Assessment of Antifungal Potential of Aqueous and Methanol Extracts of Cassia alata

MATERIALS AND METHODS

Plant materials: C. alata leaves were collected from the premises of the Federal Government College in Warri, Delta State-Nigeria and authenticated at the Department of Pharmacognosy, Faculty of Pharmacy, University of Lagos. The collected leaves were cleaned of unwanted foreign materials, cut up into small pieces and dried in sunlight for a week, ground and weighed. The dried material was coarsely milled, packed into a brown paper bag and stored at room temperature in the laboratory until used.

Chemicals and reagents: Sterile petri dishes, Sabouraud Dextrose agar (SDA) were purchased in Lagos. Cup borer, diameter 6 mm, Hot air oven, Cooling incubator C1-10S (REMI Instruments Ltd., Mumbai, India), Incubator (Astell Hearson, England), Uniscope SM801A Laboratory Water bath (Surgifield medicals, England), Vertical Heating pressure steam sterilizer LDZX-30FB (Labnet international Inc., Woodbridge, USA), Mettler P1210 balance (Gallen Horup) was provided by the Department of Pharmaceutics and Pharmaceutical Technology, University of Lagos.

Extraction of plant materials
Methanol extraction: The leave material was soaked in a Winchester bottle with methanol for 48 h (maceration). The extract was concentrated using a Buchi V-801 rotary evaporator at 35°C.

Aqueous extraction: The leave material was boiled with water in a 2000 mL (2 L) of pyrex beaker at 60°C in a water bath for an hour twice, filtered and concentrated on a water bath at 70°C. The coarsely milled leaves and stem bark of C. alata were extracted separately using water and methanol as solvents. About 140 g of the powdered sample was continuously extracted with a particular solvent by use of a Soxhlet extraction apparatus for 24 h. The extracts were filtered and concentrated to dryness under reduced pressure and controlled temperature (50-55°C) to obtain solvent-free semisolid extracts. The solvent-free semisolid extracts obtained were used for the antimicrobial studies.

Test microorganism and growth media: The microorganisms C. albicans, T. mentagrophyte, A. niger and Penicillium used for the study were obtained from the stocks of the Pharmaceutical Microbiology laboratory of the Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmacy, University of Lagos.

Assay of organism: The fungal strains were grown and maintained on Sabouraud Dextrose Agar (SDA) at 30°C. With the use of SDA, all spore formers among the fungi were cultivated and incubated at room temperature until spores developed. The spores were harvested and suspended in 1% tween 80. The turbidity was adjusted to about 10⁸ SFU mL^-1 using the serial dilution and plate count method. Candida was cultivated in SDA until growth was seen. Candida was harvested and suspended in sterile normal saline and then turbidity was adjusted to about 10⁸ CFU mL^-1.

All standardized assay organisms were kept for the assay.

Antifungal activity evaluation: The antifungal activity of aqueous and methanol extracts of the leaves of C. alata at concentrations of 50, 100, 150 and 200 mg mL^-1 were determined using the cup plate method. A molten Sabouraud Dextrose agar also stabilized at 45°C was seeded with 0.1 mL of test organism (C. albicans, T. mentagrophytes, A. niger and Penicillium) containing approximately 10⁸ SFU mL^-1 in a sterile petri dish and allowed to set.

This method depends on the diffusion of the various extracts from a cavity through the solidified agar layer of Petri dish. Strains sensitive to the antimicrobial are inhibited at a distance from the disc whereas, resistant strains have smaller zones of inhibition or grow up to the edge of the disc.

Statistical analysis: The experiments were run in duplicate and the mean recorded. The zones of inhibition were determined and also recorded (mean, n = 2). The different effects of methanol and aqueous extract of C. alata extracts on the test organisms were analyzed using ANOVA.

Minimum inhibition concentration (MIC): The Agar dilution technique was used to determine the Minimum Inhibition Concentration (MIC) of the extract of C. alata against the test organisms. A stock concentration of 500 mg mL^-1 of extract was prepared by dissolving 15 g of extract in 30 mL of propylene glycol. Ten working concentration were subsequently prepared from the stock.

Ten different concentration of the extract was used for this determination ranging from 0.625-320 mg mL^-1. Sterile petri dishes containing varying volumes of extract and molten agar (total volume 20 mL in each petri dish) depending on the concentration of extract intended were inoculated with 0.2 mL of the test organisms previously diluted to contain approximately 10⁵ SFU mL^-1 for fungi. A plate without an extract and another without a test organism were used as controls. The plates were incubated at 30°C for 72 h and observed for growth. The experiments were conducted in duplicate. The plate with the lowest concentration of the extract which showed no growth after incubation was taken and recorded as the MIC.

RESULTS AND DISCUSSION

Crude methanol and aqueous extract of leaves and barks from C. alata were assessed for selected fungi (C. albicans, T. mentagrophytes, A. niger and Penicillium).

All the crude extracts inhibited the growth of C. albicans, T. mentagrophytes, A. niger (Table 1, 2) as indicated by the zones of inhibition but did not show any activity against Penicillium. This is in line with the observations of Palanichamy and Nagarajan (1990), Ibrahim and Osman (1995), Reezal et al. (2002), Idu et al. (2007), Abubacker et al. (2008) and Adnan et al. (2011). The results showed that the extracts demonstrated a concentration-dependent antifungal activity with higher concentrations of 150 and 200 mg mL^-1 showing greater zones of inhibition than with lower concentrations of 50 and 100 mg mL^-1. High MIC values are indication of low activity while low MIC values are indication of high activity. In this study, C. albicans, T. mentagrophytes and A. niger had low MIC values thus suggesting higher activity against the corresponding organisms (Table 3).

Table 1:	In vitro antifungal activity of methanol extract of C. alata

Table 2:	In vitro antifungal activity of aqueous extract of C. alata

Table 3:	Minimum Inhibitory Concentration (MIC) profile of C. alata against test (mg mL-1)
+: High values; -: Low values

This research has shown that methanol and aqueous extracts of C. alata is a potential antifungal agent. This result is in line with (Palanichamy and Nagarajan, 1990; Ibrahim and Osman, 1995; Reezal et al., 2002; Abubacker et al., 2008; Krishnan et al., 2010; Adnan et al., 2011).

CONCLUSION

The finding of this study provides an insight into the usage of these plants in traditional medicine for the treatment of common fungal infections. This plant can be locally sourced since it grows well in any Nigerian soil. However, the effect of this plant against a wider range of bacteria and fungi and toxicological studies of the extracts is recommended. Further work on preformulation testing, pharmaceutical dosage formulation and development, pharmacokinetics which can be tested in vivo for safety and efficacy in patients is ongoing.

ACKNOWLEDGMENT

We are grateful to Mr. A. R Usman of the Pharmaceutical Microbiology Laboratory, Faculty of Pharmacy, University of Lagos, for his technical assistance in carrying out this study.