STUDIES ON ANTIBACTERIAL

FINISHING OF COTTON TEXTILES

USING NEEM EXTRACT

by

ROLI PURWAR

Department of Textile Technology

Submitted

In fulfillment of the requirements of the degree of

Doctor of Philosophy
to the

Indian Institute of Technology, Delhi

December 2005
deVicateete mycpeire.nt6
 CERTIFICATE

This is to certify that the thesis entitled "Studies on Antibacterial Finishing of Cotton

Textiles using Neem Extract" being submitted by Mrs. Roli Purwar, to the Indian

Institute of Technology, Delhi, for the award of the degree of Doctor of Philosophy

in the department of Textile Technology, is a record of bonafide research work carried

out by her. Mrs. Purwar has worked under my guidance and supervision and fulfilled

all the requirements for the submission of the thesis.

The results contained in this thesis have not been submitted, in part or full, to any

other university or Institute for the award of any degree or diploma.

Dr. anga a Joshi,

Assistant Professor,
Department of Textile Technology,
Indian Institute of Technology, Delhi
 ACKNOWLEDGMENT

I wish to express my heartiest gratitude and sincere regards to my respectable

supervisor, "Dr. Mangala Joshi" for inspiration, guidance, valuable suggestions,

constant encouragement and liberty provided in planning of work and implementation

of ideas.

I am very thankful to my SRC members, Prof. M L Gulrajani, Prof. R B Chavan and

Dr. Prashant Mishra for their constructive criticism and valuable suggestions.

I gratefully acknowledge the help and suggestions by Prof. B L Deopura and Prof. A

K Gupta as and when required.

I am thankful to Dr. S S Pillai, Vice President E I D Parry (I) Ltd for allowing me to

work in their Bangalore R&D Centre. My special thanks to Dr. Sreenivasa Rao D,

Head & Scientist, Natural Product Division, E I D Parry (I) Ltd, for his continuous

guidance and support in isolation and chromatography work. I am also thankful to

Balamurugan, Hariharan, Priya, Devendra Kumar and other colleagues at EID, for

their help and cooperation.

I extend my sincere thanks to Dr. Prashant Mishra for allowing me to work in the

Microbiology Laboratory of the Department of Biochemical Engineering and

Biotechnology, IIT Delhi and for his continuous guidance and suggestion for

conducting microbiology work. I am also thankful to Mrs. Gunjan Prakash for her

help in conducting the HPLC work.

ii
I gratefully acknowledge the help and cooperation given by Department of Anatomy,

AIIMS, New Delhi for TEM facility.

I am thankful to the staff member of all the laboratories of Textile Department, for

extending a helping hand whenever needed. In the same breath, I thank the staff

members of the laboratories of the Department of Biochemical Engineering and

Biotechnology, NMR, SEM and AIIMS.

I would like to express my heartiest thanks and gratitude to all my friends and

colleagues for their direct and indirect help. I am especially thankful to Mrs. Mahuya

Ghosh, Ms. Muthulaxami, Ms. Nimisha Aggrawal, Mrs. Rhea Pandita, Mr. B S

Butola, Mr. Shirish, Mrs. Parul Priya, Ms Richa Aggrawal, Mr. Chaitra, Mrs. Archana

Vyas, Ms. Rashmi Sharma and Mr. Wazid Ali.

I am grateful to my parents, sister, brother and other relatives for their continuous

help, support and encouragement.

I gratefully acknowledge the help and cooperation by my uncle, Mr. B L Shah and

aunty, Usha Shah, as and when required.

My heartfelt thanks to my husband, Mr. Amit Singh Rathod, who patiently stood

beside me during entire research work. It would have been impossible to complete

this task without his consistent support and cooperation.

Roli Purwar

iii
 ABSTRACT

The use of natural antimicrobial agents for bioactive textiles is an emerging area of

research. Neem (Azadirachta indica), an evergreen tree of India, is one of the

promising sources of biologically active compounds chemically known as limonoids

which are bitter in taste. The most important quality of neem extract and its active

compounds is that they are skin friendly and less toxic to warm-blooded animals like

human. The aim of this work is to explore the possibility of using neem extract as an

antibacterial finishing agent for cotton fabric.

