BIOINNOVATE

A RESEARCH PROJECT ON

"THE POTENTIAL OF BIOTIC AND ABIOTIC COMPOUNDS AS ALTERNATIVE

TO ANTI-FUNGAL AGAINST MDR CANDIDA SPECIES"

Submitted by

RAMIJ RAJA

SAMMILANI MAHAVIDYALAYA

(AFFILIATED BY UNIVERSITY OF CALCUTTA)

Reg No.: 513-1115-0348-21

Roll No.: 213513-21-0140

A.M EDUCARE
 ACKNOWLEDGEMENT

I would like to thank all those who have guided and supported me during the duration of
my project entitled "The Potential of Biotic and Abiotic Compounds as Alternatives to
Antifungal Against MDR Candida Species" under the Bioinnovate program at AM
Educare.

Above all, I am deeply indebted to Dr. Anirban Mukherjee and Prof. Aniruddha Mukherjee
for their precious guidance, continuous motivation, and thought-provoking advice, which
were instrumental in the successful fulfillment of my study. I am also thankful to Mr.
Diptesh Chakraborty, Ms. Saborni Biswas, Ms. Bipasha Saha, and Ms. Joyeeta Chatterjee
for their continuous support, professional inputs, and for sharing their ideas and expertise
with me throughout the project.

I would also like to thank AM Educare for furnishing a very stimulating research context
and the material resources necessary for conducting this endeavor. Lastly, I would also like
to gratefully acknowledge the support and motivation of my friends and family all along
the way.
 ABSTRACT

The efficiency of traditional antifungal treatments has been hampered by the emergence of
multidrug-resistant (MDR) Candida species, which has prompted a quest for new
substitutes. By breaking down membranes, preventing the formation of biofilms, and
interfering with fungal metabolism, biotic substances such phytochemical extract,
microbial metabolites, and antimicrobial peptides have antifungal properties. By causing
oxidative stress and destroying fungal structures, abiotic agents—such as metal
nanoparticles and synthetic polymers—have antifungal effects. These substances exhibit
potential against Candida tropicalis and other MDR strains. Combining biotic and abiotic
substances may increase effectiveness and reduce the likelihood of resistance. While
encouraging, more study is required to evaluate toxicity, improve delivery, and confirm
clinical uses. Investigating these options may result in novel treatments to counter the rising
risk of MDR fungal infections.
 INTRODUCTION

Multidrug resistance (MDR) refers to the ability of an organism, such as bacteria, fungi,
or cancer cells, to resist the effects of multiple drugs that are typically used to treat
infections or diseases. This resistance occurs when the organism adapts to survive exposure
to several different classes of drugs, rendering standard treatments ineffective.
In the fungus Candida sp. This MDR developed through mechanisms like overexpression
of efflux pumps, mutations in drug target enzymes, biofilm formation, and alterations in
the fungal cell membrane.

Curcumin, a natural compound derived from the root of turmeric plant (Curcuma longa)
has bioactive molecule called curcumin, which has drawn a lot of interest due to its ability
to treat fungal infections, notably those brought on by Candida species. Curcumin has
several bioactive qualities, such as antibacterial, anti-inflammatory, and antioxidant
actions. Curcumin may be able to stop fungal growth and biofilm formation, which is a
major cause of fungal cells' resistance to antifungal medications, according to research.

Nanobiotechnology is an emerging interdisciplinary field combining nanotechnology and

biotechnology, focusing on the use of nanoscale materials for biological and medical
applications. It makes use of the special qualities of nanoparticles, namely their large
surface area and reactivity, for uses in biosensors, medication delivery, and diagnostics. The
production of nanoparticles such as nickel nanoparticles (NiNPs) is an important
component of nanobiotechnology. These particles are created utilizing a variety of
approaches, such as chemical reduction, green synthesis (using microbes or plant extracts),
and physical methods including laser ablation. They are known for their unique magnetic
and catalytic capabilities. Green synthesis is especially appealing because of its
environmentally friendly methodology. Nickel nanoparticles are useful in both industrial
and biomedical domains since they can be used in sensors, energy storage, catalysis, and
even as antibacterial agents.

