MEDICINAL FUNGI Naranmandakh Sh, Oulen B, Bayarmaa E

MEDICINAL FUNGI

Naranmandakh Sh, Oulen B, Bayarmaa E

This research study was undertaken to evaluate the antibacterial activity of some Mongolian medicinal fungi. Antibacterial activity was tested by the disc diffusion method [8]. Only the fungi Fomitopsis officinalis has showed antibacterial activity. However, the ethanolic extract showed an activity against gram-positive (Staph. aureus ATCC 25923, inhibition zone Ø (IZD) 12mm, M. luteus ATCC 9341 IZD 13mm, B. cereus ATCC 10987 IZD 13mm) and gram-negative (E. coli ATCC 25922 IZD 10mm, E. faecalis ATCC 14506 IZD 14mm) bacteria. The extract failed to inhibit the growth of Ps. aeruginosa ATCC 27853. 2 fractions, isolated from this fungi showed a pronounced effect against M. luteus (IZD 17.5mm and 16.4mm, resp), fraction 2 also against Staph. aureus (IZD 15.2mm). Disc Ø: 9 mm. All fractions were active against the tested H. pylori strains. Fraction 1 was separated by different chromatographic methods. Bio-assay guided fractionation resulted in ursolic acid which could be responsible for the antibacterial activity of Fomitopsis officinalis.

Basidiomycetes produce a large number of secondary metabolites which show antibacterial, antifungal, antiviral, cytotoxic and hallucinogenic activity [1,2]. The antimicrobial activity of basidiomycetes, 317 isolates, representing 204 species collected in Spain were screened against a range of human clinical pathogens and laboratory controls. Extracts from 45% isolates, representing 109 species, showed antimicrobial activity [3].

In Mongolia many species of basidiomycetes are growing, which are widely used in traditional medicine. In Mongolian tradition a wood-rotting fungi mainly is used as a tea. According on this reason we have chosen wood-rotting fungi. The antibacterial activity of the Mongolian medicinal fungi has never been tested up to now. Therefore, the aim of this study was the evaluation of this activity.
Key words. wood-rotting basidiomycetes, antibacterial activity, Fomitopsis officinalis,

Materials and methods

Fungi. The basidiomycetes Fomitopsis officinalis, Fomes fomentarus, Fomitopsis pinicola, Laetiporus sulphureus and Inonotus obliquus were collected on November, 2008, at Eruu soum, Selenge province, Mongolia and identified by Prof. Ch.Sanchir, Botany Institute of Mongolian Academy of Sceinces. And air dried, powdered.

Sample preparation. Dried species of fungi were extracted with ethanol and water. Extracts were evaporated and samples for antibacterial testing were prepared in ethanol with concentration 10mg/ml. Only the ethanolic extract of fungi F. officinalis was separated into 3 fractions. A white powder (Fraction 1 [Fo1.]) precipitated from the ethanolic extract at 4°C. The concentrated extract (Fraction 2 [Fo2.]) was suspended in water and extracted with petroleum ether to give an oily fraction 3.

Test of antibacterial activity. Antibacterial activity was tested by the disc diffusion method [5] using following bacterial strains: Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, Bacillus cereus ATCC 11778, Escherichia coli ATCC 25922, Micrococcus luteus ATCC 9341, Pseudomonas aeruginosa ATCC 27853, Helicobacter pylori lab. strain 105.659, Helicobacter pylori lab. strain 106.278 and Helicobacter pylori DSM 10242. Suspensions of laboratory strains were spread on Petri dishes (Mueller-Hinton Agar (BioMerieux Inc) +5% FBS). The turbidity of the suspensions was McFarland 3 in Brucella–Broth. 50µl of samples (10mg/ml) were applied on sterile paper discs (Ø 9mm) and placed on agar. Incubation: 1-3 days at 35⁰C. Inhibition was determined by measuring the diameter of the clear zone around the disc. References: Kanamicin and ciprofloxacin.

Column chromatography. The fraction 1 and the fraction 2 were purified on a silica gel (0.015-0.040 mm) column eluting with CHCI₃:MeOH (95:5).

