**3.2 Antimicrobial activity**

Antibacterial activity of the methanolic extracts from endophytes of *S. terebinthifolius* was evaluated (Table 2). From the twenty isolates selected to fermentation, three released

Antimicrobial Activity of Endophytes from Brazilian Medicinal Plants 247

*K.pneumonia P. aeruginosa*

*Phomopsis* sp. 0 0 nt 0 0 nt 0 + nt 0 nt 0 nt

*Alternaria* sp. 0 0 nt 0 0 nt 0 + nt 0 nt 0 nt

*Streptomyces* sp. 0 0 nt 0 0 nt 0 0 nt 0 nt + nt

N43B2 nt 0 0 nt 0 0 nt 0 0 0 0 + 0

N2P10 nt 0 0 nt 0 0 nt 0 0 0 0 0 +

N4P61 nt 0 0 nt + 0 nt 0 0 0 0 + 0

A3F5 nt 0 0 nt 0 0 nt 0 0 0 0 + +

A1F10 nt 0 0 nt 0 0 nt 0 0 0 0 + 0

N34C1 nt 0 0 nt 0 0 nt 0 + 0 + 0 0

N5P3 nt 0 + nt 0 + nt 0 + 0 + 0 +

*S. aureus C. albicans P. aeruginosa*

NOTE: **O**: no inhibition; **Tr**: traces of inhibition; **+** inhibition zone between 4 – 5 mm in diameter. Nt: not tested

*Phomopsis* **sp. (LGMF694)** X X

Table 2. Antibacterial activity of the methanolic (ME) and aqueous (AE) extracts

*Penicillium roseopurpureum* **(LGMF698)** X X *Phomopsis* **sp. (LGMF627)** X

*Streptomyces* **sp. (LGMF696)** X X **Not identify (LGMF673)** X X

Table 3. Endophytes with active metabolites on their cell structures

*Alternaria* **sp. (LGMF626)** X *Alternaria* **sp. (LGMF692)** X **Basideomycete (LGMF713)** X

X = represent the pathogen that was inhibited by the extract

*M. luteus*

**ME ME AE ME ME AE ME ME AE ME AE ME AE** 

*S. aureus*

*E. coli* *C. albicans*

*E. faecalis*

*S. terebinthifolius*

*V. divergens*

**MRSA**

bioactive compounds in the medium culture: *Phomopsis* sp. (LGMF655) and *Alternaria* sp. (LGMF692) released active metabolites against *S. aureus*; and *Streptomyces* sp. (LGMF696) released active metabolites against *C. albicans.* Eight isolates had secondary metabolites with antimicrobial activity on their cell structures (Table 3). Thirteen endophytic *Pestalotiopsis*  spp. Isolates obtained from *M. ilicifolia* were used for evaluation of the antimicrobial activity. *Pestalotiopsis* sp. (14JES) was effective in inhibiting MRSA, *K. pneumoniae*, *M. luteus*, *S. aureus*, and *E. coli*; and the isolate *Pestalotiopsis microspora* showed similar results, except that it was unable to inhibit *K. pneumonia*. *P. vismae* showed traces of inhibition against *S. aureus* and *E. coli*; and 2 isolates of *Pestalotiopsis* sp. (10JAES and 11JAES) showed inhibition against *S. aureus* and *M. luteus,* respectively (Table 2).

The extract of endophytic actinomycetes isolated from *V. divergens* showed activity against pathogenic bacteria (Table 2). The *Microbispora* genus isolates showed activity against several clinical strains. The isolate (N4P61) inhibited *P. aeruginosa* while the isolate N34C1 had activity against *S. aureus* and *E. coli*. The isolate N5P3 was effective in inhibiting *S.aureus* MRSA, *E. coli*, *P. aeruginosa* including *C. albicans*. Besides that two isolates identified as *Microbispora* sp. (N43B2 e N4P61) and two isolates of *Streptomyces sampsonii* (A3F5 e A1P10) showed activity against *C. albicans*.

The crude extract from all the plants studied presented antimicrobial activity. So far, we have got detailed data of the *Schinus* plant's extract fractionation, and the minimum inhibitory concentration (MIC) of the crude extract from leaves and the fractions were evaluated (Table 4).


bioactive compounds in the medium culture: *Phomopsis* sp. (LGMF655) and *Alternaria* sp. (LGMF692) released active metabolites against *S. aureus*; and *Streptomyces* sp. (LGMF696) released active metabolites against *C. albicans.* Eight isolates had secondary metabolites with antimicrobial activity on their cell structures (Table 3). Thirteen endophytic *Pestalotiopsis*  spp. Isolates obtained from *M. ilicifolia* were used for evaluation of the antimicrobial activity. *Pestalotiopsis* sp. (14JES) was effective in inhibiting MRSA, *K. pneumoniae*, *M. luteus*, *S. aureus*, and *E. coli*; and the isolate *Pestalotiopsis microspora* showed similar results, except that it was unable to inhibit *K. pneumonia*. *P. vismae* showed traces of inhibition against *S. aureus* and *E. coli*; and 2 isolates of *Pestalotiopsis* sp. (10JAES and 11JAES) showed inhibition against *S.* 

