expression levels This might had caused minimizing of bone erosion inflammation

and formation of pannus These consequences are in line with the previous study (2)

IL-6 was considered a major instigator of joint inflammation in RA Earlier studies showed

that IL-1β and TNF-α are the major inducers of IL-6 and these are further responsible for

synovial inflammation and cartilage destruction in arthritic patients (75) It was a pleiotropic

cytokine maintaining hematopoiesis and bone metabolism It also regulated many immune

responses Increased production of IL-6 results into systemic inflammation and auto-immune

disorders It is responsible for osteoporosis and joint destruction in RA patients Inhibition of

IL-6 can prevent osteoclast activity in RA patients (76) IL-1β is more abundant in the

destructive tissues of RA patients (77) It is over expressed in inflamed synovial tissue

particularly in synovial lining and is elevated in lymphatic drainage of affected joints In

addition it is localized in affected joint thus causing pannus formation in the arthritic patients

(78) An increase in IL-1β levels in joints has been associated with the histological features of

RA There is a reason to believe that IL-1β is an essential cytokine that promotes inflammation

and joint destruction in RA (6) J regia and T gharuensis attenuated the mRNA levels Our

outcomes of this research study are similar with the inferences of already published study

which established that attenuated levels of IL-1β and IL-6 reduced cartilage and bone

destruction (79)

IL-4 mediates anti-inflammatory response and repression of macrophage activation (80)

Moreover it is responsible for negative regulation of NF-ĸB by increasing IКB IL-4 inhibits

osteoclasts which is liable for destruction of cartilage and bone erosion occur in arthritic

patients (81) Therapy with recombinant IL-4 have resulted in the inhibition of cytokine

production in patients suffering from RA (82) J regia and T gharuensis extracts significantly

170

increased IL-4 levels thus reducing RA development by upregulating anti-inflammatory

response

COX-1 and COX-2 enzymes possess effective physiological roles in biological signaling

pathways COX-1 entails homeostatic and inflammation-modulating potentials while COX-2

induces generation of prostaglandins to carry out inflammatory and pain outcomes (83) COX-

2 is over expressed in RA resulting in the over-production of PGE2 (84 85) Various studies

describe functions of COX-1 in maintaining the gastric mucosa integrity facilitating normal

platelet function and regulating renal blood flow The COX-1 and COX-2 levels were

significantly reduced by J regia and T gharuensis which could result in the inhibition of PGE2

levels However the extent of COX-1 suppression did not significantly counter the constitutive

outcomes of COX-1 which is in line with the results of other studies that the constitutive

functions of this enzyme are hardly affected in COX-1 knock-out mice (83)

We evaluated the levels of PGE2 and found high levels in arthritic control group (86) Binding

of PGE2 to prostaglandin receptors initiate the process of erosion of juxta-articular cartilage

(58) Treatment with J regia and T gharuensis attenuated PGE2 levels These outcomes are

in line with the study of (60 73) who showed that suppression of PGE2 was involved in bone

remodeling and suppression of inflammation caused by arthritis The considerable reduction of

TNF-α IL-6 IL-1β and increased levels of IL-4 in extracts treated groups confirm

immunomodulatory role of both extracts (87)

Hb content and RBC counts were reduced in arthritic control group which indicate anemic

condition in rats The anemia might be associated to low plasma iron levels that is negatively

correlated with significantly higher IL-6 levels (88 89) Bone marrow produced inadequate

cells in anemia and aberrant deposits of iron in synovial tissues (12) J regia and T gharuensis

171

nearly normalized these altered hematological parameters which are in accordance with the

result of the previous study (10) On the other hand WBC and platelet counts were found

increased in blood samples IL-6 could be responsible for high rise in leukocyte levels and

platelets (90 91) Treatment with J regia and T gharuensis restored white blood cell counts

and platelets Increased levels of ALP may be responsible for bone destruction and

mineralization (58) Both plant extracts and piroxicam significantly reduced ALP levels in

treatment groups To find out the safety of extracts we measured different biochemical

parameters for instance urea creatinine ALT and AST levels The results explained that no

significant difference was observed among all groups Hence validating the safety of extracts

in term of liver and renal parameters

Inflammation is a normal body response to harmful agents in order to get rid of noxious stimuli

but uncontrolled inflammation may lead to tissue damage (92) Modern scientific research is

inclined towards treatment of inflammation with natural products having least side effects and

being cheapest source (18) Investigation of anti-inflammatory activities of J regia and T

gharuensis extracts were performed using carrageenan as phlogistic agents while various other

agents were used to determine the autacoids inhibition as one the anti-inflammatory

mechanisms by extracts of plants Different supporting models like dextran-induced paw

edema castor oil-induced diarrhea and xylene-induced ear edema were also developed to

strengthen the inferences (93)

An excellent model was used for elucidation of the cascade of complex events such as

carrageenan-induced edema especially the interactions of autacoids as an anti-inflammatory

mechanism The first phase (1-2 h) started with the release of histamine serotonin and kinin

while the second phase was ascribed with the release of prostaglandin and bradykinins (12)

Extracts of J regia and T gharuensis inhibited paw edema at first stage and prevention was

172

also observed till fifth hours indicating its efficacy at second stage Results were authenticated

with the inferences of reported literature (56 94)

To determine possible inhibitory effect on markers involved in first phase we further

developed serotonin- as well as histamine- induced paw edema models Histamine and

serotonin increase vascular permeability and are potent vasodilators which in turn accumulate

fluids and leukocytes imparting edema and inflammation (95) All these events contribute

towards principal signs of inflammation It was previously suggested that inhibitory effect of

plant extracts determined using both serotonin- and histamine- induced paw edema models

might be ascribed to the inhibition of releasesynthesis of these autacoids (12) Significant

inhibitions were observed in both models when they were pre-treated with extracts of plants

Similar to the results of carrageenan-induced paw edema pre-treatment with ethanolic and n-

hexane extracts caused significantly higher prevention of paw edema These results were

further validated through dextran-induced paw edema model

Dextran-induced paw edema was commonly taken as an experimental model for inflammation

This model was accompanied by different mechanism of inflammation such as vascular

permeation which was increased due to release of serotonin and histamine followed by

activated kinins further promoted accumulation of neutrophils and osmotic edema having low

protein levels (96) The ethanolic as well as n-hexane extracts of J regia and T gharuensis

prevented dextran-induced paw edema significantly thus strengthening the inhibitory effects

of J regia and T gharuensis found using two different paw edema models induced by

histamine and serotonin

Xylene-induced ear edema was another model used to further strengthen inhibitory effects of

J regia and T gharuensis extracts on autacoids inhibitions Xylene mediates the release of

substance P an undecapeptide neurotransmitter which is widely distributed in central as well

173

as peripheral nervous system responsible for many physiological processes Vasodilation and

plasma extravasation caused by the release of substance P result into neurogenic inflammation

(97) Other studies described that mechanism of xylene induced inflammation was also in part

involved with the release of histamine and other mediators (98) The two suggested

mechanisms of inflammation are interlinked as substance P and histamine both induce the

release of each other Thus inhibition of substance P can negatively influence the release of

histamine and vice versa (99) Current study revealed that inhibition of paw edema induced by

histamine further validates attenuation of xylene-induced ear edema Thus inhibition of ear

edema further validated autacoids inhibition was another unique anti-inflammatory mechanism

of J regia and T gharuensis extracts

Castor-oil induced diarrhea model was used to evaluate role of prostaglandins in inflammatory

conditions (100) Castor oil is metabolized to ricinoleic acid which irritates the gastric mucosa

The irritation of mucosa in turn releases prostaglandin which stimulates intestinal motility and

mucus secretion (101) Both ethanolic and n-hexane extracts of J regia and T gharuensis

significantly prevented diarrhea Inhibition of castor oil-induced diarrhea validated inhibitory

effect of plants extracts in 2nd stage of carrageenan-induced paw edema Thus plants extracts

inhibited prostaglandin which was also suggested along with inhibition of histamine and

serotonin as underlying mechanisms of anti-inflammatory activity Plant extracts having

important biological compounds such as tannin flavonoids and alkaloids had been described

to have considerable anti-diarrheal activity (102 103) Flavonoids especially played an

imperative part in inhibiting synthesis of prostaglandin (104 105) Qualitative analysis of

plants extracts revealed that these contained flavonoids alkaloids and tannins in both plant

extracts which might be attributed anti-diarrheal activity and prostaglandin inhibitory activity

Anti-inflammatory activities are also frequently reported with detected phytochemical

constituents in current study (106)

174

Experimental studies of acute toxicity of plants extracts revealed that extracts were found

nontoxic at 2 gkg for J rgia and 16 gkg for T gharuensis The extract did not show any sign

of mortality and behavior changes This is in line with findings of Ruijun et al (53)

The GC-MS method is considered a direct and precise technique to evaluate the presence of

medicinally important components in plant extracts The analysis showed that J regia and T

gharuensis possesses different constituents with previously reported anti-inflammatory and

anti-oxidant properties such as methyl linoleate phytol along with squalene (107 108)

Presence of phytol ethyl palmitate and nonacosane impart anti-inflammatory activity to EETG

and to n-hexane NHTG extract Costunolide (224-trimethy-1233a68a-hexahydroazulene-

57-dicarbaldehyde) present in n-hexane extract of J regia significantly reduced

inflammation by inhibiting NF-kB (Chaturvedi et al 2011) and inhibits IL-1β expression

levels (109) hence validating the anti-inflammatory properties of J regia and T gharuensis

extracts The 388-Trimethoxy-3-piperidyl-22-binaphthalene-1144-tetrone present in n-

hexane extract of J regia also attenuated inflammation because it possessed considerable anti-

inflammatory and anti-arthritic properties (110) Ezhilan and Neelamegam had stated that 12-

Benzenedicarboxylic acid mono (2-ethylhexyl) ester retained considerable anti-inflammatory

in addition to antioxidant properties and interestingly we have found this compound in our

extract (111)

175

CHAPTER 6 CONCLUSION

The current study validates the traditional use of J regia and T gharuensis in inflammatory

disorders and RA Amelioration of joint inflammation in both acute and chronic models of

inflammation highlighted that J regia and T gharuensis extracts possessed significant

immunomodulatory and anti-inflammatory properties The attenuation of RA might be

ascribed to downregulation of pro-inflammatory markers such as TNF-α IL- -6 NF-kB COX-

1 and COX-2 and up-regulation of anti-inflammatory IL-4 Moreover PGE2 levels were also

found reduced after treatment with plant extracts The inhibition of autacoids in different

models of acute inflammation is suggested as one of the possible mechanisms of anti-

inflammatory activity Further studies are warranted to isolate active phytochemicals which

are responsible for anti-arthritic and anti-inflammatory effects

176

FUTURE PERSPECTIVE

J regia and T gharuensis possess considerable anti-arthritic compounds and anti-

inflammatory compounds which should be separated on the basis of activity-based

fractionation and isolation of compounds

Plants extracts should be administered through specific targeted drug delivery system

J regia and T gharuensis attenuated FCA induced arthritis by downregulating of pro-

inflammatory cytokines and upregulating of anti-inflammatory cytokine therefore

plants extracts may be effective in treatment of other inflammatory disorders such as

asthma ulcer gout and acute hepatitis etc

There is a need to further evaluate reproductive mutagenic teratogenic and toxicity

studies of J regia and T gharuensis extracts so as to determine their safe use in special

population groups such as animals of childbearing potential pregnant females and

neonates

Clinical studies are required to evaluate safety and efficacy of extracts on human

population

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arthritis Chinese Journal of Cell Biology 20092

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Biomed Pharmcother 20181212-21

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rats Inflammation 201235(4)1314-21

58 Shabbir A Shahzad M Ali A Zia-ur-Rehman M Discovery of New Benzothiazine

Derivative as Modulator of Pro-and Anti-inflammatory Cytokines in Rheumatoid Arthritis

Inflammation 201639(6)1918-29

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dihydroxyphenyl) methylidene]-2-(3 4-dimethyl-5 5-dioxidopyrazolo [4 3-c][1 2]

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rats by downregulating PGE2 COX2 IL-1β IL-6 TNF-α NF-kB and upregulating IL-4 and

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and Toxicology 20121(2)14-7

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phytology 2011

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pathogenesis of rheumatoid arthritis and as a novel therapeutic strategy Endocrine Metabolic

amp Immune Disorders-Drug Targets (Formerly Current Drug Targets-Immune Endocrine amp

Metabolic Disorders) 20066(4)383-94

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Freundrsquos complete adjuvant-induced arthritis in rats via inhibiting P2X7NF-κB pathway

Chemico-biological interactions 201523641-6

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production of IL-6 and IL-8 in rheumatoid arthritis synovial fibroblasts via NF-κB-and PI3-

kinaseAkt-dependent pathways Arthritis Res Ther 20046(2)R120

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(SAMURAI) evidence of clinical and radiographic benefit from an x ray reader-blinded

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7

77 Smeets T Barg E Kraan M Smith M Breedveld F Tak P Analysis of the cell infiltrate

and expression of proinflammatory cytokines and matrix metalloproteinases in arthroscopic

synovial biopsies comparison with synovial samples from patients with end stage destructive

rheumatoid arthritis Annals of the rheumatic diseases 200362(7)635-8

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effects of magnolol in human interleukin 1β-stimulated fibroblast-like synoviocytes and in a

rat arthritis model PLoS One 20127(2)e31368

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extracts of Circaea mollis Sieb amp Zucc(whole plant) in rodents Journal of

ethnopharmacology 2018225359-66

80 Aslam H Shahzad M Shabbir A Irshad S Immunomodulatory effect of thymoquinone

on atopic dermatitis Molecular immunology 2018101276-83

81 Lubberts E Joosten LA Chabaud M van den Bersselaar L Oppers B Coenen-de Roo

CJ et al IL-4 gene therapy for collagen arthritis suppresses synovial IL-17 and osteoprotegerin

ligand and prevents bone erosion The Journal of clinical investigation 2000105(12)1697-

710

82 Fraser D Thoen J Reseland J Foslashrre Oslash Kjeldsen-Kragh J Decreased CD4+

lymphocyte activation and increased interleukin-4 production in peripheral blood of

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401

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inflammatory cytokines gene expression by moringa leaves and its aqueous extract in aspirin-

induced gastric ulcer rats Molecular biology reports 201946(4)4213-24

84 Myers LK Kang AH Postlethwaite AE Rosloniec EF Morham SG Shlopov BV et

al The genetic ablation of cyclooxygenase 2 prevents the development of autoimmune arthritis

Arthritis amp Rheumatism 200043(12)2687-93

85 Woods JM Mogollon A Amin MA Martinez RJ Koch AE The role of COX-2 in

angiogenesis and rheumatoid arthritis Experimental and molecular pathology

200374(3)282-90

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factor-kappaB pathways potential implications for autoimmunity and rheumatoid arthritis

Arthritis research amp therapy 200810(4)212

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immunomodulatory potential and their therapeutic use in rheumatoid arthritis Int J Pharm Sci

Res 201910(5)2087-100

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evaluation of the effects of treatment with tocilizumab and TNF-α inhibitors on serum hepcidin

anemia response and disease activity in rheumatoid arthritis patients Arthritis research amp

therapy 201315(5)R141

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arthritisndashlike syndrome in mice following stimulation of the immune system with Freunds

complete adjuvant regulation by interferon‐γ Arthritis amp rheumatology 201466(5)1340-51

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and TNF-α sustain neutrophil recruitment during inflammation through synergistic effects on

endothelial activation The Journal of Immunology 20121200385

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platelet volume levels and inflammation in SLE patients presented with arthritis African health

sciences 201414(4)919-24

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of Strophanthus hispidus DC(Apocynaceae) International Journal of Applied Research in

Natural Products 20124(4)7-14

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protocols Springer 2003 p 115-21

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Biological activities of Juglans regia flowers

Brazilian Journal of Pharmacognosy

3(21)465-70

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and anti-nociceptive properties of Blepharis maderaspatensis leaves Revista Brasileira de

Farmacognosia 201323(5)830-5

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derivatives on formaldehyde- carrageenan- and dextran-induced acute paw edema in rats

Biological and Pharmaceutical Bulletin 200124(10)1133-6

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activities of total extract and individual fractions of Chinese medicinal plants Polyrhachis

lamellidens Biol Pharm Bull 200528(1)176-80

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mechanisms of action of the petroleum ether fraction of Rosa multiflora Thunb hips J

Ethnopharmacol 2011138(3)717ndash22

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Pharmacol 170(1)38ndash45

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gigantea R Br in experimental animals J Pharm Pharm Sci 20047(1)70-5

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theophylline and cholera exotoxin upon transmucosal water and electrolyte movement in the

canine jejunum Gastroenterology 197160(1)22-32

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Intestinal Motility and Secretion by Flavonoids in Mice and Rats Structure‐activity

Relationships Journal of pharmacy and pharmacology 199345(12)1054-9

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Antidiarrhoeal activity of an ethanol extract of the stem bark of Piliostigma reticulatum

(Caesalpiniaceae) in rats African Journal of Traditional Complementary and Alternative

Medicines 20129(2)242-9

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lipophilic flavonol from Tanacetum parthenium Phytochemistry 199538(1)267-70

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mammalian cells implications for inflammation heart disease and cancer Pharmacological

reviews 200052(4)673-751

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from Abrus precatorius Linn Int J Pharm Bio Sci 20134(3)8

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ETHANOL LEAF EXTRACTS FROM ABRUS PRECATORIUS LINN 2013

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extract Asian J Pharm Clin Res 20125(2)90-4

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expression by down-regulation of AP-1 and MAPK activity in LPS-stimulated RAW 2647

cells Biochemical and biophysical research communications 2004313(1)171-7

110 Al-Tameme HJ Hameed IH Idan SA Hadi MY Biochemical analysis of Origanum

vulgare seeds by fourier-transform infrared (FT-IR) spectroscopy and gas chromatography-

mass spectrometry (GC-MS) Journal of Pharmacognosy and Phytotherapy 20157(9)221-37

111 Ezhilan BP Neelamegam R GC-MS analysis of phytocomponents in the ethanol

extract of Polygonum chinense L Pharmacognosy Research 20124(1)11

In the name of ALLAH

The Most Beneficial

The Most Merciful









Aisha Mobashar

October 2019





Shabbir




Student Name


Supervisor Name


Associate Professor

Co-Supervisor Name


Professor



Signature Date

Name of HOD


Signature Date

i

DEDICATION



ii

ACKNOWLDGEMENTS





















iii









my dreams

Aisha Mobashar

iv

TABLE OF CONTENTS

DEDICATION i

ACKNOWLDGEMENTS ii


LIST OF TABLES xi



ABSTRACT xix


11 Hypothesis 3






21 Epidemiology 4


23 Treatments 7

24 Inflammation 10


26 Plants used 12






v


















buffer (50X) 22


349 Densitometry 23









vi




carrageenan 28


histamine 28


serotonin 28


dextran 29



















vii






421 At Day 8 39

422 At Day 15 40

423 At Day 22 41

424 At Day 28 42







marker 57


levels of TNF-α 57



levels 59


levels of IL-1β 60






levels of IL-4 63

viii


64


parameters 66


count 66


content 67


count 68


platelet count 69


71



levels 72






gharuensis 77



451 At 1st hr 78

452 At 2nd hr 79

453 At 3rd hr 80

454 At 4th hr 81

ix

455 At 5th hr 82



461 At 1st hr 85

462 At 2nd hr 86

463 At 3rd hr 87



471 At 1st hr 90

472 At 2nd hr 91

473 At 3rd hr 92



481 At 1st hr 95

482 At 2nd hr 96

483 At 3rd hr 97

484 At 4th hr 98

485 At 5th hr 99











x





REFERENCES 177

xi

LIST OF TABLES

Page






40





66




T gharuensis

78



induced paw edema

84

xii



88



92



98



104






xiii

LIST OF FIGURES

Page

















xiv






of TNF-α

58


of NF-κB

59


of IL-6

60


of IL-1β

61


of COX-1

62


of COX-2

63


IL-4

64


xv


















xvi














xvii













Hb Haemoglobin

RBC Red blood cells






IL-6 Interleukin 6

IL-4 Interleukin 4



PXM Piroxicam

xviii

IND Indomethacin

DPH Diphenhydramine

Dexa Dexamethasone

LOP Loperamide



I2 Iodine


KI Potassium iodide


DW Distilled water

SM

bw

PO

Synovial membrane

Body weight

Per oral

xix



ABSTRACT




respectively
















xx
























xxi






1
























2

























3
















11 Hypothesis

111 Null hypothesis


properties



4














21 Epidemiology










5



22 Pathophysiology






















6

























7


















23 Treatments







8









cyclooxygenase (42)

















9

























10
















24 Inflammation









11









(45)







immunologic (46)






12










26 Plants used















13








(51)














14

Aim of the study



Objective of study













GC-MS analysis

15


31 Plant collection





name was checked




05














16





57)



















17









FCA (59)








342 (12)


2 and IL-4

351 RNA extraction


18









separated






3514 Washing of RNA







19






GAPDH


pairs sequence








20



IL-4


231 (58)


IL-6


247 (5)


TNF-α


202 NM_0126753


NF-КB


207 (58)


COX-1


200 (59)


COX-2


237 (58)


IL-1β


293 (58 60)


GAPDH


197 NM_0170084


21

353 cDNA synthesis





















22












Each one litre

Tris base 242 g


05M EDTA 100 ml


600ml




23




buffer











358 Densitometry






experiment

24




















immediately




25




five times










1800i)







26












Models Groups



EEJR (500 mgkg)

NHJR (500 mgkg )

EETG (400 mgkg)

NHTG (400 mgkg)




EEJR (500 mgkg)

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)



