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1 ABSTRACT

The art of dyeing is as old as our civilization. Natural dyes are derived from natural
resources and based upon their source of origin; these are broadly classified as plant,
animal, mineral, and microbial dyes although plants are the major sources of natural dyes.
Different plant materials were collected such as Henna (Lawsonia inermis), Hibiscus
(Hibiscus rosasinesis), Neem (Azadirachta indica), Tea leaves (Camellia sinesis),
Turmeric (Curcumalonga), Beetroot (Betavulgaris), Carrot (Daucus carota), Orange
(Citrus sinensis) were collected and aqueous extraction was carried out for extraction of
dye. Different cloth samples like cotton, muslin, spun and net were dyed using the
extracted dye. Antibacterial activity of the extracted dye against Escherichia coli,
Staphylococcus, Bacillus sp were carried out. The extracted dye was used for staining
bacteria. The residual matter after extraction of the dye was used as biofertilizer. The
aqueous extract of Hibiscus and Turmeric was found to be effective against Bacillus and
that of Henna was found to be effective against Staphylococcus and Tea was against
Escherichia coli. The morphology of bacteria were visible as blue and pink rods under
the microscope. Plants supplemented with flower residual extract showed an increase in
length of 10 cm when compared to the control 6 cm. The colouring property of the cloth
is due to the pigment in the flowers xanthophylls, anthocyanin, carotenes. Antibacterial
activity of the flower extract is because of presence of phytochemicals phenol,
flavanoids, tannin. The phytochemicals and other minerals support the growth of the
plants.

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2.0 INTRODUCTION

Dyeing is an ancient art, which predates written records. It was practiced during the
Bronze Age in Europe. Primitive dyeing techniques included sticking plants to fabric or rubbing
crushed pigments into cloth. The methods became more sophisticated over time and techniques
were developed using natural dyes from crushed fruits, berries and other plants, which were
boiled into the fabric and gave light and water fastness [resistance]. In many of the worlds
developing countries, however, natural dyes can offer not only a rich and varied source of
dyestuff, but also the possibility of an income through sustainable harvest and sale of these dye
plants. Many dyes are available from tree waste or can be easily grown in market gardens. In
areas where synthetic dyes, mordants [fixatives] and other additives are imported and are
therefore relatively expensive, natural dyes can offer an attractive alternative. Presently there is
an excessive use of synthetic dyes, estimated at around 10,000,000 tons per annum, the
production and application of which release vast amounts of waste and unfixed colorants,
causing serious health hazards and disturbing the eco-balance of nature. Currently, ecological
considerations are becoming important factors in the selection of consumer goods all over the
world. Since the mid-1980s, more interest has been shown in the use of natural dyes and a
limited number of commercial dyes, and small businesses have started to look at the possibility
of using natural dyes for coloration. At present, large and small-scale industries have begun
exploring the use of natural colorants as a possible means of producing an ecologically sound
product which would also appeal to the Green-minded consumer.

In the present study, Flowers from four different plant samples Marigold (Tagetes erecta),
Rose (Rosa indica), Butterfly pea (Clitoria ternatea), Cocks comb (Celosia cristata) were
collected and aqueous extraction was carried out for extraction of dye. Natural dyes are friendly
and satisfying to use. They are familiar substances that can spark creative ideas and widen your
view of the world. Try experimenting with them. Colour can be coaxed from all kinds of natural
sources. Once the cloth or fibre is prepared for dyeing it will soak up the colour, yielding a range
of shades from vibrant jewel tones to dusky heathers and pastels. Variations are easily achieved
by changing dyestuff, recipes, and methods

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SCIENTIFIC CLASSIFICATION
FLOWER MARIGOLD ROSE BUTTERFLY PEA COCKSCOMB
Kingdom Plantae Plantae Plantae Plantae
Phylum Magnoliophyta Magnoliophyta Magnoliophyta Magnoliophyta
Class Magnoliopsida Magnoliopsida Eudicots Magnoliopsida
Order Asrerales Rosales Fabales Caryophyllales
Family Calenduleae Rosaceae Fabaceae Amaranthacea
Genus Tagetes Rosa Clitoria Celosia
Species T.erecta R.indica C.ternatea C.cristata
Scientific name Tagetes erecta Rosa indica Clitoria ternatea Celosia cristata

2.1 NATURAL FLORAL DYE CHARACTERISTICS:


Majorities of the natural dyes are having the hydroxyl group in its nucleus & they are
sparingly soluble in water. Increase in solubility may be achieved by adding alkali such as
Sodium carbonate to the aqueous solution. Some of the natural colourants dont have a
solubilising group in which case a temporary solubility group is generated at time of application.
For a substance to act as dye, certain condition must be fulfilled:-
i) It must have a suitable colour.
ii) It must be able to fix or must be capable of being fixed to fabric.
iii) It must not be fugitive after fixing on fabric to be dyed.

2.2 ADVANTAGES AND DISADVANTAGES OF NATURAL DYES


In the recent years, there has been a trend to revive the art of natural dyeing. This is mainly
because in some aspects natural colourants are advantageous against synthetic dyes. Some of
these advantages along with some limitations are listed below:
2.2.1 ADVANTAGES
i. The shades produced by natural dyes/colourants are usually soft, lustrous and soothing to the
human eye.
ii. Natural dyestuff can produce a wide range of colours by mix and match system
iii. Natural dyestuffs produce rare colour ideas and are automatically harmonizing.

