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3.

MATERIALS AND METHODS


The increase in prevalence of multiple drug resistance has slowed
down the development of new synthetic antimicrobial drugs, and has
necessitated the search for new antimicrobials from alternative sources.
Natural compounds are a source of numerous therapeutic agents. Recent
progress to discover drugs from natural sources has resulted in compounds
that are being developed to treat cancer, resistant bacteria and viruses and
immunosuppressive disorders (Amghalia et al., 2009).
Phytochemicals

from

medicinal

plants

showing

antimicrobial

activities have the potential of filling this need, because their structures are
different from those of the more studied microbial sources, and therefore
their mode of action are also very likely to differ. There is growing interest
in correlating the phytochemical constituents of a medicinal plant with its
pharmacological activity (Prachayasittikul et al., 2008; Nogueira et al.,
2008). Screening the active compounds from plants has lead to the discovery
of new medicinal drugs which have efficient protection and treatment roles
against various diseases (Roy et al., 2009).
The experimental procedure employed in the present study to analyze
the various parts of Couroupita guianensis for their antimicrobial and
antioxidant properties, is presented in this chapter.
PHASE I
In Phase I, the antibacterial and antifungal activity of the leaves, bark,
flowers and fruit pulp of Couroupita guianensis were assayed.

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COLLECTION OF PLANT MATERIAL


The leaves, bark, flowers and fruit pulp of Couroupita guianensis
were collected from Perur temple, Coimbatore and the plant specimens were
identified, certified and the voucher specimen number (2430) was deposited
at the Botanical Survey of India, Southern Circle, Coimbatore.
PREPARATION OF THE EXTRACTS
The plant extracts were prepared using the solvents water, methanol
and chloroform. 10g of the samples were taken and homogenized with
100ml of the respective solvents. The crude preparation was left overnight in
the shaker at room temperature and then centrifuged at 4000rpm for 20mins.
The supernatant containing the plant extract was then transferred to a preweighed beaker and the extract was concentrated by evaporating the solvent
at 60C. The crude extract was weighed and dissolved in a known volume of
dimethyl sulphoxide, to obtain a final concentration of 20mg / 5l.
TEST MICRORGANISMS
The seven bacterial strains and the six fungal strains used in the
present study were the clinical isolates obtained from P.S.G. Hospitals,
Coimbatore. The bacteria used were Escherichia coli, Staphylococcus
aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Shigella flexneri,
Salmonella typhi and Proteus vulgaris.
The fungal strains used were Aspergillus niger, Aspergillus flavus,
Aspergillus fumigatus, Candida albicans, Rhizopus oryzae and Mucor
indicus.

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ANTIBACTERIAL ASSAY
The effect of various plant extracts on the several bacterial strains
were assayed by Agar well diffusion method and further confirmed by Disc
diffusion method. The minimum concentrations of the plant extracts to
inhibit the microorganisms were also determined by microdilution method
using plant fractions serially diluted in sterile nutrient broth.
AGAR- WELL DIFFUSION METHOD
PRINCIPLE
The antimicrobials present in the plant extract are allowed to diffuse
out into the medium and interact in a plate freshly seeded with the test
organisms. The resulting zones of inhibition will be uniformly circular as there
will be a confluent lawn of growth. The diameter of zone of inhibition can be
measured in millimeters.
REAGENTS
1. Muller Hinton Agar Medium (1 L)
The medium was prepared by dissolving 33.9 g of the commercially
available Muller Hinton Agar Medium (HiMedia) in 1000ml of distilled
water. The dissolved medium was autoclaved at 15 lbs pressure at 121C
for 15 minutes. The autoclaved medium was mixed well and poured onto
100mm petriplates (25-30ml/plate) while still molten.
2. Nutrient broth (1L)
One litre of nutrient broth was prepared by dissolving 13 g of
commercially available nutrient medium (HiMedia) in 1000ml distilled
water and boiled to dissolve the medium completely. The medium was

43

dispensed as desired and sterilized by autoclaving at 15 lbs pressure


(121C) for 15 minutes.
3. Chloramphenicol disc (standard antibacterial agent)
PROCEDURE
Petriplates containing 20ml Muller Hinton medium were seeded with
24hr culture of bacterial strains. Wells were cut and 20 l of the plant
extracts (namely aqueous, methanol and chloroform extracts) were added.
The plates were then incubated at 37C for 24 hours. The antibacterial
activity was assayed by measuring the diameter of the inhibition zone
formed around the well (NCCLS, 1993). Chloramphenicol disc was used as
a positive control.
DISC DIFFUSION METHOD
PRINCIPLE
Paper discs impregnated with specific antibiotics or the test substances
are placed on the surface of the Muller Hinton agar medium inoculated with
the target organisms, which is recommended for the diffusion of
antimicrobial agents as described in NCCLS approved standard. The plates
are incubated and the zones of inhibition around each disc are measured.
PROCEDURE
Muller Hinton Agar plates were prepared and the test microorganisms
were inoculated by the spread plate method. Filter paper discs approximately
6mm in diameter were soaked with 15l of the plant extract and placed in
the previously prepared agar plates. Each disc was pressed down to ensure
complete contact with the agar surface and distributed evenly so that they are
no closer than 24 mm from each other, center to center. The agar plates were

44

then incubated at 37C. After 16 to 18 hours of incubation, each plate was


examined. The resulting zones of inhibition were uniformly circular with a
confluent lawn of growth. The diameters of the zones of complete inhibition
were measured, including the diameter of the disc where the chloramphenicol
was used as control (NCCLS, 1997).
MICRODILUTION METHOD
PRINCIPLE
Dilution susceptibility testing methods are used to determine the
minimal concentration of antimicrobial needed to inhibit or kill the
microorganism. This can be achieved by dilution of antimicrobial in either
agar or broth media. Antimicrobials are tested in log2 serial dilutions (two
fold).
PROCEDURE
The minimum inhibitory concentration (MIC) was determined by
micro dilution method using serially diluted plant extracts according to the
NCCLS protocol (NCCLS, 2000). The aqueous, methanol and chloroform
extracts were diluted to get series of concentrations from 6.25mg/ml to
100mg/ml in sterile nutrient broth. The microorganism suspension of 50l
was added to the broth dilutions. These were incubated for 18 hours at 37C.
MIC of each extract was taken as the lowest concentration that did not give
any visible bacterial growth.
ANTIFUNGAL ASSAY
The activity of the plant extracts on various fungal strains were
assayed by agar plug method and spore germination inhibition assay.

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AGAR PLUG METHOD


PRINCIPLE
The fungicidal effect of the plant extracts can be assessed by the
inhibition of mycelial growth of the fungus and is observed as a zone of
inhibition near the disc or the wells.
REAGENTS
1. Potato Dextrose Agar medium
The commercially available (HiMedia) potato dextrose agar medium (39
g) was suspended in 1000ml of distilled water. The medium was
dissolved completely by boiling and was then autoclaved at 15 lbs
pressure (121C) for 15 minutes.
2. Nystatin (standard antifungal agent)
PROCEDURE
Potato Dextrose Agar medium was prepared and poured on to the
petriplates. A fungal plug was placed in the center of the plate. Sterile discs
immersed in the three plant extracts were also placed in the plates. Nystatin
was used as antifungal control. The antifungal effect was seen as crescent
shaped zones of inhibition (Schlumbaum et al., 1986).
SPORE GERMINATION ASSAY
(Rana et al., 1997)
PRINCIPLE
Lactophenol cotton blue stains the fungal cytoplasm and provides a
light blue background, against which the walls of the hyphae can readily be
seen. It contains four constituents: phenol which serves as a fungicide, lactic

