Antioxidant and Cytotoxic Activities of Myrtaceae Essential Oils Rich in Terpenoids From Brazil

Abstract

This work analyzed the chemical compositions and evaluated the antioxidant and cytotoxic activities of essential oils (EO) of Eugenia patrisii (Epat), Eugenia stipitata (Esti), Myrcia splendens (Mspl), Myrcia sylvatica (Msyl), Psidium guajava (Pgua), and Psidium guineense (Pgui-1 and Pgui-2) from the Brazilian Amazon. Sesquiterpenoids were found in high concentrations in the oils of E. patrisii and M. splendens, which were rich in E-caryophyllene (32.0% and 45.8%); E. stipitata and M. sylvatica, which displayed germacrene D (11.8%) and germacrene B (24.5%); and P. guajava that showed epi-β-bisabolol (16.1%) as the main compound. However, P. guineense samples (Pgui-1 and Pgui-2) were rich in monoterpenoids such as limonene (Pgui-1: 30.2%; Pgui-2 30.4%) and α-pinene (Pgui-1: 22.5%; Pgui-2: 17.7%). The samples showed a weak and moderate antioxidant activities in the DPPH assay, displaying inhibition rates from 11.5% to 38.6% (at 10 mg/mL). All samples were cytotoxic against human cancer cells by the MTT method. Epat oil showed higher activity against melanoma (SKMEL-19, IC₅₀5.8 µg/mL), gastric (AGP01, IC₅₀3.2 µg/mL), and colon (HCT116, IC₅₀6.7 µg/mL). Meanwhile, the samples Pgua and Pgui were more active against breast cancer cells (MCF7, IC₅₀12.4 µg/mL and 11.6 µg/mL, respectively).

Keywords

Eugenia Myrcia sesquiterpenes monoterpenes DPPH MTT

Cancer represents a significant public health problem and affects around 18 million people worldwide, and caused more than 9.6 million deaths in 2018.¹ In Brazil, the greatest number of deaths are related to cancers of the breast, stomach, colon, and rectum.² Together they represent about 23% of the total of more than 224 thousand deaths caused by cancer.²

The cause of uncontrolled multiplication of the healthy cells is mainly related to oxidative stress, a state generated by an imbalance of reactive oxygen species (ROS) released during mitochondrial respiration. There is a higher concentration of ROS compared to cellular antioxidants.³

The loss of redox homeostasis by cells can trigger destructive processes in biomolecules that are influenced by superoxide (O₂ ^-), hydroxyl (OH^●), and hydrogen peroxide (H₂O₂), such as the breakdown of nucleic acids (DNA and RNA), enzymatic inactivation, polysaccharide depolymerization, lipid peroxidation, in addition to also causing oxidation of carbohydrates and proteins.^3,4

Despite advances and improvements in diagnosis and monitoring, overall cancer survival rates remain elevated. There has been an increase in targeted molecular therapies to develop better clinical outcomes for cancer patients. Unfortunately, these strategies did not provide substantial improvement due to the development of resistance to therapies. In this context, phytochemicals have received considerable attention due to their diverse pharmacological properties, including cytotoxicity, with minimal or no toxicity in normal cells as well as providing cancer chemopreventive effects.⁵

Therefore, the anticancer potential of essential oils of Myrtaceae from Brazil has been widely studied, aiming to mitigate the resistance development to multiple drugs and side effects of antitumor drugs currently used.⁶ Essential oils extracted from Eugenia egensis DC., Eugenia flavescens DC., Eugenia patrisii Vahl, Eugenia polystachya Rich., Eugenia uniflora L., and Psidium myrtoides O. Berg showed cytotoxic activity against human cancer cell lines.^6,7

Myrtaceae are generally woody shrubs or tree species, mostly perennial, and commonly distributed in the tropics and subtropics.⁸ Among the 132 Myrtaceae genera, Eugenia, Myrcia, and Psidium are the largest in the American and African continents, with more than 1400 occurring in different biomes.^6,9,10

Eugenia and Psidium fruits are consumed in nature or used in desserts, juices, creams, and ice creams. They are also used in traditional Brazilian medicine, such as Psidium guineense Sw. against inflammatory diseases and Eugenia stipitata McVaugh to treat intestinal, urinary disorders, and relieve cold symptoms.^11,12 On the other hand, Myrcia species are commonly used as phytopharmaceuticals to prepare teas to treat diabetes, hypertension, diarrhea, and compresses form to treat inflammation and skin infections.^13,14

Essential oils have been shown to possess a wide range of anticancer properties and mechanisms. Moreover, the ability of essential oils to demonstrate anticancer effects through action on various pathways and cellular mechanisms has aroused interest in searching for possible phytopharmaceuticals against cancer. Thus, more studies are necessary to expand the present knowledge of these mechanisms to promote cell-specific and individualized cancer therapy.¹⁵

Due to the great phytotherapeutic use of Eugenia, Psidium, and Myrcia species and the use of their essential oils in traditional medicine, this study is aimed to analyze the chemical composition and evaluate the antioxidant and cytotoxic activities of essential oils of some Myrtaceae species from the Brazilian Amazon.

