Essential Oil from Piper aduncum: Chemical Analysis, Antimicrobial Assessment, and Literature Review

Background: The challenge in antimicrobial chemotherapy is to find safe and selective agents with potency that will not be compromised by previously developed resistance. Terrestrial plants could provide new leads to antibacterial, antifungal, or antiprotozoal activity. Methods: The essential oil (EO) of Piper aduncum L. (Piperaceae) from Cuba was analyzed by gas chromatography—mass spectrometry (GC-MS). A cluster analysis of P. aduncum EO compositions reported in the literature was carried out. The EO was screened against a panel of microorganisms (bacteria, fungi, parasitic protozoa) as well as for cytotoxicity against human cells. In addition, a review of scientific literature and a bibliometric study was also conducted. Results: A total of 90 compounds were identified in the EO, of which camphor (17.1%), viridiflorol (14.5%), and piperitone (23.7%) were the main components. The cluster analysis revealed at least nine different chemotypes. The EO did not show notable activity against bacteria or fungi, but was active against parasitic protozoa. Conclusions: The results from this study indicate P. aduncum from Cuba is a unique chemotype, support the importance of P. aduncum EOs as medicines, and demonstrate the promise of Cuban P. aduncum EO as a chemotherapeutic agent against parasitic protozoal infections.


Introduction
The challenge in antimicrobial chemotherapy is to find safe and selective agents with potency that will not be compromised by previously developed resistance. In this context, the search for new antimicrobial compounds from terrestrial plants could provide new leads to antibacterial, antifungal, or antiprotozoal activity. The World Health Organization also advocates the use of natural medicines for the treatment of microbial infectious agents. The search for new medicinal agents has become extremely important, and higher plants are a very rich source of new and selective substances with therapeutic potential against these ailments. There are approximately 250,000 plant species worldwide, of which only a fraction has so far been studied [1,2].
The Piperaceae family is distributed in tropical and subtropical regions in the northern and southern hemispheres and comprises around 3600 species. The Piper genus constitutes the most represented, with around 2000 species [3]. In particular, biological properties of essential oils (EOs) from Piper aduncum L. (Figure 1) have been widely studied, including antiprotozoal [4,5], anthelminthic [6], from Piper aduncum L. (Figure 1) have been widely studied, including antiprotozoal [4,5], anthelminthic [6], antioxidant [7], and insecticidal [8,9] activities. Considering these previously documented effects and the potentialities as therapeutic agents, we have focused on the EO from P. aduncum growing in Cuba. In this sense, chemical characterization of the EO was performed and the EO was also screened against a selection of microorganisms, including a Gram-negative (Escherichia coli) and a Gram-positive (Staphylococcus aureus) bacterium, a yeast (Candida albicans), and protozoa that are responsible for human parasitic infections: malaria (Plasmodium falciparum), sleeping sickness (Trypanosoma brucei), Chagas disease (T. cruzi), cutaneous (Leishmania amazonensis) and visceral (L. donovani and L. infantum) leishmaniasis. For comparison, in vitro cytotoxicity screening was performed on MRC-5 (normal human lung) cells. Finally, a summary of relevant aspect in the cited scientific literature of EO from P. aduncum was reviewed and a bibliometric study was also conducted.

Plant Material
Aerial parts of P. aduncum were collected in the early morning of June 2010 from the Institute of Pharmacy and Food, Havana, Cuba. The plant was identified by M.Sc. Ramón Scull following the guidelines of the National Flora of Cuba; a voucher specimen was deposited in the Experimental Station of Medicinal Plants "Dr. Juan Tomás Roig", Mayabeque, Cuba, under registration voucher number 4649. The fresh plant sample was manually crushed and the EO was immediately hydrodistilled by the conventional method using a Clevenger type apparatus for 5 h, the oil was separated, and stored in an amber-colored glass bottle under refrigeration (4 °C) prior to analysis. Solutions of the EO in dimethylsulfoxide (DMSO) were prepared (20 mg/mL) and used for biological evaluations (see below).

