2019

NPC Natural Product Communications Vol. 14

No. 1
Essential Oil of Piper oradendron from the Pacific Slope of Guatemala 79 - 81
José V. Martínez-Arévaloa, Sully M. Cruzb, Miriam A. Apelc, Amélia T. Henriquesc and Armando Cáceresb*,
a
Facultad de Agronomía, and bFacultad de Ciencias Químicas y Farmacia, Universidad de San Carlos de Guatemala
c
Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brasil

acaceres@farmaya.gt

Received: June 18th, 2018; Accepted: September 5th, 2018

Essential oils of leaves of the endemic species Piper oradendron Trel. & Standl. (Piperaceae) were obtained from three sites of the Guatemalan Pacific slope.
The yields of the essential oils obtained by hydrodistillation varied from the site of collection (0.4-1.4%). The GC/MS analysis showed that sample A
(Samayac) had up to 53 peaks that were identified, B (Popoyá) showed 31 peaks and C (Bulbuxyá) showed 22; major constituents were similar in the three
samples, including - and β-pinene (28.3-46.9%), germacrene D (10.7-22.7%), and iso-spathulenol (10.2-22-3%). This is the first report on the chemical
composition of the essential oil of samples of P. oradendron from different provenances of Guatemala, suggesting little variability in its main components.

Keywords: Germacrene D, iso-Spathulenol, Pinenes, Cordoncillo, Chiral analysis.

Piper is a Neotropical genus showing great diversity and economic
importance. In the Americas, there are three biogeographic
provinces of this genus: Central America-Mexico, Colombia-
Amazonas basin, and Atlantic forest in Brazil [1]. In Guatemala 88
species have been described [2], some of them endemic, popularly
used, but never been studied. This study is part of a national effort
to detect and evaluate native Piper species used in traditional
medicine, culinary or phytocosmetic for their potential
agroindustrial utilization [3].
Piper oradendron Trel. & Standl. (Piperaceae) is a shrub 1-2.5 m
high, densely hispidulous; leaf blades are thin, lustrous,
penninerved; peduncles opposite, spikes slender, obtuse; bracts
densely pubescent. Common but endemic to foothills and plains of
the Pacific slope of Guatemala, temperatures 23-34°C and relative
humidity 51-85% [2]. Distribution is restricted to Guatemalan
Pacific basin lowlands, with only two specimens reported from
Oaxaca and Veracruz, Mexico [4]. Considered class 2 in Cites list
of in-danger species [5]. Known as cordoncillo and used for its
flavor, aroma and medicinal properties.
Little information is available on international databases about the
composition of this species. No specific compounds have been
isolated from this plant, there is only a phytochemical screening of
the leaves containing alkaloids, flavonoids, anthraquinones,
saponins, bitter principles, coumarins [6a] and essential oil (yield
0.5-0.6%, 30-35 peaks, major constituents β-pinene 20-30%,
germacrene D 10-14% and iso-spathulenol 12-17%) [6b]. The
methanol extract showed antioxidant activity in leaves (DPPH IC50
0.306  0.005 mg/ml, ABTS IC50 0.049  0.001 mg/ml) and roots
(DPPH IC50 0.153  0.001 mg/ml, ABTS IC50 0.043  0.077
mg/ml) [7], as well as tyrosinase inhibition activity (IC50 6.5  0.3
g/ml) [8].
(7.8-11.7%) as well as the sesquiterpenes germacrene D (10.7-
22.7%) and iso-spathulenol (10.2-22-3%).
Enantiomers distribution is important in flavors and fragrances,
where enantiomeric excess (ee) and/or ratio (er) determination is
essential for quality control; to correlate composition to
organoleptic properties; biosynthesis of a compound; or botanical
and geographic origin. Analysis by a DB5 column allowed the
characterization of P. oradendron oil and was used as analytical
reference. Sample from Bulbuxyá was selected for enantiomeric
composition of terpenoids due to high pinenes content, showing
three enantiomeric pairs viz., α- and β-pinene with significant
enantiomeric variability. Table 2 outlines the terpenes identified by
enantio-GC. Enantiomeric excess for iso-spathulenol enantiomers
was very similar while an enantiomeric proportion of 62:38 was
accounted for (1S) (-)-α-pinene and 78:22 for (1S) (-)-β-pinene.
The major constituents were - and β-pinene, germacrene D, and
iso-spathulenol, showing similar composition in all samples. - and
β-pinene are important constituents of essential oils of Piper spp.
[9], the highest amount found in Piper rivinoides Kunth (32.9-
73.2%) [10]. Other Latin America studies demonstrated both
pinenes in species from Panama with diverse content (0.2-6.6%)
[11]. In seven out of 10 species from Brazil Atlantic forest small
amounts (0.2-0.8%) of -pinene with high amount in P. dilatatum
L.C. Rich (17.8%) and P. amplum Kunth (16.8%), and five
contained small amounts of -pinene (0.2-1.7%) [12]. In 12 species
from Costa Rica, -pinene was found in all samples being the main
component in P. aequale Vahl (39.3%) and P. glabrescens (Miq.)
C. DC. (26.0%), β-pinene was detected in seven species in small
amounts [13]. In Guatemala 13 out of 15 species, contained
moderate amount of - and β-pinene (0.3-6.4%), with the highest
amount in P. oradendron (20.5%) [6].

