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The chemical composition and antibacterial activity of eleven Piper species from distinct rainforest areas in Southeastern Brazil
Industrial Crops and Products 94 (2016) 528–539
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Industrial Crops and Products journal homepage: www.elsevier.com/locate/indcrop
The chemical composition and antibacterial activity of eleven Piper species from distinct rainforest areas in Southeastern Brazil Crislene Vaz Perigo a , Roseli B. Torres a , Luís C. Bernacci a , Elsie F. Guimarães d , Lenita L. Haber c , Roselaine Facanali a , Maria A.R. Vieira a , Vera Quecini b,∗ , Márcia. Ortiz M. Marques a a
Instituto Agronômico – Av. Barão de Itapura, 1481, Campinas, SP 13020-902, Brazil Embrapa Uva e Vinho – Av. Livramento, 515, Bento Gonc¸alves, RS, 95701-008, Brazil c Embrapa Hortalic¸as, Rodovia BR-060, Km 09, Brasília, DF 70359-970, Brazil d Instituto de Pesquisas Jardim Botânico do Rio de Janeiro, Rua Pacheco Leão, 915, Rio de Janeiro, RJ 22460-030, Brazil b
a r t i c l e
i n f o
Article history: Received 30 April 2016 Received in revised form 30 July 2016 Accepted 12 September 2016 Keywords: Antimicrobial Bacteria Essential oil Monoterpene Rainforest Sesquiterpene
a b s t r a c t Piper is a large and highly diverse genus of plants with medicinal and aromatic use. It is widely distributed in tropical forests occupying distinct environments. We have determined the chemical composition of the essential oils from 11 species of Piper from seven locations of Atlantic rainforest in Brazil. The essential oils were isolated by hydrodistillation and their composition, determined by GC–MS. The chemical composition of the oils exhibits qualitative and quantitative differences among the species and intraspecifically for distinct locations. Mono and sesquiterpenes were the most abundant compounds, although P. aduncum also contained phenylpropanoids. The oils of P. xylosteoides, P. umbellatum and P. leptorum from coastal and inland regions consisted exclusively of oxygenated sesquiterpenes. In contrast, monoterpene was the sole chemical class in the oils extracted from P. rivinoides and P. solmsianum. Hierarchical clustering analyses demonstrated the chemical phenotype is associated to species-specific and environmental factors. The essential oils from the majority of the investigated Piper species exhibit inhibitory activity against pathogenic bacteria in vitro, reaching up to 30% of the inhibition levels of commercial antibiotics. The contents of bicyclogermacre and ␥-muurolene were positively associated to E. coli inhibition, whereas, levels of germacrene D and trans-caryophyllene were associated to the inhibitory activity against all tested bacteria. Limonene and cis--ocymene were associated with Staphylococci inhibition. Higher contents of -phellandrene were positively correlated to wide spectrum antibacterial activity. Taken together, our results demonstrate the chemical diversity of Piper essential oils and their potential as novel antibacterial agents for several industrial applications. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Piperaceae is a highly diverse family of small trees, shrubs or lianas distributed throughout the tropical regions in the Americas, Asia, Africa and Southern Pacific. It consists of more than 3000 species, recently attributed to three large sub-families: Verhuellioideae, Piperoideae and Zippelideae (Semain et al., 2008). Although the family distribution is pantropical, Central and South America, along with Malaysia, are considered the diversity centers (Quijano-Abril et al., 2006). In Brazil, species of the Piperaceae
∗ Corresponding author. E-mail address: [email protected] (V. Quecini). http://dx.doi.org/10.1016/j.indcrop.2016.09.028 0926-6690/© 2016 Elsevier B.V. All rights reserved.
family are spread throughout a wide range of primary and secondary rainforest areas, from the sea level up to 2000 m in altitude. Besides the commercial importance of black pepper (Piper nigrum L.), several other species from the Piper genus are known for their aromatic and medicinal properties (Picard et al., 2014; Raut and Karuppayil, 2014). The specialized metabolism of the genus Piper is diverse and include alkaloids, terpenoids and flavonoids (Scott et al., 2008). The essential or volatile oils are complex mixture of chemical compounds produced by plants from terpenes and their oxygenated derivatives, terpenoids, aromatic and aliphatic acid esters and phenols (Solórzano-Santos and Miranda-Novales, 2012). In nature, the chemical properties of plant terpenoids make them important biological components in defense and signaling processes, helping their adaptation to complex environments (Gershenzon and Dudareva, 2007; Pichersky and Lewinsohn, 2011).
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The hydrophobicity and reactivity of these compounds allow the partition with the lipids from biological membranes, leading to disturbances in its structure, thus, impairing cellular functions. These properties of plant volatile compounds make them potentially interesting for antibacterial, antifungal and antiviral activity in medical, cosmetic and technological applications (SolórzanoSantos and Miranda-Novales, 2012). In the current work, we have chemically characterized the essential oils of 11 Piper species from seven distinct rainforest locations in the State of São Paulo, Brazil. The influence of the species and location on the chemical composition was investigated by multivariate analyses. The biological activity of the essential oils was tested against four bacterial species, pathogenic to mammals, and the inhibitory action of the individual monoterpene, sesquiterpene and phenylpropanoid compounds was assessed by correlation analyses.
2. Material and methods 2.1. Collection of biological samples Seven independent locations of Atlantic rainforest in the State of São Paulo, Brazil (Fig. 1) were sampled for plants of the Piper genus, comprehending 11 species. The aerial part of the plants was collected and used for botanical identification, composition of herbarium mounts and chemical analyses. The sampled plants were marked and coordinate reference locations were determined by Global Positioning System (GPS). Marked plants were resampled, throughout the year, in two subsequent years. The individuals failing to be located or in poor condition were excluded from the analyses. The names of the identified species are presented according to the list of species of the Brazilian flora (Lista de Espécies da Flora do Brasil, Jardim Botânico do Rio de Janeiro (2015), available at http:// floradobrasil.jbrj.gov.br). Voucher specimens were deposited at the Herbarium of the Instituto Agronômico (IAC) (http://herbario.iac. sp.gov.br/) and the voucher numbers are given in Table 1. 2.2. Essential oil extraction Leaves were separated from woody stalks and air dried at room temperature, under diffuse light. Fresh plant material ranging from 54 to 1870 g, depending on availability, was used to extract the essential oils using a Clevenger-type apparatus until total recovery of oil. The oils were transferred to hermetically closed vials and stored at −20 ◦ C until further use. 2.3. Chemical characterization and quantification of essential oils The chemical composition of the essential oils was determined by gas chromatography coupled to mass spectrometry (GC–MS) using a Shimadzu QP-5000 equipped with fused silica capillary column OV − 5 (30 m × 0.25 mm × 0.25 m, Ohio Valley Specialty Chemical, Inc., USA), operating with electron impact (70 eV), using Helium (1.0 mL/min.) as carrier, injector and detector at 240 ◦ C and 230 ◦ C, respectively, split ratio of 1/30 and the following temperature program: 60 ◦ C–240 ◦ C, 3 ◦ C/min. Area normalization method of triplicate readings was used to quantify the extracted oils by gas chromatography with flame ionization detection (GC–FID) at a Shimadzu equipment, model GC-2010, under the previously described operating conditions. The compounds were identified by comparative analyses of the mass spectra against the database of the GC/MS system (Nist 62.lib) along with retention indices (Adams, 2007), obtained from the injection of a mixture of
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n-alcanes (C9 H20 –C25 H52 , Sigma Aldrich, 99%) using Van Den Dool and Kratz (1963) equation. 2.4. Bacterial strains and growth conditions Certified cultures of Escherichia coli (ATCC 8739), Staphylococcus aureus (ATCC 6538), S. epidermidis (ATCC 12228), Corynebacterium xerosis (ATCC 373) and Pseudomonas aeruginosa (ATCC 9027) were provided as lyophilized stocks from Instituto Adolfo Lutz, São Paulo, Brazil. Cultures were reactivated, grown in liquid media and kept as 50% (w/v) glycerol stocks at −80 ◦ C. For biological activity assays, fresh bacteria cultures were initiated from isolated colonies on Agar Nutrient Broth (Oxoid, UK). Bacterial concentration was estimated based on spectrophotometric absorbance readings at 600 nm for E. coli and P. aeruginosa, 490 nm for S. epidermidis and P. aeruginosa, 530 nm for S. aureus and 578 nm for C. xerosis. 2.5. Antibacterial activity analyses Antibacterial activity of the oils was investigated by growth inhibition in agar diffusion assays against four bacterial species. Growth inhibition positive controls were Melaleuca essential oil and the antibiotics cefotaxime and negative control was sterile mineral oil. A single colony was inoculated to 20 mL of liquid TSB (Oxoid, UK) and grown to saturation (approximately 14 h) with 200 rpm shaking. An aliquot of 1 mL was transferred to 20 mL of fresh TSB and the procedure was repeated twice, so that bacterial cultures were replicated three times prior to biological activity analyses. For each bacterial species, 400 mL of fused Nutrient Agar medium (Oxoid, UK), supplemented with 1.5 mL of a 2% (w/v) solution of 2,3,5-triphenyl tetrazolium chloride (TTC), were kept at 45 ◦ C and added with 1.0 mL of the saturated bacterial culture. The mixture was poured on 9 mm sterile Petri dishes containing five, evenly distributed aluminum rings of 6 mm of diameter. The rings were removed from the solidified medium and 300 uL of essential oil at 5% (w/v) in sterile mineral oil were added to the wells. The plates were incubated horizontally in a bacteriological oven at 37 ± 2 ◦ C for 48 h and the inhibition halos were measured using a digital caliper (Mitutoyo America Corp., Illinois, USA). Plates were also digitalized and halo measures obtained by ImageJ software (Abramoff et al., 2004). Halo measurements from the actual plates and digitalized images were equivalent. Bacterial concentrations on the saturated suspension were corrected to 108 colony forming unit (CFU) mL−1 by absorbance readings. 2.6. Statistical analyses The assays of antimicrobial activity were performed in triplicate, in independent experiments for oil samples from independent extractions and collections. Chemical composition results were analyzed by ANOVA, honest significant difference (HSD) Tukey’s test and multifactorial analyses using the R project for statistical computing (R Core Team, 2014) and the package FactoMineR (Lê et al., 2008). Antibacterial activity was correlated to the contents of the major components in the oils using the function cor and the method Pearson in R. Graphs were plotted using the package corrgram (Wright, 2015). The function rcor from the package Hmisc (Harrell, 2016) was used to determine the correlations/covariances and significance levels. Chemical composition results were compared and submitted to multivariate statistical analyses (MANOVA) using the software MINITAB Statistical. Hierarchical clustering analyses were performed in R using the hclust function on composition data standardized by a vector of means (function center) and a vector of standard deviations (function scale).
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Table 1 Main (≥5%) and minor components of the essential oil extracted from Pipearaceae of the Atlantic rainforest. Essential oil yield is presented as percentage from dry weight (w/v). Retention indices of the metabolites are given in Table S1. Species
Sample/Herbarium Code
Location (decimal coordinates, elevation)
Yield (%)
Main Components (%)
Minor Components (%)
Piper aduncum L.
Lp52911/IAC 47090
Monte Alegre do Sul (−46.672500, −22.70010, 743m)
0.51
spathulenol (10.6), valencene (9.7), ␣-pinene (6.4) asaricin (14.9), safrole (13.3)
Piper aduncum L.
Lp6101606/IAC 47958
Votuporanga (−50.064750, −20.46286, 458m)
1.52
asaricin (80.1), safrole (10.8)
Piper aduncum L.