Gas Chromatography-Mass Spectrometry
Chemical characterization of the EO from P. aduncum was performed by gas chromatography/mass spectrometry using an Agilent 6890 gas chromatography (Agilent Technologies, Palo Alto, CA, USA) coupled with an Agilent 5973 mass selective detector (MSD) (Agilent Technologies, Palo Alto, CA, USA), operated in the electron impact (EI) mode with electron

Plant Material
Aerial parts of P. aduncum were collected in the early morning of June 2010 from the Institute of Pharmacy and Food, Havana, Cuba. The plant was identified by M.Sc. Ramón Scull following the guidelines of the National Flora of Cuba; a voucher specimen was deposited in the Experimental Station of Medicinal Plants "Dr. Juan Tomás Roig", Mayabeque, Cuba, under registration voucher number 4649. The fresh plant sample was manually crushed and the EO was immediately hydrodistilled by the conventional method using a Clevenger type apparatus for 5 h, the oil was separated, and stored in an amber-colored glass bottle under refrigeration (4 • C) prior to analysis. Solutions of the EO in dimethylsulfoxide (DMSO) were prepared (20 mg/mL) and used for biological evaluations (see below).

Gas Chromatography-Mass Spectrometry
Chemical characterization of the EO from P. aduncum was performed by gas chromatography/mass spectrometry using an Agilent 6890 gas chromatography (Agilent Technologies, Palo Alto, CA, USA) coupled with an Agilent 5973 mass selective detector (MSD) (Agilent Technologies, Palo Alto, CA, USA), operated in the electron impact (EI) mode with electron energy = 70 eV, a scan range of 40-400 amu, and a scan rate of 3.99 scans/s, and operated through an Agilent ChemStation data system (G1701CA, version C.00.01.080, Agilent Technologies, Palo Alto, CA, USA). The GC column was an HP-5ms fused silica capillary column (Agilent Technologies, Palo Alto, CA, USA), with (5% phenyl)-polymethylsiloxane stationary phase, a film thickness of 0.25 µm, a length of 30 m, and an internal diameter of 0.25 mm. The carrier gas was helium with a column head pressure of 48.7 kPa and a flow rate of 1.0 mL/min. The inlet temperature was 200 • C, the interface temperature was 280 • C, and the following GC oven temperature program was used: 40 • C initial temperature, which was held for 10 min; increased at 3 • C/min to 200 • C; increased 2 • C/min to 220 • C. A 1% w/v solution of the EO in CH 2 Cl 2 was prepared and 1 µL was injected using a splitless injection technique. Identification of the EO components was achieved using their retention indices (calculated relative to a homologous series of normal alkanes, and by comparison of their mass spectral fragmentation patterns with those reported in the literature [11] and our own in-house database. The percentages of the constituents were calculated from total ion current without standardization.

