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Piper diospyrifolium Kunth.: Chemical analysis and antimicrobial (intrinsic and combined) activities
Microbial Pathogenesis 136 (2019) 103700
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Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Piper diospyrifolium Kunth.: Chemical analysis and antimicrobial (intrinsic and combined) activities
T
Joara Nályda Pereira Carneiroa, Rafael Pereira da Cruza, Josefa Carolaine Pereira da Silvaa, Janaína Esmeraldo Rochaa, Thiago Sampaio de Freitasa, Débora Lima Salesa, Camila Fonseca Bezerraa, Waltécio de Oliveira Almeidaa, José Galberto Martins da Costaa, Luiz Everson da Silvab, Wanderlei do Amaralb, Ricardo Andrade Rebeloc, Ieda Maria Begninic, Henrique Douglas Melo Coutinhoa,∗, Maria Flaviana Bezerra Morais-Bragaa a
Universidade Regional do Cariri, URCA, Cel Antônio Luis, 1161, 63105-000, Pimenta, Crato, CE, Brazil Universidade Federal do Paraná, UFPR, XV de Novembro, 1299, 80.060-000, Centro, Curitiba, PR, Brazil c Universidade Regional de Blumenau, FURB, Antônio da Veiga, 140, 89030-903, Itoupava Seca, Blumenau, SC, Brazil b
ARTICLE INFO
ABSTRACT
Keywords: Fluconazole Virulence Escherichia coli Z-Carpacin Synergism Cell viability
The secular use of plants in popular medicine has emerged as a source for the discovery of new compounds capable of curing infections. Among microbial resistance to commercial drugs, species such as Piper diospyrifolium Kunth, which are used in popular therapy, are targets for pharmacological studies. With this in mind, antimicrobial experiments with the essential oil from the P. diospyrifolium (PDEO) species were performed and its constituents were elucidated. The oil compounds were identified by gas chromatography coupled to mass spectrometry (GC/MS). The broth microdilution method with colorimetric readings for bacterial tests (Escherichia coli and Staphylococcus aureus) and spectrophotometric readings for fungal tests (Candida albicans and Candida tropicalis), whose data were used to create a cell viability curve and calculate its IC50 against fungal cells, were used to determine the minimum inhibitory concentration of the oil and its combined action with commercial drugs. The oil's minimal fungicidal concentration and its action over fungal morphological transition were analyzed by subculture and microculture, respectively. Chemical analysis revealed Z-Carpacin, Pogostol and E-Caryophyllene as the most abundant compounds. Results from the intrinsic analysis were considered clinically irrelevant, however the oil presented a synergistic effect against multiresistant E. coli and S. aureus strains when associated with gentamicin, and against the standard and isolated C. tropicalis strains with fluconazole. A fungicidal effect was observed against the C. albicans isolate. Candida spp. hyphae inhibition was verified for all strains at the highest tested concentrations. The P. diospyrifolium essential oil presented a promising effect when associated with commercial drugs and against a fungal virulence factor. Thus, the oil presented active compounds which may help the development of new drugs, however, new studies are needed in order to clarify the oil's mechanism of action, as well as to identify its active constituents.
1. Introduction Plants are traditionally used in popular medicine and have been an alternative worth exploring, especially as a source of natural antimicrobials [1]. The richness of their compounds, termed secondary metabolites, has been shown to be effective at combating the most varied of infections [2]. The increase in number of microbial infections is directly associated with the irrational use of antimicrobials, this being one of the major ∗
causes of the emergence of multiresistant microorganisms [3]. Antimicrobial resistance increases morbidity and mortality for patients with infections, while a considerable increase in costs for healthcare institutions also exists [4]. The Piperaceae family possesses species distributed across tropical and subtropical regions in the northern and southern hemispheres, including herbaceous and shrubby plants. The Piper genus is the largest and most well-known for its production of plant extracts, including essential oils abundant in secondary metabolites, specifically those
Corresponding author. E-mail address: [email protected] (H.D. Melo Coutinho).
https://doi.org/10.1016/j.micpath.2019.103700 Received 19 July 2019; Received in revised form 27 August 2019; Accepted 28 August 2019 Available online 28 August 2019 0882-4010/ © 2019 Elsevier Ltd. All rights reserved.
Microbial Pathogenesis 136 (2019) 103700
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derived from the shikimate pathway such as neolignans, phenylpropanoids and flavonoids, among others [5,6]. Species from the Piper genus are used in popular medicine for the treatment of diarrhea [7], gastrointestinal disorders, stomach aches [8], throat diseases, coughs [9], gynecological and urological infections, as well as flatulence [10,11]. The studied species, Piper diospyrifolium, possesses antibacterial [5,12], antifungal [13,14] and leishmanicidal activity [5], among others, that have been scientifically proven. While faced with the resistance phenomenon and with the search for new antimicrobial agents, the scientific community has turned its attention to the use of natural products, especially those originating from plants with medicinal indications [15]. Although P. diospyrifolium has been widely used over many generations, its chemical and pharmacological properties have not yet been fully characterized [3]. The objective of this study was to investigate the chemical composition of the P. diospyrifolium Kunth. essential oil, thus elucidating its chemical profile, and to validate its pharmacological properties and antimicrobial effects against standard and resistant Escherichia coli and Staphylococcus aureus bacteria, as well as Candida albicans and Candida tropicalis yeasts.
2.2. Microbiological test 2.2.1. Microbial strains and solution preparations Standard strains obtained from the American Type Culture Collection (ATCC) and clinical isolates obtained from the Laboratory of Microbiology of the Federal University of Paraíba, namely E. coli ATCC 25922, E. coli 06, S. aureus ATCC 6538 and S. aureus 10, were used. The C. albicans INCQS 40006, C. albicans URM 4127, C. tropicalis INCQS 40042 and C. tropicalis URM 4262 fungal strains were used, where the standard strains were obtained from the Collection of Reference Microorganisms in Sanitary Surveillance - CMRVS, from FIOCRUZ -INCQS (National Institute of Quality Control in Health), Rio de Janeiro, while clinical isolates were obtained from the library of the Federal University of Recife (URM - University Recife Mycology). Bacterial and fungal cultures were revived in Petri dishes containing their respective culture media: HIA (Heart Infusion Agar) for bacteria and SDA (Sabouraud Dextrose Agar) for fungi, which were then placed in an incubator at 37 °C for 24 h. Following this period, a suspension was made by removing one border of each microbial culture and placing it in tubes with 4 mL of 0.9% Physiological Serum (PS). The inoculum concentration was standardized using the 0.5 McFarland scale turbidity (1 × 108 CFU/mL) [19]. Stock solutions from the essential oil were obtained by weighing 10 mg (10,000 μg) and 5 mg (50,000 μg) of the oil and diluting these in 1 mL of dimethyl sulfoxide (DMSO). Subsequently, these solutions were diluted in distilled sterile water to prevent the DMSO from exerting an activity over the tested cells, until reaching the 1024 μg/mL concentration for bacteria and 4096 μg/mL for fungi [20]. The reference drugs (norfloxacin, gentamicin, erythromycin and fluconazole) were diluted in distilled sterile water until norfloxacin, gentamicin and erythromycin reached an initial concentration of 1024 μg/mL and fluconazole a concentration of 4096 μg/mL.
