Colourimetric analysis and antimicrobial study of natural dyes and dyed silk

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Journal of Cleaner Production 18 (2010) 1750e1756

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Journal of Cleaner Production

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Colourimetric analysis and antimicrobial study of natural dyes and dyed silk

A.K. Prusty, Trupti Das, A. Nayak, N.B. Das*

Institute of Minerals & Materials Technology, Council for Scientific and Industrial Research (CSIR), Bhubaneswar 751013, India

a r t i c l e i n f o

Article history:Received 4 December 2009Received in revised form21 May 2010Accepted 23 June 2010Available online 30 June 2010

Keywords:Natural dyeHuman pathogensAntimicrobial activityYarnDyeingMordant

* Corresponding author. Tel.: þ91 674 2581635/6x3E-mail addresses: [email protected], nalinbiharee

0959-6526/$ e see front matter � 2010 Elsevier Ltd.doi:10.1016/j.jclepro.2010.06.020

a b s t r a c t

Colourimetric and antimicrobial activities of natural colourants and dyed silk have been studied. Thecolour strength (K/S) values were increased with increase in dye absorbance. The absorption of dyes onsilk yarns was obtained from 10.56 to 39.48% at 5% concentration of dye from different plant extracts. Thecolourimetric parameters L*, a*, b*, C, and H were measured for depth of the colour. The dyed silk withnatural colourants displayed excellent antimicrobial activity (reduction rate: 25e65%) against thebacteria Escherichia coli and (reduction rate: 3e68%) against the fungal strain Aspergillus niger. The dyedsilk exhibited good and durable fastness properties.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

The greatest challenge of the present era is to develop naturefriendly sustainable technologies that would make life easy andproductive for the current as well as future generations. Thisprovides an opportunity for reintroduction of natural dyes thatcould be considered as a suitable alternative to synthetic dyes,which have been known to cause health hazards due to theircarcinogenic effects (Sewekow, 1988). A wide spread interest hasemerged (Bechtold et al., 2003; Kenneth, 1973; Gulrajani andGupta, 1992; Komboonchoo and Bechtold, 2009; Feng et al.,2007) in the dyeing of textile fibers using natural colourants onaccount of their better biodegradability and higher compatibilitywith the environment. India has an abundance of dye yielding plantspecies in different parts of the country. Traditionally, the ruralfolks of different region dyed their materials from leaves, roots,flowers and bark of the plants, mostly by boiling to get the desiredcolour. It is well known that the problems in dyeing with naturalcolourant are the low exhaustion colours and the poor fastnessproperties of dyed fabrics. These problems have been dealt with byusing the metallic salts as mordants, which are used to improvefastness properties and to develop different shades with the samedyes (Hwang et al., 1998; Cristea and Vilarem, 2006; Lee and Kim,2004; Vankar et al., 2007).

30; fax: þ91 674 [email protected] (N.B. Das).

All rights reserved.

In recent years, there has been an increasing tendencytowards the evaluation of microbial pigments and prevention ofmicrobial attack on textiles (Ham Lyn, 1995; Singh et al., 2005).Textile materials are known to be susceptible to microbial attack,as these fabrics provide large surface area and absorb moisture,thus generating a suitable environment for microbial growth andmultiplication. Natural dyes from Punica granatum and manyothers are reported (Gerson, 1975; Machado et al., 2002; Hanand Yang, 2005; Hussein et al., 1997) as potent antimicrobialagents owing to the presence of a large amount of anthraqui-nones, tannins, naphthoquinones etc. However, despite the factthat there are many natural antimicrobial agents againstcommon human pathogens, very few studies have been reportedin the literature regarding the antimicrobial properties on textilematerials, especially with respect to the human pathogenicstrains.

In the present investigation, various plant waste materials fromfour plant species such as, Terminalia catappa, Artocarpus hetero-phyllus, Tectona grandis and Morinda citrofolia, have been selectedfor the solvent extraction of their natural colourants followed bythe application of dyeing techniques on mulberry silk yarn.Adequate precautions have been taken not to cause any harm to theindividual plants and no uprooting has been done. M. citrifolia isa widely grown plant in the state of Orissa, India. The root bark hasbeen collected from certain parts of the lateral roots withoutcausing any harm to the primary (tap) root as well as to the entireplant. Further, the plant propagation is easily possible through rootcuttings (Nelson, 2006).

