CHAPTER 1 INTRODUCTION

For centuries, cultures have used plant-based colorants for textiles, food, and cosmetics, a
practice known as natural dyeing. However, because synthetic dyes were more affordable,
produced more color, and were easier to use, they largely replaced natural dyes when they were
introduced in the 19th century (Geetha and Sumathy, 2013). Synthetic dyes, which are widely
used in textile manufacturing, are mainly derived from petrochemicals and contain hazardous
substances like azo compounds, heavy metals, and aromatic amines. The textile industry is one
of the largest industrial sectors worldwide, but it is also a significant source of environmental
pollution (Handa, 2008). When untreated or partially treated dye effluents are released into rivers
and other bodies of water, these chemicals can seriously pollute the water. It has been stated that
roughly 20% of global water pollution occurs from textile dyeing and finishing operations,
making it one of the most polluting sectors in the world (Bhandari et al., 2020).
The environmental concerns posed by synthetic dyes are not restricted to water pollution. High
energy inputs and carbon emissions are required for the production of artificial dyes, which
exacerbate climate change (Hamdy et al., 2021). Furthermore, exposure to synthetic dyes over an
extended period has been associated with skin allergies, respiratory issues, and even cancer.
Many synthetic dyes are carcinogenic, mutagenic, and allergic, posing major health concerns to
both humans and animals (Adeel et al., 2022). Researchers and industries are searching for
sustainable, environmentally friendly dye substitutes, especially from natural sources like plants,
fungi, and microbes, as a result of these considerations (Kıcık and Gökbulut, 2023).
Natural dyes provide special qualities like UV protection, antibacterial activities, and therapeutic
advantages in addition to their positive effects on the environment. According to studies, natural
extracts- dyed textiles may absorb up to 80% of UV rays, which makes them ideal for
applications requiring protective apparel (Bhandari et al., 2020). Furthermore, herbal textiles,
which incorporate plant-based colorants into textiles for therapeutic and health purposes, make
extensive use of natural dyes (Verma and Gupta, 2017). Natural dyes, particularly non-toxic,
biodegradable, and renewable resources, are gaining popularity again as environmental concerns
increase. Therefore, investigating plant-based dyes for textile applications is an essential first
step towards environmentally conscious and sustainable dyeing methods (Tuli et al., 2015).
Natural dyes have many benefits, but their commercialization is hindered by several issues that
prevent their widespread use. Among the main challenges are:
As natural dyes are frequently found in trace amounts in plant tissues, extraction is laborious and
ineffective (Handa, 2008). High production costs result from the Cuse of traditional extraction
techniques like solvent extraction and maceration, which frequently call for huge amounts of
plant material and lengthy processing durations (Tuli et al., 2015). Furthermore, temperature,
solvent choice, and pH all have a significant impact on extraction efficiency and are not usually
optimized for optimal yield (Kumari et al., 2024).
Poor wash, light, and rubbing resistance are some of the main drawbacks of employing natural
dyes in textile applications (Sufian et al., 2016). Natural dyes frequently need mordants, or metal
salts like alum, to improve their fixation and durability on textiles, in contrast to synthetic dyes,
which create strong chemical interactions with textile fibers (Geetha and Sumathy, 2013).
Researchers are searching for environmentally acceptable mordanting alternatives because the
usage of metal-based mordants may conflict with the sustainability objectives of natural dyeing
(Verma and Gupta, 2017).
Without the standardization and scalability required for industrial uses, the majority of natural
dyeing techniques continue to be small-scale or artisanal (Adeel et al., 2022). It is challenging to
create consistent colors, which is a crucial requirement in the textile business, due to color
variances brought on by variations in plant species, growing circumstances, and extraction
techniques (Hamdy et al., 2021). Natural dyes are derived from a range of sources, such as fungi,
micro-organisms, and the flowers, leaves, bark, roots, and fruits of plants. They are a desirable
substitute for synthetic dyes because they are typically non-toxic, biodegradable, and devoid of
dangerous chemicals (Geetha and Sumathy, 2013).
High-quality plant-based dyes may be obtained from natural sources such as Carissa carandas
(Karonda) and Alkanna tinctoria (Ratan jot). Alkannins, a type of red naphthalene pigment found
in Alkanna tinctoria, can give fabrics vivid red tones. Beyond its use in dyeing, it has also been
utilized historically for its anti-bacterial and anti-oxidant qualities (Shaheen et al., 2020).
However, the use of Carissa carandas for dye extraction is not only environment-friendly but
also supports sustainable agriculture and rural economic development by giving these plant
species an alternative use. Carissa carandas being rich in anthocyanin and flavonoids, can
produce red, blue, and purple hues depending on the pH conditions (Alino et al., 2014).
1.1 Carissa carandas
A perennial shrub with substantial economic, commercial, medicinal, and nutritional
potential, Carissa carandas(C. carandas) is a member of the Apocynaceae family
(Thiyagarajan et al., 2005). It is also called Crane berry, Karonda, Karamcha, and Ci-Huang-
Guo. It is abundantly found in tropical and subtropical areas of Bangladesh, Nepal, India, Sri
Lanka, Myanmar, and Pakistan (Begum et al., 2013). This plant features white flowers,
rhombus and conical leaves, and clusters (3-10) with berry-like fruits (Dalal et al., 2010).
When fully matured, the pinkish-white unripe fruit becomes scarlet to dark purple. Scurvy is
prevented by drinking fresh fruit juice, which is high in iron and vitamin C, and is low in iron
(Mahajan et al., 2022). In India, the visually appealing berry-sized fruits are typically used in
pickles and spices. The fruit of C. carandas is a great option for preparing drinks, jams,
jellies, and curries since it includes a significant amount of macro and micro nutrients,
organic acids, and pectin (Jayakumar and Muthuraman, 2018). A significant amount of macro
(carbohydrates, proteins, fat, and fiber) and micro (zinc, copper, and manganese) nutrients
are present (Jayakumar and Muthuraman, 2018). Carissa carandas is a deciduous, evergreen
shrub that belongs to the Apocynaceae family of dogbane and is typically 2-4 meters tall.
White latex is abundant on the stem, and the branches have angular spines. The flowers are
white and tiny, with a diameter of 3-5cm. the fruit is a berry that grows in clusters of three to
ten fruits. It is glabrous, has five to seven wings, five hard angles that curve upward, and is
woody and fibrous (Bhosale et al., 2020).
Taxonomical Classification
Kingdom Plantae
Division Angiospermae
Class Eudicots
Order Genitanales
Family Apocynaceae
Genus Carissa
Specie Carandas

1.2 Alkanna tinctoria

The Boraginacea family includes the perennial Alkanna tinctoria, also known as dyer's alkanet,
dyer's bugloss, orchanet, or Ratanjot. It is indigenous to Europe, the Mediterranean area, and
Asia, especially the Middle East (Alshamar and Dapson, 2023). The plant roots have a long
history of use in the food and dyeing industries, as natural colors in cosmetics and fabrics, and as
food additives for coloring wines and confections, due to the synthesis of alkannin pigment, a
naphthoquinone.The napthoquinone enantiomers, alkannin and shikonin, have demonstrated
strong antioxidant, wound healing, antimicrobial, and anti-inflammatory qualities. More recently,
their antitumor properties have been well understood through extensive research, suggesting that
these metabolites can be utilized as active ingredients in various pharmaceutical and cosmetic
preparations. Because of their many applications, they have been synthesized effectively and
manufactured in large quantities using cell tissue cultures (Assimopoulou et al., 2006).
Alkannin and Shikonin are the two main substances present in the plant roots. Plant cells
generate the red naphthoquinone pigment known as shikonin. Alkanin is the initial pigment
identified as the primary constituent of dark red pigments was extracted from the plant roots. It
was discovered in 1935 that shikonin and alkannin extracts were enantiomers of one another.
They have been recognized for the past 25 years to have anticancer action, which stops the
development of some skin malignancies during cancer therapy (Kaynar et al., 2014) The typical
phytochemical analysis was used to examine secondary metabolites, including alkaloids,
bufadienolides, carbohydrates, flavonoids, proteins, resins, saponins, tannins, phenols,
triterpenoids, steroids, gallotannins, and pseudotannins (Alshamar and Dapson, 2023).

Taxonomical Classification
Kingdom Plantae
Division Magnoliophyta
Class Dicotyledons
Order Boraginales
Family Boraginaceae
Genus Alkanna
Specie Tinctoria
1.3 Microbial-Assisted Extraction as a Novel Approach
Researchers are currently investigating microbial-assisted extraction as a cutting-edge and
environmentally friendly substitute for conventional extraction techniques to overcome their
drawbacks. By using bacterial enzymes to break down plant cell walls, this technique more
effectively releases bioactive dye chemicals (Sharma et al., 2018).
Plant cell wall components like cellulose, hemicellulose, and lignin are broken down by
hydrolytic enzymes produced by several bacterial strains, including Bacillus cereus and Bacillus
megaterium. By facilitating the release of phytochemicals, anthocyanins, and flavonoids, this
enzymatic degradation improves the effectiveness of dye extraction (Sohrabi et al., 2025).

1.4 Benefits of Microbial-Assisted Extraction

 Compared to traditional solvent extraction, enzymatic degradation of plant tissues gives
better yields because it permits a bigger release of color molecules (Sharma et al., 2018).
 By removing the need for harsh chemicals and organic solvents, microbial-assisted
extraction makes the process more environmentally friendly and sustainable (Tuli et al.,
2015).
 Dye molecules can be stabilized by microbiological treatment, increasing their color-
fastness and shelf life (Hamdy et al., 2021).
 Microbial-assisted extraction is scalable and economically feasible for industrial
applications since microorganisms may be grown in inexpensive culture media (Kıcık
and Gökbulut, 2023).

1.5 LIMITATIONS OF THIS RESEARCH

Notwithstanding the encouraging possibilities of microbially assisted plant dye extraction, it is
important to recognize the various limitations of this study. First, the bacterial strains, growing
circumstances, and extraction parameters can all affect how well microbial-assisted dye
extraction works, which could result in inconsistent yield and color intensity. Furthermore,
reproducibility may be impacted by the seasonal availability and regional differences in the
chemical composition of Alkanna tinctoria and Carissa carandas, although they were chosen for
their rich phytochemical content.
The reliance on particular bacterial strains, Bacillus cereus and Bacillus megaterium, which need
to be optimized for optimal effectiveness, is another drawback. Additionally, microbial extraction
could take longer than traditional techniques, which could be problematic for large-scale
commercial applications. Additionally, there are still issues with the stability of natural dyes in
textiles, particularly their fastness characteristics like wash and light fastness, which may call for
additional adjustments through mordanting or other processes.
For phytochemical Characterization, analytical procedures including FTIR, spectrophotometry,
and HPLC will be employed; nonetheless, these techniques might not fully comprehend all dye
components. Furthermore, life cycle evaluations are required to confirm the environmental
advantages of this technique and fully compare its sustainability to traditional dyeing techniques.
Finally, even though microbial-assisted extraction is a promising environmentally benign
substitute, more research is needed to determine its economic viability and industrial scalability.
Its immediate commercial feasibility may be limited by the intricacy of microbial cultures and
the requirement for specialized handling and monitoring. These drawbacks highlight the need for
more study to improve techniques and broaden the use of natural dyeing in the textile sector.
The potential of microbial-assisted extraction for dye extraction from Alkanna tinctoria and
Carissa carandas is still mainly unknown, even though it has been investigated in other
applications (Tuli et al., 2015). Prior research has concentrated chiefly on solvent-based
extraction techniques, ignoring the contribution of microbial bioconversion to improving dye
quality and production (Kıcık and Gökbulut, 2023). Furthermore, nothing is known about these
dyes' functional characteristics and how well they work on textile materials in terms of
colorfastness, homogeneity, and environmental friendliness (Adeel et al., 2022). By examining
the microbially assisted extraction of plant dyes and assessing the potential for sustainable textile
applications, the study seeks to close these research gaps.
1.6 Aims and Objectives
The main goal of this study is to use Carissa carandas and Alkanna tinctoria to create an
environmentally acceptable microbially assisted dye extraction method. The study’s particular
goals are as follows
 Extraction of Phytochemicals
Utilizing Bacillus cereus and Bacillus megaterium, the dye components of Alkanna tinctoria and
Carissa carandas were separated and extracted.
 Sustainable textile dyeing
To use phytochemicals collected from plants to create a sustainable textile dyeing method.
 Characterization of Dyes
Sophisticated spectroscopic methods like HPLC, NMR, and FTIR must be employed to examine
the extracted phytochemicals.
 Performance Evaluation:
To evaluate the dyed cotton fabric's general quality, colorfastness, and UV protection.

