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Antimicrobial effect of natural dyes on some pathogenic bacteria

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Abstract

In this study we have detected the antimicrobial activity of four natural dyes (obtained from Rubia tinctorum, Allium cepa, Punica granatum L and Mentha sp.) on Staphylococcus aureus ATCC 25923, Shigella sonnei RSKK 877, Escherichia coli ATCC 35218, Bacillus megaterium RSKK 5117, Bacillus subtilis RSKK 244, Bacillus cereus RSKK 863, Pseudomonas aeruginosa ATCC 29212, Streptococcus epidermidis, Salmonella 21.3 and P. aeruginosa ATCC 27853. P. granatum dye was most effective against the test bacteria except E. coli and S. epidermidis. The textile material impregnated with four natural dyes and maximum inhibition rates (respectivelly, 80, 86, 52%) were obtained against B. subtilis of wool samples dyed with P. granatum, A. cepa and R. tinctorum while maximum inhibition rates (91%) was found against P. aeruginosa of wool sample dyed with R. tinctorum.
African Journal of Biotechnology Vol. 8 (2), pp. 291-293, 19 January, 2009
Available online at http://www.academicjournals.org/AJB
ISSN 1684–5315 © 2009 Academic Journals
Short Communication
Antimicrobial effect of natural dyes on some
pathogenic bacteria
Ayfer Çalı
1
, Gökçen Yuvalı Çelik
1
* and Hikmet Katırcıoglu
2
*
1
Department of Biology, Faculty of Science and Arts, University of Nigde, Campus, 51200, Nigde, Turkey.
2
Gazi University, Faculty of Education, Department of Biology Education, 06500, Teknikokullar-Ankara, Turkey.
Accepted 1 August, 2008
In this study we have detected the antimicrobial activity of four natural dyes (obtained from Rubia
tinctorum, Allium cepa, Punica granatum L and Mentha sp.) on Staphylococcus aureus ATCC 25923,
Shigella sonnei RSKK 877, Escherichia coli ATCC 35218, Bacillus megaterium RSKK 5117, Bacillus
subtilis RSKK 244, Bacillus cereus RSKK 863, Pseudomonas aeruginosa ATCC 29212, Streptococcus
epidermidis, Salmonella 21.3 and P. aeruginosa ATCC 27853. P. granatum dye was most effective
against the test bacteria except E. coli and S. epidermidis. The textile material impregnated with four
natural dyes and maximum inhibition rates (respectivelly, 80, 86, 52%) were obtained against B. subtilis
of wool samples dyed with P. granatum, A. cepa and R. tinctorum while maximum inhibition rates (91%)
was found against P. aeruginosa of wool sample dyed with R. tinctorum.
Key words: Antimicrobial activity, natural dyes, textile.
INTRODUCTION
In recent decades, there has been an increasing
tendency towards the prevention of microbial attack on
textiles (Han and Yang, 2005). Natural fibres have keratin
and cellulose, etc., that provide important requirements
such as oxygen, moisture, nutrients and temperature for
the bacterial growth (Singh et al., 2005). A variety of
antimicrobial textile agents are reported such as organo-
metalics, phenols, quaternary ammonium salts and
organo-silicones (Yang et al., 2000). Synthetic com-
pounds are more complex and it will take a long time for
them to complete their natural cycles and return to
nature; thus causing a lot of environmental pollution. Due
to the fact that natural dyes can often inhibit the growth of
microorganisms traditionally, different plants have been
used as natural dyes in textile and carpet industries and it
is believed that these dyes are less allergic and more
stable than the chemical ones (Mehrabian et al., 2000).
