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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

nt6 .deVicateete mycpeire.

for the award of the degree of Doctor of Philosophy in the department of Textile Technology. Delhi. is a record of bonafide research work carried out by her. Indian Institute of Technology. CERTIFICATE This is to certify that the thesis entitled "Studies on Antibacterial Finishing of Cotton Textiles using Neem Extract" being submitted by Mrs. Department of Textile Technology. Assistant Professor. Delhi . Dr. to the Indian Institute of Technology. to any other university or Institute for the award of any degree or diploma. in part or full. The results contained in this thesis have not been submitted. Purwar has worked under my guidance and supervision and fulfilled all the requirements for the submission of the thesis. Roli Purwar. anga a Joshi. Mrs.

I am very thankful to my SRC members. ACKNOWLEDGMENT I wish to express my heartiest gratitude and sincere regards to my respectable supervisor. Head & Scientist. Prof. valuable suggestions. Vice President E I D Parry (I) Ltd for allowing me to work in their Bangalore R&D Centre. R B Chavan and Dr. A K Gupta as and when required. I gratefully acknowledge the help and suggestions by Prof. I am also thankful to Balamurugan. I extend my sincere thanks to Dr. Sreenivasa Rao D. Prof. My special thanks to Dr. Natural Product Division. S S Pillai. I am thankful to Dr. Prashant Mishra for their constructive criticism and valuable suggestions. constant encouragement and liberty provided in planning of work and implementation of ideas. Gunjan Prakash for her help in conducting the HPLC work. "Dr. Priya. for their help and cooperation. E I D Parry (I) Ltd. ii . I am also thankful to Mrs. guidance. M L Gulrajani. B L Deopura and Prof. 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. Devendra Kumar and other colleagues at EID. Mangala Joshi" for inspiration. Hariharan. for his continuous guidance and support in isolation and chromatography work.

Archana Vyas. Ms. Ms Richa Aggrawal. Wazid Ali. Mrs. Mr. sister. Mrs. Parul Priya.I gratefully acknowledge the help and cooperation given by Department of Anatomy. I am especially thankful to Mrs. brother and other relatives for their continuous help. Mahuya Ghosh. Mr. I thank the staff members of the laboratories of the Department of Biochemical Engineering and Biotechnology. Shirish. NMR. In the same breath. I gratefully acknowledge the help and cooperation by my uncle. It would have been impossible to complete this task without his consistent support and cooperation. Mrs. New Delhi for TEM facility. Mr. Ms. My heartfelt thanks to my husband. I am grateful to my parents. Rashmi Sharma and Mr. as and when required. B S Butola. Amit Singh Rathod. for extending a helping hand whenever needed. SEM and AIIMS. B L Shah and aunty. Mr. support and encouragement. I would like to express my heartiest thanks and gratitude to all my friends and colleagues for their direct and indirect help. Muthulaxami. Chaitra. Mr. I am thankful to the staff member of all the laboratories of Textile Department. Roli Purwar iii . Rhea Pandita. Ms. Usha Shah. who patiently stood beside me during entire research work. Nimisha Aggrawal. AIIMS.

e. is one of the promising sources of biologically active compounds chemically known as limonoids which are bitter in taste.e. The antibacterial activity of neem extracts and the isolated limonoids was investigated qualitatively by using disc diffusion and parallel streak methods. The mechanism of bactericidal action of neem seed and bark extract active compounds on Staphylococcus aureus was iv . Neem (Azadirachta indica). The minimum inhibitory concentration for seed and bark extract are 8-11 mg/ml and 3. The characterization of neem seed and bark extracts in terms of azadirachtin A. The identification of compounds was done by using thin layer chromatography (TLC). Escherichia coli. infrared spectroscopy (IR) and 1H nuclear magnetic resonances (NMR) techniques.e. nimbin and salannin has been carried out using high performance liquid chromatography (HPLC). Neem seed and bark extract were prepared in methanol using solvent extraction method. The aim of this work is to explore the possibility of using neem extract as an antibacterial finishing agent for cotton fabric. Staphylococcus aureus. The isolation of major limonoids of neem seed and bark extract i. azadirachtin A.3-10 mg/ml respectively. Bacillus subtilis as well as gram negative bacteria i. 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. an evergreen tree of India. nimbin and salanin was performed using column chromatography. ABSTRACT The use of natural antimicrobial agents for bioactive textiles is an emerging area of research. Both seed and bark extract showed antibacterial activity against gram positive bacteria i.