Neem seed and bark extract were prepared in methanol using solvent

extraction method. The isolation of major limonoids of neem seed and bark extract i.e.

azadirachtin A, nimbin and salanin was performed using column chromatography.

The identification of compounds was done by using thin layer chromatography

(TLC), infrared spectroscopy (IR) and 1H nuclear magnetic resonances (NMR)

techniques. The characterization of neem seed and bark extracts in terms of

azadirachtin A, nimbin and salannin has been carried out using high performance

liquid chromatography (HPLC).

The antibacterial activity of neem extracts and the isolated limonoids was

investigated qualitatively by using disc diffusion and parallel streak methods. Both

seed and bark extract showed antibacterial activity against gram positive bacteria i.e.

Staphylococcus aureus, Bacillus subtilis as well as gram negative bacteria i.e.

Escherichia coli. The minimum inhibitory concentration for seed and bark extract are

8-11 mg/ml and 3.3-10 mg/ml respectively. The mechanism of bactericidal action of

neem seed and bark extract active compounds on Staphylococcus aureus was

iv
investigated using transmission electron microscopy (TEM). Both neem seed and bark

extract treated cells show the rupture of cell wall and the loss of cytoplasmic content.

The integrity of cell membrane of Staphylococcus aureus was studied by determining

the release of material absorbing at 260nm. A significant loss of 260nm absorbing

material in Staphylococcus aureus suspension treated with neem seed and bark extract

suggests that nucleic acids are lost because of damage of cell wall. The time for

damage of cells was lower in case of bark extract (20 min) as compared to seed

extract (40 min).

The neem seed and bark extract were integrated on the cotton fabric

substrate along with non formaldehyde, glyoxal/glycol based crosslinking agent. The

fabrics were cured using two different curing methods conventional and microwave

for crosslinking. The major advantage of using microwave curing method is that it

reduces the time of processing as well as retains the mechanical properties of the

crosslinked fabric.

The curing conditions of microwave were optimized on the basis of power

and time. Low microwave power 200 W and 1 min curing time was sufficient to

impart high crease recovery property to the fabric. Comparisons were made for

conventionally and microwave cured cotton fabrics treated with glyoxal/glycol.

Crosslinking efficiency in conventionally and microwave cured samples were

evaluated on the basis of increase in crease recovery angle and retention of tensile

strength and tearing strength after crosslinking. Conventional cured samples showed

higher crosslink efficiency than microwave cured samples. FTIR and TGA results

also support the higher crosslinking efficiency in conventional curing technique.

V
 The attachment of neem seed and bark extract on to the fabric was confirmed

by using IR analysis. Major peaks at 1724 / 1750cm-1(ester and acetate), 1586 / 1582

cm' and 878 cm' ((3 substituted furan ring), 1261cm-1 (ether linkage), 1071 / 1025

cm 1 (oxide/ether function) for neem seed and bark extract treated fabric after washing

clearly indicates that neem seed and bark extract are chemically attached on to the

cotton fabric. The range of wt. add-on percent for unwashed samples is 11-22 wt%

and for washed samples it decreased to 1-8 wt%.

The antibacterial activity of finished fabric was evaluated qualitatively using

parallel streak method and quantitatively using modified colony counting method in

which the percent reduction in number of bacterial colonies in treated sample as

compared to the untreated sample gives the antibacterial activity of the fabric. The

unwashed samples were subjected to parallel streak method to evaluate the

antibacterial activity. In general, it was found that as the concentration of neem seed

and bark extract increases, the zone of inhibition increases but at still higher

concentration it starts decreasing. The washed samples were subjected to the modified

colony counting method. It is found that as the concentration of extract on to the

fabric increases, the antibacterial activity increases. The concentration (10%w/v) of

both the extracts i.e. seed and bark showed high (>94%-99%) antibacterial activity in

conventional as well as microwave cured samples as compared to 8-42% antibacterial

activity for control samples.

