Chemical compositions and herbicidal (phytotoxic) activity of essential oils of three Copaifera species (Leguminosae-Caesalpinoideae) from Amazon-Brazil

Industrial Crops & Products 142 (2019) 111850

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Chemical compositions and herbicidal (phytotoxic) activity of essential oils of three Copaifera species (Leguminosae-Caesalpinoideae) from AmazonBrazil

T

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Ely Simone Cajueiro Gurgela,c, Mozaniel Santana de Oliveirab, , Marília Caldas Souzac, Sebastião Gomes da Silvab, Maria Silvia de Mendonçad, Antônio Pedro da Silva Souza Filhoe a

Paraense Museum Emílio Goeldi, Postal Box: 399. Av. Magalhães Barata, 376, 66040-170, São Braz, Belem, Pará, Brazil Federal University of Para, Rua Augusto Corrêa S/N, Guamá, 66075-900 Belém, Pará, Brazil Postgraduate Program in Biological Sciences - Tropical Botany, Federal Rural University of Amazonia / Paraense Museum Emilio Goeldi, Av. Perimetral 1901, Terra Firme, 66077-830, Belém, PA, Brazil d Federal University of Amazonas (UFAM), Faculty of Agricultural Sciences (FCA), Av. General Rodrigo Otávio Jordão, N. 3000, 69.077-000, Japiim, Manaus, Amazonas, Brazil e Brazilian Agricultural Research Corporation, Embrapa Eastern Amazon, Postal Box: 48. Trav. Dr. Eneas Pinheiro, S/N, 66095-100, Marco, Belem, Pará, Brazil b c

A R T I C LE I N FO

A B S T R A C T

Keywords: Natural products Bioactive compounds Allelochemicals Weed

The rich and diversified Amazonian flora represents an excellent resource of new chemical structures with biological activities. This study aims to contribute with information about the phytochemical profile and phytotoxic activity of the Copaifera species essential oils: Copaifera duckei (Dwyer), Copaifera martii (Hayne), and Copaifera reticulata (Ducke) (Leguminosae - Caesalpinioideae). In this study, essential oils were extracted from leaves and stems by hydrodistillation process in an adapted Clevenger type device. The identification of the essential oils chemical components was performed by gas chromatography coupled mass spectrometry (GC/MS) and gas chromatography (GC–FID). Statistical analyses of the results were obtained by the data were analyzed using F-test and the mean values were compared by the Tukey test at 5% and Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA). The phytotoxic activity of these extracted essential oils was tested against two species of invasive plants native to the Brazilian Amazon, namely, Mimosa pudica L. and Senna obtusifolia (L.) Irwin & Barneby). The essential oils effects on seed germination and elongation of the radicle and hypocotyl were observed. The compounds with the highest concentrations in the essential oils were sesquiterpene hydrocarbons, including germacrene D, β-caryophyllene, α-humulene, δ-elemene, and δ-cadinene. Inhibitory effects of the essential oils were greater on root development than on seed germination. Mimosa pudica L. tended to be more sensitive to the phytotoxic effects than Senna obtusifolia (L.) Irwin & Barneby). Leaf oils presented greater inhibitory effects on root and hypocotyl development, whereas stem oils showed greater inhibition of seed germination, although in some cases, these differences were not statistically significant. The leaves essential oils had a greater number of constituents than those of the stem; this was especially observed in Copaifera martii (Hayne). These variations could justify the differences observed in the phytotoxic effects between the oils from the stems and the leaves. The recipient specie Mimosa pudica L. was most affected by the effects of essential oils.

1. Introduction

issues, pest resistance, animal death, water contamination, harvest loss, and environmental and economic losses reaching up to US dollars 10 billion in countries such as the United States (Pimentel and Burgess, 2014). Despite the large volume of agrochemicals used annually in farming, yearly losses attributed to biotic agents have been high (Wang et al., 2015), which reflects negatively on the efficiency of these

Over the past decades, agriculture has become increasingly dependent on the use of fertilizers and agrochemicals (e.g., herbicides, fungicides, insecticides). This has led to social problems, mainly due to environmental damage caused by these products (Carvalho, 2017; Mahmood et al., 2016). Agrochemicals have caused public health ⁎

Corresponding author. E-mail address: [email protected] (M.S. de Oliveira).

https://doi.org/10.1016/j.indcrop.2019.111850 Received 9 July 2019; Received in revised form 5 September 2019; Accepted 7 October 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.