Lp6101608/IAC 47960
Votuporanga (−50.064750, −20.46294, 465m)
1.55
asaricin (73.4), safrole (10.5)
Piper amalago L.
Lp06091202/IAC 32056
Campinas (−47.067306, −22.86491, 664m)
0.20
-phellandrene (39.3), ␣-pinene (14.8), germacrene D (11.7)
Piper amalago L.
Lp06091206/IAC 46823
Campinas (−50.064750, −20.46286, 458m)
0.36
-phellandrene (15.9), ␣-pinene (6.7), sabinene (6.3), bicyclogermagrene (20.8), spathulenol (9.1)
Piper amalago L.
R1763/IAC 47512
Mococa (−46.981013, −21.44825, 600m)
0.26
-phellandrene (33.1), ␣-pinene (11.7), bicyclogermagrene (15.0)
Piper amalago L.
Lp6101821/IAC 47987
Adamantina (−51.152194, −21.66311, 349m)
0.23
-phellandrene (12.3), sabinene (8.2), myrcene (6.8), bicyclogermagrene (19.4), ␥-muurolene (5.9), spathulenol (5.6)
Piper amplum Kunth.
R1740/IAC 7267
Pariquera-Ac¸u (−47.880212, −24.61345, 25m)
0.38
Piper cernuum Vell.
L51904/IAC 7068
Ubatuba (−45.127511, −23.42181, 30m)
0.32
␣-pinene (18.1), cis--ocimene (10.5), limonene (8.6), trans-caryophyllene (8.8), germacrene D (5,5) ␣-pinene (10.0), camphene (6.3), dihydro-agarofuran (28.7), 10epi- ␥-eudesmol (13.5), 4-epi-cis-dihydroagarofuran (10.8)
 − pinene (4.5), ␥-cadinene (3.9), trans-caryophyllene (3.6), caryophyllene oxide (3.6), ␦-cadinene (2.6), trans--ocimene (2.7), linalool (2.2), epi-␣-cadinol (1.7), limonene (1.3), cis--ocimene (1.0), ␣-copaene (0.8), germacrene D (0.8), ␣-muurolene (0.6), dehydro-aromadrene (0.3), ␥-gurjunen (0.4), camphene (0.3), germacrene B (0.2) trans--ocimene (1.9), ␥-cadinene (1.2), spathulenol (1.0), germacrene B (0.9), germacrene D (0.9), valencene (0.8), cis--ocimene (0.6), trans-caryophyllene (0.4), ␦-cadinene (0.2),  − pinene (0.2) trans--ocimene (5.0), valencene (3.1), cis--ocimene (1.9), ␥-cadinene (1.0), spathulenol (1.0), ␣-muurolene (0.7), germacrene D (0.6), germacrene B (0.5), trans-caryophyllene (0.5), ␦-cadinene (0.3),  − pinene (0.3), ␣-pinene (0.1), ␣-humulene (0.1) myrcene (3.2), trans-caryophyllene (2·8), -bourbonene (2.6), ␣-phellandrene (1.9), ␣-copaene (1.4), -pinene (1.4), bicyclogermacrene (1.0), spathulenol (1.0), cis--ocimene (0.8), ␦-elemene (0.6), linalool (0.7), germacrene B (0.5), sabinene (0.4), ␣-muurulene (0.3), ␦-cadinene (0.3) ␦-cadinene (4.6), myrcene (4.3), epi-␣-cadinol (4.1), caryophyllene oxide (3.0), trans-nerolidol (2.5), trans-caryophyllene (2.4), cis--ocimene (1.3), linalool (1.3), -pinene (1.1), ␥-cadinene (1.0), ␣-muurulene (0.9), ␣-phellandrene (0.9), ␣-muurulol (0.5), o-cymene (0.5), trans--ocimene (0.5), 10-epi-␥-eudesmol (0.4), ␣-thujene (0.2), -elemene (0.2) myrcene (2.8), 1-epi-cubenol (2.6), trans-caryophyllene (2.1), ␦-cadinene (1.9), ␣-phellandrene (1.9), cis--ocimene (1.9), ␦-elemene (1.7), trans-nerolidol (1.5), epi-␣-cadinol (1.2), ␥-cadinene (1.1), -pinene (1.0), linalool (0.8), o-cymene (0.8), caryophyllene oxide (0.5), germacrene D (0.5), trans--ocimene (0.5), ␣-muurulene (0.3), sabinene (0.3), ␣-terpinine (0.2), -elemene (0.2) ␣-pinene (4.8), ␦-cadiene (3.6), trans-caryophyllene (3.6), trans-nerolidol (2.0), linalool (1.9), 2-decanol (1.8), germacrene D (1.8), epi-␣-cadinol (1.7), cis--ocimene (1.6), ␦-elemene (1.5), ␥-cadiene (1.3), ␣-muurulene (1.2), -elemene (1.2), ␣-copaene (1.1), ␣-phellandrene (1.0), caryophyllene oxide (0.9), 1-epi-cubenol (0.8), ␣-humulene (0.8), -pinene (0.9), trans--ocimene (0.4), ␣-muurulol (0.3) bicyclogermacrene (2.6), ␣-copaene (1.7), trans--ocimene (1.5), germacrene A (1.1), ␣-humulene (1.0), -pinene (0.9), ␦-cadinene (0.9), camphene (0.5), aromadrene (0.4), cis--guaiene (0.2), myrcene (0.2) -pinene (3.8), trans-caryophyllene (2.0), ␣-muurulol (1.8), ␥-cadinene (1.6), ␣-agarofuran (1.2), germacrene D (0.7), ␥-eudesmol (0.7), limonene (0.7), trans-nerolidol (0.7), ␥-terpinene (0.5), cis--guaiene (0.4), myrcene (0.4), ␣-terpinene (0.3), ␦-cadinene (0.3), germacrene A (0.3), spathulenol (0.3), ␣-copaene (0.2), ␣-humulene (0.2), -elemene (0.2), caryophyllene oxide (0.2), methyl-geranate (0.2), germacrene B (0.1), sabinene (0.1), terpinolene (0.1)
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Table 1 (Continued) Species
Sample/Herbarium Code
Location (decimal coordinates, elevation)
Yield (%)
Main Components (%)
Minor Components (%)
Piper cernuum Vell.
R1741/IAC 7268
Pariquera-Ac¸u (−47.880212, −24.61345, 25m)
1.84
␣-pinene (11.8), camphene (8.7), dihydro--agarofuran (33.8), 10-epi-␥-eudesmol (12.2)
Piper crassinervium Kunth.
R1764/IAC 7513
Mococa (−46.998695, −21.42348, 568m)
0.53
-pinene (11.6), ␣-pinene (11.5), germacrene D (9.2), trans-caryophyllene (7.8), guaiol (5.5), bicyclogermacrene (5.1)
Piper gaudichaudianum Kunth.
R1738/IAC 7265
Pariquera-Ac¸u (−47.880212, −24.61345, 25m)
0.16
␣-pinene (12.2), -pinene (7.0), trans-nerolidol (17.5), caryophyllene oxide (8.5), trans-caryophyllene (8.2), trans--guaiene (6.9)
Piper leptorum Kunth.
Lp052903/IAC 7085
Monte Alegre do Sul (−46.665000, −22.70370, 778m)
0.60
seychellene (34.7), caryophyllene oxide (12.5)
Piper rivinoides Kunth.
L52007/IAC 47078
Ubatuba (−45.121121, −23.42121, 30m)
0.63
␣-pinene (73.2), -pinene (5.2)
Piper solmsianum C.DC.
R1633/IAC 46832
Ubatuba (−45.119111, −23.40902, 40m)
0.39
␦-3-carene (66.9), myrcene (26.1), ␣-pinene (22.7), ␣-selinene (5.5)
Piper umbellatum (L.)
R4169/IAC 46978
Campinas (−47.067972, −22.86516, 667m)
0.18
germacrene D (55.8), bicyclogermacrene (11.8), trans-caryophyllene (6.3)
Piper xylosteoides (Kunth.) Steud.
L52004/IAC 47075
Ubatuba (−45.127123, −23.42112, 30m)
1.04
spathulenol (12.3), germacrene B (10.6), -copaen-4-␣-ol (9.4), trans-nerolidol (8.2), trans--guaiene; (7.8)
-pinene (2.8), ␥-cadinene (1.7), trans-caryophyllene (1.6), ␣-agarofuran (1.4), ␣-muurulol (1.3), -phellandrene (0.7), trans-nerolidol (0.6), ␣-copaene (0.5), cis--guaiene (0.4), ␥-eudesmol (0.4), germacrene D (0.3), myrcene (0.3), methyl-geranate (0.2), trans-nerolidol (5.0), ␦-cadinene (4·2), ␥-muurolene (3.3), 6-methyl-5-hepten-2-one (2.4), ␥-cadinene (1.7), 1-epi-cubenol (1.5), spathulenol (1.4), ␣-eudesmol (1.1), aromadrene (1.0), -phellandrene (1.0), linalool (1.0), ␣-copaene (0.7), ␣-muurolene (0.7), ␣-humulene (0.6), ␣-calacorene (0.5), -elemene (0.3), camphene (0.3), -gurjunene (0.2), -bourbonene (0.1) 7-epi-␣-selinene (4.5), limonene (3.9), cis--guaiene (3.8), ␥-cadiene (2.4), linalool (2.7), aromadrene (1.8), germacrene D (1.5), ␣-humulene (1.4), ␦-cadinene (1.1), spathulenol (0.9), ␣-phellandrene (0.8), trans--ocimene (0.6), ␥-muurulene (0.5), myrcene (0.5), o-cymene (0.3), camphene (0.2), spathulenol (4.7), ␣-pinene (3.2), ␣-selinene (3.0), myrcene (2.8), germacrene D (2.7), -pinene (2.2), cis--guaiene (1.9), ␣-humulene (1.6), germacrene B (1.6), 7-epi-␣-selinene (1.4), ␥-muurolene (1.3), guaiol (1.0), limonene (0.9), -elemene (0.6), ␥-gurjunene (0.6), germacrene A (0.5), ␥-cadinene (0.3), sabinene (0.3), terpinolene (0.3), ␦-cadinene (0.2), ␣-copaene (0.1), o-cymene (0.1), trans-nerolido (0.1) linalool (4.0), bicyclogermacrene (3.9), caryophyllene oxide (2.2), aromadrene (1.6), camphene (1.6), spathulenol (1.5), trans-caryophyllene (1.5), limonene (1.2), myrcene (1.2), cis-calamene (0.6), 10-␥-epi-eudesmol (0.4), o-cymene (0.4), dehydro-aromadrene (0.3), ␥-cadinene (0.2), ␣-cubebene (0.1), ␣-phellandrene (0.1) -pinene (3.5), ␣-phellandrene (3.4), -phellandrene (3.0), dehydro-aromadrene (2.2), germacrene D (1.5), trans-caryophyllene (1.5), terpinolene (0.7), sabinene (0.6), camphene (0.3), germacrene B (0.2), p-cymene (0.1), trans--ocimene (0.1) -cubebene (3.2), caryophyllene oxide (2.5), ␦-cadinene (2.0), ␣-copaene (1.5), ␦-elemene (1.5), ␥-cadinene (1.4), germacrene A (1.3), ␥-muurolene (0.5), ␣-humulene (0.7), ␣-muurulol (0.6), -bourbonene (0.6), ␣-cubebene (0.4), trans-nerolidol (0.4), -gurjunene (0.2) dehydro-aromadrene (4.8), trans-caryophyllene (4.5), -elemene (4.1), cis-calamenene (3.7), limonene (3.5), germacrene A (2.0), ␣-humulene (1.9), ␥-elemene (0.4), germacrene D (0.3), -bourbonene (0.1)
3. Results 3.1. Chemical composition of Piper essential oils The essential oil yield from Piper species ranged from 1.84 to 0.18% (average 0.63 ± 0.40%), with higher production from P. aduncum (1.19 ± 0.45%) and P. cernuum (1.10 ± 0.76%), although exhibiting great influence of exogenous factors, as demonstrated by the high standard deviation values (Table 1, Fig. 2A). In contrast, the yield of essential oils from P. amalago was smaller but less influenced by the environment (0.26 ± 0.05%) (Table 1, Fig. 2A). Terrain
elevation and harvest season did not significantly affect the yield of essential oils by the investigated Piper species (Table 1, Fig. 2A). Twenty four (5%) distinct terpenoids were identified in essential oils from 11 Piperaceae species, collected from seven rainforest locations in the state of São Paulo (Table 1). The most frequent components from the essential oils were mono- and oxygenated sesquiterpenes (Table 1, Fig. 2B). Qualitative and quantitative differences in the terpene composition were found among and within the Piper species (Table 1, Fig. 2B). The main components of the essential oils from P. leptorum, P. xylosteoides and P. umbellatum from coastal and inland regions had only oxygenated
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Fig. 1. Biogeographically defined domain of the Atlantic rainforest in Brazil (A) and plant collection sites in the state of São Paulo (B). Satellite images obtained from Google Earth.
