G. changii were obtained from a Pantai Morib, Selangor, authenticated by by Prof. Phang Siew Moi (Institute of Biological Sciences, Faculty of Science, University Malaya, Malaysia). The sun-dried algae were cut into small pieces. Approximately 100 g of dried algae was added to 400 mL of methanol and soaked for 4 days. Removal of the algae from solvents was done by filtration through cheesecloth, and refiltered through a Whatman filter no 4. The filtrate was concentrated using a rotary evaporator and stored at 4°C in a sterile tube until use.

Candida albicans (B3648) was used as the test organism and was obtained from a laboratory stock culture. The yeast was cultured on Sabouraud dextrose agar at 30 ° C for 24 h. The stock culture was maintained on Sabouraud dextrose agar slants at 4 ° C.

Swiss albino Mice (male) weighing between 25 and 35 g were used. The cages with the mice were placed in a room (temperature 26 ± 2 ° C) with controlled cycles of 12 h of light and 12 h of darkness; light went on at 7 am and relative humidity 45-55%. Water and food were provided to animals ad libitum. The experimental protocols were approved by the Institutional Animal Ethics Committee (IAEC) of School of Biological Sciences, Universiti Sains Malaysia. Experiments were conducted in accordance with the internationally accepted principles for laboratory animal use and care (EEC Directive of 1986; 86/609/EEC).

The fungicidal activity of the extract was determined following the method described by NCCLS ₅ with slight modifications.

The test microbe was removed aseptically with an inoculating loop and transferred to a test tube containing 5mL sterile distilled water. Sufficient inoculums were added until the

turbidity was equal to 0.5 McFarland (10 ⁸ colony-forming units mL ^-1 ) standard (bioMerieux, Marcy Petoile, France). One milliliter of the test tube suspension was added to 15– 20 mL of Sabouraud dextrose agar before setting aside the seeded agar plate (9 cm in diameter) to solidify for 15 min. Nine Whatman's filter paper no. 1 disks of 6 mm diameter were used to screen the fungicidal activity. Each sterile disk was impregnated with 20 µL of extract (corresponding to 100 mg/mL of crude extract); miconazole nitrate (30 µg/mL, as positive control); 10% DMSO (v/v) (as negative control). The disks were placed on the surface of the seeded plates, incubated at 37 ° C overnight, and examined for zones of growth inhibition.

A 16-h culture was diluted with a sterile physiologic saline solution [PS; 0.85% (w/v) sodium chloride] with reference to the 0.5 McFarland standard to achieve inoculums of approximately 106 CFU mL ^-1 . A serial dilution was carried out to give final concentrations between 1.563 and 200.00 mg crude extract per milliliter. The tubes were inoculated with 20 µL yeast suspension per milliliter nutrient broth, homogenized, and incubated at 37 ° C. After incubation, 50 µL was withdrawn from each tube, inoculated on agar plates, and incubated at 37 ° C for 24 h. The MIC value was determined as the lowest concentration of the crude extract in the broth medium that inhibited the visible growth of the test microorganism.

The standard intravenous (i.v.) inoculation of C. albicans used in this study was 1x10 ⁷ viable cells/mL PBS, of which 0.1 mL was injected into the lateral tail vein of mice ₆ . Animals were divided into three groups of 10 mice each and received treatment as described in Table 1. All mice were killed by cervical dislocation on day 7 after i.v. C. albicans inoculation.

Figure 1

Table 1: Details of experimental groups

The kidneys of each of these animals were removed aseptically, and 0.1 ml blood was withdrawn from renal artery before shipped to 0.1 ml hepharine 25U/ml. The kidneys than, transferred into sterile centrifuge tubes and homogenized in 5 mL of sterile PBS. Aliquots from each homogenate and blood samples were serially diluted and plated on Sabouraud dextrose agar plates, and incubated at 37 oC for 24 h. All cultures were done in triplicate. The colonies were then enumerated and the colony forming units (CFU) were calculated per gram of each organ and per ml of blood sample respectively.

Kidneys were processed for histopathological examination. Formalin fixed slides were prepared and stained with periodic asid–Schiff (PAS).

The values are expressed as mean ± SEM (standard errors of means). The student's t-test for significance of two independent sample means was employed. The difference between means was considered significant when p was <0.05.

The results of antiyeast activity of the extract against C. albicans are given in Table 2. The extract exhibited a favorable activity against the yeast tested. The zone of clearance produce by the commercial antibiotic disk was larger than that produced by the extract disk. The agar dilution method recorded the MIC value of 3.13 mg/mL.

Figure 2

Table 2: Antiyeast activity (zone of inhibition and MIC ) of extract compared with commercial antibiotic miconazole nitrate.

The intravenous inoculum of C. albicans (1x10 ⁷ viable organisms/mL) produced a colony of abscesses in different organs with a maximum concentration in the kidneys as expected (Figure 1, a).

Figure 3

Figure 1: Kidney pictures of mice

a. present abscesses in group 1 kidney ( → )

b. kidney withou infection

c. Section through the kidney of group 3.

d, e & f. Section through the kidney of group 1. The picture showed massive infection of C. albicans with the formation of blastospore and clamydospore

On gross examination of the specimens there were fewer abscesses present in the animals of group 2 and 3 compared with group 1. The histopathological examination showed that the kidney was massively infected by the C. albicans with the formation of blastospore and Clamydospore (Figure 1, d, e and f) compared to treated group 3 (Figure 1, c).

Figure 2 show the mean of CFU/g organ from the three groups. In group 2 and 3 animals, that received the plant extract (2.5 g/kg body weight) and ketoconazole, 10 mg/kg body weight, a significant (p>0.05) reduction in CFU was observed compared to the group 1 animal that received PBS. There was no C. albicans colony was formed by the blood sample. Figure 3 showed the mortality (%) of the animal in the group 1 to 3. The mortality (%) was decreased from 50 to 20% when the animals group received the treatment of G. changii extract or ketoconazole compared with PBS.

Figure 4

Figure 2: Effect of methanol extract of on recovered from kidney of mice

All values are colony forming units (cfu/g kidneys) mean ± SEM

Figure 5

Figure 3: Mortality rate of mice in different group of treatment for seven days