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2.3. Analysis of volatile compounds

products. In addition, biotic agents induce tolerance to currently available pesticides, a problem which has emerged in different countries (Torres-Acosta et al., 2012; González-Torralva et al., 2012). In Brazil, for example, several cases of weed tolerance have been reported recently (Barcellos Junior et al., 2017; Lopez Ovejero et al., 2017). Thus, economic, efficient and innovative weed control strategies need to be established. Sustainable methods of environmental control are an alternative that has gained the attention of the academic community to minimize synthetic herbicides in weed management (Hamdi et al., 2017; Lim et al., 2017). The use of natural products such as essential oils may be a viable alternative for the control of invasive plants (de Oliveira et al., 2016). Among the Leguminosae, Copaifera L., stands out from the rest because of its various uses, consisting in 28 species (16 found in Brazil and 9 only in the Brazilian Amazon) (Gebara et al., 2016). All species in this study are found in the state of Pará; moreover, Copaifera duckei (Dwyer) can also be found in Maranhão, Copaifera martii (Hayne) in Maranhão and Tocantins, and Copaifera reticulate in Amapá and Mato Grosso, these species have been the target of studies that demonstrated different biological activities through fractions of essential oils, oil resins, and Copaifera extracts, due to their antibacterial property (Bardají et al., 2016). They have been used for the treatment of monogenean infections (da Costa et al., 2017), as an anti-inflammatory agent, and as an inhibition agent of tyrosinase and lipoxygenase. They also present hemolytic activity, cytotoxicity, inhibition of nitric oxide production (Vargas et al., 2015), and neuroprotective (Botelho et al., 2015), antinociceptive, and antiparasitic effects (Arruda et al., 2019). However, in the last ten years, few literature has addressed phytotoxic activity (Franco et al., 2016; Linhares Neto et al., 2014). In this context, this study presents the use of essential oils of three Copaifera species (Copaifera martii (Hayne), Copaifera duckei (Dwyer), and C. reticulate) for the control of the invasive plant species Mimosa pudica L. and Senna obtusifolia (L.) Irwin & Barneby), which compete with other species, in addition to cause damage to animals by intoxication and infection of the oral mucosa (Caldas et al., 2016; Furlan et al., 2014).

The chemical composition of the essential oils was evaluated by gas chromatography/mass spectrometry (GC/MS) according to (Silva et al., 2019) using a Shimadzu, QP-2010 plus system under the following conditions: silica capillary column Rtx-5MS (30 m ×0.25 mm, 0.25 μm film thickness); program temperature of (60–240)°C 3 °C/min; injector temperature of 250 °C; carrier gas: helium (linear velocity of 32 cm/s, measured at 100 °C); splitless injection (1 μl of a 2:1000 hexane solution). Ionization was obtained by the electronic impact technique at 70 eV, and the temperature of the ion source and the other parts was set at 200 °C. The quantification of volatile compounds was determined by gas chromatography with a flame ionization detector (FID; Shimadzu, QP 2010 system) under the same conditions as gas chromatography coupled to mass spectrometry (GC–MS), except that hydrogen was used as the carrier gas. The retention index was calculated for all volatile constituents using a homologous series of n-alkanes (C8 - C20), and were identified by comparing the experimentally obtained mass spectra and retention indices to those found in literature (Adams, 2007; Maia et al., 2000; Stein; et al., 2011). 2.4. Bioassays for phytotoxicity The bioassays were carried out based on the protocol of (Batista et al., 2016). Seeds were selected from receptor species of Mimosa pudica L. – Leguminosae-Mimosoideae and Senna obtusifolia (L.) Irwin & Barneby)– Leguminosae-Caesalpinioideae. It was ensured that chosen seeds had the same size, shape, and coloration. These were collected at the Experimental Field of Embrapa Amazônia Oriental, located in Belém, state of Pará. The seeds were cleaned and then immersed in sulfuric acid in order to disrupt seed dormancy. A 9.0 cm Petri dish was inoculated with 20 seeds from each receptor species. Seed germination was monitored for 10 days; seed counting and removal were also performed on a daily basis. The bioassays were performed in BOD chambers set at 25 °C with a 12 -h photoperiod. Seeds were considered germinated when the roots were equal to or greater than 2.0 mm in length. Assessments of root and hypocotyl development were performed under the same conditions as the germination assays; however, these were done with a 24 -h photoperiod. For these assays, each 9.0 cm Petri dish was covered with a qualitative filter paper and inoculated with two pregerminated seeds that had germinated approximately 3 days prior to inoculation.

2. Material and methods 2.1. Botanical materials The leaves and stems were collected from plants growing wild in Belém “Mosqueiro” (Mari-Mari farm, km 28 of PA 391 highway) and in the city of Barcarena. Voucher specimens were deposited in the herbariums of Museu Paraense Emílio Goeldi (MG) and Embrapa Amazônia Oriental (IAN): Copaifera duckei (Dwyer) (IAN 175,605), Copaifera martii (Hayne) (IAN 176,276), and Copaifera reticulata (Ducke) (MG 186,090). The leaves and stems obtained were dried for 7 days in an airconditioned room at 20 °C and low humidity and then ground in a Willey-Mill plant grinder. The moisture content was analyzed in a moisture analyzer (model IV2500 - GEHAKA, Duquesa de Góias, Real Parque, São Paulo - Brazil).