Turkey has a rich flora because of its geographical
position and climate. It is well known that plant based
dyes were widely used by the Turks in both centre Asia
and Anatolia throught history. Rubia tinctorium L. is
*Corresponding author. E-mail: hturk@gazi.edu.tr.
known for dyestufs which orginate from root material of
field grown madder including anthraquinone derivates,
the most important one is alizarin (Merck, 1996). It is
known that anthraquinone derivates have been used as
antiinflammatory, antimicrobic, antibacterial and anti-
diuretic drugs (Swain, 1996). Punica granatum dye and
many other common natural dyes are reported as potent
antimicrobial agents owing to the presence of a large
amount of tannins (Machado et al., 2002). Mentha
piperita L. contains -terpinene, isomenthone, menthol,
trans-carveol,pipertitinone oxide, -caryophyllene, carv-
one and limonene (Yadegarinia et al 2006). Flavanoids
are a second class of health enhancing compound pro-
duced by Allium cepa L., with quercetin as an example.
Flavanoids are active against microorganisms (Ekwenye
et al., 2005)
At present, many of the plants used for dye extraction
are classified as medicinal, and some of these have
been shown to posses significantly antimicrobial effect.
The antimicrobial activity of some of these dyes are
reported as potent owing to the existence of phenol,
tannin and quinone in their extracts. The antimicrobial
effects of some plants used in dye industries contribute to
the longer life of the products they are used in (Hussein
et al., 1997, Mehrabian et al., 2000).
292 Afr. J. Biotechnol.
The purpose of this research is to study the
antimicrobial effect of four commercially available dye
powders, obtained from R. tinctorum L., A. cepa L., P.
granatum L., M. piperita L. against some common
microbes. We also focused on the antimicrobial activity of
wool fabric treated with these naturel dyes.
EXPERIMENTAL
Naturel dyes
The optimized commercial naturel dye powders of R. tinctorum L.,
A. cepa L., P. granatum L. and M. piperita L. were obtained from
Mugla and Nigde, Turkey.
Naturel dyes application
To prepare aqueous dye solutions of these plants, 10 g of each of
the powders were added to 300 ml of distilled water and boiled for
60 min 100
o
C. The hot solution was filtered and any plant material
left in the filter paper were discarded.
Wool was dyed by the standard method prescribed for natural
dyes. The dyeing was carried out at 1:30 MLR (material to liquor
ratio), for 30 min at 80
o
C at neutral pH. Dyed samples were further
treated with the aluminum potassium sulfate (KA
l
(SO4)
12 H
2
O) as
mordant (0.5 gl
-1
) at 60
o
C for 20 min, and rinsed in hot and then
cold water.
Test bacteria
Cultures of following microorganisms were used in the study:
Bacillus megaterium RSKK 5117, B. megaterium RSKK 5117, B.
subtilis RSKK 244, P. aeruginosa ATCC 29212, S. epidermidis, B.
cereus RSKK 863, P. aeruginosa ATCC 27853, S. sonnei RSKK
877, Salmonella sp. 21.3, S. aureus ATCC 25923, E. coli ATCC
35218.
Antimicrobial screening test
Susceptibility of the bacterial strains to the natural dyes was
investigated using the disc diffusion method (NCCLS, 1997). The
culture suspensions were prepared and adjusted against 1
McFarland turbidity standard tubes. Muller Hinton Agar medium (15
mL) was poured into each sterile petri dish. Then the surface of the
agar medium dish was inoculated with 100 µl cultures. Dyes were
sterilized by filtration through 0.45 µm membrane filters. Empty
sterilized discs of 6 mm were each impregnated with 100 µl natural
dyes. Discs were placed on agar plates, and the plates were incu-
bated at 37
o
C for 24 h. The inhibition zones formed on the medium
were evaluated in mm. All experiments were performed in duplicate.
In the next set of experiments the antimicrobial activity of dyed
wool specimens was tested. The 1 g fabric (dyed and undyed) was
introduced into the 100 ml nutrient broth inoculated with the desired
microbe and incubated at 37
o
C overnight for 16 h. The reduction of
bacterial growth by dye was expressed as follows:
R = 100(A-B)/A
Where R = % reduction in bacterial population; A = absorbance
(660 nm) of the media inoculated with bacteria and undyed fabric; B
= absorbance (660 nm) of the media inoculated with bacteria and
dyed fabric (Singh et al., 2005).