46

acid as cleaning agent, cotton blue to stain the cytoplasm of the fungus and
glycerol to give a semi-permeable preparation.
REAGENTS
Lactophenol cotton blue stain
Phenol crystals (20g)
Cotton blue (0.05g)
Lactic acid (20ml)
Glycerol (20ml)
Distilled water (20ml)
The stain was prepared by dissolving the chemicals with gentle heating for
complete dissolution.
PROCEDURE
Aliquots of spore were prepared by mixing loopful of fungal spores in
sterile distilled water. 25l of spore suspension was added to 10l of the
plant extracts and placed in separate glass slides. Slides with 25l of spore
suspension alone served as the controls. Slides were then incubated in moist
chamber at 25 20oC for 24 hours. Each slide was fixed in lactophenol
cotton blue stain. The mold was mixed gently with the stain using two
teasing needles. A coverslip was placed on the preparation and examined
under the phase contrast microscope (Kozo XJS500T, Japan) for spore
germination.
The results of the Phase I of the study (presented in the next chapter)
revealed that the methanolic extract of Couroupita guianensis exhibited
maximum bioactivity. Therefore, only the methanolic extracts of the leaves,
flower and fruit pulp of the candidate plant were taken for all the subsequent
analyses in this study.

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PHASE II
In this phase, after testing the antimicrobial activity, in order to check
the other medicinal value of Couroupita guianensis Aubl., the antioxidant
property of the candidate plant was analysed. Based on the results of phase I,
the leaves, flowers and fruit pulp of Couroupita guianensis were taken for
further study and both the enzymic and non-enzymic antioxidants were
analyzed in them. The methodology adopted for analyzing these parameters
is given below.
ENZYMIC ANTIOXIDANTS
The enzymic antioxidants analysed were superoxide dismutase,
catalase, peroxidase, glutathione S-transferase and polyphenol oxidase.
ASSAY OF SUPEROXIDE DISMUTASE (SOD)
The

activity

of

superoxide

dismutase

was

assayed

spectrophotometrically by the method of Misra and Fridovich (1972) in the


leaves, flower and fruit pulp of Couroupita guianensis.
PRINCIPLE
Superoxide dismutase uses the photochemical reduction of riboflavin
as oxygen generating system and catalyses the inhibition of Nitroblue
tetrazolium (NBT) reduction, the extent of which can be assayed
spectrophotometrically at 600nm.
REAGENTS
1.

Potassium phosphate buffer (500 mM, pH 7.8)

2.

Methionine (450 M)

3.

Riboflavin (53 mM)

4.

Nitro Blue Tetrazolium (NBT) (840 M)

5.

Potassium cyanide (200 M)

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PROCEDURE
Couroupita guianensis leaves, flowers and fruit (0.5g) were ground
separately with 3.0 ml of potassium phosphate buffer. The homogenates
were centrifuged at 2000 rpm for 10 minutes and the supernatants were used
for the assay. The incubation medium contained, in a final volume of 3.0 ml,
50 mM potassium phosphate buffer (pH 7.8), 45 M methionine, 5.3 mM
riboflavin, 84 M NBT and 20 M potassium cyanide. The amount of
homogenate added to this medium was kept below one unit of enzyme to
ensure sufficient accuracy.
The tubes were placed in an aluminium foil-lined box maintained at
25C and equipped with 15W fluorescent lamps. After exposure to light for
10 minutes, the reduced NBT was measured spectrophoto-metrically at
600nm. The maximum reduction was observed in the absence of the enzyme.
One unit of enzyme activity was defined as the amount of enzyme giving a
50% inhibition of the reduction of NBT. The values were calculated as
units/mg protein.
ASSAY OF CATALASE
Catalase activity in the selected plant samples were determined by
adopting the method of Luck (1974).
PRINCIPLE
The UV light absorption of hydrogen peroxide can be easily measured
between 230 250 nm. On decomposition of hydrogen peroxide by catalase,
the absorption decreases with time. The enzyme activity can be estimated by
this decrease in absorption.

49

REAGENTS
1. Phosphate buffer : 0.067 M (pH 7.0)
2. Hydrogen peroxide in phosphate buffer (2mM)
PROCEDURE
A 20% homogenate of the plant samples were prepared in phosphate
buffer (0.067M, pH 7.0) and the homogenate was employed for the assay.
The samples were read against a control without homogenate, but containing
the H 2O2-phosphate buffer.
To the experimental cuvette, 3 ml of H2O2-phosphate buffer was
added, followed by the rapid addition of 40l enzyme extract and mixed
thoroughly. The time interval required for a decrease in absorbance by 0.05
units was recorded at 240nm. The enzyme solution containing H 2O2-free
phosphate buffer served as control.
One enzyme unit was calculated as the amount of enzyme required to
decrease the absorbance at 240nm by 0.05 units.
ASSAY OF PEROXIDASE
The activity of peroxidase in the plant samples was assessed by the
method of Reddy et al., (1995).
PRINCIPLE
Peroxidase catalyses the conversion of H2O2 to H2O and O2, in the
presence of the hydrogen donor pyrogallol. The oxidation of pyrogallol to a
coloured product called purpurogalli can be measured spectrophotometrically at 430nm with the specified time interval. The intensity of the
product is proportional to the activity of the enzyme.

50

REAGENTS
1.

Pyrogallol (0.05 M in 0.1 M phosphate buffer, pH 6.5)

2.

H2O2 (1% in 0.1M phosphate buffer, pH 6.5)

PROCEDURE
The plant samples were prepared as 20% homogenate in 0.1M
phosphate buffer (pH 6.5) and used for the assay. Pyrogallol solution (3.0
ml) and enzyme extract (0.1 ml) were pipetted out into a cuvette. The
spectrophotometer was adjusted to read zero at 430nm followed by the
addition of 0.5 ml of 1% H2O2 and mixed. The change in absorbance was
recorded every 30 seconds up to 3 minutes.
One unit of peroxidase activity is defined as the change in absorbance
per minute at 430nm.
ASSAY OF GLUTATHIONE S-TRANSFERASE
The assay of glutathione S-transferase activity was performed by the
method of Habig et al. (1974).
PRINCIPLE
Glutathione S-transferase conjugates GSH with CDNB and the extent
of conjugation is used as a measure of enzyme activity from the
proportionate change in the absorption at 340 nm.
REAGENTS
1. Chloro-2,4-dinitrobenzene (CDNB) (1mM in ethanol)
2. Reduced glutathione (1mM)
3. Phosphate buffer (0.1M, pH 6.5)

51

PROCEDURE
Couroupita guianensis leaves, flowers and fruit pulp (0.5g) were
homogenized with 5.0 ml of phosphate buffer. The homogenate was
centrifuged at 5000rpm for 10 minutes and the supernatant was used for the
assay. The enzyme activity was determined by monitoring the change in
absorbance at 340 nm in a spectrophotometer. The assay mixture contained
0.1ml of GSH, 0.1 ml of CDNB and phosphate buffer in a total volume of
2.9 ml. The reaction was started by the addition of 0.1ml of enzyme extract
to this mixture and the readings were recorded against distilled water blank
for a minimum of three minutes. The complete assay mixture without the
enzyme served as the control to monitor non-specific binding of the
substrates.
One unit of GST activity is defined as the nmoles of CDNB
conjugated per minute.
ASSAY OF POLYPHENOL OXIDASE (PPO)
The activity of polyphenol oxidase, comprising of catechol oxidase
and laccase, can be simultaneously assayed by the spectrophotometric
method proposed by Esterbauer et al. (1977).
PRINCIPLE
Phenol oxidases are copper proteins of wide occurrence in nature,
which catalyse the aerobic oxidation of phenolic substrates to quinones,
which are autooxidized to dark brown pigments generally known as
melanins, which can be estimated spectrophotometrically at 495nm.

