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 DOI: 10.1177/0040517512447585

2013 83: 462 originally published online 27 September 2012Textile Research JournalMingbo Ma, Rongxia Li, Yangyang Du, Zhirong Tang and Wenlong Zhou

Analysis of antibacterial properties of naturally colored cottons  

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Original article

Analysis of antibacterial properties ofnaturally colored cottons

Mingbo Ma, Rongxia Li, Yangyang Du, Zhirong Tang andWenlong Zhou

Abstract

The antibacterial activity of naturally colored cottons (NCCs) is evaluated. Compared to the conventional white cotton,

brown NCC was found to exhibit excellent antibacterial activity with a reduction rate of 89.1% and 96.7%, when in

contact with two species of bacteria, Staphylococcus aureus and Klebsiella pneumoniae, respectively, while the antibacterial

effect of green NCC was faint. To investigate the antibacterial mechanism of NCCs, pigments have been extracted from

NCC fibers for further determination of their antibacterial activity using the disk diffusion method, and the chemical

nature of extracted pigments was also analyzed. The results are as follows: the pigment from brown cotton fibers

showed significant inhibition against the two challenge bacteria, while the resistance capacity of the pigment from green

cotton fiber was insignificant, indicating that the antibacterial efficacy of NCC fibers was highly related to the chemical

nature of the pigment. Diagnostic results indicated that the pigment from brown cotton belonged to condensed tannins,

and the pigment from green cotton fiber was identified as flavonoids. The effect of high-temperature treatment on

antibacterial activity of the pigment extracted from brown cotton was also examined. It showed that the general textile

high-temperature curing conditions decrease its antibacterial activity, but the antibacterial activity still has a satisfactory

retention, indicating a durable natural antibacterial performance of brown NCC to the high-temperature curing

technology.

Keywords

Antibacterial property, condensed tannins, flavonoids, naturally colored cotton, pigment

Introduction

Naturally colored cottons (NCCs) are cottonvarieties endowed with natural colors during theirdeveloping period. Since 1988, when Sally Fox devel-oped two hybrids with a fiber quality suitable to bemachine-spun, NCCs have attracted a great deal ofattention for their ecological and economical advan-tages. Compared to conventional white cotton, theyneed not be dyed with synthetic dyes, not only savingthe dyeing cost, but also avoiding health hazards andenvironmental pollution. Although more than tencotton varieties with different colors have been reportedbreeding successfully worldwide,1,2 only brown cottonand green cotton have been marketed due to their rela-tively better fiber quality.3,4 NCCs have been theconcern of the textile materials, due to the world-wide requirements to develop the sustainable textileindustry.

Except for the existence of pigment, the fundamentalphysical structures and main chemical compositions ofNCCs are similar to that of white cotton fiber, whichshow natural convolution, a cuticle, secondary wall,lumen, and consist of pectin, wax, cellulose, hemicellu-lose and ash.5,6 However, the content of the chemicalcompositions of NCCs is significantly different.5 Thewax content of NCC fibers is much higher than that

Key Laboratory of Advanced Textile Materials and Manufacturing

Technology (Ministry of Education), Zhejiang Sci-Tech University, China

Corresponding author:

Wenlong Zhou, Key Laboratory of Advanced Textile Materials and

Manufacturing Technology (Ministry of Education), Zhejiang Sci-Tech

University, No.2 Street, Xiasha Higher Education District, Hangzhou,

Zhejiang 310018, China.

Email: wzhou@zstu.edu.cn

Textile Research Journal

83(5) 462–470

! The Author(s) 2013

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of white cotton fiber, and the cellulose content is rela-tively lower than that of white cotton fiber, leading tothe lower moisture regain and tensile strength of fiber.4

Hence, the textile processing properties of NCCs areinferior to the white cotton. But fortunately, thehigher content of metal ions in ash gives NCCs anti-flammability properties with the Limiting OxygenIndex (LOI) value from 22.4 to 25.4 depending on thespecies of NCCs.7,8 The LOI of NCCs is much higherthan that of white cotton, which is about 19.0. Naturalpigments, as the source substance of the colors ofNCCs, render NCCs UV radiation protective proper-ties, which permit NCCs to be processed into naturalsun-protection textiles possible.7 The pigments, prob-ably together with the wax, have also been found toserve as a defense against mildew and soil-borne patho-gens, resulting in much lower rate of biodegradation ofNCC fibers than that of white cotton and conventionaldyed cotton.9

However, the effect of pigments in NCCs on theactivity of anti-skin-pathogenic bacteria is stillunknown. The purpose of this work was to investigatethe antibacterial performance properties of twocommon NCCs (brown and green cotton), to determinethe antibacterial effects after heat treatment, and toevaluate whether the general high-temperature curingfinishing process decreases their antibacterial activityor not. The chemical composition of pigments wasalso analyzed according to their UV spectra and chem-ical properties.