Antimicrobial Tests
The integrated panel of microbial screens for the present study and the standard screening methodologies were adopted as described by Cos et al. [12].
Escherichia coli and S. aureus were cultured in Mueller Hinton broth (MHB) medium, while C. albicans was cultured in RPMI medium. Assays were carried out by adding 5 × 10 3 CFU/well. After 17 h incubation with different EO concentrations at 37 • C, bacterial or fungal viability was determined fluorimetrically after adding resazurin [13] for 30 min at 37 • C to E. coli and S. aureus or 4 h at 37 • C to C. albicans. Fluorescence was measured using a GENios Tecan fluorimeter (Tecan Group Ltd., Männedorf, Switzerland) (excitation 530 nm, emission 590 nm).
Plasmodium parasites were cultured in human erythrocytes A+ at 37 • C under an atmosphere of 3% O 2 , 4% CO 2 , and 93% N 2 [14] in RPMI-1640 culture medium, supplemented with 0.5 (g/v)% AlbumaxTM. Suspensions of infected human red blood cells (1% parasitemia, 2% hematocrit) were added to each well with the test EO and incubated for an additional 72 h. The plate was then frozen at −20 • C and parasite multiplication was measured by the Malstat method [15]. One hundred microliters of Malstat TM reagent was transferred in a new plate and mixed with 20 µL of the hemolysed parasite suspension for 15 min at room temperature. Then, 20 µL of nitro blue tetrazolium chloride (NBT) at 2 mg/mL/PES at 0.1 mg/mL solution was added and the plate was incubated again for 2 h at room temperature in the dark. Absorbance was read at 655 nm in a Bio-Rad 3550-UV microplate reader (Bio-Rad Laboratories, Hercules, CA, USA).
Antitrypanosomal activity was performed using trypomastigotes of T. brucei, cultured at 37 • C and 5% CO 2 in Hirumi-9 medium [16], supplemented with 10% inactivated fetal calf serum (FCSi). Assays were performed by adding 1.5 × 10 4 trypomastigotes/well. After 72 h incubation, parasite growth was assessed fluorimetrically by adding resazurin for 24 h at 37 • C. In parallel, the EO at different concentrations was added to 4 × 10 4 amastigotes of T. cruzi in 4 × 10 3 MRC-5 cells maintained in minimal essential medium (MEM) supplemented with 20 mM L-glutamine, 16.5 mM sodium bicarbonate, and 5% FCSi. After incubation at 37 • C and 5% CO 2 for 7 days, parasite growth was assessed by adding the β-galactosidase substrate chlorophenol red β-D-galactopyranoside [17] for 4 h at 37 • C and the color reaction was read at 540 nm.
Antileishmanial activity against the promastigote form of L. amazonensis and L. donovani were performed with exponentially growing cells (10 5 promastigotes/mL in 199 µL) in 96-well plates. One microliter of EO at different concentrations were added and then incubated at 26 • C for 72 h. After 72 h of incubation, 20 µL of p-nitrophenyl phosphate at 20 mg/mL and dissolved in 1 M sodium acetate buffer, 1% Triton X-100, pH 5.5 were added to each well and the plate was incubated at 37 • C for 3 h. The absorbance was determined in an EMS Reader MF Version 2.4-0 (Labsystems Oy, Helsinki, Finland) at a wavelength of 405 nm [18]. In the case of experiment with L. infantum, amastigotes were obtained from an infected hamster and were used to infect primary peritoneal mouse macrophages (PMM). Then, 3 × 10 4 macrophages were transferred to each well of a 96-well plate, and the plate was incubated for 48 h at 37 • C and 5% CO 2 . The cells were then washed and infected with L. infantum amastigotes at a density of 15 parasites per cell. After 2 h of infection, pre-diluted concentrations of EO were added to the plates, which were then incubated for an additional 120 h under the same conditions. The supernatant was then discarded, the cells were fixed with methanol, and stained with 10% Giemsa for microscopic analysis. The total parasite burden (equal to the average number of amastigotes per cell) in treated wells was determined for each well.

Cytotoxicity Assay
MRC-5 cells were cultivated in MEM, supplemented with L-glutamine (20 mM), 16.5 mM sodium hydrogen carbonate, and 5% FCSi at 37 • C and 5% CO 2 . For the assay, 10 4 MRC-5 cells/well were seeded onto the test plates containing the pre-diluted EO at different concentrations and incubated at the same conditions for 72 h. Cell viability was determined fluorimetrically 72 h after the addition of resazurin, as previously described (see above).

Statistical Analysis
In each case, the percentage growth inhibition for each concentration of EO was calculated compared to the untreated controls. The median inhibitory (IC 50 ) and median cytotoxicity (CC 50 ) concentration values were determined from the lineal concentration-response curves. Statistical differences between values were determined using Mann-Whitney with Statistica for Windows Program, Release 4.5, 1993 (StatSoft, Inc., Tulsa, OK, USA).

Bibliometric Study
With the objective of obtaining reports about EOs from P. aduncum, an electronic search in PubMed [19] database was performed. Online access was on 30 January 2017 and the study period was from 1 January 1945 to 30 January 2017. The subject content analysis of records was conducted according to the Medical Subject Headings (MeSH), using the MeSH terms or descriptors 'Piper aduncum' and 'essential oil'. We did not restring any language or document type, in order to analyze publication patterns of all publications in the studied thematic.
The number of papers by year was obtained, as well as the language used and journals according to main topic. In addition, productivity by country was analyzed, considering only the institutional affiliation for the first participating author.

