Babassu (
A number of biological activities have been attributed to babassu mesocarp, such as anti-inflammatory [
Sepsis is a complex syndrome and still continues to be a major cause of morbidity and mortality among critically ill patients and at the intensive care units worldwide [
Antibiotic drugs generally interfere with the infection but not with the inflammatory response, a fact that might explain the high mortality rate observed in patients with septic shock [
The progressive resistance of pathogenic microorganisms to multiple drugs [
All assays were carried out using flour prepared from
The babassu mesocarp flour (500 g) was air-dried at room temperature, powdered, and extracted in 2000 mL ethanol PA (Merck, Brazil), for 72 h. The extract was filtered and concentrated under low pressure at 24°C. The extract obtained was stored (10°C) prior to antimicrobial studies. The final yield was 7.9% (w/w).
The analysis of polyphenols and flavonoids was performed accordingly as previously described [
The flavonoid concentration was measured with aluminum chloride at 425 nm using quercetin as standard. All samples were tested in triplicate. The phenolic acids concentration was determined from the difference between the flavonoids and total phenol concentration [
The dry EE was dissolved in phosphate-buffered saline, pH 7.2, to a final concentration of 500 mg/mL, sterilized by filtration (0.22
The bacterial inoculum was adjusted to final concentration of 1.5 × 108 CFU/mL (0.5, McFarland) and seeded onto a Mueller-Hinton agar plate. Sterile filter paper disks (6 mm) were placed on the plate and impregnated with 10
For the determination of MIC, a serial dilution of EE ranging from 500 to 0.9 mg/mL was added to tubes containing broth cultures of each strain, prepared in brain heart infusion [BHI (1.5 × 108 UFC/mL, 0.5 on McFarland scale)], and incubated at 35°C, for 24 h. Tubes containing only BHI plus bacteria were used as positive controls and those ones containing BHI plus EE were considered as negative controls. The MIC is defined as the lowest concentration of the EE at which the microorganism tested does not demonstrate visible growth [
Female Swiss mice weighing 25 ± 5 g were obtained from the Central Animal House of the Federal University of Maranhão.
Polymicrobial sepsis was induced by cecal ligation and puncture (CLP). Briefly, following anesthesia with sodium pentobarbital (50–65 mg/Kg, by intraperitoneal route i.p.), a small mid-abdominal incision was made and the cecum was exposed. A distended portion of the cecum just distal to the ileocecal valve was isolated and ligated with a silk suture in a manner not to disrupt bowel continuity. The ligated portion of the cecum was punctured eight times with an 18-gauge hypodermic needle. The abdomen was then closed in two layers and the animals were allowed to recover [
The animals were randomly assigned to the experimental groups. Sham: the cecum was not perforated and the mice were not treated. The other 3 groups were given subcutaneously NaCl solution (CLP), EE 125 mg/Kg (EE125), or EE 250 mg/Kg (EE250). Animals were cared for in accordance with the guidelines of the Brazilian College of Animal Experimentation and the experimental protocol was approved by the Ethics Committee (protocol 23115011476/2007-50).
To evaluate the lifespan, the number of remaining animals was recorded every 12 h until the 5th day.
Bacterial counts were performed on aseptically obtained peritoneal fluid. At 12 h after CLP, mice were sacrificed and the skin of abdomen was cut open in the midline without injury to the muscle. Sterile phosphate-buffered saline (PBS) (2 mL) was injected into and aspirated out of the peritoneal cavities. Samples were serially diluted in PBS and cultured on Mueller-Hinton agar dishes (Difco Laboratories, Detroit). Colony-forming units were counted after overnight incubation at 37°C. The results were expressed as log10 of the number of colony-forming units per peritoneal cavity.
Serum TNF-
All data were expressed as mean ± standard error (
The EE at the two tested concentrations inhibited the growth of
Antimicrobial activity of babassu mesocarp ethanolic extract evaluated by disk diffusion assay.
Bacterial strains | Zones of inhibition (mm) | |
---|---|---|
EE250 | EE500 | |
| 12.4 ± 0.2 | 14.4 ± 0.4 |
| 15.0 ± 0.3 | 18.5 ± 0.9 |
MRSA (hospital strain) | 15.3 ± 0.3 | 17.4 ± 0.3 |
| 0 | 0 |
| 0 | 0 |
The MIC was determined only for the effective EE doses and bacteria strains. The MIC was 31.2 mg/mL for
Minimum inhibitory concentration of babassu mesocarp ethanolic extract.
