Antimicrobial potentials of crude fractions of polysaccharides of Ganoderma spp

1 Department of Microbiology, Medical Microbiology Research Center, School of Medicine, Yasuj University of Medical Sciences, Yasuj, Iran. 2 Alborz University of Medical Sciences, Karaj, Iran. 3 Farhangian Bahonar Univesity of Shiraz, Iran. 4 Department of Parasitology and Mycology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran. 5 Department of Pathobiology, School of Medicine, Alborz University of Medical Sciences. Karaj, Iran.


INTRODUCTION
One of the most apparent influences that modern times have brought to people is their realization of going back to natural products.Although the advances brought by technology has made life easier to people, many are still looking for better alternatives that are proven to be more effective in their most natural form, like mushrooms, in particular Ganoderma spp.; a fungus known for hundreds or even thousands of years, recognized as powerful medicinal fungi because it has properties often associated with health and healing, long life and etc (Bhagwan et al., 2009).
In the last decades, the wide range of pharmaceutically interesting metabolites from mushrooms has been one of the most attractive groups of natural products studied (Smania Júnior et al., 2006;Shrestha et al., 2006).Ganoderma spp.has been an economically important, particularly in the Far East countries, for over 4000 years GA-11 Ganoderma praelongum (Murrill) GA-37 Ganoderma sp.
GA-K, GA-S Ganoderma stipitatum (Murrill) GA-07 and used as antitumor activity (Mizushina et al., 1998;Chiu et al., 2000).Several antimicrobial metabolites have been isolated from mushrooms like Ganoderma spp., which have potent antiviral, bacterial and fungal activity (Jonathan et al., 2008;Jonathan and Awotona, 2010).This report elucidates interesting chemical compounds extracted (polysaccharide), purified and identified from fruit bodies of Ganoderma spp. as a bioactive agents tested against a selected pathogenic microorganisms.Therefore, the present study aims to screen antimicrobial activity of polysaccharide extracts of Ganoderma spp. on a variety of pathogenic microorganisms.

Sample collection
During March to August 2010, Ganoderma (Table 1) were collected from different parts of Mazandaran province (North of Iran), and identified using keys and morphological characters mentioned by Steyaert (1972) and Ryvarden (2000) and then brought to laboratory, air-dried, grounded and maintained in airtight plastic bag for further use.(Mizuno, 2000) 3 g of Ganoderma powder was first extracted overnight with 80% ethanol at room temperature.The next day, the solution was filtered through Whatman's filter paper no. 1 and the supernatant was discarded.The residue was then extracted with hot water on a water bath for 3 h.The solution was cooled to room temperature and filtered.The supernatant was measured and four times the volume of ethanol was mixed drop wise in cold condition.The solution was mixed and kept at 4°C overnight.The next day, solution was centrifuged at 6000 g for 30 min, the supernatant was discarded and the pellet was collected.This served as fraction I.

Polysaccharides extraction
To obtain fraction II, the residue left over after hot water extraction was extracted with 1% ammonium oxalate solution for 6 h on water bath.Then, the solution was cooled and centrifuged at 2500 to 3000 g, the supernatant was collected, measured and reduced to half.This served as fraction II.
The residue was obtained after fraction II extraction, and then extracted with 5% sodium hydroxide for 6 h on water bath.The solution was then cooled, and centrifuged at 2500 g. supernatant was collected and pH of this solution adjusted to 5 to 6 with acetic acid.The mixture was stirred vigorously at room temperature and allowed to stand for some time and centrifuged at 2500 g.The residue was collected and served as fraction IIIA.
The supernatant was measured and 80% ethanol, four times the volume of supernatant, added drop wise in cold condition.The resultant solution was mixed vigorously and kept in ice overnight.The residue was served as fraction IIIB.Thus, residue of fraction I, solution of fraction II, residue of IIIA and IIIB was served as test and was tested against target cultures.Similarly, antibiotic sensitivity testing was done by disk diffusion procedure (Mizuno, 2000).

Minimum inhibitory concentration (MIC)
The lowest concentration of the antimicrobial extract inhibiting the visible growth after overnight incubation is denoted as MIC.For bacteria, MIC of the extract was determined using broth dilution method (Bylka et al., 2004).
To determine minimum bactericidal concentration (MBC), a known quantity of inoculum from each of the tubes of broth that showed no visible turbidity is sub-cultured to solid agar plate.The lowest concentration of antimicrobial agent that allowed less than 0.1% of the original inoculum to survive is said to be the minimum bacterial concentration (MBC).
For bacteria extract in Nutrient Broth (NB) and for fungi, Yeast Potato Dextrose Broth (YPDB) was used.The solutions were serially diluted in respective media to obtain concentrations of 128, 64, 32, 16, 8, 4 and 2 mg ml -1 .The experiments were performed in triplicate and analyzed by ANOVA test.