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iv. Unlike non-renewable basic raw materials for synthetic dyes, the natural dyes are usually
renewable , being agro-renewable/vegetable based and at the same time biodegradable.
v. Application of natural dyes has potential to earn carbon credit by reducing consumption of
fossil fuel (petroleum) based synthetic dyes.
vi. Some of its constituents are anti-allergens, hence prove safe for skin contact and are mostly
non-hazardous to human health.
vii. Some of the natural dyes are enhanced with age, while synthetic dyes fade with time.
2.2.2 ADVANTAGES OF USING NATURAL DYES OVER SYNTHETIC DYES
In the present scenario, natural dyes confer several advantages over synthetic dyes. These are
as follows:
i. Biodegradable
ii. Non-toxic
iii. Environment friendly
iv. Aesthetically appealing
v. Employment generation and utilization of wasteland.
vi. Easy extraction of colors by boiling the plants, berries, leaves, bark or flower heads in water.
vii. Most of the synthetic dyes such as azo dyes are carcinogenic and can produce toxic and
allergic reactions.
viii. Higher UV absorption by the fabric which contains natural dye can result in reduced
incidence of melanoma.
Despite these advantages, natural dyes do carry some inherent disadvantages, which are
responsible for the decline of this ancient art of dyeing textiles.
2.2.3 DISADVANTAGES
i. It is difficult to reproduce shades by using natural dyes/, as these agro products vary from one
crop season to another crop season, place to place and species to species, maturity period etc
ii. It is difficult to standardize a recipe for the use of natural dyes, as the natural dyeing process
and its colour development depends not only on colour component but also on materials.
iii. Scientific backup of a large part of the science involved in natural dyeing is still need to be
explored.
iv. Lack of availability of precise technical knowledge on extraction and dyeing techniques.
v. The dyed textile may change colour when exposed to the sun, sweat and air.

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vi. Nearly all-natural dyes with a few exceptions require the use of mordants to fix them on to
the textile substrate. While dyeing, a substantial portion of the mordant remains unexhausted
in the residual dye bath and may pose serious effluent disposal problem.
2.3 EXTRACTION PROCESS OF COLOUR FROM NATURAL DYES
Extraction of colour component from source natural dye material is important step for
dyeing any textile substrate to maximize the colour yield. Moreover, standardization of
extraction process and optimizing the extraction variables both, for a particular source natural
dye material have technical and commercial importance on colour yield and cost of extraction
process as well as dyeing cost. The natural dyes can be taken from various vegetable sources like
flowers, stem or wood, roots, bark, etc. as well as animal sources and mineral sources. The
colour component present in these sources needs to be extracted so that it can be applied suitably
on textiles. Natural dyes of different origin can be extracted using aqueous method i.e. by using
water for the extraction with or without addition of salt/acid/alkali/alcohol in the extraction bath,
or soxhlet extraction method.
2.4ANTIMICROBIAL PROPERTY
Nowadays, the interest for antimicrobial textiles has significantly increased due to their
potential to provide a higher level of hygiene in home areas and safety benefits to people. Textile
materials provide ideal environment for growth and multiplication of pathogenic microbes
leading to unpleasant odor, dermal infection, weakening of the substrate, discoloration, allergies,
and other related diseases. For this reason, there is an urgent need for a potentially effective
means to control and/or inhibit microbiological growth to protect both the wearer and textiles.
Antimicrobial textiles can be used to produce many goods such as sportswear, working clothes,
undergarments, shoes linings, carpets, upholstery, hospital linens, wound care wraps, towels, and
toys for children. Antibacterial finishes can have potential benefits to contain the spread of
disease, avoid the danger of injury-induced infection, and prevent the deterioration of textiles
and have other advantages. Pure cultures of bacterial strains Escherichia coli, Enterobacter
aerogenes and Pseudomonas sp were used for the present study.

2.5 STATUS OF NATURAL DYES IN INDIA

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Indians have been considered as forerunners in the art of natural dyeing. Natural dyes find
use in the colouring of textiles, drugs, cosmetics, etc. Owing to their nontoxic effects, they are
also used for colouring various food products. In India, there are more than 450 plants that can
yield dyes. Though there is a large plant resource base, little has been exploited so far. Due to
lack of availability of precise technical knowledge on the extracting and dyeing technique, it has
not commercially succeeded like the synthetic dyes. Although indigenous knowledge system has
been practiced over the years in the past, the use of natural dyes has diminished over generations
due to lack of documentation. Also there is not much information available on databases of either
dye-yielding plants or their products.
2.6 FUTURE PROSPECTS IN THE INDIAN TEXTILE INDUSTRY
Dyes currently used for dyeing textile material are classified as soluble, disperse, and
pigments. These are all synthetic compounds, which are environmentally unfriendly compounds,
as their degradation by organisms is not carried out naturally. The industry has to design
expensive ways to remove these harmful compounds from the environment. Nowadays,
fortunately, there is increasing awareness among people towards natural products. Availability of
natural dyes is a desired technology for dyeing fabrics with naturally produce compounds. Thus,
natural dyes provide a reasonable solution to the problems posed by synthetic dyes if the
technology behind application of natural dyes can be formulated. Due to environmental
awareness the natural dyes obtained from plants are the dyes of 21st century. Their production as
well as trade is anticipated to increase manifold in the times to come.