In this investigation the essential oil yields from leaves by hydro-
distillation varied from site of collection from 0.4% (Bulbuxyá) to
1.5% (Samayac). The GC/MS analysis showed differences,
although major constituents were the same. Sample A (Samayac)
had up to 53 peaks, B (Popoyá) showed 31, and C 22 (Bulbuxyá)
(Table 1); major constituents were similar in the three essential oils,
including the monoterpenes β-pinene (20.5-35.2%) and -pinene
Germacrene D, a sesquiterpenoid with insecticidal, antimicrobial,
cytotoxic and antioxidant activities, is present in P. arboreum
Aublet var. latifolium (C. DC.) Yuncker with a concentration up to
72.9%, followed by P. fimbriulatum C. DC. (32.9%), P. bredmeyeri
J. Jac. (21%) [14] and other species with lesser amount, such as P.
cubeba L., P. guineense Schumac., and P. kadsura Trel. & Yunck.
[9,10]. In an analysis of 10 species from the Brazilian Atlantic
80 Natural Product Communications Vol. 14 (1) 2019 Martínez-Arévalo et al.

Table 1: Composition of the essential oil of flowering aerial parts of Piper oradendron forest, all species contained germacrene D in a range of 0.4-28.9%
from three sites of the Guatemalan Pacific slope.
[12]. In Central American species, it was found in all species
A: B: C:
Compound Retention index
Samayac Popoyá Bulbuxyá studied from Costa Rica, being the main constituent (30.7-32.9%) in
DB-5 LM-20 (%) leaves of P. bisasperatum Trel. and P. amalago L. (28.9-29.4%)
Essential oil yield (%) 1.5 ± 0.3 0.4 ± 0.1 0.7 ± 0.2 [13,15]. It was detected in 11 out of 15 species from Guatemala
Monoterpene hydrocarbons 32.3 34.2 49.3
-Pinene 922 1012 9.2 7.8 11.7
(range 0.7-18.4%), P. oradendron containing 17.6% [6]; and from
-Fenchene 934 1053 0.2 0.3 0.1 Panama in all three native species (P. arboreum, P. fimbriulatum,
Sabinene 958 1111 0.5 0.5 and P. oblioquum Ruiz & Pavon) in a range of 3.9-12.8% [11]. It
β-Pinene 964 1104 20.5 23.7 35.3
Myrcene 976 1144 0.5 0.5 0.3 was also demonstrated in leaves of P. lanceaefolium HBK (12.5%)
-Phellandrene 996 1132 0.7 0.8 1.0 [16], and, recently in six of 11 species of Piper in a range from 1.1
-Terpinene 1003 1158 0.1 to 3.3%, with the exception of P. trigonum C. DC. which yielded
o-Cymene 1010 1247 0.1
Limonene 1014 1175 0.6 0.6 0.4 19.7% [17].
E-β-Ocimene 1032 1229 0.1
-Terpinene 1042 1222 0.2 Iso-spathulenol is a sesquiterpene alcohol with virucidal activity,
Terpinolene 1071 1259 0.1
Oxigenated monoterpenes 3.9 1.1 0.8 which has demonstrated to be present in small amount in P.
1,8-Cineole 1016 1184 0.1 aduncum L., P. auritum Kunth and P. guineense [9]. In Brazil [12]
Linalool 1085 1518 0.1 0.5
Nopinone 1118 1544 0.1
and Costa Rica [13], several species contained spathulenol, but
trans-Pinocarveol 1121 1629 1.0 0.8 none contained iso-spathulenol. In Central America it was reported
cis-Verbenol 1124 0.1 in two species of Piper (0.7-2.9%) with highest percentage (10.6%)
trans-Verbenol 1128 0.5
Pinocarvone 1142 1598 0.4 in P. obliquum from Panama [11], as well as in seven of 11 species
Borneol 1148 1550 0.1 in a range of 0.7-6.1% [16]. In Guatemala, two of 15 species
Terpinen-4-ol 1159 1573 0.2 contained moderate amount (0.6-3.6%), with high amount in P.
-Terpineol 1173 1675 0.4
Myrtenol 1179 0.5 oradendron (17.6%) [6].
Bornyl acetate 1268 1679 0.4 0.6
Sesquiterpene hydrocarbons 21.6 42.4 20.7
The composition of P. oradendron oil, as well as the chiral
-Elemene 1320 1438 0.8 0.4 0.7
Cyclosativene 1348 1666 0.1 isometric analysis, demonstrated that there seems to be little
-Copaene 1356 0.1 variability among samples from different provenance from this
β-Bourbonene 1364 0.1
β-Elemene 1372 1562 0.4 0.5 0.4
