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Journal of Cleaner Production 27 (2012) 42e50

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Journal of Cleaner Production

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Assessment of colorimetric, antibacterial and antifungal properties of woollenyarn dyed with the extract of the leaves of henna (Lawsonia inermis)

Mohd Yusuf a, Aijaz Ahmad b, Mohammad Shahid a, Mohd Ibrahim Khan a, Shafat Ahmad Khan a,Nikhat Manzoor b, Faqeer Mohammad a,*

aDepartment of Chemistry, Jamia Millia Islamia (Central University), Jamia Nagar, New Delhi 110025, IndiabDepartment of Biosciences, Jamia Millia Islamia (Central University), New Delhi, India

a r t i c l e i n f o

Article history:Received 21 July 2011Received in revised form21 December 2011Accepted 3 January 2012Available online 10 January 2012

Keywords:Colorimetric propertiesAntimicrobial activityWoolNatural dyeLawsonia inermis

* Corresponding author. Tel.: þ91 9350114878.E-mail address: faqeermohammad@rediffmail.com

0959-6526/$ e see front matter � 2012 Elsevier Ltd.doi:10.1016/j.jclepro.2012.01.005

a b s t r a c t

The extract of leaves of henna was applied on woollen yarn to investigate the dyeing characteristics andantimicrobial efficacy against common human pathogens such as Escherichia coli MTCC 443, Staphylo-coccus aureus MTCC 902 and Candida albicans ATCC 90028. Bioactivity of henna dyed woollen yarn wascompared with commercial antibacterial (Ampicillin) and antifungal (Fluconazole) agents. Lawsoniainermis dyed woollen yarn samples were found considerably active against tested microorganisms. Dyedwool yarns were tested for fastness toward light, washing and crocking (dry and wet). Fastness prop-erties of dyed woollen yarn samples were found considerably good. Effect of eco-friendly metallic saltmordants on bioactivity and color characteristics of dyed woollen yarn samples were also investigated.The results proved that mordanted wool yarn showed increase in dye uptake resulting in high colorstrength and better fastness properties but considerable decrease in antimicrobial activity and slightdecrease in the case of antifungal activity were observed with the application of mordants.

The results indicate that extract of leaves of henna can be applied on woollen yarns to produce coloredclothings and textiles (sportswear, clothings for hospitals and babies) with semidurable antimicrobialproperties.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

During last few decades, there has been a growing interest in thedevelopment of bioactive textile materials and clothings withantimicrobial properties. The rapid growth in technical textiles andtheir end-uses has generated many opportunities for the applica-tion of innovative finishes of textile materials. Novel finishes of highvalue added apparel fabrics are greatly appreciated by morediscerning and demanding consumer market for health andhygiene. Textile materials composed of proteinous materials suchas wool & silk, cellulosic material such as cotton, jute, flax and otherfibers in contact with body provide ideal environment for growthand multiplication of pathogenic microbes leading to objectionableodor, dermal infection, product deterioration, allergies, and otherrelated diseases (Khan et al., 2011). These factors necessitates thedevelopment of methods to impart microbial resistance to textileswith all usual desirable characteristics of textiles, as these textilematerials find extensive use in different sectors related to hygienic

(F. Mohammad).

All rights reserved.

and healthy life style apart from the conventional apparel usage(Sathianarayanan et al., 2010; Velmurugan et al., 2009). Majorknown antibacterial agents for textiles are oxidizing agents, halo-gens, metal based complexes, phenolic compounds and quaternaryammonium salts (Ramchandaran et al., 2003). Although thesynthetic antimicrobial agents are very effective against a range ofmicrobes and give durable effect onto textile materials but thecause of concern in its use is due to the associated side effects,action on non-target microorganisms and growing-up environ-mental issues (Gupta et al., 2004). Most of the natural colorants arebelieved to be safe because of their non-toxic, non-allergic andbiodegradable nature (Ali et al., 2009), which are used in colorationof textile materials (Khan et al., 2010b; Mirjalili et al., 2011; Nateri,2011), cosmetics (Kole et al., 2005) and in food coloration(MacDougall, 2002).

