**2.2 Isolation of endophytes from plant**

242 Antimicrobial Agents

An important medicinal plant in this context is *Maytenus ilicifolia*, commonly known as espinheira santa. *M. ilicifolia* is native to South America, being most commonly found in southern Brazil, and is widely used in the treatment of stomach ulcers and other gastric problems. The heavy exploitation of this plant because of its medicinal properties led it to be included in the current list of endangered species (SEMA, 1995; Bittencourt, 2000). *Schinus terebinthifolius* Raddi (peppertree) is other important medicinal plant in Argentina, Brazil and Paraguay (Mytinger & Williamson, 1987). In Brazil the bark, leaves and fruits have been used in popular medicine due to their medicinal properties (Guerra et al., 2000; Lorenzi, 2002; Dgáspari et al., 2005; Ribas et al., 2006). Actions anti-inflammatory and antiseptic for treatment of wounds, urinary and respiratory infections are listed as medicinal properties popularly known (Lima et al., 2006). Studies showed antimicrobial (Degáspari et al., 2005; Schmourlo et al., 2005; Fenner et al., 2006; Ribas et al., 2006; Johann et al., 2007; Soares et al., 2007) and antitumor activities (Queires et al., 2006). *Vochysia divergens*, popularly known as cambará, is a tree commonly found in wet soils of "Pantanal Matogrossense" in Brazil. This tree has great economic importance for the local population, especially in the production of wood. Despite the economic interest and broad popular medicinal usage of the *V. divergens*, there are very few reports on the chemical composition and biological activity of this plant. In respect to the biological activities related to this species, it was verified that the etanolic extract of *V. divergens* barks presented bactericide activity against *Staphylococcus aureus* and antinociceptive activity. Leaves and barks are used in popular medicine against respiratory

Looking forward to find a solution for the advance of multi-resistant bacteria the present study made a comparison between the composts with antimicrobial activity produced by the leaves of the medicinal plants *S. terebinthifolius* and from its endophytes and from the plants *M. ilicifolia* and *V. divergens*. The antimicrobial activity and the chemical composition of the crude extract and fractions of the *S. terebenthifolius* leaves, were analyzed. Parallel with it, endophytes from the same tree were isolated and selected in order to extract its active secondary metabolites. Those extracts with positive result were also chemist evaluated and compared with the extract and fractions from the leaves. Endophytes were isolated from these 3 medicinal plants and selected in order to extract their active compounds. Similarities and differences between active compounds produced by S.

The peppertree (*S. terebinthifolius*) leaves were collected from a tree found at the latitude - 25º26.827S, longitude – 49º13.997O. The botanical identification has been made at the herbarium of the Botanical Department of Federal University of Parana (UFPR - UPCB), where a specimen of the plant can be found under the registration: UPCB-30848. *M. ilicifolia*  leaves were collected from Centro Nacional de Pesquisa de Floresta (CNPF) of EMBRAPA (Empresa Brasileira de Pesquisa Agropecuária), Paraná, Brazil (latitude -25.369227, longitude -49.189301). The *V. divergens* leaves were collected from 10 tree from Pantanal,

and gastrointestinal problems (Hess et al., 1995).

Brazil (latitude -19.254108, longitude -57.030029).

**2. Methods** 

**2.1 Plant material** 

terebinthifolius leaves and some of their endophytes were analised.

To the endophytic isolation, preference was given to leaves with no marks, scratches or wounds, according to methodology described by Petrini (1991). The leaves were washed in running water. The petioles were paraffin-embedded and went through this battery of solutions: sterile distilled water for 1 minute, ethanol 70% for 1 minute, sodium hypochlorite 3% for 4 minutes, ethanol 70% for 30 seconds and sterile distilled water for 6 minutes. The leaves were cut in fragments that were later cultivated for 20 days at 28ºC in a potato-dextrose-agar medium or selective agar for actinomycete (AC) (Küster & Williams, 1964). To eliminate the epiphytic microorganisms of V. divergens leaves we used the purification protocol of six steps (Bettiol, 2008), in medium AC added of Tetracycline (100 µg/mL) and Cycloheximide (50 µg/mL). The living cultures were deposited in the LabGeM collection, Federal University of Paraná, Curitiba, Paraná, Brazil (http://www.labgem.ufpr.br/).