52

REAGENTS
1. Tris HCl (50 mM, pH 7.2)
2. Sorbitol (0.4 M)
3. NaCl (10 mM)
4. Catechol (0.01 M) in phosphate buffer (0.1 M, pH 6.5)
PROCEDURE
The leaves, flowers and fruit pulp of Couroupita guianensis (5g) were
homogenized in about 20ml medium containing 50mM Tris HCl, pH 7.2,
0.4M sorbitol and 10 mM NaCl. The homogenate was centrifuged at
2000rpm for 10 minutes and the supernatant was used for the assay. The
assay mixture contained 2.5ml of 0.1M phosphate buffer and 0.3ml of
catechol solution (0.01M). The spectrophotometer was set at 495nm. The
enzyme extract (0.2ml) was added to the same cuvette and the change in
absorbance was recorded every 30 seconds up to 5 minutes.
One unit of either catechol oxidase or laccase is defined as the amount
of enzyme that transforms 1 mole of dihydrophenol to 1 mole of quinone
per minute under the assay conditions.
The activity of PPO was calculated using the formula,
Enzyme unit

K x (A/min)

K for catechol oxidase

0.272

K for laccase

0.242

where,

NON-ENZYMIC ANTIOXIDANTS
The non-enzymic antioxidants analysed in the leaves, flowers and fruit
pulp of Couroupita guianensis were ascorbic acid, tocopherol, total
53

carotenoids, lycopene, reduced glutathione, total phenols, total flavonoids


and chlorophyll.
ESTIMATION OF ASCORBIC ACID
The amount of ascorbic acid present in the leaves, flowers and fruit
pulp of Couroupita guianensis was estimated by the method of Roe and
Keuther (1943).
PRINCIPLE
Ascorbate is converted to dehydroascorbate by treatment with
activated charcoal or bromine. Dehydroascorbic acid then reacts with 2,4dinitrophenyl hydrazine to form osazones, which dissolves in sulphuric acid
to give an orange coloured solution. The coloured product can be measured
spectrophotometrically at 540nm.
REAGENTS
1. Trichloroacetic acid (4%)
2. Sulphuric acid (9N)
3. 2,4-dinitrophenylhydrazine reagent (2% in 9N sulphuric acid)
4. Thiourea solution (10%)
5. Sulphuric acid (85%)
6. Standard ascorbate solution: 10mg ascorbate in 100ml of 4% TCA.
PROCEDURE
The plant samples of 1g were taken and homogenized with 4% TCA
to extract the ascorbate and the final volume was made up to 10ml with 4%
TCA. The supernatant obtained after centrifugation at 2000 rpm for 10
minutes was treated with a pinch of activated charcoal, shaken well and kept

54

for 10 minutes. Centrifugation was repeated once again to remove the


charcoal residue. The volumes of the clear supernatants obtained were noted.
Two different aliquots of the supernatant were taken for the assay
(0.5ml and 1.0 ml). The assay volumes were made up to 2.0 ml with 4%
TCA. A range of 0.2 to 1.0ml of the working standard solution containing
20-100g of ascorbate respectively were pipetted into clean dry test tubes,
the volumes of which were also made up to 2.0 ml with 4% TCA.
DNPH reagent (0.5ml) was added to all the tubes, followed by two
drops of 10% thiourea solution. The osazones formed after incubation at
37C for 3 hours, were dissolved in 2.5ml of 85% H 2SO4, in cold conditions,
to avoid an appreciable rise in temperature. To the blank alone, DNPH
reagent and thiourea were added after the addition of H2SO 4. After
incubation for 30 minutes at room temperature, the samples were read at 540
nm and the levels of ascorbic acid in the samples were determined using the
standard graph constructed on an electronic calculator set to the linear
regression mode and expressed as mg ascorbate /g leaf.
ESTIMATION OF TOCOPHEROL
The levels of tocopherol in the plant samples were estimated
spectrophotometrically by the method reported by Rosenberg (1992).
PRINCIPLE
The estimation of tocopherols can be done using Emmerie-Engel
reaction, based on the reduction of ferric to ferrous ions by tocopherols,
which forms a red colour with 2, 2-dipyridyl. Tocopherols and carotenes are
first extracted with xylene and read at 460nm to measure carotenes. A
correction is made for this after adding ferric chloride and read at 520nm.

55

REAGENTS
1. Absolute alcohol
2. Xylene
3. 2,2-dipyridyl (1.2g in 1 litre of n-propanol)
4. Ferric chloride (1.2g in one litre of ethanol stored in brown bottle)
5. Standard solution of D,L- tocopherol, 10mg/L in absolute alcohol
(91mg of -tocopherol is equivalent to 100mg of tocopherol acetate).
6. Sulphuric acid (0.1N)
PROCEDURE
The plant samples (2.5g) were homogenized in a small volume of
0.1N sulphuric acid and the volume was finally made up to 50 ml by adding
0.1N sulphuric acid slowly, without shaking and the contents were allowed
to stand overnight. The contents of the flask were shaken vigorously on the
next day and filtered through Whatman No.1 filter paper. Aliquots of the
filtrate were used for the estimation of tocopherol. The plant extract,
standard and water of 1.5ml were pipetted out into three centrifuge tubes
namely test, standard and blank respectively. To all the tubes, 1.5ml each of
ethanol and xylene were added, stoppered, mixed well and centrifuged.
After centrifugation, the xylene layer was transferred into another
tube, taking care not to include any ethanol or protein. To 1.0 ml of xylene
layer, 1.0ml of 2,2-dipyridyl reagent was added, stoppered and mixed. This
reaction mixture was taken in the spectrophotometric cuvettes and the
extinctions of the test and the standard were read against the blank at 460nm.
Then, in turn, beginning with the blank, 0.33ml of ferric chloride solution
was added, mixed well and after exactly 15 minutes, the test and the standard
were read against the blank at 520nm.

56

The levels of tocopherol were calculated using the formula


Tocopherol (g) =

A 520 A 450
x 0.29x15
StdA 520

ESTIMATION OF TOTAL CAROTENOIDS AND LYCOPENE


The estimation of total carotenoids and lycopene was done by the
method described by Zakaria et al. (1979).
PRINCIPLE
The total carotenoids and lycopene in the sample are extracted in
petroleum ether. The total carotenoids are estimated in UV/visible
spectrophotometer at 450nm and the same extract can be used for estimating
lycopene at 503nm. At 503nm, lycopene has a maximum absorbance, while
carotenes have only negligible absorbance.
REAGENTS
1. Petroleum ether (40C - 60C)
2. Anhydrous sodium sulphate
3. Calcium carbonate
4. Alcoholic potassium hydroxide (12%)
PROCEDURE
All the steps subsequent to the saponification were carried out in the
dark to avoid photolysis of carotenoids. Saponification was done with 5g of
the plant samples using 2.5ml of 12% ethanolic potassium hydroxide in a
water bath at 60C for 30 minutes. The saponified extract was then
transferred into a separating funnel (packed with glass wool and calcium
carbonate) containing 10-15ml of petroleum ether and mixed gently. The

57

lower aqueous phase was transferred to another separating funnel and the
upper petroleum ether containing the carotenoid pigment was collected. The
extraction was repeated until the aqueous phase became colourless. To the
petroleum ether extract a small quantity of anhydrous Na2SO4 was added to
remove excess moisture, if any. The final volume of the petroleum ether
extract was noted and diluted if needed by a known dilution factor.
The absorbance of the yellow colour was read at 450nm and 503nm in
a spectrophotometer using petroleum ether as a blank.
The amount of total carotenoids and lycopene was calculated using the
formula,
P x 4 x V x 100
Amount of total carotenoids

mg
W

where,
P = optical density of the sample
V = Volume of the sample
W = Weight of the sample
3.12 x ODsample x Vol of sample x dilution x 100
Lycopene

=
1 x weight of the sample x 1000

The total carotenoids and lycopene are expressed as mg/g tissue.