RESULTS AND DISCUSSION
Antimicrobial activity of natural dyes in solution
Four natural dyes were screened for their antimicrobial
activity against ten test bacteria and the first screening
showed that P. granatum was effective against most of
the test bacteria except E. coli and S. epidermidis.
Mentha sp. was effective against six test bacteria (B.
megaterium, B. subtilis, P. aeruginosa, S. epidermidis, B.
cereus). R. tinctorum has shown inhibition effect against
B. megaterium, S. aureus, and B. subtilis. A. cepa exhi-
bited activity against only two Bacillus strain (Table 1).
Singh et al. (2005) investigated antimicrobial activity of
four natural dyes against some microorganisms and they
found that Quercus infectoria was effective against all the
test microorganisms (Singh et al., 2005). Other research-
es studied antimicrobial properties of eleven natural dyes
against three types of gram-negative bacteria and they
reported that seven of the dyes showed activity against
one or more of the bacteria (Gupta et al., 2004).
Table 1 has also indicated that natural dyes had signi-
ficant antibacterial activities towards the gram-positive
bacteria and had less inhibition effect on the gram-
negative test bacteria (S. sonnei, E. coli, P. aeruginosa
and Salmonella sp.). The cell wall structure of the gram-
negative bacteria is constructed essentially with LPS that
avoids the accumulation of the antimicrobial agents on
the cell membrane (Rabe and Staden, 1997; Ali-Shtayeh
et al., 1998; Bezi, et al., 2003). The above reports have
confirmed our results.
Antimicrobial activity of natural dyes on substrate
It was important to study the antimicrobial activity on
dyed textile substrate (wool fabric) because the natural
dyes showed inhibition effect against test bacteria in
solution. The results are shown in Table 2. A reduction of
4-80% in bacterial growth is seen on a wool sample dyed
with P. granatum and a reduction of 53-86% on wool
samples dyed with A. cepa. A reduction of 32-52% in
bacterial growth is seen on a wool sample dyed with R.
tinctorum and a reduction of 28-91% in bacterial growth is
seen on a wool sample dyed with Mentha sp. Also
maximum inhibition rates (respectivelly, 80, 86, 52%)
were obtained against B. subtilis of wool samples dyed
with P. granatum, A. cepa, R. tinctorum while maximum
inhibition rates (91%) was found against P. aeruginosa of
wool sample dyed with R. tinctorum.
Han and Yang (2005) also observed an inhibition rate
of 70% against S. aureus when 0.01% of curcumin was
applied to the fabric and also 70% inhibition rate against
E. coli with 0.05% of curcumin. However, inhibition rate of
more than 95% was obtained against both S. aureus and
E. coli when 0.2% of curcumin was applied to the fabric
(Han and Yang, 2005). The results from these experi-
ments indicated that these natural dyes had antimicrobial
Çalı et al. 293
Table 1. The antimicrobial activity of natural dyes against test bacteria (inhibition zone, mm).
Table 2. Antimicrobial activity (% reduction) of textile materials dyed with natural dyes.
activity both on solutions and substrate. However, further
research is needed to determine the effect of dye
structure on inhibition.
