molecules Chemical Composition and Antioxidant Activity of Essential Oils from Eugenia patrisii Vahl, E. punicifolia (Kunth) DC., and Myrcia tomentosa (Aubl.) DC., Leaf of Family Myrtaceae

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Article
Chemical Composition and Antioxidant Activity of Essential
Oils from Eugenia patrisii Vahl, E. punicifolia (Kunth) DC.,
and Myrcia tomentosa (Aubl.) DC., Leaf of Family Myrtaceae
Celeste de Jesus Pereira Franco 1, Oberdan Oliveira Ferreira 2,Ângelo Antônio Barbosa de Moraes 1,
Everton Luiz Pompeu Varela 2,3, Lidiane Diniz do Nascimento 4, Sandro Percário 2,5 ,
Mozaniel Santana de Oliveira 3, 4, * and Eloisa Helena de Aguiar Andrade 1,2,3,4


Citation: Franco, C.d.J.P.; Ferreira,
O.O.; Antônio Barbosa de Moraes, Â.;
Varela, E.L.P.; Nascimento, L.D.d.;
Percário, S.; de Oliveira, M.S.;
Andrade, E.H.d.A. Chemical
Composition and Antioxidant
Activity of Essential Oils from
Eugenia patrisii Vahl, E. punicifolia
(Kunth) DC., and Myrcia tomentosa
(Aubl.) DC., Leaf of Family
Myrtaceae. Molecules 2021,26, 3292.
https://doi.org/10.3390/
molecules26113292
Academic Editors: Henryk H. Jele ´n
and Félix Tomi
Received: 26 March 2021
Accepted: 14 May 2021
Published: 29 May 2021
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Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
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conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1Faculdade de Química, Universidade Federal do Pará, Rua Augusto Corrêa S/N, Guamá,
Belém 66075-900, PA, Brazil; celeste.frango12@gmail.com (C.d.J.P.F.);
angeloquimica17@gmail.com (Â.A.B.d.M.); eloisa@museu-goeldi.br (E.H.d.A.A.)
2Programa de Pós-Graduação em Biodiversidade e Biotecnologia- Rede Bionorte, Universidade Federal do
Pará, Rua Augusto Corrêa S/N, Guamá, Belém 66075-900, PA, Brazil; oberdan@museu-goeldi.br (O.O.F.);
evertonlpvalerla@gmail.com (E.L.P.V.); percario@ufpa.br (S.P.)
3Programa de Pós-Graduação em Química, Universidade Federal do Pará, Rua Augusto Corrêa S/N,
Guamá, Belém 66075-900, PA, Brazil
4
Laboratório Adolpho Ducke–Coordenação de Botânica, Museu Paraense Emílio Goeldi, Av. Perimetral, 1901,
Terra Firme, Belém 66077-830, PA, Brazil; lidianenascimento@museu-goeldi.br
5Laboratório de Pesquisas em Estresse Oxidativo, Universidade Federal do Pará, Rua Augusto Corrêa S/N,
Guamá, Belém 66075-900, PA, Brazil
*Correspondence: mozanieloliveira@museu-goeldi.br; Tel.: +55-91-988657823
Abstract:
Essential oils (EOs) were extracted from Eugenia patrisii,E. punicifolia, and Myrcia tomentosa,
specimens A and B, using hydrodistillation. Gas chromatography coupled with mass spectrometry
(GC/MS) was used to identify the volatile constituents present, and the antioxidant capacity of
EOs was determined using diphenylpicryl-hydrazyl (DPPH) and trolox equivalent antioxidant
capacity (TEAC) assays. For E. patrisii, germacrene D (20.03%), bicyclogermacrene (11.82%), and
(E)-caryophyllene (11.04%) were identified as the major constituents of the EOs extracted from
specimen A, whereas specimen B primarily comprised
γ
-elemene (25.89%), germacrene B (8.11%),
and (E)-caryophyllene (10.76%). The EOs of E. punicifolia specimen A contained
β
-Elemene (25.12%),
(E)-caryophyllene (13.11%), and bicyclogermacrene (9.88%), while specimen B was composed of
(E)-caryophyllene (11.47%), bicyclogermacrene (5.86%),
β
-pinene (5.86%), and
γ
-muurolene (5.55%).
The specimen A of M.tomentosa was characterized by
γ
-elemene (12.52%), germacrene D (11.45%),
and (E)-caryophyllene (10.22%), while specimen B contained spathulenol (40.70%),
α
-zingiberene
(9.58%), and
γ
-elemene (6.89%). Additionally, the chemical composition of the EOs was qualitatively
and quantitatively affected by the collection period. Furthermore, the EOs of the studied specimens,
especially specimen A of E. punicifolia, showed a greater antioxidant activity in DPPH rather than
TEAC, as represented by a significantly high inhibition percentage (408.0%).
