50

CHAPTER 3

MATERIALS AND METHODS

3.1 Plant Sample

Seeds of Psoralea corylifolia were collected from Coimbatore District,

Tamil Nadu. Seeds were shade dried at room temperature (28 ± 2°C) for 3-4 days.
The dried seeds were ground using a pulverizer and stored in airtight containers at
room temperature in the dark (Plate 3.1).

3.2 Certification of the Medicinal Plant Psoralea corylifolia Linn.

Herbarium was prepared and submitted to the Botanical Survey of India,

Coimbatore, Tamil Nadu, India for authentication of the plant sample.

a b

Plate 3.1. Plant sample collected for the study (a) natural habitat of
Psoralea corylifolia (b) Seeds of Psoralea corylifolia
 67

3.15 Preparation of Botanical Formulation using PDP [386]

Principle

In botanical formulation, emulsifying agent helps in uniform dispersal of

formulation, glycerol for the stability of the active fraction in the formulation and
solvent is used for the solubility of the active fraction and these together helpful in
uniform dispersion upon application for the disease management in crops.

Materials required

1. PDP

2. Emulsifying agent Unitox 60

3. Glycerol

4. Ethanol

5. Glassware

Procedure

Five grams of the active fraction obtained from petroleum ether extract of
seeds of Psoralea corylifolia seeds was mixed with 70 mL Unitox 60, and 10mL
glycerol and swirled to obtain an uniform composition. The formulation was namd
as PDP 5EC i.e. 5% emulsfiable concenrate of phenyl derivative of pyranocoumarin.

3.15.1 Effect of Botanical Formulation on Radial Mycelial Growth of the

Pathogens - Poisoned Food Technique [387]

Principle

This technique involves the poisoning of the fungal growth medium using
antifungal agent and then measuring the reduction of growth of the organism on the
medium. The decrease in mycelial growth indicates the inhibition of fungal growth
by the antifungal substance.
 68

Materials required

1. PDP

2. Potato dextrose agar (Himedia)

3. Mancozeb

4. Glass ware

Procedure

Potato dextrose agar medium was prepared and amended with different
concentrations of the formulation. The different concentrations of the formulation
include 0.5% (T1), 1% (T2), 1.5% (T3), 2% (T4), 0.2% Mancozeb (T5), and control
(T6). Mycelial discs of selected pathogens namely A. solani, F. oxysporum f.sp.
lycopersici, F. moniliforme and F. gramenearum were placed at the centre of
petriplate and incubated at 25 ± 2 oC for ten days. The plates without the formulation
served as control. The radial growth (mm) of mycelium was measured. All the
experiments were carried out in triplicates and percent reduction of mycelial growth
over control was calculated using the following formula.

Dc Dt
Percent decrease over control = x 100
Dc
Where,
Dc = Average diameter of fungal growth in control.
Dt = Average diameter of fungal growth in treatment.

3.15.2 Determination of Stability for PDP 5EC

Materials required

1. PDP 5EC

2. Glassware
 69

Procedure

The formulation was poured in to a glass bottle until three fourth was
filled. The bottle was closed airtight with colloid oil sealing wax to avoid any loss of
volatile solvents and kept in the thermostat at varying temperatures of 10°C, 20°C,
30°C, 40°C and 50°C. After 7 days, the volume of the cream at the top and the
sediment at the bottom was measured [388]

3.15.3 Effect of Storage of PDP 5EC on Growth of the Pathogens -Poisoned

Food Technique

The botanical formulation was stored for 30, 60, 90, 120, 150 and 180
days at room temperature and tested for antifungal activity against the selected
pathogens according to the procedure described under section 3.15.

3.15.4 Determination of Phytotoxicity of PDP 5EC - Leaf Scorching Test [389].

Principle

The leaf scorching test indicates whether the botanical formulation has
any phytotoxicity effect upon spraying on the crops. Phytotoxicity results include
abnormal discolorition and leaf necrosis.

Materials required

1. PDP 5EC

2. Hand sprayer

3. Glass ware

Procedure

The formulation at various concentrations including 0.5%, 1.0%, 1.5%

and 2.0 % were sprayed on to 45 day old healthy Lycopersicon esculentum (PKM1)
leaves grown in green house. After 48 h the leaves were observed for colour
change, curling or flaming and the changes were recorded
 70

3.15.5 Effect of PDP 5EC on Infection and Germination of Seeds of

Lycopersicon esculentum Challenged with A. solani and F. oxysporum
f.sp. lycopersici

3.15.5.1 Seed Infection - Blotter Method [390]

Principle

Blotter test is done for analyzing the seed infection due to pathogenic
fungi. Seeds are placed on blotter paper and incubated under conditions that promote
the fungal growth of the pathogens present in the seed.