1.7 Goal of this research

The goal of this research is to create a scalable, sustainable, and effective natural dye extraction
process by combining microbial technology with natural dye sources. The results of this study
may open the doors for the widespread commercialization of microbial-assisted dyeing,
providing a competitive substitute for synthetic dyes in the textile sector (Geetha and Sumathy,
2013).
By encouraging the use of renewable plant-based dyes and lessening the environmental impact
of textile dyeing, this work supports global sustainability goals (Gnanavelu et al., 2021). By
providing a practical substitute for synthetic colors and resolving environmental concerns, the
textile industry may embrace natural dyes broadly if microbial-assisted extraction techniques are
successfully implemented (Patel et al., 2011). This research supports industry's innovation and
sustainability by improving the effectiveness and sustainability of natural dyes, which advances
the larger goal of green textile manufacturing.
This study uses microbial-assisted extraction methods for natural dyes to answer the pressing
need for sustainable textile dyeing solutions. This study has the potential to transform the
manufacturing of natural dyes by addressing the shortcomings of conventional extraction
techniques and addressing current research gaps, hence increasing its efficiency, scalability, and
environmental friendliness (Geetha and Sumathy, 2013). The results of this study will not only
advance scholarly understanding but also have real-world applications in the textile sector,
opening the door to more sustainable and environmentally friendly techniques.
CHAPTER 2 REVIEW OF LITERATURE
Dyes are the color-containing compounds in textiles, foods, and medicine. The properties of dyes
are different from those of pigments, as the particles of dyes are finer and have high solubility.
With the advancement in human society and overpopulation, the need for dyes has increased
with people's growing desire for colors. Documentations prove that the use of dyes initiated
when human society came into being. Using pigments and natural dyes provides continuous
coloring material, if these materials are obtained from a natural environmental method, as they
are inexpensive and produce a yield with better performance. Most synthetic dyes are not in use,
and natural dyes have supplanted many synthetic dyes in today's world, which is why many
companies are currently looking for natural dyes to utilize in their color goods to improve the
quality of their output. Natural dyes are still widely utilized in many nations, and their use has
not diminished. In conclusion, natural dyes have become more and more popular than synthetic
ones, which has raised demand from businesses looking to use natural dyes in their goods for
superior outcomes. In many nations, natural dyes are still commonly utilized despite the passage
of time (Yadav et al., 2023).

2.1 EXTRACTION SOURCES OF NATURAL DYES

Baths that are alkaline, neutral, or acidic can be used for dyeing. Natural dyes may be used to
color a wide range of cellulose, synthetic, and protein fibers. By utilizing various mordants,
colors can vary from brown to black and from yellow to orange. On the other hand, natural dyes
give textiles a smooth, eco-friendly finish and have lower production costs. Natural dyes are
being employed more and more. Studies have indicated that henna and marigold can be used to
color silk and cotton. Furthermore, flowers like Bixa, China rose, and Indigo are utilized for
coloring (Geetha and Sumathy, 2013).

Since we extract natural dyes from various plant sources, the main justification for doing so is
their provenance. Many plants have roots, leaves, stems, and bark that may be used to make
natural colors. Flowers may provide us with a wide range of hues. Sappan-wood yields the bright
pink dye that is most well-known. Another source is the exoskeleton of a little insect. Lime juice
is combined with shells to create a crimson or pink hue. Natural dyes are still employed in
businesses today despite the labor and time-intensive nature of this procedure; their hues are
incredibly calming and velvety (Vankar and Shukla, 2019). The primary usage of plants is in
extraction. The plant components that are extracted include the hulls, bark, fruits, flowers, leaves,
stalks, roots, and twigs. Indigofera tinctoria is the plant from which the well-known indigo dye is
also derived. Traditional medical systems, food, and medications all employ plant-

Natural dyes are extracted from natural materials such as plants (indigo, saffron), insects(lac
scale insects, cochineal beetles), animals(shellfish and some species of molluscs), minerals
(ferrous sulphate and clay). Natural dyes are obtained without the use of any chemical(Kadolph,
2008). For the coloration of fabrics, different chemicals are used in the textile industry. In this
case, synthetic dyes resuscitated the interest of consumers in natural dyes during the 20 th century.
The total share of natural dyes on textiles is 1% due to some technical issues involved, and they
are not available in the standard form. Overutilization of sources to manufacture dyes may result
in deforestation. To prevent this, the Global Organic Textile Standard(GOTS) allows the safe use
of synthetic dyes and not natural dyes. Various efforts are made to avoid the shortcomings of
natural dyes (Saxena and Raja, 2014). Colorants are obtained from various natural sources like
plants, insects, microbes, and animals have been explored for their use in the past. Research in
different applications using natural dye sources, being environment-friendly, robust, and cheap
techniques for processing, helps revolutionize the use of natural dyes and their scope in different
fields and for various applications (Shahid and Mohammad, 2013).

Baths that are alkaline, neutral, or acidic can be used for dyeing. Natural dyes may be used to
color a wide range of cellulose, synthetic, and protein fibers. By utilizing various mordants,
colors can vary from brown to black and from yellow to orange. On the other hand, natural dyes
give textiles a smooth, eco-friendly finish and have lower production costs. Natural dyes are
being employed more and more. Studies have indicated that henna and marigold can be used to
color silk and cotton. Furthermore, flowers like Bixa, China rose, and Indigo are utilized for
coloring (Geetha and Sumathy, 2013).
Reducing pollution is the primary goal of utilizing dyes derived from natural sources. Due to
growing environmental concerns and the demand for eco-friendly, biodegradable products,
natural dyes for the textile industry are currently the subject of numerous studies. Pollution from
synthetic dyes occurs not only when they are used to make textiles but also when they are
manufactured and used to make raw materials. The usage of natural colors drastically decreased
after the invention of synthetic dyes in 1856. However, cotton textiles were used to test natural
dyes to see how well the color held up after washing. The bark of Eucalyptus camaldulensis,
both with and without gamma radiation treatment, is used to make one such natural dye. Cotton
fabric was dyed using this to observe how the radiation impacted the color's intensity. To
maintain the color on the fabric, various mordants- chemicals that aid in fixing dye to fabric were
also tested (Yusuf et al., 2017).
Numerous chemicals and colors are used daily in the textile production process. The quantity and
composition of wastewater from textile factories make it one of the most hazardous types of
industrial waste. A significant amount of this hazardous effluent is frequently discharged directly
into rivers, streams, and canals. This is regarded as a substantial source of pollution that damages
crops, the environment, and farming land (Repon et al., 2016).
To put it simply, the chromophore, auxochrome, and dye matrix are the three essential
components of a dye molecule. The portion of the molecule that absorbs the light energy is called
a chromophore. Even in the medical area, chromophores are frequently found in synthetic colors.
Groups like azo, nitroso, thiocarbonyl, and nitro are examples of common chromophores. These
groups cause the molecule to exhibit color and absorb light energy. Another set of atoms that can
alter the dye's color is called an auxochrome. These groups might be acidic (SO 3, OH, COOH) or
basic (NH2, NR2, NHR). Auxochrome helps the dye attach to fabrics and modify its color. Other
atoms in the molecule interact with the dye's structure, called the matrix, which is the third
important part of the dye. Amino, azo, and carboxylic groups are difficult to handle using
standard techniques in textiles. The addition of auxochrome groups (such as hydroxyl, alkoxyl,
and amino groups) to a particular kind of chemical system known as a conjugated aromatic
system causes the molecules to interact and aid in the absorption of various light types, and
darker hues are developed as a result of this absorption (Benkhaya et al., 2020).
The organic substances known as dyes are soluble in water. Particularly simple, acidic, or basic
dyes dissolve readily in water, making it challenging to remove them using conventional
techniques. This is due to the chromophoric groups that give them their color in their chemical
structure. Because they are polar (have a small charge) and readily attach to textile fibers, the
auxochromic groups aid in the dye's adhesion (Lellis et al., 2019).
2.1 CLASSIFICATION OF DYES BASED ON CHEMICAL STRUCTURES
The primary method used to categorize natural dyes is their chemical structure. Based on their
unique characteristics, this makes it is simple to identify dyes that are part of a certain group.
Carotenoids, indigoids, quinonoids, and flavonoids are a few examples.

2.1.1 Indigoids
The two most significant categories of natural dyes are known as indigoids, namely Indigo and
Tyrian. They are some of the first dyes that people have ever utilized. One of the most significant
and precious coloring ingredients is natural indigo, a blue dye with a lengthy history. Plants such
as Polygonum tinctorium, Indigofera tinctoria, and Isatis tinctoria are used to make indigo.
Today, however, the majority of indigo is produced synthetically, with an annual production of
around 1000 tons. Indigo is renowned for its exceptional light fastness, or resistance to fading in
the presence of light.

2.1.2 Carotenoids
Nearly every plant has carotenoids, which are naturally occurring pigments. Chromoplasts and
chloroplasts are two examples of plant components that contain these tetraterpenoids. In addition
to plants, several bacteria and fungi may also produce carotenoids. Carotenoids' lengthy chain of
interconnected double bonds is what gives them their vibrant hues. They are red, orange, and
yellow because to their absorption of visible light, primarily between 400-500 nm. Among the
plants that generate carotenoids are Cedrela Toona, Bixa Orellana, Curcuma longa, and Crocus
sativus.

2.1.3 Flavonoids
Flavonoids are the most widely used plant dyes. Flavonoids may produce a variety of hues,
including orange, red, blue, and light yellow. Common sources of these dyes include plants such
as Artocarpus Heterophyllus (jackfruit), Allium Cepa (onion), Paterocarpus santalinus (red
sandalwood), and Cannabis Sativa (Hemp).

2.2.4 Quinonoids
Quinonoids are widely distributed in nature and range from yellow to red. They are a broad and
diverse class of pigments found in plants. They are separated into anthraquinones,
benzoquinones, and naphthoquinones based on their color and chemical makeup. Quinonoid-
containing plants include henna, garlic, safflower, walnut, and sundew (Yusuf et al., 2017). In the
past few decades, a wide variety of colorants have been derived from natural sources, including
plants, animals, microorganisms, and insects. These natural dyes have been researched and
applied in a wide range of disciplines and traditions. Natural dyes may be used more widely
since they are long-lasting, economical, and environmentally benign. Pomegranate peels were
gathered and crushed for this investigation. Equal parts crushed peels and a solvent were
combined to create the color, which was then heated for 60 minutes. The solution was then
filtered. The natural dyes were applied to cotton cloth using a water-ethanol mixture in a
specialized device known as a Soxhlet extractor. A substance known as a mordant was applied to
the cotton fabric before dyeing, since the colors made from pomegranate peels are light-
sensitive. To assess the dye's strength, researchers conducted a variety of studies, such as light
fastness and washing fastness tests, to see how well the dye holds up after washing
(Satyanarayana and Chandra, 2013).

