Chemical Composition and In Vitro Antimicrobial Activity of the Essential Oil Obtained from Eugenia pyriformis Cambess. (Myrtaceae)

Abstract

Our study aimed to evaluate the chemical composition of the essential oil from the leaves of Eugenia pyriformis Cambess., belonging to the Myrtaceae family and native to the Brazilian Atlantic forest. The volatile compounds in the essential oil were extracted by hydrodistillation and analyzed using GC-MS; 36 compounds accounted for 78.80% of the total oil content. The major compounds were β-caryophyllene, bicyclogermacrene, globulol, and (δ-cadinene. We evaluated their antimicrobial potential of the essential oil and toxicity to Artemia salina. The antimicrobial activity of the essential oil was evaluated against 12 microorganisms using the broth microdilution method. Our results showed moderate inhibition of Staphylococcus aureus and methicillin-resistant S. aureus (MIC, 250 and 125 μg.mL^-1, respectively) and toxicity to A. salina (LC50, 125.64 μg.mL^-1). Our results establish the biological activity of the essential oil obtained from E. pyriformis.

Keywords:
Eugenia pyriformis; chemical composition; essential oil; antimicrobial activity

GRAPHICAL ABSTRACT

HIGHLIGHTS

β-caryophyllene, bicyclogermacrene, globulol, and δ-cadinene were de major constituents.

Antimicrobial activity was tested with twelve microorganisms of interest.

Promissing antibacterial activity of essential oil against several gram-positive bacteria.

The essential oil showed marked toxicity to A. salina.

INTRODUCTION

The genus Eugenia are the fourth largest producers of essential oils among the plants of the Myrtaceae family [¹,2]. The Myrtaceae family are found in the Americas and Oceania and in the tropical and subtropical regions of the world [³]. Twenty three genera and 1000 species to the Myrtaceae family are found in Brazil [⁴].

Most plants of the genus Eugenia have been used ethnopharmacologically for the treatment of various diseases such as infectious diseases, intestinal infections, gastrointestinal disorders, for the treatment of wounds, or as repellents or insecticides against domestic and agricultural pests [⁵,⁶]. Previous studies have shown positive antibacterial and antifungal effects of the essential oils obtained from plants belonging to this genus. Eugenia axillaris exerts antibacterial and antifungal effects with a minimum inhibitory concentration (MIC) of 625 μg.mL^-1 against Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli and Candida albicans [⁷]. In addition, the essential oils obtained from E. brasiliensis, E. beaurepaireana, and E. umbelliflora were active against strains of S. aureus, P. aeruginosa, and E. coli with MICs ranging from 156.2 to 624.9 μg.mL^-1 [⁸]. These effects have been attributed to the presence of active constituents, particularly the cyclic sesquiterpenes and monoterpenes in smaller amounts. Typically, the monoterpene α-pinene and the sesquiterpene β-caryophyllene are present in high amounts in plants of this genus [⁹].

This study focuses on the E. pyriformis Cambess., which is known as uvaia, uvaieira, uvalha, uvalheira, or uvalha-do-campo; it is a fruit tree native to Brazil and is found in the states of São Paulo, Paraná, Santa Catarina, and Rio Grande do Sul [¹⁰]. The essential oil obtained from this tree has not been extensively investigated; some studies have reported the antimicrobial activity of the extracts and fractions obtained from the stem, leaves, fruits, and seeds of this tree against various strains of bacteria and fungi. Souza et al. [⁴] showed that the ketone extract obtained from the stem and leaves of E. pyriformis had a marked antimicrobial effect against Enterococcus faecalis and S. aureus (MIC = 62.5-125 μg.mL^-1) and an antifungal effect against the leveduriformes species of fungi C. albicans, C. parapsilosis, and C. krusei (MIC = 7.81 μg.mL^-1). Our study aims to evaluate the composition of the essential oil obtained from E. pyriformis Cambess. and investigate its antimicrobial activity and preliminary toxicity and thus provide information about the therapeutic effects of this species.

MATERIAL AND METHODS

Plant material

Aerial parts of E. pyriformis Cambess. were collected from Curitiba, Paraná State (25°26'51.83" S, 49°20'47.92" W) in October of 2014. The plant identification was performed by José Tadeu Weidlich Motta, at the Botanical Garden of Curitiba (MBM) Herbariums and voucher was deposited under the number MBM 320419. The research was authorized by the National Council for Scientific and Technological Development for access to Genetic Heritage under the number 02001.001165/2013-47.

