Antimicrobial and antibiofilm properties of essential oils from Piper marginatum Jacq

A low shrub growing in the Amazonian region, Piper marginatum Jacq. has been related to the treatment of a disease variety in folk medicine, however, still lacking scientific support. This study aimed to describe the composition of essential oils obtained from leaves (EOL) and branches (EOB) of P. marginatum and their antimicrobial effects on six relevant pathogenic bacteria. A combination of GC-FID and GC-MS was used to identify the phytochemical constituents. As antimicrobial assays, the oils were screened at the minimum inhibitory concentration (MIC) of 3 μg/ml for planktonic and biofilm inhibition. EOL revealed the presence of trans–nerolidol, o–cymene, spathulenol, elemicin, and α–copaene, while EOB composition was mainly of myristicin, trans-caryophyllene, trans-nerolidol, caryophyllene oxide, α–copaene, γ–muurolene and spathulenol. The strongest inhibition of planktonic growth was achieved against Pseudomonas aeruginosa (EOB) and Escherichia coli (EOB). Overall, Gram negative bacteria were more sensitive to both EOB/EOL showing less ability of growth and biofilm formation. The Gram-positive strains seemed to react to the essential oils by massive adhesion. Our results corroborate the relevance of Piperaceae and indicate the possible use of P. marginatum in future developments of antimicrobials.


Introduction
Upper and aromatic plants are widely used in folk medicine, since they present a wide spectrum of activity and proven inhibition against microorganisms (Duarte et al. 2005). Among many other families, Piperaceae has been extensively studied with special interest in Piper and Potomorphe representatives (Mesquita et al. 2004, Oliveira et al. 2013. The well known white and black culinary peppers (Piper nigrum L.) with high economic value are exceptions, since the majority of Piperaceae species are popularly used for their biological properties (Oliveira et al. 2013). Anti-parasitic activity of Piperaceae oils has been demonstrated against Leishmania spp., Leishmania amazonensis, Trypanosoma cruzi and Plasmodium falciparum (Marques et al. 2010;Flores et al. 2019).
Essential oils obtained from 10 species of Piperaceae were analyzed identifying 71 different compounds with prevalence of sesquiterpenes and monoterpenes (Santos et al. 2001). These findings were corroborated by other authors studying P. claussenianum (Marques et al. 2010), P. officinarum (Salleh et al. 2012) and P. aduncum (Oliveira et al. 2013).
Piper marginatum Jacq., called "malvarisco" or "caapeba cheirosa" in Amazonas State, Brazil, has been cited as sedative and anti-inflammatory in the treatment of snake bites (D'Angelo et al. 1997;Guimarães & Giordano, 2004). In a recent study, Pereira et al. (2020) indicated its use against erysipelas, urinary and dermatological infections, among others. The present study gives a contribution to enhance the knowledge about the composition and demonstrates evidence of antimicrobial effects of essential oils from P. marginatum by screening six different pathogenic bacteria growing in suspension and adhered to polystyrene microplates. 2.2 Obtention and analysis of essential oils -Dry samples of branches and leaves (100 g each) were processed by hydrodistillation in a Clevenger modified system for a period of 3.5 hours at constant temperature of 100°C. Subsequently, they were filtered using anhydrous sodium sulfate (Na2SO4) to remove water traces. The obtained oils were transferred to amber vials, sealed and kept under refrigeration to maintain the integrity of their volatile chemical constituents.

Methodology
Each essential oil was diluted in hexane and the solutions were submitted to gas chromatography (CG-FID, model CG 2010, SHIMADZU CORPORATION, Kyoto, Japan) for quantitative analysis and to determine the retention indexes. A CP-Sil 5 CB (100% dimethylpolysiloxane) column from Varian (lenght = 15 m, i. d.: 0.25 mm, and film thickness = 0.25 µm) was employed. A flow rate of 2.0 mL/min of Helium was applied as gas carrier. The injection was set to split mode 1:20 and performed at 250 o C. The detector was set to 240 o C and the oven programmed to a temperature ranging from 60ºC to 180ºC at 3 ºC/min. Pattern linear hydrocarbons were co-injected to determine the retention indexes. To obtain the mass spectrograms, the essential oils were analyzed by gas chromatography with mass spectrometer detector ( The retention indices were calculated relative to the elution times of the substances and the elution times of a series of linear hydrocarbons (C9-C30) which were co-injected with the sample GC-FID. The identification of the components was obtained with a set of retention indices and mass spectra data, compared with literature data (Adams, 2009) and Wiley's spectra library 7.0. Each 96-microtiter plate was prepared as follows: 100 µl trypticase soy broth enriched with 1% dextrose (TSB-D) was added to each well followed by 100 µl of each essential oil (EOB or EOL), and last of all, 100 µl of bacterial suspension (10 8 BC/ml) was inoculated. Five experimental controls were used as follows: TSB-D + inoculum: as negative control, the target strain should grow without interference; chlorhexidine digluconate (CHX 2%)as positive control of inhibition of bacterial growth; and ethylenediaminetetraacetic acid (EDTA 17%)as positive control of biofilm inhibition and TSB-D solely as a sterility control. All plates were incubated at 35 ± 2 o C for 24h. Each essential oil and control were tested in triplicate.