Lawsonia inermis (Henna) belongs to the family Lythraceae. It isa perennial shrub up to 2e5 m. in height. Powdered leaves of thisplant (aqueous paste) used as cosmetic for staining palm, hands,hairs and other body parts (Dweck, 2002; Rao et al., 2008). Lawsoneis the chief coloring component of henna leaves and chemically themolecule of Lawsone is 2-hydroxy-1,4-naphthoquinone shown inFig. 1. Lawsone is described as Natural Orange 6 (CI 75480) (Color

O

OHO

Fig. 1. Structure of Lawsone.

M. Yusuf et al. / Journal of Cleaner Production 27 (2012) 42e50 43

Index, 1971), which acts as a substantive dye for keratin andimparts orange color due to the presence of eOH (auxochrome)group in naphthoquinone structure (Bechtold, 2009; Gulrajaniet al., 1992; Mayer and Cook, 1943; Mondal et al., 2009).

L. inermis is found to have several pharmacological uses such asantitumor (Ozaslan et al., 2009), anthelmintic (Bairagi et al., 2011),antioxidant, immunomodulatory (Mikhaeil et al., 2004), burnwound healing (Muhammad andMuhammad, 2005), UV protective(Dweck, 2002) and antimicrobial properties (Saadabi, 2007). Due tothe wide application of henna in hair coloration and body paints,numerous studies about potential health hazards have been per-formed. As a general result the studies demonstrate that henna isa natural product with low health risk potential. From an assess-ment of the genotoxicity of 2-hydroxy-1,4-naphthoquinone, dyeingredient of henna, Kirkland and Marzin suggested from theirstudies that henna or 2-hydroxy-1,4-naphthoquinone pose nogenotoxic risk to the consumers (Kirkland and Marzin, 2003, 2004;Kirkland et al., 2005). A number of researchers around the globeworked on the applications of natural coloring materials to thetextiles but most of the studies focused on standardization ofmordanting and dyeingmethods, study of colorimetric and fastnessproperties (Bechtold et al., 2003; Samanta and Agarwal, 2009). Afew studies were made on bioactive properties of naturally dyedtextile materials (Han and Yang, 2005; Prusty et al., 2010), but stillthere is dearth of systematic studies on bioactivity retention ofnatural dyes after the application on textile materials. Conse-quently, it is necessary to ascertain whether L. inermis can be thenatural source of the producer of orange color and keep its anti-microbial activity after the application on textile materials.

Present study aims to evaluate dyeing characteristics and anti-microbial efficacy of woollen yarn dyed with the extract of leaves ofhenna against Gram (�ve) bacterium Escherichia coli, Gram (þve)bacterium Staphylococcus aureus and fungus Candida albicans.Colorimetric properties of dyed woollen yarns were obtained interms of CIELab and K/S values. Color fastness (light exposure, washand rub) were also assessed. Alum and ferrous sulfate were chosenfor use as mordants in this research work due to their eco-friendliness as compared to other commonly used metal saltmordants (Burkinshaw and Kumar, 2009; Chariat et al., 2011).

2. Materials and methods

2.1. Yarn, mordants and dye

Commercially available 100% pure NZ semi-worsted woollenyarn was purchased and used for the study. Commercially availablepowdered henna leaves powder was used for dyeing. All otherchemicals including mordants (ferrous sulfate and potash alum)used were of Laboratory grade.

2.2. Strains and media

Stock cultures of the microorganisms were maintained on agarslants and stored at 4 �C. E. coli MTCC 443, S. aureus MTCC 902 andC. albicans ATCC 90028 were grown and sub-cultured in Muller-Hinton broth, Luria-Bertani broth and YPD media respectively at37 �C in orbital shaker at 200� rpm (REMI CIS 24 BL).

2.3. Mordanting

Mordanting was performed by pre-mordanting method usingferrous sulfate and alum as mordants. Desired percentages of eachmordant, 5% ferrous sulfate and 20% alum on the weight of fabric/fibre (owf), were added to water in separate and the temperature ofthe mordant solution was raised to 40 �C and then water soakedwoollen yarns were put in baths. The temperature of the mor-danting bath was brought to simmering point (92e93 �C) for 1 hwith continuous stirring. Mordant baths were cooled and mor-danted samples were taken out of the mordanting baths and rinsedwith water.