Extraction of the essential oil

Hydrodistillation using water vapor drag was performed using a modified Clevenger apparatus [¹¹] to extract the essential oil from the leaves dry of E. pyriformis. The extracted oils were stored in tightly closed dark bottles under refrigeration at 4 °C until analyzed and tested.

Gas chromatography-mass spectrometry analysis

Gas chromatography mass spectrometry (GC-MS) (GC-MS-QP 2010 Plus, Shimadzu) was performed using a capillary column RTX - 5MS (30 m × 0.25 mm × 0.25 µm). The samples were diluted at 1% (v/v) in methylene chloride. Injector in splitless mode at 250 °C, ion source and interface at 300 °C. The mass window was analyzed from m/z 40 and m/z 350 using helium as the carrier gas at a flow rate of 1.02 mL/min, in 1:90 split mode, injection volume = 0.1 µL (split ratio of 1:10). Ramp injection for analysis injector temperature 250 °C, pressure of 20 psi column, starting at 50 °C for 5 min increasing to 200 °C at a rate of 5 °C/min.

Identification and quantification of essential oil constituents

The relative amount of individual components in the total oil is expressed as a percentage peak area relative to total peak area. The Kovats indices (KI) were calculated by comparing the retention times of the eluting peaks with those of C₅-C₂₈ n-alkanes, injected under the same conditions as the samples. Identification of the oil components was performed by comparing their KI and mass spectra with those reported in the NIST library and in the literature [¹²].

Antibacterial activity

The antibacterial activity tests were performed using the following strains: Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 25923, MRSA ATCC 33591, Staphylococcus epidermidis ATCC 12228, Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 700603, Pseudomonas aeruginosa ATCC 27853, Salmonella typhimurium ATCC 14028, and Enterobacter aerogenes ATCC 13048.

The MIC was determined using the broth microdilution technique [¹³]. Bacterial suspensions were prepared at a concentration of 1.0 × 10⁸ CFU.mL^-1 with saline corresponding to 0.5 tube of the McFarland scale, and 5 μL of the suspension was inoculated into each well at a final concentration of 10⁴ CFU.mL^-1. The negative control of inhibitory activity of the diluent was prepared by adding 100 μL of 5% Polysorbate 80 in 100 μL of Mueller-Hinton broth (MHB) and 5 μL of bacterial inoculum. For controlling the sterility, 100 μL of MHB and 100 μL of the samples were used. Bacterial viability or positive controls were prepared with 100 μL of MHB and 5 μL of bacterial inoculum.

The microplates were incubated at 35 °C for 16 to 20 h. After this interval, 20 μL of triphenyltetrazolium chloride aqueous solution (TTC; Merck, Darmstadt, Germany) were added to the microplates and then reincubated for 3 h at 35 °C. Development of red color was interpreted as the absence of antimicrobial activity.

The results were classified as good inhibitory activity (above 100 μg.mL^-1), moderate inhibitory activity (between 100 and 500 μg.mL^-1), weak inhibitory activity (between 500 and 1000 μg.mL^-1), and absence of inhibitory activity (above 1000 μg.mL^-1) [¹⁴].

Antifungal activity

The antifungal activity was evaluated using Candida albicans ATCC 14053, Candida krusei ATCC 6258, and Candida parapsilosis ATCC 22019. Serial dilutions of the sample were prepared in a concentration range of 7.81-1000 μg.mL^-1 in RPMI 1640 liquid medium (Gibco/Invitrogen, New York, USA) [¹⁵] The different fungal suspensions were prepared in physiological saline at an initial concentration of 1.0 × 10⁸ CFU.mL^-1. These were diluted in a liquid medium to a final concentration of 1.0 to 5.0 × 10³ CFU.mL^-1 and then inoculated with 100 μL into wells. The microplates were incubated for 48 h at 35 °C. Then, 20 μL of 0.5% TTC was added and the plates were reincubated for 3 h at 35 °C. The results were analyzed according to the same method as that used for determination of the antibacterial activity.

Brine shrimp lethality assay

This toxicity assay, which uses the larvae of the brine shrimp A. salina Leach., was first described by Meyer et al. [¹⁶] The cysts were placed to hatch in a saline solution for 48 h, producing a larger number of larvae. The essential oil diluted in 0.5% of Polysorbate 80 and saline solution was examined at concentrations of 10, 100, and 1000 μg.mL^-1, followed by a positive control prepared with saline solution and sodium dodecylsulfate (SDS), and negative control with saline solution and Polysorbate 80. The assay was performed in triplicate. The tubes were incubated in the oven (27-30 °C) for 24 h and subsequently the nauplii were counted. The data were statistically analyzed using the Probitos method, which provided LC50 with 95% reliability. The degree of toxicity was classified as low toxicity, LC50 > 500 μg.mL^-1; moderate toxicity, LC50 between 100 μg.mL^-1 and 500 μg.mL^-1; and high toxicity, LC50 < 100 μg.mL^-1 [¹⁷].

