Chapter 3. Spirulina platensis has been selected for the present study on the basis of the following

Transcription

1 Chapter 3 DESIGNING OF EXPERIMENT AND METHODOLOGY 3.1 Selecting a cyanobacteria (Spirulina platensis) Spirulina platensis has been selected for the present study on the basis of the following points: It is rich in secondary metabolites. It contains an extensive fatty acid profile. These essential fatty acids also play a key role in the production of antimicrobial compounds. Preliminary studies showed the existence of antimicrobial properties in this alga. It has motivated us to carry out detailed investigation on the antimicrobial activity of Spirulina platensis because during the literature survey it was found out that less focus has been given by the scientists and researcher towards its against dermatophytic and other microbes 47

2 3.2 Collection of Spirulina platensis Spirulina platensis cultures were isolated from four different habitats. The isolated culture was then grown in an incubator at 30 C with 500 lux light intensity for 30 days. Samples were then shade dried and ground in pulverization to get coarse powder. The powdered samples were used to check the antimicrobial activity and to find out the most effective strain of Spirulina platensis amongst the four isolated strains of Spirulina platensis. Identification will be done using morphological variation, studies and taxonomical approaches (Anagnostidis and Komarek, 1988 and Desikachary, 1959). The four strains of Spirulina platensis collected from four different habitats are: (i) (ii) (iii) (iv) S. platensis Jal Mahal Lake strain (Jaipur) S. platensis Ramgarh Lake strain (Jaipur) S. platensis Dayalbagh strain (Agra) S. platensis Rajkot strain (Ahmedabad) 3.3 CULTURING OF SPIRULINA PLATENSIS Culture isolated was then further grown in CFTRI (Central Food and Technology Research Institute, Mysore) medium for further study. [Plate 2 and 3] 48

3 Table 5: Composition of CFTRI medium for culturing Spirulina platensis Chemical Name Gram/Liter Sodium bicarbonate 4.5 Dipotassium hydrogen phosphate 0.05 Sodium nitrate 1.5 Potassium sulphate 1.0 Sodium chloride 1.0 Magnesium sulphate 0.2 Calcium chloride 0.04 Iron sulphate

4 Mass production of Spirulina platensis 50

5 3.4: THE EXTRACTION METHOD Variation in extraction methods are usually depend on the Length of the extraction period, Solvent used, ph of the solvent, Temperature, Particle size of the plant tissues and the solvent-to-sample ratio : Microwave-assisted extraction 10 gram of algae powder was added to 150 ml of respective solvent. The extracts obtained after microwave heating (at 720 W, for 300, 120, 50, 70, 180 second, with intermittent cooling for avoiding overheating) were cooled, centrifuged at 10,000 rpm for 15 min and filtered through Whatman filter paper 1 (Whatman International Ltd., England). The supernatant were evaporated at the respective boiling points of the solvents in rotary evaporator. Dried extracts were then reconstituted in respective solvents. 3.5: ANTIMICROBIAL ACTIVITY TESTS 3.5.1: Preparation of various concentrations Different amount of each dry crude extract was weighed and placed in a 10 ml volumetric flask. The respective solvents was then added up make up the 10ml solution of different concentrations (250ppm-7000ppm) 3.5.2: Selection of Fungal Species 51

6 On account of increasing fungal skin infection mainly cutaneous and invasive fungal infections are found responsible. Following fungal species [Plate 4] have been selected as test pathogens: Candida albicans [MTCC 227] Microsporum fulvum [MTCC 7675] Microsporum canis [MTCC 3270] 52

7 3.5.3: Selection of the bacterial species In recent years, some commonly encountered pathogens have been associated with some of the human diseases like septice mias, wound infection, boils, abcesses, toxic shock syndrome etc. Therefore, following bacterial species [Plate 5] have been selected as test pathogens: Salmonella typhimurium [ MTCC 98] Staphylococcus aureus [MTCC 96] 53