2. Materials and methods 2.1. Essential oil extraction and chemical composition The essential oil was kindly gifted by professor Dr. Luiz Everson da Silva from the Federal University of Paraná - UFPR - Brazil, where the oil was extracted and subjected to gas chromatography coupled to mass spectrometry (GC-MS) for further quantification and identification of its constituents. Initially, the plant material was collected from the coast of Paraná, Brazil (S 25° 13.644′, W 48° 34.985’ and altitude of 6 m). Exsiccates from the collected specimens were produced and taken to the Herbarium of the Municipal Botanical Museum of Curitiba (MBM), where they were herborized (HFIE) under number MBM 396413 [16,17]. The leaves were selected and dried (electric dryer model FANEM Mod. 320 SE with air circulation at 40 °C for 24 h) for the essential oil preparation. After weighing 50 g of the leaves, the plant material was subjected to hydrodistillation in 1 L of distilled water in a Clevenger type apparatus for 4.5 h. This process was performed in triplicates, based on the Wasicky method (1963) [18]. Following extraction, the samples were kept in a freezer until needed for analysis. The total essential oil mass produced in relation to the botanical material dry mass used in the extraction process was measured to determine the dry essential oil content. For the GC/MS chemical constituent identification analysis, the oil solution and 1% dichloromethane were injected with a 1:20 flow division in an Agilent 6890 (Palo Alto, CA) chromatograph coupled to an Agilent 5973 N selective mass detector at 250 °C. The HP-5MS capillary column (5%-phenyl-95%-dimethylpolysiloxane, 30 m × 0.25 mm x 0.25 μm) was used with helium as the carrier gas (1.0 mL min−1). The chamber temperature was programmed from 60 to 240 °C at a rate of 3 °C min−1. The mass detector operated in the electronic ionization mode (70 eV) at a rate of 3.15 s−1 scans with a mass range of 40–450. The transfer line was maintained at 260 °C, the ion source at 230 °C and the analyzer (quadrupole) at 150 °C. For quantification, the sample was injected into an Agilent 7890A chromatograph equipped with a flame ionization detector (FID) operating at 280 °C under the same analytical conditions, except for the carrier gas where hydrogen was used instead at a flow rate of 1.5 mL min−1. The percentage composition was obtained by FID signal electronic integration, dividing the area of each component by the total area (area %).
2.2.2. Minimal inhibitory concentration determination Test eppendorfs were prepared containing 1350 μL of 10% BHI (Brain Heart Infusion Broth) and double concentrated SDB (Sabouraud Dextrose Broth) plus 150 μL of the inoculum (corresponding to 10% of the total solution) for bacterial and fungal strains, respectively. This solution was then distributed in microdilution plates (100 μL/well) before serial dilutions were performed with 100 μL of the oil solution per column with concentrations ranging from 512 μg/mL to 0.5 μg/mL for bacteria, and from 2048 μg/mL to 2 μg/mL for fungi. The microdilutions were performed in triplicates and quadruplicates, respectively. The plates were taken to an incubator at 37 °C/24 h. The bacterial plates were read by adding 20 μL resazurin (colorimetric reagent) in each well and visually observing these after 1 h [21]. As for the fungal plates, spectrophotometric readings were performed using an ELISA (Termoplate®) (λ: 630 nm) where the results obtained were used to create the cell viability curve and determine IC50 values. Dilution controls with 0.9% sodium chloride instead of the inoculum, media sterility and growth controls were also performed [22]. 2.2.3. Modulatory effect of the compounds on the activity of clinically used drugs To verify the combined action of the natural product with commercial drugs, the method proposed by Coutinho et al. (2008) [23] was used, in which the product is tested at a sub-inhibitory concentration (MIC/8 – bacteria and MFC/16 – fungi). Eppendorf tubes containing the oil +10% BHI medium and the double concentrated SDB +150 μL of the microbial suspension (corresponding to 10% of the solution) were prepared. Growth and dilution controls were also prepared. The plate was filled by adding 100 μL of the solutions into each well, followed by serial microdilution with 100 μL of the drug. The plates were then incubated at 37 °C/24 h. For these bacterial tests, only the multiresistant
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strains were used. Readings were performed as described in the previous subsection. If an interaction between the drug and the oil occurred such that it reduced the MIC of the drug, the effect is said to be synergistic, otherwise it is said to be antagonistic.
Table 1 Chemical composition of the P. diospyrifolium leaf essential oil.
2.2.4. Minimal fungicidal concentration determination From the microdilution tests, a sterile rod was inserted into each well of the plate, with the medium being homogenized and subcultured in Petri dishes containing SDA (Sabouraud Dextrose Agar) and a guide of the tested concentrations. The plates were incubated at 37 °C for 24 h and the reading was performed by observing the growth of Candida colonies or their absence thereof, thus observing fungal viability (Ernst et al. [24] modified). 2.2.5. Evaluation of the fungal virulence effect With this test, the action of the oil over one of the fungal virulence factors, the formation of hyphae, can be observed. Humidified chambers were mounted and sterilized. Inside each chamber, 3 mL of depleted PDA (Potato Dextrose Agar) media containing the oil at concentrations defined by the MFC (MFC/2, MFC/4 and MFC/8) were poured onto a microscopic (sterile) slide. The inoculum was removed from the previously cultivated Petri dishes and two parallel grooves were made on the already solidified medium with a sterile cover plate being deposited over the medium. The chambers were incubated and visualized with an optical microscope (AXIO IMAGER M2-3525001980 - ZEISS - Germany) using a 20 X objective after 24 h (37 °C). Images were taken to verify the emission or inhibition of hyphae filaments. Growth and antifungal controls were also performed. Afterwards, the images were analyzed using the Zen 2.0 software in which the total length of the inoculum striae and the length of the hyphae filaments were verified for statistical analysis according to the results and sample concentrations.
Constituent
RICal
%
α – pinene α-copaene β-elemene (E)-caryophyllene γ-elemene α-guaiene α-humulene 9-epi-(E)-caryophyllene α-amorphene Germacrene D α-muurolene Germacrene A α-bulnesene (Z)-carpacin Germacrene B β-copaen-4-α-ol Globulol Guaiol 1-epi-cubenol α-muurolol Pogostol Total identified
935 1381 1397 1426 1439 1444 1459 1468 1483 1488 1502 1506 1512 1532 1566 1587 1593 1601 1638 1652 1665
1.13 4.45 1.71 7.16 1.01 2.94 1.39 1.08 1.45 5.28 2.89 6.87 3.37 24.30 3.70 1.79 1.85 1.04 2.01 1.92 9.85 87.19
RICal: Calculated Retention Index.
majority is Z-carpacin (24.30%), followed by pogostol (9.85%), E-caryophyllene (7.16%), germacrene A (6.87%), germacrene D (5.28%), αcopaene (4.45%) and germacrene B (3.70%) are displayed. Fig. 1 presents the chemical structures of the most abundant constituents in the oil. 3.2. Antibacterial activity The Minimum Inhibitory Concentration (MIC) assays did not present clinically relevant results for any of the tested strains. The concentration value obtained was ≥1024 μg/mL, meaning the oil alone presented no inhibitory action on bacterial growth at the highest tested concentration. The action of the oil at a subinhibitory concentration in association with commercial antibiotics is shown in Fig. 2. An antagonistic action was observed with the antibiotic norfloxacin against the two multiresistant strains, increasing the mean MIC of the drug from 50.79 to 101.59 for S. aureus and from 0.0 to 10.7 for E. coli. No significant action was observed for erythromycin. The association with gentamicin showed synergism against both strains since the combined action of the oil and drug obtained a reduction of the drug mean MIC from 16 to 6.34 for S. aureus and from 59.79 to 25.39 for E. coli.
2.3. Statistical analysis Data were analyzed using the GraphPad Prism 6.0 statistical program. A two-way ANOVA was applied to the sample. For bacteria the ANOVA test used the geometric mean of triplicates as the central data and the mean standard deviation. Furthermore, Bonferroni's post hoc test was performed, in which p < 0.05 and p < 0.0001 are considered significant and p > 0.05 is not significant. For the fungal data obtained by spectrophotometric readings, its normal distribution was verified and further analyzed by an ANOVA comparing the values from each natural product concentration with a Bonferroni's post hoc test. IC50 values were obtained through nonlinear regression for value interpolation from standard curves. To evaluate virulence, the total area of the striae and areas where the hyphae grew were measured. Measurements of all hyphae filaments were then taken from five randomly selected areas at each striae for each concentration. The mean length of the hyphae filaments was calculated and analyzed by an ANOVA followed by Bonferroni's correction for multiple comparisons comparing the values according to the concentration of the product.
