3. MATERIALS AND METHODS

Transcription

1 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. 41

2 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 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. 42

3 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 43

4 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 44

5 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 log 2 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. 45

6 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 46

7 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 ± 20 o C 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. 47

8 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) 48

9 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 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

10 REAGENTS 1. Phosphate buffer : 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 2 O 2 -phosphate buffer. To the experimental cuvette, 3 ml of H 2 O 2 -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 H 2 O 2 -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 H 2 O 2 to H 2 O and O 2, 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

11 REAGENTS 1. Pyrogallol (0.05 M in 0.1 M phosphate buffer, ph 6.5) 2. H 2 O 2 (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% H 2 O 2 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

12 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

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

14 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 54

15 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 µg 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% H 2 SO 4, in cold conditions, to avoid an appreciable rise in temperature. To the blank alone, DNPH reagent and thiourea were added after the addition of H 2 SO 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

16 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. 56

17 The levels of tocopherol were calculated using the formula A Tocopherol (µg) = 520 A 450 x0.29x15 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 (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 57

18 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 Na 2 SO 4 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. formula, The amount of total carotenoids and lycopene was calculated using the 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 Lycopene = 3.12 x OD sample x Vol of sample x dilution x 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

19 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. 59

20 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

21 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 61

22 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 H 2 O 2. 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. 62

23 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 A 518 [sample] A 518 [blank] Scavenging activity (%) = 100 x 100 A 518 [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) (ABTS 2 ), 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

24 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 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 Inhibition (%) = (Control - test) x 100 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 H 2 O 2 scavenging ability. 64

25 REAGENTS 1. Phosphate buffer (40mM, ph 7.4) 2. H 2 O 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/10µl were added to 0.6ml of H 2 O 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 2 O 2. The percentage of H 2 O 2 scavenging by the plant extracts was calculated as % scavenged hydrogen peroxide = where, A o - Absorbance of control A o - A 1 x 100 A 1 - 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 A o 65

26 radicals generated with Fe 3+ / ascorbate / EDTA / H 2 O 2 system. The hydroxyl radicals attack deoxyribose, which eventually result in TBARS formation, which can be quantified spectrophotometrically. REAGENTS 1. Deoxyribose (28mM) 2. FeCl 3 (1mM) 3. EDTA (1mM) 4. H 2 O 2 (10mM) 5. Ascorbate (1mM) 6. KH 2 PO 4 -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 FeCl 3, 0.1mM EDTA, 1mM H 2 O 2, 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 (H 2 O 2 ) 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

27 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 λ 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. H 2 O 2 (30%) 3. FeCl 2 (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% H 2 O 2 and 67

28 5µl of 500µM FeCl 2 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 H 2 O 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. H 2 O 2 (30%) 3. MgCl 2 (5mM) 4. FeCl 3 (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 H 2 O 2, 0.05ml of MgCl 2, 0.05ml of FeCl 3 (50µM) and the plant 68

29 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 H 2 O 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 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 69

30 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 H 2 O 2 (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. 70

31 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 (FeSO 4 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 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 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 H 2 O 2 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. 71

32 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. 72

33 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. 73

34 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% FeCl 3 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 74

35 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 1/4 th 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% NH 4 OH and filtered. The residue that contained alkaloids was then weighed, dissolved in ethanol and stored at 4 C. 75

36 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. 76

37 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 2 O 2. 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 100 o C 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

38 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% H 2 SO 4 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-h 2 SO 4 spray reagent (10% vanillin in ethanol: conc. H 2 SO 4 in 2:1 ratio). The R f values of the spots were calculated by the formula, R f = Distance travelled by the sample 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 78