ESTIMATION OF REDUCED GLUTATHIONE
The levels of reduced glutathione were estimated by the method
proposed by Moron et al. (1979).

58

PRINCIPLE
Reduced glutathione (GSH) is measured by its reaction with DTNB
(5,5-dithiobis-2-nitrobenzoic acid) (Ellmans reaction) to give a yellow
coloured product that absorbs at 412 nm.
REAGENTS
1. Phosphate buffer (0.2M, pH 8.0)
2. DTNB (0.6mM in 0.2M phosphate buffer)
3. TCA (5% and 25%)
4. Standard GSH (10 nmoles/ml in 5% TCA)
PROCEDURE
A 20% homogenate was obtained by homogenizing 0.5g of the plant
sample in 2.5 ml of 5% TCA. The homogenate was immediately acidified by
adding 125l of 25% TCA to prevent aerial oxidation of glutathione. The
precipitated protein was centrifuged at 1000rpm for 10 minutes. The
homogenate was cooled on ice and 0.1ml of the supernatant was taken for
the estimation. The supernatant was made up to 1 ml with 0.2M sodium
phosphate buffer (pH 8.0). Two ml of freshly prepared DTNB solution was
added to the tubes and the intensity of the yellow colour formed was read at
412 nm in a spectrophotometer after 10 minutes.
A standard curve of GSH was prepared using concentrations ranging
from 2-10 nano moles of GSH in an electronic calculator set to the linear
regression mode and the values of the samples were read off it. The values
are expressed as nmoles of GSH /g tissue.

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DETERMINATION OF TOTAL PHENOLS


Total phenols were assayed by the method proposed by Mallick and
Singh (1980) in the samples of the candidate plant.
PRINCIPLE
Phenols react with phosphomolybdic acid in Folin-Ciocalteau reagent
in alkaline medium to produce a blue-coloured complex (molybdenum blue)
which can be estimated spectrophotometrically at 650 nm.
REAGENTS
1. Ethanol (80%)
2. Folin-Ciocalteau reagent (1N)
3. Sodium carbonate (20%)
4. Standard solution - 10 mg catechol in 100ml of distilled water
PROCEDURE
The homogenate was prepared with 0.5g of the leaves, flower and fruit
pulp of Couroupita guianensis in 10X volumes of 80% ethanol. The
homogenate was centrifuged at 10,000 rpm for 20 minutes. The residue was
re-extracted with 80% ethanol. The supernatants were pooled and evaporated
to dryness. The residue was then dissolved in a known volume of distilled
water. Different aliquots (0.2 to 2.0ml) were pipetted out into test tubes. The
volume in each tube was made up to 3.0ml with water. To all the tubes, 0.5
ml of Folin-Ciocalteau reagent was added and mixed. After 3 minutes, 2.0ml
of 20% sodium carbonate solution was added to each tube. After mixing the
tubes thoroughly, all the tubes were kept in a boiling water bath for exactly 1
minute, and allowed to cool. The absorbance was measured at 650 nm
against a reagent blank.

60

ESTIMATION OF FLAVONOIDS
Flavonoids were estimated by the method of Cameron et al. (1943) in
the leaves, flowers and fruit pulp of Couroupita guianensis.
PRINCIPLE
Flavonoids react with vanillin reagent to produce a colored product
which can be measured spectrophotometrically at 340nm.
REAGENTS
1. Vanillin reagent (1% in 70% sulphuric acid)
2. Catechin standard (110g/ml)
PROCEDURE
The plant samples (0.5g) were extracted first with methanol: water
mixture (2:1) and secondly with the same mixture in the ratio 1:1. The
extracts were shaken well and allowed to stand overnight, pooled the
supernatants and measured the volume. This was concentrated and then used
for the assay. An aliquot of the extract was pipetted out and evaporated to
dryness. Vanillin reagent (4.0) ml was added and the tubes were heated for
15minutes in a boiling water bath. Varying concentrations of the standard
were also treated in the same manner. The optical density was read at
340nm. The standard curve was constructed and the concentration of
flavonoids was calculated. The values are expressed as mg flavonoids/g
sample.
RADICAL SCAVENGING EFFECTS OF Couroupita guianensis
The effects of the selected parts of Couroupita guianensis in
scavenging / neutralizing free radicals and oxidants were analysed against a

61

battery of known standard radicals and oxidants, in vitro. Since the


methanolic extract of the plant parts exhibited the maximum antimicrobial
effect in the first phase, only the methanolic extract was taken for these
analyses.
PREPARATION OF PLANT EXTRACTS
The leaves, flowers and fruit pulp of Couroupita guianensis were
weighed and extracted with methanol (10g/100ml). The methanol extract
was dried at 60C protected from light. The residue was weighed and
dissolved in dimethyl sulfoxide (DMSO) to obtain a final concentration of
20mg/5l. The free radical scavenging and DNA and lipid protective effects
of the extracts of these plant parts were analysed as given below.
EVALUATION

OF

RADICAL

SCAVENGING

EFFECTS

OF

Couroupita guianensis EXTRACTS


The antioxidant effects of the leaves, flower and fruit pulp were
assessed by the ability to scavenge a battery of free radicals and oxidants
namely DPPH, ABTS, hydroxyl and H2O2.
DPPH SCAVENGING EFFECT
The ability of the plant extracts to scavenge the stable free radical
DPPH was assayed by the method of Mensor et al. (2001).
PRINCIPLE
DPPH (2,2-diphenyl-2-picryl hydrazyl), a stable free radical, when
acted upon by an antioxidant, is converted into diphenyl-picryl hydrazine
with a colour change from deep violet to light yellow colour. This can be
quantified spectrophotometrically at 518 nm to indicate the extent of DPPH
scavenging activity by the plant extracts.
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REAGENTS
1. DPPH (0.3 mM in methanol)
2. Methanol
PROCEDURE
The extracts of Couroupita guianensis parts (25l) and 0.48 ml of
methanol were added to 0.5 ml of methanolic solution of DPPH. The mixture
was allowed to react at room temperature for 30 minutes. Methanol alone
served as blank and DPPH in methanol, without the plant extracts, served as
positive control. After 30 minutes of incubation, the discolourisation of the
purple colour was measured at 518nm. The radical scavenging activity was
calculated as follows
A518 [sample] A518 [blank]
Scavenging activity (%) = 100

x 100
A518 [blank]

ABTS SCAVENGING EFFECT


The ability of Couroupita guianensis to scavenge the free radical
ABTS (2,2-azino-bis 3-ethyl benz thiazoline-6-sulfonic acid) was studied
using the method adopted by Shirwaikar et al. (2006).
PRINCIPLE
In this decolourisation assay, ABTS, the oxidant, is generated by
persulphate oxidation of 2,2-azinobis(3-ethylbenzoline-6-sulphonic acid)
(ABTS2), based on the inhibition of the absorbance of the radical cation
ABTS+, which has a characteristic long wavelength absorption spectrum.
This can be measured spectrophotometrically at 745nm to analyse the ABTS
scavenging ability of the plant extracts.

