06 Chapter 3

General laboratory techniques recommended by Purvis et al.
(1966) and
Tuite (1969) were followed for the preparation of media, inoculation and maintenance of
cultures.
Cleaning solution (Mahadevan and Sridhar, 1996)

Potassium dichromate : 60 g
Sulphuric acid (Conc.) : 60 mL
Distilled water : 1000 mL
Potassium dichromate was dissolved in warm water, cooled and

concentrated sulphuric acid was added slowly. It was mixed thoroughly and used for
cleaning glassware.
Cleaning of glasswares
Borosil and Schott Duran glassware were used for all the laboratory
experiments. All the glassware were immersed for 24 h in chromic acid cleaning solution
and washed thoroughly in tap water, followed by detergent solution and again washed
thoroughly with distilled water. The cleaned glasswares were dried in an oven.
Sterilization
Glassware and culture media were sterilized in an autoclave at 121˚C for
20 min at 15 psi.
Chemicals
Analytical grade chemicals supplied by Hi-Media, SD Fine Chemicals, E-
Merck, Qualigens and Sigma Chemicals (USA) were used.
Media and their composition

Name of the media used and their composition are given below. All the
media were prepared in distilled water.
Jaya Prakash Priya, A. 2019. Isolation, purification and characterization of anticandidal compounds from
Zanthoxylum limonella (Dennst.) Alston. Ph. D. Thesis. University of Madras, Chennai, India. 120 p.
32
Nutrient agar (NA) (g/L)
Beef extract : 1.0
Yeast extract : 2.0
Peptone : 5.0
NaCl : 5.0
Agar : 20.0
pH : 7.0 ± 0.2
Potato dextrose agar (PDA) (g/L)

Potato : 200
Dextrose : 20.0
Agar : 20.0
pH : 6.8± 0.2
Mueller Hinton agar (MHA) (g/L)

Beef extract : 4.0
Acid hydrolysate of casein : 17.5
Starch : 1.5
Agar : 15.0
pH : 7.4 ± 0.2
Yeast peptone dextrose agar (g/L)

Yeast extract : 10.0
Peptone : 20.0
Dextrose : 20.0
Agar : 20.0
pH : 7.0± 0.2
33
Sabouraud dextrose agar (SDA) (g/L)
Dextrose : 40.0
Peptone (mycological grade) : 10.0
Agar : 20.0
pH : 5.6 ± 0.2
Egg yolk agar (g/L)

Dextrose : 40.0
Peptone (mycological grade) : 10.0
NaCl : 57.3
CaCl2 : 0.55
Agar : 20.0
Sterile egg yolk emulsion : 8%
pH : 7.6 ± 0.2
Bovine serum albumin agar (g/L)

Carbon base : 11.7
Yeast extract : 0. 1
BSA : 2.0
Agar : 20.0
pH : 5.0 ± 0.2
34
Plant sources
A total of 121 rare and important herbal plants were collected from the
Eastern Ghats, Western Ghats and other nearby locations of Tamil Nadu, Kerala,
Puducherry and Odisha states in south India by Dr. S. Aroumougame. The collected plant
parts were air dried at room temperature. The dried plants were ground to fine powder
and extracted using different solvents. The filtrates were condensed with the help of a
rotary evaporator at 40°C. And the extracts were weighed and stored. The percentage of
yield of crude extracts was calculated. A total of 180 plant crude extracts (Fig. 1; Table 1)
were prepared and evaluated.
Fig. 1. Crude extracts of medicinal plants
Details of crude extracts of medicinal plants

1. Total no.of plants 121
2. Locations Tamil Nadu, Kerala, Puducherry, Odisha
3. Plant parts used Roots, Stems, Bark, Leaves, Fruits
4. Total no.of extracts 180
35
MDR human pathogens
Methicilin resistant S. aureus (MRSA), Vancomycin resistant S. aureus
(VRSA), Bacillus cereus, Micrococcus luteus, Pseudomonas aeruginosa, Salmonella
typhi, Klebsiella pneumoniae, Enterobacter aerogens, Candida albicans (ATCC 90028)
and Candida tropicalis (ATCC 750) were obtained from Tuberculosis Research Centre,
Chennai, Tamil Nadu, India (Figs 2 & 3). The cultures were maintained in the respective
sterile medium and the plates were incubated at 37°C throughout the study.
Fig. 2. Multidrug resistant bacterial pathogens
36
Candida albicans Candida tropicalis
ATCC 90028 ATCC 750
Fig. 3. Multidrug resistant yeast pathogens
Characterization of antibiotic susceptibility for multi drug resistant human

pathogens
Antibiotic resistant test for Gram positive MDR pathogens
The antibiotic resistance profile was determined by the disc-agar diffusion
technique (Bauer et al., 1966) using the discs contained 20 different antibiotics. The
commercial antibiotics discs [IcosaUniversal-1; 1C001] were procured from the Hi-
media Laboratory, Mumbai, India. The Mueller-Hinton agar (MHA) medium was
prepared, poured into Petriplates and allowed to solidify. The methicilin resistant S.
aureus (MRSA), Vancomycin resistant S. aureus (VRSA), B. cereus and M. luteus strains
were inoculated evenly on the surface of agar medium by swabbing the culture and kept
for 5 min to dry. Then, the commercial antibiotic discs IC001 were placed separately on
the surface of culture with the help of sterile forceps and slightly pressed to adhere the
agar surface in Petriplates. The plates were incubated at 37˚C for 24 h and the zone of
inhibition around each antibiotic disc was recorded.
Antibiotic resistant test for Gram negative MDR pathogens