Results and Discussion

Yield and Chemical Composition of Essential Oil

The descending order of leaf essential oil yields of Myrtaceae samples are Myrcia sylvatica (G. Mey.) DC. (0.7%), Psidium guineense (Pgui-1: 0.5%; Pgui-2: 0.6%), Eugenia patrisii (0.4%), Psidium guajava L. (0.4%), Eugenia stipitata (0.2%), and Myrcia splendens (0.1%). In all samples, 173 compounds were identified by GC-FID and GC-MS, mainly terpenoids representing an average of 94.18% of total composition in oils (Table 1). Eugenia and Myrcia species showed a predominance of sesquiterpene hydrocarbons: E. patrisii (85.7%), M. splendens (76.5%), M. sylvatica (71.5%), and E. stipitata (61.7%). Sesquiterpene hydrocarbons (43.6%) and oxygenated sesquiterpenoids (44.7%) were the major terpenoid classes in P. guajava oil. Meanwhile, the two P. guineense oil samples showed monoterpene hydrocarbons as predominant (Pgui-1: 58.1%; Pgui-2: 54.3%).

Table 1

Leaf Essential Oil Yield (%) and Constituents (%) of Myrtaceae Species.

Oil yield (%)			Epat0.4	Esti0.2	Mspl0.1	Msyl0.7	Pgua0.4	Pgui-10.5	Pgui-20.6
IR_(C)	IR_(L)	Constituent	(%)
847	846^a	2E-Hexenal		tr		0.1	tr	0.1	tr
849	850^a	3Z-Hexenol	tr	tr		0.1	0.3	0.1
862	863^a	n-Hexanol	tr			0.1	0.1	tr
919	921^a	Tricyclene						0.1	tr
934	932^a	α-Pinene	0.2	0.5	2.4		0.1	22.5	17.7
946	945^a	α-Fenchene						0.1	0.1
948	946^a	Camphene						0.5	0.6
957	952^a	Benzaldehyde					0.8	0.7	1.1
977	974^a	β-Pinene	tr	0.1	1.4			1.4	1.4
985	981^a	6-Methyl-5-hepten-2-one					0.3	0.1	0.2
990	988^a	Myrcene			7.9		tr	1.3	1.2
1005	1003^a	p-Mentha-1(7),8-diene						0.6	0.9
1024	1020^a	p-Cymene					0.1	0.7	1.0
1029	1024^a	Limonene	tr	tr	0.6		0.2	30.3	30.4
1031	1032^a	1,8-Cineole					2.1	0.1	0.1
1035	1032^a	Z-β-Ocimene					0.3	tr	0.1
1045	1044^a	E-β-Ocimene					tr	0.1	0.2
1057	1054^a	γ-Terpinene						0.5	0.7
1088	1086^a	Terpinolene			0.5			0.1	0.2
1099	1095^a	Linalool	0.2	0.1			0.1	0.1	0.1
1113	1114^a	endo-Fenchol						0.1	0.1
1116	1113^a	4,8-Dimethyl-E-nona-1,3,7-triene						tr	0.1
1120	1122^b	trans-p-Mentha-2,8-dien-1-ol						0.1	0.1
1125	1122^a	α-Campholenal						0.1	0.1
1138	1135^a	trans-Pinocarveol						0.3	0.4
1147	1145^a	Camphene hydrate						0.1	0.1
1165	1165^a	Borneol						0.1	0.2
1166	1165^a	1,3-Dimethoxybenzene		0.1
1161	1165^a	Hydrocinnamaldehyde							0.9
1176	1174^a	Terpinen-4-ol						0.2	0.2
1186	1187^a	trans-p-Mentha-1(7),8-dien-2-ol						0.2
1190	1186^a	α-Terpineol		0.1	3.8		0.4	0.9	1.3
1187	1189^a	trans-Isocarveol							0.4
1193	1190^a	Methyl salicylate						0.1	0.1
1218	1215^a	trans-Carveol						0.1	0.2
1220	1218^a	endo-Fenchyl acetate						0.4	0.4
1226	1227^a	cis-p-Mentha-1(7),8-dien-2-ol						0.1	0.4
1230	1235^a	E-Ocimenone							0.2
1243	1239^a	Carvone							0.1
1266	1261^a	cis-Chrysanthenyl acetate							0.1
1285	1287^a	Bornyl acetate			0.1			0.9	0.7
1299	1298^a	trans-Pinocarvyl acetate						0.7	0.3
1323	1322^a	Methyl geranate						0.3	0.6
1338	1335^a	δ-Elemene	0.9	1.9		0.4	0.1	0.1	tr
1337	1339^a	trans-Carvyl acetate						0.1	0.1
1351	1345^a	α-Cubebene	0.2	0.5
1364	1359^a	Neryl acetate						0.1	0.1
1367	1369^a	Cyclosativene						0.1	0.1
1372	1373^a	α-Ylangene		0.2
1374	1374^a	Isoledene	0.1			tr
1376	1374^a	α-Copaene	4.8	1.7	4.5	0.1	2.8	6.6	8.1
1380	1380^a	2-epi-α-Funebrene					0.1
1381	1378^a	3Z-Hexenyl hexanoate					0.2	0.1
1383	1379^a	Geranyl acetate						0.9	1.0
1385	1383^a	3Z-Hexenyl-3Z-hexenoate					0.1	0.1
1386	1387^a	β-Bourbonene		0.2		0.2
1391	1387^a	β-Cubebene		0.2
1393	1389^a	β-Elemene	6.9	0.6		1.3
1402	1410^a	α-Cedrene					0.3
1404	1405^a	Italicene					0.6
1407	1405^a	Sesquithujene					0.1
1408	1400^a	β-Longipinene	0.1
1411	1409ª	α-Gurjunene	0.9	0.3		0.1
1413	1410ª	α-Cedrene					0.8
1415	1415^b	β-Maaliene	0.1
1417	1416^b	cis-α-Bergamotene	0.1
1420	1417^a	E-Caryophyllene	32.0	5.0	45.8	10.0	5.1	2.1	0.1
1428	1428^a	E-α-Ionone		0.2
1430	1430^a	β-Copaene	0.3			0.2	0.1		tr
1433	1431^a	β-Gurjunene	0.3	1.4			0.2
1434	1432^b	γ-Elemene		0.2	2.1	12.5
1435	1435^b	Perillyl acetate						0.1	0.1
1437	1432^b	trans-α-Bergamotene	1.5	1.1		0.6	0.3
1439	1439ª	Aromadendrene	1.8	0.6		0.4		0.1	tr
1444	1442^a	6,9-Guaiadiene		0.6
1444	1445^b	Selina-5,11-diene	0.5			0.1