3.0 REVIEW OF LITERATURE

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India has a rich biodiversity and it is not only one of the worlds twelve mega diversity
countries, but also one of the eight major centers of origin and diversification of domesticated
taxa. It has approximately 4, 90,000 plant species of which about 17, 500 are Angiosperms; more
than 400 are domesticated crop species and almost an equal number their wild relatives. Thus,
India harbours a wealth of useful germplasm resources and there is no doubt that the plant
kingdom is a treasure-house of diverse natural products. One such product from nature is the
dye. Dyes are one of the most important uses of the plants, as they are related with cultural
practices, rituals, arts and crafts, fabrics and to satisfy personal embodiment, however, dye
yielding plants have not received significant attention. Recently, interest in the use of natural
dyes has been growing rapidly due to the result of stringent environmental standards imposed by
many countries in response to toxic and allergic reactions associated with synthetic dyes.
Research has shown that synthetic dyes are suspected to release harmful chemicals that are
allergic, carcinogenic and detrimental to human health. On the other hand, natural dyes are
environment-friendly; for example, turmeric, the brightest of naturally occurring yellow dyes is a
powerful antiseptic which revitalizes the skin, while indigo gives a cooling sensation. Though,
dyes have been discovered accidentally, their use has become so much a part of mans customs
that it is difficult to imagine a modern world without dyes. The art of dyeing spread widely as
civilization advanced. (Iqbal Sanjeeda and Ansari Taiyaba N 2014)

3.1 Extraction Methods


As natural dye-bearing materials contain only a small percentage of coloring matter or dye
along with a number of other plant and animal constituents such as water-insoluble fibers,
carbohydrates, protein, chlorophyll, and tannins, among others, extraction is an essential step not
only for preparing purified natural dyes but is also required to be carried out by users of crude
dye-bearing materials. As natural coloring materials are not a single chemical entity and the plant
matrix also contains a variety of non-dye plant constituents, extraction of natural dyes is a
complex process. The nature and solubility characteristics of the coloring materials need to be
ascertained before employing an extraction process. The different methods for extraction of
coloring materials are
Aqueous extraction
Alkali or acid extraction

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Microwave and ultrasonic assisted extraction
Fermentation
Enzymatic extraction
Solvent extraction
Super critical fluid extraction.

3.2 Aqueous Extraction


Aqueous extraction was traditionally used to extract dyes from plants and other materials. In
this method, the dye-containing material is first broken into small pieces or powdered and sieved
to improve extraction efficiency. It is then soaked with water in earthen, wooden, or metal
vessels (preferably copper or stainless steel) for a long time usually overnight to loosen the cell
structure and then boiled to get the dye solution which is filtered to remove non-dye plant
remnants. The process of boiling and filtering is repeated to remove as much dye as possible.
When the extraction is to be carried out on a larger scale for preparation of purified dye
powders, stainless steel vessels are used and the time of soaking the materials in water may be
reduced by boiling the solution for an extended time period. Generally, centrifuges are used to
separate residual matter. Use of trickling filters can ensure removal of fine plant material
particles and ensure better solubility of the purified natural dye.
As most of the dyeing operations are carried out in aqueous media, the extract obtained by
this method can be easily applied to the textile materials. Disadvantages of this extraction
method are long extraction time, large water requirement, use of high temperature, and low dye
yield as only water-soluble dye components get extracted whereas many dyes have low water
solubility. Also, along with dye, other water-soluble substances such as sugars and the like get
extracted that may have to be removed if the extract is to be concentrated and converted to a
powder form. Yield of heat-sensitive dye substances gets reduced at boiling temperature,
therefore a lower temperature should be used for extraction in such instances.

3.3 Acid and Alkali Extraction Process

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As many dyes are in the form of glycosides, these can be extracted under dilute acidic or
alkaline conditions. The addition of the acid or alkali facilitates the hydrolysis of glycosides
resulting in better extraction and higher yield of coloring materials. An acid hydrolysis process is
used for the extraction of dye from Buteamonosperma flowers. Acidified water is also used for
extracting some flavone dyes to prevent oxidative degradation. Alkaline extraction is suitable for
dyes having phenolic groups as they are soluble in alkali, which improves the dye yield. Dyes
can be later precipitated by the use of acids. Dye from annatto seeds can be extracted by this
technique. This process is also used to extract lac dye from lac insect secretions and red dye from
safflower petals. The disadvantage of this process is that some coloring materials may be
destroyed under alkaline conditions considering the fact that some of the natural dyes are pH
sensitive. Inasmuch as natural dyes are usually a mixture of different chemical constituents,
changing the pH of the extraction medium by adding acid or alkali can lead to the extraction of
different dye constituents which can lead to different hues upon subsequent dyeing and
differences in colorfastness properties. Many researchers have studied the extraction of natural
dyes under various pH conditions and compared the color and fastness properties of dyed fabric
to find out the optimum dye extraction conditions and further additions to this information
continue to be made every year in the scientific literature.