Low microwave power —200 W and 1 min curing time was sufficient to impart high crease recovery property to the fabric. glyoxal/glycol based crosslinking agent. Comparisons were made for conventionally and microwave cured cotton fabrics treated with glyoxal/glycol. The fabrics were cured using two different curing methods conventional and microwave for crosslinking. The curing conditions of microwave were optimized on the basis of power and time. The time for damage of cells was lower in case of bark extract (20 min) as compared to seed extract (40 min). FTIR and TGA results also support the higher crosslinking efficiency in conventional curing technique. 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. The neem seed and bark extract were integrated on the cotton fabric substrate along with non formaldehyde.investigated using transmission electron microscopy (TEM). The integrity of cell membrane of Staphylococcus aureus was studied by determining the release of material absorbing at 260nm. V . 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. Both neem seed and bark extract treated cells show the rupture of cell wall and the loss of cytoplasmic content. 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. Conventional cured samples showed higher crosslink efficiency than microwave cured samples.

It is found that as the concentration of extract on to the fabric increases. it was found that as the concentration of neem seed and bark extract increases. the antibacterial activity increases. The washed samples were subjected to the modified colony counting method.e. The concentration (10%w/v) of both the extracts i. 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. clearly indicates that the neem seed and bark extract act as an effective bactericidal agent on the fabric vi . 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). 1586 / 1582 cm' and 878 cm' ((3 substituted furan ring). 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. In general. The unwashed samples were subjected to parallel streak method to evaluate the antibacterial activity. Major peaks at 1724 / 1750cm-1(ester and acetate). the zone of inhibition increases but at still higher concentration it starts decreasing. The attachment of neem seed and bark extract on to the fabric was confirmed by using IR analysis. The range of wt. SEM micrographs. 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. add-on percent for unwashed samples is 11-22 wt% and for washed samples it decreased to 1-8 wt%. 1261cm-1 (ether linkage).

crease recovery angle. The durability of neem seed and bark extract treated fabric was evaluated in term of its retention of antibacterial activity on washing. 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. 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. 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. 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. tensile strength. appearance. 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. there was sharp decrement in antibacterial activity of bark extract treated fabric as the washing progressed. The neem seed and bark extract treated samples were tested for various fabric properties like. neem finished fabric show improved tearing strength as compared to the control sample. vii .

6.5. Outline of the Thesis 9 2.1.4.3.4. Representative Microorganisms on Textiles 12 2.4. Bacterial Cell Organization/Morphology 19 2. Size.3. Types of Antimicrobial Agents Used For Textiles 22 2. Mode of Action of Antimicrobial Agents 21 2. An Overview of Bacteria Cell Structure 18 2.3.4. Chapter I: Introduction and Objective of the Work 1-10 1.2.1. Shape and Arrangement 18 2. CONTENTS Page No. Motivation 7 1. Introduction 11 2.2.1.2. Metals & Their Salts 22 viii . Definitions 16 2.4.6. Objective of the Work 8 1. Certificate Acknowledgement ii Abstract iv Contents viii-xiv List of Figures xv-xviii List of Tables xix-xx 1.1. Gram Positive and Gram Negative Bacteria 20 2. General 1 1. Chapter II: Literature Survey 1 1 -54 2.

7.2.2. Eco Friendly Antimicrobial Agents Used In Textiles 29 2.5.9. 2.10. Regeneration Principle 36 2.10.3. Biologically Active Ingredients 49 ix . Extraction 47 2.8.7.9.6. Taxonomy 45 2.6.6. Barrier or Blocking Mechanism 39 2.4.1. Cationising Agents 25 2.2.6. Mechanisms for Antimicrobial Protection of Textile 34 2.1. Antimicrobial Finishes in Combination with Other Finishes: 29 2.8.1. Triclosan 27 2.7. Controlled Release 35 2. Dyes: 23 2. Chitosan 30 2.10.5. Magnesium Peroxide 32 2.4.6.6.10.7. Extraction Techniques 48 2. Test Methods for Evaluation of Antibacterial Activity 40 2. Polyhexamethylene Biguanide (PHMB) 28 2. Antimicrobial Additives 26 2.2.10.10.8.3. Natural Products 33 2.8. Botanical Characteristics 46 2.8. Polyaniline 27 2.6. Neem (Azadirachta Indica) 45 2.7.3. Antibiotic Treatment on Textiles 28 2.6.6.3.