Materials required

1. PDP 5EC

2. Seeds of Lycopersicon esculentum (PKM1)

3. Blotting paper

Procedure

Seeds of Lycopersicon esculentum (PKM1) were preinfected with

A. solani and F. oxysporum f.sp. lycopersici by soaking the seeds in spore
suspensions of the respective fungi. The seeds were then soaked in different
concentrations of botanical formulation including 0.5 % (T1), 1.0 % (T2), 1.5 %
(T3), 2.0 % (T4), 0.2% Mancozeb (T5) for 2 h. Twenty five seeds of each treatment
were placed on moist blotters in petriplate and incubated (20 ± 2°C) for seven days.
The seeds were examined for growth of seed borne pathogens. Untreated preinfected
seeds (T6) served as control. Experiments were carried out in triplicate and the seed
infection was expressed as percentage reduction when compared to the control.
 71

3.15.5.2 Seed Germination - Roll Towel Method

Principle

Roll towel method is used for analyzing the seed germination percentage.

Materials required

1. PDP 5EC

2. Seeds of Lycopersicon esculentum (PKM1)

3. Roll Towel

4. Glass ware

Procedure

Seeds were prinfected accroding to the procedure described under

3.15.5.1. Pre infected seeds were soaked in different concentrations of the PDP 5EC
as given in 3.15.5.1 for 18 h and then shade dried. Three replicates of 100 seeds
were uniformly placed on standard germination paper roll-towel medium and incubated
at 25 ± 2°C and 90 ± 2 per cent relative humidity. After 14 days, the percent
germination was recorded. Ten normal seedlings per replication from roll towel
were carefully removed at random from each treatment. The root length measured
from the base of the shoot to the tip of the primary root and shoot length was
measured from the base of the shoot to tip of primary leaf [390]. Values were
expressed as mean ± SD. Further the Vigour Index (VI) was calculated based on the
following formula and expressed as whole number [391].

VI = Germination (%) x Mean total length of seedling in cm

 72

3.15.6 Pot Culture Studies

3.15.6.1 Effect of “PDP 5EC” on A. solani and F. oxysporum f.sp. lycopersici

Challenged Lycopersicon esculaentum

The ability of the botanical formulation to prevent the infection of crop

plant introduced with pathogenic fungi under pot culture conditions is measured
using percent disease index for A. solani and Percent disease incidence for
F. oxysporum f.sp. lycopersici.

Materials required

1. PDP 5EC

2. 45 days old Lycopersicon esculentum (PKM1)

3. Sprayers

4. Glassware

3.15.6.2 Efect of PDP 5EC on A. solani Challenged Lycopersicon esculentum [392].

Fourty five days old tomato plants (PKM1) were inoculated with A. solani
at concentration of the 10 6 spores/mL. The leaves were injured with sterilized pin
and the mycelial disc of pathogen was placed over the injured leaf portion and
covered with moist cotton and incubated inside the moist chamber. After 48 h, the
plants were sprayed with PDP 5EC as per the treatments 0.50 % PDP 5EC (T1), 1.0
% PDP 5EC (T2), 1.5 % PDP 5EC (T3), 2.0 % PDP 5EC (T4), 0.2% Mancozeb (T5),
Control (T6). Second spraying was done after 15 days interval. The plants were
watered every alternate day. Experiments were carried out in triplicates. The percent
disease index of A. solani was scored at 30th day after inoculation using intensity
scale of 0-5 in Table 3.2.
 73

Table 3.2 Disease Score Card

Disease severity
Disease score (Score)
(% of leaf area affected)
0 No infection
1 0.1 to 5
2 5.1 to 15
3 15.1 to 30
4 30.1 to 50
5 50.1 to 100

Percent disease index (PDI) was calculated based on the following

formula

Sum of all numerical grdes

PDI x 100
Total number of leaves Counted x maximum grade

3.15.6.3 Efect of PDP 5EC on F. oxysporum f.sp. lycopersici Challenged

Lycopersicon esculentum [393]

Fourty five days old tomato plants (PKM1) were inoculated with the
F. oxysporum f.sp. lycopersici at the concentration of 106cfu /ml. After 48 h, in each
treatment pots, soil drenching was done with PDP 5EC as per the treatments 0.50 %
PDP 5EC (T1), 1.0 % PDP 5EC (T2), 1.5 % PDP 5EC (T3), 2.0 % PDP 5EC (T4), 0.2%
Mancozeb (T5), Control (T6). Second drenching was done after 15 days interval and the
disease incidence was recorded on the 30th day after inoculation. The plants were watered
every alternate day. The experiment was carried out in triplicates.

Percent disease Incidence was calculated based on the formula

Numberof infected plants

Percent disease Incidence x 100
Total number of plants
 74

3.15.7 Biochemical Changes Induced by PDP 5EC on Lycopersicon esculentum

The plant samples, leaf in case of A. solani and roots in case of

F. oxysporum f.sp. lycopersici were collected at specific time intervals viz., 0th , 2nd
day, 4th day, 6th day, 8th day and 10th day for studying the induced changes in defense
enzymes including peroxidase, polyphenol oxidase, phenylalanine ammonia lyase and
the total phenolic content.

3.15.7.1 Assay of Peroxidase [394]

Principle

Peroxidase catalyses the conversion of H2O2 to H2O and O2, in the

presence of the hydrogen donor pyrogallol according to the reaction.