3.4 Ultrasonic and Microwave Extraction


These are actually microwave- and ultrasound-assisted extraction processes where extraction
efficiency is increased by the use of ultrasound or microwaves thus reducing the quantity of
required solvent, time, and temperature of extraction. When the natural dye containing plant
materials is treated with water or any other solvent in the presence of ultrasound, very small
bubbles or cavitations are formed in the liquid. These increase in size but upon reaching a certain
size, they cannot retain their shape. When this happens, the cavity collapses or the bubbles burst
creating high temperature and pressure. Millions of these bubbles form and collapse every
second. The creation of very high temperature and pressure during extraction increases the
extraction efficiency within a short time. Also the process can be performed at lower temperature
and therefore extraction of heat-sensitive dye molecules is better. As exploration of new dye
sources and attempts to optimize the dye extraction process is continuing, use of this extraction
technique has been recently reported by many researchers.

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In microwave extraction, the natural sources are treated with a minimum amount of solvent
in the presence of microwave energy sources. Microwave increases the rate of the processes so
the extraction can be completed in a shorter time with better yield. (Sinha et al.) have reported
extraction of annatto colorant with microwave energy. Earlier their group had reported
microwave-assisted extraction of blue pigment from the butterfly pea. Microwave and ultrasound
extractions can be considered as green processes due to reduction of extraction temperature,
solvent usage, and time which results in lower consumption of energy.

3.5 Fermentation
This method of extraction uses the enzymes produced by the microorganisms present in the
atmosphere or those present in the natural resources for assisting the extraction process. Indigo
extraction is the most common example for this type of extraction. Freshly harvested indigo
leaves and twigs are soaked in warm water (about 32C). Fermentation sets in and the colorless
indigo containing glucosideindican present in the leaves is broken down into glucose and indoxyl
by the indimulsin enzyme also present in the leaves. Fermentation is complete in about 1015 h
and the yellow liquor containing indoxyl is then taken to beating vats where indoxyl gets
oxidized by air to the blue-colored insoluble indigotin which settles down at the bottom. It is
collected, washed, and after removing excess water is pressed into cakes. Extraction of indigo
from other indigo-containing plants such as woad is also carried out by fermentation. This
process can also be used for extraction of certain other colorants such as annatto. The
fermentation method is similar to aqueous extraction with the exception that this method does
not require high temperatures. The microorganisms disintegrate the coloring matter binding
substances in natural way. Long extraction time, need for immediate extraction of pigments after
harvesting, foul smell due to microbial action, and so on, are some of the disadvantages of this
method.

3.6 Enzymatic Extraction


As plant tissues contain cellulose, starches, and pectins as binding materials,commercially
available enzymes including cellulase, amylase, and pectinase have been used by some
researchers to loosen the surrounding material leading to the extraction of dye molecules under

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milder conditions. This process may be beneficial in the extraction of dye from hard plant
materials such as bark, roots, and the like.
3.7 Solvent Extraction
Natural coloring matters depending upon their nature can also be extracted by using organic
solvents such as acetone, petroleum ether, chloroform, ethanol, methanol, or a mixture of
solvents such as mixture of ethanol and methanol, mixture of water with alcohol, and so on. The
water/alcohol extraction method is able to extract both water-soluble and water-insoluble
substances from the plant resources. The extraction yield is thus higher as compared to the
aqueous method as a larger number of chemicals and coloring materials can be extracted.
Acid or alkali can also be added to alcoholic solvents to facilitate hydrolysis of glycosides
and release of coloring matter. Purification of extracted color is easier as solvents can be easily
removed by distillation and reused. Extraction is performed at a lower temperature thus chances
of degradation are fewer. The disadvantages of the method are the presence of toxic residual
solvents and their greenhouse effect. Another disadvantage of this method is that the extracted
material is not readily soluble in water and the subsequent dyeing process has to be carried out in
an aqueous medium. Co-extraction of substances such as chlorophylls and waxy materials also
creates problem.

3.8 Supercritical Fluid Extraction


Supercritical fluid extraction is an emerging area in natural product extraction and
purification. A gas functions as a supercritical fluid above its critical values of temperature and
pressure. Such a fluid has physical properties somewhere between those of a liquid and a gas.
They are able to spread out along a surface more easily than a true liquid because they have
much lower surface tension than liquids. As their viscosity is also low, they have very good
diffusivity and thus better interaction with the substrate. At the same time, a supercritical fluid is
able to dissolve many substances like a liquid as solubility of a substance in any solvent is higher
at higher pressure and temperature and such conditions are needed to maintain a gas in the
supercritical state. Supercritical fluid extraction using carbon dioxide (CO2) is a good alternative
to solvent extraction as it is nontoxic, cheap, easily available, and does not leave residues.
Critical temperature and pressure values for carbon dioxide are 31.4 C and 1,070 pounds per
square inch (psi) or 73.8 bars, respectively. Supercritical extractions using CO2 typically operate

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at temperatures between 32 and 49 C and pressures between 1,070 and 3,500 psi. As the CO2 is
a non-polar molecule it behaves as does a non-polar organic solvent. A co-solvent or a modifier
may be added to improve the solubility of slightly polar solutes. The advantage of the process is
that the extract is free from residual solvent traces and heavy metals and is light colored due to
the absence of polar polymerizing substances hence the process has gained popularity in
extraction of purified natural products for food and pharmaceutical applications. The
disadvantage of the method is the high cost of the equipment and poor extraction of polar
substances.