The fish gut hosts a diverse and dynamic community of microorganisms that play vital
roles in digestion, nutrient absorption, immunity, and overall fish health. Understanding
these intricate relationships and investigating their possible uses in aquaculture and
biotechnology require the isolation of fish gut microorganisms. To produce pure microbial
isolates, the procedure usually entails the aseptic collection of gut samples, followed by
culture on selective media under controlled conditions. Researching these microorganisms
aids in the discovery of advantageous bacteria that can improve fish growth, act as
probiotics, and fend against infections. Additionally, isolated gut microorganisms are
useful for studying the synthesis of siderophores and other antimicrobial compounds. The
goal of this research is to identify the biological and functional characteristics of microbial
strains from fish gut habitats by isolating, growing, and characterizing them.
Biopolymers are naturally occurring polymers that have drawn interest due to their
biological and environmentally beneficial qualities. According to recent research, several
biopolymers can affect the growth and activity of fungi. By breaking down fungal cell
walls, slowing spore germination, and stopping hyphal development, certain biopolymers,
like chitosan, demonstrate potent antifungal effects. Others may serve as antifungal drugs'
transporters, increasing their potency. On the other hand, several fungi have the ability to
break down biopolymers such as starch and cellulose and use them as fuel. In industries
like agriculture, where biopolymer-based coatings can shield crops, and medicine, where
biopolymer scaffolds help fend against fungal infections, the relationship between
biopolymers and fungus is crucial. The dual function of biopolymers in either inhibiting
fungal growth or acting as substrates for fungal metabolism is examined in this work.

Computational drug design is a recent strategy that finds, creates, and optimizes novel
medicinal molecules using computer-based techniques. It involves designing the molecular
interactions between pharmaceuticals and biological targets, including proteins or enzymes,
in order to forecast the efficacy and safety of possible medications. Researchers can find
interesting candidates more quickly and affordably than they could with conventional
experimental approaches by using techniques like molecular docking, molecular dynamics
simulations, and quantitative structure-activity relationship (QSAR) modeling. Deeper
comprehension of disease pathways is made possible by computational drug design, which
also speeds up the drug development process. It is commonly utilized in pharmaceutical
research to create medications that are less harmful, more potent, and more selective.
 MATERIALS AND METHODS

 GLASSWARES
1. Test Tubes
2. Beakers
3. Petri Plates
4. Conical Flasks
5. Measuring Cylinders
6. Capillary Tube
7. 96 Well Plates
8. MCT Tubes
9. Thermometer
10. Mortar and Pestle
11. Falcon

 CHEMICALS

1. Agar powder
2. Chloroform
3. n-Hexane
4. Ethanol
5. Methanol
6. Nickel sulfate hexahydrate
7. Crystal Violet
8. Curcumin Boil
9. Sodium Hydroxide
10. Sabouraud Dextrose Agar
11. Nutrient Broth
12. TE Buffer
13. Trise HCL
14. Ethylene diamine tetra acetic acid (EDTA)
15. Lyticase
16. Potassium Acetate
17. Phenol
18. Isoamyl alcohol
19. Isopropanol
20. RNase
21. Acetic acid
22. TAE Buffer
23. Ethidium bromide (EtBr)
24. MTT reagent
25. Crystal Violet
 26. Sodium Dodecyl Sulphate (SDS)
27. Acrylamide
28. Bisacrylamide
29. Ammonium per sulphate
30. TEMED
31. Glycerol
32. β -mercapto ethanol
33. Coomasie Brilliant Blue
34. Running Buffer
35. Glycine
36. Trichloro acetic acid (TCA)
37. Dimethyl Sulfoxide (DMSO)
38. Antifungal discs
39. Distilled water

 INSTRUMENTS

1. Magnetic Stirrer (LYZER)

2. Laminar Air- flow (ARG SCIENTIFIC)
3. Gel Apparatus (GeNei)
4. Autoclave (Indfos)
5. Weighing Balance (WENSAR)
6. UV Spectrophotometer
7. Vortex Mixture (REMI CM- 12C)
8. Dry Bath
9. Microwave (BAJAJ)
10. Centrifuge (REMI RM- 12C)
11. SDS PAGE Apparatus (BIO-RAD)
12. Silica Plates
13. Micro pipette
14. Incubator
15. Refrigerator
16. Cork borer
17. Elisa Reader
18. PCR
 ISOLATION OF CANDIDA FROM SOIL.

 10gm of soil sample was dissolved in 90ml 0f distilled water.

 Serial dilution was performed up to 10 dilution.
 From the tube of 10 dilution 100 µl sample were collect and spread on a Sabouraud
Dextrose Agar plate.
 Incubate at room temperature for 48 hours.
 MDR PROFILING OF CANDIDA.