Experimental

Materials

The NCCs were Caixuan series supplied by ZhejiangAcademy of Agricultural Sciences, Zhejiang, China.Gram-positive S. aureus (ATCC No. 6538) andGram-negative K. pneumoniae (ATCC No. 4352),obtained commercially from Hangzhou TianheMicroorganism Reagent Co., Ltd., Zhejiang, China,were chosen as representative species. All of thereagents were analytical grade and were purchasedfrom Hangzhou Mike Chemical Instrument Co., Ltd.,Zhejiang, China.

Antibacterial assessment

The degree of antibacterial activity of NCCs was eval-uated according to AATCC Test Method 100-2004.The control specimen was the conventional whitecotton. The test and the control fibers first underwentextraction with petroleum ether for 24 h using a Soxhletextractor in order to remove the surface wax, and thenwere sterilized with ethylene oxide and were desiccated

at 60 �C after being tidied up in a fiber bundle by hand-drawing. Sterilization of the NCCs should be avoidedin the autoclave because they would suffer a significantcolor change when the heat temperature exceeds90 �C,10 with the probable chemical structure changeof pigment due to the heat. The sterilized swatches(0.6 g each) were placed separately in sterile Petridishes and inoculated evenly with 1.0 mL of bacteriasuspension. These inoculated swatches were then asep-tically transferred to the 250 mL wide-mouth glass jar.The bacteria suspension should contain 1–2�105

colony forming units (cfu) of the bacteria/mL dilutedfrom a 24 h broth culture of the test bacteria in nutrientbroth. After inoculated cotton swatches were incubatedat 37 �C for 18 h, 100 mL of sterile normal saline waspoured into jars and was vigorously shaken for oneminute. Then, 1 mL of eluted bacteria liquids wereplated on nutrient agar after being suitably dilutedand incubated at 37 �C for 48 h. Afterward, theviable colonies of bacteria on the agar plate werecounted and the percent reduction of bacteria was cal-culated by following formula:

Reductionrate ð%Þ ¼ 100� ðB� AÞ=B

where A is the number of bacteria recovered from theinoculated test specimen swatches in the jar incubatedover 18 h and B is the number of bacteria recoveredfrom the inoculated test specimen swatches in the jarimmediately after inoculation (at ‘0’ contact time).

Extraction and purification of pigments

5 g of brown cotton fiber were put into 450 mL ofdistilled water in a beaker and sonicated at 60 �C,40 kHz and 70 W for 2 h using an ultrasonic cleaner(JK-2200DB model, Hefei Kinic MachineryManufacture Co., Ltd, China). The liquid was wrungout of the fiber and was filtered through filter paper(Whatman No.1) using a Buchner funnel. Filtrateswere vacuum evaporated at 50 �C with an R-201Drotary evaporator (Zhengzhou Yaron Instrument Co.,Ltd, China) until the solution became turbid.Subsequently, one-quarter volume of methanol wasadded to precipitate the pigment. The solid precipitateand the upper clear solution were well-separated aftercentrifuged at 8000 rpm by an Anke TDLSO-2B cen-trifuge (ShangHai Anting Scientific InstrumentFactory, China). The solid pigment was re-dissolvedin water and re-precipitated twice. The obtained solidpigment was then lyophilized in a CoolSafe 110-4PROfreeze-dryer (Labogene, Denmark) to obtain the pig-ment powder, for the following investigation.

The pigment powder from the green cotton was pre-pared by the same process as that used for brown

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cotton, except that the pigment of the green cotton wasextracted with chloroform and was crystallized byadding methanol. Since the extracts of white cotton,when using either water or chloroform as the solvents,had nearly nothing left after being evaporated to dry-ness, the small amount (10 mL) of concentrated solu-tion evaporated from the extracts served as the control.

