Relationships Between Chemical Structure and Activity of Triterpenes Against Gram-Positive and Gram-Negative Bacteria

Bacteria are non-chlorophyllated unicellular organisms that reproduce by fission and do not present nuclear envelope. Gram s stain is a staining technique used to classify bacteria based on the different characteristic of their cell walls. Gram-positive or Gram-negative bacteria are determined by the amount and location of peptidoglycan in the cell wall, exhibiting different chemical compositions and structures, cell-wall permeabilities, physiologies, metabolisms, and pathogenicities.


Introduction
Bacteria are non-chlorophyllated unicellular organisms that reproduce by fission and do not present nuclear envelope. Gram´s stain is a staining technique used to classify bacteria based on the different characteristic of their cell walls. Gram-positive or Gram-negative bacteria are determined by the amount and location of peptidoglycan in the cell wall, exhibiting different chemical compositions and structures, cell-wall permeabilities, physiologies, metabolisms, and pathogenicities.
Microbial diseases present a significant clinical interest because some species of bacteria are more virulent than other ones and show alteration in sensibility to the conventional antimicrobial drugs, mainly species of the genera Staphylococcus, Pseudomonas, Enterococcus, and Pneumococcus. The extensive use of the penicillin since the Second World War promoted the appearance of the first strains of penicillin-resistant Gram-positive bacteria (Silveira et al., 2006). Vancomicin and methicillin showed a large spectrum of bactericidal actions against many Gram-positive bacteria. However, some strains also presented resistance to these compounds, as observed to the drugs vancomycin-resistant Enterococcus (VRE) and methicillin-resistant Staphylococcus aureus (MRSA), respectively. As a consequence, the resistance that pathogenic microorganisms build against antibiotics has stimulated the search of new antimicrobial drugs (Al-Fatimi et al., 2007;Rahman et al., 2002).
In the last few decades, the ethnobotanical search has been the subject of very intense pharmacological studies about drug discovery as potential sources of new compounds of therapeutic value in the treatment of bacterial diseases (Matu & Staden, 2003). The importance of secondary metabolites for the antimicrobial activity has been observed to triterpenoid compounds (Geyid et al., 2005). The triterpenes are widely distributed in the plant and animal kingdoms and occur in either a free state or in a combined form, mainly in the form of esters and glycosides (Ikan, 1991). Triterpenes present a carbon skeleton based on six isoprene units, being biosynthetically derived from the squalene, which may usually yield the pentacyclic triterpenes with six-membered rings. These pentacyclic triterpenes (PCTTs) present a basic skeleton which provides a large amount of derivative structures because different positions on their skeleton may be substituted. As result, there are at least 4000 known PCTTs (Dzubak et al., 2006), exhibiting a large spectrum of biological activities (James & Dubery, 2009). Some classes of triterpenes present other skeleton, such as fernane-and lupane-type triterpenes. The literature describes the isolation of triterpenes from the vegetal species which exhibit bactericidal activity (Katerere et al., 2003;Sunitha et al., 2001;Ryu et al., 2000;Yun et al., 1999). Table 1 shows the most recent studies relating plant that exhibit bactericidal activity and contain triterpenes. The activity against Gram-negative bacteria has been few studied in relation to Gram-positive ones. The Gram-positive bacteria more studied are S. aureus, B. subtilis, B. cereus, and S. faecalis (24, 11, 7, and 6 occurrences, respectively). On the other hand, the Gram-negative bacteria more studied are P. aeroginosa, E. coli, K. pneumoniae, and S. typhi (15, 13, 9, and 6 occurrences, respectively).

Species
Isolated compound Activity against Grampositive bacteria Activity against Gramnegative bacteria Ref.

Abies sachalinensis
Triterpenes Some plants exhibit a broad spectrum of activity against both Gram-positive and Gramnegative bacteria and contain other chemical classes, such as coumarins, flavonoids, phenolic compounds, and alkaloids. However, there is an expressive quantity of vegetal species that only triterpenes were isolated, suggesting an intrinsic relationship between this chemical class and the bactericidal activity of these plants. Thus, the present work provides an extensive search in original and review articles addressing the bactericidal activity of triterpenes, which may inspire new biomedical applications, considering atom economy, the synthesis of environmentally benign products without producing toxic by-products, the use of renewable sources of raw materials, and the search for processes with maximal efficiency of energy. To systematization of the results, it was considered that the biological activities are related to the presence of functionalized sites on the chemical structure of each triterpene. Obviously the obtained data do not make them possible the comparison of the intensity of bactericidal activities among the active triterpenes. Moreover, many triterpenes were tested against few species of bacteria, and as a consequence this work only records biological positive test. Table 2 shows the bactericidal activity of oleanane-type triterpenes isolated from vegetal species and fungi (Compounds 1 to 43 shown in Figure 1). In the case of Gram-positive bacteria, oleananes with different functionalizations exhibit activity against S. aureus and a relationship between chemical structure and bactericidal activity could not established. The oleananes 6, 20, 21, 35, and 36 exhibit activity against E. faecalis. All these compounds present functional groups on the alpha side of the triterpene skeleton (hydroxyl group at C-1 and oxygenated group at C-20 or C-16). Compounds 1 to 5, and 42 exhibit activity against M. luteus and present carboxyl group at C-17 or C-20 and oxygenated group at C-3. The presence of a functional group at C-17 is an important criterion to the activity against B. subtilis, except compounds 29 and 43, which are carboxyl group funcionalized at other positions (i.e. C-3 and C-20, respectively). The activity against S. mutans is exhibited by the compounds 14, 15, 17, 18, and 24, which present oxygenated group at C-3 and carboxyl group at C-17. Few oleanane-type triterpenes were tested against S. pneumoniae and B. pumilus, and as a consequence, relationships between chemical structure and activity against these Gram-positive bacteria were not possible.
Moreover, the bactericidal activity attributed to the C-3 site is not influenced by the steric effects, because very active compounds contain groups that present large volumes at C-3, such as compounds 23-25, 28, 36-42, 64, 81, 99, 101, 102, 111-116, 127, and 134-137. A carboxyl group at C-17 on the beta side is also important ⎯ 78% and 81% of the triterpenes active against Gram-positive and Gram-negative bacteria, respectively, present this functional group. The same analysis can be made for the compounds containing functionality at C-20 on the alpha-or beta-side.
The majority of the active triterpenes presents π-bonding at positions C-5, C-6, C-9, C-11, C-12, and C-13 (i.e., Δ 5,6 Δ 9,11 , and Δ 12,13 , respectively), few of them present Δ 20,30 and Δ 20,21 , and π-bondings are absent in few active triterpenes. The bactericidal activities are mainly related to functional groups at the rings A and E of the triterpene skeleton. Considering a great quantity of active triterpenes containing π-bonding at the ring C, it may be proposed that this functionalization is also important to the bactericidal activity. Antimicrobial and anti-inflammatory activity of four known and one new triterpenoid from Combretum imberbe (Combretaceae). Journαl of Ethnophαrmαcology, Vol. 110, pp. 56-60.