sesquiterpenes, whereas only monoterpenes were found in oils extracted from P. rivinoides and P. solmsianum from a coastal location (Table 1, Fig. 2B). The phenylpropanoid composition of the oils was more homogeneous, with asaricin and safrole as the most abundant compounds of the class (Table 1, Fig. 2B). Phenylpropanoids were solely present in P. aduncum (Table 1, Fig. 2B), reaching up to 80% of the main components in accessions from the northernmost location in the state (Table 1, Fig. 1). The levels of phenylpropanoids, mainly asaricin, were greatly reduced (to approximately 15% of the main components) in P. aduncum from higher altitudes (Table 1). In order to gain further insight on the factors affecting the chemical composition of the essential oils from Piperaceae, we have employed cluster analyses to investigate the hierarchical relationships among the contents of sesqui-, monoterpenes and all identified metabolites (Fig. 3). Hierarchical clustering was distinct for the chemical profiles based on sesqui-, monoterpenes or all compounds (Fig. 3). The analyses of the sesquiterpene profile produced five significant clusters (Fig. 3A). The first group (Cluster I) comprised oils from P. cernuum from coastal areas (Ubatuba and Pariquera-Ac¸u) characterized by the presence of sesquiterpenes based on tricyclic 5,11-epoxy-5-,10-␣-eudesman-4-(14)-ene skeleton. The absence of sesquiterpenes from the main components was characteristic for the oils from Cluster II (Fig. 3A). The chemical profile of oils in Clusters III and IV is variable with the prevalence of bicyclogermacrene and ␥-muurolene in III and trans--guaiene and trans-nerolidol in IV, respectively (Fig. 3A). Cluster V is characterized by the presence of high contents of germacrene and trans-caryophyllene among the main components (Fig. 3A).
Based on monoterpene composition, the essential oils were grouped in three main clusters (Fig. 3B); the first one (Cluster I) characterized by the presence camphene, limonene or cis-ocymene in addition to ␣- and -pinene (Fig. 3B). Cluster II consisted exclusively of oils from P. amalago, containing myrcene, sabinene or -phellandrene along with pinene forms (Fig. 3B). The absence of monoterpenes was characteristic of the species in Cluster III (Fig. 3B), although the oil of P. aduncum from Monte Alegre do Sul consisted of approximately 6% of the monoterpene ␣-pinene (Table 1). Similarly, three clusters were formed using all identified major components; the first one characterized by the species containing phenylpropanoids asaricin and safrole (cluster I), a second large group (cluster II), with higher contents of ␣-pinene and/or biclyclogermacrene, and a third cluster (cluster III) comprising species with caryophyllene oxide, trans--guaiene, trans-nerolidol, germacrene B or -copaen-4-␣-ol (Fig. 3C). Cluster II consists of three subclusters, the first one comprising oil samples exclusively from P. cernuum (subcluster IIa), whereas, the other two consist of samples from several species containing monoterpene myrcene and sabinene and sesquiterpenes bicyclogermacrene (subcluster IIb). The last subcluster in group II is also composed of several species containing sesquiterpene germacrene B (subcluster IIc). 3.2. Factors influencing the chemical diversity of the essential oils from Piper species In order to reduce the dimensionality of the chemical data, we have employed a second exploratory unsupervised approach, the Multiple Factor Analysis (MFA), since the metabolite contents
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Fig. 2. Schematic representation of the yield (A) and main mono-, sesquiterpenes and phenylpropanoid components (B) of the essential oils from 11 Piperaceae species from seven locations of Atlantic rainforest in the State of São Paulo, Brazil. Plant species and location names are abbreviated by three or four letters as follows: Pad − P. aduncum, Pama − P. amalago, Pamp − P. amplum, Pcer − P. cernuum, Pcrass − P. crassinervium, Pgau − P. gaudichaudianum, Plep − P. leptorum, Priv − P. rivinoides, Psol − P. solmsianum, Pxyl − P. xylosteoides and Pumb −P. umbellatum and Ada − Adamantina, Camp − Campinas, MAS − Monte Alegre do Sul, Moc − Mococa, PqA − Pariquera-Ac¸u, Uba − Ubatuba, Vot − Votuporanga.
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Fig. 3. Hierarchical cluster analyses of the chemical composition of sesquiterpenes (A), monoterpenes (B) and all compounds (C) from 11 Piperaceae species, from seven locations of the Atlantic rainforest in the state of São Paulo. Contents of the chemical compounds are represented as color scale. Clustering was performed using Spearman rank correlation and is displayed as association trees. Columns represent (from left to right): (A) germacrene, trans-caryophyllene, guaiol, bicyclogermacrene, ␥-muurolene, caryophylene oxide, trans--guaiene, trans-nerolidol, valencene, spathulenol, germacrene B, -copaen-4-␣-ol, dihydro--agarofuran, 10-epi-␥-eudesmol, 4-epi-cis-dihydroagarofuran, (B) mircene, ␥-3-carene, sabinene, -phellandrene, camphene, ␣-pinene, -pinene, limonene and cis--ocymene and (C) ␣-pinene, limonene, cis--ocymene, germacrene D, trans-caryophyllene, -pinene, guaiol, dihydro--agarofuran, 10-epi-␥-eudesmol, 4-epi-cis-dihydro-agarofuran, myrcene, bicyclogermacrene, sabinene, ␥muurolene, caryophylene oxide, seychellene, trans--guaiene, trans-nerolidol, germacrene B, -copaen-4-␣-ol, spathulenol, valencene, asaricin and safrole. Cluster distance is represented vertically for the compounds and horizontally for oil samples.
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variables resulting from MFA is presented in Fig. S1. The first component is more closely correlated to the contents of -pinene, whereas, the percentages of germacrene B, -copaen-4-␣-ol, 10-epi-␥-eudesmol, 4-epi-cis-dihydro-agarofuran and dihydro-agarofuran are slightly more associated to the second dimension (Fig. S1). In agreement with hierarchical clustering data, the coordinates of the chemical composition of the samples for two main dimensions was not sufficient to group the species, with the exception of P. cernuum (Fig. 4A), indicating the presence of intraspecific variation in the chemistry of the essential oils. The confidence interval for P. aduncum samples is almost completely overlapped by the broader P. amalago interval, meaning that the chemical composition of the samples from these two species are not significantly different, differing by less than four standard errors where the confidence interval are plus/minus twice the standard error (Fig. 4A). Employing the collection location as categorical variable, MFA analyses have shown that the coastal locations Pariquera-Ac¸u and Ubatuba, which are the richest in species diversity, encompassed larger amounts of chemical variation (Fig. 4B). In contrast, the essential oils of plants from Campinas and Votuporanga exhibited the smallest range of chemical variation (Fig. 4B). The chemical composition of the essential oils isolated from plants harvested in the Autumn and Winter was more divergent with most of the variation associated to the second dimension (Fig. 4C). The higher variance in the chemical profile of the cooler seasons is probably associated to the higher number of species sampled during these seasons, reaching a total of 10 (P. aduncum, P. amalago, P. amplum, P. cernuum, P. crassinervium, P. gaudichaudianum, P. leptorum, P. rivinoides, P. umbellatum, P. xylosteoides), whereas, only three species (P. aduncum, P. amalago and P. solmsianum) were found in the Spring/Summer collections. Moreover, the chemical profile of the essential oils extracted from P. aduncum and P. amalago was shown to be less divergent (Fig. 4A). 3.3. Biological activity of Piper essential oils
Fig. 4. Multiple factor analyses of the chemical composition of the essential oils from 11 Piper species from the Atlantic rainforest in seven locations in the state of São Paulo with plant species (A), collection location (B) and sampling season (C) as categorical variables. Ellipses represent confidence level of 0.95, bootstrapped 100 times. Barycenter coordinates for each categorical variable is represented by color coded squares. Variable factor map for the oil metabolites is presented in Fig. S1.
(variable) constitute structured subsets of descriptive attributes, namely the botanical species, collection locations, and harvesting seasons. Approximately one-third (31.9%) of the variance in the chemical composition of the essential oils from the investigated Piper species was explained by the first two principal components (16.41% for PC1, and 15.50% for PC2) (Fig. 4). However, as expected for highly dimensional large-scale data, approximately 70% (68.09%) of the total variance remained unexplained by the first two components. The vector map for the metabolite
The agar diffusion method was employed to investigate the antibacterial activity of the essential oils isolated from 11 species from the Piper family sampled from seven distinct locations of Atlantic rainforest in the State of São Paulo. Three skin-associated Gram positive (S. epidermidis, S. aureus and C. xerosis) and one Gram negative (E. coli) bacterial pathogens were investigated. The vast majority of the essential oils exhibited inhibitory activity against one or more bacteria, in comparison to a commercial wide-spectrum antibiotic, with the exception of the oil from P. xylosteoides (Fig. 5). The biological activity of the volatile components of Piper species has been reported for a wide range of applications (Almeida et al., 2009; Raut and Kuruppayil, 2014), although P. nigrum remains the sole with potential application (Raut and Kuruppayil, 2015; Nikolic´ et al., 2015). The levels of bacterial growth inhibition were similar among the investigated essential oils, in spite of their distinct chemical composition (Fig. 5, Fig. 6, Fig. S2). The variable factor map from principal component analysis demonstrated that the composition of the bacterial cell wall is the most important factor determining the biological activity of the essential oils (Fig. S2). The essential oils from P. amalago from Adamantina and Mococa promoted the most significant levels of bacterial growth inhibition (Fig. 5, Fig. 6), whereas the oil isolated from P. amplum from Pariquera-Ac¸u exhibited the widest spectrum of growth inhibition, reaching at least 10% of growth inhibition for all tested bacterial strains (Fig. 5, Fig. 6). We have employed Pearson correlation analyses to investigate the association between the chemical composition of the tested essential oils and their biological activity (Fig. 7; Fig. S3). High levels of bicyclogermacre and ␥-muurolene were positively
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Fig. 5. Percentage growth inhibition of the essential oil from 11 species of plants from Piperaceae family from the Atlantic rainforest in the state of São Paulo against one Gram negative (E. coli) and three Gram-positive (S. epidermidis, S. aureus and C. xerosis) bacterial pathogens. Inhibition percentage was calculated based on the recommended concentration (300 g mL−1 ) of wide spectrum commercial antibiotic cefotaxime. Sterile mineral oil was used as negative control.
associated to strong inhibition of E. coli growth (Fig. 7A). In contrast, the presence of germacrene D and trans-caryophyllene was associated to the inhibitory activity against all tested bacteria (Fig. 7A). The presence of caryophyllene oxide was associated to the growth inhibition against the investigated Gram positive bacteria (Fig. 7A). Among the monoterpene, limonene and cis--ocymene were the
compounds with the highest correlation to bacterial growth inhibition, especially against Staphylococcus species (Fig. 7B). Higher contents of -phellandrene were positively correlated to wide spectrum inhibitory activity (Fig. 7B). In contrast, sabinene and myrcene were able to selectively inhibit the growth of E. coli and S. aureus, and E. coli and C. xerosis, respectively (Fig. 7B). The contents
Fig. 6. Principal component analyses of antibacterial activity of the essential oils from 11 species of plants from Piperaceae family from the Atlantic rainforest in the state of São Paulo against one Gram negative (E. coli) and three Gram-positive (S. epidermidis, S. aureus and C. xerosis) bacterial pathogens.