2.2 Autoclave: The process of sterilization

The autoclave was developed in 1879 by the French microbiologist Charles Chamberland for use
in therapeutic settings. Robert Koch conducted studies on the disinfecting qualities of hot air and
steam. He explained how steam may pierce materials more effectively than dry heat. In 1933, the
first pressure steam sterilizer was finally released, controlling operation by measuring the
chamber drain lines' temperature (Blessing et al.). An autoclave is a massive pressure cooker that
sterilizes items by sealing the lid and forcing steam under pressure. Because high pressure allows
steam to reach a higher temperature, steam has a higher heat content and sterilizing power
(Talekar and Pise, 2023). When steam comes into contact with a colder surface, it rapidly
condenses to water and creates folds as a substitute; hence, by lowering the steam content, steam
may seep through objects with a drop in temperature. This causes negative pressure at the
condensation locations, which draws in more steam for greater condensation. A saturated steam
environment is created when the condensing surface is lower than the temperature of the steam
present, until temperature equilibrium is reached. Since steam can transfer more heat when there
is more moisture present, it is one of the most effective heat transporters. Moist heat destroys
microorganisms by making the cell non-viable by causing the coagulation of vital protein
components, such as the cytoplasmic membrane and nucleus. The pace at which bacterial cells
are thermally inactivated depends on the temperature and duration of heat exposure (Judelson,
2004).
The autoclave is the most efficient and successful sterilizing technique. In every autoclave, the
relationship between temperature and time is linear. It is necessary to establish these two
variables via verification, since they are essential. A greater temperature ensures quicker death.
Standard operating temperatures and pressures include 115 °C/10 psi. 121C/15p.s.i. and
132C/27p.s.i. for example. Greater weights, higher liquid volumes, and denser materials need
longer timeframes. A variety of microbiologic media, liquids, surgical dressings, glassware, and
many other objects may all be sanitized using an autoclave. When the proper conditions and
duration are met, no living things will make it through an autoclave (Talekar and Pise, 2023).

2.2.1 Working principle of an autoclave

Autoclave sterilizes materials inside the chamber using the wet heat sterilization concept, which
uses steam under pressure. The high pressure raises the water's boiling point, which aids in
raising the sterilizing temperature. Under normal atmospheric pressure (760 mm Hg), water
typically boils around 100 °C; however, if the pressure is raised, the boiling point of water rises.
Similarly, the high pressure makes it easier for heat to quickly enter deeper areas of the material,
and the moisture in the steam causes proteins to coagulate, which results in an irreversible loss of
microbial activity and function. An autoclave is typically operated with saturated steam at a
pressure of at least 30 minutes at a temperature of 121C °C. This idea is used in an autoclave,
where water boils at 121 °C while being compressed by 15 psi, or 775 mm Hg. When this steam
releases latent heat as it comes into contact with the surface, it kills the microorganisms. The wet
destruction of the microorganisms is guaranteed by the condensed liquid. The whistle allows the
pressure inside the chamber to be released when the sterilizing process is finished, which is
contingent upon the degree of contamination of the material inside. After that, the chamber's
internal pressure is returned to the surrounding air pressure while the internal components
continue to heat up for a while (Blessing et al.)

2.2 Microbial maceration

The maceration process involves utilizing liquids to soften and break up the tissue. The tissue
softens during maceration, and the substance within the tissue leaches out into the liquid
(extract). Numerous metabolites, including phenols, terpenes, flavonoids, and pigments, are
present in the resulting extract (Azwanida, 2015). Many researchers have employed this method
to produce wine from a variety of fruits, in which the chemicals leak out into the must (Joshi et
al., 2009).
Researchers have recently used a variety of microbes to extract different bioactive compounds,
including phenols, flavonoids, antioxidants, tannins, and saponins, from a wide range of
substrates, including fruits, vegetables, legumes, cereals, pulses, and agro-industrial waste. The
benefit of employing microorganisms is their ability to extract compounds from a variety of
sources and their ease of use in obtaining large yields, as well as their affordability and minimal
energy consumption (Demain and Fang, 2000). Several input elements in microbial maceration,
including duration, temperature, humidity, inoculum concentration, and other circumstances,
determine the effectiveness of the process. Furthermore, the effective development of the
bacteria to macerate and ferment various sources and improve the extraction efficiency depends
on sustaining microbial maceration conditions (Sharma et al., 2018).
Bacillus cereus
As a gram-positive, aerobic, or facultative anaerobic motile pathogen and opportunistic
bacterium, Bacillus cereus (B. cereus) can produce resistant endospores when oxygen is present.
The spores of B. cereus are persistent in unfavorable conditions and are widely dispersed in the
soil. Although B. cereus can thrive in a variety of temperatures (8- 55 °C), it is not well suited to
withstand low water content (minimum water activity 0.95) or pH values (minimum 5-6). The
hydrophilic properties of B. cereus endospores allow them to adhere to processing equipment
and withstand cleaning methods, and they are resistant to heat, radiation, disinfectants, and
desiccation. These microorganisms commonly contaminate food production, biotechnological
operations, and hospital settings. Both immunocompromised and immunocompetent patients are
susceptible to a variety of opportunistic infections caused by B. cereus. These infections include
severe eye infections, bacteremia, endocarditis, septicemia, severe meningitis, pneumonia,
gastritis, and skin infection, in addition to two different foodborne illness syndromes: diarrhea
and emesis. Diarrheal food poisoning can result from enterotoxins produced in the human small
intestine by B. cereus infection. When Bacillus cereus is intoxicated, a toxin called cereulide is
produced, which results in vomiting (Parihar, 2014).

Role of B. cereus in fabric dyeing

Bacillus cereus plays a crucial role in fabric dyeing due to its dye degradation and decolorization
ability. It decolorizes and degrades synthetic dyes, promoting the production of eco-friendly
pigments. The enzymatic and pigment production activity of B. cereus provides an alternative to
toxic and conventional dyeing techniques.

Dye degradation

Textile dyes decolorize in anoxic environments, producing colorless but poisonous metabolites,
as most of the research explains. However, aerobic degradation provides a simpler way to break
down azo dyes. Even though aerobic degradation has a lot of potential, it is still necessary to
determine how much hazardous intermediates that these dyes produce under aerobic
circumstances have degraded. The isolation and identification of a bacterial strain can break
down azo dyes, specifically Reactive Orange 16 and Reactive Black 5, in an aerobic
environment. To completely describe the biological breakdown of compounds, the impact of
several environmental factors, such as pH, temperature, and concentration of dye, was also
investigated. The rate of dye decolorization was examined using cell immobilization in contrast
to bacterial cells that were freely floating. Cell immobilization preserved a high biomass
concentration, shielded the cells from the deadly effects of dyes and their byproducts, was
economical, and could be reused (Pandey et al., 2020).

Phytochemical constituents in Carissa carandas

The chemical constituents present in Carissa carandas are terpenes, steroids, benzonoids,
lignin, sesquiterpenes, phenylpropanoids, and coumarins. Karonda fruits, primarily in the
maturation and ripening state, are rich in calcium, proteins, carbohydrates, fibers, and other
biologically active substances, which have anti-cancerous and antioxidant properties
(Patathananone et al., 2024).
The organic acids present in the unripe karonda fruits are fumaric acid, oxalates, succinic
acid, citric acid, and quinic acid. Out of all the organic acids, the maximum number is of
oxalates(5.88 mg/100g). The total number of carotenoids present was reported to be
55.89µg/100g sample. The carotenoids present in the unripe fruit are zeaxanthin and lutein.
The phenolic acids present in karonda are reported to be 3,4-dihydroxybenzoic acid,
protocatechuic acid, 2,4-dihydroxybenzoic acid, ferulic acid, vanillic acid, and o-coumaric
acid. Other phenolic acids, quercetin, naringenin, luteolin, (-)-epicatechin, (+)-epicatechin,
epigallocatechin-3-gallate, were reported to be present only in unripe fruits. The phytosterol
constituents in unripe fruits indicated the presence of stigmasterol, phytate, and campesterol
(Mishra et al., 2024).

CHAPTER 3
MATERIALS AND METHODS
Materials required
Sr # Materials
1 Distilled water
2 Bacteria
3 Nutrient broth

Equipments
Sr # Equipment
1 Funnels
2 Flask
 3 Beakers
4 Stirrer
5 Water bath
Equipment used
 Weighing balance
 Incubator
 UV-Visible spectrophotometer
 Grinding machine
 Water bath
 Dyeing machine
 Spatula
 Tongs or forceps
 Strainer
Glass wears
 Funnels
 Beakers
 Conical flasks
 Glass rod
 Test tubes
 Columns
 Thermometer
 Petri dishes
Chemicals required
Gallic acid
Quercetin
Sodium carbonate

Collection of samples
Fruits of Karonda (Carissa carandas) and roots of Ratanjot (Alkanna tinctoria) were gathered
from various locations in Faisalabad. After the plant roots and fruits were cleaned of impurities
 with water, they were allowed to dry in the shade before being pulverized into a fine powder
(Chauhan et al., 2015).

Dye extraction process

Extracting bioactive chemical compounds from plant sources is a crucial step in the process.
Various extraction techniques are employed to enhance the yield of bioactive chemicals, and the
sample should be carefully selected and have sufficient solubility to yield the desired results
(Jeyaraj et al., 2021). The effectiveness of extracting phytochemicals from natural sources, such
as plants and animals, depends on the type of media used, which can be acidic, basic, or organic;
other factors that must be considered include time, pH, temperature, and the mother liquor
(Samanta and Agarwal, 2009).

Plant powder Mordant Bacteria Conditions

Ratan Jot plant Salt +FeSO4+ Na2CO3 Bacillus cereus Autoclave
Neem+ Guava Temp. 121ºC
Neem+ Pomegranate Dyeing temp: 90°C

Salt +FeSO +Na2CO3 Bacillus megaterium Autoclave

Neem+ Guava Temp 121ºC
 Neem+ Pomegranate Dyeing temp: 90°C
Karonda Salt + FeSO4+Na2CO3 Bacillus cereus Autoclave
Neem+ Guava Temp. 121ºC
Neem+ Pomegranate Dyeing temp: 90°C

Salt + FeSO4 + Na2CO3 Bacillus megaterium Autoclave

Neem+ Guava Temp. 121ºC
Neem+ Pomegranate Dyeing temp: 90°C

3.5.1 Optimum conditions for incubation

Time 24 hours
Temperature 37ºC
Ratio 1:10
3.5.2 Scouring of fabric

Ready-to-use scoured fabric was provided by the NATIONAL TEXTILES UNIVERSITY,

FAISALABAD.
3.5.3 Application to cotton fabric

The cotton cloth was treated with mordants and dyes for that goal, and several procedures were
followed to guarantee superior outcomes
Time 1 hour
Temperature 90º C
Mordanting method Pre-mordanting method

The procedure of pre-mordanting was applied. Different mordants were applied to the cloth, and
it was left at 90°C for an hour. To determine which had the greatest outcomes, various natural
mordants (neem, guava, pomegranate peels) and synthetic ones (Sodium carbonate, Ferrous
sulphate, salt) were utilized.
3.5.4 Conditions for dyeing
Time 1 hour
Temperature 80º C
Ratio 1:1
The dyeing procedure was carried out at 80°C for one hour. The cloth was left in the solution to
get a uniform color using a 1:1 water-to-dye ratio.
3.6 Characterization of compounds
Various quantities of the same solvent were combined with the floral extract. Different
phytochemicals were then identified by testing at different wavelengths (Dhivya and Kalaichelvi,
2017). A variety of active compounds with various characteristics are often present in plant
extracts. It is difficult to distinguish and identify them. Scientists employ methods such as High-
performance liquid chromatography(HPLC) and column chromatography to do this. These
techniques aid in the isolation of pure substances for application in biological and chemical
research (Sasidharan et al., 2011).
 Characterization of plant extract
 Characterization of dyed fabric

