Antimicrobial activity and chemical composition of fixed oil extracted from the body fat of the snake Spilotes pullatus

Abstract

Context: Ethnozoological studies have shown that Spilotes pullatus Linn. (Colubridae: Ophidia), is associated with medicinal and magic-religious uses in Brazil.

Objectives: This study was designed to determine the chemical composition of the oil extracted from the body fat of S. pullatus and to test its antimicrobial properties, alone and in association with aminoglycosides, against fungi and bacterial strains in concentrations ranging between 1024 and 0.5 µg/mL.

Material and methods: The snakes were collected in the Chapada do Araripe, county of Crato, Ceará State, Brazil. The oil was extracted in a Soxhlet apparatus using hexane. The methyl esters of the fatty acids present in the samples were identified using GC-MS. The antimicrobial and drug modulatory activities of oil were tested by microdilution against fungal and bacterial strains.

Results: The chemical composition of the fixed oils of S. pullatus identified 10 constituents representing 94.97% of the total sample. The percentages of saturated and unsaturated fatty acids were 33.59 and 61.38%, respectively, with the most abundant components being elaidic (37.26%). The oil did not demonstrate any antimicrobial or antifungal activity when tested alone, presenting MIC values ≥ 1024 µg/mL. However, when associated with antibiotics, it demonstrated synergistic effects with gentamicin against all the bacterial lineages assayed, and antagonistic effects with amikacin and neomycin against strains of Escherichia coli.

Conclusions: Oil extracted from the body fat of S. pullatus did not demonstrate any inhibitory effects on bacterial or fungal activities, but was effective in modulating the effects of certain antibiotics.

• Aminoglycosides
• antibiotic
• ethnozoology
• modulatory antibiotic activity

Introduction

Spilotes pullatus Linn. (Colubridae), popularly known as “cobra caninana” in Brazil, is a diurnal and semi-arboreal snake that preys on small rodents and birds and is widely distributed throughout of South America to Mexico (Vanzolini et al., 1980).

Globally, 123 snake species are used in traditional folk medicine for therapeutic purposes (Alves et al., 2013) to treat asthma, rheumatism, wounds, and thrombosis. Some studies of ethnomedicinal practices have reported the use of S. pullatus to alleviate pain caused by the bites of insects and other snakes, although there is no proof yet of its efficiency in these situations (Alves & Pereira Filho, 2007; Alves & Rosa, 2007; Alves et al., 2007a, 2009). In addition to its medicinal uses, Alves et al. (2010, 2012) reported the use of S. pullatus in magic-religious circumstances, as well as the persecution of these snakes in situations of direct contact with humans.

The use of animal resources constitutes an important therapeutic alternative for many populations (Alves et al., 2007b), and they have been cited being used against illnesses apparently caused by pathogenic microorganisms (Aguiar et al., 2008; Lima et al., 2006; Salvagnini et al., 2008). Associated with this fact, microbiological studies indicate the natural products as a weapon against the microbial resistance to drugs (Sousa et al., 2010). Due this fact, zootherapeutic products can be effective in treating human illnesses directly, or in association with synthetic antibiotics – to amplify their action spectrum and minimize undesirable side effects (Gibbons, 2004; Gurib-Fakim, 2006; Salvat et al., 2001; Shin & Pyun, 2004; Sousa et al., 2010). With this objective, several studies using the body fat of animals, as demonstrated in the work of Dias et al. (2013), Cabral et al. (2013), Ferreira et al. (2011, 2009), and Falodun et al. (2008).

Due to the huge use of the animal resources and the traditional medicine and due to the aspects involved in the conservation of species against the massive exploitation, the bioprospection of natural products starting from the traditional knowledge represent an important activity to isolate and validate the pharmacological properties of the animal resources (Alves & Rosa, 2005; Hunt & Vincent, 2006), for a possible therapeutic usage (Pieroni et al., 2002) and demonstrate the efficacy or risks in the usage of these products (Alves, 2009).

The present work undertook chemical analyses of the oil extracted from the body fat of S. pullatus, tested its antimicrobial activity against standard lines of bacteria and fungi, and examined its effects against multiresistant bacterial strains when combined with antibiotics.

Materials and methods

Zoological material

Four specimens of the snake S. pullatus were collected on the slopes of the Chapada do Araripe Range (7 °07′ to  °49′ S and 38 °30′ to 40 °55′ W), in the municipality of Crato, in the Cariri region of southern Ceará State, Brazil, between the months of August and October, 2011. The region has a semiarid climate, with average temperatures varying from 24 to 26 °C (IPECE, 2012). S. pullatus is distributed on all studied region, occurring in the biomes called “caatinga”, “cerrado”, and wet-forest (Ribeiro et al., 2012).

