**1.6 Extraction methods**

374 Antimicrobial Agents

**Hirsutic acid C**  Complicatic acid

**Hypnophillin**  Pleurotelic acid Pleurotellol

Coriolin A, B, C **Hypnophillin**  1-desoxyhipnophyilin

**Gloeosteretriol Incarnal** 

**Cucumins A-H**

**Merulidial** 

Meruliolactone

Stereopolide Dihydrostereopolide

*G lucidum*

Triterpenoid lactons **Fomlactons A, B, C** *Fomes cajanderi*

Lanostane-type, fatty acids lanostane and ergostane derivatives

**Fuscoatroside Enfumafungin** - WF11605

as a aglyconic component

(with variable cyclic structure and method of substitution)

Table 3. Antimicrobial effects of sesquiterpenoids and triterpenoids from macrofungi

**Favolon** 

 

TRITERPENES Lanostane-type *G. applanatum*

Triterpenic glycosides **Kolocosides A, B, C, D** 

Triterpenoid saponins glycosides with *betulin* 

(according to Abraham, 2001)

(dehydro-hirsutanol A)

**ORIGIN EFFECT** (ACTIVITY)

*Stereum hirsutum* 

*Coriolus consors* 

*Lentinus crinitus*-

*Macrocystidia cucumis* 

*Merulius tremellosus - culture*

*Stereum purpureum* – culture,

common in plants and lichens, so far only three representatives found: *Xylaria* from Hawaiian Islands

*Merulius tremellosus*

(de Silva et al., 2006)

*Fomes*

*P. ostreatus*

*Favolaschia*

*Stereum complicatum - culture Pleurotus hypnophilus*

*Gloeostereum incarnatum - culture* 

**Compound Name or** 

**PLEUROTELANES** pleurotelic

modification of hypnophillin

 **HIRSUTANES** 

 **CUCUMANES** 

MERULANES ISOLACTARANES **TRITERPENOIDES** 

skeleton, created by

**chemical structure**

Extraction procedures are important in assessing good antibacterial activities of extracts. Macrofungi are commonly collected either randomly or by locals in geographical areas or forest habitats where the fruiting bodies are found. Initial screenings of fungi for possible antibacterial activities usually begin by using crude aqueous or alcohol extractions. Since the majority of the identified components of mushrooms are active against microorganisms, they are mostly obtained through initial ethanol or methanol extraction.

Water-soluble compounds, such as polysaccharides and polypeptides, including lectins, are commonly more effective as inhibitors of virus adsorption and cannot be identified in the screening techniques commonly used. Tannins and terpenoids are occasionally obtained by treatment with less polar solvents.

For alcoholic extraction, the intact mature fruiting bodies or their segments are brush cleaned, air-dried to constant mass and pulverized, and then soaked in methanol or ethanol for extended periods (24-72h). The resultant filtrated extracts are then filtered and washed, concentrated under reduced pressure at low temperature to avoid destroying of any thermo-labile antimicrobial agents present in the extract and redissolved in the alcohol (or 5% DMSO) to a determined concentration. Water extractions, generally used distilled water, blending of slurry, filtration and centrifugation (approximately 15,000 for 30 min) multiple times for clarification.


Table 4. Other compounds from macrofungi with antimicrobial activity

Antibacterial Agents from Lignicolous Macrofungi 377

et al., 1991). In the bioautography agar overlay method, the drug to be evaluated is adsorbed onto the TLC plate and the inoculum is laid onto the plate as a very thin layer (1 mm). The advantage of this method is that the amount of sample being used is very small and that the fractionalisation of the crude extracts on its different components simplifies the

In our recent work, the TLC chemical profile of the analyzed species of lignicolous macrofungi showed that they are rich in phenols, although the differences in the number and quality of the extracted compounds have been noticed. Comparing the TLC profiles, fungi can be classified into three groups according to the obtained retention factor e.g. Rf values representing the distance traveled by the compound divided by the distance traveled by the solvent: 1) three species: *C. versicolor, G. lucidum* and *G. applanatum* contain compounds with similar (Rf = 0.68, Rf = 0.69, Rf = 0.70, respectively), 2) five species *M. giganteus, L. sulphureus, F. velutipes, F. hepatica* and *P. ostreatus* showed a small amount of eluated compounds and intense fluorescence at the start line after the spraying, 3) the species *P. betulinus* expressed with three spots in the MeOH extracts (Rf = 0.62, Rf = 0.65,

Furthermore we made slight modifications of the standard procedure of bioautography in the same study using the following: soft (top) agar (0.7% Nutrient agar) which was mixed with freshly prepared inoculum of bacteria (0.5Mac Farland optical density) and with the aqueous solution of tetrazolium red dye 0.1% w/v (1mg/ml)- 2,3,5-triphenyltetrazolium chloride (TTC, Sigma) (3:1:0.1). The strain S*. aureusan* was used as the indicator organism. Amoxicillin (64µg/ml) was used as positive control. Approximately 10µl of the solution of each extract was applied on a TLC plates (silica gel 60, F 254, DC-Plastikfolien, 0.2 mm thick, Merck, Germany) for about 2h, equally prepared as a reference plate for chemical analysis. Bioautography test plate was developed in the same tank using the pre-determined mobile phase which was removed from the plate by drying with a stream of cool air from a heating gun. Separated spots were visualised under UV light and marked by pencil (Figure 2A). Developed plates were placed upside-down in the petri dishes containing bottom agar (nutrient agar, Torlak, Belgrade). Soft agar (07% Nutrient agar) was melted and poured into sterile tubes (100 ml) in which the dye and bacteria were added quickly. That mixture was flowed over the chromatograms in the petri dishes. After the agar has solidified, the plates were inverted and incubated at 35ºC for 24h. The clear zones on the chromatogram indicate areas of inhibition zones on the red background where bacteria are present. Comparing clearing zones with reference TLC plate according to Rf values the most active components

Bioautography results showed many antibacterial compounds against animal strain of *S. aureus* that were mostly present in the polar region of the bioautogram. According to detected clearing zones, chloroform extracts were more active corresponding to more detected UV absorptive substances along the chromatogram. However, these substances were not active in methanolic extracts on bioautogram for *C. versicolor* and *P. betulinus.*

**Developing system:** toluene-ethyl acetate – 90% formic acid (5:4:1 v/v/v). **Detection:** 366 nm UV light without spraying. **Extracts:** lane 1- *M. giganteus* (MeOH), lane 2- *L. sulphureus*  (MeOH), lane 3- *C. versicolor* (MeOH), lane 4- *F. velutipes* (MeOH), lane 5- *G. lucidum* (EtOH), lane 6- *G. applanatum* (MeOH)*,* lane 7- *P. tigrinus* (MeOH)*,* lane 8- *P. betulinus* (MeOH)*,* lane 9- *P. ostreatus* (MeOH), lane 10- *F. hepatica* (MeOH), lane 2´- *L. sulphureus* (CHCl3),

Rf = 0.68), which extinguished fluorescence in the UV 254th (Karaman, 2009c).

of crude fungal extracts could be approximately detected (Fig. 1B).

identification of active compound.26