RESULTS AND DISCUSSION

Chemical composition of the essential oil

The yield of the oil essential from leaves of E. pyriformis Cambess. obtained by hydrodistillation was 0.14%. Analysis of the essential oil indicated the presence of 36 constituents, which accounted for 78.80% of the total oil composition. The constituents identified, their retention indices, and their relative amounts are summarized in Table 1.

Table 1
Composition of the essential oils from leaves of E. pyriformis Cambess.

No.a	tR [min.] b	Compound name	RI c	RIt d	(%) e	Id. f
1	5.27	Hexadienol	908	916	0,71	RI, MS
2	5.82	Isocitronellene	927	923	0.24	RI, MS
3	6.37	Heptenol	947	954	1.07	RI, MS
4	6.73	Cumene	960	924	0.37	RI, MS
5	7.20	β-Pinene	976	974	0.16	RI, MS
6	7.72	Mesitylene	995	994	0.60	RI, MS
7	7.76	n-Decane	996	1000	0.91	RI, MS
8	11.45	n-Undecane	1097	1100	0.28	RI, MS
9	14.94	Terpinen-4-ol	1178	1174	0.34	RI, MS
10	15.54	α-Terpineol	1193	1186	0.35	RI, MS
11	21.92	(-Elemene	1337	1335	0.39	RI, MS
12	23.61	α-copaene	1376	1374	3.83	RI, MS
13	24.02	β-Bourbonene	1385	1387	0.35	RI, MS
14	24.32	β-Elemene	1392	1389	1.97	RI, MS
15	25.11	α-Gurjunene	1411	1409	1.29	RI, MS
16	25.54	β-caryophyllene	1421	1417	17.82	RI, MS
17	25.93	β-Copaene	1430	1430	0.68	RI, MS
18	26.36	Aromandrene	1440	1439	2.21	RI, MS
19	26.54	α-Guaiene	1445	1437	0.37	RI, MS
20	26.99	α-Humulene	1455	1452	2.69	RI, MS
21	27.30	9-epi-(E)-Caryophyllene	1463	1464	2.17	RI, MS
22	27.94	(-Muurolene	1478	1478	0.70	RI, MS
23	28.14	Germacrene-D	1483	1484	2.79	RI, MS
24	28.37	β-Selinene	1488	1489	0.25	RI, MS
25	28.82	Bicyclogermacrene	1499	1500	12.84	RI, MS
26	28.95	α-Muurolene	1502	1500	1.06	RI, MS
27	29.52	(-Cadinene	1516	1513	0.54	RI, MS
28	29.97	(-Cadinene	1525	1522	4.33	RI, MS
29	31.43	Maaliol	1564	1567	0.35	RI, MS
30	32.18	Esphatulenol	1582	1577	3.24	RI, MS
31	32.43	Globulol	1588	1590	5.96	RI, MS
32	32.75	Viridiflorol	1596	1592	3.52	RI, MS
33	33.15	Rosifoliol	1606	1600	1.18	RI, MS
34	33.96	5-epi-7-epi-α-Eudesmol	1627	1607	0.91	RI, MS
35	34.70	Tau-muurolol	1647	1640	1.03	RI, MS
36	34.93	α-Cadinol	1660	1652	1.30	RI, MS
		Total identification [%]			78.80
		Aromatic hydrocarbons [%]			0.97
		Alcohols [%]			0.71
		Aldehydes [%]			1.07
		Hydrocarbon alkane [%]			0.28
		Monoterpene hydrocarbons [%]			0.40
		Monoterpene oxygenated [%]			0.69
		Sesquiterpene hydrocarbons [%]			57.19
		Oxigenated sesquiterpenes [%]			17.49

• ^a
  Order of elution is given on apolar column (Rtx-5MS). Bold types refer to main compounds. ^b Time retention on the apolar Rtx-5MS column. ^c Retention indices on the polar Rtx-5MS column. ^d Kovats retention index relative to n-alkanes (C8 - C30). ^e Percentages of compounds ^f RI: Retention Indices; MS: Mass Spectrometry in electronic impact mode.