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A.K. Prusty et al. / Journal of Cleaner Production 18 (2010) 1750e1756 1751

The current study deals with laboratory scale extraction process.It has been reported (Kaur and Benipal, 2006; Lal et al., 2000;Tejada and Gonzalez, 2004; Okoroigwe, 2007; Tejada et al., 2009)that the plant based residue is utilized in the form of bio-fertilizer/farm-yard manure for enhancing crop productivity. Hence, furtherstudy is being envisaged for the utility of huge amount of biomassgenerated during large scale extraction in the form of bio-fertilizer/farm-yard manure.

The four different plants under study have been reported (Arunget al., 2010; Elevitch, 2006; Mian-Ying et al., 2002; Kinoshita et al.,2007; Varier, 1997; Singh et al., 1996) to exhibit brilliant medicinalproperties against various common human diseases linked withskin, cardiac, urinary, pulmonary systems, etc. Various parts ofthese plants have also been reported to be strong anti-oxidants andthus exhibit potential anti cancer properties.

These natural dyes as well as the dyed silk yarn have beenexplored for their potential antimicrobial properties againstcommon human pathogens, such as Klebsiella pneumoniae,Escherichia coli, Candida albicans and Aspergillus niger. The studyenvisages the potential use of these naturally dyed silk yarns indeveloping protective clothing against common infections. Thecolourimetric parameters such as colour strength (K/S) value andthe Hunter coordinate (L*, a*, b*) values of dyed samples for colourdepth have been examined in this work. The dye absorption (%) hasalso been determined and the fastness properties of the dyed silkyarn have been examined for their durability.

2. Experimental

2.1. Materials

The plant materials such as root bark (from the lateral rootsystem without causing any harm to the tap root) of M. citrifolia(family: Rubuaceae), waste leaves of T. catappa (family: Com-bretaceae), T. grandis (family: Verbenaceae) and waste leaves andheart wood of A. heterophyllus (family: Moraceae) were selected forthe present study and different parts of plants were collected fromthe nearby area of Bhubaneswar (20� 110 0600e20� 110 4500 N latitudeand 80� 500 5200e85� 510 5300 E longitude) and Koraput district (17�

400e20� 70 N latitude and 81� 240e84� 20 E longitude) of Orissa,India. The collected plant samples were washed, shade dried, pro-cessed and grinded to powder form in an electrically operated

% Dye absorbance ¼ Absorbance before dyeing� Absorbance after dyeingAbsorbance before dyeing

� 100 (1)

grinder. Heating mantle, soxhlet apparatus, condenser and roundbottom flasks were used for extraction of the natural colourants.Water bath with automatic temperature control was used fordyeing silk yarn.

2.1.1. Textile materialDe-gummy mulberry Silk (20/22 Denier) was used for dyeing.

The material was supplied by Weavers Service Center, Bhuba-neswar, India for this study.

2.1.2. Chemicals usedMordants such as alum [K2SO4Al2 (SO4)3.24H2O], copper

sulphate (CuSO4.5H2O), stannous chloride (SnCl2.2H2O), ferroussulphate (FeSO4.7H2O) and solvents such as petroleum ether, ethylacetate, acetone and methanol were procured from E. Merck, Indiafor dyeing the silk yarn.

2.1.3. Test organism usedLyophilized cultures of K. pneumoniae (ATCC 35657), E. coli

(ATCC 14948), C. albicans (ATCC 24433) and A. niger (ATCC 16404)were purchased from Microbiologics, USA through HIMEDIABioSciences. MicroBioLogics, Inc is licensed to use the ATCC trade-marks and to sell products derived from ATCC� cultures.

2.2. Methods

2.2.1. Extraction of colourantsThe processed powder materials (500 g) were subjected to

solvent extraction along with 10% aqueous methanol (2500 ml) ina soxhlet extractor and heated for 24 h to extract the colouringmaterials until the colour in the extract was negligible. Theresulting extract was distilled up to obtain the pasty mass by theprocess of distillation. The pasty extract was washed with non-polar solvent such as petroleum ether and freeze dried to yieldsolid/semi-solid colouring materials for testing.