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Dye
Test bacteria
P. granatum R. tinctorum M. piperita A. cepa
B. cereus RSKK 863
17.9 ± 0.0 - 10.8 ± 0.6 10.0 ± 0.0
B. megaterium RSKK 5117
12.4 ± 0.1 8.5±0 11.8 ± 0.2 13.5 ± 0.0
Shigella sonnei RSKK 877
13.8 ± 0.1 - - -
S. aureus ATCC 25923
13.7 ± 0.3 9.7±0 - -
B. subtilis RSKK 244
17.6 ± 1.7 10.2±0 13.5 ± 1.6 9.9 ± 0.0
P. aeruginosa ATCC 29212
15.8 ± 2.0 - 12.2 ± 0.6 -
Salmonella sp. 21.3
10.5 ± 1.5 - - -
P. aeruginosa ATCC 27853
11.6 ± 0.5 - 10.2 ± 0.9 -
S. epidermidis
- - 10.7 ± 0.6 -
Dye
Test bacteria
P. granatum A. cepa R. tinctorum
M. piperita
B. cereus RSKK 863
61 53 75
B. megaterium RSKK 5117
35
Shigella sonnei RSKK 877
24
S. aureus ATCC 25923
4 32
B. subtilis RSKK 244
80 86 52 88
P. aeruginosa ATCC 29212
79 91
Salmonella sp. 21.3
P. aeruginosa ATCC 27853
12 46
S. epidermidis 28
E. coli ATCC 35218
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This work investigated improving the durability of antibacterial textiles. Four common methods to produce antibacterial textiles at different conditions were examined. Using some of the processes developed in this work, the antibacterial response of the treated fabrics was maintained after 50 home launderings.
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Leaves of Punica granatum contain the new gallotannins, 1,2,4-tri-O-galloyl-beta-glucopyranose and 1,3,4-tri-O-galloyl-beta-glucopyranose together with the hitherto unknown ellagitannins, 1,4-di-O-galloyl-3,6-(R)-hexahydroxydiphenyl-beta-glucopyranose and brevifolin carboxylic acid 10-monopotassium sulphate. Structures were established by conventional methods of analysis and confirmed by H-1, C-13 NMR, 2D-chemical shift correlation NMR and ESI-MS (negative mode) spectrometric analysis. (C) 1997 Elsevier Science Ltd.
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There is increasing interest in adding value to textiles by the use of natural products. Many of the plants from which natural dyes are obtained are, for example, also known to have medicinal properties. In the current study, the antimicrobial properties of eleven natural dyes against three types of Gram-negative bacteria were studied experimentally. Seven of the dyes showed activity against one or more of the bacteria. The minimum inhibitory concentration for three selected dyes was determined. The results demonstrate that certain dyes are able to reduce microbial growth almost completely in the case of Escherichia coli and Proteus vulgaris. Selected dyes would therefore be valuable for the dyeing of sheets and gowns for hospital use, and on articles which are less suitable for laundering such as mattresses and upholstery. The dyes examined exhibited good wash fastness and the antibacterial effect is therefore likely to be durable.
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In our study we have detected the antimicrobial effects of the aqueous, methanolic and chloroformic extracts of Rubia tinctorum, Carthamus tinctorius and Juglans regia on some airborne microorganisms; using the usual methods which are used routinely for this purpose in microbiology. Previous investigations have shown that extracts of these plants have antimicrobial effects on some bacteria such as Bacillus subtilis, Bacillus cereus, Bacillus mycoides, and also on some fungi specially Aspergillus niger, Penicillium expansum and Geotrichum candidum.The antimicrobial effects we have detected are ''microbicidal''. The aqueous and the chloroformic extracts had the most and the least microbicidal effects, respectively. The aqueous extracts of Carthamus tintorious and Juglans regia had the most and the least microbicidal effects respectively.
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Curcumin, a common natural dye used for fabric and food colorations, was used as an antimicrobial finish due to its bactericidal properties on dyed textiles. A common dyeing process, either batch or continuous, could provide textiles with colour as well as antimicrobial properties. The relationship between the sorption of an interesting natural colorant onto wool and the antimicrobial ability of the dyed wool were investigated. Relations between the bacterial inhibition rate and curcumin concentration, and inhibition rate and K/S value were developed. Antimicrobial activity of wool fabric finished with curcumin can be predicted without antimicrobial testing based on the developed relationships. Durability of antimicrobial activity to laundering and to light is also discussed.