41

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.

42

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 pre-

weighed beaker and the extract was concentrated by evaporating the solvent

at 60°C. The crude extract was weighed and dissolved in a known volume of

dimethyl sulphoxide, to obtain a final concentration of 20mg / 5µl.

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.

43

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 121°C

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

44

dispensed as desired and sterilized by autoclaving at 15 lbs pressure

(121ºC) 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 µl of the plant

extracts (namely aqueous, methanol and chloroform extracts) were added.

The plates were then incubated at 37°C 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 15µl 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

45

then incubated at 37ºC. 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 50µl

was added to the broth dilutions. These were incubated for 18 hours at 37ºC.

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.

46

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 (121ºC) 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

47

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. 25µl of spore suspension was added to 10µl of the

plant extracts and placed in separate glass slides. Slides with 25µl 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.

48

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 µM)

3. Riboflavin (53 mM)

4. Nitro Blue Tetrazolium (NBT) (840 µM)

5. Potassium cyanide (200 µM)

49

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 µM methionine, 5.3 mM

riboflavin, 84 µM NBT and 20 µM 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

25°C 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.

50

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 H2O2-phosphate buffer.

To the experimental cuvette, 3 ml of H2O2-phosphate buffer was

added, followed by the rapid addition of 40µl 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 H2O2-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 spectrophoto-

metrically at 430nm with the specified time interval. The intensity of the

product is proportional to the activity of the enzyme.

51

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)

52

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.

53

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 µmole of dihydrophenol to 1 µmole of quinone

per minute under the assay conditions.

The activity of PPO was calculated using the formula,

Enzyme unit = K x (∆A/min)

where,

K for catechol oxidase = 0.272

K for laccase = 0.242

NON-ENZYMIC ANTIOXIDANTS

The non-enzymic antioxidants analysed in the leaves, flowers and fruit

pulp of Couroupita guianensis were ascorbic acid, tocopherol, total

54

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 2,4-

dinitrophenyl 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

55

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

20-100µg 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

37°C for 3 hours, were dissolved in 2.5ml of 85% H2SO4, in cold conditions,

to avoid an appreciable rise in temperature. To the blank alone, DNPH

reagent and thiourea were added after the addition of H2SO4. 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.

56

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-α tocopherol, 10mg/L in absolute alcohol

(91mg of α-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.

57

The levels of tocopherol were calculated using the formula

Tocopherol (µg) = 15x29.0x520StdA

450A520A −

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 (40°C - 60°C)

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 60°C 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

58

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).

59

PRINCIPLE

Reduced glutathione (GSH) is measured by its reaction with DTNB

(5,5′-dithiobis-2-nitrobenzoic acid) (Ellman’s 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 125µl 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.

60

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.

61

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 (110µg/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

62

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 60°C protected from light. The residue was weighed and

dissolved in dimethyl sulfoxide (DMSO) to obtain a final concentration of

20mg/5µl. 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.

63

REAGENTS

1. DPPH (0.3 mM in methanol)

2. Methanol

PROCEDURE

The extracts of Couroupita guianensis parts (25µl) 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.

64

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.

65

REAGENTS

1. Phosphate buffer (40mM, pH 7.4)

2. H2O2 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/10µl were added

to 0.6ml of H2O2 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 H2O2. 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

66

radicals generated with Fe3+

/ ascorbate / EDTA / H2O2 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. H2O2 (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. 20µl of plant extract was added such that the final volume

was 1ml. The reaction mixture was then incubated for one hour at 37°C.

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 (H2O2) 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

67

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 (λ DNA) and of animal origin

(herring sperm DNA).

EFFECT OF Couroupita guianensis ON λλλλ DNA

The extent of DNA damage induced in λ 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. H2O2 (30%)

3. FeCl2 (500µM)

4. 1X TAE buffer (pH 8.0) – Tris 40mM, EDTA 10mM

PROCEDURE

The reaction was conducted in a total volume of 30µl containing 5µl

of 50mM tris buffer (pH 7.4), λ DNA (2µg concentration) and 5µl of tris

buffer or plant extract prepared in tris buffer. Then 10µl of 30% H2O2 and

68

5µl of 500µM FeCl2 were added and incubated at 37°C 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 H2O2 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. H2O2 (30%)

3. MgCl2 (5mM)

4. FeCl3 (50µm)

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 (50µM) and the plant

69

extract (10µl containing 10mg of extract diluted in tris buffer. The mixture

was incubated at 37°C 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 37°C

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 H2O2) 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 µM, 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

70

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 37°C

for one hour. The lysate was then centrifuged at 5000 rpm for 10 minutes at

4°C. 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 50µl 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 50µl 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 (Hank’s

Balanced Salt Solution, HiMedia) and treated with H2O2 (5µl of 30%

solution), with or without 20µl of the leaf extract prepared in HBSS

(corresponding to an extract concentration of 20mg). The slices were

incubated at 37°C 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.

71

LPO ASSAY

Control tubes were prepared for each sample containing the respective

plant extract (50µl corresponding to 20mg), membrane aliquot (RBC ghosts

or liver homogenate) and TBS to make a final volume of 500µl. Pro-oxidant

(FeSO4 at 10µmoles final concentration) was added to all the tubes except

the control tubes. A blank containing no leaf extract, no membrane aliquots,

but only FeSO4 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 37°C for one hour.

At the end of the incubation, the samples, along with the homogenates

prepared from the liver slices incubated with the oxidant H2O2 and extracts

were subjected to the TBARS quantification. The LPO reaction in all the

tubes was arrested by the addition of 500µl 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, 500µl 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.

72

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 4°C 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 20µl were added and kept at incubation for 1 hour at 37°C. 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.

73

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) Mayer’s test: A fraction of the extract was treated with Mayer’s 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.

74

b) Dragendroff’s test: An aliquot of the extract was treated with

Dragendroff’s reagent and observed for the formation of reddish orange

coloured precipitate.

c) Wagner’s test: A fraction of the extract was treated with Wagner’s 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) Schinodo’s 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

Salkowski’s test: A small amount of sample was dissolved in 2ml of

chloroform taken in a dry test tube. Equal volume of concentrated sulphuric

75

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 4°C.

76

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 (40-60°C). 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.

77

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 H2O2. 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

78

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 120°C for the detection of

organic components. The alkaloids were detected by spraying with

Dragendroff’s reagent, phenolics were detected with Folin-Ciocalteau

reagent and flavonoids with vanillin-H2SO4 spray reagent (10% vanillin in

ethanol: conc. H2SO4 in 2:1 ratio). The Rf values of the spots were calculated

by the formula,

Distance travelled by the sample

R f =

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 a

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 a

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

79

minutes. The supernatant was collected and used as test solution for HPTLC

analysis. 3µl 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 120ºC 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%

80

aqueous Folin Ciocalteau reagent and heated at 120ºC 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 120ºC 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/µl concentration and filtered through a 0.22µ 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). 20µl 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

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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 Schrödinger 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,

82

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 (alpha-

hemolysin) that facilitate tissue invasion occurs postexponentially. The

coordinated synthesis of cell wall proteins in the exponential phase and

83

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

84

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 Schrödinger. 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 Schrödinger. 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 molecule’s properties

85

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 Schrödinger.

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 Schrödinger.

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.