Bacterial strains | MIC |
---|---|
| 7.8 |
| 32.1 |
MRSA (hospital strain) | 32.1 |
The predominance of phenolic compounds was detected. The extract contained 56% total polyphenols, including 55% phenolic acids and 1% flavonoids.
As shown in Figure
Effect of treatment with ethanolic extract (EE) of babassu mesocarp on the survival of mice with lethal sepsis induced by cecal ligation and puncture (CLP). The animals were treated with EE at doses of 125 mg/Kg (EE125) or 250 mg/Kg (EE250) 6 h after the induction of sepsis by cecal ligation and puncture and compared to animals that have received saline (CLP). The animals were examined at intervals of 12 h until day 10. The results are expressed as mean ± SEM (5 animals/group). (
Sepsis by cecal perforation frequently induces an expressive increase in cell migration to the peritoneal cavity, as it was shown by the comparison between the group without sepsis (Sham) and group CLP, but the treatment with 250 mg/Kg of EE inhibited this cellular influx, in comparison to the other groups with sepsis (CLP and EE125) (Figure
Effect of treatment with ethanolic extract (EE) of babassu mesocarp on the number of lymphoid cells. The animals were treated with EE at doses of 125 (EE125) or 250 mg/Kg (EE250) 6 h after the induction of sepsis by cecal ligation and puncture, sacrificed 12 h after the procedure, and compared to the untreated animals without (Sham) or with sepsis (CLP). The number of cells in the peritoneum (a), lymph nodes (b), bone marrow (c), and spleen (d) was quantified. The results are expressed as mean ± SEM (5 animals/group). (
Treatment with EE inhibited the production of TNF-
Effect of treatment with ethanolic extract (EE) of babassu mesocarp on the production of cytokines. The animals were treated with EE at doses of 125 (EE125) or 250 mg/Kg (EE250) 6 h after the induction of sepsis by cecal ligation and puncture and compared to the untreated group also submitted to the inductions of sepsis (CLP) or not (Sham). The animals were sacrificed 12 h after the procedure and serum was obtained for the measurement of IL-6 (a), TNF-
The medicinal potential of babassu (
The EE showed an effective antimicrobial
The MIC and minimum bactericidal concentration (MBC) were only determined for strains against which antimicrobial activity was observed in the disk diffusion assay. A higher MIC was observed for
Antimicrobial compounds of plant origin with a restricted action against specific bacteria species and/or strains are desired not only due to their efficacy in the infection control, but also because they permit the maintenance of the normal microbiota. One important finding of this study was the sensitivity of the hospital strain of MRSA to the babassu mesocarp extract, since therapeutic options for patients infected with MRSA are limited. MRSA strains are always resistant to all cephalosporins, including fourth-generation drugs, as well as to carbapenems, irrespective of the result of the antibiogram [
An increasing resistance of
The presence of phenolic acids in the extract suggests a direct relationship with its antimicrobial activity since an antimicrobial action of these compounds has been attributed to their ability to form complexes with extracellular polysaccharides and proteins, rupturing the bacterial cell wall and inhibiting the enzymatic systems responsible for the synthesis of cell wall components [
In view of the restricted number of active products available for the control of MRSA and of the growing resistance of
Due to this antibacterial action, the effect of EE treatment on
The CLP model reproduces a type of infection mainly caused by
Treatment with EE was initiated 6 h after the induction of sepsis to evaluate the therapeutic effect of the extract. The results showed that treatment with the babassu extract did not affect bacterial counts but increased the survival of the animals and immunoregulated the proinflammatory cytokine production. Similar results were previously described by Maciel et al. [
Mice from EE250 group showed inhibition of peritoneal cell migration and a lower production of TNF-
Proinflammatory cytokines such as TNF-
Babassu mesocarp flour has a potent activating effect on macrophages as shown previously [
Taken together, the present results show that the efficacy of EE in increasing the lifespan in mice with sepsis by CLP is related to an immunomodulatory effect on the inflammatory process, mediated by cytokines. The immunomodulatory effect of babassu mesocarp on inflammation has been reported in other studies [
In conclusion the present study showed that the EE has a relevant and selective bacteriostatic action
The authors declare that they have no competing interests.
The authors thank CNPq for the financial support (Proc. no. 562248/2008) and for the researcher fellowship to Flávia R. F. Nascimento and Rosane N. M. Guerra and master fellowship to Wanderson S. Pereira. The authors thank CAPES for the master fellowship to Dayanna S. Prado, Priscila S. Barcellos, Tonicley A. Silva, and Lucilene A. Silva. The authors are also grateful to FAPEMA and UFMA.