The MHA medium was prepared, poured into Petriplates and allowed to
solidify. P. aeruginosa, S. typhi, K. pneumoniae and E. aerogens cultures were inoculated
separately on the surface of agar medium by swabbing the cultures and kept for 5 min to
dry. Then, the commercial Hi-media antibiotic discs, IC003 and Icosa G -1 minus were
placed separately on the surface of culture with the help of sterile forceps and slightly
37
pressed to adhere the agar surface. The plates were incubated at 37˚C for 24 h and the
zone of inhibition around each antibiotic disc was recorded.
Antibiotic resistant test for MDR yeast pathogens

solidify. C. albicans (ATCC 90028) and C. tropicalis (ATCC 750) strains were
inoculated evenly on the surface of agar medium by swabbing the cultures and kept for 5
min to dry. Then, the commercial Hi-media antibiotic discs, HX104 and Hexa Antimyco-
01 were placed separately on the surface of culture with the help of sterile forceps and
slightly pressed to adhere the agar surface. The plates were incubated at 37˚C for 24 h,
and the zone of inhibition around each antibiotic disc was recorded.
In vitro antibacterial activity of plant crude extracts against human pathogens by

disc diffusion method
A modified standard agar diffusion method of Bauer et al. (1966) was
used to determine the antibacterial activity. The human pathogens, Methicillin resistant S.
aureus (MRSA), Vancomycin resistant S. aureus (VRSA), B. cereus, M. luteus, P.
aeruginosa, S. typhi, K. pneumoniae and E. aerogens were inoculated separately into 5
mL of sterile MHB and incubated at 37˚C for 16 h (0.5 O.D cultures). The cultures were
swabbed separately on the surface of sterile MHA plates using a sterile cotton swab.
Sterile filter paper discs of 6 mm diameter (The commercial sterile discs were procured
from the Hi-media Laboratory, Mumbai, India) were impregnated with 20 µL extract
solution dissolved in 10% DMSO (equivalent to 1 mg of the dried extract) and after
evaporation, placed on the surface of the inoculated agar plates. Methicillin and
streptomycin were used as the positive controls for Gram positive and Gram negative
bacteria, respectively and 10% DMSO was used as the negative control. The plates were
incubated at 37°C for overnight. After 24 h of incubation, the bacterial growth was
observed and the zone of inhibition was measured. All tests were conducted in triplicates
(NCCLS, 1990).
38
In vitro anticandidal activity of plant crude extracts against human pathogens by
disc diffusion method
used to determine the antiCandidal activity. The human pathogens C. albicans ATCC
90028 and C. tropicalis ATCC 750 were inoculated separately into 5 mL of sterile MHB
and incubated at 37˚C for 16 h (0.5 O.D cultures). The cultures were swabbed separately
on the surface of sterile MHA plates using a sterile cotton swab. A totally 180 crude
extracts prepared using different parts of 121 herbal plants in this study. Sterile filter
paper discs of 6 mm diameter (The commercial sterile discs were procured from the Hi-
media Laboratory, Mumbai, India) were impregnated with 20 µL of each extract
dissolved in 10% DMSO (equivalent to 1 mg of the dried extract) and after evaporation
placed on the surface of the inoculated agar plates. Fluconazole was used as positive
control and 10% DMSO was used as negative control. The plates were incubated at 37°C
for overnight. After 24 h of incubation, the culture growth was observed and the zone of
inhibition was measured. All tests were conducted in triplicates (NCCLS, 1990).
Selection of potent plant crude extracts

All the 180 plant crude extracts showed less antibacterial activity but
promising antiCandidal activity against two tested yeast pathogens. Therefore, among
180 plant crude extracts, 26 plant crude extracts which showed prominent anticandidal
activity were selected for further studies.
Characterization of antibiotic resistance/susceptibility for multidrug resistant

yeast human pathogens
Clinical isolates
Seven clinical isolates tentatively identified as Candida spp. (Candida. sp.
-1, Candida sp. -2, Candida sp. -3, Candida sp. -4, Candida sp. -5, Candida sp. -6, and
Candida sp. -7) were obtained from the Kilpauk Medical College, Kilpauk, Chennai,
Tamil Nadu, India (Fig. 4). The cultures were maintained in the respective sterile medium
and the plates were incubated at 37°C throughout the study.
39
Fig. 4. Colony morphology of different clinical isolates of Candida spp.
Antibiotic resistant test for clinical isolates