Only the fungi Fomitopsis officinalis has showed antibacterial activity (Table 2). The ethanolic and water extracts of the fungi Fomitopsis officinalis showed activity against gram-positive (S. aureus, M. luteus, B. cereus) and gram-negative (E. coli, E. faecalis) bacteria (Table 2). The activity of ethanolic extract was higher than water extract.

Table1. Antibacterial activity of some wood-rotting fungi

	Fungi species			Inhibition zonea Ø, mm
		Micro-coccus luteus			Entero-coccus faecalis		Staphylo-coccus aureus		Bacillus cereus		Escherichia coli		Pseudo-monas aerugi-nosa
	Extractsb	EtOH	H2O		EtOH	H2O	EtOH	H2O	EtOH	H2O	EtOH	H2O	EtOH	H2O
1	Fomitopsis officinalis	13	11		14	12	12		13	10	10	-	-	-
2	Fomes fomentarus	-	-		11	-	10	-	10	-	-	-	-	-
3	Fomitopsis pinicola	-	-		-	-	-	-	-	-	-	-	-	-
4	Inonotus obliquus	11	-		-	-	-	-	-	-	-	-	-	-
5	Laetiporus sulphureus	-	-		-	-	-	-	-	-	-	-	-	-
6	Kanamycin (Reference)	10			13		19		12		12		15

^a Disc Ø: 9mm ^-No inhibition

^bConcentration of extracts: 1 mg/disc

According on this result, the ethanolic extract was chosen on further study and this extract was separated into 3 fractions. Fractions 1 and 2 showed a pronounced effect against M. luteus (Figure 2), fraction 2 also against S. aureus (Figure 1).

Fig.1 Activity against S. aureus Fig.2 Activity against M. luteus

Table 2. Antibacterial activity of the fungi Fomitopsis officinalis

	Extractb	Inhibition zone Ø a, mm
		Micrococcus luteus ATCC 9341	Enterococcus faecalis ATCC 14506	Staphylococcus aureus ATCC 25923	Bacillus cereus ATCC 10987	Escherichia coli ATCC 25922	Pseudomonas aeruginosa ATCC 27853
1	Water extract	11	12.5	-	10	-	-
2	Ethanolic extract	13	14	12	13	10	-
3	Fraction 1	17.5	-	-	11.0	9.7	11.2
3	Fraction 2	16.4	11.8	15.2	11.5	-	-
4	Ethanolc	-	-	-	-	-	-
5	Kanamycin (reference)	9	11.6	9	12	15	16

^a Disc Ø: 9mm ^c solvent

^b concentration of extract: 1 mg/disc ^- no inhibition
All fractions of the Fomitopsis officinalis were active against the tested H. pylori strains. Fraction 1 has showed higher activity than other fraction (Table 3). Fraction 1 was separated by Column chromatographic method and fractions were purified by Preparative TLC. Results showed in Table 3. R_f of the Fraction 1.1 was similar to ursolic acid.
Table 3. Antihelicobacterial activity of the fungi Fomitopsis officinalis

	Extractb	Inhibition zone Ø a, mm
		H. pylori 105.659	H. pylori 106.278	H. pylori DSM10242
1	Fraction 1	12	15	15
2	Fraction 2	12	12	12
4	Petroleum ether extract	12	10	10
5	DMSO c	-	-	-
6	MeOH c	-	-	-
5	Ciprofloxacin (ref.5µg/disc)	48	56	58

Table 3. TLC results of fractions of fungi F.officinalis.

Conclusion

Only the fungi Fomitopsis officinalis has showed antibacterial activity from five wood-rotting fungi growing in Mongolia.

The ethanolic and water extracts of the Fomitopsis officinalis have showed an activity against human clinical pathogens (S. aureus, M. luteus, B. cereus, E. coli, E. faecalis). Fractions 1 and 2 from the ethanolic extract of Fomitopsis officinalis showed a pronounced effect against M. luteus (Fig.1), fraction 2 also against S. aureus.

All fractions of the Fomitopsis officinalis were active against the tested H. pylori strains.
The Chromatographic analysis resulted in Fraction 1.1 of the Fomitopsis officinalis is a compound like with ursolic acid. Investigations on the chemical structure are still in progress.
Acknowledgements This research was supported by the Mongolian Foundation for Science and Technology.

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