“IN-VITRO STUDY OF ANTIMICROBIAL POTENTIAL BY

NATURAL DYES EXTRACTED FROM FLOWERS”

A
Dissertation
Thesis
Submitted to
BABASAHEB BHIMRAO AMBEDKAR UNIVERSITY

2019-2021
For the degree of
Master of Science In

Food Microbiology and Toxicology

Submitted By:
Mohd. Javed Khan
Roll No-197603

Under the Guidance of :

Prof. Rajesh Kumar
DEPARTMENT OF ENVIRONMENTAL MICROBIOLOGY
SCHOOL OF EARTH & ENVIRONMENTAL SCIENCES
BABASAHEB BHIMRAO AMBEDKAR UNIVERSITY
(A CENTRAL UNVIVERSITY)
VIDYA VIHAR, RAEBARELI ROAD, LUCKNOW-226025, INDIA

1
Dedicated to My
Beloved Parents

2
 Babasaheb Bhimrao Ambedkar University

(A Central University)

Vidya Vihar, Raebareli Road Lucknow - 226025

बाबासाहे बभीमरावअ बेडकर व व ालय

व ा वहार, रायबरे लीरोड, लखनऊ– 226025

Prof. Rajesh Kumar Letter No.:-----

Coordinator of Integrated Basic Science

(Environmental Microbiology) Date:

CERTIFICATE

This is to certify that the thesis entitled “In-vitro study of antimicrobial potential by natural
dyes extracted from flowers” submitted in partial fulfillment of the requirement for the
degree of M.Sc. in Food Microbiology and Toxicologyis a record of bona fide research
carried out by “Mohd Javed Khan” under my guidance and no part of this dissertation/thesis
has been submitted for any other degree or diploma. This is also to certify that the work has
been carried out in the ‘Rhizosphere Biology Laboratory’ of the Department of
Environmental Microbiology, (School of Earth and Environmental Sciences).

(Prof. Rajesh Kumar)

3
 DECLARATION

I, Mohd Javed Khan, hereby declare that the dissertation work entitled “In-vitro study of antimicrobial
potential by natural dyes extracted from flowers” is my own work carried under the guidance of Prof.
Rajesh Kumar, Department of Environmental Microbiology, Babasaheb Bhimrao Ambedkar
University, (A Central University) Vidya-Vihar, Raebareli Road Lucknow. The matter embodied in
this dissertation work is written by me and has not been submitted to any other university for the
fulfillment of the requirement of any other Degree or Diploma.

MOHD JAVED KHAN

Place-Lucknow

Date

4
 ACKNOWLEDEGEMENT

I express my heartiest veneration and indeed feel my privilege to work under the talented and inspiring
guidance, supervision, conspicuous ability, and constructive criticism of my supervisor Prof. Rajesh
Kumar, Department of Environmental Microbiology, BBAU, Lucknow. for allowing me permission to
conduct my study at ( Regional Food Research & Analysis Centre, Lucknow). I am overwhelmed with
his talented and inspiring guidance, invaluable suggestion, unending zeal, and sympathetic attitude
rendering during the study and while writing the dissertation.
My sincere thanks are also due to Prof. Ram Chandra , Head of Department of Environmental
Microbiology, BBAU, Lucknow for constant guidance and extending valuable support, wherever needed.
Dr. Sangeeta Dhawan, and Dr. Suman Upadhyay for their unterminated help, invaluable suggestion.
Words are lacking to express my heartiest thanks to Dr. S.K.Chauhan (Director, Regional Food
Research & Analysis Centre, Lucknow ) for sharing his knowledge and experience with me along with the
endearment, support, and cooperation, during the whole period of this study. Any attribute will be less for
him.
I would also like to thank Mr. Udit Jaiswal and Ms. Archana for their insightful comments and
encouragement.
I am also grateful to my brother Adv. Mohd Rizwan Khan, Highcourt Lucknow. This work was enriched
by his expert comments. He helped me in overcoming certain logical inconsistencies in my arguments.
My thanks and appreciation also go to my some best friends Mariyam Fatima, Anupriya Sharma, Alima
Anwar .
I heartily thankful for Mr. Zobair Iqbal for unconditional support and help.

Last but not least, I would like to acknowledge moral support and comfort extended to me by my family –
Parents ,Mr. Umar Mohammad and Mrs. Noor Jahan , Brothers, Shamim Khan, Shaheryar Sultan,
MohdJavvad khan and Mohd Shan.

Date: Mohd Javed Khan

5
 List of Figures

S.No. Figure name

1 Plant Sample

2 Extraction of plant sample

3 Qualitative estimation of Saponine test from plant sample

4 Qualitative estimation of Tannin test from plant sample

5 Qualitative estimation of Flavonoid test from plant sample

6 Qualitative estimation of Fat test from plant sample

7 Qualitative estimation of Cardiac glycoside test from plant sample

8 Qualitative estimation of Starch test from plant sample

9 Qualitative estimation of Carbohydrate test by fehling’s test from plant sample

10 Qualitative estimation of Protein test from plant sample

11 Media preparation for Antifungal activity (PDA)

12 Inoculation of Fungus on PDA media with plant sample

13 Media preparation for Antibacterial activity

14 Inoculation of Bacteria on NA media with plant sample

6
 Contents

S.No. Chapter Page no.