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Fig. 7. Pearson r correlation analysis between the most abundant mono and sesquiterpene components of the essential oils from 11 species of Piperaceae from the Atlantic rainforest in the state of São Paulo against one Gram negative (E. coli) and three Gram-positive (S. epidermidis, S. aureus and C. xerosis) bacterial pathogens. Pearson r is represented as colorscale. The correlation and p-values are given in Table S2.
of camphene, ␣- and -pinene exhibit negative correlation with the antibacterial activity of the essential oils for all tested bacteria (Fig. 7B). Essential oils containing phenylpropanoids asaricin and safrole exhibit no significant antibacterial activity (Fig. 7C). In contrast, asaricin and safrole contents were positively correlated to the growth inhibition against S. epidermidis and C. xerosis (Fig. 7C). The presence of asaricin was also positively correlated with the activity against S. aureus (Fig. 7C). Pearson r correlations between the bacteria and the compounds, along with their p values, are given in Table S2 and presented as percentage in Fig. S3. 4. Discussion The high botanical diversity of the genus Piper is reflected in its specialized metabolism, since twenty four distinct terpenoids (5%) were identified from 11 species. Accordingly, terpene composition was quantitative and qualitatively different among the investigated species. In plants, terpenoid biosynthesis depends on 5-carbon compounds, namely isopentenyl diphosphate (IPP) and dimethyl allyl diphosphate (DMAPP), produced by the mevalonate (MVA) and the 2-C-methyl-d-erythritol-4-phosphate (MEP) dependent pathway (Vranová et al., 2013). The precursors DMAPP and IPP are fused to form the 10-carbon monoterpenoids, which, in turn, are further coupled to other molecule of IPP to produce the 15carbon sesquiterpenoids. The natural diversity of plant terpenoids is associated to the complex network controlling the biosynthesis and biological function of these compounds (Kitaoka et al., 2015). The complex interactions between environmental and endogenous factors is responsible for intraspecific differences in the specialized metabolism (Schuman et al., 2016). In the current study, quantitative differences in the chemical profile were found in P.
aduncum, P. amalago and P. cernuum, whereas significant qualitative differences were present in P. aduncum. The interspecific variation in the oil composition was smaller for P. aduncum and P. amalago, although the chemical composition of the essential oils only allowed clustering of P. cernuum individuals. The oils from P. aduncum sampled from deforested and intact areas were demonstrated to have distinct chemical profiles, with oils from plants grown in intact forest areas enriched in terpene compounds (Almeida et al., 2009). Similarly, terpenes were the most abundant chemical class in P. aduncum from Monte Alegre do Sul, indicating that the specialized metabolism of the species in the region is similar to that of plants from intact forests. Phenylpropanoids were solely present in P. aduncum, reaching up to 80% of the main components, however its levels were reduced in plants from higher altitudes. Phenylpropanoids are involved in a wide range of developmental and adaptation processes in plants, thus, a complex network of factors regulates their biosynthesis and accumulation (Gaquerel et al., 2014). Abiotic factors, such as light and nutrients, are considered the main modulators of the expression of genes in the phenylpropanoid biosynthesis pathway and could contribute for the observed decreased levels in plants from higher altitudes, along with possible differences in the genetic background of the plants. Hierarchical clustering was employed to investigate the relationships between the chemical composition of the essential oils and Piper species growing in different locations, sampled at different times of the year. The use of distinct chemical classes resulted in differential clustering of the species, in agreement with the distinct intraspecific chemical profile of the essential oils from P. aduncum, P. amalago and P. cernuum. Our data suggest that the sesquiterpene profile based on compounds with tricyclic 5,11-epoxy-5-,10-␣-eudesman-4-(14)-ene skeleton is useful to
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identify P. cernuum. Similarly, the monoterpene profile based on the presence of myrcene, sabinene or -phellandrene may provide auxiliary tools to identify P. amalago. The multivariate and clustering analyses demonstrate the strong influence of environmental factors on the production of phenylpropanoid or terpenoid compounds of interest in Piper species. In agreement with the analyses of the chemical composition of the essential oils, botanical studies investigating the morphology of Piper flowers and inflorescences were unable to resolve infrageneric taxonomy (Jaramillo and Manos, 2001; Tebbs, 1993). More recently, the use of molecular markers based on internal transcribed spacers (ITS) of nuclear ribosomal DNA provided a consistent separation between the South Pacific/Asia and the Neotropical (American) clade, although several infrageneric recognized groups of the American cluster remained monophyletic (Jaramillo and Manos, 2001). Other studies have also demonstrated that the agreement among chemical, morphological and genetic data is variable and highly dependent on the plant material (Barra, 2009). Thus, the association of several characterization techniques may be required to discriminate precisely Piper species of interest. Despite the differences in their chemical profile, the vast majority of the essential oils exhibited inhibitory activity against one or more bacteria. The biological activity of the volatile components of Piper species has been reported for a wide range of applications (Almeida et al., 2009; Raut and Kuruppayil, 2014), although P. nigrum remains the sole with potential application (Raut and Kuruppayil, 2015; Nikolic´ et al. , 2015). Bacterial cell wall composition is the most important factor controlling the biological activity of the Piper oils. The antibacterial effect of plant essential oils is hypothesized to be dependent on the disruption of the organization of lipids in bacterial cellular and mitochondria membranes, causing the leakage of cell contents (Solórzano-Santos and Miranda-Novales, 2012). The thicker, theicoic acids containing cell wall of Gram positive bacteria make them more resistant to essential oils (Solórzano-Santos and Miranda-Novales, 2012). The determination of the compound(s) responsible for biological activity of essential oils require extensive purification procedures, which makes the process expensive and ineffective. In contrast, the assumption that the most abundant component in the oil is responsible for the biological activity may be misleading but can provide a starting point for further, more detailed investigations. We have employed Pearson correlation analyses to investigate the association between the chemical composition of the tested essential oils and their biological activity. The presence of high levels of germacrene D and trans-caryophyllene was associated to the inhibitory activity against all tested bacteria, whereas caryophyllene oxide was associated to the growth inhibition against the investigated Gram positive bacteria. Among the monoterpene, limonene and cis--ocimene were the compounds with the highest correlation to bacterial growth inhibition, especially against Staphylococcus species. The contents of camphene, ␣- and -pinene exhibit negative correlation with the antibacterial activity of the essential oils for all tested bacteria. Interestingly, camphene isolated from P. cernuum has been demonstrated to promote in vivo antitumor activity, by inducing intrinsic apoptosis of melanoma cells due to stress of the endoplasmic reticulum, followed by the release of Ca2+ along with HmgB1 and calreticulin, loss of mitochondrial membrane potential and up regulation of caspase-3 activity (Girola et al., 2015). The distinct features between prokaryotic bacterial and eukaryotic cells may account for the distinct biological action of the compound. Volatile plant terpenes are collectively associated to defensive roles against pathogens and herbivores (Richter et al., 2015; Matarese et al., 2014; Menzel et al., 2014; Trowbridge et al., 2014). However, the individual roles of the compounds in distinct biotic stresses is likely to differ. In general, the deleterious
effect of terpenes on microbial or herbivore pathogens is thought to involve oxidative damage of cell membranes and disrupting cell metabolism and energy production (Chan et al., 2013; Chueca et al., 2014). Our in vitro assays have demonstrated that several abundant monoterpenes in plant essential oils, such as pinene forms and camphene do not appear to have a role against the investigated bacteria. In contrast, as demonstrated for other species, including cultivated rice, limonene appears to have an important defensive role against bacterial pathogens (Lee et al., 2015). In other species, pinene has been demonstrated to be associated to biological activity against fungus, insects and protozoans (Pontin et al., 2015; Rodrigues et al., 2015). Although in vitro analyses may help to investigate the biological function of natural products, the complex mixture of compounds usually produced by the plants prevents the definite determination of their biological function. The chemical profile of the essential oils from Piper species in the Atlantic rainforest results from the complex interactions among physiological, genetic and environmental factors, and contribute to generate the metabolic plasticity required for the adaptation. In order to profit from the promising bioactive compounds from Piper species, further studies on the genus genetics, development and physiology are required. 5. Conclusions The essential oils from Piper species in the Atlantic rainforest are mainly composed by mono- and sesquiterpenes, with phenylpropanoids restricted to P. aduncum. The chemical composition of the oils from the investigated species is influenced, but not determined, by the plant species and environmental conditions. The chemical composition of the oils based on mono-, sesquiterpenes and/or phenylpropanoids generates distinct clustering of the Piper species, thus, chemotaxonomic data should be coupled with genetic and botanical analyses. The vast majority of the isolated essential oils exhibit in vitro biological activity against pathogenic Gram negative and positive bacteria. Higher contents of biclyclogermacrene and ␥-muurolene are associated with strong inhibitory activity against E. coli, whereas the levels of limonene and cis--ocymene are positively correlated to antibacterial activity against Staphylococci. Acknowledgments The authors would like to thank Fundac¸ão de Apoio à Pesquisa do Estado de São Paulo (FAPESP) and Natura Inovac¸ão e Tecnologia de Produtos Ltda for financial support (grant 03/08896-1 to MOMM). The authors also would like to express their gratitude to Prof. Dr. Lin Chau Ming (Universidade Estadual Paulista Júlio de Mesquita Filho − UNESP) for his help during the conception of the work. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.indcrop.2016.09. 028. References Abramoff, M.D., Magalhaes, P.J., Ram, S.J., 2004. Image processing with image. J. Biophot. Int. 11, 36–42. Adams, R.P., 2007. Identification of Essential Oil Components by Gas Cromatography/Mass Spectroscopy. Allured Publ. Corp, Carol Stream. Almeida, R.R.P., Souto, R.N.P., Bastos, C.N., Silva, M.H.L., Maia, J.G.S., 2009. Chemical variation in Piper aduncum and biological properties of its dillapiole-rich essential oil. Chem. Biodivers. 6, 1427–1434, http://dx.doi.org/10.1002/cbdv. 200800212. Barra, A., 2009. Factors affecting chemical variability of essential oils: a review of recent developments. Nat. Prod. Commun. 4, 1147–1154.