1451	1451^a	trans-Muurola-3,5-diene		0.2		0.1
1452	1449^a	α-Himachalene					0.1
1453	1453^a	Geranyl acetone						0.1	0.1
1454	1452^a	α-Humulene	5.6	1.4	5.0	1.4	0.9	0.4	0.3
1457	1454^a	E-β-Farnesene	0.4	1.2			0.5
1461	1458^a	allo-Aromadendrene		0.9		0.6		0.2	0.3
1461	1457^a	β-Santalene		0.4			1.2
1462	1464ª^,b	9-epi-E-Caryophyllene	7.6
1464	1463^b	cis-Cadina-1(6),4-diene		0.2
1465	1464^a	α-Acoradiene					1.9
1468	1469^a	β-Acoradiene					0.5
1475	1474^a	10-epi-β-Acoradiene					0.6
1475	1475^a	trans-Cadina-1(6),4-diene	0.2	0.4		0.2
1478	1478^a	γ-Muurolene	1.5	2.2	0.5	0.6	0.4	0.3	0.4
1480	1481^a	γ-Curcumene					0.1
1481	1483^a	α-Amorphene			0.1
1482	1480^b	Germacrene D	0.8	11.9		3.8
1484	1480^b	ar-Curcumene					9.8
1486	1481^a	γ-Himachalene	0.4					0.9
1492	1489^b	δ-Selinene		0.7		0.3
1495	1492^b	β-Selinene	1.2		0.1	0.6	1.2	0.8	1.0
1496	1498ª	α-Selinene					1.1		0.9
1497	1496^a	Viridiflorene	2.2	1.3	0.9	2.0
1498	1500^a	Bicyclogermacrene	10.0	4.2		5.0
1499	1500^a	α-Muurolene	0.6	2.1	0.4	0.5	0.2	0.4	0.5
1508	1511^a	δ-Amorphene				0.4
1506	1509^a	α-Bulnesene	0.3
1507	1501^a	epi-zonarene						0.1
1503	1503^a	Z-α-Bisabolene		8.4			0.8
1509	1505^a	α-E,E-Farnesene			0.5
1510	1508^a	β-Bisabolene	0.4	2.6			9.2
1513	1514^a	β-Curcumene		0.1			0.6
1515	1513^a	γ-Cadinene	0.3	2.0	0.4	0.4		0.3	0.3
1519	1520^a	7-epi-α-Selinene	0.0						tr
1523	1522ª	δ-Cadinene	3.4	5.7	2.7	1.8	2.2	1.2	1.7
1527	1528ª	Zonarene		0.3	3.2
1532	1533ª	trans-Cadina-1,4-diene	0.1			0.1		0.1	0.1
1533	1529ª	E-γ-Bisabolene					0.7
1535	1536^a	Italicene ether					0.4
1536	1540^b	Selina-4(15),7(11)-diene			1.7	1.6
1539	1538^b	α-Cadinene	0.1	0.6					tr
1542	1545ª	Selina-3,7(11)-diene			2.7	1.9
1544	1540^b	E-α-Bisabolene	0.2				0.5
1544	1544^a	α-Calacorene		0.2				0.3	0.2
1558	1559^a	Germacrene B	0.2	0.3	5.9	24.5
1564	1561^a	E-Nerolidol		0.8			4.5	0.2	0.4
1568	1567^a	Palustrol	0.4			0.8
1570	1570^a	Caryophyllenyl alcohol			0.3
1570	1571^a	Caryolan-8-ol					0.5	0.1
1570	1565^a	3Z-Hexenyl benzoate		0.2					tr
1580	1582^a	ar-Tumerol					0.4
1584	1582^a	Caryophyllene oxide	4.1	2.6	1.3	3.3	4.5	1.0
1585	1586^a	Gleenol							0.3
1587	1590^a	β-Copaen-4-α-ol							0.5
1587	1577^a	Spathulenol	2.2	1.8		2.9		0.3
1588	1590^a	Globulol		0.5
1593	1592^a	Viridiflorol	0.9	1.4		1.5		0.1	0.2
1595	1595^a	Cubeban-11-ol	0.4	0.6		0.7			tr
1602	1600^a	Cedrol					0.5
1603	1600^a	Rosifoliol		0.5		0.9
1604	1602^a	Ledol	1.0					1.0
1605	1613^b	Copaborneol					0.8
1610	1608^a	Humulene epoxide II	0.2				1.1	0.1	0.1
1614	1618^a	epi-Cedrol					0.8
1616	1618^a	1,10-di-epi-Cubenol		0.3
1619	1618^a	Junenol		0.7
1628	1632^b	Muurola-4,10(14)-dien-1-β-ol						3.6	3.6
1629	1631^b	1-epi-Cubenol	0.4	1.8	0.2
1629	1632^a,b	α-Acorenol				0.8	2.8
1632	1632^b	γ-Eudesmol			0.1	0.3
1634	1636^a	β-Acorenol					0.5
1636	1639^a	Caryophylla-4(12),8(13)-dien-5b-ol						0.6
1638	1638^b	Gossonorol					1.6
1638	1639^a	allo-Aromadendrene epoxide							tr
1642	1640^b	epi-α-Cadinol (τ-cadinol)	0.6	1.7	0.3	0.4		1.8	1.7
1643	1645^b	epi-α-Murrolol (τ-muurolol)		5.0	0.8	1.2	0.6
1647	1651^b	α-Muurolol (torreyol)	0.2	2.2	0.1	0.4	2.2	1.0	1.2
1656	1651^a	Pogostol					2.0
1655	1652^a	α-Cadinol	0.5	1.1	1.5	1.9		1.6	1.8
1656	1659^b	Cadin-4-en-10-ol		6.1
1666	1665^a	Intermedeol				0.9
1670	1679ª	Khusinol							0.3
1672	1670ª	epi-β-Bisabolol					16.1
1680	1674^a	β-Bisabolol					0.2
1684	1683^a	epi-α-Bisabolol	tr				1.5
1686	1684^a	Longiborneol acetate				0.1
1686	1685^a	Germacra-4(15),5,10(14)-trien-1-α-ol		0.6					0.1
1687	1685^a	α-Bisabolol		0.5			3.7
1696	1696^b	Juniper camphor			0.9	1.5
1714	1713^a	2E, 6Z-Farnesal						1.0	1.5
1721	1722^a	2Z,6E-Farnesol					0.1	1.1	3.7
1741	1740^a	2E,6E-Farnesal						1.4	2.1
1767	1768^a	β-Bisabolenal						0.1	0.2
1782	1779^a	14-hydroxy-α-Muurolene		0.1
1841	1832^b	Farnesyl acetate						0.1	0.2
1844	1841^b	Phytone		0.1
1962	1958^a	Geranyl benzoate						0.1	0.1
Monoterpene hydrocarbons			0.2	0.6	12.7	0.0	0.7	58.1	54.3
Oxygenated monoterpenoids			0.2	0.1	3.9	0.0	2.6	6.2	7.5
Sesquiterpene hydrocarbons			85.7	61.7	76.5	71.5	42.6	14.0	14.1
Oxygenated sesquiterpenoids			10.9	28.4	5.5	17.4	44.7	15.2	17.8
Others			0.1	0.4	0.0	0.3	1.8	1.2	2.3
Total identified			97.1	91.3	98.6	89.2	92.4	94.7	96.0