3.6.1 HPLC (High Performance Liquid Chromatography)

One technique for removing particular components from a mixture is HPLC. It aids in
identifying various substances within a sample. HPLC is beneficial for the isolation of natural
chemicals. In chromatography, this method is regarded as one of the most dependable and widely
applied approaches (Sasidharan et al., 2011). Molecules can be swiftly purified and their
structure and function studied using high-performance liquid chromatography (HPLC). At room
temperature, an HPLC UV-Visible detector tuned to 280nm was utilized for detection. The range
of the gradient HPLC was 125 mV. A CLC-ODS(C-18) type column measuring 25 cm by 4.6mm
and having a particle size of 5 micrometers was employed. Throughout the run, the mobile phase
gradient- the liquid moving through the column- changed. There were two liquids used;
Liquid A has a pH of 2.27 and is composed of water and acetic acid (ratio 9.4:6)
Solution B: 1ml/min of 100% acetonitrile used during 0-15 minutes.
3.6.2 FTIR (Fourier Transform Infrared Spectroscopy)
The majority of molecules absorb infrared light, which makes it easier to determine the kinds of
chemical bonds they have. These chemical bonds are discovered using FTIR, which examines
the molecules' light absorption. It is possible to identify the various chemical groups in a material
by reviewing the FTIR spectrum. FTIR was used to evaluate the sample, and the peaks in the
spectrum were used to identify the sorts of substances present. The presence of compounds such
as alcohols, carboxylic acids, ketones, aldehydes, and alkanes was verified by FTIR (Shalini and
Sampathkumar, 2012).
3.6.3 Determination of antimicrobial activity
Samples were tested against a variety of microorganisms, including the beneficial Bacillus
subtilis and the dangerous E. coli. The bacteria used in the tests were verified by the University
of Agriculture's Microbiology Department in Faisalabad, Pakistan. On nutrient agar, the bacteria
were cultured for the entire night at 37°C.
The diffusion method was employed to test the antibacterial effect. The agar was covered with a
solution of 10^7 CFU/mL, which is a measure of the quantity of bacteria. The agar plates
containing the bacteria were covered with filter paper discs (6mm) containing the sample
(Balouiri et al., 2016).
A disc containing the antibiotic rifampicin (30g/dish) served as the positive control, while a disc
containing no sample served as the negative control. After two hours at 4°C, the plates where the
bacteria did not grow around the discs were compared to the control; the antibacterial impact was
quantified (Carvalho et al., 2023).

3.7 Spectroscopic Analysis

3.7.1 Analysis of total phenolic content
The Folin-Ciocalteu technique was used to quantify the quantity of phenolic compounds in
methanol extracts (Naz et al., 2016). To create a standard curve, several gallic acid
concentrations (ranging from 0.01 to 0.10 mg/ml) were employed. Five milliliters of ten-fold
diluted Folin-Ciocalteu reagent were combined with one milliliter of each gallic acid solution,
and an hour later, four milliliters of 20% sodium carbonate were added. After an hour, the
absorbance, or light absorption, was measured at 765nm. Plotting a standard curve was aided by
this measurement. One milliliter of a plant extract (0.01g/ml) was combined with the same
substances, and the absorption at 765 nm was also measured to determine the phenolic content of
the extract. Every test was conducted three times (Sharif et al., 2018).
Gallic acid equivalents (GAE), a measure of phenolic content, were calculated using the
following formula:
T= C*V/M
Where,
C = gallic acid concentration calculated from the calibration curve in mg/ml.
T = total contents of phenolic compounds in mg GAE/g plant extract.
M = the weight of plant extract in grams
V = the volume of extract in ml.

3.7.2 Total flavonoid content

The detailed procedure for the total flavonoid content of extracted plants was evaluated by using
the method described by (Hossain et al., 2013).
Initially, 0.15 ml of a 10% sodium nitrite (NaNO 2) solution, 2 ml of distilled water, and 0.5 ml of
the plant extract were combined. Six minutes were spent with this concoction. After that, 0.15
mL of a 4% aluminum chloride (AlCl 3) solution was added, and the mixture was then well
agitated before methanol was added to bring the total amount up to 5 mL. After the combination
was left for 15 minutes, a spectrophotometer was used to measure the absorbance, or color
intensity, at 510nm (Ayub et al., 2017). TFC of the extracts was expressed as μg catechin
equivalents per mL of the plant extracts from the linear regression curve of the standard catechin
used.
3.7.3 Antioxidant activity (DPPH)
The antioxidant activity of the plant extracts developed by (Shahid Chatha et al., 2014) was
tested using the 2,2-diphenyl-1-picrylhydrazine (DPPH) radical scavenging assay method. In this
method, 3mL of the plant extract was combined with 1mL of freshly prepared 0.004% DPPH
solution in methanol. For 30 minutes, this combination was kept in the dark. Its absorbance at
517nm was then measured. A sample with high antioxidant (radical-scavenging) activity has a
low absorption. For comparison, ascorbic acid was used as a standard and was also examined.
There was a control (without plant extract and bacterial extract) sample (Naseem et al., 2020).
Every experiment was conducted in triplicate. The following formula was used to compute the %
inhibition of DPPH radical samples.
DPPH inhibition (%) = absorbance of blank- absorbance of sample/ Blank absorbance
Where
A1 absorbance of the sample
A0 = absorbance of the sample
From the graph of inhibition% % versus sample concentration, the concentration of the sample
providing 5% (IC50) was computed.

3.8 Biological oxygen demand

The quantity of oxygen required by microbes to decompose biodegradable organic matter in
water is known as BOD. It is evaluated in particular settings, such as in the dark to prevent algae
from producing oxygen, for five days. This test aids in determining the level of organic waste
pollution in the water.

3.9 Chemical oxygen demand

The COD is the quantity of oxygen needed to use chemicals to oxidize and break down organic
molecules in water. It displays the quantity of organic materials or pollution. COD provides a
more accurate picture of overall pollution and is consistently greater than BOD. It aids in
estimating the amount of organic matter released into the environment. COD can be calculated
using two methods.
 Closed reflux method ( it requires only a minute amount of metallic salt reagents and
produces a small amount of hazardous waste)
 Open reflux method ( a wide range of wastes requires a large amount of sample)
The chemical oxygen demand will be performed within 24 hours. COD was performed through
the reflux method.
A series of known standards is prepared using potassium hydrogen phthalate (KHP) for the
completion of the chemical oxygen demand test, as wastewater samples are commonly prepared
in a high standard range of 100, 250, 500, and 1000 mg/L. The COD heating block is both
activated to allow the instruments to stabilize. The COD test requires pre-prepared high-range (3-
50 ppm) or low-range (20-150 ppm) vials, based on the expected results. If the expected results
are unknown, either range can be used.

CHAPTER 4
RESULTS AND DISCUSSIONS
4.1 HPLC (High Performance Liquid Chromatography)
For the identification and isolation of natural compounds, the high liquid performance
chromatography technique is used, which is the most vigorous and widely used technique. This
technique is used for the extraction or separation of targeted components from other compounds.
HPLC identification of various compounds is a crucial step in the HPLC assay (Sasidharan et al.,
2011).
By using High Pressure Liquid Chromatography(HPLC), it is possible to investigate the
functional and structural analysis, as well as the rapid purification of molecules. This technique
is also helpful in the separation and identification of carbohydrates, lipids, nucleic acids,
proteins, amino acids, steroids, and other biologically active compounds. At a high pressure of
10- 400 atm and a high rate of (0.1-5cm/ sec), the mobile phase flows through the HPLC column.
The rate of solvent flow and the use of tiny particles in this technique increase the separation
power of HPLC, and the analysis is also completed in a short time.
For the retardation of sample components, HPLC column chromatography is designed in such a
way that the molecules of sample have enough opportunities to contact the solid phase. Many
columns possess a volume of more than 1 liter and are a few meters long. Various columns are a
few centimeters, and some have a volume of less than 10 milliliters. Moreover, there are limited
advantages of changing the column dimension. The efficiency of the column is improved when
the size of the particle decreases from 250 micrometers to 5 micrometers for two reasons. The
solvent volume and sample molecules are exposed by the solid phase area( for the volume of
any column) so the diffusion time decreases between the particle size. With these changes, there
is an increase in sample molecule dissolved in solvent and the solvent will come in contact wih
solid phase for retardation. However, there are some practical issues with the reduction in the
size of particles of solid phase, and the back of column increases with the reduction in particle
size and space among the particles shrinks at normal pressure with better physiochemical
properties. Various solid phases like cellulose or organic resins(005kpa) cannot provide a high
performance when there is a reduction in particle size because they would collapse at increased
working pressure of 3-5kpa (Bird, 1989).
The HPLC equipment is composed of several parts like solvent reservoir that contain the mobile
phase, a pressure gauge for monitoring the pressure of the pump, a high pressure for pushing the
mobile phase through a packed, tight column, an injector device for sample introduction into the
moving mobile phase normally a sample loop, a column 10-30 cm long tube consisting of 3-4nm
stainless steel packed (-10/an) of material for separation, detector usually a variable wavelength
UV- spectrophotometer, which is used to measure the concentration of sample components as
they are eluted from the column (McNair, 1984).
4.2 FTIR (Fourier Transform Infrared Spectroscopy)
The absorption or emission infrared spectrum of a liquid, gas, or solid can be achieved using
FTIR spectroscopy. The FTIR spectrometer involves high-resolution information over a wide
spectral range between (4000 and 400cm-1) at the same time, which is the most important
advantage over a dispersive spectrophotometer that calculates power over a narrow range of
frequencies at the same time. The main goal of spectroscopy technique FTIR-UV visible is to
investigate how much the sample absorbs light and then recalculating it for each frequency.
Dispersive spectroscopy mainly focusses on monochromatic beam of light at the sample,
calculating the amount of light absorbed and then calculating it for each frequency. FTIR is a less
responsive method for obtaining comparable data. FTIR tells us that most molecules absorb light
in the infrared region of electromagnetic spectrum. This type of absorbance is based on the
molecules bond. We can also confirm the functional components present in the plant extracts,
identifying the medicinal constituents from the adulterants and even the evaluation of medicinal
component qualities using FTIR. Based on the peak values in the infrared radiation region, FTIR
can be used to identify the functional groups of the bioactive components. The powder of
different plants was evaluated and the functional group of various components was separated on
peak ratio.
At start, it was hoped for using mordant fibers, and precursors of dye would result in better
library matches. When applied to freshly dyed fabrics, the overall degree of sensitivity was not
enough to obtain any good identification. The challenges and concerns explained by the extent of
chemical bonding between dye and fabric, which was obtained through the use of mordants. As a
conclusion, library matches under such conditions was not good. To address this issue, many
combinations of fibers mordants were used. The system was continuously tested to compare the
obtained matched values.
4.2.1 FTIR of extracts
Ratanjot Bacillus cereus
Sr# Wavenumber Wavenumber Functional Expected
cm-1(test cm-1(for group compound
sample) reference) assessment
1 1640 cm-1 16450-1600 cm-1 C=O stretch Ketone
2 3324cm-1 3200-3600 cm-1 O-H stretch Alcohols,
N-H stretch phenols, amines,
and amides
3 514 cm-1 1200-700 cm-1 C-C bond Alkanes, esters
C-N stretch Amines
C-O stretch

In the FTIR analysis of Ratanjot B. cereus, 3 different peaks were observed. The peak that was
observed at 1640 cm-1 shows the expected compound of a ketone. The peak that was observed at
3324 cm-1 indicates Alcohols, phenols, amines, and amides with O-H and N-H stretch. The
observed peak of 514 cm-1 showed a C-C bond along with C-N and C-O stretch, indicating
alkanes, esters, and amines.
Karonda Bacillus cereus

Sr# Wavenumber Wavenumber Functional group Expected

cm-1 cm-1 assessment compound
Test sample For reference
s1 1632 cm-1 1650-1600 cm-1 C=O stretch Ketone group
2 2996 cm-1 2850-3000 cm-1 C-H stretch Alkanes
3 3362 cm-1 3200-3600 cm-1 O-H, N-H Alcohols,
stretch phenols, amines
4 566 cm-1 500-600 cm-1 C-Br stretch Alkyl halide( Br)
5 574 cm-1 500-600 cm-1 C-Br/ C-Cl Alkyl halide
stretch
6 611 cm-1 600-800 cm-1 C-Cl/ C-Br Alkyl-halide
 stretch (chloride/
bromide)
In the FTIR analysis of Karonda B. cereus, 6 different peaks were observed. The peak that was
observed at 1632 cm-1 shows expected compounds of the ketone group. The peak that was
observed at 2996 cm-1 shows the expected compound to be Alkanes. The third peak at 3362 cm -1
shows that the compounds are alcohols, phenols, and amines. The fourth peak at 566 am -1
indicates that the compounds expected are C-Br (bromides). The fifth peak observed at 574 cm -1
shows that the expected compounds are alkyl halides. The sixth peak observed at 611 cm -1 shows
that the expected compounds are alkyl halides (chloride/ bromides).

