Chemical identification and evaluation of the antimicrobial activity of fixed oil extracted from Rhinella jimi

Abstract

Context: The toad Rhinella jimi (Stevaux, 2002) (Bufonidae) is used in traditional medicine to treat a number of illnesses (inflammation, infections, and wounds) in humans as well as animals.

Objectives: The present work examined the antimicrobial actions of the extracted oils from the body fat of R. jimi (ORJ) against fungi and standard and multi-resistant lines of bacteria, as well as their effects when combined with aminoglycosides.

Materials and methods: The toads were collected in the municipality of Exu in Pernambuco State, Brazil, and their body fat oils extracted in a Soxhlet apparatus using hexane. A gas chromatograph coupled to a mass spectrometer was used to identify the fatty acids, based on their methyl esters. The antimicrobial activities of the oil were analyzed against standard and multi-resistant lines of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa, as well as against fungal lines of Candida albicans and Candida krusei using the broth micro-dilution method.

Results: The minimum inhibitory concentrations (MIC) of ORJ were 512 µg/mL for Candida krusei and ≥1024 µg/mL for the other microorganisms. When associated with amikacin, ORJ demonstrated an increase in its ability to inhibit E. coli growth (from 156.25 to 39.06 µg/mL), indicating synergistic interaction. In the same way, when allied with amikacin, gentamicin, and neomycin, the ORJ reduced the MICs meaningly, against P. aeruginosa.

Conclusions: These data will enable searches to be made to obtain new products in combination with antibiotics, enhancing the efficacy of these drugs against drug-resistant microorganisms.

• Conservation
• ethnobiology
• modulation of aminoglycosides
• zootherapy

Introduction

Many natural products are used in traditional medical systems, and while plants and plant parts represent the majority of products utilized, animals (whole animals, their parts, or derived products) are well-represented (Alves & Rosa, 2005; Scarpa, 1981). Alves and Alves (2011) listed 584 animal species distributed among 13 distinct taxonomic categories being used for therapeutic purposes in Latin America. A total of 354 animal species have been recorded as being used in medicinal practices in Brazil, which demonstrates the importance of the regional fauna to traditional populations (Alves et al., 2013a).

A large number of animal species are utilized for medicinal purposes throughout the world, including 47 amphibian species, of which six are cited for Brazil (Alves et al., 2013a,b). According to Alves and Albuquerque (2013), these animals gained the attention of researchers because of their pharmaceutical potential and have shown interesting physiological activities that include vasoconstriction, and antitumor cells, anti-inflammatory, cardiotoxic, neurotoxic, antimicrobial, and hallucinogenic activities.

Due to the use of both natural resources and allopathic medicinal substances by many traditional communities, there are now numerous examples of the utilization of synthetic medicines combined with natural products. While there is a growing concern about the increasing resistance of infectious agents to commonly used antimicrobial products, which limits their efficiency against many types of infections (De Backer et al., 2008; Deurenberger et al., 2007; Varaldo, 2002), a number of authors have reported the use of multi-drug treatments (Keith et al., 2005) composed of combinations between antibiotics, or their association with natural products, to amplifying their action spectra and minimize toxic effects (Salvat et al., 2001; Shin & Pyun, 2004; Sousa et al., 2010).

The amphibian Rhinella jimi (Stevaux, 2002) (Bufonidae), popularly known as “sapo cururu”, is distributed throughout the entire northeastern region of Brazil (Stevaux, 2002). This toad is utilized in traditional medicinal practices for treating illnesses in both humans and animals (inflammation, infections, and wounds) (Ferreira et al., 2009a). Recent studies have reported microbiological activities in extracts obtained from these animals, with demonstrations of antileishmanial and antitrypanossomal actions, in addition to showing antimicrobial effects against different bacterial lines, and cytotoxic properties (Brito et al., 2012a,b; Tempone et al., 2008).