2.4. Dyeing procedure

2.4.1. Preparation of extract of henna (L. inermis) leavesIn order to carry out the dyeing process, the colorant must be

extracted from the powdered henna leaves. Required quantity (10%and 20% owf) of powdered leaves of henna dye was mixed with 2%aqueous solution of sodium carbonate (Na2CO3) at pH 8.5e9,keeping material to liquour (M:L) ratio of 1:20 and boiled for 1 hwith occasional stirring. The extracted henna dye was then filteredthrough clean cotton cloth; the solution obtained is reddish orangein color. This process was repeated three to four times, until therewas no colorant left. Then, the dye extract was neutralized by theaddition of HCl. The filtrate obtained is extracted liquid dye andwasused for dyeing.

2.4.2. Dyeing with extract of henna (L. inermis) leavesThe dyeing was carried out by using 10% and 20% owf of dye

concentrations, material to liquor (M:L) ratio of 1:40 maintainingneutral pH (z7). Dyebath containing extracted liquid dye washeated up to 40 �C and woollen yarn samples were drenched. Thetemperature of dyebath was raised to simmering point and main-tained at that level for 1 h. Finally the dye solution was allowed tocool down. Dyed samples were washed with the 5 ml/l non-ionicdetergent (safewash, Wipro), rinsed with tap water and dried inshade.

2.5. Determination of exhaustion of dye

Dye uptake was determined bymeasuring the absorbance of thedyebath solution containing extracted henna dye at thewavelengthof maximum absorbance (lmax, 430 nm) before and after dyeing.The % dye exhaustion was calculated by using the given formula:

% Dye exhaustion ¼ ½ðA0 � A1Þ=A0� � 100 (1)

where A0 and A1 are absorbance of dyebath before and after dyeingrespectively.

2.6. Color measurement

The colorimetric properties of the dyed woollen yarn sampleswere obtained with Gretag Macbeth Color-Eye 7000 A Spectro-photometer in terms of CIELab color coordinates (L*, a*, b*, c*, ho)and color strength values (K/S). The color strength value (K/S) in the

Fig. 2. Effect of mordants on dye exhaustion.

M. Yusuf et al. / Journal of Cleaner Production 27 (2012) 42e5044

visible region of the spectrum (400e700 nm) was calculated basedon the KubelkaeMunk equation:

K=S ¼ ð1� RÞ2=2R (2)

where (K) is the absorption coefficient, (R) is the reflectance of thedyed sample and (S) is the scattering coefficient.

The relative color strength and color difference of dyed woollenyarn samples were obtained using following relationships:

Relative color strength ð%Þ ¼ K=S of mordanted sampleK=S of unmordanted sample

�100

(3)

Color Difference ðEÞ ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiðDLÞ2þðDaÞ2þðDbÞ2

q(4)

whereDL ¼ L*mordanted � L*unmordanted;Da ¼ a*mordanted �a*unmordanted;Db ¼ b*mordanted � b*unmordanted; ‘L’ describes lightness (100¼white,0¼ black), ‘a’ measures redness (þve), greenness (�ve) and ‘b’measures yellowness (þve), blueness (�ve).

2.7. Color fastness properties

2.7.1. Light fastnessThe light fastness of the dyed woollen yarn samples was con-

ducted on Digi light NxTM having water cooled Mercury BlendedTungsten lamp, as per the test method AATCC 16e-1993 (2004)similar to ISO 105-B02:1994 (Amd.2:2000).

2.7.2. Wash fastnessThe wash fastness of the dyed woollen yarn samples was

measured in Digi wash SSTM (Launder-o-meter) as per the ISO 105-C06:1994 (2010) specifications. The samples were also assessed forstaining on white adjacent fabric (cotton and wool).

2.7.3. Rub fastnessDry andwet rub fastness of the dyedwoollen yarn samples were

tested using a Digi crockTM (Crockmeter) as per Indian standard IS766:1988 (reaffirmed in 2004) based on ISO 105-X12:2001 bymounting the fabric on panel and giving ten strokes for both dryand wet rub fastness tests.

2.8. Minimum inhibitory concentration

The Minimum Inhibitory Concentration (MIC) was defined asthe lowest concentration of the dye that causes inhibition of visiblegrowth of test microorganisms. In vitro susceptibility tests wereperformed to evaluate MICs using our previous method (Khan et al.,2011). The effect of the dye concentration (0.01e5% w/v) on theantimicrobial activity was assessed.