FTIR of Ratanjot Bacillus megaterium

 Sr # Wavenumber Wavenumber Functional Expected
cm-1 cm-1 group compound
Test sample For reference assessment
1 1647 cm-1 1600-1700 cm-1 C=O Ketone,
aldehyde
2 2124cm-1 2100-2260 cm-1 C=C Alkyne,
C=N Nitrile
3 3317 cm-1 3200-3600 cm-1 O-H, N-H Alcohol,
Phenol
4 644 cm-1 600-800 cm-1 C-Cl Alkyl halide (Cl)

In the FTIR analysis of Ratanjot B. megaterium, a total of 4 peaks are observed. The peak at
1647 cm-1 shows the C=O group and is expected to be a ketone and an aldehyde. The other peak
at 2124 cm-1 shows C=-C and C=-N, showing alkyne and nitrile compounds. The third peak at
3317 cm-1 shows O-H and N-H groups with expected compounds, alcohol and phenols. The last
peak at 644 cm-1 shows alkyl halides, mainly the C-Cl bond.
Karonda Bacillus Megaterium
Sr # Wavenumber Wavenumber Functional Expected
cm-1 cm-1(for group compound
(Test sample) reference) assessment
1 1632 cm-1 1600- 1680 cm-1 C=C stretch Alkenes
C=O stretch Amides
2 3309 cm-1 3300-3500 cm-1 N-H Alcohol/
O-H phenol

3 424 cm-1 400-500 cm-1 M-O/ M-N Fe/Cl

4 551 cm-1 500-600 cm-1 C-Cl/ C-Br Alkyl/aryl
halide
in the FTIR analysis of Karonda B. megaterium, a total of 4 peaks are observed. The first peak at
1632 cm-1 corresponds to the C=C stretch and C=O stretch, as expected for alkenes and amides.
The next peak at 3309 cm-1 shows N-H and O-H stretch with alcohols and phenols as expected
compounds. The next peak at 424 cm -1 shows metal-oxides, mainly Fe/Cl. The next peak at 551
cm-1 indicates the presence of C-Cl/C-Br and the expected compounds are alkyl or aryl halides.
Ratan Jot only
Sr # Wavenumber Wavenumber Functional Expected
cm-1 cm-1 group compound
Test sample For reference assessment

1 1640.28 cm-1 1680-1640 cm-1 C=C Conjugated

C=O alkene or
Amide
carbonyl
2 3309.87 cm-1 3400-3250 cm-1 N-H Amine /alcohol
O-H
3 439.8 cm-1 400-600 cm-1 M-X /M-O Metal ligand
4 514.3 cm-1 690-515 cm-1 C-Br Alkyl bromide

In the FTIR analysis of the Ratanjot plant only, a total of 4 peaks are observed. The first peak at
1640.2 cm-1 shows conjugated alkene or amide carbonyl. The second peak at 3309.87 cm -1
indicates the presence of N-H and O-H bond stretches, suggesting the presence of an amine or
alcohol. The third peak at 439.8 cm-1 indicates the presence of metal oxides or metal ligands. The
fourth peak shows alkyl bromide at 514.3 cm-1.
Bacillus megaterium only
Sr # Wavenumber Wavenumber Functional Expected
cm-1 cm-1(for group compound
(test sample) reference) assessment
1 1632.5 cm-1 1630-1650 cm-1 C=O Stretch/ Amides,
C=C phenols
2 2132.72 cm-1 2100-2200 cm-1 C=-C Alkyne group
3 3332.2 cm-1 3300-3500 cm-1 O-H Alcohols,
N-H stretch phenols,
amines
4 574.01 cm-1 500-600 cm-1 C-Br stretch Halogens(Br)
5 596.3 cm-1 500-600 cm-1 C-Cl bending Halogenated
aromatic
 compounds

In the FTIR analysis of B. megaterium, a total of 5 peaks are observed. The first peak at 1632
cm-1 shows amides or phenols with C=O and C=C stretching. The second peak at 2132.74 cm -1
shows the alkyne group. The third peak at 3332.2 cm -1 shows O-H and N-H stretch with expected
compounds to be alcohols, phenols, or amines. The fourth peak at 574.01 cm -1 shows halogens
with C-Br stretching. The fifth peak at 596.3 cm -1 shows Halogenated aromatic compounds,
mainly metal-complex interactions with C-Cl bending.

4.3 FTIR of dyed fabrics

4.3.1 The fabric of Ratanjot Bacillus cereus(NG)
 Sr # Wavenumber Wavenumber Functional Expected
test sample reference group compound
assesment
1 1028.8 cm-1 1020-1100 cm-1 C-O Stretch Alcohol,
phenols, ethers
2 1088.8 cm-1 1080-1100 cm-1 C-O stretch Polysaccharides
3 1180.5 cm-1 1170-1190 cm-1 C-O stretch Esters, ethers
1312.3 cm-1 1300-1330 cm-1 C-N, Amines,
4 O-H bending phenolic O-H
bending
5 1495.7 cm-1 1450-1500 cm-1 C=C stretch Aromatic rings
6 289.1 cm-1 300-400 cm-1 M-X bond Metal ligand
7 3312.3 cm-1 3000-3300 cm-1 O-H Alcohols,
N-H stretch phenols
8 644.7 cm-1 400-600 cm-1 C-Cl stretch Alkyl halide or
bending

The FTIR of Ratanjot B. cereus and mordants Neem and guava shows a total of 8 peaks. The first
peak at 1028.8 cm-1 shows the functional group to be C-O stretching and the expected
compounds to be alcohols, phenols and ethers. The next peak at 1088.8 cm -1 shows the
wavenumber range to be 1080-1100 cm -1 which shows C-O stretch as functional group
assessment and the expected compound to be polysaccharides. The third peak at 1180 shows a
wavenumber range of 1170-1190 cm-1, showing the functional group to be C-O stretch and the
expected compounds to be esters and ethers. The fourth peak at 1312.5 cm -1 shows a
wavenumber range of 1300-1330 cm-1 with the functional groups to be C-N and O-H bending,
and the expected compounds to be amines, phenolic O-H bending. The fifth peak at 1495.7 cm -1
shows the functional group to be C=C, a C-C double bond of the wavenumber range of 1450-
1500 cm-1 and the expected compounds to be aromatic ring compounds. The next peak at 289.1
cm-1 shows 300-400 cm-1 wavenumber range which shows M-X bond and compounds expected
to be are the metal ligand bonds. The next peak at 3312.3 cm -1 shows the functional groups to be
O-H or N-H stretching with the expected compounds to be alcohols and phenols. The last peak at
644.7 cm-1 shows the wavenumber range of 400-600 cm -1 shows C-Cl stretching and expected
compound to be alkyl halide.

RBC-Np
 Sr # Wavenumber Wavenumber Functional Expected
sample reference group compound
assessment
1 1025.8 cm-1 1300-1000 cm-1 C-O stretch Alcohol, ether
2 1091.7 cm-1 1300-1000 cm-1 C-N stretch Amine
3 1189.1 cm-1 1470-1450 cm-1 CH2 bend Alkane/ ester
4 1312.3 cm-1 1020-1320 cm-1 C-O stretch Alcohol/ ester
5 1461.3 cm-1 1320-1000 cm-1 C-O stretch Alcohol
6 1742.1 cm-1 1750-1735 cm-1 C=O Ester
7 2851 cm-1 3000-2850 cm-1 C-H stretch Saturated
hydrocarbons
8 2922.6 cm-1 3000-2850 cm-1 C-H stretch Alkanes
9 3346.7 cm-1 3200-3550 cm-1 Broad O-H Alcohol,
phenol
10 916.9 cm-1 900-650 cm-1 =C-H / O-H Alkene

The FTIR of Ratanjot B. cereus and mordants Neem and Pomegranate shows a total of 10 peaks.
The first peak at 1025.8 cm-1 corresponds to the C-O stretch and is expected to be characteristic
of alcohols and ethers. The next peak at 1091.7 cm-1 corresponds to the C-N stretch, and the
expected compound is an Amine. The third peak at 1189 cm-1 shows CH2 bending with the
expected compound to be alkane/ ester. The fourth peak at 1312 cm-1 shows the functional group
to be C-O stretch and the expected compound to be alcohol/ ester. The fifth peak at 1461 cm-1
shows the wavenumber range of 1320-1000 cm-1, and the functional group is C-O stretch, and
the expected compound is alcohol. The next peak at 1742 cm-1 shows the wavenumber range
1750-1735 cm-1 with the functional group to be C=O and expected compound ester. The next
peak at 2851 cm-1 with C-H stretch and is expected to be saturated hydrocarbons. The next peak
at 2922.6 cm-1 with a 3000-2850 cm-1 wavenumber range shows a C-H stretch and is expected to
be alkanes. The peak at 3346 cm-1 shows the functional group to be a broad O-H stretch, with the
expected compounds to be alcohol or phenols. The last peak at 916 cm-1 shows a wavenumber
range of 900-650 cm-1, with the expected compounds to be alkene and =C-H or O-H stretch.
RBC-SALT

Sr # Wavenumber Wavenumber Functional Expected

sample reference group compound
assessment
1 1031.5 cm-1 1020-1060 cm-1 C-O stretch Phenols, ethers
2 1085.9 cm-1 1080-1100 cm-1 C-O stretch Alcohols, esters
3 1214.9 cm-1 1200-1220 cm-1 C-O stretch/ C-N Esters
stretch
4 1318 cm-1 1300-1330 cm-1 O-H bending Amines
5 1398.3 cm-1 1380-1410 cm-1 O-H bending / Alcohols,
C-H deformation alkanes
6 2166.2 cm-1 2100-2260 cm-1 C=- C stretch Alkynes
7 2378.2 cm-1 2350-2390 cm-1 CO2 Plant compound
interference
8 2908.3 cm-1 2850-2960 cm-1 C-H stretch Alkanes
9 3343.8 cm-1 3300-3400 cm-1 O-H broad Alcohol/ phenols
stretch
The FTIR of Ratanjot B. cereus and mordants, Salt, FeSO4, and Na2CO3 shows a total of 9 peaks.
The first peak at 1031.5 cm-1 corresponds to the C-O functional group and is expected to be
phenols and ethers at a wavenumber range of 1020-1060 cm -1. The second peak at 1085.9 cm-1
shows C-O stretch, with the expected compounds to be alcohols and esters. The third peak at
1214.9 cm-1 shows C-O stretch and C-N stretch, with expected compounds to be ester. The fourth
peak at 1318 cm-1 in the range of 1300-1330 cm-1 is associated with the functional group to be O-
H bending with the expected compound to be Amines. The fifth peak at 1398.3 cm -1 is associated
with the functional group to be O-H bending or C-H deformation with expected compounds to be
alcohols and alkanes. The sixth peak at 2166.2 cm -1 shows a C-C triple bond, with the expected
compound to be an alkyne. The next peak at 2378.2 cm-1 shows a wavenumber range of 2350-
2390 cm-1, with the expected compound to be a plant compound; CO2 interference. The next
peak at 2908.3 cm-1 shows C-H stretch and is expected to be alkanes, and the wavenumber range
of 2850-2960 cm-1. The last peak at 3343.8 cm-1 shows a wavenumber range of 3300-3400 cm-1
and functional group to be O-H broad stretching, and expected compounds to be alcohol and
phenols.
KBC-NG
Sr # Wavenumber Wavenumber Functional group Expected
sample reference assessment compound
1 1034.4 cm-1 1075-1020 cm-1 C-O Stretch Alcohol,
aliphatic amine
2 1191.9 cm-1 1200-1185 cm-1 C-O, C-N Alcohol, ester,
ether
3 1389.7 cm-1 1390-1380 cm-1 C-H bending Aldehyde
C-H stretch Amine
4 3332.4 cm-1 3400-3200 cm-1 O-H Alcohol
N-H Phenols
5 851 cm-1 900-675 cm-1 Alkyl halide, C- Chloride or
H bending bromide