EEJR (500 mgkg)

27


water

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)




EEJR (500 mgkg)

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)




EEJR (500 mgkg)

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)




EEJR (500 mgkg)

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)

Loperamide (5 mgkg)



bw = body weight

28























29




















30




















31








(68)





glycosides (70)


3841 Salkowski test







red colour (71)

32






















33

CHAPTER 4 RESULTS


model








34









35









36









37

Figure 5 Paw edema



38










39


421 At Day 8








40

422 At Day 15









group

41

423 At Day 22









control group

42

424 At Day 28




0153)





43





44




Days







45











46









47











48


Parameters

Arthritic control

Piroxicam

EEJR

NHJR

EETG

NHTG

Mean plusmn SEM

Infiltration of






49







50





(Fig 15)

51





52





53





54





55





56



57











control groups

58






groups




control groups

59










60










61










62










63










64










65


marker

Markers

Vehicle

Control

Arthritic

Control Piroxicam

EEJR

NHJR

EETG

NHTG

Mean plusmn SEM


NF-κB


IL-6


IL-1β


COX-1







66







group





67











68





(1512 plusmn 0256)





69










70


Parameters


Mean plusmn SEM

RBC


Hb

(gdl)


WBC

(103Ul)


Platelets

(103Ul)




two control groups

71









in all groups

72







creatinine levels

73





other



creatinine levels

74





other



levels

75











76


Biochemical

Parameters

Vehicle


Mean plusmn SEM

Urea


Creatinine


AST


ALT




77









gharuensis


distress Diarrhea

Motor

activity

Posture


Group-1 - - - - - -

Group-1I - - - - - -

Group-1II - - - - - -

Group-1V - - - - - -

Group-V - - - - - -

Group-VI - - - - - -

Group-VII - - - - - -

Group-

VIII - - - - - -

GROUP-

IX - - - - - -

78


carrageenan

451 At 1st hr









79

452 At 2nd hr









80

453 At 3rd hr









81

454 At 4th hr









82

455 At 5th hr









83



84



Groups




EEJR

(500 mgkg)

0210 plusmn0003

(2708)

0195 plusmn 0002

(3275)

0172 plusmn0003

(4591)

0156 plusmn0004

(5411)

0125 plusmn 0003

(6508)

NHJR

(500 mgkg)

0235 plusmn 0002

(1840)

0218 plusmn 0003

(2482)

0206 plusmn0003

(3522)

0193 plusmn0003

(4323)

0175 plusmn 0003

(5111)

EETG

(400 mgkg)

0226 plusmn0004

(21387)

0236 plusmn 0005

(1866)

0231 plusmn0006

(2735)

0210 plusmn0006

(3823)

0195 plusmn 0008

(4553)

NHTG

(400 mgkg)

0255 plusmn0003

(1145)

0250 plusmn 0002

(1379)

0280 plusmn0010

(1194)

0260 plusmn0010

(2352)

0236 plusmn 0011

(3407)

Piroxicam

(10 mgkg)

0208 plusmn0007

(2777)

0213 plusmn 0004

(2655)

0210 plusmn0003

(3396)

0183 plusmn0004

(4617)

0165 plusmn 0003

(5391)



85


edema

461 At 1st hr









86

462 At 2nd hr









87

463 At 3rd hr









88



89


edema model

Groups

1sthr 2ndhr 3rdhr

(Mean plusmnSEM)

(ml)

Inhibition

(Mean plusmnSEM)

(ml)

Inhibition

(Mean plusmnSEM)

(ml)

Inhibition


- 0318 plusmn 0001 - 0321 plusmn 0004 -

EEJR

( 500 mgkg)

0235 plusmn 0002

2192

0220 plusmn 0003

3081

0186 plusmn 0004

4205

NHJR

( 500 mgkg)

0251 plusmn 0003

1860

0240 plusmn 0002

2452

0225 plusmn 0008

2990

EETG


NHTG


Indomethacin

(10 mgkg)

0238 plusmn 0003

2093

0223 plusmn 0002

2987

0203 plusmn 0004

3676



90


471 At 1st hr









91

472 At 2nd hr





second hour




92

473 At 3rd hr









93




94


Groups

1sthr 2ndhr 3rdhr

(Mean plusmn SEM)

(ml) Inhibition

(Mean plusmn SEM)

(ml) Inhibition

(Mean plusmn SEM)

(ml) Inhibition


- 0331 plusmn 0004 - 0340 plusmn 0004 -

EEJR

( 500 mgkg)

0236 plusmn 0005

2411

0223 plusmn 0004

3262

0206 plusmn 0003

3941

NHJR

( 500 mgkg)

0258 plusmn 0004

1704

0236 plusmn 0004

2870

0225 plusmn 0005

3382

EETG


NHTG



2347

0230 plusmn 0005

2051

0195 plusmn 0003

4264



95


481 At 1st hr









96

482 At 2nd hr





at second hour




97

483 At 3rd hr









98

484 At 4th hr





fourth hour




99

485 At 5th hr









100



101


Groups

1sth

2ndh

3rdh

4thh

5thh




EEJR

(500 mgkg)

0216 plusmn 0004

(28)

0206 plusmn 0004

(4801)

0193 plusmn 0004

(4255)

0175 plusmn 0002

(4927)

0173 plusmn 0004

(5099)

NHJR

(500 mgkg)

0238 plusmn 0006

(2066)

0225 plusmn 0002

(2879)

0205 plusmn 0006

(3898)

0193 plusmn 0004

(4405)

0183 plusmn 0009

(4815)

EETG

(400 mgkg)

0243 plusmn 0005

(19)

0226 plusmn 0004

(2848)

0213 plusmn 0004

(3660)

0195 plusmn 0002

(4347)

0183 plusmn 004

(4815)

NHTG

(400 mgkg)

0266 plusmn 0005

(1133)

0245 plusmn0005

(2246)

0223 plusmn 0006

(3363)

0218 plusmn 0005

(3681)

0205 plusmn 0006

(4192)

DPH

(60 mgkg)

0220 plusmn 0004

(2667)

0195 plusmn 0004

(3829)

0176 plusmn 0003

(4761)

0168 plusmn 0003

(5130)

0161 plusmn0 006

(5443)



102





induced by xylene




103










104








105








106








107


Parameters


Mean plusmn SEM

Time of onset of








108







Alkaloids + + + +

Glycosides _ _ _ _

Saponins _ _ _

Phenols + + + +

Terpenoids + + + +

Steroids _ _ _ _

Flavonoids + + + +

Tannins + _ + +

109















110


Sr

No

Retention

Time

(Minute)

Total

Percentage

Name of Identified

Compound

Molecular

Formula

Molecular

Weight

(gmol)

Structures


2 5350 3956 Decane C10H22 142

3 6255 1169 (E)-3(10)-Caren-4-ol C10H16O 152

OH

CH2

CH3

CH3

4 6795 2182 Undecane C11H24 156

CH3

CH3

CH3 Cl

CH3

CH3

111

5 8311 0859 Dodecane C12H26 170

CH3

CH3


CH3

CH3


CH3

CH3


CH3CH3


CH3

112


CH3

CH3


CH3

CH3


CH3

CH3


CH3

CH3


CH3

CH3

113


CH3

CH3


CH3

CH3


CH3

CH3

18 20512 3754

Benzene (1-

methylundecyl)-

C18H30 246

CH3

CH3


CH3

CH3

114


CH3

CH3


CH3 CH3


CH3

CH3

23 22506 2311

Benzene (1-

methyldodecyl)-

C19H32 260

CH3 CH3


CH3

O

O CH3


CH3

O

O

CH3

115


O

O

CH3

CH3


O

O

CH3

28 30178 0212



ester

C16H22O4 278

O

OH

O

O

CH3

CH3

29 32303 1309 Squalene C30H50 410

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

30 32738 0380 Heptacosane C27H56 380

CH3

CH3

116

117


2 Decane

30( E )-3 (10)-Caren-4-ol

118

4 Undecane

5 Dodecane


119




120




121




122




123


20



124




125



27 Heptacosane

126













127


Sr

No

Retention

Time

(Minute)

Total

Percentage

Name of Identified

Compound

Molecular

Formula

Molecular

Weight

(gmol)

Structures


CH3

CH3

2 17498 4905

Velleral

C15H20O2 232 CH3

CH3

CH3O

O

3 20372 2361

Benzene (1-

methylundecyl)-

C18H30 246

CH3

CH3

128

4 20773 2105

Benzene (1-

pentylheptyl)

C18H30 246

5 20930 2199

ZZZ-1469-

Nonadecatetraene

C19H32 260

CH3 CH2


CH3

O

O

CH3

7 25737 4597 Phytol C20H40O 296

OH CH3

CH3

CH3

CH3

129


CH3

O

CH3


O

CH3


CH3

O

CH3

11 30178 2740

388-Trimethoxy-3-

piperidyl-22-

binaphthalene-1144-

tetrone

C28H25NO7 487

N

O

O

O

O

O

O

CH3CH3

O

CH3

130

12 32294 15138 Squalene C30H50 410

CH3

CH3

CH3

CH3CH3

CH3

CH3

CH3

13 32730 4184


ethylbutyl)-

C26H54 366 CH3

CH3


CH3

15 34759 6222 Vitamin E C29H50O2 430 O

CH3

CH3

OH

CH3

CH3

CH3

CH3

CH3

131

132




133




134

7 Phytol



135



6 Squalene

136


8 Heptacosane

9 Vitamin E

137










(Table 19)

138


SrNo Retention

Time (mins)

Total

(age)

Name of Identified


MW

(gmol) Structures


O

CH3

2 12186 15848 1H-2-Benzopyran-1-


OO



ester C14H28O2 228

5 16175 1580

Tetracyclo

[33101(39)]decan-10-

one

C10H12O 148 OO

139


ester C16H32O2 256

CH3

O

CH3

O


OH O


CH3

O

O

CH3

9 25737 0614 Phytol C20H40O 296 OH CH3

CH3

CH3

CH3



O

CH3

O

CH3

140



O CH3

O

CH3


CH3

O

CH3

13 32764 6519 Nonacosane C29H60 408 CH3

CH3



CH3


CH3

141

142




143




144




145

10 Phytol



146


14 Nonacosane


147

16 Hexatriacontane

148












20)

149


Sr

No

Retention

Time

(mins)

Total


Mol

Formula

MW

(gmol) Structures


CH3

CH3

2 5358 3731 Decane C10H22 142 CH3

CH3


CH3

CH3

4 6264 1048 (E)-3(10)-Caren-4-ol C10H16O 152

OH

CH2

CH3

CH3

150

5 6804 1990 Undecane C11H24 156 CH3

CH3

6 8311 0750 Dodecane C12H26 170 CH3

CH3



CH3

CH3


CH3

CH3


CH3

151


CH3


CH3

CH3


CH3

CH3


CH3


CH3

152


CH3

CH3


CH3

CH3


CH3

CH3


CH3


CH3

CH3


CH3

CH3

153


CH3 CH3


CH3

CH3



O

CH3


CH3

O

O

CH3



O

O

CH3

CH3

154


ester C20H36O2 308

O

CH3

O

CH3



O CH3

O

CH3


CH3

O

CH3



O

OH

O

O

CH3

CH3

155

32 32799 2525 Nonacosane C29H60 408

CH3

CH3


CH3

156

157

1 Decane

2 Decane 4-methyl-

3 (E)-3(10)-caren-4-ol

158

3 Undecane

4 Dodecane


159




160




161




162




163




164




165




166




167

31 Nonacosane

32 Tetratriacontane


168
























169


















destruction (79)






170


response






















171

























172

























173


























174


















extract (111)

175












176

FUTURE PERSPECTIVE












neonates


population

REFERENCES


201262(8)1085-93





therapeutics 2019













of Medicine 2011365(23)2205-19



20191614-11









21















Medica 201682(05)PC63



of Pain 201822(3)455-70



201771955






reviews 201711(22)145




of Nutrition 200899(4)715-22














201933(10)1531-4


199123(1)90-114















reviews 20054(3)130-6
























Toxicology 2003188(1)49-71



198136(6)435-6










200155(4)204





201113(1)27



201547(2)177






200389(1)123-9






































phytology 2011



2010439-44

















7
















710




401









200374(3)282-90






Res 201910(5)2087-100




therapy 201315(5)R141









sciences 201414(4)919-24









3(21)465-70


























Medicines 20129(2)242-9





reviews 200052(4)673-751























Aisha Mobashar

October 2019





Shabbir




Student Name


Supervisor Name


Associate Professor

Co-Supervisor Name


Professor



Signature Date

Name of HOD


Signature Date

i

DEDICATION



ii

ACKNOWLDGEMENTS





















iii









my dreams

Aisha Mobashar

iv

TABLE OF CONTENTS

DEDICATION i

ACKNOWLDGEMENTS ii


LIST OF TABLES xi



ABSTRACT xix


11 Hypothesis 3






21 Epidemiology 4


23 Treatments 7

24 Inflammation 10


26 Plants used 12






v


















buffer (50X) 22


349 Densitometry 23









vi




carrageenan 28


histamine 28


serotonin 28


dextran 29



















vii






421 At Day 8 39

422 At Day 15 40

423 At Day 22 41

424 At Day 28 42







marker 57


levels of TNF-α 57



levels 59


levels of IL-1β 60






levels of IL-4 63

viii


64


parameters 66


count 66


content 67


count 68


platelet count 69


71



levels 72






gharuensis 77



451 At 1st hr 78

452 At 2nd hr 79

453 At 3rd hr 80

454 At 4th hr 81

ix

455 At 5th hr 82



461 At 1st hr 85

462 At 2nd hr 86

463 At 3rd hr 87



471 At 1st hr 90

472 At 2nd hr 91

473 At 3rd hr 92



481 At 1st hr 95

482 At 2nd hr 96

483 At 3rd hr 97

484 At 4th hr 98

485 At 5th hr 99











x





REFERENCES 177

xi

LIST OF TABLES

Page






40





66




T gharuensis

78



induced paw edema

84

xii



88



92



98



104






xiii

LIST OF FIGURES

Page

















xiv






of TNF-α

58


of NF-κB

59


of IL-6

60


of IL-1β

61


of COX-1

62


of COX-2

63


IL-4

64


xv


















xvi














xvii













Hb Haemoglobin

RBC Red blood cells






IL-6 Interleukin 6

IL-4 Interleukin 4



PXM Piroxicam

xviii

IND Indomethacin

DPH Diphenhydramine

Dexa Dexamethasone

LOP Loperamide



I2 Iodine


KI Potassium iodide


DW Distilled water

SM

bw

PO

Synovial membrane

Body weight

Per oral

xix



ABSTRACT




respectively
















xx
























xxi






1
























2

























3
















11 Hypothesis

111 Null hypothesis


properties



4














21 Epidemiology










5



22 Pathophysiology






















6

























7


















23 Treatments







8









cyclooxygenase (42)

















9

























10
















24 Inflammation









11









(45)







immunologic (46)






12










26 Plants used















13








(51)














14

Aim of the study



Objective of study













GC-MS analysis

15


31 Plant collection





name was checked




05














16





57)



















17









FCA (59)








342 (12)


2 and IL-4

351 RNA extraction


18









separated






3514 Washing of RNA







19






GAPDH


pairs sequence








20



IL-4


231 (58)


IL-6


247 (5)


TNF-α


202 NM_0126753


NF-КB


207 (58)


COX-1


200 (59)


COX-2


237 (58)


IL-1β


293 (58 60)


GAPDH


197 NM_0170084


21

353 cDNA synthesis





















22












Each one litre

Tris base 242 g


05M EDTA 100 ml


600ml




23




buffer











358 Densitometry






experiment

24




















immediately




25




five times










1800i)







26












Models Groups



EEJR (500 mgkg)

NHJR (500 mgkg )

EETG (400 mgkg)

NHTG (400 mgkg)




EEJR (500 mgkg)

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)



EEJR (500 mgkg)

27


water

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)




EEJR (500 mgkg)

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)




EEJR (500 mgkg)

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)




EEJR (500 mgkg)

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)

Loperamide (5 mgkg)



bw = body weight

28























29




















30




















31








(68)





glycosides (70)


3841 Salkowski test







red colour (71)

32






















33

CHAPTER 4 RESULTS


model








34









35









36









37

Figure 5 Paw edema



38










39


421 At Day 8








40

422 At Day 15









group

41

423 At Day 22









control group

42

424 At Day 28




0153)





43





44




Days







45











46









47











48


Parameters

Arthritic control

Piroxicam

EEJR

NHJR

EETG

NHTG

Mean plusmn SEM

Infiltration of






49







50





(Fig 15)

51





52





53





54





55





56



57











control groups

58






groups




control groups

59










60










61










62










63










64










65


marker

Markers

Vehicle

Control

Arthritic

Control Piroxicam

EEJR

NHJR

EETG

NHTG

Mean plusmn SEM


NF-κB


IL-6


IL-1β


COX-1







66







group





67











68





(1512 plusmn 0256)





69










70


Parameters


Mean plusmn SEM

RBC


Hb

(gdl)


WBC

(103Ul)


Platelets

(103Ul)




two control groups

71









in all groups

72







creatinine levels

73





other



creatinine levels

74





other



levels

75











76


Biochemical

Parameters

Vehicle


Mean plusmn SEM

Urea


Creatinine


AST


ALT




77









gharuensis


distress Diarrhea

Motor

activity

Posture


Group-1 - - - - - -

Group-1I - - - - - -

Group-1II - - - - - -

Group-1V - - - - - -

Group-V - - - - - -

Group-VI - - - - - -

Group-VII - - - - - -

Group-

VIII - - - - - -

GROUP-

IX - - - - - -

78


carrageenan

451 At 1st hr









79

452 At 2nd hr









80

453 At 3rd hr









81

454 At 4th hr









82

455 At 5th hr









83



84



Groups




EEJR

(500 mgkg)

0210 plusmn0003

(2708)

0195 plusmn 0002

(3275)

0172 plusmn0003

(4591)

0156 plusmn0004

(5411)

0125 plusmn 0003

(6508)

NHJR

(500 mgkg)

0235 plusmn 0002

(1840)

0218 plusmn 0003

(2482)

0206 plusmn0003

(3522)

0193 plusmn0003

(4323)

0175 plusmn 0003

(5111)

EETG

(400 mgkg)

0226 plusmn0004

(21387)

0236 plusmn 0005

(1866)

0231 plusmn0006

(2735)

0210 plusmn0006

(3823)

0195 plusmn 0008

(4553)

NHTG

(400 mgkg)

0255 plusmn0003

(1145)

0250 plusmn 0002

(1379)

0280 plusmn0010

(1194)

0260 plusmn0010

(2352)

0236 plusmn 0011

(3407)

Piroxicam

(10 mgkg)

0208 plusmn0007

(2777)

0213 plusmn 0004

(2655)

0210 plusmn0003

(3396)

0183 plusmn0004

(4617)

0165 plusmn 0003

(5391)



85


edema

461 At 1st hr









86

462 At 2nd hr









87

463 At 3rd hr









88



89


edema model

Groups

1sthr 2ndhr 3rdhr

(Mean plusmnSEM)

(ml)

Inhibition

(Mean plusmnSEM)

(ml)

Inhibition

(Mean plusmnSEM)

(ml)

Inhibition


- 0318 plusmn 0001 - 0321 plusmn 0004 -

EEJR

( 500 mgkg)

0235 plusmn 0002

2192

0220 plusmn 0003

3081

0186 plusmn 0004

4205

NHJR

( 500 mgkg)

0251 plusmn 0003

1860

0240 plusmn 0002

2452

0225 plusmn 0008

2990

EETG


NHTG


Indomethacin

(10 mgkg)

0238 plusmn 0003

2093

0223 plusmn 0002

2987

0203 plusmn 0004

3676



90


471 At 1st hr









91

472 At 2nd hr





second hour




92

473 At 3rd hr









93




94


Groups

1sthr 2ndhr 3rdhr

(Mean plusmn SEM)

(ml) Inhibition

(Mean plusmn SEM)

(ml) Inhibition

(Mean plusmn SEM)

(ml) Inhibition


- 0331 plusmn 0004 - 0340 plusmn 0004 -

EEJR

( 500 mgkg)

0236 plusmn 0005

2411

0223 plusmn 0004

3262

0206 plusmn 0003

3941

NHJR

( 500 mgkg)

0258 plusmn 0004

1704

0236 plusmn 0004

2870

0225 plusmn 0005

3382

EETG


NHTG



2347

0230 plusmn 0005

2051

0195 plusmn 0003

4264



95


481 At 1st hr









96

482 At 2nd hr





at second hour




97

483 At 3rd hr









98

484 At 4th hr





fourth hour




99

485 At 5th hr









100



101


Groups

1sth

2ndh

3rdh

4thh

5thh




EEJR

(500 mgkg)

0216 plusmn 0004

(28)

0206 plusmn 0004

(4801)

0193 plusmn 0004

(4255)

0175 plusmn 0002

(4927)

0173 plusmn 0004

(5099)

NHJR

(500 mgkg)

0238 plusmn 0006

(2066)

0225 plusmn 0002

(2879)

0205 plusmn 0006

(3898)

0193 plusmn 0004

(4405)

0183 plusmn 0009

(4815)

EETG

(400 mgkg)

0243 plusmn 0005

(19)

0226 plusmn 0004

(2848)

0213 plusmn 0004

(3660)

0195 plusmn 0002

(4347)

0183 plusmn 004

(4815)

NHTG

(400 mgkg)

0266 plusmn 0005

(1133)

0245 plusmn0005

(2246)

0223 plusmn 0006

(3363)

0218 plusmn 0005

(3681)

0205 plusmn 0006

(4192)

DPH

(60 mgkg)

0220 plusmn 0004

(2667)