3.3. Antifungal activity 3.3.1. Cell viability and 50% inhibitory concentration (IC50) of Candida spp. The C. albicans and C. tropicalis cellular viability can be visualized in Figs. 3 and 4, where decreases in microorganism counts as a function of concentration increases are verified. The graphs show the MIC was clinically irrelevant [30] with an inhibitory effect at a concentration of 2048 μg/mL against all tested strains. The oil at subinhibitory concentrations (MFC/16: 128 μg/mL for CA URM 4127 and 256 μg/mL for the remainder) in association with the antifungal fluconazole presented a similar viability curve to that of the drug alone against the CA INCQS 40006 strain. An antagonism was observed against the CA URM 4127 strain, where the association of the substances impaired the isolated effect of the drug over fungal growth (Fig. 3).
3. Results 3.1. Chemical constituent identification The yield from the essential oil extraction was of 0.49%. The identification of the chemical constituents was obtained by comparing their mass spectrum and their linear retention index with data from the apparatus’ library and the literature [25–27]. The constituents, their retention indices and percentages are shown in Table 1, where 21 compounds including monoterpenes and sesquiterpenes, of which the
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Fig. 1. Chemical structures of the major compounds present in the Piper diospyrifolium essential oil. Source: [28,29].
Fig. 2. Modulatory action of the Piper diospyrifolium oil (PDEO) in association with commercial antibiotics. p < 0.0001.
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Fig. 3. Antifungal action of fluconazole (FCZ) in association with the PDEO (Piper diospyrifolium essential oil) against Candida albicans (CA) strains. INCQS: National Institute of Quality Control in Health; URM: Recife Mycology University. p < 0.0001.
For C. tropicalis, concentrations ranging from 4 to 512 μg/mL against the standard strain (CT INCQS 40042) presented a synergistic effect obtaining better results than the isolated fluconazole MIC. A similar behavior can also be seen in the viability curve for the isolated strain (CT URM 4262) where the association also improved the drug's antifungal action at concentrations ranging from 64 to 2018 μg/mL, as shown in Fig. 4. When analyzing the 50% inhibitory effect over the fungal population (Table 2), the oil's potentiating effect stood out against CA INCQS 40006 and CT URM 4262, where it showed significant antifungal activity when in association with fluconazole (IC50: 94.12 μg/mL for C. albicans and 103.74 μg/mL for C. tropicalis). The IC50 values (Table 2) against CA URM 4127 and CT URM 4262, when compared to the isolated fluconazole values, emphasize the decrease or increase in antifungal action when the drug is associated with the oil. The intrinsic action of the oil did not present a relevant IC50 only inhibiting 50% of the microorganism population at high concentrations.
was observed when the oil was associated with the drug. 3.3.3. Piper diospyrifolium essential oil action over a fungal virulence factor In the fungal morphology analysis, the PDEO was tested at MFC/ 2–1024 μg/mL, MFC/4–512 μg/mL, MFC/8–256 μg/mL concentrations against the CA URM 4127 strain, while the remaining strains were tested with the MFC/2–2048 μg/mL, MFC/4–1024 μg/mL and MFC/ 8–512 μg/mL concentrations. The graphs in Fig. 5 show the oil reduced morphological transition at the highest concentrations (MFC/2 and MFC/4) for the standard and isolated C. albicans strains, respectively, by 81.7% and 96.8% of the filament growth at the full striae border seen in the growth control. At the lowest concentration, a reduction of 25.3% for CA INCQS 40006 and 12.2% for CA URM 4127 was observed. For the C. tropicalis isolate, complete filament inhibition was only seen at the highest concentration, with the MFC/4 and MFC/8 concentrations obtaining an inhibition of 14.4% and 1%, respectively, compared to the 100% mycelial growth. The CT INCQS 40042% inhibitions were: 0.97% for MFC/8; 1.7% for MFC/4; 13.4% for MFC/ 2, showing the C. tropicalis strains are more resistant to the oil's actions. The best results for fluconazole were against the CA INCQS 40006 strain completely inhibiting the fungal virulence factor at the highest concentrations (MFC/2 and MFC/4), while for CT INCQS 40042 a 63.3% inhibition and a 51.5% inhibition for CT URM 4262 were observed, both at the MFC/2 concentration. For CA URM 4127, the drug acted best with the MFC/8 concentration obtaining 57.1% inhibition compared to the growth control. In general terms, the oil presented a better effect than fluconazole against Candida albicans.
3.3.2. Minimal fungicidal concentration (MFC) The MFC was considered as the concentration which caused the absence (fungicidal effect) or inhibition (fungistatic effect) of colony growth. The oil presented a fungicidal effect only against the CA URM 4127 strain with a MFC value of 2048 μg/mL, obtaining fungistatic actions (MFC ≥ 4096 μg/mL) for the other tested strains. When the oil was associated with fluconazole against the CT URM 4262 strain, this combination became sensitized and a fungicidal effect was observed at the 2048 μg/mL concentration. A similar effect was not observed for the other tested strains. Moreover, for CA URM 4127 an antagonistic effect
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Fig. 4. Antifungal action of fluconazole (FCZ) in association with the PDEO (Piper diospyrifolium essential oil) against Candida tropicalis (CT) strains. INCQS: National Institute of Quality Control in Health; URM: Recife Mycology University. p < 0.0001.
from both the leaves and the fruits in the aforementioned study have compounds similar to those obtained in this study in their composition (presented in Table 1), however at different quantities, having a greater amount of the compounds in common and therefore being richer. The specimens tested by Morandim et al. (2010) [1] were collected in São Paulo where the collection area, climate, soil and different annual seasons may be the cause of the distinct chemical variety present in the species [31,32]. Divergent results were also observed by Vieira et al. (2011) [14], whose leaves collected in Maringá, PR, Brazil, possessed the sesquiterpene (E)-eudesma-6,11-diene as its major compound, as well as by Bernuci et al. (2016) [5] who collected leaves from AntoninaPR where the Selin-11-en-4-α-ol sesquiterpene was the major compound. Carpacin is found in the plant kingdom and studies report its use in the manufacture of insecticides due to its toxicity to insect eggs [33–35]. Additionally, antimicrobial activities can also be verified, not only for carpacin, but also for the compounds pogostol and E-Caryophyllene [36–40], found in abundance in the P. diospyrifolium oil, with some studies being discussed below. Abdollahnejad et al. (2016) [41] used the Artemisia dracunlus essential oil, which is rich in carpacin, against bacterial strains obtaining significant results for Streptococcus pyogenes (12 mm) and E. coli (6 mm) when compared to the amikacin control (10.5 mm, 24.5 mm) with the disk diffusion method. The action of pogostol and E-caryophyllene was seen by Adhavan et al. (2017) [39] when working with the Pogostemon cablin and Pogostemon heyneanus essential oils, where their action was promising, obtaining MIC values ranging from 3.2 to 25 mg/mL against Shigella flexneri, S. aureus, S. mutans and C. albicans strains. Nafis et al.
Table 2 The 50% inhibitory effect (IC50) (μg/mL) of the Piper diospyrifolium essential oil over fungal populations against different Candida spp. strains.
