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INTERNATIONAL JOURNAL OF SCIENCE FOR GLOBAL SUSTAINABILITY

(A publication of Faculty of Science, Federal University Gusau, Nigeria)

Justicia Carnea Dyes: Extraction, Characterization, Application and

Evaluation of Antimicrobial Activity on Cotton Fabric
Gboyega Oluwaseun Oyeleke1*, Rasheed Tunde Lawal2, Fatai Afolabi3, Ismaila Jide Olawale4, Ibraheem
Abimbade Abdulazeez4 and Ajisola Agnes Adebisi4
1
Department of Science Laboratory Technology, Osun State Polytechnic, Iree, Nigeria
2
Department of Biological Sciences, University of Ilesa, Osun State, Nigeria
3
Department of Physical and Chemical Sciences, Federal University of Health Sciences, Ila Orangun, Osun State, Nigeria
4
Department of Applied Sciences, Osun State Polytechnic, Iree, Nigeria

*
Corresponding author’s Email: [email protected]
Received on: January, 2024 Revised and Accepted on: March 2024 Published on: March 2024
ABSTRACT
This study aims at extracting natural dyes from fresh and dried leaves of Justicia carnea. Application of the aqueous
extracts of these leaves on fabrics with mordants (cobalt chloride and lime) and without mordants at a dyeing
temperature of 800 C was carried out using pre-mordanting, simultaneous- mordanting and post- mordanting
processes. The extracts show a good antimicrobial activity to bacteria and fungi. The improvement in fastness
properties of the dyed cotton fabrics to rubbing and perspiration showed that mordants as well as mordanting
methods have influence on colour efficiency. The FTIR and UV spectroscopy analysis of the dyes revealed some
important peaks and functional groups related to colour formation. The phytochemical screening of the extracts
revealed the presence of alkaloids, flavonoids, tannins, anthraquinones, steroids and phenols in the fresh and dried
samples. This research work reported a successful extraction of ecofriendly dyes from Justicia carnea that can meet
the current yearning for industrial development in the developing countries of the world where biosources materials
from nature abounds in large quantities.
Keywords: Antimicrobial, dyes, ecofriendly, extraction, fastness, mordants
1.0 INTRODUCTION more expensive due to seasonal limitation of source,
The production and utilization of synthetic dyes problems of shades and blending, non uniformity and
increased at the expense of natural dyes due to low cost non reproducibility of shades, poor colour fastness and
of production, high dye uptake and better fastness low affinity due to formation of weak bonds with the
properties. Most of the synthetic dyes are however made fibre (Bhandari et al., 2020; Jose et al., 2020). The
from non renewable petroleum based chemical by- quality and quantity of dyes from any natural sources
products and coal tar (Berhanu and Ratnapandian, depends on age, climate and processing methods of the
2017). Non-biodegradable wastes from excessive use of raw materials.
synthetic dyes are major pollutants to the environment
(Sohini and Shyamala, 2017) and a serious concern to Colour fastness is the property of the pigment or dye to
human existence because they are carcinogenic, led to retain its original hue especially without fading or
destruction of ecosystem and causes environmental bleeding under the influence of various conditions they
pollution due to increase in population and may likely be exposed to in textile manufacturing and
industrialization. With these shortcomings associated use (Thet et al., 2020). Most natural dyes have low
with synthetic dyes, many people worldwide are colour fast with fibres, hence mordants which have the
anxiously demanding for natural dyes because they can ability to enhance the chemical bonding of the dyes and
easily overcome major defects of synthetic dyes of fibres with a view of increasing dye - fibre fixation
harmfulness to human body, pollution and wastewater (Ahmed et. al., 2019) are required. Mordants can either
(Thet et al., 2020). be chemical (usually the metallic salts of aluminium,
chromium, iron, copper and tin) or natural products like
Natural dyes are coloured materials obtained from wood ash, lime, stale urine, cow dung etc (Bhandari et
natural sources without synthesizing and can be al., 2020). Aside applying natural dyes as colourants in
obtained from plants and animals which are textiles, they are also useful in colouration of food and
biodegradable, anti-allergic, non-toxic, deodorizing and cosmetic materials.
colours soothing to human eyes (Ahmed et al., 2019;
Thet et al., 2020) with little or no environmental impact. Microorganisms are considered to be present almost
Despite these advantages, natural dyes are generally everywhere and textile materials could easily provide a