1. Introduction 9-12

2. Objectives 13
3. Review of literature 14-18
4. Material & method 19-25

5. Result & discussion 26-28

6. Conclusion 29
7. References 30-33

7
\

Abstract:
The present study has been focused on the extraction of eco- friendly dyes from Rose and
Marigold
flowers and investigation of their antimicrobial characteristics against pathogenic strains. Dyes
were extracted by aqueous extraction technique. Further qualitative method was performed to
check the presence of saponin, tannin, flavonoid, fat, protein, carbohydrates, and starch like
secondary metabolites in the extracted material from Rose and Marigold flowers. In-vitro dyes
were tested for antimicrobial activity and found to posses for both antibacterial as well as
antifungal potential against E.coli, Staphyloccocus aureus bacteria and Aspergillus niger fungus
respectively.

Keywords: Extraction, Saponin, Tannin, Flavonoid, Secondary metabolites.

8
Chapter- 1
INTRODUCTION

Dyes are one of the most important uses of the plants. Recently, interest in the use of natural
dyes has been growing rapidly due to the result of stringent environmental standards imposed by
many countries in response to toxic and allergic reactions associated with synthetic dyes. As a
result with a distinct lowering in synthetic dyestuff costs, the natural dyes were virtually unused
at the beginning of twentieth century (Kumaresan et. al., 2011).
Nowadays in most of the countries, natural dyeing is practiced only as a handcraft and synthetic
dyes are being used in all commercial dyeing processes. However with the worldwide concern
over the use of eco-friendly and biodegradable materials, the use of natural dyes has once again
gained interest (Agarwal A, Goel A & Gupta K C, 1992). Dyeing can be carried out in an
alkaline bath, acidic bath or in a neutral bath. There are various reports available on different
methods of mordanting on different fibers such as cellulosic, protenic and synthetic for dyeing
with different natural dyes. Various kinds of shades like black to brown, green to yellow to
orange, etc can be obtained by application of different mordants. Dyeing of cotton and silk with
henna, indigo, marigold etc is reported. (Gulrajani et al, 1992).
There are primarily four sources from which natural dyes are available. Specialized plant and
animal sources - Many plants and some animals have been identified as potentially rich in
natural dye contents, and some of them have been used for natural dyeing. Normally natural dyes
are extracted from the roots, stems, leaves, flowers, fruits of various plants, dried bodies of
certain insects and minerals. The shade of the color a plant produces will vary according to time
of the year the plant is picked, how it was grown, soil conditions, etc. By-products (especially lac
dye) - The lac industry gives lac dye as a by-product, which is extracted from the effluent.
Chemical synthesis - This involves synthesis of dyes with molecular structures identical to those
of natural dyes.Tissue or cell cultures by DNA transfer biotechnology - Certain fungi such as
Drechslera and Trichoderma produce anthraquinone derivatives as secondary metabolites (Salam
M A, et.al., 2006).
Floral sources of natural dyes: Many natural dyestuff and stains were obtained mainly from
plants and dominated as sources of natural dyes, producing different colours like red, yellow,
blue, black, brown and a combination of these. Almost all parts of the plants like root, bark, leaf,

9
fruit, wood, seed, flowers, etc. produce dyes. Dyeing can be carried out in an alkaline bath,
acidic bath or in a neutral bath. There are various reports available on different methods of
mordanting on different fibers such as cellulosic, protenic and synthetic for dyeing with different
natural dyes. Various kinds of shades like black to brown, green to yellow to orange, etc can be
obtained by application of different mordants. Dyeing of cotton and silk with henna, indigo,
marigold etc is reported. (Gulrajani et al, 1992). There is a growing interest in the revival of
natural dyes in textile colouration (Mehanta z. & Osman et al, 2003 & 2004). In contrast, natural
dyes are environmental friendly, exhibit better biodegradability and generally have a higher
compatibility with the environment than synthetic dyes. (Ahlstrom et al, 2005). The process is
economically viable as the raw materials are available at low cost and so cost of production is
also very low. Similar findings were reported in Marigold, China rose and Bixa flower (Ibrahim
et al, 1997) There is a growing interest in the revival of natural dyes in textile colouration
(Mehanta z. & Osman et al, 2003 & 2004). In contrast, natural dyes are environmental friendly,
exhibit better biodegradability and generally have a higher compatibility with the environment
than synthetic dyes. (Ahlstrom et al, 2005). The process is economically viable as the raw
materials are available at low cost and so cost of production is also very low. Similar findings
were reported in Marigold, China rose and Bixa flower (Ibrahim et al, 1997). Natural
dyes/colorants derived from flora and fauna are believed to be safe because of its nontoxic,
noncarcinogenic and biodegradable in nature (Cristea & Vilarem, 2003). Many tribes of
Arunachal Pradesh have been using this plant species traditionally in combination with other
plants for extraction and preparation of dyes utilizing indigenous processes (Mahanta & Tiwari,
2005).
Natural dyes are now a days in demand not only in textile industry but in cosmetics, leather, food
and pharmaceuticals. The rich biodiversity of our country has provided us plenty of raw
materials, yet sustainable linkage must be developed between cultivation, collection and their use
(Gokhale et al, 2004).
ANTIMICROBIAL PROPERTIES OF NATURAL DYES:
Tagetes erecta, also known as African marigold has numerous medicinal values. With the rising
need to explore better antifungal, anticancer agents in therapeutics, we have done this study to
evaluate the antifungal and anticancer properties of Tagetes erecta petal extract. Antifungal
activity against was evaluated against Candida albicans, Aspergillus niger, Aspergillus flavus,
and Penicillium.