Abbreviations: Epat, Eugenia patrisii; Esti, Eugenia stipitata; Mspl, Myrcia splendens; Msyl, Myrcia sylvatica; Pgua, Psidium guajava; Pgui, Psidium guineense; RI_(C), calculated retention index; RI_(L), literature retention index; tr, trace (<0.1%).

^aAdams (2007)¹⁶

^bMondello (2011)¹⁷

Among Eugenia species, E. patrisii presented E-caryophyllene (32.0%) and bicyclogermacrene (10.0%) as major compounds, whereas E. stipitata showed germacrene D (11.8%) and Z-α-bisabolene (8.38%). It is known that E. patrisii leaves oils were characterized by the presence of cyclic sesquiterpenes with cadinane and caryophyllane skeletons, including trans-cadin-1,4-diene (16.5%), trans-muurola-3,5-diene (13.3%), and E-caryophyllene (11.1%).¹⁸ However, E. patrisii aerial parts (leaves and fine stems) had a mixture of acyclic and cyclic oxygenated sesquiterpenes as 2E,6E-farnesol (34.5%), 2E,6Z-farnesol (23.2%) and caryophylla-4(12), 8(13)-dien-5β-ol (15.6%) as major constituents.^6,19

Eugenia stipitata leaves essential oil collected in Portugal was characterized by E-caryophyllene (22.7%) and caryophyllene oxide (15.4%).²⁰ Likewise, the EO of the leaves from the Brazilian Northeast was characterized by guaiol (13.7%) and E-caryophyllene (11.3%).²¹ The variability in the chemical compositions of Eugenia spp. may be related to the environmental differences of the collection sites since this is a factor that can influence chemotypes varieties.^6,19