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Industrial Crops and Products journal homepage: www.elsevier.com/locate/indcrop
The chemical composition and antibacterial activity of eleven Piper species from distinct rainforest areas in Southeastern Brazil Crislene Vaz Perigo a , Roseli B. Torres a , Luís C. Bernacci a , Elsie F. Guimarães d , Lenita L. Haber c , Roselaine Facanali a , Maria A.R. Vieira a , Vera Quecini b,∗ , Márcia. Ortiz M. Marques a a
Instituto Agronômico – Av. Barão de Itapura, 1481, Campinas, SP 13020-902, Brazil Embrapa Uva e Vinho – Av. Livramento, 515, Bento Gonc¸alves, RS, 95701-008, Brazil c Embrapa Hortalic¸as, Rodovia BR-060, Km 09, Brasília, DF 70359-970, Brazil d Instituto de Pesquisas Jardim Botânico do Rio de Janeiro, Rua Pacheco Leão, 915, Rio de Janeiro, RJ 22460-030, Brazil b
a r t i c l e
i n f o
Article history: Received 30 April 2016 Received in revised form 30 July 2016 Accepted 12 September 2016 Keywords: Antimicrobial Bacteria Essential oil Monoterpene Rainforest Sesquiterpene
a b s t r a c t Piper is a large and highly diverse genus of plants with medicinal and aromatic use. It is widely distributed in tropical forests occupying distinct environments. We have determined the chemical composition of the essential oils from 11 species of Piper from seven locations of Atlantic rainforest in Brazil. The essential oils were isolated by hydrodistillation and their composition, determined by GC–MS. The chemical composition of the oils exhibits qualitative and quantitative differences among the species and intraspecifically for distinct locations. Mono and sesquiterpenes were the most abundant compounds, although P. aduncum also contained phenylpropanoids. The oils of P. xylosteoides, P. umbellatum and P. leptorum from coastal and inland regions consisted exclusively of oxygenated sesquiterpenes. In contrast, monoterpene was the sole chemical class in the oils extracted from P. rivinoides and P. solmsianum. Hierarchical clustering analyses demonstrated the chemical phenotype is associated to species-specific and environmental factors. The essential oils from the majority of the investigated Piper species exhibit inhibitory activity against pathogenic bacteria in vitro, reaching up to 30% of the inhibition levels of commercial antibiotics. The contents of bicyclogermacre and ␥-muurolene were positively associated to E. coli inhibition, whereas, levels of germacrene D and trans-caryophyllene were associated to the inhibitory activity against all tested bacteria. Limonene and cis--ocymene were associated with Staphylococci inhibition. Higher contents of -phellandrene were positively correlated to wide spectrum antibacterial activity. Taken together, our results demonstrate the chemical diversity of Piper essential oils and their potential as novel antibacterial agents for several industrial applications. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Piperaceae is a highly diverse family of small trees, shrubs or lianas distributed throughout the tropical regions in the Americas, Asia, Africa and Southern Pacific. It consists of more than 3000 species, recently attributed to three large sub-families: Verhuellioideae, Piperoideae and Zippelideae (Semain et al., 2008). Although the family distribution is pantropical, Central and South America, along with Malaysia, are considered the diversity centers (Quijano-Abril et al., 2006). In Brazil, species of the Piperaceae
∗ Corresponding author. E-mail address: [email protected] (V. Quecini). http://dx.doi.org/10.1016/j.indcrop.2016.09.028 0926-6690/© 2016 Elsevier B.V. All rights reserved.
family are spread throughout a wide range of primary and secondary rainforest areas, from the sea level up to 2000 m in altitude. Besides the commercial importance of black pepper (Piper nigrum L.), several other species from the Piper genus are known for their aromatic and medicinal properties (Picard et al., 2014; Raut and Karuppayil, 2014). The specialized metabolism of the genus Piper is diverse and include alkaloids, terpenoids and flavonoids (Scott et al., 2008). The essential or volatile oils are complex mixture of chemical compounds produced by plants from terpenes and their oxygenated derivatives, terpenoids, aromatic and aliphatic acid esters and phenols (Solórzano-Santos and Miranda-Novales, 2012). In nature, the chemical properties of plant terpenoids make them important biological components in defense and signaling processes, helping their adaptation to complex environments (Gershenzon and Dudareva, 2007; Pichersky and Lewinsohn, 2011).
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The hydrophobicity and reactivity of these compounds allow the partition with the lipids from biological membranes, leading to disturbances in its structure, thus, impairing cellular functions. These properties of plant volatile compounds make them potentially interesting for antibacterial, antifungal and antiviral activity in medical, cosmetic and technological applications (SolórzanoSantos and Miranda-Novales, 2012). In the current work, we have chemically characterized the essential oils of 11 Piper species from seven distinct rainforest locations in the State of São Paulo, Brazil. The influence of the species and location on the chemical composition was investigated by multivariate analyses. The biological activity of the essential oils was tested against four bacterial species, pathogenic to mammals, and the inhibitory action of the individual monoterpene, sesquiterpene and phenylpropanoid compounds was assessed by correlation analyses.
2. Material and methods 2.1. Collection of biological samples Seven independent locations of Atlantic rainforest in the State of São Paulo, Brazil (Fig. 1) were sampled for plants of the Piper genus, comprehending 11 species. The aerial part of the plants was collected and used for botanical identification, composition of herbarium mounts and chemical analyses. The sampled plants were marked and coordinate reference locations were determined by Global Positioning System (GPS). Marked plants were resampled, throughout the year, in two subsequent years. The individuals failing to be located or in poor condition were excluded from the analyses. The names of the identified species are presented according to the list of species of the Brazilian flora (Lista de Espécies da Flora do Brasil, Jardim Botânico do Rio de Janeiro (2015), available at http:// floradobrasil.jbrj.gov.br). Voucher specimens were deposited at the Herbarium of the Instituto Agronômico (IAC) (http://herbario.iac. sp.gov.br/) and the voucher numbers are given in Table 1. 2.2. Essential oil extraction Leaves were separated from woody stalks and air dried at room temperature, under diffuse light. Fresh plant material ranging from 54 to 1870 g, depending on availability, was used to extract the essential oils using a Clevenger-type apparatus until total recovery of oil. The oils were transferred to hermetically closed vials and stored at −20 ◦ C until further use. 2.3. Chemical characterization and quantification of essential oils The chemical composition of the essential oils was determined by gas chromatography coupled to mass spectrometry (GC–MS) using a Shimadzu QP-5000 equipped with fused silica capillary column OV − 5 (30 m × 0.25 mm × 0.25 m, Ohio Valley Specialty Chemical, Inc., USA), operating with electron impact (70 eV), using Helium (1.0 mL/min.) as carrier, injector and detector at 240 ◦ C and 230 ◦ C, respectively, split ratio of 1/30 and the following temperature program: 60 ◦ C–240 ◦ C, 3 ◦ C/min. Area normalization method of triplicate readings was used to quantify the extracted oils by gas chromatography with flame ionization detection (GC–FID) at a Shimadzu equipment, model GC-2010, under the previously described operating conditions. The compounds were identified by comparative analyses of the mass spectra against the database of the GC/MS system (Nist 62.lib) along with retention indices (Adams, 2007), obtained from the injection of a mixture of
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n-alcanes (C9 H20 –C25 H52 , Sigma Aldrich, 99%) using Van Den Dool and Kratz (1963) equation. 2.4. Bacterial strains and growth conditions Certified cultures of Escherichia coli (ATCC 8739), Staphylococcus aureus (ATCC 6538), S. epidermidis (ATCC 12228), Corynebacterium xerosis (ATCC 373) and Pseudomonas aeruginosa (ATCC 9027) were provided as lyophilized stocks from Instituto Adolfo Lutz, São Paulo, Brazil. Cultures were reactivated, grown in liquid media and kept as 50% (w/v) glycerol stocks at −80 ◦ C. For biological activity assays, fresh bacteria cultures were initiated from isolated colonies on Agar Nutrient Broth (Oxoid, UK). Bacterial concentration was estimated based on spectrophotometric absorbance readings at 600 nm for E. coli and P. aeruginosa, 490 nm for S. epidermidis and P. aeruginosa, 530 nm for S. aureus and 578 nm for C. xerosis. 2.5. Antibacterial activity analyses Antibacterial activity of the oils was investigated by growth inhibition in agar diffusion assays against four bacterial species. Growth inhibition positive controls were Melaleuca essential oil and the antibiotics cefotaxime and negative control was sterile mineral oil. A single colony was inoculated to 20 mL of liquid TSB (Oxoid, UK) and grown to saturation (approximately 14 h) with 200 rpm shaking. An aliquot of 1 mL was transferred to 20 mL of fresh TSB and the procedure was repeated twice, so that bacterial cultures were replicated three times prior to biological activity analyses. For each bacterial species, 400 mL of fused Nutrient Agar medium (Oxoid, UK), supplemented with 1.5 mL of a 2% (w/v) solution of 2,3,5-triphenyl tetrazolium chloride (TTC), were kept at 45 ◦ C and added with 1.0 mL of the saturated bacterial culture. The mixture was poured on 9 mm sterile Petri dishes containing five, evenly distributed aluminum rings of 6 mm of diameter. The rings were removed from the solidified medium and 300 uL of essential oil at 5% (w/v) in sterile mineral oil were added to the wells. The plates were incubated horizontally in a bacteriological oven at 37 ± 2 ◦ C for 48 h and the inhibition halos were measured using a digital caliper (Mitutoyo America Corp., Illinois, USA). Plates were also digitalized and halo measures obtained by ImageJ software (Abramoff et al., 2004). Halo measurements from the actual plates and digitalized images were equivalent. Bacterial concentrations on the saturated suspension were corrected to 108 colony forming unit (CFU) mL−1 by absorbance readings. 2.6. Statistical analyses The assays of antimicrobial activity were performed in triplicate, in independent experiments for oil samples from independent extractions and collections. Chemical composition results were analyzed by ANOVA, honest significant difference (HSD) Tukey’s test and multifactorial analyses using the R project for statistical computing (R Core Team, 2014) and the package FactoMineR (Lê et al., 2008). Antibacterial activity was correlated to the contents of the major components in the oils using the function cor and the method Pearson in R. Graphs were plotted using the package corrgram (Wright, 2015). The function rcor from the package Hmisc (Harrell, 2016) was used to determine the correlations/covariances and significance levels. Chemical composition results were compared and submitted to multivariate statistical analyses (MANOVA) using the software MINITAB Statistical. Hierarchical clustering analyses were performed in R using the hclust function on composition data standardized by a vector of means (function center) and a vector of standard deviations (function scale).
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Table 1 Main (≥5%) and minor components of the essential oil extracted from Pipearaceae of the Atlantic rainforest. Essential oil yield is presented as percentage from dry weight (w/v). Retention indices of the metabolites are given in Table S1. Species
Sample/Herbarium Code
Location (decimal coordinates, elevation)
Yield (%)
Main Components (%)
Minor Components (%)
Piper aduncum L.
Lp52911/IAC 47090
Monte Alegre do Sul (−46.672500, −22.70010, 743m)
0.51
spathulenol (10.6), valencene (9.7), ␣-pinene (6.4) asaricin (14.9), safrole (13.3)
Piper aduncum L.
Lp6101606/IAC 47958
Votuporanga (−50.064750, −20.46286, 458m)
1.52
asaricin (80.1), safrole (10.8)
Piper aduncum L.