solidify. All the seven clinical isolates (Candida. sp. -1, Candida sp. -2, Candida sp. -3,
Candida sp. -4, Candida sp. -5, Candida sp. -6, and Candida sp. -7) were inoculated
separately on the surface of agar medium by swabbing the cultures and kept for 5 min to
dry. Then, the commercial Hi-Media antibiotic discs, HX104 and Hexa Antimyco–01
were placed separately on the surface of culture with the help of sterile forceps and
slightly pressed to adhere the agar surface. The plates were incubated at 37˚C for 24 h,
and the zone of inhibition around each antibiotic disc was recorded.
40
Molecular characterization of clinical isolates of Candida spp. (Kannan et al.,
2015)
Solutions and buffers
Stock solutions were prepared as follows: 1 M Tris HCl, 4 M NaCl, 10%
SDS, 0.5% EDTA. 100 mL GBL working buffer solution contained 1 mL 1 M Tris, 10
mL 4 M NaCl, 0.4 mL 0.5% EDTA, 6.8 mL 10% SDS and 80.2 mL DDH2O). TE Buffer
contained 10 mM Tris HCl and 1 mM EDTA (pH 7.5).
Isolation of genomic DNA

The culture pellet was collected from 48 h old culture by centrifugation at
10,000 rpm for 15 min. The pellet was resuspended in 300 µL of GBL buffer by pipetting
and vortexing. Immediately, 200 µL of chloroform and 150 µL of 6 M NaCl were added
and the tubes were inverted 10x. The mixture was centrifuged at 10,000 rpm for 15 min.
The supernatant was transferred to a fresh 1.5 mL Eppendorf tube. An equal volume of
absolute ethanol was added carefully down the wall of the tube. The mixture was
centrifuged again at 10,000 rpm for 15 min and the supernatant was discarded.
Subsequently, the precipitated DNA was pipetted out to a new Eppendorf tube and
washed twice with 70% ethanol by centrifugation at 5000 rpm for 15 min. The pellet was
air dried at room temperature for 10 min. Finally the pellet was dissolved in 50 - 100 µL
of TE buffer (pH8) and it was stored at −20°C until further use. The obtained DNA
sample was loaded in 1.0% agarose gel electrophoresis to identify the concentration of
DNA and its purity.
DNA amplification (White et al., 1990)

The ITS region of the genomic DNA of each clinical isolate, including
ITS1, ITS4 and the inverting 5.8S coding rDNA were amplified using the primers of
ITS1 and ITS4.
Primers
ITS1: 5’-TCC GTA GGT GAA CCT GCG G-3’
ITS4: 5’-TCC TCC GCT TAT TGA TAT GC- 3’
41
Each amplification reaction included 12.5 µL of premix (2x Master mix
red) containing 2.5 U Taq DNA polymerase, PCR buffer, 1.5 mM MgCl2 and 200 µM
dNTPs (Ampliqon, Denmark), 1 µL of template DNA, 5 µL (20 pmol) of each primers
and 1.5 µL of sterile double distilled water in a final volume of 25 µL. Polymerase chain
reaction (PCR) was performed in an automated MyGene TM Peltier Thermal Cycler
(MG96G) with the following conditions:
PCR conditions
Initial denaturation - 94°C for 4 min
Denaturation - 94°C for 1 min - 30 cycles
Annealing - 55°C for 30 sec
Extension - 72°C for 2 min
Each PCR product was analyzed on a 1.2% agarose gel with ethidium
bromide (0.5 µg mL-1) and 1× TAE buffer. Electrophoresis was carried out at 100 V, until
the tracking dye migrated to the end of the gel. Ethidium bromide stained DNA bands
were viewed under UV transilluminator and photographed for documentation. PCR
products were sequenced after purification with a support of a service provider, Eurofins
Genomics India Pvt Ltd. Bangalore, India.
ITS regions sequencing and BLAST analysis

The amplified ITS regions of all the seven clinical isolates were sequenced
for both stands using an automated DNA sequencer at Eurofins Genomics India Pvt. Ltd.,
Bangalore. The nucleotide sequences obtained were checked for sequence similarity on-
line with the BLAST search program at the National Centre for Biotechnology
Information website (NCBI, http://www.ncbi.nlm.nih.gov), and the sequences found to
share a high level of similarity were used for phylogenetic studies.
GenBank submission and nucleotide sequence accession numbers

The nucleotide sequences of all the isolates were submitted to the
GenBank databases of NCBI and EMBL and accession numbers were obtained.
42
Confirmation of the pathogenicity of Candida spp.
Phospholipase activity
All the nine Candida strains were evaluated for phospholipase production
using the egg yolk agar plate method described by Price et al. (1982) and Pereira et al.
(2011). The SDA plates contained 57.3 g NaCl, 0.55 g CaCl2 and 8% sterile egg yolk
emulsion. The test strains were spot inoculated separately (~6 mm), and the plates were
incubated at 37ºC for up to 2 days. Each culture was tested in duplicates. The diameter of
the colony and the surrounding precipitation zone (Pz) were measured, and phospholipase
activity was scored as per the described method. The Pz value represented the ratio of the
colony diameter to the diameter of the colony plus the precipitation zone. The results
were classified as follows: no activity (Pz=1), moderate activity (0.64>Pz<1) and strong
activity (Pz<0.64). By this classification, a high Pz value indicates low enzymatic
activity.
Proteinase activity
All the nine Candida spp. were tested for proteinase activitty in bovine
serum albumin (BSA) agar using the method of Ruchell et al. (1982). The medium was
adjusted to pH 5.0, sterilized by filtration and added to autoclaved 2% agar. The test
strains were spot inoculated separately (~6 mm) and the plates were incubated at 37 ºC
for up to 2 days. Each culture was tested in duplicate. The post-incubation clearance zone
around the colony was recorded, and the Pz value for proteinase activity was calculated
as described above.
In vitro anticandidal activity of selected 26 plant crude extracts against different

