http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, Early Online: 1–5 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2014.922587
SHORT COMMUNICATION
Anti-infective effects of Brazilian Caatinga plants against pathogenic bacterial biofilm formation
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Laura Nunes Silva1*, Danielle da Silva Trentin1,2*, Karine Rigon Zimmer3, Janine Treter1,2, Clara Lia Costa Brandelli1,2, Amanda Piccoli Frasson2, Tiana Tasca2, Alexandre Gomes da Silva4, Ma´rcia Vanusa da Silva4, and Alexandre Jose´ Macedo1,2 1
Centro de Biotecnologia do Estado do Rio Grande do Sul, Porto Alegre, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil, 2Faculdade de Farma´cia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil, 3Escola de Quı´mica e Alimentos, Universidade Federal do Rio Grande, Rio Grande, Rio Grande do Sul, Brazil, and 4Departamento de Bioquı´mica, Universidade Federal de Pernambuco, Recife, Pernanbuco, Brazil Abstract
Keywords
Context: The local communities living in the Brazilian Caatinga biome have a significant body of traditional knowledge on a considerable number of medicinal plants used to heal several maladies. Objective: Based on ethnopharmacological data, this study screened 23 aqueous plant extracts against two well-known models of biofilm-forming bacteria: Staphylococcus epidermidis and Pseudomonas aeruginosa. Materials and methods: Crystal violet assay and scanning electron microscopy (SEM) were used to evaluate the effect of extracts on biofilm formation and measurements of the absorbance at 600 nm to assess bacterial growth. Selected extracts were investigated regarding the cytotoxicity by MTT assay using mammal cells and the qualitative phytochemical fingerprint by thin layer chromatography. Results: Harpochilus neesianus Mart. ex Nees. (Acanthaceae) leaves, Apuleia leiocarpa Vogel J. F. Macbr. (Fabaceae), and Poincianella microphylla Mart. ex G. Don L. P. Queiroz (Fabaceae) fruits showed non-biocidal antibiofilm action against S. epidermidis with activities of 69, 52, and 63%, respectively. SEM confirmed that biofilm structure was strongly prevented and that extracts promoted overproduction of the matrix and/or bacterial morphology modification. Poincianella microphylla demonstrated toxicity at 4.0 mg/mL and 2.0 mg/mL, A. leiocarpa presented toxicity only at 4.0 mg/mL, whereas H. neesianus presented the absence of toxicity against Vero cell line. Preliminary phytochemical analysis revealed the presence of flavonoids, terpenoids, steroids, amines, and polyphenols. Discussion and conclusions: This work provides a scientific basis which may justify the ethnopharmacological use of the plants herein studied, indicating extracts that possess limited mammal cytotoxicity in vitro and a high potential as a source of antibiofilm drugs prototypes.
Antibiofilm, antibacterial, cytotoxicity, medicinal plants
Introduction Plants have long been used by humans to maintain and recover health, and several approaches have addressed the selection of plants as candidates for drug discovery (Fabricant & Farnsworth, 2001). Ethnopharmacology contributes with an interdisciplinary view in the search for human health and it has been proven to be a powerful tool in the discovery of natural products with therapeutic action (Reyes-Garcı´a, 2010). The Brazilian Caatinga, located in northeast region of Brazil, is subjected to a hot, dry, semi-arid climate, resulting in xerophyte vegetation with significant floristic *These authors are first authors. Correspondence: Alexandre Jose´ Macedo, Faculdade de Farma´cia, Universidade Federal do Rio Grande do Sul, Av. Ipiranga 2752, Porto Alegre 90610-000, Brazil. Tel: +55 51 33086082. Fax: +55 51 33087309. E-mail: [email protected]
History Received 23 January 2014 Revised 22 April 2014 Accepted 29 April 2014 Published online 3 December 2014
diversity (Rodal & Nascimento, 2006). This semi-arid biome is the only major region fully inserted in Brazilian national territory and has a richness of traditional knowledge accumulated by local communities. Ethnopharmacological reports have been confirmed by recent studies showing that Caatinga plants present potential to prevent bacterial adherence and growth (Trentin et al., 2011, 2013), to significantly decreasing flagellate protozoan viability (Frasson et al., 2012), and to reduce inflammation, lipoperoxidation, and hyperalgesia in rats (Santana et al., 2012). In recent years, biofilms have attracted considerable attention, especially due to their enormous impact on medicine and public health. Bacteria in the biofilm form contribute to chronicity of persistent infections such as those associated with implanted medical devices. This important lifestyle allows pathogens to escape from host immune defenses and resist antibacterial treatments (Høiby et al.,
2
L. N. Silva et al.
2010). The pressure to find novel antibacterials with new modes of action, including antiadherent compounds, drives the discovery of innovative antimicrobials and emphasizes the potential of plant-derived molecules as a source of biofilm control products. As part of our ongoing ethno-directed research on antibiofilms from Caatinga plants, the present study aimed to investigate 23 aqueous plant extracts from 14 medicinal plants against Staphylococcus epidermidis and Pseudomonas aeruginosa. In addition, the most active extracts were submitted to a cytotoxic evaluation and to a preliminary phytochemical screening.
developed using butanol, acetic acid, and water (5:1:4) as mobile phase. The plates were visualized under UV light (254 and 365 nm, Handheld UV Lamp Model 9403E, BioAmerica Inc., Miami, FL) and revealed with different chemical sprays. Natural reagent followed by polyethylene glycol was used to detect flavonoids; ferric chloride for polyphenols; ninhydrin for amines and amino acids; anisaldehyde sulfuric for steroids, terpenoids, and saponins; and Dragendorff for alkaloids and heterocyclic nitrogen compounds (Wagner & Bladt, 1996).