Bacteria growth inhibition by pigments

Lyophilized powders of the brown cotton pigment andgreen cotton pigment were dissolved in distilled waterand methanol, respectively, to prepare pigment solu-tions at the concentration of 5 mg/mL. The concen-trated white cotton extracts served as the control. Theantibacterial activity of pigment solutions was carriedout according to disk diffusion method11,12 with twotest bacteria, S. aureus and K. pneumoniae. Briefly, ster-ilized filter papers (Whatman No.1, cut with a diameterof 6 mm) were soaked in prepared pigment solutionsand then dried. The paper disks obtained were thenplaced on the agar plates, on which the bacterial sus-pension in sterilized normal saline was uniformlyspread. After 5 min standing, the plates were turnedupside down and incubated at 37 �C for 18 h. Finally,the diameter of the clear zone of inhibition shown onthe plates was measured in millimeters, and the datawas the average of five measurements.

The color of NCCs is easily changed by heat treat-ment indicating the probable structure change of pig-ments by heat.10,13 So it is essential to investigate theinfluence of the heat treatment on the antibacterialactivity of the pigment. The bacterial growth inhibitionof the brown cotton pigment after being treated withhigh temperature was conducted as above. For thehigh-temperature treatment process, the pigment waspretreated at 100 �C for 3 min and 180 �C for 2 min,typically the drying and curing conditions in NCCsprocessing, respectively.14 In this test, samples were pre-pared in quintuplicate, and mean values of the diameterof the inhibition zone were calculated.

Qualitative analysis of pigments

Various methods including chemical analysis and spec-troscopic analyses were tried to analyze the chemicalnature of the pigments extracted from the NCCs, andthe effective methods were reported. For the greencotton pigment, drops of the shift reagents including1% (w/v) methanol solution of sodium acetate,sodium hydroxide, aluminum chloride and aluminumchloride-hydrochloric acid were added into the metha-nol solution of green cotton pigment, respectively, andthe UV-Vis spectra (by a Perkin-Elmer Lambda 900UV-visible spectrophotometer) of these solutions were

measured. Similarly, 1 mL of reagents including 1%(w/v) aqueous solution of sodium borohydride, magne-sium acetate, boric acid, ferric chloride, lead acetate,gelatin (pH 4) as well as HCl/magnesium, formalde-hyde-HCl and anisaldehyde-H2SO4 solution wereadded into about 4 mL of the aqueous solution ofbrown cotton pigment and the methanol solution ofgreen cotton pigment, respectively. After 5 min atroom temperature, the reaction appearance andchanges in the mixed solution such as solid precipita-tion or color change was observed.

A ThermoElectron Nicolet 5700 FT-IR spectropho-tometer was used to collect the transmission spectra.The infrared spectra of the pigment powders were col-lected using KBr pellets in a frequency range between4000 and 450 cm�1. The resolution was 4 cm�1 andthere were 100 scans for each spectrum.

Results and discussion

Antibacterial properties of NCCs

After 18 h incubation, the bacterial population densitiesof three kinds of cotton fibers inoculated with K. pneu-moniae and S. aureus are shown in Figure 1. Comparedto the control (white cotton), the population density ofthe two challenge bacteria on green cotton fiber didshow a slight but insignificant decrease, and the reduc-tion rates were both less than 20%. As for the browncotton, the population density of the two challenge bac-teria decreased significantly, indicating great antibac-terial activity, with the percentage of reduction up to96.4% against K. pneumoniae and 89.1% against S.aureus. The significantly different responses for thetwo challenge bacteria to the three cotton varietiesmay be due to the differences of the chemical compos-ition of fibers such as pectin, wax, sugars, metal ionsand, especially, the pigments in the fiber.5 However, itwas unlikely, and also unnecessary, to investigate all ofthe components influencing the antibacterial activity ofcottons since most of these substances, except the pig-ments, would be minimized or removed during thescouring process before utilization. Pigments, the spe-cial component of NCC fibers, are different in thebrown and green cotton, and may contribute to thedifferent antibacterial performances due to their pos-sibly different active constituents. It is necessary toextract pigment compounds from fibers for the investi-gation of their antibacterial activity.

Antibacterial activity of the pigments

Extraction efforts have attempted to obtain the pig-ments from fibers by using various solvents with differ-ent polarities, including petroleum ether, n-hexane,

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benzene, ether, ethyl acetate, chloroform, methanol anddistilled water. Shown in Figure 2(d), are the appear-ances of the dissolved cotton pigments in different solv-ents after putting 0.2 g of brown and green cotton fiber,respectively, into 8 mL of various solvents in a test tubefor 10 days at room temperature. It was evident that thepigments could be most easily dissolved out in distilledwater and chloroform, for brown and green cotton,respectively. The method used for extraction and puri-fication of pigments, in this paper, was the optimizedmethod. As shown in Figure 2(c), the hue of the pig-ment powder extracted was in agreement with that ofthe corresponding fiber, and the color of the extractedbrown and green cotton were much lighter than theircorresponding original fibers, indicating the successfulextraction of pigment from the NCCs. The investiga-tion suggested that the pigment compounds in the twocolored cottons may be quite different.