Candida spp. by disc diffusion method
used to determine the anticandidal activity. The human pathogens C. albicans ATCC
90028, C. tropicalis ATCC 750, C. albicans KT315910, C. tropicalis KT315901, C.
albicans KT831886, Candida sp. KT831887, C. dubliniensis KT831888, C. albicans
KT831889 and C. albicans KT315909 were inoculated into 5 mL of sterile MHB and
incubated at 37˚C for 16 h (0.5 O.D cultures). The cultures were swabbed on the surface
43
of sterile MHA plates using a sterile cotton swab. Sterile filter paper discs of 6 mm
diameter (Hi-media Laboratory, Mumbai, India) were impregnated with 20 µL crude
extract solution dissolved in 10% DMSO (equivalent to 1 mg of the dried extract) and
after evaporation placed on the surface of the inoculated agar plates. Fluconazole (25
mcg/unit) was used as positive control and 10% DMSO was used as negative control.
The plates were incubated at 37°C for overnight. After 24 h, the culture growth was
observed and the zone of inhibition was measured. All tests were conducted in triplicates
(NCCLS, 1990).
Determination of percentage of inhibition by MTT assay against different

Candida spp.
The percentage of inhibition was determined using the modified method of
Gabrielson et al. (2002). In the microtitre plate, 5 µl (5 × 105 cfu/mL) culture of C.
albicans ATCC 90028, C. tropicalis ATCC 750, C. albicans KT315910, C. tropicalis
KT315901, C. albicans KT831886, Candida sp. KT831887, C. dubliniensis KT831888,
C. albicans KT831889 and C. albicans KT315909, then 195 µl of the freshly prepared
Muller Hinton Broth and selected 12 plant crude extracts at 100 µg concentration
dissolved in 10% DMSO were added and the plates were kept for incubation at 37ºC for
24 h. Fluconazole (25 mcg/unit) was used as positive control and 10% DMSO was used
as negative control. Finally, to each well 10 µL (5 mg/mL) of 2, 3-bis [2-methoxy-4-
nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt (MTT) indicator solution
was added and incubated for 4 h. The contents were collected and centrifuged at 8000
rpm for 15 min, and the pellets were dissolved with 100 µL of 10% DMSO. Then the
contents were transferred to the appropriate well and read at 570 nm in an ELISA reader
(Thermo Scientific Multiskan Ex). After incubation, the colour change was assessed
visually. This MTT (tetrazolium salt) assay measures the cells ability to convert the
tetrazolium salt to formazan product. Any colour change from yellow to purple was
recorded as positive. The purple colour indicated the growth of pathogens and percentage
of inhibition was calculated, using the following formula.
44
% of Inhibition = Control O.D - Test O.D X 100
Control O.D
Determination of minimum inhibitory concentration (MIC) by MTT assay