Peroxidase
H2O2 + Pyrogallol 2H2O + Purpurogallin

The oxidation of pyrogallol to a coloured product purpurogallin can be

measured spectrophotometrically at 420 nm with the specified time interval. The
intensity of the product is proportional to the activity of the enzyme.

Materials Required

1. 0.1M potassium phosphate buffer, pH 6.5 at 20ºC

27.2 g of KH2PO4 was dissolved in 1000mL of distilled water and

34.8 g of K2HPO4 was dissolved in 1000mL of distilled water. 68.5
mL of KH2PO4 and 31.5mL of K2HPO4 were made up to 200mL to
give 0.1M Potassium Phosphate buffer.

2. Enzyme extract

Leaves/ roots were macerate in 5mL of 0.1 M phosphate buffer in a

homogeniser. The homogenate was centrifuged at 16 000 g at 4 C
for 15 min and the supernatant was used the enzyme source.

3. 0.5% (w/w) H2O2 (freshly prepared)

1.7mL of H2O2 was made up to 100mL with distilled water.

 75

4. Pyrogallol solution (freshly prepared)

0.05 M in 0.1M phosphate buffer (pH 6.5). 0.630 g of pyrogallol

was dissolved in100mL of 0.1 M phosphate buffer and stored in
dark condition.

Procedure

Peroxidase assay was carried out according to Hammerschmidt et al.

(1982) [394] The reaction mixture consisted 1.5 ml of 0.05 M pyrogallol, 0.5 ml of
enzyme extract and 0.5 ml 1 per cent H2O2. The changes in absorbance at 420 nm
were recorded at 30 seconds interval for 3 min. The enzyme activity was expressed
as changes in the absorbance per min per g of sample.

3.15.7.2 Assay of Polyphenoloxidase [395]

Principle

Catechol is oxidised initially to the orange compound benzoquinone by

Polyphenoloxidase which is then converted to melanins. The conversion to melanin
is spontaneous but slow.

Polyphenoloxidase (slowly)
Catechol + Oxygen Benzoquinone + Water Melanins

The oxidation of catechol to a coloured product benzoquinone can be

measured spectrophotometrically at 495 nm with the specified time interval. The
intensity of the product is proportional to the activity of the enzyme.

Materials required

1. 0.1M sodium phosphate buffer (pH 6.5)

27.6 g NaH2PO4.H2O was dissolved in 1000mL of distilled water

and 53.65 g Na2HPO4.7H2O was dissolved in 1000mL of distilled
water. 68.5 ml of NaH2PO4.H2O and 31.5 ml of Na2HPO4.7H2O
were made up to 200mL to give 0.1M sodium phosphate buffer (pH
6.5).
 76

2. Enzyme extract

As described in 3.15.7.1

3. 0.01M Catechol

0.110 g of catechol was dissolved in 100ml of distilled water.

Procedure

Polyphenoloxidase assay was carried out according to Mayer et al. (1965)

[395]. The reaction mixture consisted of 200 µl of enzyme extract and 1.5 ml of 0.1
M sodium phosphate buffer. To start the reaction, 200 µl of 0.01 M catechol was
added and the activity was expressed as changes in absorbance at 495 nm per min
per g of sample.

3.15.7.3 Assay of Phenylalanine Ammonia Lyase [396]

Principle

Phenylalanine ammonia lyase (PAL) is responsible for the conversion of

L-Phenylalanine to trans-cinnamic acid. Cinnamic acid serves as a precursor for the
biosynthesis of coumarins, isoflavanoids and lignin. These compounds play an
important role in pest and disease resistance mechanism

PAL
L-Phenylalanine Trans-Cinnamate + NH3

The conversion of of L-Phenylalanine to trans-cinnamic acid can be

measured spectrophotometrically at 290 nm . The intensity of the product is
proportional to the activity of the enzyme.

Materials required

1. 0.1M Sodium borate buffer, pH 7

Dissolve 3.815 g of sodium borate Na2B4O7 10H2O in 90mL of

water and make up the volume to 100mL.
 77

2. 1.4 mM of 2- mercaptoethanol

1.992 mg in 100 mL of distilled water.

3 0.1 g of polyvinyl pyrrolidone

4. 0.05 M Tris- HCl buffer pH 8.8

Dissolve 0.606 g of Tris (hydroxymethyl) aminomethane in 90

mL of water. Adjust the pH with hydrochloric acid and make up
the volume to 100mL with water.

5. 12 mM L- phenylalanine

Dissolve 0.198 g of L-phenylalanine in 80 mL of 0.05M Tris- HCl

buffer and make up the volume to 100 mL

6. Extract preparation

Leaves / Root (250 mg) samples were homogenized in 5 mL of ice

cold 0.1 M sodium borate buffer, pH 7.0 containing 1.4 mM of
2-mercaptoethanol and 0.1 g of insoluble polyvinyl pyrrolidone.
The extract was filtered through cheese cloth and the filtrate was
centrifuged at 15000g for 15 min.