0195 plusmn 0004

(3829)

0176 plusmn 0003

(4761)

0168 plusmn 0003

(5130)

0161 plusmn0 006

(5443)



102





induced by xylene




103










104








105








106








107


Parameters


Mean plusmn SEM

Time of onset of








108







Alkaloids + + + +

Glycosides _ _ _ _

Saponins _ _ _

Phenols + + + +

Terpenoids + + + +

Steroids _ _ _ _

Flavonoids + + + +

Tannins + _ + +

109















110


Sr

No

Retention

Time

(Minute)

Total

Percentage

Name of Identified

Compound

Molecular

Formula

Molecular

Weight

(gmol)

Structures


2 5350 3956 Decane C10H22 142

3 6255 1169 (E)-3(10)-Caren-4-ol C10H16O 152

OH

CH2

CH3

CH3

4 6795 2182 Undecane C11H24 156

CH3

CH3

CH3 Cl

CH3

CH3

111

5 8311 0859 Dodecane C12H26 170

CH3

CH3


CH3

CH3


CH3

CH3


CH3CH3


CH3

112


CH3

CH3


CH3

CH3


CH3

CH3


CH3

CH3


CH3

CH3

113


CH3

CH3


CH3

CH3


CH3

CH3

18 20512 3754

Benzene (1-

methylundecyl)-

C18H30 246

CH3

CH3


CH3

CH3

114


CH3

CH3


CH3 CH3


CH3

CH3

23 22506 2311

Benzene (1-

methyldodecyl)-

C19H32 260

CH3 CH3


CH3

O

O CH3


CH3

O

O

CH3

115


O

O

CH3

CH3


O

O

CH3

28 30178 0212



ester

C16H22O4 278

O

OH

O

O

CH3

CH3

29 32303 1309 Squalene C30H50 410

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

30 32738 0380 Heptacosane C27H56 380

CH3

CH3

116

117


2 Decane

30( E )-3 (10)-Caren-4-ol

118

4 Undecane

5 Dodecane


119




120




121




122




123


20



124




125



27 Heptacosane

126













127


Sr

No

Retention

Time

(Minute)

Total

Percentage

Name of Identified

Compound

Molecular

Formula

Molecular

Weight

(gmol)

Structures


CH3

CH3

2 17498 4905

Velleral

C15H20O2 232 CH3

CH3

CH3O

O

3 20372 2361

Benzene (1-

methylundecyl)-

C18H30 246

CH3

CH3

128

4 20773 2105

Benzene (1-

pentylheptyl)

C18H30 246

5 20930 2199

ZZZ-1469-

Nonadecatetraene

C19H32 260

CH3 CH2


CH3

O

O

CH3

7 25737 4597 Phytol C20H40O 296

OH CH3

CH3

CH3

CH3

129


CH3

O

CH3


O

CH3


CH3

O

CH3

11 30178 2740

388-Trimethoxy-3-

piperidyl-22-

binaphthalene-1144-

tetrone

C28H25NO7 487

N

O

O

O

O

O

O

CH3CH3

O

CH3

130

12 32294 15138 Squalene C30H50 410

CH3

CH3

CH3

CH3CH3

CH3

CH3

CH3

13 32730 4184


ethylbutyl)-

C26H54 366 CH3

CH3


CH3

15 34759 6222 Vitamin E C29H50O2 430 O

CH3

CH3

OH

CH3

CH3

CH3

CH3

CH3

131

132




133




134

7 Phytol



135



6 Squalene

136


8 Heptacosane

9 Vitamin E

137










(Table 19)

138


SrNo Retention

Time (mins)

Total

(age)

Name of Identified


MW

(gmol) Structures


O

CH3

2 12186 15848 1H-2-Benzopyran-1-


OO



ester C14H28O2 228

5 16175 1580

Tetracyclo

[33101(39)]decan-10-

one

C10H12O 148 OO

139


ester C16H32O2 256

CH3

O

CH3

O


OH O


CH3

O

O

CH3

9 25737 0614 Phytol C20H40O 296 OH CH3

CH3

CH3

CH3



O

CH3

O

CH3

140



O CH3

O

CH3


CH3

O

CH3

13 32764 6519 Nonacosane C29H60 408 CH3

CH3



CH3


CH3

141

142




143




144




145

10 Phytol



146


14 Nonacosane


147

16 Hexatriacontane

148












20)

149


Sr

No

Retention

Time

(mins)

Total


Mol

Formula

MW

(gmol) Structures


CH3

CH3

2 5358 3731 Decane C10H22 142 CH3

CH3


CH3

CH3

4 6264 1048 (E)-3(10)-Caren-4-ol C10H16O 152

OH

CH2

CH3

CH3

150

5 6804 1990 Undecane C11H24 156 CH3

CH3

6 8311 0750 Dodecane C12H26 170 CH3

CH3



CH3

CH3


CH3

CH3


CH3

151


CH3


CH3

CH3


CH3

CH3


CH3


CH3

152


CH3

CH3


CH3

CH3


CH3

CH3


CH3


CH3

CH3


CH3

CH3

153


CH3 CH3


CH3

CH3



O

CH3


CH3

O

O

CH3



O

O

CH3

CH3

154


ester C20H36O2 308

O

CH3

O

CH3



O CH3

O

CH3


CH3

O

CH3



O

OH

O

O

CH3

CH3

155

32 32799 2525 Nonacosane C29H60 408

CH3

CH3


CH3

156

157

1 Decane

2 Decane 4-methyl-

3 (E)-3(10)-caren-4-ol

158

3 Undecane

4 Dodecane


159




160




161




162




163




164




165




166




167

31 Nonacosane

32 Tetratriacontane


168
























169


















destruction (79)






170


response






















171

























172

























173


























174


















extract (111)

175












176

FUTURE PERSPECTIVE












neonates


population

REFERENCES


201262(8)1085-93





therapeutics 2019













of Medicine 2011365(23)2205-19



20191614-11









21















Medica 201682(05)PC63



of Pain 201822(3)455-70



201771955






reviews 201711(22)145




of Nutrition 200899(4)715-22














201933(10)1531-4


199123(1)90-114















reviews 20054(3)130-6
























Toxicology 2003188(1)49-71



198136(6)435-6










200155(4)204





201113(1)27



201547(2)177






200389(1)123-9






































phytology 2011



2010439-44

















7
















710




401









200374(3)282-90






Res 201910(5)2087-100




therapy 201315(5)R141









sciences 201414(4)919-24









3(21)465-70


























Medicines 20129(2)242-9





reviews 200052(4)673-751



















Shabbir




Student Name


Supervisor Name


Associate Professor

Co-Supervisor Name


Professor



Signature Date

Name of HOD


Signature Date

i

DEDICATION



ii

ACKNOWLDGEMENTS





















iii









my dreams

Aisha Mobashar

iv

TABLE OF CONTENTS

DEDICATION i

ACKNOWLDGEMENTS ii


LIST OF TABLES xi



ABSTRACT xix


11 Hypothesis 3






21 Epidemiology 4


23 Treatments 7

24 Inflammation 10


26 Plants used 12






v


















buffer (50X) 22


349 Densitometry 23









vi




carrageenan 28


histamine 28


serotonin 28


dextran 29



















vii






421 At Day 8 39

422 At Day 15 40

423 At Day 22 41

424 At Day 28 42







marker 57


levels of TNF-α 57



levels 59


levels of IL-1β 60






levels of IL-4 63

viii


64


parameters 66


count 66


content 67


count 68


platelet count 69


71



levels 72






gharuensis 77



451 At 1st hr 78

452 At 2nd hr 79

453 At 3rd hr 80

454 At 4th hr 81

ix

455 At 5th hr 82



461 At 1st hr 85

462 At 2nd hr 86

463 At 3rd hr 87



471 At 1st hr 90

472 At 2nd hr 91

473 At 3rd hr 92



481 At 1st hr 95

482 At 2nd hr 96

483 At 3rd hr 97

484 At 4th hr 98

485 At 5th hr 99











x





REFERENCES 177

xi

LIST OF TABLES

Page






40





66




T gharuensis

78



induced paw edema

84

xii



88



92



98



104






xiii

LIST OF FIGURES

Page

















xiv






of TNF-α

58


of NF-κB

59


of IL-6

60


of IL-1β

61


of COX-1

62


of COX-2

63


IL-4

64


xv


















xvi














xvii













Hb Haemoglobin

RBC Red blood cells






IL-6 Interleukin 6

IL-4 Interleukin 4



PXM Piroxicam

xviii

IND Indomethacin

DPH Diphenhydramine

Dexa Dexamethasone

LOP Loperamide



I2 Iodine


KI Potassium iodide


DW Distilled water

SM

bw

PO

Synovial membrane

Body weight

Per oral

xix



ABSTRACT




respectively
















xx
























xxi






1
























2

























3
















11 Hypothesis

111 Null hypothesis


properties



4














21 Epidemiology










5



22 Pathophysiology






















6

























7


















23 Treatments







8









cyclooxygenase (42)

















9

























10
















24 Inflammation









11









(45)







immunologic (46)






12










26 Plants used















13








(51)














14

Aim of the study



Objective of study













GC-MS analysis

15


31 Plant collection





name was checked




05














16





57)



















17









FCA (59)








342 (12)


2 and IL-4

351 RNA extraction


18









separated






3514 Washing of RNA







19






GAPDH


pairs sequence








20



IL-4


231 (58)


IL-6


247 (5)


TNF-α


202 NM_0126753


NF-КB


207 (58)


COX-1


200 (59)


COX-2


237 (58)


IL-1β


293 (58 60)


GAPDH


197 NM_0170084


21

353 cDNA synthesis





















22












Each one litre

Tris base 242 g


05M EDTA 100 ml


600ml




23




buffer











358 Densitometry






experiment

24




















immediately




25




five times










1800i)







26












Models Groups



EEJR (500 mgkg)

NHJR (500 mgkg )

EETG (400 mgkg)

NHTG (400 mgkg)




EEJR (500 mgkg)

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)



EEJR (500 mgkg)

27


water

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)




EEJR (500 mgkg)

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)




EEJR (500 mgkg)

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)




EEJR (500 mgkg)

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)

Loperamide (5 mgkg)



bw = body weight

28























29




















30




















31








(68)





glycosides (70)


3841 Salkowski test







red colour (71)

32






















33

CHAPTER 4 RESULTS


model








34









35









36









37

Figure 5 Paw edema



38










39


421 At Day 8








40

422 At Day 15









group

41

423 At Day 22









control group

42

424 At Day 28




0153)





43





44




Days







45











46









47











48


Parameters

Arthritic control

Piroxicam

EEJR

NHJR

EETG

NHTG

Mean plusmn SEM

Infiltration of






49







50





(Fig 15)

51





52





53





54





55





56



57











control groups

58






groups




control groups

59










60










61










62










63










64










65


marker

Markers

Vehicle

Control

Arthritic

Control Piroxicam

EEJR

NHJR

EETG

NHTG

Mean plusmn SEM


NF-κB


IL-6


IL-1β


COX-1







66







group





67











68





(1512 plusmn 0256)





69










70


Parameters


Mean plusmn SEM

RBC


Hb

(gdl)


WBC

(103Ul)


Platelets

(103Ul)




two control groups

71









in all groups

72







creatinine levels

73





other



creatinine levels

74





other



levels

75











76


Biochemical

Parameters

Vehicle


Mean plusmn SEM

Urea


Creatinine


AST


ALT




77









gharuensis


distress Diarrhea

Motor

activity

Posture


Group-1 - - - - - -

Group-1I - - - - - -

Group-1II - - - - - -

Group-1V - - - - - -

Group-V - - - - - -

Group-VI - - - - - -

Group-VII - - - - - -

Group-

VIII - - - - - -

GROUP-

IX - - - - - -

78


carrageenan

451 At 1st hr









79

452 At 2nd hr









80

453 At 3rd hr









81

454 At 4th hr









82

455 At 5th hr









83



84



Groups




EEJR

(500 mgkg)

0210 plusmn0003

(2708)

0195 plusmn 0002

(3275)

0172 plusmn0003

(4591)

0156 plusmn0004

(5411)

0125 plusmn 0003

(6508)

NHJR

(500 mgkg)

0235 plusmn 0002

(1840)

0218 plusmn 0003

(2482)

0206 plusmn0003

(3522)

0193 plusmn0003

(4323)

0175 plusmn 0003

(5111)

EETG

(400 mgkg)

0226 plusmn0004

(21387)

0236 plusmn 0005

(1866)

0231 plusmn0006

(2735)

0210 plusmn0006

(3823)

0195 plusmn 0008

(4553)

NHTG

(400 mgkg)

0255 plusmn0003

(1145)

0250 plusmn 0002

(1379)

0280 plusmn0010

(1194)

0260 plusmn0010

(2352)

0236 plusmn 0011

(3407)

Piroxicam

(10 mgkg)

0208 plusmn0007

(2777)

0213 plusmn 0004

(2655)

0210 plusmn0003

(3396)

0183 plusmn0004

(4617)

0165 plusmn 0003

(5391)



85


edema

461 At 1st hr









86

462 At 2nd hr









87

463 At 3rd hr









88



89


edema model

Groups

1sthr 2ndhr 3rdhr

(Mean plusmnSEM)

(ml)

Inhibition

(Mean plusmnSEM)

(ml)

Inhibition

(Mean plusmnSEM)

(ml)

Inhibition


- 0318 plusmn 0001 - 0321 plusmn 0004 -

EEJR

( 500 mgkg)

0235 plusmn 0002

2192

0220 plusmn 0003

3081

0186 plusmn 0004

4205

NHJR

( 500 mgkg)

0251 plusmn 0003

1860

0240 plusmn 0002

2452

0225 plusmn 0008

2990

EETG


NHTG


Indomethacin

(10 mgkg)

0238 plusmn 0003

2093

0223 plusmn 0002

2987

0203 plusmn 0004

3676



90


471 At 1st hr









91

472 At 2nd hr





second hour




92

473 At 3rd hr









93




94


Groups

1sthr 2ndhr 3rdhr

(Mean plusmn SEM)

(ml) Inhibition

(Mean plusmn SEM)

(ml) Inhibition

(Mean plusmn SEM)

(ml) Inhibition


- 0331 plusmn 0004 - 0340 plusmn 0004 -

EEJR

( 500 mgkg)

0236 plusmn 0005

2411

0223 plusmn 0004

3262

0206 plusmn 0003

3941

NHJR

( 500 mgkg)

0258 plusmn 0004

1704

0236 plusmn 0004

2870

0225 plusmn 0005

3382

EETG


NHTG



2347

0230 plusmn 0005

2051

0195 plusmn 0003

4264



95


481 At 1st hr









96

482 At 2nd hr





at second hour




97

483 At 3rd hr









98

484 At 4th hr





fourth hour




99

485 At 5th hr









100



101


Groups

1sth

2ndh

3rdh

4thh

5thh




EEJR

(500 mgkg)

0216 plusmn 0004

(28)

0206 plusmn 0004

(4801)

0193 plusmn 0004

(4255)

0175 plusmn 0002

(4927)

0173 plusmn 0004

(5099)

NHJR

(500 mgkg)

0238 plusmn 0006

(2066)

0225 plusmn 0002

(2879)

0205 plusmn 0006

(3898)

0193 plusmn 0004

(4405)

0183 plusmn 0009

(4815)

EETG

(400 mgkg)

0243 plusmn 0005

(19)

0226 plusmn 0004

(2848)

0213 plusmn 0004

(3660)

0195 plusmn 0002

(4347)

0183 plusmn 004

(4815)

NHTG

(400 mgkg)

0266 plusmn 0005

(1133)

0245 plusmn0005

(2246)

0223 plusmn 0006

(3363)

0218 plusmn 0005

(3681)

0205 plusmn 0006

(4192)

DPH

(60 mgkg)

0220 plusmn 0004

(2667)

0195 plusmn 0004

(3829)

0176 plusmn 0003

(4761)

0168 plusmn 0003

(5130)

0161 plusmn0 006

(5443)



102





induced by xylene




103










104








105








106








107


Parameters


Mean plusmn SEM

Time of onset of








108







Alkaloids + + + +

Glycosides _ _ _ _

Saponins _ _ _

Phenols + + + +

Terpenoids + + + +

Steroids _ _ _ _

Flavonoids + + + +

Tannins + _ + +

109















110


Sr

No

Retention

Time

(Minute)

Total

Percentage

Name of Identified

Compound

Molecular

Formula

Molecular

Weight

(gmol)

Structures


2 5350 3956 Decane C10H22 142

3 6255 1169 (E)-3(10)-Caren-4-ol C10H16O 152

OH

CH2

CH3

CH3

4 6795 2182 Undecane C11H24 156

CH3

CH3

CH3 Cl

CH3

CH3

111

5 8311 0859 Dodecane C12H26 170

CH3

CH3


CH3

CH3


CH3

CH3


CH3CH3


CH3

112


CH3

CH3


CH3

CH3


CH3

CH3


CH3

CH3


CH3

CH3

113


CH3

CH3


CH3

CH3


CH3

CH3

18 20512 3754

Benzene (1-

methylundecyl)-

C18H30 246

CH3

CH3


CH3

CH3

114


CH3

CH3


CH3 CH3


CH3

CH3

23 22506 2311

Benzene (1-

methyldodecyl)-

C19H32 260

CH3 CH3


CH3

O

O CH3


CH3

O

O

CH3

115


O

O

CH3

CH3


O

O

CH3

28 30178 0212



ester

C16H22O4 278

O

OH

O

O

CH3

CH3

29 32303 1309 Squalene C30H50 410

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

30 32738 0380 Heptacosane C27H56 380

CH3

CH3

116

117


2 Decane

30( E )-3 (10)-Caren-4-ol

118

4 Undecane

5 Dodecane


119




120




121




122




123


20



124




125



27 Heptacosane

126













127


Sr

No

Retention

Time

(Minute)

Total

Percentage

Name of Identified

Compound

Molecular

Formula

Molecular

Weight

(gmol)

Structures


CH3

CH3

2 17498 4905

Velleral

C15H20O2 232 CH3

CH3

CH3O

O

3 20372 2361

Benzene (1-

methylundecyl)-

C18H30 246

CH3

CH3

128

4 20773 2105

Benzene (1-

pentylheptyl)

C18H30 246

5 20930 2199

ZZZ-1469-

Nonadecatetraene

C19H32 260

CH3 CH2


CH3

O

O

CH3

7 25737 4597 Phytol C20H40O 296

OH CH3

CH3

CH3

CH3

129


CH3

O

CH3


O

CH3


CH3

O

CH3

11 30178 2740

388-Trimethoxy-3-

piperidyl-22-

binaphthalene-1144-

tetrone

C28H25NO7 487

N

O

O

O

O

O

O

CH3CH3

O

CH3

130

12 32294 15138 Squalene C30H50 410

CH3

CH3

CH3

CH3CH3

CH3

CH3

CH3

13 32730 4184


ethylbutyl)-

C26H54 366 CH3

CH3


CH3

15 34759 6222 Vitamin E C29H50O2 430 O

CH3

CH3

OH

CH3

CH3

CH3

CH3

CH3

131

132




133




134

7 Phytol



135



6 Squalene

136


8 Heptacosane

9 Vitamin E

137










(Table 19)

138


SrNo Retention

Time (mins)

Total

(age)

Name of Identified


MW

(gmol) Structures


O

CH3

2 12186 15848 1H-2-Benzopyran-1-


OO



ester C14H28O2 228

5 16175 1580

Tetracyclo

[33101(39)]decan-10-

one

C10H12O 148 OO

139


ester C16H32O2 256

CH3

O

CH3

O


OH O


CH3

O

O

CH3

9 25737 0614 Phytol C20H40O 296 OH CH3

CH3

CH3

CH3



O

CH3

O

CH3

140



O CH3

O

CH3


CH3

O

CH3

13 32764 6519 Nonacosane C29H60 408 CH3

CH3



CH3


CH3

141

142




143




144




145

10 Phytol



146


14 Nonacosane


147

16 Hexatriacontane

148












20)

149


Sr

No

Retention

Time

(mins)

Total


Mol

Formula

MW

(gmol) Structures


CH3

CH3

2 5358 3731 Decane C10H22 142 CH3

CH3


CH3

CH3

4 6264 1048 (E)-3(10)-Caren-4-ol C10H16O 152

OH

CH2

CH3

CH3

150

5 6804 1990 Undecane C11H24 156 CH3

CH3

6 8311 0750 Dodecane C12H26 170 CH3

CH3



CH3

CH3


CH3

CH3


CH3

151


CH3


CH3

CH3


CH3

CH3


CH3


CH3

152


CH3

CH3


CH3

CH3


CH3

CH3


CH3


CH3

CH3


CH3

CH3

153


CH3 CH3


CH3

CH3



O

CH3


CH3

O

O

CH3



O

O

CH3

CH3

154


ester C20H36O2 308

O

CH3

O

CH3



O CH3

O

CH3


CH3

O

CH3



O

OH

O

O

CH3

CH3

155

32 32799 2525 Nonacosane C29H60 408

CH3

CH3


CH3

156

157

1 Decane

2 Decane 4-methyl-

3 (E)-3(10)-caren-4-ol

158

3 Undecane

4 Dodecane


159




160




161




162




163




164




165




166




167

31 Nonacosane

32 Tetratriacontane


168
























169


















destruction (79)






170


response






















171

























172

























173


























174


















extract (111)