Pharmacological and Chemical Reports
The scientific reports previously obtained were screened and analyzed to identify and summarize the pharmacological and chemical studies of EOs from P. aduncum as well. The data extracted from the literature included country of collection, pharmacological application, and main chemical isolated compounds. In this case, there were also no exclusions related to publication dates or languages.

Chemical Analysis of the Essential Oil from Piper aduncum
A total of 90 compounds (Table 1) were found in the chromatogram of the EO obtained by gas chromatography coupled with mass spectrometry (GC-MS). The essential oil of P. aduncum from Cuba was dominated by oxygenated monoterpenoids (50.3%) and oxygenated sesquiterpenoids (29.2%), followed by monoterpene hydrocarbons (9.7%) and sesquiterpene hydrocarbons (8.2%), and an unidentified diterpenoid (2.0%). Piperitone, camphor, and viridiflorol ( Figure 2) were the main components with 23.7%, 17.1%, and 14.5%, respectively. With the objective of obtaining reports about EOs from P. aduncum, an electronic search in PubMed [19] database was performed. Online access was on 30 January 2017 and the study period was from 1 January 1945 to 30 January 2017. The subject content analysis of records was conducted according to the Medical Subject Headings (MeSH), using the MeSH terms or descriptors 'Piper aduncum' and 'essential oil'. We did not restring any language or document type, in order to analyze publication patterns of all publications in the studied thematic.
The number of papers by year was obtained, as well as the language used and journals according to main topic. In addition, productivity by country was analyzed, considering only the institutional affiliation for the first participating author.

Pharmacological and Chemical Reports
The scientific reports previously obtained were screened and analyzed to identify and summarize the pharmacological and chemical studies of EOs from P. aduncum as well. The data extracted from the literature included country of collection, pharmacological application, and main chemical isolated compounds. In this case, there were also no exclusions related to publication dates or languages.

Antimicrobial Screening
Several studies have indicated that EOs may be an antimicrobial alternative to antibiotics. In this sense, a wide panel of microorganisms that causes infectious diseases was included. In our study, the reference drugs caused a potent inhibition, as was expected, while different activities of P. aduncum EO were observed against the microorganisms tested. In the antibacterial and antifungal assays, the EO did not exhibit activity at 64 μg/mL (the highest concentration used), except against S. aureus, which did show some activity (Table 2). Currently, one of the widely used applications of EOs includes food preservatives, precisely due to their bactericidal and fungicidal properties [33][34][35]. However, the EO from P. aduncum in this work did not display high activity against human pathogenic bacteria and fungi.

