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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

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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) S. platensis Jal Mahal Lake strain (Jaipur)

(ii) S. platensis Ramgarh Lake strain (Jaipur)

(iii) S. platensis Dayalbagh strain (Agra)

(iv) 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]

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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 0.015

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Mass production of Spirulina platensis

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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.

3.4.1: 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

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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]

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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]

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3 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.

3.5.5: 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.

3.5.6: 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).

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Samples were inoculated on Mannitol salt agar and nutrient agar medium for 24-48 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 sub-

cultured after a time interval of 30 days.

3.5.7: Identification of S. aureus

The promising bacterial isolate was identified on the basis of their morphological and

biochemical characteristics features (Claus and Berkley, 1986).

3.5.8: 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).

3.5.9 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)

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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

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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, 1×105 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

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for alkaloids, foam test for saponins, Liberman Burchard and Salkowaski for

steroids/triterpnoids, Schinoda test for flavonoids, Fecl3 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:

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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) Fecl3 Test

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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 Fecl3 and 1ml

concentrated H2SO4 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. H2SO4 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

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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.

3.7.1 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. H2SO4 or in Iodine Chamber) on chromatograms to detect bioactive spots.

3.7.2 Column chromatography

Silica gel (60-120 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

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different fractions were eluted with different solvent ratios. The extracted fractions were

collected separately in 100 ml of volumetric flask.

3.7.3 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 (100-500 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.

3.8.1 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 40-750 amu.