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Journal of Arid Environments 76 (2012) 138e141

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Journal of Arid Environments

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Short communication

Antimicrobial activity of natural products from the skins of the semiarid livinglizards Ameiva ameiva (Linnaeus, 1758) and Tropidurus hispidus (Spix, 1825)

I.J.M. Santosa, H.D. Melo Coutinhob,*, E.F. Ferreira Matiasb, J.G. Martins da Costac, R.R. Nóbrega Alvesd,W. de Oliveira Almeidaa

a Laboratorio de Zoologia, Brazilb Laboratorio de Microbiologia e Biologia Molecular, Brazilc Laboratorio de Pesquisa de Produtos Naturais, Universidade Regional do Cariri, URCA, BrazildDepartamento de Biologia, Universidade Estadual da Paraiba, UEPB, Brazil

a r t i c l e i n f o

Article history:Received 8 April 2011Accepted 19 August 2011Available online 16 September 2011

Keywords:Antimicrobial activityZootherapyAmeiva ameiva and Tropidurus hispidus

* Corresponding author. Tel.: þ55 8386601556.E-mail address: hdmcoutinho@gmail.com (H.D. M

0140-1963/$ e see front matter � 2011 Elsevier Ltd.doi:10.1016/j.jaridenv.2011.08.005

a b s t r a c t

The reptiles are the animal species most often used in traditional medicine. Tropidurus hispidus andameiva ameiva are two species of lizards utilized as medicines in zootherapic practice in Braziliansemiarid region. In this work, we evaluated the antimicrobial activity of extracts of the skin of Ameivaameiva (MEAA) and T. hispidus (METH). The samples were tested against standard and multiresistantstrains of Escherichia coli, Staphylococus aureus and Pseudomonas aureuginosa, alone and in combinationwith aminoglycoside antibiotics. Alone, none of the samples showed significant inhibition of bacterialgrowth at clinically relevant concentrations. However, combinated with the antibiotics, MEAA potenti-ated the effect of amikacin and gentamicin, reducing their minimal inhibitory concentration (MIC) andthe growth of E. coli and S. aureus. METH lowered the MIC of gentamicin against S. aureus. Chemicalprospecting of the extracts revealed the presence of alkaloids in both, which can account for themodulatory action of the extracts, indicating a promising source of new drugs with antibiotic properties.

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Humans have utilized animals for producing drugs and to treatdiseases and injuries since ancient times. Wild animals and theirproducts constitute ingredients essential in the preparation oftraditional medicines. For many years, ethnobiological studies havebeen directed more to medicinal plants, but recently, variousinvestigators demonstrated that in culture/nature interaction thereis notable utilization of fauna for medicinal purposes in differenthuman societies (Alves and Rosa, 2010). In Brazil, there are reportsdocumenting at least 290 species are used in popular medicine.Particularly for the Northern and Northeastern regions, variousworks report that animals are widely utilized and sold for thera-peutic purposes in both rural and urban areas (Alves et al., 2008).

Reptiles are among the animal species most often utilized intraditional medicine. Alves et al. (2008) reported that a total of 165species of reptiles belonging to104genera and30 families are used inpopularmedicine indifferent countries of theworld. Thegroupswiththe largest number of species utilized are snakes (60 species), fol-lowed by lizards (51), turtles and tortoises (43) and crocodilians (11).

elo Coutinho).

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According to Ferreira et al. (2009, 2010), studies with extracts ofreptiles have been conducted with the aim of demonstrating theirantimicrobial and pharmacological activities. T. hispidurs and A.Ameiva are among the species utilized in traditional medicine bycommunities living in the Northeastern region, a semiaridecosystem, which have not yet been studied from a pharmacolog-ical point of view.

Tropidurus hispidus, also known as the lava lizard or “catenga”,lives in 14 Brazilian states, of which 9 are in the Northeast region,and its occurrence has been recorded for the semiarid (Abreu et al.,2002). It is found in diverse habitats, mainly on tree trunks, rocksand walls and in open areas (heliophiles) (Abreu et al., 2002). Thislizard is diurnal and is territorial, and it is a sedentary and oppor-tunistic forager that feeds mostly on arthropods, some plants,basically flowers, small vertebrates and needs constant sun expo-sure and is therefore found generally in open areas (Abreu et al.,2002). According to Alves et al. (2008), T. Hispidus (Spix, 1825), itis used in the treatment of boils in rural areas in the semiaridregion. Besides, this species is used in the treatment of sore throat,tonsillitis and pharyngitis. Costa Neto (2000) pointed out thepopular medicine use of Tropidurus torquatus in the treatment ofchickenpox.