175












176

FUTURE PERSPECTIVE












neonates


population

REFERENCES


201262(8)1085-93





therapeutics 2019













of Medicine 2011365(23)2205-19



20191614-11









21















Medica 201682(05)PC63



of Pain 201822(3)455-70



201771955






reviews 201711(22)145




of Nutrition 200899(4)715-22














201933(10)1531-4


199123(1)90-114















reviews 20054(3)130-6
























Toxicology 2003188(1)49-71



198136(6)435-6










200155(4)204





201113(1)27



201547(2)177






200389(1)123-9






































phytology 2011



2010439-44

















7
















710




401









200374(3)282-90






Res 201910(5)2087-100




therapy 201315(5)R141









sciences 201414(4)919-24









3(21)465-70


























Medicines 20129(2)242-9





reviews 200052(4)673-751















i

DEDICATION



ii

ACKNOWLDGEMENTS





















iii









my dreams

Aisha Mobashar

iv

TABLE OF CONTENTS

DEDICATION i

ACKNOWLDGEMENTS ii


LIST OF TABLES xi



ABSTRACT xix


11 Hypothesis 3






21 Epidemiology 4


23 Treatments 7

24 Inflammation 10


26 Plants used 12






v


















buffer (50X) 22


349 Densitometry 23









vi




carrageenan 28


histamine 28


serotonin 28


dextran 29



















vii






421 At Day 8 39

422 At Day 15 40

423 At Day 22 41

424 At Day 28 42







marker 57


levels of TNF-α 57



levels 59


levels of IL-1β 60






levels of IL-4 63

viii


64


parameters 66


count 66


content 67


count 68


platelet count 69


71



levels 72






gharuensis 77



451 At 1st hr 78

452 At 2nd hr 79

453 At 3rd hr 80

454 At 4th hr 81

ix

455 At 5th hr 82



461 At 1st hr 85

462 At 2nd hr 86

463 At 3rd hr 87



471 At 1st hr 90

472 At 2nd hr 91

473 At 3rd hr 92



481 At 1st hr 95

482 At 2nd hr 96

483 At 3rd hr 97

484 At 4th hr 98

485 At 5th hr 99











x





REFERENCES 177

xi

LIST OF TABLES

Page






40





66




T gharuensis

78



induced paw edema

84

xii



88



92



98



104






xiii

LIST OF FIGURES

Page

















xiv






of TNF-α

58


of NF-κB

59


of IL-6

60


of IL-1β

61


of COX-1

62


of COX-2

63


IL-4

64


xv


















xvi














xvii













Hb Haemoglobin

RBC Red blood cells






IL-6 Interleukin 6

IL-4 Interleukin 4



PXM Piroxicam

xviii

IND Indomethacin

DPH Diphenhydramine

Dexa Dexamethasone

LOP Loperamide



I2 Iodine


KI Potassium iodide


DW Distilled water

SM

bw

PO

Synovial membrane

Body weight

Per oral

xix



ABSTRACT




respectively
















xx
























xxi






1
























2

























3
















11 Hypothesis

111 Null hypothesis


properties



4














21 Epidemiology










5



22 Pathophysiology






















6

























7


















23 Treatments







8









cyclooxygenase (42)

















9

























10
















24 Inflammation









11









(45)







immunologic (46)






12










26 Plants used















13








(51)














14

Aim of the study



Objective of study













GC-MS analysis

15


31 Plant collection





name was checked




05














16





57)



















17









FCA (59)








342 (12)


2 and IL-4

351 RNA extraction


18









separated






3514 Washing of RNA







19






GAPDH


pairs sequence








20



IL-4


231 (58)


IL-6


247 (5)


TNF-α


202 NM_0126753


NF-КB


207 (58)


COX-1


200 (59)


COX-2


237 (58)


IL-1β


293 (58 60)


GAPDH


197 NM_0170084


21

353 cDNA synthesis





















22












Each one litre

Tris base 242 g


05M EDTA 100 ml


600ml




23




buffer











358 Densitometry






experiment

24




















immediately




25




five times










1800i)







26












Models Groups



EEJR (500 mgkg)

NHJR (500 mgkg )

EETG (400 mgkg)

NHTG (400 mgkg)




EEJR (500 mgkg)

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)



EEJR (500 mgkg)

27


water

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)




EEJR (500 mgkg)

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)




EEJR (500 mgkg)

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)




EEJR (500 mgkg)

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)

Loperamide (5 mgkg)



bw = body weight

28























29




















30




















31








(68)





glycosides (70)


3841 Salkowski test







red colour (71)

32






















33

CHAPTER 4 RESULTS


model








34









35









36









37

Figure 5 Paw edema



38










39


421 At Day 8








40

422 At Day 15









group

41

423 At Day 22









control group

42

424 At Day 28




0153)





43





44




Days







45











46









47











48


Parameters

Arthritic control

Piroxicam

EEJR

NHJR

EETG

NHTG

Mean plusmn SEM

Infiltration of






49







50





(Fig 15)

51





52





53





54





55





56



57











control groups

58






groups




control groups

59










60










61










62










63










64










65


marker

Markers

Vehicle

Control

Arthritic

Control Piroxicam

EEJR

NHJR

EETG

NHTG

Mean plusmn SEM


NF-κB


IL-6


IL-1β


COX-1







66







group





67











68





(1512 plusmn 0256)





69










70


Parameters


Mean plusmn SEM

RBC


Hb

(gdl)


WBC

(103Ul)


Platelets

(103Ul)




two control groups

71









in all groups

72







creatinine levels

73





other



creatinine levels

74





other



levels

75











76


Biochemical

Parameters

Vehicle


Mean plusmn SEM

Urea


Creatinine


AST


ALT




77









gharuensis


distress Diarrhea

Motor

activity

Posture


Group-1 - - - - - -

Group-1I - - - - - -

Group-1II - - - - - -

Group-1V - - - - - -

Group-V - - - - - -

Group-VI - - - - - -

Group-VII - - - - - -

Group-

VIII - - - - - -

GROUP-

IX - - - - - -

78


carrageenan

451 At 1st hr









79

452 At 2nd hr









80

453 At 3rd hr









81

454 At 4th hr









82

455 At 5th hr









83



84



Groups




EEJR

(500 mgkg)

0210 plusmn0003

(2708)

0195 plusmn 0002

(3275)

0172 plusmn0003

(4591)

0156 plusmn0004

(5411)

0125 plusmn 0003

(6508)

NHJR

(500 mgkg)

0235 plusmn 0002

(1840)

0218 plusmn 0003

(2482)

0206 plusmn0003

(3522)

0193 plusmn0003

(4323)

0175 plusmn 0003

(5111)

EETG

(400 mgkg)

0226 plusmn0004

(21387)

0236 plusmn 0005

(1866)

0231 plusmn0006

(2735)

0210 plusmn0006

(3823)

0195 plusmn 0008

(4553)

NHTG

(400 mgkg)

0255 plusmn0003

(1145)

0250 plusmn 0002

(1379)

0280 plusmn0010

(1194)

0260 plusmn0010

(2352)

0236 plusmn 0011

(3407)

Piroxicam

(10 mgkg)

0208 plusmn0007

(2777)

0213 plusmn 0004

(2655)

0210 plusmn0003

(3396)

0183 plusmn0004

(4617)

0165 plusmn 0003

(5391)



85


edema

461 At 1st hr









86

462 At 2nd hr









87

463 At 3rd hr









88



89


edema model

Groups

1sthr 2ndhr 3rdhr

(Mean plusmnSEM)

(ml)

Inhibition

(Mean plusmnSEM)

(ml)

Inhibition

(Mean plusmnSEM)

(ml)

Inhibition


- 0318 plusmn 0001 - 0321 plusmn 0004 -

EEJR

( 500 mgkg)

0235 plusmn 0002

2192

0220 plusmn 0003

3081

0186 plusmn 0004

4205

NHJR

( 500 mgkg)

0251 plusmn 0003

1860

0240 plusmn 0002

2452

0225 plusmn 0008

2990

EETG


NHTG


Indomethacin

(10 mgkg)

0238 plusmn 0003

2093

0223 plusmn 0002

2987

0203 plusmn 0004

3676



90


471 At 1st hr









91

472 At 2nd hr





second hour




92

473 At 3rd hr









93




94


Groups

1sthr 2ndhr 3rdhr

(Mean plusmn SEM)

(ml) Inhibition

(Mean plusmn SEM)

(ml) Inhibition

(Mean plusmn SEM)

(ml) Inhibition


- 0331 plusmn 0004 - 0340 plusmn 0004 -

EEJR

( 500 mgkg)

0236 plusmn 0005

2411

0223 plusmn 0004

3262

0206 plusmn 0003

3941

NHJR

( 500 mgkg)

0258 plusmn 0004

1704

0236 plusmn 0004

2870

0225 plusmn 0005

3382

EETG


NHTG



2347

0230 plusmn 0005

2051

0195 plusmn 0003

4264



95


481 At 1st hr









96

482 At 2nd hr





at second hour




97

483 At 3rd hr









98

484 At 4th hr





fourth hour




99

485 At 5th hr









100



101


Groups

1sth

2ndh

3rdh

4thh

5thh




EEJR

(500 mgkg)

0216 plusmn 0004

(28)

0206 plusmn 0004

(4801)

0193 plusmn 0004

(4255)

0175 plusmn 0002

(4927)

0173 plusmn 0004

(5099)

NHJR

(500 mgkg)

0238 plusmn 0006

(2066)

0225 plusmn 0002

(2879)

0205 plusmn 0006

(3898)

0193 plusmn 0004

(4405)

0183 plusmn 0009

(4815)

EETG

(400 mgkg)

0243 plusmn 0005

(19)

0226 plusmn 0004

(2848)

0213 plusmn 0004

(3660)

0195 plusmn 0002

(4347)

0183 plusmn 004

(4815)

NHTG

(400 mgkg)

0266 plusmn 0005

(1133)

0245 plusmn0005

(2246)

0223 plusmn 0006

(3363)

0218 plusmn 0005

(3681)

0205 plusmn 0006

(4192)

DPH

(60 mgkg)

0220 plusmn 0004

(2667)

0195 plusmn 0004

(3829)

0176 plusmn 0003

(4761)

0168 plusmn 0003

(5130)

0161 plusmn0 006

(5443)



102





induced by xylene




103










104








105








106








107


Parameters


Mean plusmn SEM

Time of onset of








108







Alkaloids + + + +

Glycosides _ _ _ _

Saponins _ _ _

Phenols + + + +

Terpenoids + + + +

Steroids _ _ _ _

Flavonoids + + + +

Tannins + _ + +

109















110


Sr

No

Retention

Time

(Minute)

Total

Percentage

Name of Identified

Compound

Molecular

Formula

Molecular

Weight

(gmol)

Structures


2 5350 3956 Decane C10H22 142

3 6255 1169 (E)-3(10)-Caren-4-ol C10H16O 152

OH

CH2

CH3

CH3

4 6795 2182 Undecane C11H24 156

CH3

CH3

CH3 Cl

CH3

CH3

111

5 8311 0859 Dodecane C12H26 170

CH3

CH3


CH3

CH3


CH3

CH3


CH3CH3


CH3

112


CH3

CH3


CH3

CH3


CH3

CH3


CH3

CH3


CH3

CH3

113


CH3

CH3


CH3

CH3


CH3

CH3

18 20512 3754

Benzene (1-

methylundecyl)-

C18H30 246

CH3

CH3


CH3

CH3

114


CH3

CH3


CH3 CH3


CH3

CH3

23 22506 2311

Benzene (1-

methyldodecyl)-

C19H32 260

CH3 CH3


CH3

O

O CH3


CH3

O

O

CH3

115


O

O

CH3

CH3


O

O

CH3

28 30178 0212



ester

C16H22O4 278

O

OH

O

O

CH3

CH3

29 32303 1309 Squalene C30H50 410

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

30 32738 0380 Heptacosane C27H56 380

CH3

CH3

116

117


2 Decane

30( E )-3 (10)-Caren-4-ol

118

4 Undecane

5 Dodecane


119




120




121




122




123


20



124




125



27 Heptacosane

126













127


Sr

No

Retention

Time

(Minute)

Total

Percentage

Name of Identified

Compound

Molecular

Formula

Molecular

Weight

(gmol)

Structures


CH3

CH3

2 17498 4905

Velleral

C15H20O2 232 CH3

CH3

CH3O

O

3 20372 2361

Benzene (1-

methylundecyl)-

C18H30 246

CH3

CH3

128

4 20773 2105

Benzene (1-

pentylheptyl)

C18H30 246

5 20930 2199

ZZZ-1469-

Nonadecatetraene

C19H32 260

CH3 CH2


CH3

O

O

CH3

7 25737 4597 Phytol C20H40O 296

OH CH3

CH3

CH3

CH3

129


CH3

O

CH3


O

CH3


CH3

O

CH3

11 30178 2740

388-Trimethoxy-3-

piperidyl-22-

binaphthalene-1144-

tetrone

C28H25NO7 487

N

O

O

O

O

O

O

CH3CH3

O

CH3

130

12 32294 15138 Squalene C30H50 410

CH3

CH3

CH3

CH3CH3

CH3

CH3

CH3

13 32730 4184


ethylbutyl)-

C26H54 366 CH3

CH3


CH3

15 34759 6222 Vitamin E C29H50O2 430 O

CH3

CH3

OH

CH3

CH3

CH3

CH3

CH3

131

132




133




134

7 Phytol



135



6 Squalene

136


8 Heptacosane

9 Vitamin E

137










(Table 19)

138


SrNo Retention

Time (mins)

Total

(age)

Name of Identified


MW

(gmol) Structures


O

CH3

2 12186 15848 1H-2-Benzopyran-1-


OO



ester C14H28O2 228

5 16175 1580

Tetracyclo

[33101(39)]decan-10-

one

C10H12O 148 OO

139


ester C16H32O2 256

CH3

O

CH3

O


OH O


CH3

O

O

CH3

9 25737 0614 Phytol C20H40O 296 OH CH3

CH3

CH3

CH3



O

CH3

O

CH3

140



O CH3

O

CH3


CH3

O

CH3

13 32764 6519 Nonacosane C29H60 408 CH3

CH3



CH3


CH3

141

142




143




144




145

10 Phytol



146


14 Nonacosane


147

16 Hexatriacontane

148












20)

149


Sr

No

Retention

Time

(mins)

Total


Mol

Formula

MW

(gmol) Structures


CH3

CH3

2 5358 3731 Decane C10H22 142 CH3

CH3


CH3

CH3

4 6264 1048 (E)-3(10)-Caren-4-ol C10H16O 152

OH

CH2

CH3

CH3

150

5 6804 1990 Undecane C11H24 156 CH3

CH3

6 8311 0750 Dodecane C12H26 170 CH3

CH3



CH3

CH3


CH3

CH3


CH3

151


CH3


CH3

CH3


CH3

CH3


CH3


CH3

152


CH3

CH3


CH3

CH3


CH3

CH3


CH3


CH3

CH3


CH3

CH3

153


CH3 CH3


CH3

CH3



O

CH3


CH3

O

O

CH3



O

O

CH3

CH3

154


ester C20H36O2 308

O

CH3

O

CH3



O CH3

O

CH3


CH3

O

CH3



O

OH

O

O

CH3

CH3

155

32 32799 2525 Nonacosane C29H60 408

CH3

CH3


CH3

156

157

1 Decane

2 Decane 4-methyl-

3 (E)-3(10)-caren-4-ol

158

3 Undecane

4 Dodecane


159




160




161




162




163




164




165




166




167

31 Nonacosane

32 Tetratriacontane


168
























169


















destruction (79)






170


response






















171

























172

























173


























174


















extract (111)

175












176

FUTURE PERSPECTIVE












neonates


population

REFERENCES


201262(8)1085-93





therapeutics 2019













of Medicine 2011365(23)2205-19



20191614-11









21















Medica 201682(05)PC63



of Pain 201822(3)455-70



201771955






reviews 201711(22)145




of Nutrition 200899(4)715-22














201933(10)1531-4


199123(1)90-114















reviews 20054(3)130-6
























Toxicology 2003188(1)49-71



198136(6)435-6










200155(4)204





201113(1)27



201547(2)177






200389(1)123-9






































phytology 2011



2010439-44

















7
















710




401









200374(3)282-90






Res 201910(5)2087-100




therapy 201315(5)R141









sciences 201414(4)919-24









3(21)465-70


























Medicines 20129(2)242-9





reviews 200052(4)673-751















ii

ACKNOWLDGEMENTS





















iii









my dreams

Aisha Mobashar

iv

TABLE OF CONTENTS

DEDICATION i

ACKNOWLDGEMENTS ii


LIST OF TABLES xi



ABSTRACT xix


11 Hypothesis 3






21 Epidemiology 4


23 Treatments 7

24 Inflammation 10


26 Plants used 12






v


















buffer (50X) 22


349 Densitometry 23









vi




carrageenan 28


histamine 28


serotonin 28


dextran 29



















vii






421 At Day 8 39

422 At Day 15 40

423 At Day 22 41

424 At Day 28 42







marker 57


levels of TNF-α 57



levels 59


levels of IL-1β 60






levels of IL-4 63

viii


64


parameters 66


count 66


content 67


count 68


platelet count 69


71



levels 72






gharuensis 77



451 At 1st hr 78

452 At 2nd hr 79

453 At 3rd hr 80

454 At 4th hr 81

ix

455 At 5th hr 82



461 At 1st hr 85

462 At 2nd hr 86

463 At 3rd hr 87



471 At 1st hr 90

472 At 2nd hr 91

473 At 3rd hr 92



481 At 1st hr 95

482 At 2nd hr 96

483 At 3rd hr 97

484 At 4th hr 98

485 At 5th hr 99











x





REFERENCES 177

xi

LIST OF TABLES

Page






40





66




T gharuensis

78



induced paw edema

84

xii



88



92



98



104






xiii

LIST OF FIGURES

Page

















xiv






of TNF-α

58


of NF-κB

59


of IL-6

60


of IL-1β

61


of COX-1

62


of COX-2

63


IL-4

64


xv


















xvi














xvii













Hb Haemoglobin

RBC Red blood cells






IL-6 Interleukin 6

IL-4 Interleukin 4



PXM Piroxicam

xviii

IND Indomethacin

DPH Diphenhydramine

Dexa Dexamethasone

LOP Loperamide



I2 Iodine


KI Potassium iodide


DW Distilled water

SM

bw

PO

Synovial membrane

Body weight

Per oral

xix



ABSTRACT




respectively
















xx
























xxi






1
























2

























3
















11 Hypothesis

111 Null hypothesis


properties



4














21 Epidemiology










5



22 Pathophysiology






















6

























7


















23 Treatments







8









cyclooxygenase (42)

















9

























10
















24 Inflammation









11









(45)







immunologic (46)






12










26 Plants used















13








(51)














14

Aim of the study



Objective of study













GC-MS analysis

15


31 Plant collection





name was checked




05














16





57)



















17









FCA (59)








342 (12)


2 and IL-4

351 RNA extraction


18









separated






3514 Washing of RNA







19






GAPDH


pairs sequence








20



IL-4


231 (58)


IL-6


247 (5)


TNF-α


202 NM_0126753


NF-КB


207 (58)


COX-1


200 (59)


COX-2


237 (58)


IL-1β


293 (58 60)


GAPDH


197 NM_0170084


21

353 cDNA synthesis





















22












Each one litre

Tris base 242 g


05M EDTA 100 ml


600ml




23




buffer











358 Densitometry






experiment

24




















immediately




25




five times










1800i)







26












Models Groups



EEJR (500 mgkg)

NHJR (500 mgkg )

EETG (400 mgkg)

NHTG (400 mgkg)




EEJR (500 mgkg)

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)



EEJR (500 mgkg)

27


water

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)




EEJR (500 mgkg)

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)




EEJR (500 mgkg)

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)




EEJR (500 mgkg)

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)

Loperamide (5 mgkg)



bw = body weight

28























29




















30




















31








(68)





glycosides (70)


3841 Salkowski test







red colour (71)

32






















33

CHAPTER 4 RESULTS


model








34









35









36









37

Figure 5 Paw edema



38










39


421 At Day 8








40

422 At Day 15









group

41

423 At Day 22









control group

42

424 At Day 28




0153)





43





44




Days







45











46









47











48


Parameters

Arthritic control

Piroxicam

EEJR

NHJR

EETG

NHTG

Mean plusmn SEM

Infiltration of






49







50





(Fig 15)

51





52





53





54





55





56



57











control groups

58






groups




control groups

59










60










61










62










63










64










65


marker

Markers

Vehicle

Control

Arthritic

Control Piroxicam

EEJR

NHJR

EETG

NHTG

Mean plusmn SEM


NF-κB


IL-6


IL-1β


COX-1







66







group





67











68





(1512 plusmn 0256)





69










70


Parameters


Mean plusmn SEM

RBC


Hb

(gdl)


WBC

(103Ul)


Platelets

(103Ul)




two control groups

71









in all groups

72







creatinine levels

73





other



creatinine levels

74





other



levels

75











76


Biochemical

Parameters

Vehicle


Mean plusmn SEM

Urea


Creatinine


AST


ALT




77









gharuensis


distress Diarrhea

Motor

activity

Posture


Group-1 - - - - - -

Group-1I - - - - - -

Group-1II - - - - - -

Group-1V - - - - - -

Group-V - - - - - -

Group-VI - - - - - -

Group-VII - - - - - -

Group-

VIII - - - - - -

GROUP-

IX - - - - - -

78


carrageenan

451 At 1st hr









79

452 At 2nd hr









80

453 At 3rd hr









81

454 At 4th hr









82

455 At 5th hr









83



84



Groups




EEJR

(500 mgkg)

0210 plusmn0003

(2708)

0195 plusmn 0002

(3275)

0172 plusmn0003

(4591)

0156 plusmn0004

(5411)

0125 plusmn 0003

(6508)

NHJR

(500 mgkg)

0235 plusmn 0002

(1840)

0218 plusmn 0003

(2482)