8 3.5.4: Maintenance of microorganisms The media used for culturing the organisms were Sabouraud s Dextrose Agar (SDA) for C. albicans, M. canis, M. fulvum and Nutrient Agar media (NAM) for S. aureus and S. typhimurium. SDA media was prepared by dissolving 10g peptone, 40g dextrose and 20g agar in one liter of sterile water with ph range of 5.6. NAM was prepared by dissolving 5g peptone, 3g beef extract, 5g NaCl and 20g agar in one litre of sterile water with 7.0 ph. The culture media were autoclaved at 121 C for 15 min. and allowed to cool to body temperature, the medium was dispersed into petri-dishes and it was flamed to remove air bubbles : Microbial strains confirmation and test for purity Microbial cultures used were obtained from Microbiology Lab., Department of Botany, Dayalbagh Educational Institute, Agra. Fungus cultures were picked up from the stock cultures with the help of needle and transferred to the petridishes containing SDA medium directly and incubated at 28±2 C for 3 to 5 days. In petridish, when fungal colonies appeared on SDA medium, it was transferred to other dishes or slants for experiment. Confirmation of fungi was carried out using manual [A Color Atlas of Pathogenic Fungi (Frey et al., 1986)] from Department of Botany, Dayalbagh Educational Institute, Agra. Confirmation of S. aureus was carried out by gram staining procedures and some biochemical tests and by re-isolating in nutrient agar medium : Isolation of Staphylococcus aureus In the present investigation different samples of pus of surgical wounds were collected from Microbiology section of medical college by cotton swab technique (Aneja, 2001). 54

9 Samples were inoculated on Mannitol salt agar and nutrient agar medium for hrs at 37 C. The developed bacterial colonies were isolated by streaking them on fresh plates containing nutrient agar medium. To avoid repeated isolation of same bacterial cultures, the colonies showing similar cultural characteristics, were obtained by repeated streaking of diluted suspension prepared from isolated bacterial colonies. The purified bacterial cultures were preserved on nutrient agar slants at 4 C. All bacterial cultures were subcultured after a time interval of 30 days : Identification of S. aureus The promising bacterial isolate was identified on the basis of their morphological and biochemical characteristics features (Claus and Berkley, 1986) : Aseptic conditions: The aseptic chamber (laminar air flow chamber with HEPA filters) was cleaned with 70% ethanol and irradiated with short wave UV light (from a lamp) Bioassay (Assessment of antimicrobial activity of S.platensis extract against fungal and bacterial species): Bioassays play an important role in evaluation of a particular bioactivity. A bioassay which is applied to large numbers of initial samples to determine whether or not they have any bioactivity of the desired type is referred to as a prescreen assay. A bioassay used to select materials for detailed individual study is referred to as screen assay. Bioassays are also used to guide fractionation of a crude material towards isolation of the pure bioactive compounds, which is referred to as bioassay guided fractionation (Pieckova et al., 1999) 55

10 For these purposes, bioassay tests must be simple, rapid, reliable, reproducible, sensitive, meaningful and, most importantly, predictive. The in vitro assessment of antimicrobial susceptibility is done by two methods: Diffusion Assay and Broth Assay. The antimicrobial activity of extract S.platensis against the targeted microbes was studied in terms of: (a) Reduction in weight of fungal colony (Kunert 1972): In sterilized conical flasks, crude extract was taken in suitable concentrations. Broth medium (40 gms dextrose, 10 gms peptone in a liter solution) was added in conical flasks in fixed amount (40ml). It was inoculated with the help of needle. Conical flasks were incubated at 28±2ºC. The observation was considered as a fraction of time, by taking weights before and after. Mycelia growth (control) Mycelial growth (treatment) % Mycelial inhibition= X 100 Mycelial growth (control) (b) Paper disc diffusion method (Pelczar et al., 1993) The SDA media was poured into sterile petri- dishes (diameter 9.0 cm) and allowed to set. The Paper disc Diffusion method was employed for the antimicrobial susceptibility testing. Whatman filter paper discs (No. 1, Diameter 10mm) saturated with different extracts containing varying concentrations (250ppm-7000ppm) were placed on culture medium seeded with the test organism. Disc fed with corresponding solvent alone served 56