Procedure

Phenylalanine ammonia lyase assay was carried out according to

Dickerson et al. (1984) [396]. The reaction mixture containing 0.4 ml of enzyme
extract was incubated with 0.5 ml of 0.1 M borate buffer and 0.5 ml of 12 mM
L-phenylalanine in the same buffer for 30 min at 30°C. The amount of trans-cinnamic
acid synthesized was calculated using its extinction coefficient of 9630 M 1.
Enzyme activity was expressed as nmol trans cinnamic acid per min per g of sample.

3.15.7.4 Estimation of Total Phenol Content [397]

Principle

Total phenolic content of an extract is evaluated spectrophotometrically

using Phosphotungstat-phosphomolibdenum complex commonly referred to as
 78

Folin - Ciocalteu reagent. Oxidation and reduction reaction of phenolat ion takes
place in alkaline conditions. The reduction of Folin-Ciocalteu reagent by phenolat
ion causes color change from yellow to blue which is measured by
spectrophotometrically at 650 nm.

Material Required

1. Extract preparation

Leaves / root sample was ground in 5 mL of 80% ethanol. The

homogenate was centrifuged at 10000 rpm for 10 minutes and the
supernatant was used for the assay.

2. Folin - Ciocalteau reagent - Diluted 10-fold with distilled water

3. 20% Sodium carbonate : 20 g o f sodium carbonate was dissolved

in 100ml of distilled water

4. 80% Ethanol : 80 mL ethanol volume was made upto 100mL with

distilled water

5. Catechol 10mg of catechol was dissolved in 100mL of distilled

water

Procedure

A sample quantity of 0.1 ml was added to 2.8 ml of water and 0.25 ml of

Folin Ciocalteau reagent and the solution was kept at 25°C. After 3 min, 1 ml of 20
per cent sodium carbonate was added. Mixed thoroughly, placed the tubes in a
boiling water both for 1 minutes, and then cooled. The absorbance of developed blue
colour was measured using spectrophotometer at 650 nm. Catechol was used as the
standard. The amount of phenol was expressed as µg catechol per g of sample using
a standard graph [397].
 79

3.15.8 Field Study [398]

The effect of PDP 5EC on disease control of Alternaria solani infected

Lycopersicon esculentum (PKM1) carried out under field conditions at
Kariyanampatti, Theni District of Tamil Nadu, India. The experiment was laid out in
a randomized block design with 5 treatments replicated thrice. The field lay out and
randomization of treatments in the plot size of 4 x 3 m was carried out.

3.15.8.1 Treatment Details

Twenty five day old seedlings of were planted in the field and were
acclimatized for 20 days. The plants were infected with A. solani using the pin prick
method. Field studies were carried out using randomized block design of the five
treatments including 2 % PDP 5EC (T1), 1.5% PDP5EC (T2), Petroleum ether
extract of seeds of P. corylifolia L. (10mg/mL) (T3), mancozeb (0.2%) (T4) and
control (T5) in triplicates in a plot area of 4 X 3 m for each treatment. Foliar
spraying PDP 5EC was carried out at thirty day interval on 25th, 55th and 85 th days
after transplantation. The field was irrigated on every alternate day. Biomass (kg),
plant height (cm), No. of Flowers/plant, No. of fruits/plant, and percent disease
index according to proceudre described in 3.15.6.1 were recorded for randomly
selected five plants at 30 day interval after transplantation up to 90 days.

3.16 Statistical Analysis

Comparisons between treatments and control for a given microorganism

were performed with the Duncan’s Multiple Range Test. Data for in vitro and in
vivo antifungal activity was expressed as mean ± S.D. Comparisons between treated
groups and control groups were performed with the Sidak’s Multiple Comparison
Test.
 51

3.3 Preparation of Extracts

Principle

The bioactive principles present in the plants are extracted using organic
solvents. Extraction is based on the relative solubility of these components in the
organic solvents. The organic solvents of varying polarities percolated into the plant
material thereby causing dissolution of the active components. The extracts are filtered
then the solvents are vapourized to obtain a concentrated form 0of extract.

Materials required

1. Powdered seed of Psoralea corylifolia

2. Organic solvents - petroleum ether, chloroform and ethanol

Procedure

The dried and powdered seed sample was extracted at room temperature
by overnight percolation separately with petroleum ether, chloroform and ethanol at
1:5 ratio. The extracts were then filtered and concentrated under vacuum in rotary
evaporator. The yield of the extracts was determined and the extracts were kept in
tightly stoppered bottle in a refrigerator until further use [378].

3.4 Fungal Pathogens used for this Study

Fungal phytopathogens were obtained from the Department of Plant

Pathology, Tamil Nadu Agricultural University, Coimbatore. The fungal
phytopathogens used in this study are listed in Table 3.1. Plate 3.2 depicts the
morphology of the fungal phytopathogens used in the present study.