The oils of Myrcia samples also displayed sesquiterpenoids as main constituents: M. splendens presented E-caryophyllene (45.8%), M. sylvatica showed an abundance of germacrene B (24.5%) and γ-elemene (12.5%). M. splendens EOs extracted from its leaves exhibited different chemical profiles.²² The EO from M. splendens collected in Amazonian Ecuador was characterized by E-nerolidol (67.8%) and α-bisabolol (17.5%).²² Similarity, α-bisabolol (83.8%) and α-bisabolene (80%) were identified as primary compound in 2 samples collected in the southern and southeastern regions of Brazil, respectively.^23,24 On the other hand, its EO from the aerial parts (leaves and stems) collected in three regions of Brazilian amazon (I-III) showed different chemical profile characterized by selin-11-en-4α-ol (24.7%), caryophyllene oxide (16.6%), and spathulenol (13.8%) (sample I); cis-calamenene (30.1%), spathulenol (18.7%) and α-calacorene (11.5%) (sample II); and spathulenol (40.2%) and β-bisabolene (14.7%) (sample III).²⁵ M. sylvatica EOs presented a chemical composition influenced by seasonal factors.⁹ The monthly monitoring of EO composition during 1 year of a specimen from northern Brazil showed variability in its major constituents β-selinene (6.2 to 10.5%) and 1-epi-cubenol (5.9 to 9.8%).⁹

Psidium guajava (Pgua) showed the sesquiterpenes epi-β-bisabolol (16.1%), ar-curcumene (9.8%), β-bisabolene (9.2%), E-caryophyllene (5.1%), and caryophyllene oxide (4.5%) as the main compounds. While P. guineense samples (Pgui-1 and Pgui-2) showed as the main compound the monoterpenes limonene (Pgui-1: 30.2% and Pgui-2 30.4%) and α-pinene (Pgui-1: 22.5% and Pgui-2: 17.7%).

The chemical composition of 22 genotypes of P. guajava from Brazil were evaluated and showed an abundance of E-caryophyllene (5.1% to 32.3%), caryophyllene oxide (1.8% to 20.9%), α-humulene (1.7% to 19.9%), β-bisabolol (2.2% to 19.4%), E-nerolidol (2.1% to 13.7%), β-selinene (0.5% to 12.8%), hinesol (3.2% to 12.4%) epi-α-cadinol (1.1% to 12.0%), α-selinene (0.5% to 11.2%), and limonene (0.1% to 11.0%). These quantitative changes were attributed to environmental factors.²⁶ Leaves of P. guineense EO from the Brazilian Midwest showed high amounts of spathulenol (80.7%).¹¹ However, P. guineense from Brazilian Amazon displayed β-bisabolol (17.4%), limonene (6.8%), and epi-α-bisabolol (6.7%) as main constituents.²⁷

Antioxidant Activity

DPPH method is based on reducing the alcoholic DPPH solution in the presence of hydrogen donating antioxidants through the formation of the non-radical form (DPPH-H). As the EO reduces the DPPH radical, the solution changes color from purple to yellow (arising of diphenylpicrylhydrazine). The reduction capability of the DPPH radical is determined by the decrease in its absorbance at 517 nm.²⁸ All Myrtaceae samples showed antioxidant activity against the DPPH method as shown in Table 2.

Table 2

Antioxidant Capacity of the Oils of Myrtaceae Species.

Sample	Inhibition (%)^a	TEAC^a (mgTE/mL)
Epat	28.9 ± 4.8^a,b	146.2 ± 24.5^a,b
Mspl	28.4 ± 7.1^a,b	144.1 ± 35.8^a,b
Msyl	18.5 ± 3.5^a,c	93.9 ± 17.7^a,c
Pgua	38.6 ± 7.0^b	195.7 ± 35.6^b
Pgui-1	11.5 ± 2.0^c	70.4 ± 11.8^c
Pgui-2	27.7 ± 2.3^a,b	166.5 ± 13.7^a,b

Abbreviations: Epat, Eugenia patrisii; Est, Eugenia stipitata; Mspl, Myrcia splendens; Msyl, Myrcia sylvatica; Pgua, Psidium guajava; Pgui, Psidium guineense.

^aEssential oils at 10 mg/mL. Values are expressed as means ± standard deviations (n = 3). Values with the same letters in the column do not differ statistically in the Tukey test (P > 0.05).

Psidium guajava essential oil (Pgua) showed the highest antioxidant activity, inhibiting 38.4% of the DPPH radical, this oil was rich in the sesquiterpenes epi-β-bisabolol (16.1%) and ar-curcumene (9.8%). Total antioxidant capacity was expressed as Trolox equivalent (TE), and Pgua sample showed a value of 195.7 mgTE/mL, only five times smaller than the Trolox Standard. On the other hand, the least active sample was Pgui-1, rich in limonene (30.2%) and α-pinene (22.5%), which presented an inhibition rate of 11.5% of DPPH radical (70.4 mgTE/mL). However, the Pgui-2 samples were also rich in limonene (30.4%) and α-pinene (17.7%) and presented inhibition of 27.7% (166.49 TE/mL), 6 times less active against DPPH compared to Trolox. The sample Pgua presents the highest content of oxygenated sesquiterpenes (44.7%), which can be attributed to its significant antioxidant activity, as this compound’s class generally exert good free radical scavenging activities.²⁹