2.2.2. Method of mordantingThe dyeing experiments were carried out with silk yarn by

adopting pre-mordanting technique (Gulrajani and Gupta, 2001)i.e. the samples were treated with different metal salt solutionsbefore dyeing. Silk yarn (0.5 g) was dipped in 20 ml of prepared 4%mordant solution at 1:30 MLR (material to liquor ratio) at 60e70 �Cfor 30e45 min. Then the mordanted yarn was air dried for 15 min.The dye solution was prepared 1:30 MLR (material to liquor ratio)with 5% dye and the mordanted silk yarn was then dipped in 20 mlof dye solution for 30e45 min at 60e70 �C. The dyed yarn was leftfor 15 min for air oxidation. The dyed yarns were washed with coldwater followed by soap solution and then washed thoroughly withwater. The wet samples were dried at room temperature.

2.2.3. Absorbance measurementsThe ultraviolet/visible (UV/Vis) absorption measurement was

recorded for the determination of absorption (%) on Perkin Elmer,Lambda 35 UV/Vis spectrophotometer in the wavelength range200e800 nm. The absorbance of 5% dye solution was recordedbefore and after dyeing the silk yarn at an average of threemeasurements. The amount of dye absorbed was calculated(Mathur and Bhandari, 2001) by using the relation:

2.2.4. Colour strength and colour depth measurementsThe colour strength and colour depth of both dyed and mor-

danted samples were determined colourimetrically (colourspace:CIELab(1976)/D65) by light reflectance technique using PremierColour Scan machine. The colour strength (K/S) value of both dyedand mordanted samples was evaluated (Bhuyan and Saikia, 2005)using the Kubelka-Munk equation :

K=S ¼ ð1� RÞ2=2R (2)

where R is the reflectance of the dye sample; K is the absorptioncoefficient and S is the scattering coefficient.

The CIELab coordinates (L*, a*, b*) of the dyed samples werecalculated using Premier Colour Scan. The colours are given in CIELabcoordinates: L* corresponding to the brightness (100¼white,0¼ black), a* to the redegreen coordinate (þve¼ red, �ve¼ green)and b* to the yelloweblue coordinate (þve¼ yellow, �ve¼ blue).

A.K. Prusty et al. / Journal of Cleaner Production 18 (2010) 1750e17561752

2.2.5. Colour fastness testThe dyed silk yarns were tested according to ISO standard

methods. The specific tests were for colour fastness to light (ISO:686 / 1985), washing (ISO: 3361 / 1979) and rubbing (ISO: 766 /1988) by using the instruments like Paramount digiLIGHT�, Para-mount digiWASH I Nx� (Launder-O-meter) and ParamountcrockMETER I� respectively. The fastness ratings were evaluatedby comparing with Grey Scale standard ISO-1976. The changes inshadeswere related to the standard Grey Scale rating (rating is 1e5;1 e poor; 5 e excellent).

2.2.6. Antimicrobial screening testInvestigations were carried out to determine the antibacterial

activity of the dyes by using five different concentrations of eachdye solution i.e. 10, 40, 60, 80 and 100 mg/ml which is equivalent to1, 4, 6, 8 and 10% dye solution respectively.

The antimicrobial (both bacterial and fungal) studies were carriedout in triplicates using standard methods (AATCC method 30) anda control set was run along with each test. The test bacteria (K. pneu-moniaeandE. coli)were streaked closelyandgentlyon sterilenutrientagarplates. 30 ml of dye solutionwasaddedon the sterilizedfilterdisc,which is placed on the top of the seeded medium. After overnightincubationat37 �C, the zoneof inhibitionwasmeasuredby taking theaverage of the zones obtained from the triplicate plates.

Anti-fungal activities of the dyes against C. albicans and A. nigerhave been detected on sterile Potato Dextrose Agar plates. Experi-ments were carried out at five different concentrations of each dyein a similar way as described above. After incubation at roomtemperature (ranging between 25 and 30 �C) for 72 h, the zone ofinhibition was measured. The average value of the triplicates hasbeen reported as the final zone of inhibition.