against different Candida spp.
The MIC was determined using the modified method of Gabrielson et al.
(2002). In the microtitre plate, 5 µl (5 × 105 cfu/mL) culture of C. albicans ATCC 90028,
C. tropicalis ATCC 750, C. albicans KT315910, C. tropicalis KT315901, C. albicans
KT831886, Candida sp. KT831887, C. dubliniensis KT831888, C. albicans KT831889
and C. albicans KT315909 and 195 µl of the freshly prepared Muller Hinton Broth
(MHB) and different concentrations (10 µg, 20 µg……100 µg) of the selected
Z. limonella crude extract dissolved in 10% DMSO were added separately and the plates
were kept for incubation at 37ºC for 24 h. Fluconazole (25 mcg/unit) was used as positive
control and 10% DMSO was used as negative control. Finally, to each well 10 µL (5
mg/mL) of 2, 3-bis [2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide
inner salt (MTT) indicator solution was added and incubated for 4 h. The contents were
collected and centrifuged at 8000 rpm for 15 min, and the pellets were dissolved with 100
µL of 10% DMSO. Then the contents were transferred to the appropriate well and read at
570 nm in an ELISA reader (Thermo Scientific Multiskan Ex). After incubation, the
colour change was assessed visually. This MTT (tetrazolium salt) assay measures the
cells ability to convert the tetrazolium salt to formazan product. Any colour change from
yellow to purple was recorded as positive. The purple colour indicated the growth of
pathogens and percentage of viable cells was calculated and plotted versus test
concentration for MIC.
Mode of action
Inhibition of ergosterol biosynthesis in MDR Candida spp. by Z. limonella
Total Ergosterol extraction and quantitation
A single Candida colony of all the nine strains from an overnight potato
dextrose agar plate culture was used to inoculate 5 ml of potato dextrose broth for control
and for various concentrations of Z. limonella crude extract. All the nine strains were
45
incubated for 16 h and harvested by centrifugation at 5,000 rpm (856 × g) for 15 min.
The net weight of the cell pellet was determined. Three mL of 25% alcoholic potassium
hydroxide solution was added to each pellet and vortex mixed for one min. Cell
suspensions were transferred to sterile borosilicate glass screw-cap tubes and were
incubated in a water bath for one hour at 85°C. Following incubation, the tubes were
allowed to cool. Sterols were then extracted by addition of a mixture of 1 ml of sterile
distilled water and 3 mL of n-heptane followed by vigorous vortex mixing for 3 min. The
heptane layer was transferred to a clean borosilicate glass screw-cap tube and stored at
−20°C. Prior to analysis, 0.6 mL aliquot of sterol extract was diluted 5-fold in 100%
ethanol and scanned spectrophotometrically between 240 and 300 nm with a
spectrophotometer (Shimadzu UV-Visible Spectrophotometer). The presence of
Ergosterol and the late sterol intermediate 24(28) dehydroergosterol (DHE) in the
extracted sample, resulted in a characteristic four-peaked curve. The absence of
detectable Ergosterol in the extracts was indicated by a flat line. A dose-dependent
decrease in the height of the absorbance peaks was evident and corresponded to
decreased Ergosterol concentration. Ergosterol content was calculated as the percentage
of the wet weight of the cell by the following equations:
% Ergosterol + % 24 (28) DHE = [(A281:5 / 290) x F] / pellet weight,
%24 (28) DHE = [(A230 / 518) x F] / pellet weight,
% Ergosterol = [% Ergosterol + % 24 (28) DHE] - % 24 (28) DHE
Where F is the factor for dilution in ethanol and 290 and 518 are the E values (in
percentage per centimeter) determined for crystalline Ergosterol and 24 (28) DHE,
respectively (Arthington-Skaggs et al., 1999).
Detection of ERG11 biosynthetic genes (Jones et al., 2004)

PCR amplification of ERG11 gene
The genomic DNA isolated from all the nine Candida pathogens were
used for the detection of biosynthetic gene that encode for the production of Ergosterol.
Gene-specific primers were used to detect the ERG11 biosynthetic genes using Gradient
PCR.
46
All the PCR amplifications were performed in a 25 µL reaction mixture
containing 1 µL (20 ng) of template DNA, 5 µL (20 pmol) of each primer, 12.5 µL of
premix (2x master mix red) containing 2.5 U Taq DNA polymerase, PCR buffer, 1.5 mM
MgCl2 and 200 µM dNTPs (Ampliqon, Denmark) and 1.5 µL of sterile double distilled
water. PCR reactions were performed in a Nexsus gradient AG (22331) Hamburg master
Cycler. The amplified products were analyzed using 1.2% agarose with 1 kb DNA ladder
as marker (Thermo Scientific GeneRuler, USA).
Primers
Primer sequences were designed by oligo architect primer and probe
designer from Sigma Aldrich. For ERG11 gene amplification, different annealing
temperature was checked for primer amplification and the following cycle conditions
were standardized.
F5'-CATACTTCTGCTTCTACT-3'
R5'-CCACCATTGAACTATAATC-3'
Reaction mixture
Initial denaturation : 95°C for 15 sec
Denaturation : 94°C for 45 sec - 35 cycles
Annealing : 50°- 60°C for 45 sec
Extension : 72°C for 90 sec
Final extension : 72°C for 10 min
Different annealing temperature optimized
Gradient 1 2 3 4 5 6 7 8 9 10 11 12
Temperature 50 50.3 50.9 51.8 53.1 54.4 55.6 56.9 58.2 59.1 59.7 60
47
Gene sequencing
The amplified ERG11 gene products were analyzed using 1.2% agarose
gel electrophoresis with the 1 kb DNA ladder (Thermo Scientific GeneRuler,USA). The
PCR products were sequenced after purification by the support of the service provider,
Eurofins Genomics India Pvt.Ltd., Bangalore, India and analyzed using the NCBI
BLAST program to confirm their identity.
Detection of virulence factors

Inhibition of Yeast-Hyphal (Y-H) transition
Inhibition of Y-H transition in different Candida spp. were determined by
the method Toenjes et al. (2005) with slight modifications. The pathogens were seeded in
complete yeast peptone dextrose broth (YPDB) supplemented with 20% heat-inactivated
foetal bovine serum in a total volume of 100 µL in 96-well microtitre plates. The crude
extract of Z. limonella was dissolved in 10% DMSO and added to the wells at different
concentration range between 10 - 80 µg/ml; whereas the control contained the same
amount of 10% DMSO but without crude extract. Assay plates were incubated at 37°C in
CO2 for 6 h. Morphology of the Candida spp. was scored using a Leica DMIL inverted
microscope equipped with DC300F digital imaging system. The minimum inhibitory
concentration (MIC) of Y-H transition (MICY-H) was determined as the minimum
concentration at which no hypha was observed. The assay was performed in duplicate.
Inhibition of biofilm formation by Crystal violet assay