10
Rose acts against bacterial infections and relieves skin irritation and purifie
s it. It improves blood
circulation and promotes hair growth, as well as controls dandruff. These are effective in
soothing tired and fatigued eyes. Drinking rose water or tea removes inner body heat and brings
down fever. It acts as an antiseptic, antispasmodic, astringent, bactericidal (against typhoid,
diarrhoea, cholera, food poisoning), antiviral (cold or influenza), antiphlogistic. It helps to
remove scars and marks. In the treatment of dreadful cancers patients are often subjected to
severe anti-cancer medicines, and results in immune-suppression. In these patients oppourtunistic
infections are more predominant. Rose extract contains 80% essential fatty acids and
antioxidants, thus helps to regenerate skin cells. In case of oral diseases, fungal infections mainly
candidiasis is often use of rose extract in curing the diseases is much effective. According to
studies done in 2007 and 2013 found that roses have high levels of polyphenols [Nowak R, et al].
Oral bacteria like Streptococcus mutans and Lactobacillus are the main microorganisms
implicated in the initiation and progression of caries, respectively. Immune-suppression allows
the formation of mucosal biofilms, leading to the clinical appearance of thrush [De Repentigny
L, et al].
This study was done to deeply investigate the antibacterial and antifungal action of rose and
marigold on various pathogenic bacteria and fungus. By proving so, roses can become a vital
part of dentistry in the near future and extensive studies on its components and properties will
enable us to utilize its benefits.

11
 OBJECTIVE

1. Collection of Rose and Marigold flowers samples from Lucknow.

2. Extraction of dye from flowers by aqueous extraction method.
3. Procurement of pathogenic strain of E.coli and Staphylcoccus aureus from MTCC
Chandigarh.
4. Antimicrobial property of extracted natural dye against pathogenic strains.

12
Chapter- 2

REVEIW OF LITERATURE

India has a rich plant biodiversity which is ranked 11th as biggest biodiversity in the world. It
has approximately 490,000 plant species and there is no doubt that the plant kingdom is a
treasure-house of diverse natural products (Neha Grover et al., (2011)). One such product from
nature is the dye. Pigment from leaves, fruits, seed, wood and roots were used as dye stuff for
textiles and as paint in art and craft. Natural dyes are environmental friendly, hygenic, user
friendly and permanent than other colorant. The replacement of natural dyes could happen until
the introduction of synthetic dyes due to feasible coloring property of natural dyes (Kumaresan et
al., (2011)). Certain problems with the use of natural dyes in textile dyeing are color yield,
complexity of dyeing process, reproducibility results, limited shades, blending problems and
inadequate fastness properties (Sachan and Kapoor (2007); Siva (2007)). But these problems can
be overcome by using chemicals called as mordants. Mordants are metal salts which produce an
affinity between the fabric and the dye (Vankar et al., (2009); Samanta and Agarwal, (2009)).
One of the pleasures of using natural dye is that no two baths will ever give exactly the same
result, there will be an element of surprise with variation accordingly to the season, the weather,
the maturity of the plant, its position in the sun or shade and the quality of the water used for
dyeing will determine the dyeing quality. Recently, interest in the use of natural dyes has been
growing rapidly due to the result of stringent environmental standards imposed by environmental
board and pollution control board of many countries in response to toxic and allergic reactions
associated with synthetic dyes (Kamel et al., (2005). Research has shown that synthetic dyes are
suspected to release harmful chemicals that are considered to be high pollutant in both water and
land which would be allergic, carcinogenic and detrimental to human health. Plants are not the
only source for the basic needs of human life, such as food, fibre, fuel, and shelter, but they can
also be used as natural colourants. These natural colours are exhibited due to the absorption of
light in the visible region of 400–800 nm by various organic and inorganic molecules and their
mixture in plants (Chengaiah, Rao Kumar, Alagusundaram, & Chetty, et al., 2010). India has a
rich wealth of genetic plant resources, of which more than500 species produce natural colours,
obtained from leaves, fruits, seeds, flowers, barks and roots of these plant spe-cies. This
abundant natural resource gives scope to explore new opportunities for commercialisation in
several indus-tries, such as, textiles, cosmetics, pharmaceuticals and paper industries in

13
India.Dye textiles found during archaeol ogical excavation at different global loca tions , provide
evidence of the practice of dying in ancient civilisations. From historical records, it is understood
that natural colourants were available during Greco-Roman periods. The dyeing process was also
practised during the Indus river valley civilization at Mohenjodaro and Harappa (3500 BC), for
mer Egyptian, and China pe riod (Siva, 2007). Moldenke (I.C) reports thatan orange or yellow
impermanent dye is made from corolla-tubes of Nyctanthes ar bor-tristis Linn, for Buddhistrobes
in Sri Lanka (Panigrahi & Murti, 1989). In Indian Vedas (the oldest written religious scriptures,
originatingfrom ancient Indi a and consists of four units namely, Rig Veda, Sama Veda, Yajur
Veda and Athar va Veda), theAtharva Veda also carries the description of natural dyes. The use
of natural dyeing materials is also evident withthe wall paintings of Ajanta, Ellora and
Sithannvasal and they still demonstrate the efficacy of dyeing craft that hadbeen inherited from
ancient times in India (Upadhayay and Choudhery, 2012). In fact, it is evident that someimpo
rtant dyes namely Madder, Indigo and Kermes, were used globally, introduced to worldwide by
Indians duringearly civilisations.Natural dyes from plants (roots, stem bark and leaves), animals
(insects, mollusks), lichens, rocks and soils werequite commonly used throughout the world, but
this method of dye production started to change about 160 years ago (since 1856) with the
introduction of a synthetic dye made from aniline by Sir William Henry Perkins in In Indian
Vedas (the oldest written religious scriptures, originatingfrom ancient Indi a and consists of four
units namely, Rig Veda, Sama Veda, Yajur Veda and Athar va Veda), theAtharva Veda also
carries the description of natural dyes. The use of natural dyeing materials is also evident withthe
wall paintings of Ajanta, Ellora and Sithannvasal and they still demonstrate the efficacy of
dyeing craft that hadbeen inherited from ancient times in India (Upadhayay and Choudhery,
2012). In fact, it is evident that someimpo rtant dyes namely Madder, Indigo and Kermes, were
used globally, introduced to worldwide by Indians duringearly civilisations.Natural dyes from
plants (roots, stem bark and leaves), animals (insects, mollusks), lichens, rocks and soils
werequite commonly used throughout the world, but this method of dye production started to
change about 160 years ago (since 1856) with the introduction of a synthetic dye made from
aniline by Sir William Henry Perkins in England(Adrosko, 1971), because of non-availability of
viable alternatives, the synthetic dyes were being manufactured and used as a matter of necessity.
Moreover, these man-made dyes were cheaper, more readily available, had greater col-our
fastness, and were more stable; this caused the downward spiral of natural dye use. However,
during recent years, the issue of natural dyes has to be taken seriously because synthetic dyes are