Materials and methods
The toxicity of extracts against the mammal Vero cell line was investigated using the MTT (Thiazolyl Blue Tetrazolium Bromide, Sigma-Aldrich, EUA, Bellefonte, PA) assay (Mosmann, 1983). In the untreated cells, the extracts were replaced by water and represented 100% of viability, while 1% Triton X-100 solution was used as positive control.
Plant material and extracts Pharmaceutical Biology Downloaded from informahealthcare.com by University of Liverpool on 01/02/15 For personal use only.
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The plants used in this study have medicinal applications by the Caatinga communities (Agra et al., 2008 and local community information) as summarized in Table 1. Plant samples were collected in Parque Nacional do Catimbau (PARNA do Catimbau), Pernambuco, Brazil, in 2010. The species were identified and a voucher was deposited. Aqueous extracts, prepared to reproduce their traditional usage, were obtained according to Trentin et al. (2011), and the powder was stored at 20 C. Assays were performed with 4.0 mg/mL as final concentration.
Cytotoxicity study
Statistical analysis Experiments were carried out in triplicate and data are presented as percentage mean ± standard deviation. Differences between groups were evaluated by Student’s t-test (p value 0.05).
Bacterial strain and culture conditions
Results and discussion
Pseudomonas aeruginosa ATCC 27853 and S. epidermidis ATCC 35984 were grown in Mueller Hinton (MH) agar (Oxoid Ltd., England) overnight, at 37 C. A bacterial suspension in 0.9% sterile saline, corresponding to 3 108 CFU/mL, was used in the assays.
Twenty-three extracts from 14 different plants were screened in order to determine their antibiofilm and antibacterial activities against two important bacterial species (Table 1). Considering S. epidermidis, the most important cause of medical device-associated infections (Otto, 2009), the results revealed that 10 extracts have potential antibiofilm activity (they allowed 50% of biofilm formation): Apuleia leiocarpa Vogel J. F. Macbr. (Fabaceae) fruits, Byrsonima gardneriana A. Juss. (Malpighiaceae) leaves, Harpochilus neesianus Mart. ex Nees. (Acanthaceae) mixture, H. neesianus Mart. ex Nees. (Acanthaceae) leaves, Jacaranda rugosa A. H. Gentry (Bignoniaceae) leaves, Piriqueta guaianensis N. E. Br. (Turneraceae) roots, Poincianella microphylla Mart. ex G. Don L. P. Queiroz (Fabaceae) fruits, Sideroxylon obstusifolium Roem. & Schult. T. D. Penn (Sapotaceae) branches and leaves, and Turnera melochioides Cambess. (Turneraceae) leaves. The S. epidermidis growth was inhibited by only three extracts: B. gardneriana leaves, S. obstusifolium branches, and leaves. Regarding P. aeruginosa, a species intrinsically resistant to several types of antibiotics (Strateva & Yordanov, 2009), our results demonstrated that the extracts exhibited a different activity profile, in which the biofilm prevention seems to be associated to the inhibition of the bacterial growth, as observed to B. gardneriana leaves, J. rugosa leaves, M. lewessia leaves, P. microphylla fruits, P. guianensis roots, and S. obtusifolium branches and leaves. These findings may explain why all the plants described above, with an exception of P. guianensis and T. melochioides, are used by Caatinga communities against illness that resemble inflammations and infections (Table 1). Three active extracts were selected for further investigation: leaves of H. neesianus, fruits of A. leiocarpa, and fruits
Antibiofilm activity and bacterial growth assays The antibiofilm activity assay was performed as established by Trentin et al. (2011), employing the crystal violet technique. The bacterial growth was evaluated by the difference between initial (t ¼ 0) and final (t ¼ 6 h for P. aeruginosa or t ¼ 24 h for S. epidermidis) absorbance values at 600 nm in 96-well microtiter plates (Costar 3599, Corning, Inc., Corning, NY). Values higher than 100% represent a stimulation of bacterial growth or biofilm formation in comparison with the control sample (untreated), in which the extracts were replaced by water. Scanning electron microscopy (SEM) Biofilms were grown in the 96-well microtiter plates with a piece of PermanoxÔ slide (Nalge Nunc International, Rochester, NY), in order to mimic the hydrophobic polystyrene surface, exposing bacteria to the plant extracts or to water (untreated control). Samples were processed, the slides were dried using the CO2 critical point technique and examined in a JEOL JSM-6060 (JOEL, Peabody, MA) scanning electron microscope. Phytochemical screening The extracts were applied in thin layer chromatography (TLC) plates (Silica gel 60 F254, Merck, Darmstadt, Germany) and