Antibacterial properties of neem seed and bark extract treated fabric and the

bacterial growth on the fabric was further examined by scanning electron microscopy

(SEM). SEM micrographs, clearly indicates that the neem seed and bark extract act as

an effective bactericidal agent on the fabric

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 The durability of neem seed and bark extract treated fabric was evaluated in

term of its retention of antibacterial activity on washing. It was found that the seed

extract retains the antibacterial activity up to the five machine washes which is

equivalent to approximately 25 home launderings. However, there was sharp

decrement in antibacterial activity of bark extract treated fabric as the washing

progressed. The semi durable nature of the antibacterial neem extract based finish is

attributed to the gradual loss of the crosslinking effect of glyoxal/glycol system after

ten washes.

The neem seed and bark extract treated samples were tested for various fabric

properties like, appearance, tensile strength, crease recovery angle, tearing strength,

bending length etc. The bark extract treated samples were wood colored but the seed

extract treated fabric shows no significant change in appearance. Crease recovery

angle of neem extract treated fabric slightly decreases but there is no significant

change in tensile strength of the treated fabric as compared to the control sample.

However, neem finished fabric show improved tearing strength as compared to the

control sample.

This study thus reveals that neem seed and bark extracts have a good potential

to be used as an ecofriendly and effective agent for antibacterial finishing of cotton

based textiles.

vii
 CONTENTS
Page No.