14
polluting the environment and the carcinogenicity of certain diazo dyes makes synthetic dyes
highly unpopular and are poorly accepted. As a result,there has recently been a global ban
imposed, including the EU, USA and India on the use of some synthetic dyes. In contrast, natural
dyes are biodegradable and highly compatible with the environment and are also free from the
defects associated with synthetic dyes. These transitions have resulted in a need to investigate the
natural, eco-friendly dyes with the view to increase their commercialization.
In Indian Vedas (the oldest written religious scriptures, originatingfrom ancient Indi a and
consists of four units namely, Rig Veda, Sama Veda, Yajur Veda and Athar va Veda),
theAtharva Veda also carries the description of natural dyes. The use of natural dyeing materials
is also evident withthe wall paintings of Ajanta, Ellora and Sithannvasal and they still
demonstrate the efficacy of dyeing craft that hadbeen inherited from ancient times in India
(Upadhayay and Choudhery, 2012). In fact, it is evident that someimpo rtant dyes namely
Madder, Indigo and Kermes, were used globally, introduced to worldwide by Indians duringearly
civilisations.Natural dyes from plants (roots, stem bark and leaves), animals (insects, mollusks),
lichens, rocks and soils werequite commonly used throughout the world, but this method of dye
production started to change about 160 years ago (since 1856) with the introduction of a
synthetic dye made from aniline by Sir William Henry Perkins in In Indian Vedas (the oldest
written religious scriptures, originatingfrom ancient Indi a and consists of four units namely, Rig
Veda, Sama Veda, Yajur Veda and Athar va Veda), theAtharva Veda also carries the description
of natural dyes. The use of natural dyeing materials is also evident withthe wall paintings of
Ajanta, Ellora and Sithannvasal and they still demonstrate the efficacy of dyeing craft that
hadbeen inherited from ancient times in India (Upadhayay and Choudhery, 2012). In fact, it is
evident that someimpo rtant dyes namely Madder, Indigo and Kermes, were used globally,
introduced to worldwide by Indians duringearly civilisations.Natural dyes from plants (roots,
stem bark and leaves), animals (insects, mollusks), lichens, rocks and soils werequite commonly
used throughout the world, but this method of dye production started to change about 160 years
ago (since 1856) with the introduction of a synthetic dye made from aniline by Sir William
Henry Perkins in England(Adrosko, 1971), because of non-availability of viable alternatives, the
synthetic dyes were being manufactured and used as a matter of necessity. Moreover, these man-
made dyes were cheaper, more readily available, had greater col-our fastness, and were more
stable; this caused the downward spiral of natural dye use. However, during recent years, the
issue of natural dyes has to be taken seriously because synthetic dyes are polluting the

15
environment and the carcinogenicity of certain diazo dyes makes synthetic dyes highly
unpopular and are poorly accepted. As a result,there has recently been a global ban imposed,
including the EU, USA and India on the use of some synthetic dyes. In contrast, natural dyes are
biodegradable and highly compatible with the environment and are also free from the defects
associated with synthetic dyes. These transitions have resulted in a need to investigate the
natural, eco-friendly dyes with the view to increase their commercialization.

Mordant dyes
Some protein fabrics such as silk and wool can be coloured simply by being dipped in dye. In
contrast, cellulosic material such as cotton requires a mordant, which improves the fastness of
the dye against water, light and perspiration. If the dye needs no mordants, they are called
substantive dyes, such as lichens and walnut hulls. I f they require a mordant, they are called
adjective dyes. Mordants can be used to enhance the colour characteristics and fastness of natural
dyes o n a textile substrate (Vankar &Shankar, 2008). The choice of mordant is crucial because
these dyes for m complexes with the mordant and different mordants can change t he final colour
significantly. Three types of mordants are used:
1. Metallic mordants – some of the important mordants used are alum, potassium
dichromate, ferrous sulphate, copper sulphate, zinc sulphate, tannin, and tannic acid (Maulik &
Pradhan, 2005; Nalankilli, 1997). Although these metal mordants contribute to developing a
wide range of hues after complexing with the natural colouring compounds. Most of these metals
are toxic and due to increased environmental awareness, the use of certain metallic mordants has
been restricted.
Bio mordants – in the recent past, bio mordants have been used as another alternative to
chemical mordants to solve the fastness problem of natural dyes. Bio mordants are those
substances that can obtain from natural sources (i.e., plants, animals, etc.) such as myrobolan
(Terminalia chebula), tannin, tannic acid, guava, and banana leaves ash. The use of bio mordant
also is a possible eco-friendly approach to improve dye uptake and colour fast-ness (Paul,
Solans, & Eerra, 2005; Vankar & Shankar, 2008; Vankar, Shanker, & Verma, 2007). However,
bio mor- dant showed a better result than alum for light, wash and perspiration fastness
properties. The most common non-metallic mordants are tannins and tannic acid, but metal
hyper-accumulating plants and chlorophylls have also been applied (Guesmi, Ladhari, Ben