63

REAGENTS
1. ABTS solution (7mM with 2.45 mM ammonium persulfate).
2. Ethanol
PROCEDURE
ABTS radical cations (ABTS+) were produced by reacting ABTS
solution (7 mM) with 2.45 mM ammonium per sulphate. The mixture was
allowed to stand in the dark at room temperature for 12-16 hours before use.
All the three different extracts (each 0.5 ml) were added to 0.3 ml of ABTS
solution and the final volume was made up to 1ml with ethanol. The
absorbance was read at 745 nm and the per cent inhibition by the plant
extracts was calculated using the formula
(Control - test) x 100
Inhibition (%) =
Control
HYDROGEN PEROXIDE SCAVENGING EFFECT
The scavenging activity of hydrogen peroxide by the plant extracts
was determined by the method of Ruch et al. (1989).
PRINCIPLE
The UV light absorption of hydrogen peroxide can be easily measured
at 230nm. On scavenging of hydrogen peroxide by the plant extracts, the
absorption decreases at this wavelength, which property can be utilized to
quantify their H2O2 scavenging ability.

64

REAGENTS
1.

Phosphate buffer (40mM, pH 7.4)

2.

H2O 2 in phosphate buffer (40mM)

PROCEDURE
A solution of hydrogen peroxide (40 mM) was prepared in phosphate
buffer (pH 7.4). Plant extracts at the concentration of 10mg/10l were added
to 0.6ml of H2O 2 solution. The total volume was made up to 3ml with
phosphate buffer. The absorbance of the reaction mixture was recorded at
230nm. The blank solution contained phosphate buffer without H 2O2. The
percentage of H2O2 scavenging by the plant extracts was calculated as
Ao- A1 x 100
% scavenged hydrogen peroxide =
Ao
where,
Ao - Absorbance of control
A1 - Absorbance in the presence of plant extract
HYDROXYL RADICAL SCAVENGING EFFECT
The DNA damage induced in vitro by hydroxyl radicals generated by
hydrogen peroxide in the presence and the absence of plant extracts was
quantified by the production of TBARS (thiobarbituric acid reactive
substances) spectrophotometrically as per the procedure given by Elizabeth
and Rao (1990).
PRINCIPLE
The hydroxyl radical scavenging activity can be measured by studying
the competition between deoxyribose and the plant extracts for hydroxyl

65

radicals generated with Fe3+/ ascorbate / EDTA / H2O 2 system. The hydroxyl
radicals attack deoxyribose, which eventually result in TBARS formation,
which can be quantified spectrophotometrically.
REAGENTS
1. Deoxyribose (28mM)
2. FeCl3 (1mM)
3. EDTA (1mM)
4. H2O 2 (10mM)
5. Ascorbate (1mM)
6. KH2PO4-KOH buffer (200 mM, pH 7.4)
7. Thio barbituric acid (10%)
8. HCl (25%)
PROCEDURE
The reaction mixture contained in a final volume of 0.98ml, 2.8mM
deoxy ribose, 0.1mM FeCl3, 0.1mM EDTA, 1mM H2O2, 0.1mM ascorbate
and 20mM buffer. 20l of plant extract was added such that the final volume
was 1ml. The reaction mixture was then incubated for one hour at 37C.
After the incubation, 0.5 ml of TBA and 0.5 ml of HCl were added and
heated in a boiling waterbath for 20 minutes. It was then allowed to cool and
the absorbance was measured at 532 nm. The per cent TBARS produced for
positive control (H 2O2) was fixed as 100% and the relative per cent TBARS
was calculated for the plant extract treated groups.
Following the assays that established the parts of Couroupita
guianensis as a rich source of antioxidants, the effect of the extracts against
oxidative damage inflicted to biomolecules was analyzed. When a cell is
assaulted by oxidation, the immediate targets that take the brunt of the attack

66

are the lipid molecules present in the membranes, both plasma membrane as
well as the internal organelle membranes. However, the ultimate damage is
inflicted to DNA. Therefore, in the present study, the effect of Couroupita
guianensis was analyzed against oxidative damage inflicted to both DNA
and lipids.
EFFECT OF Couroupita guianensis ON OXIDANT INDUCED DNA
DAMAGE
The DNA damage was assessed in vitro in commercially available
preparations of DNA. The DNA was selected in such a way that they were
from different hierarchies of evolutionary development. The commercially
available preparations included viral DNA ( DNA) and of animal origin
(herring sperm DNA).
EFFECT OF Couroupita guianensis ON DNA
The extent of DNA damage induced in DNA was followed by the
variation in relocated pattern of migration in agarose (Chang et al., 2002).
REAGENTS
1. Tris buffer (50mM, pH 7.4)
2. H2O 2 (30%)
3. FeCl2 (500M)
4. 1X TAE buffer (pH 8.0) Tris 40mM, EDTA 10mM
PROCEDURE
The reaction was conducted in a total volume of 30l containing 5l
of 50mM tris buffer (pH 7.4), DNA (2g concentration) and 5l of tris
buffer or plant extract prepared in tris buffer. Then 10l of 30% H2O 2 and

67

5l of 500M FeCl2 were added and incubated at 37C for 30 minutes. The
reaction mixture was then placed in 1% agarose gel and run at 100V for 15
minutes in a submarine gel electrophoretic apparatus using 1X TAE as the
running buffer. The DNA was visualized and photographed using an Alpha
Digidoc gel documentation system (Alpha Innotech, UK).
EFFECT OF Couroupita guianensis ON HERRING SPERM DNA
The biomolecular protective effect of the plant extracts on the
damaged DNA was studied by the method reported by Aeschlach et al.
(1994).
PRINCIPLE
The H 2O 2 induced damage to herring sperm DNA results in the
production of TBARS. The extent of DNA damage can be measured
spectrophotometrically at 532nm.
REAGENTS
1. Herring sperm DNA (0.5mg/ml in 500mM tris buffer)
2. H2O 2 (30%)
3. MgCl2 (5mM)
4. FeCl3 (50m)
5. EDTA (0.1M)
7. TBA (1% w/v)
8. HCl (25%)
9. Tris buffer (10mM, pH 7.4)
PROCEDURE
The assay mixture (0.5 ml) contained 0.05ml of herring sperm DNA,
0.167ml of H2O2, 0.05ml of MgCl2, 0.05ml of FeCl3 (50M) and the plant
68

extract (10l containing 10mg of extract diluted in tris buffer. The mixture
was incubated at 37C for 1 hour. The reaction was terminated by the
addition of 0.05ml of 0.1M EDTA. The colour was developed by adding 0.5
ml of thiobarbituric acid and 0.5ml of HCl, followed by incubation at 37C
for 15 minutes. After centrifugation, the extent of DNA damage was
measured by the increase in absorbance at 532nm.
EFFECT OF Couroupita guianensis ON LIPID PEROXIDATION
Oxidizing agents (ferrous ions and ascorbate, or H2O 2) impose a stress
on membrane lipids which can be quantified as the extent of thiobarbituric
acid reactive substances (TBARS) formed. The extent of inhibition of LPO
by the plant extracts in three diverse membrane preparations, namely goat
RBC ghosts (plasma membrane preparation), goat liver homogenate
(mixture of plasma membrane and internal membranes) and goat liver slices
(intact cells) were determined (Dodge et al. 1963; Okhawa et al., 1979).
REAGENTS
1. Isotonic KCl (1.15%)
2. Hypotonic KCl (0.3%)
3. Tris buffered saline (TBS) (10 mM Tris, 0.5 M NaCl, pH 7.4)
4. Ferrous sulphate (10 M, prepared fresh in TBS)
5. Thiobarbituric acid (TBA) (1% in TBS)
6. Alcohol (70%)
7. Acetone
PREPARATION OF MEMBRANE SYSTEMS
GOAT RBC GHOSTS
Goat blood (50ml) was collected from a slaughterhouse and the fresh
blood was immediately defibrinated using acid-washed stones. The
69