Certificate

Acknowledgement ii

Abstract iv

Contents viii-xiv

List of Figures xv-xviii

List of Tables xix-xx

1. Chapter I: Introduction and Objective of the Work 1-10

1.1. General 1

1.2. Motivation 7

1.3. Objective of the Work 8

1.4. Outline of the Thesis 9

2. Chapter II: Literature Survey 1 1 -54

2.1. Introduction 11

2.2. Representative Microorganisms on Textiles 12

2.3. Definitions 16

2.4. An Overview of Bacteria Cell Structure 18

2.4.1. Size, Shape and Arrangement 18

2.4.2. Bacterial Cell Organization/Morphology 19

2.4.3. Gram Positive and Gram Negative Bacteria 20

2.5. Mode of Action of Antimicrobial Agents 21

2.6. Types of Antimicrobial Agents Used For Textiles 22

2.6.1. Metals & Their Salts 22

viii
 2.6.2. Dyes: 23

2.6.3. Cationising Agents 25

2.6.4. Antimicrobial Additives 26

2.6.5. Triclosan 27

2.6.6. Polyaniline 27

2.6.7. Antibiotic Treatment on Textiles 28

2.6.8. Polyhexamethylene Biguanide (PHMB) 28

2.6.9. Antimicrobial Finishes in Combination with Other

Finishes: 29

2.7. Eco Friendly Antimicrobial Agents Used In Textiles 29

2.7.1. Chitosan 30

2.7.2. Magnesium Peroxide 32

2.7.3. Natural Products 33

2.8. Mechanisms for Antimicrobial Protection of Textile 34

2.8.1. Controlled Release 35

2.8.2. Regeneration Principle 36

2.8.3. Barrier or Blocking Mechanism 39

2.9. Test Methods for Evaluation of Antibacterial Activity 40

2.10. Neem (Azadirachta Indica) 45
2.10.1. Taxonomy 45

2.10.2. Botanical Characteristics 46

2.10.3. Extraction 47

2.10.4. Extraction Techniques 48

2.10.5. Biologically Active Ingredients 49

ix


2.10.6. Antibacterial Activity of Neem Extracts and Its

Compounds 51

2.10.7. Application of Neem on Textiles 53

3. Chapter III: Neem Active Ingredients: Extraction,

Isolation, Characterization And Antibacterial Activity 55-104

3.1. General 55

3.2. Neem Active Ingredients: Extraction, Isolation, Characterization

and antibacterial activity 56

3.2.1. Introduction 56

3.2.2. Materials And Methods 58

3.2.1. Materials 58

3.2.2. Preparation of Bark Extract 58

3.2.3. Thin Layer Chromatography (TLC) Analysis 58

3.2.4. High Performance Liquid Chromatography (HPLC)

Analysis 59

3.2.5. Nuclear Magnetic Resonance (NMR) Analysis 60

3.2.6. Infrared Analysis 60

3.2.7. Preparatory Chromatography 60

3.2.8. Isolation of Azadirachtin A 61

3.2.9. Isolation of Nimbin and Salannin 66

3.2.3. Results 11

3.2.1. Characterization of Limonoids 71

3.2.2. Thin Layer Chromatography 71

3.2.3. Infrared Analysis 73

 3.2.4. NMR Analysis 75

3.2.5. Characterization of Neem Extracts 75

3.2.4. Discussion 81

3.2.5. Summary 83

3.3. Antibacterial Activity of Neem Extracts and Its Limonoids 84

3.3.1. Introduction 84

3.3.2. Materials And Methods 85

3.3.1. Test Organisms 85

3.3.2. Antibacterial Screening Test 85

3.3.2.1. Disc Diffusion Method: 85

3.3.2.2. Parallel Streak Method: 86

3.3.2.3. Minimum Inhibitory Concentration 86

3.3.3. Mode of Action of Neem Extracts on Staphylococcus

aureus 87

3.3.3.1. Transmission Electron Microscopy 87

3.3.3.2. Integrity of Cell Membrane 87

3.3.3. Results and Discussion 88

3.3.1. Antibacterial Screening Test 88

3.3.1.1. Disc Diffusion Method: 88

3.3.1.2. Parallel Streak Method: 91

3.3.1.3. Minimum Inhibitory Concentration 92

3.3.2. Mode of Action of Neem Extracts on Staphylococcus

aureus 96

3.3.2.1. Transmission Electron Microscopy 96

3.3.2.2. Integrity of Cell Membrane 102

xi


3.3.4. Summary 104

4. Chapter IV: Antibacterial Finishing of Cotton Fabric

Using Neem Extracts 105-157

4.1. General 105

4.2. Optimization of Easy Care Finish 107

4.2.1. Introduction 107

4.2.2. Experimental 110

4.2.1. Materials 110

4.2.2. Method of Fabric Treatment 110

4.2.3. Performance Evaluation and Testing 111

4.2.3. Results and Discussion 111

4.2.1. Effect of Catalyst Concentration 112

4.2.2. Effect of Crosslinking Agent 112

4.2.3. Effect of Microwave Curing Conditions 114

4.2.4. Microwave Curing vs Conventional Cure 115

4.2.5. Performance Properties 115

4.2.5.1. Thermogravimetric Analysis 117

4.2.5.2. Infrared Analysis 118

4.2.6. Washing Durability 120

4.2.4. Summary 121

4.3. Integration of Neem Extracts on Cotton Fabric and Evaluation

of Antibacterial Activity of the Finished Fabric 123

4.3.1. Introduction 124

124

xii
 124
4.3.2. Material and Methods

4.3.1. Materials

4.3.2. Methods 124

4.3.2.1. Integration of Neem Seed and Bark Extract on

Cotton Fabric 124

4.3.2.2. Sample Coding 125

4.3.2.3. Infrared Analysis 12:5

4.3.2.4. Antibacterial Activity Test 126

4.3.2.2.4.1.Parallel Streak Method 126

4.3.2.2.4.2.Colony Counting Static Test method: 126

4.3.2.5. Scanning Electron Microscopy 127

4.3.2.6. Performance Properties 127

4.3.3. Results and Discussion 128

4.3.3.1. Infrared Analysis of Neem Extract Treated

Fabric 128

4.3.3.2. Weight add on of Seed Extract Treated Fabric 133

4.3.3.3. Weight add on of bark extract treated fabric 1. 33

4.3.3.4. Antibacterial Activity 134

4.3.3.5. Antibacterial activity against gram negative

bacteria 147

4.3.3.6. SEM Analysis 148

4.3.3.7. Performance Properties of Neem Finished

Fabrics 150

4.3.4. Summary 156

5. Chapter V : Conclusion and Future Scope of the Work 158-164

5.1. Conclusion 158

5.2. Future Scope of the Work 164

6. References 165-177

Annexure I 178

Annexure II 179

Curriculum vitae 184

xiv