Target microorganisms and microbiological procedures -
After the incubation period, each microplate had the results recorded by a microplate reader. The supernatant was carefully washed out (3x with sterile saline 0.9%) and the plates were left at 60 o C for 1 hour in a Pasteur oven. After this, 120 µl of a crystal violet solution (0.06%) was added to each well and kept at room temperature for 5 minutes. The plates were washed gently and 40 µL of Dimetilsulfoxyde (DMSO) was added to each well in order to perform the last screening by the microplate reader.   , Society andDevelopment, v. 10, n. 11, e514101119967, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i11.19967 -T0 for planktonic growth and T24b-T0 for biofilm formation). To relate the results of the potential inhibitors to the untreated control, the latter was considered as 100% growth/biofilm and a percentage value for each essential oil and controls was calculated (Trentin et al. 2011). Any value lower than 100% was considered inhibition of growth/biofilm formation. A comparison between means gave the significance between each essential oil and controls (EDTA, CHX and untreated control) using a Student's t-test (GraphPad® Software) considering p ≤ 0.01. (Table 2).

Results and Discussion
Previous studies have discussed the properties of Piperaceae oils and extracts as antibacterial, antifungal (Holetz et al. 2002;Santos et al. 2012) and anti-parasitic (Carmo et al. 2012) among other biological activities. In this study, the efficacy of the essential oils of P. marginatum was demonstrated by both planktonic and biofilm cultivation.
Leaf samples of P. marginatum were collected in different areas of the Brazilian Amazon and classified under seven chemotypes based on the main components of essential oils identified by GC and GC/MS. The main constituents were sesquiterpenes such as (E)-beta-ocimene, beta-caryophyllene, bicyclogermacrene, alpha-copaene and gamma-terpinene and aromatic compounds such as safrole, 3,4-(methylenedioxy) propiophenone, 2-methoxy-4,5-(methylenedioxy) propiophenone, myristicin, (E)-isoosmorhizole, (E)-anethole and (E)-asarone (Andrade et al. 2008). Although 4 of their samples came from the same city as those used in the present work, they did not find trans-nerolidol and ocimene which were the 2 major compounds in EOL (29.20% and 27.49%, respectively). Differences in oil components probably resulted from different environmental conditions for plant development (Simas et al. 2004). However, the data showed here could suggest the existence of another chemotype not described before.
Both essential oils were more effective in inhibiting the Gram-negative strains, being P. aeruginosa the most sensitive microorganism. E. faecalis, S. aureus and S. sanguinis responded to the presence of the essential oils by enhancing the biofilm formation. The results are summarized in Table 2.
The reduction of growth and biofilm formation in response to the essential oils was statiscally significant against the Gram-negative strains, highlighting EOB. On the contrary, in contact with the Gram-positive targets, both EOB/EOL enhanced the bacterial proliferation and strongly stimulated the biofilm formation. This contradiction might be due to the differences in the cell wall structures combined with the essential oil chemical composition. EOB showed higher percentage of phenylpropanoids than EOL which were described before as inhibitors of Gram-negative species (Hyldgaard et al. 2012) including E. coli and Listeria monocytogenes (Gill & Holley, 2004) as well as S. enteritidis (Lanfranchi et al. 2010).
The inhibition of Gram-positive strains corresponded inversely to an increase in the measurements of attached cells, with the exception of E. faecalis. This behavior was also observed by all Gram-positive targets in the samples treated with EDTA. It was expected that EDTA, as a typical metal chelator would have more strongly affected the biofilm formation. This chemical property helped to attribute to EDTA the capacity of avoiding and/or removing biofilms, being one of the reasons for its application in the treatment of endodontic compromised teeth (Zehnder 2006, Dotto et al. 2020. In this particular case, Research, Society andDevelopment, v. 10, n. 11, e514101119967, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i11.19967 5 however, it promoted a higher percentage of adhesion rather than planktonic growth with exception of the Gram-negative bacteria. *Kovats index determined on the DB-5 capillary column referring to n-alkanes (Adams, 2009); Kovats index determined on the CP-Sil 5 CB capillary column referring to n-alkanes IK1(leaves) IK2 (branches); SIsimilarity index. Source: Authors.
Comparing the results obtained for CHX, EOB achieved the best performance, inhibiting growth and biofilm formation of all Gram-negative strains. EOL was not able to avoid growth at a significant level but was very effective in preventing biofilm formation by E. coli and P. aeruginosa. Two probable explanations would be, first, an interaction of small molecules present in EOL with some bacterial wall structures or, second, an interference with the exoenzimes involved in quorum sensing mechanisms which are essential to build biofilms (Nazzaro et al. 2013).

Conclusion
In this work, the essential oils obtained from leaves and branches of Piper marginatum Jacq. were described concerning their chemical composition and antimicrobial effect against six pathogenic bacteria. Myristicin and trans-nerolidol were the main components detected in branches and leaves essential oils, respectively. Gram negative bacteria were the most susceptible targets, growing planktonic or in biofilms, highlighting the inhibition of Pseudomonas aeruginosa by the branches oil. These results indicate P. marginatum as a promising Piperaceae representative in further developments of phytochemical based antimicrobial products.