Introduction
The use of natural compounds coming from plants for the treatment of diseases originates from previous millennia. It is understood that Neanderthal men that would occupy present day Iraq more than 60,000 years ago used plants for medical purposes (Stockwell, 1998). There has been documentation by early scientists and thinkers such as Hippocrates in ancient Greek medicine, Avicena in early Arab medicine, and Paracelsius in early Center-European culture. All of them were specialists in treating different diseases with medicinal plants (Gómez, 1990).
The Croton genus is frequently utilized by South America’s indigenous communities. It belongs to the Euphorbiaceae family. From these trees, the latex colloquially named "Dragon’s blood" or "Drago’s blood" is extracted to treat different diseases (Jones, 2003; Risco et al., 2005; INDECOCPI, 2007; Suárez et al., 2012). The first written documentation of its curative properties dates back to the XVII century when the explorer and naturalist Bernabe Cobo described the uses that the indigenous tribes from Mexico, Ecuador and Peru gave to this sap (Joyce, 1994). Among the best known applications as a healing agent are due to its anti-inflammatory properties. Furthermore, it is may be used as an antiseptic and homostatic, in addition to other uses in traditional South American medicine, such as the treatment of gastrointestinal ulcers, colic, diarrhea and cancer (Sandoval et al., 2002; Suarez et al., 2003; Tamariz et al., 2003; Suarez et al., 2006; Cevallos-Verdesoto et al., 2006); Gupta et al., 2008; Suarez et al., 2009A).
Croton is includes up to more than 1,300 species that include herbs, bushes, trees and unusually lianas widely distributed in tropical forests (Berry et al., 2005). The chemistry and pharmacological composition of Venezuelan species have been described by Suárez et al. in different studies (Suarez et al., 2003; Suarez et al., 2009B; Suarez et al., 2012; Suarez et al., 2013). Specifically, the characterization observed from the latex of Croton gossypiifolius demonstrates that the majority of secondary metabolites that are present in the cortex of this tree correspond to diterpenes like the kaurenoic acid, grandiflorenic acid, ent-15β-Ecinamoil-16- kauren-19-oic acid, 7-desoxogeayin and quercitrin (Suarez et al., 2013).
The presence of these type of secondary metabolites such as the tapsin, proanthocyanidin SP-303 and other fenolic compounds such as the chlorequinic acid, coberins A and B, 1,3,5-trimetoxibenzene, and 2,4,6-trimetoxifenol, contribute to the "dragon’s blood" medicinal and pharmacological properties described previously, as well as anti-microbial characteristics (Jones, 2003; Risco et al., 2005). In this regard, it has been shown that the bioactive compounds present in the Croton lechleri latex has anti- microbial effects against Helicobacter pylori, Staphylococcus aureus, Pseudomona aeruginosa and Streptococcus mutans (Tamariz et al., 2003; Huapaya et al., 2003; Risco et al., 2005; Cayo & Barrera, 2014; Avilés et al., 2018) and against the herpes virus, hepatitis, and influenza (Gilbrty et al., 1993; Sidwell et al., 1994). Nevertheless, it has shown little to no activity against bacteria such as Escherichia coli and Bacillus subtilis, as well as against the human cytomegalovirus (Risco et al., 2005).
Currently there is a growing need to implement the use of this type of herbal product for the treatment of different types of infections and to meet the health needs of the population, especially given the problems caused by bacterial resistance (Chinin & Cisneros, 2018). Nosocomial infections are a public health issue of great economic and social importance. The association of these infections with the increase in microbial resistance represent a challenge for health institutions and medical personnel due to high rates of morbidity and mortality, the increase in hospitalization days, as well as the costs of care (Cervantes-García et al., 2014). Therefore, it is important to explore natural alternatives for the control of this type of microorganisms.
In regards to the above information and of the arsenal of secondary metabolites present in the latex of these plants, an exploratory study was performed to determine the antimicrobial capacity of the Croton gossypiifolius latex against strains associated with common infections in humans such as Klebsiella pneumoniae, Pseudomona aeruginosa, Escherichia coli and Staphylococcus aureus, and the fungi Aspergillus niger. These microbial species were chosen, particularly because many are associated with nosocomial infections.
Materials and methods
Biologic material
The "dragon’s blood" latex was collected from C. gossypiifolius trees located in the forest zone of Cerro el Volcán (10°25’27.10" N, 66°51’35.81" O) at 1164 meters above sea level, which belongs to the Chacao municipality (Miranda- Venezuela). The process for collecting latex or bleeding from the tree was done according to what was traditionally established, with oblique incisions or V-cuts in the bark of the tree (Risco et al., 2005). The exudate was collected in sterile plastic containers, which were well sealed to prevent solidification of the latex until later use.
Microorganisms: we worked with three certified strains of bacteria Klebsiella pneumoniae ATCC 700603, Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923, donated by the National Hygiene Institute "Rafael Rangel". Clinical isolates of Pseudomona aeruginosa and Aspergillus niger were also used, donated by the Microbiology laboratory from the Microbiology Department of the Health Sciences Faculty, Carabobo University. The strains were stored under freezing temperatures until used in the experiments at the Biomedical Research Institute facilities "Dr. Francisco Javier Triana Alonso" of the Carabobo University in Maracay, Venezuela.