16
Hamadi, & Sakli, 2012; Moiteiro, Gaspar, Rodrigues, Lopes, &Carnide, 2008). Some effecti
ve bio mordants were reported by many workers such as oxalic acid and citric acid (Lohtander,
Arola, & Laaksonen, 2020); Acacia catechu (Yusuf, Mohammad, Shabbir, & Khan, 2016) and
Eurya acuminate (Vankar & Shankar, 2008).

2. Oil mordants – oil mordants make a complex with the alum used in mordanting
treatment. Metal atom combined with carboxylic groups of oil and bound metal then makes a
bond with the dye molecu les to get superior wash fastness (Mansour, 2018). O il mordants are
used in the dyeing of madder. Previously, castorand til (sesame) oils were used as oil mordants,
but later they were replaced by a sulponate dcastor o il, tha tis, Turkey Red Oil (TRO).
Different parts of the plants were used for the extraction of dyes such as leaves, flowers,
vegetables, etc and different types of mordants were used for fixing the dye into the fabric. In
ancient days people have used natural dyes to paint their caves. Over 15,000 BC man began to
produce those natural dyes which have been used in textiles as well. In order to understand the
art and history of dyeing, we must first understand the process of dyeing itself. Natural dyes can
be broken down into two categories: substantive and adjective. Most ancient and medieval dyers
mordanted their yarns and fabrics before dyeing them. Different fibers also have different
tendencies to absorb natural and synthetic dyes. Wool, a protein-based fiber, has been found in
Europe dating back to 2000 BC. In Europe the art of dyeing rose to new heights with the
diversity of climate, culture and migration or invasion waves. Eventually, the old natural dyes
lost popularity in favor of the newer synthetic ones.
The alchemy of colors started from early time. With the modern phases of development, dyes
have become the most important resources, owing to their multifarious utilization, including an
emerging branch of medicine i.e., Chromotherapy which greatly depends on natural coloring
dyes. Usually, methods of collection and extraction of dyes are still crude and traditional with
only a few experts related to cottage industries being well versed with dyeing procedures.
Indigenous traditional knowledge on various resources including dye yielding plants is very
essential for rural based development and future bioprospecting, provided proper precautionary
measures are considered for sustainability, conservation and value based selection of use pattern.
Nowadays most of the natural dyers are interested to use natural dye materials in the same ways
used for synthetic dyes. Textile dyers must know the chemistry of these natural colors and its
added advantages of medicinal values. Use of suitable binary or ternary mixtures of similar or

17
compatible natural dyes for coloring natural eco-friendly textiles in variety of soothing /
uncommon shades with eco-friendly mordants and finishing agents are the most desirable
product of the customers for future. So, a textile dyer must know the effects of variability for
extraction, mordanting and

dyeing and should follow only the standardized recipe for selection fiber mordant natural dye
system to get reproducible color yield and color matching besides to follow different eco-
friendly ways to improve color fastness to a possible extent. Thus with the worldwide concern
over the use of eco-friendly and biodegradable materials, the use of natural dyes has undoubtedly
once again gained interest and momentum.(Geeta B et al ).

18
Chapter- 3
MATERIAL AND METHODS

3.1. Experimental Material

All the ingredients and media used in these experiments were purchased from Hi-media
Laboratories Pvt Ltd (India) and Rankem Pvt Ltd (India).The glassware used in experiment was
washed with 6N HCl to remove residual iron and rinsed with pure water after analysis.
3.2. Requirements:
Petri dishes, Autoclavable Bag, Inoculation Loop, Forceps, Transfer Needle, Micropipette,
Scissor, sprit lamp, centrifuge tube, beaker, conical flask, Test Tubes, Boiling Tubes, Durham
tubes, Measuring Cylinder , Glass microscope slides, Cover slips , Dropper, spatula ,Immersion
Oil etc.
3.3. Instrument Used:
Autoclave, pH meter, laminar, shaking incubator, refrigerator, microscope, oven, chemical
balance, spectrophotometer, centrifuge, hot plate etc.
3.4. Miscellaneous:
Test tube stand, culture media, cotton, cotton plug, sprit, spreader, parafilm, muslin cloth, filter
paper, disinfectant etc.
3.5. Chemical Used:
Qualitative test for identification of secondary metabolites in plant sample
• Saponine test
Sample1 ml
Distilled water 6 ml

• Tanine test
Sample1 ml
FeCl3 (5%) 1 ml

19
• Flavonoid test
Sample100µl

NaOH 2 ml

• Fat test

Sample1 ml
NaOH 1 ml
Phenoptheline 1 drop

• Cardiac glycoside test

Sample1 ml
Glacial acetic acid 0.5 ml
Ferric chloride soln 1 drop Sulphuric acid 0.2 ml

• Starch test
Sample1 ml
Iodine 1 ml

• Carbohydrate test
Sample1 ml
CuSO4 0.7 gm.
NaK tatrate 3.5 gm.
NaOH 1.2 gm.
Dextrose 0.1 gm.
Distilled water

20
• Protein test

Sample1 ml
CuSO4 (1%) 4 ml
NaOH (5%) 4 ml
Milk powder 0.1 gm. Distilled water

Media used for the test :

• Plate Count Agar (PCA): Plate Count Agar is recommended for the determination of
plate counts of microorganisms in food, water, waste water and also from clinical samples.

