In vitro and In vivo antifungal activity of the methanol extract from Gracilaria changii
S Sasidharan, I Darah, K Jain
antifungal activity, marine algae
S Sasidharan, I Darah, K Jain. In vitro and In vivo antifungal activity of the methanol extract from Gracilaria changii . The Internet Journal of Pharmacology. 2007 Volume 6 Number 1.
that the methanol extract of
There is currently an enormous surge of interest in the use, development and conservation of marine algae throughout the world 12 . Malaysia is endowed naturally with a very rich algae life and the use of some of these in traditional medicines needs to be well documented. Among the algae with therapeutic properties in Malaysia, the
There is still much that is not known about the
Materials and Methods
samples and extraction
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).
In vitro anticandudal activity
The fungicidal activity of the extract was determined following the method described by NCCLS 5 with slight modifications.
Disk diffusion technique
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
Determination of minimum inhibitory concentration (MIC)
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
In vivo antifungal assay
The standard intravenous (i.v.) inoculation of
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
In vitro anticandidal activity
The results of antiyeast activity of the extract against
In vivo anticandidal activity
The intravenous inoculum of
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
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
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
All values are colony forming units (cfu/g kidneys) mean ± SEM
In recent 20 years, the risk of opportunistic fungal infections has significantly increased in patients who are severely immunocompromised due to cancer chemotherapy, organ or bone marrow transplantation and human immunodeficiency virus infection 678 . Despite advances in antifungal therapies, many problems remain to be solved for most antifungal drugs available. Algae provide rich resources of anticandidal compounds and have been used for years to inhibit fungal growth. Hence, in this study we evaluate the
In this study the methanol extract of
In the present report, we particularly concentrate on dermatophytic fungi
In conclusion, the methanol extract of