Psidium guajava oil from Malaysia displayed DPPH radical scavenging with an IC₅₀ value of 460.4 µg/mL and a high phenolic compound (495.9 mgGAE/g), activity related to the concentration of phenols that the species presents.²⁸ Psidium guineense rich in spathulenol (80.7%), presented inhibition of DPPH radical with IC₅₀ of 63.08 µg/mL, being lass active of 2,6-di-tert-butyl-4-methylphenol standard (BHT, IC₅₀ 16.7 µg/mL).¹¹

The E. patrisii essential oil (Epat), rich in E-caryophyllene (32.0%) and bicyclogermacrene (10.0%), presented 28.9% of DPPH radical scavenging (146.2 mgTE/mL), about six times less activity than Trolox standard. Another E. patrisii sample from Brazilian Amazon, rich in 2E,6E-farnesol (34.5%), showed DPPH inhibition of 40.9%, about 10 times less active than Trolox standard (111.2 mgTE/mL).¹⁹

Myrcia splendens essential oil (Mspl), rich in E-caryophyllene (45.8%) presented DPPH radical scavenging of 28.4%, about seven times less than Trolox (144.1 mgTE/mL). On the other hand, M. sylvatica oil sample (Msyl), rich in the sesquiterpene hydrocarbons germacrene B (24.5%), γ-elemene (12.5%) and E-caryophyllene (10.0%), presented the inhibition value of 18.5%, being about ten times less active than Trolox.

Oils from Myrcia species have been reported as antioxidants. For example, M. splendens demonstrated antioxidant activity with an IC₅₀ of 43.5 µg/mL, rich in the E-nerolidol sesquiterpene (67.81%).¹³ Moreover, EO of Myrcia oblongata DC., rich in caryophyllene oxide (22.0%) and trans-verbenol (11.9%), was identified as an antioxidant (IC₅₀ 2.8 µg/mL) compared to the synthetic antioxidant BHT (IC₅₀ 1.4 µg/mL).³⁰

Cytotoxic Activity

The cytotoxic activity of the seven Myrtaceae essential oil samples were evaluated by the MTT method and showed antiproliferative activity against the 4 hoursuman cancer cell lines, breast (MCF7), melanoma (SKMEL-19), gastric (AGP01), colon (HCT116), and a non-malignant human lung fibroblast cell (MRC5), see Table 3. It has been suggested IC₅₀ values below 100 µg/mL show relevant activity.³¹

Table 3

Cytotoxic Activity of the Myrtaceae Species Essential Oils on Cell Lines.

Oilsamples	IC₅₀ (μg/mL)/SI^a
Oilsamples	MCF7Breast	SKMEL-19Melanoma	AGP01Gastric	HCT116Colon	MRC5Human Fibroblast
Epat	ND	5.80^.6 (4.4, 7.6)	3.21^.1 (2.5, 4.1)	6.70^.5 (5.2, 8.6)	3.5 (2.9, 4.3)
Esti	19.10^.7 (16.4, 22.1)	17.20^.8 (15.2, 19.5)	ND	ND	13.8 (11.8, 16.0)
Mspl	ND	8.50^.8 (6.6, 11.0)	4.71^.4 (3.1, 7.2)	8.80^.7 (7.3, 10.5)	6.5 (5.6, 7.6)
Msyl	>25	20.01^.2 (17.5, 22.9)	17.31^.3 (13.7, 22.0)	ND	23.3 (20.8, 26.0)
Pgua	12.41^.7 (9.1, 17.1)	15.31^.4 (13.8, 17.0)	16.31^.3 (14.0, 19.0)	ND	20.8 (19.6, 22.1)
Pgui-1	11.60^.7 (9.2, 14.6)	11.10^.7 (8.5, 14.5)	8.21^.0 (7.2, 9.4)	ND	8.27 (7.3, 9.4)
Pgui-2	18.21^.3 (11.6, 28.6)	19.11^.3 (16.10, 22.63)	15.71^.5 (14.4, 17.1)	ND	24.0 (21.5, 26.7)
Doxorubicin(μM)	0.9 µM(0.7, 1.2)	0.1 µM(0.0, 0.3)	0.2 µM(0.2, 0.3)	0.1 µM(0.1, 0.2)	0.2 µM(0.2, 0.3)

Abbreviations: Epat, Eugenia patrisii; Esti, Eugenia stipitata; Mspl, Myrcia splendens; Msyl, Myrcia sylvatica; Pgua, Psidium guajava; Pgui, Psidium guineense.

^aData are presented as IC₅₀ values and 95% CIs obtained by nonlinear regression for all cell lines from three independent experiments. ND = not determined due to insufficient essential oil. SI = IC₅₀ in MRC-5 cells/IC₅₀ in a cancer cell (superscript value).

Among the samples tested, the oil of E. patrisii (Epat) showed the highest activity against melanoma (SKMEL-19) and colon (HCT116) cells with IC₅₀ values of 5.8 and 6.7 µg/mL, respectively. Likewise, gastric ascites (AGP-01) was the most sensitive human cancer cell line against Epat oil and showing an IC₅₀ value of 3.2 µg/mL and SI of 1.1. The SI value reveals the differential activity; therefore, the higher the SI value is, the more selective it is. On the other hand, an SI value <2 suggests general toxicity to compounds.³² In contrast to our findings in this study, E. patrisii EO from Brazilian Amazon, rich in (2E,6Z)-farnesol (23.2%) and (2E,6E)-farnesol (34.5%), sesquiterpene alcohols that have shown anti-neoplastic effects in various human cancers,³³ did not show activity against gastric ascites cancer cells.¹⁹

Eugenia stipitata essential oil (Esti) displayed cytotoxic activity against melanoma (IC₅₀ 17.2 µg/mL), and breast (IC₅₀ 19.1 µg/mL) human cancer cells; and the normal human fibroblast (IC₅₀ 13.8 µg/mL). As far as we are aware, there have been no reports of cytotoxic activity of essential oils of E. stipitata reported in the literature.