2.5. Other experimental procedures In all bioassays, the test concentration was set at 1.0% using diethyl ether as eluent. At the beginning of each bioassay, 3.0 ml of test solution was added once to each 9.0 cm Petri dish. The solvent was allowed to evaporate, and distilled water was added as needed to replace the evaporated liquid in order to maintain the original concentration. 2.6. Experimental design and statistical data analysis A completely randomized design was used for all bioassays with three replicates, using a two-factor hierarchical model consisting of (a) the plant parts (leaves and stems) and (b) the Copaifera species (Copaifera duckei (Dwyer), Copaifera martii (Hayne), and Copaifera reticulata (Ducke). Distilled water was used as the control treatment. The data were analyzed using F-test and the mean values were compared by the Tukey test at 5%. The data were transformed to arc sine √x. The multivariate analysis was performed using Minitab® software (free version 390, Minitab Inc., State College, PA, USA) to analyze the chemical constituents of the essential oils (concentration of compounds ≥ 3%) of the leaves and stems of the three Copaifera species. The raw data were standardized to have the same "weight" in order to correct possible discrepancies between the variables. For this, the mean of the

2.2. Extraction procedures (Hydrodistillation) The oils were extracted from 100 g of each sample by hydrodistillation using Clevenger-type apparatus for 3 h. For the experiment, a 2 l flask was used. The water volume had ratio of 1:10 (w/v), with the water condensation maintained between 12 and 15 °C using the same refrigeration system (de Oliveira et al., 2019) for all experiments. The resulting oils were centrifuged for 5 min at 3000 rpm, dried using anhydrous sodium sulfate (Na2SO4), then centrifuged again under the same conditions, after which the solutions for chromatographic analysis were immediately prepared. Total oil yields were expressed as essential oil (g) / dried material (g). 2

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for the major compounds observed in Table 1 are β-caryophyllene, Germacrene D, α-humulene, bicyclogermacrene, and δ-cadinene. Regarding the species Copaifera martii (Hayne), the main compounds identified in the present work were β-caryophyllene, Germacrene D, and bicyclogermacrene. These results are different from those obtained by (Zoghbi et al., 2007), who identified α-copaene and δ-cadinene (36.4% and 51.2%; 3.7% and 17.3%, respectively), as the major compounds in the sample of a specimen collected in the city of Moju, State of Pará, Brazil. A specimen of Copaifera reticulata (Ducke) collected from Moju, state of Pará, Brazil, presented as major constituents β-Caryophyllene (25.1–50.2%), trans-α-Bergamotene (6.4–12%) and β-Bisabolene (5.2–17.4%) (Sachetti et al., 2011). In another specimen collected in Belterra-Pará-Brazil, there was predominance of the compounds β-bisabolene (0.92%), trans-α-bergamotene (21.8%), and β-caryophyllene (17.4%) (Herrero-Jáuregui et al., 2011). (Zoghbi et al., 2009) identified, as major compounds, β-bisabolene (18.4–42.4%) and trans-αbergamotene (11.8–29.6%) in samples collected in the state of Pará, whereas samples collected in the state of Amapá, the substances βcaryophyllene (27.8–68.0%), β-selinene (0.2–20.6%) and β-bisabolene (3.7–17.8%) predominated. In this work, the predominance of the compounds β-caryophyllene, germacrenne D, β-selinene, and α-selinene were observed in different parts of the plant (Table 1). Moreover, it was observed in this study and previous works that the chemical composition varies among specimens of the same species of Copaifera. These different chemical profiles may be associated with the environmental conditions and relationships that exist in the locations, from which these samples were collected (Silva et al., 2018). Since essential oils are secondary metabolites biosynthesized by aromatic plants, they can be directly influenced by multiple factors such as genetics, anthropic action, environmental conditions, geographical origin, circadian regime, seasonality, stage of development and others (Maia et al., 2003; Moghaddam and Mehdizadeh, 2017; Raposo et al., 2018; Stashenko et al., 2010). The principal components analysis (PCA) (Fig. 1) and HCA (Fig. 2) were performed to analyze the variability in the chemical constituents of the essential oils (those that had concentrations ≥ 3%) obtained from the leaves and branches of three Copaifera species. The PC1 component comprised 50.5% of the chemical variance and showed positive correlations with α-humulene, germacrene D, δ-elemene, βelemene, β-caryophyllene, and β-copaene, and negative correlations with trans-α-bergamotene, β-farnesene, allo-aromadendrene, δ-muurolene, β-cubebene, α-copaene, cyperene, and spathulenol. The PC2 component comprised 29.2% of the variance and presented a positive correlation, especially with γ-muurolene, β-caryophyllene, β-selinene, α-selinene, valencene, caryophyllene oxide, and α-humulene, and a negative correlation with α-muurolol, α-cadinol, bicyclogermacrene, δelemene, β-copaene, germacrene D, and δ-cadinene. The PC1 (50.5%) and PC2 (29.2%) components represent approximately 80% of the total sample variability of the chemical constituents of the essential oils obtained from the leaves and branches of Copaifera duckei (Dwyer), Copaifera reticulata (Ducke), and Copaifera martii (Hayne). These samples were divided into three groups (Fig. 2). LeafCd, Leaf-Cm, and Leaf-Cr samples were used in group I, which comprised germacrene D, bicyclogermacrene, β-copaene, α-cadinol, δ-elemene, and α-humulene. Group II comprised the Stem-Cd and Stem-Cr samples, which had β-caryophyllene, β-selinene, α-selinene, α-humulene, γ-muurolene, valencene, β-elemene, and caryophyllene oxide. Group III comprised the Stem-Cr sample and the chemical compounds α-copaene, cyperene, δ-cadinene, trans-α-bergamotene, β-cubebene, δmuurolene, spathulenol, β-allo-aromadendrene, and (Z)-β-farnesene. The groups formed by PCA were confirmed by HCA analysis, which generated a dendrogram that clustered the data into three groups with differing similarity levels (Fig. 2).