Antimicrobial Screening
Several studies have indicated that EOs may be an antimicrobial alternative to antibiotics. In this sense, a wide panel of microorganisms that causes infectious diseases was included. In our study, the reference drugs caused a potent inhibition, as was expected, while different activities of P. aduncum EO were observed against the microorganisms tested. In the antibacterial and antifungal assays, the EO did not exhibit activity at 64 µg/mL (the highest concentration used), except against S. aureus, which did show some activity (Table 2). Currently, one of the widely used applications of EOs includes food preservatives, precisely due to their bactericidal and fungicidal properties [33][34][35]. However, the EO from P. aduncum in this work did not display high activity against human pathogenic bacteria and fungi. In contrast, against protozoal parasites, the P. aduncum EO was able to inhibit all pathogens (Table 3). Parasitic protozoal infections cause much human morbidity and mortality. The prevalence of these diseases is higher in the tropics, where a significant number of deaths is attributed to malaria, leishmaniasis, African and American trypanosomiasis [36]-protozoal parasites that are included in this study. The best antiprotozoal activity of P. aduncum EO was observed against P. falciparum, the causal agent of malaria. Discovering new drugs in this field is a health priority because malaria is the major parasitic infection in many tropical and subtropical regions. Currently, more than one million deaths occur each year, and between 400 and 500 million new cases are reported annually; it is estimated that 40% of the world's population is at risk of malaria [37]. In addition to the Plasmodium parasite, flagellated parasitic protozoa belonging to the genera Trypanosoma and Leishmania (Trypanosomatidae) were also included, which are considered Neglected Tropical Diseases (NTDs) by the World Health Organization [38]. In this sense, T. brucei is a causative agent of African trypanosomiasis or sleeping sickness, while T. cruzi causes American trypanosomiasis, commonly known as Chagas disease. Leishmaniasis exhibits a range of clinical appearances including cutaneous (CL), mucocutaneous (MCL), and visceral (VL) forms. In these human pathogenic trypanosomes, the management of the infections is currently based on chemotherapeutics that are not ideal due to cost, availability, toxicity, and resistance [39].
Among trypanosomes, both species of Trypanosoma were inhibited at the same concentration range with IC 50 values around 2 µg/mL. African trypanosomiasis invariably leads to coma and death if left untreated, with two stages. The first phase is characterized by nonspecific clinical symptoms, and trypanosomes are restricted to the lymphatic and circulatory systems, whereas in the second stage severe neurological symptoms appear, and parasites can be found in the brain and in cerebrospinal fluid [40]. It has been estimated that 60 million people in sub-Saharan Africa countries are currently at risk, and around 50,000 to 70,000 people are infected [39]. Chagas disease is present in Central and South America, with an annual death toll of about 50,000 people in 18 endemic countries, while more than 8 million people are infected, while nearly 90 million people are at risk of infection [41]. Recently, Villamizar and collaborators also demonstrated the antitrypanosomal activity of EO from P. aduncum collected in Brazil, showing an IC 50 of 2.8, 12.1, and 9 µg/mL against cell-derived, metacyclic trypomastigotes, and intracellular amastigotes, respectively [5]. In addition, they suggested that the probable mechanism of action was related with decreases in the mitochondrial membrane potential of the parasite after treatment [5].
Leishmania spp. showed variable susceptibilities to P. aduncum EO, with the species that cause visceral leishmaniasis (L. infantum and L. donovani) showing lower IC 50 values. In the clinical form of VL, the parasites colonize the bone marrow, liver, and spleen, resulting in host immunosuppression and death in the absence of treatment [39]. Finally, the species that causes CL (L. amazonensis), in which parasites remain localized in the epithelial tissues, the EO displayed a higher IC 50 value. Currently, leishmaniasis affects 12 million people in 98 countries where the disease is endemic and 350 million people live at risk of infection. In particular, the number of cases of VL is calculated to be as high as 0.2-0.4 million people per year, with mortality estimated at 10-20%, especially in poor areas [42]. Notably, Bernuci and collaborators reported that the EO from P. aduncum growing in Brazil displayed activity against L. amazonensis [43].
The antiprotozoal potential of P. aduncum essential oil has been demonstrated in this study. The activity may be attributed to the major components piperitone, camphor, and viridiflorol. Both piperitone and camphor have shown antiprotozoal activity against the bloodstream forms of Trypanosoma brucei brucei [44,45]. To our knowledge, however, there have been no reports on the antiprotozoal activity of viridiflorol. Nevertheless, the antiprotozoal activity of the Cuban P. aduncum chemotype EO shows promise, complements the reports of activity for other chemotypes of P. aduncum, and underscores the importance of EOs as potential treatment options for neglected tropical diseases.
In this present study, the cytotoxicity of P. aduncum EO was also studied using MRC-5 (Human fetal lung fibroblast) cells as a model. The EO inhibited cell growth, with an IC 50 value of 5.1 µg/mL. Other authors also reported the cytotoxicity of EO from P. aduncum, in particular the EO collected in Brazil [5,43]. In this study, the reference drug tamoxifen also showed a high cytotoxic effect (IC 50 = 4.3 µg/mL).