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better and conducive environment for multiplication of reported to be used in the management of many health
pathogenic microbes to thrive. Presence of conditions. Investigation by the authors from local users
microorganisms on textile materials among other things of the leaves revealed that most anaemic patients,
can cause discoloration, develop stains, break yarns, pregnant and women that have just completed their
cause smell and loss of tear and strength (Ijaz, et al., menstrual cycle use the leaf extract obtained either by
2020). Since most plants are source of natural herbs, soaking or boiling in water to replenish their blood.
they have the ability to produce many natural Ajuru et al., (2022) and Orjiakor et al., (2019) reported
antibacterial due to the presence of certain bioactive that its application as blood booster has not been
compounds such as phenols, saponins, tannins, certified and without chemical tests.
terpenoids etc in them. Antibacterial materials were
reported by Mirjalili and Karim, (2013) to have the The aim of the research work was to evaluate the
ability to eliminate or reduce the spread of disease and performance of dyes extracted from Justicia carnea leaf
prevent deterioration of textiles. The use of natural in cotton fabric dyeing in order to explore the
colourants from plants as a means of producing possibilities of obtaining better colours using different
antimicrobial agents has been documented (Alam et al., mordants and mordanting processes and also to
2022). Several researchers (Sutrisna et al., 2020; determine the antimicrobial activity of the dyed cotton
Vaghela and Upasai, 2023; Oforghor et al., 2023) have fabrics against two common pathogenic bacteria
reported their findings on extraction, characterization (Bacilus spp and Streptococcus) and a fungi (Candida
and fastness properties of dyes extracted from different albican) with a view to get a cheap, safe, fascinating
bio sources. and easily available colourants for the expansion of
dyeing industries.
Nature has blessed Nigeria with many green plants from
where dyes could be extracted. Among these is the 2.0 MATERIALS AND METHODS
Justicia carnea plant. The genus Justicia, belongs to the 2.1 Sample collection
large family of Acanthaceae consisting of about 600 Justicia carnea was collected from Molasuru area in
species distributed in both hemisphere in the tropics and Ore, Odigbo Local Government area, Ondo State,
temperate regions and widely used traditionally as blood Nigeria. The plant leaves were identified in Biology
tonic for years past in Nigeria (Okocha et al., 2023). Laboratory of Osun State Polytechnic, Iree, Nigeria by a
Justicia carnea is a flowering plant widely distributed in herbarium staff as Justicia carnea with voucher number
various parts of Africa especially Nigeria. The shrubs of 0143
Justicia carnea are grown around homesteads as fence
(Onochie et al., 2020). The plant is called “Ogwu 2.2 Samples pretreatment
obara” and “Ewe eje” meaning blood leaf in Igbo and The fresh leaves were washed and divided into two
Yoruba speaking parts of Nigeria respectively. Some parts. One part was air dried at room temperature for 7
tribes in Nigeria have given it names like “Hospital too days and pulverized using a locally made laboratory
far”, “Blood of Jesus”, “Blood leaf” and Jehovah grinding machine. The powdered sample was stored in
Witness leaf” (Okocha et al., 2023). airtight containers until used while dye was extracted
from the other part immediately.
Traditionally, several species of Justicia have been