98.1 ± 2.1 241.8 ± 5.6* 91.4 ± 13.8 26.5 ± 5.7* 152.5 ± 14.3* 182.0 ± 30.8*
Roots Branches Leaves Leaves Branches Branches
118.0 ± 1.0*
13.7 ± 1.3* 15.0 ± 1.5*
Branches Leaves Entire plant
156.8 ± 2.4* 26.6 ± 1.6* 86.3 ± 3.2* 36.7 ± 6.3* 237.0 ± 7.4*
Leaves Roots Branches Fruits Leaves
129.0 ± 10.7*
Leaves
31.3 ± 12.1* 150.1 ± 14.3* 37.2 ± 3.8*
143.2 ± 3.2* 217.3 ± 5.6*
19.6 ± 3.2*
Mixture of leaves, fruits and branches Leaves Mixture of branches and leaves Leaves
46.2 ± 7.1*
85.4 ± 11.3
Leaves
249.6 ± 2.4*
161.5 ± 4.4* 202.0 ± 2.6* 82.5 ± 1.6 190.4 ± 34.2* 252.2 ± 11.3*
170.8 ± 5,2*
57.7 ± 6.7* 51.6 ± 1.7*
285.2 ± 7.0* 70.5 ± 0.7* 118.7 ± 1.9 163.3 ± 1.6* 243.7 ± 3.3*
199.9 ± 28.4*
198.9 ± 26.5*
91.8 ± 2.4
162.0 ± 34.0*
16.0 ± 3.8*
Leaves
121.4 ± 6.4* 209.3 ± 5.5*
185.7 ± 3.3* 47.9 ± 2.0*
Leaves Fruits
Part used
Bacterial growth (%)
S. epidermidis Biofilm formation (%)
82.5 ± 15.9*
72.7 ± 1.6* 94.5 ± 3.4* 78.5 ± 7.1* 73.1 ± 8.7* 104.8 ± 8.7
114.1 ± 2.4
30.6 ± 7.6* 41.9 ± 3.6*
80.7 ± 10.4* 12.8 ± 8.8* 78.4 ± 8.3* 1.6 ± 4.8* 111.0 ± 1.7*
45.2 ± 8.3*
26.6 ± 10.0*
103.1 ± 13.8 60.7 ± 17.1*
78.9 ± 5.4*
87.2 ± 2.5*
31.7 ± 5.08*
99.6 ± 16.0 82.4 ± 4.8*
86.7 ± 1.0
81.4 ± 0.2 106.4 ± 1.8 119.3 ± 0.8* 107.5 ± 2.1 121.2 ± 0.7
164.0 ± 1.7*
57.0 ± 2.9* 58.0 ± 2.8*
161.0 ± 2.5* 9.9 ± 0.2* 88.4 ± 2.4 0 ± 0* 202.5 ± 7.2*
0 ± 0*
0 ± 0*
109.6 ± 3.4 103.3 ± 1.4
118.2 ± 2.3
125.9 ± 3.6
44.5 ± 2.3*
134.9 ± 2.5* 83.4 ± 1.3
Bacterial growth (%)
P. aeruginosa Biofilm formation (%)
Anti-infective Brazilian Caatinga plants
PC, personal communication. Results represent mean ± standard deviation of three experiments. *Significant difference in relation to the untreated control (p 0.05).
IPA 84965
IPA 85873
Turnera subulata Sm. – Turneraceae
IPA 85911
Poincionella microphylla Mart. ex G. Don L. P. Queiroz – Fabaceae Sideroxylon obtusifolium Roem. & Schult. T.D. Penn – Sapotaceae
IPA 84959
IPA 84869
Piriqueta guianensis N.E.Br. – Turneraceae
Turnera melochioides Cambess. – Turneraceae
IPA 85902
Mimosa lewisii Barneby – Fabaceae
IPA 84962
IPA 85710
Jacaranda rugosa A.H.Gentry – Bignoniaceae
Turnera hermannioides Cambess. – Turneraceae
IPA 85701
Ipomea brasiliana Choisy Meisn – Convolvulaceae
IPA 85698
Against ovarian inflammations and diabetes. A decoction or maceration of a handful is prepared in a liter of water (Agra et al., 2008) Infusions of a spoonful in a cup of water are consumed as stomachic after meals (PC) Against amenorrhea and dysmenorrhea. A decoction of a handful is prepared in a liter of water. It is consumed as tea (PC) Against amenorrhea and dysmenorrhea. A decoction of a handful is prepared in a liter of water. It is consumed as tea (PC) Against amenorrhea and dysmenorrhea. A decoction of a handful is prepared in a liter of water. It is consumed as tea (Agra et al., 2008)
IPA 85766
Harpochilus neesianus Mart. ex Nees. – Acanthaceae
Stylosanthes viscosa Sw. – Fabaceae
The decoction is drunk as digestive and sedative (Agra et al., 2008)
IPA 85697
Croton heliotropiifolius Kunth – Euphorbiaceae
Decoctions are used against external ulcers and inflammations (Agra et al., 2008) The decoction is used against intestinal pain, influenza, asthma, and bronchitis (Agra et al., 2008) Drink. The syrup is used against asthmas, cough, bronchitis, and as expectorant (PC) Against dermatitis, scabies, syphilis, skin ulcers and external. It is used to bathe or wash the affected parts (PC) An infusion in water or maceration in alcohol. It is use against syphilis and ulcers (PC) As expectorant and against respiratory diseases. A small piece in a cup of water is prepared as syrup and symptoms disappear (PC) A decoction is used as topical emollient (PC)
IPA 85917
Byrsonima gardneriana A. Juss. – Malpighiaceae
The decoction is used against external ulcers. It is consumed as tonic (PC)
IPA 87902
Voucher
Usage forms, preparation and therapeutic indication
Apuleia leiocarpa Vogel J. F. Macbr. – Fabaceae
Plant scientific name and family
Table 1. Ethnopharmacological data from 14 Caatinga medicinal plants and the biological activity of their aqueous extracts (4.0 mg/mL) against biofilm formation and growth of Staphylococcus epidermidis ATCC 35984 and Pseudomonas aeruginosa ATCC27853.