0206 plusmn0003

(3522)

0193 plusmn0003

(4323)

0175 plusmn 0003

(5111)

EETG

(400 mgkg)

0226 plusmn0004

(21387)

0236 plusmn 0005

(1866)

0231 plusmn0006

(2735)

0210 plusmn0006

(3823)

0195 plusmn 0008

(4553)

NHTG

(400 mgkg)

0255 plusmn0003

(1145)

0250 plusmn 0002

(1379)

0280 plusmn0010

(1194)

0260 plusmn0010

(2352)

0236 plusmn 0011

(3407)

Piroxicam

(10 mgkg)

0208 plusmn0007

(2777)

0213 plusmn 0004

(2655)

0210 plusmn0003

(3396)

0183 plusmn0004

(4617)

0165 plusmn 0003

(5391)



85


edema

461 At 1st hr









86

462 At 2nd hr









87

463 At 3rd hr









88



89


edema model

Groups

1sthr 2ndhr 3rdhr

(Mean plusmnSEM)

(ml)

Inhibition

(Mean plusmnSEM)

(ml)

Inhibition

(Mean plusmnSEM)

(ml)

Inhibition


- 0318 plusmn 0001 - 0321 plusmn 0004 -

EEJR

( 500 mgkg)

0235 plusmn 0002

2192

0220 plusmn 0003

3081

0186 plusmn 0004

4205

NHJR

( 500 mgkg)

0251 plusmn 0003

1860

0240 plusmn 0002

2452

0225 plusmn 0008

2990

EETG


NHTG


Indomethacin

(10 mgkg)

0238 plusmn 0003

2093

0223 plusmn 0002

2987

0203 plusmn 0004

3676



90


471 At 1st hr









91

472 At 2nd hr





second hour




92

473 At 3rd hr









93




94


Groups

1sthr 2ndhr 3rdhr

(Mean plusmn SEM)

(ml) Inhibition

(Mean plusmn SEM)

(ml) Inhibition

(Mean plusmn SEM)

(ml) Inhibition


- 0331 plusmn 0004 - 0340 plusmn 0004 -

EEJR

( 500 mgkg)

0236 plusmn 0005

2411

0223 plusmn 0004

3262

0206 plusmn 0003

3941

NHJR

( 500 mgkg)

0258 plusmn 0004

1704

0236 plusmn 0004

2870

0225 plusmn 0005

3382

EETG


NHTG



2347

0230 plusmn 0005

2051

0195 plusmn 0003

4264



95


481 At 1st hr









96

482 At 2nd hr





at second hour




97

483 At 3rd hr









98

484 At 4th hr





fourth hour




99

485 At 5th hr









100



101


Groups

1sth

2ndh

3rdh

4thh

5thh




EEJR

(500 mgkg)

0216 plusmn 0004

(28)

0206 plusmn 0004

(4801)

0193 plusmn 0004

(4255)

0175 plusmn 0002

(4927)

0173 plusmn 0004

(5099)

NHJR

(500 mgkg)

0238 plusmn 0006

(2066)

0225 plusmn 0002

(2879)

0205 plusmn 0006

(3898)

0193 plusmn 0004

(4405)

0183 plusmn 0009

(4815)

EETG

(400 mgkg)

0243 plusmn 0005

(19)

0226 plusmn 0004

(2848)

0213 plusmn 0004

(3660)

0195 plusmn 0002

(4347)

0183 plusmn 004

(4815)

NHTG

(400 mgkg)

0266 plusmn 0005

(1133)

0245 plusmn0005

(2246)

0223 plusmn 0006

(3363)

0218 plusmn 0005

(3681)

0205 plusmn 0006

(4192)

DPH

(60 mgkg)

0220 plusmn 0004

(2667)

0195 plusmn 0004

(3829)

0176 plusmn 0003

(4761)

0168 plusmn 0003

(5130)

0161 plusmn0 006

(5443)



102





induced by xylene




103










104








105








106








107


Parameters


Mean plusmn SEM

Time of onset of








108







Alkaloids + + + +

Glycosides _ _ _ _

Saponins _ _ _

Phenols + + + +

Terpenoids + + + +

Steroids _ _ _ _

Flavonoids + + + +

Tannins + _ + +

109















110


Sr

No

Retention

Time

(Minute)

Total

Percentage

Name of Identified

Compound

Molecular

Formula

Molecular

Weight

(gmol)

Structures


2 5350 3956 Decane C10H22 142

3 6255 1169 (E)-3(10)-Caren-4-ol C10H16O 152

OH

CH2

CH3

CH3

4 6795 2182 Undecane C11H24 156

CH3

CH3

CH3 Cl

CH3

CH3

111

5 8311 0859 Dodecane C12H26 170

CH3

CH3


CH3

CH3


CH3

CH3


CH3CH3


CH3

112


CH3

CH3


CH3

CH3


CH3

CH3


CH3

CH3


CH3

CH3

113


CH3

CH3


CH3

CH3


CH3

CH3

18 20512 3754

Benzene (1-

methylundecyl)-

C18H30 246

CH3

CH3


CH3

CH3

114


CH3

CH3


CH3 CH3


CH3

CH3

23 22506 2311

Benzene (1-

methyldodecyl)-

C19H32 260

CH3 CH3


CH3

O

O CH3


CH3

O

O

CH3

115


O

O

CH3

CH3


O

O

CH3

28 30178 0212



ester

C16H22O4 278

O

OH

O

O

CH3

CH3

29 32303 1309 Squalene C30H50 410

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

30 32738 0380 Heptacosane C27H56 380

CH3

CH3

116

117


2 Decane

30( E )-3 (10)-Caren-4-ol

118

4 Undecane

5 Dodecane


119




120




121




122




123


20



124




125



27 Heptacosane

126













127


Sr

No

Retention

Time

(Minute)

Total

Percentage

Name of Identified

Compound

Molecular

Formula

Molecular

Weight

(gmol)

Structures


CH3

CH3

2 17498 4905

Velleral

C15H20O2 232 CH3

CH3

CH3O

O

3 20372 2361

Benzene (1-

methylundecyl)-

C18H30 246

CH3

CH3

128

4 20773 2105

Benzene (1-

pentylheptyl)

C18H30 246

5 20930 2199

ZZZ-1469-

Nonadecatetraene

C19H32 260

CH3 CH2


CH3

O

O

CH3

7 25737 4597 Phytol C20H40O 296

OH CH3

CH3

CH3

CH3

129


CH3

O

CH3


O

CH3


CH3

O

CH3

11 30178 2740

388-Trimethoxy-3-

piperidyl-22-

binaphthalene-1144-

tetrone

C28H25NO7 487

N

O

O

O

O

O

O

CH3CH3

O

CH3

130

12 32294 15138 Squalene C30H50 410

CH3

CH3

CH3

CH3CH3

CH3

CH3

CH3

13 32730 4184


ethylbutyl)-

C26H54 366 CH3

CH3


CH3

15 34759 6222 Vitamin E C29H50O2 430 O

CH3

CH3

OH

CH3

CH3

CH3

CH3

CH3

131

132




133




134

7 Phytol



135



6 Squalene

136


8 Heptacosane

9 Vitamin E

137










(Table 19)

138


SrNo Retention

Time (mins)

Total

(age)

Name of Identified


MW

(gmol) Structures


O

CH3

2 12186 15848 1H-2-Benzopyran-1-


OO



ester C14H28O2 228

5 16175 1580

Tetracyclo

[33101(39)]decan-10-

one

C10H12O 148 OO

139


ester C16H32O2 256

CH3

O

CH3

O


OH O


CH3

O

O

CH3

9 25737 0614 Phytol C20H40O 296 OH CH3

CH3

CH3

CH3



O

CH3

O

CH3

140



O CH3

O

CH3


CH3

O

CH3

13 32764 6519 Nonacosane C29H60 408 CH3

CH3



CH3


CH3

141

142




143




144




145

10 Phytol



146


14 Nonacosane


147

16 Hexatriacontane

148












20)

149


Sr

No

Retention

Time

(mins)

Total


Mol

Formula

MW

(gmol) Structures


CH3

CH3

2 5358 3731 Decane C10H22 142 CH3

CH3


CH3

CH3

4 6264 1048 (E)-3(10)-Caren-4-ol C10H16O 152

OH

CH2

CH3

CH3

150

5 6804 1990 Undecane C11H24 156 CH3

CH3

6 8311 0750 Dodecane C12H26 170 CH3

CH3



CH3

CH3


CH3

CH3


CH3

151


CH3


CH3

CH3


CH3

CH3


CH3


CH3

152


CH3

CH3


CH3

CH3


CH3

CH3


CH3


CH3

CH3


CH3

CH3

153


CH3 CH3


CH3

CH3



O

CH3


CH3

O

O

CH3



O

O

CH3

CH3

154


ester C20H36O2 308

O

CH3

O

CH3



O CH3

O

CH3


CH3

O

CH3



O

OH

O

O

CH3

CH3

155

32 32799 2525 Nonacosane C29H60 408

CH3

CH3


CH3

156

157

1 Decane

2 Decane 4-methyl-

3 (E)-3(10)-caren-4-ol

158

3 Undecane

4 Dodecane


159




160




161




162




163




164




165




166




167

31 Nonacosane

32 Tetratriacontane


168
























169


















destruction (79)






170


response






















171

























172

























173


























174


















extract (111)

175












176

FUTURE PERSPECTIVE












neonates


population

REFERENCES


201262(8)1085-93





therapeutics 2019













of Medicine 2011365(23)2205-19



20191614-11









21















Medica 201682(05)PC63



of Pain 201822(3)455-70



201771955






reviews 201711(22)145




of Nutrition 200899(4)715-22














201933(10)1531-4


199123(1)90-114















reviews 20054(3)130-6
























Toxicology 2003188(1)49-71



198136(6)435-6










200155(4)204





201113(1)27



201547(2)177






200389(1)123-9






































phytology 2011



2010439-44

















7
















710




401









200374(3)282-90






Res 201910(5)2087-100




therapy 201315(5)R141









sciences 201414(4)919-24









3(21)465-70


























Medicines 20129(2)242-9





reviews 200052(4)673-751















iii









my dreams

Aisha Mobashar

iv

TABLE OF CONTENTS

DEDICATION i

ACKNOWLDGEMENTS ii


LIST OF TABLES xi



ABSTRACT xix


11 Hypothesis 3






21 Epidemiology 4


23 Treatments 7

24 Inflammation 10


26 Plants used 12






v


















buffer (50X) 22


349 Densitometry 23









vi




carrageenan 28


histamine 28


serotonin 28


dextran 29



















vii






421 At Day 8 39

422 At Day 15 40

423 At Day 22 41

424 At Day 28 42







marker 57


levels of TNF-α 57



levels 59


levels of IL-1β 60






levels of IL-4 63

viii


64


parameters 66


count 66


content 67


count 68


platelet count 69


71



levels 72






gharuensis 77



451 At 1st hr 78

452 At 2nd hr 79

453 At 3rd hr 80

454 At 4th hr 81

ix

455 At 5th hr 82



461 At 1st hr 85

462 At 2nd hr 86

463 At 3rd hr 87



471 At 1st hr 90

472 At 2nd hr 91

473 At 3rd hr 92



481 At 1st hr 95

482 At 2nd hr 96

483 At 3rd hr 97

484 At 4th hr 98

485 At 5th hr 99











x





REFERENCES 177

xi

LIST OF TABLES

Page






40





66




T gharuensis

78



induced paw edema

84

xii



88



92



98



104






xiii

LIST OF FIGURES

Page

















xiv






of TNF-α

58


of NF-κB

59


of IL-6

60


of IL-1β

61


of COX-1

62


of COX-2

63


IL-4

64


xv


















xvi














xvii













Hb Haemoglobin

RBC Red blood cells






IL-6 Interleukin 6

IL-4 Interleukin 4



PXM Piroxicam

xviii

IND Indomethacin

DPH Diphenhydramine

Dexa Dexamethasone

LOP Loperamide



I2 Iodine


KI Potassium iodide


DW Distilled water

SM

bw

PO

Synovial membrane

Body weight

Per oral

xix



ABSTRACT




respectively
















xx
























xxi






1
























2

























3
















11 Hypothesis

111 Null hypothesis


properties



4














21 Epidemiology










5



22 Pathophysiology






















6

























7


















23 Treatments







8









cyclooxygenase (42)

















9

























10
















24 Inflammation









11









(45)







immunologic (46)






12










26 Plants used















13








(51)














14

Aim of the study



Objective of study













GC-MS analysis

15


31 Plant collection





name was checked




05














16





57)



















17









FCA (59)








342 (12)


2 and IL-4

351 RNA extraction


18









separated






3514 Washing of RNA







19






GAPDH


pairs sequence








20



IL-4


231 (58)


IL-6


247 (5)


TNF-α


202 NM_0126753


NF-КB


207 (58)


COX-1


200 (59)


COX-2


237 (58)


IL-1β


293 (58 60)


GAPDH


197 NM_0170084


21

353 cDNA synthesis





















22












Each one litre

Tris base 242 g


05M EDTA 100 ml


600ml




23




buffer











358 Densitometry






experiment

24




















immediately




25




five times










1800i)







26












Models Groups



EEJR (500 mgkg)

NHJR (500 mgkg )

EETG (400 mgkg)

NHTG (400 mgkg)




EEJR (500 mgkg)

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)



EEJR (500 mgkg)

27


water

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)




EEJR (500 mgkg)

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)




EEJR (500 mgkg)

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)




EEJR (500 mgkg)

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)

Loperamide (5 mgkg)



bw = body weight

28























29




















30




















31








(68)





glycosides (70)


3841 Salkowski test







red colour (71)

32






















33

CHAPTER 4 RESULTS


model








34









35









36









37

Figure 5 Paw edema



38










39


421 At Day 8








40

422 At Day 15









group

41

423 At Day 22









control group

42

424 At Day 28




0153)





43





44




Days







45











46









47











48


Parameters

Arthritic control

Piroxicam

EEJR

NHJR

EETG

NHTG

Mean plusmn SEM

Infiltration of






49







50





(Fig 15)

51





52





53





54





55





56



57











control groups

58






groups




control groups

59










60










61










62










63










64










65


marker

Markers

Vehicle

Control

Arthritic

Control Piroxicam

EEJR

NHJR

EETG

NHTG

Mean plusmn SEM


NF-κB


IL-6


IL-1β


COX-1







66







group





67











68





(1512 plusmn 0256)





69










70


Parameters


Mean plusmn SEM

RBC


Hb

(gdl)


WBC

(103Ul)


Platelets

(103Ul)




two control groups

71









in all groups

72







creatinine levels

73





other



creatinine levels

74





other



levels

75











76


Biochemical

Parameters

Vehicle


Mean plusmn SEM

Urea


Creatinine


AST


ALT




77









gharuensis


distress Diarrhea

Motor

activity

Posture


Group-1 - - - - - -

Group-1I - - - - - -

Group-1II - - - - - -

Group-1V - - - - - -

Group-V - - - - - -

Group-VI - - - - - -

Group-VII - - - - - -

Group-

VIII - - - - - -

GROUP-

IX - - - - - -

78


carrageenan

451 At 1st hr









79

452 At 2nd hr









80

453 At 3rd hr









81

454 At 4th hr









82

455 At 5th hr









83



84



Groups




EEJR

(500 mgkg)

0210 plusmn0003

(2708)

0195 plusmn 0002

(3275)

0172 plusmn0003

(4591)

0156 plusmn0004

(5411)

0125 plusmn 0003

(6508)

NHJR

(500 mgkg)

0235 plusmn 0002

(1840)

0218 plusmn 0003

(2482)

0206 plusmn0003

(3522)

0193 plusmn0003

(4323)

0175 plusmn 0003

(5111)

EETG

(400 mgkg)

0226 plusmn0004

(21387)

0236 plusmn 0005

(1866)

0231 plusmn0006

(2735)

0210 plusmn0006

(3823)

0195 plusmn 0008

(4553)

NHTG

(400 mgkg)

0255 plusmn0003

(1145)

0250 plusmn 0002

(1379)

0280 plusmn0010

(1194)

0260 plusmn0010

(2352)

0236 plusmn 0011

(3407)

Piroxicam

(10 mgkg)

0208 plusmn0007

(2777)

0213 plusmn 0004

(2655)

0210 plusmn0003

(3396)

0183 plusmn0004

(4617)

0165 plusmn 0003

(5391)



85


edema

461 At 1st hr









86

462 At 2nd hr









87

463 At 3rd hr









88



89


edema model

Groups

1sthr 2ndhr 3rdhr

(Mean plusmnSEM)

(ml)

Inhibition

(Mean plusmnSEM)

(ml)

Inhibition

(Mean plusmnSEM)

(ml)

Inhibition


- 0318 plusmn 0001 - 0321 plusmn 0004 -

EEJR

( 500 mgkg)

0235 plusmn 0002

2192

0220 plusmn 0003

3081

0186 plusmn 0004

4205

NHJR

( 500 mgkg)

0251 plusmn 0003

1860

0240 plusmn 0002

2452

0225 plusmn 0008

2990

EETG


NHTG


Indomethacin

(10 mgkg)

0238 plusmn 0003

2093

0223 plusmn 0002

2987

0203 plusmn 0004

3676



90


471 At 1st hr









91

472 At 2nd hr





second hour




92

473 At 3rd hr









93




94


Groups

1sthr 2ndhr 3rdhr

(Mean plusmn SEM)

(ml) Inhibition

(Mean plusmn SEM)

(ml) Inhibition

(Mean plusmn SEM)

(ml) Inhibition


- 0331 plusmn 0004 - 0340 plusmn 0004 -

EEJR

( 500 mgkg)

0236 plusmn 0005

2411

0223 plusmn 0004

3262

0206 plusmn 0003

3941

NHJR

( 500 mgkg)

0258 plusmn 0004

1704

0236 plusmn 0004

2870

0225 plusmn 0005

3382

EETG


NHTG



2347

0230 plusmn 0005

2051

0195 plusmn 0003

4264



95


481 At 1st hr









96

482 At 2nd hr





at second hour




97

483 At 3rd hr









98

484 At 4th hr





fourth hour




99

485 At 5th hr









100



101


Groups

1sth

2ndh

3rdh

4thh

5thh




EEJR

(500 mgkg)

0216 plusmn 0004

(28)

0206 plusmn 0004

(4801)

0193 plusmn 0004

(4255)

0175 plusmn 0002

(4927)

0173 plusmn 0004

(5099)

NHJR

(500 mgkg)

0238 plusmn 0006

(2066)

0225 plusmn 0002

(2879)

0205 plusmn 0006

(3898)

0193 plusmn 0004

(4405)

0183 plusmn 0009

(4815)

EETG

(400 mgkg)

0243 plusmn 0005

(19)

0226 plusmn 0004

(2848)

0213 plusmn 0004

(3660)

0195 plusmn 0002

(4347)

0183 plusmn 004

(4815)

NHTG

(400 mgkg)

0266 plusmn 0005

(1133)

0245 plusmn0005

(2246)

0223 plusmn 0006

(3363)

0218 plusmn 0005

(3681)

0205 plusmn 0006

(4192)

DPH

(60 mgkg)

0220 plusmn 0004

(2667)

0195 plusmn 0004

(3829)

0176 plusmn 0003

(4761)

0168 plusmn 0003

(5130)

0161 plusmn0 006

(5443)



102





induced by xylene




103










104








105








106








107


Parameters


Mean plusmn SEM

Time of onset of








108







Alkaloids + + + +

Glycosides _ _ _ _

Saponins _ _ _

Phenols + + + +

Terpenoids + + + +

Steroids _ _ _ _

Flavonoids + + + +

Tannins + _ + +

109















110


Sr

No

Retention

Time

(Minute)

Total

Percentage

Name of Identified

Compound

Molecular

Formula

Molecular

Weight

(gmol)

Structures


2 5350 3956 Decane C10H22 142

3 6255 1169 (E)-3(10)-Caren-4-ol C10H16O 152

OH

CH2

CH3

CH3

4 6795 2182 Undecane C11H24 156

CH3

CH3

CH3 Cl

CH3

CH3

111

5 8311 0859 Dodecane C12H26 170

CH3

CH3


CH3

CH3


CH3

CH3


CH3CH3


CH3

112


CH3

CH3


CH3

CH3


CH3

CH3


CH3

CH3


CH3

CH3

113


CH3

CH3


CH3

CH3


CH3

CH3

18 20512 3754

Benzene (1-

methylundecyl)-

C18H30 246

CH3

CH3


CH3

CH3

114


CH3

CH3


CH3 CH3


CH3

CH3

23 22506 2311

Benzene (1-

methyldodecyl)-

C19H32 260

CH3 CH3


CH3

O

O CH3


CH3

O

O

CH3

115


O

O

CH3

CH3


O

O

CH3

28 30178 0212



ester

C16H22O4 278

O

OH

O

O

CH3

CH3

29 32303 1309 Squalene C30H50 410

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

30 32738 0380 Heptacosane C27H56 380

CH3

CH3

116

117


2 Decane

30( E )-3 (10)-Caren-4-ol

118

4 Undecane

5 Dodecane


119




120




121




122




123


20



124




125



27 Heptacosane

126













127


Sr

No

Retention

Time

(Minute)

Total

Percentage

Name of Identified

Compound

Molecular

Formula

Molecular

Weight

(gmol)

Structures


CH3

CH3

2 17498 4905

Velleral

C15H20O2 232 CH3

CH3

CH3O

O

3 20372 2361

Benzene (1-

methylundecyl)-

C18H30 246

CH3

CH3

128

4 20773 2105

Benzene (1-

pentylheptyl)