In the second set of experiments (AATCC method 100), dyed andun-dyed yarns of each dye,were introduced into 20ml nutrient brothand inoculated with the respective bacterial strains followed byovernight (16 h) incubation at 37 �C. Growths of the bacterial strainswere determined in presence of both the dyed and un-dyed yarns,spectrophotometrically (OD660) against a blank of uninoculatedsterile medium (Singh et al., 2005). Similarly the fungal strains wereinoculated into potato dextrose broth and incubated for 48 h at 28 �Cin a shaker incubator followed by measurement of OD450 againsta blank of uninoculated sterile medium (Banerjee et al., 1993). Beforerecording theODof the respectivemedia after incubation, the culturetubes were thoroughly shaken in order to bringmicroorganisms intosuspension. Optical density is directly proportional to the number ofmicroorganism(bacteria and fungi) in themedium. Thepercentage ofreduction of the microorganism by dye was expressed as follows.

R ¼ ðB� AÞ=B� 100 (3)

where R¼ % of reduction of microbial population; B¼Absorbanceof the media inoculated with microbes and un-dyed fabric; andA¼Absorbance of the media inoculated with microbes and dyedfabric.

The unmordanted dyed yarns were washed for five times withsoap solution followed by water and air dried. These washed yarnswere then subjected for the antimicrobial study.

3. Results and discussion

3.1. Optimization of solvent extraction

Experiments were carried out for dye extraction from fourdifferent plant species using organic solvents such as petroleumether, ethyl acetate, acetone, methanol and 10% aqueous methanol.From several sets of experiments, it was observed that the yield of

the dye extract was better in 10% aqueous methanol in comparisonwith other solvents. The quantity of resulting extract in each casefrom respective plants might vary depending on the pigmentspresent in the plant materials taken. Due to an enhancement of theproduct yield by solvent extraction, the later is believed to be anefficient technique for the extraction of dyes. The efficiency ofsolvent extraction at laboratory scale is supported by our repeatedexperiments with the recovered solvent. In all our experiments90e95% solvent is recovered which has been recycled subse-quently. The minor loss of solvent is having negligible effect on eco-efficiency.

3.2. Dye components

The most important class of pigments from the plant resourcesis basically flavonoids, quinonoids, indigoids, tannins, etc., whichare responsible for dyeing the textiles (Gulrajani and Gupta, 1999).The known flavonoid pigments, such as artocarpesin, nor-arto-carpesin, cycloartocarpesin, dihydrocycloartocarpesin, artocarpe-tin, nor-artocarpetin, etc. from the plant A. heterophyllus werecharacterized and reported (Parthasarathy et al., 1969;Radhakrishnan et al., 1965; Lin et al., 1995).

The leaves of T. catappa revealed the presence of pigments, viz.violanxanthin, lutein and zeaxanthin and b-cryptoxanthine (Lopez-Hernandez and Cruz-Sosa, 2001), tannins (Tanaka et al., 1986) andflavone glycosides (Lin et al., 2000).

The quinonoid colouring matters such as tectoquinone, tectog-randone, tectoleafquinone, 1,5-dihydroxy-2-methyl-9,10-anthra-quinone and 5-hydroxy-2-methyl-9,10-anthraquinone wereidentified and characterized (Agarwal et al., 1965; Aguinaldo et al.,1993; Bhargava et al., 1991) from the leaves of T. grandis.

The main pigments such as morindone, alizarin, rubiadin, luci-din, 2-methyl-3,5,6-trihydroxyanthraquinone and 3-hydroxymor-indone isolated from the plant M. citrifolia were identified andcharacterized (Eckhard, 1975; Inoue et al., 1981; Bowie and Cooke,1962)

3.3. Effect of mordant with colour hue changes

It was observed from different experiments that the pre-mor-danting method imparted better shades on silk yarn than the post-mordanting method. A variety of colour hues were obtained withrespect to the nature of mordants such as alum, CuSO4, SnCl2 andFeSO4 adopting pre-mordanting technique. It was observed fromthe colour fastness data that the extracted dyes from M. citrifolia,T. grandis and T. catappa furnished different colour hues with verygood affinity for silk yarn in presence of metal salts as mordants.The colour intensity was found to be maximumwhen treated withCu(II) and Fe(II) salts as compared to Al(III) and Sn(II) salts for thesilk yarn. The brightness of the shades on silk yarn might be due tothe better absorption of dye and easy formation of metal complexeswith the yarn. The overall results indicatewell to very good fastnessproperties as shown in Table 1. The concentration of Fe(II) and Cu(II)ions in the mordant solution was estimated by Atomic AbsorptionSpectrophotometer, before and after mordanting. Similarly, theconcentration of Al(III) and Sn(II) was estimated through ICPMS(Inductively Coupled Plasma Mass Spectrometry). The metal ionconcentration consumed during mordanting is given in Table 2,before and after treatment. The left-over mordant solution, aftermakeup of desired concentration, has been re-used continuously insubsequent mordanting of fresh silk yarns.