Keywords: myrtaceae; natural products; essential oils; antioxidant capacity
1. Introduction
Aromatic and medicinal plants have been used in food, agriculture, and the treatment
of diseases for many years [
1
]. They are known for producing essential oils (EOs) and
impart fragrances or aromas that stimulate the sense of smell. Usually a product of
secondary metabolism, EOs are of great economic importance and have applications in
several fields such as pharmaceuticals, cosmetics, and food. They are present in different
parts of the plant including flowers, leaves, stems, fruits, branches, and seeds [2–4].
EOs are complex, hydrophobic mixtures primarily composed of monoterpenes, sesquiter-
penes, and their oxygenated derivatives [
5
,
6
]. They are high-value products with a wide variety
Molecules 2021,26, 3292. https://doi.org/10.3390/molecules26113292 https://www.mdpi.com/journal/molecules

Molecules 2021,26, 3292 2 of 12
of interesting biological properties. These include antifungal, antibacterial, anticancer, cytotoxic,
and allelopathic properties with profound effects on animals, humans, and even other plants [
7
].
The Myrtaceae family of angiosperms includes approximately 130 genera and 5671
species, distributed in tropical and subtropical regions of the planet, with centers of di-
versity in South America, Australia, and tropical Asia [
8
]. In Brazil, the Myrtaceae family
comprises 27 genera and 1026 species and is distributed across five regions and different
phytogeographic domains [
9
]. Scattered in the Brazilian forests, the species of this family
are economically important and cultivated not only for their edible fruits but also for
ornamental and purposes and as a source of timber [
10
]. In addition, they are sources
of EOs that have insecticidal, parasiticidal, antifungal, antibacterial, antimicrobial, and
antioxidant properties [11,12].
Eugenia is one of the most important genera of the Myrtaceae family, with edible
fruits, wood, and EOs being commercially exploited in addition to its use in traditional
medicine [
13
,
14
]. In Brazil, this genus is represented by 392 species distributed across
all regions [
15
]. Eugenia patrisii, popularly known as Ubaía-rubí, predominantly grows
in the Amazon [
15
] and produces edible fruits that are used to make juice, jam, and ice
cream [
16
]. Eugenia punicifolia (Kunth), DC. is a member of the Pedra-ume-caágenus and
is used in traditional medicine to treat diabetes, fever, and other ailments in the form of
infusions [17,18].
Myrcia is also one of the largest genera within the Myrtaceae family, with over 400
species found in different biomes from the south to the north of Brazil [
19
]. Members
of the genus Myrcia exhibit several biological activities, including antinociceptive, anti-
inflammatory, antioxidant, antimicrobial, hypoglycemic, and anti-hemorrhagic activities.
Many Myrcia species also produce EOs with a high concentration of mono- and sesquiter-
penes, as well as extracts rich in phenolic compounds and flavonoids, responsible for a
wide range of biological activities [10].
The primary aim of this study was to determine the chemical composition of EOs
extracted from Eugenia patrisii,E.punicifolia, and Myrcia tomentosa specimens, and evaluate
their antioxidant activity, to contribute to the studies on aromatic plants found in the
Amazon region, particularly in the state of Pará, Brazil.
2. Results and Discussion
2.1. Yields of Essential Oils
The EOs content of E.patrisii, was 0.24% for specimen A and 0.77% for specimen
B when calculated on a dry basis. Specimens A and B of E.punicifolia had 0.26% and
0.14%, of EOs, respectively, while specimens A and B of M.tomentosa had EO contents of
0.35%, and 0.41%, respectively. These findings corroborate with those of several previous
studies, which have suggested that the yields of EOs from different Myrtaceae species vary
according to the studied species and the season of collection [20–24].
2.2. Chemical Composition of Essential Oils
The EOs of the specimens under study were obtained through hydrodistillation, which
yielded a total of 107 chemical constituents. The hydrocarbon sesquiterpenes accounted
for 70.64%, 76.79%, 66.14%, 56.74%, 75.82%, 51.2%, while the oxygenated sesquiterpenes
accounted for 24.63%, 16.50%, 22.26%, 15.09%, 16.83%, 40.7% in the specimens E. patrisii (A,
B), E. punicifolia (A, B), and M. tomentosa (A, B), respectively.
Germacrene D (20.03%), bicyclogermacrene (11.82%), and (E)-caryophyllene (11.04%),
were identified as the major compounds in the EOs extracted from specimen A of E. patrisii.