3.9 Drying Techniques


Coloring matter extract obtained from natural sources is mostly in aqueous media. If it is to be
immediately used for dyeing as in many small-scale or cottage-dyeing establishments, it can be
used as such after adjustment of concentration according to the shade requirements. However, if
it is to be used at a later date for dyeing or in dye extract producing units, it has to be converted
into either powder form or concentrated solid rich form for long-term storage and ease of
transport. This also ensures uniformity of shade for the entire batch of dye powder or concentrate
produced and natural dyes thus converted into powder form or liquid concentrates can be used
like synthetic dyes by the industries. The following techniques are generally used for converting
natural dye extracts into powder form or concentrates.
Spray drying
Drying under vacuum
Freeze drying.
Spray drying is the most commonly used technique for converting natural dye extracts into
powder form as it is simple and cost effective. The plant extract is sprayed into the spray
chamber as fine droplets through an atomizer or spray nozzle. These droplets come into contact
with hot air flowing into the chamber which removes the solvent; the resulting dry particles
aggregate and fall to the bottom of the chamber where this powder can be collected. Natural dyes
in ready to- use dry powder form are mostly produced by this method. Dye molecules should be
sufficiently stable to heat to follow this method of drying as dry powder is exposed to dry heat
from the hot air. Also many fine dye particles that are too small to settle at the chamber bottom
are carried away by the hot air and are lost resulting in lower recovery. Also higher dye content is

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needed to get larger size particles and lower losses that may be achieved by pre concentration of
the extract or addition of inert compounds such as lactose and so on. Extracts can also be
concentrated under vacuum by use of a rotary evaporator or tray dryers and the concentrated dye
can be purified further by the use of various solvents. Another drying technique, lyophilization or
freeze-drying can also be used for preparation of natural dye powders. In this process, the natural
dye extract is subjected to freezing and water is removed from the frozen extract through
sublimation by reducing the pressure. The equipment is costly and operating cost is also higher
but heat-sensitive dyes can also be converted into dry powders as the process takes place at low
freezing temperatures. Any one of the above techniques can be used for producing natural dye
powder depending upon the dye extract properties and costs. Very pure dye extracts are now
available for use in many countries especially the United States, but these are very costly and are
mainly used by hobby groups for their uniqueness.

3.10 Application of Natural Dyes on Textiles


Natural dyes are mostly employed for dyeing of natural fiber textiles to enhance their eco-
friendly characteristics. They are usually applied to textiles by dyeing. Apart from indigo, other
natural dyes are usually not used for printing directly. For producing printed fabrics, the printing
is usually done with mordant and the whole material is dyed whereby only the area printed with
mordants picks up the color.
Natural dyes, like synthetic dyes, can also be used to dye textiles at all stages such as fiber,
yarn, or fabric. Fiber dyeing has the advantage that any shade variation can be easily adjusted by
blending and therefore has been practiced at industrial scale also but is costly due to problems in
spinning and loss of dyed fibers. Wool is generally dyed in yarn form and traditional dyers prefer
yarn dyeing for all materials as it offers versatility in designing during weaving. Dyeing in hank
form is preferred by traditional dyers operating at the cottage level due to its simplicity and low
investment and also its compatibility with their usage of crude dye-bearing natural resources for
reasons of authenticity as well as cost effectiveness. Dyeing is normally carried out by these
artisans by hand in large vessels. Iron, stainless steel, copper, and aluminum vessels are used.
Dyeing in copper vessels is considered to produce bright shades. Aluminum vessels are normally
stained with a particular dye hence should be used if only one type of dye is used. Stainless steel
vessels are most preferred for the natural dyeing process. On a larger scale, hank-dyeing

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machines have been successfully used. Fine purified powders or concentrates are needed for
package dyeing as otherwise dyeing is uneven. Fabric dyeing is also carried out in metal vessels
at the cottage level. Machines such as the jigger and winch have been employed for dyeing larger
lots. Suitable dyes or dye-bearing resources are selected based on the color requirement.
Information about some dye sources has been provided earlier. In general, tannin-containing
barks are used to produce brown and grey colors. Flowers and leaves containing flavonoids are
used to produce yellow color shades. Anthraquinone dyes from both animal and plant resources
can be used for red color. Indigo is normally used to produce blue color shades. Secondary colors
such as orange can be obtained by proper selection of dye and mordant or mixing two compatible
dyes. However to get a green color or wherever blue color is needed to make a secondary color,
material is first dyed with indigo and then over dyed with the other dye. If raw plant materials
are used (which is usually the case at the cottage level), dye has to be extracted prior to dyeing.
Details of various dye extraction methods have already been discussed. Aqueous-based
extraction methods are employed. If purified natural dyes are used, these can be directly used for
dyeing. The process for the dyeing of textiles with natural dyes differs from the synthetic dye
application process because only some natural dyes can be applied directly to textiles. In most of
the cases the dye is not substantive to the fiber on which it is being dyed therefore an additional
step of mordanting is involved, making it a two-step process.

3.11 Food Colourant

Additives have been used to color food for hundreds of years and governments have
attempted to regulate the usage nearly as long. In the 1200s, Englands King Edward I decreed
that any baker who colored his bread white would be dragged through the streets with the
fraudulent loaf hanging around his neck. Todays rhetoric about food dyes is much tamer, as is
the regulation, and, as a result, exposure is rising. In the past 60 years, in fact, the amount of
artificial dye used in food in the United States has increased fivefold. The first inkling that
artificial food dyes have a downside came in the 1970s, when pediatric allergist Ben Feingold
asserted that hyperactive kids who eliminated artificial flavors and colors from their diets
showed a remarkable improvement in behavior. Since then, other researchers have confirmed

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that many kids with hyperactivity experience a worsening of symptoms when they consume
artificial dyes.