The FTIR analysis of Karonda B. cereus with the mordants neem and guava shows a total of 5
peaks. The first peak at 1034.4 cm-1 shows a wavenumber range of 1075-1020 cm -1 and
functional group assessment to be C-O stretching, and the expected compound to be alcohol and
aliphatic amine. The second peak at 1191.9 cm -1 shows a wavenumber range of 1200-1185 cm -1
with the functional group assessment to be C-O and C-N stretch, and expected compound to be
alcohol, ester, or ethers. The third peak at 1389.7 cm -1 shows a wavenumber range of 1390-1380
cm-1 with functional group assessment to be C-H bending and C-H stretch, and expected
compound to be aldehyde or amine. The fourth peak at 3332.4 cm -1 shows a wavenumber range
of 3400-3200 cm-1 and functional group to be O-H or N-H stretching, with the expected
compounds to be alcohols or phenols. The fifth peak at 851 cm -1 shows a wavenumber range of
900-675 cm-1 with the functional groups to be alkyl halide or C-H bending, and the expected
compound to be chloride or bromide.
KBC-NP
Sr # Wavenumber Wavenumber Functional group
Expected
sample reference range assessment compound
1 1034.4 cm-1 1020-1220 cm-1 C-O stretch Alcohol, ether
2 1220.6 cm-1 1020-1260 cm-1 S=O, C-N Aromatic ether,
sulfonate
3 1521.5 cm-1 1500-1600 cm-1 C=C Nitro, aromatic
compound
4 1991.4 cm-1 2000-1900 cm-1 C=C=C, C=C=N Allene,
ketenimide
5 2160 cm-1 2160-2120 cm-1 =-C-H, N3 Azide, alkyne

The FTIR analysis of Karonda B. cereus and mordants, including neem and pomegranate, reveals
a total of 5 peaks. The first peak at 1034.4 cm -1 shows the C-O stretch with a wavenumber range
of 1020-1220 cm-1 and is expected to be an alcohol or ether. The second peak at 1220.6 cm -1
shows a wavenumber reference range of 1220-1260 cm-1 with the functional group to be S=O
and C-N, and the expected compound to be aromatic ether and sulfonate. The third peak at
1521.5 cm-1 shows a wavenumber range of 1500-1600 cm-1 and functional group assessment to
be C=C and expected compound to be nitro and aromatic compound. The fourth peak at 1991.4
cm-1 corresponds to a wavenumber range of 2000-1900 cm -1, indicating functional groups of
C=C=C and C=C=N. This suggests the expected compounds are allene and ketenimide. The last
peak at 2160 cm-1 shows the functional groups to be =-C-H and N3, with the expected compound
to be azide or alkyne.

KBC-SALT
Sr # Wavenumber Wavenumber Functional Expected
sample reference range group compound
assessment
1 1022.9 cm-1 1000-1100 cm-1 C-O stretch Alcohols, ethers
2 1163.3 cm-1 1150-1200 cm-1 C-O stretch Ether or tertiary
alcohol
3 1220.6 cm-1 1220-1260 cm-1 C-O stretch Ether or ester
4 1426.9 cm-1 1450-1370 cm-1 C-H bending Alkane bending
5 2160.4 cm-1 2150-2250 cm-1 C=-C or N3 Alkyne, azide
6 2372.5 cm-1 2400-2000 cm-1 O=C=O CO2
contamination
7 2899.7 cm-1 2850-2950 cm-1 C-H stretch Alkane
8 3315.2 cm-1 3310-3350 cm-1 =-C-H /N-H Alkyne, amine/
amide
9 908 cm-1 900-915 cm-1 =C-H bend Vinyl/alkene
 bending

The FTIR analysis of Karonda B. cereus with mordant salt, FeSO4, and Na2CO3 shows a total of
9 peaks. The first peak at 1022.9 cm-1 shows a 1000-1100 cm-1 reference wavenumber range with
functional group to be C-O stretch and expected compounds to be alcohols and ethers. The
second peak at 1163.3 cm-1 shows the expected compound to be ether or tertiary alcohol, and the
functional group to be C-O stretching. The next peak at 1220.6 cm -1 shows a wavenumber range
of 1220-1260 cm-1 and the functional group to be C-O stretch, and is expected to be ether or
ester. The next peak at 1426.9 cm -1 shows C-H bending at wavenumber range 1450-1370 cm -1,
and the expected compound is alkanes. The next peak at 2160.4 cm -1 shows C=-C or N3 shows
the expected compound to be an Alkyne or an azide. The next peak at 2372.5 cm -1 shows the
functional group to be O=C=O, and the compound may have CO 2 contamination. The next peak
at 2899.7 cm-1 shows the functional group to be C-H stretch and the expected compound to be
alkanes. The next peak at 3315.2 cm-1 shows =-C-H or N-H functional group and is expected to
be alkyne, amine, or amide. The last peak at 908 cm -1 shows the functional group to be =C-H
bending, with the expected compound to be vinyl or alkene bending.

RBM-NG
 Sr # Wavenumber Wavenumber Functional group Expected
sample reference assessment compound
1 1025.8 cm-1 1100-950 cm-1 C-O stretch Alcohol/ ether
2 1085.9 cm-1 1100-950 cm-1 C-O stretch Alcohol/ ether
3 1140.4 cm-1 1150-1100 cm-1 C-O stretch Alcohol/ ether
4 1154.7 cm-1 1150-1100 cm-1 C-O stretch Alcohol/ ether
5 1191.9 cm-1 1200-1185 cm-1 S=O stretch Sulphate
6 1309.4 cm-1 1372-1290 cm-1 N-O stretch Nitro
compounds
7 1401 cm-1 1400-1330 cm-1 O-H bending Phenols/ C-H
aromatic
8 2031.5 cm-1 2140-2100 cm-1 C-=C stretch Terminal alkyne
9 2169 cm-1 2260-2222 cm-1 C-=C stretch Disubstituted
alkyne
10 2598.8 cm-1 2600-2550 cm-1 S-H stretch Thiol
11 3309.5 cm-1 3310-3200 cm-1 -=C-H stretch Terminal alkyne
12 905 cm-1 915-905 cm-1 C-H bending Mono-
substituted
alkene

The FTIR analysis of Ratanjot B. megaterium and mordants Neem and Guava shows a total of
12 peaks. The first peak at 1025.8 cm-1 shows C-O stretch with the expected compound to be
alcohol or ether. The second peak at 1085.9 cm -1 shows C-O stretch with the expected compound
to be alcohol or ether. The third peak at 1140.4 cm -1 with the wavenumber range of 1150-1100
cm-1 shows C-O stretch, and the functional group is C-O stretch, and the expected compound is
alcohol or ether. The fourth peak at 1154.7 cm -1 shows C-O stretch, with the expected compound
to be alcohol or ethers. The fifth peak at 1191.9 cm -1 shows S=O stretch and is expected to be
sulphates. The sixth peak shows the functional group is N-O stretch as it is 1309.4 cm -1, and
expected compounds to be Nitro compounds with the wavenumber range 1372-1290 cm -1. The
next peak at 1401 cm-1 shows a wavenumber range of 1400-1330 cm -1 shows O-H bending with
phenols and aromatic C-H compounds to be expected. The next peak at 2031.5 cm -1 shows 2140-
2100 cm-1 with a functional group to be C=-C stretch, with the expected compound to be
terminal alkyne. The next peak at 2169 cm -1 shows a 2600-2222 cm-1 wavenumber range, and the
functional group is C=-C stretch, and the expected compound is disubstituted alkyne. The next
peak at 2598.8 cm-1 shows a wavenumber range and functional group is S-H stretch, with the
expected compound to be thiol. The next peak at 3309.5 cm -1 shows a wavenumber range of
3310-3200 cm-1, and the functional group is =-C-H stretch, and the expected compound is
terminal alkyne. The last peak at 905 cm -1 shows a wavenumber range is 3310-3200 cm -1 and a
functional group is C-H bending, and the expected compound is Mono-substituted alkene.
RBM-NP
Sr # Wavenumber Wavenumber Functional Expected
sample reference group compounds
assessment
1 1022.9 cm-1 1000-1060 cm-1 C-O stretch Alcohol, ether
2 1512.9 cm-1 1550-1475 cm-1 -NO2 Nitro-aromatic
compound
3 2157.6 cm-1 2260-2100 cm-1 C-=C / C-=N Alkyne
stretch
4 2401.1 cm-1 2500-2000 cm-1 N-=N Ketene
5 3747.9 cm-1 3640-3610 cm-1 Free hydroxyl Alcohol or
stretch phenol
The FTIR analysis of Ratanjot B. megaterium with mordants neem and pomegranate shows a
total of 5 peaks. The first peak at 1022.9 cm-1 shows a wavenumber range of 1000-1060 cm-1 and
functional group to be a C-O stretch, and shows the expected compound is alcohol or ether. The
second peak at 1512.9 cm-1 shows a wavenumber range of 1550-1475 cm-1 with the functional
group assessment to be -NO2 group, with the expected compound to be a nitro-aromatic ring
compound. The next peak at 2157.6 cm-1 exhibits a wavenumber range of 2260-2100 cm-1,
indicating a functional group of C=C or C=N stretch, consistent with the expected compound
being an alkyne. The next peak at 2401.1 cm-1 exhibits a wavenumber range of 2500-2000 cm-1,
corresponding to the functional group N=-N, and the expected compound is ketene. The last peak
at 3747.9 cm-1 shows the wavenumber range is 3640-3610 cm-1 with the functional group to be a
free hydroxyl stretch and the expected compound to be alcohol or phenol.

RBM-SALT
Sr # Wavenumber Wavenumber Functional Expected
sample reference range group compound
assessment
1 1032.4 cm-1 1000-1200 cm-1 C-O stretch Carbohydrates,
phenolic ethers
2 1392.5 cm-1 1370-1410 cm-1 CH3 bending Aliphatic
 methyl group
3 2103.3 cm-1 2100-2260 cm-1 C-=N/ C-=C Nitriles or
terminal
alkynes
4 2896.8 cm-1 2860-2935 cm-1 C-H stretch Alkane chain
5 3343.8 cm-1 3200-3550 cm-1 O-H/N-H Alcohol/
stretch phenols

The FTIR analysis of Ratanjot B. megaterium with mordant salts, FeSO4 and Na2CO3 shows a
total of 5 peaks. The first peak at 1032.4 cm-1 falls within the wavenumber range of 1000-1200
cm-1, indicating a C-O stretch characteristic of carbohydrates or phenolic ethers. The next peak
at 1392.5 cm-1 corresponds to a wavenumber range of 1370-1410 cm-1, indicating a CH3
bending mode, which is characteristic of an aliphatic methyl group. The next peak at 2103.3 cm -1
shows the wavenumber range is between 2100-2260 cm-1 with the functional group to be a C=-N
or C=-C stretching, and determines the expected compound to be nitriles or terminal alkynes.
The fourth peak at 2896.8 cm-1 shows the wavenumber range is 2860-2935 cm-1, with the
functional group assessment showing there's a C-H stretching, and thus determines the expected
compound to be an alkane chain. The last peak at 3343.8 cm-1 shows the wavenumber range is
3200-3550 cm-1, and the functional group assessment here shows an O-H or N-H stretching, thus
the expected compound is alcohols or phenols.
KBM-NG