This is in contrast to the findings reported by Silva et al. [
25
] wherein (2E, 6E)-farnesol
(34.5%), (2E, 6Z)-farnesol (23.2%), and a mixture of caryophylla-4(12),8(13)-dien-5
α
-ol,
and caryophylla-4(12),8(13)-dien-5
β
-ol (15.6%) were identified as the major compounds
in the EOs extracted from a specimen of E. patrisii collected in São Geraldo do Araguaia,
Pará-Brazil. Germacrene D has been described in the literature as having antimicrobial
properties [
26
]. Besides, both germacrene D and (E)-caryophyllene have immunomod-

Molecules 2021,26, 3292 3 of 12
ulatory activity in human neutrophils, inhibiting Ca
2+
mobilization, chemotaxis, and
production of reactive oxygen species (ROS) [
27
]. This sesquiterpene constituent also has
antioxidant and cytotoxic activity against melanoma cancer cells, which are responsible
for skin cancer, breast adenocarcinoma, and colon carcinoma [
28
]. Bicyclogermacrene
has been associated with larvicidal activity [
29
] and antiviral activity against SARS-CoV-
2 [
30
]. (E)-caryophyllene, on the other hand, has been reported to exhibit antiprotozoal
activity against the parasite Leishmania amazonensis, which causes leishmaniasis [
31
]. Addi-
tionally, sesquiterpenes have anticonvulsant [
32
], antifungal [
33
], and anti-inflammatory
properties [34].
The EOs of E. patrisii specimen B were characterized by
γ
-elemene (25.89%), germa-
crene B (8.11%), and (E)-caryophyllene (10.76%), which was slightly lower than that of
specimen A. Because it has larvicidal activity against Spodoptera litura,
γ
-elemene has the
potential to be developed into biopesticides for pest control [
35
]. The compound is highly
effective against the larvae of the mosquito species Anopheles subpictus,Aedes albopictus, and
Culex tritaeniorhynchus, as well as having antioxidant activity and cytotoxic activity against
melanoma cells [
36
,
37
]. The antibiotic [
37
] and antiproliferative activity [
38
] of Germacrene
B has also been reported in the literature.
The hydrocarbon sesquiterpenes
β
-elemene (25.12%), (E)-caryophyllene (13.11%),
selin-11-en-4
α
-ol (9.16%), and bicyclogermacrene (9.88%) were the major constituents E.
punicifolia EOs extracted from specimen A. The main constituents of specimen B EOs
were (E) -caryophyllene (11.47%),
β
-pinene (5.86%), bicyclogermacrene (5.86%), and
γ
-
muurolene (5.55%). A sample of E. punicifolia, collected in the municipality of Maracanã,
Pará, revealed a predominance of (E)-caryophyllene (9.87%), bicyclogermacrene (8.75%),
and (E)-
β
-ocimene (5.50%). Low concentrations of
β
-pinene and
γ
-muurolene were also
discovered at 3.91% and 2.08%, respectively [
39
]. Sesquiterpenes were also found to be the
predominant constituents in a sample of E.punicifolia collected from the Atlantic Forest
in Rio de Janeiro by Ramos et al. [
40
]. Conversely, the EOs extracted from E.punicifolia
specimens in this study were found to be primarily composed of
α
-cadinol (10.6%), 10-epi-
γ
-eudesmol (10.2%), and paradisiol (9%). The use of sesquiterpenes can help patients with
squamous cell carcinoma of the esophagus have a better prognosis. Furthermore, it has
the potential to reduce the adverse effects of chemoradiotherapy [
41
].
δ
-cadinene was also
found in the EOs of E. patrisii and E. punicifolia, specimens A and B, at low concentrations
(1.39–6.64%), and has been reported for acaricidal activity against Psoroptes cuniculi [
42
] as
well as antimicrobial activity [43].
The EOs of M. tomentosa, specimen A, was characterized by the presence of the hydro-
carbon sesquiterpenes
γ
-elemene (12.52%), germacrene D (11.45%), and (E)-caryophyllene
(10.22%). For specimen B, the oxygenated sesquiterpene spathulenol had the highest con-
centration (40.70%), followed by the hydrocarbon sesquiterpenes
α
-zingiberene (9.58%) and
γ
-elemene (6.89%), which is significantly different from the findings previously reported in
the literature [
44
]. Spathulenol has been reported to harbor insecticidal, insect repellent,
antioxidant, anti-inflammatory, antiproliferative, and antimycobacterial properties [
45
–
48
],
while
α
-zingiberene is a known inhibitor for aflatoxin and fungal mycotoxins as well as
having antihyperlipidemic and anti-inflammatory properties [49,50].