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DOI: 10.3109/13880209.2014.922587
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Pharmaceutical Biology Downloaded from informahealthcare.com by University of Liverpool on 01/02/15 For personal use only.
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L. N. Silva et al.
Pharm Biol, Early Online: 1–5
Figure 1. (A–D) Scanning electron microscopy images of staphylococcal biofilm on PermanoxÔ. (A) Untreated S. epidermidis (control) and bacteria treated with three selected extracts: (B) H. neesianus leaves extract, (C) A. leiocarpa fruits extract, and (D) P. microphylla fruits extract. Scales bar: 30 000 magnification (in the images: inset (1) – 2000 magnification and inset (2) – 10 000 magnification). Solid arrows: cell deformation. Dotted arrows: matrix overproduction. (E) The viability of mammal Vero cells and the biofilm formation by S. epidermidis according to different concentrations of extracts. Untreated cells and bacteria were considered as presenting 100% of viability and biofilm formation, respectively. *Significant difference in relation to untreated samples regarding the results about mammal cell viability and **the results about bacterial biofilm formation.
of P. microphylla. Harpochilus neesianus and P. microphylla are arboreal shrub endemics species of Caatinga while A. leiocarpa is a leguminous widely distributed in Brazil. However, Caatinga forest are becoming increasingly sparse due to the devastation (Leal et al., 2005). These species were able to prevent biofilm formation by S. epidermidis without inhibiting bacterial growth (Table 1). The inhibition of biofilm formation by such way that bacterial growth is not negatively affected comprises an alternative and attractive approach that may hamper the rapid development of selective pressure for bacterial resistance (Rasko & Sperandio, 2010).
The SEM images confirm the results obtained by the colorimetric assay, showing that the selected extracts strongly prevented attachment and biofilm formation by S. epidermidis and also induced overproduction of the matrix and/or bacterial morphology modification (Figure 1A–D). In order to evaluate these extracts regarding their cytotoxicity against mammal cells and to correlate with their ability to prevent S. epidermidis biofilm formation, two lower extract concentrations were included (2.0 and 0.4 mg/mL). We observed that H. neesianus leaves extract shows no mammal cytotoxicity; A. leiocarpa fruits extract presents cytotoxicity
Anti-infective Brazilian Caatinga plants
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DOI: 10.3109/13880209.2014.922587
only at the higher concentration tested and P. microphylla fruits extract significantly decreased mammal cell viability at 4.0 and 2.0 mg/mL, but it was not cytotoxic at 0.4 mg/mL (Figure 1, panel E). To the best of our knowledge, this is the first study in the literature evaluating the toxicity of these species. Concerning the antibiofilm activity of H. neesianus and A. leiocarpa extracts, no important variation was observed even when a 10-fold lower extract concentration was used. Differently, P. microphylla extract demonstrated such a dosedependent profile of biofilm formation inhibition, being active in the highest concentration and not significantly active at the intermediary and lowest concentrations (Figure 1, panel E). The preliminary qualitative phytochemical screening of these extracts indicated the presence of flavonoids, terpenoids, steroids, and amines while polyphenols were detected only in P. microphylla (data not shown). These findings will direct our future efforts on the purification of the bioactive compounds and on studies about their pathways of action. This in vitro screening using the ethnopharmacological approach is important for validating the traditional use of herbal remedies by Caatinga communities, to stimulate an active preservation politic of plants and also to guide new bioprospection studies. The study highlights the valuable ethnopharmacological knowledge preserved in this region, since all plants used by the communities against illness that resemble inflammations and infections fitted with our antiinfective investigation. Moreover, we identified plants with high potential for antibiofilm drugs displaying limited cytotoxicity in vitro against the important biofilm-forming pathogen S. epidermidis.
Acknowledgements The authors thank the curator of the Herbarium IPA and the Instituto Chico Mendes de Conservac¸a˜o da Biodiversidade for authorizing collections (Sisbio 16.806); and to the Centro de Microscopia Eletroˆnica (CME/UFRGS) for technical assistance in electron microscopy.
Declaration of interest The authors have declared that there is no conflict of interest. The authors thank NANOBIOTEC-Brasil/CAPES,
5
CNPq, and FAPERGS for financial support and for fellowships.