Table 3.1. List of fungal phytopathogens

S. No Fungal pathogens
1. Alternaria solani
2. Fusarium oxysporum f.sp. lycopersici
3 F. moniliforme
4. F. graminearum
5. Pythium aphanidermatum
6. Rhizoctonia solani
7 Phytophthora capsici
8 Colletotrichum musae
Alternaria solani F.moniliforme Fusariun oxysporum f.sp. F.graminearun
lycopersici

Phytophthron capsici Pythiun aphanidermatum Rhizoctonia solani Colletrichum musae

Plate 3.2 Plate cultures of the selected fungal phytopathogens

52
 53

3.5 Maintenance of Pathogens

The plant pathogens obtained were subcultured in sterilized potato

dextrose agar (Himedia) plates and stored for further study.

3.6 Antifungal Susceptibility Tests

Antifungal susceptibility tests were carried out using the agar well
diffusion assay, followed by the determination of the minimal concentrations of the
extract that could inhibit the growth of fungal pathogens.

3.6.1 Agar Well Diffusion Assay [379]

Principle

Antimicrobial susceptibility tests measure the ability of an antibiotic or

other antimicrobial agent to inhibit fungal growth in vitro. This can be estimated by
the agar well diffusion method. When the antimicrobial agent is loaded into the
wells it diffuses freely in the solid nutrient medium and as a result of its inhibitory
activity does not allow the growth of microorganism in the diffused regions of the
nutrient medium. The inhibition of growth surrounding the wells is a clear indication
of the microbial susception.

Materials required

1. Petroleum ether, chloroform and ethanol extracts of seeds of

P. corylifolia (100mg/mL)

2. Potato dextrose broth (Himedia)

3. Potato dextrose agar (Himedia)

4. Ketoconazole (Positive control)(1mg/mL)

5. Dimethyl Sulfoxide (DMSO) (Negative control)

 54

Procedure

The potato dextrose agar (Himedia) was sterilized and poured into
petriplates and allowed to solidify. Pathogens were subcultured in sterile potato
dextrose broth to a density of 106 cfu / mL and were swabbed on the plates. Using a
sterile cork borer, wells were cut. 20 µl of the petroleum ether, chloroform and
ethanol extracts were loaded in the wells separately. Ketoconazole and DMSO were
used as positive and negative controls respectively. The plates were incubated under
temperature 25 ± 2 ºC for 4 days. Diameters of inhibition zone (DIZ) were
measured for each of the organism and each extract. Experiments were carried out in
triplicates and the values expressed as mean ± S.D.

3.6.2 Determination of Minimal Inhibitory Concentration (MIC)

For quantitative estimates of antibiotic activity, antibiotics with different

dilutions are added to the broth and inoculated with the test organism. The least
concentration of the antibiotic that prevents the growth after incubation is taken as
the MIC of the extract.

Materials required

1. Petroleum ether extract of seeds of P. corylifolia

2. Potato dextrose broth (Himedia)

3. Potato dextrose agar (Himedia)

4. Ketoconazole (Positive control)

5. Dimethyl Sulfoxide (DMSO)

 55

Procedure

The tube dilution assay was carried out as per Kumar et al. (1999) [380].
Four different concentrations of the petroleum ether extract, namely, 100 mg/mL, 10
mg/mL, 1 g/mL and 0.1 mg/mL were prepared by serially diluting the extract taken
in sterilized eppendorf tubes and to this 900 µl of 10 -4 diluted pathogens were
added. After incubation, 20 µl from each of the tubes were spotted on the petriplates.
The plates were then covered and incubated at 25 ± 2 ºC for 48 hrs. The growth of
the organism for each dilution was observed and the minimum concentration of the
extract inhibiting the growth of the fungi was obtained.

3.7 Fractionation of Extract using Silica Gel Column Chromatography

3.7.1 Primary Fractionation

Principle

When a mixture of mobile phase and sample to be separated are

introduced from top of the column, the individual components of mixture move at
different rates. Those with lower adsorption to stationary phase move faster and are
eluted out first while those with greater adsorption move slowly and get eluted out
last. The solute molecules adsorb to the column in a reversible manner and can be
eluted using solvents of varying polarities.