In 2012, a meta-analysis of 24 studies showed that as many as 33 percent of kids with


ADHD may benefit from diets free of artificial food colors and additives. Some experts even
think that these dyes may push borderline kids over the threshold toward a diagnosis of ADHD in
the first place. In other words, they say, some children would not develop ADHD if they werent
consuming artificially colored foods.

Parents need to be aware that some children, at the amounts found in everyday kinds of food
items, respond with aberrant behaviors, Weiss says. Should such behaviors become a common
occurrence, parents might undertake a little experiment in order to determine the possibility of a
connection.

Even more alarming, the Southampton Study showed for the first time conclusively and
scientifically that artificial food colors and additives can affect the behavior of kids who dont
have any proclivities toward ADHD. Researchers examined the hyperactivity levels of about 300
kids selected from the general population to represent the full range of behavior, from normal to
hyperactive. Some kids ate a totally additive-free diet, including additive-free fruit juice, while
the rest were given a drink containing artificial food colors and sodium benzoate preservative.

The families werent told which drink their kids were getting. Reports from the childrens
teachers and parents on the childrens impulsivity, attention, and movement showed that the kids
were more hyperactive when they consumed the drinks containing artificial colors and additives.

That transformed it from an ADHD issue to a public-health issue, says Gene Arnold, MD,
MEd, Ohio State University professor emeritus of psychiatry. The results prompted the European
Union (EU) to mandate warning labels on foods and drinks that contain any of the six colors
used in the study. (The Southampton team chose to study those six colors because they are
commonly found in childrens food, but the EU is also currently reassessing all the other
artificial food colors it permits.)

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The amount of dyes given to children in the Southampton Study was actually well below
what many American children consume, according to Laura Stevens, MS, a Purdue nutritional
science research associate who studies the link between food dyes and hyperactivity.

While the behavioral effects are most often discussed, artificial food dyes may have
adverse physical effects as well, the Center for Science in the Public Interest (CSPI) documented
through a review of various studies. Some food dyes including the three most common ones can
provoke hypersensitivity in certain children and adults.

Animal studies, meanwhile, have shown a link to other health problems such as
reproductive issues and kidney disease. This prompted the European Food Safety Authority to
further decrease recommended daily allowances of several artificial dyes and recommend further
safety tests.

Natural Colours as Health Cure:

Besides colouring food, several natural dyes possess bioactive properties and have been
used as therapeutic agents and as diagnostic tools. Some of the dyes have been reported for
following curative effects; analgesics, antibacterial, antifungal, antileprotic, antiviral and anti-
inflammatory.

Turmeric contains phytochemical compounds (curcuminoids), which give bright yellow-


orange colour. These curcuminoids have been the focus of numerous clinical studies
investigating their long term safety, antimicrobial and anti-inflammatory activities. Turmerics
most active curcuminoid is curcumin. Preliminary studies have examined the possible role of
turmeric (specifically curcumin) in reducing the risk or severity of several types of cancer .
Recent research has examined curcumins ability to block the effects of homocysteine on the
vascular system. This could potentially benefit those with HIV as well as protect the
cardiovascular system.

Turmeric is also used as a dye in textile industry, in cosmetics, preparation of medicinal oils
and textile industry, in cosmetics, preparation of medicinal oils and ointments. It is a Stomachic,
a carminative, a tonic, a blood purifier and an antiseptic. Presently, there has been much interest

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in carotenoids, especially betacarotene (carrots, mango, papaya etc.) which besides natural
orange pigment is converted in body to vitamin A and has antioxidant powers. Similarly, there is
trend towards the use of anthocyanins (red grapes, red cabbage, elderberries, sweet potatoes etc.)
and betacyanins (red potatoes, beet, amaranth etc.), which contribute positive health effect.

3.12 Antimicrobial Activity

The increase in prevalence of multiple drug resistance has slowed down the development of
new synthetic antimicrobial drugs, and has necessitated the search for new antimicrobials from
alternative sources. Natural compounds are a source of numerous therapeutic agents. Recent
progress to discover drugs from natural sources has resulted in compounds that are being
developed to treat cancer, resistant bacteria and viruses and immunosuppressive disorders
(Amghalia et al., 2009). Phytochemicals from medicinal plants showing antimicrobial activities
have the potential of filling this need, because their structures are different from those of the
more studied microbial sources, and therefore their mode of action are also very likely to differ.
There is growing interest in correlating the phytochemical constituents of a medicinal plant with
its pharmacological activity (Prachayasittikul et al., 2008; Nogueira et al., 2008). Screening the
active compounds from plants has lead to the discovery of new medicinal drugs which have
efficient protection and treatment roles against various diseases (Roy et al., 2009).

The agar diffusion test is a test of the antibiotic sensitivity of bacteria. It uses Wells to test
the extent to which bacteria are affected by those antibiotics. In this test, waters containing
antibiotics are placed on an agar plate where bacteria have been placed, and the plate is left to
incubate. If an antibiotic stops the bacteria from growing or kills the bacteria, there will be an
area around the wafer where the bacteria have not grown enough to be visible. This is called a
zone of inhibition.

