**1. Introduction**

238 Antimicrobial Agents

Xu, K.D., Franklin, M.J., Park, C.H., McFeters, G.A. & Stewart, P.S. (2001). Gene expression

Zgurskaya, H.I. & Nikaido, H. (2000). Multidrug resistance mechanism: drug efflux across

two membranes. *Molecular Microbiology*, Vol. 37, No. 2, pp. 219-225

71, ISSN 1574-6968

and protein levels of the stationary phase sigma factor, RpoS, in continuously-fed *Pseudomonas aeruginosa* biofilms. *FEMS Microbiology Letters*, Vol. 199, No. 1, pp. 67-

> The increased use of antibiotics has become the bacteria resistant. Currently, there are increasing problems worldwide with multiresistant bacteria. Examples of the resistance problems on a global scale are the methicillin-resistant *Staphylococcus aureus* (MRSA), vancomycin-resistant enterococci and *Enterobacteriaceae* producing beta-lactamases. A study of the *World Health Organization (WHO)* revealed that 90% of the bacteria strains are resistant to drugs of first choice. Bioprospecting studies of endophytic microorganisms for pharmaceutical and biotechnological purposes are fundamental for the discovery of new substances for human therapeutics including antibiotics, antimalarials, and anticarcinogenics (Strobel & Long 1998; Strobel 2002; Strobel & Daisy 2003). Endophytic fungi of medicinal plants are currently being widely studied in the search for new potentially useful secondary metabolites. The production of bioactive secondary metabolites by medicinal plants and by the endophytes provided countless drugs selected as important therapeutic options for innumerable disease. The endophytes still have wide potential to be explored what could expand even more the phenomenal contribution to health and well being. Aware of the reality of multi-resistant pathogenic microorganisms and the producing capacity of antimicrobial compounds by endophytes it is indispensable the search of antibiotic substances with new mechanisms of action, less toxic effect and/or medication enhance through this apparently inexhaustible bioactive metabolites source (Demain &

 \* Corresponding Author

Antimicrobial Activity of Endophytes from Brazilian Medicinal Plants 241

hydroxymellein, which exhibited detection limits of 5.00 and 10.0 *µ*g against *Cladosporium cladosporioides* and *C*. *sphaerospermum*. Silva et al. (2005 and 2006) reported a isolate of *Phomopsis cassiae* endophytic from *Cassia spectabilis* (Fabaceae) producer of ethyl 2,4 dihydroxy-5,6-dimethylbenzoate and phomopsilactone. Both displayed strong antifungal activity against the phytopatogenic fungi *Cladosporium cladosporioides* and *C. sphaerospermum*, as well as cytotoxicity against human cervical tumor cell line (HeLa), in *in vitro* assays. *Bacillus pumilus* was isolated from cassava (*Manihot esculenta*) cultivated by Brazilian Amazon Indian tribes, which produces other metabolite with antifungal activity,

**Medicinal plant Endophytes fungi References**

*Cassia spectabilis Phomopsis cassiae* Silva et al., 2005 and

*Maytenus ilicifolia Pestalotipsis sp., Pestalotiopis vismae and Pestalotiopsis microspora* Figueiredo et al., 2007 *Melia azedarach* L. *Penicillium* sp. and *P. janthinellum* Marinho et. al., 2005 *Murraya paniculata Penicillium* sp. Pastre et al., 2007 *Ocotea corymbosa Curvalaria* sp Teles et al., 2006

*Piper aduncum Xylaria* sp. Silva et al., 2010 *Piptadenia adiantoides Arthrium* sp. *Gibberella* sp. Rosa et al., 2010 *Smallanthus sonchifolius Papulaspora immersa and Arthrinium arundinis* Ramos et al., 2010

*Spondias mombin Guignardia* sp. Rodrigues-Heerklotz et

*Tithonia diversifolia Phoma sorghina* Borges & Pupo, 2006 *Trixis vauthieri Arthrium* sp. and *Xylaria* sp. Rosa et al., 2010 *Viguiera robusta*, *Chaetomium globosum* Momesso et al., 2008 *Virola michelii Pestalotiopsis guepinii* Oliveira et al., 2011