Materials required

1. Petroleum ether extract of seeds of P. corylifolia L.

2. Glass column

3. Glass wool

4. Silica gel (60 – 120 mesh)

5. Petroleum ether

6. Ethyl acetate
 56

Procedure

Glass column of height 50 cm and diameter 9 cm was taken. Small plug of

cotton was placed at the bottom of the column. Acid washed sand was layered to a
height of 2-4 mm on top of the cotton and gently tapped to obtain a flat surface. The
stopcock was closed and 100 mL of petroleum ether was gently poured along the
walls into the column to wash the sand off the walls. Slurry of 150 g of silica gel
(60 - 120 mesh) was prepared in 300mL of petroleum ether. The stopcock was
opened and the silica slurry was poured into the column using a funnel. The column
was gently tapped to allow tight packing of the silica as it settles in the column.
Petroleum ether was drained out up to 1 cm above the top of the silica. Slurry of
extract was loaded on the column and eluted with a mixture of petroleum ether and
ethyl acetate as mobile phase. The stopcock was opened and the flow rate was set as
2.5 mL /min. Fractions of 100 mL each were collected using solvents of increasing
polarity petroleum ether (9.5): ethyl acetate (0.5) to petroleum ether (8): ethyl
acetate (2). A total of 210 fractions were collected and successive fraction revealing
identities on TLC plates were pooled. Based on identity on TLC plates fractions 1 to
15 were pooled and are here after referred to as F1, similarly fractions 16 to 50 are
referred to as F2, fractions 51 to 80 are referred to as F3, fractions 81 to 110 as F4,
fractions 111 to 140 as F5, fractions 141 to 160 as F6, fractions 161 to 180 as F7 and
fractions 181 to 210 as F8. Eight different primary fractions were obtained and were
tested for in vitro antifungal activity [381].

3.8 Evaluation of Antifungal Activity of Primary Fractions (F1-F8)

Agar well diffusion assay of the isolated fractions F1 to F8 was performed

for all eight fungal phytopathogens listed under in Table 3.1, according to the
procedure described under 3.6.1.
 57

3.9 Secondary Fractionation

Based on the result obtained primary fraction F7 was sub fractionated

using column chromatography according to the procedure described under 3.7.1.
Fractions of 100 mL each were collected using solvents of increasing polarity
petroleum ether (9.5): ethyl acetate (0.5) to petroleum ether (9): ethyl acetate (1). A
total of 120 fractions were collected and successive fractions revealing identities on
TLC plates were pooled and are reffered to as secondary fractions. Based on identity
on TLC plates fractions 1 to 20 were pooled and are here after referred to as SF1,
similarly fractions 21 to 52 are referred to as SF2, fractions 53 to 70 as SF3,
fractions 71 to 96 as SF4 and fractions 97 to 120 as SF5. All the five secondary
fractions obtained were tested for in vitro antifungal activity.

3.10 Antifungal studies

3.10.1 Evaluation of Antifungal Activity of Secondary Fractions (SF1 - SF5)

Agar well diffusion assay of the isolated secondary fractions SF1 to SF5
was performed for selected fungal pathogens listed in Table 3.1, according to the
procedure described under 3.6.1. Secondary fraction SF4 revealing clear zone of
inhibition in the agar well diffusion assay of selected fungal phytopathogens here
after referred to as active fraction was taken up for further studies.

3.10.2 Minimum Inhibitory Concentration

The minimum concentration at which the selected fungal pathogens were

inhibited was analyzed for the active fraction SF4 as per the procedure described
under section 3.6.2. The pathogens used for the study include Alternaria solani,
Fusarium oxysporum f.sp. lycopersici, F. moniliforme and F. gramenearum.
 58

3.11 Qualitative Tests for Secondary Metabolites

3.11.1 Dragendorff’s Test for Detection of Alkaloids

Principle

A solution of potassium bismuth iodide when added to an alkaloid

containing solution gives a orange red precipitate.

Materials required

1. Active Fraction (SF4)

2. Dragendorff’s reagent (Potassium bismuth iodide solution)

Procedure

To 1 mL of compound, 1 mL of Potassium bismuth iodide solution was

added. The color change was observed and recorded.

3.11.2 Liebermann’s-Burchard Test For Detection of Terpenoids

Principle

Lieberman-Burchard is a reaction used in a colorimetric test to detect

terpenoids, which gives a deep green colour. This colour begins as a purplish, pink
colour and progresses through to a light green then very dark green colour. The
colour is due to the hydroxyl group (-OH) of terpenoids reacting with the reagents
and increasing the conjugation of the un-saturation in the adjacent fused ring.

Materials required

1. Active Fraction
2. Acetic anhydride
3. Concentrated H2SO4
4. Chloroform
 59

Procedure

To 1 mL of active fraction , 1 mL of chloroform, 2-3 mL of acetic

anhydride and 1 to 2 drops of concentrated H2SO4 were added. The color change
was observed and recorded.

3.11.3 Shinoda Test for Detection of Flavanoids

Principle

The Shinoda test involves a reductive transformation of colourless or pale

yellow coloured flavones and flavonols into deeply coloured products. Pink
coloration of the solution indicates the presence of flavonoids.

Materials required

1. Active Fraction

2. Ethanol

3. Magnesium ribbon

4. Concentrated Hydrochloric acid (HCl)

Procedure

To a small amount of active fraction dissolved in ethanol, magnesium

ribbon and 1 mL of concentrated HCl were added and the color change was
observed and recorded.

3.11.4 Folin-Ciocalteau Spray Test for Detection of Phenols

Principle

The reduction of Folin-Ciocalteu reagent by phenols causes color change

from yellow to blue.
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Materials required

1. Precoated silicagel sheet (Merk)

2. Active fraction

3. Folin-Ciocalteu reagent

Procedure

In pre-coated silica gel sheets, active fraction was spotted on the sheet
was kept in room temperature for drying. The TLC sheets were sprayed with Folins
reagent. The colour change was observed and recorded.