References Agra MF, Silva KN, Bası´lio IJLD, et al. (2008). Survey of medicinal plants used in the region Northeast of Brazil. Rev Bras Farmacog 18: 472–508. Fabricant DS, Farnsworth NR. (2001). The value of plants used in traditional medicine for drug discovery. Environ Health Perspect 109:69–75. Frasson AP, Santos O, Duarte M, et al. (2012). First report of antiTrichomonas vaginalis activity of the medicinal plant Polygala decumbens from the Brazilian semi-arid region, Caatinga. Parasitol Res 110:2581–7. Høiby N, Bjarnsholt T, Givskov M, et al. (2010). Antibiotic resistance of bacterial biofilms. Int J Antimicrob Agents 35:322–32. Leal IR, Da Silva JMC, Tabarelli M, Lacher TE. (2005). Changing the course of biodiversity conservation in the Caatinga of northeastern Brazil. Conserv Biol 19:701–6. Mosmann T. (1983). Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63. Otto M. (2009). Staphylococcus epidermidis – The ‘accidental’ pathogen. Nat Rev Microbiol 7:555–67. Rasko DA, Sperandio V. (2010). Anti-virulence strategies to combat bacteria-mediated disease. Nat Rev Drug Discov 9:117–28. Reyes-Garcı´a V. (2010). The relevance of traditional knowledge systems for ethnopharmacological research: Theoretical and methodological contributions. J Ethnobiol Ethnomed 6:32. doi:10.1186/ 1746-4269-6-32. Rodal MJN, Nascimento LM. (2006). The arboreal component of a dry forest in Northeastern Brazil. Braz J Biol 66:479–91. Santana DG, Santos CA, Santos ADC, et al. (2012). Beneficial effects of the ethanol extract of Caesalpinia pyramidalis on the inflammatory response and abdominal hyperalgesia in rats with acute pancreatitis. J Ethnopharmacol 142:445–55. Strateva T, Yordanov D. (2009). Pseudomonas aeruginosa – A phenomenon of bacterial resistance. J Med Microbiol 58: 1133–48. Trentin DS, Giordani RB, Zimmer KR, et al. (2011). Potential of medicinal plants from the Brazilian semi-arid region (Caatinga) against Staphylococcus epidermidis planktonic and biofilm lifestyles. J Ethnopharmacol 137:327–35. Trentin DS, Silva DB, Amaral MW, et al. (2013). Tannins possessing bacteriostatic effect impair Pseudomonas aeruginosa adhesion and biofilm formation. PLoS One 8:e66257. doi:10.1371/journal. pone.0066257. Wagner HH, Bladt S. (1996). Plant Drug Analysis: A Thin Layer Chromatography Atlas. Berlin Heidelberg: Springer-Verlag.
SHORT COMMUNICATION
Anti-infective effects of Brazilian Caatinga plants against pathogenic bacterial biofilm formation
Pharmaceutical Biology Downloaded from informahealthcare.com by University of Liverpool on 01/02/15 For personal use only.
Laura Nunes Silva1*, Danielle da Silva Trentin1,2*, Karine Rigon Zimmer3, Janine Treter1,2, Clara Lia Costa Brandelli1,2, Amanda Piccoli Frasson2, Tiana Tasca2, Alexandre Gomes da Silva4, Ma´rcia Vanusa da Silva4, and Alexandre Jose´ Macedo1,2 1
Centro de Biotecnologia do Estado do Rio Grande do Sul, Porto Alegre, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil, 2Faculdade de Farma´cia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil, 3Escola de Quı´mica e Alimentos, Universidade Federal do Rio Grande, Rio Grande, Rio Grande do Sul, Brazil, and 4Departamento de Bioquı´mica, Universidade Federal de Pernambuco, Recife, Pernanbuco, Brazil Abstract
Keywords
Context: The local communities living in the Brazilian Caatinga biome have a significant body of traditional knowledge on a considerable number of medicinal plants used to heal several maladies. Objective: Based on ethnopharmacological data, this study screened 23 aqueous plant extracts against two well-known models of biofilm-forming bacteria: Staphylococcus epidermidis and Pseudomonas aeruginosa. Materials and methods: Crystal violet assay and scanning electron microscopy (SEM) were used to evaluate the effect of extracts on biofilm formation and measurements of the absorbance at 600 nm to assess bacterial growth. Selected extracts were investigated regarding the cytotoxicity by MTT assay using mammal cells and the qualitative phytochemical fingerprint by thin layer chromatography. Results: Harpochilus neesianus Mart. ex Nees. (Acanthaceae) leaves, Apuleia leiocarpa Vogel J. F. Macbr. (Fabaceae), and Poincianella microphylla Mart. ex G. Don L. P. Queiroz (Fabaceae) fruits showed non-biocidal antibiofilm action against S. epidermidis with activities of 69, 52, and 63%, respectively. SEM confirmed that biofilm structure was strongly prevented and that extracts promoted overproduction of the matrix and/or bacterial morphology modification. Poincianella microphylla demonstrated toxicity at 4.0 mg/mL and 2.0 mg/mL, A. leiocarpa presented toxicity only at 4.0 mg/mL, whereas H. neesianus presented the absence of toxicity against Vero cell line. Preliminary phytochemical analysis revealed the presence of flavonoids, terpenoids, steroids, amines, and polyphenols. Discussion and conclusions: This work provides a scientific basis which may justify the ethnopharmacological use of the plants herein studied, indicating extracts that possess limited mammal cytotoxicity in vitro and a high potential as a source of antibiofilm drugs prototypes.
Antibiofilm, antibacterial, cytotoxicity, medicinal plants
Introduction Plants have long been used by humans to maintain and recover health, and several approaches have addressed the selection of plants as candidates for drug discovery (Fabricant & Farnsworth, 2001). Ethnopharmacology contributes with an interdisciplinary view in the search for human health and it has been proven to be a powerful tool in the discovery of natural products with therapeutic action (Reyes-Garcı´a, 2010). The Brazilian Caatinga, located in northeast region of Brazil, is subjected to a hot, dry, semi-arid climate, resulting in xerophyte vegetation with significant floristic *These authors are first authors. Correspondence: Alexandre Jose´ Macedo, Faculdade de Farma´cia, Universidade Federal do Rio Grande do Sul, Av. Ipiranga 2752, Porto Alegre 90610-000, Brazil. Tel: +55 51 33086082. Fax: +55 51 33087309. E-mail: [email protected]
History Received 23 January 2014 Revised 22 April 2014 Accepted 29 April 2014 Published online 3 December 2014
diversity (Rodal & Nascimento, 2006). This semi-arid biome is the only major region fully inserted in Brazilian national territory and has a richness of traditional knowledge accumulated by local communities. Ethnopharmacological reports have been confirmed by recent studies showing that Caatinga plants present potential to prevent bacterial adherence and growth (Trentin et al., 2011, 2013), to significantly decreasing flagellate protozoan viability (Frasson et al., 2012), and to reduce inflammation, lipoperoxidation, and hyperalgesia in rats (Santana et al., 2012). In recent years, biofilms have attracted considerable attention, especially due to their enormous impact on medicine and public health. Bacteria in the biofilm form contribute to chronicity of persistent infections such as those associated with implanted medical devices. This important lifestyle allows pathogens to escape from host immune defenses and resist antibacterial treatments (Høiby et al.,
