CHAPTER 3

METHODOLOGY

3.1 Preparation of plant materials

Fresh leaves of Cassia alata were collected around the district of Alor Gajah,

Melaka and quickly brought to the laboratory to dry the leaves. The leaves were dried

in the drying oven at 40°C for five days (Fernand et al., 2008) to make sure that the

leaves were dry enough and make the grinding process a lot easier. The oven-dried

leaves were ground by using laboratory grinder in order to get a very fine powder.

The powdered samples were stored in airtight containers and kept at room

temperature until required.

3.2 Extraction of C. alata leaves

The extraction process was carried out by using Soxhlet extractor and 95%

ethanol (v/v) was employed as the solvent. 100 ml of solvent was used for 10 g of

plant powder. The extraction process took three hours to complete. The extracts

obtained were kept in a beaker and covered with parafilm before dried under reduced
pressure by using rotary evaporator at a temperature of 40°C (Ibrahim and Osman,

1995), while the pressure employed was 115 mmHg (Aureggi et al., 2008).

3.3 Phytochemical screening of C. alata leaves extract

A small portion of the concentrated extract was subjected to the

phytochemical screening as described by Adegboye et al. (2008) and Gritsanapan and

Mangmeesri (2009) to identify the presence of alkaloids, anthraquinones, cardiac

glycosides, flavonoids, steroids and tannins.

3.3.1 Qualitative analysis

3.3.1.1 Alkaloids

Exactly 0.5 g of the plant extract was dissolved in 5 ml of 1% hydrochloric

acid, HCl on steam bath. 1 ml of the filtrate was treated with drops of Dragendorff’s

reagent. Turbidity or precipitation was taken as indicative of the presence of alkaloids.

3.3.1.2 Anthraquinones

Borntrager’s reaction was used to detect anthraquinone aglycones in the

extract. HCl (2M) was added to the sample and the mixture was heated on a hot water

bath for 15 minutes, then cooled and filtered. The filtrate was extracted with

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chloroform. The chloroform layer was separated and shaken with 10% potassium

hydroxide solution. The upper aqueous layer became pink-red.

3.3.1.3 Cardiac glycosides

About 0.5 g of the extract was dissolved in 2 ml of glacial acetic acid

containing one drop of 1% ferric chloride, FeCl3. This was underlaid with

concentrated sulphuric acid, H2SO4. A brown ring obtained at the interface indicated

the presence of a deoxy sugar, a characteristic of cardiac glycosides. A violet ring

appeared below the ring.

3.3.1.4 Flavonoids

A 0.2 g of the extract was dissolved in 2 ml of methanol and heated. A chip of

magnesium metal was added to the mixture followed by the addition of a few drops of

concentrated HCl. The mixture then became red.

3.3.1.5 Steroids

About 0.5 g of the extract was dissolved in 3 ml of chloroform, CHCl 3 and

filtered. To the filtrate was added concentrated H2SO4 to form a lower layer. A

reddish brown colour was taken as positive for steroid ring.

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3.3.1.6 Tannins

About 1 g of the extract was dissolved in 20 ml of distilled water and filtered.

Two to three drops of 10% of FeCl3 was added to 2 ml of the filtrate. The production

of a blackish-blue or blackish-green colouration was indicative of tannins.

3.3.2 Quantitative analysis

3.3.2.1 Total phenolic content

Total phenolic content of the leaves extract was determined by using method

described by Pourmorad et al. (2006) and Amornlerdpison et al. (2007). 0.5 ml of the

sample solution was mixed with 2.5 ml of 10% Folin-Ciocalteu solution and 2 ml of

7.5% sodium carbonate solution. The mixture was incubated for an hour at room

temperature and the total phenolic content was determined by colorimetry at 765 nm.

The standard curve was prepared using 0, 50, 100, 150, 200 and 250 mg/L solutions

of gallic acid in ethanol:water (50:50, v/v). The absorbance obtained at 765 nm was

converted to phenolic contents according to the calibration curve of gallic acid and

expressed in terms of gallic acid equivalent, GAE (mg/g of dry mass).

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3.4 Development of microorganisms

The microorganisms used in this study consisted of two strains of bacteria

which were Escherichia coli and Bacillus subtilis. One strain of fungi was also used

namely Aspergillus niger. The purpose of selecting different microorganisms used in

this study was mainly to compare the action of the C. alata leaves extract against

different microorganisms.

3.4.1 Activation of stock culture

The stock culture of the microorganisms mentioned previously, which were in

the form of agar slant were transferred to a newly-formed agar slant in order to

activate and also to subculture the microorganisms. Subculture is quite important

because it enables the reproduction of microorganisms, so that it can be used

whenever needed. The bacterial strains were maintained grown and maintained on

nutrient agar slants while fungi strain on Potato Dextrose agar slants. The inoculated

agar slants were incubated at 37°C for bacteria and 30°C for fungi.

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3.4.2 Broth culture

After the microorganisms were grown on agar slant, they were then grown in

broth culture. Universal bottles were used to contain the broth while inoculation loop

was used to transfer the culture from agar slant into broth culture. The bacterial strains

were grown in nutrient broth while fungi strain in Potato Dextrose broth at 37°C and

30°C respectively. The turbidity of the broth culture was compared with 0.5

McFarland standard solution after six hour of incubation. This was performed mainly

to ensure that the growth of the culture reached approximately 10 8 cells/ml. The

culture was then used in the antibacterial susceptibility assays.

3.5 Antibacterial susceptibility assays

3.5.1 Well diffusion method

One milliliter (1 ml) of microorganisms from the broth culture was inoculated

into Petri plates. Then 19 ml of molten nutrient agar (for bacterial strains) and Potato

Dextrose agar (for fungi) were poured into the Petri plates and the plates were shaken

gently for even mixing of the contents. The agar was allowed to solidify on a flat

bench. Four wells (6 mm diameter and 4 mm deep) were punched in the agar with the

aid of sterile cork borer. Two of the holes were filled with the leaves extract and the

other two filled with commercial antibiotics namely chloramphenicol as positive

control and distilled water as negative control respectively. The leaves extract (500

mg/ml), chloramphenicol (50 mg/ml) and pure solvent were pippetted into the holes

bored from the agar. The plates were left on a flat bench for an hour to dry, before

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incubated at 37°C for bacteria and 30°C for fungi. Antibacterial activity was

evaluated by measuring the diameters of zones of growth inhibition.

3.5.2 Minimum inhibitory concentration (MIC)

` The crude extracts were diluted with distilled water to 50, 100, 150, 200, 250,

300, 350, 400, 450 and 500 mg/ml and put into the universal bottles that contained

nutrient broth for bacterial strains and Potato Dextrose broth for fungi. 1.0 ml (108

cells/ml) of microorganisms was added to the universal bottles and the bottles were

incubated at 37°C for bacteria and 30°C for fungi. The extracts were replaced with the

solutions of chloramphenicol (50 mg/ml) and the solutions of pure solvent (95%

ethanol v/v), where both solutions were used as positive and negative control

respectively. The MIC was taken as the lowest concentration of the extracts that did

not permit any visible growth.

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