The capture of specimens of S. pullatus was authorized by the Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio) by the System of Authorization and Information about Biodiversity (SISBIO), with number 29838-1 (19 August 2011). The project was submitted and approved by the Committee of Ethics in Animal Research of Universidade Regional do Cariri (URCA), with number 16/2012.

Collections were made by actively searching microenvironments favorable to the occurrence of these reptiles (active capture in trees and bushes). The collected specimens were sacrificed using lidocaine and the fat located in the ventral region of their bodies was removed. The animal specimens were then fixed using formol 10% and conserved in 70% alcohol, and subsequently deposited in the Herpetological Collection of the Zoology Laboratory of the Regional University at Cariri (record number URCA-2096).

Extraction of the fixed oils of S. pullatus

Oil extraction was performed for 4 h using hexane as a solvent in a Soxhlet apparatus, using 119.56 g of body fat. The hexane was removed in a rotary evaporator and the material subsequently held in a water bath (60 °C) for 2 d. The final recovery was 27.39%, and this material was maintained in a freezer until used.

Fatty acid determinations

The fatty acid content of the fixed oil of S. pullatus (FOSP) was determined indirectly using their corresponding methyl esters. The oil was saponified for 30 min by refluxing with a solution of potassium hydroxide in methanol, following the methodology described by Hartman and Lago (1973). After this treatment, the pH was adjusted, and the free fatty acids were methylated with methanol through acid catalysis to obtain the respective methyl esters.

Analyses of the fixed oils of S. pullatus using gas chromatography coupled to a mass spectrometer (GC/MS)

Analyses of the volatile constituents of the FOSP were undertaken using a gas chromatograph coupled to a mass spectrometer (GC/MS) (Hewlett-Packard, model 5971, Los Angeles, CA) using a non-polar DB-1 capillary column of fused silica (30 m × 0.25 mm internal diameter); the carrier gas was helium with a flux velocity of 0.8 mL/min. The injector and detector temperatures were 250 °C and 200  °C, respectively. The column temperature was programmed from 35 °C to 180 °C at 4 °C/min, and followed by 180–250 °C at 10 °C/min. The mass spectra were recorded from 30 to 450 m/z. The individual components were identified by comparing their corresponding mass spectra (70 eV) to an accumulated database for the spectrometer (Wiley, 229, San Diego, CA) and with two other databases using retention indices as the pre-selection criteria (Alencar et al., 1984, 1990), as well as by visual comparisons of the fragmentation patterns with those reported in the literature (Adams, 2001; Stenhagen, 1974).

Bacterial material

Minimum inhibitory concentration trials used standard lines of Escherichia coli ATCC 10536, Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 15442, and Klebsiella pneumoniae ATCC 4362. Modulation trials used multiresistant clinical isolates of E. coli (EC27), S. aureus (SA358), and P. aeruginosa (P03), whose origins and profile resistances are indicated in Table 1. All the strains were maintained in Heart Infusion Agar (HIA, Difco, St. Louis, MO); before the trials, they were transferred to Brain Heart Infusion (BHI, Difco) for 24 h at 37 °C (Coutinho et al., 2005; Freitas et al., 1999).

Table 1. Origins of the bacterial strains and their antibiotic resistance profiles.

Bacteria	Origin	Resistance profiles
Escherichia coli 27	Surgical wound	Ast, Ami, Amox, Ca, Cfc, Cf, Caz, Cip, Clo, Imi, Can, Szt, Tet, Tob
Staphylococcus aureus 358	Surgical wound	Oxa, Gen, Tob, Ami, Can, Neo, Para, But, Sis, Net
Pseudomonas aeruginosa 03	Uroculture	Cpm, Ctz, Imi, Cip, Ptz, Lev, Mer, Ami

Ast, aztreonam; Ami, amicillin; Amox, amoxicillin, Ca, cefadroxil; Cfc, cefaclor; Cf, cefalotin; Caz, ceftazidime; Cip, ciprofloxacin; Clo, chloramphenicol; Imi, imipenem; Can, kanamycin; Szt, sulfatrim; Tet, tetracycline; Tob, tobramycin; Oxa, oxacillin; Gen, gentamicin; Neo, neomycin; Para, paromomycin; But, butirosin; Sis, sisomicin; Net, netilmicin; Cpm, cfepime; Ctz, ceftazidime; Ptz, piperacillin-tazobactam; Lev, levofloxacin; Mer, meropene.