individual value of each variable was subtracted from the raw data, and the results were divided by the standard deviation. Principal Component Analysis (PCA) was performed using the "correlation" option of the matrix type. In the Hierarchical Cluster Analysis (HCA) of the samples, Euclidean distance and complete binding were used. 3. Results and discussion 3.1. Humidity and yields The moisture content in the leaves was calculated to be 9.4% for Copaifera duckei (Dwyer), 10% for Copaifera martii (Hayne), and 12% for Copaifera reticulata (Ducke). The essential oil yields had little variation, with the Copaifera duckei (Dwyer) sample having the highest volume yield at 0.9 ml of oil, which is equivalent to 0.8 g of oil, therefore the yield was 0.88% on a dry basis, while the Copaifera martii (Hayne) and Copaifera reticulata (Ducke) samples yielded 0.7 g and 0.5 g respectively, which equates to 0.77% and 0.56% yield on a dry basis. The moisture content in stems was by Copaifera duckei (Dwyer) 11%, Copaifera martii (Hayne) 13% and Copaifera reticulata (Ducke) 10.7%. The yields of essential oils from the stems were 0.3 g 0.33%, 0.23 g 0.26%, and 0.1 g 0.11% for Copaifera duckei (Dwyer), Copaifera martii (Hayne), and Copaifera reticulata (Ducke), respectively; these values were lower than those obtained for the leaves. The yields obtained in the present study were higher than those obtained in previous studies (de Almeida et al., 2014, 2016; Gramosa and Silveira, 2005) for Copaifera langsdorffii (Desf). 3.2. Chemical composition In total, 70 constituents with varying concentrations were present in the essential oil fractions obtained from the three species studied (Table 1). Oxygenated monoterpenes and oxygenated diterpenes were identified as the classes of compounds with the lowest concentrations, ranging from 0.38 to 3.72% and 0.68 to 1.86%, respectively. This results were similar to those reported by other studies on copaiba species (Carvalho et al., 2015; Soares et al., 2013). The compounds occurring at the highest concentrations were oxygenated sesquiterpenes and sesquiterpenic hydrocarbons ranging from 1.62 to 12.9% and 93.69 to 77.74%, respectively. Moreover, germacrene D, β-caryophyllene, αhumulene, δ-elemene, and δ-cadinene were identified in all essential oil fractions of the three plant samples studied (Table 1), whereas the transα-bergamotene was identified only in the stem of Copaifera martii (Hayne) at a concentration of 6.76%, and α-selinene was only found in the stems of Copaifera reticulata (Ducke) at 11.57% and Copaifera martii (Hayne) at 4.06%. In general, copaiba species are observed to be rich in sesquiterpenic hydrocarbons; however, in some cases, the concentrations of compounds such as β-caryophyllene, α-selinene, and germacrene D vary due to differences in collection period and the part of the plant from which the compound was isolated (do Nascimento et al., 2012; Sachetti et al., 2011). In the study performed by (Lameira et al., 2009), the authors identified the main substances present in Copaifera duckei (Dwyer), collected in Moju city, state of Pará: β-caryophyllene (13–50.2%), transα-bergamotene (8.3–12%), β-selinene (1.8–15.2%), and β-bisabolene (5.2–33.6%). In the study performed by (Cascon and Gilbert, 2000), in samples collected in Amapá state-Brazil, the major component found was kaur-16-en-19-oic (19.8–24.5%). In Caxiuanã city, state of Pará, Brazil, the major compounds were β-elemene (2.2%), β-caryophyllene (5.0%), α-bergamotene (14.7%), and β-bisabolene (27.4%); and the diterpenes kaur-16-en19-oic (13.5%), kauran-19-oic (8.3%), copalic (4.1%), polyalthic (6.9%) and eperu-8(17)-en-15,18-dioic (2.6%) (Carvalho et al., 2005), whereas (Gomes dos Santos et al., 2013), in samples collected in Moju (Pará), identified β-Bisabolene (40.94%), trans-α-bergamotene, and β-Caryophyllene (9.16%). However, the results of the present work, for the same species, show some differences, 3

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Table 1 Qualitative and quantitative results of the chemical composition of the essential oils of Copaifera duckei(Dwyer), Copaifera martii(Hayne), and Copaifera reticulata (Ducke) (Leguminosae - Caesalpinioideae) expressed as a relative percentage (%). Constituents