PDEO FLUCONAZOLE PDEO + FCZ
CA INCQS 40006
CA URM 4127
CT INCQS 40042
CT URM 4262
950.85 111.95 94.12
1206.93 12.18 74.95
1520.99 1314.13 1373.46
1260.97 149.3 103.74
FCZ: Fluconazole; PDEO: Piper diospyrifolium essential oil; CA: Candida albicans; CT: Candida tropicalis; INCQS: National Institute of Quality Control in Health; URM: Recife Mycology University.
Fig. 6 shows the progressive effect of the oil on fungal morphology. The CA URM 4127 clinical isolate growth control is shown in photo A where mycelial formation is apparent, with extensive hyphae as indicated by the arrows. Photo B demonstrates the effect of the MFC/8 concentration where a significant filament reduction is noted. Photos C and D correspond to the MFC/4 and MFC/2 concentrations where in these mycelia formation is no longer observed, verifying the fungus has remained in its yeast form. 4. Discussion The yield obtained for the essential oil (0.49%) in this study was lower than those obtained by Morandim et al. (2010) [1] (1.46%), where both studies used fresh leaves. It is noteworthy the author also studied the P. diospyrifolium fruit essential oil (1.40%). The essential oils
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The antibacterial activity of the P. diospyrifolium leaf essential oil was evaluated by Bernuci et al. (2016) [5], where the results against the Mycobacterium tuberculosis bacterium displayed growth inhibition from the 125 μg/mL concentration. An antifungal activity was also seen by Vieira et al. (2011) [14] who evaluated the action of the pure oil extracted from the leaves of the same plant. The results seen using the disc diffusion method were promising against C. albicans, C. parapsilosis and C. tropicalis strains presenting inhibition halos ranging from 8 mm to 15 mm in diameter. The susceptibility of a microorganism to an essential oil depends on the properties of the oil and the characteristics of the microorganism itself. The effect of the P. diospyrifolium oil may be attributed to its interaction with amphiphilic peptides in the cell membrane destabilizing these and resulting in cellular lysis, damages which have been observed in studies using essential oils [43–49]. The quantities of each compound present in an oil displaying antimicrobial activity may also play an important role in the effectiveness of the oil [50]. The Piper genus has been widely studied as a source of active compounds with antimicrobial activity, as seen in the studies cited below. Salleh et al. (2011) [51] proved the effective action of P. caninum essential oils (leaves and stems) against B. subtilis, S. aureus, P. aeruginosa, Pseudomonas putida, E. coli, and its moderate activity against C. albicans and Aspergillus niger fungi. The Piper divaricatum species was analyzed by Barbosa et al. (2012) [52], who used the leaf and fruit essential oils against E. coli, P. aeruginosa, Salmonella enterica Ser. Typhimurium, Listeria monocytogenes, Bacillus cereus and S. aureus bacterial strains obtaining inhibitory results against all strains. Gasparetto et al. (2016) [53] described promising P. cernuum essential oil activity against S. aureus, Streptococcus pyogenes, B. subtilis bacteria and Microsporum gypseum, Trichophyton mentagrophytes, Trichophyton rubrum, Epidermophyton flocosum and Criptococcus neoformans fungi. Morandim et al. (2010) [1] also obtained an effective action with the P. cernuum fruit oil and the P. solmsianum leaf oil against Cladosporium sphaerospermum and Cladosporium cladosporioides phytopathogenic fungi. Fungal morphology analysis permits the observation of the oil against an important virulence factor in the development of candidiasis. Candida species belong to an individual's natural microbiota and in situations of homeostatic imbalance they become responsible for the development of various infections, especially in individuals with immunosuppressive diseases [54]. Essential oils are known in the literature for their activity against Candida spp. virulence attributes. The Piper rivinoides leaf oil (4096 μg/ mL) was evaluated against C. albicans and C. tropicalis mycelial formation, obtaining complete inhibition of filament emission [55]. Macêdo et al. (2018) [31] observed C. albicans hyphal and pseudo-hyphal inhibition using the Psidium guajava essential oil (8192 μg/mL). The Eugenia uniflora oil (8192 μg/mL) was studied with respect to C. albicans, C. tropicalis and C. krusei morphological transitions, displaying an inhibitory effect against C. albicans and C. tropicalis, whilst being inefficient against C. krusei [56]. Using a method similar to that of this study, Sousa et al. (2016) [57] evaluated the effect of the Coriandrum sativum leaf essential oil (1024 μg/mL) over C. albicans germinal tube inhibition, where an inhibition was observed with the 512 μg/mL concentration. It is extremely important to inhibit virulence processes without the death of the commensal microorganism such that evolutionary pressures, which contribute to microbial resistance, are smaller and the ecological balance of the microbiota is not impaired [58]. Thus, studies searching for drugs containing fungal resistance without causing damages to the host have intensified. In this study the PDEO has been shown as an alternative for the development of new drugs due to its associated action and its ability to contain fungal pleomorphism.
Fig. 5. Inhibitory effect of the antifungal fluconazole and of the Piper diospyrifolium essential oil (PDEO) on Candida spp. mycelial growth. CA: Candida albicans; CT: Candida tropicalis; INCQS: National Institute of Quality Control in Health; URM: Recife Mycology University; MFC/2–2048 μg/mL, MFC/ 4–1024 μg/mL, MFC/8–512 μg/mL, p < 0.0001.
(2019) [42] evaluated the antimicrobial effect of the Cannabis sativa oil, which presents E-Caryophyllene (35%) as its major compound, where the best inhibitory effects were obtained against E. coli (11.1 mm), Micrococcus luteus (11 mm), Bacillus subtilis (13 mm), S. aureus (13 mm), C. albicans (12 mm) and C. krusei (12.5 mm) compared to the drugs ciprofloxacin (12 mm, 26.3 mm, 35 mm, 27.5 mm) and fluconazole (20 mm, 24 mm), respectively, when using the disc diffusion method.
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Fig. 6. Demonstration of the progressive inhibitory effect of the Piper diospyrifolium essential oil on Candida spp. morphological transition. The arrows indicate the formation of hyphae in the growth control (A), and its progressive reduction (B, C, D) as the concentration of the oil increases (B- MFC/8, C- MFC/4, DMFC/2). Scale: 50 μm.
5. Conclusion
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The antimicrobial action of the P. diospyrifolium essential oil was not promising against the studied strains. The combined action of the oil with commercial drugs presented a synergistic action against bacteria, although fungi did not obtain clinically relevant results. Inhibitory actions were observed against the formation of invasive filaments, an important factor of fungal pathogenicity for both Candida species. The oil was predominantly composed of mono- and sesquiterpenes, compounds already known in the literature for their antimicrobial effects, which may have collectively contributed to the oil's effect. Further research is needed to understand the action of the oil on microorganisms and the metabolic and genetic processes associated with fungal virulence inhibition. Declaration of interest The authors declare that there are no conflicts of interest regarding the publication of this paper. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.micpath.2019.103700. References [1] A.D.A. Morandim, A.R. Pin, N.A. Pietro, A.C. Alecio, M.J. Kato, C.M. Young, J.E. Oliveira, M. Furlan, Composition and screening of antifungal activity against Cladosporium sphaerospermum and Cladosporium cladosporioides of essential oils of leaves and fruits of Piper species, Afr. J. Biotechnol. 9 (37) (2010) 6135–6139. [2] R.J. Pereira, M.G. Cardoso, Metabólitos secundários vegetais e benefícios antioxidantes, J. Biotechnol. Biodiversity 3 (4) (2012). [3] K.S. Moresco, A.K. Silveira, F. Zeidán-Chuliá, A.P.F. Correa, R.R. Oliveria, A.G. Borges, L. Grun, F. Barbé-Tuana, A. Zmonzinski, A. Brandelli, M.G.R. Vale, D.P. Gelain, V.L. Bassani, J.C.F. Moreira, Effects of Achyrocline satureioides inflorescence extracts against pathogenic intestinal bacteria: chemical characterization, in vitro tests, and in vivo evaluation, Evid. Based Complement Altern. Med.