2.7.1.8. Antibacterial Activity of Neem Extracts and Its Compounds 51 2.2. Neem Active Ingredients: Extraction. Preparatory Chromatography 60 3. Isolation. Characterization of Limonoids 71 3. Nuclear Magnetic Resonance (NMR) Analysis 60 3. Materials 58 3.2. Thin Layer Chromatography 71 3.10.2.1. Isolation of Azadirachtin A 61 3. Characterization And Antibacterial Activity 55-104 3.3.2.1.2. Introduction 56 3. Chapter III: Neem Active Ingredients: Extraction. Infrared Analysis 73 .2.1.6.2. Isolation of Nimbin and Salannin 66 3.3. Isolation.2.7. 2. Preparation of Bark Extract 58 3.4.2.10. Characterization and antibacterial activity 56 3.5. Infrared Analysis 60 3.2.2. Thin Layer Chromatography (TLC) Analysis 58 3. Results 11 3. Materials And Methods 58 3.3.2.2.2.9.2. General 55 3. High Performance Liquid Chromatography (HPLC) Analysis 59 3.6.2.2. Application of Neem on Textiles 53 3.2.

2. Mode of Action of Neem Extracts on Staphylococcus aureus 96 3.1.3. Test Organisms 85 3.2.1.1.1.2.1.2. Summary 83 3.3. Antibacterial Screening Test 88 3.2.3. NMR Analysis 75 3. Minimum Inhibitory Concentration 86 3.3.3. Discussion 81 3.4.3.3.1. Transmission Electron Microscopy 87 3.3.2.2.1.1.3.3.2.2.3. Antibacterial Screening Test 85 3.3.3. Characterization of Neem Extracts 75 3. Parallel Streak Method: 86 3.2.3.2. Results and Discussion 88 3.3. Disc Diffusion Method: 88 3. Antibacterial Activity of Neem Extracts and Its Limonoids 84 3. Materials And Methods 85 3.3.5. Integrity of Cell Membrane 102 xi .3.3.3. Mode of Action of Neem Extracts on Staphylococcus aureus 87 3.3. Disc Diffusion Method: 85 3.3.1. Integrity of Cell Membrane 87 3.3.2. 3.3.2. Transmission Electron Microscopy 96 3. Parallel Streak Method: 91 3.1.5. Introduction 84 3.3. Minimum Inhibitory Concentration 92 3.2.3.4.2.2.

2. Microwave Curing vs Conventional Cure 115 4.2.2.3.6. Summary 121 4. Introduction 107 4.5. Summary 104 4. Effect of Microwave Curing Conditions 114 4.2. Washing Durability 120 4. Effect of Crosslinking Agent 112 4. Thermogravimetric Analysis 117 4.1.2. Introduction 124 124 xii .5.2. Method of Fabric Treatment 110 4.2.1. General 105 4.2.2.2.4. Performance Evaluation and Testing 111 4.1.2.2.2. Chapter IV: Antibacterial Finishing of Cotton Fabric Using Neem Extracts 105-157 4.1.4.1. Effect of Catalyst Concentration 112 4.3.2. Results and Discussion 111 4. Infrared Analysis 118 4. Integration of Neem Extracts on Cotton Fabric and Evaluation of Antibacterial Activity of the Finished Fabric 123 4.1. Experimental 110 4.2.3.4.2.2.3. 3. Materials 110 4. Performance Properties 115 4.2.3.3. Optimization of Easy Care Finish 107 4.5.2.2.

3. Infrared Analysis 12:5 4.2.3.2.3.3. Infrared Analysis of Neem Extract Treated Fabric 128 4. Sample Coding 125 4.1.1.1.2.2.3.3. 124 4.2. Summary 156 .3.3.2.3.3. Weight add on of Seed Extract Treated Fabric 133 4.3.3.2. Results and Discussion 128 4.3.7.3.2.2.3.3.6.3.3. Performance Properties 127 4.4.3.3.Colony Counting Static Test method: 126 4.2.3. Performance Properties of Neem Finished Fabrics 150 4.3. Weight add on of bark extract treated fabric 1.2.3.5.3.5. Scanning Electron Microscopy 127 4.3.4. Integration of Neem Seed and Bark Extract on Cotton Fabric 124 4. Methods 124 4. 33 4. Materials 4. Antibacterial activity against gram negative bacteria 147 4.2.4.2.2. Antibacterial Activity Test 126 4.3.4. SEM Analysis 148 4. Antibacterial Activity 134 4.3.3.4.3.1.3. Material and Methods 4.2.6.Parallel Streak Method 126 4.

Future Scope of the Work 164 6.2. Conclusion 158 5. References 165-177 Annexure I 178 Annexure II 179 Curriculum vitae 184 xiv .1. Chapter V : Conclusion and Future Scope of the Work 158-164 5.5.