Piper Essential Oils Inhibit Rhizopus oryzae Growth, Biofilm Formation, and Rhizopuspepsin Activity

Piper is the largest genus of the Piperaceae family. The species of this genus have diverse biological activities and are used in pharmacopeia throughout the world. They are also used in folk medicine for treatment of many diseases in several countries including Brazil, China, India, Jamaica, and Mexico. In Brazil, Piper species are distributed throughout the national territory, making this genus a good candidate for biological activity screening. During our studies with Piper essential oils, we evaluated its activity against Rhizopus oryzae, the main agent of mucormycosis. The main compounds of seven Piper essential oils analyzed were Piper callosum—safrole (53.8%), P. aduncum—dillapiole (76.0%), P. hispidinervum—safrole (91.4%), P. marginatum—propiopiperone (13.2%), P. hispidum—γ-terpinene (30.9%), P. tuberculatum—(E)-caryophyllene (30.1%), and Piper sp.—linalool (14.6%). The minimum inhibitory concentration of Piper essential oils against R. oryzae ranged from 78.12 to >1250 μg/mL. The best result of total inhibition of biofilm formation was obtained with Piper sp. starting from 4.88 μg/mL. Considering the bioactive potential of EOs against planktonic cells and biofilm formation of R. oryzae could be of great interest for development of antimicrobials for therapeutic use in treatment of fungal infection.


Introduction
Piper is the largest genus of the Piperaceae family. e species of this genus have diverse biological activities and are used in pharmacopeia throughout the world. ey are also used in folk medicine for treatment of many diseases in several countries including Brazil, China, India, Jamaica, and Mexico. In Brazil, Piper species are distributed throughout the national territory. Among the aromatic flora of the Amazon region, there are more than a dozen species that provide essential oils that are used by the population for therapeutic purposes. e tea of the decoction of Piper hispidum leaves is useful for the treatment of malaria. Piper marginatum is used as a tonic, carminative, stimulant, diuretic, and sudorific agent against stomach, liver and gallbladder pain, toothaches, and snake and insect bites [1]. Regasini et al. [2] related trypanocidal activity of the Piper tuberculatum extract. Zygomycosis, also referred to as phycomycosis or mucormycosis, is an aggressive and rapidly progressive infection that primarily occurs in immunocompromised patients. Members of the genera Rhizopus, Mucor, and Absidia are the organisms most commonly isolated from patients with zygomycosis. Rhizomucor, Cunninghamella, Apophysomyces, and Saksenaea are other zygomycetes that have been implicated in human diseases. Amphotericin B, as well as its lipid formulation, has been essential for treatment for several decades [3]. e purpose of the present work was to evaluate the anti-Rhizopus oryzae activity of Piper aduncum, P. hispidinervum, P. callosum, P. hispidum, P. tuberculatum, P. marginatum, and Piper sp. essential oil of leaves.

Plant Material and Essential Oil
Extraction. Plant material was obtained from EMBRAPA Experimental Farm, Amazonas, Brazil. A voucher of each specimen was deposited at Federal Agrotechnical School of Machado Herbarium ( Table 1). Leaves of Piper species were collected between 8 and 9 a.m., dried at room temperature, and coarsely ground into powder just before distillation. e oil was obtained by hydrodistillation in a modified Clevenger apparatus for 5 h [4].

Essential Oil Analyses.
Sample of each Piper essential oil was analyzed in an Agilent 6890 N gas chromatograph fitted with a 5% diphenyl-95% dimethylpolysiloxane capillary column (DB-5MS, 30 m × 0.25 mm × 0.25 μm). e results were compared to data from the literature [5].

Antifungal Activity Assay.
e antifungal activity of Piper essential oils was evaluated against R. oryzae (UCP1506). e strain used belongs to the culture collection of the "Universidade Católica de Pernambuco," located in the Nucleus of Research in Environmental Sciences, Catholic University of Pernambuco, Brazil, NPCIAMB/UNICAP. e culture collection is registered in the WFCC. e microdilution broth method was used according to CLSI reference document M38-A [6] for filamentous fungi. Briefly, the cells were grown in RPMI-MOPS (pH 7.2) for 18 h at 30°C in the presence of different concentrations (1.22 to 1250 μg/mL) of each essential oil. Positive and negative growth controls were performed. Amphotericin B (Sigma) was used as a reference drug, and stock solution was made at 20 mg/mL in sterile distilled water. All experiments were performed in duplicate and repeated twice.
In order to evaluate the fungicide/fungistatic properties of Piper essential oils, a 10 μL aliquot was collected from the inhibited cultures and dropped on the surface of potato dextrose agar. e absence or presence of growth in the solid medium was evaluated after 48 h incubation period at 30°C.

Biofilm Formation.
e influence of Piper essential oils on biofilm formation was determined as described by Singh et al. [7]. Briefly, spores of R. oryzae were put in 96-well microtiter plate at 5 × 10 4 cells per mL in RPMI and treated with twofold serial dilution of each Piper essential oil. After incubation for 18 h at 30°C, the culture media was removed and the wells were washed twice with PBS 0.01 M and pH 7.2. Biofilms were stained with 200 µL of 0.1% safranin for 5 min.
en, the supernatants were removed, and the wells were washed twice with PBS. Finally, 200 µL of 30% glacial acetic acid was added to the microplates in order to elute safranine from the matrix. Biofilm formation was estimated by spectrophotometry (SpectraMax M5) at 490 nm.