RI(c)

RI(L)

C. duckei Leaf

linalool (3Z)-hexenyl butanoate hexyl butanoate (3Z)-hexenyl 2-metylbutanoate δ-elemene α-cubebene cyclosativene α-ylangene α-copaene (3Z)-hexenyl hexanoate β-bourbonene β-cubebene β-elemene cyperene sesquithujene α-gurjunene cis-α-bergamotene β-caryophyllene β-copaene γ-elemene trans-α-bergamotene aromadendrene (Z)-β-farnesene cis-muurola-3,5-diene trans-muurola-3,5-diene α-humulene allo-aromadendrene cis-cadina-1(6),4-diene 4,5-di-epi-aristolochene δ-muurolene γ-gurjunene γ-muurolene germacrene D β-selinene trans-muurola-4(14),5-diene viridiflorene valencene α-selinene Constituents

1103 1189 1194 1234 1340 1353 1369 1375 1379 1384 1389 1394 1395 1404 1409 1415 1420 1425 1432 1437 1439 1444 1446 1450 1455 1458 1466 1468 1474 1472 1479 1482 1486 1491 1497 1498 1500 1501 RI(c)

1095a 1184a 1191a 1145b 1335a 1345a 1369a 1373a 1374a 1378a 1387a 1387a 1389a 1398a 1405a 1409a 1411a 1417a 1430a 1434a 1432a 1439a 1440a 1448a 1451a 1452a 1458a 1461a 1471a 1468c 1475a 1478a 1484a 1489a 1493a 1496a 1496a 1498a RI(L)

bicyclogermacrene α-muurolene β-bisabolene δ-amorphene γ-cadinene 7-epi-α-selinene δ-cadinene trans-cadina-1,4-diene α-cadinene α-calacorene elemol germacrene B (E)-nerolidol β-calacorene spathulenol caryophyllene oxide β-copaen-4α-ol rosifoliol humulene epoxide II 1,10-di-epi-cubenol 1-epi-cubenol γ-eudesmol cubenol epi-α-cadinol α-muurolol α-cadinol 14-hydroxy-9-epi-β-caryophyllene mustakone eudesma-4(15),7-dien-1β-ol pentadecanal kaurene

1502 1504 1511 1512 1518 1522 1527 1535 1540 1546 1552 1560 1563 1565 1579 1583 1592 1603 1611 1617 1631 1635 1636 1646 1648 1659 1665 1685 1693 1717 2051

1500a 1500a 1505a 1511a 1513a 1520a 1522a 1533a 1537a 1544a 1548a 1559a 1561a 1564a 1577a 1582a 1590a 1600a 1608a 1618a 1627a 1630a 1645a 1638a 1644a 1652a 1668a 1676a 1687a 1702b 2042a

C. reticulata Stem

Leaf

C. martii Stem

0.38

3.35 0.5

1.37

0.73 1.01

0.57 2.21

0.37

1.93 0.46 0.3 0.46 1.15

1.46 1.6 0.68 1.97

Leaf 0.53 2.81 0.38 0.14 3.47 0.36 0.41 3.18 0.41

Stem

0.51

14.41

0.42 5.19

1.81 0.29

1.35 1.8 0.16

1.67 2.44

4.18 1.54

2.5 2.19

8.25

0.19 13.92 4.45

0.25 33.45 1.47 1.16

20.06 2.22 0.56

24.77 1.34 0.8

0.59

0.54

0.7

0.57

19.9 2.12 0.84

9.2 0.47 6.76 3.03 0.82

4.81

7.63

0.49 4.35

1.32

0.62

0.71

4.97

4.9

0.11 0.33

1.32

1.85 3.07

3.9 5.91 12 1.03 4.79

23.37

1.13 4.76 17.53 1.96

4.79 10.61 14.36

2.98 0.83

1.11 1.72 15.82 0.56

1.21 11.57

C. duckei Leaf 9.15

Stem

C. reticulata Leaf 3.16 1.98

Stem

4.06 C. martii Leaf 8.86

Stem

1.06

1.09 1.36 2.5

4.92 2.15

1.01

2.53

0.92 2.35

6.18 0.53 0.72 0.25 0.15 0.41

5.26 0.17 0.34 0.27

6.61 0.37 0.48 0.18

0.12 1.36 1.95

0.13 0.67 3.71

0.61 1.67 0.4 3.74 0.12 0.17 0.08

1.08 1.04 5.19 0.34 0.36

1.12 7.19 0.58 1.04

0.13 0.71 1.08 1.34

0.06 0.15 1.47

0.3

3.32 1.16 0.8

1.11

0.65

0.43 0.53 0.09 0.56 0.26 0.99

0.19 1.78 0.57 2.41 0.19

3.09 4.08 0.28 0.26 0.31 0.69

0.13

0.13

1.52

1.35

2.98 3.3

1.12 0.16 0.95

1.54

0.18

1.68

(continued on next page) 4

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Table 1 (continued) Constituents