The different mixtures of these natural colourants have beensubjected for dyeing silk yarns and have revealed synergetic impacton colours. The mixture of various colourants imparted brightyellow shade without mordant, golden yellow shade with alum,

Table 1Shades and fastness properties of dyed and mordent silk yarn.

Dye from plantspecies

Mordant Shade Lightfastness

Washingfastness

Rubbingfastness

Dry Wet

Tc Direct Lightyellow

2 1/2 3 2/3

Alum Goldenyellow

4/5 4 4/5 4

CuSO4 Brown 4 4 4/5 4FeSO4 Slate grey 4/5 3/4 4 3/4SnCl2 Golden

yellow4/5 4 4 4/5

Mc Direct Goldenyellow

4/5 4 4/5 4/5

Alum Orange red 4 3/4 5 4/5CuSO4 Brick red 4 4 4/5 3/4SnCl2 Golden

yellow4/5 4 4/5 4

Tg Direct Maroon 3 2/3 2 1/2Alum Brown 4 4/5 4 5CuSO4 Brick red 4 4/5 4/5 5FeSO4 Slate grey 4/5 4 4 3

Ah Direct Lightyellow

2 1/2 2/3 2

Alum Goldenyellow

2/3 3 3/4 4

FeSO4 Slate grey 4 3/4 3/4 3

Tc, Terminalia catappa; Mc, Morinda citrofolia; Tg, Tectona grandis; Ah, Artocarpusheterophyllus.

Table 3Absorption (%) and K/S values of 5% concentration of natural dye with dyed andmordanted yarns.

Plantspecies

Wavelength(nm)

Mordant Absorbance Absorption(%)

K/S

Beforedyeing

Afterdyeing

Tc 396 Direct 2.358 1.961 16.83 10.360Alum 2.358 1.698 27.98 19.793CuSO4 2.358 1.638 30.53 24.352FeSO4 2.358 1.427 39.48 27.515SnCl2 2.358 1.659 29.64 21.738

Mc 384 Direct 1.019 0.803 21.19 19.061Alum 1.019 0.822 19.33 15.610CuSO4 1.019 0.794 22.08 20.601SnCl2 1.019 0.853 16.29 11.133

Tg 382 Direct 1.486 1.329 10.56 6.569Alum 1.486 1.120 24.62 9.923CuSO4 1.486 1.104 25.70 10.946FeSO4 1.486 0.947 36.27 22.386

Ah 398 Direct 5.750 4.800 16.52 4.385Alum 5.750 3.530 38.60 27.961FeSO4 5.750 4.340 24.52 19.959


Table 4Colourimetric data of the dyed and mordanted silk yarns.

Plant species Mordant Colour coordinates ofdyed yarn

C H Mean DE

L* a* b*

Tc Direct 57.522 6.277 33.067 33.657 79.220 3.440Alum 66.685 11.904 59.002 60.191 78.562 9.843CuSO4 35.641 8.979 26.086 27.588 70.978 1.699FeSO4 19.241 0.196 2.478 2.486 85.443 1.839SnCl2 58.776 7.826 61.607 62.102 82.727 2.331

Mc Direct 62.157 22.581 70.639 74.160 72.244 8.691Alum 45.376 28.818 40.789 49.942 54.736 3.840


brown shade with CuSO4 and brownish grey shade with FeSO4 asmordant, on silk yarn.