Biofilm formation was quantitated using crystal violet according to the
methods of Chauhan et al. (2011) and Raut et al. (2013). Crystal violet solution of 0.02%
was prepared and stored at -20°C. The wells containing biofilms were washed with PBS
to remove non-adhered cells and incubated with 99% methyl alcohol for 15 min. Wells
without crude extract of Z. limonella were considered as the control, and those without
biofilm were the blank. Supernatant was removed and wells were air-dried. One hundred
microliters of 0.02% crystal violet was added to each well and allowed to stand for 15
min for staining of the biofilm. Excessive crystal violet was removed by washing the
wells for 2-3 times with sterile distilled water. Absorbed stain was released by addition of
48
150 µL of 33% of acetic acid and transferred to the fresh wells. Optical density was read
at 620 nm using a microplate reader. Percentage biofilm formation was calculated by
comparing the optical density of treatment with that of control biofilm. More than 50%
reduction in absorbance of crystal violet was considered as significant inhibition.
Cytotoxicity of crude extract of Z. limonella

MTT – based cytotoxicity assay
The cytotoxic effect of crude extract of Z. limonella against normal human
cell line was determined by the modified rapid colorimetric assay using 3-(4, 5
dimethylthiazol-2-yl)-,5-diphenyl tetrazolium bromide (MTT) and compared with
untreated controls (Mosmann, 1983). Normal human Vero cell line was cultured in
Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% of fetal bovine
serum and seeded into 96-wells microtitre plates (BD Falcon, Germany) at plating
density of 10,000 cells/well and incubated at 37°C, 5% CO2, 95% air and relative
humidity (100%) for 24 h prior to addition of crude extract. The crude extract of Z.
limonella was solubilized in PBS and diluted in serum free medium. After 24 h, 100 µL
of the medium containing the crude extract at 1 mg concentration was added and
incubated at 37°C, 5% CO2, 95% air and 100% relative humidity for 48 h. Triplicates
were maintained and the medium without the sample was served as control.
After 48 h, the medium was discarded and the morphology of the cells was
observed using an inverted microscope. After the microscopic observation, 15 µL of
MTT (3 mg/mL) in phosphate buffered saline (PBS) and 200 µL of medium was added to
each well and incubated at 37°C for 4 h. The medium with MTT was then flicked-off and
then the formed formazan crystals were solubilized in 200 µL of 10% DMSO and then
measured the absorbance at 570 nm using a Microplate ELISA reader (Biotech
Instruments, USA). The cell viability was calculated using the following formula.
Test OD
Viable cell (%) = × 100
Control OD
49
IC50 value of crude extract of Z. limonella against normal cell line
The IC50 (50% inhibition concentration) values were defined as the
concentration of a sample result in 50% growth inhibition of the normal cells. The
percentage of cell inhibition was determined using the following formula.
100 – Absorbance (Sample)

Cell inhibition (%) =
Absorbance (Control)
Phytochemical analysis of Z. limonella

Collection of plant materials
The fresh plant materials of Z. limonella (collection code no. 404) roots
were collected from the Western Ghats, India (Fig. 5). The collected roots were air dried
at room temperature. The dried roots were ground to fine powder and extracted using
methanol (1:10 ratio). The filtrates were condensed with the help of a rotary evaporator at
40°C, the extract was weighed and stored at -20°C for further studies.
Fig. 5. Zanthoxylum limonella
50
Quantitative phytochemical determination of crude extract of Z. limonella
Different quantitative chemical tests were performed for establishing the
profile of Z. limonella crude extract for its phytochemical composition.
Estimation of total phenolic content (Folin-Ciocalteau method)

The total phenolic content in the crude extract was determined with the
Folin-Ciocalteau’s reagent (FCR). In brief, 0.5 mL of test sample (equivalent to 100 mg
dried extract) was mixed with 2.5 mL of FCR (diluted 1:10 V/V) followed by 2 mL of
Na2CO3 (7.5% W/V) solution. The tubes were vortexed and allowed to stand for 90 min
at room temperature. Absorbance of sample was measured against a blank at 750 nm
using a spectrophotometer (DU-40). A Calibration curve was constructed using gallic
acid as standard and total phenolic content of the crude extract was expressed in terms of
gram of gallic acid (g GAE) per 100 gram dry weight (Mahnaz Khanavi et al., 2009).
Estimation of flavanoids (Aluminium chloride colorimetric technique)

Total flavanoid content of the extract of Z. limonella was determined with
1 mL of test sample (equivalent to 100 mg dried extract) and 4 mL of water was added to
a volumetric flask (10 mL volume). After 5 min, 0.3 mL of 5% NaNO2 and 0.3 mL of
10% AlCl3.6H2O were added. After 6 min incubation at room temperature, 2 mL of 1 M
NaOH was added to the reaction mixture. Immediately the final volume was made up to
10 mL with distilled water. The absorbance was measured against a blank at 510 nm
using a spectrophotometer (Shimadzu, UV-1800). A calibration curve was constructed,
using Quercetin equivalent (gram quercetin per 100 g dry weight) (Atanassova et al.,
2011).
Estimation of protein (Bradford’s method)