*Penicillium griseofulvum* Rosa et al., 2010

*Penicillium citrinum* Rosa et al., 2010

*Candida tropicalis* Rocha et al., 2009

*bidwelli and Phomopsis archeri* Souza-Motta et al., 2011

2006

2010

al., 2001

Bernardi-Wenzel et al.,

*Ageratum myriadenia Alternaria arborescens, Bipolaris* sp.*, Penicillium citrinum and* 

*Lippia sidoides Colletotrichum gloeosporioides, Alternaria alternata, Guignardia* 

*and Phomopsis*

*Luehea divaricata Alternaria*,*Cochliobolus, Diaporthe*, *Epicoccum, Guignardia, Phoma* 

*Palicourea tetraphylla Arthrium* sp. *, Fusarium oxysporum, Penicillium griseofulvum and* 

*Symphytumofficinale* <sup>L</sup> *Trichophyton* sp., *Chrysosporium* sp., *Candida pseudotropicalis and* 

Table 1. Some Brazilian medicinal plants and endophytic fungi associated

the pumilacidin (Melo et al., 2009).

Sanchez, 2009). Some studies show a relation between the endophyte secondary metabolites producing and the plant where it is found increasing the interest in endophytic microbiota of medicinal plants. Gene transference from the plant to the endophyte and the other way around is said to happen allowing common secondary metabolites production (Peixoto Neto et al., 2004). The classical example was the isolation of the fungi *Taxomyces andreanae* from the plant *Taxus brevifolia*, both taxol producers, making possible the use of this antitumor and the preservation of the medicinal plant (Stierle et al., 1993).

Recent and promising reports of the some brazilian medicinal plant and endophytes fungi as a source of important bioactive compounds and novel structures (Table 1). Endophytic fungi from *Lippia sidoides* demonstrate pharmaceutical potential and can be seen as an attractive source of biologically active compounds (Souza-Motta et al., 2011). One isolate of *Penicillium janthinellum*, endophytic from fruits of *Melia azedarach* (Meliaceae) producers of polyketides citrinin, emodin, 1,6,8-trihydroxy-3-hydroxymethylanthraquinone, and a new modified anthraquinone, named janthinone. The authors reported citrinin inhibited 100% of *Leishmania* growth after 48h at a concentration of 40 mg mL-1 (Marinho et al., 2005). Oliveira et al. (2010), in study with metabolites produced by the fungus *Pestalotiopsis guepinii* isolated from *Virola michelii* reported a new anthraquinone derivative, named guepinone, along with the known substances isosulochrin and chloroisosulochrin. *In vitro* quantitative and qualitative information obtained in study with endophytic fungi isolated from comfrey (*Symphytum officinale* L*.*) leaves indicates potential against the phytopathogenic fungus *S. sclerotiorum* (Rocha et al., 2009). Therefore, the use of bioactive products from the endophytic strains and/or the biological control with *S. sclerotiorum* needs investigation. Two novel benzopyrans have been isolated from *Curvularia* sp., an endophytic fungus from *Ocotea corymbosa* showed weak in vitro antifungal activity against *Cladosporium sphaerospermum* and *C. cladosporioides* (Teles et al., 2006). Endophytic fungi recovered from leaves of the bioactive Brazilian plant species *Ageratum myriadenia, Palicourea tetraphylla, Piptadenia adiantoides* and *Trixis vauthieri* could be a promising source of antitumoral, leishmanicidal and trypanocidal secondary metabolites, which could be used for the development of new drugs (Rosa et al., 2010). Dendryol E and dendryol F, two novel anthraquinone derivatives, have also been isolated from *Phoma sorghina*, endophyte found in association with the medicinal plant *Tithonia diversifolia* (Borges & Pupo, 2006). *Chaetomium globosum* was isolated as an endophytic fungus from the healthy leaves of *Viguiera robusta* (Momesso et al., 2008) and were identified genera *Alternaria*, *Cochliobolus, Diaporthe*, *Epicoccum, Guignardia, Phoma,* and *Phomopsis* from *Luehea divaricata*, known popularly in Brazil as açoita-cavalo (Bernardi-Wenzel et al., 2010). Crude extracts of endophytic fungi isolated from *Smallanthus sonchifolius* also showed antimicrobial effectiveness (Ramos et al., 2010) The antibacterial activity of the azaphylones, citrinin and citrinin H-1, were identified in *Penicillium* species isolated as endophytic fungi from *Melia azedarach* and *Murraya paniculata* (Pastre et al., 2007). *Xylaria* sp., an endophytic fungus from *Piper aduncum* were evaluated against the fungi *C. cladosporioides* and *C. sphaerospermum* and cytotoxicity *in vitro* against HeLA and CHO cells lines were investigated, the cytochalasins showed a strong activity against HeLA (Silva et al., 2010). Oliveira et al. (2009) isolated as endophytes two strains of *Penicillium* sp. from *Alibertia macrophylla* (Rubiaceae), producers of orcinol and 4-