3.4. Evaluation of absorption (%) and colour strength

Maximum absorption (%) was 39.48 for T. catappa and 36.27 forT. grandis using FeSO4 as mordant, whereas the adsorption (%) forM. citrifolia and A. heterophyllus was 22.08 and 38.60 using CuSO4and alum as mordants respectively. Similarly, the K/S values werefound to be maximum for T. catappa (27.515) and for T. grandis(22.386) using FeSO4 as mordant, whereas the K/S values weremaximum for M. citrifolia (20.601) and for A. heterophyllus (27.961)using CuSO4 and alum as mordant respectively. A synchronizedincrease in colour strength (K/S) values has been observed withincrease in absorbance (Table 3) of the above mordant systems. Theabsorption (%) and K/S values were observed to beminimum in caseof unmordanted dyed yarn, except for the plant M. citrifolia. Theminimum absorption (%) and K/S value forM. citrifoliawas obtainedusing SnCl2 as mordant. Therefore the colour strength has a directcorrelation with the dye absorbance.

3.5. Determination of colour coordinates of dyed yarn

The colourimetric data (Table 4) indicated the depth and naturaltone of dyed and mordanted silk yarns. The chroma (C) of the yarn

Table 2Metal ion concentration of the mordanting solution before and after treatment.

Metalion

Concentration of freshmetal ion solution(mg/l)

Concentration of metal ion insolution after mordanting(mg/l)

Loss of metal ionduringmordanting (%)

Cu 1058 1046 1.13Fe 11,440 11,340 1.04Al 7035 6810 3.19Sn 25,400 25,245 0.61

dyed with FeSO4 in case of T. catappa (2.486), A. heterophyllus(9.514) and T. grandis (14.259) and with CuSO4 for M. citrifolia(32.039) was found to be low indicating their natural tones. Themean DE values were determined by comparison with the nearestequivalent shades of standard Pantone shade cards. Further, the L*

values were found to be lower using FeSO4 as mordant for T. cata-ppa and A. heterophyllus, using CuSO4 for M. citrifolia and usingalum for T. grandis corresponding to deeper shades. The L* valueswere found to be higher in case of unmordanted dyed yarn for M.citrifolia and T. grandis corresponding to lighter shades. Similarly, byusing alum as mordant for T. catappa and A. heterophyllus, the L*

values were also higher corresponding to lighter shades. The highervalues of a* and b* indicated the brightness, which were more dueto redness and yellowness respectively. Thus, Cu(II), Fe(II) and Al(III) might be effectively used as mordant salts for the dyes fromT. catappa and T. grandis. Similarly salts of Al(III), Cu(II) and Fe(II)

CuSO4 29.521 25.136 19.867 32.039 38.307 7.410SnCl2 52.330 22.783 46.435 51.723 63.840 2.033

Tg Direct 50.515 7.664 20.014 21.431 69.019 3.978Alum 32.745 13.908 7.902 15.996 29.592 4.348CuSO4 33.138 13.355 8.486 15.823 32.420 5.745FeSO4 35.890 5.564 13.129 14.259 67.006 5.433

Ah Direct 65.336 5.329 24.019 24.603 77.459 7.089Alum 70.577 6.805 82.803 83.082 85.267 3.403FeSO4 26.321 1.680 9.364 9.514 79.797 2.174



can be used as effective mordants for M. citrifolia whereas Al(III)and Fe(II) for A. heterophyllus respectively.

Fig. 1. Effect of different concentrations of Terminallia catappa dye on growth ofAspergillus niger.

3.6. Screening of natural dyes for antimicrobial activity

K. pneumoniae, E. coli, C. albicans and A. niger showed goodinhibition properties in the presence of four natural dyes as shownin Table 5. In case ofM. citrifolia, themaximum zonewidth for E. coli(1.76 cm) and A. niger (2.4 cm) was obtained by the application of6% of the dye solution, whereas with increase in concentration upto 8% and 10%, the inhibition zones were observed to decreaseslightly. This might be due to the fact that at higher concentrationsthe dye became thicker and gummy. Therefore, proper diffusionwas hampered in the nutrient agar plates thus decreasing itseffectiveness. The MIC (Minimum Inhibitory Concentration) forE. coli, C. albicans and A. niger was 1%, whereas it was 4% concen-tration for K. pneumoniae. In case of C. albicans, the inhibition zonewas more or less similar (1.45e1.5 cm) in presence of all thedifferent dye concentrations. Thus M. citrifolia was highly effectivedue to significant inhibitory effects against all the bacterial as wellas fungal strains.