The fats extracted from wild animals have been cited in a number of studies in traditional treatments of many illnesses involving inflammatory processes caused (or not) by infections (Costa-Neto & Alves, 2010; Ferreira et al., 2009a, 2011, 2012). Rhinella jimi fat has been specifically reported in the literature as a treatment for a number of infectious and inflammatory illnesses of possible microbial origin, such as infections, toothaches, sore throats, ear infections, and for treating wounds in animals (Costa-Neto & Alves, 2010; Ferreira et al., 2009a, 2011, 2012).

In light of the popular use of this toad species, and of scientific investigations of their biological properties, the present work examined extracts of the body fat of the Bufonidea Rhinella jimi to evaluate its antimicrobial action, both alone and in association with aminoglycoside antibiotics.

Materials and methods

Animal collections

Toad specimens were collected between April and June 2011 in the municipality of Exu (7°30′S × 39°43′W), Pernambuco State in Brazil (Figure 1). The 15 live specimens collected were sacrificed by freezing and the fat from the ventral region of their bodies was subsequently removed. After their identification, the animals were fixed in 10% formalin and subsequently conserved in 70% alcohol; testimonial specimens were deposited in the Herpetological Collection of the Regional University of Cariri (collection number 3132). The ethical approval for the study was obtained from the Ethics Committee of Universidade de Fortaleza – UNIFOR (No. of protocol: 006/2011-CEUA).

Figure 1. The municipality of Exu (7°30′S × 39°43′W), Pernambuco State in Brazil.

Extraction and analysis of the fixed oil composition

The fixed oil from the body fat of Rhinella jimi (ORJ) were extracted for 4 h in a Soxhlet apparatus using the solvent hexane. The extracted oil was heated in a water bath at 70 °C for 6 h to evaporate the solvent which was then stored in a freezer for subsequent analyses.

The ORJ was saponified by refluxing in a solution of potassium hydroxide and methanol for 2 h, following the method described by Hartman and Lago (1973). The residue was mixed with water and ethyl ether, the aqueous phase separated, and its pH subsequently adjusted by the addition of sulfuric acid. The free fatty acids were then methylated with methanol through acid catalysis to obtain their respective methyl esters (subsequently used to identify the original fatty acids). The analyses of the chemical composition of the ORJ were undertaken using a Shimadzu gas chromatography system coupled to a QP5050A selective mass spectrometer (using an ionizing energy of 70 eV). A DB-5HT capillary column was used (30 m × 0.25 mm internal diameter) with the following specifications: temperatures of 270 °C at the injector and 290 °C at the detector, using helium as a carrier gas (1.0 mL/min); a linear velocity of 47.3 cm/s; a total flux of 24 mL/min; a carrier flux of 24 mL/min; a pressure of 107.8 kPa; the column temperature was program for 60 °C (2 min) to 180 °C (1 min) at 4 °C/min, and 180–260 °C at 10 °C/min (10 min). Component identifications were made by comparisons of their respective mass spectra with standards in the database of the Wiley 229 library, and by comparing their calculated retention indices with indices published in the specialized literature (Adams, 2001; Alencar et al., 1984, 1990; Stenhagen et al., 1974).

Bacterial and fungal lineages

The experiments were undertaken using multi-resistant clinical isolates of Escherichia coli 27, Staphylococcus aureus 358, and Pseudomonas aeruginosa 24. The standard bacterial lineages were E. coli ATCC 10536, S. aureus ATCC 25923, P. aeruginosa ATCC 15442, and Klebisiella pneumonie ATCC 4362; the fungi used were Candida albicans ICB 12 and Candida krusei ATCC6258. All the isolates were provided by the Universidade Federal da Paraíba, and were maintained in Heart Infusion Agar (HIA, Difco) until tested, at which time they were transferred into Brain Heart Infusion (BHI, Difco, Detroit, MI) growth media for 24 h at 37 °C (Coutinho et al., 2008; Freitas et al., 1999).