Table 2Fresh weight, dry weight and yield of methanolic extracts of Tropidurus hispidus andAmeiva ameiva (g).

Species Fresh weight Dry weight Solvent Crude extract Yield

Tropidurus hipidusSkin 89.14 44.5275 Methanol (METH) 1.9672Ameiva ameivaSkin 26.00 10.3998 Methanol (MEAA) 1.303

METH- Methanol extract of Tropidurus hispidus; MEAA- Methanol extract of Ameivaameiva.

I.J.M. Santos et al. / Journal of Arid Environments 76 (2012) 138e141 139

Ameiva ameiva (Linnaeus, 1758) is a species of lizard commonlyknown as “calango verde” or “calango” and has a one of thebroadest geographical distributions among the neotropical lizards,occurring in Brazil, it is found in most of the country, including inthe semiarid (Vanzolini et al., 1980). According to Huey and Pianka(1981) it is considered a species with a feeding strategy of the activeforaging type, providing it was a variety of prey. According to Alveset al. (2010), these lizards are used in traditional medicine forinflammation, dermatitis, venereal diseases, and snake bites.

Some diseases treated by communities using the traditionalknowledge report the utilization of these lizards against pathogenicbacteria and fungi, suggesting that these species are used aspotential antimicrobial agents, which shows the need for studiesaimed at determining the medicinal efficacy of these naturalproducts based on these species of lizards.

The use of zootherapeutic products is also of importance fromthe point of view of public health, because diseases can be trans-mitted through their medicinal use (Alves and Rosa, 2007; Ferreiraet al., 2009). As Alves et al. (2008) have noted, the social use of thebiodiversity in countries such as Brazil is crucial for biologicalconservation, public health, sustainable use of natural resourcesand biological prospecting. According to Still (2003), there are fewstudies assessing the true role of animal remedies, but areextremely necessary. Knowledge about the true role of these zoo-therapeutic agents from a chemical point of view is scarce. There-fore, the aim of this work was to determine the antimicrobialactivity of the methanolic extract of the skin of T. hispidus andAmeiva ameiva, alone or in combination with antibiotics.

The animals were collected (Permission for collection: 154/2007no. 23544-1 process no. 17842812) in the municipality of Crato(7�1400300S � 39�240340’W), Ceara, Brazil, in April 2010. They werecaught manually, besides by active collections by rummagingthrough habitats where these animals can be found. Once thelizards were collected and sacrificed, their skins were removed anddried in a drying oven to prepare extracts. Control specimens werefixed in 70% alcohol and deposited in the zoology collection of theUniversidade Regional do Cariri/LAZ-URCA (Table 1).

The extracts of A. ameiva and T. hispidus were prepared bysubmersing the skins (which were perforated to increase surfacearea) for 72 h in methanol (cold extraction). Afterward, they werefiltered and concentrated using a rotary evaporator (Soxhlet)(model Q-344B e Quimis, Brazil), and completely dried in waterbath at 70 �C for 2 h (model Q-214M2 e Quimis, Brazil). The yieldsfor the crude extracts are given in Table 2. The extracts were thenstored in a freezer until later analyses. The experiments werecarried out with clinical isolates of Escherichia coli (EC27), Staphy-lococcus aureus 358 (SA358) and Pseudomonas aureuginosa (PARB1),as wells as the standard strains E. coli ATCC 10536, Staphylococcusaureus ATCC 25923 and P. aureuginosa ATCC 15692 (Coutinho et al.,2008). All strains were maintained on heart infusion agar (HIA,Difco) slants, and before the assays, the cells were grown overnightat 37 �C in brain heart infusion broth (BHI, Difco). The drugs usednamely gentamicin, kanamycin, amikacin and neomycin were ob-tained from Sigma Chemical Corp, St. Louis, MO, USA. All drugswere dissolved in sterile water just before use.