Multidrug resistance (MDR) profiling of Candida involves determining how susceptible it

is to various antifungal medications. This is accomplished by subjecting Candida isolates
to various antifungals by techniques such as disk diffusion, broth microdilution, or the E-
test. To find the minimum inhibitory concentration (MIC) or inhibition zones, growth
inhibition is measured. To categorize susceptibility, results are analyzed using CLSI or
EUCAST breakpoints. MDR is indicated by resistance to two or more antifungal classes,
which aids in treatment guidance and resistance trend monitoring in clinical settings.

 Antifungal agents used

1. Nystain.
2. Amphotericin B.
3. Itraconazole.
4. Clotrimazole.
5. Fluconazole.
6. Miconazole.
7. Ketoconazole.

 Procedure

 Media was prepared by 50gms of Sabouraud Dextrose Agar and 2% of agar agar
(1gm)
 The media was Poured in a sterile petri plate and wait for solidify.
 Isolated culture of Candida tropicalis was spread in this petri plate.
 The antifungal agents were placed in the plate, and incubate for 48 hours.
  FACTOR PREPARETION

 EXTRACTION OF CURCUMIN.

Curcumin, a natural compound derived from the root of turmeric plant (Curcuma longa)

10% Methanolic extract of curcumin produced through few steps:

 5gm of turmeric were paste in a mortar pestle.

 50ml of methanol were added.
 Stire in a magnetic stirrer and volume was reduced in half.
 The extract was collected in a falcon.

 CHARACTERIZATION OF CURCUMIN EXTRACT.

 THIN LAYER CHROMATOGRAPHY(TLC)

Thin Layer Chromatography (TLC) is based on the separation of components in a mixture
due to differences in their affinity toward a stationary phase (a thin layer of adsorbent like
silica gel) and a mobile phase (solvent). Components move at different rates, depending on
their solubility and interaction with the stationary phase, resulting in distinct spots on the
TLC plate.

The extract was spotted onto a TLC plate. Solvent front migration was allowed under
normal and UV light. Retention Factor (Rf) values were calculated to confirm the presence
of Plumbagin.

Rf =

 Procedure
 Solvent for mobile phase was made with n hexane: Ethanol: Chloroform
 49: 10: 41
 Solvent required 10 ml.;
n hexane - 4.9 ml
Ethanol - 1ml
Chloroform- 4.1 ml
 Solvent was kept in a Beaker and covered with a paper
  Silica plates were cut in size.
 With help of a capillary Tube, Methanolic extract of Curcumin was placed in
TLC plate. 3 dots each in 3 points in the TLC plate were applied.
 TLC plate was transferred to the mobile phase, and leave few times.
 When the solved travelled 80 % of the silica plate, the silica
Plate was removed from solvent and the distance travelled by the solvent was measured.
 Silica plate was observed under UV transilluminator and Distance travelled
by the bands was measured.
 The Rf values of the three bands was calculated.

 EXTRACTION OF PURIFIED CURCUMIN BY COLUMN CHROMATOGRAPHY

The differential adsorption and partitioning of substances between a stationary phase

and a mobile phase forms the basis of column chromatography's basic idea. A column
filled with an adsorbent (silica gel, for example) is loaded with a mixture. Depending
on their polarity and affinity for the stationary phase, the components move through the
mobile phase (solvent) at varying rates. The components of a mixture can be separated
and purified because weakly adsorbed compounds elute more slowly than firmly
adsorbed ones.

 Procedure

 Glasswool was poured in the column and stack well.

 Stationary phase was made by silica gel. Silica gel was poured in the column and
stack with a glass rod.
 For mobile phase Solvent was prepared with n hexane, Ethanol, Chloroform in the ratio
of [Link]
 Curcumin was pored into the column and for creating a pressure, solvent was poured
into the column.
 Stopcock was opened and purified curcumin samples according to their colour gradient
was collected in different test tubes.
 N ANOPARTICLE SYNTHESIS.

The production of nanoparticles such as nickel nanoparticles (NiNPs) is an important

component of nanobiotechnology. These particles are created utilizing a variety of
approaches, such as chemical reduction, green synthesis (using microbes or plant
extracts), and physical methods including laser ablation. They are known for their
unique magnetic and catalytic capabilities. Green synthesis is especially appealing
because of its environmentally friendly methodology. Nickel nanoparticles are useful
in both industrial and biomedical domains since they can be used in sensors, energy
storage, catalysis, and even as antibacterial agents.