3.11.5 Flourescence Test for Detection of Coumarins

Principle

Coumarins absorb ultraviolet light and emits as blue fluorescence. Some

of the energy absorbed is lost to the solvent. The molecule returns to the lower state
by emitting a photons with less energy than was absorbed..

Materials required

1. Active Fraction (SF4)

2. 10% NH4OH

Procedure

Three millilitres of the active fraction was evaporated to dryness in a test

tube and the residue was dissolved in hot distilled water. It was then cooled. To the
test tube, 0.5ml of 10% NH4OH was added. The presence or absence of fluorescence
under UV light was recorded.
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3.12 Spectroscopic Studies of the Active Fraction

3.12.1 UV-Visible spectrometry

Principle

When radiation interacts with matter, a number of processes occur,

including absorbance. Absorption of light in UV-visible region increases the energy
of the molecules (or atoms).

Procedure

The fraction containing the bioactive compound was scanned in the

wavelength ranging from 190 - 1200 nm using Chemito Spectrophotometer.

3.12.2 Gas Chromatography - Mass Spectrometry

Principle

Gas chromatography mass spectrometry (GC/MS) is an instrumental

technique, comprising a gas chromatograph (GC) coupled to a mass spectrometer
(MS), by which complex mixtures of chemicals may be separated, identfied and
quantified. The sample solution is injected into the GC inlet where it is vaporized
and swept onto a chromatographic column by the carrier gas (usually helium). The
sample flows through the column and the compounds comprising the mixture of
interest are separated by virtue of their relative interaction with the coating of the
column (stationary phase) and the carrier gas (mobile phase). The latter part of the
column passes through a heated transfer line and ends at the entrance to ion source
where compounds eluting from the column are converted to ions. The next
component is a mass analyser (filter), which separates the positively charged ions
according to various mass related properties. After the ions are separated they enter
a detector the output from which is amplified to boost the signal. The detector sends
information to a computer that records all of the data produced, converts the
electrical impulses into visual displays and hard copy displays. In addtion, the
computer also controls the operation of the mass spectrometer.
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Procedure

The purity of the active fraction was analyzed using GC MS Thermo

GC - Trace Ultra Ver: 5.0, Thermo Ms Dsq II unit. The mass of the compound was
also determined.

3.12.3 Fourier Transform Infra Red Spectrometry (FT-IR)

Principle

In infrared spectroscopy, IR radiation is passed through a sample. Some of

the infrared radiation is absorbed by the sample and some of it is passed through
(transmitted). The resulting spectrum represents the molecular absorption and
transmission, creating a molecular ngerprint of the sample. An infrared spectrum
represents a ngerprint of a sample with absorption peaks, which correspond to the
frequencies of vibrations between the bonds of the atoms making up the material.

Procedure

FTIR spectrum was recorded on BRUKER Optik GmbH, TENSOR 27 via

KBr pellet method or dissolving in CH2Cl2 using OPUS TM software.

3.12.4 Nuclear Magnetic Resonance Spectroscopy (NMR)

Principle

Nuclear magnetic resonance spectroscopy exploits the magnetic properties

of certain atomic nuclei. It relies on the phenomenon of nuclear magnetic resonance
and can provide detailed information about the structure, dynamics, reaction state,
and chemical environment of molecules. When placed in a magnetic field, NMR
active nuclei (such as 1H or 13
C) absorb electromagnetic radiation at a frequency
characteristic of the isotope. The resonant frequency, energy of the absorption, and
the intensity of the signal are proportional to the strength of the magnetic field.
 63

Procedure

1 13
H and C NMR spectra were recorded on Bruker AM-500 NMR
spectrometer operating at 400 MHz. The solvent used to dissolve the compound was
CDCl3.

Based on the chromatographic and spectral studied the compound was

identified as phenyal derivative of pyranocoumarin (PDP) and is here after refferred
in the methodology as PDP.

3.13 In Vitro Antifungal Studies

3.13.1 Effect of PDP Spore Germination-Cavity Slide Method

Principle

Cavity slide method involves the incubation of spores of the test organism
and antifungal substance in a cavity slide and observation of germination under
microscope. Decrease in germination of the spores indicates the inhibition of fungal
growth by the antifungal substance.

Materials required

1. PDP
2. Potato dextrose broth (Himedia)

3. Ketoconazole

4. Cavity Slide
5. Glass wares

Procedure

The effect of the bioactive fraction purified from the petroleum ether
extract of seeds of P. corylifolia on germination of spores of the pathogens
Alternaria solani, Fusarium oxysporum f.sp. lycopersici, F. moniliforme and
F. gramenearum was tested using cavity slide. Spores of the selected pathogens
 64

were transferred using a glass rod to separate test tubes containing sterile distilled
water. The spore suspension was adjusted to a density of 10 6 spores / mL using a
haemocytometer. Ten microlitres of each of the concentrations namely, 0.02, 0.04,
0.06, 0.08 and 0.1 % of the active fraction SF4 was placed on separate cavity slides.
Ten microliters of each spore suspension was added separately to the cavity slides
and incubated in moist chamber at 28 ± 2°C. Cavities which did not receive any
active fraction served as control. Three replications were maintained and percentage
of spore germination was recorded after 24 h under an optical microscope [382].