The test solution of extracts of the two species was prepared bydissolving 10 mg of the samples in 1 mL of dimethylsulfoxide(DMSO- Merck, Darmstadt, Germany), obtaining an initial

Table 1Species utilized in the antimicrobial analyses.

Species University/Archive no.

Tropidurus hispidus Universidade Regional do Carri-URCA LZ-847Ameiva ameiva Universidade Regional do Carri-URCA LZ-746

concentration of 10 mg/mL; this solution was then diluted to1024 mg/mL. The minimal inhibitory concentrations (MIC) of theextracts were determined bymicrodilution assays in BHI brothwithbacterial suspensions of 105 CFU/mL and drug concentrationsvarying from 1 mg/mL to 1024 mg/mL (in 2-fold serial dilutions)(Javadpour et al., 1996). MIC was defined as the lowest concentra-tion of drug at which no bacterial growth was observed. For theevaluation of extracts for antibiotic-modifying activity, MICs of theantibiotics were determined in the presence of each extract at sub-inhibitory concentrations, and the plates were incubated for 24 h at37 �C.

The chemical tests to determine the presence of heterosides,saponins, tannins, flavonoids, steroids, triterpenes, cumarins,quinones, organic acids and alkaloids were performed according tothe method described by Matos (1997). The tests are based on thevisual observation of a color change or formation of a precipitateafter the addition of specific reagents.

Methanolic extracts of the skin of A. ameiva and T. hispidus didnot show antibacterial activity at clinically relevant concentrations,with MIC�1024 for all bacterial strains tested, thus suggesting thatthe medicinal use of the lizards is ineffective against bacterialinfections.

METH and MEAA were then tested for antibacterial activity insynergy with antibiotics of common use. When combined with theaminoglycosides kanamycin and neomycin, the extracts did notdemonstrate any modulatory effect on antibiotic activity againstthe three strains tested (EC27, SA358 and PARB1). However, whencombined with the aminoglycoside amikacin, MEAA showedsynergism, reducing the MIC of amikacin from 156.25 to 19.53against the multiresistant strain EC27. In combination with genta-micin, MEAA also demonstrated synergism, reducing theMIC of theaminoglycoside from 39.06 to 9.76 against the strain EC27, andwithSA358, a reduction in MIC was also observed, from 39.06 to 9.76.The samewas seenwithMETH plus gentamicin, where theMICwaslowered from 39.06 to 9.76 against SA358. An antagonistic effectwas found with METH when combined with amikacin, increasingits MIC from 39.06 to 156.25 against the strain SA358 (Table 3). Thetests for chemical prospecting showed the presence of alkaloids inthe extracts of the lizards.

The present work showed a non-clinically relevant MIC for theextracts of T. hispidus and A. ameiva, �1024 in all cases, like otherstudies that have demonstrated antimicrobial inefficiency in themedicinal use of some natural products tested. Ferreira et al. (2009)demonstrated the ineffectiveness of the body fat of Tupinambismerianae as an antimicrobial, a product that is used in popularmedicine for infections caused by bacteria such as E. coli and S.aureus, also obtaining a MIC �1024 mg/mL. On the other hand,a synergistic effect was observed with the extracts tested incombination with particular aminoglycosides, reducing the MIC ofthe antibiotics against the bacterial strains used, which has alsobeen found in other studies on natural products of animal as well asplant origin. Therefore, there is a need to understand how thesesubstances act in order to increase the activity of conventionalantibiotics, since a substantial decrease in the concentration of

Table 3MIC (mg/ml) of aminoglycosides in the absence and presence of 128 mg/mL of methanolic extracts of skin of T. hispidus or A. ameiva against Escherichia coli 27, Staphylococcusaureus 358 and Pseudomonas aeuruginosa RB1.

EC 27 SA 358 PA RB1

MIC MIC combined MIC MIC combined MIC MIC combined

Antibiotic METH MEAA METH MEAA METH MEAAKanamycin 156.2 156.25 156.2 625 625 625 1250 1250 1250Amikacin 156.2 156.25 19.5 39.0 156.2 39.0 625 625 625Neomycin 78.1 78.12 78.1 156.2 156.2 156.2 1250 1250 1250Gentamicin 39.0 39.06 9.8 39.0 9.8 9.8 312.5 312.5 312.5

METH e Methanolic extract of Tropidurus hispidus; MEAA e Methanolic extract of Ameiva ameiva.

