**4. Discussion**

Resistance to antimicrobial drugs today, remains a major problem in modern health care, about the impact on treatment options, mortality, infection control and economic issues. All identified taxon in this study, *Alternaria* (Kjer et al., 2009), *Phomopsis* (Du et al., 2008), *Penicillium* (Bertinetti et al., 2009), *Pestalotiopsis* (Liu, 2011), basidiomycete (Suay et al., 2000) and *Streptomyces* (Maruna et al., 2010) had already been described as producers of metabolite with antimicrobial activity. Although the TLC method is only qualitative, it is a simple and relatively cheap technique to recognize the inhibitory activity of a large quantity of organic extracts. However, our study has shown that fungal endophytes isolated from Brazilian native plant species have chemical and biochemical properties potentially useful. In the Table 1 there is a list of other Brazilian medicinal plant and endophytes fungi with antimicrobial activity. Further investigation may yield novel compounds with practical applications in a variety of biotechnological areas, with countless useful drugs as important therapeutics options for innumerable disease. The mechanical removal of cell metabolites from the inside part of their structure amplified the action spectrum of the extracted compounds besides revealing a higher number of endophytes producing active substances in relation to the attempt on releasing bioactive compounds on the culture medium. Apparently the microorganisms store these compounds for a future competition situation. The pursuit of cell release of these substances by different fermentation means would provide better biotechnological conditions for production of these compounds in higher amount for a more detailed study to be carried out.

The results of the bioautographic TLC agar overlay assay are at present only qualitative; however, it indicates that the endophytic extracts have antimicrobial potential, suggesting the need for further investigations to elucidate the chemical structure of the secondary metabolites that provide the antimicrobial properties to these endophytic isolates. Owing to the high genetic variability among *Pestalotiops* species found in the present study, new isolation efforts of "espinheira santa" endophytes should be carried out with the goal of bioprospecting, given the importance of the genus *Pestalotiopsis* in the biotechnological study of secondary metabolites, in particular, the inhibition of the activity of cells in gastric tumors (Lee et al. 1996; Strobel et al. 1998).

In the present study, isolates 6JAES and 29JES, which showed antimicrobial activity and were suggested as belonging to the species *P. microspora,* are of special importance, given that this species has become important in the past few years in the production of taxol and other secondary metabolites of antifungical, anticarcinogenic, and antioxidant properties (Strobel 2002; Strobel & Daisy 2003).

Results of the TLC suggest the presence of phenolic and anthraquinone compounds at the crude extract of the leaves of peppertree and active fractions. Yet, according to the present

The chemical analysis of the compounds with antimicrobial activity extracted from the endophytes of the peppertree leaves suggest the presence of alkaloids in all the tested extracts. Two microorganisms (*Alternaria* sp. - LGMF692 and *Streptomyces* sp. - LGMF696) also produced anthraquinones while one of them (*Phomopsis* sp. - LGMF655) produced terpenoids.

Resistance to antimicrobial drugs today, remains a major problem in modern health care, about the impact on treatment options, mortality, infection control and economic issues. All identified taxon in this study, *Alternaria* (Kjer et al., 2009), *Phomopsis* (Du et al., 2008), *Penicillium* (Bertinetti et al., 2009), *Pestalotiopsis* (Liu, 2011), basidiomycete (Suay et al., 2000) and *Streptomyces* (Maruna et al., 2010) had already been described as producers of metabolite with antimicrobial activity. Although the TLC method is only qualitative, it is a simple and relatively cheap technique to recognize the inhibitory activity of a large quantity of organic extracts. However, our study has shown that fungal endophytes isolated from Brazilian native plant species have chemical and biochemical properties potentially useful. In the Table 1 there is a list of other Brazilian medicinal plant and endophytes fungi with antimicrobial activity. Further investigation may yield novel compounds with practical applications in a variety of biotechnological areas, with countless useful drugs as important therapeutics options for innumerable disease. The mechanical removal of cell metabolites from the inside part of their structure amplified the action spectrum of the extracted compounds besides revealing a higher number of endophytes producing active substances in relation to the attempt on releasing bioactive compounds on the culture medium. Apparently the microorganisms store these compounds for a future competition situation. The pursuit of cell release of these substances by different fermentation means would provide better biotechnological conditions for production of these compounds in higher