Determination of the minimum inhibitory concentration (MIC)

The minimum inhibitory concentrations (MIC) of the animal oils were determined by microdilution using bacterial suspensions containing 10⁵ cells/mL. To prepare the test solutions, 10 mg of the original oil sample was mixed into 1 mL of dimethylsulfoxide (DMSO – Merck, Darmstadt, Germany) resulting in an initial concentration of 10 mg/mL. This initial solution was successively diluted with sterile water until reaching a concentration of 1024 µg/mL. After preparing wells with the inoculates, 100 µL of ORJ were added to initiate the serial dilutions, and the plates were then incubated for 24 h at 37 °C (Javadpour et al., 1996). The MIC was defined as the lowest concentration of ORJ that inhibited bacterial growth. Bacterial growth was observed using an indicator solution of resazurin, which turns from blue to pink in the presence of growing bacteria (due to the reduction of that dye) (Palomino et al., 2002). Fungal growth was visualized by noting turbidity of the culture solutions.

Drug susceptibility tests

The putative zootherapeutic extracted oils were also tested in association with antibiotics and antifungal compounds to determine their effects on fungi and multi-resistant bacteria. The examination of drug activity modulation was based on the results of the MIC, making it necessary to reduce the test concentrations of ORJ to sub-inhibitory levels (MIC/8). The antibiotics tested were gentamicin, neomycin, and amikacin, all at concentrations of 5000 µg/mL. The antifungal agents used were mebendazol, amphotericin B, nystatin and benzoilmetronidazole at concentrations of 1024 µg/mL. Aliquots (100 µL) of each antibiotic were added, in serial dilutions, to the plates containing the inoculums in 10% BHI with added ORJ, or just the inoculums in 10% BHI (control). The plates were maintained at 37 °C for 24 h, after which growth was measured using turbidity evaluation or by adding resazurine (for anti-fungal and antibacterial tests, respectively).

Checkerboard method

The best results obtained in the drug modulation experiments were retested using the checkerboard method (Eliopoulos & Moellering, 1991), using combinations of the antibiotics (an initial concentration of 5000 µg/mL) and ORJ (an initial concentration of 1024 µg/mL). The results were evaluated by adding 20 µL of resazurine and evaluating any color changes after 1 h. The fractionated inhibitory concentration (FIC) was calculated as the sum of FIC_A + FIC_O, where A represents the antibiotic and O represents the substance tested (ORJ). The parameters for the interpretation of the FIC are: synergistic when the FIC is less than 0.5; additive when between 0.5 and 1.0; indifferent when greater than 1.0; and antagonistic when greater than 4.0.

Results and discussion

The analyses of the chemical composition of the ORJ by GC/MS allowed the identification of 12 constituents responsible for 93.7% of the total fatty acid methyl esters present (Table 1). Of this percentage, 55.39% were unsaturated acids, notably oleic, palmitoleic, and linoleic acids; saturated acids represented 44.61% of the total percentage of acids identified, notably palmitic, stearic, and myristic acids.

Table 1. Methyl esters and fatty acids identified in the fixed oil extracted from Rhinella jimi specimens collected in Exu – PE (ORJ) using a gas chromatograph coupled to a mass spectrometer (CG/MS), in increasing order of retention/min (RT).

Constituents	RT (min)	(%)	Equivalent fatty acid
Saturated
Methyl dodecanoate	17.5	0.6	Lauric acid
Methyl tetradecanoate	21.9	2.4	Myristic acid
Methyl pentadecanoate	23.2	1.9	Pentadecanoic acid
Methyl eicosanoate	33.9	0.4	araquídico acid
Methyl octadecanoate	30.6	8.1	Steric acid^a
Methyl hexadecanoate	25.9	26.8	Palmitic acid^a
Methyl heptadecanoate	27.8	1.6	Heptadecanoic acid
Unsaturated
Methyl (Z,Z)-octadeca-9, 12-dienoate	29.5	6.2	Linoleic acid^a
Methyl (E)-octadec-10-enoate	29.8	38.0	Oleic acid^a
Methyl (Z)-octadec-13-enoate	29.9	1.4	Oleic acid^a
Methyl hexadec-11-enoate	25.3	1.4	Palmitoleic acid
Methyl (Z)-hexadec-9-enoate	25.4	4.9	Palmitoleic acid
Total		93.7

^aPrincipal fatty acids.