Sr # Wavenumber Wavenumber Functional Expected

sample reference range group compound
assessment
1 1020.07 cm-1 1250-1020 cm-1 C-N stretch Aliphatic
amine
2 1375.3 cm-1 1370-1350 cm-1 C-H bending Alkane
or
aldehyde
3 1636.1 cm-1 1670-1600 cm-1 C=C stretch Alkene
4 2888.3 cm-1 3000-2850 cm-1 C-H stretch Saturated
alkane
5 3260.8 cm-1 3330-3250 cm-1 -=C-H stretch Terminal
alkyne
 6 3343 cm-1 3400-3250 cm-1 N-H stretch Primary amine
7 564.4 cm-1 540-760 cm-1 C-Cl stretch Alkyl chloride
8 813.7 cm-1 850-550 cm-1 C-Cl / C-H Alkyl
aromatic chloride/
bend aromatic C-H
bend

The FTIR analysis of Karonda B. megaterium and mordants Neem and Guava shows a total of 8
peaks. The first peak at 1020.07 cm-1 shows the wavenumber range is between 1250-1020 cm-1,
and functional group assessment is a C-N stretch, and the expected compound is an aliphatic
amine. The next peak at 1375.3 cm-1 shows the wavenumber range is between 1370-1350 cm-1,
and the functional group assessment shows C-H bending, and the expected compound is an
alkane or aldehyde. The third peak at 1636.1 cm-1 shows the wavenumber range is between 1670-
1600 cm-1, and the functional group shows a C=C stretch, and the expected compound is an
alkane. The fourth peak at 2888.3 cm-1 shows a wavenumber range between 3000-2850 cm-1, and
the functional group is a C-H stretch, with the expected compound to be a saturated alkane. The
next peak at 3260.8 cm-1 shows the wavenumber range is 3330-3250 cm-1 and the functional
group is a =-C-H stretch, with the expected compound to be terminal alkyne. The next peak at
3343cm-1 shows the wavenumber range is between 3400-3250 cm-1, and the functional group
shows N-H stretch with the expected compound to be a primary amine. The next peak at 564.4
cm-1 shows the wavenumber range is between 540-760 cm-1 and shows a C-X stretch, which
shows the expected compound is an alkyl chloride. The last peak at 813.7 cm-1 shows the
wavenumber range is between 850-550 cm-1 and shows the functional group is a C-Cl or a C-H
aromatic bend, with the expected compound to be an alkyl chloride or an aromatic bend of
hydrocarbons.
KBM-NP
Sr # Wavenumber Wavenumber Functional group Expected
sample reference range assessment compound
1 1025.8 cm-1 1020-1220 cm-1 C-N/ C-O Amine, alcohol
Stretch
2 1149 cm-1 1100-1300 cm-1 C-O/C-OH Ester, ether
3 2014.3 cm-1 2000-2200 cm-1 C-=C Terminal
alkyne
4 2163.3 cm-1 2160-2120 cm-1 C-=C or Disubstituted
cumulene alkyne
5 2363.9 cm-1 2400-2000 cm-1 -N3 or isothiocyanate
N=C=O or azide
6 2916.9 cm-1 2850-2960 cm-1 C-H stretch Alkane
The FTIR analysis of Karonda B. megaterium and mordants Neem and pomegranate shows a
total of 6 peaks. The first peak at 1025.8 cm-1 shows C-N or C-O- stretch with the expected
compound to be amines or alcohols with a wavenumber range of 1020-1220 cm-1. The next peak
at 1149 cm-1 shows C-O or C-OH stretch, with the expected compound to be ester or ether. The
next peak at 2014.3 cm-1 shows the wavenumber range is between 2000-2200 cm-1, and the
functional group is a C=-C bond, which determines it is a terminal alkyne. The next peak at
2163.3 cm-1 shows the wavenumber range is between 2160-2120 cm-1, and the functional group
assessment shows a C=-C bond or cumulene, with the expected compound to be a disubstituted
alkyne. The next peak at 2363.9 cm-1 shows the wavenumber range is between 2400-2000 cm-1,
and the functional group assessment shows -N3 group or N=C=O group, which determines the
expected compound to be isothiocyanate or azide. The last peak at 2916.9 cm-1 shows the
wavenumber range is between 2850-2960 cm-1, with the functional group assessment to be a C-H
stretch, and the expected compound is an alkane.

KBM-SALT
Sr # Wavenumber Wavenumber Functional Expected compound
sample reference range group
assessment
1 1048.7 cm-1 1000-1060 cm-1 C-O stretch Alcohol or ether
 2 1977 cm-1 2000-1900 cm-1 C=C=C/ Allene or ketene
C=C=N
-1 -1
3 2157 cm 2260-2220 cm C-=N Nitrile
4 802.3 cm-1 840-790 cm-1 C=C bend or C-Cl or
C-X substituted
benzene

The FTIR analysis of Karonda B. megaterium and mordant is Salt, FeSO4, and Na2CO3.
shows a total of 4 peaks. The first peak at 1048.7 cm-1 shows C-O stretch, and the expected
compound is alcohol or ether. The second peak at 1977 cm-1 shows a wavenumber range of 2000-
1900 cm-1 with C=C=C or C=C=N functional groups, and the expected compound is allene or
ketene. The third peak at 2157 cm-1 shows the functional group of C=-N, and the expected
compound is Nitrile with the wavenumber range of 2260-2220 cm-1. The last peak at 802.3 cm-1
shows C=C bend or C-X bond, which shows the expected compound is C-Cl or a substituted
benzene with the wavenumber range of 840-790 cm-1.

FTIR of undyed fabric

Sr # Wavenumber Wavenumber Functional Expected
 sample reference range group compound
assessment
1 452.7 cm-1 440-460 cm-1 C-O-C bending Cellulose
2 484.2 cm-1 470-490 cm-1 C-O ring bend Hemicellulose
ring

The FTIR analysis of undyed cotton fabric showed a total of 2 peaks. One peak at 452.7 cm -1
shows a reference range between 440-460 cm-1, indicating the functional group is a C-O-C
bending, consistent with the expected compound being cellulose or a skeletal deformation. The
next peak at 484.2 cm-1 shows a wavenumber range between 470-490 cm-1, indicating the
functional group to be a C-O ring bending, and the expected compound is a hemicellulose ring.
Analysis of total phenolic content
The total phenolic content in various plant extracts of Ratanjot and Karonda, along with Bacteria
(B. cereus and B. megaterium) was analyzed by the Folin-Ciocalteu method using Gallic acid as
a standard. The calibration curve was built from the absorbance value obtained at various Gallic
acid concentrations. The regression equation of TPC was computed from the given equation (Y =
0.0055x + 0.0987, R2 = 0.9968) that was reported as mg gallic acid as equivalent (GAE) per
gram of given sample in dry weight (mg/g). As per the results and total phenolic content values
of various plant samples along with different bacterial strains, the TPC values show that the
highest TPC value is shown by Ratanjot Bacillus cereus, and the lowest TPC value is shown by
Bacillus megaterium, which is 0.10 mg.

Sample Final TPC mg GAE/ mL

Ratanjot B. cereus 0.99
Ratanjot B. cereus 1.23
Ratanjot B. cereus 0.86
Ratanjot B. megaterium 0.16
Ratanjot B. megaterium 0.14
Ratanjot B. megaterium 0.17
Ratanjot 0.95
Ratanjot 0.97
Ratanjot 1.2
Karonda B. cereus 0.86
Karonda B. cereus 0.82
Karonda B. cereus 0.86
Karonda B. megaterium 0.17
Karonda B. megaterium 0.25
Karonda B. megaterium 0.13
Bacillus cereus 0.9
Bacillus cereus 0.92
Bacillus cereus 0.89
Bacillus megaterium 0.1
Bacillus megaterium 0.16
Bacillus megaterium 0.14
Karonda 0.95
Karonda 1.02
Karonda 0.14
The antioxidant activity of any plant is directly linked to the phenolic concentration. The
hydrogen donors or reducing agents, along with the free-radical scavengers, are all the functions
of phenolic compounds. The presence of massive amounts of phenolics in plants may have a
powerful impact on the antioxidant qualities. These plant extracts can be used in various
medicinal remedies because of their beneficial qualities. Depending upon the chemical phenolic
substrate constituents, it possesses a wide range of antioxidant characteristics. Ascorbic acid and
sugar are two chemical constituents in the extract that can cause some effects. Plants possess
secondary metabolites termed as polyphenolic and phenolic compounds because of their ability
to neutralize the radicals. Phenolic compounds are antioxidants.

Figure 1: Standard curve of Gallic acid

ANOVA: Single Factor

SOV SS Df MS F P-value F crit
Between 3.37224 0.22481 3.00365 0.06026 3.21840
Groups 7 15 6 7 8 6
Within 0.59878 0.07484
Groups 1 8 8

3.97102
Total 8 23
Analysis of Total Flavonoid Content
From the regression calibration curve equation (Y= 0.0038x + 0.0285)
R2 = 0.9835. The total flavonoids of the extracts were analyzed, expressed as quercetin
equivalents (QE) per gram of dry sample weight. Similar trends will be shown in total flavonoid
content and total phenolic content. In the table given below, various plant extracts along with
different bacterial strains, along with different total flavonoid content, are determined, which
shows that Ratanjot Bacillus cereus shows the highest values of TFC. The lowest total flavonoid
content is given by Ratanjot Bacillus megaterium.

Sample Final TFC mg GAE/ mL

Ratanjot B. cereus 0.91
Ratanjot B. cereus 1.02
Ratanjot B. cereus 0.93
Ratanjot B. megaterium 0.21
Ratanjot B. megaterium 0.21
Ratanjot B. megaterium 0.66
Ratanjot 0.92
Ratanjot 0.93
Ratanjot 0.98
Karonda B. cereus 0.36
Karonda B. cereus 0.34
Karonda B. cereus 0.34
Karonda B. megaterium 0.76
Karonda B. megaterium 0.76
 Karonda B. megaterium 0.71
Bacillus cereus 0.83
Bacillus cereus 0.88
Bacillus cereus 0.81
Bacillus megaterium 0.64
Bacillus megaterium 0.64
Bacillus megaterium 0.65
Karonda 0.98
Karonda 0.96
Karonda 0.91

The polarity of the extraction solvents has a significant impact on flavonoids and phenols
concentrations as well. The polyphenol and flavonoid compounds in plants that have benzo-
pyrone structures colorimetrically with aluminum chloride reagent, which can be used to
calculate the total flavonoid content of the extracts.

Figure 2: Standard curve of Quercetin

Anova: Single Factor
Source of
Variation SS Df MS F P-value F cri
Between 1.34091 0.10314 6.8249 0.00226 2.88717
Groups 7 13 7 3 8 5
Within 0.15113 0.01511
Groups 3 10 3

Total 1.49205 23
Antibacterial Activity
Clothing and textiles are particularly resistant to bacterial attack. Due to their high surface area
and ability to retain moisture, which both promote the growth of bacteria. Bacteria grow on
protein (like keratin) and cellulose that are found in natural fibers like cotton. Bacteria are aided
in their growth by essential elements such as moisture, oxygen, nutrients, and temperature. This
may also result in allergies, various odors, fabric degradation and deterioration, and other issues.
In the past decades, a variety of antimicrobial textile materials have been reported. A detailed
study was undertaken to investigate if some of the dyes have inherited antimicrobial activity,
with the main purpose of developing protective fabric and clothing from these Acacia catechu,
Rubia cordifolia, Kerria Lacca, Quercus infectoria, and Rumex maritimus. The natural dye
extracts were tested against various species of microbes, such as Bacillus subtilis, E. coli,
Pseudomonas aeruginosa, and Proteus Vulgaris. The Quercus infectoria was the most effective
and had the highest zone of inhibition, indicating the best antimicrobial activity among all tested
microbes. The minimum concentration of inhibition ranges from 5 to 40 mg. In fact, due to the
uptake of these dyes in the textile material, the Minimum Inhibitory concentration, the
antimicrobial activity of the textile material colored with these natural dyes was low (Singh et
al., 2005).
In this research, various natural dyes are tested against several bacterial strains, including gram-
negative Escherichia coli (E. coli ATCC 25922) and gram-positive Bacillus subtilis (B. subtilis).
Bacterial strains are cultured in the nutrient broth overnight at 37ºC. The method of diffusion was
used to determine the antimicrobial activity of the compound. The effect of the concentration of
dye on antibacterial activity was further assessed in each case, and the zone of inhibition
diameter was determined. It was determined that increasing the dye concentration causes
inhibition, as assessed by an increase in diameter. And as for negative control with no samples
were used. As a positive bacterial reference, rifampicin was used. The antibacterial activity was
assessed using the diameter of organism inhibition zone (zone reader) in millimeters and
compared to the control. The highest zone of inhibition is shown by Ratanjot only, in which E.
coli has a zone of inhibition of 32 mm, and S. aureus shows a 33 mm zone of inhibition. The
lowest value of anti-microbial activity is shown by Ratanjot B. cereus, Karonda B. cereus,
Ratanjot B. megaterium, Karonda B. megaterium, Bacillus megaterium only, and Bacillus cereus
only, as they show a 0 zone of inhibition. A zero zone of inhibition refers to the absence of
antimicrobial activity seen (Mumtaz et al., 2021).