C18H30 246

5 20930 2199

ZZZ-1469-

Nonadecatetraene

C19H32 260

CH3 CH2


CH3

O

O

CH3

7 25737 4597 Phytol C20H40O 296

OH CH3

CH3

CH3

CH3

129


CH3

O

CH3


O

CH3


CH3

O

CH3

11 30178 2740

388-Trimethoxy-3-

piperidyl-22-

binaphthalene-1144-

tetrone

C28H25NO7 487

N

O

O

O

O

O

O

CH3CH3

O

CH3

130

12 32294 15138 Squalene C30H50 410

CH3

CH3

CH3

CH3CH3

CH3

CH3

CH3

13 32730 4184


ethylbutyl)-

C26H54 366 CH3

CH3


CH3

15 34759 6222 Vitamin E C29H50O2 430 O

CH3

CH3

OH

CH3

CH3

CH3

CH3

CH3

131

132




133




134

7 Phytol



135



6 Squalene

136


8 Heptacosane

9 Vitamin E

137










(Table 19)

138


SrNo Retention

Time (mins)

Total

(age)

Name of Identified


MW

(gmol) Structures


O

CH3

2 12186 15848 1H-2-Benzopyran-1-


OO



ester C14H28O2 228

5 16175 1580

Tetracyclo

[33101(39)]decan-10-

one

C10H12O 148 OO

139


ester C16H32O2 256

CH3

O

CH3

O


OH O


CH3

O

O

CH3

9 25737 0614 Phytol C20H40O 296 OH CH3

CH3

CH3

CH3



O

CH3

O

CH3

140



O CH3

O

CH3


CH3

O

CH3

13 32764 6519 Nonacosane C29H60 408 CH3

CH3



CH3


CH3

141

142




143




144




145

10 Phytol



146


14 Nonacosane


147

16 Hexatriacontane

148












20)

149


Sr

No

Retention

Time

(mins)

Total


Mol

Formula

MW

(gmol) Structures


CH3

CH3

2 5358 3731 Decane C10H22 142 CH3

CH3


CH3

CH3

4 6264 1048 (E)-3(10)-Caren-4-ol C10H16O 152

OH

CH2

CH3

CH3

150

5 6804 1990 Undecane C11H24 156 CH3

CH3

6 8311 0750 Dodecane C12H26 170 CH3

CH3



CH3

CH3


CH3

CH3


CH3

151


CH3


CH3

CH3


CH3

CH3


CH3


CH3

152


CH3

CH3


CH3

CH3


CH3

CH3


CH3


CH3

CH3


CH3

CH3

153


CH3 CH3


CH3

CH3



O

CH3


CH3

O

O

CH3



O

O

CH3

CH3

154


ester C20H36O2 308

O

CH3

O

CH3



O CH3

O

CH3


CH3

O

CH3



O

OH

O

O

CH3

CH3

155

32 32799 2525 Nonacosane C29H60 408

CH3

CH3


CH3

156

157

1 Decane

2 Decane 4-methyl-

3 (E)-3(10)-caren-4-ol

158

3 Undecane

4 Dodecane


159




160




161




162




163




164




165




166




167

31 Nonacosane

32 Tetratriacontane


168
























169


















destruction (79)






170


response






















171

























172

























173


























174


















extract (111)

175












176

FUTURE PERSPECTIVE












neonates


population

REFERENCES


201262(8)1085-93





therapeutics 2019













of Medicine 2011365(23)2205-19



20191614-11









21















Medica 201682(05)PC63



of Pain 201822(3)455-70



201771955






reviews 201711(22)145




of Nutrition 200899(4)715-22














201933(10)1531-4


199123(1)90-114















reviews 20054(3)130-6
























Toxicology 2003188(1)49-71



198136(6)435-6










200155(4)204





201113(1)27



201547(2)177






200389(1)123-9






































phytology 2011



2010439-44

















7
















710




401









200374(3)282-90






Res 201910(5)2087-100




therapy 201315(5)R141









sciences 201414(4)919-24









3(21)465-70


























Medicines 20129(2)242-9





reviews 200052(4)673-751















iv

TABLE OF CONTENTS

DEDICATION i

ACKNOWLDGEMENTS ii


LIST OF TABLES xi



ABSTRACT xix


11 Hypothesis 3






21 Epidemiology 4


23 Treatments 7

24 Inflammation 10


26 Plants used 12






v


















buffer (50X) 22


349 Densitometry 23









vi




carrageenan 28


histamine 28


serotonin 28


dextran 29



















vii






421 At Day 8 39

422 At Day 15 40

423 At Day 22 41

424 At Day 28 42







marker 57


levels of TNF-α 57



levels 59


levels of IL-1β 60






levels of IL-4 63

viii


64


parameters 66


count 66


content 67


count 68


platelet count 69


71



levels 72






gharuensis 77



451 At 1st hr 78

452 At 2nd hr 79

453 At 3rd hr 80

454 At 4th hr 81

ix

455 At 5th hr 82



461 At 1st hr 85

462 At 2nd hr 86

463 At 3rd hr 87



471 At 1st hr 90

472 At 2nd hr 91

473 At 3rd hr 92



481 At 1st hr 95

482 At 2nd hr 96

483 At 3rd hr 97

484 At 4th hr 98

485 At 5th hr 99











x





REFERENCES 177

xi

LIST OF TABLES

Page






40





66




T gharuensis

78



induced paw edema

84

xii



88



92



98



104






xiii

LIST OF FIGURES

Page

















xiv






of TNF-α

58


of NF-κB

59


of IL-6

60


of IL-1β

61


of COX-1

62


of COX-2

63


IL-4

64


xv


















xvi














xvii













Hb Haemoglobin

RBC Red blood cells






IL-6 Interleukin 6

IL-4 Interleukin 4



PXM Piroxicam

xviii

IND Indomethacin

DPH Diphenhydramine

Dexa Dexamethasone

LOP Loperamide



I2 Iodine


KI Potassium iodide


DW Distilled water

SM

bw

PO

Synovial membrane

Body weight

Per oral

xix



ABSTRACT




respectively
















xx
























xxi






1
























2

























3
















11 Hypothesis

111 Null hypothesis


properties



4














21 Epidemiology










5



22 Pathophysiology






















6

























7


















23 Treatments







8









cyclooxygenase (42)

















9

























10
















24 Inflammation









11









(45)







immunologic (46)






12










26 Plants used















13








(51)














14

Aim of the study



Objective of study













GC-MS analysis

15


31 Plant collection





name was checked




05














16





57)



















17









FCA (59)








342 (12)


2 and IL-4

351 RNA extraction


18









separated






3514 Washing of RNA







19






GAPDH


pairs sequence








20



IL-4


231 (58)


IL-6


247 (5)


TNF-α


202 NM_0126753


NF-КB


207 (58)


COX-1


200 (59)


COX-2


237 (58)


IL-1β


293 (58 60)


GAPDH


197 NM_0170084


21

353 cDNA synthesis





















22












Each one litre

Tris base 242 g


05M EDTA 100 ml


600ml




23




buffer











358 Densitometry






experiment

24




















immediately




25




five times










1800i)







26












Models Groups



EEJR (500 mgkg)

NHJR (500 mgkg )

EETG (400 mgkg)

NHTG (400 mgkg)




EEJR (500 mgkg)

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)



EEJR (500 mgkg)

27


water

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)




EEJR (500 mgkg)

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)




EEJR (500 mgkg)

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)




EEJR (500 mgkg)

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)

Loperamide (5 mgkg)



bw = body weight

28























29




















30




















31








(68)





glycosides (70)


3841 Salkowski test







red colour (71)

32






















33

CHAPTER 4 RESULTS


model








34









35









36









37

Figure 5 Paw edema



38










39


421 At Day 8








40

422 At Day 15









group

41

423 At Day 22









control group

42

424 At Day 28




0153)





43





44




Days







45











46









47











48


Parameters

Arthritic control

Piroxicam

EEJR

NHJR

EETG

NHTG

Mean plusmn SEM

Infiltration of






49







50





(Fig 15)

51





52





53





54





55





56



57











control groups

58






groups




control groups

59










60










61










62










63










64










65


marker

Markers

Vehicle

Control

Arthritic

Control Piroxicam

EEJR

NHJR

EETG

NHTG

Mean plusmn SEM


NF-κB


IL-6


IL-1β


COX-1







66







group





67











68





(1512 plusmn 0256)





69










70


Parameters


Mean plusmn SEM

RBC


Hb

(gdl)


WBC

(103Ul)


Platelets

(103Ul)




two control groups

71









in all groups

72







creatinine levels

73





other



creatinine levels

74





other



levels

75











76


Biochemical

Parameters

Vehicle


Mean plusmn SEM

Urea


Creatinine


AST


ALT




77









gharuensis


distress Diarrhea

Motor

activity

Posture


Group-1 - - - - - -

Group-1I - - - - - -

Group-1II - - - - - -

Group-1V - - - - - -

Group-V - - - - - -

Group-VI - - - - - -

Group-VII - - - - - -

Group-

VIII - - - - - -

GROUP-

IX - - - - - -

78


carrageenan

451 At 1st hr









79

452 At 2nd hr









80

453 At 3rd hr









81

454 At 4th hr









82

455 At 5th hr









83



84



Groups




EEJR

(500 mgkg)

0210 plusmn0003

(2708)

0195 plusmn 0002

(3275)

0172 plusmn0003

(4591)

0156 plusmn0004

(5411)

0125 plusmn 0003

(6508)

NHJR

(500 mgkg)

0235 plusmn 0002

(1840)

0218 plusmn 0003

(2482)

0206 plusmn0003

(3522)

0193 plusmn0003

(4323)

0175 plusmn 0003

(5111)

EETG

(400 mgkg)

0226 plusmn0004

(21387)

0236 plusmn 0005

(1866)

0231 plusmn0006

(2735)

0210 plusmn0006

(3823)

0195 plusmn 0008

(4553)

NHTG

(400 mgkg)

0255 plusmn0003

(1145)

0250 plusmn 0002

(1379)

0280 plusmn0010

(1194)

0260 plusmn0010

(2352)

0236 plusmn 0011

(3407)

Piroxicam

(10 mgkg)

0208 plusmn0007

(2777)

0213 plusmn 0004

(2655)

0210 plusmn0003

(3396)

0183 plusmn0004

(4617)

0165 plusmn 0003

(5391)



85


edema

461 At 1st hr









86

462 At 2nd hr









87

463 At 3rd hr









88



89


edema model

Groups

1sthr 2ndhr 3rdhr

(Mean plusmnSEM)

(ml)

Inhibition

(Mean plusmnSEM)

(ml)

Inhibition

(Mean plusmnSEM)

(ml)

Inhibition


- 0318 plusmn 0001 - 0321 plusmn 0004 -

EEJR

( 500 mgkg)

0235 plusmn 0002

2192

0220 plusmn 0003

3081

0186 plusmn 0004

4205

NHJR

( 500 mgkg)

0251 plusmn 0003

1860

0240 plusmn 0002

2452

0225 plusmn 0008

2990

EETG


NHTG


Indomethacin

(10 mgkg)

0238 plusmn 0003

2093

0223 plusmn 0002

2987

0203 plusmn 0004

3676



90


471 At 1st hr









91

472 At 2nd hr





second hour




92

473 At 3rd hr









93




94


Groups

1sthr 2ndhr 3rdhr

(Mean plusmn SEM)

(ml) Inhibition

(Mean plusmn SEM)

(ml) Inhibition

(Mean plusmn SEM)

(ml) Inhibition


- 0331 plusmn 0004 - 0340 plusmn 0004 -

EEJR

( 500 mgkg)

0236 plusmn 0005

2411

0223 plusmn 0004

3262

0206 plusmn 0003

3941

NHJR

( 500 mgkg)

0258 plusmn 0004

1704

0236 plusmn 0004

2870

0225 plusmn 0005

3382

EETG


NHTG



2347

0230 plusmn 0005

2051

0195 plusmn 0003

4264



95


481 At 1st hr









96

482 At 2nd hr





at second hour




97

483 At 3rd hr









98

484 At 4th hr





fourth hour




99

485 At 5th hr









100



101


Groups

1sth

2ndh

3rdh

4thh

5thh




EEJR

(500 mgkg)

0216 plusmn 0004

(28)

0206 plusmn 0004

(4801)

0193 plusmn 0004

(4255)

0175 plusmn 0002

(4927)

0173 plusmn 0004

(5099)

NHJR

(500 mgkg)

0238 plusmn 0006

(2066)

0225 plusmn 0002

(2879)

0205 plusmn 0006

(3898)

0193 plusmn 0004

(4405)

0183 plusmn 0009

(4815)

EETG

(400 mgkg)

0243 plusmn 0005

(19)

0226 plusmn 0004

(2848)

0213 plusmn 0004

(3660)

0195 plusmn 0002

(4347)

0183 plusmn 004

(4815)

NHTG

(400 mgkg)

0266 plusmn 0005

(1133)

0245 plusmn0005

(2246)

0223 plusmn 0006

(3363)

0218 plusmn 0005

(3681)

0205 plusmn 0006

(4192)

DPH

(60 mgkg)

0220 plusmn 0004

(2667)

0195 plusmn 0004

(3829)

0176 plusmn 0003

(4761)

0168 plusmn 0003

(5130)

0161 plusmn0 006

(5443)



102





induced by xylene




103










104








105








106








107


Parameters


Mean plusmn SEM

Time of onset of








108







Alkaloids + + + +

Glycosides _ _ _ _

Saponins _ _ _

Phenols + + + +

Terpenoids + + + +

Steroids _ _ _ _

Flavonoids + + + +

Tannins + _ + +

109















110


Sr

No

Retention

Time

(Minute)

Total

Percentage

Name of Identified

Compound

Molecular

Formula

Molecular

Weight

(gmol)

Structures


2 5350 3956 Decane C10H22 142

3 6255 1169 (E)-3(10)-Caren-4-ol C10H16O 152

OH

CH2

CH3

CH3

4 6795 2182 Undecane C11H24 156

CH3

CH3

CH3 Cl

CH3

CH3

111

5 8311 0859 Dodecane C12H26 170

CH3

CH3


CH3

CH3


CH3

CH3


CH3CH3


CH3

112


CH3

CH3


CH3

CH3


CH3

CH3


CH3

CH3


CH3

CH3

113


CH3

CH3


CH3

CH3


CH3

CH3

18 20512 3754

Benzene (1-

methylundecyl)-

C18H30 246

CH3

CH3


CH3

CH3

114


CH3

CH3


CH3 CH3


CH3

CH3

23 22506 2311

Benzene (1-

methyldodecyl)-

C19H32 260

CH3 CH3


CH3

O

O CH3


CH3

O

O

CH3

115


O

O

CH3

CH3


O

O

CH3

28 30178 0212



ester

C16H22O4 278

O

OH

O

O

CH3

CH3

29 32303 1309 Squalene C30H50 410

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

30 32738 0380 Heptacosane C27H56 380

CH3

CH3

116

117


2 Decane

30( E )-3 (10)-Caren-4-ol

118

4 Undecane

5 Dodecane


119




120




121




122




123


20



124




125



27 Heptacosane

126













127


Sr

No

Retention

Time

(Minute)

Total

Percentage

Name of Identified

Compound

Molecular

Formula

Molecular

Weight

(gmol)

Structures


CH3

CH3

2 17498 4905

Velleral

C15H20O2 232 CH3

CH3

CH3O

O

3 20372 2361

Benzene (1-

methylundecyl)-

C18H30 246

CH3

CH3

128

4 20773 2105

Benzene (1-

pentylheptyl)

C18H30 246

5 20930 2199

ZZZ-1469-

Nonadecatetraene

C19H32 260

CH3 CH2


CH3

O

O

CH3

7 25737 4597 Phytol C20H40O 296

OH CH3

CH3

CH3

CH3

129


CH3

O

CH3


O

CH3


CH3

O

CH3

11 30178 2740

388-Trimethoxy-3-

piperidyl-22-

binaphthalene-1144-

tetrone

C28H25NO7 487

N

O

O

O

O

O

O

CH3CH3

O

CH3

130

12 32294 15138 Squalene C30H50 410

CH3

CH3

CH3

CH3CH3

CH3

CH3

CH3

13 32730 4184


ethylbutyl)-

C26H54 366 CH3

CH3


CH3

15 34759 6222 Vitamin E C29H50O2 430 O

CH3

CH3

OH

CH3

CH3

CH3

CH3

CH3

131

132




133




134

7 Phytol



135



6 Squalene

136


8 Heptacosane

9 Vitamin E

137










(Table 19)

138


SrNo Retention

Time (mins)

Total

(age)

Name of Identified


MW

(gmol) Structures


O

CH3

2 12186 15848 1H-2-Benzopyran-1-


OO



ester C14H28O2 228

5 16175 1580

Tetracyclo

[33101(39)]decan-10-

one

C10H12O 148 OO

139


ester C16H32O2 256

CH3

O

CH3

O


OH O


CH3

O

O

CH3

9 25737 0614 Phytol C20H40O 296 OH CH3

CH3

CH3

CH3



O

CH3

O

CH3

140



O CH3

O

CH3


CH3

O

CH3

13 32764 6519 Nonacosane C29H60 408 CH3

CH3



CH3


CH3

141

142




143




144




145

10 Phytol



146


14 Nonacosane


147

16 Hexatriacontane

148












20)

149


Sr

No

Retention

Time

(mins)

Total


Mol

Formula

MW

(gmol) Structures


CH3

CH3

2 5358 3731 Decane C10H22 142 CH3

CH3


CH3

CH3

4 6264 1048 (E)-3(10)-Caren-4-ol C10H16O 152

OH

CH2

CH3

CH3

150

5 6804 1990 Undecane C11H24 156 CH3

CH3

6 8311 0750 Dodecane C12H26 170 CH3

CH3



CH3

CH3


CH3

CH3


CH3

151


CH3


CH3

CH3


CH3

CH3


CH3


CH3

152


CH3

CH3


CH3

CH3


CH3

CH3


CH3


CH3

CH3


CH3

CH3

153


CH3 CH3


CH3

CH3



O

CH3


CH3

O

O

CH3



O

O

CH3

CH3

154


ester C20H36O2 308

O

CH3

O

CH3



O CH3

O

CH3


CH3

O

CH3



O

OH

O

O

CH3

CH3

155

32 32799 2525 Nonacosane C29H60 408

CH3

CH3


CH3

156

157

1 Decane

2 Decane 4-methyl-

3 (E)-3(10)-caren-4-ol

158

3 Undecane

4 Dodecane


159




160




161




162




163




164




165




166




167

31 Nonacosane

32 Tetratriacontane


168
























169


















destruction (79)






170


response






















171

























172

























173


























174


















extract (111)

175












176

FUTURE PERSPECTIVE












neonates


population

REFERENCES


201262(8)1085-93





therapeutics 2019













of Medicine 2011365(23)2205-19



20191614-11









21















Medica 201682(05)PC63



of Pain 201822(3)455-70



201771955






reviews 201711(22)145




of Nutrition 200899(4)715-22














201933(10)1531-4


199123(1)90-114















reviews 20054(3)130-6
























Toxicology 2003188(1)49-71



198136(6)435-6










200155(4)204





201113(1)27



201547(2)177






200389(1)123-9






































phytology 2011



2010439-44

















7
















710




401









200374(3)282-90






Res 201910(5)2087-100




therapy 201315(5)R141









sciences 201414(4)919-24









3(21)465-70


























Medicines 20129(2)242-9





reviews 200052(4)673-751















v


















buffer (50X) 22


349 Densitometry 23









vi




carrageenan 28


histamine 28


serotonin 28


dextran 29



















vii






421 At Day 8 39

422 At Day 15 40

423 At Day 22 41

424 At Day 28 42







marker 57


levels of TNF-α 57



levels 59


levels of IL-1β 60






levels of IL-4 63

viii


64


parameters 66


count 66


content 67


count 68


platelet count 69


71



levels 72






gharuensis 77



451 At 1st hr 78

452 At 2nd hr 79

453 At 3rd hr 80

454 At 4th hr 81

ix

455 At 5th hr 82



461 At 1st hr 85

462 At 2nd hr 86

463 At 3rd hr 87



471 At 1st hr 90

472 At 2nd hr 91

473 At 3rd hr 92



481 At 1st hr 95

482 At 2nd hr 96

483 At 3rd hr 97

484 At 4th hr 98

485 At 5th hr 99











x





REFERENCES 177

xi

LIST OF TABLES

Page






40





66




T gharuensis

78



induced paw edema

84

xii



88



92



98



104






xiii

LIST OF FIGURES

Page

















xiv






of TNF-α

58


of NF-κB

59


of IL-6

60


of IL-1β

61


of COX-1

62


of COX-2

63


IL-4

64


xv


















xvi














xvii













Hb Haemoglobin

RBC Red blood cells






IL-6 Interleukin 6

IL-4 Interleukin 4



PXM Piroxicam

xviii

IND Indomethacin

DPH Diphenhydramine

Dexa Dexamethasone

LOP Loperamide



I2 Iodine


KI Potassium iodide


DW Distilled water

SM

bw

PO

Synovial membrane

Body weight

Per oral

xix



ABSTRACT




respectively
















xx
























xxi






1
























2

























3
















11 Hypothesis

111 Null hypothesis


properties



4














21 Epidemiology










5



22 Pathophysiology






















6

























7


















23 Treatments







8









cyclooxygenase (42)

















9

























10
















24 Inflammation









11









(45)







immunologic (46)






12










26 Plants used















13








(51)














14

Aim of the study



Objective of study













GC-MS analysis

15


31 Plant collection





name was checked




05














16





57)



















17









FCA (59)








342 (12)


2 and IL-4

351 RNA extraction


18









separated






3514 Washing of RNA







19






GAPDH


pairs sequence








20



IL-4


231 (58)