Earlier research, as well as the results of the present study, established that unsaturated fatty acids compose the bulk of animal fat (Cabral et al., 2013; Ferreira et al., 2009b, 2011; McCue, 2008), and our results also corroborated these previous research papers in terms of the notable presence of oleic, linoleic, palmitic, and stearic acids.

The application of resazurine to the bacterial culture wells, and the observation of turbidity in the fungal cultures, established the MIC of ORJ as being 512 µg/mL for Candida krusei and ≥1024 µg/mL for the other fungi. The MIC results demonstrated that although ORJ is indicated in traditional medicine for treating numerous infections (Costa-Neto & Alves, 2010; Ferreira et al., 2009a, 2012) it did not demonstrate any clinically relevant antimicrobial activity; similar results were found for the body oils of Tupinambis merianae (Duméril & Bibron, 1839) (Teiidae) (Ferreira et al., 2009b). However, when ORJ was combined with aminoglycosides, the positive results (Tables 2 and 3) were significantly different from the use of oils extracted from T. merianae (which did not demonstrate any increase in anti-microbial efficiency when combined with aminoglycosides).

Table 2. Test results of ORJ concentrations as effective modulators of micro-diluted antibiotics. Control = without ORJ. All results in µg/mL.

	S. aureus 358		P. aeruginosa 24		E. coli 27
Antibiotics	ORJ	Control	ORJ	Control	ORJ	Control
Amikacin	78.12	39.06	39.06	312.50^a	39.06	156.25^a
Neomycin	9.76	9.76	39.06	156.25^a	39.06	78.12
Gentamicin	2.44	4.88	9.76	39.06^a	9.76	19.53

^aMost expressive results.

Table 3. Test results of the use of ORJ concentrations as effective modulators of the actions of micro-diluted anti-fungal agents. Control = without ORJ.

	C. albicans ICB 12		C. krusei ATCC6258
Anti-fungal agent	ORJ	Control	ORJ	Control
Mebendazol	≥1024	≥1024	≥1024	≥1024
Amphotericin B	≥1024	≥1024	64^a	512^a
Nystatin	≥1024	≥1024	≥1024	≥1024
Benzoilmetronidazole	≥1024	≥1024	≥1024	≥1024

All results in µg/mL.

^aSignificant results.

ORJ did not demonstrate any modulation by antibiotics when acting against S. aureus (Table 2). When associated with amikacin, however, ORJ demonstrated a four-fold increase in its ability to inhibit E. coli growth, indicating a synergistic interaction. In the same way, when allied with amikacin, gentamicin, and neomycin, the MICs of ORJ were reduced eight-, four-, and four-fold, respectively, against P. aeruginosa.

There are very few effective treatments against opportunist infections caused by species of Candida, and many antifungal agents have undesirable side effects or can induce fungal resistance (principally in immune-depressed individuals; see Fica, 2004), which can result in superficial or even systemic infections (Shao et al., 2007).

There are numerous reports in the literature of the use of industrialized medicines in association with natural products (Calvet-Mir et al., 2008; Shin et al., 2008; Vandebroek, et al., 2008), as well as evidence that animal and plant products can modulate the effects of commercial medicines by increasing or reducing their efficiency (Coutinho et al., 2008; Santos et al., 2012a,b).

Table 3 presents the results of the association of ORJ in sub-inhibitory concentrations (MIC/8) with antifungal agents (mebendazol, amphotericin B, nystatin, and benzoilmetronidazole) against species of Candida. No modulation was seen against C. albicans, although ORJ reduced by eight the MIC of amphotericin B against C. kusei, indicating a synergistic effect between them.