Lp6101608/IAC 47960
Votuporanga (−50.064750, −20.46294, 465m)
1.55
asaricin (73.4), safrole (10.5)
Piper amalago L.
Lp06091202/IAC 32056
Campinas (−47.067306, −22.86491, 664m)
0.20
-phellandrene (39.3), ␣-pinene (14.8), germacrene D (11.7)
Piper amalago L.
Lp06091206/IAC 46823
Campinas (−50.064750, −20.46286, 458m)
0.36
-phellandrene (15.9), ␣-pinene (6.7), sabinene (6.3), bicyclogermagrene (20.8), spathulenol (9.1)
Piper amalago L.
R1763/IAC 47512
Mococa (−46.981013, −21.44825, 600m)
0.26
-phellandrene (33.1), ␣-pinene (11.7), bicyclogermagrene (15.0)
Piper amalago L.
Lp6101821/IAC 47987
Adamantina (−51.152194, −21.66311, 349m)
0.23
-phellandrene (12.3), sabinene (8.2), myrcene (6.8), bicyclogermagrene (19.4), ␥-muurolene (5.9), spathulenol (5.6)
Piper amplum Kunth.
R1740/IAC 7267
Pariquera-Ac¸u (−47.880212, −24.61345, 25m)
0.38
Piper cernuum Vell.
L51904/IAC 7068
Ubatuba (−45.127511, −23.42181, 30m)
0.32
␣-pinene (18.1), cis--ocimene (10.5), limonene (8.6), trans-caryophyllene (8.8), germacrene D (5,5) ␣-pinene (10.0), camphene (6.3), dihydro-agarofuran (28.7), 10epi- ␥-eudesmol (13.5), 4-epi-cis-dihydroagarofuran (10.8)
 − pinene (4.5), ␥-cadinene (3.9), trans-caryophyllene (3.6), caryophyllene oxide (3.6), ␦-cadinene (2.6), trans--ocimene (2.7), linalool (2.2), epi-␣-cadinol (1.7), limonene (1.3), cis--ocimene (1.0), ␣-copaene (0.8), germacrene D (0.8), ␣-muurolene (0.6), dehydro-aromadrene (0.3), ␥-gurjunen (0.4), camphene (0.3), germacrene B (0.2) trans--ocimene (1.9), ␥-cadinene (1.2), spathulenol (1.0), germacrene B (0.9), germacrene D (0.9), valencene (0.8), cis--ocimene (0.6), trans-caryophyllene (0.4), ␦-cadinene (0.2),  − pinene (0.2) trans--ocimene (5.0), valencene (3.1), cis--ocimene (1.9), ␥-cadinene (1.0), spathulenol (1.0), ␣-muurolene (0.7), germacrene D (0.6), germacrene B (0.5), trans-caryophyllene (0.5), ␦-cadinene (0.3),  − pinene (0.3), ␣-pinene (0.1), ␣-humulene (0.1) myrcene (3.2), trans-caryophyllene (2·8), -bourbonene (2.6), ␣-phellandrene (1.9), ␣-copaene (1.4), -pinene (1.4), bicyclogermacrene (1.0), spathulenol (1.0), cis--ocimene (0.8), ␦-elemene (0.6), linalool (0.7), germacrene B (0.5), sabinene (0.4), ␣-muurulene (0.3), ␦-cadinene (0.3) ␦-cadinene (4.6), myrcene (4.3), epi-␣-cadinol (4.1), caryophyllene oxide (3.0), trans-nerolidol (2.5), trans-caryophyllene (2.4), cis--ocimene (1.3), linalool (1.3), -pinene (1.1), ␥-cadinene (1.0), ␣-muurulene (0.9), ␣-phellandrene (0.9), ␣-muurulol (0.5), o-cymene (0.5), trans--ocimene (0.5), 10-epi-␥-eudesmol (0.4), ␣-thujene (0.2), -elemene (0.2) myrcene (2.8), 1-epi-cubenol (2.6), trans-caryophyllene (2.1), ␦-cadinene (1.9), ␣-phellandrene (1.9), cis--ocimene (1.9), ␦-elemene (1.7), trans-nerolidol (1.5), epi-␣-cadinol (1.2), ␥-cadinene (1.1), -pinene (1.0), linalool (0.8), o-cymene (0.8), caryophyllene oxide (0.5), germacrene D (0.5), trans--ocimene (0.5), ␣-muurulene (0.3), sabinene (0.3), ␣-terpinine (0.2), -elemene (0.2) ␣-pinene (4.8), ␦-cadiene (3.6), trans-caryophyllene (3.6), trans-nerolidol (2.0), linalool (1.9), 2-decanol (1.8), germacrene D (1.8), epi-␣-cadinol (1.7), cis--ocimene (1.6), ␦-elemene (1.5), ␥-cadiene (1.3), ␣-muurulene (1.2), -elemene (1.2), ␣-copaene (1.1), ␣-phellandrene (1.0), caryophyllene oxide (0.9), 1-epi-cubenol (0.8), ␣-humulene (0.8), -pinene (0.9), trans--ocimene (0.4), ␣-muurulol (0.3) bicyclogermacrene (2.6), ␣-copaene (1.7), trans--ocimene (1.5), germacrene A (1.1), ␣-humulene (1.0), -pinene (0.9), ␦-cadinene (0.9), camphene (0.5), aromadrene (0.4), cis--guaiene (0.2), myrcene (0.2) -pinene (3.8), trans-caryophyllene (2.0), ␣-muurulol (1.8), ␥-cadinene (1.6), ␣-agarofuran (1.2), germacrene D (0.7), ␥-eudesmol (0.7), limonene (0.7), trans-nerolidol (0.7), ␥-terpinene (0.5), cis--guaiene (0.4), myrcene (0.4), ␣-terpinene (0.3), ␦-cadinene (0.3), germacrene A (0.3), spathulenol (0.3), ␣-copaene (0.2), ␣-humulene (0.2), -elemene (0.2), caryophyllene oxide (0.2), methyl-geranate (0.2), germacrene B (0.1), sabinene (0.1), terpinolene (0.1)
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Table 1 (Continued) Species
Sample/Herbarium Code
Location (decimal coordinates, elevation)
Yield (%)
Main Components (%)
Minor Components (%)
Piper cernuum Vell.
R1741/IAC 7268
Pariquera-Ac¸u (−47.880212, −24.61345, 25m)
1.84
␣-pinene (11.8), camphene (8.7), dihydro--agarofuran (33.8), 10-epi-␥-eudesmol (12.2)
Piper crassinervium Kunth.
R1764/IAC 7513
Mococa (−46.998695, −21.42348, 568m)
0.53
-pinene (11.6), ␣-pinene (11.5), germacrene D (9.2), trans-caryophyllene (7.8), guaiol (5.5), bicyclogermacrene (5.1)
Piper gaudichaudianum Kunth.
R1738/IAC 7265
Pariquera-Ac¸u (−47.880212, −24.61345, 25m)
0.16
␣-pinene (12.2), -pinene (7.0), trans-nerolidol (17.5), caryophyllene oxide (8.5), trans-caryophyllene (8.2), trans--guaiene (6.9)
Piper leptorum Kunth.
Lp052903/IAC 7085
Monte Alegre do Sul (−46.665000, −22.70370, 778m)
0.60
seychellene (34.7), caryophyllene oxide (12.5)
Piper rivinoides Kunth.
L52007/IAC 47078
Ubatuba (−45.121121, −23.42121, 30m)
0.63
␣-pinene (73.2), -pinene (5.2)
Piper solmsianum C.DC.
R1633/IAC 46832
Ubatuba (−45.119111, −23.40902, 40m)
0.39
␦-3-carene (66.9), myrcene (26.1), ␣-pinene (22.7), ␣-selinene (5.5)
Piper umbellatum (L.)
R4169/IAC 46978
Campinas (−47.067972, −22.86516, 667m)
0.18
germacrene D (55.8), bicyclogermacrene (11.8), trans-caryophyllene (6.3)
Piper xylosteoides (Kunth.) Steud.
L52004/IAC 47075
Ubatuba (−45.127123, −23.42112, 30m)
1.04
spathulenol (12.3), germacrene B (10.6), -copaen-4-␣-ol (9.4), trans-nerolidol (8.2), trans--guaiene; (7.8)
-pinene (2.8), ␥-cadinene (1.7), trans-caryophyllene (1.6), ␣-agarofuran (1.4), ␣-muurulol (1.3), -phellandrene (0.7), trans-nerolidol (0.6), ␣-copaene (0.5), cis--guaiene (0.4), ␥-eudesmol (0.4), germacrene D (0.3), myrcene (0.3), methyl-geranate (0.2), trans-nerolidol (5.0), ␦-cadinene (4·2), ␥-muurolene (3.3), 6-methyl-5-hepten-2-one (2.4), ␥-cadinene (1.7), 1-epi-cubenol (1.5), spathulenol (1.4), ␣-eudesmol (1.1), aromadrene (1.0), -phellandrene (1.0), linalool (1.0), ␣-copaene (0.7), ␣-muurolene (0.7), ␣-humulene (0.6), ␣-calacorene (0.5), -elemene (0.3), camphene (0.3), -gurjunene (0.2), -bourbonene (0.1) 7-epi-␣-selinene (4.5), limonene (3.9), cis--guaiene (3.8), ␥-cadiene (2.4), linalool (2.7), aromadrene (1.8), germacrene D (1.5), ␣-humulene (1.4), ␦-cadinene (1.1), spathulenol (0.9), ␣-phellandrene (0.8), trans--ocimene (0.6), ␥-muurulene (0.5), myrcene (0.5), o-cymene (0.3), camphene (0.2), spathulenol (4.7), ␣-pinene (3.2), ␣-selinene (3.0), myrcene (2.8), germacrene D (2.7), -pinene (2.2), cis--guaiene (1.9), ␣-humulene (1.6), germacrene B (1.6), 7-epi-␣-selinene (1.4), ␥-muurolene (1.3), guaiol (1.0), limonene (0.9), -elemene (0.6), ␥-gurjunene (0.6), germacrene A (0.5), ␥-cadinene (0.3), sabinene (0.3), terpinolene (0.3), ␦-cadinene (0.2), ␣-copaene (0.1), o-cymene (0.1), trans-nerolido (0.1) linalool (4.0), bicyclogermacrene (3.9), caryophyllene oxide (2.2), aromadrene (1.6), camphene (1.6), spathulenol (1.5), trans-caryophyllene (1.5), limonene (1.2), myrcene (1.2), cis-calamene (0.6), 10-␥-epi-eudesmol (0.4), o-cymene (0.4), dehydro-aromadrene (0.3), ␥-cadinene (0.2), ␣-cubebene (0.1), ␣-phellandrene (0.1) -pinene (3.5), ␣-phellandrene (3.4), -phellandrene (3.0), dehydro-aromadrene (2.2), germacrene D (1.5), trans-caryophyllene (1.5), terpinolene (0.7), sabinene (0.6), camphene (0.3), germacrene B (0.2), p-cymene (0.1), trans--ocimene (0.1) -cubebene (3.2), caryophyllene oxide (2.5), ␦-cadinene (2.0), ␣-copaene (1.5), ␦-elemene (1.5), ␥-cadinene (1.4), germacrene A (1.3), ␥-muurolene (0.5), ␣-humulene (0.7), ␣-muurulol (0.6), -bourbonene (0.6), ␣-cubebene (0.4), trans-nerolidol (0.4), -gurjunene (0.2) dehydro-aromadrene (4.8), trans-caryophyllene (4.5), -elemene (4.1), cis-calamenene (3.7), limonene (3.5), germacrene A (2.0), ␣-humulene (1.9), ␥-elemene (0.4), germacrene D (0.3), -bourbonene (0.1)
3. Results 3.1. Chemical composition of Piper essential oils The essential oil yield from Piper species ranged from 1.84 to 0.18% (average 0.63 ± 0.40%), with higher production from P. aduncum (1.19 ± 0.45%) and P. cernuum (1.10 ± 0.76%), although exhibiting great influence of exogenous factors, as demonstrated by the high standard deviation values (Table 1, Fig. 2A). In contrast, the yield of essential oils from P. amalago was smaller but less influenced by the environment (0.26 ± 0.05%) (Table 1, Fig. 2A). Terrain
elevation and harvest season did not significantly affect the yield of essential oils by the investigated Piper species (Table 1, Fig. 2A). Twenty four (5%) distinct terpenoids were identified in essential oils from 11 Piperaceae species, collected from seven rainforest locations in the state of São Paulo (Table 1). The most frequent components from the essential oils were mono- and oxygenated sesquiterpenes (Table 1, Fig. 2B). Qualitative and quantitative differences in the terpene composition were found among and within the Piper species (Table 1, Fig. 2B). The main components of the essential oils from P. leptorum, P. xylosteoides and P. umbellatum from coastal and inland regions had only oxygenated
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Fig. 1. Biogeographically defined domain of the Atlantic rainforest in Brazil (A) and plant collection sites in the state of São Paulo (B). Satellite images obtained from Google Earth.