2
L. N. Silva et al.
2010). The pressure to find novel antibacterials with new modes of action, including antiadherent compounds, drives the discovery of innovative antimicrobials and emphasizes the potential of plant-derived molecules as a source of biofilm control products. As part of our ongoing ethno-directed research on antibiofilms from Caatinga plants, the present study aimed to investigate 23 aqueous plant extracts from 14 medicinal plants against Staphylococcus epidermidis and Pseudomonas aeruginosa. In addition, the most active extracts were submitted to a cytotoxic evaluation and to a preliminary phytochemical screening.
developed using butanol, acetic acid, and water (5:1:4) as mobile phase. The plates were visualized under UV light (254 and 365 nm, Handheld UV Lamp Model 9403E, BioAmerica Inc., Miami, FL) and revealed with different chemical sprays. Natural reagent followed by polyethylene glycol was used to detect flavonoids; ferric chloride for polyphenols; ninhydrin for amines and amino acids; anisaldehyde sulfuric for steroids, terpenoids, and saponins; and Dragendorff for alkaloids and heterocyclic nitrogen compounds (Wagner & Bladt, 1996).
Materials and methods
The toxicity of extracts against the mammal Vero cell line was investigated using the MTT (Thiazolyl Blue Tetrazolium Bromide, Sigma-Aldrich, EUA, Bellefonte, PA) assay (Mosmann, 1983). In the untreated cells, the extracts were replaced by water and represented 100% of viability, while 1% Triton X-100 solution was used as positive control.
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The plants used in this study have medicinal applications by the Caatinga communities (Agra et al., 2008 and local community information) as summarized in Table 1. Plant samples were collected in Parque Nacional do Catimbau (PARNA do Catimbau), Pernambuco, Brazil, in 2010. The species were identified and a voucher was deposited. Aqueous extracts, prepared to reproduce their traditional usage, were obtained according to Trentin et al. (2011), and the powder was stored at 20 C. Assays were performed with 4.0 mg/mL as final concentration.
Cytotoxicity study
Statistical analysis Experiments were carried out in triplicate and data are presented as percentage mean ± standard deviation. Differences between groups were evaluated by Student’s t-test (p value 0.05).
Bacterial strain and culture conditions
Results and discussion
Pseudomonas aeruginosa ATCC 27853 and S. epidermidis ATCC 35984 were grown in Mueller Hinton (MH) agar (Oxoid Ltd., England) overnight, at 37 C. A bacterial suspension in 0.9% sterile saline, corresponding to 3 108 CFU/mL, was used in the assays.
Twenty-three extracts from 14 different plants were screened in order to determine their antibiofilm and antibacterial activities against two important bacterial species (Table 1). Considering S. epidermidis, the most important cause of medical device-associated infections (Otto, 2009), the results revealed that 10 extracts have potential antibiofilm activity (they allowed 50% of biofilm formation): Apuleia leiocarpa Vogel J. F. Macbr. (Fabaceae) fruits, Byrsonima gardneriana A. Juss. (Malpighiaceae) leaves, Harpochilus neesianus Mart. ex Nees. (Acanthaceae) mixture, H. neesianus Mart. ex Nees. (Acanthaceae) leaves, Jacaranda rugosa A. H. Gentry (Bignoniaceae) leaves, Piriqueta guaianensis N. E. Br. (Turneraceae) roots, Poincianella microphylla Mart. ex G. Don L. P. Queiroz (Fabaceae) fruits, Sideroxylon obstusifolium Roem. & Schult. T. D. Penn (Sapotaceae) branches and leaves, and Turnera melochioides Cambess. (Turneraceae) leaves. The S. epidermidis growth was inhibited by only three extracts: B. gardneriana leaves, S. obstusifolium branches, and leaves. Regarding P. aeruginosa, a species intrinsically resistant to several types of antibiotics (Strateva & Yordanov, 2009), our results demonstrated that the extracts exhibited a different activity profile, in which the biofilm prevention seems to be associated to the inhibition of the bacterial growth, as observed to B. gardneriana leaves, J. rugosa leaves, M. lewessia leaves, P. microphylla fruits, P. guianensis roots, and S. obtusifolium branches and leaves. These findings may explain why all the plants described above, with an exception of P. guianensis and T. melochioides, are used by Caatinga communities against illness that resemble inflammations and infections (Table 1). Three active extracts were selected for further investigation: leaves of H. neesianus, fruits of A. leiocarpa, and fruits