The results of the bioautographic TLC agar overlay assay are at present only qualitative; however, it indicates that the endophytic extracts have antimicrobial potential, suggesting the need for further investigations to elucidate the chemical structure of the secondary metabolites that provide the antimicrobial properties to these endophytic isolates. Owing to the high genetic variability among *Pestalotiops* species found in the present study, new isolation efforts of "espinheira santa" endophytes should be carried out with the goal of bioprospecting, given the importance of the genus *Pestalotiopsis* in the biotechnological study of secondary metabolites, in particular, the inhibition of the activity of cells in gastric

In the present study, isolates 6JAES and 29JES, which showed antimicrobial activity and were suggested as belonging to the species *P. microspora,* are of special importance, given that this species has become important in the past few years in the production of taxol and other secondary metabolites of antifungical, anticarcinogenic, and antioxidant properties

results, the crude extract and the ethyl acetate fraction had alkaloids and terpenoids.

**3.3 Chemical comparison of active compounds** 

amount for a more detailed study to be carried out.

tumors (Lee et al. 1996; Strobel et al. 1998).

(Strobel 2002; Strobel & Daisy 2003).

**4. Discussion** 

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).

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

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

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

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.

Antimicrobial Activity of Endophytes from Brazilian Medicinal Plants 251

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#### **5. References**


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**1. Introduction** 

**12** 

**Quinolones:** 

Pintilie Lucia

*Romania* 

**Synthesis and Antibacterial Activity** 

After the concept of selective toxicity in chemotherapy was introduced at the beginning of the 20th century, (Ehrlich, 1913), classes of substances with antibacterial properties, produced by microorganisms or created through synthesis were obtained. After the discovery of penicillin, the first antibiotic introduced in clinical use in man in 1940s, a large number of different types of antibiotics were produced. Antibiotics such as beta-lactams, macrolides, aminoglycozides and tetracyclines were discovered and introduced during an extremely short period. These were obtained either by isolation from fungi or by chemically modification of the naturally isolated substrates. These dominated the antimicrobial industry, while synthetically obtained

In 1962, G. Y. Lesher and his collaborators introduced the first quinolone derivative, nalidixic acid **(**1-ethyl-1,4-dihydro-7-methyl-4-oxo-1,8-naphthyridine-3-carboxilyc acid), (1, Lesher et al. 1962) which had moderate activity against gram-negative organisms and was used for treating urinary tract infections. In the following years, a large gamma of derivatives from common elements were synthesized, which could be grouped by: cinoline (cinoxacin**),** pyrido-pyrimidine (pipemidic acid; piromidic acid), naphthyridine (nalidixic acid) and quinolones (oxolinic acid, miloxacin , tioxacin, etc.). These derivatives, with differentiated structures, have 2 common pharmacological properties, which allowed them to be classified as first generation biologically active derivatives with quinolone structure.


O

N N

C2H5

Cinoxacine

COOH

substances only played a minor role. (Chu & Fernandes, 1991)

The two common characteristics for first generation quinolones are:

O

O

only allowing them to be used as urinary antiseptics.

N N

CH3

O

C2H5

Nalidixic Acid

COOH


*National Institute for Chemical-Pharmaceutical* 

*Research and Development, Bucharest* 

N

Oxolinic Acid

O

O

C2H5

O

COOH

from *Curvularia* sp., an endophytic fungus associated with *Ocotea corymbosa*  (Lauraceae). *Phytochemistry*. Vol. 66, pp. 2363-2367.

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