11 as control. These Agar plates were incubated at 27±2 C for 3-5 days for fungi and 37±2 C for bacteria. After incubation, the zone of inhibition around the disc was measured in mm diameter and the mean value of triplicate was recorded. Griseofulvin was the standard antifungal used and Chloramphenicol used as antibacterial agent. (c) Agar-well diffusion method (Shanmuga et al., 2002) Briefly, spores/ml of microbes was prepared and 0.2 ml spore suspension was spread over the agar surface of the plates. The plates were placed at 27±2 C for 30 min in order to make the agar surface dry. Different conc. of the algal extract was added into the well with the help of sterilized micropipette. The plates were kept in an upright position in an incubator until the extracts diffused in the agar at least for 3-4 hr. These plates were then inverted and further incubated at 27 c for 3-5days for fungal culture and 37 C for bacterial cultures. The plates were observed for zone of inhibition (mm) around the wells. (d) Minimum inhibitory concentration (MIC): The minimum inhibitory concentration (MIC) of S.platensis extract was performed by modified disc diffusion method using paper discs in different concentrations as described by (Shanmuga et al.,2002). 3.6 PRELIMINARY PHYCOCHEMICAL SCREENING OF SPIRULINA PLATENSIS EXTRACTS The Acetonic and Methanolic extracts were separately subjected to preliminary phycochemical tests using standard methods by following the procedure of Sofawara, Trease and Evans, and Harborne. The Mayer s/ Hagner s/ Wagner s and Tannic acid test 57

12 for alkaloids, foam test for saponins, Liberman Burchard and Salkowaski for steroids/triterpnoids, Schinoda test for flavonoids, Fecl 3 solution test for tannins and Keller-Kiliani test for cardiac glycosides. 1. Alkaloids: The qualitative identification of alkaloids was done by cream colour precipitate formation on adding small amount of Mayer s reagent/ characteristic crystalline precipitate with Hagner s reagent/ Brownish red precipitate Wagner s reagent. (a) Mayer s Test To a few drops of the Mayer s reagent, 2 mg of extract was added. Formation of white or pale yellow precipitate. Indicate the presence of alkaloids. (b) Hagner s test 2 mg of extract taken in a test tube, a few drops of Hager s reagent was added. Formation of yellow precipitate confirms the presence of alkaloids. (c) Wagner s test 2 mg of extract was acidified with 1.5 % v/v of hydrochloric acid and a few drops of Wagner s reagent was added. A yellow or brown ppt. indicates the presence of alkaloids. (d) Tannic Acid Test A freshly prepared tannic acid solution (5% w/v) gives precipitate with most of the alkaloids. 2. Saponins Foam Test: 58

13 The extract was diluted with 20 ml of distilled water and it was agitated in a graduated cylinder for 15 minutes. The formation of 1cm layer of foam showed the presence of saponins. 3. Steroids / Terpenoids (a) Liebermann-Burchard s test: This is based on the formation of a series of colors (as pink to blue to green) with acetic an-hydride in the presence of concentrated sulfuric acid. (b) Salkowaski reaction 2 mg of dry extract was shaken with chloroform, to the chloroform layer sulphuric acid was added slowly by the sides of test tube. Formation of red colour indicated the presence of steroids. 4. Flavonoids (a) Shinoda s test 2 mg of extract was dissolved in 5ml of ethanol and to this 10 drops of dilute hydrochloric acid followed by a small piece of magnesium were added. Formation of pink, reddish or brown colour indicates the presence of flavonoids. (b) Sodium hydroxide test One ml of the extract, a few drops of dil. Sodium hydroxide was added. An intense yellow colour was produced in the extract, which become colourless on addition of a few drops of dilute acid indicates the presence of flavonoids. 5. Tannins (a) Fecl 3 Test 59