Red Blood Cell Lysis Assay.
e hemolytic activity was evaluated by Franca Rodrigues et al. [8] by mixing 80 μL of a 5% suspension of fresh human red blood cells (O + ) in PBS with 20 μL of different concentrations of Piper sp. essential oil and incubating at 37°C for 1 h. e reaction was slowed by adding 200 μL of PBS, and the suspension was centrifuged (1000 g for 10 min). e supernatant was transferred to a 96-well plate, and cell lysis was quantified by spectrophotometrical measurement of absorbance at 540 nm, as previously described. e maximal lysis and blank control were obtained by replacing the extract sample with an equal volume of PBS or distilled water, respectively.

Rhizopuspepsin Inhibition.
In order to evaluate a possible mode of action of the Piper essential oils, the inhibition of rhizopuspepsin (Sigma) activity was determined as previously described by Buroker-Kilgore and Wang with some modifications [9]. First, 59 μL of the rhizopuspepsin solution was mixed with 1 μL inhibitor, 20 μL BSA (1 mg/mL), and 20 μL buffer (pH 3.0). After 1 h incubation at 37°C, 100 μL of Bradford solution (0.025% Coomassie Blue G-250, 11.75% ethanol, and 21.25% phosphoric acid) previously diluted (1 : 1) was added. Negative control was performed by adding the substrate immediately after the incubation period. Finally, the plate was read on a spectrophotometer (SpectraMax M5) at 595 nm. One unit of enzyme activity was defined as the total enzyme that causes an increase of 0.001 in unit of absorbance under the conditions of the standard assay. e inhibitors tested were Piper essential oils (48 μg/mL) and 10 mM Pepstatin A (standard inhibitor).

Antioxidant Activity of Piper spp. Essential Oils.
e antioxidant activity was evaluated qualitatively [10,11] by application of 0.5 μL of each essential oil and 7-hydroxycalamenene (as standard) on a plate of silica gel 60 F 254 and eluted with hexane-ethyl acetate (9 : 1). e plates were treated with a 0.2% methanolic solution of DPPH and read just after spraying and after 45 min.

Results and Discussion
e average oil yield obtained was 0.65% (dry wt.). e compounds present in the essential oils from Piper species used are shown in Table 2. Quantitative and/or qualitative variations were observed among samples of Piper.
e essential oils of P. aduncum, P. hispidinervum, P. callosum, P. marginatum, P. hispidum, P. tuberculatum, and Piper sp. were analyzed by GC and GC-MS, and the percentage of identified components is given in Table 2.
Sartoratto et al. [22] considered MIC values between 50 and 500 µg/mL as strong activity, MIC values between 600 and 1500 µg/mL as moderate activity, and above 1500 µg/mL as weak activity [21]. According to this classification, it could be stated that Piper sp., P. marginatum, and P. hispidum essential oils present high activity, P. tuberculatum and P. hispidinervum present moderate activity, and P. aduncum and P. callosum against R. oryzae planktonic cells present weak activity.
Based on previous MIC results, the essential oils tested on biofilm formation were P. hispidum, P. marginatum, and P. tuberculatum; Piper sp. Rhizopus oryzae biofilm formation in the presence of each Piper essential oil was inhibited in  Figure 1). In their natural environments, most of bacteria and fungi change from a planktonic to a sessile state forming the socalled biofilms. Biofilms are sessile microbial and fungal communities that are strongly attached to surfaces and to each other; in such phase, they are protected by a polymeric extracellular matrix (ECM), constituted primarily of polysaccharides. According to Singh et al. [7], the major compounds of biofilm matrix are GlcN and GlcNAc. e cell wall of zygomicetes is also mainly formed by GlcN and GlcNAc polymer constituents of chitosan and chitin, respectively.
en, our results on MIC and inhibition of biofilm formation could be associated with each other. e essential oil of Piper sp. showed the most active agent against the two cell forms, planktonic and biofilm [7,23].

Canadian Journal of Infectious Diseases and Medical Microbiology
Piper sp. essential oil was the most active agent against planktonic cells and biofilm formation (78.12 and 4.88 μg/mL, resp.). However, this essential oil displayed hemolytic activity ( Figure 2) at higher concentration (2500 μg/mL), making it a promising antifungal candidate.
Other important mechanism of action is the inhibition of rhizopuspepsin and/or saps, a class of enzymes secreted for R. oryzae and other Rhizopus species [24]. e results in Figure 3 showed inhibition of proteolytic activity of rhizopuspepsin when Piper essential oils were used, mainly P. hispidum and P. tuberculatum which inhibited 11.8% and 12.05% of enzymatic activity, respectively. e antioxidant activity was evaluated after TLC of Piper essential oils. It was not possible to identify regions containing substances with activity even after 45 min of application of DPPH. Terpenes are the most significant class of compounds present in essential oils. Among them, several monoterpene hydrocarbons, oxygenated monoterpenes, and sesquiterpenes are often reported as weak antioxidant agents [25]. However, due to the complexity of essential oils' composition, some antioxidant activity was expected. us, further investigation will be necessary in order to evaluate other antioxidant methods.

Conclusion
is study showed the promising anti-Rhizopus oryzae activity of Piper tuberculatum, P. hispidum, and Piper sp. against planktonic cells, biofilm formation, and rhizopuspepsin which makes these essential oils useful in formulating strategies to limit the growth of R. oryzae.

Conflicts of Interest
e authors declare that they have no conflicts of interest concerning this article.