Antibiofilm activity and bacterial growth assays The antibiofilm activity assay was performed as established by Trentin et al. (2011), employing the crystal violet technique. The bacterial growth was evaluated by the difference between initial (t ¼ 0) and final (t ¼ 6 h for P. aeruginosa or t ¼ 24 h for S. epidermidis) absorbance values at 600 nm in 96-well microtiter plates (Costar 3599, Corning, Inc., Corning, NY). Values higher than 100% represent a stimulation of bacterial growth or biofilm formation in comparison with the control sample (untreated), in which the extracts were replaced by water. Scanning electron microscopy (SEM) Biofilms were grown in the 96-well microtiter plates with a piece of PermanoxÔ slide (Nalge Nunc International, Rochester, NY), in order to mimic the hydrophobic polystyrene surface, exposing bacteria to the plant extracts or to water (untreated control). Samples were processed, the slides were dried using the CO2 critical point technique and examined in a JEOL JSM-6060 (JOEL, Peabody, MA) scanning electron microscope. Phytochemical screening The extracts were applied in thin layer chromatography (TLC) plates (Silica gel 60 F254, Merck, Darmstadt, Germany) and
98.1 ± 2.1 241.8 ± 5.6* 91.4 ± 13.8 26.5 ± 5.7* 152.5 ± 14.3* 182.0 ± 30.8*
Roots Branches Leaves Leaves Branches Branches
118.0 ± 1.0*
13.7 ± 1.3* 15.0 ± 1.5*
Branches Leaves Entire plant
156.8 ± 2.4* 26.6 ± 1.6* 86.3 ± 3.2* 36.7 ± 6.3* 237.0 ± 7.4*
Leaves Roots Branches Fruits Leaves
129.0 ± 10.7*
Leaves
31.3 ± 12.1* 150.1 ± 14.3* 37.2 ± 3.8*
143.2 ± 3.2* 217.3 ± 5.6*
19.6 ± 3.2*
Mixture of leaves, fruits and branches Leaves Mixture of branches and leaves Leaves
46.2 ± 7.1*
85.4 ± 11.3
Leaves
249.6 ± 2.4*
161.5 ± 4.4* 202.0 ± 2.6* 82.5 ± 1.6 190.4 ± 34.2* 252.2 ± 11.3*
170.8 ± 5,2*
57.7 ± 6.7* 51.6 ± 1.7*
285.2 ± 7.0* 70.5 ± 0.7* 118.7 ± 1.9 163.3 ± 1.6* 243.7 ± 3.3*
199.9 ± 28.4*
198.9 ± 26.5*
91.8 ± 2.4
162.0 ± 34.0*
16.0 ± 3.8*
Leaves
121.4 ± 6.4* 209.3 ± 5.5*
185.7 ± 3.3* 47.9 ± 2.0*
Leaves Fruits
Part used
Bacterial growth (%)
S. epidermidis Biofilm formation (%)
82.5 ± 15.9*
72.7 ± 1.6* 94.5 ± 3.4* 78.5 ± 7.1* 73.1 ± 8.7* 104.8 ± 8.7
114.1 ± 2.4
30.6 ± 7.6* 41.9 ± 3.6*
80.7 ± 10.4* 12.8 ± 8.8* 78.4 ± 8.3* 1.6 ± 4.8* 111.0 ± 1.7*
45.2 ± 8.3*
26.6 ± 10.0*
103.1 ± 13.8 60.7 ± 17.1*
78.9 ± 5.4*
87.2 ± 2.5*
31.7 ± 5.08*
99.6 ± 16.0 82.4 ± 4.8*
86.7 ± 1.0
81.4 ± 0.2 106.4 ± 1.8 119.3 ± 0.8* 107.5 ± 2.1 121.2 ± 0.7
164.0 ± 1.7*
57.0 ± 2.9* 58.0 ± 2.8*
161.0 ± 2.5* 9.9 ± 0.2* 88.4 ± 2.4 0 ± 0* 202.5 ± 7.2*
0 ± 0*
0 ± 0*
109.6 ± 3.4 103.3 ± 1.4
118.2 ± 2.3
125.9 ± 3.6
44.5 ± 2.3*
134.9 ± 2.5* 83.4 ± 1.3
Bacterial growth (%)
P. aeruginosa Biofilm formation (%)
Anti-infective Brazilian Caatinga plants
PC, personal communication. Results represent mean ± standard deviation of three experiments. *Significant difference in relation to the untreated control (p 0.05).
IPA 84965
IPA 85873
Turnera subulata Sm. – Turneraceae
IPA 85911
Poincionella microphylla Mart. ex G. Don L. P. Queiroz – Fabaceae Sideroxylon obtusifolium Roem. & Schult. T.D. Penn – Sapotaceae
IPA 84959
IPA 84869
Piriqueta guianensis N.E.Br. – Turneraceae
Turnera melochioides Cambess. – Turneraceae
IPA 85902
Mimosa lewisii Barneby – Fabaceae
IPA 84962
IPA 85710
Jacaranda rugosa A.H.Gentry – Bignoniaceae
Turnera hermannioides Cambess. – Turneraceae
IPA 85701
Ipomea brasiliana Choisy Meisn – Convolvulaceae
IPA 85698
Against ovarian inflammations and diabetes. A decoction or maceration of a handful is prepared in a liter of water (Agra et al., 2008) Infusions of a spoonful in a cup of water are consumed as stomachic after meals (PC) Against amenorrhea and dysmenorrhea. A decoction of a handful is prepared in a liter of water. It is consumed as tea (PC) Against amenorrhea and dysmenorrhea. A decoction of a handful is prepared in a liter of water. It is consumed as tea (PC) Against amenorrhea and dysmenorrhea. A decoction of a handful is prepared in a liter of water. It is consumed as tea (Agra et al., 2008)
IPA 85766
Harpochilus neesianus Mart. ex Nees. – Acanthaceae
Stylosanthes viscosa Sw. – Fabaceae
The decoction is drunk as digestive and sedative (Agra et al., 2008)
IPA 85697
Croton heliotropiifolius Kunth – Euphorbiaceae
Decoctions are used against external ulcers and inflammations (Agra et al., 2008) The decoction is used against intestinal pain, influenza, asthma, and bronchitis (Agra et al., 2008) Drink. The syrup is used against asthmas, cough, bronchitis, and as expectorant (PC) Against dermatitis, scabies, syphilis, skin ulcers and external. It is used to bathe or wash the affected parts (PC) An infusion in water or maceration in alcohol. It is use against syphilis and ulcers (PC) As expectorant and against respiratory diseases. A small piece in a cup of water is prepared as syrup and symptoms disappear (PC) A decoction is used as topical emollient (PC)
IPA 85917
Byrsonima gardneriana A. Juss. – Malpighiaceae
The decoction is used against external ulcers. It is consumed as tonic (PC)
IPA 87902
Voucher
Usage forms, preparation and therapeutic indication
Apuleia leiocarpa Vogel J. F. Macbr. – Fabaceae
Plant scientific name and family
Table 1. Ethnopharmacological data from 14 Caatinga medicinal plants and the biological activity of their aqueous extracts (4.0 mg/mL) against biofilm formation and growth of Staphylococcus epidermidis ATCC 35984 and Pseudomonas aeruginosa ATCC27853.