IL-6


247 (5)


TNF-α


202 NM_0126753


NF-КB


207 (58)


COX-1


200 (59)


COX-2


237 (58)


IL-1β


293 (58 60)


GAPDH


197 NM_0170084


21

353 cDNA synthesis





















22












Each one litre

Tris base 242 g


05M EDTA 100 ml


600ml




23




buffer











358 Densitometry






experiment

24




















immediately




25




five times










1800i)







26












Models Groups



EEJR (500 mgkg)

NHJR (500 mgkg )

EETG (400 mgkg)

NHTG (400 mgkg)




EEJR (500 mgkg)

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)



EEJR (500 mgkg)

27


water

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)




EEJR (500 mgkg)

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)




EEJR (500 mgkg)

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)




EEJR (500 mgkg)

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)

Loperamide (5 mgkg)



bw = body weight

28























29




















30




















31








(68)





glycosides (70)


3841 Salkowski test







red colour (71)

32






















33

CHAPTER 4 RESULTS


model








34









35









36









37

Figure 5 Paw edema



38










39


421 At Day 8








40

422 At Day 15









group

41

423 At Day 22









control group

42

424 At Day 28




0153)





43





44




Days







45











46









47











48


Parameters

Arthritic control

Piroxicam

EEJR

NHJR

EETG

NHTG

Mean plusmn SEM

Infiltration of






49







50





(Fig 15)

51





52





53





54





55





56



57











control groups

58






groups




control groups

59










60










61










62










63










64










65


marker

Markers

Vehicle

Control

Arthritic

Control Piroxicam

EEJR

NHJR

EETG

NHTG

Mean plusmn SEM


NF-κB


IL-6


IL-1β


COX-1







66







group





67











68





(1512 plusmn 0256)





69










70


Parameters


Mean plusmn SEM

RBC


Hb

(gdl)


WBC

(103Ul)


Platelets

(103Ul)




two control groups

71









in all groups

72







creatinine levels

73





other



creatinine levels

74





other



levels

75











76


Biochemical

Parameters

Vehicle


Mean plusmn SEM

Urea


Creatinine


AST


ALT




77









gharuensis


distress Diarrhea

Motor

activity

Posture


Group-1 - - - - - -

Group-1I - - - - - -

Group-1II - - - - - -

Group-1V - - - - - -

Group-V - - - - - -

Group-VI - - - - - -

Group-VII - - - - - -

Group-

VIII - - - - - -

GROUP-

IX - - - - - -

78


carrageenan

451 At 1st hr









79

452 At 2nd hr









80

453 At 3rd hr









81

454 At 4th hr









82

455 At 5th hr









83



84



Groups




EEJR

(500 mgkg)

0210 plusmn0003

(2708)

0195 plusmn 0002

(3275)

0172 plusmn0003

(4591)

0156 plusmn0004

(5411)

0125 plusmn 0003

(6508)

NHJR

(500 mgkg)

0235 plusmn 0002

(1840)

0218 plusmn 0003

(2482)

0206 plusmn0003

(3522)

0193 plusmn0003

(4323)

0175 plusmn 0003

(5111)

EETG

(400 mgkg)

0226 plusmn0004

(21387)

0236 plusmn 0005

(1866)

0231 plusmn0006

(2735)

0210 plusmn0006

(3823)

0195 plusmn 0008

(4553)

NHTG

(400 mgkg)

0255 plusmn0003

(1145)

0250 plusmn 0002

(1379)

0280 plusmn0010

(1194)

0260 plusmn0010

(2352)

0236 plusmn 0011

(3407)

Piroxicam

(10 mgkg)

0208 plusmn0007

(2777)

0213 plusmn 0004

(2655)

0210 plusmn0003

(3396)

0183 plusmn0004

(4617)

0165 plusmn 0003

(5391)



85


edema

461 At 1st hr









86

462 At 2nd hr









87

463 At 3rd hr









88



89


edema model

Groups

1sthr 2ndhr 3rdhr

(Mean plusmnSEM)

(ml)

Inhibition

(Mean plusmnSEM)

(ml)

Inhibition

(Mean plusmnSEM)

(ml)

Inhibition


- 0318 plusmn 0001 - 0321 plusmn 0004 -

EEJR

( 500 mgkg)

0235 plusmn 0002

2192

0220 plusmn 0003

3081

0186 plusmn 0004

4205

NHJR

( 500 mgkg)

0251 plusmn 0003

1860

0240 plusmn 0002

2452

0225 plusmn 0008

2990

EETG


NHTG


Indomethacin

(10 mgkg)

0238 plusmn 0003

2093

0223 plusmn 0002

2987

0203 plusmn 0004

3676



90


471 At 1st hr









91

472 At 2nd hr





second hour




92

473 At 3rd hr









93




94


Groups

1sthr 2ndhr 3rdhr

(Mean plusmn SEM)

(ml) Inhibition

(Mean plusmn SEM)

(ml) Inhibition

(Mean plusmn SEM)

(ml) Inhibition


- 0331 plusmn 0004 - 0340 plusmn 0004 -

EEJR

( 500 mgkg)

0236 plusmn 0005

2411

0223 plusmn 0004

3262

0206 plusmn 0003

3941

NHJR

( 500 mgkg)

0258 plusmn 0004

1704

0236 plusmn 0004

2870

0225 plusmn 0005

3382

EETG


NHTG



2347

0230 plusmn 0005

2051

0195 plusmn 0003

4264



95


481 At 1st hr









96

482 At 2nd hr





at second hour




97

483 At 3rd hr









98

484 At 4th hr





fourth hour




99

485 At 5th hr









100



101


Groups

1sth

2ndh

3rdh

4thh

5thh




EEJR

(500 mgkg)

0216 plusmn 0004

(28)

0206 plusmn 0004

(4801)

0193 plusmn 0004

(4255)

0175 plusmn 0002

(4927)

0173 plusmn 0004

(5099)

NHJR

(500 mgkg)

0238 plusmn 0006

(2066)

0225 plusmn 0002

(2879)

0205 plusmn 0006

(3898)

0193 plusmn 0004

(4405)

0183 plusmn 0009

(4815)

EETG

(400 mgkg)

0243 plusmn 0005

(19)

0226 plusmn 0004

(2848)

0213 plusmn 0004

(3660)

0195 plusmn 0002

(4347)

0183 plusmn 004

(4815)

NHTG

(400 mgkg)

0266 plusmn 0005

(1133)

0245 plusmn0005

(2246)

0223 plusmn 0006

(3363)

0218 plusmn 0005

(3681)

0205 plusmn 0006

(4192)

DPH

(60 mgkg)

0220 plusmn 0004

(2667)

0195 plusmn 0004

(3829)

0176 plusmn 0003

(4761)

0168 plusmn 0003

(5130)

0161 plusmn0 006

(5443)



102





induced by xylene




103










104








105








106








107


Parameters


Mean plusmn SEM

Time of onset of








108







Alkaloids + + + +

Glycosides _ _ _ _

Saponins _ _ _

Phenols + + + +

Terpenoids + + + +

Steroids _ _ _ _

Flavonoids + + + +

Tannins + _ + +

109















110


Sr

No

Retention

Time

(Minute)

Total

Percentage

Name of Identified

Compound

Molecular

Formula

Molecular

Weight

(gmol)

Structures


2 5350 3956 Decane C10H22 142

3 6255 1169 (E)-3(10)-Caren-4-ol C10H16O 152

OH

CH2

CH3

CH3

4 6795 2182 Undecane C11H24 156

CH3

CH3

CH3 Cl

CH3

CH3

111

5 8311 0859 Dodecane C12H26 170

CH3

CH3


CH3

CH3


CH3

CH3


CH3CH3


CH3

112


CH3

CH3


CH3

CH3


CH3

CH3


CH3

CH3


CH3

CH3

113


CH3

CH3


CH3

CH3


CH3

CH3

18 20512 3754

Benzene (1-

methylundecyl)-

C18H30 246

CH3

CH3


CH3

CH3

114


CH3

CH3


CH3 CH3


CH3

CH3

23 22506 2311

Benzene (1-

methyldodecyl)-

C19H32 260

CH3 CH3


CH3

O

O CH3


CH3

O

O

CH3

115


O

O

CH3

CH3


O

O

CH3

28 30178 0212



ester

C16H22O4 278

O

OH

O

O

CH3

CH3

29 32303 1309 Squalene C30H50 410

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

30 32738 0380 Heptacosane C27H56 380

CH3

CH3

116

117


2 Decane

30( E )-3 (10)-Caren-4-ol

118

4 Undecane

5 Dodecane


119




120




121




122




123


20



124




125



27 Heptacosane

126













127


Sr

No

Retention

Time

(Minute)

Total

Percentage

Name of Identified

Compound

Molecular

Formula

Molecular

Weight

(gmol)

Structures


CH3

CH3

2 17498 4905

Velleral

C15H20O2 232 CH3

CH3

CH3O

O

3 20372 2361

Benzene (1-

methylundecyl)-

C18H30 246

CH3

CH3

128

4 20773 2105

Benzene (1-

pentylheptyl)

C18H30 246

5 20930 2199

ZZZ-1469-

Nonadecatetraene

C19H32 260

CH3 CH2


CH3

O

O

CH3

7 25737 4597 Phytol C20H40O 296

OH CH3

CH3

CH3

CH3

129


CH3

O

CH3


O

CH3


CH3

O

CH3

11 30178 2740

388-Trimethoxy-3-

piperidyl-22-

binaphthalene-1144-

tetrone

C28H25NO7 487

N

O

O

O

O

O

O

CH3CH3

O

CH3

130

12 32294 15138 Squalene C30H50 410

CH3

CH3

CH3

CH3CH3

CH3

CH3

CH3

13 32730 4184


ethylbutyl)-

C26H54 366 CH3

CH3


CH3

15 34759 6222 Vitamin E C29H50O2 430 O

CH3

CH3

OH

CH3

CH3

CH3

CH3

CH3

131

132




133




134

7 Phytol



135



6 Squalene

136


8 Heptacosane

9 Vitamin E

137










(Table 19)

138


SrNo Retention

Time (mins)

Total

(age)

Name of Identified


MW

(gmol) Structures


O

CH3

2 12186 15848 1H-2-Benzopyran-1-


OO



ester C14H28O2 228

5 16175 1580

Tetracyclo

[33101(39)]decan-10-

one

C10H12O 148 OO

139


ester C16H32O2 256

CH3

O

CH3

O


OH O


CH3

O

O

CH3

9 25737 0614 Phytol C20H40O 296 OH CH3

CH3

CH3

CH3



O

CH3

O

CH3

140



O CH3

O

CH3


CH3

O

CH3

13 32764 6519 Nonacosane C29H60 408 CH3

CH3



CH3


CH3

141

142




143




144




145

10 Phytol



146


14 Nonacosane


147

16 Hexatriacontane

148












20)

149


Sr

No

Retention

Time

(mins)

Total


Mol

Formula

MW

(gmol) Structures


CH3

CH3

2 5358 3731 Decane C10H22 142 CH3

CH3


CH3

CH3

4 6264 1048 (E)-3(10)-Caren-4-ol C10H16O 152

OH

CH2

CH3

CH3

150

5 6804 1990 Undecane C11H24 156 CH3

CH3

6 8311 0750 Dodecane C12H26 170 CH3

CH3



CH3

CH3


CH3

CH3


CH3

151


CH3


CH3

CH3


CH3

CH3


CH3


CH3

152


CH3

CH3


CH3

CH3


CH3

CH3


CH3


CH3

CH3


CH3

CH3

153


CH3 CH3


CH3

CH3



O

CH3


CH3

O

O

CH3



O

O

CH3

CH3

154


ester C20H36O2 308

O

CH3

O

CH3



O CH3

O

CH3


CH3

O

CH3



O

OH

O

O

CH3

CH3

155

32 32799 2525 Nonacosane C29H60 408

CH3

CH3


CH3

156

157

1 Decane

2 Decane 4-methyl-

3 (E)-3(10)-caren-4-ol

158

3 Undecane

4 Dodecane


159




160




161




162




163




164




165




166




167

31 Nonacosane

32 Tetratriacontane


168
























169


















destruction (79)






170


response






















171

























172

























173


























174


















extract (111)

175












176

FUTURE PERSPECTIVE












neonates


population

REFERENCES


201262(8)1085-93





therapeutics 2019













of Medicine 2011365(23)2205-19



20191614-11









21















Medica 201682(05)PC63



of Pain 201822(3)455-70



201771955






reviews 201711(22)145




of Nutrition 200899(4)715-22














201933(10)1531-4


199123(1)90-114















reviews 20054(3)130-6
























Toxicology 2003188(1)49-71



198136(6)435-6










200155(4)204





201113(1)27



201547(2)177






200389(1)123-9






































phytology 2011



2010439-44

















7
















710




401









200374(3)282-90






Res 201910(5)2087-100




therapy 201315(5)R141









sciences 201414(4)919-24









3(21)465-70


























Medicines 20129(2)242-9





reviews 200052(4)673-751















vi




carrageenan 28


histamine 28


serotonin 28


dextran 29



















vii






421 At Day 8 39

422 At Day 15 40

423 At Day 22 41

424 At Day 28 42







marker 57


levels of TNF-α 57



levels 59


levels of IL-1β 60






levels of IL-4 63

viii


64


parameters 66


count 66


content 67


count 68


platelet count 69


71



levels 72






gharuensis 77



451 At 1st hr 78

452 At 2nd hr 79

453 At 3rd hr 80

454 At 4th hr 81

ix

455 At 5th hr 82



461 At 1st hr 85

462 At 2nd hr 86

463 At 3rd hr 87



471 At 1st hr 90

472 At 2nd hr 91

473 At 3rd hr 92



481 At 1st hr 95

482 At 2nd hr 96

483 At 3rd hr 97

484 At 4th hr 98

485 At 5th hr 99











x





REFERENCES 177

xi

LIST OF TABLES

Page






40





66




T gharuensis

78



induced paw edema

84

xii



88



92



98



104






xiii

LIST OF FIGURES

Page

















xiv






of TNF-α

58


of NF-κB

59


of IL-6

60


of IL-1β

61


of COX-1

62


of COX-2

63


IL-4

64


xv


















xvi














xvii













Hb Haemoglobin

RBC Red blood cells






IL-6 Interleukin 6

IL-4 Interleukin 4



PXM Piroxicam

xviii

IND Indomethacin

DPH Diphenhydramine

Dexa Dexamethasone

LOP Loperamide



I2 Iodine


KI Potassium iodide


DW Distilled water

SM

bw

PO

Synovial membrane

Body weight

Per oral

xix



ABSTRACT




respectively
















xx
























xxi






1
























2

























3
















11 Hypothesis

111 Null hypothesis


properties



4














21 Epidemiology










5



22 Pathophysiology






















6

























7


















23 Treatments







8









cyclooxygenase (42)

















9

























10
















24 Inflammation









11









(45)







immunologic (46)






12










26 Plants used















13








(51)














14

Aim of the study



Objective of study













GC-MS analysis

15


31 Plant collection





name was checked




05














16





57)



















17









FCA (59)








342 (12)


2 and IL-4

351 RNA extraction


18









separated






3514 Washing of RNA







19






GAPDH


pairs sequence








20



IL-4


231 (58)


IL-6


247 (5)


TNF-α


202 NM_0126753


NF-КB


207 (58)


COX-1


200 (59)


COX-2


237 (58)


IL-1β


293 (58 60)


GAPDH


197 NM_0170084


21

353 cDNA synthesis





















22












Each one litre

Tris base 242 g


05M EDTA 100 ml


600ml




23




buffer











358 Densitometry






experiment

24




















immediately




25




five times










1800i)







26












Models Groups



EEJR (500 mgkg)

NHJR (500 mgkg )

EETG (400 mgkg)

NHTG (400 mgkg)




EEJR (500 mgkg)

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)



EEJR (500 mgkg)

27


water

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)




EEJR (500 mgkg)

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)




EEJR (500 mgkg)

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)




EEJR (500 mgkg)

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)

Loperamide (5 mgkg)



bw = body weight

28























29




















30




















31








(68)





glycosides (70)


3841 Salkowski test







red colour (71)

32






















33

CHAPTER 4 RESULTS


model








34









35









36









37

Figure 5 Paw edema



38










39


421 At Day 8








40

422 At Day 15









group

41

423 At Day 22









control group

42

424 At Day 28




0153)





43





44




Days







45











46









47











48


Parameters

Arthritic control

Piroxicam

EEJR

NHJR

EETG

NHTG

Mean plusmn SEM

Infiltration of






49







50





(Fig 15)

51





52





53





54





55





56



57











control groups

58






groups




control groups

59










60










61










62










63










64










65


marker

Markers

Vehicle

Control

Arthritic

Control Piroxicam

EEJR

NHJR

EETG

NHTG

Mean plusmn SEM


NF-κB


IL-6


IL-1β


COX-1







66







group





67











68





(1512 plusmn 0256)





69










70


Parameters


Mean plusmn SEM

RBC


Hb

(gdl)


WBC

(103Ul)


Platelets

(103Ul)




two control groups

71









in all groups

72







creatinine levels

73





other



creatinine levels

74





other



levels

75











76


Biochemical

Parameters

Vehicle


Mean plusmn SEM

Urea


Creatinine


AST


ALT




77









gharuensis


distress Diarrhea

Motor

activity

Posture


Group-1 - - - - - -

Group-1I - - - - - -

Group-1II - - - - - -

Group-1V - - - - - -

Group-V - - - - - -

Group-VI - - - - - -

Group-VII - - - - - -

Group-

VIII - - - - - -

GROUP-

IX - - - - - -

78


carrageenan

451 At 1st hr









79

452 At 2nd hr









80

453 At 3rd hr









81

454 At 4th hr









82

455 At 5th hr









83



84



Groups




EEJR

(500 mgkg)

0210 plusmn0003

(2708)

0195 plusmn 0002

(3275)

0172 plusmn0003

(4591)

0156 plusmn0004

(5411)

0125 plusmn 0003

(6508)

NHJR

(500 mgkg)

0235 plusmn 0002

(1840)

0218 plusmn 0003

(2482)

0206 plusmn0003

(3522)

0193 plusmn0003

(4323)

0175 plusmn 0003

(5111)

EETG

(400 mgkg)

0226 plusmn0004

(21387)

0236 plusmn 0005

(1866)

0231 plusmn0006

(2735)

0210 plusmn0006

(3823)

0195 plusmn 0008

(4553)

NHTG

(400 mgkg)

0255 plusmn0003

(1145)

0250 plusmn 0002

(1379)

0280 plusmn0010

(1194)

0260 plusmn0010

(2352)

0236 plusmn 0011

(3407)

Piroxicam

(10 mgkg)

0208 plusmn0007

(2777)

0213 plusmn 0004

(2655)

0210 plusmn0003

(3396)

0183 plusmn0004

(4617)

0165 plusmn 0003

(5391)



85


edema

461 At 1st hr









86

462 At 2nd hr









87

463 At 3rd hr









88



89


edema model

Groups

1sthr 2ndhr 3rdhr

(Mean plusmnSEM)

(ml)

Inhibition

(Mean plusmnSEM)

(ml)

Inhibition

(Mean plusmnSEM)

(ml)

Inhibition


- 0318 plusmn 0001 - 0321 plusmn 0004 -

EEJR

( 500 mgkg)

0235 plusmn 0002

2192

0220 plusmn 0003

3081

0186 plusmn 0004

4205

NHJR

( 500 mgkg)

0251 plusmn 0003

1860

0240 plusmn 0002

2452

0225 plusmn 0008

2990

EETG


NHTG


Indomethacin

(10 mgkg)

0238 plusmn 0003

2093

0223 plusmn 0002

2987

0203 plusmn 0004

3676



90


471 At 1st hr









91

472 At 2nd hr





second hour




92

473 At 3rd hr









93




94


Groups

1sthr 2ndhr 3rdhr

(Mean plusmn SEM)

(ml) Inhibition

(Mean plusmn SEM)

(ml) Inhibition

(Mean plusmn SEM)

(ml) Inhibition


- 0331 plusmn 0004 - 0340 plusmn 0004 -

EEJR

( 500 mgkg)

0236 plusmn 0005

2411

0223 plusmn 0004

3262

0206 plusmn 0003

3941

NHJR

( 500 mgkg)

0258 plusmn 0004

1704

0236 plusmn 0004

2870

0225 plusmn 0005

3382

EETG


NHTG



2347

0230 plusmn 0005

2051

0195 plusmn 0003

4264



95


481 At 1st hr









96

482 At 2nd hr





at second hour




97

483 At 3rd hr









98

484 At 4th hr





fourth hour




99

485 At 5th hr









100



101


Groups

1sth

2ndh

3rdh

4thh

5thh




EEJR

(500 mgkg)

0216 plusmn 0004

(28)

0206 plusmn 0004

(4801)

0193 plusmn 0004

(4255)

0175 plusmn 0002

(4927)

0173 plusmn 0004

(5099)

NHJR

(500 mgkg)

0238 plusmn 0006

(2066)

0225 plusmn 0002

(2879)

0205 plusmn 0006

(3898)

0193 plusmn 0004

(4405)

0183 plusmn 0009

(4815)

EETG

(400 mgkg)

0243 plusmn 0005

(19)

0226 plusmn 0004

(2848)

0213 plusmn 0004

(3660)

0195 plusmn 0002

(4347)

0183 plusmn 004

(4815)

NHTG

(400 mgkg)

0266 plusmn 0005

(1133)

0245 plusmn0005

(2246)

0223 plusmn 0006

(3363)

0218 plusmn 0005

(3681)

0205 plusmn 0006

(4192)

DPH

(60 mgkg)