endemic species of the Guatemalan Pacific basin, suggesting a
β-Caryophyllene 1397 1567 1.9 2.9 2.2 relatively homogeneous composition. It is confirmed the association
β-Copaene 1407 0.2 of the main compounds (- and -pinene, germacrene D, and iso-
-Humulene 1431 1642 0.5 0.9
Z-β-Farnesene 1438 0.1 spathulenol) from the three accessions of P. oradendron.
Germacrene D 1463 1690 10.7 22.7 12.3
β-Selinene 1466 0.1
Bicyclogermacrene 1476 1713 2.1 8.4 2.1
Experimental
-Muurolene 1480 1705 1.2 0.7 1.5 Plant material: Leaves and inflorescences of P. oradendron were
Germacrene A 1484 1741 0.6 1.4 0.3
-Cadinene 1496 1922 1.0 1.5 0.5 collected from three sites of the Pacific slope of Guatemala [A:
β-Cadinene 1501 0.8 0.3 Ecoparcela El Kakawatal, Samayac (14°33’05.6” N, 91° 27’59.6”
Germacrene B 1537 1807 0.9 2.7 0.7
Oxygenated sesquiterpenes 30.2 20.7 26.1
W, altitude 459 m), B: Finca Bulbuxyá, San Miguel Panan
Elemol 1531 2065 0.1 0.5 (14°29’37.1” N, 91°21’36.2” W, altitude 288 m], both from
E-Nerolidol 1549 2032 1.4 2.8 Suchitepéquez, and C: Finca Popoyá, Santa Lucia Cotzumalguapa,
Spathulenol 1561 2108 3.5 6.2 4.1
Germacrene D-4-ol 1564 2034 1.2 1.3 from Escuintla (14°23’20.0” N, 91°06’29,7” W, altitude 378 m) in
Globulol 1565 1964 1.3 the same season (March-April 2013). David Mendieta, curator at
Caryophyllene oxide 1566 1963 1.2 1.2 the Herbarium of the School of Agronomy, University of San
β-Copaen-4-alpha-ol 1583 0.3
Humulene epoxide II 1593 1.1 Carlos, identified the samples (voucher # 1,160, 1,161, 1,162) and
τ-Cadinol 1612 2162 0.3 1.2 0.7 deposited at Farmaya Laboratories Ethnobotanical Herbarium
Iso-spathulenol 1624 2245 17.5 3.9 19.5
Eudesmol* 1625 1.8
(CFEH). Plant materials (500-900 g) were shade-dried at 35°C for
τ-Muurolol 1632 2170 0.7 0.5 0.5 5-7 days. Powdered material was submitted to hydrodistillation in a
-Muurolol 1633 2183 0.6 0.3 Clevenger-type apparatus for 3 h in triplicate [18], measured to
β-Eudesmol 1636 2226 0.2
-Cadinol 1644 2215 1.8 0.8 calculate yield, transferred to glass vials, and kept at -18°C for
14-Hydroxy--muurolene 1748 2407 0.1 further analysis.
Nonadecanal 2106 2568 0.4
Total identified 88.0 98.4 96.9 Analysis of essential oils: Samples were analysed on a Shimadzu
*isomer not identified LM-20, Carbowax LM-20
gas chromatographer (GC-17A) with injector, quantified in
duplicate by electronic integration by normalization technique. For
Table 2: Enantiomeric excess (ee in %) of predominant enantiomer of chiral terpenes in
separation of constituents, an apolar Durabond-DB5 column (30 m
Piper oradendron (Bulbuxyá) essential oil by enantioselective gas chromatography x 0.25 mm of I.D.) with polydimethylphenyl siloxane containing
(eGC-MS) using permethylated β-cyclodextrin as stationary phase. 5% of phenyl groups in a 0.25 µm thick film (John Wiley & Sons
Enantiomersa Percentage ee Scientific, USA); a polar column (LM-20, coated with polyethylene
(1S) (-)-α-pinene 3.1 61.8
(1R) (+)-α-pinene 0.7
glycol by L&M, San Carlos, SP, Brazil), filled with propylene
(1R) (+)-β-pinene 1.2 glycol; and, a flame ionization detector (FID) was used. Conditions
(1S) (-)-β-pinene 9.7 78.7 included: temperature program 60-300°C, 3°C/min; analysis time
Iso-spathulenol* 13.7
Iso-spathulenol* 14.5 2.7 80 min; injector temperature 220°C; FID/EM interface: 250°C;
a
Compounds are listed as per elution order in Supelco β-CEDEX 120 capillary amount of sample injected: 1 µL diluted in diethyl ether (2%) with
column, enantiomeric excess (in %) calculated only for predominant enantiomer. 1:50 split ratio.
*Enantiomer not identified.
Essential oil of Piper oradendron from Guatemala Natural Product Communications Vol. 14 (1) 2019 81