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Before drying After drying

Plate 1: Fresh and dried Justicia carnea leaf samples

2.0 MATERIALS AND METHODS

The cotton fabric was purchased in Alamisi market Mordanting and dyeing processes
Ikirun, Ifelodun Local Government Area of Osun State, Using an analytical weighing scale, 2 g of the pre-
Nigeria while the chemicals (lime and cobalt chloride) mordanting mixture - cobalt chloride and lime were put
used were of analytical grade. into a 250 ml beaker containing 50 ml of water along
with the cotton fabrics. The beaker was then placed in a
3.1 Methods water bath for 20 minutes. Following the mordanting
3.1.1 Extraction of dye from the samples process, the materials were taken out and 100 ml of
Aqueous extraction of dye from fresh and dried leaves each extracted dye sample was measured, poured into
of Justicia carnea was done by weighing 8 g each of the the beaker containing the mordanted fabrics, and the
samples in different 500 ml beaker. 400 ml of distilled beaker was submerged in water for 30 minutes. The
water was added and the beakers were heated in water fabric was dyed, rinsed with water and allowed to air
bath for 1 h at temperature of 80 0C. After extraction, dry. For the dye that was removed from the dried
the solutions were allowed to cool and thereafter filtered sample, the process was repeated. In post-mordanting,
separately using muslin cloth and stored for dyeing the materials were mordanted after dyeing, whereas in
process. simultaneous mordanting, the fabric, mordants, and dye
extracts were all combined in the beaker utilizing the
3.1.2 Phytochemical analysis same method as previously described.
The phytochemical screening of the extracted dyes was
determined using standard methods of Wanyama et al., Antibacterial and antifungal analysis
(2011) and Divya and Ravi, (2013). Bacterial (Bacillus and Streptococcus) and fungi
(Candida albican) species used for the tests were
3.1.3 UV-Vis spectroscopy analysis obtained from department of Applied Science, Osun
I ml of the total aqueous dye extract was diluted in 100 State Polytechnic, Iree, Nigeria. Agar disc diffusion
ml of distilled water. An aliquot was introduced in a 1 method was used where 0.1 ml of 1 in 100 dilutions of
cm path way quartz cell and analyzed in Philip Harris the overnight broth culture of each bacterium and fungi
spectrophotometer. A scan from 300 to 600 nm was cells was added to sterile molten nutrient agar for
performed in order to generate the characteristics bacteria and potatoes dextrose agar for fungi cell was
absorption spectra of the sample. maintained at 37o C. The plates were allowed to
solidify, then 40 g/ml and 80 g/ml of aqueous extract of
Fourier transform infrared spectral analysis the Justicia carnea were added into appropriate wells (7
5 mg of the dye extracts were mixed thoroughly mm diameter) made in sealed plate which were
with195 mg of KBr until homogenized in an agate incubated at 37o C for 24 hr.
mortar. The mixture was placed into the sample
compartment of the Fourier Transform Infrared Colour fastness
Spectrophotometer (PerkinElmer system 2000 FTIR Dyed fabrics were evaluated for different colour
spectrophotometer) where the transmission spectra of all fastness properties according to ISO standards such as
powder samples in the wave number range 400-4000 ISO 105-B02 for perspiration (alkaline and acidic) and
cm-1 were collected at a resolution of 4 cm-1 with 100 ISO 105 X 12 for rubbing (wet and dry) (Avinc et al.,
scans for each spectrum. 2013).

3.0 RESULTS AND DISCUSSION present. These bioactive components have been reported
The phytochemical screening (Table 1) revealed the in the leaf extract of Justicia carnea (Ajuru et al., 2022;
presence of flavonoids, steroids, phenols, Asiwe et al., 2023; Andrew et al., 2024) and these
anthraquinones, tannins and alkaloids. Phenols and contributes to various physiologic and protective effects
alkaloids were found to be significantly present in the attributed to the plant (Shomali et al., 2022). The
fresh sample while only anthraquinones provided presence of flavonoids and tannins among other
similar results with dried sample. Anthraquinones, phytochemicals are considered to be important
flavonoids and tannins were moderately present in fresh characters for textile dyes (Sohini and Shyamala, 2017)
samples relative to dried sample where they were fairly to enhance better fastness. Flavonoids are the largest

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group of plant phenols that are capable of providing the greenish nature of J. carnea leaves when boiled for
flavour and colour to fruits and vegetables. This may consumption (Orjiakor et al., 2019).
explain the strong flavour and deep red colour despite

Table 1: Results of Phytochemical Screening for Bioactive Components in the Dye Extracts
Parameter Fresh Dried
Anthraquinones ++ +++ Steroids
+ +
Alkaloids +++ ++ Flavonoids
++ +
Phenols +++ +
Tannins ++ +
Key:
+++ denotes significantly present
++ denotes moderately present
+ denotes present

Table 2: Antibacterial and Antifungal Activities of the Dye Extracts

Organisms Fresh Dried
Bacteria
Bacillus sp. 4 mm 10 mm
Streptococcus sp. 8 mm 6 mm
Fungi
Candida albican 8 mm Resistant

The antimicrobial sensitivity test (Table 2) of the antimicrobial agent as the tested organism related
aqueous extract of fresh and dried leaves of Justicia diseases are inhibited with extract from Justicia carnea
carnea showed (4 mm and 8 mm) and (10 mm and 6 leaves. Antimicrobial properties of dye extracts from
mm) zones of inhibition for Bacillus spp and bio sources have been reported (Imohiosen, 2023;
Streptococcus spp respectively. This indicates that Asiwe, et al., 2023) and the mechanism of this
bacillus and streptococcus spp are averagely resistant to behaviour among others could be attributed to
dye extract from fresh leaves but only resistant to hydrogen-bonding and hydrophobic reactions between
streptococcus spp using dye extract from dried leaves the polyphenols from bio source and protein of the
which means it is susceptible to bacillus spp. The bacterial cell wall causing the leakage of cell fluids and
sensitivity test against Candida albican (Fungi) showed ultimately killing the bacterial (Shafiq et al., 2021).
8 mm zone of inhibition against the extract from fresh Tannins which are naturally astringent in nature along
leaves while it was completely resistant against dye flavonoids and other bioactive compounds have been
extract from dried leaves. The difference in resistance of reported to have antibacterial property (Orjiakor et al.,
these microorganisms could be related to differences in 2019) which is an expected characteristic of dyed
their cell membranes (Abu-Zaid et al., 2021). The textiles in order to prevent damage to the surface of the
results obtained indicate the presence of bioactive fabric and prolong shelf life.
ingredients which are considered to serve as