 Procedure
 1.3142 grams of Nickel sulfate hexahydrate was added to 50 ml of distilled
water in a magnetic stirrer.
 Temperature should be maintained at 45°C.
 20ml of curcumin boil was added drop wise,
 Stir for 30 minutes.
 5M NaOH was added to maintained pH 8- 9. We have added 1600 µL to
maintain pH 8- 9.
 Stir for 3 hours.
 The gel like material was collected in a petri plate and incubate until it turns in
solid form.
 The solid particle was collected in a mortar and make fine powder.
 The powder was collected into an eppendorf tube.
 UV SPECTRA OF SYNTHESIZED NANOPARTICLE.
The foundation of the UV-Vis absorbance method is the idea that molecules, depending
on their structure, absorb light at particular wavelengths. Certain wavelengths of visible
or ultraviolet light are absorbed by a sample, resulting in electronic changes in the
molecules. The concentration of the absorbing material is directly correlated with the
optical density, or O.D., or amount of light absorbed.

The absorbance of various substances (such as NiSO₄, turmeric extract, and nickel
nanoparticles) is measured over a range of wavelengths (200–1000 nm) using an ELISA
(microplate) reader in spectrum mode. Characteristic peaks that show the presence and
production of substances like nanoparticles or bioactive extracts can be found in the
ensuing absorbance spectra.

 Procedure
1. Sample Preparation
 The following liquid samples were prepared:
I. Nickel sulfate (NiSO₄) solution
II. Turmeric extract (methanolic extract, with curcumin preferred)
III. Synthesized nickel nanoparticles (NiNPs)
 The solutions were filtered using either Whatman filter paper or a 0.22 um
membrane to remove particulates.

2. Blank Preparation
 The appropriate solvent is filled into a clean quartz cuvette. (distilled water or
methanol).
 The blank cuvette is placed into the spectrophotometer to calibrate (zero) the
instrument.

3. Sample Measurement
 A clean cuvette is filled with the first sample (typically the NiSO₄ solution).
 The cuvette is placed into the sample holder.
 The wavelength range is selected (typically 200-1000 nm).
 The absorbance spectrum is found.
 This process is repeated for turmeric extract and nickel nanoparticles.

4. Data Collection
 A clean cuvette is filled with the first sample (typically the NiSO₄ solution).
 The cuvette is placed into the sample holder.
 The wavelength range is selected (typically 200-1000 nm).
 The absorbance spectrum is found.
 This process is repeated for turmeric extract and nickel nanoparticles.
 ISOLATION OF SIDEROPHORES.
Siderophores are small, high-affinity molecules produced by microorganisms to bind
and transport iron from the environment into their cells, especially under iron-limited
conditions.

 ISOLATION FROM FISH GUT.

 Intestine of a Tilapia (Oreochromis niloticus) was taken out.
 0.9% saline water was injected through the intestine.
 The saline water passed through the intestine was collected to a clean falcon.
 30 ml of NA broth was prepared in a culture tube.
 The broth was inoculated with the isolated fish gut microbes.
 Incubate for overnight.

 ISOLATION FROM PROBIOTICS.

 30ml of NA broth was prepared in a culture tube.
 One capsule of probiotic bacteria Lacticaseibacillus rhamnosus. Put into this broth.
 The culture was incubated for overnight.
 After overnight incubation, bacterial culture was filtered with membrane filter.
 BIOPOLYMER SOLUTION.

A biopolymer solution's basic idea is to dissolve natural polymers, such as proteins or

polysaccharides, in an appropriate solvent to create a stable, viscous medium. These
solutions have special qualities such gel formation, high viscosity, and biocompatibility.
Applications in food stabilization, medication delivery, and biodegradable coatings are
made possible by their interactions with other molecules. Biopolymer solutions are
useful for long-term, practical applications in the industrial, medicinal, and
environmental domains because they are non-toxic, environmentally benign, and can
form films or hydrogels.

 Biopolymer was prepared with hyperbranched polymer PEG (Polyethylene

Gycol) and citric acid in the ratio of 1:5
 Prepared biopolymer was collected in a falcon and kept in refrigerator for future
use.
 CUP PLATE ASSAY.

The cup plate assay's basic idea is to measure a substance's antimicrobial activity by
allowing it to diffuse through a solid substrate. This technique involves creating wells,
or cups, on an agar plate that has been infected with a test microbe. After being added
to the wells, the antimicrobial solution diffuses into the nearby agar. If the material is
active, it prevents the growth of microorganisms, forming a distinct zone of inhibition
surrounding the well. The sample's antibacterial efficacy is directly correlated with the
diameter of this zone.