Bibliometric Study
To carry out the bibliometric study we used the PubMed database, which is one of the most used databases by academics, medical students, and primary care practitioners according to several surveys. In addition, it is easily accessible and widely used, and it uses a controlled vocabulary for indexing and recovering documents with a certain criterion for quality [46][47][48][49].
A total of 20 articles were obtained, of which one was repeated, thus resulting in a total of 19 original reports about studies of EO from P. aduncum. Results in this field appear in the last decade, with a particular increase after 2010 ( Figure 4). The language used was English in 15 scientific journals. The most used journal was Pest Management Science with three articles (16%) followed by Molecules and Chemistry & Biodiversity with two articles (11%). The remaining journals published only one article each (5%): Annals of Academia Brasileira Ciencia, Bulletin of Entomological Research, Experimental and Applied Acarology, Iranian Journal of Arthropod-Borne Diseases, Journal of the American Mosquito Control Association, Memórias do Instituto Oswaldo Cruz, Natural Product Communications, Neotropical Entomology, Pharmaceutical Biology, Revista Brasileira de Parasitologia Veterinária, Tropical Biomedicine and Veterinary Parasitology. The source journals mainly include the fields of vectors > biology > parasitology > biomedicine. The first author's institutional address displayed that only four countries are involved in these studies, with different research groups. Brazil was the predominant country (63%), followed by Cuba and Malaysia (16%), and finally New Guinea (5%).
This study has shown an increase in the number of publications on EOs from P. aduncum in recent years. A high representation in journals related to control of vectors was observed, which is in concordance with the main applications reported for Piper essential oils. In addition, the most representative country was Brazil, probably due to the exuberant flora, traditional use of natural products and the technical development of several research groups in the country in the field of chemistry and pharmacology. The bibliometric methodology used may present some limitations and further databases such as Science Direct and Scielo could be analyzed to search for additional studies. Nevertheless, this study represents a useful tool for scientists that are planning to study the EO from P. aduncum and could be used to help organize research in the field of EO and natural products in general.

Pharmacological and Chemical Reports
The earlier analyses of scientific reports about the EO from P. aduncum indicated that insecticidal and antiparasitic activities have been the most reported of plants collected in tropical countries (Table 4). In the case of vectors, useful applications were found against Aedes aegypti mosquitoes, the vector of dengue and yellow fever. Antiparasitic effects were more studied against kinetoplastids, including Trypanosoma and Leishmania.  [57] Insect repellent against A. aegypti [9] Several different components have been identified in EOs from P. aduncum. In general, between 23 and 68 compounds have been reported, with a higher percentage of monoterpenes. Unfortunately, many pharmacological studies did not include a chemical characterization of the EO. Some chemical compounds have been identified, isolated, and studied ( Table 5). The most representative was the dill apiole, a phenylpropanoid, which showed antifungal, insecticidal, and anti-inflammatory activities.

Pharmacological and Chemical Reports
The earlier analyses of scientific reports about the EO from P. aduncum indicated that insecticidal and antiparasitic activities have been the most reported of plants collected in tropical countries (Table 4). In the case of vectors, useful applications were found against Aedes aegypti mosquitoes, the vector of dengue and yellow fever. Antiparasitic effects were more studied against kinetoplastids, including Trypanosoma and Leishmania. Several different components have been identified in EOs from P. aduncum. In general, between 23 and 68 compounds have been reported, with a higher percentage of monoterpenes. Unfortunately, many pharmacological studies did not include a chemical characterization of the EO. Some chemical compounds have been identified, isolated, and studied ( Table 5). The most representative was the dill apiole, a phenylpropanoid, which showed antifungal, insecticidal, and anti-inflammatory activities. Anti-inflammatory Brazil [58] a Pharmacological activity was only included if pure compound was tested.

Conclusions
The results of this study support the importance of the EO from P. aduncum, in particular in regard to antiparasitic activity. Its effect against P. falciparum indicates that this EO could be a promising antimalarial agent. Further studies including in vivo bioassays are needed to validate the in vitro results and to ascertain the safety of the EO. In addition, the antimicrobial evaluation of the major components, camphor, viridiflorol, and piperitone, will be useful and will surely provide complementary data about antiparasitic active compounds as well as the mechanism(s) of action. There is significant chemical diversity in the EOs of P. aduncum, and this, no doubt, will affect the biological activities. Thus, there is a need to characterize the EOs of this plant and correlate this information with bioactivities.