2.9. Disc diffusion assay

Strains were inoculated into liquid YPD medium and grownovernight at 35 �C. The cells were then pelleted and washed thricewith distilled water. Approximately 105 cells/ml were inoculated inmolten agar media at 40 �C and poured into 90-mm-diameterpetri-plates. Filter discs were kept on solid agar and dye wasspotted on the disc. Test compound dissolved in double distilledwater with final concentrations of 1%, 5% and 10% (w/v) or control(distilled water) was pipetted onto 4 mm diameter filter disc. Thediameter of zone of inhibition was recorded in mm after 48 h andwas compared with that of control. The experiment was performedfor both the Gram (�ve) & Gram (þve) bacterial and yeast strains.

Values were shown in terms of mean� standard error of all threerespective categories.

Index of sensitivity defined as:X

Zone diameter ðmmÞ=concentration ðmg=mlÞ¼ clearing ðmm=mgÞ

2.10. Growth studies

Growth studies of the tested microorganisms were done asdescribed (Khan et al., 2010a) earlier with slight modifications.Prior to testing, the test microorganisms were sub-cultured at leasttwice and grown for 24 h at 35 �C on SDA plates. For growth studies,106 cells (optical density A600¼ 0.1) of test strains were grownaerobically in 50 ml media on automated shaker set at 35 �C withagitation of 200� rpm. Extracted L. inermis dye with the concen-trations of 10% and 20% w/v along with negative and positivecontrols (1% w/v of ampicillin for both bacteria and 1% w/v of flu-conazole for fungus) for each test isolate were also added to thecultures. At pre-determined time points (after every 2 h) for 24 h,aliquots were removed and growth was followed turbido-metrically at 595 nm using LABOMED Spectrophotometer (USA).Optical density was recorded for each concentration against time.

2.11. Determination of antimicrobial activity of henna (L. inermis)dyed woollen yarn

To determine antimicrobial activity of dyed woollen yarn spec-imens, 1 inch2 yarn was introduced in the 10 ml nutrient brothinoculated with a desired microbe and incubated overnight at37 �C. The reduction of the microbial growth by the dyed woollenyarn was expressed as follows:

R ¼ B� A=A� 100 (5)

where R¼ % reduction in microbial population; B¼ absorbance(595 nm) of the media inoculated with microbe and un-dyed yarn;A¼ absorbance (595 nm) of themedia inoculatedwithmicrobe anddyed woollen yarn.

The greater the growth, the higher is the turbidity, and also theoptical density. Turbidity and optical density are therefore, directlyproportional to the number of microbial cells in the media.

Table 1Colorimetric properties.

Dye (L. inermis) Mordant L* a* b* c* ho K/S Relative color strength DE

10% Unmordanted 69.58 4.36 22.4 22.82 78.98 1.84 100 e

20% Alum 66.97 7.49 30.41 31.31 76.16 2.99 162 8.985% Iron 60.61 5.34 18.9 19.63 74.22 2.62 142 9.67

20% Unmordanted 65.45 6.03 25.14 25.85 76.51 2.70 100 e

20% Alum 60.88 8.91 31.53 32.76 74.22 4.68 173 8.365% Iron 56.25 6.32 20.36 21.31 72.75 3.79 140 10.37

M. Yusuf et al. / Journal of Cleaner Production 27 (2012) 42e50 45

2.12. Determination of durability of antimicrobial activity towashing

Antimicrobial activity of dyed yarns was evaluated after severalwashing cycles and durability of antimicrobial finishing wascalculated in terms of percent retention of antimicrobial activityusing the formula given below:

% Retention of antimicrobial activity ¼ RnR0

� 100 (6)

where R0¼ % microbial reduction before washing and Rn¼ %microbial reduction after n wash cycles.

2.13. Statistical analysis

Each experiment was performed twice and in triplicate. Resultsobtained were expressed in terms of mean� standard error.Statistical analyses were performed considering P value £ 0.05 assignificant.

3. Results and discussion

3.1. Dye exhaustion

The amount of dye uptake by woollen yarn samples wasexpressed as % dye exhaustion and results are shown in Fig. 2. Themaximum exhaustion was observed in case of alum mordantedsamples followed by iron and unmordanted woollen yarn samples.The difference in exhaustion rates was because of difference ininteraction between fiberemordantedye.

Fig. 3. a*eb* plot of dyed woollen yarns. (1) 20% henna, (2) 10% henna, (3) 5% ferroussulphateþ 20% henna, (4) 5% ferrous sulphateþ 10% henna, (5) 20% alumþ 20% henna,(6) 20% alumþ 10% henna.