Multivariate Analysis
To assess the similarity between the EO samples obtained by hydrodistillation, hierar-
chical cluster analysis (HCA) (Figure 1) was applied to the chemical compounds identified
and quantified by CG/MS and CG-FID. The HCA shows that the samples E.patrisii (B)
and M.tomentosa (A) have the greatest similarity (40.98%), sufficient to form a group.
The chemical composition of the EOs was found to be directly influenced by the collection
periods of the Amazon winter and summer samples, with a strong influence on compounds
with concentrations ≥3% (Table 2).

Molecules 2021,26, 3292 4 of 12
Figure 1. Dendrogram representing the similarity ratio of samples of EO from the leaves
of E. patrisii (A), E. patrisii (B), E. punicifolia (A), E. punicifolia (B), M. tomentosa (A), and M.
tomentosa (B).
Table 1.
Chemical composition of EOs extracted from leaves of Eugenia patrisii,E. punicifolia, and Myrcia tomentosa by (HD)
hydrodistillation; concentration values are expressed in (%).
E. patrisii E. punicifolia M. tomentosa
RILRICConstituents A B A B A B
932 a932 α-Pinene - - 0.11 4.35 - -
974 a974 β-Pinene - - 0.11 5.86 - -
988 a989 Myrcene - - 0.08 - - -
1001 a1016 δ-2-Carene - - 0.01 0.03 - -
1025 a1028 Sylvestrene - - 0.15 0.66 - -
1026 a1030 1,8-Cineole - - 0.12 - - -
1032 a1036 (Z)-β-Ocimene - - 1.11 1.98 - -
1044 a1048 (E)-β-Ocimene - - 3.09 4.96 - -
1054 a1057 γ-Terpinene - - 0.02 0.06 - -
1086 a1088 Terpinolene - - - 0.09 - -
1095 a1099 Linalool - - 0.03 0.06 - -
1128 a1128 allo-Ocimene - - 0.36 1 - -
1174 a1177 Terpinen-4-ol - - 0.05 0.1 - -
1186 a1190 α-Terpineol - - 0.04 0.52 - -
1335 a1339 δ-Elemene 3.31 0.66 1.42 3.5 4.57 -
1345 a1351 α-Cubebene 0.08 0.09 0.03 0.16 0.03 -
1373 a1374 α-Ylangene - 0.08 - 0.12 0.06 -
1374 a1375 Isoledene - - 0.01 0.05 - -
1374 a1379 α-Copaene 3.38 1.61 0.44 1.77 0.35 -
1387 a1387 β-Bourbonene 0.59 0.53 - 0.24 0.31 -
1389 a1394 β-Elemene 3.01 5.52 25.12 2.38 5.79 1.73
1409 a1413 α-Gurjunene - - - 0.26 - -
1411 a1417 cis-α-bergamotene 0.14 - - - - -
1417 a1424 (E)-Caryophyllene 11.04 10.76 13.11 11.47 10.22 4.27
1430 a1432 β–Copaene 1.05 - 0.29 0.97 0.86 -
1434 a1435 γ-Elemene 0.29 25.89 - 4.22 12.52 6.89
1439 a1442 Aromadendrene 0.46 - 0.52 - 0.42 -
1437 a1446 α-Guaiene - 0.65 - 1.06 - -
1447 c1447 Isogermacrene D 0.31 - - - - -
1442 a1455 6,9-Guaiadiene - 0.2 - 0.37 0.31 -
1451 a1453 trans-Muurola-3,5-diene 0.2 - - 0.61 - -
1440 a1457 (Z)-β-Farnesene - - - - - 0.96

Molecules 2021,26, 3292 5 of 12
Table 1. Cont.