To 1 mL of test sample (equivalent to 100 mg dried extract) 5 mL of
Coomassie brilliant blue G250 was added and mixed thoroughly. The absorbance was
read at 595 nm using a spectrophotometer against blank. The amount of protein was
51
calculated using standard graph with different concentrations of bovine serum albumin
(BSA) (Bradford, 1976).
Estimation of lipids (Folch et al., 1957)

To 1 mL of test sample (equivalent to 100 mg dried extract), 0.5 mL of
concentrated sulphuric acid was added and mixed well. The tubes were kept in a boiling
water bath for 10 min and allowed to cool at room temperature. To 0.2 mL of sample, 5
mL of phosphovanillin reagent was added, mixed well and kept for 30 min. After
incubation period, the color developed was read at 520 nm. Standard graph was
constructed using cholesterol and values are expressed in terms of cholesterol (gCE) per
100 g dry weight.
Estimation of carbohydrate (Dubois et al., 1956)

To 1 mL of test sample (equivalent to 100 mg dried extract), 1 mL of 5%
phenol and 5 mL of concentrated sulphuric acid were added and the content was mixed
thoroughly by shaking. Then the solution was allowed to stand for 15 min at boiling
water bath. After cooling, the OD of the solution was read at 490 nm in a
spectrophotometer. The amount of total carbohydrate was calculated using a standard
graph prepared from D-glucose and values are expressed in terms of glucose (gGE) per
100 g dry weight.
Thin layer chromatography (TLC) of crude extract of Z. limonella

TLC was performed on a pre-coated silica gel TLC plates grade F254 (E-
Merck, Darmstadt, Germany) with 10% methanol in chloroform as solvent system. The
crude extract of 1 mg/mL concentration was spotted on the TLC plates using capillary
tubes and dried. Development of the chromatogram was done in closed tanks, in which
the atmosphere has been saturated with eluent solvents’s vapour by wetting a filter paper
lining. The chromatogram was visualized under UV light (365 nm and 254 nm) and
iodine vapour. Then the Rf values were calculated.
52
Gas chromatography-mass spectrometry (GC-MS) analysis of crude extract of Z.
limonella (Dennst.) Alston.
The GC-MS analysis was performed for the crude extract of Z. limonella.
Further, the crude extract was quantified using a gas chromatograph (GCMS-QP 2010
Shimadzu) equipped with a DB-5ms column (mm inner diameter 0.25 mm, length 30.0
m, film thickness 0.25 µm) mass spectrometer (ion source 200˚C, RI -70 eV)
programmed (50-300˚C) with a rate 3 min. Injector temperature was 240˚C; carrier gas
was He (20 psi), column flow rate was 1.4 mL/min and the injection mode was split. The
chemical structures of the compounds were referred using MS library – NIST08s,
WILEY8, FAME.
Purification of anticandidal compounds from the crude extract of Z. limonella by

column chromatography
The concentrated crude extract (5 g) of Z. limonella was mixed with silica
gel (230 - 400 mesh) to prepare the metabolites-silica gel slurry and air dried. A glass
column measuring 3 cm diameter × 90 cm height was packed with 230 - 400 mesh silica
gel up to 58 cm height. The metabolites-silica gel slurry was loaded on to the column and
eluted initially with chloroform followed by different ratio of chloroform: methanol [9:1,
8:2, 7:3, 6:4, 5:5 (v/v)] and finally with methanol. About 300 mL of each solvent system
was used for elution of the column and fractions of 100 mL each were collected. The
separation was done by gradient elution with low polar/high polar using the flow rate of 2
mL/min. The eluted fractions were further subjected for anticandidal activity against nine
Candida spp., by well diffusion assay. The presence of compounds was analyzed by thin
layer chromatography (TLC). The fractions showing similar single spot on TLC were
pooled together and concentrated.
Anticandidal activity of eluted fractions by well diffusion method

A modified standard agar well diffusion method (Bauer et al., 1966) was
used to determine the anticandidal activity. The human pathogens C. albicans ATCC
90028, C. tropicalis ATCC 750, C. albicans KT315910, C. tropicalis KT315901, C.
albicans KT831886, Candida sp. KT831887, C. dubliniensis KT831888, C. albicans
53
incubated at 37˚C for 16 h (0.5 O.D cultures). The cultures were swabbed on the surface
of sterile MHA plates using a sterile cotton swab. Agar wells were prepared with the help
of sterilized cork borer (6 mm diameter) and loaded with 100 µL of eluted fractions
(equivalent to 100 µg of the dried extract). Fluconazole was used as positive control;
methanol, chloroform, ethyl acetate and DMSO were used as negative control. The plates
were incubated at 37°C for overnight. After 24 h of incubation, the culture growth was
observed and the zone of inhibition was measured. All tests were conducted in duplicates
(NCCLS, 1990).
3.14.5. Preparative TLC for purification of compounds

A streak of eluted fraction No. 9 was applied manually on a preparative
TLC glass plate (20 cm × 20 cm; 1500 m thickness) with inorganic fluorescent indicator
binder (Analtech, Sigma-Aldrich, Steinheim, Germany). After air drying, the plate was
developed, using the same mobile phase as used in the analytical TLC, in a pre-saturated
glass chamber. The bands separated were scraped-off carefully from the plates of each set
of experiment. The scratched sample was dissolved in HPLC grade chloroform and
centrifuged at 12000 rpm for 15 min in order to remove silica. The supernatant was
collected, filtered using a 0.22 m filter, and dried under reduced pressure. Further, all
the dried compounds were then dissolved in chloroform for further characterization and
bioactivity analysis.
Characterization of purified compound -1