Oxygenated monoterpenes Hydrocarbons sesquiterpenes Oxygenated sesquiterpenes Oxygenated diterpene Total

RI(c)

RI(L)

C. duckei

C. reticulata

C. martii

Leaf

Stem

Leaf

Stem

Leaf

Stem

0.38 77.74 12.9 0.69 91.71

86.1 5.04 1.52 92.66

83.16 8.41 1.35 92.92

93.69 1.62 1.54 96.85

3.72 78.67 8.59 0.18 91.71

79.53 8.16 1.68 89.37

Italic: main constituents above 5%. RI(C): Calculated Retention Index; RI(L): Literature Retention Index. aAdams (Adams, 2007); bNist (Stein et al., 2011); (Maia et al., 2000)c.

3.3. Phytotoxic effect The use of crude extracts such as essential oils in bioassays to evaluate phytotoxic activity requires special attention to the effects of osmotic potential on the analyzed material, otherwise, phytotoxic activity may be overestimated or observed in cases where it is non-existent, thus creating false positives (Reigosa et al., 2006). For this study, the concentration used is 1.0%, because at this concentration (and at concentrations slightly above 1.0%), the contribution of the osmotic potential effects may be disregarded (Batista et al., 2016). Thus, the results of this study may be attributed to the effects of phytotoxic activity of the oils on seed germination and on root and hypocotyl development. Both Mimosa pudica L. and Senna obtusifolia (L.) Irwin & Barneby) were subjected to the seed germination bioassay, and the effects of the donor plant versus the fraction from which the oils were obtained are presented in (Fig. 3 (a) and (b). The data indicate extremely low inhibition, not exceeding 17.3% for Mimosa pudica L. and 18% for Senna obtusifolia (L.) Irwin & Barneby). In all three donor plants, the essential oils obtained from the stem fractions had the greatest inhibiting potential. Seed germination of Mimosa pudica L. was more susceptible to inhibition than Senna obtusifolia (L.) Irwin & Barneby). Among the three species of donor plants, Copaifera reticulata (Ducke) exhibited the greatest inhibition of both Mimosa pudica L. and Senna obtusifolia (L.) Irwin & Barneby) seed germination. The capacity to inhibit seed germination observed in this study is lower than that which was observed using essential oils from Citrus

Fig. 2. Dendrogram representing the similarity relationship of the oil’s composition of (C d) Copaifera duckei (Dwyer), (C m) Copaifera martii (Hayne), and (c r) Copaifera reticulata (Ducke) (Leguminosae - Caesalpinioideae).

aurantiifolia (Fagodia et al., 2017). Previous studies emphasized the potential of essential oils to inhibit seed germination (de Oliveira et al., 2016; Pinheiro et al., 2015); however, the low inhibition values observed for the essential oils in this study may be attributed to differences in the chemical compositions and concentrations of these essential oils, since complex mixtures can generate acute, synergistic and antagonistic effects (Pavela, 2014). The analysis of variance for the essential oil effects on root development showed a significant relationship (p < 0.05) between the

Fig. 1. The bidimensional plot of the two components (PC1 and PC2) obtained in the PCA analysis of the oils of (C d) Copaifera duckei (Dwyer), (C m) Copaifera martii (Hayne), and (c r) Copaifera reticulata (Ducke) (Leguminosae - Caesalpinioideae). 5

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Fig. 3. Inhibitory effect of the essential oils of Copaifera duckei, C. martii and C. reticulata on seed germination of Mimosa pudica and Senna obtusifolia. Same letters (capital for fractions within each species); small letters (for fractios between species) do not differ as per Tukey test (p > 0.05). Leaves and Stems.

Fig. 4. (a) and (b). (a) Inhibitory effect of the fractions of the essential oils of Copaifera duckei, C. martii and C. reticulata on root development of Mimosa pudica. Same letters (capital for fractions within each species); small letters (for fractios between species) do not differ as per Tukey test (p > 5%). Leaves and Stems. (b) Comparative analyses of Phytotoxic effects of the essential oils of Copaifera duckei, C. martii and C. reticulata on root development of Senna obtusifolia. Same letters (capital for fractions within each species); small letters (for fractios between species) do not differ as per Tukey test (p > 0.05).

donor plant species versus fraction of donor plant used in the assessment of the inhibition of hypocotyl development of both receiver species. All oils (Copaifera duckei (Dwyer), Copaifera martii (Hayne) and Copaifera reticulata (Ducke) exhibited high inhibition of Mimosa pudica L. hypocotyl development, with values above 69%, the most significant of which is Copaifera reticulata (Ducke) which exhibited an inhibition value above 76%. The inhibitory effects of these oils on Senna obtusifolia (L.) Irwin & Barneby) were lower than the observed effects on Mimosa pudica L. For Senna obtusifolia (L.) Irwin & Barneby), the oils obtained from Copaifera martii (Hayne) led to the highest inhibitory effects at 47.2% (Fig. 5 (b)). These data clearly show that Mimosa pudica L. was more sensitive to essential oil effects. Leaves and stems did not show statistical differences (p > 0.05) when comparing the inhibitory effects on Mimosa pudica L. and Senna obtusifolia (L.) Irwin & Barneby) hypocotyl development. However, the oils from the leaves showed greater inhibitory capacity (Fig. 6). Mimosa pudica L. hypocotyl development was more intensely inhibited than that of Senna obtusifolia (L.) Irwin & Barneby). This information confirms that Mimosa pudica L. has the greatest sensitivity to the phytotoxic effects of the oils from three Copaifera species, regardless of plant fraction. Considering all the potentially phytotoxic effects caused by the