E. patrisii E. punicifolia M. tomentosa
RILRICConstituents A B A B A B
1452 a1457 α-Humulene 3.29 2.48 3.88 4.41 2.21 -
1458 a1464 allo-Aromadendrene - - - 0.34 0.47 -
1464 a1464 9-epi-(E)- Caryophyllene 0.38 - - - - -
1460 a1466 dehydro-Aromadendrane - - 0.14 - - -
1471 a1473 4,5-di-epi-Aristolochene - 0.04 0.09 - - -
1471 a1477 Dauca-5,8-diene 0.2 - - 0.13 - -
1475 a1477 trans-Cadina-1(6),4-diene - - - 0.74 - -
1475 a1479 γ-Gurjunene - 0.79 2 - - -
1478 a1480 γ- Muurolene - 1.4 - 5.55 - -
1479 a1484 ar-Curcumene - - - - - 18.54
1476 a1487 β-Chamigrene - 0.27 - - - -
1484 a1487 Germacrene D 20.03 - 2.05 - 11.45 0.13
1489 a1490 β–Selinene 0.56 1.77 4.96 1.02 1.34 -
1492 a1494 δ-Selinene 0.46 0.63 - 0.96 - -
1492 a1494 cis-β-Guaiene - - - - 0.99 -
1493 a1496 α- Zingiberene - - - - - 9.58
1498 a1499 α-Selinene - 2.87 - - - -
1500 a1501 Bicyclogermacrene 11.82 9.88 5.86 6.4 -
1509 a1502 α-Bulnesene - 0.44 - - -
1500 a1503 α- Muurolene - - - 1.04 1.33 -
1511 a1509 δ-Amorphene - 1.19 - 1.33 -
1502 a1510 trans-β-Guaiene - - - 1.62 - -
1505 a1510 β–Bisabolene 2.33 - - - - 1.83
1505 a1514 (E,E)-α-Farnesene 0.07 - - - - -
1513 a1517 γ-Cadinene - 0.28 - 0.63 0.41 -
1520 a1521 7-epi-α-selinene - 1.56 0.31 0.29 - -
1521 a1525 β–Sesquiphellandrene - - - - - 3.22
1522 a1527 δ-Cadinene 6.64 1.39 1.76 4.01 3.54 -
1528 a1529 Zonarene - - - 0.33 - -
1529 a1534 (E)- γ-Bisabolene 0.84 0.53 - - 0.45 3.15
1533 a1537 trans-Cadina-1,4-diene - - 0.08 0.52 0.36 -
1537 a1540 α-Cadinene 0.16 0.4 0.05 - - -
1540 b1542 Selina-4(15),7(11)-diene - 3.58 - 0.72 1.21 -
1546 b1546 Selina-3,7(11)-diene - 2.16 - 0.29 0.58 -
1556 a1549 (E)-Dauca-4(11),7-diene - 0.12 - - - -
1548 a1553 Elemol - 0.51 - - - -
1554 a1558 β-Vetivenene - 0.33 - - - -
1559 a1564 Germacrene B - 8.11 - 1.1 8.31 0.9
1561 a1565 (E)-Nerolidol 0.37 - - - - -
1570 a1575 Dendrolasin - 0.35 - 0.06 - -
1577 a1582 Spathulenol 5.37 - 5.43 2.24 2.04 40.7
1582 a1587 Caryophyllene oxide - 1.62 - - - -
1590 a1589 Globulol 3.69 - - 3.53 2.87 -
1592 a1591 Viridiflorol 1.68 0.31 3.86 2.38 2.6 -
1596 a1594 Fokienol - 0.56 - - - -
1608 a1600 β-Atlantol - 0.15 - - - -
1595 a1602 Cubeban-11-ol - - 0.7 - - -
1600 a1606 Rosifoliol 0.38 - 0.43 0.61 0.39 -
1608 a1613 Humulene epoxide II 0.3 - 0.22 - - -
1630 a1623 γ-Eudesmol - 2.52 - - - -
1618 a1626 Junenol - - 0.06 - 0.48 -
1629 a1629 Eremoligenol 0.38 - - - - -
1627 a1634 1-epi-Cubenol - - 0.21 - - -
1635 a1636 cis-Cadin-4-en-7-ol 0.17 0.52 0.38 - -
1645 a1638 Cubenol - - 0.11 - 2.74 -
1639 a1640 allo-Aromadendrene epoxide - 0.06 - - - -
1640 a1647 epi-α-muurolol 4.09 - - - - -
1642 a1648 Selina-3,11-dien-6α-ol - 0.73 - - - -

Molecules 2021,26, 3292 6 of 12
Table 2.
Chemical composition of EOs extracted from leaves of Eugenia patrisii,E. punicifolia, and Myrcia tomentosa by (HD)
hydrodistillation; concentration values are expressed in (%).
E. patrisii E. punicifolia M. tomentosa
RILRICConstituents A B A B A B
1652 a1650 Himachalol 1.13 0.32 0.14 - - -
1644 a1655 α-Muurolol - - - 3.15 - -
1651 a1659 Pogostol - 1.95 - - - -
1652 a1660 α-Cadinol 7.12 - 1.91 2.44 4.38 -
1668 a1666 14-hydroxy-9-epi-(E)-Caryophyllene - 0.07 - - - -
1658 a1669 Selin-11-en-4α-ol - - 9.16 - - -
1658 a1670 neo-Intermedeol - 0.48 - - - -
1670 a1671 Bulnesol - - - 0.34 - -
1687 a1681 Eudesma-4(15)-dien-1β-ol - 0.16 - - - -
1685 a1686 α-Bisabolol 0.05 - - - - -
1679 a1692 Khusinol - - 0.03 - - -
1700 a1700 Eudesm-7(11)-en-4-ol - 2.34 - - 1.29 -
1709 a1706 Mayurone - 0.08 - - - -
1708 a1713 cis-Thujopsenal - 0.12 - - - -
1714 a1717 Nootkatol - 1.93 - - - -
1775 a1773 2-α-hydroxy-Amorpha-4,7(11)-diene - 2.18 - - - -
2026 a2030 (E,E)-Geranyl linalool - - - - 0.04 -
Hydrocarbon monoterpenes - - 5.04 18.99 - -
Oxygenated monoterpenes - - 0.24 0.68 - -
Hydrocarbon sesquiterpenes 70.64 76.79 66.14 56.74 75.82 51.2
Oxygenated sesquiterpenes 24.63 16.50 22.26 15.09 16.83 40.7
Oxygenated diterpenes - - - - 0.04 -
Others - - - 0.04 - -
Total 95.37 93.29 93.68 91.54 92.65 91.9
RI
C
: calculated from a series of n-alkanes (C
8
–C
40
) in a DB-5MS column capillar column, RI
L
): Literature
a
Adams [
51
],
b
Mondello [
52
] and
cNist [53].