 Factors used
1. Curcumin.
2. Nickel Nanoparticles.
3. Fish gut microbes.
4. Probiotics.
5. Biopolymer.

 Procedure

 Media was prepared by 50gms of Sabouraud Dextrose Agar and 2% of agar agar
(1gm)
 The media was Poured in a sterile petri plate and wait for solidify.
 Isolated culture of Candida tropicalis was spread in this petri plate.
 5 wells were created with a cork borer.
 75µl of each 5 factors were poured in 5 different wells.
 Incubate at room temperature for 48 hours.
 96 WELL PLATE ASSASY.

The 96-well plate assay's basic idea is to perform several simultaneous measurements or
reactions in a microplate with 96 separate wells, enabling high-throughput screening.
Samples, reagents, or cells can be added to each well, which functions as a separate reaction
vessel. Measurable signals like color, fluorescence, or luminescence are produced by
reactions (such enzyme activity, cell growth, or binding assays). These signals are picked
up by a microplate reader, which quantifies the outcomes for every well. Rapid, parallel
analysis with tiny quantities and high efficiency is made possible by this technology.

 Experiments

1. Biofilm Eradication Assay.

2. Anti Biofilm Assay.
3. MIC (Minimal Inhibitory Concentration)
4. MTT (Cell Viability Assay)

Control C N FG PR BP C N F PR BP
1 2 3 4 5 6 7 8 9 10 11 12
Only - Cr Cr Cr Cr Cr
cell
- - 1/2 1/2 1/2 1/2 1/2
Only - 1/4 1/4 1/4 1/4 1/4
cell
- - 1/8 1/8 1/8 1/8 1/8
Only - 1/16 1/16 1/16 1/16 1/16
cell
- - 1/32 1/32 1/32 1/32 1/32
SBD C N F PR BP - - - - - -
H2O - - - - - - - - - - -
 5 Factors
 C = Curcumin.
 N = Nickel Nanoparticle.
 F = Fish gut.
 PR = Probiotic.
 BP = Biopolymer.
 Procedure

 Day 1:
 Cell was added in wells for Biofilm Eradication Assay.
 Control well was prepared.

 Day 2:
 Cells and factors were added for Anti-biofilm assay
 Well for control was done.

 Day 3:
 5 different factors were added in the wells for biofilm eradication except control
well.
 200µl call and 20µl of factors of different dilution (½, ¼, 1/8, 1/16, 1/32) were
added in wells for MIC assay.
 100µl call and 10µl of factors of different dilution (½, ¼, 1/8, 1/16, 1/32) were
added in wells for MTT assay.

 Day 4:
 96 well plate was inserted in ELISA reader and the result of MTT & MIC was taken.
 Cells was discarded.
 1% crystal violet was added tom the wells of biofilm eradication and antibiofilm
assay and incubate for 15 minutes.
 After 15 minutes, crystal violet was discarded and 30% acetic acid was added for
destaining.
 96 well plate was inserted in ELISA reader and the result of biofilm and antibiofilm
was taken.
  GENOMIC DNA EXTRACTION.

The basic idea behind genomic DNA extraction is to separate pure DNA from cells by
first releasing the DNA by the rupture of nuclear and cell membranes, then filtering out
proteins, lipids, and other impurities. Cell lysis, protein degradation by enzymes or
chemicals, and DNA separation from other constituents are the methods used to
accomplish this. Alcohol (ethanol or isopropanol) is then used to precipitate DNA, then
washing is used to purify it. Finally, the recovered DNA is dissolved in a buffer for
molecular biology applications or additional analysis.

 Procedure

i. AGAROSE GEL PREPARATION.

1% agarose gel for 30ml was prepared

 0.3 grams of Agarose powder was dissolved in 30 ml of 1X TAE buffer.