Fresh Sample Dried Sample

UV analysis of the extracts

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Fresh Sample Dried Sample

Fig 1: The results of UV (up) and FTIR (down) spectroscopy analysis of dye extracts from fresh and dried leaves of J.
carnea indicating important peaks and functional groups related to color formation.

The absorption peaks of the UV analysis of the extracts at 3300 cm-1 which is a broad absorption attributable to
from the fresh and dried leaves were shown on Fig 1. A free - OH of hydrogen bonded alcohol. The second
broad band at 600 nm indicates weak absorbing species absorption at 1638 cm-1 is a sharp stretching vibration
that resulted in low absorbance of 0.25 that can be that characterized aromatic -C C- unsaturated group
attributed to the -C C-. An absorbance of 0.68 at 300 and C=O group.. The absorption at about 3300 cm-1 is a
nm substantiates the presence of low chromophoric OH bond stretching peak for N-H. While absorption
component due to the absence of conjugation in the between 2200-2300 cm-1 indicated C-N or C-C triple
structure as revealed in the IR spectrum. Other bond. These functional groups, C=O, C=C, C-N, OH are
spectrum values at 0.68, 0.9 and 1.2 showed that found in alkanoid compounds present in J. carnea such
extracted dyes are mixtures of different compounds as Hordenine, Cytisine; flavonoids (which usually
which may include phytochemicals such as tannins, contained carbonyl and hydroxyl group) such as
phenols and flavonoids. The peaks within the range 200 Isoquercetin, Rutin, Hesperidin and phenols such as
-300 nm are related to tannin compounds while the peak ferulic acid (Obazeh, 2023). The spectral revealed a
at 320 nm and above indicate flavonoids derivatives. degree of purity owing to the absence of background
interference absorptions as a result of impurity. The
The results of IR Spectra of dye extracts from the fresh presence of these functional groups in the dye extracts
and dried leaves of J. carnea also on (Figs 1) revealed which are electron rich species when in conjugation are
identical spectra with two distinctive stretching very good chromophores and can enhance its
vibrations each. The two dye extracts showed vibrations application in dyeing of fabrics.

Table 3: Rubbing Fastness of Dyed Cotton Fabrics using Selected Mordants and Mordanting ethods
Mordanting Method Mordant Rubbing
Fresh Dried
Wet Dry Wet Dry

PRM Lime 3 3 3 3
SIM Lime 4 3 3 3
POM Lime 3 3 3 3
PRM Cobalt Chloride 3 3 3 4

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SIM Cobalt Chloride 4 4 4 4

POM Cobalt Chloride 4 3 4 3
NM 1 2 2 2

Scales for the Tables (3 & 4): 5- excellent, 4- very good, 3- good, 2- moderate, 1- poor
Key:
PRM – pre mordanting SIM – simultaneous mordanting
POM - post mordanting NM - no mordanting

The results of fastness property due to rubbing (wet and extracted from catechu plant. Rub fastness is a process
dry) and perspiration (alkali and acid) were presented on related to the unfixed dye deposit on the fabric surface
Tables 3 and 4 which revealed that fastness due to which could be removed easily by washing and this
perspiration and rubbing were improved by mordants implies that the method of washing whether intensive or
and mordanting processes. The improved fastness of otherwise will affect the rubbing fastness performance.
mordanted cotton fabrics (metallic and bio - source) has
been reported (Asiagwu et al., 2021) and could be Using SIM and lime as mordant, the fastness due to
explained in term of the fact that the dye was able to perspiration (Table 4) of dyed cotton fabric using fresh
form complex with the fibre. dye extract was 4 (very good) and 3/4 (good/ very good)
SIM process was found to produce better perspiration in alkaline and acidic conditions respectively while it
and rubbing fastness results than the POM and PRM was 4 (very good) in both conditions with dye extract
processes. The rubbing fastness of the dyed cotton from dried leaf. With SIM and cobalt chloride as
fabrics with lime and cobalt chloride as mordants and mordant, the perspiration fastness value of 4 (very
SIM process are presented on Table 3. The values of 4 good) was obtained for alkaline and acidic conditions
(wet) and 3 (dry) were obtained for fabric dyed with with dye extract from fresh leaf while it was 4 -4/5
extract from fresh leaf while a value of 4 was obtained (very good and very good/excellent) with extract from
for both rubbing fastness conditions (wet and dry) with dried leaf. The synthetic mordant (cobalt chloride) and
dye extract from dried leaf. From these results, wet natural mordants (lime) exhibit similar rubbing and
rubbing fastness gave better results for both dye extracts perspiration fastness which differs from the submission
and may not require any after treatment (Jain, 2017). of Alam et al., (2022) that synthetic mordanted fabric
Chaudhari et al., (2019) however reported overall good has less colour fastness to rubbing than bio- mordanted
dry rubbing fastness properties for natural dyes fabric.