2.3. Antioxidant Activity
The EOs from specimen A of E.patrisii showed inhibition of 31.4% (ABTS
•
+) and
99.0% (DPPH
•
) (Table 3). Conversely, EOs from specimen B showed inhibition of 17.9%
(ABTS
•
+) and 204.0% (DPPH
•
) (Table 3). While specimens A and B had a lower antioxidant
capacity than Trolox (ABTS
•
+), in the DPPH assay, specimens A and B had shown an
antioxidant capacity equivalent to that of the Trolox standard, with specimen B exhibiting
better antioxidant activity than Trolox. The profound antioxidant activity observed in
specimen B may be associated with the high content of sesquiterpenes present in its
chemical composition.
Table 3.
Activity of elimination of the radicals ABTS
•
+ and DPPH
•
(%) of EOs from leaves of the specimens of Eugenia and Myrcia.
Species Specimen Collection Period CA-ABTS•+ (%) CA-DPPH•(%)
Eugenia patrisii A May 31.4 ±0.1 99.0 ±0.099
B September 17.9 ±0.069 204.0 ±0.877
Eugenia punicifolia A May 9.5 ±0.034 408.0 ±0.10
B September 37.7 ±0.035 285.0 ±0.028
Myrcia tomentosa A May 53.6 ±0.150 213.0 ±0.905
B September 0.333 ±0.247 208.5 ±0.940
Values are expressed as mean and standard deviation (n= 3) of the percentage of inhibition.
The EOs extracted from specimen A of E. punicifolia showed inhibition of 9.5%
(ABTS
•
+) and 408.0% (DPPH
•
), while that of specimen B showed inhibition of 37.7%
(ABTS
•
+) and 285.0% (DPPH
•
) (Table 3). In the ABTS
•
+ assay, specimen B showed a
higher oxidizing capacity compared to specimen A but was lower than that of the Trolox
standard. In the DPPH
•
assay, specimen A exhibited a superior antioxidant capacity com-

Molecules 2021,26, 3292 7 of 12
pared to both Trolox standard and specimen B. This may be attributed to the abundance
of cyclic sesquiterpene compounds such as Germacrene D and (E)-caryophyllene in speci-
men A of E. punicifolia., reported to offer strong antioxidant and free radicals neutralizing
properties in earlier studies [54].
The EO of specimen A of M.tomentosa inhibited ABTS •and DPPH•+ by 53.6% and
213%, respectively (Table 3). The inhibition of ABTS
•
+ and DPPH
•
in specimen B of the
aforementioned species was 0.333% and 208.5%, respectively (Table 3). In both assays,
specimen A of M.tomentosa outperformed specimen B in terms of antioxidant activity. This
is the first study to report the antioxidant activity of EOs extracted from E.punicifolia and M.
tomentosa. Furthermore, specimen A of E.punicifolia exhibited higher antioxidant activity
in the DPPH assays compared to the other specimens studied. This may be attributed to
the presence of oxygenated compounds in their composition, as DPPH is more sensitive to
polar substances [
55
]. Additionally, synergistic action between the chemical constituents
may have contributed to the higher antioxidant activity observed [56].
3. Materials and Methods
3.1. Botanical Material
The aerial parts of E. patrisii,E. punicifolia, and M.tomentosa were collected in May
(A) and September (B) 2019, during the Amazon winter and summer, respectively. These
collections took place in the Vila Nova district, located in the municipality of Magalhães
Barata, in the State of Pará, Brazil (0
◦
48
0
7.1” S 47
◦
33
0
50.3” W). All samples were collected
from fertile plants, and incorporated into the Herbário MG João Murça Pires collection
of aromatic plants at the Museu Paraense Emílio Goeldi, Belém, Pará, Brazil. They were
subsequently recorded as E.patrisii A (MG237487) and B (MG237497), E. punicifolia A
(MG237519), B (MG237496), and M.tomentosa A (MG237518) and B (MG237478).