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Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Piper diospyrifolium Kunth.: Chemical analysis and antimicrobial (intrinsic and combined) activities
T
Joara Nályda Pereira Carneiroa, Rafael Pereira da Cruza, Josefa Carolaine Pereira da Silvaa, Janaína Esmeraldo Rochaa, Thiago Sampaio de Freitasa, Débora Lima Salesa, Camila Fonseca Bezerraa, Waltécio de Oliveira Almeidaa, José Galberto Martins da Costaa, Luiz Everson da Silvab, Wanderlei do Amaralb, Ricardo Andrade Rebeloc, Ieda Maria Begninic, Henrique Douglas Melo Coutinhoa,∗, Maria Flaviana Bezerra Morais-Bragaa a
Universidade Regional do Cariri, URCA, Cel Antônio Luis, 1161, 63105-000, Pimenta, Crato, CE, Brazil Universidade Federal do Paraná, UFPR, XV de Novembro, 1299, 80.060-000, Centro, Curitiba, PR, Brazil c Universidade Regional de Blumenau, FURB, Antônio da Veiga, 140, 89030-903, Itoupava Seca, Blumenau, SC, Brazil b
ARTICLE INFO
ABSTRACT
Keywords: Fluconazole Virulence Escherichia coli Z-Carpacin Synergism Cell viability
The secular use of plants in popular medicine has emerged as a source for the discovery of new compounds capable of curing infections. Among microbial resistance to commercial drugs, species such as Piper diospyrifolium Kunth, which are used in popular therapy, are targets for pharmacological studies. With this in mind, antimicrobial experiments with the essential oil from the P. diospyrifolium (PDEO) species were performed and its constituents were elucidated. The oil compounds were identified by gas chromatography coupled to mass spectrometry (GC/MS). The broth microdilution method with colorimetric readings for bacterial tests (Escherichia coli and Staphylococcus aureus) and spectrophotometric readings for fungal tests (Candida albicans and Candida tropicalis), whose data were used to create a cell viability curve and calculate its IC50 against fungal cells, were used to determine the minimum inhibitory concentration of the oil and its combined action with commercial drugs. The oil's minimal fungicidal concentration and its action over fungal morphological transition were analyzed by subculture and microculture, respectively. Chemical analysis revealed Z-Carpacin, Pogostol and E-Caryophyllene as the most abundant compounds. Results from the intrinsic analysis were considered clinically irrelevant, however the oil presented a synergistic effect against multiresistant E. coli and S. aureus strains when associated with gentamicin, and against the standard and isolated C. tropicalis strains with fluconazole. A fungicidal effect was observed against the C. albicans isolate. Candida spp. hyphae inhibition was verified for all strains at the highest tested concentrations. The P. diospyrifolium essential oil presented a promising effect when associated with commercial drugs and against a fungal virulence factor. Thus, the oil presented active compounds which may help the development of new drugs, however, new studies are needed in order to clarify the oil's mechanism of action, as well as to identify its active constituents.
1. Introduction Plants are traditionally used in popular medicine and have been an alternative worth exploring, especially as a source of natural antimicrobials [1]. The richness of their compounds, termed secondary metabolites, has been shown to be effective at combating the most varied of infections [2]. The increase in number of microbial infections is directly associated with the irrational use of antimicrobials, this being one of the major ∗
causes of the emergence of multiresistant microorganisms [3]. Antimicrobial resistance increases morbidity and mortality for patients with infections, while a considerable increase in costs for healthcare institutions also exists [4]. The Piperaceae family possesses species distributed across tropical and subtropical regions in the northern and southern hemispheres, including herbaceous and shrubby plants. The Piper genus is the largest and most well-known for its production of plant extracts, including essential oils abundant in secondary metabolites, specifically those
Corresponding author. E-mail address: [email protected] (H.D. Melo Coutinho).
https://doi.org/10.1016/j.micpath.2019.103700 Received 19 July 2019; Received in revised form 27 August 2019; Accepted 28 August 2019 Available online 28 August 2019 0882-4010/ © 2019 Elsevier Ltd. All rights reserved.
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derived from the shikimate pathway such as neolignans, phenylpropanoids and flavonoids, among others [5,6]. Species from the Piper genus are used in popular medicine for the treatment of diarrhea [7], gastrointestinal disorders, stomach aches [8], throat diseases, coughs [9], gynecological and urological infections, as well as flatulence [10,11]. The studied species, Piper diospyrifolium, possesses antibacterial [5,12], antifungal [13,14] and leishmanicidal activity [5], among others, that have been scientifically proven. While faced with the resistance phenomenon and with the search for new antimicrobial agents, the scientific community has turned its attention to the use of natural products, especially those originating from plants with medicinal indications [15]. Although P. diospyrifolium has been widely used over many generations, its chemical and pharmacological properties have not yet been fully characterized [3]. The objective of this study was to investigate the chemical composition of the P. diospyrifolium Kunth. essential oil, thus elucidating its chemical profile, and to validate its pharmacological properties and antimicrobial effects against standard and resistant Escherichia coli and Staphylococcus aureus bacteria, as well as Candida albicans and Candida tropicalis yeasts.
2.2. Microbiological test 2.2.1. Microbial strains and solution preparations Standard strains obtained from the American Type Culture Collection (ATCC) and clinical isolates obtained from the Laboratory of Microbiology of the Federal University of Paraíba, namely E. coli ATCC 25922, E. coli 06, S. aureus ATCC 6538 and S. aureus 10, were used. The C. albicans INCQS 40006, C. albicans URM 4127, C. tropicalis INCQS 40042 and C. tropicalis URM 4262 fungal strains were used, where the standard strains were obtained from the Collection of Reference Microorganisms in Sanitary Surveillance - CMRVS, from FIOCRUZ -INCQS (National Institute of Quality Control in Health), Rio de Janeiro, while clinical isolates were obtained from the library of the Federal University of Recife (URM - University Recife Mycology). Bacterial and fungal cultures were revived in Petri dishes containing their respective culture media: HIA (Heart Infusion Agar) for bacteria and SDA (Sabouraud Dextrose Agar) for fungi, which were then placed in an incubator at 37 °C for 24 h. Following this period, a suspension was made by removing one border of each microbial culture and placing it in tubes with 4 mL of 0.9% Physiological Serum (PS). The inoculum concentration was standardized using the 0.5 McFarland scale turbidity (1 × 108 CFU/mL) [19]. Stock solutions from the essential oil were obtained by weighing 10 mg (10,000 μg) and 5 mg (50,000 μg) of the oil and diluting these in 1 mL of dimethyl sulfoxide (DMSO). Subsequently, these solutions were diluted in distilled sterile water to prevent the DMSO from exerting an activity over the tested cells, until reaching the 1024 μg/mL concentration for bacteria and 4096 μg/mL for fungi [20]. The reference drugs (norfloxacin, gentamicin, erythromycin and fluconazole) were diluted in distilled sterile water until norfloxacin, gentamicin and erythromycin reached an initial concentration of 1024 μg/mL and fluconazole a concentration of 4096 μg/mL.