defibrinated blood was diluted with saline and transported to the laboratory
on ice. The RBCs were collected by centrifugation at 3000 rpm for 10
minutes and washed thrice with isotonic (1.15%) KCl. The cells were then
treated with hypotonic (0.3%) KCl and allowed to lyse completely at 37C
for one hour. The lysate was then centrifuged at 5000 rpm for 10 minutes at
4C. The pellet obtained was washed several times with hypotonic KCl until
most of the hemoglobin was washed off and a pale pink pellet was obtained.
The pellet was suspended in 1.5ml of TBS (Tris buffered saline 10mM tris,
0.15M NaCl, pH 7.4) and 50l aliquots were used for the assay.
GOAT LIVER HOMOGENATE
Goat liver was obtained fresh from the slaughterhouse and transported
to the laboratory on ice. A 20% homogenate of the liver was prepared in cold
TBS. The homogenate was centrifuged at low speed to remove debris and
other particulate matter and 50l aliquots were used for the assay.
GOAT LIVER SLICES
The liver was placed on a watch glass held on ice and cut into thin
(1mm thick) slices using a sharp sterile scalpel. 250mg portions of the slices
were used for the assay. The slices were taken in 1ml of HBSS (Hanks
Balanced Salt Solution, HiMedia) and treated with H2O 2 (5l of 30%
solution), with or without 20l of the leaf extract prepared in HBSS
(corresponding to an extract concentration of 20mg). The slices were
incubated at 37C in a water bath for one hour. At the end of the incubation
period, the slices were taken into a homogenizer tube along with the
incubated HBSS and homogenized. The homogenate was clarified using low
speed centrifugation and an aliquot was taken for the assay of TBARS
formed.

70

LPO ASSAY
Control tubes were prepared for each sample containing the respective
plant extract (50l corresponding to 20mg), membrane aliquot (RBC ghosts
or liver homogenate) and TBS to make a final volume of 500l. Pro-oxidant
(FeSO4 at 10moles final concentration) was added to all the tubes except
the control tubes. A blank containing no leaf extract, no membrane aliquots,
but only FeSO 4 and TBS was also prepared. An assay medium
corresponding to 100% oxidation was prepared by adding all the other
constituents except leaf extracts. The experimental medium corresponding to
autooxidation contained only the membrane preparation. All the tubes were
incubated at 37C for one hour.
At the end of the incubation, the samples, along with the homogenates
prepared from the liver slices incubated with the oxidant H2O 2 and extracts
were subjected to the TBARS quantification. The LPO reaction in all the
tubes was arrested by the addition of 500l of 70% ethanol. 1ml of 1% TBA
was added to all the tubes and treated in a boiling water bath for 20 minutes.
After cooling to room temperature, 500l of acetone was added and the
TBARS measured at 535nm in a spectrophotometer.
ANTIOXIDANT STATUS USING AN in vitro MODEL
The in vitro model used in the present study was goat liver slices as
the earlier studies in our laboratory have proved the liver slices to be the best
alternative to live animals (Varier, 2002; Kiruthika, 2003; Saraswathi, 2006;
Sumathi, 2007; Vidya, 2007). The enzymic and non-enzymic antioxidants
were assessed in the goat liver slices, following exposure to hydrogen
peroxide in the presence and the absence of the different extracts.

71

PREPARATION OF GOAT LIVER SLICES


Liver was the organ of choice because it is the metabolic organ and it
is responsible for the metabolic clearance of many xenobiotics. The goat
liver was collected fresh from a slaughter house, plunged into cold, sterile
PBS and maintained at 4C till the assay. Very thin (1mm) slices of the goat
liver were cut accurately using sterile scalpel.
TREATMENT GROUPS
One gram of goat liver slice was taken in 4.0ml of sterile PBS in broad
flat bottomed flasks. Hydrogen peroxide, at 0.2M concentration, was used as
an oxidant for the induction of oxidative stress in the liver slices. The plant
extracts of 20l were added and kept at incubation for 1 hour at 37C. The
treatment groups for antioxidant assays were as follows:
Group 1 Untreated liver slice (Negative control)
Group 2 Liver slice + Hydrogen peroxide (Positive control)
Group 3 Liver slice + Methanol extract of leaf
Group 4 Liver slice + Methanol extract of flower
Group 5 Liver slice + Methanol extract of fruit pulp
Group 6 Liver slice + Methanol extract of leaf+ Hydrogen peroxide
Group 7 Liver slice + Methanol extract of flower+ Hydrogen peroxide
Group 8 Liver slice + Methanol extract of fruit pulp+ Hydrogen peroxide
After the incubation period, a homogenate was prepared from the slices
using the same incubation solution (PBS). The homogenate was centrifuged at
1500rpm for 5 minutes to clarify the debris and the supernatant was used for
the analyses of various enzymic and non-enzymic antioxidants.

72

ASSAY OF ENZYMIC ANTIOXIDANTS


The activities of enzymic antioxidants namely superoxide dismutase,
catalase, peroxidase and glutathione S-transferase in the liver slices were
determined. These enzymes were assayed by the same protocols used earlier
in this study. An aliquot of the liver slice homogenate was used as the
enzyme source instead of the plant samples.
DETERMINATION OF NON-ENZYMIC ANTIOXIDANTS
The non-enzymic antioxidants estimated were vitamin A, ascorbic
acid, tocopherol and reduced glutathione. The non-enzymic antioxidant
levels in the different treatment groups were estimated following the same
procedures used for the plant extract analyses. An aliquot of the slice
homogenate was used instead of plant tissue in all the assays.
PHASE III
In order to identify the chemical nature of the active component
present in the plants, a preliminary phytochemical screening was done
followed by TLC.
PHYTOCHEMICAL ANALYSIS
(Khandelwal et al., 2002).
DETECTION OF ALKALOIDS
a) Mayers test: A fraction of the extract was treated with Mayers reagent
(1.36g of mercuric chloride and 5g of potassium iodide in 100ml of
distilled water) and observed for the formation of cream coloured
precipitate.

73

b) Dragendroffs test: An aliquot of the extract was treated with


Dragendroffs reagent and observed for the formation of reddish orange
coloured precipitate.
c) Wagners test: A fraction of the extract was treated with Wagners reagent
(1.27g of iodine and 2g of potassium iodide in 100ml distilled water) and
observed for the formation of reddish brown coloured precipitate.
DETECTION OF PHENOLICS
a) Ferric chloride test: A fraction of the extract was treated with 5% FeCl3
reagent and observed for the formation of deep blue-black colour.
b) Lead acetate test: A fraction of the extract was treated with 10% lead
acetate solution and observed for the formation of white precipitate.
DETECTION OF FLAVONOIDS
a) Aqueous sodium hydroxide test: A fraction of the extract was treated
with 1N aqueous NaOH solution and observed for the formation of
yellow-orange colouration.
b) Sulphuric acid test: A fraction of the extract was treated with
concentrated sulphuric acid and observed for the formation of orange
colour.
c) Schinodos test: A fraction of the extract was treated with a piece of
magnesium turnings followed by a few drops of concentrated HCl, heated
slightly and observed for the formation of dark pink colour.
DETECTION OF STEROIDS AND TERPENOIDS
Salkowskis test: A small amount of sample was dissolved in 2ml of
chloroform taken in a dry test tube. Equal volume of concentrated sulphuric
74

acid was added. The tube was shaken gently. The presence of steroids and
terpenoids was indicated by the upper layer of chloroform turning red and
lower layer showing yellow green fluorescence.
DETECTION OF SAPONINS
Sodium bicarbontate test: In a test tube, about 5ml of extract was added
and a drop of sodium bicarbonate was added. The mixture was shaken
vigorously and kept for 3minutes. The formation of a honey comb like froth
showed the presence of saponins.
EXTRACTION OF ALKALOIDS, PHENOLICS AND FLAVONOIDS
(Harborne, 1973)
The preliminary phytochemical analysis of the leaves, flower and fruit
pulp indicated the presence of the secondary metabolites namely alkaloids,
phenolics and flavonoids. These plant fractions were isolated and assessed
for their bioactivity.
Extraction of Alkaloids
Fresh leaves, flowers and fruit pulp (5g each) were crushed in a mortar
and pestle with 10% acetic acid in ethanol (200ml) and incubated for 4 hours
in the dark. After incubation, the extract was filtered and the solution was
concentrated to 1/4th volume in a boiling water bath. To the extract, 25%
ammonium hydroxide or 25% ammonia was added until a precipitate was
formed and then centrifuged at 2500 rpm for 5 minutes. The residue obtained
was washed with 1% NH4OH and filtered. The residue that contained
alkaloids was then weighed, dissolved in ethanol and stored at 4C.