Samples E. coli Staphylococcus aureus

Zone of inhibition in mm Zone of inhibition in mm
Ratanjot Bacillus cereus 0 0
Karonda Bacillus cereus 0 0
Ratanjot Bacillus megaterium 0 0
Karonda Bacillus 0 0
megaterium
Positive control 32 33
Ratanjot only 0 11
Bacillus megaterium only 0 0
Bacillus cereus only 0 0
Karonda only 0 8
Positive control 31 37
Antioxidant activity (DPPH)
Antioxidant activity of various plant extracts was determined by DPPH free radical scavenging
assay. In a concentration-dependent way, the radical scavenging activity of several extracts
increased. The highest antioxidant activity was given by, and the lowest antioxidant activity was
shown by. The radical scavenging activity of various plant extracts (Ratanjot, Karonda) along
with bacteria (Bacillus cereus and Bacillus megaterium) may be attributed to the presence of
flavonoids and phenolic content, with most of the activity being due to the presence of phenols.
The destructive effect of oxidative stress is avoided by the naturally occurring antioxidant
potential of plants, among many, is the DPPH assay.
Sample % DPPH inhibition
Ratanjot B. cereus 0.093
Ratanjot B. cereus 0.094
Ratanjot B. cereus 0.09501
Ratanjot B. megaterium 0.156
Ratanjot B. megaterium 0.157
Ratanjot B. megaterium 0.157
Ratanjot 0.101
Ratanjot 0.101
Ratanjot 0.102
Karonda B. cereus 0.107
Karonda B. cereus 0.106
Karonda B. cereus 0.107
Karonda B. megaterium 0.128
Karonda B. megaterium 0.129
Karonda B. megaterium 0.129
Bacillus cereus 0.105
Bacillus cereus 0.105
Bacillus cereus 0.106
Bacillus megaterium 0.108
Bacillus megaterium 0.109
Bacillus megaterium 0.108
Karonda 0.105
Karonda 0.106
Karonda 0.104

As DPPH is the more accurate, reliable, and repeatable way to search in vitro, it is employed
frequently to calculate and determine the antioxidant activity of an antioxidant agent in plants as
well as pure compounds (Koleva et al., 2002). Antioxidants may occur naturally in plants,
microbes, and animals, and they can also be created by artificial techniques. Natural antioxidants
like polyphenols and tocopherols are present in massive amounts in herbs, vegetables, spices,
fruits, and cereals are present in large quantities in higher plants and their components.
Antioxidants from marine origin, such as fish, algae, and marine microbes, have also been
considered (AMAROWICZ et al., 1999). Antioxidants are extremely essential molecules that can
protect organisms from free-radical oxidative stress because of the hydrogen bonding ability of
their hydroxyl(O-H) groups, which have the ability to donate hydrogen. Polyphenols in plants
have anti-oxidant reducing properties.
 Fastness properties of the fabric
A very important step in dyeing technique is the extraction and isolation of variable colorants
because of the dye solubility that varies in various extraction media used in studies to extract the
pigments from the extracts. Dyeing of fabric is a long and time-consuming process. The shorter
time duration means less energy, cost, efficiency, and less labor involvement normally preferred.
The dyeing bath temperature plays a vital role in textile processing. Fastness testing, rubbing,
washing, and light properties are valued in the dyed textile field.
In this research, the dyed fabric was tested using bio-mordants (Neem, pomegranate peels,
guava) and synthetic mordants(NaCl, FeSO4, Na2CO3). A combination of Neem and guava,
Neem and pomegranate, and a synthetic mordant combination of NaCl, FeSO 4, Na2CO3. The
optimum temperature for dyeing fabric was 90ºC for obtaining a better color strength, but the
results of testing showed that Ratanjot B. megaterium with a mordant combination of neem and
pomegranate showed poor results in washing, and Karonda B.megaterium and Karonda B.
cereus, along with the same mordant combination of neem and guava, showed poor washing
fastness results, while the shades obtained from Karonda B. megaterium with a mordant
combination neem and pomegranate peels showed fair washing fastness results.
Furthermore, applying different mordants for dyeing, various shades were obtained on fabrics.
From this research, it was concluded that natural dyes, along with two different strains of
bacteria, are successful in obtaining various shades on cotton fabric. Only the karonda with
Bacillus megaterium and Bacillus cereus with mordants neem and guava (natural mordants) and
Ratanjot B.megaterium with natural mordants neem and pomegranate peels showed poor
washing results on cotton fabric, as the fabric shade faded away upon washing. Otherwise, all the
other shades of fabric with bio-mordants and synthetic mordant combinations showed moderate
results upon washing. Moreover, the crocking results of almost all the fabrics were good with 4-5
ratings. The wet crocking result of Karonda B.megaterium with a mordant combination of NaCl,
FeSO4, and Na2CO3 was poor with 1-2 ratings.
Sam Neem and pomegranate Neem and guava NaCl, FeSO4, Na2CO3
ple
Karo
nda
B.
cere
us

Karo
nda
B.
meg
ateri
um

Rata
njot
B.
cere
us

Rata
njot
B.
meg
ateri
um
Analysis of color strength
Fastness properties
Dyes are classified according to their attachment to the fiber. Cotton fibers are classified with
respect to their adhesion, surface bonding, and covalent bonding mechanisms. Pigments are used
to color the cotton fabric, but they are not considered dyes as they have no affinity for the cotton
fibers and are not soluble in water. To adhere them to the cotton fibers, various kinds of resins,
adhesives, and bonding agents are used. The colorfastness of cotton textiles might be a difficult
topic to understand.

Washing
The fabric of cotton was colored and dyed using a pre-mordanting procedure, and various grey
scale ratings (4-5 excellent, 3-4 Moderate, 2-3 Fair, and 1-2 poor) were noted. Moreover, 4-5
staining color on cotton fabric was also observed on all samples.

Crocking
The crocking of the Karonda plant along with bacterial strain Bacillus cereus and mordants
(NaCl, FeSO4, and Na2CO3) shows that Dry fastness (4) shows better results than wet fastness(2-
3). The crocking results of Ratanjot Bacillus cereus with mordants Neem and Guava indicate that
Dry fastness (4-5) yields better results than wet fastness (3-4). The crocking of Ratanjot Bacillus
cereus and mordants Neem and pomegranate shows dry fastness (3) and wet fastness (3). The
crocking of Karonda Bacillus cereus and mordants Neem and pomegranate shows dry fastness
(4-5), better results than wet fastness (3-4). The crocking results of Ratanjot B. cereus and
mordants NaCl, FeSO4, Na2CO3 show that dry fastness result (4) is better than wet fastness(3).
Crocking of Karonda B. megaterium and mordants Neem and Pomegranate shows better dry
fastness (4-5) than wet fastness(3-4). Ratanjot B. megaterium with mordants Neem and
pomegranate shows better dry fastness (4-5) than wet fastness (4). Crocking of Ratanjot B.
megaterium with mordants Neem and guava shows better dry fastness (4-5) than wet fastness
(4). Crocking of Ratanjot B. megaterium with mordants NaCl, FeSO4, Na2CO3 shows better dry
fastness (4) than wet fastness (3). The crocking test of Karonda B. megaterium with mordants
neem and guava shows better dry fastness results (4-5) than wet fastness (4). Crocking test of
Karonda B. megaterium with mordants NaCl, FeSO4, and Na2CO3 shows better dry fastness result
(3-4) than wet fastness (2), which is considered poor. The crocking test of Karonda B. cereus and
mordants Neem and Guava shows better dry fastness results (4-5) than wet fastness (4).

Pigments are commonly used to dye the fabric of cotton, but they are not considered dyes as
they have no affinity for the fibers of cotton and are not entirely insoluble in water. To attach
them to the fabric of cotton, different kinds of resins, adhesives, or binding agents are used. The
color durability of cotton textile can be a tough topic to understand. Consumer particles, Yarn
formation, and Fabric construction, Textile wet processes, along with the quality of fabric, may
have an impact on the characteristics and performance of fabric. The selection choices made
during the textile wet processing have a huge impact on the fastness properties and dye
selection. The dimensional stability of cotton fabric is also strained by consumer practices and
procedures, such as washing techniques and selection of detergents.

In the past, various researchers practiced cotton fabric dyeing using various mordants. The
Alkanna tinctoria root extract was used to dye the cotton fabric, and different mordants were
used, such as iron or aluminum, which produced violet and grey to violet shades on cotton, and
they improved the color fastness on cotton (Assimopoulou et al., 2006). Similarly, natural
colorants were produced on cotton, yarn, and silk fabric using Carissa carandas fruit extract and
various mordants such as lemon juice, acetic acid, alum, and copper sulfate, and the results
were compared, and the fastness properties were examined (Manicketh et al., 2021).

Colorant source Mordant Washing fastness Crocking

Stain of Shade of Dry Wet
cotton cotton
Ratanjot B. cereus Neem and guava 4-5 3 4-5 3-4
Neem+ pomegranate 4-5 3 3 3
NaCl, FeSO4, Na2CO3 4-5 3-4 4 3

Ratanjot B. Neem and guava 4-5 2 4-5 4

megaterium Neem+ pomegranate 4-5 1-2 4-5 4
NaCl, FeSO4, Na2CO3 4-5 3-4 4 3

Karonda B. cereus Neem and guava 4-5 2 4-5 4

Neem+ pomegranate 4-5 1-2 4-5 3-4
NaCl, FeSO4, Na2CO3 4-5 3 4 2-3

Karonda B. Neem and guava 4-5 2 4-5 4

megaterium Neem+ pomegranate 4-5 2-3 4-5 3-4
NaCl, FeSO4, Na2CO3 4-5 3 3-4 2

Biological Oxygen Demand(BOD)

The biological oxygen demand is a laboratory test that determines how much oxygen is required
to break down and decompose waste in water. It tells us how much oxygen is needed to
decompose the organic matter ( such as plant wastes and food) and some inorganic effluents,
such as iron and sulfides. It also includes the oxygen which is required for the breakdown of
nitrogenous compounds unless a chemical is added to seize it(Liu and Mattiasson, 2002).
Assimopoulou, A., I. Karapanagiotis, A. Vasiliou, S. Kokkini and V. Papageorgiou. 2006. Analysis
of alkannin derivatives from Alkanna species by high‐performance liquid
chromatography/photodiode array/mass spectrometry. Biomedical Chromatography.
20(12): 1359-1374.
Liu, J. and B. Mattiasson. 2002. Microbial BOD sensors for wastewater analysis. Water research.
36(15): 3786-3802.
Manicketh, T. J., M. S. Francis and G. Joseph. 2021. Extraction of natural colourants from
Mussaenda hybrid (M. philippica× M. luteola), Carissa carandas L. & Syzygium cumini L.
for textile colouration. Natural Product Research. 35(21): 4159-4163.

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