Gas chromatography-mass spectrometry: Qualitative analyses
were carried out on the same equipment coupled to a Shimadzu
mass spectrophotometer (GC/MS-QP5000), connected with
cylindrical quadruple and operated by ionization energy of 70 eV,
obtained by electron impact technique. Chromatographic conditions
were similar to that described above. The mass spectra were
recorded over 40–500 a.m.u. range. Identification of essential oils
components was achieved based on the retention indices,
determined relative to the retention times of a series of n-alkane
standards (C8-C32, Sigma), by comparison of their mass spectral
fragmentation patterns (National Institute of Standards and
Technology (NIST) database), and with reference to previously
published data [6,19].
Chiral isometric analysis: The chiral isomeric ratios of the main
monoterpene components of the essential oils were studied by enan-
tioselective gas chromatography (eGC-MS) using permethylated β-
cyclodextrin as stationary phase using a Supelco B-CEDEX120
fused-silica column (30 m x 0.25 mm x 0.25 m film thickness).
The oven temperature was programmed from 60-250ºC at 3ºC/min.
Acknowledgments - This research was supported by National
Council for Science and Technology (Fodecyt grants 114-2006 and
27-2011) and the cooperation linkages established within Ribiofar
Network (Red 0284) of the Ibero-American Program of Science and
Technology for Development (CYTED). Technical support by Luis
Álvarez and Ana Gómez is kindly appreciated.

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