75

Extraction of phenolics
Leaf, flower and fruit pulp samples (1g) were taken and crushed using
a mortar and pestle. To the crushed sample, 20ml of 80% ethanol was added.
The conical flask was plugged and placed in a boiling water bath for 15
minutes with occasional shaking. The content was then centrifuged and the
supernatant thus collected was the phenolic extract.
Extraction of flavonoids
Approximately half the volume of the phenolic fraction was
transferred to a 50ml separating funnel. The sample was then extracted with
petroleum ether (40-60C). The aqueous layer thus obtained was the
flavonoid extract.
These phytochemical fractions isolated were then assessed for their
antimicrobial activity and free radical scavenging activity.
ANTIMICROBIAL ACTIVITY OF THE ISOLATED FRACTIONS
The isolated phytochemical fractions, namely the alkaloids, phenolics
and flavonoids, were assessed for their antibacterial activity against the
pathogenic bacteria used in Phase I namely Escherichia coli, Staphylococcus
aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Shigella flexneri,
Salmonella typhi and Proteus vulgaris. The antifungal activity of the
fractions was assayed against the fungal pathogenic strains namely
Aspergillus niger, Aspergillus flavus, Aspergillus fumigatus, Candida
albicans, Rhizopus oryzae and Mucor indicus. All the procedures adopted for
the antimicrobial assays were as described in Phase I. The minimum
inhibitory concentration of the fractions against the test microorganisms
were also determined as explained in Phase I.

76

FREE RADICAL SCAVENGING ACTIVITY OF THE ISOLATED


FRACTIONS
The alkaloid, phenolic and flavonoid fractions were analysed for their
effectiveness in counteracting an array of free radicals namely DPPH,
ABTS, hydroxyl and H 2O2. The free radical scavenging activity of the
isolated fractions were performed by the various methods as described in
Phase II.
Among the three parts of Couroupita guianensis, the flower extracts
were found to be have better antimicrobial and antioxidant properties. Hence
only the flowers were subjected to further spectral analyses.
TLC SEPARATION OF THE PHYTOCHEMICALS
(Harborne, 1973)
The plant extracts were subjected to thin layer chromatography in
order to separate the active compounds present. The plates were prepared
using a slurry of silica gel G in distilled water. Silica gel G (20g) was added
to 40ml of distilled water and a thick slurry was made. All solid particles
were blended well and the uniform silica gel slurry was applied onto the
TLC plate at a thickness of 0.25mm. The plate was allowed to dry at room
temperature. The dried plate was placed in the oven at 100oC for 30 minutes
to activate the silica gel. The plate was taken from the oven and kept at room
temperature for 15 minutes.
Using a microcapillary tube, a small drop of methanolic extract of the
flowers was placed on the TLC plate, 3cm above the bottom. This spot was
allowed to dry and the TLC plate was placed into the TLC chamber which
was saturated with the solvent mixture carefully to have uniform solvent

77

level. When the solvent reached 2 cm below the top, the plates were taken
out of the chamber and detected with the respective spraying reagents.
The chromatogram was developed with chloroform: methanol (9:1)
and sprayed with 10% H2SO4 and heated at 120C for the detection of
organic components. The alkaloids were detected by spraying with
Dragendroffs reagent, phenolics were detected with Folin-Ciocalteau
reagent and flavonoids with vanillin-H2SO 4 spray reagent (10% vanillin in
ethanol: conc. H2SO 4 in 2:1 ratio). The Rf values of the spots were calculated
by the formula,
Distance travelled by the sample
Rf =
Distance travelled by the solvent
UV ABSORPTION SPECTRAL ANALYSIS
A preliminary spectral analysis was done by a survey scan of the
methanolic

extract

of

flowers

of

Couroupita

guianensis

in

nanospectrophotometer (Optizen, Korea). The absorption spectra of the


components present in the methanolic extracts of Couroupita guianensis
flowers as well as the isolated fractions (alkaloids, phenolics and flavonoids)
from

flowers

were

studied.

The

fractions were

evaluated

in

nanospectrophotometer (Optizen 3220bio, Korea). The instrument was set to


the scan mode and the absorption spectrum was obtained in the range of
190nm-350nm.
HPTLC ANALYSIS
PROCEDURE
The methanolic residue (100mg) of the flowers of Couroupita
guianensis, was dissolved in 1ml methanol and centrifuged at 3000rpm for 5

78

minutes. The supernatant was collected and used as test solution for HPTLC
analysis. 3l of the test solution was loaded as a 8mm band in the 5 x 10
Silica gel 60 F254 TLC plate using a Hamilton syringe and CAMAG
INOMAT 5 instrument. The flower extract and reference loaded plate was
kept in TLC twin trough developing chamber (after saturation with solvent
vapour) with the respective mobile phase and the plate was developed up to
90mm.
The developed plate was dried in hot air to evaporate the solvents
from the plate. The plate was kept in Photo-documentation chamber
(CAMAG REPROSTAR 3) and the images were captured in white light, UV
254nm and UV366nm. After derivatization with the appropriate reagents, the
plate was photo-documented at daylight for alkaloids and phenolics and at
UV 366nm for flavonoids using the Photo-documentation chamber. Finally,
the plate was fixed in the scanner stage and scanned at 500nm for alkaloids
and phenolics and at UV 366nm for flavonoids. The peak table, peak display
and peak densitogram of alkaloids, phenoilcs and flavonoids were noted.
ALKALOID PROFILE
Nicotine was used as the reference standard for the analysis of
alkaloids. The mobile phase used was ethylacetate:methanol:water
(10:1.35:1). For derivatization of alkaloids, the developed plate was sprayed
with Dragondorff's reagent, followed by 10% ethanolic sulfuric acid reagent
and heated at 120C for 5 minutes in a hot air oven.
PHENOLIC PROFILE
Quercetin was used as the reference standard for the analysis of
phenolics. The mobile phase used was toluene:chloroform:acetone
(4:2.5:3.5). For derivatization, the developed plate was sprayed with 25%

79

aqueous Folin Ciocalteau reagent and heated at 120C for 5 minutes in a hot
air oven.
FLAVONOID PROFILE
Rutin was used as the reference standard for flavonoid analysis. The
mobile

phase

used

for

development

of

flavonoids

was

ethylacetate:butanone:formic acid:water (5:3:1:1). For derivatization, the


developed plate was sprayed with 1% ethanolic aluminium chloride reagent
and heated at 120C for 5 minutes in a hot air oven.
HPLC ANALYSIS OF THE METHANOLIC EXTRACT OF THE
FLOWER SAMPLE
The methanolic extract of Couroupita guianensis flowers was
prepared for High Performance Liquid Chromatography (HPLC) by
dissolving the shade dried flower samples in HPLC grade methanol at 0.1
mg/l concentration and filtered through a 0.22 Millipore membrane filter.
It was then subjected to HPLC analysis on RP C-18 column as mentioned
below and the fractions corresponding to particular maximum peaks with
specific retention time were collected using a fraction collector.
HPLC analysis was performed with two LC-6AD pumps (Shimadzu)
with CTO-10 AS VP column oven (Shimadzu), SPD-M20A diode array
detector (Prominence) and CBM-20A communications bus module
(Prominence) with Luna 5 micron C-18 (2) Phenomenex reverse phase
column (250 x 4.6 mm). The HPLC was equipped with software class VP
series version 6.1 (Shimadzu). 20l of the methanolic extract of the flowers
was injected using Rheodyne injector and the column temperature was
maintained at 40oC. The solvent system was set in binary mode, using