3.2. Preparation of Botanical Material
The leaves of E. patrisii,E. punicifolia, and M. tomentosa were dried for 5 days at 35
◦
C
in an oven with air circulation before being crushed in a knife mill. The moisture content
was analyzed using an ID50 infrared humidity determiner in the temperature range of
60–180 ◦C, with a 1 ◦C increment and bidirectional RS-232 ◦C output.
3.3. Essential Oil Isolation
The samples were hydrodistilled for 3 h in a modified Clevenger-type glass system
coupled to a refrigeration system, to maintain condensation water at ~12
◦
C [
57
]. After
extraction, the oils were centrifuged at 3000 rpm for 5 min, dehydrated with anhydrous
sodium sulfate (Na
2
SO
4
), and centrifuged again under the same conditions. The oil yield
was calculated as mL/100 g. The collected EOs were stored in amber glass ampoules,
sealed with flame, and stored in a freezer at
−
15
◦
C. The EO yield was calculated on a dry
basis (db) [58].
3.4. Chemical Composition Analysis
The chemical compositions of the EOs of E.patrisii,E.punicifolia, and M.tomentosa,
were analyzed using a Shimadzu QP-2010 (Kyoto, Japan) plus gas chromatography system
equipped with an Rtx-5MS capillary column (Restek Corporation, Bellefonte, PA USA)
(30 m
×
0.25 mm; 0.25
µ
m film thickness) coupled to a mass spectrometer (GC/MS)
(Shimadzu, Kyoto, Japan). The program temperature was maintained at 60–240
◦
C at a
rate of 3
◦
C/min, with an injector temperature of 250
◦
C, helium as the carrier gas (linear
velocity of 32 cm/s, measured at 100
◦
C) and a splitless injection (1
µ
L of a 2:1000 hexane
solution) using the same operating conditions as described in the literature [
59
,
60
]. Except
for the carrier hydrogen gas, the components were quantified using gas chromatography
(CG) on a Shimadzu QP-2010 system (Kyoto, Japan), equipped with a flame ionization
detector (FID), under the same operating conditions as before. The retention index for all
volatile constituents was calculated using a homologous series of n-alkanes (C
8
–C
40
) Sigma-

Molecules 2021,26, 3292 8 of 12
Aldrich (San Luis, AZ, USA), according Van den Dool and Kratz [
61
]. The components
were identified by comparison i) of the experimental mass spectra with those compiled in
libraries (reference) and ii) their retention indices to those found in the literature [51–53].
3.5. Antioxidant Capacity Equivalent to Trolox
The antioxidant potential of the substances under investigation was calculated by com-
paring them to Trolox (6-hydroxy-2,5,7,8-tetramethylchromono-2-carboxylic acid; Sigma-
Aldrich; 23881-3; São Paulo / Brazil), a water-soluble synthetic analog of vitamin E.
The trolox equivalent antioxidant capacity (TEAC) was determined according to the
methodology adapted from [
62
] modified by [
63
]. TEAC was based on the antioxidant
inhibition of the radical cation ABTS+
•
. ABTS+
•
is a blue-green chromophore formed by
the reaction between 2,2
0
-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium
salt (ABTS; Sigma-Aldrich; A1888; São Paulo / Brazil) and potassium persulfate (K
2
O
8
S
2
;
Sigma-Aldrich; 216224; São Paulo/Brazil). When antioxidants are added to this preformed
cation radical, it is reduced to ABTS on a scale that depends on the antioxidant capacity,
antioxidant concentration, and reaction time. Both TEAC and DPPH assays were used to
determine the antioxidant capacities of the specimens’ EOs. Trolox (1 mM) was used as a
standard for the calibration curves (TEAC: concentration = [absorbance + 0.0023] / 0.4162,
r = 0.9789; DPPH: Concentration = [Absorbance + 0.0046] / 0.1346, r = 0.9851), and the
antioxidant capacity was expressed as a percentage of inhibition.
The cuvette was first filled with 2970
µ
L of the ABTS+ working solution, followed by
the first reading (T0). Subsequently, 30
µ
L of the sample was transferred to a cuvette contain-
ing the ABTS+
•
radical, and the second reading (T5) was recorded after 5 min. The reaction
was measured using spectrophotometry by observing the change in absorbance at 734
nm for 5 min (Spectrophotometer; Bioespectro SP22; São Paulo /Brazil). Thus, the total
antioxidant activity of the sample was determined, and its relationship to the reactivity of
Trolox as a standard was calculated through the realization of a standard curve under the
same conditions.