Fungal material

Antifungal activity was evaluated using the fungal strains Candida tropicalis (6526), Candida albicans (40046), and Candida krusei (6258), which were maintained in HIA and kept at 4 °C. Before the trials, the cells were cultivated for growth in BHI for 24 h at 37 °C.

Drugs

The antifungal agents, nystatin, amphotericin B, mebendazole, and benzoilmetronidazol, were used at concentrations of 1024 µg/mL. The antibiotics amikacin, gentamicin, and neomycin were used at concentrations of 5000 µg/mL. All the antimicrobial agents were prepared according to the manufacturer's instructions.

Testing for antibacterial and antifungal activities

Testing was undertaken using a solution with 100 mg/mL of the extracted oil solubilized in 1 mL of dimethyl sulfoxide (DMSO – Merck, Darmstadt, Germany), which was then subsequently diluted with distilled water to 1024 µg/mL.

BHI suspensions (100 µL) of each of the microbial lineages were placed in each well of a microdilution plate (96-well plates), followed by 100 µL of the oil solution in serial dilutions – with the final oil concentrations varying from 8 to 512 µg/mL. The negative control wells received only DMSO. The microdilution plates were then incubated for 24 h at 37 °C (Javadpour et al., 1996). To determine the MIC of the bacterial strains, resazurin indicator solution (20 µg) was added to each well. Color changes from blue to pink indicated bacterial growth. Fungal growth was determined by observing the formation of turbidity in the wells (Palomino et al., 2002).

Drug susceptibility testing

Suspensions (100 µL) of the microbial lineages were placed in each well of a microdilution plate, followed by 100 µL of serially diluted drugs corresponding to each type of microorganism. The concentrations of the antifungal agents varied from 0.5 to 1024 µg/mL, and antibiotic concentrations varied from 1.22 to 2500 µg/mL. The solutions of fixed oil were subsequently added at the MIC concentrations determined for each microorganism. The plates were then incubated for 24 h at 37 °C (Javadpour et al., 1996). The results were evaluated using the same techniques applied for the MIC.

Results

The analyses of the chemical composition of the fixed oil of S. pullatus (FOSP) by GC/MS allowed the identification of the methyl esters of 10 fatty acids that constituted 94.97% of the sample (Table 2). Unsaturated fatty acids represented 61.38% of the 10 principal methyl esters identified, with elaidic acid (37.26%) being the most prevalent, followed by linoleic acid (17.28%). Saturated fatty acids represented 33.59% of the principal methyl esters identified, with palmitic (19.01%) and stearic (10.58%) being the most prevalent.

Table 2. Methyl esters of the fatty acids identified in the fixed oil extracted from the body fat of Spilotes pullatus by gas chromatography coupled to a mass spectrometer.

Fatty acid equivalent	RT (min)	(%)	Constituents
Saturated
Myristic acid	19.48	1.28	Methyl myristate
Palmitic acid	22.97	19.01^a	Methyl palmitate
Stearic acid	25.37	10.58^a	Methyl stearate
Nonadecanoic acid	27.07	2.72	Methyl nonadecanoate
Unsaturated
Palmitoleic acid	22.64	4.02	Methyl palmitoleate
Linoleic acid	25.09	17.28^a	Methyl linoleate
Elaidic acid	25.12	37.26^a	Methyl elaidate
Arachidonic acid	26.68	1.48	Methyl arachidonate
Eicosanoic acid	26.85	0.88	Methyl eicosa-7,10,13- trienoate
Docosahexaenoic acid	28.40	0.46	Methyl docosahexaenoate
Total saturated		33.59
Total unsaturated		61.38
General total		94.97

^aPrincipal fatty acids.

FOSP did not inhibit fungal or bacterial growth in any of the strains tested, indicating that this product alone was not efficient in controlling microorganisms. Likewise, FOSP in association with antimicrobial agents did not show any inhibitory effect against multiresistant bacterial strains, or inhibitory effects against fungi in association with antifungal agents (using a MIC ≥ 1024 µg/mL for all of the microbial strains tested) (Table 3).

Table 3. Results of the trials using FOSP as a modulator of the actions of micro-diluted antifungal agents and controls (without FOSP); all results in µg/mL.