Physical properties
The physical appearance of the purified compound-1 was determined
visually. Solubility was checked with methanol, ethyl acetate, chloroform, hexane,
DMSO and water.
IR spectrum
IR spectrum of the purified compound-1 was recorded using Perkin-
Elmer FTIR spectrometer model spectrum two (Singapore) using KBr pellets.
54
ESI-Mass spectrum
The ESI-MS spectrum was recorded using a LC-MS 2020 Shimadzu
(Japan) mass spectrometer.
Nuclear magnetic resonance (NMR) spectrum

1
H NMR and 13C NMR spectra of the purified compound-1 were recorded
using CDCl3 as solvent and tetramethyl silane as an internal standard using a Bruker
AVIII 400 NMR instrument (Japan).
Structural elucidation of the purified compound-1 of Z. limonella

Based on various chemical and spectral analyses, the purified compound-1
of Z. limonella was identified as a (1E, 4E) - undeca -1, 4 - dienyl acetate. The molecular
structure and molecular formula of the purified compound of Z. limonella were
elucidated.
Biological properties of the purified compound-1

Anticandidal activity of purified compounds by well diffusion method
A modified standard agar well diffusion method of Bauer et al. (1966) was
used to determine the anticandidal activity. The human pathogenic strains of C. albicans
ATCC 90028, C. tropicalis ATCC 750, C. albicans KT315910, C. tropicalis KT315901,
C. albicans KT831886, Candida sp. KT831887, C. dubliniensis KT831888, C. albicans
incubated at 37˚C for 16 h (0.5 O.D cultures). The cultures were swabbed separately on
the surface of sterile MHA plates using sterile cotton swabs. Five agar wells were
prepared with the help of sterilized cork borers (6 mm diameter) and loaded with 100 µL
of crude extract (1 mg), Fraction-9 (100 µg), purified compound-1 (25 µg), purified
compound-2 (25 µg) and Chloroform + Methanol. Chloroform and methanol were used
as negative control. The plates were incubated at 37°C for overnight. After 24 h of
incubation, the culture growth was observed and the zone of inhibition was measured. All
tests were conducted in duplicates (NCCLS, 1990).
55

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General laboratory techniques recommended by Purvis et al.

Cleaning solution (Mahadevan and Sridhar, 1996)

Potassium dichromate was dissolved in warm water, cooled and

Media and their composition

Potato dextrose agar (PDA) (g/L)

Mueller Hinton agar (MHA) (g/L)

Yeast peptone dextrose agar (g/L)

Egg yolk agar (g/L)

Bovine serum albumin agar (g/L)

Fig. 1. Crude extracts of medicinal plants

Details of crude extracts of medicinal plants

2. Locations Tamil Nadu, Kerala, Puducherry, Odisha

3. Plant parts used Roots, Stems, Bark, Leaves, Fruits

4. Total no.of extracts 180

Fig. 2. Multidrug resistant bacterial pathogens

Fig. 3. Multidrug resistant yeast pathogens

Characterization of antibiotic susceptibility for multi drug resistant human

Antibiotic resistant test for Gram negative MDR pathogens

Antibiotic resistant test for MDR yeast pathogens

In vitro antibacterial activity of plant crude extracts against human pathogens by

Selection of potent plant crude extracts

Characterization of antibiotic resistance/susceptibility for multidrug resistant

Antibiotic resistant test for clinical isolates

Isolation of genomic DNA

DNA amplification (White et al., 1990)

ITS regions sequencing and BLAST analysis

GenBank submission and nucleotide sequence accession numbers

In vitro anticandidal activity of selected 26 plant crude extracts against different

Determination of percentage of inhibition by MTT assay against different

Determination of minimum inhibitory concentration (MIC) by MTT assay

Detection of ERG11 biosynthetic genes (Jones et al., 2004)

Different annealing temperature optimized

Detection of virulence factors

Inhibition of biofilm formation by Crystal violet assay

Cytotoxicity of crude extract of Z. limonella

100 – Absorbance (Sample)

Phytochemical analysis of Z. limonella

Fig. 5. Zanthoxylum limonella

Estimation of total phenolic content (Folin-Ciocalteau method)

Estimation of flavanoids (Aluminium chloride colorimetric technique)

Estimation of protein (Bradford’s method)

Estimation of lipids (Folch et al., 1957)

Estimation of carbohydrate (Dubois et al., 1956)

Thin layer chromatography (TLC) of crude extract of Z. limonella

Purification of anticandidal compounds from the crude extract of Z. limonella by

Anticandidal activity of eluted fractions by well diffusion method

3.14.5. Preparative TLC for purification of compounds

Characterization of purified compound -1

Nuclear magnetic resonance (NMR) spectrum

Structural elucidation of the purified compound-1 of Z. limonella

Biological properties of the purified compound-1

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