Antibacterial activity
Classic microbiology techniques were used to determine whether or not the extract affected the growth of the selected bacteria. The study was conducted in two stages; the first one was qualitative, in which two Petri dishes with nutritive agar were prepared per bacteria, one impregnated with the "Dragon´s blood" latex and the other one without it in order to compare the growth of bacteria, which were cultured with the swab technique. The second quantitative stage in which the bacteria susceptible to the latex were selected and cultured in a liquid medium at different concentrations thereof for a period of time to subsequently sow them on agar plates and quantify the number of viable cells; this was done in order to determine if the effect depended on the concentration used.
For the development of the qualitative stage a pre-inoculum of the bacteria was performed in fiolas with 20 mL of liquid Luria-Bertani medium incubated at 37ºC under continuous agitation to 150 rpm during 24 h inside an incubator Labnet model 211DS. After that time the turbidity was determined at 600 nm in a spectrophotometer Beckman DU 650, and the bacterial solution was adjusted to 0.5 absorbance units per mL. In parallel, four Petri dishes with nutritive agar were prepared for each bacteria, two impregnated with Dragon’s blood and two without it to serve as control. Sowing was performed using the diffusion technique with sterile swabs, which were impregnated with the different bacterial solutions. The dishes were incubated at 37 °C with an inverted position during 24 - 48 h.
In the quantitative stage, as in the previous case, a pre-inoculum of the bacteria was made and a volume equivalent to 0.5 absorbance units was taken which was incubated in flasks with liquid LB medium in the presence of the latex with different concentrations (0%; 0.6%; 1.25%; 1.9%; 2.5%; and 3.3 %) in a final volume of 20mL. It was incubated during a 6h period at 37 °C under continuous agitation at 150 rpm. After the incubation time, serial dilutions were made which were seeded in the Petri dishes with nutritive agar by the immersion technique and incubated for 24h at 37 ºC. Accounting plaques were considered to be those that ranged from 10-299 colonies. This procedure was performed in duplicate for each concentration tested.
Antifungal activity
To determine if the latex interferes in the growth of the Aspergillus niger strain, a procedure similar to the one developed by Falco et al. (2011), with Aspergillus oryzae was done. Unlike the antibacterial procedure, saboraud medium was used in the Petri dish. A 0.5 cm portion of the mycelium of the fungus was seeded in the center of the Petri dish with medium plus latex. The plates were incubated at 30 °C for 7 to 10 days and growth was observed and compared with a sample of mycelium grown under the same conditions, without the presence of the latex.
Results and discussion
Antibacterial activity
When comparing the growth of the bacteria in the dishes with solid LB medium impregnated with the C. gossypiifolius latex with its respective control (medium without latex), it was observed that it did not cause growth inhibition of E. coli, K. pneumoniae y P. aeruginosa (Figure 1). Nor did it cause inhibition on the growth of A. niger (Figure 3). In the case of S. aureus a significant reduction in growth was observed in the presence of latex (Figure 2).
This result coincides with Chininin and Cisneros (2018), findings, whom used 100% Croton lechleri latex with the disk diffusion test and achieved a 54.75% inhibition of the in vitro growth of S. aureus ATCC 25923 strain.
Based on these results, S. aureus was grown in LB medium with different concentrations of the latex for a period of 6 hours and then cultured in agar in order to quantify the number of viable bacteria (cfu / mL). An evident dependence on the concentration used for the inhibitory effect was observed. The highest bacterial counts were reached when the lowest (0.6%) latex concentration was used; while at 3.3% the growth was minimal (Figure 4). With these data, a linear regression analysis was carried out to determine the magnitude of this effect of the concentration on the number of viable bacteria. A negative slope of -430 was obtained (r2 = 0.94), which implies that there was a reduction of 430 CFU / mL per unit of added concentration of the latex in the culture medium. As seen in the figure there is a strong tendency to inhibition, showing a concentration-dependent behavior.
All these results are very important due to the relevance of this bacterium in daily medical practice. Since its discovery in 1880 by the doctor Alexander Ogston, S. aureus is considered a pathogen with great potential to cause multiple infections in humans and animals, which affect the skin and soft tissues and can sometimes be fatal (Cervantes- García et al., 2014; Chinin & Cisneros, 2018). It is one of the most prevalently isolated bacteria in communities as well as nosocomial infections at the skin level, central nervous system, respiratory system, urinary tract; capable of producing localized abscesses, osteomyelitis, endocarditis, food poisoning and septicemia (Dorante et al., 2013). One of the main issues associated with treating these infections is antibiotic resistance. The first oxacillin resistant S. aureus strains appeared in 1960, shortly after the introduction of this antimicrobial in clinical practice (Gómez-Gamboa et al., 2016) and until now, resistance mechanisms continue to evolve at the same rate as new drugs are modified or created.
Consequently, the findings of this work on the effectiveness of C. gossypiifolius latex to inhibit the growth of this bacterium profile this natural product as a therapeutic alternative to meet the health needs of the population. It is necessary to deepen the investigation performing standard sensitivity tests on these bacteria, including strains from clinical isolates.
Conclusion
The latex of Croton gossypiifolius did not cause any inhibition in the growth of the bacteria E. coli, K. pneumoniae y P. aeruginosa, nor the fungi A. niger but it did inhibit the growth of S. aureus and the effect was dose dependent. Consequently, it is important on deepen the study focused on S. Aureus infectious agent.