The antibacterial activity of the obtained pigmentswas tested against K. pneumoniae and S. aureus with theconcentrated extracts from white cotton serving as acontrol. The antibacterial activity was achieved by mea-suring the diameter of the zone of inhibition. Figure 3shows the inhibition by pigments extracted from greencotton and brown cotton, as well as the extract fromwhite cotton, toward two challenge bacteria. It was evi-dent that around the paper soaked in brown cottonpigment aqueous solution a significant inhibition zoneagainst K. pneumoniae and S. aureus was exhibited,with the mean diameter of the inhibition zone of14.4 mm and 12.8 mm, respectively, however therewas no evident inhibition for the extracts from greencotton and white cotton against the challenge bacteria.

It proved that the extracts obtained from white cottonand green cotton possessed no antibacterial effect.

Influences of high-temperature treatment onantibacterial activity of pigments

The antibacterial properties of the brown pigment com-pound after being treated at 100 �C for 3 min and180 �C for 2 min were also investigated. The resultsare shown in Figure 4. The inhibition zones of thepapers soaked with heat-treated concentrated whitecotton extracts (the control) were still invisible, indicat-ing no antibacterial performance. The antibacterialactivity of the pigment after high-temperature treat-ment had all suffered a certain level of decrease, andthe mean diameter of the inhibition zones were13.8 mm and 11.0 mm for brown cotton pigment trea-ted at 100 �C and at 180 �C against K. pneumoniae, aswell as 12.2 mm and 9.8 mm against S. aureus, respect-ively. The antibacterial activity of the pigment had aslight decrease after being treated at 100 �C, while theantibacterial effect decreased much more for that trea-ted at 180 �C. The results indicated that the presentdrying and curing process such as in durable press fin-ishing would result in an inevitable decrease in antibac-terial activity of brown cotton fabrics, but the retentionof the antibacterial activity was not as bad.

Chemical composition of pigment ingreen cotton fiber

Currently, little is known on the exact structure of pig-ments whether from green cotton or from brown cotton

K. pneu

moniae

S.

aureus

White cotton Green cotton Brown cotton

Figure 1. Antibacterial results of white cotton, green cotton and brown cotton against K. pneumoniae and S. aureus.

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b c da

Figure 2. Pictures of the original and pigment extracted cottons, and extracted pigment powder, as well as the appearance of the

pigment in cottons dissolved out in different solvents. (a) Original fibers; (b) extracted fibers; (c) extracted pigments; (d) dissolved out

appearance of the pigments in cottons in the following solvents: distilled water, methanol, ethyl acetate, chloroform, ether, benzene,

n-hexane and petroleum ether (from left to right), respectively.

c b

a

a

c b

a

a

b c

b c

Figure 3. Bacteria growth inhibition against K. pneumoniae (left) and S. aureus (right) by the extracts of fibers (a) white cotton,

(b) green cotton and (c) brown cotton.

(a)

control

100 °C

180 °C

(b)

control

100 °C 180 °C

Figure 4. Antibacterial activity of pigments treated with high temperature against (a) K. pneumoniae and (b) S. aureus.

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even though much effort has been employed in thisarea. The pigment in NCCs is far from an easymatter to be isolated and identified. There is a reportthat the methanol extracts from green cotton andbrown cotton both consist of flavones or flavonols.15

As the previous extraction assay indicated that the pig-ments in brown cotton were hydrophilic compoundsand that the extracting efficiency by methanol waslow, it was unreasonable to identify the brown cottonpigment using the alcohol extracts. In this work, thepigments used for identification were all extractedusing the solvent in which they were most soluble andpurified by crystallization.