I.J.M. Santos et al. / Journal of Arid Environments 76 (2012) 138e141140

aminoglycosides would be a promising improvement in thechemotherapy of infections. According to Matias et al. (2010) themechanisms by which animal- or plant-derived natural productscan inhibit the growth of microorganisms differ and can be partlydue to the hydrophobic nature of some components. Variouscomponents of the extracts can act as permeabilizers of the cellmembrane, increasing the cellular uptake of antibiotics (Helanderet al., 1998). The interference on bacterial enzyme systems canalso be a potential mechanism of action (Wendakoon andSakaguchi, 1995). These mechanisms of action can be involved inthe combination of an antibiotic with a natural product extract ata sub-inhibitory concentration added directly to the culturemedium (Coutinho et al., 2010).

The presence of alkaloids in the extracts used in this antimi-crobial assay can be a strong indication that these substancesaccount for the biological activity shown in the antibiotic-modifying assay, since these extracts when combined with ami-noglycosides significantly reduced the MIC of the latter. Manystudies have demonstrated the pharmacological activity of alkaloids(Paulo et al., 1992). Alkaloids with antimicrobial activity were alsodemonstrated in the extract of amphibian skin (Gomes et al., 2007).

In cases where antagonism is seen between the test substancesand antibiotics, as demonstrated in the present work, it is veryimportant to understand the causes underlying this process, sincethe combined use of antibiotics with other substances can lead todiverse effects, positive as in the cases of synergism and negativewith antagonism, also observed in this study. According toGranowitz and Brown (2008), an antagonistic effect due thecombined use of antibiotics probably results from a binding inter-action. This is likely an indication of an antagonistic effect of theinteraction between METH and the aminoglycosides, reducingantibiotic activity.

It is important to note that the use of natural productscombined with commercial drugs has been previously reported.Calvet-Mir et al. (2008) reported the use of traditional medicineproducts combined with Eastern medicine for the treatment ofdiarrhea, vomiting and stomach ache. The combination of naturalproducts with conventional drugs can be very beneficial asdemonstrated in this study, where the MIC of particular antibioticswas significantly reduced due their interaction with the extractstested. On the other hand, such combinations may pose a healthrisk when the effects of natural products are not known, as couldbe observed here, where the combination of METH with amikacinresulted in a significant decrease in antibiotic efficacy. Shin et al.(2007) made a survey in 304 persons who had had a stroke inthe city of Kwangju in Korea, where 34% of those interviewedacknowledged using traditional medicine combined with easternmedicine. Alves and Rosa (2010), who studied the popularmarketing of medicinal animals in North and Northeast Brazil,pointed out the need of considering their interaction with allo-pathic medications (which the zootherapeutic product user can betaking) and their possible negative effects.

According to Ferreira et al. (2009, 2010) studies of substancesfrom reptiles have been conducted to determine their pharma-cological activities. The venom of the snake Bothrops jararacapossesses various medicinal properties. Ciscotto et al. (2009)described the antibacterial and antiparasitic activities of L-aminoacid oxidase from the venom of this snake. Products from otherspecies of reptiles were also studied in attempt to elucidatetheir pharmacological proprieties. Liu et al. (2007) demonstratedthe anti-tumor effect of extracts of the lizard Gecko japonicus,which is widely utilized in Chinese traditional medicine. Thelysozymes of the turtles Trionyx sinensis, Amyda cartilagenea andChelonia mydas demonstrated a strong antibacterial activity(Thammasirirak et al., 2006).

The extracts of T. hispidus and A. ameiva alone did not showclinically relevant effects against the bacterial strains tested, but incombination with the aminoglycosides used, they mostly demon-strated a significant synergistic effect, whereas an antagonisticeffect with METH in combination with amikacin. A likely explana-tion for the biological effects of these extracts is the presence ofalkaloids in the extracts of these lizards from semiarid. Moredetailed studies are warranted in order to identify these alkaloidsand to determine their relation to the antibacterial activity of theextracts of the lizards T. hispidus and A. ameiva and demonstrate theefficacy of these natural resource used in the traditional medicineby semiarid living communities.

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