It was observed in case of T. catappa dye that the zone of inhi-bition gradually increased with the increase in its concentration upto 8% and beyond that, the zone diameter was more or less similar.The maximum zonewidth (2.36 cm) was obtained for C. albicans bythe application of 8% concentration of the dye. The MIC wasdetermined to be 1% of the dye for all the four different pathogens.A typical example is shown in Fig. 1 where A. niger is shown to havesimilar zone diameter beyond 1% of the dye. Thus, dye obtainedfrom T. catappa plant also inhibits microbial growth and forms clearzone around the disc. Overall, the dye was highly effective againstall the pathogenic strains.

Preliminary screening showed that the dye from A. heterophyllusplant was also effective against all the microbes. The zone of inhi-bition was clear at 6, 8, 10% concentrations of the dye whereas at 1and 4% concentration, mild growth was observed inside the zone ofinhibition. Therefore, theMIC for E. coliwas 4% and 1% for rest of the

Table 5Zone of inhibition obtained during incubation of the pathogenic strains with theapplication of the four different dyes.

Plant species Conc Average zone of inhibition in cm

K. pneumoniae E. coli C. albicans A. niger

Mc 10% 1.63 1.43 1.5 28% 1.63 1.4 1.5 2.336% 1.36 1.76 1.53 2.44% 1.23 1.53 1.45 2.11% e 1.2 1.45 1.93

Tc 10% 2.03 2.1 2.26 1.768% 2.06 2.23 2.36 1.836% 1.93 1.83 2.1 1.764% 1.9 1.7 1.73 1.71% 1.06 1.3 1.7 1.43

Ah 10% 1.7 1.96 1.7 1.838% 1.76 2.4 1.74 2.436% 1.73 1.96 1.63 1.864% 1.5 1.66 1.56 1.531% 0.56 e 1.33 1.5

Tg 10% 1.66 1.5 1.8 1.88% 2.33 1.6 1.83 2.16% 2.2 1.43 1.66 1.334% 1.93 1.5 1.6 1.51% 1.96 0.46 1.43 1.4


microbes. The maximum zone width (2.43 cm) was obtained forA. niger in 8% concentration of the dye.

Screening test for T. grandis showed that the dye was effectiveagainst all the four microbes as a clear zone of inhibition wasobtained at 6, 8, 10% concentrations. The maximum zone width(2.33 cm) was obtained for K. pneumoniae in 8% concentration ofthe dye. The zone occurred with mild growth inside it at 1 and 4%concentrations that implying the effectiveness of the dye againstthe microbes is reduced at lower concentrations. The MIC for all thepathogens was determined to be 1%.

The antimicrobial study of the various dye mixtures has alsobeen undertaken and the results are given in Table 6. The abovemixtures were prepared in 1:1 proportion. It can be observed thatall the dyes, in various mixtures, depicted brilliant inhibition zonefor both the bacterial and fungal strains.

Table 6Zone of inhibition obtained during incubation of the pathogenic strains with theapplication of the mixture of different dyes (prepared in 1:1 proportion).

Mixture of dyes Conc. Average zone of inhibition in cm

K. pneumoniae E. coli C. albicans A. niger

Mcþ Tg 10% 1.8 1.7 1.5 1.68% 1.6 1.5 1.5 1.56% 1.4 1.3 1.5 1.44% 1.3 1.1 1.4 1.31% e e 1.2 1.1

Ahþ TcþMc 10% 2.6 2.1 1.5 1.58% 2.4 1.8 1.4 1.46% 2.3 1.7 1.4 1.34% 1.7 1.5 1.4 1.21% 1.9 1.2 1.2 1.0

TgþMcþAhþ Tc 10% 2.4 1.9 1.5 1.68% 2.3 1.7 1.4 1.46% 2.2 1.6 1.4 1.34% 2.0 1.4 1.3 1.21% 1.6 1.3 e 1.1


0

10

20

30

40

50

60

70

80

K pneumoniae E coli C albicans A niger

Microorganisms

% r

educ

tion

Morinda citrifolia

Terminalia catappa

Artocarpus heterophyllus

Tectona grandis

Fig. 3. Percentage reduction of the four pathogenic strains in presence of therespective dyed yarns without mordant after five times washing.


