Isolation of phytosterols of Dalbergia ecastophyllum (L.) Taub. (Leguminosae) and modulation of antibiotic resistance by a possible membrane effect

1 Introduction

Improper use of some antimicrobials is causing the drugs to lose their effectiveness very quickly, which makes necessary for the development of new drugs and effective management techniques. Resistance is a phenomenon characterized not only by evolutionary pressure, but especially by the indiscriminate and irrational use of antimicrobial therapeutics (Coutinho et al., 2008).

The species Pseudomonas aeruginosa is responsible for a variety of infections, such as those affecting the skin, urinary tract, eye and ear. The wide distribution of Pseudomonas in the environment is ensured by its non-fastidious growth requirements, and Pseudomonas possess many structural factors, enzymes and toxins that enhance their virulence, and make them resistant to most common antibiotics (Murray et al., 2004). Staphylococcus aureus is often found colonizing the natural microbiota, especially the skin, and with the breakdown of skin barriers or decrease of immunity can become pathogenic. It causes a variety of infections, such as infections of the skin and subcutaneously, post-surgical infections, osteomyelitis, pneumonia, abscesses, endocarditis and bacteremia (Gelatti et al., 2009). Escherichia coli is a gram negative bacterium belonging to the family Enterobacteriaceae normally found in the intestine of being endothermic, becoming recognized as a pathogen and a harmless commensal versatile (Vogt, 2005).

Another problem associated with microorganisms is increased opportunistic fungal infections. Although a number of commercially available antimycotics have increased in recent years, they are still at a disadvantage when compared to antibacterial drugs. Additionally, resistance to antifungal drugs has represented a major challenge for clinical investigations (Batista et al., 1999).

The phenomena of antibiotic resistance are increasing despite the advances in the development of new drugs (Keith et al., 2005) and the combination with natural products of plants that can alter the effect of antibiotics, either increasing or decreasing the antibiotic activity can be an interesting alternative to overlap this problem (Coutinho et al., 2008).

Most of the biological activities exhibited by the medicinal plant are provided by the products of secondary metabolism (Adam et al., 1998). Through phytochemical screening of plant extracts, it is possible to identify the presence of several classes of secondary metabolites that exhibit a wide variety of biological activities such as antimicrobial, antioxidant, antitumor and anti-ophidian (Matias et al., 2010; Daferera et al., 2003).

Dalbergia ecastophyllum (L.) Taub. (Leguminosae), popularly known as rabo-de-bugio, has a traditional use of its roots and barks combating uterine inflammation and anemia. Studies show that propolis from D. ecastophyllum has demonstrated several biological activities (Daugsch et al., 2008). Because of that, this study was to evaluate the in vitro antimicrobial activity and modulate the ethanol extract and hexane phase of D. ecastophyllum against bacterial strains in order to identify this natural product as a new strategy to combat resistance of microorganisms to antimicrobials (see Fig. 1).

Close

Figure 1

Phytocompounds identified from the hexane fraction of Dalbergia ecastophyllum. De–1 = Friedelan-3-one, (friedelin). De–2 (a/b) = mixture of β-sitosterol (De–2a) and stigmasterol (De–2b). De–3–(a/b) = mixture of sitosterol-3- O-β-D-glucopyranoside (De-3a) and Stigmasterol 3- O-β-D-glucopyranoside (De-3b). De–4 (a/b/c) = mixture of lup-20 (29)-en-3-ol (lupeol), olean-12-en-3-ol (β-amyrin) and olean-18-en-3-ol (germanicol).

Export to PPT

2 Experimental

3 Results and discussion

As shown in Table 2, the ethanol extract and hexane fraction showed no clinically relevant antibacterial activity against the assayed strains (MIC ⩾ 1024). As can be seen in Table 2, the ethanol extract (EE) had success when combined with aminoglycosides against E. coli 27, there was a MIC lowering of 31 and 8 times when combined with gentamicin and neomycin respectively.

The mechanisms by which the extracts could inhibit the growth of microorganisms are varied and may be in part due to the hydrophobic nature of some components. These components can interact with lipid bilayer of the cell membrane and affect the respiratory chain and energy production (Nicolson et al., 1999), or even make the cell more susceptible to antibiotics, leading to disruption of vital cellular activity (Burt, 2004). Interference with the enzyme systems of bacteria can also be a potential mechanism of action (Wendakoon and Sakaguchi, 1995).

With S. aureus 358 in association extract antibiotic, an increase of 3 points of the Neomycin MIC, featuring an antagonism. No effect was seen when done association with hexane fraction (FH). The main mechanism of resistance to aminoglycosides in staphylococci is drug inactivation by cellular enzymes modifying aminoglycosides. Several distinct loci encoding such modifying enzymes were characterized in staphylococci. Clinically, the most important of these region coding for enzyme activities acetyltransferase (AAC), adenylyltransferase (ANT) and phosphotransferase (APH). Aminoglycosides modified into amino groups by the enzyme AAC or hydroxyl groups or by enzyme ANT APH, lose the ability to bind to ribosomes and therefore not further inhibit protein synthesis of the bacterial cells (Paulsen et al., 1997). When was done the phytochemical screening of the hexane fraction, was identified some classes of secondary metabolites present in this fraction: friedelin as terpenes and mixture of lupeol, b-amyrin and germanicol and steroids as a mixture of b-sitosterol and stigmasterol and B-sitosterol and stigmasterol glycosylated. According Bresciani et al. (2000) and Carvalho and Carvalho, (2001), nonpolar solvents such as ether and hexane allow the extraction of groups steroids, coumarin esters, sesquiterpene lactones and terpenoids.

Table 2 shows the action of the hexane fraction (FH), with Ec 27 the MIC of Neomycin was lowered 8 times and the Gentamicin 128 times. In the case of Pa-22, the fraction combined with amikacin and gentamicin enhanced 8 times the antibiotic activity of these drugs. This favorable result for gram negative modulation can be justified because these bacteria present a higher fat content (Vargas et al., 2004). This allows a greater affinity with the components identified in this fraction. The mechanism of action of antimicrobial activity of terpenes is not fully understood but it is speculated that involves the disruption of the cell membrane by lipophilic compounds (Cowan, 1999). This break allows for greater penetration of the antibiotic.

Terpenes or terpenoids are active against bacteria (Ahamd et al., 1993). The isolation and identification of various terpenes with antinociceptive effect, anti-inflammatory, antimicrobial, antiparasitic, trypanocidal, larvicide, have been demonstrated in preliminary studies (Sartori, 2005).

Studies involving this species has been reported, according to Daugsch et al. (2008) D. ecastophyllum is the botanical origin of propolis (propolis group 13) and has antimicrobial activity, and the ethanol extracts of the propolis group 13 showed antibacterial activity against S. aureus, Salmonella typhimurium, Streptococcus mutans. Results The results show a broad spectrum against various microorganisms (fungi, bacteria, viruses, protozoa). Although important biological antioxidant properties, and immunomodulatory and cytotoxic activities has been proven (Bankova et al., 2000). Silva (2008) showed that both the ethanol extracts of propolis as the ethanol extracts of D. ecastophyllum resin exhibited high antimicrobial activity.

The verified data point extract and fraction of D. ecastophyllum were used as a potent modulator of resistance to bacteria, since they reduced the MIC of the antibiotic assayed. Research is to be developed further that can support in vivo testing and therapeutic use in future. According with the results, these natural products can be an alternative against the rise of bacterial drugs resistance, with the positive point that, by the use of lower concentrations of natural products and drugs, the risk of toxicity is lower.