Ingredients:

Casein enzymic hydrolysate 5.000

Yeast extract 2.500
Dextrose 1.000
Agar 15.00

3.6. Sample collection of flowers:

Sample is collected from the flower shop, Chowk, Lucknow city. The name of the sample is
Rose and Marigold. The sample is extracted by the Aqueous Extraction Method.
3.7. Extraction of natural dye from flowers:
Aquoeous extraction method 10 gm fresh petals boiled in 100 ml distilled water at 100 c for 30
minutes. The decolorized petals taken out from extraction solvent.

3.8. Qualitative test:

• Saponine test
Procedure:-
1. Take 1ml plant extract in a test tube.

21
2. Add 6ml of distilled water in the same test tube.
3. Mixed gently.
4. If the forth is formed, then saponine is present.

• Tannine test
Procedure:-
1. Take 1ml of plant extract in a test tube.
2. Make 1ml of 5% FeCl3.
3. Mix 1 ml of plant extract and 1ml of 5% FeCl3.
4. If dark color like brown, green, & black will be shown in the solution, then it show that
tannin is present.
• Flavonoid test
Procedure:-
1. Take 2 ml of NaOH in a test tube.
2. Take 1.5µl of plant extract in an append drop tube and centrifuge it.
3. After centrifugation, take 100ul of supernatant from the append drop tube.

4. Mix 100 µl of supernatant in 2 ml of NaOH.

5. If the mixing solution shows yellow color then, it seems flavonoid is present.
• Fattest: Procedure:-
1. Take 1ml of diluted NaOH in a test tube.
2. Add 1 drop of phenopthaline with diluted NaOH.
3. Mix it well until the pink colour is form.
4. Add 1ml of sample in the solution.
5. After adding the sample in the solution, if pink colour will be disappear then it seems fat
is present

• Cardiac glycoside test

Procedure:-
1. Take 1ml of sample in a test tube.
2. Add 0.5ml of Glacial acetic acid in the test tube.

22
3. Add 1 drop of ferric chloride solution
4. Mix them well.
5. Then, add 0.2 ml of concentrated sulphuric acid.
6. After adding Sulphuric acid, a brown will be formed at the top of the layer.

7. Then, a ring of voilet colour will be formed.

8. A greenish layer formed in acitic acid layer.
9. If rings were formed it seems that, cardiac glycoside is present in the sample.

• Starch test
Procedure:-
1. Take 1ml of sample in a test tube.
2. Add 1ml of Iodine in the sample.
3. After mixing it well, if blue color will be formed, it seems starch is present.
• Carbohydrate test
Procedure:-
1. Firstly, make Fehling’s solution A by adding – 0.7 gm of CuSO4 with 5 ml of distilled
water.
2. Then, make Fehling’s solution B by adding – 3.5 gm of Sodium potassium nitrate and 1.2
gm of NaOH in 5 ml of distilled water.
3. Mix Fehling’s solution A and B in 1:1 ratio.
4. Now, take 3 test tubes and labelled them.
5. In test tube no. 1, take 1 ml of sample with 1ml of mixture of Fehling’s solution A and B.
6. In test tube no. 2 i.e., -ve control, add 1ml of distilled water with 1ml mixture of
Fehling’s
solution A and B.
7. In test tube no. 3 i.e. , +ve control, add 1 ml of distilled water with 1ml mixture of
Fehling’s solution A and B with a pinch of Dextrose.
8. Give 10mins of water bath to all 3 test tubes.
9. After water bath, if red precipitate will be form in the sample, it will showed that
Carbohydrate is present in the sample, if not then Carbohydrate is absent.

23
• Protein test
Procedure:-
1. Firstly, make 1% of CuSO4 solution in 4 ml of distilled water.
2. Then, make 5 % of NaOH solution in 4 ml of distilled water.
3. Now, take 3 test tubes and labelled it properly.

4. In test tube number 1, take 1ml of sample.

5. In test tube number 2 and 3, take 1 ml of distilled water.
6. In test tube number 3, add pinch of milk protein
7. Then, add 1 ml of 1% of CuSO4 and 5% of NaOH in each test tube.
3.9. Gram staining of pathogens:
Principle: Gram’s staining technique is used to differentiate bacteria on the basis of their cell
wall composition. The bacterial culture which appeared purple in colors is known as Gram’s
positive while the bacteria which appear pink in color are Gram’s negative. Gram positive cells
have a thick peptidoglycan cell wall that is able to retain the crystal violet – iodine complex that
occurs during staining, while gram negative cells have only thin layer of peptidoglycan. Thus
grampositive cell do not decolorize with ethanol, and gram-negative cells do decolorize, this
allowed the Gram-negative cells to accept the counter stain safranin. Gram-positive cells appears
blue to purple, while Gram-negative cells appears pink to red.
Procedure:
• Prepare the smear and heat fix.
• Stain the slide by flooding it with crystal violet for 2-3 minutes.
• Pour off excess dye and wash gently in tap water and drain the slide against a filter paper.
• Add Gram’s iodine for 1-2 minutes and leave it on the smear until the smear is over.
• Wash with 95% alcohol for 30 second.
• Wash with tap water at the end of 30 second to stop decolorization and drain.
• Counter stain with 0.25% safranin for 2-4 minutes.