14 About 0.5g of dried sample was boiled in 20ml of water in a test tube then filtered. Few drops of 0.1 ferric chloride was added and observed for brownish green or blue black colouration. (b) Lead Acetate test Five ml of the extract and a few drops of 1% lead acetate were added. Yellow precipitate was formed, indicates the presence of tannins. 6. Cardiac glycoside Keller-Killani Test: Five ml extract was treated with 2ml glacial acetic acid. Then one drop of Fecl 3 and 1ml concentrated H 2 SO 4 was added. Appearance of brown ring of interface indicates deoxysugar characteristic of cardenolides. A violet ring appeared below the brown ring while in the acetic acid layer, a greenish ring appeared. 7. Anthraquinones Five ml of the extract solution was hydrolysed with diluted conc. H 2 SO 4 extracted with benzene. 1 ml of dil. Ammonia was added to it. Rose pink colouration suggested the positive response for anthraquinones. 3.7 ANALYTICAL METHODS (CHROMATOGRAPHIC FRACTIONATION OF EXTRACT): Isolation of pharmacologically active constituents from crude methanolic algal extract remains a long and tedious process. For this reason, it is necessary to have methods available, which eliminate unnecessary separation procedures. Chemical screening is thus performed to allow localization and targeted isolation of new or useful types of constituents with potential activities. This procedure enables recognition of known 60

15 metabolites in extracts or at the earliest stages of separation and is thus economically very important. Analytical methods like Thin Layer Chromatography and Column Chromatography are the economical methods Thin layer chromatography Thin-layer chromatography (TLC) is the simplest and cheapest method of detecting components of algal extracts because the method is easy to run, reproducible and requires little equipment i.e. does not require expensive instrumentation, nor do samples generally require extensive cleanup prior to analysis. Compounds can be separated with good resolution, and methods are readily adaptable for applications ranging from high throughput to preparative-scale work. Adsorbent layer of Silica gel-g (0.25 mm thick) have been used with a variety of mobile-phase solvent systems. Substances are visualized by UV absorption, chromogenic reaction with spray reagents (Universal reagent Conc. H 2 SO 4 or in Iodine Chamber) on chromatograms to detect bioactive spots Column chromatography Silica gel ( mesh) was used as a stationary phase in column. To prepare the column a glass tube of 5.0 cm diameter and 1200 cm length was clamped vertically. The lower end of the column was fitted with a stopper and plugged with glass wool as a support. Slurry of silica gel was prepared in solvent used for separation. The slurry was added to the column gradually and with gentle tapping to avoid cracks. This process was continued till a uniform column of desired length was obtained. 15 g of the crude extract obtained from soxhlet extraction was shaken with 625g of silica gel and 750 ml of methanol to obtain homogenous mixture. This mixture was poured into the column and 61

16 different fractions were eluted with different solvent ratios. The extracted fractions were collected separately in 100 ml of volumetric flask Antimicrobial bioassay of the fractions The compound recovered from methanol extract was screened for antimicrobial activity against different microbes by Disc diffusion technique. The fraction was taken in suitable concentration range ( ppm). Compound was found to be significantly effective against dermatophyte, bacteria and therefore, considered for its chemical characterization. 3.8 INSTRUMENTAL ANALYSIS Complete and rapid characterization of the separated compound requires various spectrometric methods GC MS (Gas Chromatography Mass Spectroscopy) analysis GC-MS analysis was obtained on a Shimadzu Mass Spectrometer-2010 series system using Helium was used as carrier gas. Inlet pressure with FID and AB inno-wax column (60 m X 0.25 mm id, film thickness 0.25 µm). Injector and detector temperatures were 270 and 280 C, respectively. Column temperature programmed from 50 to 180 C at 3 C/min with hold time of 2 min and from 180 to 250 C at 5 C/min with hold time 20 min respectively. EI source and mass range were 70 ev and amu. 62