According to Granowitz and Brown (2008), antagonistic effects are often observed with antibiotic combinations, probably due to their mutual chelation – quite different from the present results indicating synergism. The data reported here corroborate Agoramoorthy et al.(2007) and Nobre et al. (2002) who noted that fatty acids can often inhibit microbial activity and that unsaturated varieties are the most effective because they affect the endogenous bacterial synthesis of their own fatty acids (Zheng et al., 2005) (with ORJ being 55.39% unsaturated).

Evidence of drug modulation also was also assessed by FIC – in which it was possible to determine with precision the types of interactions occurring (indifferent, additive, synergistic, or antagonistic). As can be seen in Table 4, modulation was confirmed. ORJ in association with gentamicin demonstrated and additive effect (FIC index of 0.51) against P. aeruginosa, indifferent against the same bacterial line when tested with amikacin and neomycin, and synergistic when associated with amikacin (FIC index of 0.25) against E. coli.

Table 4. Results of the checkerboard experiments using ORJ with amikacin, gentamicin, and neomycin against the multi-resistant bacteria Escherichia coli 27 and Pseudomonas aeruginosa 24, and the types of interactions of ORJ with aminoglycosides.

	E. coli 27		P. aeruginosa 24
	MIC	MIC combined	MIC	MIC combined
ORJE	1024	4	1024	64
Amikacin	78.12	19.53	39.06	78,12
CIF and type of interaction	0.25 – synergistic		2.06 – indifferent
ORJE			1024	8
Gentamicin			39.06	19,53
CIF and type of interaction			0.51 – additive
ORJE			1024	16
Neomycin			39.06	39,06
CIF and type of interaction			1.01 – indifferent

MIC, minimum inhibitory concentration; ORJ_E, Rhinella jimi oil collected in Exu – PE; FIC, fractionated inhibitory concentration.

Clinical evaluations of the bio-activities of animal fats have yielded varying results. The fat of Tupinambis merianae, for example, did not demonstrate any clinically relevant antimicrobial activity (Ferreira et al., 2009b), while the body fat of Boa constrictor Linnaeus, 1758 (Boidae) demonstrated antimicrobial and anti-inflammatory effects (Falodun et al., 2008) as well as synergism in modulating antibiotic activities (Ferreira et al., 2011).

Conclusions

The results presented here indicated that the fixed oils extracted from the body fat of Rhinella jimi (ORJ) did not demonstrate clinically relevant antimicrobial activity against the microbial lineages used when tested alone. However, the use of ORJ in the traditional medicine can be explained by the symbolic nature of this animal by the traditional communities. According with the work of Ârhem (1989) and Heinrich (1994), there is an important relationship between the communities and the natural resources. This symbolism together with the emotional situation of the patient can be more easy to cure the process and the symbolic efficacy (Tesser & Luz, 2008).

However, when ORJ was tested in combination with antibiotics and antifungal agents, it modulated the action of amphotericin B against C. krusei, the action of amikacinin a synergistic manner against Escherichia coli 27, and gentamicin in an additive manner against Pseudomonas aeruginosa 24. These data will permit new searches for new products to be used in new formulations in association with antibiotics, enhancing the efficacy of these drugs against drug-resistant microorganisms.

It should be noted that the present study examined the effects of ORJ through in vitro testing – so that similar studies should be undertaken with live organisms to verify the systemic efficiency and toxicity of the oils extracted from R. jimi during continuous use, and elucidate additional related clinical questions.

Acknowledgements

The authors would like to thank the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – CAPES for the study grants awarded to Mario E.S. Cabral and Diógenes Q. Dias; the Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico – FUNCAP for the study grants awarded to Débora L. Sales, Olga P. Oliveira, and the Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq for the productivity grants awarded to Waltécio O. Almeida (No. 311713/2012-2); and IBAMA for the collecting permit (SISBIO – IBAMA: 154/2007 no. 27486-1, process identification code: 62956869).

Declaration of interest

The authors report no conflicts of interest.