80

methanol: water (75:25 v/v) at a flow rate of 1 ml/min and UV detection in


the range of 190-350 nm at 1000 psi.
IR SPECTRAL ANALYSIS
The infra red spectrum of the methanolic extract of Couroupita
guianensis was recorded in Shimadzu FT-IR spectrophotometer using KBr
pellet method. The IR spectrum obtained was compared with the HPLC and
GC-MS spectra for interpretation.
GC-MS ANALYSIS
The powdered plant material was analysed using an Agilant-5 gas
chromatography-MS spectrometer using a HP-5 column equipped with SEM
detector with helium as a carrier gas at a flow rate of 1.5 psi. The compounds
were identified using the database available in the light of the available
literature in the journals and books.
PHASE IV
The spectral studies indicated the presence of isatin and indirubin
derivatives, both of which are alkaloids in nature. Hence in this phase, these
two components were subjected to in silico studies for their efficacy against
the target proteins of the most susceptible organisms namely the bacterial
strains Shigella flexneri and Staphylococcus aureus and the fungal strain of
Candida albicans. The modules of the drug designing and modeling
software of Schrdinger Inc. was used for this phase of the study.
SELECTION OF THE TARGET PROTEINS
Shigella infections alone result in over a million deaths annually. The
initial steps of Shigella infection include their attachment to and subsequent
penetration of the epithelial cells of the intestinal mucosa. After infection,
81

the bacteria multiply intracellularly and then spread to adjacent host


cells.This spreading is accomplished by destabilization of the cytoplasmic
network of the host and thereby results in the destruction of tissues (Parsot,
2005). Virulent species of Shigella rely on a type III secretion system (T3SS)
to deliver a small number of proteins, termed effectors, into the cytosol of
host cells where they subvert mechanisms that control the actin cytoskeleton
so as to promote invasion and cell-to-cell spreading (Schroeder and Hilbi,
2008). One of these effectors is the 45-kDa protein VirA, which creates a
path that enables the bacteria to move through the dense, organized
cytoplasmic network of the host cell (Ogawa et al., 2008). Shigella variants
that lack a functional virA gene are unable to move through the cytoplasm,
and the invasiveness of these virA mutants is attenuated, suggesting that
VirA is essential for Shigella virulence (Davis et al., 2008). Hence the VirA
protein was taken as a target for Shigella flexneri. The structure of the
protein was downloaded from the RCSB protein databank. The PDB ID for
Vir A is 3EB8.
Staphylococcus aureus is a gram-positive bacterium that normally
colonizes the epithelial surface in 30 to 40% of humans. Despite advances in
antimicrobial therapy, S. aureus remains a major cause of infections in the
hospital setting. Many of these infections begin locally (skin and catheters)
and subsequently spread to the bloodstream. The pathogenicity of S. aureus
is a complex process involving the spatial-temporal production of a diverse
array of virulence factors. Many cell wall components that act as adhesins
(e.g., fibrinogen and fibronectin binding proteins) or contribute to the
evasion of host defense (protein A) are produced primarily during the
exponential phase while the production of toxins and enzymes (alphahemolysin) that facilitate tissue invasion occurs postexponentially. The
coordinated synthesis of cell wall proteins in the exponential phase and

82

extracellular proteins during the postexponential phase suggests that many of


these virulence determinants are governed by global regulatory elements.
Members of these regulatory systems include the SarA protein family and a
number of two-component regulatory systems. The first member of the third
SarA subfamily, MgrA, was originally identified as an important regulator of
autolytic activity in S. aureus. Hence, interference with MgrA may be a
reasonable antiinfective strategy, since this approach would promote
autolysis, an important regulator of virulence determinants in S. aureus
(Ingavale et al., 2005). In view of these facts, MgrA was selected as the
target protein for Staphylococcus aureus. The PDB ID for MgrA is 2BV6.
A well-known virulence attribute of the human-pathogenic yeast
Candida albicans is the secretion of aspartic proteases (SAPs), which may
contribute to the colonization and infection of different host niches by
degrading tissue barriers, destroying host defence molecules, or digesting
proteins for nutrient supply. The 10 different SAP genes may have distinct
roles at different times of the infection process and during different types of
infection. SAP1, SAP2, and SAP3 contribute significantly to tissue damage
and invasion of oral epithelium and cutaneous epidermis, while SAP4, SAP5,
and SAP6 are important for systemic infections. Among these, SAP2 and
SAP5 are the most important for the virulence as proved by several
experimental models (Schaller et al., 2003; Lermann and Morschhauser,
2008). Therefore, SAP2 and SAP5 were taken as the targets for Candida
albicans. The PDB ID for SAP2 is 1EAG and SAP5 is 2QZX.
PREPARATION OF THE TARGET PROTEINS
The Protein Preparation Wizard accepts a protein from its raw state
(which may include missing hydrogen atoms, incorrect bond order
assignments, charge states or orientations of various groups), to a state in

83

which it is properly prepared for calculations. The refined target proteins


were prepared using the protein preparation wizard and the results were
saved in .png format.
PREPARATION OF THE LIGAND
Isatin and indirubin were chosen as the small molecule compounds to
be docked to the target proteins. The structures of these compounds (ligands)
were obtained from NCBI-PubChem Compound (http://www.ncbi.nlm.nih.
gov/pubchemcompound) and were saved in a Word document.
DRAWING OF THE LIGANDS
The structures of isatin and indirubin were drawn using the tools
available on the Maestro window of Schrdinger. The refined structures
were then saved as new entries in the project table.
LIGAND PREPARATION
The preparation of the ligand was done using LigPrep 2.1, a module
on the Maestro window of Schrdinger. LigPrep produces a number of
structures for each input structure of the ligand with various ionization states,
tautomers, stereochemistry and ring conformations and eliminates molecules
using various criteria including molecular weight or specified numbers and
types of functional groups present. The prepared ligands can be used for
docking.
ADME STUDIES
The QikProp 3.0 module predicts physically significant descriptors
and pharmaceutically relevant properties of organic molecules, either
individually or in batches. In addition to predicting molecular properties,
Qikprop provides ranges for comparing a particular molecules properties
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with those of 95% of known drugs. The Absorption, Distribution,


Metabolism and Excretion (ADME) studies of the prepared ligands were
done using QikProp 3.0 of Schrdinger.
MOLECULAR DOCKING USING GLIDE
Glide uses a hierarchical series of filters to search for possible
locations of the ligand in the active-site region of the receptor. The receptor
grid was generated at the receptor site bound by a ligand. The ligands were
then docked to the target proteins using Glide 4.5 module of Schrdinger.
The docking was done in Standard Precision Mode (SP). The docked protein
and the ligands were viewed with Glide Pose Viewer. The images of the best
docked poses of the ligand and the protein were saved as .jpg files.
STATISTICAL ANALYSIS
The parameters analysed in all the phases of the study were subjected
to statistical treatment using SigmaStat statistical package (version 3.1).
Statistical significance was determined by one-way analysis of variance with
p<0.05 considered as significant.
The results obtained for the bioactivity of Couroupita guianensis in all
the four phases of the study and the significant observations made during the
study are presented in the next chapter.

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