The essential oil of leaves of E. pyriformis is mainly composed of sesquiterpene hydrocarbons (57.19%) and oxygenated sesquiterpenes (17.49%), with smaller amounts of aldehydes (1.07%), aromatic hydrocarbon (0.97%), alcohols (0.71%), monoterpene oxygenated (0.69%), monoterpene hydrocarbons (0.40%) and hydrocarbon alkane (0.28%). The major constituents were found to β-Caryophyllene (17,82%), Bicyclogermacrene (12,84%), Globulol (5,96%) and δ-Cadinene (4,33%). Other representative components of the oil were identified as α-Copaene (3.83%), Esphatulenol (3.24%), Germacrene-D (2.79%), α-Humulene (2.69%), Aromandrene (2.21%) and 9-epi-(E)-Caryophyllene (2.17%) (Figure 1).

Figure 1
Chemical structures of the major compounds of essential oils from leaves of Eugenia pyriformis Cambess. β-Caryophyllene (1), Bicyclogermacrene (2), Globulol (3), δ-Cadinene (4), α-Copaene (5), Esphatulenol (6), Germacrene-D (7), α-Humulene (8), Aromandrene (9) and 9-epi-(E)-Caryophyllene (10).

The essential oil obtained from the plants of Eugenia species have high levels of cyclic sesquiterpenes and low levels of monoterpenes. A few species produce aliphatic and aromatic compounds. The sesquiterpene β-caryophyllene and the monoterpene α-pinene are the most abundant compounds in the essential oil plants of the Eugenia species [⁹].

Previous studies have shown that β-caryophyllene and bicyclogermacrene are present in the essential oil obtained from species other than E. pyriformis. β-Caryophyllene is a major component of essential oil of E. klotzschiana [¹⁸], E. moraviana [¹⁹], E. argentea [⁵] and E. involucrata [²⁰]; it is abundantly present in the essential oil of E. stipitata [²¹] and E. umbeliflora [¹⁹].

β-Caryophyllene and bicyclogermacrene are present in moderate amounts in the essential oil obtained from E. burkartiana [¹⁹], E. ramboi [¹⁹], E. repanda [²²], E. cuprea, E. pitanga [²³], E. beaurepaireana [²³], E. hyemalis [²³], E. mattosii [²⁴] and E. klotzschiana [¹⁸,²⁵].

Sesquiterpene compounds are widely found in the essential oils of medicinal plants and fruits and are used for the treatment of various conditions [²⁶]. β-Caryophyllene has several properties such as anti-edema, phagorepullic, anti-inflammatory, antitumor, insecticidal, spasmolytic, bactericidal properties [¹⁸], and bicyclogermacrene has antifungal activity [²⁷].

Antimicrobial activities

The MIC of the essential oil of E. pyriformis obtained using the antimicrobial activity test are shown in Table 2.

Table 2
Antimicrobial activity of leaves essential oil of E. pyriformis Cambess.

Microorganisms	MIC (µg.mL-1)
Gram-positive
S. aureus ATCC 25923	250
MRSA ATCC 33591	125
S. epidermidis ATCC 12228	1000
E. faecalis ATCC 29212	>1000
Gram-negative
E. coli ATCC 25922	>1000
K. pneumoniae ATCC 700603	>1000
P. aeruginosa ATCC27853	>1000
E. aerogenes ATCC 13048	>1000
S. typhimurium ATCC 14028	1000
Leveduriform fungi
C. albicans ATCC 14053	>1000
C. krusei ATCC 6258	>1000
C. parapsilosis ATCC 22019	>1000

• MIC, minimum inhibitory concentration (given as µg.mL^-1).

To validate the technique and methodological control, vancomycin and fluconazole were used, and the confidence interval was stipulated using the Clinical and Laboratory Standards Institute (CLSI) method [¹³].

The essential oil of E. pyriformis showed moderate antibacterial potential against the gram-positive bacteria S. aureus and methicillin-resistant S. aureus (MRSA) with MIC values of 250 and 125 μg.mL^-1, respectively. A previous study using the essential oil obtained from E. umbelliflora and E. brasiliensis showed an MIC of 119 and 156 μg.mL^-1, respectively, against S. aureus [²⁴]. The essential oil from E. axillaris had an MIC of 625 µg.mL^-1 for S. aureus. In addition, we evaluated the antimicrobial activities of 4-terpineol, α-terpineol, β-caryophyllene, α-humulene, and germacrene-D; the MIC of these compounds was 39-1250 µg.mL^-1 [⁷].