3.2. Color measurement

CIELab system was used to evaluate the color parameters andthe color difference, where L* refers to lightness� darkness values,a* values run from negative (green) to positive (red), b* values runfrom negative (blue) to positive (yellow), c* values represents thechroma and ho corresponds to hue angle. L*, a*, b*, c* and ho valuesof henna dyed woollen yarn samples are given in Table 1. Resultsobtained indicate that Lightness (L*) values are found to be lesser incase of mordanted samples. Ferrous sulfate mordant has compar-ativelymore darkening/saddening effect than alum as evident fromL* value data. Highest chroma (c*) values were obtained in case ofalum mordanted samples. The a*eb* plot (Fig. 3) reveal that allhenna dyed woollen yarn samples found to be little inclined towardyellow coordinate of yellowered zone. K/S value graph is given inFig. 4 to examine the effect of mordant on color strength. Highest K/S values were obtained in case of alum mordanted woollen yarnsamples indicate that alum mordant has much more pronouncedeffect on color strength than ferrous sulfate mordant. The colordifference (DE) data is also given in Table 1 and it is clearly observedthat there is a significant color difference between the unmor-danted and mordant treated samples. The color difference is moreprominent in case of ferrous sulfate mordanted woollen yarnsamples.

3.3. Color fastness properties

Color fastness properties (light exposure, washing and rubbing)of the henna dyed woollen yarn samples are given in Table 2. Lightfastness of dyed woollen yarn samples improved after mordantingwith metallic salts (ferrous sulfate and potash alum) compared to

Fig. 4. Effect of mordants on color strength.

Table 2Color fastness properties.

Dye (L. inermis) Mordant Light fastness Wash fastness Rub fastness

c.c. c.s. c.w. Dry Wet

10% Unmordanted 4e5 4 5 5 4 320% Alum 5 4e5 5 5 4e5 3e45% Iron 5 4e5 5 5 5 3e4

20% Unmordanted 4 3e4 5 5 3e4 320% Alum 5 4e5 5 5 4 35% Iron 5 5 5 5 4e5 3

c.c.¼ color change, c.s.¼ color staining on cotton, c.w.¼ color staining on wool.

Table 3Sensitivity index of inhibition zone (mm) to the concen-tration (mg/ml) for the L. inermis dye in all tested isolates.

Isolates L. inermis dye

Sensitivity index

E. coli 1.8� 0.01S. aureus 2.82� 0.07C. albicans 4.4� 0.03

M. Yusuf et al. / Journal of Cleaner Production 27 (2012) 42e5046

unmordanted woollen yarn samples. The wash fastness propertiesof mordanted samples have been improved and found to be good tovery good ratings of 4e5 on gray scale whereas unmordantedsamples have been found to have fairly good to good wash fastness

Fig. 5. Disc diffusion assay of E. coli (Panel-A), S. aureus (Panel-B) and C. albicans (Pa

ratings of 3e4. No staining on adjacent fabrics (cotton and wool)was observed. Dry andWet rub fastness of mordantedwoollen yarnsamples have been found to be good to very good rating of 4e5 andfairly good to good ratings of 3e4 on gray scale respectivelywhereas dry and wet rub fastness of unmordanted samples werefound to be fairly good to good ratings of 3e4.

3.4. Antimicrobial activity of L. inermis in solution

3.4.1. Minimum inhibitory concentration (MIC)The MIC of the dye was determined against two bacterial and

one fungal species using micro-broth dilution method. Theminimum inhibitory concentration of the dye against the fungi wasobserved to be as low as 0.35% w/v whereas in case of bacteria itwas observed to be 0.86% w/v.

3.4.2. Disc diffusion assayExtract of leaves of henna was screened for antimicrobial

activities against selected bacteria (E. coli and S. aureus) and fungus(C. albicans). The results summarized in Table 3 give the sensitivityassay, using discs of L. inermis dye. The tested microorganisms(E. coli, S. aureus and C. albicans) showed high degree of sensitivity.It is evident from zone of clearance (Fig. 5).

The index of sensitivity is greater (4.4� 0.03) for C. albicans andis least (1.8� 0.01) for E. coli isolates. The most important thingwhich has been noticed, is that the dye was slightly more effective

nel-C) treated with different concentrations of L. inermis dye (1%, 10% and 20%).