2. Materials and methods 2.1. Essential oil extraction and chemical composition The essential oil was kindly gifted by professor Dr. Luiz Everson da Silva from the Federal University of Paraná - UFPR - Brazil, where the oil was extracted and subjected to gas chromatography coupled to mass spectrometry (GC-MS) for further quantification and identification of its constituents. Initially, the plant material was collected from the coast of Paraná, Brazil (S 25° 13.644′, W 48° 34.985’ and altitude of 6 m). Exsiccates from the collected specimens were produced and taken to the Herbarium of the Municipal Botanical Museum of Curitiba (MBM), where they were herborized (HFIE) under number MBM 396413 [16,17]. The leaves were selected and dried (electric dryer model FANEM Mod. 320 SE with air circulation at 40 °C for 24 h) for the essential oil preparation. After weighing 50 g of the leaves, the plant material was subjected to hydrodistillation in 1 L of distilled water in a Clevenger type apparatus for 4.5 h. This process was performed in triplicates, based on the Wasicky method (1963) [18]. Following extraction, the samples were kept in a freezer until needed for analysis. The total essential oil mass produced in relation to the botanical material dry mass used in the extraction process was measured to determine the dry essential oil content. For the GC/MS chemical constituent identification analysis, the oil solution and 1% dichloromethane were injected with a 1:20 flow division in an Agilent 6890 (Palo Alto, CA) chromatograph coupled to an Agilent 5973 N selective mass detector at 250 °C. The HP-5MS capillary column (5%-phenyl-95%-dimethylpolysiloxane, 30 m × 0.25 mm x 0.25 μm) was used with helium as the carrier gas (1.0 mL min−1). The chamber temperature was programmed from 60 to 240 °C at a rate of 3 °C min−1. The mass detector operated in the electronic ionization mode (70 eV) at a rate of 3.15 s−1 scans with a mass range of 40–450. The transfer line was maintained at 260 °C, the ion source at 230 °C and the analyzer (quadrupole) at 150 °C. For quantification, the sample was injected into an Agilent 7890A chromatograph equipped with a flame ionization detector (FID) operating at 280 °C under the same analytical conditions, except for the carrier gas where hydrogen was used instead at a flow rate of 1.5 mL min−1. The percentage composition was obtained by FID signal electronic integration, dividing the area of each component by the total area (area %).
2.2.2. Minimal inhibitory concentration determination Test eppendorfs were prepared containing 1350 μL of 10% BHI (Brain Heart Infusion Broth) and double concentrated SDB (Sabouraud Dextrose Broth) plus 150 μL of the inoculum (corresponding to 10% of the total solution) for bacterial and fungal strains, respectively. This solution was then distributed in microdilution plates (100 μL/well) before serial dilutions were performed with 100 μL of the oil solution per column with concentrations ranging from 512 μg/mL to 0.5 μg/mL for bacteria, and from 2048 μg/mL to 2 μg/mL for fungi. The microdilutions were performed in triplicates and quadruplicates, respectively. The plates were taken to an incubator at 37 °C/24 h. The bacterial plates were read by adding 20 μL resazurin (colorimetric reagent) in each well and visually observing these after 1 h [21]. As for the fungal plates, spectrophotometric readings were performed using an ELISA (Termoplate®) (λ: 630 nm) where the results obtained were used to create the cell viability curve and determine IC50 values. Dilution controls with 0.9% sodium chloride instead of the inoculum, media sterility and growth controls were also performed [22]. 2.2.3. Modulatory effect of the compounds on the activity of clinically used drugs To verify the combined action of the natural product with commercial drugs, the method proposed by Coutinho et al. (2008) [23] was used, in which the product is tested at a sub-inhibitory concentration (MIC/8 – bacteria and MFC/16 – fungi). Eppendorf tubes containing the oil +10% BHI medium and the double concentrated SDB +150 μL of the microbial suspension (corresponding to 10% of the solution) were prepared. Growth and dilution controls were also prepared. The plate was filled by adding 100 μL of the solutions into each well, followed by serial microdilution with 100 μL of the drug. The plates were then incubated at 37 °C/24 h. For these bacterial tests, only the multiresistant
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strains were used. Readings were performed as described in the previous subsection. If an interaction between the drug and the oil occurred such that it reduced the MIC of the drug, the effect is said to be synergistic, otherwise it is said to be antagonistic.
Table 1 Chemical composition of the P. diospyrifolium leaf essential oil.
2.2.4. Minimal fungicidal concentration determination From the microdilution tests, a sterile rod was inserted into each well of the plate, with the medium being homogenized and subcultured in Petri dishes containing SDA (Sabouraud Dextrose Agar) and a guide of the tested concentrations. The plates were incubated at 37 °C for 24 h and the reading was performed by observing the growth of Candida colonies or their absence thereof, thus observing fungal viability (Ernst et al. [24] modified). 2.2.5. Evaluation of the fungal virulence effect With this test, the action of the oil over one of the fungal virulence factors, the formation of hyphae, can be observed. Humidified chambers were mounted and sterilized. Inside each chamber, 3 mL of depleted PDA (Potato Dextrose Agar) media containing the oil at concentrations defined by the MFC (MFC/2, MFC/4 and MFC/8) were poured onto a microscopic (sterile) slide. The inoculum was removed from the previously cultivated Petri dishes and two parallel grooves were made on the already solidified medium with a sterile cover plate being deposited over the medium. The chambers were incubated and visualized with an optical microscope (AXIO IMAGER M2-3525001980 - ZEISS - Germany) using a 20 X objective after 24 h (37 °C). Images were taken to verify the emission or inhibition of hyphae filaments. Growth and antifungal controls were also performed. Afterwards, the images were analyzed using the Zen 2.0 software in which the total length of the inoculum striae and the length of the hyphae filaments were verified for statistical analysis according to the results and sample concentrations.
Constituent
RICal
%
α – pinene α-copaene β-elemene (E)-caryophyllene γ-elemene α-guaiene α-humulene 9-epi-(E)-caryophyllene α-amorphene Germacrene D α-muurolene Germacrene A α-bulnesene (Z)-carpacin Germacrene B β-copaen-4-α-ol Globulol Guaiol 1-epi-cubenol α-muurolol Pogostol Total identified
935 1381 1397 1426 1439 1444 1459 1468 1483 1488 1502 1506 1512 1532 1566 1587 1593 1601 1638 1652 1665
1.13 4.45 1.71 7.16 1.01 2.94 1.39 1.08 1.45 5.28 2.89 6.87 3.37 24.30 3.70 1.79 1.85 1.04 2.01 1.92 9.85 87.19
RICal: Calculated Retention Index.
majority is Z-carpacin (24.30%), followed by pogostol (9.85%), E-caryophyllene (7.16%), germacrene A (6.87%), germacrene D (5.28%), αcopaene (4.45%) and germacrene B (3.70%) are displayed. Fig. 1 presents the chemical structures of the most abundant constituents in the oil. 3.2. Antibacterial activity The Minimum Inhibitory Concentration (MIC) assays did not present clinically relevant results for any of the tested strains. The concentration value obtained was ≥1024 μg/mL, meaning the oil alone presented no inhibitory action on bacterial growth at the highest tested concentration. The action of the oil at a subinhibitory concentration in association with commercial antibiotics is shown in Fig. 2. An antagonistic action was observed with the antibiotic norfloxacin against the two multiresistant strains, increasing the mean MIC of the drug from 50.79 to 101.59 for S. aureus and from 0.0 to 10.7 for E. coli. No significant action was observed for erythromycin. The association with gentamicin showed synergism against both strains since the combined action of the oil and drug obtained a reduction of the drug mean MIC from 16 to 6.34 for S. aureus and from 59.79 to 25.39 for E. coli.
2.3. Statistical analysis Data were analyzed using the GraphPad Prism 6.0 statistical program. A two-way ANOVA was applied to the sample. For bacteria the ANOVA test used the geometric mean of triplicates as the central data and the mean standard deviation. Furthermore, Bonferroni's post hoc test was performed, in which p < 0.05 and p < 0.0001 are considered significant and p > 0.05 is not significant. For the fungal data obtained by spectrophotometric readings, its normal distribution was verified and further analyzed by an ANOVA comparing the values from each natural product concentration with a Bonferroni's post hoc test. IC50 values were obtained through nonlinear regression for value interpolation from standard curves. To evaluate virulence, the total area of the striae and areas where the hyphae grew were measured. Measurements of all hyphae filaments were then taken from five randomly selected areas at each striae for each concentration. The mean length of the hyphae filaments was calculated and analyzed by an ANOVA followed by Bonferroni's correction for multiple comparisons comparing the values according to the concentration of the product.