The size of this zone depends on how effective the antibiotic is at stopping the growth of the
bacterium. A stronger antibiotic will create a larger zone, because a lower concentration of the
antibiotic is enough to stop growth.

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In general, larger zones correlate with smaller minimum inhibitory concentration (MIC) of
antibiotic for that bacteria. Inhibition produced by the test is compared with that produced by
known concentration of a reference compound. This information can be used to choose
appropriate antibiotics to combat a particular infection.

3.13 Staining

The Gram staining method, named after the Danish bacteriologist who originally devised it in
1882 (published 1884), Hans Christian Gram, is one of the most important staining techniques in
microbiology. It is almost always the first test performed for the identification of bacteria. The
primary stain of the Gram's method is crystal violet. Crystal violet is sometimes substituted with
methylene blue, which is equally effective. The microorganisms that retain the crystal violet-
iodine complex appear purple brown under microscopic examination. These microorganisms that
are stained by the Gram's method are commonly classified as Gram-positive or Gram non-
negative. Others that are not stained by crystal violet are referred to as Gram negative, and
appear red.

Gram staining is based on the ability of bacteria cell wall to retaining the crystal violet dye
during solvent treatment. The cell walls for Gram-positive microorganisms have a higher
peptidoglycan and lower lipid content than gram-negative bacteria. Bacteria cell walls are
stained by the crystal violet. Iodine is subsequently added as a mordant to form the crystal violet-
iodine complex so that the dye cannot be removed easily. This step is commonly referred to as
fixing the dye. However, subsequent treatment with a decolorizer, which is a mixed solvent of
ethanol and acetone, dissolves the lipid layer from the gram-negative cells. The removal of the
lipid layer enhances the leaching of the primary stain from the cells into the surrounding solvent.
In contrast, the solvent dehydrates the thicker Gram-positive cell walls, closing the pores as the
cell wall shrinks during dehydration. As a result, the diffusion of the violet-iodine complex is
blocked, and the bacteria remain stained. The length of the decolorization is critical in
differentiating the gram-positive bacteria from the gram-negative bacteria. A prolonged exposure
to the decolorizing agent will remove all the stain from both types of bacteria. Some Gram-

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positive bacteria may lose the stain easily and therefore appear as a mixture of Gram-positive
and Gram-negative bacteria (Gram-variable).

Finally, a counterstain of basic fuchsin is applied to the smear to give decolorized gram-negative
bacteria a pink color. Some laboratories use safranin as a counterstain instead. Basic fuchsin
stains many Gram-negative bacteria more intensely than does safranin, making them easier to
see. Some bacteria which are poorly stained by safranin, such
as Haemophilus spp., Legionella spp., and some anaerobic bacteria, are readily stained by basic
fuchsin, but not safranin. The polychromatic nature of the gram stain enables determination of
the size and shape of both Gram-negative and Gram-positive bacteria. If desired, the slides can
be permanently mounted and preserved for record keeping.

Besides Gram's stain, there are a wide range of other staining methods available. By using
appropriate dyes, different parts of the bacteria structures such as capsules, flagella, granules, and
spores can be stained. Staining techniques are widely used to visualize those components that are
otherwise too difficult to see under a light microscope. In addition, special stains can be used to
visualize other microorganisms not readily visualized by the Gram stain, such as mycobacteria,
rickettsia, spirochetes, and others. In addition, there are modifications of the Gram stain that
allow morphologic analysis of eukaryotic cells in clinical specimens.

3.14 Bio-dye Degradation

The contamination of soils and waters by dye-containing effluents is of environmental concern.


Due to the increasing awareness and concern of the global community over the discharge of
synthetic dyes into the environment and their persistence there, much attention has been
focused on the remediation of these pollutants. Among the current pollution control
technologies, biodegradation of synthetic dyes by different microbes is emerging as an
effective and promising approach. The bioremediation potentials of many microbes for
synthetic dyes have been demonstrated and those of others to be explored in future. The
biodegradation of synthetic dyes is an economic, effective, biofriendly, and environmentally
benign process. Bioremediation of xenobiotics including synthetic dyes by different microbes

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will hopefully prove a green solution to the problem of environmental soil and water pollution
in future.

3.15 Biofertilizer

Biofertilizers are defined as preparations containing living cells or latent cells of efficient
strains of microorganisms that help crop plants uptake of nutrients by their interactions in the
rhizosphere when applied through seed or soil. They accelerate certain microbial processes in
the soil which augment the extent of availability of nutrients in a form easily assimilated by
plants.

Very often microorganisms are not as efficient in natural surroundings as one would expect
them to be and therefore artificially multiplied cultures of efficient selected microorganisms play
a vital role in accelerating the microbial processes in soil.

Use of biofertilizers is one of the important components of integrated nutrient management,


as they are cost effective and renewable source of plant nutrients to supplement the chemical
fertilizers for sustainable agriculture. Several microorganisms and their association with crop
plants are being exploited in the production of biofertilizers.