Table 4: Perspiration Fastness of Dyed Cotton Fabrics using Selected Mordants and Mordanting Methods
Mordanting Method Mordant Perspiration
Fresh Dried
Acid Alkali Acid Alkali
PRM Lime 3 1 2 2
SIM Lime ¾ 4 4 4
POM Lime 3 3 3 2
PRM3 Cobalt Chloride 4 ¾ 3 3
SIM 3 Cobalt Chloride 4 4 4/5 4
Cobalt Chloride 3 3 3 3
NM 1 1 1 1

Cobalt Chloride
D

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Lime D

No mordant D

Plate 2: Cotton fabrics dyed with aqueous dye extracts of J. carnea using different mordants and mordanting methods
where F and D designate Fresh and dried leaves respectively.

From plate 2, it was observed that cotton fabrics could Abu-Zaid, A. A., Al-Barty, A., Morsy, K. & Hamdi, H.
be dyed directly from the extracted dyes without (2022). In vitro study of antimicrobial activity of
mordants but more variety shades of colours were some plant seeds against bacterial strains causing
obtained using chemical and natural mordants. This is in food poisoning diseases. Brazilian Journal of Biology,
line with the work of Kumar et al., (2019) and Alam et 82, e256409. https://doi.org/10.1590/1519-
al., (2022) that reported different colours and shades 6984.256409
with the use of mordants. The influence of mordanting Ahmed, M., Islam, T., Karim, Md. R., Kaiser, S. &
techniques on colour formation despite using the same Barua, P. (2019). Assessment of fastness properties
mordant was also established. The ability of the of knitted cotton fabric dyed with natural dyes: a
extracted dyes to produce colours with the cotton fabric sustainable approach of textile coloration. J. Text.
without mordants could be attributed to the presence of Eng. & Fashion Technol. 5(3): 177-182.
bioactive polyphenols in the dyes that acts as natural Ajuru, M. G., Kpekot, K. A., Robinson, G. E. &
mordants to sustain colouring matters (Win et al., Amutadi, M. C. (2022). Proximate and phytochemical
2020). These polyphenolic compounds especially analysis of the leaves of Justicia carnea Lindi. and
tannins are naturally occurring water soluble materials Justicia secunda Vahl and its taxonomic implications.
containing phenolic hydroxyl group which enable them Journal of Biomedicine and Biosensors. 2(1): 1-12.
to form effective crosslink’s (Win et al., 2020) and in Alam, S.M.M., Islam, S. & Akter, S. (2020). Reviewing
this case between the dye and fiber. the sustainability of natural dyes. Advance Research
Conclusion in Textile Engineering, 5(2): 01 – 06.
Andrew C. A., Nwaoguikpe, R. N., Ujowundu, C. O. &
The aqueous extracts from fresh and dried leaves of J. Ujowundu, F. N. (2024). Biochemical analysis of
carnea produced different colours on the cotton fabric Justicia carnea leaves used as a hematinic. GSC
with the help of mordants. This result indicates the Biological and Pharmaceutical Sciences, 26(02): 105–
presence of bioactive ingredients which serves as 114. Article DOI:
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organism related diseases were inhibited with the 4.
Justicia carnea dye extracts. The mordants and Asiagwu, A. K., Otutu J. O., Onyesom, I. E. & Ebigwai,
mordanting methods produced better choice for cotton J. K. (2021). Extraction and application of natural
fabric dyeing because of the improvement in fastness colourant from the bark of Berlinia grandiflora on
properties. SIM process was found to produce better cotton and polyamide 6 fabrics. Iraqi Journal of
perspiration and rubbing fastness results than the POM Science, 62 (6): 1745-1760. DOI:
and PRM processes. 10.24996/ijs.2021.62.6.1
Conflict of interest: None Asiwe, E. S., Ejiogu, P. C., Enenebeaku, U. E.,
Ugenyi, A. U., Opara, P. O. & Mbakwe, I. E. (2023).
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