S. aureus is an important etiological agent associated with acquired infections, both in the community and in hospitals. S. aureus is the most frequently isolated bacterial pathogen associated with several serious clinical infections, including endovascular and soft tissue infections, pneumonia, and sepsis, and has a high prevalence and a high degree of pathogenicity [²⁸,²⁹]. MRSA is a well-known nosocomial pathogen, often associated with health care and acquired in the community. MRSA is resistant to penicillin and other available β-lactam antibiotics, limiting potential treatment options with standard antibiotic therapy and increasing the risk of more unfavorable clinical outcomes for patients [²⁹].

S. epidermidis and Salmonella typhimurium showed MICs of 1000 μg.mL^-1, which indicated a weak inhibitory potential according to the established scale. The MICs of other microorganisms were above 1000 μg.mL^-1. S. epidermidis is a human pathogen that colonizes body surfaces, is prevalent in moist areas such as the armpits, inguinal and perineal area, nostrils, and conjunctivae [³⁰]. S. typhimurium is a food-borne pathogen that causes salmonellosis, a gastrointestinal disease of public health importance, which affects humans and animals [³¹].

Resistance to antibiotics available in the market is increasing at alarming levels, whereas the development of new antimicrobial agents is taking place at a slow rate. The development of resistance and the emergence of new bacterial pathogens warrants the development of new drugs, and thus new discovery, surveillance, and control programs must be implemented on a priority basis. Medicinal plants and fungi have been widely used in the past to combat human and animal pathogens and may be the key to the production of new antibiotics [³²].

Brine shrimp lethality bioassay

The brine shrimp bioassay is used to evaluate the acute toxicity and is a preliminary assay in the study of compounds with potential biological activities [³³]. This assay is an economical method for investigating the activities of compounds obtained from plants, particularly in countries where plant-based medicines are commonly used [³⁴]. The results obtained from this bioassay correlate with those of cytotoxicity to 9Kb and 9PS cells (leukemia); thus, the brine shrimp lethality bioassay may be used for the preliminary determination of antitumor activity [¹⁶,³⁵].

The essential oil from E. pyriformis was toxic with a lethal concentration required to kill 50% of the population (LC50) of 125.64 (μg.mL^-1). Previous studies have reported the toxic effects of essential oils from species of plants to Artemia salina. Studies using the essential oil from E. uniflora showed an LC50 of 253.43 μg.mL^-1 [³⁶]. E. uniflora is used in folk medicine as a diuretic, antirheumatic, antipyretic, and anti-inflammatory agents and for the diseases of the stomach [³⁷].

The in vitro cytotoxic effects of the essential oil obtained from the leaves of E. zuchowskiae was examined using the MCF-7 (mammary adenocarcinoma), MDA-MB-468 (mammary adenocarcinoma), and UACC-257 (malignant melanoma) human tumor cell lines; the leaf oil was cytotoxic with 100% kill at a concentration of 100 µg.mL^-1 on the cell lines tested. The cytotoxic effects of the major components α-pinene, α-copaene, β-caryophyllene, and α-humulene of the essential oil on MCF-7 cells were similar to those of the anticancer agent doxorubicin [³⁸].

CONCLUSION

Our results indicate that the essential oil of E. pyriformis Cambess. predominantly consists of hydrocarbon sesquiterpenes, and β-caryophyllene, bicyclogermacrene, globulol, and δ-cadinene are present in high levels. The essential oil obtained from E. pyriformis shows marked toxicity to A. salina and antibacterial activity against several gram-positive bacteria. The essential oil was effective against MRSA. Further in vitro cytotoxicity studies should be performed to identify new drugs with an anticancer potential. The promising results observed using this oil provide insights for developing an alternative to commercially available drugs for treating infections caused by drug-resistant bacteria. Further studies using this species should be performed to identify new molecules for the treatment of diseases associated with microbial pathogens and malignancies.

Acknowledgements

The authors are grateful to the Postgraduate Program in Pharmaceutical Sciences, Department of Chemistry of the Federal University of Paraná, Brazil for the assistance in GC-MS analysis.

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Edited by

Editor-in-Chief:

Paulo Vitor Farago

Associate Editor:

Paulo Vitor Farago

Publication Dates

• Publication in this collection
  05 July 2021
• Date of issue
  2021

History

• Received
  15 Oct 2020
• Accepted
  24 Feb 2021