M. Yusuf et al. / Journal of Cleaner Production 27 (2012) 42e50 47

against fungus as compared to both the bacteria. The resultsshowed that in case of control disc, no zone of inhibition wasobserved. In our study distilled water was used as control (solvent)and having no effect on the tested organisms, hence it is effectivelyconcluded here that whole of the antimicrobial effect is because ofthe dye only.

3.4.3. Growth studiesBy the use of growth curve studies the effect of increasing

concentrations of the dye on the growth of tested microbes hasbeen studied. Fig. 6 depicts the growth rates of E. coli, S. aureus andC. albicans in the presence of L. inermis dye at 10% and 20% w/v. Theabsorbance obtained for the growth control (only organism)showed that the test cultures reached the stationary growth phaseafter 16e18 h showing a normal growth pattern. The curve depictsa lag phase in the initial phase of growth, active log phase andstationary phase. All the test microorganism isolates were found tobe susceptible to the test dye at lower value (10%) of the dye, the

Fig. 6. The effect of various concentrations of the dye on the growth E. coli, S. aureus and obacteria and 1% w/v of fluconazole for yeasts (Control þive), 10% dye and 20% dye.

test microorganisms show the extension of the lag phase by 2e6 hand growth was suppressed with respect to the control. More than90% inhibition in case of all tested microbes was observed whencells were treated with 20% of the dye. It was worth to note that atthe higher percentage, the dye showed more inhibitory effect inC. albicans than the used commercial antimicrobials.

3.5. Antimicrobial activity of L. inermis dyed woollen yarn

Since extract of henna leaves showed good antimicrobialactivity in solution against all the tested microbes, it was worth-while to study antimicrobial activity after application of henna dyeon woollen yarn. Antimicrobial activity of dyed woollen yarnsamples against E. coli, S. aureus and C. albicans was assessedquantitatively in the terms of % microbial reductions and given inTable 4. The antimicrobial activities of henna treated samples alongwith untreated wool and commercial antimicrobial agents (Ampi-cillin and fluconazole) in terms of their % reduction values against

f C. albicans. The cells were grown with 0% dye (Control �ive), 1% w/v of ampicillin for

Table 4Antimicrobial activity of henna dyed woollen yarn.

Microbe % Microbial reduction

Blank Ampicillin/fluconazole Untreated wool 10% Henna 20% Henna

Unmordanted 20% Alum 5% FeSO4 Unmordanted 20% Alum 5% FeSO4

E. coli 0 94 1 82 47 48 91 74 66S. aureus 0 98 4 85 42 46 94 70 57C. albicans 0 98 7 90 80 62 93 94 70

M. Yusuf et al. / Journal of Cleaner Production 27 (2012) 42e5048

all the tested microbes are given in Fig. 7. From Fig. 7 it is observedthat untreated woollen yarn samples showed zero % reductionwhereas dyed substrate showed significant activity against all thetested microbes. Highest activities (82e94% microbial reductions)were observed when dye (10% and 20% owf) was applied alone onthe woollen yarn without the use of mordant. Lower concentrationof henna extracts (10% owf) was found more effective against yeast(62e90% fungal reduction) than bacteria (42e85% bacterialreduction). Significant increase in microbial reduction wasobserved when higher concentration (20% owf) of dye was used.Fig. 7 shows that the maximum concentration of dye (20% owf)

Fig. 7. Antimicrobial activity of the woollen yarn treated with L. inermis dye. Bar 1 representrespective known available antimicrobial agent; 3 is untreated wool; 4e6 represent 10% HeHenna, 20% alumþ 20% henna & 5% FeSO4þ 20% henna respectively.

showed highest % reduction in microbial growth corresponding toexcellent antimicrobial activity with all tested microbes: 91% forE. coli, 94% for S. aureus and 93% for C. albicans. From the data inTable 4 it is clear that woollen yarns dyed with henna alone wasproved to be most effective in inhibiting microbial growth. This isdue to the inherent antimicrobial characteristics of the lawsonedye. As mentioned before, henna has an orange-colored pigmentlawsone which is responsible for both its dyeing as well as anti-microbial characteristics (Dev et al., 2009).