 Heat the mixture in a microwave (30 sec intervals) or on a hot plate.
 Swirl until the solution becomes clear (no particles).
 Let it cool to ~50–60°C (warm but not hot to touch).
 Place the comb into the casting tray to form wells.
 Pour the warm agarose solution into the tray evenly.
 Let it set at room temperature (~20–30 min) until firm.
 Carefully pull out the comb to form wells.
 Place in Electrophoresis Chamber
 Cover with 1X running buffer until the gel is submerged.
 Now it's ready for sample loading and electrophoresis.

ii. DNA ISOLATION

 1.5 ml of candida culture is taken in micro centrifugal tube and centrifuged at 10000
RPM for 2 minutes.
 Take the pellet and add 60 µL of TE buffer (10 mM tris HCL of pH 8 + 1mM EDTA)
and add 30 µL of lyticase (1mg/ml)
 Incubate for 10 minutes at room temperature.
 Add 30µL of proteinase k (1mg/ml), 50 µL of 10% SDS (anionic detergent) and
incubate for 20 minutes at 55°C
 Add 100µL of 3M potassium acetate and mix well
 Incubator on ice for 10 minutes.
 Centrifuge on 10000 RPM for 10 minutes at 4°C
 Transfer the clear supernatant to a new MCT.
 To the clear supernatant add equivalent of phenol: chloroform: isoamyl alcohol
([Link])
 invert mix 14 minutes then centrifuged at 10000 RPM for 10 minutes.
 Collect the aqueous layer to it add equal volume of chloroform: isoamyl alcohol (24:1)
 Invert means for 10 minutes and then centrifuge at 10000 RPM for 2 minutes.
 Collect the aqueous layer and to it at equal volume of chilled isopropanol
 Incubate at -20°C for 30 minutes.
 Centrifuge at 10000 RPM for 10 minutes.
 Discard the isopropanol
 Add 1ml of 70% ethanol to the pellet
 Centrifuge at 10000 RPM for 10 minutes
 Discard the ethanol and air dry the pellet.
 Add 20µL of 1X TE buffer.
 Add 5µL of RNase and incubate at 42°C for 45 minutes.
 5µL of Ethidium Bromide (EtBr) and 10 µL of DNA sample was mix well
 Load in prepared agarose gel and leave for electrophoresis.
 Observed the gel under UV transilluminator.
 STRESS PROFILING.

The idea behind PCR stress profiling is to identify alterations in the expression of genes
linked to stress when under stress. Stress (such as heat, oxidative, or salt stress) causes
some genes to either upregulate or downregulate in cells or organisms. RNA isolation,
complementary DNA (cDNA) synthesis, and PCR (or quantitative PCR) amplification
of target genes allow for the measurement of stress-responsive gene expression levels.
This makes it possible to profile biochemical reactions to stress and find genes that are
involved in signaling pathways or stress tolerance.

 Procedure:

 1µl each of template DNA was taken in 5 different PCR tube.

 12.5µl of Master mix (Taq polymerase + DNTPs + Mg2+) was added to the PCR
tube.
 1.5µl of primer was added.
 5 different factors were added to 5 different PCR tubes. Each tube contains 10µl of
factors.
 Tubes were insert into the PCR machine.
 PCR was run for approx. 1 hour and 40 minutes.
 On the other side, 1% agarose gel was prepared for electrophoresis.
 After PCR, 5µl loading dye was mixed with 25µl of the sample and load in wells.
 Leave for electrophoresis.
 COMPUTATIONAL DRUG DESIGN

Computational drug design is founded on the premise that biocomputational methods can
find, and improve, bioactive molecules that bind selectively to protein targets that relate to
a particular disease. The aim is to model the interaction between the proposed drug (ligand)
and its biological target (receptor) usually via docking and similar molecular modeling and
simulation approaches.

Two common approaches that illustrate how drug design strategies can vary are:
(SBDD) Structure-Based drug design, which utilizes proteins' three-dimensional structure
(often from X-ray crystallography or NMR) to also design small molecules to occupy the
same active site in the protein.

(LBDD) Ligand-Based drug design, which starts with known active compounds to build
models that will allow predictions of the activity of new compounds.

Molecular docking, which is one commonly-used method to predict the best orientation
and position of a ligand to the binding site on a protein, and predict approximate binding
affinity (in terms of free energy) and important binding interactions (e.g. hydrogen bonds,
hydrophobic interactions, etc.).

By representing drug-target interactions in silico, computational drug design will lessen the
amount of time, cost, and rate of failure in experimental drug design, and will assist in
focusing on which candidates to test in the laboratory.
  OBSERVATION & RESULTS

 ISOLATION OF CANDIDA FROM SOIL:

After 48 hours of incubation, there were different types of candida colony were observed
in Sabouraud Dextrose Agar plate.

Among those colonies, blue colony were picked by a pipette tip and inoculate to Sabouraud
Dextrose broth.