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4.0. AIM AND OBJECTIVE

The aim of the present study was to extract natural dyes from plant materials and to
evaluate its applications. The study was carried out with the following objectives:

To extract dyes from natural source (plant source i.e. Flowers, leaves, fruits and

roots)
Determination of its dyeing properties in different fabrics
Determination of efficiency of fabrics dyed with natural dyes
Determination of its antimicrobial activity
Determination of its staining properties in microbes
Evaluation of the effect of residual matter as an bio fertilizer
Determination of biodegradation of natural dye
Natural dye as food colorant

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5.0 MATERIALS AND METHODS

5.1 Plant materials

The following plant materials were collected

Flower Hibiscus(Hibiscus rosa sinesis)


Leaves Henna (Lawsonia inermis), Neem (Azadirachta indica), Tea(Camellia sinesis)
Fruit orange(Citrus sinensis)
Root Carrot (Daucus carota), Beetroot(Beta vulgaris),Turmeric(Curcuma longa)
5.2 Extraction of natural dyes

The natural dye was prepared as follows;

Aqueous extraction: Dye from different plant materials were extracted by preparing an aqueous
solution (10g in 100 ml distilled water) and the extraction process was carried out at a
temperature range of 80-85C for 1hour. After the extraction procedure is complete, the residue
were taken out from the liquor (Neha Grover and Vidya Patni,2011)
3 Dyeing of different fabrics
Chemicals and reagents
Sodium hydroxide
Sodium chloride
Copper sulphate
Vinegar

Fabrics : Cotton, Muslin,Spun,Net

Procedure
Cloth used for dyeing was boiled in NaOH solution (10%) for 15 minutes to remove starch
from the cloth, then washed with cold distilled water. This cloth was then transferred in mordant
(copper sulphate) for 30 minutes followed by treatment in the dye bath for one hour. Then the

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cloth was treated with vinegar (colour fixative) and dried in sunlight (Neha Grover and Vidya
Patni,2011)
5.4 Checking the efficiency of fabrics dyed with natural dyes

Efficiency of fabrics [ Cotton, Muslin, Spun, Net]dyed with natural dyes was checked by washing it
with detergents.

5.5Antimicrobial activity of natural dye

Procedure

Well Diffusion

Mueller Hinton agar was prepared and poured into each plate and kept for solidification.
Culture (E.coli, Staphylococcus, Bacillus) were swabbed using sterile cotton swab. Well was
punctured in agar using gel puncture.Natural dyes obtained (Carrot- with and without
preservative,Beetroot-with and without preservative,Neem andTurmeric) were loaded into
respective wells. It was then Kept for incubation at 37c for 24 hours.

5.6 Simple staining of bacteria

Procedure

Prepare bacterial smear and heat fix by gently warming over flame. Flood the smear with
natural dye for five minutes. Air dry the slide. Examine the stained slide under microscope.

5.7 Effect of residual matter as an bio fertilizer

Two grow bags were taken. First one was taken as control. In second one , soil is mixed with
flower residue obtained after extraction. Chilly plant of 4cm length were planted in each grow
bags. It was watered daily. Result observed after 2 weeks.

5.8 Biodegradation of natural dye

Biodegradation of different dyes (Henna, Tea, Hibiscus, Turmeric) were tested using three
different strains(E coli, Staphylococcus ,Bacills)by incubating them at 37c for 72 hrs and the %

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of decolourisation of natural dye were compared with decolourisation of synthetic dye (congo
red) were examined using UV vis spectrophotometer at600nm.The results were tabulated.

5.9 Bio dye as food colorant

250g of dough was prepared by adding 25ml natural dye (Orange, Carrot, Beetroot) with
1% citric acid as preservative and were baked at 180c for 20 mins

5.10 Microbial quality checking of bio dye in biscuits

The biscuits were crushed using motor and pestle.1g of biscuit sample were weighed and
were serially diluted upto 107dilutions.The even dilutions (2,4,6) were plated and kept for
incubation at 37c for 24hrs.

6.0. RESULTS AND DISCUSSION


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6.1 Extraction of natural dyes

Dyes were extracted from plant materials such as flowers, leaves fruits and roots using
aqueous extraction. Obtained dyes were labeled and stored for further studies.

6.2 Dyeing and checking the efficiency of dyed fabrics

Cloth samples[cotton,Muslin,Spun and Net] were dyed with natural dyes obtained from
different plant materials. The efficiency of dyeing is determined by washing the fabrics with
detergent and there was no fade in colour.

6.3 Antimicrobial activity

Zone of inhibition for different natural dyes (mm)

Organism Rose Marigold Butterfly Pea Cockscomb

Escherichia coli 15 11 18 19

Enterobacter aerogenes 13 19 16 15

Pseudomonas sp 15 16 11 10

Zone of inhibition were obtained for the organisms (Escherichia coli,


Enterobacter aerogenes, Psuedomonas sp) Antibacterial activity of the flower extract is because
of presence of phenol, flavanoids, tannin etc

6.4 Staining of bacteria


Bacteria were stained with dyes obtained from flowers and they were appeared as pink and
blue rods under microscope. Natural dyes can be used for simple staining of bacteria

6.5 Natural dyes as pH indicator

Natural dyes can be used as pH indicator. Natural dyes are weak acid so addition of alkali
leads to change in colour.

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6.6 Effect of residual matter as an bio fertilizer

Initial height of chilly plant Height of chilly plant after 3 weeks


Samples

Control plant 4cm 6cm

Plant supplied with flower 4cm 10cm


residue

Flower residue and effluent obtained after dyeing with natural dye can be used as bio
fertilizer, since it is rich in secondary metabolites such as phenol, flavanoids, antioxidants which
assist in the growth of plant. The plant supplied with flower residue showed more growth than
control.

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