Myrcia splendens (Mspl) showed good cytotoxic activity against gastric (IC₅₀ 4.7 µg/mL), melanoma (IC₅₀ 8.6 µg/mL), and colon (IC₅₀ 8.8 µg/mL) human cancer cells. However, Mspl oil was more selective against AGP01 (SI 1.4). Myrcia sylvatica oil (Msyl) did not show activity against breast cells, but displayed activity against melanoma (IC₅₀ 20.0 µg/mL) and gastric (IC₅₀ 17.3 µg/mL) cancer cells. Previous studies have reported the cytotoxic activity of M. splendens EO. A sample rich in E-nerolidol (67.8%), α-bisabolol (17.5%), and E-caryophyllene (4.2%) displayed cytotoxic potential against breast (MCF7, IC₅₀ 5.6 µg/mL) and normal human keratinocytes (HaCaT, IC₅₀ 21.6 µg/mL).¹³ However, there have been no reports about the cytotoxic activity of M. sylvatica EO.

Psidium guajava (Pgua) showed activity against melanoma (IC₅₀ 15.3 µg/mL), gastric (IC₅₀ 16.3 µg/mL), breast (IC₅₀ 12.4 µg/mL) human cancer cells with a SI of 1.7 against MCF7 cells. Likewise, P. guineense samples (Pgui-1 and Pgui-2) displayed activity against breast (IC₅₀ 12.6 and 18.2 µg/mL), melanoma (IC₅₀ 11.1 and 19.1 µg/mL), gastric (IC₅₀ 8.2 and 15.7 µg/mL), and a normal human fibroblast (IC₅₀ 8.27 and 24.0 µg/mL). Pgui-1 sample was more active than Pgui-2 against all cell lines. Pgui-1 and Pgui-2 sample have similar chemical compositions of their essential oils, except for the concentration of E-caryophyllene (Pgui-1 2.1%; Pgui-2 0.1%).

Literature data have already demonstrated the feasibility of applying P. guajava OE in the pharmacological sector due to its variability of organic compounds, as well as its efficiency in preliminary tests of anti-inflammatory activity, indicating possible viability in combating gastric ulcers.^26,34 In addition, significant cytotoxic activity was reported by the P. guineense essential oil against ovarian cancer cells (OVCAR-3) with GI50 of 0.9 µg/m.³⁵

Moreover, literature data indicate that E-caryophyllene, a compound detected in all samples of Myrtaceae essential oils in this study, has a potent anticancer activity, affecting the growth and proliferation of different types of cancer cells.³⁶ For example, E-caryophyllene showed cytotoxic active against MCF7 cells (LC₅₀ 190 µM).³⁷ In addition, this compound has shown synergistic enhancement with other essential oil components.³⁸ Thus, a synergistic effect between E-caryophyllene with various substances found in these essential oils likely contribute to the cytotoxic activities and may explain the different activities of Pgui-1 compared to Pgui-2.

Conclusions

The essential oil of Myrtaceae samples showed significant antioxidant activities in comparison to Trolox, and notable cytotoxic activity against the lung, colon, stomach, and melanoma cells, with a real prospect to their subsequent phytotherapeutic development. However, due to the presence of different chemical profiles requires their standardization to consider development of new anticancer drugs.

Materials and Methods

Plant Material

The leaves of seven Myrtaceae specimens were collected in Pará state (PA), Brazil, during the dry season (August-December). The collection site, voucher number, geographic coordinates for these specimens are listed in Table 4. After identification, plant vouchers were deposited in the Herbaria of Museu Paraense Emílio Goeldi (MG) and Embrapa Amazônia Oriental (IAN) at the city of Belém (PA), Brazil. The leaves were dried for 2 days at room temperature, ground and then submitted to essential oil extraction.

Table 4

Collection Site, Voucher Number, and Coordinates for the Myrtaceae Specimens.

Species	Code	Collection site	Voucher number	Coordinates latitude/longitude
Eugenia patrisii	Epat	Belém, PA, Brazil	MG-227357	1°16'0.15’'S / 48°28'18.65'’W
Eugenia stipitata	Esti	Belém, PA, Brazil	MG-228737	1°16'0.15'’S / 48°28'18.65'’W
Myrcia splendens	Mspl	Curuçá, PA, Brazil	NR^a	0°72'49'’S / 47°84'31'’W
Myrcia sylvatica	Msyl	Belém, PA, Brazil	MG-228738	1°15'52.65'’S / 48°28'12.85'’W
Psidium guajava	Pgua	Curuçá, PA, Brazil	MG-235601	0°43'48'’S / 47°51'17'’W
Psidium guineense	Pgui-1	Curuçá, PA, Brazil	IAN-195397	0°43'40'’S / 47°50'58'’W
Psidium guineense	Pgui-2	Curuçá, PA, Brazil	IAN-197141	0°72'67'’S / 47°85'13'’W

Abbreviation: NR, not registered.