Candida tropicalis

Fig: different types of Candida sp. isolated in SB Agar plate.

 MDR PROFILING OF CANDIDA

After 48 hour of incubation, zone of inhibition was appeared more or less in every
single antifungal disc.
The zone of inhibition of antifungal discs are given below in millimetres.

Antifungal discs Zone of inhibition

1. Nystain. 17 mm
2. Amphotericin B. 10 mm
3. Itraconazole. 15 mm
4. Clotrimazole. 25 mm
5. Fluconazole. 30 mm
6. Miconazole. 27 mm
7. Ketoconazole. 25 mm

Fig: MDR Profiling of candida sp. With different antifungal discs.

 CHARACTERIZATION OF CURCUMIN EXTRACT.

 THIN LAYER CHROMATOGRAPHY(TLC)

After the solvent was travelled approx. 80% of the TLC plate, the plate was observed under UV
transilluminator and Rf values were calculated.

 Calculation:

Under UV transilluminator, 3 bands were observed, let’s suppose, 3 bands was named band ‘A’ band ‘B’
and band ‘C’.

Total distance travelled by the solvent= 3.8 cm.

Distance travelled by band ‘A’= 2.8 cm.
Distance travelled by band ‘B’= 3 cm
Distance travelled by band ‘C’= 3.2 cm

Now
.
Rf of band A = = 0.736
.
Rf of band B = = 0.789
.
.
Rf of band C = = 0.842
.

Fig: TLC plate under UV transilluminator.

 N ANOPARTICLE SYNTHESIS.

After those processes, mentioned in the procedure part, nickel nanoparticle was
synthesized.

Fig: synthesized nickel Nanoparticles.

 UV SPECTRA OF SYNTHESIZED NANOPARTICLE.

Characteristic U.V. Absorbance of NiSO₄, Turmeric Extract, and Ni Nanoparticles

The UV-Vis absorbance spectra of three different samples Nickel sulfate (NiSO₄),
turmeric extract, and nickel nanoparticles (NiNPs) were plotted from 200–1000 nm.

1. NiSO₄ (light blue line):

 There is a distinct peak at around 390–400 nm.
 This is characteristic absorbance due to d–d transition of Ni²⁺ ions in aqueous
solution.
 -he spectrum is sharp and narrow examining the shape of the peak, indicating a
pure ionic state without the formation of complex.

2. Turmeric Extract (Orange Line):

 The spectrum characterizes a strong, broad peak at around 420–450 nm with
maximum O.D. approaching 1.7 (high absorbance).
 This peak correlates directly with curcumin, the main bioactive component of
turmeric.
 The strong absorbance present at this region confirms the presence of
conjugated double bonds and aromatic rings in curcumin.

3. Ni Nanoparticles (Green Line):

 The spectrum displays multiple smaller peaks but is notably displayed at
approximately 420 nm and at 460 nm with O.D. values ~0.7.
 These peaks signify the surface plasmon resonance (SPR) of the nickel
nanoparticles confirming their successful synthesis.
 The distinct difference observed from NiSO₄ indicates that nanoparticles were
produced as opposed to ionic Ni²⁺ ions in solution.
 Result/Conclusion

The UV-Vis spectrum validates the occurrence and successful synthesis of nickel
nanoparticles (Ninp) since their absorbance peaks have moved and broadened
compared to NiSO₄.

Turmeric extract has high absorbance within the visible spectrum, supporting the idea
that curcumin is present in the extract and this curcumin was successfully used in the
green synthesis of nanoparticles.

The differences in spectra between NiSO₄, turmeric extract, and Ninp, indicates the
materials have changed from the raw material to the nano-form, and that curcumin was
likely interacting with nickel ions during the nanoparticle synthesis process.
 ISOLATION OF SIDEROPHORES.

 Fish gut microbes were collected in a falcon by injecting 0.9% saline through
intestine of fish. And collected microbes were inoculated in nutrient agar broth.

Fig: saline water injected through fish gut.

 Capsules of Lacticaseibacillus rhamnosus was poured into nutrient broth

Fig: capsules of probiotic Lacticaseibacillus rhamnosus.

Fig: fish gut and probiotic culture in nutrient broth.

 BIOPOLYMER SOLUTION.

 Biopolymer was prepared with hyperbranched polymer PEG (Polyethylene

Gycol) and citric acid in the ratio of 1:5
 Prepared biopolymer was collected in a falcon and kept in refrigerator for future
use.