The green cotton pigments were identified from theultraviolet spectrum. Figure 5 shows the ultravioletspectrum of green cotton pigment dissolved in metha-nol with or without the addition of shift reagents. Thesolutions revealed two UV absorption bands at 235 to280 nm (band II) and 300 to 400 nm (band I), whichwas very similar to the characteristic ultraviolet spectraof flavonoids.16,17 Various shift reagents with exclusivespecificity toward flavonoids were added to the analysisof the position of phenolic hydroxyl groups. Whenexcess sodium acetate solution was added to the pig-ment solution, the maximum absorption of band II wasshifted from 240 nm to 260 nm in its ultraviolet spec-trum, which indicated the presence of a 7-hydroxyl ofthe flavonoid. The peak of band I shifted 50 nm towardthe longer wavelengths after an excess of sodium

hydroxide solution was added, indicating the 4-hydro-xyl existence. The spectrum of the pigment solutionafter adding with aluminum chloride-hydrochloricacid was almost the same as that of the untreated.Compared to the spectrum of pigment solution afteradding only aluminum chloride, however, its band Ihad shifted 23 nm toward shorter wavelengths, indicat-ing that the 3-hydroxyl and 5-hydroxyl do not exist orare substituted by O-glucosides or methoxyls, respect-ively, and also indicating pyrocatechol on the B-ring. Inconclusion, the pigment of green cotton could be a classof flavones or 3-O-glucoside flavonoids.

The FT-IR spectrum of the pigment from greencotton is presented in Figure 6(a). The spectrum ofthe pigment from green cotton revealed a stretchingband of an intermolecular bonded hydroxyl group at3388 cm-1 with very strong methylene and methylstretching frequencies appearing at 2918 cm-1 and2850 cm-1, respectively. Close to a strong carbonylstretching band at 1709 cm-1, two medium intensitychromattic bands of skeletal stretching vibration ofaromatic ring were observed at 1608 cm-1 and1383 cm-1. The band at 1467 cm-1 can be attributedto the C–H deformation of methylene and methyl. Abroad, strong band at 1223 cm-1 was due to the stretch-ing vibration of carbon single bonded oxygen that con-nected the aromatic rings and substituents, such asmethoxyl, glycosyl and other groups. The strong bandappearing at 1165 cm-1 may be the other stretching

250 300 350 400 450 500 550

Wavelength/nm

Abs

orba

nce

untreated

added NaAc

added NaOH

added AlCl3

added AlCl3 / HCl

Figure 5. Effect of shift reagents on UV spectrum of green cotton pigment dissolved in methanol.

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band of carbonyl which usually absorbs in the 1210–1163 cm-1 region. The medium shoulder appearing at1108 cm-1 may arise from the aromatic ether connectingthe A ring and B ring. The absorption at 804 cm-1 and720 cm-1 were assigned to the out of plane bendingvibration of the saturated C–H bond and aromaticC–H bond, respectively. The group functionalities andmolecular structure of the pigment from green cotton,which revealed in the spectrum, were basically in con-formity with the flavonoids.

Chemical composition of pigment inbrown cotton fiber

It is unacceptable to consider the pigment of browncotton as flavonoids. The brown cotton pigmentextracted by water revealed two UV-Vis absorptionpeaks (Figure 7), which were at 202 nm and 275 nm,and without an obvious peak in the 300–400 nm range,the standardized UV spectroscopy of flavonoids. Fromthe observation of the reaction with diagnosticreagents, the chemical properties of brown cotton pig-ment were analyzed, and the results (Table 1) did not fitthat of the flavonoids. Added with the aqueous solutionof ferric chloride, the pigment solution presented anobviously blackish-green color, and added to lead acet-ate solution a flesh-colored precipitate was observed.This evidence indicated that the pigment of browncotton could be preliminarily characterized as phenols.When HCl/magnesium, sodium borohydride and boricacid were added to the pigment solution, respectively,there were no obvious reactions. These negative

reactions, as well as the data presented in the UV spec-trum suggested the absence of flavonoids, such as fla-vones, flavonols, dihydroflavones, dihydroflavonolsand chalcones.18,19 The magnesium acetate was addedto the pigment solution and no change was observed,indicating the lack of existence of the ingredients with atypical anthraquinone structure. A drop of pigmentsolution was added on the silica gel plate and sprayedwith anisaldehyde-H2SO4 solution, and an orange spotwas observed. The aqueous solution of pigment became

804

720

1108

1223

11

65

1383

14

67

1608

1709

2850

2918

3388

a

1404

1618

3415

2933

b

cm–1

%T

500 1000 1500 2000 3000 4000

1116

Figure 6. FT-IR spectra of pigments from (a) green cotton and (b) brown cotton.

200 250 300 350 400 450 500 550

Wavelength/nm

Abs

orba

nce

Figure 7. UV-Vis spectrum of brown cotton pigment dissolved

in distilled water.