It is observed from Table 4 that both the mordants (alum andiron) decreased % inhibition rate of microbial growth, however,

s the control cells without any treatment; 2 represents the treatment of cells with theirnna, 20% alumþ 10% henna & 5% FeSO4þ10% henna respectively; 7e9 represent 20%

Fig. 8. % Retention of antimicrobial activity to washing against (a) E. coli, (b) S. aureus and (c) C. albicans.

M. Yusuf et al. / Journal of Cleaner Production 27 (2012) 42e50 49

decrease in antibacterial activity is more pronounced than anti-fungal activity which indicates that henna extract is more effectiveagainst fungal strain than bacterial strains. Alum mordantedsamples showed better microbial reduction potential (42e94%)than iron mordanted samples (46e70% microbial reduction). Thesharp decline of antimicrobial activity in case of mordantedsamples could be the consequence of complex formation betweenactive functional groups of the dye with the metal salt mordants.Results indicated that, 10% henna extract, is the effective concen-tration for inhibiting fungal growth whereas in case of bacterialreduction, 20% henna extract concentration is more effective for thecontrol on bacterial growth. Microbial % reduction values shown byhenna dyed woollen yarn samples were considerably good and itcan be used to develop clothings for protection against commonpathogens normally growing in home textiles.

3.6. Wash durability of antimicrobial activity

Fig. 8 represents the durability of antimicrobial finish in terms of% retention of bioactivity after washing. Microbial % reduction ofdyed woollen yarn samples were examined over three differentlaundering cycles of 1, 5 and 10. Woollen yarn samples dyedwithout mordants were found most effective against all testedmicrobes but this finish is found to be least durable to washing,after 5 washing cycles it was reduced to less than 50% of the initialbioactivity against both bacteria as well as fungus. Both mordantsproved helpful in increasing washing durability to a significantextent. Comparatively, antifungal activity was found to be durable

to washing than antibacterial activity. Existence of higher anti-fungal activity than antibacterial activity after several washingcycles indicated that a relatively high concentration of extract ofhenna leaves is needed to control bacteria (E. coli and S. aureus)than fungus (C. albicans). Lastly it is observed that the concentrationof henna dye used for microbial inhibition depends on the type ofmicrobes.

4. Conclusion

The study mainly focused on colorimetric investigation andantimicrobial properties of woollen yarn dyed with the extract ofhenna leaves. From the study, the effect of metallic salt mordants onthe color, fastness and antimicrobial properties of woollen yarnswas successfully investigated, and the following conclusions weremade:

� L. inermis (henna), a non-toxic natural dye producing beautifulorangeebrown to light yellow color, exhibits antimicrobialactivity in solution and retains its activity when applied onwoollen yarn substrate.

� When woollen yarns were dyed with extract of henna leaveswithout mordanting, dyeings displayed good light fastness,very good wash fastness, fairly good to good rub fastnessproperties. As anticipated, dyeing of mordanted woollen yarnsamples considerably improved overall fastness values in withslight change in hue and tone of the shade.

M. Yusuf et al. / Journal of Cleaner Production 27 (2012) 42e5050

� Dyed woollen yarn samples exhibit reasonable durability withgood fastness properties. It is assumed that the antimicrobialeffect would be durable in practice and naturally occurringlawsone in L. inermis could be a promising antimicrobial dye forclothings.

� Results clearly demonstrate that utilizing extracted naturalcolorants from L. inermis as dye significantly improved thequality antibacterial fabrics having good soothing effect.

At the end it can be concluded that dyeing with extract of hennaleaves is a very promising, simple and practical method for devel-oping color as well as antimicrobial effect onwoollen yarn, and canbe proved an eco-friendly alternative source for expensive,synthetic and toxic antimicrobial agents available in the markettoday. A combination of henna leaves extract and mordants hasa good scope to produce protective clothings and other textilematerials for their use in value added products for makingsportswear, clothings for hospitals and babies. Future work will beaimed to test against more microbe types and to investigate thepossibility of imparting antimicrobial activities to the other types offabrics/clothings by treatment with extract of henna leaves.

Acknowledgments

The authors are grateful to Dr B.S. Butola, Department of TextileTechnology, IIT Delhi, for extending the facility of recording CIELabvalues. Financial support provided by University Grant Commis-sion, Govt. of India; through Fellowship to Central University Ph.D.Scholars (Mohd Yusuf, Shafat A Khan) and BSR Research Fellowshipin Science for Meritorious Students (Mohammad Shahid) is alsoacknowledged.

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