3.10. Antifungal activity:

1. Prepare Potato dextrose agar media.
2. Take a culture bottle and put dextrose in the bottle with starch and distilled water.
3. Maintain the pH of the solution.

24
4. Now add agar to the solution and allow to autoclave for 20mins.
5. After autoclave, leave the media until it get slightly cool.
6. Now pour the media into the petri-plate and allowed to cool it.
7. Then spread the fungus on culture plate.
8. Now bisect the plate in four section.(S1,S2,-ve control and +ve control)
9. Pour the disc into the four different sections after dissolving them into given sample.

10. Leave for a week for result.

3.11. Antibacterial activity: Procedure:-
1. Prepare Nutrient agar media.
2. Take a culture bottle and put peptone, beef extract, and NaCl.
3. Dissolve them in distilled water and its pH.
4. Add agar in the mixture and allow to autoclave.
5. After autoclave, leave the media until it get slightly cool.
6. Now pour the media into the petri-plate and allowed to cool it.
7. Then spread the bacteria on culture plate.
8. Now bisect the plate in two parts.
9. Pour the disc into the two different sections after dissolving them into given sample.
10. Leave for 24 hrs for result.

For extraction of dye, one part of powder was mixed in 10 parts of DDW at 30 ± 1 °C and 60 ± 1
°C for 1 h. The obtained extract was boiled at 100 ± 1 °C at atmospheric pressure for 1 h
followed by soaking at 30 ± 1 °C for 1 h with constant stirring at 50 rpm. Finally, the extract was
separated from the biomass by filtration through cotton cloth to remove insoluble residues
(Shahid Adeel 2009).

25
Chapter- 4
RESULT AND DISCUSSION
4.1 Sample collection of flower of Rose and Marigold :
Collection was done of fresh leaves of Rose and Marigold flower to perform the experimental
study.

Fig.1. Freshly collected leaves of Rose and Marigold flowers

4.2. Extraction of natural dye from collected flowers:

Filtrate obtained light pink in color as showed in fig.2

Fig.2. Extracted material from flowers

4.3. Qualitative test for the detection of secondary metabolites:

1. Saponin test Present

2. Tannin test Present
3. Flavonoid test Present
4. Fat test Present
5. Cardiac glycoside test Present
6. Starch test Present
7. Carbohydrate test Present
8. Protein test Present

26
4.4. Antimicrobial potential of flowers extract:

Antibacterial Activity of Rose extract:

8 mm diameter of zone was measured by the use of Rose extract which revealed that extract of
rose has antibacterial property against E.coli whereas 19 mm zone was observed for S.aureus. As
showed in fig.3. Similar study was reported by Mohamed Shohayeb 2014 where they were used
Rose extract for the control of Staphylococcus aureus, Baccilus subtilis and found that the extract
showed antibacterial activity against these bacterial strains.

Fig.3. Antibacterial activity of E.coli and S. aureus by Rose extract

Antibacterial Activity of Marigold extract:

13 mm of inhibition zone was seen by the use of Marigold extract to check the antibacterial
potential of E.coli while 18mm zone was found in S.aureus as depicted in fig.4

Fig.4. Antibacterial activity of E.coli and S. aureus by Marigold extract

Antifungal activity of Rose extract:
Due to the presence of some secondary metabolites which were essential for the antifungal
activity, 17 mm of inhibition zone was examined after the use of Rose extract for Aspergillus
niger. Similar study was done by Mohamed Shohayeb 2014 where they observed that Rose

27
extract has antifungal activity against Aspergillus niger, and Penecillium notatum fungal strains.

Antifungal activity of Marigold extract:

16.5 mm of inhibition zone was found to suppress the growth of Aspergillus niger which ultimately
confirmed the presence of potential secondary metabolites in the marigold extract.

28
Chapter- 5
CONCLUSION

Natural dyes, generally supposed to be cheap, non-toxic, worthwhile, and sustainable resource
with minimal environmental impact, have attracted the attention of the scientific community to
use them in a variety of traditional and newly discovered application disciplines. We found in
our study that Rose and Marigold extract have antimicrobial property which can serves as a
natural preservatives instead the use of chemical preservatives. Flowers extract has organoleptic
properties especially in Rose extract which may use a flavor enhancer as well as for strong
odour. These days food industries using chemical preservatives as well as flavor enhancer in a
large scale which ultimately shows their toxic effects in the body and sometimes it may cause
severe allergic symptoms also.

29
Chapter- 6
REFRENCES

Mirjalili, M., & Karimi, L. (2013). Extraction and characterization of natural dye from green
walnut shells and its use in dyeing polyamide: Focus on antibacterial properties. Journal of
Chemistry, 2013.

Mirjalili, M., & Abbasipour, M. (2013). Comparison between antibacterial activity of some
natural dyes and silver nanoparticles. Journal of Nanostructure in Chemistry, 3(1), 1-3.

Grover, N., & Patni, V. (2011). Extraction and application of natural dye preparations from the
floral parts of Woodfordia fruticosa (Linn.) Kurz.

Pargai, D., Jahan, S., & Gahlot, M. (2020). Functional properties of natural dyed textiles. In
Chemistry and technology of natural and synthetic dyes and pigments. IntechOpen.

Calis, A., Çelik, G. Y., & Katircioglu, H. (2009). Antimicrobial effect of natural dyes on some
pathogenic bacteria. African Journal of Biotechnology, 8(2).