	C.T. 13803		C.A. 40046		C.K. 6258
Antifungal agents	FOSP	Control	FOSP	Control	FOSP	Control
Benzoilmetronidazol	≥1024	≥1024	≥1024	≥1024	≥1024	≥1024
Anfotericina B	≥1024	≥1024	≥1024	≥1024	≥1024	≥1024
Mebendazol	≥1024	≥1024	≥1024	≥1024	≥1024	≥1024
Nistatina	≥1024	≥1024	≥1024	≥1024	≥1024	≥1024

CT, Candida tropicalis; CK, Candida krusei; CA, Candida albicans.

FOSP did demonstrate synergistic effects in association with gentamicin against all the microbial lines tested. No clinically significant interaction with S. aureus (SA 358) or P. aeruginosa (PA 03) was seen when FOSP was combined with the antibiotics amikacin and neomycin. Antagonistic effects were observed, however, with these aminoglycosides against E. coli (EC 27) (Table 4).

Table 4. CIM values (µg/mL) of aminoglycosides in the absence and presence of the fixed oil from S. pullatus, against Staphylococcus aureus 358 (SA 358), Escherichia coli 27 (EC 27), and Pseudomonas aeruginosa 03 (PA 03).

Antibiotics	SA 358		EC 27		PA 03
	Control	FOSP	Control	FOSP	Control	FOSP
Amikacin	39.06	39.06	9.76	78.125 ^a	156.25	78.125
Gentamicin	39.06	2.44 ^a	39.06	9.76 ^a	39.06	9.76 ^a
Neomycin	9.76	19.53	39.06	156.25 ^a	625	312.5

^aSignificant results.

Discussion

Fatty acids can inhibit microbial activity (Agoramoorthy et al., 2007; Nobre et al., 2002). According to Zheng et al. (2005), unsaturated fatty acids are especially effective in this type of microbial control as they impact endogenous bacterial fatty acid synthesis. From this perspective, higher unsaturated fat contents may indicate greater therapeutic efficiencies.

The presence of myristic, palmitic, stearic, palmitoleic, and linoleic acids in the body fat of the reptiles Crotalus atrox, Elaphe obsoleta, Python regius, Boa constrictor, Bitis gabonica, and Varanus exanthematicus has been reported in other studies (McCue, 2008). According to Agoramoorthy et al. (2007), palmitic, linoleic, stearic, and myristic acids are known for their antimicrobial activities, and even though FOSP did not show any antimicrobial activity when used alone, the positive results seen when used in combination with aminoglycosides may be related to the presence of these fatty acids.

Although FOSP did not demonstrate any inhibitory effects on bacterial or fungal activities, the results of the interactions of this oil with antimicrobial compounds indicated that it negatively influenced treatments to control E. coli using the antibiotics amikacin and neomycin. In contrast, FOSP did appear to be clinically effective when associated with gentamicin, positively boosting its activity.

The inefficiency of FOSP in controlling microbial activity when used alone corroborates the results of Ferreira et al. (2009), who undertook a microbiological study of the oil extracted from the body fat of Tupinambis merianae – with no modulations of the activities of antibiotics being observed in the presence of that oil. Research with Phrynops geoffroanus perfomed by Dias et al. (2013) indicates that the oil did not demonstrate clinical efficacy against the bacterial strains alone; however, a MIC = 128 µg/mL was observed against C. krusei. When the oil was associated with antibiotics, the oil from P. geoffroanus presented a similar behavior with the oil of S. pullatus, demonstrating a synergism against P. aeruginosa when associated with gentamin. Falodun et al. (2008) tested the fixed oil of B. constrictor in similar manners but did not report any antimicrobial activity. Ferreira et al. (2011) complemented this latter study and did report the modulation of antibiotic activity by the oils extracted from B. constrictor, but only in terms of the action of gentamicin against E. coli. This study demonstrated a higher proportion of unsaturated fatty acids, as observed in the work of Ferreira et al. (2011).

While the oil extracted from the body fat of S. pullatus was effective in modulating the effects of certain antibiotics, natural products with few studies or with unsafely manipulating practices, can be important agents of diseases, causing serious health problems (Alves & Rosa, 2005). So, more studies are necessary to evaluate this and other natural products from animals before their usage as possible new drugs or pharmaceutical formulations.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. The authors would like to thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the research productivity grant awarded to Dr. Waltécio de Oliveira Almeida; 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 Olga P. Oliveira and Débora L. Sales.

Acknowledgements

The authors would like to thank the ICMBio for the collecting permit; ao com; the Universidade Federal da Paraíba (UFPB) for donating the microbial strains; and Prof. Dr. Robson Waldemar Ávila for identification and catalogation of this zoological material.