sesquiterpenes, whereas only monoterpenes were found in oils extracted from P. rivinoides and P. solmsianum from a coastal location (Table 1, Fig. 2B). The phenylpropanoid composition of the oils was more homogeneous, with asaricin and safrole as the most abundant compounds of the class (Table 1, Fig. 2B). Phenylpropanoids were solely present in P. aduncum (Table 1, Fig. 2B), reaching up to 80% of the main components in accessions from the northernmost location in the state (Table 1, Fig. 1). The levels of phenylpropanoids, mainly asaricin, were greatly reduced (to approximately 15% of the main components) in P. aduncum from higher altitudes (Table 1). In order to gain further insight on the factors affecting the chemical composition of the essential oils from Piperaceae, we have employed cluster analyses to investigate the hierarchical relationships among the contents of sesqui-, monoterpenes and all identified metabolites (Fig. 3). Hierarchical clustering was distinct for the chemical profiles based on sesqui-, monoterpenes or all compounds (Fig. 3). The analyses of the sesquiterpene profile produced five significant clusters (Fig. 3A). The first group (Cluster I) comprised oils from P. cernuum from coastal areas (Ubatuba and Pariquera-Ac¸u) characterized by the presence of sesquiterpenes based on tricyclic 5,11-epoxy-5-,10-␣-eudesman-4-(14)-ene skeleton. The absence of sesquiterpenes from the main components was characteristic for the oils from Cluster II (Fig. 3A). The chemical profile of oils in Clusters III and IV is variable with the prevalence of bicyclogermacrene and ␥-muurolene in III and trans--guaiene and trans-nerolidol in IV, respectively (Fig. 3A). Cluster V is characterized by the presence of high contents of germacrene and trans-caryophyllene among the main components (Fig. 3A).
Based on monoterpene composition, the essential oils were grouped in three main clusters (Fig. 3B); the first one (Cluster I) characterized by the presence camphene, limonene or cis-ocymene in addition to ␣- and -pinene (Fig. 3B). Cluster II consisted exclusively of oils from P. amalago, containing myrcene, sabinene or -phellandrene along with pinene forms (Fig. 3B). The absence of monoterpenes was characteristic of the species in Cluster III (Fig. 3B), although the oil of P. aduncum from Monte Alegre do Sul consisted of approximately 6% of the monoterpene ␣-pinene (Table 1). Similarly, three clusters were formed using all identified major components; the first one characterized by the species containing phenylpropanoids asaricin and safrole (cluster I), a second large group (cluster II), with higher contents of ␣-pinene and/or biclyclogermacrene, and a third cluster (cluster III) comprising species with caryophyllene oxide, trans--guaiene, trans-nerolidol, germacrene B or -copaen-4-␣-ol (Fig. 3C). Cluster II consists of three subclusters, the first one comprising oil samples exclusively from P. cernuum (subcluster IIa), whereas, the other two consist of samples from several species containing monoterpene myrcene and sabinene and sesquiterpenes bicyclogermacrene (subcluster IIb). The last subcluster in group II is also composed of several species containing sesquiterpene germacrene B (subcluster IIc). 3.2. Factors influencing the chemical diversity of the essential oils from Piper species In order to reduce the dimensionality of the chemical data, we have employed a second exploratory unsupervised approach, the Multiple Factor Analysis (MFA), since the metabolite contents
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Fig. 2. Schematic representation of the yield (A) and main mono-, sesquiterpenes and phenylpropanoid components (B) of the essential oils from 11 Piperaceae species from seven locations of Atlantic rainforest in the State of São Paulo, Brazil. Plant species and location names are abbreviated by three or four letters as follows: Pad − P. aduncum, Pama − P. amalago, Pamp − P. amplum, Pcer − P. cernuum, Pcrass − P. crassinervium, Pgau − P. gaudichaudianum, Plep − P. leptorum, Priv − P. rivinoides, Psol − P. solmsianum, Pxyl − P. xylosteoides and Pumb −P. umbellatum and Ada − Adamantina, Camp − Campinas, MAS − Monte Alegre do Sul, Moc − Mococa, PqA − Pariquera-Ac¸u, Uba − Ubatuba, Vot − Votuporanga.
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Fig. 3. Hierarchical cluster analyses of the chemical composition of sesquiterpenes (A), monoterpenes (B) and all compounds (C) from 11 Piperaceae species, from seven locations of the Atlantic rainforest in the state of São Paulo. Contents of the chemical compounds are represented as color scale. Clustering was performed using Spearman rank correlation and is displayed as association trees. Columns represent (from left to right): (A) germacrene, trans-caryophyllene, guaiol, bicyclogermacrene, ␥-muurolene, caryophylene oxide, trans--guaiene, trans-nerolidol, valencene, spathulenol, germacrene B, -copaen-4-␣-ol, dihydro--agarofuran, 10-epi-␥-eudesmol, 4-epi-cis-dihydroagarofuran, (B) mircene, ␥-3-carene, sabinene, -phellandrene, camphene, ␣-pinene, -pinene, limonene and cis--ocymene and (C) ␣-pinene, limonene, cis--ocymene, germacrene D, trans-caryophyllene, -pinene, guaiol, dihydro--agarofuran, 10-epi-␥-eudesmol, 4-epi-cis-dihydro-agarofuran, myrcene, bicyclogermacrene, sabinene, ␥muurolene, caryophylene oxide, seychellene, trans--guaiene, trans-nerolidol, germacrene B, -copaen-4-␣-ol, spathulenol, valencene, asaricin and safrole. Cluster distance is represented vertically for the compounds and horizontally for oil samples.
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variables resulting from MFA is presented in Fig. S1. The first component is more closely correlated to the contents of -pinene, whereas, the percentages of germacrene B, -copaen-4-␣-ol, 10-epi-␥-eudesmol, 4-epi-cis-dihydro-agarofuran and dihydro-agarofuran are slightly more associated to the second dimension (Fig. S1). In agreement with hierarchical clustering data, the coordinates of the chemical composition of the samples for two main dimensions was not sufficient to group the species, with the exception of P. cernuum (Fig. 4A), indicating the presence of intraspecific variation in the chemistry of the essential oils. The confidence interval for P. aduncum samples is almost completely overlapped by the broader P. amalago interval, meaning that the chemical composition of the samples from these two species are not significantly different, differing by less than four standard errors where the confidence interval are plus/minus twice the standard error (Fig. 4A). Employing the collection location as categorical variable, MFA analyses have shown that the coastal locations Pariquera-Ac¸u and Ubatuba, which are the richest in species diversity, encompassed larger amounts of chemical variation (Fig. 4B). In contrast, the essential oils of plants from Campinas and Votuporanga exhibited the smallest range of chemical variation (Fig. 4B). The chemical composition of the essential oils isolated from plants harvested in the Autumn and Winter was more divergent with most of the variation associated to the second dimension (Fig. 4C). The higher variance in the chemical profile of the cooler seasons is probably associated to the higher number of species sampled during these seasons, reaching a total of 10 (P. aduncum, P. amalago, P. amplum, P. cernuum, P. crassinervium, P. gaudichaudianum, P. leptorum, P. rivinoides, P. umbellatum, P. xylosteoides), whereas, only three species (P. aduncum, P. amalago and P. solmsianum) were found in the Spring/Summer collections. Moreover, the chemical profile of the essential oils extracted from P. aduncum and P. amalago was shown to be less divergent (Fig. 4A). 3.3. Biological activity of Piper essential oils
Fig. 4. Multiple factor analyses of the chemical composition of the essential oils from 11 Piper species from the Atlantic rainforest in seven locations in the state of São Paulo with plant species (A), collection location (B) and sampling season (C) as categorical variables. Ellipses represent confidence level of 0.95, bootstrapped 100 times. Barycenter coordinates for each categorical variable is represented by color coded squares. Variable factor map for the oil metabolites is presented in Fig. S1.
(variable) constitute structured subsets of descriptive attributes, namely the botanical species, collection locations, and harvesting seasons. Approximately one-third (31.9%) of the variance in the chemical composition of the essential oils from the investigated Piper species was explained by the first two principal components (16.41% for PC1, and 15.50% for PC2) (Fig. 4). However, as expected for highly dimensional large-scale data, approximately 70% (68.09%) of the total variance remained unexplained by the first two components. The vector map for the metabolite
The agar diffusion method was employed to investigate the antibacterial activity of the essential oils isolated from 11 species from the Piper family sampled from seven distinct locations of Atlantic rainforest in the State of São Paulo. Three skin-associated Gram positive (S. epidermidis, S. aureus and C. xerosis) and one Gram negative (E. coli) bacterial pathogens were investigated. The vast majority of the essential oils exhibited inhibitory activity against one or more bacteria, in comparison to a commercial wide-spectrum antibiotic, with the exception of the oil from P. xylosteoides (Fig. 5). The biological activity of the volatile components of Piper species has been reported for a wide range of applications (Almeida et al., 2009; Raut and Kuruppayil, 2014), although P. nigrum remains the sole with potential application (Raut and Kuruppayil, 2015; Nikolic´ et al., 2015). The levels of bacterial growth inhibition were similar among the investigated essential oils, in spite of their distinct chemical composition (Fig. 5, Fig. 6, Fig. S2). The variable factor map from principal component analysis demonstrated that the composition of the bacterial cell wall is the most important factor determining the biological activity of the essential oils (Fig. S2). The essential oils from P. amalago from Adamantina and Mococa promoted the most significant levels of bacterial growth inhibition (Fig. 5, Fig. 6), whereas the oil isolated from P. amplum from Pariquera-Ac¸u exhibited the widest spectrum of growth inhibition, reaching at least 10% of growth inhibition for all tested bacterial strains (Fig. 5, Fig. 6). We have employed Pearson correlation analyses to investigate the association between the chemical composition of the tested essential oils and their biological activity (Fig. 7; Fig. S3). High levels of bicyclogermacre and ␥-muurolene were positively
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Fig. 5. Percentage growth inhibition of the essential oil from 11 species of plants from Piperaceae family from the Atlantic rainforest in the state of São Paulo against one Gram negative (E. coli) and three Gram-positive (S. epidermidis, S. aureus and C. xerosis) bacterial pathogens. Inhibition percentage was calculated based on the recommended concentration (300 g mL−1 ) of wide spectrum commercial antibiotic cefotaxime. Sterile mineral oil was used as negative control.
associated to strong inhibition of E. coli growth (Fig. 7A). In contrast, the presence of germacrene D and trans-caryophyllene was associated to the inhibitory activity against all tested bacteria (Fig. 7A). The presence of caryophyllene oxide was associated to the growth inhibition against the investigated Gram positive bacteria (Fig. 7A). Among the monoterpene, limonene and cis--ocymene were the
compounds with the highest correlation to bacterial growth inhibition, especially against Staphylococcus species (Fig. 7B). Higher contents of -phellandrene were positively correlated to wide spectrum inhibitory activity (Fig. 7B). In contrast, sabinene and myrcene were able to selectively inhibit the growth of E. coli and S. aureus, and E. coli and C. xerosis, respectively (Fig. 7B). The contents
Fig. 6. Principal component analyses of antibacterial activity of the essential oils from 11 species of plants from Piperaceae family from the Atlantic rainforest in the state of São Paulo against one Gram negative (E. coli) and three Gram-positive (S. epidermidis, S. aureus and C. xerosis) bacterial pathogens.