RESULTS
By disc diffusion agar, the polysaccharide crude fractions IIIa and IIIb of G. multicornum, G. multiplicatum, G. perzonatum, and G. stipitatum spp., from Mazandaran, Iran, showed a good antimicrobial activity with a zone diameter between 6 to 40 mm against the target cultures.Data represented in Table 2 showed that, G. stipitatum (fraction III a and IIIb) had a very high activity on the growth of E. coli, P. mirabilis and C. albicans with the inhibition zone diameter of 10 to 30 mm, 15 to 20 mm, and 28 to 40 mm, respectively.Similarly, the effect of commonly used antibiotics was also tested against these microorganisms and showed that they were highly resistant to at least one antibiotic (P < 0.01).
The MIC value of fraction IIIb of G. stipitatum against P. mirabilis was 32 µg/ml, while the maximum MIC of this fraction was 64 µg/ml as tested against E. coli, K. pneumoniae and C. albicans (Table 3).Our results also indicate that the minimum lethal concentration (MLC) values for P. mirabilis and C. albicans were 64 and 128 µg/ml, respectively.Moreover, the target cultures were highly resistant to at least two or more antibiotics particularly penicillin (Table 4).

DISCUSSION
As the microorganisms have developed resistance to most of the antibiotics, the research for novel antibiotic utmost important and for this very reason, the present work was carried out using extract from 13 Ganoderma spp.Although, a few reports (Tawfik et al., 2011;Smania et al., 2001)   exhibited an inhibitory effect against bacteria and fungi.These findings are in concomitant with other reports (Abdel-Mallek et al., 1995;Wasser and Weis, 1997), who reported that G. stipitatum produces steroides compounds which are active against G-negative and Gpositive bacteria.Apparently, the polysacharide fraction IIIa and IIIb of Ganoderma multicornum, Ganoderma multiplicatum, G. perzonatum, and G. stipitatum were potent and effective against the fungal isolates (C.albicans) since growth inhibition zone of 35 to 40 mm was observed, which is in contrast with the earlier findings (Wasser and Weis, 1997).However, Smala et al. (2003) stated that Ganoderma annulare produces applanoxidic acid compound which showed a weak activity against the dermatophyte Trichophyton mentagrophyes.
According to Gao et al. (2003), Ganoderma lucidum and other Ganoderma species more potent often in combination with chemotherapeutic agents have been used to treat various bacterial diseases.They have suggested that the polysaccharide components play important role in its bioactive principle.Furthermore, Chen et al. (2006) in an extensive paper, mentioned that many ''herbal'' medicines are used widely as immunomodulators in Asian countries.G. lucidum is one of the most used ''herbs'' in Asia and preclinical studies have established that the polysaccharide fractions have potent effects.Furthermore, Muller et al. (2006) emphasize that over many centuries, ''herbal'' remedies have treated a variety of ailments and the empirical/observational approach has produced a number of leads for formulated medicines.
Therefore, it could be concluded from our results that the polysaccharide fraction IIIA and IIIB are equally compatible with the standard antibiotics as tested against variety of resistant bacteria.Resistance to antibiotics is emerging in a wide variety of organism and multiple drug resistant organisms pose a serious threat to the treatment of infectious diseases.Hence, mushroom derived antimicrobial metabolites could be considered as an alternative for microbial infection.It is apparent from the present study that mushroom extracts from Ganoderma spp.could be employed to combat several diseases caused by pathogenic microorganisms.Nevertheless, more mushrooms need to be examined for their potentiality against bacteria and pathogenic fungi.

Table 2 .
Antimicrobial activity of polysaccharides extracts fractions from Ganoderma spp.against the target cultures.

Table 3 .
Determination of the minimum inhibitory concentration (MIC) of Ganoderma fraction III a and IIIb against selected target cultures.
on the bioactive compounds of Ganoderma spp.are available; the results obtained in our study clearly indicate that extracts of medicinal mushroom belonging to Ganoderma possess potent antimicrobial activity.

Table 4 .
Antimicrobial resistance pattern of the selected commercial antibiotic against the target cultures.