3.7. Determination of antimicrobial activities of dyed yarn

Having studied the antimicrobial activity of the dyes in solutionagainst selected microbes, the next step was to assess their effec-tiveness on the dyed yarn (silk). Investigations were carried out tostudy the influence of the unmordanted dyed yarn (after washingwith soap solution followed by running water) and mordantedyarns followed by thorough washing, in order to avoid the inter-ference of the metal salts used as mordants on the growth andmetabolism of the pathogens.

Effectiveness of the unmordanted dyed yarn (after one timewashing) against the chosen microorganism is shown in Fig. 2. Itwas observed that the yarn dyed with T. grandis and M. citrofoliashowed maximum inhibitory effect (>50%) against both thebacterial strains. Growth of A. niger was most affected in presenceof yarn dyed with M. citrifolia (68% inhibition) followed by T. cata-ppa (51%), A. heterophyllus (44%) and T. grandis (38%). The fourdifferent dyed yarns showed comparatively less effectivenessagainst C. albicans (<40%), T. catappa being the least effective (<3%).Moreover, the inhibition rate was <45% in case of yarn dyed withA. heterophyllus against all the four pathogens.

It is essential to detect, the retention of antimicrobial property ofthe yarn after subjection to several rounds of washing, whichmightbe the case when the yarn is made into real fabrics. Hence theunmordanted dyed yarns were initially subjected to washing withsoap solution followed by running water for five times. The resultsindicated a �10% reduction rate, on an average, in comparison tothe yarns that were subjected to one time washing as describedabove. The growth inhibition of the bacterial as well as fungalstrains in the presence of the washed yarns is shown in Fig 3.

However, another set of experiment that was conducted withthe dyed yarn treated with the mordant, successfully inhibited thegrowth of the pathogens. The % in reduction of growth of the fourpathogenic strains in presence of the respective dyed yarns withmordant (after washing) showed little variation (�0.5%) from theresults of the dyed yarns without mordant (after one timewashing).

It is well known that the metallic salts used as mordants exhibittoxic effects against the pathogens. But in the current study, thedyed yarns were thoroughly washed with soap solution followedby water resulting in subsequent removal of the metallic salts.Therefore, there was least chance of interference of the mordants inthe growth inhibition of the pathogens.

0

10

20

30

40

50

60

70

80

K pneumoniae E coli C albicans Aspergillus nigerMicroorganisms

noitcuder

%

Morinda citrofoliaTerminalia catappaArtocarpus heterophyllus Tectona grandis

Fig. 2. Percentage reduction of growth of the four pathogenic strains in presence of therespective dyed yarns without mordant after one time washing.

4. Conclusion

The extraction of plant colourants is a sustainable techniquetowards waste utilization. The plants under study are widelydistributed in the tropical and subtropical climatic conditionshence special harvesting techniques could be avoided. The silk yarndyed with natural colourants exhibit reasonably good fastnessproperties and durability; therefore, the colourants might be analternative source to synthetic dyes. The colourants portrayedpromising results to obstruct the growth of the pathogenic bacte-rial and fungal strains even after subjection to five times washing.The antimicrobial tests demonstrate an exciting opportunity for thedyed textile as a potential perspective in developing protectiveclothing against common infections in medicals and hotels. Basedon the results presented in this article, recently we are provided theopportunity to initiate a project for scaling up of the above process,with financial assistance from the Government of India. The hugeamount of biomass which would be generated during pilot scalestudies would have a multiple utility in the form of bio-fertilizer aswell as farm-yard manure. The study envisages developinga sustainable technology for utilization of bio-resources to upliftthe rural weavers societies in India for their socio-economicgrowth.

Acknowledgements

The authors are grateful to Prof. B.K. Mishra, Director, Institute ofMinerals & Materials Technology, Bhubaneswar, India for providingthe necessary facilities to carry out the work. The support providedbyMs R. Pati andMs B. Majhi, trainees for antimicrobial screening ishighly acknowledged. One of the authors (NBD) is grateful to CSIR,New Delhi for the financial support given as “Emeritus Scientist,CSIR”.

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Colourimetric analysis and antimicrobial study of natural dyes and dyed silk