Sanchez, 2009). Some studies show a relation between the endophyte secondary metabolites producing and the plant where it is found increasing the interest in endophytic microbiota of medicinal plants. Gene transference from the plant to the endophyte and the other way around is said to happen allowing common secondary metabolites production (Peixoto Neto et al., 2004). The classical example was the isolation of the fungi *Taxomyces andreanae* from the plant *Taxus brevifolia*, both taxol producers, making possible the use of this antitumor

Recent and promising reports of the some brazilian medicinal plant and endophytes fungi as a source of important bioactive compounds and novel structures (Table 1). Endophytic fungi from *Lippia sidoides* demonstrate pharmaceutical potential and can be seen as an attractive source of biologically active compounds (Souza-Motta et al., 2011). One isolate of *Penicillium janthinellum*, endophytic from fruits of *Melia azedarach* (Meliaceae) producers of polyketides citrinin, emodin, 1,6,8-trihydroxy-3-hydroxymethylanthraquinone, and a new modified anthraquinone, named janthinone. The authors reported citrinin inhibited 100% of *Leishmania* growth after 48h at a concentration of 40 mg mL-1 (Marinho et al., 2005). Oliveira et al. (2010), in study with metabolites produced by the fungus *Pestalotiopsis guepinii* isolated from *Virola michelii* reported a new anthraquinone derivative, named guepinone, along with the known substances isosulochrin and chloroisosulochrin. *In vitro* quantitative and qualitative information obtained in study with endophytic fungi isolated from comfrey (*Symphytum officinale* L*.*) leaves indicates potential against the phytopathogenic fungus *S. sclerotiorum* (Rocha et al., 2009). Therefore, the use of bioactive products from the endophytic strains and/or the biological control with *S. sclerotiorum* needs investigation. Two novel benzopyrans have been isolated from *Curvularia* sp., an endophytic fungus from *Ocotea corymbosa* showed weak in vitro antifungal activity against *Cladosporium sphaerospermum* and *C. cladosporioides* (Teles et al., 2006). Endophytic fungi recovered from leaves of the bioactive Brazilian plant species *Ageratum myriadenia, Palicourea tetraphylla, Piptadenia adiantoides* and *Trixis vauthieri* could be a promising source of antitumoral, leishmanicidal and trypanocidal secondary metabolites, which could be used for the development of new drugs (Rosa et al., 2010). Dendryol E and dendryol F, two novel anthraquinone derivatives, have also been isolated from *Phoma sorghina*, endophyte found in association with the medicinal plant *Tithonia diversifolia* (Borges & Pupo, 2006). *Chaetomium globosum* was isolated as an endophytic fungus from the healthy leaves of *Viguiera robusta* (Momesso et al., 2008) and were identified genera *Alternaria*, *Cochliobolus, Diaporthe*, *Epicoccum, Guignardia, Phoma,* and *Phomopsis* from *Luehea divaricata*, known popularly in Brazil as açoita-cavalo (Bernardi-Wenzel et al., 2010). Crude extracts of endophytic fungi isolated from *Smallanthus sonchifolius* also showed antimicrobial effectiveness (Ramos et al., 2010) The antibacterial activity of the azaphylones, citrinin and citrinin H-1, were identified in *Penicillium* species isolated as endophytic fungi from *Melia azedarach* and *Murraya paniculata* (Pastre et al., 2007). *Xylaria* sp., an endophytic fungus from *Piper aduncum* were evaluated against the fungi *C. cladosporioides* and *C. sphaerospermum* and cytotoxicity *in vitro* against HeLA and CHO cells lines were investigated, the cytochalasins showed a strong activity against HeLA (Silva et al., 2010). Oliveira et al. (2009) isolated as endophytes two strains of *Penicillium* sp. from *Alibertia macrophylla* (Rubiaceae), producers of orcinol and 4-

and the preservation of the medicinal plant (Stierle et al., 1993).