3.13.2 Effect of PDP on Mycelial Growth of Fungal Pathogens [383]

Principle

Mycelial dry weight is considered to be basic measure of fungal growth.

This technique involves the cultivation of the test organism in a medium containing
antifungal substance and measuring its growth. Decrease in mycelial growth
indicates the inhibition of fungal growth by the antifungal substance.

Materials required

1. PDP

2. Potato dextrose broth (Himedia)

3. Ketoconazole

4. Whatman filter paper

Procedure

Mycelial discs (9 mm) of the selected pathogens were inoculated into

potato dextrose broth separately containing 0.02, 0.04, 0.06, 0.08 and 0.1% of active
fraction SF4 isolated from the petroleum ether extract of seeds of P. corylifolia L.
and incubated at 25 ± 2 oC for 21 days. The mycelium was then harvested through
filtration using Whatman filter paper, oven dried at 70°C for 24 h and weight was
recorded. Potato dextrose broth without active fraction served as control. Triplicates
were maintained and the values are expressed as mean ± SD.
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3.13.3 Effect of PDP on morphology of Fusarium spp. [384]

One week old culture of selected Fusarium spp. was serially diluted up to
10 6 CFU/ mL. 900 µl of culture was transferred to eppendorf tubes containing 100
µl of 1 mg/mL of PDP. After 48hrs the mycelia of both the samples were harvested
and specimens were the observed under ESEM (Quanta 250 30KV with EADX).
The cultures without any active fraction served as control.

3.14 In silico Docking Studies [385]

Induced Fit Docking studies were carried out using GLIDE software v5.5,
developed by Schrodinger, running on Red Hat Enterprise Linux 5 (RHEL5)
workstation and Maestro v9.0 Graphical User Interface (GUI) workspace was used
for all the steps involved in ligand preparation, protein preparation, and Induced Fit
Docking (IFD). The ligand used in this study, the bioactive fraction was prepared
using Ligprepmodule of v2.3 of Schrodinger Suite 2009. Ligprep follows OPLS-AA
(Optimized Potential Liquid Simulations for All Atoms) force fields for energy
minimization. The proteins taken for the study were T2 mycotoxin with
trichothecene 3O- acetyl transferase enzyme, deoxyvelonol with trichothecene 3O-
acetyl transferase enzyme, Nep1-like proteins (NLPs), endopolygalacturonase (PG)
from Fusarium moniliforme, 1CZF Endopolygalacturonase II from Aspergillus
niger, pectin lyase and cutinase with respective PDB IDs as 2RKV, 3B2S, 3GNU,
1HG8, 1IDK, 1CZF and 1CEX.

3.14.1 Preparation of the Ligand

The ligand used i,e PDP was prepared using Ligprepmodule of v2.3 of
Schrodinger Suite 2009. Ligprep follows OPLS-AA (Optimized Potential Liquid
Simulations for All Atoms) force fields for energy minimization.The optimized
structure was then energy minimized to remove the steric clashes between the
atoms. The energy minimization was done till it reached a Root Mean Square
Deviation (RMSD) cut-off of 0.18 Å and the resulting structure was used for
docking.
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3.14.2 Induced Fit Docking (IFD)

IFD of the prepared ligand with the prepared protein was performed using
IFD protocol of GLIDE v5.5 from Schrodinger Suite 2009. Both the ligand and the
receptor were flexible which enabled the ligand to dock at the receptor’s binding site
and generate multiple poses of the receptor-ligand complex. Each docking included
unique structural conformations of the receptor needed to fit the ligand pose. The
best structure of the docked complex based on the Glide score (G-score) of the
dockings were recorded.

3.14.3 Induced Fit Docking Protocol

The prepared protein is docked using induced fit protocol using the
following system.

Constrained minimization of the receptor (Glide protein

preparation, refinement only) with an RMSD cutoff of 0.0018
Initial Glide docking of each ligand using a softened potential (Van
der Waals radii scaling). By default, a maximum 20 poses per ligand
are retained, and by default poses to be retained must have a
Coulombic-vdW score less than 100 and an H-bond score is less
than – 0.05.
One round of Prime side-chain prediction for each protein/ligand
complex, on residues within a given distance of any ligand pose
(default 5 Å).
Prime minimization of the same set of residues and the ligand for
each protein/ligand complex pose. The receptor structure in each
pose now reflects an induced fit to the ligand structure and
conformation.
Glide re-docking of each protein/ligand complex structure within a
specified energy of the lowest-energy structure (default 30
kcal/mol). The ligand is now rigorously docked, using default Glide
settings, into the induced-fit receptor structure.
Minimization of the re-docked ligand within the protein.
Estimation of the binding energy (Glide Energy) for each output pose.