0220 plusmn 0004

(2667)

0195 plusmn 0004

(3829)

0176 plusmn 0003

(4761)

0168 plusmn 0003

(5130)

0161 plusmn0 006

(5443)



102





induced by xylene




103










104








105








106








107


Parameters


Mean plusmn SEM

Time of onset of








108







Alkaloids + + + +

Glycosides _ _ _ _

Saponins _ _ _

Phenols + + + +

Terpenoids + + + +

Steroids _ _ _ _

Flavonoids + + + +

Tannins + _ + +

109















110


Sr

No

Retention

Time

(Minute)

Total

Percentage

Name of Identified

Compound

Molecular

Formula

Molecular

Weight

(gmol)

Structures


2 5350 3956 Decane C10H22 142

3 6255 1169 (E)-3(10)-Caren-4-ol C10H16O 152

OH

CH2

CH3

CH3

4 6795 2182 Undecane C11H24 156

CH3

CH3

CH3 Cl

CH3

CH3

111

5 8311 0859 Dodecane C12H26 170

CH3

CH3


CH3

CH3


CH3

CH3


CH3CH3


CH3

112


CH3

CH3


CH3

CH3


CH3

CH3


CH3

CH3


CH3

CH3

113


CH3

CH3


CH3

CH3


CH3

CH3

18 20512 3754

Benzene (1-

methylundecyl)-

C18H30 246

CH3

CH3


CH3

CH3

114


CH3

CH3


CH3 CH3


CH3

CH3

23 22506 2311

Benzene (1-

methyldodecyl)-

C19H32 260

CH3 CH3


CH3

O

O CH3


CH3

O

O

CH3

115


O

O

CH3

CH3


O

O

CH3

28 30178 0212



ester

C16H22O4 278

O

OH

O

O

CH3

CH3

29 32303 1309 Squalene C30H50 410

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

30 32738 0380 Heptacosane C27H56 380

CH3

CH3

116

117


2 Decane

30( E )-3 (10)-Caren-4-ol

118

4 Undecane

5 Dodecane


119




120




121




122




123


20



124




125



27 Heptacosane

126













127


Sr

No

Retention

Time

(Minute)

Total

Percentage

Name of Identified

Compound

Molecular

Formula

Molecular

Weight

(gmol)

Structures


CH3

CH3

2 17498 4905

Velleral

C15H20O2 232 CH3

CH3

CH3O

O

3 20372 2361

Benzene (1-

methylundecyl)-

C18H30 246

CH3

CH3

128

4 20773 2105

Benzene (1-

pentylheptyl)

C18H30 246

5 20930 2199

ZZZ-1469-

Nonadecatetraene

C19H32 260

CH3 CH2


CH3

O

O

CH3

7 25737 4597 Phytol C20H40O 296

OH CH3

CH3

CH3

CH3

129


CH3

O

CH3


O

CH3


CH3

O

CH3

11 30178 2740

388-Trimethoxy-3-

piperidyl-22-

binaphthalene-1144-

tetrone

C28H25NO7 487

N

O

O

O

O

O

O

CH3CH3

O

CH3

130

12 32294 15138 Squalene C30H50 410

CH3

CH3

CH3

CH3CH3

CH3

CH3

CH3

13 32730 4184


ethylbutyl)-

C26H54 366 CH3

CH3


CH3

15 34759 6222 Vitamin E C29H50O2 430 O

CH3

CH3

OH

CH3

CH3

CH3

CH3

CH3

131

132




133




134

7 Phytol



135



6 Squalene

136


8 Heptacosane

9 Vitamin E

137










(Table 19)

138


SrNo Retention

Time (mins)

Total

(age)

Name of Identified


MW

(gmol) Structures


O

CH3

2 12186 15848 1H-2-Benzopyran-1-


OO



ester C14H28O2 228

5 16175 1580

Tetracyclo

[33101(39)]decan-10-

one

C10H12O 148 OO

139


ester C16H32O2 256

CH3

O

CH3

O


OH O


CH3

O

O

CH3

9 25737 0614 Phytol C20H40O 296 OH CH3

CH3

CH3

CH3



O

CH3

O

CH3

140



O CH3

O

CH3


CH3

O

CH3

13 32764 6519 Nonacosane C29H60 408 CH3

CH3



CH3


CH3

141

142




143




144




145

10 Phytol



146


14 Nonacosane


147

16 Hexatriacontane

148












20)

149


Sr

No

Retention

Time

(mins)

Total


Mol

Formula

MW

(gmol) Structures


CH3

CH3

2 5358 3731 Decane C10H22 142 CH3

CH3


CH3

CH3

4 6264 1048 (E)-3(10)-Caren-4-ol C10H16O 152

OH

CH2

CH3

CH3

150

5 6804 1990 Undecane C11H24 156 CH3

CH3

6 8311 0750 Dodecane C12H26 170 CH3

CH3



CH3

CH3


CH3

CH3


CH3

151


CH3


CH3

CH3


CH3

CH3


CH3


CH3

152


CH3

CH3


CH3

CH3


CH3

CH3


CH3


CH3

CH3


CH3

CH3

153


CH3 CH3


CH3

CH3



O

CH3


CH3

O

O

CH3



O

O

CH3

CH3

154


ester C20H36O2 308

O

CH3

O

CH3



O CH3

O

CH3


CH3

O

CH3



O

OH

O

O

CH3

CH3

155

32 32799 2525 Nonacosane C29H60 408

CH3

CH3


CH3

156

157

1 Decane

2 Decane 4-methyl-

3 (E)-3(10)-caren-4-ol

158

3 Undecane

4 Dodecane


159




160




161




162




163




164




165




166




167

31 Nonacosane

32 Tetratriacontane


168
























169


















destruction (79)






170


response






















171

























172

























173


























174


















extract (111)

175












176

FUTURE PERSPECTIVE












neonates


population

REFERENCES


201262(8)1085-93





therapeutics 2019













of Medicine 2011365(23)2205-19



20191614-11









21















Medica 201682(05)PC63



of Pain 201822(3)455-70



201771955






reviews 201711(22)145




of Nutrition 200899(4)715-22














201933(10)1531-4


199123(1)90-114















reviews 20054(3)130-6
























Toxicology 2003188(1)49-71



198136(6)435-6










200155(4)204





201113(1)27



201547(2)177






200389(1)123-9






































phytology 2011



2010439-44

















7
















710




401









200374(3)282-90






Res 201910(5)2087-100




therapy 201315(5)R141









sciences 201414(4)919-24









3(21)465-70


























Medicines 20129(2)242-9





reviews 200052(4)673-751















vii






421 At Day 8 39

422 At Day 15 40

423 At Day 22 41

424 At Day 28 42







marker 57


levels of TNF-α 57



levels 59


levels of IL-1β 60






levels of IL-4 63

viii


64


parameters 66


count 66


content 67


count 68


platelet count 69


71



levels 72






gharuensis 77



451 At 1st hr 78

452 At 2nd hr 79

453 At 3rd hr 80

454 At 4th hr 81

ix

455 At 5th hr 82



461 At 1st hr 85

462 At 2nd hr 86

463 At 3rd hr 87



471 At 1st hr 90

472 At 2nd hr 91

473 At 3rd hr 92



481 At 1st hr 95

482 At 2nd hr 96

483 At 3rd hr 97

484 At 4th hr 98

485 At 5th hr 99











x





REFERENCES 177

xi

LIST OF TABLES

Page






40





66




T gharuensis

78



induced paw edema

84

xii



88



92



98



104






xiii

LIST OF FIGURES

Page

















xiv






of TNF-α

58


of NF-κB

59


of IL-6

60


of IL-1β

61


of COX-1

62


of COX-2

63


IL-4

64


xv


















xvi














xvii













Hb Haemoglobin

RBC Red blood cells






IL-6 Interleukin 6

IL-4 Interleukin 4



PXM Piroxicam

xviii

IND Indomethacin

DPH Diphenhydramine

Dexa Dexamethasone

LOP Loperamide



I2 Iodine


KI Potassium iodide


DW Distilled water

SM

bw

PO

Synovial membrane

Body weight

Per oral

xix



ABSTRACT




respectively
















xx
























xxi






1
























2

























3
















11 Hypothesis

111 Null hypothesis


properties



4














21 Epidemiology










5



22 Pathophysiology






















6

























7


















23 Treatments







8









cyclooxygenase (42)

















9

























10
















24 Inflammation









11









(45)







immunologic (46)






12










26 Plants used















13








(51)














14

Aim of the study



Objective of study













GC-MS analysis

15


31 Plant collection





name was checked




05














16





57)



















17









FCA (59)








342 (12)


2 and IL-4

351 RNA extraction


18









separated






3514 Washing of RNA







19






GAPDH


pairs sequence








20



IL-4


231 (58)


IL-6


247 (5)


TNF-α


202 NM_0126753


NF-КB


207 (58)


COX-1


200 (59)


COX-2


237 (58)


IL-1β


293 (58 60)


GAPDH


197 NM_0170084


21

353 cDNA synthesis





















22












Each one litre

Tris base 242 g


05M EDTA 100 ml


600ml




23




buffer











358 Densitometry






experiment

24




















immediately




25




five times










1800i)







26












Models Groups



EEJR (500 mgkg)

NHJR (500 mgkg )

EETG (400 mgkg)

NHTG (400 mgkg)




EEJR (500 mgkg)

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)



EEJR (500 mgkg)

27


water

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)




EEJR (500 mgkg)

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)




EEJR (500 mgkg)

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)




EEJR (500 mgkg)

NHJR (500 mgkg)

EETG (400 mgkg)

NHTG (400 mgkg)

Loperamide (5 mgkg)



bw = body weight

28























29




















30




















31








(68)





glycosides (70)


3841 Salkowski test







red colour (71)

32






















33

CHAPTER 4 RESULTS


model








34









35









36









37

Figure 5 Paw edema



38










39


421 At Day 8








40

422 At Day 15









group

41

423 At Day 22









control group

42

424 At Day 28




0153)





43





44




Days







45











46









47











48


Parameters

Arthritic control

Piroxicam

EEJR

NHJR

EETG

NHTG

Mean plusmn SEM

Infiltration of






49







50





(Fig 15)

51





52





53





54





55





56



57











control groups

58






groups




control groups

59










60










61










62










63










64










65


marker

Markers

Vehicle

Control

Arthritic

Control Piroxicam

EEJR

NHJR

EETG

NHTG

Mean plusmn SEM


NF-κB


IL-6


IL-1β


COX-1







66







group





67











68





(1512 plusmn 0256)





69










70


Parameters


Mean plusmn SEM

RBC


Hb

(gdl)


WBC

(103Ul)


Platelets

(103Ul)




two control groups

71









in all groups

72







creatinine levels

73





other



creatinine levels

74





other



levels

75











76


Biochemical

Parameters

Vehicle


Mean plusmn SEM

Urea


Creatinine


AST


ALT




77









gharuensis


distress Diarrhea

Motor

activity

Posture


Group-1 - - - - - -

Group-1I - - - - - -

Group-1II - - - - - -

Group-1V - - - - - -

Group-V - - - - - -

Group-VI - - - - - -

Group-VII - - - - - -

Group-

VIII - - - - - -

GROUP-

IX - - - - - -

78


carrageenan

451 At 1st hr









79

452 At 2nd hr









80

453 At 3rd hr









81

454 At 4th hr









82

455 At 5th hr









83



84



Groups




EEJR

(500 mgkg)

0210 plusmn0003

(2708)

0195 plusmn 0002

(3275)

0172 plusmn0003

(4591)

0156 plusmn0004

(5411)

0125 plusmn 0003

(6508)

NHJR

(500 mgkg)

0235 plusmn 0002

(1840)

0218 plusmn 0003

(2482)

0206 plusmn0003

(3522)

0193 plusmn0003

(4323)

0175 plusmn 0003

(5111)

EETG

(400 mgkg)

0226 plusmn0004

(21387)

0236 plusmn 0005

(1866)

0231 plusmn0006

(2735)

0210 plusmn0006

(3823)

0195 plusmn 0008

(4553)

NHTG

(400 mgkg)

0255 plusmn0003

(1145)

0250 plusmn 0002

(1379)

0280 plusmn0010

(1194)

0260 plusmn0010

(2352)

0236 plusmn 0011

(3407)

Piroxicam

(10 mgkg)

0208 plusmn0007

(2777)

0213 plusmn 0004

(2655)

0210 plusmn0003

(3396)

0183 plusmn0004

(4617)

0165 plusmn 0003

(5391)



85


edema

461 At 1st hr









86

462 At 2nd hr









87

463 At 3rd hr









88



89


edema model

Groups

1sthr 2ndhr 3rdhr

(Mean plusmnSEM)

(ml)

Inhibition

(Mean plusmnSEM)

(ml)

Inhibition

(Mean plusmnSEM)

(ml)

Inhibition


- 0318 plusmn 0001 - 0321 plusmn 0004 -

EEJR

( 500 mgkg)

0235 plusmn 0002

2192

0220 plusmn 0003

3081

0186 plusmn 0004

4205

NHJR

( 500 mgkg)

0251 plusmn 0003

1860

0240 plusmn 0002

2452

0225 plusmn 0008

2990

EETG


NHTG


Indomethacin

(10 mgkg)

0238 plusmn 0003

2093

0223 plusmn 0002

2987

0203 plusmn 0004

3676



90


471 At 1st hr









91

472 At 2nd hr





second hour




92

473 At 3rd hr









93




94


Groups

1sthr 2ndhr 3rdhr

(Mean plusmn SEM)

(ml) Inhibition

(Mean plusmn SEM)

(ml) Inhibition

(Mean plusmn SEM)

(ml) Inhibition


- 0331 plusmn 0004 - 0340 plusmn 0004 -

EEJR

( 500 mgkg)

0236 plusmn 0005

2411

0223 plusmn 0004

3262

0206 plusmn 0003

3941

NHJR

( 500 mgkg)

0258 plusmn 0004

1704

0236 plusmn 0004

2870

0225 plusmn 0005

3382

EETG


NHTG



2347

0230 plusmn 0005

2051

0195 plusmn 0003

4264



95


481 At 1st hr









96

482 At 2nd hr





at second hour




97

483 At 3rd hr









98

484 At 4th hr





fourth hour




99

485 At 5th hr









100



101


Groups

1sth

2ndh

3rdh

4thh

5thh




EEJR

(500 mgkg)

0216 plusmn 0004

(28)

0206 plusmn 0004

(4801)

0193 plusmn 0004

(4255)

0175 plusmn 0002

(4927)

0173 plusmn 0004

(5099)

NHJR

(500 mgkg)

0238 plusmn 0006

(2066)

0225 plusmn 0002

(2879)

0205 plusmn 0006

(3898)

0193 plusmn 0004

(4405)

0183 plusmn 0009

(4815)

EETG

(400 mgkg)

0243 plusmn 0005

(19)

0226 plusmn 0004

(2848)

0213 plusmn 0004

(3660)

0195 plusmn 0002

(4347)

0183 plusmn 004

(4815)

NHTG

(400 mgkg)

0266 plusmn 0005

(1133)

0245 plusmn0005

(2246)

0223 plusmn 0006

(3363)

0218 plusmn 0005

(3681)

0205 plusmn 0006

(4192)

DPH

(60 mgkg)

0220 plusmn 0004

(2667)

0195 plusmn 0004

(3829)

0176 plusmn 0003

(4761)

0168 plusmn 0003

(5130)

0161 plusmn0 006

(5443)



102





induced by xylene




103










104








105








106








107


Parameters


Mean plusmn SEM

Time of onset of








108







Alkaloids + + + +

Glycosides _ _ _ _

Saponins _ _ _

Phenols + + + +

Terpenoids + + + +

Steroids _ _ _ _

Flavonoids + + + +

Tannins + _ + +

109















110


Sr

No

Retention

Time

(Minute)

Total

Percentage

Name of Identified

Compound

Molecular

Formula

Molecular

Weight

(gmol)

Structures


2 5350 3956 Decane C10H22 142

3 6255 1169 (E)-3(10)-Caren-4-ol C10H16O 152

OH

CH2

CH3

CH3

4 6795 2182 Undecane C11H24 156

CH3

CH3

CH3 Cl

CH3

CH3

111

5 8311 0859 Dodecane C12H26 170

CH3

CH3


CH3

CH3


CH3

CH3


CH3CH3


CH3

112


CH3

CH3


CH3

CH3


CH3

CH3


CH3

CH3


CH3

CH3

113


CH3

CH3


CH3

CH3


CH3

CH3

18 20512 3754

Benzene (1-

methylundecyl)-

C18H30 246

CH3

CH3


CH3

CH3

114


CH3

CH3


CH3 CH3


CH3

CH3

23 22506 2311

Benzene (1-

methyldodecyl)-

C19H32 260

CH3 CH3


CH3

O

O CH3


CH3

O

O

CH3

115


O

O

CH3

CH3


O

O

CH3

28 30178 0212



ester

C16H22O4 278

O

OH

O

O

CH3

CH3

29 32303 1309 Squalene C30H50 410

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

30 32738 0380 Heptacosane C27H56 380

CH3

CH3

116

117


2 Decane

30( E )-3 (10)-Caren-4-ol

118

4 Undecane

5 Dodecane


119




120




121




122




123


20



124




125



27 Heptacosane

126













127


Sr

No

Retention

Time

(Minute)

Total

Percentage

Name of Identified

Compound

Molecular

Formula

Molecular

Weight

(gmol)

Structures


CH3

CH3

2 17498 4905

Velleral

C15H20O2 232 CH3

CH3

CH3O

O

3 20372 2361

Benzene (1-

methylundecyl)-

C18H30 246

CH3

CH3

128

4 20773 2105

Benzene (1-

pentylheptyl)

C18H30 246

5 20930 2199

ZZZ-1469-

Nonadecatetraene

C19H32 260

CH3 CH2


CH3

O

O

CH3

7 25737 4597 Phytol C20H40O 296

OH CH3

CH3

CH3

CH3

129


CH3

O

CH3


O

CH3


CH3

O

CH3

11 30178 2740

388-Trimethoxy-3-

piperidyl-22-

binaphthalene-1144-

tetrone

C28H25NO7 487

N

O

O

O

O

O

O

CH3CH3

O

CH3

130

12 32294 15138 Squalene C30H50 410

CH3

CH3

CH3

CH3CH3

CH3

CH3

CH3

13 32730 4184


ethylbutyl)-

C26H54 366 CH3

CH3


CH3

15 34759 6222 Vitamin E C29H50O2 430 O

CH3

CH3

OH

CH3

CH3

CH3

CH3

CH3

131

132




133




134

7 Phytol



135



6 Squalene

136


8 Heptacosane

9 Vitamin E

137










(Table 19)

138


SrNo Retention

Time (mins)

Total

(age)

Name of Identified


MW

(gmol) Structures


O

CH3

2 12186 15848 1H-2-Benzopyran-1-


OO



ester C14H28O2 228

5 16175 1580

Tetracyclo

[33101(39)]decan-10-

one

C10H12O 148 OO

139


ester C16H32O2 256

CH3

O

CH3

O


OH O


CH3

O

O

CH3

9 25737 0614 Phytol C20H40O 296 OH CH3

CH3

CH3

CH3



O

CH3

O

CH3

140



O CH3

O

CH3


CH3

O

CH3

13 32764 6519 Nonacosane C29H60 408 CH3

CH3



CH3


CH3

141

142




143




144




145

10 Phytol



146


14 Nonacosane


147

16 Hexatriacontane

148












20)

149


Sr

No

Retention

Time

(mins)

Total


Mol

Formula

MW

(gmol) Structures


CH3

CH3

2 5358 3731 Decane C10H22 142 CH3

CH3


CH3

CH3

4 6264 1048 (E)-3(10)-Caren-4-ol C10H16O 152

OH

CH2

CH3

CH3

150

5 6804 1990 Undecane C11H24 156 CH3

CH3

6 8311 0750 Dodecane C12H26 170 CH3

CH3



CH3

CH3


CH3

CH3


CH3

151


CH3


CH3

CH3


CH3

CH3


CH3


CH3

152


CH3

CH3


CH3

CH3


CH3

CH3


CH3


CH3

CH3


CH3

CH3

153


CH3 CH3


CH3

CH3



O

CH3


CH3

O

O

CH3



O

O

CH3

CH3

154


ester C20H36O2 308

O

CH3

O

CH3



O CH3

O

CH3


CH3

O

CH3



O

OH

O

O

CH3

CH3

155

32 32799 2525 Nonacosane C29H60 408

CH3

CH3


CH3

156

157

1 Decane

2 Decane 4-methyl-

3 (E)-3(10)-caren-4-ol

158

3 Undecane

4 Dodecane


159




160




161




162




163




164




165




166




167

31 Nonacosane

32 Tetratriacontane


168
























169


















destruction (79)






170


response






















171

























172

























173


























174


















extract (111)

175












176

FUTURE PERSPECTIVE












neonates


population

REFERENCES


201262(8)1085-93





therapeutics 2019













of Medicine 2011365(23)2205-19



20191614-11









21















Medica 201682(05)PC63



of Pain 201822(3)455-70



201771955






reviews 201711(22)145




of Nutrition 200899(4)715-22














201933(10)1531-4


199123(1)90-114















reviews 20054(3)130-6
























Toxicology 2003188(1)49-71



198136(6)435-6










200155(4)204





201113(1)27



201547(2)177






200389(1)123-9






































phytology 2011



2010439-44

















7
















710




401









200374(3)282-90






Res 201910(5)2087-100




therapy 201315(5)R141









sciences 201414(4)919-24









3(21)465-70


























Medicines 20129(2)242-9





reviews 200052(4)673-751















evaluation of anti-inflammatory and anti-arthritic

Documents