3.6. Antioxidant Capacity by the DPPH Method
The antioxidant capacity of the EOs was assessed according to the method proposed by
Blois [
64
]. This method evaluates the ability of synthetic or natural substances to eliminate
or neutralize 1,1-diphenyl-2-picrylhydrazyl (DPPH
•
; Sigma-Aldrich; D9132; São Paulo
/Brazil) free and stable free radical. The free radical, purple or violet in color, exhibits
absorbance in 515–520 nm, in ethanol or methanol solution. An antioxidant can donate a
hydrogen atom or transfer an electron to the DPPH radical
•
, resulting in its reduced form
DPPH-H, which is a stable diamagnetic molecule. This is accompanied by the loss of violet
color over time to pale yellow or light violet. The change in color from dark violet to light
violet, resulting from a decrease in the absorbance of the DPPH radical
•
, was monitored
using a UV/visible spectrophotometer (517 nm; Spectrophotometer; Biospectrum SP22;
São Paulo /Brazil) to determine the antioxidant capacity of the EOs. A standard curve was
constructed using Trolox as a standard curve.
3.7. Statistical Analysis
The results are expressed as the average of three repetitive assessments
±
the standard
deviation. The activity of EOs from E. patrisii,E. punicifolia, and M. tomentosa leaves was
analyzed by the Student0st-test, with a p-value < 0.05.
Multivariate Analysis
Multivariate analysis was performed according to the methodology described by
Silva et al. [
65
] and de Oliveira et al. [
66
], using the Minitab 17
®
software (free version
number 17, Minitab Inc., State College, PA, USA). The chemical constituents of the EOs
extracted from the leaves of E. patrisii (A), E. patrisii (B), E. punicifolia (A), E. punicifolia (B),
M. tomentosa (A), and M. tomentosa (B) (
≥
0.3%), were affixed as the experimental variables,

Molecules 2021,26, 3292 9 of 12
thus forming a matrix of 6 (samples)
×
26 (variables). The Euclidean distance options were
used for distance measurement in the HCA of the samples, and the connection method
was complete.
4. Conclusions
The chemical composition of the studied species was not found to differ significantly,
which can be explained by the location and collection periods. The chemical profile of
the EOs of the studied specimens, characterized by the terpenic class, showed a predomi-
nance of hydrocarbon sesquiterpenes
β
-elemene, (E)-caryophyllene, bicyclogermacrene,
germacrene D, and
γ
-elemene), and oxygenated sesquiterpenes spathulenol and selin-11-
4
α
-ol. The chemical composition of the EOs studied was not significantly influenced by
the climate at the time of sample collection, as evident from the cluster analysis of the
experimental variables. The results of the antioxidant activity suggested that the Myrtaceae
specimens, assessed in the study, may be natural sources of antioxidants. The differences
in the chemical profiles of the EOs influenced the antioxidant potential of the specimens.
Specimens A of E. punicifolia and E. patrisii showed the highest and lowest antioxidant
capacities, respectively, using the DPPH method. In the TEAC method, specimens A and B
of M. tomentosa showed the highest and lowest antioxidant potentials, respectively. The
antioxidant activity of the main compounds found in the EOs of the specimens has not
been reported in the literature. However, the observed antioxidant effect may be due to a
synergistic action between the various components.
Author Contributions:
Conceptualization, C.d.J.P.F., Â.A.B.d.M. and O.O.F.; methodology, E.L.P.V.,
L.D.d.N., S.P. and M.S.d.O., software, M.S.d.O.; formal analysis, E.H.d.A.A.; investigation, C.d.J.P.F.;
writing—original draft preparation, C.d.J.P.F., O.O.F.; writing—review and editing, M.S.d.O. and
E.H.d.A.A.; visualization, E.H.d.A.A.; supervision, E.H.d.A.A.; project administration, E.H.d.A.A.;
funding acquisition, E.H.d.A.A. All authors have read and agreed to the published version of the
manuscript.
Funding: This research received no external funding.
Acknowledgments:
The authors C.d.J.P.F. and Â.A.B.d.M. thank CNPq for the scientific initiation
scholarship. The author M.S.d.O., thanks PCI-MCTIC/MPEG, as well as CNPq for the scholarship
process number: [301194/2021-1]. The authors would like to thank the Universidade Federal do
Pará—Propesp/PAPQ—Programa de Apoio àPublicação Qualificada.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability:
Samples of Eugenia patrisii Vahl, E. punicifolia (Kunth) DC., and Myrcia tomentosa
(Aubl.) DC. The essential oil of the Museu Paraense Emílio Goeldi is available from the authors.
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