3.3. Antifungal activity 3.3.1. Cell viability and 50% inhibitory concentration (IC50) of Candida spp. The C. albicans and C. tropicalis cellular viability can be visualized in Figs. 3 and 4, where decreases in microorganism counts as a function of concentration increases are verified. The graphs show the MIC was clinically irrelevant [30] with an inhibitory effect at a concentration of 2048 μg/mL against all tested strains. The oil at subinhibitory concentrations (MFC/16: 128 μg/mL for CA URM 4127 and 256 μg/mL for the remainder) in association with the antifungal fluconazole presented a similar viability curve to that of the drug alone against the CA INCQS 40006 strain. An antagonism was observed against the CA URM 4127 strain, where the association of the substances impaired the isolated effect of the drug over fungal growth (Fig. 3).
3. Results 3.1. Chemical constituent identification The yield from the essential oil extraction was of 0.49%. The identification of the chemical constituents was obtained by comparing their mass spectrum and their linear retention index with data from the apparatus’ library and the literature [25–27]. The constituents, their retention indices and percentages are shown in Table 1, where 21 compounds including monoterpenes and sesquiterpenes, of which the
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Fig. 1. Chemical structures of the major compounds present in the Piper diospyrifolium essential oil. Source: [28,29].
Fig. 2. Modulatory action of the Piper diospyrifolium oil (PDEO) in association with commercial antibiotics. p < 0.0001.
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Fig. 3. Antifungal action of fluconazole (FCZ) in association with the PDEO (Piper diospyrifolium essential oil) against Candida albicans (CA) strains. INCQS: National Institute of Quality Control in Health; URM: Recife Mycology University. p < 0.0001.
For C. tropicalis, concentrations ranging from 4 to 512 μg/mL against the standard strain (CT INCQS 40042) presented a synergistic effect obtaining better results than the isolated fluconazole MIC. A similar behavior can also be seen in the viability curve for the isolated strain (CT URM 4262) where the association also improved the drug's antifungal action at concentrations ranging from 64 to 2018 μg/mL, as shown in Fig. 4. When analyzing the 50% inhibitory effect over the fungal population (Table 2), the oil's potentiating effect stood out against CA INCQS 40006 and CT URM 4262, where it showed significant antifungal activity when in association with fluconazole (IC50: 94.12 μg/mL for C. albicans and 103.74 μg/mL for C. tropicalis). The IC50 values (Table 2) against CA URM 4127 and CT URM 4262, when compared to the isolated fluconazole values, emphasize the decrease or increase in antifungal action when the drug is associated with the oil. The intrinsic action of the oil did not present a relevant IC50 only inhibiting 50% of the microorganism population at high concentrations.
was observed when the oil was associated with the drug. 3.3.3. Piper diospyrifolium essential oil action over a fungal virulence factor In the fungal morphology analysis, the PDEO was tested at MFC/ 2–1024 μg/mL, MFC/4–512 μg/mL, MFC/8–256 μg/mL concentrations against the CA URM 4127 strain, while the remaining strains were tested with the MFC/2–2048 μg/mL, MFC/4–1024 μg/mL and MFC/ 8–512 μg/mL concentrations. The graphs in Fig. 5 show the oil reduced morphological transition at the highest concentrations (MFC/2 and MFC/4) for the standard and isolated C. albicans strains, respectively, by 81.7% and 96.8% of the filament growth at the full striae border seen in the growth control. At the lowest concentration, a reduction of 25.3% for CA INCQS 40006 and 12.2% for CA URM 4127 was observed. For the C. tropicalis isolate, complete filament inhibition was only seen at the highest concentration, with the MFC/4 and MFC/8 concentrations obtaining an inhibition of 14.4% and 1%, respectively, compared to the 100% mycelial growth. The CT INCQS 40042% inhibitions were: 0.97% for MFC/8; 1.7% for MFC/4; 13.4% for MFC/ 2, showing the C. tropicalis strains are more resistant to the oil's actions. The best results for fluconazole were against the CA INCQS 40006 strain completely inhibiting the fungal virulence factor at the highest concentrations (MFC/2 and MFC/4), while for CT INCQS 40042 a 63.3% inhibition and a 51.5% inhibition for CT URM 4262 were observed, both at the MFC/2 concentration. For CA URM 4127, the drug acted best with the MFC/8 concentration obtaining 57.1% inhibition compared to the growth control. In general terms, the oil presented a better effect than fluconazole against Candida albicans.
3.3.2. Minimal fungicidal concentration (MFC) The MFC was considered as the concentration which caused the absence (fungicidal effect) or inhibition (fungistatic effect) of colony growth. The oil presented a fungicidal effect only against the CA URM 4127 strain with a MFC value of 2048 μg/mL, obtaining fungistatic actions (MFC ≥ 4096 μg/mL) for the other tested strains. When the oil was associated with fluconazole against the CT URM 4262 strain, this combination became sensitized and a fungicidal effect was observed at the 2048 μg/mL concentration. A similar effect was not observed for the other tested strains. Moreover, for CA URM 4127 an antagonistic effect
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Fig. 4. Antifungal action of fluconazole (FCZ) in association with the PDEO (Piper diospyrifolium essential oil) against Candida tropicalis (CT) strains. INCQS: National Institute of Quality Control in Health; URM: Recife Mycology University. p < 0.0001.
from both the leaves and the fruits in the aforementioned study have compounds similar to those obtained in this study in their composition (presented in Table 1), however at different quantities, having a greater amount of the compounds in common and therefore being richer. The specimens tested by Morandim et al. (2010) [1] were collected in São Paulo where the collection area, climate, soil and different annual seasons may be the cause of the distinct chemical variety present in the species [31,32]. Divergent results were also observed by Vieira et al. (2011) [14], whose leaves collected in Maringá, PR, Brazil, possessed the sesquiterpene (E)-eudesma-6,11-diene as its major compound, as well as by Bernuci et al. (2016) [5] who collected leaves from AntoninaPR where the Selin-11-en-4-α-ol sesquiterpene was the major compound. Carpacin is found in the plant kingdom and studies report its use in the manufacture of insecticides due to its toxicity to insect eggs [33–35]. Additionally, antimicrobial activities can also be verified, not only for carpacin, but also for the compounds pogostol and E-Caryophyllene [36–40], found in abundance in the P. diospyrifolium oil, with some studies being discussed below. Abdollahnejad et al. (2016) [41] used the Artemisia dracunlus essential oil, which is rich in carpacin, against bacterial strains obtaining significant results for Streptococcus pyogenes (12 mm) and E. coli (6 mm) when compared to the amikacin control (10.5 mm, 24.5 mm) with the disk diffusion method. The action of pogostol and E-caryophyllene was seen by Adhavan et al. (2017) [39] when working with the Pogostemon cablin and Pogostemon heyneanus essential oils, where their action was promising, obtaining MIC values ranging from 3.2 to 25 mg/mL against Shigella flexneri, S. aureus, S. mutans and C. albicans strains. Nafis et al.
Table 2 The 50% inhibitory effect (IC50) (μg/mL) of the Piper diospyrifolium essential oil over fungal populations against different Candida spp. strains.
PDEO FLUCONAZOLE PDEO + FCZ
CA INCQS 40006
CA URM 4127
CT INCQS 40042
CT URM 4262
950.85 111.95 94.12
1206.93 12.18 74.95
1520.99 1314.13 1373.46
1260.97 149.3 103.74
FCZ: Fluconazole; PDEO: Piper diospyrifolium essential oil; CA: Candida albicans; CT: Candida tropicalis; INCQS: National Institute of Quality Control in Health; URM: Recife Mycology University.