donor source and the fraction of the donor plant in the case of Mimosa pudica L.; however, this was insignificant (p > 0.05) for Senna obtusifolia (L.) Irwin & Barneby). (Fig. 4 (a)) shows the effects of interaction between these two factors for Mimosa pudica L. The inhibition of root development observed was of a much greater magnitude than that observed for seed germination. Contrary to what was observed in the seed germination studies, essential oils from leaves showed greater inhibiting potential than those from stems, with values consistently above 42%. As for the stems, the effects were more significant for Copaifera martii (Hayne), with inhibition potential values above 43%. For Senna obtusifolia (L.) Irwin & Barneby), there was a lack of significance (p > 0.05) observed in the interactions between these factors; thus, these data are presented separately. No significant difference (p > 0.05) was observed in the inhibitory effects of the essential oils from the three donor plants on Senna obtusifolia (L.) Irwin & Barneby) root development (Fig. 4 (b)). However, when comparing the fractions of the donor plant used (Fig. 5 (a)), it was observed that essential oils from the leaves caused inhibition above 60%, while inhibition by those from stems was below 40%. These results confirm the greater ability of essential oil from leaves to inhibit root development on both receiver plants. No significant relationships (p > 0.05) were observed between 6

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Fig. 5. (a) and (b). (a) Comparative analyses of Phytotoxic effects from two fractions of three Copaifera species on root development of Senna obtusifolia. Similar letters do not differ by the Tukey test (p > 0.05). (b) Phytotoxic effects of the essential oils from different plants on hypocotyl development of Mimosa pudica and Senna obtusifolia compared to control treatment. Means followed by similar letters, capital letters between species, do not differ by the Tukey test (5%). C. duckei, C. martii and C. reticulate.

phytotoxic potential of Copaifera species were also observed; however, in some instances, this difference was not statistically significant (p > 0.05). Plants produce and store metabolites as defenses which are then distributed differently throughout organs. Phytotoxic activity and different allelochemicals have already been identified in fruits, seeds, flowers, rhizomes, and in other plant parts (Delgado-Adámez et al., 2017; Kordali et al., 2016; SHAH et al., 2017). The phytotoxic effects of essential oils should be considered as a synergy of the different components. In this study, the chemical composition of essential oils varied considerably among the species and between the two fractions of the plants (Table 1). The oils of the leaves had a greater number of constituents than those of the stem, especially in Copaifera martii (Hayne). These differences may explain, in part, the superior inhibitory activities of leaves over stems, especially in terms of the effects caused on root and hypocotyl development. However, small variations in phytotoxic activity observed among the three species were not correlated to the variations in major chemical constituents, which indicates the role of other non-major constituents in phytotoxic activity of the oils. Phytotoxic activities were reported in essential oils containing germacrene D, δ-cadinene, β-caryophyllene, and linalool (Amri et al., 2017; Mota et al., 2017; Silva et al., 2012). Based on this, the phytotoxic activities of the essential oils of Copaifera duckei (Dwyer), Copaifera reticulata (Ducke), and Copaifera martii (Hayne) can be attributed to these compounds. In the present study, the variations in inhibitory capacity are due to sources of the oils (leaves and stems) rather than the species from which were extraction their ned. Moreover, the oils obtained from the leaves more effectively inhibited root and hypocotyl development, while oils from stems caused greater inhibition on seed germination. The phytotoxic effects of the oils were observed to be greater on Mimosa pudica L. than on Senna obtusifolia (L.) Irwin & Barneby). Hypocotyl development was more sensitive to phytotoxic effects, whereas seed germination was less affected. Differences in the oil chemical compositions may partially explain the differences observed, especially the presence of δ-cadinene and linalool, which are molecules with proven phytotoxic activities (Singh et al., 2002; Ens et al., 2009). The results show that the Amazon flora has biological and economic importance, as it contains important sources of chemical molecules with possible uses in agricultural activity. However, more studies should be performed to determine the inhibitory activities of the different constituents in the oil of each species, as there are currently no studies on individual effects.

Fig. 6. Phytotoxic effects of the essential oils of two fractions of donating plants, on the hypocotyl development of Mimosa pudica and Senna obtusifolia compared to control treatment. Means followed by similar letters for each receiving species do not differ by the Tukey test (p > 0.05). Leaves and Stems.