hydroxymellein, which exhibited detection limits of 5.00 and 10.0 *µ*g against *Cladosporium cladosporioides* and *C*. *sphaerospermum*. Silva et al. (2005 and 2006) reported a isolate of *Phomopsis cassiae* endophytic from *Cassia spectabilis* (Fabaceae) producer of ethyl 2,4 dihydroxy-5,6-dimethylbenzoate and phomopsilactone. Both displayed strong antifungal activity against the phytopatogenic fungi *Cladosporium cladosporioides* and *C. sphaerospermum*, as well as cytotoxicity against human cervical tumor cell line (HeLa), in *in vitro* assays. *Bacillus pumilus* was isolated from cassava (*Manihot esculenta*) cultivated by Brazilian Amazon Indian tribes, which produces other metabolite with antifungal activity, the pumilacidin (Melo et al., 2009).


Table 1. Some Brazilian medicinal plants and endophytic fungi associated

Antimicrobial Activity of Endophytes from Brazilian Medicinal Plants 243

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

An analysis based on a polyphasic approach integrating taxonomic information, morphological traits and the sequencing of the ITS1–5.8S–ITS2 of the rDNA or 16S was used, as described by Gomes-Figueiredo et al. (2007). Isolates were initially identified based on their microscopic and macroscopic characteristics including their morphology and characteristics when grown on the following culture media: PDA, oatmeal agar (OA) (20 g l-1 oat, 20 g l-1 glucose, 15 g l-1 agar), malt extract agar (MEA), and complete medium (CM) (Pontecorvo et al., 1953). Isolates were incubated for 7 days at 22 or 280C and a 12 h light: 12 h dark photoperiod. The experimental design was completely randomized with 3 replicates. Colonies were analyzed with respect to their average diameter (cm), the aspect of their borders, the aspect and coloration of the mycelium, sporulation, mycelium characteristics, the production of acervuli, the coloration of the reverse of the Petri dish, the viscosity and coloration of the medium, and the size and coloration of the conidia. A total of 20 conidia from each culture medium were observed under light microscopy (x 1000 magnification) after being grown for 7, 14, and 21 days. Conidia were assessed with respect to their width and length and the length of the apical appendages. The coloration of the median cells was also recorded. For actinomycetes identification, characteristics of colonies were used, after growth in AC medium. The isolates Gram-stained were observed under light microscopy (x

The fungi isolates were randomly selected as morphotypes according to Arnold et al. (2000), and the endophytes that presented at least one of the extracts with antimicrobial activity were submitted to identification using ITS sequences of the rDNA. DNA extraction followed method described by Raeder & Broda (1985), modified by Glienke-Blanco et al. (2002). For the fungi, the primers V9G (De Hoog et al., 2003) and ITS4 (White et al., 1990) were used to amplify the ITS1-5.8S-ITS2 of the nuclear ribosomal RNA, in the following reaction mixture (50 µl): 0,2 mM of each dNTP, 1X Tris/HCl, 1.5 mM MgCl2, 1.5 U Taq polymerase, 0.06 µM each primer and 50ng of DNA; the PCR was processed in a Mastercycler Gradient (Eppendorf®) with the following program: 94 °C for 2 min at the start followed by 35 cycles of 94 °C for 30 s, 55 °C for 1 min and 72 °C for 1 min and a final extension of 72 °C for 3 min. For the actinomycete the primers Sm6F

**2.2 Isolation of endophytes from plant** 

(http://www.labgem.ufpr.br/).

**2.3 Endophytes identification** 

1000 magnification).

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

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. terebinthifolius leaves and some of their endophytes were analised.