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DOI: 10.3109/13880209.2014.922587
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L. N. Silva et al.
Pharm Biol, Early Online: 1–5
Figure 1. (A–D) Scanning electron microscopy images of staphylococcal biofilm on PermanoxÔ. (A) Untreated S. epidermidis (control) and bacteria treated with three selected extracts: (B) H. neesianus leaves extract, (C) A. leiocarpa fruits extract, and (D) P. microphylla fruits extract. Scales bar: 30 000 magnification (in the images: inset (1) – 2000 magnification and inset (2) – 10 000 magnification). Solid arrows: cell deformation. Dotted arrows: matrix overproduction. (E) The viability of mammal Vero cells and the biofilm formation by S. epidermidis according to different concentrations of extracts. Untreated cells and bacteria were considered as presenting 100% of viability and biofilm formation, respectively. *Significant difference in relation to untreated samples regarding the results about mammal cell viability and **the results about bacterial biofilm formation.
of P. microphylla. Harpochilus neesianus and P. microphylla are arboreal shrub endemics species of Caatinga while A. leiocarpa is a leguminous widely distributed in Brazil. However, Caatinga forest are becoming increasingly sparse due to the devastation (Leal et al., 2005). These species were able to prevent biofilm formation by S. epidermidis without inhibiting bacterial growth (Table 1). The inhibition of biofilm formation by such way that bacterial growth is not negatively affected comprises an alternative and attractive approach that may hamper the rapid development of selective pressure for bacterial resistance (Rasko & Sperandio, 2010).
The SEM images confirm the results obtained by the colorimetric assay, showing that the selected extracts strongly prevented attachment and biofilm formation by S. epidermidis and also induced overproduction of the matrix and/or bacterial morphology modification (Figure 1A–D). In order to evaluate these extracts regarding their cytotoxicity against mammal cells and to correlate with their ability to prevent S. epidermidis biofilm formation, two lower extract concentrations were included (2.0 and 0.4 mg/mL). We observed that H. neesianus leaves extract shows no mammal cytotoxicity; A. leiocarpa fruits extract presents cytotoxicity
Anti-infective Brazilian Caatinga plants
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DOI: 10.3109/13880209.2014.922587
only at the higher concentration tested and P. microphylla fruits extract significantly decreased mammal cell viability at 4.0 and 2.0 mg/mL, but it was not cytotoxic at 0.4 mg/mL (Figure 1, panel E). To the best of our knowledge, this is the first study in the literature evaluating the toxicity of these species. Concerning the antibiofilm activity of H. neesianus and A. leiocarpa extracts, no important variation was observed even when a 10-fold lower extract concentration was used. Differently, P. microphylla extract demonstrated such a dosedependent profile of biofilm formation inhibition, being active in the highest concentration and not significantly active at the intermediary and lowest concentrations (Figure 1, panel E). The preliminary qualitative phytochemical screening of these extracts indicated the presence of flavonoids, terpenoids, steroids, and amines while polyphenols were detected only in P. microphylla (data not shown). These findings will direct our future efforts on the purification of the bioactive compounds and on studies about their pathways of action. This in vitro screening using the ethnopharmacological approach is important for validating the traditional use of herbal remedies by Caatinga communities, to stimulate an active preservation politic of plants and also to guide new bioprospection studies. The study highlights the valuable ethnopharmacological knowledge preserved in this region, since all plants used by the communities against illness that resemble inflammations and infections fitted with our antiinfective investigation. Moreover, we identified plants with high potential for antibiofilm drugs displaying limited cytotoxicity in vitro against the important biofilm-forming pathogen S. epidermidis.
Acknowledgements The authors thank the curator of the Herbarium IPA and the Instituto Chico Mendes de Conservac¸a˜o da Biodiversidade for authorizing collections (Sisbio 16.806); and to the Centro de Microscopia Eletroˆnica (CME/UFRGS) for technical assistance in electron microscopy.
Declaration of interest The authors have declared that there is no conflict of interest. The authors thank NANOBIOTEC-Brasil/CAPES,
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CNPq, and FAPERGS for financial support and for fellowships.
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