^aThe botanical identification was made by comparison with authentic samples.

Extraction and Chemical Composition Analysis of the Essential Oil

The plant material was air-dried (2 days) at room temperature after collection. Then, it was ground and submitted to hydrodistillation using a Clevenger-type apparatus (3 hours).³⁹ The oils obtained were dried over anhydrous sodium sulfate and total oil yields were expressed as mL/100 g of the dried material.⁴⁰ The chemical composition analysis was performed by GC-MS, using a Shimadzu instrument Model QP 2010 ultra (Shimadzu, Tokyo, Japan), equipped with a Rtx-5MS (30 m × 0.25 mm; 0.25 µm film thickness) fused silica capillary column (Restek, Bellefonte, USA). Helium was used as carrier gas adjusted to 1.0 mL/min at 57.5 KPa; split injection (split ratio 1:20) of 1 µL of hexane solution (oil 5 µL : hexane 500 µL); injector and interface temperature were 250 °C; oven temperature programmed was 60 to 240 °C (3 °C/min), followed by an isotherm of 10 minutes. EIMS (Electron Impact Mass Spectrometry): electron energy, 70 eV; ion source temperature was 200 °C. The mass spectra were obtained by automatic scanning every 0.3 s, with mass fragments in the range of 35‐400 m/z. The compounds present in the samples were identified by comparison of their mass spectrum and retention index calculated for all volatile components using a linear equation of Van Den Dool and Kratz⁴¹ with data present in commercial libraries of FFNSC 2¹⁷ and Adams.¹⁶ Retention indices were calculated using n-alkane standard solutions (C8–C40, Sigma-Aldrich, St. Louis, MO, USA), in the same chromatographic conditions. The GC-FID analysis was carried out on a Shimadzu QP-2010 instrument, equipped with FID detector, in the same conditions, except that hydrogen was used as the carrier gas. The percentage composition of the oil samples was computed from the GC-FID peak areas.

DPPH Radical Scavenging Assay

A stock solution of DPPH radical (0.5 mM) in ethanol was prepared. After dilution in ethanol (60 µM approx.), the solution measured an initial absorbance of 0.62 ± 0.02 in 517 nm at room temperature. The reaction mixture was composed by 1000 µL of DPPH solution, 5 µL of the oil sample, 900 µL of Tris-HCl buffer (100 mM, pH 7.4), 45 µL of ethanol, and 50 µL of Tween 20 solution (0.5%, w/w). For each sample, an ethanol blank was also measured. The absorbance was measured at the beginning of the reaction (time zero), each 5 minutes during the first 20 minutes and then at continuous intervals of 30 minutes up to constant absorbance.

Standard curves were prepared using Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) (1.0–10.0 mM). The DPPH inhibition percentage calculated the radical scavenging activity of each sample according to the Equation, Inhibition = 100[(A-B)/A], where A and B are the blank and sample absorbance values in the end reaction. The total antioxidant activity was expressed as milligrams of Trolox, calculated utilizing the Equation, TE(mg/)g = [(A-B)/(A-C)]*[25/1000]*[250.29/1000]*[1000/10]*D, where A, B, and C are the blank, sample, and Trolox absorbance values in the end reaction, and D is the dilution factor. All experiments were triplicated.⁴²

Cell Viability Assay (MTT Assay)

The cell viability was determined by reduction of the yellow dye 3-(4,5-dimethyl-2-thiazol)−2,5-diphenyl-2H-tetrazolium bromide (MTT) to a blue formazan product as previously described by Mosmann (1983).⁴³ Oils (0.4, 25 µg/mL) were tested for cytotoxic activity against the cancer cell lines HCT116 (colon), AGP01 (malignant gastric ascites), MCF7 (breast), and SKMEL-19 (melanoma), as well as, against the normal non-malignant human lung fibroblast (MRC-5) cells. Cell lines were provided by Núcleo de Pesquisa e Desenvolvimento de Medicamentos (Drugs Research and Development Center) of Universidade Federal do Ceará. All cell lines were maintained in DMEM (Dulbecco’s Modified Eagle Medium) supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, at 37 °C and with 5% CO₂. The DMSO final concentration in the culture medium was kept constant (<0.1%). The oils were incubated with the cells for 72 hours, and the negative control received the same amount of DMSO (0.001% in the highest concentration). Doxorubicin was used as positive control.^44,45 SI (selectivity index) values were measured using the ratio between IC₅₀ of the essential oil against MRC-5 and IC₅₀ of the same oil essential against each cancer cell line.

Statistical Analyses

The antioxidant experiments were performed in triplicate and their data analyzed are expressed as mean ± SD. Statistical differences were evaluated by Tukey’s test (P < 0.05) using the software GraphPad Prism 6.0. The IC50’s values of MTT assay were calculated by nonlinear regression using GraphPad 6.0 software (Intuitive Software for Science, San Diego, CA).⁴²

Footnotes

Acknowledgments

We are grateful to CNPq and CAPES, institutions of support for scientific research of the Brazilian Government. WNS and JKRS participated in this work as part of the activities of the Aromatic Plant Research Center (APRC, ).

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID IDs

William N. Setzer

Pablo Luis B. Figueiredo

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