The extract of endophytic actinomycetes isolated from *V. divergens* showed activity against pathogenic bacteria (Table 2). The *Microbispora* genus isolates showed activity against several clinical strains. The isolate (N4P61) inhibited *P. aeruginosa* while the isolate N34C1 had activity against *S. aureus* and *E. coli*. The isolate N5P3 was effective in inhibiting *S.aureus* MRSA, *E. coli*, *P. aeruginosa* including *C. albicans*. Besides that two isolates identified as *Microbispora* sp. (N43B2 e N4P61) and two isolates of *Streptomyces sampsonii* (A3F5 e A1P10)

The crude extract from all the plants studied presented antimicrobial activity. So far, we have got detailed data of the *Schinus* plant's extract fractionation, and the minimum inhibitory concentration (MIC) of the crude extract from leaves and the fractions were

*E. faecalis*

**MRSA**

*K.pneumonia* 

*Pestalotiopsis sp.* (10JAES) 0 0 nt 0 0 nt 0 Tr nt 0 nt 0 nt

*Pestalotiopsis sp.* (11JAES) 0 0 nt 0 0 nt Tr 0 nt 0 nt 0 nt

*Pestalotiopsis sp.* (14JAES) 0 + nt + 0 nt + + nt + nt 0 nt

*P. microspora* 0 + nt 0 0 nt + + nt + nt 0 nt

*P. vismae* 0 Tr nt 0 0 nt 0 0 nt Tr nt 0 nt

*P. aeruginosa*

*M. luteus*

**ME ME AE ME ME AE ME ME AE ME AE ME AE** 

*S. aureus*

*E. coli*

*C. albicans*

*aureus* and *M. luteus,* respectively (Table 2).

showed activity against *C. albicans*.

evaluated (Table 4).

*M. ilicifolia* 


NOTE: **O**: no inhibition; **Tr**: traces of inhibition; **+** inhibition zone between 4 – 5 mm in diameter. Nt: not tested Table 2. Antibacterial activity of the methanolic (ME) and aqueous (AE) extracts


X = represent the pathogen that was inhibited by the extract

Table 3. Endophytes with active metabolites on their cell structures

Antimicrobial Activity of Endophytes from Brazilian Medicinal Plants 249

Despite the economic interest and broad popular medicinal usage of the *Vochysia divergens* plant 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 ethanolic extract of *V. divergens* barks presented bactericide activity against *Staphylococcus aureus* and antinociceptive activity (Hess et al., 1995). The endophytic actinomycetes of the V. divergens plant showed activity against *C. albicans, S. aureus, E. coli, P. aeruginosa* and MRSA, suggesting a higher potential to the antimicrobial activity than the one found on the plant by Hess (1995). Bioprospecting studies of endophytic actinomycetes for pharmaceutical and biotechnological purposes are fundamental for the discovery of new substances for human

therapeutics including antibiotics, antimicotic, and anticarcinogenics (Bi et al. 2011).

interfering, mainly the in the dichloromethane: ethyl acetate fraction.

*terebinthifolius* bark, however not in the leaves extract (Lima et al., 2006).

Degáspari et al., 2005; Queires & Rodrigues, 1998).

The peppertree crude methanolic extract present higher activity against *C. albicans* followed by *S. aureus*, and less active against *P. aeruginosa.* The fractions dichloromethane: ethyl acetate and ethyl acetate were more active against the Gram positive microorganism followed by the Gram negative and with less action against the yeast tested. There is a difference between the antimicrobial activities found to the crude extract of the plant in relation to their fractions, probably due to the existence of an interaction of compounds on the crude extract, what would enhance the activity against *C. albicans.* Therefore, when the extract is fractionated these compounds are put apart, reducing their potential to act. According to another study about peppertree antimicrobial activity it was verified that the aqueous extract when fractionated would lose activity against *C. albicans* (Schmourlo et al., 2005), confirming the importance of synergism in this case. Apparently compound interactions that help crude extract activity in relation to fractions against yeast do not show the same effect to the bacteria tested. It indicates a higher concentration of active compounds against these microorganisms or an elimination or decrease of compounds

It is suggested that most the active extracts of endophytes studied are compound by alkaloids. Other compound classes were also revealed in these extracts, however less frequent, two endophytes, an isolate from *Alternaria* sp. and another from de *Streptomyces*  sp. had produced anthraquinones and an isolate from *Phomopsis* sp. had produced terpenoids. Results of TLC reveal there are strong evidences that phenolic compounds present on peppertree, found either on the crude extracts as well as the two active fractions, were responsible for the antimicrobial activity of the plant. Other authors also address the activity of the plant to a group of phenolic compounds, the polifenoles (Ceruks et al., 2008;

Yet, the crude extract and active fractions of the plant also presented anthraquinones (Table 4), creating a new hypothesis that the antimicrobial substances linked to the peppertree could be connected to this group of compounds. Another study had identified anthraquinones, fenoles and triterpenes in the extract with antimicrobial activity of *S.* 

With data obtained it was not found direct connection between the secondary metabolites with antimicrobial activity produced by the plant with the ones produced by the studied endophytes, once none endophytic chemical profiles studied showed the presence of phenolic compounds. This fact shows the enormous diversity of secondary metabolites present on nature and the importance of looking for active substances in medicinal plants and their endophytes.