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Fig. 7. Pearson r correlation analysis between the most abundant mono and sesquiterpene components of the essential oils from 11 species of Piperaceae from the Atlantic rainforest in the state of São Paulo against one Gram negative (E. coli) and three Gram-positive (S. epidermidis, S. aureus and C. xerosis) bacterial pathogens. Pearson r is represented as colorscale. The correlation and p-values are given in Table S2.
of camphene, ␣- and -pinene exhibit negative correlation with the antibacterial activity of the essential oils for all tested bacteria (Fig. 7B). Essential oils containing phenylpropanoids asaricin and safrole exhibit no significant antibacterial activity (Fig. 7C). In contrast, asaricin and safrole contents were positively correlated to the growth inhibition against S. epidermidis and C. xerosis (Fig. 7C). The presence of asaricin was also positively correlated with the activity against S. aureus (Fig. 7C). Pearson r correlations between the bacteria and the compounds, along with their p values, are given in Table S2 and presented as percentage in Fig. S3. 4. Discussion The high botanical diversity of the genus Piper is reflected in its specialized metabolism, since twenty four distinct terpenoids (5%) were identified from 11 species. Accordingly, terpene composition was quantitative and qualitatively different among the investigated species. In plants, terpenoid biosynthesis depends on 5-carbon compounds, namely isopentenyl diphosphate (IPP) and dimethyl allyl diphosphate (DMAPP), produced by the mevalonate (MVA) and the 2-C-methyl-d-erythritol-4-phosphate (MEP) dependent pathway (Vranová et al., 2013). The precursors DMAPP and IPP are fused to form the 10-carbon monoterpenoids, which, in turn, are further coupled to other molecule of IPP to produce the 15carbon sesquiterpenoids. The natural diversity of plant terpenoids is associated to the complex network controlling the biosynthesis and biological function of these compounds (Kitaoka et al., 2015). The complex interactions between environmental and endogenous factors is responsible for intraspecific differences in the specialized metabolism (Schuman et al., 2016). In the current study, quantitative differences in the chemical profile were found in P.
aduncum, P. amalago and P. cernuum, whereas significant qualitative differences were present in P. aduncum. The interspecific variation in the oil composition was smaller for P. aduncum and P. amalago, although the chemical composition of the essential oils only allowed clustering of P. cernuum individuals. The oils from P. aduncum sampled from deforested and intact areas were demonstrated to have distinct chemical profiles, with oils from plants grown in intact forest areas enriched in terpene compounds (Almeida et al., 2009). Similarly, terpenes were the most abundant chemical class in P. aduncum from Monte Alegre do Sul, indicating that the specialized metabolism of the species in the region is similar to that of plants from intact forests. Phenylpropanoids were solely present in P. aduncum, reaching up to 80% of the main components, however its levels were reduced in plants from higher altitudes. Phenylpropanoids are involved in a wide range of developmental and adaptation processes in plants, thus, a complex network of factors regulates their biosynthesis and accumulation (Gaquerel et al., 2014). Abiotic factors, such as light and nutrients, are considered the main modulators of the expression of genes in the phenylpropanoid biosynthesis pathway and could contribute for the observed decreased levels in plants from higher altitudes, along with possible differences in the genetic background of the plants. Hierarchical clustering was employed to investigate the relationships between the chemical composition of the essential oils and Piper species growing in different locations, sampled at different times of the year. The use of distinct chemical classes resulted in differential clustering of the species, in agreement with the distinct intraspecific chemical profile of the essential oils from P. aduncum, P. amalago and P. cernuum. Our data suggest that the sesquiterpene profile based on compounds with tricyclic 5,11-epoxy-5-,10-␣-eudesman-4-(14)-ene skeleton is useful to
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identify P. cernuum. Similarly, the monoterpene profile based on the presence of myrcene, sabinene or -phellandrene may provide auxiliary tools to identify P. amalago. The multivariate and clustering analyses demonstrate the strong influence of environmental factors on the production of phenylpropanoid or terpenoid compounds of interest in Piper species. In agreement with the analyses of the chemical composition of the essential oils, botanical studies investigating the morphology of Piper flowers and inflorescences were unable to resolve infrageneric taxonomy (Jaramillo and Manos, 2001; Tebbs, 1993). More recently, the use of molecular markers based on internal transcribed spacers (ITS) of nuclear ribosomal DNA provided a consistent separation between the South Pacific/Asia and the Neotropical (American) clade, although several infrageneric recognized groups of the American cluster remained monophyletic (Jaramillo and Manos, 2001). Other studies have also demonstrated that the agreement among chemical, morphological and genetic data is variable and highly dependent on the plant material (Barra, 2009). Thus, the association of several characterization techniques may be required to discriminate precisely Piper species of interest. Despite the differences in their chemical profile, the vast majority of the essential oils exhibited inhibitory activity against one or more bacteria. The biological activity of the volatile components of Piper species has been reported for a wide range of applications (Almeida et al., 2009; Raut and Kuruppayil, 2014), although P. nigrum remains the sole with potential application (Raut and Kuruppayil, 2015; Nikolic´ et al. , 2015). Bacterial cell wall composition is the most important factor controlling the biological activity of the Piper oils. The antibacterial effect of plant essential oils is hypothesized to be dependent on the disruption of the organization of lipids in bacterial cellular and mitochondria membranes, causing the leakage of cell contents (Solórzano-Santos and Miranda-Novales, 2012). The thicker, theicoic acids containing cell wall of Gram positive bacteria make them more resistant to essential oils (Solórzano-Santos and Miranda-Novales, 2012). The determination of the compound(s) responsible for biological activity of essential oils require extensive purification procedures, which makes the process expensive and ineffective. In contrast, the assumption that the most abundant component in the oil is responsible for the biological activity may be misleading but can provide a starting point for further, more detailed investigations. We have employed Pearson correlation analyses to investigate the association between the chemical composition of the tested essential oils and their biological activity. The presence of high levels of germacrene D and trans-caryophyllene was associated to the inhibitory activity against all tested bacteria, whereas caryophyllene oxide was associated to the growth inhibition against the investigated Gram positive bacteria. Among the monoterpene, limonene and cis--ocimene were the compounds with the highest correlation to bacterial growth inhibition, especially against Staphylococcus species. The contents of camphene, ␣- and -pinene exhibit negative correlation with the antibacterial activity of the essential oils for all tested bacteria. Interestingly, camphene isolated from P. cernuum has been demonstrated to promote in vivo antitumor activity, by inducing intrinsic apoptosis of melanoma cells due to stress of the endoplasmic reticulum, followed by the release of Ca2+ along with HmgB1 and calreticulin, loss of mitochondrial membrane potential and up regulation of caspase-3 activity (Girola et al., 2015). The distinct features between prokaryotic bacterial and eukaryotic cells may account for the distinct biological action of the compound. Volatile plant terpenes are collectively associated to defensive roles against pathogens and herbivores (Richter et al., 2015; Matarese et al., 2014; Menzel et al., 2014; Trowbridge et al., 2014). However, the individual roles of the compounds in distinct biotic stresses is likely to differ. In general, the deleterious
effect of terpenes on microbial or herbivore pathogens is thought to involve oxidative damage of cell membranes and disrupting cell metabolism and energy production (Chan et al., 2013; Chueca et al., 2014). Our in vitro assays have demonstrated that several abundant monoterpenes in plant essential oils, such as pinene forms and camphene do not appear to have a role against the investigated bacteria. In contrast, as demonstrated for other species, including cultivated rice, limonene appears to have an important defensive role against bacterial pathogens (Lee et al., 2015). In other species, pinene has been demonstrated to be associated to biological activity against fungus, insects and protozoans (Pontin et al., 2015; Rodrigues et al., 2015). Although in vitro analyses may help to investigate the biological function of natural products, the complex mixture of compounds usually produced by the plants prevents the definite determination of their biological function. The chemical profile of the essential oils from Piper species in the Atlantic rainforest results from the complex interactions among physiological, genetic and environmental factors, and contribute to generate the metabolic plasticity required for the adaptation. In order to profit from the promising bioactive compounds from Piper species, further studies on the genus genetics, development and physiology are required. 5. Conclusions The essential oils from Piper species in the Atlantic rainforest are mainly composed by mono- and sesquiterpenes, with phenylpropanoids restricted to P. aduncum. The chemical composition of the oils from the investigated species is influenced, but not determined, by the plant species and environmental conditions. The chemical composition of the oils based on mono-, sesquiterpenes and/or phenylpropanoids generates distinct clustering of the Piper species, thus, chemotaxonomic data should be coupled with genetic and botanical analyses. The vast majority of the isolated essential oils exhibit in vitro biological activity against pathogenic Gram negative and positive bacteria. Higher contents of biclyclogermacrene and ␥-muurolene are associated with strong inhibitory activity against E. coli, whereas the levels of limonene and cis--ocymene are positively correlated to antibacterial activity against Staphylococci. Acknowledgments The authors would like to thank Fundac¸ão de Apoio à Pesquisa do Estado de São Paulo (FAPESP) and Natura Inovac¸ão e Tecnologia de Produtos Ltda for financial support (grant 03/08896-1 to MOMM). The authors also would like to express their gratitude to Prof. Dr. Lin Chau Ming (Universidade Estadual Paulista Júlio de Mesquita Filho − UNESP) for his help during the conception of the work. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.indcrop.2016.09. 028. References Abramoff, M.D., Magalhaes, P.J., Ram, S.J., 2004. Image processing with image. J. Biophot. Int. 11, 36–42. Adams, R.P., 2007. Identification of Essential Oil Components by Gas Cromatography/Mass Spectroscopy. Allured Publ. Corp, Carol Stream. Almeida, R.R.P., Souto, R.N.P., Bastos, C.N., Silva, M.H.L., Maia, J.G.S., 2009. Chemical variation in Piper aduncum and biological properties of its dillapiole-rich essential oil. Chem. Biodivers. 6, 1427–1434, http://dx.doi.org/10.1002/cbdv. 200800212. Barra, A., 2009. Factors affecting chemical variability of essential oils: a review of recent developments. Nat. Prod. Commun. 4, 1147–1154.
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