Fig. 6 shows the progressive effect of the oil on fungal morphology. The CA URM 4127 clinical isolate growth control is shown in photo A where mycelial formation is apparent, with extensive hyphae as indicated by the arrows. Photo B demonstrates the effect of the MFC/8 concentration where a significant filament reduction is noted. Photos C and D correspond to the MFC/4 and MFC/2 concentrations where in these mycelia formation is no longer observed, verifying the fungus has remained in its yeast form. 4. Discussion The yield obtained for the essential oil (0.49%) in this study was lower than those obtained by Morandim et al. (2010) [1] (1.46%), where both studies used fresh leaves. It is noteworthy the author also studied the P. diospyrifolium fruit essential oil (1.40%). The essential oils
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The antibacterial activity of the P. diospyrifolium leaf essential oil was evaluated by Bernuci et al. (2016) [5], where the results against the Mycobacterium tuberculosis bacterium displayed growth inhibition from the 125 μg/mL concentration. An antifungal activity was also seen by Vieira et al. (2011) [14] who evaluated the action of the pure oil extracted from the leaves of the same plant. The results seen using the disc diffusion method were promising against C. albicans, C. parapsilosis and C. tropicalis strains presenting inhibition halos ranging from 8 mm to 15 mm in diameter. The susceptibility of a microorganism to an essential oil depends on the properties of the oil and the characteristics of the microorganism itself. The effect of the P. diospyrifolium oil may be attributed to its interaction with amphiphilic peptides in the cell membrane destabilizing these and resulting in cellular lysis, damages which have been observed in studies using essential oils [43–49]. The quantities of each compound present in an oil displaying antimicrobial activity may also play an important role in the effectiveness of the oil [50]. The Piper genus has been widely studied as a source of active compounds with antimicrobial activity, as seen in the studies cited below. Salleh et al. (2011) [51] proved the effective action of P. caninum essential oils (leaves and stems) against B. subtilis, S. aureus, P. aeruginosa, Pseudomonas putida, E. coli, and its moderate activity against C. albicans and Aspergillus niger fungi. The Piper divaricatum species was analyzed by Barbosa et al. (2012) [52], who used the leaf and fruit essential oils against E. coli, P. aeruginosa, Salmonella enterica Ser. Typhimurium, Listeria monocytogenes, Bacillus cereus and S. aureus bacterial strains obtaining inhibitory results against all strains. Gasparetto et al. (2016) [53] described promising P. cernuum essential oil activity against S. aureus, Streptococcus pyogenes, B. subtilis bacteria and Microsporum gypseum, Trichophyton mentagrophytes, Trichophyton rubrum, Epidermophyton flocosum and Criptococcus neoformans fungi. Morandim et al. (2010) [1] also obtained an effective action with the P. cernuum fruit oil and the P. solmsianum leaf oil against Cladosporium sphaerospermum and Cladosporium cladosporioides phytopathogenic fungi. Fungal morphology analysis permits the observation of the oil against an important virulence factor in the development of candidiasis. Candida species belong to an individual's natural microbiota and in situations of homeostatic imbalance they become responsible for the development of various infections, especially in individuals with immunosuppressive diseases [54]. Essential oils are known in the literature for their activity against Candida spp. virulence attributes. The Piper rivinoides leaf oil (4096 μg/ mL) was evaluated against C. albicans and C. tropicalis mycelial formation, obtaining complete inhibition of filament emission [55]. Macêdo et al. (2018) [31] observed C. albicans hyphal and pseudo-hyphal inhibition using the Psidium guajava essential oil (8192 μg/mL). The Eugenia uniflora oil (8192 μg/mL) was studied with respect to C. albicans, C. tropicalis and C. krusei morphological transitions, displaying an inhibitory effect against C. albicans and C. tropicalis, whilst being inefficient against C. krusei [56]. Using a method similar to that of this study, Sousa et al. (2016) [57] evaluated the effect of the Coriandrum sativum leaf essential oil (1024 μg/mL) over C. albicans germinal tube inhibition, where an inhibition was observed with the 512 μg/mL concentration. It is extremely important to inhibit virulence processes without the death of the commensal microorganism such that evolutionary pressures, which contribute to microbial resistance, are smaller and the ecological balance of the microbiota is not impaired [58]. Thus, studies searching for drugs containing fungal resistance without causing damages to the host have intensified. In this study the PDEO has been shown as an alternative for the development of new drugs due to its associated action and its ability to contain fungal pleomorphism.
Fig. 5. Inhibitory effect of the antifungal fluconazole and of the Piper diospyrifolium essential oil (PDEO) on Candida spp. mycelial growth. CA: Candida albicans; CT: Candida tropicalis; INCQS: National Institute of Quality Control in Health; URM: Recife Mycology University; MFC/2–2048 μg/mL, MFC/ 4–1024 μg/mL, MFC/8–512 μg/mL, p < 0.0001.
(2019) [42] evaluated the antimicrobial effect of the Cannabis sativa oil, which presents E-Caryophyllene (35%) as its major compound, where the best inhibitory effects were obtained against E. coli (11.1 mm), Micrococcus luteus (11 mm), Bacillus subtilis (13 mm), S. aureus (13 mm), C. albicans (12 mm) and C. krusei (12.5 mm) compared to the drugs ciprofloxacin (12 mm, 26.3 mm, 35 mm, 27.5 mm) and fluconazole (20 mm, 24 mm), respectively, when using the disc diffusion method.
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Fig. 6. Demonstration of the progressive inhibitory effect of the Piper diospyrifolium essential oil on Candida spp. morphological transition. The arrows indicate the formation of hyphae in the growth control (A), and its progressive reduction (B, C, D) as the concentration of the oil increases (B- MFC/8, C- MFC/4, DMFC/2). Scale: 50 μm.
5. Conclusion
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The antimicrobial action of the P. diospyrifolium essential oil was not promising against the studied strains. The combined action of the oil with commercial drugs presented a synergistic action against bacteria, although fungi did not obtain clinically relevant results. Inhibitory actions were observed against the formation of invasive filaments, an important factor of fungal pathogenicity for both Candida species. The oil was predominantly composed of mono- and sesquiterpenes, compounds already known in the literature for their antimicrobial effects, which may have collectively contributed to the oil's effect. Further research is needed to understand the action of the oil on microorganisms and the metabolic and genetic processes associated with fungal virulence inhibition. Declaration of interest The authors declare that there are no conflicts of interest regarding the publication of this paper. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.micpath.2019.103700. References [1] A.D.A. Morandim, A.R. Pin, N.A. Pietro, A.C. Alecio, M.J. Kato, C.M. Young, J.E. Oliveira, M. Furlan, Composition and screening of antifungal activity against Cladosporium sphaerospermum and Cladosporium cladosporioides of essential oils of leaves and fruits of Piper species, Afr. J. Biotechnol. 9 (37) (2010) 6135–6139. [2] R.J. Pereira, M.G. Cardoso, Metabólitos secundários vegetais e benefícios antioxidantes, J. Biotechnol. Biodiversity 3 (4) (2012). [3] K.S. Moresco, A.K. Silveira, F. Zeidán-Chuliá, A.P.F. Correa, R.R. Oliveria, A.G. Borges, L. Grun, F. Barbé-Tuana, A. Zmonzinski, A. Brandelli, M.G.R. Vale, D.P. Gelain, V.L. Bassani, J.C.F. Moreira, Effects of Achyrocline satureioides inflorescence extracts against pathogenic intestinal bacteria: chemical characterization, in vitro tests, and in vivo evaluation, Evid. Based Complement Altern. Med.
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