Gupta, V. K. (2019). Fundamentals of natural dyes and its application on textile substrates.
Chemistry and technology of natural and synthetic dyes and pigments.
Silva, N. C. C., & Fernandes Júnior, A. J. J. O. V. A. (2010). Biological properties of medicinal
plants: a review of their antimicrobial activity. Journal of venomous Animals and Toxins
including tropical diseases, 16(3), 402-413.

Mishra, A., & Gautam, S. (2020). Application of Natural Dyes for Herbal Textiles. In Chemistry
and Technology of Natural and Synthetic Dyes and Pigments. IntechOpen.

Kumar, M., Kaur, P., Garg, R., Patil, R. K., & Patil, H. C. (2020). A study on antibacterial
property of curcuma longa–herbal and traditional medicine. Adesh University Journal of Medical
Sciences & Research, 2(2), 103-108.

30
Govindarjan VS. Turmeric-chemistry, technology, and quality. CRC Crit Rev Food Sci Nutr

1980;12:199-301.

Peret-Almeida L, Cherubino AP, Alves RJ. Separation and determination of the physico-
chemical characteristics of curcumin, demethoxycurcumin, bisdemethoxycurcumin. Food Res Int
2005;38:1039-44.

Antony MB. Indigenous medicinal plants: Their extracts and isolates as a value added export
product. J Agro Bios 2003;1:39-41.

Schieffer GW. Pressurized liquid extraction of curcuminoids and curcuminoid degradation

products from turmeric (Curcuma longa) with subsequent HPLC assays. J Liq Chromatogr Relat
Technol 2002;25:3033-44.

Johnson JJ, Mukhtar H. Curcumin for chemoprevention of colon cancer. Cancer Lett
2007;255:170-81.

Ravindran P, Babu KN, Sivaraman K. Turmeric: The genus Curcuma. Boca Raton: CRC Press;
2007. p. 150-5.

Revathy S, Elumalai S, Benny M, Antony B. Evaluation of curcuminoids in turmeric rhizome

(Curcuma longa L.) collected from different places in India. Biosci Biotechnol Res Asia
2011;8:259-64.

Wichitnithad W. A simple isocratic HPLC method for the simultaneous determination of

curcuminoids in commercial turmeric extracts. Phytochem Anal 2009;20:314-9.

Janssen A, Gole TH. Thin layer chromatographic determination of curcumin from turmeric.
Chromatography 1984;18:546-9.

Cohly HH, Asad S, Das SK, Angel MF. Effect of antioxidant (turmeric, turmerin and curcumin)

31
on human immunodeficiency virus. Int J Mol Sci 2003;4:22-33.
Irving GR, Karmokar A, Berry DP, Brown K, Steward WP. Curcumin: The potential for efficacy
in gastrointestinal diseases. Best Pract Res Clin Gastroenterol 2011;25:519-34.

Sharma RA, Gescher AJ, Steward WP. Curcumin: The story so far. Eur J Cancer 2005;41:1955-
68.
Aggarwal BB, Sundaram C, Malani N, Ichikawa H. Curcumin: The Indian solid gold. Adv Exp
Med Biol 2005;595:1-75.

Jayaprakasha GK, Rao LJ, Sakariah KK. Improved HPLC method for the determination of
curcumin, demethoxycurcumin and bisdemethoxycurcumin. J Agric Food Chem 2002;50:3668-
72. Lim GP, Chu T,

Yang F, Beech W, Frautschy SA, Cole GM. The curry spice curcumin reduces oxidative damage
and amyloid pathology in an Alzheimer transgenic mouse. J Neuroscince 2001;21:8370-7.

Simon A. Inhibitory effect of curcuminoids on MCF-7 cell proliferation and structure-activity

relationship. Cancer Lett 1998;129:111-6.

Anand P, Aggarwal BB. Bioavailability of curcumin: Problems and promises. Mol Pharmacol
2007;4:807-18.
Antony B, Benny M, Rao SB. Enhancing the absorption of curcuminoids. J Spices India
2005;11:23-6.
Abdollahzadeh E, Rezaej M, Hosseini H. Antibacterial activity of plant essential oils and
extracts: The role of thyme essential oil, nisin, and their combination to control Listeria
monocytogenes inoculated in minced fish meat. Food Control
2014;35:177-83.

Aggarwal BB, Harikumar KB. Potential therapeutic effects of curcumin, the anti-inflammatory
agent, against neurodegerative, cardiovascular, pulmonary, metabolic, autoimmune and
neoplastic diseases. Int J Biochem Cell Biol
2009;41:40-59.

32
Aligiannis N, Kalpoutzakis E, Mitaku S, Chinou IB. Composition and antimicrobial activity of
the essential oils of two Origanum species. J Agric Food Chem 2001;49:4168-70.

Arora DS, Kaur J. Antimicrobial activity of spices. Int J Antimicrob Agents 1999;12:257-62.
Gupta AP, Gupta MM, Kuma SJ. Simultaneous determination of curcuminoids in curcuma
samples using high-performance thin-layer chromatography. Liq Chromatogr Real Time
1999;22:1561-9.

Paramapojn S, Gritsanapan W. Free radical scavenging activity determination and quantitative

analysis of curcuminoids in Curcuma zedoaria rhizome extracts by HPLC method. Curr Sci
2009;97:1069-73.
Naidu MM, Shyamala BN, Srinivas P. Simple HPLC method for resolution of curcuminoids
with antioxidant potential. J Food Sci 2009;74:C312-8.
Mohammed AN. In vitro studies of antimicrobial activity of (Curcuma longa L.) rhizomes
against Helicobacter pylori. Iraq Med J 2017;1:7-9.
Kapoor LD. CRC Handbook of Ayurvedic

33