Copaifera essential oils on seed germination, root development, and hypocotyl development, it was observed that the inhibitory effects were greatest on hypocotyl development, and least on seed germination. As for the two fractions of the donor plants, essential oils from stems showed higher inhibiting potential during seed germination, while those from leaves showed greater inhibition of root and hypocotyl development. However, these differences were occasionally statistically insignificant (p > 0.05), especially when comparing the essential oil effects on hypocotyl development (Fig. 6). Allelopathy studies have shown that variations on phytotoxic potential can be found when comparing different species and cultivars; however, these variations are also observed when comparing plants of the same gender and species (Gaaliche et al., 2017; Grul’ová et al., 2016; Sołtys-Kalina et al., 2019). In addition, phytotoxic effects may vary according to the recipient plant species and the oil concentration tested; literature suggests that stimulatory effects are based on the oils and extracts present in each plant (Matoušková et al., 2019; Shah, 2018), which was not observed in this study. Here, differences in 7

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4. Conclusion

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The chemical composition of the essential oils of Copaifera duckei (Dwyer), Copaifera martii (Hayne), and Copaifera reticulata (Ducke) showed that hydrocarbon sesquiterpenes are predominant in the leaves and stem, whereas germacrene D and β-caryophyllene were at high concentrations in all fractions of the plants studied. PCA and HCA statistical analyses of the chemical variability of the essential oils of leaves and branches of three Copaifera species showed the formation of three groups. Group I agglutinated the leaf samples of the three species presented in this study (Leaf-C.d, Leaf-C.m and Leaf-C.r); group II agglutinated the samples Stem-C.d and Stem-Cr, and group III, the sample Stem-Cr. Through the multivariate analysis it was possible to visualize and explain the differences among the sets of samples of the essential oils leaves and branches of the three species of Copaifera, thus facilitating the possible choices of application of this natural product based on the aggregated constituents. These data on the chemical variability of the essential oil of these three Copaifera species, with different chemical profiles from those previously reported, may have ecological, chemosystematic and taxonomic significance in the management and economic use of the species. In all cases observed, the essential oils of Copaifera duckei (Dwyer), Copaifera martii (Hayne), and Copaifera reticulata (Ducke) showed phytotoxic activity, with differing levels of intensity against Mimosa pudica L. and Senna obtusifolia (L.) Irwin & Barneby). Between the two, Mimosa pudica L. was more susceptible to the phytotoxic effects of the essential oils. The greatest phytotoxic effects were observed on the elongation of Mimosa pudica hypocotyl with value of 76%. The oils of Copaifera duckei (Dwyer), Copaifera martii (Hayne) and Copaifera reticulata (Ducke) could be indicated as candidates to the development of bioherbicides. Therefore, the results obtained in this study may support future research seeking natural products, such as essential oils, with allelopathic activity, thus contributing to the reduction of indiscriminate herbicide use, which consequently reduces environmental and human health damage. Declaration of Competing Interest The authors declare no conflict of interest. Acknowledgments The authors thank the following researchers from Embrapa Amazônia Oriental, Miguel Pastana do Nascimento, João Carlos Lima de Oliveira and Jair Freitas da Costa. The PCI internees from Museu Paraense Emílio Goeldi MSc. Raimunda Alves Pereira; Júlio Souza and Maria Maricélia Félix da Silva for thier valuable contribution from the processing of the botanical material until the extraction of essential oils. Fundação de Amparo à Pesquisa do Estado do Amazonas, FAPEAM, Brasil. References Adams, R.P., 2007. Identification of Essential Oil Components by Gas Chromatography/ Mass Spectroscopy, 4th ed. Allured Publishing Corporation, Carol Stream, IL, USA (2007), 4th ed. Allured Pub Corp; 4th edition (February 28, 2007), Carol Stream. Amri, I., Hanana, M., Jamoussi, B., Hamrouni, L., 2017. Essential oils of Pinus nigra J.F. Arnold subsp. Laricio Maire: chemical composition and study of their herbicidal potential. Arab. J. Chem. 10, S3877–S3882. https://doi.org/10.1016/j.arabjc.2014. 05.026. Arruda, C., Aldana Mejía, J.A., Ribeiro, V.P., Gambeta Borges, C.H., Martins, C.H.G., Sola Veneziani, R.C., Ambrósio, S.R., Bastos, J.K., 2019. Occurrence, chemical composition, biological activities and analytical methods on Copaifera genus—a review. Biomed. Pharmacother. 109, 1–20. https://doi.org/10.1016/j.biopha.2018.10.030. Barcellos Junior, L.H., Pereira, G.A.M., Gonçalves, V.A., Matos, C.C., Silva, A.A., 2017. Differential tolerance of sugarcane cultivars to clomazone. Planta Daninha 35. https://doi.org/10.1590/s0100-83582017350100069. Bardají, D.K.R., da Silva, J.J.M., Bianchi, T.C., de Souza Eugênio, D., de Oliveira, P.F., Leandro, L.F., Rogez, H.L.G., Venezianni, R.C.S., Ambrosio, S.R., Tavares, D.C.,

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