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turbid when gelatin solution (pH 4) and formaldehyde-HCl were added, respectively, and a flesh-colored pre-cipitate was presented after 10 min, then the mixture ofsolution was filtered, and the filtrate exhibited a faintbrown color. These were the characteristic chemicalreactions of condensed tannins, so the main componentof pigment from brown cotton most probably belongsto condensed tannins. There was no visible reactionwhen the methanol solution of green cotton pigmentwas added to formaldehyde-HCl, and the pigmentspot on silica gel plate turned red instead of orangewhen sprayed with anisaldehyde-H2SO4 solution, indi-cating no tannins existed in green cotton pigmentextracts.

As shown in Figure 6(b), the FT-IR spectrum of thepigment from brown cotton revealed a strong, intermo-lecular bonded phenolic hydroxyl group stretching at3415 cm-1, and coupled with weak bands of stretchingmethylene groups at 2933 cm-1, which may be attribut-able to the methylene in the 4-position of the endflavan-3-ol monomer unit. The main vibrationalbands that were observed in the middle of the spectrumincluded ring skeletal bands at 1618 cm-1 and 1404 cm-1

and finally an aryl alkyl ether symmetrical stretching at1116 cm-1. The spectrum of the pigment from browncotton was much similar to the spectra of condensedtannins.20

Condensed tannins, obtained from plants, havedesirable biological activities such as antioxidization,antinflammation, antimicrobial, and so on.21,22 It isgenerally accepted that the mechanism of antimicrobialactivity of these plant polyphenols is related to theirability to complex irreversibly with amino acids in

proteins through hydrogen bonding as well as covalentbonding, thus inactivating the enzymes, surface adhe-sions and cell envelop transport proteins ofmicrobes.23,24 It is reasonable that the great antibacter-ial activity of brown cotton could be attributed to itsinherent pigment, the condensed tannin-based sub-stance. However, the tannin-based pigment couldbecome deeper after being treated, probably indicatinga change in pigment structure and it may be the reasonwhy the decrease of antibacterial activity of browncotton pigment, with the heat treatment, occurred.

Conclusions

The degree of antibacterial activity of NCCs and pig-ments were evaluated. The brown cotton, as well as thepigment extract, was found to possess excellent antibac-terial activity. However, the antibacterial activity ofgreen cotton and its pigment were both insignificant.The results suggested that the antibacterial activity ofNCC fibers was relevant to their pigment compositions.The antibacterial activity of the brown cotton pigmentsuffered some decrease after heat treatment, especiallyafter being treated at 180 �C, but still had a good reten-tion. The identification results showed that the mainchemical compositions of pigments in green cottonand brown cotton belong to flavonoids and condensedtannins, respectively.

It should be noted that the identification work isbased on the crude pigments which need to be furtherisolated and identified in the future. Furthermore, ourstudy only focused on the antibacterial property ofbrown cotton pigment influenced by the high

Table 1. Chemical reactions of pigment solution of brown and green NCC with diagnostic reagents

Reaction reagents Assay type Target substance18,19Results

BCP a GCP b

Ferric chloride Color development General test for phenols Positive Positive

Lead acetate Precipitation General test for phenols with adjacent

hydroxyl groups

Positive —c

HCl/magnesium Color development Specific test for flavonols Negative Negative

Sodium borohydride Color development Specific for dihydroflavones and dihydroflavonols Negative —c

Boric acid Color development Specific for chalcones Negative Negative

Magnesium acetate Color development Specific for hydroxylated

anthraquinone, dihydroflavones,

dihydroflavonols and isoflavones

Negative Negative

Gelatin (pH 4) Precipitation Specific for condensed tannins Positive —c

Formaldehyde-HCl Precipitation Specific for condensed tannins Positive Negative

Anisaldehyde-H2SO4 Color development Specific for condensed tannins, sterols

and terpenoids

Positive d Positive e

aBrown cotton pigment; bGreen cotton pigment; cReagents precipitate out of solvent; dShowing an orange color, the condensed tannins’ reaction;eShowing a red color.

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temperature generally used in textile finishing proced-ure, the effects of the long-term home laundering, solarradiation and other conditions on its antibacterialactivity are also worth investigating.

Funding

This work was supported by the Zhejiang Provincial NaturalScience Foundation of China (Grant No.Y4090558) and

Zhejiang Provincial Top Key Academic Discipline ofApplied Chemistry and Eco-Dyeing & FinishingEngineering (Grant No.ZYG201112).

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