Fig: Biopolymer.
 CUP PLATE ASSAY

After 48 hour of incubation, zone of inhibition was appeared in same wells.

The zone of inhibition of factors are given below in millimetres

Factors Zone of inhibition

1. Curcumin. 12 mm
2. Nickel Nanoparticles. 20 mm
3. Fish gut microbes. 12 mm
4. Probiotics. No zone of inhibition.
5. Biopolymer No zone of inhibition.

Fig: Cup plate assay using 5 different factors.

 96 WELL PLATE ASSASY.

from the values getting from ELISA reader, graphs of different experiments
in 96 well plates are given below:

 Biofilm Eradication Assay:

BIOFILM ERADICATION ASSAY

1.2

0.8
Absorbance

0.6

0.4

0.2

0
Control Curcumin Nickel Np Fishgut Probiotic Biopolymer

Fig: Graph from absorbance of different factors in Biofilm eradication assay

 Anti Biofilm Assay:

ANTI BIOFILM ERADICATION ASSAY

1

0.9

0.8

0.7

0.6
Absorbance

0.5

0.4

0.3

0.2

0.1

0
Control Curcumin Nickel Np Fishgut Probiotic Biopolymer

Fig: Graph from absorbance of different factors in Anti Biofilm assay

  MIC (Minimal Inhibitory Concentration)

MIC ASSAY
1.4

1.2

1
Absorbance

0.8

0.6

0.4

0.2

0
Curcumin Nickel Np Fishgut Probiotic Biopolymer

Control 1/2 dil. 1/4 dil. 1/8 dil. 1/16 dil. 1/32 dil 1/32 dil

Fig: Graph from different absorbance vs different dilutions of factor in MIC assay.
 MTT Assay:

Fig: graph from absorbance of different factors in MTT assay.

 GENOMIC DNA EXTRACTION.
After electrophoresis, gel was placed on an UV transilluminator and observed.

Fig: GDNA bands after electrophoresis.

 In lane 1,2 and 4 show bright, high-molecular-weight bands indicating

successful genomic DNA extraction.
 The bands also appear intact and not smeared indicating limited degradation.
 Smeared bands are not present and indicate reasonable DNA integrity and
purity.
 Band intensity indicates sufficient yield of DNA from the samples.
 No RNA contamination or visible degradation products are present.
 STRESS PROFILING
After electrophoresis, gel was placed on an UV transilluminator and observed.

Fig: Visible DNA bands in electrophoresis after PCR

Observation:

 The gel places several lanes; this first lane (to the far left) appears to be the
protein molecular weight marker (ladder), showing distinct bands of decreasing
size from top to bottom.
 The other lanes contain scales of protein samples showing one or more bands at
different distances indicating the presence of proteins of different molecular
weights.
 The bands are all horizontal and well-spaced apart indicating decent
electrophoresis happened.
 A few lanes have bright single bands which indicate a dominating protein
species.
 Some lanes show multiple bands indicating a mixture of proteins.
 There is no major smearing which again suggests protean quality was decent,
and there was successful denaturation.

Interpretation:

 The distinct bands in the different lanes demonstrate successful separation of

proteins.
 The migration pattern reflects molecular weights of proteins, where higher
molecular weight proteins are near the top, and the lowest molecular weight
proteins are at the bottom.
 Lanes with single bands most likely have purified or overexpressed proteins.
 Lanes with multiple bands likely have crude extracts or partially purified
proteins.
 With the marker lane, estimates of the protein bands can be compared for
molecular weight.
 COMPUTATIONAL DRUG DESIGN
By using the software like Chemsketch, PubChem, Open Babel, CB-Doc, BIOVIA
Discovery Studio, this structure has been found.

Fig: Molecular docking or structural visualization of a protein-ligand complex.

 The 3D structure of the target protein was obtained and studied through molecular
docking methods. The docking simulations suggest proficient binding of the chosen
compound (e.g. curcumin or other bioactive molecule) with the active site of the
fungal target protein.
 The image presented shows a protein-ligand interaction in which,
 The protein backbone consists of ribbon structure (cyan β-sheets, green coils/loops).
 The ligand/drug is satisfied within the binding pocket, suggesting an active
engagement.
 Several hydrogen bonding and hydrophobic interactions are present between the
ligand and important amino acid residues of the protein, thereby reinforcing the
stability of this complex.
 This structural perspective supports the possible antifungal activity of the
compound and supports further study of the compound as a candidate against MDR
Candida species, via in vitro and in vivo studies.